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Sample records for affects lysosomal distribution

  1. Depletion of kinesin 5B affects lysosomal distribution and stability and induces peri-nuclear accumulation of autophagosomes in cancer cells

    DEFF Research Database (Denmark)

    Cardoso, Carla M P; Groth-Pedersen, Line; Høyer-Hansen, Maria

    2009-01-01

    cells. In KIF5B-depleted cells the autophagosomes formed and accumulated in the close proximity to the Golgi apparatus, whereas in the control cells they appeared uniformly distributed in the cytoplasm. CONCLUSIONS/SIGNIFICANCE: Our data identify KIF5B as a cancer relevant lysosomal motor protein...

  2. AP-3 and Rabip4' coordinately regulate spatial distribution of lysosomes.

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    Viorica Ivan

    Full Text Available The RUN and FYVE domain proteins rabip4 and rabip4' are encoded by RUFY1 and differ in a 108 amino acid N-terminal extension in rabip4'. Their identical C terminus binds rab5 and rab4, but the function of rabip4s is incompletely understood. We here found that silencing RUFY1 gene products promoted outgrowth of plasma membrane protrusions, and polarized distribution and clustering of lysosomes at their tips. An interactor screen for proteins that function together with rabip4' yielded the adaptor protein complex AP-3, of which the hinge region in the β3 subunit bound directly to the FYVE domain of rabip4'. Rabip4' colocalized with AP-3 on a tubular subdomain of early endosomes and the extent of colocalization was increased by a dominant negative rab4 mutant. Knock-down of AP-3 had an ever more dramatic effect and caused accumulation of lysosomes in protrusions at the plasma membrane. The most peripheral lysosomes were localized beyond microtubules, within the cortical actin network. Our results uncover a novel function for AP-3 and rabip4' in regulating lysosome positioning through an interorganellar pathway.

  3. Streptozotocin-induced diabetes mellitus affects lysosomal enzymes in rat liver

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    Peres, G.B. [Universidade Federal de São Paulo, Escola Paulista de Medicina, Departamento de Bioquímica, São Paulo, SP, Brasil, Departamento de Bioquímica, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, SP (Brazil); Juliano, M.A. [Universidade Federal de São Paulo, Escola Paulista de Medicina, Departamento de Biofísica, São Paulo, SP, Brasil, Departamento de Biofísica, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, SP (Brazil); Aguiar, J.A.K.; Michelacci, Y.M. [Universidade Federal de São Paulo, Escola Paulista de Medicina, Departamento de Bioquímica, São Paulo, SP, Brasil, Departamento de Bioquímica, Escola Paulista de Medicina, Universidade Federal de São Paulo, São Paulo, SP (Brazil)

    2014-05-09

    It has been previously shown that dextran sulfate administered to diabetic rats accumulates in the liver and kidney, and this could be due to a malfunction of the lysosomal digestive pathway. The aim of the present study was to evaluate the expression and activities of lysosomal enzymes that act upon proteins and sulfated polysaccharides in the livers of diabetic rats. Diabetes mellitus was induced by streptozotocin in 26 male Wistar rats (12 weeks old), while 26 age-matched controls received only vehicle. The livers were removed on either the 10{sup th} or the 30{sup th} day of the disease, weighed, and used to evaluate the activity, expression, and localization of lysosomal enzymes. A 50-60% decrease in the specific activities of cysteine proteases, especially cathepsin B, was observed in streptozotocin-induced diabetes mellitus. Expression (mRNA) of cathepsins B and L was also decreased on the 10{sup th}, but not on the 30{sup th} day. Sulfatase decreased 30% on the 30{sup th} day, while glycosidases did not vary (or presented a transitory and slight decrease). There were no apparent changes in liver morphology, and immunohistochemistry revealed the presence of cathepsin B in hepatocyte granules. The decrease in sulfatase could be responsible for the dextran sulfate build-up in the diabetic liver, since the action of sulfatase precedes glycosidases in the digestive pathway of sulfated polysaccharides. Our findings suggest that the decreased activities of cathepsins resulted from decreased expression of their genes, and not from general lysosomal failure, because the levels of glycosidases were normal in the diabetic liver.

  4. Streptozotocin-induced diabetes mellitus affects lysosomal enzymes in rat liver

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    G.B. Peres

    2014-06-01

    Full Text Available It has been previously shown that dextran sulfate administered to diabetic rats accumulates in the liver and kidney, and this could be due to a malfunction of the lysosomal digestive pathway. The aim of the present study was to evaluate the expression and activities of lysosomal enzymes that act upon proteins and sulfated polysaccharides in the livers of diabetic rats. Diabetes mellitus was induced by streptozotocin in 26 male Wistar rats (12 weeks old, while 26 age-matched controls received only vehicle. The livers were removed on either the 10th or the 30th day of the disease, weighed, and used to evaluate the activity, expression, and localization of lysosomal enzymes. A 50-60% decrease in the specific activities of cysteine proteases, especially cathepsin B, was observed in streptozotocin-induced diabetes mellitus. Expression (mRNA of cathepsins B and L was also decreased on the 10th, but not on the 30th day. Sulfatase decreased 30% on the 30th day, while glycosidases did not vary (or presented a transitory and slight decrease. There were no apparent changes in liver morphology, and immunohistochemistry revealed the presence of cathepsin B in hepatocyte granules. The decrease in sulfatase could be responsible for the dextran sulfate build-up in the diabetic liver, since the action of sulfatase precedes glycosidases in the digestive pathway of sulfated polysaccharides. Our findings suggest that the decreased activities of cathepsins resulted from decreased expression of their genes, and not from general lysosomal failure, because the levels of glycosidases were normal in the diabetic liver.

  5. Effect of Readthrough Treatment in Fibroblasts of Patients Affected by Lysosomal Diseases Caused by Premature Termination Codons.

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    Matalonga, Leslie; Arias, Ángela; Tort, Frederic; Ferrer-Cortés, Xènia; Garcia-Villoria, Judit; Coll, Maria Josep; Gort, Laura; Ribes, Antonia

    2015-10-01

    Aminoglycoside antibiotics, such as gentamicin, may induce premature termination codon (PTC) readthrough and elude the nonsense-mediated mRNA decay mechanism. Because PTCs are frequently involved in lysosomal diseases, readthrough compounds may be useful as potential therapeutic agents. The aim of our study was to identify patients responsive to gentamicin treatment in order to be used as positive controls to further screen for other PTC readthrough compounds. With this aim, fibroblasts from 11 patients affected by 6 different lysosomal diseases carrying PTCs were treated with gentamicin. Treatment response was evaluated by measuring enzymatic activity, abnormal metabolite accumulation, mRNA expression, protein localization, and cell viability. The potential effect of readthrough was also analyzed by in silico predictions. Results showed that fibroblasts from 5/11 patients exhibited an up to 3-fold increase of enzymatic activity after gentamicin treatment. Accordingly, cell lines tested showed enhanced well-localized protein and/or increased mRNA expression levels and/or reduced metabolite accumulation. Interestingly, these cell lines also showed increased enzymatic activity after PTC124 treatment, which is a PTC readthrough-promoting compound. In conclusion, our results provide a proof-of-concept that PTCs can be effectively suppressed by readthrough drugs, with different efficiencies depending on the genetic context. The screening of new compounds with readthrough activity is a strategy that can be used to develop efficient therapies for diseases caused by PTC mutations.

  6. Endo-lysosomal dysfunction in human proximal tubular epithelial cells deficient for lysosomal cystine transporter cystinosin.

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    Ekaterina A Ivanova

    Full Text Available Nephropathic cystinosis is a lysosomal storage disorder caused by mutations in the CTNS gene encoding cystine transporter cystinosin that results in accumulation of amino acid cystine in the lysosomes throughout the body and especially affects kidneys. Early manifestations of the disease include renal Fanconi syndrome, a generalized proximal tubular dysfunction. Current therapy of cystinosis is based on cystine-lowering drug cysteamine that postpones the disease progression but offers no cure for the Fanconi syndrome. We studied the mechanisms of impaired reabsorption in human proximal tubular epithelial cells (PTEC deficient for cystinosin and investigated the endo-lysosomal compartments of cystinosin-deficient PTEC by means of light and electron microscopy. We demonstrate that cystinosin-deficient cells had abnormal shape and distribution of the endo-lysosomal compartments and impaired endocytosis, with decreased surface expression of multiligand receptors and delayed lysosomal cargo processing. Treatment with cysteamine improved surface expression and lysosomal cargo processing but did not lead to a complete restoration and had no effect on the abnormal morphology of endo-lysosomal compartments. The obtained results improve our understanding of the mechanism of proximal tubular dysfunction in cystinosis and indicate that impaired protein reabsorption can, at least partially, be explained by abnormal trafficking of endosomal vesicles.

  7. Loss of β-glucocerebrosidase activity does not affect alpha-synuclein levels or lysosomal function in neuronal cells.

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    Dermentzaki, Georgia; Dimitriou, Evangelia; Xilouri, Maria; Michelakakis, Helen; Stefanis, Leonidas

    2013-01-01

    To date, a plethora of studies have provided evidence favoring an association between Gaucher disease (GD) and Parkinson's disease (PD). GD, the most common lysosomal storage disorder, results from the diminished activity of the lysosomal enzyme β-glucocerebrosidase (GCase), caused by mutations in the β-glucocerebrosidase gene (GBA). Alpha-synuclein (ASYN), a presynaptic protein, has been strongly implicated in PD pathogenesis. ASYN may in part be degraded by the lysosomes and may itself aberrantly impact lysosomal function. Therefore, a putative link between deficient GCase and ASYN, involving lysosomal dysfunction, has been proposed to be responsible for the risk for PD conferred by GBA mutations. In this current work, we aimed to investigate the effects of pharmacological inhibition of GCase on ASYN accumulation/aggregation, as well as on lysosomal function, in differentiated SH-SY5Y cells and in primary neuronal cultures. Following profound inhibition of the enzyme activity, we did not find significant alterations in ASYN levels, or any changes in the clearance or formation of its oligomeric species. We further observed no significant impairment of the lysosomal degradation machinery. These findings suggest that additional interaction pathways together with aberrant GCase and ASYN must govern this complex relation between GD and PD.

  8. Radiation-induced alterations in the distribution of lysosomal hydrolases in rat spleen homogenates. [Gamma radiation

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    Snyder, S.L.; Eklund, S.K.

    1978-07-01

    Whole-body exposure of rats to /sup 60/Co-..gamma.. radiation results in increases in the activities of two lysosomal hydrolases, ..beta..-glucuronidase and ..cap alpha..-fucosidase, found in the supernatant fraction of spleen homogenates. The redistribution of these enzymes from the ''particulate-bound'' to the ''free-supernatant'' fraction of spleen homogenates has been studied as a function of radiation dose. The response curves for the ratio of free/bound enzyme versus dose sigmoidal with maximum occurring at 300 to 400 rad.

  9. Cancer-associated lysosomal changes

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    Kallunki, T; Olsen, O D; Jaattela, Marja

    2013-01-01

    Rapidly dividing and invasive cancer cells are strongly dependent on effective lysosomal function. Accordingly, transformation and cancer progression are characterized by dramatic changes in lysosomal volume, composition and cellular distribution. Depending on one's point of view, the cancer-associated......-targeting anti-cancer drugs. In this review we compile our current knowledge on cancer-associated changes in lysosomal composition and discuss the consequences of these alterations to cancer progression and the possibilities they can bring to cancer therapy.Oncogene advance online publication, 9 July 2012; doi...

  10. Brief exposure to copper activates lysosomal exocytosis.

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    Peña, Karina; Coblenz, Jessica; Kiselyov, Kirill

    2015-04-01

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

  11. Pervasive supply of therapeutic lysosomal enzymes in the CNS of normal and Krabbe-affected non-human primates by intracerebral lentiviral gene therapy.

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    Meneghini, Vasco; Lattanzi, Annalisa; Tiradani, Luigi; Bravo, Gabriele; Morena, Francesco; Sanvito, Francesca; Calabria, Andrea; Bringas, John; Fisher-Perkins, Jeanne M; Dufour, Jason P; Baker, Kate C; Doglioni, Claudio; Montini, Eugenio; Bunnell, Bruce A; Bankiewicz, Krystof; Martino, Sabata; Naldini, Luigi; Gritti, Angela

    2016-05-02

    Metachromatic leukodystrophy (MLD) and globoid cell leukodystrophy (GLD or Krabbe disease) are severe neurodegenerative lysosomal storage diseases (LSD) caused by arylsulfatase A (ARSA) and galactosylceramidase (GALC) deficiency, respectively. Our previous studies established lentiviral gene therapy (GT) as a rapid and effective intervention to provide pervasive supply of therapeutic lysosomal enzymes in CNS tissues of MLD and GLD mice. Here, we investigated whether this strategy is similarly effective in juvenile non-human primates (NHP). To provide proof of principle for tolerability and biological efficacy of the strategy, we established a comprehensive study in normal NHP delivering a clinically relevant lentiviral vector encoding for the human ARSA transgene. Then, we injected a lentiviral vector coding for the human GALC transgene in Krabbe-affected rhesus macaques, evaluating for the first time the therapeutic potential of lentiviral GT in this unique LSD model. We showed favorable safety profile and consistent pattern of LV transduction and enzyme biodistribution in the two models, supporting the robustness of the proposed GT platform. We documented moderate inflammation at the injection sites, mild immune response to vector particles in few treated animals, no indication of immune response against transgenic products, and no molecular evidence of insertional genotoxicity. Efficient gene transfer in neurons, astrocytes, and oligodendrocytes close to the injection sites resulted in robust production and extensive spreading of transgenic enzymes in the whole CNS and in CSF, leading to supraphysiological ARSA activity in normal NHP and close to physiological GALC activity in the Krabbe NHP, in which biological efficacy was associated with preliminary indication of therapeutic benefit. These results support the rationale for the clinical translation of intracerebral lentiviral GT to address CNS pathology in MLD, GLD, and other neurodegenerative LSD.

  12. Lysosomal stress: a new player in perturbed lipid metabolism

    NARCIS (Netherlands)

    Gabriel, T.L.

    2017-01-01

    Lysosomes are involved in many different essential cellular processes, among others organelle and molecule degradation, exocytosis, cell energy metabolism, cholesterol and sphingolipid level regulation. Lysosomal stress has a strong impact on the immune system, affecting specially macrophages as the

  13. First-Generation Antipsychotic Haloperidol Alters the Functionality of the Late Endosomal/Lysosomal Compartment in Vitro.

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    Canfrán-Duque, Alberto; Barrio, Luis C; Lerma, Milagros; de la Peña, Gema; Serna, Jorge; Pastor, Oscar; Lasunción, Miguel A; Busto, Rebeca

    2016-03-18

    First- and second-generation antipsychotics (FGAs and SGAs, respectively), have the ability to inhibit cholesterol biosynthesis and also to interrupt the intracellular cholesterol trafficking, interfering with low-density lipoprotein (LDL)-derived cholesterol egress from late endosomes/lysosomes. In the present work, we examined the effects of FGA haloperidol on the functionality of late endosomes/lysosomes in vitro. In HepG2 hepatocarcinoma cells incubated in the presence of 1,1'-dioctadecyl-3,3,3,3'-tetramethylindocarbocyanineperchlorate (DiI)-LDL, treatment with haloperidol caused the enlargement of organelles positive for late endosome markers lysosome-associated membrane protein 2 (LAMP-2) and LBPA (lysobisphosphatidic acid), which also showed increased content of both free-cholesterol and DiI derived from LDL. This indicates the accumulation of LDL-lipids in the late endosomal/lysosomal compartment caused by haloperidol. In contrast, LDL traffic through early endosomes and the Golgi apparatus appeared to be unaffected by the antipsychotic as the distribution of both early endosome antigen 1 (EEA1) and coatomer subunit β (β-COP) were not perturbed. Notably, treatment with haloperidol significantly increased the lysosomal pH and decreased the activities of lysosomal protease and β-d-galactosidase in a dose-dependent manner. We conclude that the alkalinization of the lysosomes' internal milieu induced by haloperidol affects lysosomal functionality.

  14. TRPML: transporters of metals in lysosomes essential for cell survival?

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    Kiselyov, Kirill; Colletti, Grace A; Terwilliger, Austen; Ketchum, Kathleen; Lyons, Christopher W P; Quinn, James; Muallem, Shmuel

    2011-09-01

    Key aspects of lysosomal function are affected by the ionic content of the lysosomal lumen and, therefore, by the ion permeability in the lysosomal membrane. Such functions include regulation of lysosomal acidification, a critical process in delivery and activation of the lysosomal enzymes, release of metals from lysosomes into the cytoplasm and the Ca(2+)-dependent component of membrane fusion events in the endocytic pathway. While the basic mechanisms of lysosomal acidification have been largely defined, the lysosomal metal transport system is not well understood. TRPML1 is a lysosomal ion channel whose malfunction is implicated in the lysosomal storage disease Mucolipidosis Type IV. Recent evidence suggests that TRPML1 is involved in Fe(2+), Ca(2+) and Zn(2+) transport across the lysosomal membrane, ascribing novel physiological roles to this ion channel, and perhaps to its relatives TRPML2 and TRPML3 and illuminating poorly understood aspects of lysosomal function. Further, alterations in metal transport by the TRPMLs due to mutations or environmental factors may contribute to their role in the disease phenotype and cell death.

  15. The proteome of lysosomes.

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    Schröder, Bernd A; Wrocklage, Christian; Hasilik, Andrej; Saftig, Paul

    2010-11-01

    Lysosomes are organelles of eukaryotic cells that are critically involved in the degradation of macromolecules mainly delivered by endocytosis and autophagocytosis. Degradation is achieved by more than 60 hydrolases sequestered by a single phospholipid bilayer. The lysosomal membrane facilitates interaction and fusion with other compartments and harbours transport proteins catalysing the export of catabolites, thereby allowing their recycling. Lysosomal proteins have been addressed in various proteomic studies that are compared in this review regarding the source of material, the organelle/protein purification scheme, the proteomic methodology applied and the proteins identified. Distinguishing true constituents of an organelle from co-purifying contaminants is a central issue in subcellular proteomics, with additional implications for lysosomes as being the site of degradation of many cellular and extracellular proteins. Although many of the lysosomal hydrolases were identified by classical biochemical approaches, the knowledge about the protein composition of the lysosomal membrane has remained fragmentary for a long time. Using proteomics many novel lysosomal candidate proteins have been discovered and it can be expected that their functional characterisation will help to understand functions of lysosomes at a molecular level that have been characterised only phenomenologically so far and to generally deepen our understanding of this indispensable organelle.

  16. TRPML and lysosomal function.

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    Zeevi, David A; Frumkin, Ayala; Bach, Gideon

    2007-08-01

    Mucolipin 1 (MLN1), also known as TRPML1, is a member of the mucolipin family. The mucolipins are the only lysosomal proteins within the TRP superfamily. Mutations in the gene coding for TRPML1 result in a lysosomal storage disorder (LSD). This review summarizes the current knowledge related to this protein and the rest of the mucolipin family.

  17. Epidermal Growth Factor Cytoplasmic Domain Affects ErbB Protein Degradation by the Lysosomal and Ubiquitin-Proteasome Pathway in Human Cancer Cells

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    Aleksandra Glogowska

    2012-05-01

    Full Text Available The cytoplasmic domains of EGF-like ligands, including EGF cytoplasmic domain (EGFcyt, have important biological functions. Using specific constructs and peptides of human EGF cytoplasmic domain, we demonstrate that EGFcyt facilitates lysosomal and proteasomal protein degradation, and this coincided with growth inhibition of human thyroid and glioma carcinoma cells. EGFcyt and exon 22–23-encoded peptide (EGF22.23 enhanced procathepsin B (procathB expression and procathB-mediated lysosomal degradation of EGFR/ErbB1 as determined by inhibitors for procathB and the lysosomal ATPase inhibitor BafA1. Presence of mbEGFctF, EGFcyt, EGF22.23, and exon 23-encoded peptides suppressed the expression of the deubiqitinating enzyme ubiquitin C-terminal hydrolase-L1 (UCH-L1. This coincided with hyperubiquitination of total cellular proteins and ErbB1/2 and reduced proteasome activity. Upon small interfering RNA-mediated silencing of endogenously expressed UCH-L1, a similar hyperubiquitinylation phenotype, reduced ErbB1/2 content, and attenuated growth was observed. The exon 23-encoded peptide region of EGFcyt was important for these biologic actions. Structural homology modeling of human EGFcyt showed that this molecular region formed an exposed surface loop. Peptides derived from this EGFcyt loop structure may aid in the design of novel peptide therapeutics aimed at inhibiting growth of cancer cells.

  18. First-Generation Antipsychotic Haloperidol Alters the Functionality of the Late Endosomal/Lysosomal Compartment in Vitro

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    Alberto Canfrán-Duque

    2016-03-01

    Full Text Available First- and second-generation antipsychotics (FGAs and SGAs, respectively, have the ability to inhibit cholesterol biosynthesis and also to interrupt the intracellular cholesterol trafficking, interfering with low-density lipoprotein (LDL-derived cholesterol egress from late endosomes/lysosomes. In the present work, we examined the effects of FGA haloperidol on the functionality of late endosomes/lysosomes in vitro. In HepG2 hepatocarcinoma cells incubated in the presence of 1,1′-dioctadecyl-3,3,3,3′-tetramethylindocarbocyanineperchlorate (DiI-LDL, treatment with haloperidol caused the enlargement of organelles positive for late endosome markers lysosome-associated membrane protein 2 (LAMP-2 and LBPA (lysobisphosphatidic acid, which also showed increased content of both free-cholesterol and DiI derived from LDL. This indicates the accumulation of LDL-lipids in the late endosomal/lysosomal compartment caused by haloperidol. In contrast, LDL traffic through early endosomes and the Golgi apparatus appeared to be unaffected by the antipsychotic as the distribution of both early endosome antigen 1 (EEA1 and coatomer subunit β (β-COP were not perturbed. Notably, treatment with haloperidol significantly increased the lysosomal pH and decreased the activities of lysosomal protease and β-d-galactosidase in a dose-dependent manner. We conclude that the alkalinization of the lysosomes’ internal milieu induced by haloperidol affects lysosomal functionality.

  19. First-Generation Antipsychotic Haloperidol Alters the Functionality of the Late Endosomal/Lysosomal Compartment in Vitro

    Science.gov (United States)

    Canfrán-Duque, Alberto; Barrio, Luis C.; Lerma, Milagros; de la Peña, Gema; Serna, Jorge; Pastor, Oscar; Lasunción, Miguel A.; Busto, Rebeca

    2016-01-01

    First- and second-generation antipsychotics (FGAs and SGAs, respectively), have the ability to inhibit cholesterol biosynthesis and also to interrupt the intracellular cholesterol trafficking, interfering with low-density lipoprotein (LDL)-derived cholesterol egress from late endosomes/lysosomes. In the present work, we examined the effects of FGA haloperidol on the functionality of late endosomes/lysosomes in vitro. In HepG2 hepatocarcinoma cells incubated in the presence of 1,1′-dioctadecyl-3,3,3,3′-tetramethylindocarbocyanineperchlorate (DiI)-LDL, treatment with haloperidol caused the enlargement of organelles positive for late endosome markers lysosome-associated membrane protein 2 (LAMP-2) and LBPA (lysobisphosphatidic acid), which also showed increased content of both free-cholesterol and DiI derived from LDL. This indicates the accumulation of LDL-lipids in the late endosomal/lysosomal compartment caused by haloperidol. In contrast, LDL traffic through early endosomes and the Golgi apparatus appeared to be unaffected by the antipsychotic as the distribution of both early endosome antigen 1 (EEA1) and coatomer subunit β (β-COP) were not perturbed. Notably, treatment with haloperidol significantly increased the lysosomal pH and decreased the activities of lysosomal protease and β-d-galactosidase in a dose-dependent manner. We conclude that the alkalinization of the lysosomes’ internal milieu induced by haloperidol affects lysosomal functionality. PMID:26999125

  20. Lysosomal Storage Diseases

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    Joseph Alroy DVM, DACVP

    2014-03-01

    Full Text Available Lysosomal storage diseases are a group of inherited and acquired disorders. They are characterized by interruption of recycling of cellular and extracellular molecules. Clinically, they are presented as developmental and neurological symptoms similar to other inherited and acquired disorders. This article reviews the function of lysosomes, the current mechanisms that cause the interruption of recycling, the consequences that are manifested clinically, and the methods to diagnose these disorders.

  1. A potentially dynamic lysosomal role for the endogenous TRPML proteins.

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    Zeevi, David A; Frumkin, Ayala; Offen-Glasner, Vered; Kogot-Levin, Aviram; Bach, Gideon

    2009-10-01

    Lysosomal storage disorders (LSDs) constitute a diverse group of inherited diseases that result from lysosomal storage of compounds occurring in direct consequence to deficiencies of proteins implicated in proper lysosomal function. Pathology in the LSD mucolipidosis type IV (MLIV), is characterized by lysosomal storage of lipids together with water-soluble materials in cells from every tissue and organ of affected patients. Mutations in the mucolipin 1 (TRPML1) protein cause MLIV and TRPML1 has also been shown to interact with two of its paralogous proteins, mucolipin 2 (TRPML2) and mucolipin 3 (TRPML3), in heterologous expression systems. Heterogeneous lysosomal storage is readily identified in electron micrographs of MLIV patient cells, suggesting that proper TRPML1 function is essential for the maintenance of lysosomal integrity. In order to investigate whether TRPML2 and TRPML3 also play a role in the maintenance of lysosomal integrity, we conducted gene-specific knockdown assays against these protein targets. Ultrastructural analysis revealed lysosomal inclusions in both TRPML2 and TRPML3 knockdown cells, suggestive of a common mechanism for these proteins, in parallel with TRPML1, in the regulation of lysosomal integrity. However, co-immunoprecipitation assays revealed that physical interactions between each of the endogenous TRPML proteins are quite limited. In addition, we found that all three endogenous proteins only partially co-localize with each other in lysosomal as well as extra-lysosomal compartments. This suggests that native TRPML2 and TRPML3 might participate with native TRPML1 in a dynamic form of lysosomal regulation. Given that depletion of TRPML2/3 led to lysosomal storage typical to an LSD, we propose that depletion of these proteins might also underlie novel LSD pathologies not described hitherto.

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

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    Takahashi, Yusuke; Nada, Shigeyuki; Mori, Shunsuke; Soma-Nagae, Taeko; Oneyama, Chitose [Department of Oncogene Research, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871 (Japan); Okada, Masato, E-mail: okadam@biken.osaka-u.ac.jp [Department of Oncogene Research, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871 (Japan)

    2012-01-27

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

  3. Mitochondrial Dysfunction in Lysosomal Storage Disorders

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    Mario de la Mata

    2016-10-01

    Full Text Available Lysosomal storage diseases (LSDs describe a heterogeneous group of rare inherited metabolic disorders that result from the absence or loss of function of lysosomal hydrolases or transporters, resulting in the progressive accumulation of undigested material in lysosomes. The accumulation of substances affects the function of lysosomes and other organelles, resulting in secondary alterations such as impairment of autophagy, mitochondrial dysfunction, inflammation and apoptosis. LSDs frequently involve the central nervous system (CNS, where neuronal dysfunction or loss results in progressive neurodegeneration and premature death. Many LSDs exhibit signs of mitochondrial dysfunction, which include mitochondrial morphological changes, decreased mitochondrial membrane potential (ΔΨm, diminished ATP production and increased generation of reactive oxygen species (ROS. Furthermore, reduced autophagic flux may lead to the persistence of dysfunctional mitochondria. Gaucher disease (GD, the LSD with the highest prevalence, is caused by mutations in the GBA1 gene that results in defective and insufficient activity of the enzyme β-glucocerebrosidase (GCase. Decreased catalytic activity and/or instability of GCase leads to accumulation of glucosylceramide (GlcCer and glucosylsphingosine (GlcSph in the lysosomes of macrophage cells and visceral organs. Mitochondrial dysfunction has been reported to occur in numerous cellular and mouse models of GD. The aim of this manuscript is to review the current knowledge and implications of mitochondrial dysfunction in LSDs.

  4. Deletion of the highly conserved N-glycan at Asn260 of HIV-1 gp120 affects folding and lysosomal degradation of gp120, and results in loss of viral infectivity.

    Directory of Open Access Journals (Sweden)

    Leen Mathys

    Full Text Available N-linked glycans covering the surface of the HIV-1 glycoprotein gp120 are of major importance for the correct folding of this glycoprotein. Of the, on average, 24 N-linked glycans present on gp120, the glycan at Asn260 was reported to be essential for the correct expression of gp120 and gp41 in the virus particle and deletion of the N260 glycan in gp120 heavily compromised virus infectivity. We show here that gp160 containing the N260Q mutation reaches the Golgi apparatus during biosynthesis. Using pulse-chase experiments with [35S] methionine/cysteine, we show that oxidative folding was slightly delayed in case of mutant N260Q gp160 and that CD4 binding was markedly compromised compared to wild-type gp160. In the search of compensatory mutations, we found a mutation in the V1/V2 loop of gp120 (S128N that could partially restore the infectivity of mutant N260Q gp120 virus. However, the mutation S128N did not enhance any of the above-mentioned processes so its underlying compensatory mechanism must be a conformational effect that does not affect CD4 binding per se. Finally, we show that mutant N260Q gp160 was cleaved to gp120 and gp41 to a much lower extent than wild-type gp160, and that it was subject of lysosomal degradation to a higher extent than wild-type gp160 showing a prominent role of this process in the breakdown of N260-glycan-deleted gp160, which could not be counteracted by the S128N mutation. Moreover, at least part of the wild-type or mutant gp160 that is normally targeted for lysosomal degradation reached a conformation that enabled CD4 binding.

  5. The lysosome and neurodegenerative diseases

    Institute of Scientific and Technical Information of China (English)

    Lisha Zhang; Rui Sheng; Zhenghong Qin

    2009-01-01

    It has long been believed that the lysosome is an important digestive organelle. There is increasing evidence that the lysosome is also involved in pathogenesis of a variety of neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis. Abnormal protein degradation and deposition induced by lysosoreal dysfunction may be the primary contributor to age-related neurodegeneration. In this review, the possible relationship between lysosome and various neurodegenerative diseases is described.

  6. Neuroinflammatory paradigms in lysosomal storage diseases

    Directory of Open Access Journals (Sweden)

    Megan Elizabeth Bosch

    2015-10-01

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

  7. Cationic lipids delay the transfer of plasmid DNA to lysosomes.

    Science.gov (United States)

    Wattiaux, R; Jadot, M; Laurent, N; Dubois, F; Wattiaux-De Coninck, S

    1996-10-14

    Plasmid 35S DNA, naked or associated with different cationic lipid preparations was injected to rats. Subcellular distribution of radioactivity in the liver one hour after injection, was established by centrifugation methods. Results show that at that time, 35S DNA has reached lysosomes. On the contrary, when 35S DNA was complexed with lipids, radioactivity remains located in organelles whose distribution after differential and isopycnic centrifugation, is clearly distinct from that of arylsulfatase, lysosome marker enzyme. Injection of Triton WR 1339, a specific density perturbant of lysosomes, four days before 35S DNA injection causes a density decrease of radioactivity bearing structures, apparent one hour after naked 35S DNA injection but visible only after more than five hours, when 35S DNA associated with a cationic lipid is injected. These observations show that cationic lipids delay the transfer to lysosomes, of plasmid DNA taken up by the liver.

  8. Syntaxin 7 and VAMP-7 are soluble N-ethylmaleimide-sensitive factor attachment protein receptors required for late endosome-lysosome and homotypic lysosome fusion in alveolar macrophages.

    Science.gov (United States)

    Ward, D M; Pevsner, J; Scullion, M A; Vaughn, M; Kaplan, J

    2000-07-01

    Endocytosis in alveolar macrophages can be reversibly inhibited, permitting the isolation of endocytic vesicles at defined stages of maturation. Using an in vitro fusion assay, we determined that each isolated endosome population was capable of homotypic fusion. All vesicle populations were also capable of heterotypic fusion in a temporally specific manner; early endosomes, isolated 4 min after internalization, could fuse with endosomes isolated 8 min after internalization but not with 12-min endosomes or lysosomes. Lysosomes fuse with 12-min endosomes but not with earlier endosomes. Using homogenous populations of endosomes, we have identified Syntaxin 7 as a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) required for late endosome-lysosome and homotypic lysosome fusion in vitro. A bacterially expressed human Syntaxin 7 lacking the transmembrane domain inhibited homotypic late endosome and lysosome fusion as well as heterotypic late endosome-lysosome fusion. Affinity-purified antibodies directed against Syntaxin 7 also inhibited lysosome fusion in vitro but had no affect on homotypic early endosome fusion. Previous work suggested that human VAMP-7 (vesicle-associated membrane protein-7) was a SNARE required for late endosome-lysosome fusion. A bacterially expressed human VAMP-7 lacking the transmembrane domain inhibited both late endosome-lysosome fusion and homotypic lysosome fusion in vitro. These studies indicate that: 1) fusion along the endocytic pathway is a highly regulated process, and 2) two SNARE molecules, Syntaxin 7 and human VAMP-7, are involved in fusion of vesicles in the late endocytic pathway in alveolar macrophages.

  9. Physicochemical conditions in affecting the distribution of spring phytoplankton community

    Science.gov (United States)

    Wei, Yuqiu; Liu, Haijiao; Zhang, Xiaodong; Xue, Bing; Munir, Sonia; Sun, Jun

    2017-03-01

    To better understand the physicochemical conditions in affecting regional distribution of phytoplankton community, one research cruise was carried out in the Bohai Sea and Yellow Sea during 3rd and 23th May, 2010. The phytoplankton community, including Bacillariophyta (105 taxa), Pyrrophyta (54 taxa), Chrysophyta (1 taxon) and Chlorophyta (2 taxa), had been identified and clearly described from six ecological provinces. And, the six ecological provinces were partitioned based on the top twenty dominant species related with notable physicochemical parameters. In general, the regional distributions of phytoplankton ecological provinces were predominantly influenced by the physicochemical properties induced by the variable water masses and circulations. The predominant diatoms in most of water samples showed well adaptability in turbulent and eutrophic conditions. However, several species of dinoflagellates e.g., Protoperidinium conicum, Protoperidinium triestinum, Protoperidinium sp. and Gymnodinium lohmanni preferred warmer, saltier and nutrient-poor environment. Moreover, the dinoflagellates with high frequency in the Yellow Sea might be transported from the Yellow Sea Warm Current. The horizontal distribution of phytoplankton was depicted by diatoms and controlled by phosphate concentration, while the vertical distribution was mainly supported by light and nutrients availability in the subsurface and bottom layers, respectively.

  10. The Distribution of Synaptotagmin Ⅱ in RBL-2H3 and Its Regulation on Exocytosis of Lysosomes in RBL-2H3

    Institute of Scientific and Technical Information of China (English)

    Jicheng Zhang; Jianmin Wu; Shixiu Pan; Wenli Lv

    2005-01-01

    Synaptotagmin (Syt) constitutes a family of membrane-trafficking proteins, so far nearly 20 Syts have been discovered. Extensive work showed that synatotagmins were a potential Ca2+ sensor for regulated exocytosis. This study was to investigate the expression and location of synaptotagmin Ⅱ (Syt2) in RBL-2H3 (RBL) and its role in regulating exocytosis of RBL. The expression of Syt2 in RBL was confirmed by Western blot. The recombinant expression vector pEGFP-N1-Syt2 was constructed and transfected into RBL by electroporation, the stable transfectant RBL-Syt2-S expressing fusion protein Syt2-EGFP were obtained and Syt2 was highly concentrated at plasma membrane with little detected in cytoplasm. To analyze the role of Syt2 during exocytosis of RBL, the release of cathepsin D was assayed by immunoblotting. Compared with control, the release of cathepsin D by RBL-Syt2-S was markedly decreased. The results indicated that Syt2 played a negative regulation in exocytosis of lysosomes in RBL.

  11. Mental training affects distribution of limited brain resources.

    Directory of Open Access Journals (Sweden)

    Heleen A Slagter

    2007-06-01

    Full Text Available The information processing capacity of the human mind is limited, as is evidenced by the so-called "attentional-blink" deficit: When two targets (T1 and T2 embedded in a rapid stream of events are presented in close temporal proximity, the second target is often not seen. This deficit is believed to result from competition between the two targets for limited attentional resources. Here we show, using performance in an attentional-blink task and scalp-recorded brain potentials, that meditation, or mental training, affects the distribution of limited brain resources. Three months of intensive mental training resulted in a smaller attentional blink and reduced brain-resource allocation to the first target, as reflected by a smaller T1-elicited P3b, a brain-potential index of resource allocation. Furthermore, those individuals that showed the largest decrease in brain-resource allocation to T1 generally showed the greatest reduction in attentional-blink size. These observations provide novel support for the view that the ability to accurately identify T2 depends upon the efficient deployment of resources to T1. The results also demonstrate that mental training can result in increased control over the distribution of limited brain resources. Our study supports the idea that plasticity in brain and mental function exists throughout life and illustrates the usefulness of systematic mental training in the study of the human mind.

  12. Mental training affects distribution of limited brain resources.

    Science.gov (United States)

    Slagter, Heleen A; Lutz, Antoine; Greischar, Lawrence L; Francis, Andrew D; Nieuwenhuis, Sander; Davis, James M; Davidson, Richard J

    2007-06-01

    The information processing capacity of the human mind is limited, as is evidenced by the so-called "attentional-blink" deficit: When two targets (T1 and T2) embedded in a rapid stream of events are presented in close temporal proximity, the second target is often not seen. This deficit is believed to result from competition between the two targets for limited attentional resources. Here we show, using performance in an attentional-blink task and scalp-recorded brain potentials, that meditation, or mental training, affects the distribution of limited brain resources. Three months of intensive mental training resulted in a smaller attentional blink and reduced brain-resource allocation to the first target, as reflected by a smaller T1-elicited P3b, a brain-potential index of resource allocation. Furthermore, those individuals that showed the largest decrease in brain-resource allocation to T1 generally showed the greatest reduction in attentional-blink size. These observations provide novel support for the view that the ability to accurately identify T2 depends upon the efficient deployment of resources to T1. The results also demonstrate that mental training can result in increased control over the distribution of limited brain resources. Our study supports the idea that plasticity in brain and mental function exists throughout life and illustrates the usefulness of systematic mental training in the study of the human mind.

  13. The Link Between Lysosomal Storage Disorders and More Common Diseases

    Directory of Open Access Journals (Sweden)

    Michael Beck MD

    2016-12-01

    Full Text Available In the last decades, it has become more and more evident that lysosomal storage disorders and common neurodegenerative diseases such as Alzheimer and Parkinson diseases have clinical, neuropathological, and genetic features in common, including lysosomal dysfunction and impaired autophagy. Patients with Gaucher and even carriers of Gaucher disease have an increased risk to develop Parkinson disease. Likewise, individuals who are heterozygous for a mutation of a gene that causes an adult form of neuronal ceroid lipofuscinosis are more likely to be affected by a form of frontotemporal dementia in their later life. A further example is the gene NAGLU encoding the enzyme α-N-acetylglucosaminidase, which is deficient in patients with mucopolysaccharidosis type IIIB. Mutations of the NAGLU gene have been observed in patients affected by an axonal neuropathy. An interesting unexpected finding was the link between stuttering and genes that are essential for the function of all lysosomal enzymes. This review will present some example of the association of lysosomal storage disorders and neurodegenerative disease and discuss possible pathogenic mechanisms that are common to both conditions. The understanding of the pathophysiology of the endosomal–lysosomal–autophagic system may help to develop drugs, which might provide benefit not only for patients with rare lysosomal storage disorders but also for individuals affected by more common diseases.

  14. Lysosomal Storage Disorders and Malignancy

    Directory of Open Access Journals (Sweden)

    Gregory M. Pastores

    2017-02-01

    Full Text Available Lysosomal storage disorders (LSDs are infrequent to rare conditions caused by mutations that lead to a disruption in the usual sequential degradation of macromolecules or their transit within the cell. Gaucher disease (GD, a lipidosis, is among the most common LSD, with an estimated incidence of 1 in 40,000 among the Caucasian, non-Jewish population. Studies have indicated an increased frequency of polyclonal and monoclonal gammopathy among patients with GD. It has been shown that two major sphingolipids that accumulate in GD, namely, β-glucosylceramide 22:0 (βGL1-22 and glucosylsphingosine (LGL1, can be recognized by a distinct subset of CD1d-restricted human and murine type II natural killer T (NKT cells. Investigations undertaken in an affected mouse model revealed βGL1-22- and LGL1-specific NKT cells were present and constitutively promoted the expression of a T-follicular helper (TFH phenotype; injection of these lipids led to downstream induction of germinal center B cells, hypergammaglobulinemia, and the production of antilipid antibodies. Subsequent studies have found clonal immunoglobulin in 33% of sporadic human monoclonal gammopathies is also specific for the lysolipids LGL1 and lysophosphatidylcholine (LPC. Furthermore, substrate reduction ameliorated GD-associated gammopathy in mice. It had been hypothesized that chronic antigenic stimulation by the abnormal lipid storage and associated immune dysregulation may be the underlying mechanism for the increased incidence of monoclonal and polyclonal gammopathies, as well as an increased incidence of multiple myeloma in patients with GD. Current observations support this proposition and illustrate the value of investigations into rare diseases, which as ‘experiments of nature’ may provide insights into conditions found in the general population that continue to remain incompletely understood.

  15. Lysosomal cell death mechanisms in aging.

    Science.gov (United States)

    Gómez-Sintes, Raquel; Ledesma, María Dolores; Boya, Patricia

    2016-12-01

    Lysosomes are degradative organelles essential for cell homeostasis that regulate a variety of processes, from calcium signaling and nutrient responses to autophagic degradation of intracellular components. Lysosomal cell death is mediated by the lethal effects of cathepsins, which are released into the cytoplasm following lysosomal damage. This process of lysosomal membrane permeabilization and cathepsin release is observed in several physiopathological conditions and plays a role in tissue remodeling, the immune response to intracellular pathogens and neurodegenerative diseases. Many evidences indicate that aging strongly influences lysosomal activity by altering the physical and chemical properties of these organelles, rendering them more sensitive to stress. In this review we focus on how aging alters lysosomal function and increases cell sensitivity to lysosomal membrane permeabilization and lysosomal cell death, both in physiological conditions and age-related pathologies.

  16. How Required Reserve Ratio Affects Distribution and Velocity of Money

    CERN Document Server

    Xi, N; Wang, Y; Xi, Ning; Ding, Ning; Wang, Yougui

    2005-01-01

    In this paper the dependence of wealth distribution and the velocity of money on the required reserve ratio is examined based on a random transfer model of money and computer simulations. A fractional reserve banking system is introduced to the model where money creation can be achieved by bank loans and the monetary aggregate is determined by the monetary base and the required reserve ratio. It is shown that monetary wealth follows asymmetric Laplace distribution and latency time of money follows exponential distribution. The expression of monetary wealth distribution and that of the velocity of money in terms of the required reserve ratio are presented in a good agreement with simulation results.

  17. How required reserve ratio affects distribution and velocity of money

    Science.gov (United States)

    Xi, Ning; Ding, Ning; Wang, Yougui

    2005-11-01

    In this paper the dependence of wealth distribution and the velocity of money on the required reserve ratio is examined based on a random transfer model of money and computer simulations. A fractional reserve banking system is introduced to the model where money creation can be achieved by bank loans and the monetary aggregate is determined by the monetary base and the required reserve ratio. It is shown that monetary wealth follows asymmetric Laplace distribution and latency time of money follows exponential distribution. The expression of monetary wealth distribution and that of the velocity of money in terms of the required reserve ratio are presented in a good agreement with simulation results.

  18. Neuronopathic Lysosomal Storage Diseases: Clinical and Pathologic Findings

    Science.gov (United States)

    Prada, Carlos E.; Grabowski, Gregory A.

    2013-01-01

    Background: The lysosomal--autophagocytic system diseases (LASDs) affect multiple body systems including the central nervous system (CNS). The progressive CNS pathology has its onset at different ages, leading to neurodegeneration and early death. Methods: Literature review provided insight into the current clinical neurological findings,…

  19. Rainfall and temperature affect tree species distributions in Ghana

    NARCIS (Netherlands)

    Amissah, L.; Mohren, G.M.J.; Bongers, F.; Hawthorne, W.D.; Poorter, L.

    2014-01-01

    We evaluated the relative importance of annual rainfall, temperature and their seasonality to tree species distribution in Ghana. We used species presence/absence data from 2505 1-ha plots systematically distributed over Ghana's forests. Logistic regression was used to determine species responses to

  20. Humidity distribution affected by freely exposed water surfaces

    DEFF Research Database (Denmark)

    Hygum, Morten Arnfeldt; Popok, Vladimir

    2014-01-01

    Accurate models for the water vapor flux at a water-air interface are required in various scientific, reliability and civil engineering aspects. Here, a study of humidity distribution in a container with air and freely exposed water is presented. A model predicting a spatial distribution and time...

  1. Parkinson's Disease Shares the Lysosome with Gaucher's Disease

    Science.gov (United States)

    Dawson, Ted M.; Dawson, Valina L.

    2015-01-01

    Summary The second most common neurodegenerative disorder, Parkinson's disease (PD) is an age dependent progressive neurodegenerative disorder that affects movement. While many of the causes of PD remain unclear, a consistent finding in PD is the abnormal accumulation of α-synuclein that has lead to the widely held notion that PD is a synucleinopathy. In a recent Cell manuscript Mazzuli et al., provide a potential mechanistic link between Gaucher's disease, a glycolipid lysosomal storage disorder due to Glucocerebrocidase (GBA) deficiency and PD. The authors reveal a reciprocal connection between the loss of GBA activity and accumulation of α-synuclein in the lysosome establishing a bidirectional positive feed back loop with pathologic consequences. These findings should stimulate further work on role of the lysosome in PD pathogenesis and the identification of new treatment strategies for PD. PMID:21753118

  2. Mucolipidosis type IV: the effect of increased lysosomal pH on the abnormal lysosomal storage.

    Science.gov (United States)

    Kogot-Levin, Aviram; Zeigler, Marsha; Ornoy, Asher; Bach, Gideon

    2009-06-01

    Mucolipidosis type IV (MLIV) is a neurodegenerative channelopathy that is caused by the deficiency of TRPML1 activity, a nonselective cation channel. TRPML1 is a lysosomal membrane protein, and thus, MLIV is a lysosomal storage disorder. The basic, specific function of TRPML1 has not been yet clarified. A recent report (Soyombo AA, Tjon-Kon-Sang S, Rbaibi Y, Bashllari E, Bisceglia J, Muallem S, Kiselyov K: J Biol Chem 281:7294-7301, 2006) indicated that TRPML1 functions as an outwardly proton channel whose function is the prevention of overacidification of these organelles. Thus, in MLIV the lysosomal pH is lower than normal. Furthermore, attempts by these investigators to increase slightly the lysososmal pH with either Nigericin or Chloroquine suggested corrective effect of the abnormal storage in MLIV cells. We investigated this approach using these agents with cultured fibroblasts from severely affected and milder patients. Our data indicated that there was no reduction in the total number of storage vesicles by either agent, although Nigericin resulted in a change in the nature of the storage materials, reducing the presence of lamellated substances (lipids) so that the storage vesicles contained predominantly granulated substances. On the other hand, transfection with the normal MCOLN1 cDNA (the gene coding for TRPML1) resulted in the removal of almost all the storage materials.

  3. Intrathecal enzyme replacement therapy reduces lysosomal storage in the brain and meninges of the canine model of MPS I.

    Science.gov (United States)

    Kakkis, E; McEntee, M; Vogler, C; Le, S; Levy, B; Belichenko, P; Mobley, W; Dickson, P; Hanson, S; Passage, M

    2004-01-01

    Enzyme replacement therapy (ERT) has been developed for several lysosomal storage disorders, including mucopolysaccharidosis I (MPS I), and is effective at reducing lysosomal storage in many tissues and in ameliorating clinical disease. However, intravenous ERT does not adequately treat storage disease in the central nervous system (CNS), presumably due to effects of the blood-brain barrier on enzyme distribution. To circumvent this barrier, we studied whether intrathecal (IT) recombinant human alpha-L-iduronidase (rhIDU) could penetrate and treat the brain and meninges. An initial dose-response study showed that doses of 0.46-4.14 mg of IT rhIDU successfully penetrated the brain of normal dogs and reached tissue levels 5.6 to 18.9-fold normal overall and 2.7 to 5.9-fold normal in deep brain sections lacking CSF contact. To assess the efficacy and safety in treating lysosomal storage disease, four weekly doses of approximately 1 mg of IT rhIDU were administered to MPS I-affected dogs resulting in a mean 23- and 300-fold normal levels of iduronidase in total brain and meninges, respectively. Quantitative glycosaminoglycan (GAG) analysis showed that the IT treatment reduced mean total brain GAG to normal levels and achieved a 57% reduction in meningeal GAG levels accompanied by histologic improvement in lysosomal storage in all cell types. The dogs did develop a dose-dependent immune response against the recombinant human protein and a meningeal lymphocytic/plasmacytic infiltrate. The IT route of ERT administration may be an effective way to treat the CNS disease in MPS I and could be applicable to other lysosomal storage disorders.

  4. How Required Reserve Ratio Affects Distribution and Velocity of Money

    OpenAIRE

    Ning Xi; Ning Ding; Yougui Wang

    2005-01-01

    In this paper the dependence of wealth distribution and the velocity of money on the required reserve ratio is examined based on a random transfer model of money and computer simulations. A fractional reserve banking system is introduced to the model where money creation can be achieved by bank loans and the monetary aggregate is determined by the monetary base and the required reserve ratio. It is shown that monetary wealth follows asymmetric Laplace distribution and latency time of money fo...

  5. Syntaxin 7 and VAMP-7 are Soluble N-Ethylmaleimide–sensitive Factor Attachment Protein Receptors Required for Late Endosome–Lysosome and Homotypic Lysosome Fusion in Alveolar Macrophages

    Science.gov (United States)

    Ward, Diane McVey; Pevsner, Jonathan; Scullion, Matthew A.; Vaughn, Michael; Kaplan, Jerry

    2000-01-01

    Endocytosis in alveolar macrophages can be reversibly inhibited, permitting the isolation of endocytic vesicles at defined stages of maturation. Using an in vitro fusion assay, we determined that each isolated endosome population was capable of homotypic fusion. All vesicle populations were also capable of heterotypic fusion in a temporally specific manner; early endosomes, isolated 4 min after internalization, could fuse with endosomes isolated 8 min after internalization but not with 12-min endosomes or lysosomes. Lysosomes fuse with 12-min endosomes but not with earlier endosomes. Using homogenous populations of endosomes, we have identified Syntaxin 7 as a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) required for late endosome–lysosome and homotypic lysosome fusion in vitro. A bacterially expressed human Syntaxin 7 lacking the transmembrane domain inhibited homotypic late endosome and lysosome fusion as well as heterotypic late endosome–lysosome fusion. Affinity-purified antibodies directed against Syntaxin 7 also inhibited lysosome fusion in vitro but had no affect on homotypic early endosome fusion. Previous work suggested that human VAMP-7 (vesicle-associated membrane protein-7) was a SNARE required for late endosome–lysosome fusion. A bacterially expressed human VAMP-7 lacking the transmembrane domain inhibited both late endosome–lysosome fusion and homotypic lysosome fusion in vitro. These studies indicate that: 1) fusion along the endocytic pathway is a highly regulated process, and 2) two SNARE molecules, Syntaxin 7 and human VAMP-7, are involved in fusion of vesicles in the late endocytic pathway in alveolar macrophages. PMID:10888671

  6. Gaucher disease: a lysosomal neurodegenerative disorder.

    Science.gov (United States)

    Huang, W J; Zhang, X; Chen, W W

    2015-04-01

    Gaucher disease is a multisystemic disorder that affects men and woman in equal numbers and occurs in all ethnic groups at any age with racial variations and an estimated worldwide incidence of 1/75,000. It is caused by a genetic deficient activity of the lysosomal enzyme glucocerebrosidase due to mutations in the β-glucocerebrosidase gene, and resulting in lack of glucocerebroside degradation. The subsequent accumulation of glucocerebroside in lysosomes of tissue macrophages primarily in the liver, bone marrow and spleen, causes damage in haematological, skeletal and nervous systems. The clinical manifestations show a high degree of variability with symptoms that varies according to organs involved. In many cases, these disorders do not correlate with mutations in the β-glucocerebrosidase gene. Although several mutations have been identified as responsible for the deficient activity of glucocerebrosidase, mechanisms by which this enzymatic defect leads to Gaucher disease remain poorly understood. Recent reports indicate the implication of complex mechanisms, including enzyme deficiency, substrate accumulation, unfolded protein response, and macrophage activation. Further elucidating these mechanisms will advance understanding of Gaucher disease and related disorders.

  7. Inhibitors of lysosomal cysteine proteases

    Directory of Open Access Journals (Sweden)

    Lyanna O. L.

    2011-04-01

    Full Text Available The review is devoted to the inhibitors of cysteine proteinases which are believed to be very important in many biochemical processes of living organisms. They participate in the development and progression of numerous diseases that involve abnormal protein turnover. One of the main regulators of these proteinases is their specific inhibitors: cystatins. The aim of this review was to present current knowledge about endogenous inhibitors of lysosomal cysteine proteases and their synthetic analogs.

  8. Factors affecting distribution of airflow in a human tracheobronchial cast.

    Science.gov (United States)

    Cohen, B S; Sussman, R G; Lippmann, M

    1993-09-01

    Air velocity was measured at end airways of hollow replicate casts of the human tracheobronchial tree in order to determine the flow distribution within casts extending to 3 mm diameter airways. Measurements were made by hot-wire anemometry for constant inspiratory flow rates of 7.5, 15, 30 and 60 L.min-1. Average flow distribution among the lung lobes was as follows: right upper, 18.5%; right middle, 9.2%; right lower, 32.3%; left upper, 15.7%; and left lower, 24.3%. An empirical model derived from the experimental flow distribution data demonstrated the effect of various morphometric parameters of the hollow cast on the distribution of airflow. Airway cross-sectional area, branching angle and total path-length were found to have the greatest influence. As the tracheal flow rate decreased from 60 to 7.5 L.min-1, the influence of branching angle was reduced, while total path-length became more influential. These results provide evidence for the transition of flow regimes within the TB tree within normal physiological flow ranges.

  9. Factors Affecting Bacterial Growth in Drinking Water Distribution System

    Institute of Scientific and Technical Information of China (English)

    WEI LU; XIAO-JIAN ZHANG

    2005-01-01

    Objective To define the influence of some parameters, including assimilable organic carbon (AOC), chloramine residual, etc. on the bacterial growth in drinking water distribution systems. Methods Three typical water treatment plants in a northern city (City T) of China and their corresponding distribution systems were investigated. Some parameters of the water samples, such as heterotrophic plate content (HPC), AOC, CODMn, TOC, and phosphate were measured. Results The AOC in most water samples were more than 100 μg/L, or even more than 200 μg/L in some cases. The HPC in distribution systems increased significantly with the decrease of residual chlorine. When the residual chlorine was less than 0.1 mg/L, the magnitude order of HPC was 104 CFU/mL; when it was 0.5-0.7 mg/L, the HPC was about 500 CFU/mL. Conclusion For controlling the biostability of drinking water, the controlling of AOC and residual chlorine should be considered simultaneously. The influence of phosphors on the AOC tests of water is not significant. Phosphors may not be the limiting nutrient in the water distribution systems.

  10. GNeosomes: Highly Lysosomotropic Nanoassemblies for Lysosomal Delivery.

    Science.gov (United States)

    Wexselblatt, Ezequiel; Esko, Jeffrey D; Tor, Yitzhak

    2015-01-01

    GNeosomes, lysosomotropic lipid vesicles decorated with guanidinoneomycin, can encapsulate and facilitate the cellular internalization and lysosomal delivery of cargo ranging from small molecules to high molecular weight proteins, in a process that is exclusively dependent on cell surface glycosaminoglycans. Their cellular uptake mechanism and co-localization with lysosomes, as well as the delivery, release, and activity of internalized cargo, are quantified. GNeosomes are proposed as a universal platform for lysosomal delivery with potential as a basic research tool and a therapeutic vehicle.

  11. Membrane cholesterol regulates lysosome-plasma membrane fusion events and modulates Trypanosoma cruzi invasion of host cells.

    Directory of Open Access Journals (Sweden)

    Bárbara Hissa

    Full Text Available BACKGROUND: Trypomastigotes of Trypanosoma cruzi are able to invade several types of non-phagocytic cells through a lysosomal dependent mechanism. It has been shown that, during invasion, parasites trigger host cell lysosome exocytosis, which initially occurs at the parasite-host contact site. Acid sphingomyelinase released from lysosomes then induces endocytosis and parasite internalization. Lysosomes continue to fuse with the newly formed parasitophorous vacuole until the parasite is completely enclosed by lysosomal membrane, a process indispensable for a stable infection. Previous work has shown that host membrane cholesterol is also important for the T. cruzi invasion process in both professional (macrophages and non-professional (epithelial phagocytic cells. However, the mechanism by which cholesterol-enriched microdomains participate in this process has remained unclear. METHODOLOGY/PRINCIPAL FINDING: In the present work we show that cardiomyocytes treated with MβCD, a drug able to sequester cholesterol from cell membranes, leads to a 50% reduction in invasion by T. cruzi trypomastigotes, as well as a decrease in the number of recently internalized parasites co-localizing with lysosomal markers. Cholesterol depletion from host membranes was accompanied by a decrease in the labeling of host membrane lipid rafts, as well as excessive lysosome exocytic events during the earlier stages of treatment. Precocious lysosomal exocytosis in MβCD treated cells led to a change in lysosomal distribution, with a reduction in the number of these organelles at the cell periphery, and probably compromises the intracellular pool of lysosomes necessary for T. cruzi invasion. CONCLUSION/SIGNIFICANCE: Based on these results, we propose that cholesterol depletion leads to unregulated exocytic events, reducing lysosome availability at the cell cortex and consequently compromise T. cruzi entry into host cells. The results also suggest that two different pools of

  12. Nanoparticle size and combined toxicity of TiO2 and DSLS (surfactant) contribute to lysosomal responses in digestive cells of mussels exposed to TiO2 nanoparticles.

    Science.gov (United States)

    Jimeno-Romero, A; Oron, M; Cajaraville, M P; Soto, M; Marigómez, I

    2016-10-01

    The aim of this investigation was to understand the bioaccumulation, cell and tissue distribution and biological effects of disodium laureth sulfosuccinate (DSLS)-stabilised TiO2 nanoparticles (NPs) in marine mussels, Mytilus galloprovincialis. Mussels were exposed in vivo to 0.1, 1 and 10 mg Ti/L either as TiO2 NPs (60 and 180 nm) or bulk TiO2, as well as to DSLS alone. A significant Ti accumulation was observed in mussels exposed to TiO2 NPs, which were localised in endosomes, lysosomes and residual bodies of digestive cells, and in the lumen of digestive tubules, as demonstrated by ultrastructural observations and electron probe X-ray microanalysis. TiO2 NPs of 60 nm were internalised within digestive cell lysosomes to a higher extent than TiO2 NPs of 180 nm, as confirmed by the quantification of black silver deposits after autometallography. The latter were localised mainly forming large aggregates in the lumen of the gut. Consequently, lysosomal membrane stability (LMS) was significantly reduced upon exposure to both TiO2 NPs although more markedly after exposure to TiO2-60 NPs. Exposure to bulk TiO2 and to DSLS also affected the stability of the lysosomal membrane. Thus, effects on the lysosomal membrane depended on the nanoparticle size and on the combined biological effects of TiO2 and DSLS.

  13. Sensitivity to lysosome-dependent cell death is directly regulated by lysosomal cholesterol content.

    Directory of Open Access Journals (Sweden)

    Hanna Appelqvist

    Full Text Available 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-β-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.

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

    Science.gov (United States)

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

    2016-04-01

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

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

    Science.gov (United States)

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

    2015-03-17

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

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

    Science.gov (United States)

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

    2015-05-26

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

  17. Structure Dependence of Lysosomal Transit of Chitosan-Based Polyplexes for Gene Delivery.

    Science.gov (United States)

    Thibault, Marc; Lavertu, Marc; Astolfi, Mélina; Buschmann, Michael D

    2016-10-01

    Chitosan-based polyplexes are known to traffic through lysosomes for a relatively long time, independent of the degree of deacetylation (DDA) and the number average molecular weight (Mn) of the polymer, even though both of these parameters have profound effects on polyplex stability and transfection efficiency. A better understanding of the lysosomal barrier is paramount to the rational design of vectors capable of overcoming obstacles to transgene expression. The aim of the present study was to investigate if lysosomal transit affects chitosan-based polyplex transfection efficiency in a structure-dependent (DDA, Mn) manner. Toward this end, we analyzed the effects of intracellular trafficking modifying agents on transfection efficiency and intracellular vesicular trafficking of polyplexes with different structural properties and stabilities or nucleic acid binding affinity. The use of agents that modify endosome/lysosome acidification and transit processes by distinct mechanisms and their effect on cell viability, polyplex uptake, vesicular trafficking, and transfection efficiency revealed novel and strong chitosan structure-dependent consequences of lysosomal transit. Inhibiting lysosomal transit using chloroquine significantly increased the efficiency of unstable polyplexes, while having minimal effects for polyplexes with intermediate or high stability. In parallel, specifically inhibiting the acidification of vesicles abrogated transfection for all formulations, suggesting that vesicular acidification is essential to promote transfection, most probably by facilitating lysosomal escape. These results provide novel insights into the structure-performance relationship of chitosan-based gene delivery systems.

  18. TRPML cation channels regulate the specialized lysosomal compartment of vertebrate B-lymphocytes.

    Science.gov (United States)

    Song, Yumei; Dayalu, Rashmi; Matthews, Sharon A; Scharenberg, Andrew M

    2006-12-01

    B-lymphocytes possess a specialized lysosomal compartment, the regulated transformation of which has been implicated in B-cell antigen presentation. Members of the mucolipin (TRPML) family of cation channels have been implicated in regulated vesicular transport in several tissues, but a role for TRPML function in lymphocyte vesicular transport physiology has not been previously described. To address the role of TRPML proteins in lymphocyte vesicular transport, we analyzed the lysosomal compartment in cultured B-lymphocytes engineered to lack TRPML1 or after expression of N- or C-terminal GFP fusion proteins of TRPML1 or TRPML2. Consistent with previous analyses of lymphocytes derived from human patients with mutations in TRPML1, we were not able to detect abnormalities in the lysosomes of TRPML1-deficient DT40 B-lymphocytes. However, while N-terminal GFP fusions of TRPML2 localized to normal appearing lysosomes, C-terminal GFP fusions of either TRPML1 or TRPML2 acted to antagonize endogenous TRPML function, localizing to large vesicular structures, the histological properties of which were indistinguishable from the enlarged lysosomes observed in affected tissues of TRPML1-deficient humans. Endocytosed B-cell receptors were delivered to these enlarged lysosomes, demonstrating that a TRPML-dependent process is required for normal regulation of the specialized lysosome compartment of vertebrate B-lymphocytes.

  19. Endosome-lysosomes and neurodegeneration.

    Science.gov (United States)

    Mayer, R J; Tipler, C; Laszlo, L; Arnold, J; Lowe, J; Landon, M

    1994-01-01

    A number of the major human and animal neurodegenerative diseases, such as Alzheimer's disease and sheep scrapie, are characterised by deposits of amyloid, arising through incomplete breakdown of membrane proteins. Although our knowledge concerning these diseases is increasing, they remain largely untreatable. Recently, attention has focussed on the mechanisms of production of different types of amyloid and the likely involvement within cells of acid compartments called endosome-lysosomes. These organelles may be 'bioreactor' sites for the unfolding and partial degradation of membrane proteins to generate the amyloid materials. These subsequently become expelled from the cell, or are released from dead cells, and accumulate as pathological entities. Common features of the disease processes give new direction to therapeutic intervention.

  20. Network Regulation and Support Schemes - How Policy Interactions Affect the Integration of Distributed Generation

    DEFF Research Database (Denmark)

    Ropenus, Stephanie; Jacobsen, Henrik; Schröder, Sascha Thorsten

    2011-01-01

    This article seeks to investigate the interactions between the policy dimensions of support schemes and network regulation and how they affect distributed generation. Firstly, the incentives of distributed generators and distribution system operators are examined. Frequently there exists a trade......-off between the incentives for these two market agents to facilitate the integration of distributed generation. Secondly, the interaction of these policy dimensions is analyzed, including case studies based on five EU Member States. Aspects of operational nature and investments in grid and distributed...

  1. Coronavirus cell entry occurs through the endo-/lysosomal pathway in a proteolysis-dependent manner.

    Directory of Open Access Journals (Sweden)

    Christine Burkard

    2014-11-01

    Full Text Available Enveloped viruses need to fuse with a host cell membrane in order to deliver their genome into the host cell. While some viruses fuse with the plasma membrane, many viruses are endocytosed prior to fusion. Specific cues in the endosomal microenvironment induce conformational changes in the viral fusion proteins leading to viral and host membrane fusion. In the present study we investigated the entry of coronaviruses (CoVs. Using siRNA gene silencing, we found that proteins known to be important for late endosomal maturation and endosome-lysosome fusion profoundly promote infection of cells with mouse hepatitis coronavirus (MHV. Using recombinant MHVs expressing reporter genes as well as a novel, replication-independent fusion assay we confirmed the importance of clathrin-mediated endocytosis and demonstrated that trafficking of MHV to lysosomes is required for fusion and productive entry to occur. Nevertheless, MHV was shown to be less sensitive to perturbation of endosomal pH than vesicular stomatitis virus and influenza A virus, which fuse in early and late endosomes, respectively. Our results indicate that entry of MHV depends on proteolytic processing of its fusion protein S by lysosomal proteases. Fusion of MHV was severely inhibited by a pan-lysosomal protease inhibitor, while trafficking of MHV to lysosomes and processing by lysosomal proteases was no longer required when a furin cleavage site was introduced in the S protein immediately upstream of the fusion peptide. Also entry of feline CoV was shown to depend on trafficking to lysosomes and processing by lysosomal proteases. In contrast, MERS-CoV, which contains a minimal furin cleavage site just upstream of the fusion peptide, was negatively affected by inhibition of furin, but not of lysosomal proteases. We conclude that a proteolytic cleavage site in the CoV S protein directly upstream of the fusion peptide is an essential determinant of the intracellular site of fusion.

  2. Coronavirus cell entry occurs through the endo-/lysosomal pathway in a proteolysis-dependent manner.

    Science.gov (United States)

    Burkard, Christine; Verheije, Monique H; Wicht, Oliver; van Kasteren, Sander I; van Kuppeveld, Frank J; Haagmans, Bart L; Pelkmans, Lucas; Rottier, Peter J M; Bosch, Berend Jan; de Haan, Cornelis A M

    2014-11-01

    Enveloped viruses need to fuse with a host cell membrane in order to deliver their genome into the host cell. While some viruses fuse with the plasma membrane, many viruses are endocytosed prior to fusion. Specific cues in the endosomal microenvironment induce conformational changes in the viral fusion proteins leading to viral and host membrane fusion. In the present study we investigated the entry of coronaviruses (CoVs). Using siRNA gene silencing, we found that proteins known to be important for late endosomal maturation and endosome-lysosome fusion profoundly promote infection of cells with mouse hepatitis coronavirus (MHV). Using recombinant MHVs expressing reporter genes as well as a novel, replication-independent fusion assay we confirmed the importance of clathrin-mediated endocytosis and demonstrated that trafficking of MHV to lysosomes is required for fusion and productive entry to occur. Nevertheless, MHV was shown to be less sensitive to perturbation of endosomal pH than vesicular stomatitis virus and influenza A virus, which fuse in early and late endosomes, respectively. Our results indicate that entry of MHV depends on proteolytic processing of its fusion protein S by lysosomal proteases. Fusion of MHV was severely inhibited by a pan-lysosomal protease inhibitor, while trafficking of MHV to lysosomes and processing by lysosomal proteases was no longer required when a furin cleavage site was introduced in the S protein immediately upstream of the fusion peptide. Also entry of feline CoV was shown to depend on trafficking to lysosomes and processing by lysosomal proteases. In contrast, MERS-CoV, which contains a minimal furin cleavage site just upstream of the fusion peptide, was negatively affected by inhibition of furin, but not of lysosomal proteases. We conclude that a proteolytic cleavage site in the CoV S protein directly upstream of the fusion peptide is an essential determinant of the intracellular site of fusion.

  3. COOH-terminal isoleucine of lysosome-associated membrane protein-1 is optimal for its efficient targeting to dense secondary lysosomes.

    Science.gov (United States)

    Akasaki, Kenji; Suenobu, Michihisa; Mukaida, Maki; Michihara, Akihiro; Wada, Ikuo

    2010-12-01

    Lysosome-associated membrane protein-1 (LAMP-1) consists of a highly glycosylated luminal domain, a single-transmembrane domain and a short cytoplasmic tail that possesses a lysosome-targeting signal (GYQTI(382)) at the COOH terminus. It is hypothesized that the COOH-terminal isoleucine, I(382), could be substituted with any other bulky hydrophobic amino acid residue for LAMP-1 to exclusively localize in lysosomes. In order to test this hypothesis, we compared subcellular distribution of four substitution mutants with phenylalanine, leucine, methionine and valine at the COOH-terminus (termed I382F, I382L, I382M and I382V, respectively) with that of wild-type (WT)-LAMP-1. Double-labelled immunofluorescence analyses showed that these substitution mutants were localized as significantly to late endocytic organelles as WT-LAMP-1. However, the quantitative subcellular fractionation study revealed different distribution of WT-LAMP-1 and these four COOH-terminal mutants in late endosomes and dense secondary lysosomes. WT-LAMP-1 was accumulated three to six times more in the dense lysosomal fraction than the four mutants. The level of WT-LAMP-1 in late endosomal fraction was comparable to those of I382F, I382M and I382V. Conversely, I382L in the late endosomal fraction was approximately three times more abundant than WT-LAMP-1. These findings define the presence of isoleucine residue at the COOH-terminus of LAMP-1 as critical in governing its efficient delivery to secondary lysosomes and its ratio of lysosomes to late endosomes.

  4. Lysosomal enlargement and lysosomal membrane destabilisation in mussel digestive cells measured by an integrative index

    Energy Technology Data Exchange (ETDEWEB)

    Izagirre, Urtzi [Cell Biology in Environmental Toxicology Research Group, Department of Zoology and Cell Biology, School of Sciences and Technology, University of the Basque Country, P.O. box 644, E-48080 Bilbo (Spain); Marigomez, Ionan, E-mail: ionan.marigomez@ehu.e [Cell Biology in Environmental Toxicology Research Group, Department of Zoology and Cell Biology, School of Sciences and Technology, University of the Basque Country, P.O. box 644, E-48080 Bilbo (Spain)

    2009-05-15

    Lysosomal responses (enlargement and membrane destabilisation) in mussel digestive cells are well-known environmental stress biomarkers in pollution effects monitoring in marine ecosystems. Presently, in laboratory and field studies, both responses were measured separately (in terms of lysosomal volume density - Vv - and labilisation period -LP) and combined (lysosomal response index - LRI) in order to contribute to their understanding and to develop an index useful for decisions makers. LRI integrates Vv and LP, which are not necessarily dependent lysosomal responses. It is unbiased and more sensitive than Vv and LP alone and diminishes background due to confounding factors. LRI provides a simple numerical index (consensus reference = 0; critical threshold = 1) directly related to the pollution impact degree. Moreover, LRI can be represented in a way that allows the interpretation of lysosomal responses, which is useful for environmental scientists. - Lysosomal responses to pollutants measured by an integrative index.

  5. Presenilin 1 maintains lysosomal Ca2+ homeostasis by regulating vATPase-mediated lysosome acidification

    Science.gov (United States)

    Lee, Ju-Hyun; McBrayer, Mary Kate; Wolfe, Devin M.; Haslett, Luke J.; Kumar, Asok; Sato, Yutaka; Lie, Pearl P. Y.; Mohan, Panaiyur; Coffey, Erin E.; Kompella, Uday; Mitchell, Claire H.; Lloyd-Evans, Emyr; Nixon, Ralph A.

    2015-01-01

    Summary Presenilin-1 (PS1) deletion or Alzheimer’s Disease (AD)-linked mutations disrupt lysosomal acidification and proteolysis, which inhibits autophagy. Here, we establish that this phenotype stems from impaired glycosylation and instability of vATPase V0a1 subunit causing deficient lysosomal vATPase assembly and function. We further demonstrate that elevated lysosomal pH in PS1KO cells induces abnormal Ca2+ efflux from lysosomes mediated by TRPML1 and elevates cytosolic Ca2+. In WT cells, blocking vATPase activity or knockdown of either PS1 or the V0a1 subunit of vATPase reproduces all of these abnormalities. Normalizing lysosomal pH in PS1KO cells using acidic nanoparticles restores normal lysosomal proteolysis, autophagy, and Ca2+ homeostasis, but correcting lysosomal Ca2+ deficits alone neither re-acidifies lysosomes nor reverses proteolytic and autophagic deficits. Our results indicate that vATPase deficiency in PS1 loss of function states causes lysosomal/autophagy deficits and contributes to abnormal cellular Ca2+ homeostasis, thus linking two AD-related pathogenic processes through a common molecular mechanism. PMID:26299959

  6. Lipid storage disorders block lysosomal trafficking by inhibiting a TRP channel and lysosomal calcium release.

    Science.gov (United States)

    Shen, Dongbiao; Wang, Xiang; Li, Xinran; Zhang, Xiaoli; Yao, Zepeng; Dibble, Shannon; Dong, Xian-ping; Yu, Ting; Lieberman, Andrew P; Showalter, Hollis D; Xu, Haoxing

    2012-03-13

    Lysosomal lipid accumulation, defects in membrane trafficking and altered Ca(2+) homoeostasis are common features in many lysosomal storage diseases. Mucolipin transient receptor potential channel 1 (TRPML1) is the principle Ca(2+) channel in the lysosome. Here we show that TRPML1-mediated lysosomal Ca(2+) release, measured using a genetically encoded Ca(2+) indicator (GCaMP3) attached directly to TRPML1 and elicited by a potent membrane-permeable synthetic agonist, is dramatically reduced in Niemann-Pick (NP) disease cells. Sphingomyelins (SMs) are plasma membrane lipids that undergo sphingomyelinase (SMase)-mediated hydrolysis in the lysosomes of normal cells, but accumulate distinctively in lysosomes of NP cells. Patch-clamp analyses revealed that TRPML1 channel activity is inhibited by SMs, but potentiated by SMases. In NP-type C cells, increasing TRPML1's expression or activity was sufficient to correct the trafficking defects and reduce lysosome storage and cholesterol accumulation. We propose that abnormal accumulation of luminal lipids causes secondary lysosome storage by blocking TRPML1- and Ca(2+)-dependent lysosomal trafficking.

  7. Impact of high cholesterol in a Parkinson's disease model: Prevention of lysosomal leakage versus stimulation of α-synuclein aggregation.

    Science.gov (United States)

    Eriksson, Ida; Nath, Sangeeta; Bornefall, Per; Giraldo, Ana Maria Villamil; Öllinger, Karin

    2017-01-16

    Parkinson's disease is characterized by accumulation of intraneuronal cytoplasmic inclusions, Lewy bodies, which mainly consist of aggregated α-synuclein. Controversies exist as to whether high blood cholesterol is a risk factor for the development of the disease and whether statin treatment could have a protective effect. Using a model system of BE(2)-M17 neuroblastoma cells treated with the neurotoxin 1-methyl-4-phenylpyridinium (MPP(+)), we found that MPP(+)-induced cell death was accompanied by cholesterol accumulation in a lysosomal-like pattern in pre-apoptotic cells. To study the effects of lysosomal cholesterol accumulation, we increased lysosomal cholesterol through pre-treatment with U18666A and found delayed leakage of lysosomal contents into the cytosol, which reduced cell death. This suggests that increased lysosomal cholesterol is a stress response mechanism to protect lysosomal membrane integrity in response to early apoptotic stress. However, high cholesterol also stimulated the accumulation of α-synuclein. Treatment with the cholesterol-lowering drug lovastatin reduced MPP(+)-induced cell death by inhibiting the production of reactive oxygen species, but did not prevent lysosomal cholesterol increase nor affect α-synuclein accumulation. Our study indicates a dual role of high cholesterol in Parkinson's disease, in which it acts both as a protector against lysosomal membrane permeabilization and as a stimulator of α-synuclein accumulation.

  8. Hsp70 stabilizes lysosomes and reverts Niemann-Pick disease-associated lysosomal pathology

    DEFF Research Database (Denmark)

    Kirkegaard, Thomas; Roth, Anke G; Petersen, Nikolaj H T

    2010-01-01

    inhibition of ASM, effectively revert the Hsp70-mediated stabilization of lysosomes. Notably, the reduced ASM activity in cells from patients with Niemann-Pick disease (NPD) A and B-severe lysosomal storage disorders caused by mutations in the sphingomyelin phosphodiesterase 1 gene (SMPD1) encoding for ASM...

  9. Distributed Speech Recognition Systems and Some Key Factors Affecting It's Performance

    Institute of Scientific and Technical Information of China (English)

    YE Lei; YANG Zhen

    2003-01-01

    In this paper we first analyze the Distributed Speech Recognition (DSR) system and the key factors that affect it's performance and then focus on the research on the relationship between the length of testing speech and the recognition accuracy of the system. Some experimental results are given at last.

  10. Thematic and spatial resolutions affect model-based predictions of tree species distribution.

    Directory of Open Access Journals (Sweden)

    Yu Liang

    Full Text Available Subjective decisions of thematic and spatial resolutions in characterizing environmental heterogeneity may affect the characterizations of spatial pattern and the simulation of occurrence and rate of ecological processes, and in turn, model-based tree species distribution. Thus, this study quantified the importance of thematic and spatial resolutions, and their interaction in predictions of tree species distribution (quantified by species abundance. We investigated how model-predicted species abundances changed and whether tree species with different ecological traits (e.g., seed dispersal distance, competitive capacity had different responses to varying thematic and spatial resolutions. We used the LANDIS forest landscape model to predict tree species distribution at the landscape scale and designed a series of scenarios with different thematic (different numbers of land types and spatial resolutions combinations, and then statistically examined the differences of species abundance among these scenarios. Results showed that both thematic and spatial resolutions affected model-based predictions of species distribution, but thematic resolution had a greater effect. Species ecological traits affected the predictions. For species with moderate dispersal distance and relatively abundant seed sources, predicted abundance increased as thematic resolution increased. However, for species with long seeding distance or high shade tolerance, thematic resolution had an inverse effect on predicted abundance. When seed sources and dispersal distance were not limiting, the predicted species abundance increased with spatial resolution and vice versa. Results from this study may provide insights into the choice of thematic and spatial resolutions for model-based predictions of tree species distribution.

  11. [Spatial distribution pattern of soil nitrogen in Huanghuadianzi watershed and related affecting factors].

    Science.gov (United States)

    Li, Long; Yao, Yun-feng; Qin, Fu-cang; Gao, Yu-han; Zhang, Mei-li

    2015-05-01

    This research was conducted in Huanghuadianzi watershed in Aohan, Chifeng, Inner Mongolia. Geostatistic was used to study the spatial distribution of soil nitrogen and their affecting factors. The results showed that the soil nitrogen contents in all layers distributed as an island shape, and the high value areas were mainly distributed in the northwest of the watershed as an obvious fertile island shape, while the low value areas were mainly distributed in the south of the watershed. Nitrogen was mainly concentrated in the surface soil, and its content decreased with the increase of soil depth. The soil nitrogen content at first increased then decreased with the altitude, decreased with the slope, and showed the order of shady slope>semi-shady slope>semi-sunny slope> sunny slope in different aspects. The average soil nitrogen contents in different land use types ranked as cropland >woodland > grassland.

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

    Science.gov (United States)

    Lloyd-Evans, Emyr; Platt, Frances M

    2011-08-01

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

  13. [Spatiotemporal distribution of negative air ion concentration in urban area and related affecting factors: a review].

    Science.gov (United States)

    Huang, Xiang-Hua; Wang, Jian; Zeng, Hong-Da; Chen, Guang-Shui; Zhong, Xian-Fang

    2013-06-01

    Negative air ion (NAI) concentration is an important indicator comprehensively reflecting air quality, and has significance to human beings living environment. This paper summarized the spatiotemporal distribution features of urban NAI concentration, and discussed the causes of these features based on the characteristics of the environmental factors in urban area and their effects on the physical and chemical processes of NAI. The temporal distribution of NAI concentration is mainly controlled by the periodic variation of solar radiation, while the spatial distribution of NAI concentration along the urban-rural gradient is mainly affected by the urban aerosol distribution, underlying surface characters, and urban heat island effect. The high NAI concentration in urban green area is related to the vegetation life activities and soil radiation, while the higher NAI concentration near the water environment is attributed to the water molecules that participate in the generation of NAI through a variety of ways. The other environmental factors can also affect the generation, life span, component, translocation, and distribution of NAI to some extent. To increase the urban green space and atmospheric humidity and to maintain the soil natural attributes of underlying surface could be the effective ways to increase the urban NAI concentration and improve the urban air quality.

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

    Institute of Scientific and Technical Information of China (English)

    Jing Zhou; Shi-Hao Tan; Valérie Nicolas; Chantal Bauvy; Nai-Di Yang; Jianbin Zhang; Yuan Xue

    2013-01-01

    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 Torinl),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 Atg7deletion 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 antophagosome-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.

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

    Science.gov (United States)

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

    2013-04-01

    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.

  16. Amount and distribution of dietary protein affects clinical response to levodopa in Parkinson's disease.

    Science.gov (United States)

    Carter, J H; Nutt, J G; Woodward, W R; Hatcher, L F; Trotman, T L

    1989-04-01

    Reducing dietary protein improves the effectiveness of levodopa (LD) but the most effective distribution of a low-protein diet (0.8 g/kg) is unclear. We compared a 1.6 g/kg protein diet, a 0.8 g/kg diet with protein evenly distributed between meals, and a 0.8 g/kg diet with protein restricted to the evening meal in 5 parkinsonian patients with motor fluctuations. We monitored clinical response, plasma LD, and plasma large amino acids (LNAAs) hourly throughout the day. Mean "on" times were 51% (1.6 g/kg diet), 67% (0.8 g/kg evenly distributed), and 77% (0.8 g/kg restricted). Hourly averages of plasma LD did not differ between the diets. The mean plasma LNAAs were 732 nmol/ml (1.6 g/kg diet), 640 (0.8 g/kg distributed), and 542 (0.8 g/kg restricted), and the diurnal pattern reflected the distribution of protein intake. In conclusion, the amount and distribution of dietary protein affect clinical response to LD. These effects are not related to LD absorption but are explained by the variation in plasma LNAAs.

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

    Science.gov (United States)

    Oh, Doo-Byoung

    2015-08-01

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

  18. Effects of ethanol, acetaldehyde and cholesteryl esters on pancreatic lysosomes.

    OpenAIRE

    Wilson, J S; Apte, M V; Thomas, M. C.; Haber, P S; Pirola, R C

    1992-01-01

    Recent studies indicate that altered lysosomal function may be involved in the early stages of pancreatic injury. Chronic consumption of ethanol increases rat pancreatic lysosomal fragility. The aim of this study is to determine whether the lysosomal fragility observed after chronic ethanol consumption is mediated by ethanol per se, its oxidative metabolite acetaldehyde or cholesteryl esters (substances which accumulate in the pancreas after ethanol consumption). Pancreatic lysosomes from cho...

  19. Mucolipidosis type IV protein TRPML1-dependent lysosome formation.

    Science.gov (United States)

    Miller, Austin; Schafer, Jessica; Upchurch, Cameron; Spooner, Ellen; Huynh, Julie; Hernandez, Sebastian; McLaughlin, Brooke; Oden, Liam; Fares, Hanna

    2015-03-01

    Lysosomes are dynamic organelles that undergo cycles of fusion and fission with themselves and with other organelles. Following fusion with late endosomes to form hybrid organelles, lysosomes are reformed as discrete organelles. This lysosome reformation or formation is a poorly understood process that has not been systematically analyzed and that lacks known regulators. In this study, we quantitatively define the multiple steps of lysosome formation and identify the first regulator of this process.

  20. Transport of Lysosome-Related Organelles

    NARCIS (Netherlands)

    Jordens, Ingrid

    2005-01-01

    Many intracellular compartments, including (MHC class II-containing) lysosomes, melanosomes and phagosomes, move along microtubules in a bi-directional manner due to the alternating activities of the plus-end directed kinesin motor and the minus-end directed dynein-dynactin motor. However, it is lar

  1. Lysosomal proteolysis: effects of aging and insulin.

    Science.gov (United States)

    Gromakova, I A; Konovalenko, O A

    2003-07-01

    Age-related characteristics of the effect of insulin on the activity of lysosomal proteolytic enzymes were studied. The relationship between the insulin effect on protein degradation and insulin degradation was analyzed. The effect of insulin on the activities of lysosomal enzymes was opposite in young and old rats (inhibitory in 3-month-old and stimulatory in 24-month-old animals). The activities of proteolytic enzymes were regulated by insulin in a glucose-independent manner: similar hypoglycemic effects of insulin in animals of different ages were accompanied by opposite changes in the activities of lysosomal enzymes. The inhibition of lysosomal enzymes by insulin in 3-month-old rats is consistent with a notion on the inhibitory effect of insulin on protein degradation. An opposite insulin effect in 24-month-old rats (i.e., stimulation of proteolytic activity by insulin) may be partly associated with attenuation of the degradation of insulin, resulting in disturbances in signaling that mediates the regulatory effects of insulin on protein degradation.

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

    DEFF Research Database (Denmark)

    Frankel, Lisa B; Di Malta, Chiara; Wen, Jiayu;

    2014-01-01

    Sulfatases are key enzymatic regulators of sulfate homeostasis with several biological functions including degradation of glycosaminoglycans (GAGs) and other macromolecules in lysosomes. In a severe lysosomal storage disorder, multiple sulfatase deficiency (MSD), global sulfatase activity is defi...

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

    Science.gov (United States)

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

    2015-04-29

    Loss of function of FIG4 leads to Charcot-Marie-Tooth disease Type 4J, Yunis-Varon syndrome, or an epilepsy syndrome. FIG4 is a phosphatase with its catalytic specificity toward 5'-phosphate of phosphatidylinositol-3,5-diphosphate (PI3,5P2). However, the loss of FIG4 decreases PI3,5P2 levels likely due to FIG4's dominant effect in scaffolding a PI3,5P2 synthetic protein complex. At the cellular level, all these diseases share similar pathology with abnormal lysosomal storage and neuronal degeneration. Mice with no FIG4 expression (Fig4(-/-)) recapitulate the pathology in humans with FIG4 deficiency. Using a flow cytometry technique that rapidly quantifies lysosome sizes, we detected an impaired lysosomal fission, but normal fusion, in Fig4(-/-) cells. The fission defect was associated with a robust increase of intralysosomal Ca(2+) in Fig4(-/-) cells, including FIG4-deficient neurons. This finding was consistent with a suppressed Ca(2+) efflux of lysosomes because the endogenous ligand of lysosomal Ca(2+) channel TRPML1 is PI3,5P2 that is deficient in Fig4(-/-) cells. We reactivated the TRPML1 channels by application of TRPML1 synthetic ligand, ML-SA1. This treatment reduced the intralysosomal Ca(2+) level and rescued abnormal lysosomal storage in Fig4(-/-) culture cells and ex vivo DRGs. Furthermore, we found that the suppressed Ca(2+) efflux in Fig4(-/-) culture cells and Fig4(-/-) mouse brains profoundly downregulated the expression/activity of dynamin-1, a GTPase known to scissor organelle membranes during fission. This downregulation made dynamin-1 unavailable for lysosomal fission. Together, our study revealed a novel mechanism explaining abnormal lysosomal storage in FIG4 deficiency. Synthetic ligands of the TRPML1 may become a potential therapy against diseases with FIG4 deficiency.

  4. Lysosomal Acid Lipase Activity Is Reduced Both in Cryptogenic Cirrhosis and in Cirrhosis of Known Etiology.

    Directory of Open Access Journals (Sweden)

    Umberto Vespasiani-Gentilucci

    Full Text Available Liver cirrhosis is characterized by a severe acquired reduction of LAL-activity, the precise causes and consequences of which need to be further addressed. DBS-determined lysosomal enzyme activities seem to be affected by white blood cell and platelet counts, and the specificity of these tests can be reduced when applied to determined populations, such as cirrhotics.

  5. The role of VAMP7/TI-VAMP in cell polarity and lysosomal exocytosis in vivo.

    Science.gov (United States)

    Sato, Mahito; Yoshimura, Shinichiro; Hirai, Rika; Goto, Ayako; Kunii, Masataka; Atik, Nur; Sato, Takashi; Sato, Ken; Harada, Reiko; Shimada, Junko; Hatabu, Toshimitsu; Yorifuji, Hiroshi; Harada, Akihiro

    2011-10-01

    VAMP7 or tetanus neurotoxin-insensitive vesicle- associated membrane protein (TI-VAMP) has been proposed to regulate apical transport in polarized epithelial cells, axonal transport in neurons and lysosomal exocytosis. To investigate the function of VAMP7 in vivo, we generated VAMP7 knockout mice. Here, we show that VAMP7 knockout mice are indistinguishable from control mice and display a similar localization of apical proteins in the kidney and small intestine and a similar localization of axonal proteins in the nervous system. Neurite outgrowth of cultured mutant hippocampal neurons was reduced in mutant neurons. However, lysosomal exocytosis was not affected in mutant fibroblasts. Our results show that VAMP7 is required in neurons to extend axons to the full extent. However, VAMP7 does not seem to be required for epithelial cell polarity and lysosomal exocytosis.

  6. Social information affects adults' evaluation of fairness in distributions: An ERP approach.

    Science.gov (United States)

    Ishikawa, Mitsuhiko; Park, Yun-Hee; Kitazaki, Michiteru; Itakura, Shoji

    2017-01-01

    The sense of fairness has been observed in early infancy. Because many studies of fairness in adults have used economic games such as the Ultimatum Game, it has been difficult to compare fairness between adults and infants. Further, recent studies have suggested that social information about actors who behave fairly or unfairly may influence the judgement of fairness in infants. Therefore, to compare the sense of fairness between infants and adults, the study using paradigm in infant research is required. We examined how social information about two characters, either prosocial or antisocial, affects the event-related potential response (ERP) to fair or unfair resource distributions in adults. In the habituation phase, participants were informed about characters' social information through their actions. One character then distributed resources fairly or unfairly, and ERP was measured at the end of the distribution. Data from eighteen adult participants were analysed. A significant interaction of social information and fairness was found for late positive potential (LPP), but a post-hoc t test revealed a significant difference between fair and unfair conditions only for actions of the antisocial character. We found that LPP can reflect the sense of fairness affected by social information. Comparison with infant studies suggests that the sense of fairness may change during development.

  7. Social information affects adults’ evaluation of fairness in distributions: An ERP approach

    Science.gov (United States)

    Ishikawa, Mitsuhiko; Park, Yun-hee; Kitazaki, Michiteru; Itakura, Shoji

    2017-01-01

    The sense of fairness has been observed in early infancy. Because many studies of fairness in adults have used economic games such as the Ultimatum Game, it has been difficult to compare fairness between adults and infants. Further, recent studies have suggested that social information about actors who behave fairly or unfairly may influence the judgement of fairness in infants. Therefore, to compare the sense of fairness between infants and adults, the study using paradigm in infant research is required. We examined how social information about two characters, either prosocial or antisocial, affects the event-related potential response (ERP) to fair or unfair resource distributions in adults. In the habituation phase, participants were informed about characters’ social information through their actions. One character then distributed resources fairly or unfairly, and ERP was measured at the end of the distribution. Data from eighteen adult participants were analysed. A significant interaction of social information and fairness was found for late positive potential (LPP), but a post-hoc t test revealed a significant difference between fair and unfair conditions only for actions of the antisocial character. We found that LPP can reflect the sense of fairness affected by social information. Comparison with infant studies suggests that the sense of fairness may change during development. PMID:28235082

  8. Factors affecting the distribution of natural and anthropogenic radionuclides in the coastal Burullus Lake.

    Science.gov (United States)

    El-Reefy, H I; Badran, H M; Sharshar, T; Hilal, M A; Elnimr, T

    2014-08-01

    In the present study, measurements of naturally occurring radioactive materials and (137)Cs activity in sediment were conducted for locations covering the entire Burullus Lake in order to gather information about radionuclides mobility and distribution. Low-background γ-spectrometry was employed to determine the activity concentrations of water and sediment samples. The activity concentrations of (226)Ra and (232)Th are close to uniform distribution in the lake environment. Among the different physical and chemical characteristics measured for water and sediment, only salinity and total organic matter content have the potential to affect the mobility of (137)Cs and (40)K. The results suggest that these two radionuclides are attached to different mobile particulates. Increasing salinity tends to strengthen the adsorption of (137)Cs and solubilization of (40)K in sediment. On the other hand, sediment with high organic matter content traps (137)Cs and (40)K associated particulates to bottom sediment.

  9. Probability distribution functions of turbulence in seepage-affected alluvial channel

    Science.gov (United States)

    Sharma, Anurag; Kumar, Bimlesh

    2017-02-01

    The present experimental study is carried out on the probability distribution functions (PDFs) of turbulent flow characteristics within near-bed-surface and away-from-bed surfaces for both no seepage and seepage flow. Laboratory experiments were conducted in the plane sand bed for no seepage (NS), 10% seepage (10%S) and 15% seepage (15%) cases. The experimental calculation of the PDFs of turbulent parameters such as Reynolds shear stress, velocity fluctuations, and bursting events is compared with theoretical expression obtained by Gram-Charlier (GC)-based exponential distribution. Experimental observations follow the computed PDF distributions for both no seepage and seepage cases. Jensen-Shannon divergence (JSD) method is used to measure the similarity between theoretical and experimental PDFs. The value of JSD for PDFs of velocity fluctuation lies between 0.0005 to 0.003 while the JSD value for PDFs of Reynolds shear stress varies between 0.001 to 0.006. Even with the application of seepage, the PDF distribution of bursting events, sweeps and ejections are well characterized by the exponential distribution of the GC series, except that a slight deflection of inward and outward interactions is observed which may be due to weaker events. The value of JSD for outward and inward interactions ranges from 0.0013 to 0.032, while the JSD value for sweep and ejection events varies between 0.0001 to 0.0025. The theoretical expression for the PDF of turbulent intensity is developed in the present study, which agrees well with the experimental observations and JSD lies between 0.007 and 0.015. The work presented is potentially applicable to the probability distribution of mobile-bed sediments in seepage-affected alluvial channels typically characterized by the various turbulent parameters. The purpose of PDF estimation from experimental data is that it provides a complete numerical description in the areas of turbulent flow either at a single or finite number of points.

  10. A decrease in phytic acid content substantially affects the distribution of mineral elements within rice seeds.

    Science.gov (United States)

    Sakai, Hiroaki; Iwai, Toru; Matsubara, Chie; Usui, Yuto; Okamura, Masaki; Yatou, Osamu; Terada, Yasuko; Aoki, Naohiro; Nishida, Sho; Yoshida, Kaoru T

    2015-09-01

    Phytic acid (myo-inositol hexakisphosphate; InsP6) is the storage compound of phosphorus and many mineral elements in seeds. To determine the role of InsP6 in the accumulation and distribution of mineral elements in seeds, we performed fine mappings of mineral elements through synchrotron-based X-ray microfluorescence analysis using developing seeds from two independent low phytic acid (lpa) mutants of rice (Oryza sativa L.). The reduced InsP6 in lpa seeds did not affect the translocation of mineral elements from vegetative organs into seeds, because the total amounts of phosphorus and the other mineral elements in lpa seeds were identical to those in the wild type (WT). However, the reduced InsP6 caused large changes in mineral localization within lpa seeds. Phosphorus and potassium in the aleurone layer of lpa greatly decreased and diffused into the endosperm. Zinc and copper, which were broadly distributed from the aleurone layer to the inner endosperm in the WT, were localized in the narrower space around the aleurone layer in lpa mutants. We also confirmed that similar distribution changes occurred in transgenic rice with the lpa phenotype. Using these results, we discussed the role of InsP6 in the dynamic accumulation and distribution patterns of mineral elements during seed development.

  11. Evaluating how species niche modelling is affected by partial distributions with an empirical case

    Science.gov (United States)

    Carretero, Miguel A.; Sillero, Neftalí

    2016-11-01

    Ecological niche models (ENMs) will successfully identify a species' ecological niche, provided that important assumptions are fulfilled, namely environment equilibrium and niche equality across the distribution. Violations may seriously affect ENM reliability, leading to erroneous biogeographic conclusions and inappropriate conservation prioritisation. We evaluate the robustness of ENMs against incomplete knowledge of distribution with a real example, the threatened Iberian lizard Podarcis carbonelli, whose distribution was gradually discovered over a long time period. We used several ENM methods for presence-only data (Maxent, ENFA, Bioclim, and Domain) to infer the realised ecological niche at two spatial resolutions (1 km and 200 m). The distribution data were split into four partial datasets corresponding to separate subranges: Central System (CS); Viseu-Aveiro (VA); Atlantic coast (AC); and Doñana (DO). We then accumulated the datasets following the species discovery sequence: CS + VA, CS + VA + AC, and CS + VA + AC + DO. Niche equivalence and similarity between partial models were compared using Ecospat. ENMs were strongly affected by the violation of niche equilibrium; only the VA subrange forecasts the complete species range. ENMs were also sensitive to the violation of niche equality: only VA models were similar to the Iberian model, altitude being the most important variable followed by annual precipitation, maximum temperature in July, and annual radiation. When the ENMs were applied only to the first subrange discovered (CS), only the VA area was predicted, while the other subranges might have remained unknown, thus compromising conservation strategies. As assumptions of niche equilibrium and equality were violated, likely owing to the species' ecological multimodality, the models generated were biased and of limited predictive value. ENMs are useful tools in biogeography and conservation, but only if their basal assumptions are achieved. Partial

  12. Geographic distance affects dispersal of the patchy distributed greater long-tailed hamster (Tscherskia triton).

    Science.gov (United States)

    Xue, Huiliang; Zhong, Min; Xu, Jinhui; Xu, Laixiang

    2014-01-01

    Dispersal is a fundamental process in ecology influencing the genetic structure and the viability of populations. Understanding how variable factors influence the dispersal of the population is becoming an important question in animal ecology. To date, geographic distance and geographic barriers are often considered as main factors impacting dispersal, but their effects are variable depending on different conditions. In general, geographic barriers affect more significantly than geographic distance on dispersal. In rapidly expanding populations, however, geographic barriers have less effect on dispersal than geographic distance. The effects of both geographic distance and geographic barriers in low-density populations with patchy distributions are poorly understood. By using a panel of 10 microsatellite loci we investigated the genetic structure of three patchy-distributed populations of the Greater long-tailed hamster (Tscherskia triton) from Raoyang, Guan and Shunyi counties of the North China Plain. The results showed that (i) high genetic diversity and differentiation exist in three geographic populations with patchy distributions; (ii) gene flow occurs among these three populations with physical barriers of Beijing city and Hutuo River, which potentially restricted the dispersal of the animal; (iii) the gene flow is negatively correlated with the geographic distance, while the genetic distance shows the positive correlation. Our results suggest that the effect of the physical barriers is conditional-dependent, including barrier capacity or individual potentially dispersal ability. Geographic distance also acts as an important factor affecting dispersal for the patchy distributed geographic populations. So, gene flow is effective, even at relatively long distances, in balancing the effect of geographic barrier in this study.

  13. Statistical analysis of factors affecting landslide distribution in the new Madrid seismic zone, Tennessee and Kentucky

    Science.gov (United States)

    Jibson, R.W.; Keefer, D.K.

    1989-01-01

    More than 220 large landslides along the bluffs bordering the Mississippi alluvial plain between Cairo, Ill., and Memphis, Tenn., are analyzed by discriminant analysis and multiple linear regression to determine the relative effects of slope height and steepness, stratigraphic variation, slope aspect, and proximity to the hypocenters of the 1811-12 New Madrid, Mo., earthquakes on the distribution of these landslides. Three types of landslides are analyzed: (1) old, coherent slumps and block slides, which have eroded and revegetated features and no active analogs in the area; (2) old earth flows, which are also eroded and revegetated; and (3) young rotational slumps, which are present only along near-river bluffs, and which are the only young, active landslides in the area. Discriminant analysis shows that only one characteristic differs significantly between bluffs with and without young rotational slumps: failed bluffs tend to have sand and clay at their base, which may render them more susceptible to fluvial erosion. Bluffs having old coherent slides are significantly higher, steeper, and closer to the hypocenters of the 1811-12 earthquakes than bluffs without these slides. Bluffs having old earth flows are likewise higher and closer to the earthquake hypocenters. Multiple regression analysis indicates that the distribution of young rotational slumps is affected most strongly by slope steepness: about one-third of the variation in the distribution is explained by variations in slope steepness. The distribution of old coherent slides and earth flows is affected most strongly by slope height, but the proximity to the hypocenters of the 1811-12 earthquakes also significantly affects the distribution. The results of the statistical analyses indicate that the only recently active landsliding in the area is along actively eroding river banks, where rotational slumps formed as bluffs are undercut by the river. The analyses further indicate that the old coherent slides

  14. Rapid recycling of Ca2+ between IP3-sensitive stores and lysosomes.

    Directory of Open Access Journals (Sweden)

    Cristina I López Sanjurjo

    Full Text Available Inositol 1,4,5-trisphosphate (IP3 evokes release of Ca2+ from the endoplasmic reticulum (ER, but the resulting Ca2+ signals are shaped by interactions with additional intracellular organelles. Bafilomycin A1, which prevents lysosomal Ca2+ uptake by inhibiting H+ pumping into lysosomes, increased the amplitude of the initial Ca2+ signals evoked by carbachol in human embryonic kidney (HEK cells. Carbachol alone and carbachol in combination with parathyroid hormone (PTH evoke Ca2+ release from distinct IP3-sensitive Ca2+ stores in HEK cells stably expressing human type 1 PTH receptors. Bafilomycin A1 similarly exaggerated the Ca2+ signals evoked by carbachol or carbachol with PTH, indicating that Ca2+ released from distinct IP3-sensitive Ca2+ stores is sequestered by lysosomes. The Ca2+ signals resulting from store-operated Ca2+ entry, whether evoked by thapsigargin or carbachol, were unaffected by bafilomycin A1. Using Gd3+ (1 mM to inhibit both Ca2+ entry and Ca2+ extrusion, HEK cells were repetitively stimulated with carbachol to assess the effectiveness of Ca2+ recycling to the ER after IP3-evoked Ca2+ release. Blocking lysosomal Ca2+ uptake with bafilomycin A1 increased the amplitude of each carbachol-evoked Ca2+ signal without affecting the rate of Ca2+ recycling to the ER. This suggests that Ca2+ accumulated by lysosomes is rapidly returned to the ER. We conclude that lysosomes rapidly, reversibly and selectively accumulate the Ca2+ released by IP3 receptors residing within distinct Ca2+ stores, but not the Ca2+ entering cells via receptor-regulated, store-operated Ca2+ entry pathways.

  15. Main Factors Affecting the Distribution of the Macroscopic Destruction Field of Earthquake

    Institute of Scientific and Technical Information of China (English)

    Li Minfeng; Li Shengqiang; Chen Yong; Mi Hongliang

    2001-01-01

    It is proposed that some possible macroseismic epicenters can be determined quickly from the relationship that the microseismic epicenters located by instruments bear with faults. Based on these so-called macroseismic epicenters, we can make fast seismic hazard estimation after a shock by use of the empirical distribution model of seismic intensity. In comparison with the method that uses the microseismic epicenters directly, this approach can increase the precision of fast seismic hazard estimation. Statistical analysis of 133 main earthquakes in China was made. The result shows that the deviation distance between the microseismic epicenter and macroseismic epicenter falls within the range of 35 km for 88 % earthquakes of the total and within the range of 35 to 75 km for the remaining ones. Then, we can take the area that has the microseismic epicenter as its center and is 35 km in radius as the area for emphatic analysis, and take the area within 75 km around the microseismic epicenter as the area for general analysis. The relation between the 66 earthquake cases on the N-S Seismic Belt in China and the spatial distribution characteristics of faults and the results of focal mechanism solution were analyzed in detail. We know from the analysis that the error of instrumental epicenter determination is not the only factor that gives effects to the deviation of the macroseismic epicenter. In addition to it, the fault size, fault distribution, fault activity, fault intersection types, earthquake magnitude, etc. are also main affecting factors. By sorting out,processing and analyzing these affecting factors, the principle and procedures for quickly determining the possible position of the macroseismic epicenter were set up. Taking these as a basis and establishing a nationwide database of faults that contains relevant factors, it is possible to apply this method in practical fast estimation of seismic hazard.

  16. Hypoxia Affects Nitrogen Uptake and Distribution in Young Poplar (Populus × canescens Trees.

    Directory of Open Access Journals (Sweden)

    Bin Liu

    Full Text Available The present study with young poplar trees aimed at characterizing the effect of O2 shortage in the soil on net uptake of NO3- and NH4+ and the spatial distribution of the N taken up. Moreover, we assessed biomass increment as well as N status of the trees affected by O2 deficiency. For this purpose, an experiment was conducted in which hydroponically grown young poplar trees were exposed to hypoxic and normoxic (control conditions for 14 days. 15N-labelled NO3- and NH4+ were used to elucidate N uptake and distribution of currently absorbed N and N allocation rates in the plants. Whereas shoot biomass was not affected by soil O2 deficiency, it significantly reduced root biomass and, consequently, the root-to-shoot ratio. Uptake of NO3- but not of NH4+ by the roots of the trees was severely impaired by hypoxia. As a consequence of reduced N uptake, the N content of all poplar tissues was significantly diminished. Under normoxic control conditions, the spatial distribution of currently absorbed N and N allocation rates differed depending on the N source. Whereas NO3- derived N was mainly transported to the younger parts of the shoot, particularly to the developing and young mature leaves, N derived from NH4+ was preferentially allocated to older parts of the shoot, mainly to wood and bark. Soil O2 deficiency enhanced this differential allocation pattern. From these results we assume that NO3- was assimilated in developing tissues and preferentially used to maintain growth and ensure plant survival under hypoxia, whereas NH4+ based N was used for biosynthesis of storage proteins in bark and wood of the trees. Still, further studies are needed to understand the mechanistic basis as well as the eco-physiological advantages of such differential allocation patterns.

  17. Biphasic regulation of lysosomal exocytosis by oxidative stress.

    Science.gov (United States)

    Ravi, Sreeram; Peña, Karina A; Chu, Charleen T; Kiselyov, Kirill

    2016-11-01

    Oxidative stress drives cell death in a number of diseases including ischemic stroke and neurodegenerative diseases. A better understanding of how cells recover from oxidative stress is likely to lead to better treatments for stroke and other diseases. The recent evidence obtained in several models ties the process of lysosomal exocytosis to the clearance of protein aggregates and toxic metals. The mechanisms that regulate lysosomal exocytosis, under normal or pathological conditions, are only beginning to emerge. Here we provide evidence for the biphasic effect of oxidative stress on lysosomal exocytosis. Lysosomal exocytosis was measured using the extracellular levels of the lysosomal enzyme beta-hexosaminidase (ß-hex). Low levels or oxidative stress stimulated lysosomal exocytosis, but inhibited it at high levels. Deletion of the lysosomal ion channel TRPML1 eliminated the stimulatory effect of low levels of oxidative stress. The inhibitory effects of oxidative stress appear to target the component of lysosomal exocytosis that is driven by extracellular Ca(2+). We propose that while moderate oxidative stress promotes cellular repair by stimulating lysosomal exocytosis, at high levels oxidative stress has a dual pathological effect: it directly causes cell damage and impairs damage repair by inhibiting lysosomal exocytosis. Harnessing these adaptive mechanisms may point to pharmacological interventions for diseases involving oxidative proteotoxicity or metal toxicity.

  18. Regulation of lysosomal ion homeostasis by channels and transporters.

    Science.gov (United States)

    Xiong, Jian; Zhu, Michael X

    2016-08-01

    Lysosomes are the major organelles that carry out degradation functions. They integrate and digest materials compartmentalized by endocytosis, phagocytosis or autophagy. In addition to more than 60 hydrolases residing in the lysosomes, there are also ion channels and transporters that mediate the flux or transport of H(+), Ca(2+), Na(+), K(+), and Cl(-) across the lysosomal membranes. Defects in ionic exchange can lead to abnormal lysosome morphology, defective vesicle trafficking, impaired autophagy, and diseases such as neurodegeneration and lysosomal storage disorders. The latter are characterized by incomplete lysosomal digestion and accumulation of toxic materials inside enlarged intracellular vacuoles. In addition to degradation, recent studies have revealed the roles of lysosomes in metabolic pathways through kinases such as mechanistic target of rapamycin (mTOR) and transcriptional regulation through calcium signaling molecules such as transcription factor EB (TFEB) and calcineurin. Owing to the development of new approaches including genetically encoded fluorescence probes and whole endolysosomal patch clamp recording techniques, studies on lysosomal ion channels have made remarkable progress in recent years. In this review, we will focus on the current knowledge of lysosome-resident ion channels and transporters, discuss their roles in maintaining lysosomal function, and evaluate how their dysfunction can result in disease.

  19. Cyclase-associated protein is essential for the functioning of the endo-lysosomal system and provides a link to the actin cytoskeleton.

    Science.gov (United States)

    Sultana, Hameeda; Rivero, Francisco; Blau-Wasser, Rosemarie; Schwager, Stephan; Balbo, Alessandra; Bozzaro, Salvatore; Schleicher, Michael; Noegel, Angelika A

    2005-10-01

    Data from mutant analysis in yeast and Dictyostelium indicate a role for the cyclase-associated protein (CAP) in endocytosis and vesicle transport. We have used genetic and biochemical approaches to identify novel interacting partners of Dictyostelium CAP to help explain its molecular interactions in these processes. Cyclase-associated protein associates and interacts with subunits of the highly conserved vacuolar H(+)-ATPase (V-ATPase) and co-localizes to some extent with the V-ATPase. Furthermore, CAP is essential for maintaining the structural organization, integrity and functioning of the endo-lysosomal system, as distribution and morphology of V-ATPase- and Nramp1-decorated membranes were disturbed in a CAP mutant (CAP bsr) accompanied by an increased endosomal pH. Moreover, concanamycin A (CMA), a specific inhibitor of the V-ATPase, had a more severe effect on CAP bsr than on wild-type cells, and the mutant did not show adaptation to the drug. Also, the distribution of green fluorescent protein-CAP was affected upon CMA treatment in the wildtype and recovered after adaptation. Distribution of the V-ATPase in CAP bsr was drastically altered upon hypo-osmotic shock, and growth was slower and reached lower saturation densities in the mutant under hyper-osmotic conditions. Taken together, our data unravel a link of CAP with the actin cytoskeleton and endocytosis and suggest that CAP is an essential component of the endo-lysosomal system in Dictyostelium.

  20. BAX channel activity mediates lysosomal disruption linked to Parkinson disease.

    Science.gov (United States)

    Bové, Jordi; Martínez-Vicente, Marta; Dehay, Benjamin; Perier, Celine; Recasens, Ariadna; Bombrun, Agnes; Antonsson, Bruno; Vila, Miquel

    2014-05-01

    Lysosomal disruption is increasingly regarded as a major pathogenic event in Parkinson disease (PD). A reduced number of intraneuronal lysosomes, decreased levels of lysosomal-associated proteins and accumulation of undegraded autophagosomes (AP) are observed in PD-derived samples, including fibroblasts, induced pluripotent stem cell-derived dopaminergic neurons, and post-mortem brain tissue. Mechanistic studies in toxic and genetic rodent PD models attribute PD-related lysosomal breakdown to abnormal lysosomal membrane permeabilization (LMP). However, the molecular mechanisms underlying PD-linked LMP and subsequent lysosomal defects remain virtually unknown, thereby precluding their potential therapeutic targeting. Here we show that the pro-apoptotic protein BAX (BCL2-associated X protein), which permeabilizes mitochondrial membranes in PD models and is activated in PD patients, translocates and internalizes into lysosomal membranes early following treatment with the parkinsonian neurotoxin MPTP, both in vitro and in vivo, within a time-frame correlating with LMP, lysosomal disruption, and autophagosome accumulation and preceding mitochondrial permeabilization and dopaminergic neurodegeneration. Supporting a direct permeabilizing effect of BAX on lysosomal membranes, recombinant BAX is able to induce LMP in purified mouse brain lysosomes and the latter can be prevented by pharmacological blockade of BAX channel activity. Furthermore, pharmacological BAX channel inhibition is able to prevent LMP, restore lysosomal levels, reverse AP accumulation, and attenuate mitochondrial permeabilization and overall nigrostriatal degeneration caused by MPTP, both in vitro and in vivo. Overall, our results reveal that PD-linked lysosomal impairment relies on BAX-induced LMP, and point to small molecules able to block BAX channel activity as potentially beneficial to attenuate both lysosomal defects and neurodegeneration occurring in PD.

  1. Flooding affects uptake and distribution of carbon and nitrogen in citrus seedlings.

    Science.gov (United States)

    Martínez-Alcántara, Belén; Jover, Sara; Quiñones, Ana; Forner-Giner, María Ángeles; Rodríguez-Gamir, Juan; Legaz, Francisco; Primo-Millo, Eduardo; Iglesias, Domingo J

    2012-08-15

    Soil flooding has been widely reported to affect large areas of the world. In this work, we investigated the effect of waterlogging on citrus carbon and nitrogen pools and partitioning. Influence on their uptake and translocation was also studied through ¹⁵N and ¹³C labeling to provide insight into the physiological mechanisms underlying the responses. The data indicated that flooding severely reduced photosynthetic activity and affected growth and biomass partitioning. Total nitrogen content and concentration in the plant also progressively decreased throughout the course of the experiment. After 36 days of treatment, nitrogen content of flooded plants had decreased more than 2.3-fold compared to control seedlings, and reductions in nitrogen concentration ranged from 21 to 55% (in roots and leaves, respectively). Specific absorption rate and transport were also affected, leading to important changes in the distribution of this element inside the plant. Additionally, experiments involving labeled nitrogen revealed that ¹⁵N uptake rate and accumulation were drastically decreased at the end of the experiment (93% and 54%, respectively). ¹³CO₂ assimilation into the plant was strongly reduced by flooding, with δ¹³C reductions ranging from 22 to 37% in leaves and roots, respectively. After 36 days, the relative distribution of absorbed ¹³C was also altered. Thus, ¹³C recovery in flooded leaves increased compared to controls, whereas roots exhibited the opposite pattern. Interestingly, when carbohydrate partitioning was examined, the data revealed that sucrose concentration was augmented significantly in roots (37-56%), whereas starch was reduced. In leaves, a marked increase in sucrose was detected from the first sampling onwards (36-66%), and the same patter was observed for starch. Taken together, these results indicate that flooding altered carbon and nitrogen pools and partitioning in citrus. On one hand, reduced nitrogen concentration appears to

  2. How Are Distributed Groups Affected by an Imposed Structuring of their Decision-Making Process?

    DEFF Research Database (Denmark)

    Lundell, Anders Lorentz; Hertzum, Morten

    2011-01-01

    Groups often suffer from ineffective communication and decision making. This experimental study compares distributed groups solving a preference task with support from either a communication system or a system providing both communication and a structuring of the decision-making process. Results...... show that groups using the latter system spend more time solving the task, spend more of their time on solution analysis, spend less of their time on disorganized activity, and arrive at task solutions with less extreme preferences. Thus, the type of system affects the decision-making process as well...... as its outcome. Notably, the task solutions arrived at by the groups using the system that imposes a structuring of the decision-making process show limited correlation with the task solutions suggested by the system on the basis of the groups’ explicitly stated criteria. We find no differences in group...

  3. Fish in shallow areas in Moreton Bay, Queensland and factors affecting their distribution

    Science.gov (United States)

    Weng, H. T.

    1990-06-01

    Five shallow areas (sea grass, river mouth, mangrove fringe and sewage outlet. A total of 86 fish species were recorded. Five species (6%) were regarded as true marine fish but these were of rare occurrence (0·5%). Four species (5%) were highly abundant (51%) and 57 species (66%) were represented by low numbers of fish (5%). The sewage outlet and the river mouth sites were found to contain the highest number of fish with relatively low numbers of species. The sand-drifting and the sea grass sites were different in fish fauna, and both were different from the remaining three sites which were of high similarity in fish faunal groups. Food availability, preference for habitats, sea grass vegetation, juvenile/adult segregation, season of juvenile recruitment and hydrological characteristics were thought to be important factors affecting the distribution of these species of fish.

  4. Cloning and expression of mouse legumain, a lysosomal endopeptidase.

    Science.gov (United States)

    Chen, J M; Dando, P M; Stevens, R A; Fortunato, M; Barrett, A J

    1998-01-01

    Legumain, a recently discovered mammalian cysteine endopeptidase, was found in all mouse tissues examined, but was particularly abundant in kidney and placenta. The distribution in subcellular fractions of mouse and rat kidney showed a lysosomal localization, and activity was detectable only after the organelles were disrupted. Nevertheless, ratios of legumain activity to that of cathepsin B differed considerably between mouse tissues. cDNA encoding mouse legumain was cloned and sequenced, the deduced amino acid sequence proving to be 83% identical to that of the human protein [Chen, Dando, Rawlings, Brown, Young, Stevens, Hewitt, Watts and Barrett (1997) J. Biol. Chem. 272, 8090-8098]. Recombinant mouse legumain was expressed in human embryonic kidney 293 cells by use of a vector containing a cytomegalovirus promoter. The recombinant enzyme was partially purified and found to be an asparagine-specific endopeptidase closely similar to naturally occurring pig kidney legumain. PMID:9742219

  5. Does day length affect winter bird distribution? Testing the role of an elusive variable.

    Directory of Open Access Journals (Sweden)

    Luis M Carrascal

    Full Text Available Differences in day length may act as a critical factor in bird biology by introducing time constraints in energy acquisition during winter. Thus, differences in day length might operate as a main determinant of bird abundance along latitudinal gradients. This work examines the influence of day length on the abundance of wintering crested tits (Lophophanes cristatus in 26 localities of Spanish juniper (Juniperus thurifera dwarf woodlands (average height of 5 m located along a latitudinal gradient in the Spanish highlands, while controlling for the influence of food availability, minimum night temperature, habitat structure and landscape characteristics. Top regression models in the AIC framework explained 56% of variance in bird numbers. All models incorporated day length as the variable with the highest magnitude effect. Food availability also played an important role, although only the crop of ripe juniper fruits, but not arthropods, positively affected crested tit abundance. Differences in vegetation structure across localities had also a strong positive effect (average tree height and juniper tree density. Geographical variation in night temperature had no influence on crested tit distribution, despite the low winter temperatures reached in these dwarf forests. This paper demonstrates for the first time that winter bird abundance increases with day length after controlling for the effect of other environmental variables. Winter average difference in day length was only 10.5 minutes per day along the 1°47' latitudinal interval (190 km included in this study. This amount of time, which reaches 13.5 h accumulated throughout the winter season, appears to be large enough to affect the long-term energy budget of small passerines during winter and to shape the distribution of winter bird abundance under restrictive environmental conditions.

  6. The governance of natural resources: Issues affecting better management of revenues and distribution of benefits within Papua New Guinea

    Directory of Open Access Journals (Sweden)

    Hitelai Polume-Kiele

    2014-09-01

    The focus of the article’s discussion is on governance and management issues that affect the distribution of benefits, delivery of essential services to rural areas of PNG, stability within government, and the expectations of landowners.

  7. Coal fly ash effluent affects the distributions of Brachionus calyciflorus sibling species.

    Science.gov (United States)

    Zhang, Gen; Xi, Yi-Long; Xue, Ying-Hao; Xiang, Xian-Ling; Wen, Xin-Li

    2015-02-01

    Fly ash, a coal combustion residue of thermal power plants and a source of multiple pollutants, has been recognized as an environmental hazard all over the world. Although it is known that fly ash effluent affects density, diversity and distribution of rotifers in drainage systems and receiving water bodies, the effect of fly ash effluent on the distributions of highly similar rotifer species remains unknown. In this study, the mtDNA COI genes of 90 individuals in Brachionus calyciflorus complex from Lake Hui (as a fly ash discharge water pond) and other two neighboring lakes (Lake Fengming and Lake Tingtang) were sequenced and analyzed, and the responses in selected life table demographic parameters (life expectancy at hatching, net reproductive rate, intrinsic rate of population increase and proportion of sexual offspring) of different rotifer populations to fly ash effluent were investigated. Overall, 72 mtDNA haplotypes were defined, and were split into two clades by the phylogenetic trees. The divergence of COI gene sequences between the two clades ranged from 11.8% to17.8%, indicating the occurrence of two sibling species (sibling species I and sibling species II). Sibling species I distributed in all the three lakes, showing strong capabilities for dispersal and colonization, which were supported by its higher level of gene flow (2.60-4.04) between the populations from Lake Hui and each of the other two lakes, longer life expectancy at hatching (101.6-148.2 h), and higher net reproductive rate (4.4-16.4 offspring/female) and intrinsic rate of population increase (0.60-0.98/d) when cultured in aerated tap water and fly ash effluent. Sibling species II distributed in both Lake Tingtang and Lake Fengming, showing that its dispersal existed between the two lakes. Considering that the distance between Lake Hui and Lake Fengming is shorter than that between Lake Tingtang and Lake Fengming, sibling species II is able to disperse at least from Lake Fengming to Lake

  8. Scale-dependent factors affecting North American river otter distribution in the midwest

    Science.gov (United States)

    Jeffress, Mackenzie R.; Paukert, C.P.; Whittier, Joanna B.; Sandercock, B.K.; Gipson, P.S.

    2011-01-01

    The North American river otter (Lontra canadensis) is recovering from near extirpation throughout much of its range. Although reintroductions, trapping regulations and habitat improvements have led to the reestablishment of river otters in the Midwest, little is known about how their distribution is influenced by local- and landscape-scale habitat. We conducted river otter sign surveys from Jan. to Apr. in 2008 and 2009 in eastern Kansas to assess how local- and landscape-scale habitat factors affect river otter occupancy. We surveyed three to nine 400-m stretches of stream and reservoir shorelines for 110 sites and measured local-scale variables (e.g., stream order, land cover types) within a 100 m buffer of the survey site and landscape-scale variables (e.g., road density, land cover types) for Hydrological Unit Code 14 watersheds. We then used occupancy models that account for the probability of detection to estimate occupancy as a function of these covariates using Program PRESENCE. The best-fitting model indicated river otter occupancy increased with the proportion of woodland cover and decreased with the proportion of cropland and grassland cover at the local scale. Occupancy also increased with decreased shoreline diversity, waterbody density and stream density at the landscape scale. Occupancy was not affected by land cover or human disturbance at the landscape scale. Understanding the factors and scale important to river otter occurrence will be useful in identifying areas for management and continued restoration. ?? 2011, American Midland Naturalist.

  9. Evaluating How Circle of Willis Topology Affects Embolus Distribution in the Brain

    Science.gov (United States)

    Jani, Neel; Mukherjee, Debanjan; Shadden, Shawn

    2016-11-01

    Embolic stroke occurs when fragmented cellular or acellular material (emboli) travels to the brain to occlude an artery. Understanding the transport of emboli across unsteady, pulsatile flow in complex arterial geometries is challenging and influenced by a range of factors, including patient anatomy. The work herein develops the modeling and mechanistic understanding of how embolus transport is affected by the arterial connections at the base of the brain known as the Circle of Willis (CoW). A majority of the human population has an incomplete CoW anatomy, with connections either missing or ill-developed. We employ numerical simulations combining image-based modeling, computational fluid dynamics, discrete particle dynamics, and a sampling based analysis to compare collateral flow through the most prevalent CoW topologies, to determine embolus distribution fractions among vessels in the CoW, and to investigate the role of inertial effects in causing differences in flow and embolus distribution. The computational framework developed enables characterization of the complex interplay of anatomy, hemodynamics, and embolus properties in the context of embolic stroke as well as statistical analysis of embolism risks across common CoW variations.

  10. Factors affecting continued use of ceramic water purifiers distributed to Tsunami-affected Communities in Sri Lanka

    OpenAIRE

    Casanova, Lisa M.; Walters, Adam; Naghawatte, Ajith; Sobsey, Mark D.

    2012-01-01

    Objectives  There is little information about continued use of point-of-use technologies after disaster relief efforts. After the 2004 tsunami, the Red Cross distributed ceramic water filters in Sri Lanka. This study determined factors associated with filter disuse and evaluate the quality of household drinking water. Methods  A cross-sectional survey of water sources and treatment, filter use and household characteristics was administered by in-person oral interview, and household water qual...

  11. Quantitative modeling of selective lysosomal targeting for drug design

    DEFF Research Database (Denmark)

    Trapp, Stefan; Rosania, G.; Horobin, R.W.;

    2008-01-01

    Lysosomes are acidic organelles and are involved in various diseases, the most prominent is malaria. Accumulation of molecules in the cell by diffusion from the external solution into cytosol, lysosome and mitochondrium was calculated with the Fick–Nernst–Planck equation. The cell model considers....... This demonstrates that the cell model can be a useful tool for the design of effective lysosome-targeting drugs with minimal off-target interactions....

  12. Release and uptake of lysosomal enzymes : studied in cultured cells

    OpenAIRE

    1980-01-01

    textabstractThe purpose of the experimental work described in this thesiswas to investigate some aspects of the release and uptake of lysosomal enzymes. The experiments involved the use of normal human and animal fibroblasts and some other cell types such as hepatocytes and hepatoma cells as sources of hydrolytic enzymes, and fibroblasts from patients with lysosomal storage diseases associated with a single lysosomal enzyme deficiency and with "1-cell" disease as recipient cells. In a number ...

  13. Factors and processes modulating phenotypes in neuronopathic lysosomal storage diseases

    OpenAIRE

    Jakóbkiewicz-Banecka, Joanna; Gabig-Cimińska, Magdalena; Banecka-Majkutewicz, Zyta; Banecki, Bogdan; Węgrzyn, Alicja; Węgrzyn, Grzegorz

    2013-01-01

    Lysosomal storage diseases are inherited metabolic disorders caused by genetic defects causing deficiency of various lysosomal proteins, and resultant accumulation of non-degraded compounds. They are multisystemic diseases, and in most of them (>70 %) severe brain dysfunctions are evident. However, expression of various phenotypes in particular diseases is extremely variable, from non-neuronopathic to severely neurodegenerative in the deficiency of the same enzyme. Although all lysosomal stor...

  14. Different tree species affect soil respiration spatial distribution in a subtropical forest of southern Taiwan

    Science.gov (United States)

    Chiang, Po-Neng; Yu, Jui-Chu; Wang, Ya-nan; Lai, Yen-Jen

    2014-05-01

    Global forests contain 69% of total carbon stored in forest soil and litter. But the carbon storage ability and release rate of warming gases of forest soil also affect global climate change. Soil carbon cycling processes are paid much attention by ecological scientists and policy makers because of the possibility of carbon being stored in soil via land use management. Soil respiration contributed large part of terrestrial carbon flux, but the relationship of soil respiration and climate change was still obscurity. Most of soil respiration researches focus on template and tropical area, little was known that in subtropical area. Afforestation is one of solutions to mitigate CO2 increase and to sequestrate CO2 in tree and soil. Therefore, the objective of this study is to clarify the relationship of tree species and soil respiration distribution in subtropical broad-leaves plantation in southern Taiwan. The research site located on southern Taiwan was sugarcane farm before 2002. The sugarcane was removed and fourteen broadleaved tree species were planted in 2002-2005. Sixteen plots (250m*250m) were set on 1 km2 area, each plot contained 4 subplots (170m2). The forest biomass (i.e. tree height, DBH) understory biomass, litter, and soil C were measured and analyzed at 2011 to 2012. Soil respiration measurement was sampled in each subplot in each month. The soil belongs to Entisol with over 60% of sandstone. The soil pH is 5.5 with low base cations because of high sand percentage. Soil carbon storage showed significantly negative relationship with soil bulk density (p<0.001) in research site. The differences of distribution of live tree C pool among 16 plots were affected by growth characteristic of tree species. Data showed that the accumulation amount of litterfall was highest in December to February and lowest in June. Different tree species planted in 16 plots, resulting in high spatial variation of litterfall amount. It also affected total amount of litterfall

  15. A lysosome-centered view of nutrient homeostasis.

    Science.gov (United States)

    Mony, Vinod K; Benjamin, Shawna; O'Rourke, Eyleen J

    2016-01-01

    Lysosomes are highly acidic cellular organelles traditionally viewed as sacs of enzymes involved in digesting extracellular or intracellular macromolecules for the regeneration of basic building blocks, cellular housekeeping, or pathogen degradation. Bound by a single lipid bilayer, lysosomes receive their substrates by fusing with endosomes or autophagosomes, or through specialized translocation mechanisms such as chaperone-mediated autophagy or microautophagy. Lysosomes degrade their substrates using up to 60 different soluble hydrolases and release their products either to the cytosol through poorly defined exporting and efflux mechanisms or to the extracellular space by fusing with the plasma membrane. However, it is becoming evident that the role of the lysosome in nutrient homeostasis goes beyond the disposal of waste or the recycling of building blocks. The lysosome is emerging as a signaling hub that can integrate and relay external and internal nutritional information to promote cellular and organismal homeostasis, as well as a major contributor to the processing of energy-dense molecules like glycogen and triglycerides. Here we describe the current knowledge of the nutrient signaling pathways governing lysosomal function, the role of the lysosome in nutrient mobilization, and how lysosomes signal other organelles, distant tissues, and even themselves to ensure energy homeostasis in spite of fluctuations in energy intake. At the same time, we highlight the value of genomics approaches to the past and future discoveries of how the lysosome simultaneously executes and controls cellular homeostasis.

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

    Science.gov (United States)

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

    2012-01-01

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

  17. Constitutive expression of a COOH-terminal leucine mutant of lysosome-associated membrane protein-1 causes its exclusive localization in low density intracellular vesicles.

    Science.gov (United States)

    Akasaki, Kenji; Shiotsu, Keiko; Michihara, Akihiro; Ide, Norie; Wada, Ikuo

    2014-07-01

    Lysosome-associated membrane protein-1 (LAMP-1) is a type I transmembrane protein with a short cytoplasmic tail that possesses a lysosome-targeting signal of GYQTI(382)-COOH. Wild-type (WT)-LAMP-1 was exclusively localized in high density lysosomes, and efficiency of LAMP-1's transport to lysosomes depends on its COOH-terminal amino acid residue. Among many different COOH-terminal amino acid substitution mutants of LAMP-1, a leucine-substituted mutant (I382L) displays the most efficient targeting to late endosomes and lysosomes [Akasaki et al. (2010) J. Biochem. 148: , 669-679]. In this study, we generated two human hepatoma cell lines (HepG2 cell lines) that stably express WT-LAMP-1 and I382L, and compared their intracellular distributions. The subcellular fractionation study using Percoll density gradient centrifugation revealed that WT-LAMP-1 had preferential localization in the high density secondary lysosomes where endogenous human LAMP-1 was enriched. In contrast, a major portion of I382L was located in a low density fraction. The low density fraction also contained approximately 80% of endogenous human LAMP-1 and significant amounts of endogenous β-glucuronidase and LAMP-2, which probably represents occurrence of low density lysosomes in the I382L-expressing cells. Double immunofluorescence microscopic analyses distinguished I382L-containing intracellular vesicles from endogenous LAMP-1-containing lysosomes and early endosomes. Altogether, constitutive expression of I382L causes its aberrant intracellular localization and generation of low density lysosomes, indicating that the COOH-terminal isoleucine is critical for normal localization of LAMP-1 in the dense lysosomes.

  18. Presenilin 1 Maintains Lysosomal Ca(2+) Homeostasis via TRPML1 by Regulating vATPase-Mediated Lysosome Acidification.

    Science.gov (United States)

    Lee, Ju-Hyun; McBrayer, Mary Kate; Wolfe, Devin M; Haslett, Luke J; Kumar, Asok; Sato, Yutaka; Lie, Pearl P Y; Mohan, Panaiyur; Coffey, Erin E; Kompella, Uday; Mitchell, Claire H; Lloyd-Evans, Emyr; Nixon, Ralph A

    2015-09-01

    Presenilin 1 (PS1) deletion or Alzheimer's disease (AD)-linked mutations disrupt lysosomal acidification and proteolysis, which inhibits autophagy. Here, we establish that this phenotype stems from impaired glycosylation and instability of vATPase V0a1 subunit, causing deficient lysosomal vATPase assembly and function. We further demonstrate that elevated lysosomal pH in Presenilin 1 knockout (PS1KO) cells induces abnormal Ca(2+) efflux from lysosomes mediated by TRPML1 and elevates cytosolic Ca(2+). In WT cells, blocking vATPase activity or knockdown of either PS1 or the V0a1 subunit of vATPase reproduces all of these abnormalities. Normalizing lysosomal pH in PS1KO cells using acidic nanoparticles restores normal lysosomal proteolysis, autophagy, and Ca(2+) homeostasis, but correcting lysosomal Ca(2+) deficits alone neither re-acidifies lysosomes nor reverses proteolytic and autophagic deficits. Our results indicate that vATPase deficiency in PS1 loss-of-function states causes lysosomal/autophagy deficits and contributes to abnormal cellular Ca(2+) homeostasis, thus linking two AD-related pathogenic processes through a common molecular mechanism.

  19. Presenilin 1 Maintains Lysosomal Ca2+ Homeostasis via TRPML1 by Regulating vATPase-Mediated Lysosome Acidification

    Directory of Open Access Journals (Sweden)

    Ju-Hyun Lee

    2015-09-01

    Full Text Available Presenilin 1 (PS1 deletion or Alzheimer’s disease (AD-linked mutations disrupt lysosomal acidification and proteolysis, which inhibits autophagy. Here, we establish that this phenotype stems from impaired glycosylation and instability of vATPase V0a1 subunit, causing deficient lysosomal vATPase assembly and function. We further demonstrate that elevated lysosomal pH in Presenilin 1 knockout (PS1KO cells induces abnormal Ca2+ efflux from lysosomes mediated by TRPML1 and elevates cytosolic Ca2+. In WT cells, blocking vATPase activity or knockdown of either PS1 or the V0a1 subunit of vATPase reproduces all of these abnormalities. Normalizing lysosomal pH in PS1KO cells using acidic nanoparticles restores normal lysosomal proteolysis, autophagy, and Ca2+ homeostasis, but correcting lysosomal Ca2+ deficits alone neither re-acidifies lysosomes nor reverses proteolytic and autophagic deficits. Our results indicate that vATPase deficiency in PS1 loss-of-function states causes lysosomal/autophagy deficits and contributes to abnormal cellular Ca2+ homeostasis, thus linking two AD-related pathogenic processes through a common molecular mechanism.

  20. Enhanced lysosomal activity by overexpressed aminopeptidase Y in Saccharomyces cerevisiae.

    Science.gov (United States)

    Yoon, Jihee; Sekhon, Simranjeet Singh; Kim, Yang-Hoon; Min, Jiho

    2016-06-01

    Saccharomyces cerevisiae contains vacuoles corresponding to lysosomes in higher eukaryotes. Lysosomes are dynamic (not silent) organelles in which enzymes can be easily integrated or released when exposed to stressful conditions. Changes in lysosomal enzymes have been observed due to oxidative stress, resulting in an increased function of lysosomes. The protein profiles from H2O2- and NH4Cl-treated lysosomes showed different expression patterns, observed with two-dimensional gel electrophoresis. The aminopeptidase Y protein (APE3) that conspicuously enhanced antimicrobial activity than other proteins was selected for further studies. The S. cerevisiae APE3 gene was isolated and inserted into pYES2.0 expression vector. The GFP gene was inserted downstream to the APE3 gene for confirmation of APE3 targeting to lysosomes, and S. cerevisiae was transformed to pYES2::APE3::GFP. The APE3 did not enter in lysosomes and formed an inclusion body at 30 °C, but it inserted to lysosomes as shown by the merger of GFP with lysosomes at 28 °C. Antimicrobial activity of the cloned S. cerevisiae increased about 5 to 10 % against eight strains, compared to normal cells, and galactose induction is increased more two folds than that of normal cells. Therefore, S. cerevisiae was transformed to pYES2::APE3::GFP, accumulating a large amount of APE3, resulting in increased lysosomal activity. Increase in endogenous levels of lysosomes and their activity following genetic modification can lead to its use in applications such as antimicrobial agents and apoptosis-inducing materials for cancer cells, and consequently, it may also be possible to use the organelles for improving in vitro functions.

  1. Geohelminths distribution as affected by soil properties, physicochemical factors and climate in Sharkyia governorate Egypt.

    Science.gov (United States)

    Etewa, Samia E; Abdel-Rahman, Sara A; Abd El-Aal, Naglaa F; Fathy, Ghada M; El-Shafey, Mahmoud A; Ewis, A M G

    2016-06-01

    Soil-transmitted helminths are mainly a group of parasitic nematodes causing human infection through contact with parasite eggs or larvae; they survive in the warm and moist soil of the tropical and subtropical countries. This study was carried out in Sharkyia governorate from October, 2011 to October, 2013, to correlate between the prevalence and distribution of these parasites in the soil and the physicochemical factors affecting the examined samples of the soil. One hundred and twenty samples of different types of soil (clay, silt, sand) from different localities were collected and examined. Diagnosis of geohelminths was confirmed by the recovery of their eggs and larvae with other protozoa by different parasitological methods. The modified baermann method was found to be more efficient in detection of geohelminths larvae than charcoal culture method. Among the examined sites geohelminths were much more numerous in the soil of rural areas especially in the spring and summer seasons, while the contamination of canal banks by geohelminths was the worst (80 %). An insignificant correlation was reported between the soil texture and the number of positive samples in the examined areas while the relationship was directly proportional among (moisture, PH, organic). It appeared that the most common geohelminthic stage was Toxocara spp. eggs besides other types of protozoa especially Balantidium coli cysts. This suggests that factors other than soil texture are important in the prevalence of geohelminths in the soil e.g. temperature, moisture, PH and organic matter. So, to change some of these factors in a trial to control geoparasites transmission but with keeping the environment should be tried. These results also open the way to further studies to highlight the mutual affection between inhabitants of these sites and the prevalence of these geoparasites.

  2. The nuclear protein Waharan is required for endosomal-lysosomal trafficking in Drosophila.

    Science.gov (United States)

    Lone, Mohiddin; Kungl, Theresa; Koper, Andre; Bottenberg, Wolfgang; Kammerer, Richard; Klein, Melanie; Sweeney, Sean T; Auburn, Richard P; O'Kane, Cahir J; Prokop, Andreas

    2010-07-15

    Here we report Drosophila Waharan (Wah), a 170-kD predominantly nuclear protein with two potential human homologues, as a newly identified regulator of endosomal trafficking. Wah is required for neuromuscular-junction development and muscle integrity. In muscles, knockdown of Wah caused novel accumulations of tightly packed electron-dense tubules, which we termed 'sausage bodies'. Our data suggest that sausage bodies coincide with sites at which ubiquitylated proteins and a number of endosomal and lysosomal markers co-accumulate. Furthermore, loss of Wah function generated loss of the acidic LysoTracker compartment. Together with data demonstrating that Wah acts earlier in the trafficking pathway than the Escrt-III component Drosophila Shrb (snf7 in Schizosaccharomyces pombe), our results indicate that Wah is essential for endocytic trafficking at the late endosome. Highly unexpected phenotypes result from Wah knockdown, in that the distribution of ubiquitylated cargos and endolysosomal morphologies are affected despite Wah being a predominant nuclear protein. This finding suggests the existence of a relationship between nuclear functions and endolysosomal trafficking. Future studies of Wah function will give us insights into this interesting phenomenon.

  3. Distribution of CO2 in Soil Air Affected by Vegetation in the Shilin National Park

    Institute of Scientific and Technical Information of China (English)

    宋林华; 梁福源

    2001-01-01

    This paper studies the CO2 distribution of soil atmosphere in the Shilin National Park. The measurement sites were chosen according to different topographic features and different vegetations. Seven measurement sites on 3 cross sections were chosen to pass through 3 karstic depressions or on the slopes of depressions. All measurement results show soils with pH values lower than 7.0 (from 5.4 to 6.6). There are 2 cases for the pH values of soil in different topographic features: the pH values of 2 profiles on the ridges or upper slopes of depressions are lower than those in the depressions;and the pH values of 2 soil profiles on the slopes of depressions are higher than those in the depressions. Most samples show relatively low humidity and CO2 contents on the ridges or slopes of depressions compared with soil profiles in the depressions. High CO2 contents occur at depths from -40 to -80 cm and high and dense grassland shows high CO2 contents in the soil atmosphere. Grass roots may grow and are distributed mainly at depths from -20 to -40 cm; while tree roots predominantly as deep as -60 cm even -80 cm. The influences of pine, cypress and eucalyptus on soil CO2 have been studied. Soil CO2 influenced by pine and cypress are generally concentrated in an area surrounding the tree with a diameter of 1 m and the strongly influenced distance is 50 cm. Eucalyptus will strongly affect the CO2 contents in an area with a diameter of 2 m, especially 1 m distant from the tree. The highest concentration of soil CO2 at a depth of-30 and 100 cm from the tree reaches 92000 ppm.``

  4. Factors Affecting the Spatial Distribution of Oviposition Sites for Tandem Black Saddlebags Dragonflies (Odonata: Libellulidae)

    Science.gov (United States)

    Thornton, Jessica L.; Switzer, Paul V.

    2015-01-01

    Oviposition site location may be affected by (1) factors influencing the costs and benefits to the offspring (e.g., resource availability, competition, predation risk) and (2) factors influencing the costs and benefits to the female (e.g., predation risk or mate harassment). In cases in which both the male and female are involved in locating a site, costs and benefits may differ for each parent and the resulting oviposition site location may represent the outcome of selection pressures on one or both of them. We studied oviposition behavior in the black saddlebags dragonfly (Tramea lacerata Hagen), a species in which the male and female typically remain together (i.e., in tandem) while traveling among potential oviposition locations. Oviposition sites tended to be away from pond shoreline at the outer edge of the vegetation on the water’s surface. We found that tandems distributed their oviposition locations widely around the pond, and interactions with other dragonflies (typically other T. lacerata, either territorial males or tandems) led to a larger distance between consecutive oviposition locations. Interestingly, for 10% of the tandems, the female became separated from the male and oviposited solitarily multiple times. These solitary females spent significantly less time and traveled significantly smaller distances between successive oviposition sites than when in tandem. Our results indicate that while some aspects of oviposition behavior and site selection may be consistent between the male and female (e.g., the characteristics that make a site suitable), other aspects, such as the distribution of sites, may be a result of a differing benefits and costs for the two sexes, perhaps as a consequence of potential sperm competition.

  5. Diversity of riceland mosquitoes and factors affecting their occurrence and distribution in Mwea, Kenya.

    Science.gov (United States)

    Muturi, Ephantus J; Shililu, Josephat I; Jacob, Benjamin G; Mwangangi, Joseph M; Mbogo, Charles M; Githure, John I; Novak, Robert J

    2008-09-01

    Knowledge of mosquito species diversity, occurrence, and distribution is an essential component of vector ecology and a guiding principle to formulation and implementation of integrated vector management programs. A 12-month entomological survey was conducted to determine the diversity of riceland mosquitoes and factors affecting their occurrence and distribution at 3 sites targeted for malaria vector control in Mwea, Kenya. Adult mosquitoes were sampled indoors by pyrethrum spray catch and outdoors by the Centers for Disease Control and Prevention light traps. Mosquitoes were then morphologically identified to species using taxonomic keys. The characteristics of houses sampled for indoor resting mosquitoes, including number of people sleeping in each house the night preceding collection, presence of bed nets, location of the house, size of eaves, wall type, presence of cattle and distance of the house to the cowshed, and proximity to larval habitats, were recorded. Of the 191,378 mosquitoes collected, 95% were identified morphologically to species and comprised 25 species from 5 genera. Common species included Anopheles arabiensis (53.5%), Culex quinquefasciatus (35.5%), An. pharoensis (4.7%), An. coustani (2.5%), and An. funestus (1.6%). Shannon's species diversity and evenness indices did not differ significantly among the 3 study sites. There was a marked house-to-house variation in the average number of mosquitoes captured. The number of people sleeping in the house the night preceding collection, size of eaves, distance to the cowshed, and the nearest larval habitat were significant predictors of occurrence of either or both An. arabiensis and Cx. quinquefasciatus. The peak abundance of An. arabiensis coincided with land preparation and the first few weeks after transplanting of rice seedlings, and that of Cx. quinquefasciatus coincided with land preparation, late stage of rice development, and short rains. After transplanting of rice seedlings, the

  6. Environmental factors affecting the distribution of Chironomid larvae of the Seybouse wadi, North-Eastern Algeria

    Directory of Open Access Journals (Sweden)

    Nadjla Chaib

    2013-03-01

    Full Text Available A survey of the Seybouse wadi (North-Eastern Algeria between 2008 and 2011 was conducted in 26 sampling sites located on the main river and its tributaries using chironomids. From 3264 collected larvae, forty-five chironomid species were identified, and were correlated to 13 environmental variables to predict determinant factors affecting their distribution. Indicator value (IndVal analysis was first performed to determine indicator chironomid species according to several factors (sites, seasons, source distance, granulometry, conductivity, water temperature, dissolved oxygen, water velocity, pollution and the abundance of filamentous algae. Co-inertia analysis (CoIA supported the IndVal results, emphasising an upstream/downstream gradient in the first axis, while a granulometry gradient was emphasised by the second axis. A pollution gradient was also highlighted in the plane of the first two axes, separating tolerant Chironomus sp. 1, Cricotopus bicinctus and Cricotopus (Isocladius sylvestris from intolerant species as Phaenopsectra flavipes, Rheotanytarsus sp.1 and Cladotanytarsus sp. 1.

  7. Particle size distribution of rice flour affecting the starch enzymatic hydrolysis and hydration properties.

    Science.gov (United States)

    de la Hera, Esther; Gomez, Manuel; Rosell, Cristina M

    2013-10-15

    Rice flour is becoming very attractive as raw material, but there is lack of information about the influence of particle size on its functional properties and starch digestibility. This study evaluates the degree of dependence of the rice flour functional properties, mainly derived from starch behavior, with the particle size distribution. Hydration properties of flours and gels and starch enzymatic hydrolysis of individual fractions were assessed. Particle size heterogeneity on rice flour significantly affected functional properties and starch features, at room temperature and also after gelatinization; and the extent of that effect was grain type dependent. Particle size heterogeneity on rice flour induces different pattern in starch enzymatic hydrolysis, with the long grain having slower hydrolysis as indicated the rate constant (k). No correlation between starch digestibility and hydration properties or the protein content was observed. It seems that in intact granules interactions with other grain components must be taken into account. Overall, particle size fractionation of rice flour might be advisable for selecting specific physico-chemical properties.

  8. Distribution of abnormal prion protein in a sheep affected with L-type bovine spongiform encephalopathy.

    Science.gov (United States)

    Matsuura, Y; Iwamaru, Y; Masujin, K; Imamura, M; Mohri, S; Yokoyama, T; Okada, H

    2013-07-01

    To investigate the topographical distribution and patterns of deposition of immunolabelled abnormal prion protein (PrP(Sc)), interspecies transmission of atypical L-type bovine spongiform encephalopathy (BSE) to Cheviot ewes (ARQ/ARQ genotype) was performed. L-type BSE was successfully transmitted via the intracerebral route to a ewe, with an incubation period of 1,562 days. Minimal vacuolar change was detected in the basal ganglia, thalamus and brainstem, and PrP(Sc) accumulated throughout the brain. The L-type BSE-affected sheep was characterized by conspicuous fine particulate deposits in the neuropil, particulate and/or granular intraneuronal and intraglial deposits, and the absence of PrP(Sc) plaques or stellate deposits. In addition, immunohistochemical and western blot analyses revealed that PrP(Sc) accumulation was present in peripheral nervous tissues (including the trigeminal ganglia and dorsal root ganglion) and adrenal glands, but was absent in lymphoid tissues. These results suggest that L-type BSE has distinct and distinguishable characteristics as well as PrP(Sc) tissue tropism in sheep.

  9. A Comparative Study on the Alterations of Endocytic Pathways in Multiple Lysosomal Storage Disorders.

    Science.gov (United States)

    Rappaport, Jeff; Manthe, Rachel L; Solomon, Melani; Garnacho, Carmen; Muro, Silvia

    2016-02-01

    Many cellular activities and pharmaceutical interventions involve endocytosis and delivery to lysosomes for processing. Hence, lysosomal processing defects can cause cell and tissue damage, as in lysosomal storage diseases (LSDs) characterized by lysosomal accumulation of undegraded materials. This storage causes endocytic and trafficking alterations, which exacerbate disease and hinder treatment. However, there have been no systematic studies comparing different endocytic routes in LSDs. Here, we used genetic and pharmacological models of four LSDs (type A Niemann-Pick, type C Niemann-Pick, Fabry, and Gaucher diseases) and evaluated the pinocytic and receptor-mediated activity of the clathrin-, caveolae-, and macropinocytic routes. Bulk pinocytosis was diminished in all diseases, suggesting a generic endocytic alteration linked to lysosomal storage. Fluid-phase (dextran) and ligand (transferrin) uptake via the clathrin route were lower for all LSDs. Fluid-phase and ligand (cholera toxin B) uptake via the caveolar route were both affected but less acutely in Fabry or Gaucher diseases. Epidermal growth factor-induced macropinocytosis was altered in Niemann-Pick cells but not other LSDs. Intracellular trafficking of ligands was also distorted in LSD versus wild-type cells. The extent of these endocytic alterations paralleled the level of cholesterol storage in disease cell lines. Confirming this, pharmacological induction of cholesterol storage in wild-type cells disrupted endocytosis, and model therapeutics restored uptake in proportion to their efficacy in attenuating storage. This suggests a proportional and reversible relationship between endocytosis and lipid (cholesterol) storage. By analogy, the accumulation of biological material in other diseases, or foreign material from drugs or their carriers, may cause similar deficits, warranting further investigation.

  10. [The blood-brain barrier and neurodegenerative lysosomal storage diseases].

    Science.gov (United States)

    Urayama, Akihiko

    2013-02-01

    Enzyme replacement therapy has been a very effective treatment for several lysosomal storage diseases. However, correcting central nervous system (CNS) storage has been challenging due to the presence of the blood-brain barrier (BBB), which hampers the entry of circulating lysosomal enzymes into the brain. In our previous studies, we discovered that luminally expressed cation-independent mannose 6-phosphate (M6P) receptor is a universal transporter for lysosomal enzymes that contain M6P moieties on the enzyme molecule. This receptor-mediated transport of lysosomal enzymes showed developmental down-regulation that resulted in a failure of delivery of lysosomal enzymes across the BBB in the adult brain. Conceptually, if one can re-induce M6P receptor-mediated transport of lysosomal enzymes in adult BBB, this could provide a novel brain targeting approach for treating abnormal storage in the CNS, regardless of the age of subjects. We found that systemic adrenergic stimuli restored functional transport of β-glucuronidase across the adult BBB. The concept of manipulating BBB transport activity by endogenous characteristics has also been demonstrated by another group who showed effective treatment in a Pompe disease model animal in vivo. It is intriguing that lysosomal enzymes utilize multiple mechanisms for their transport across the BBB. This review explores pharmacological manipulations for the delivery of lysosomal enzymes into the CNS, and the mechanisms of their transport across the BBB, based on existing evidence from studies of β-glucuronidase, sulfamidase, acid α-glucosidase, and arylsulfatase A.

  11. Photoaffinity labeling of the lysosomal neuraminidase from bovine testis

    NARCIS (Netherlands)

    G.T.J. van der Horst (Gijsbertus); U. Rose (Ursula); R. Brossmer (Reinhard); F.W. Verheijen (Frans)

    1990-01-01

    markdownabstractAbstract ASA-NeuAc2en, a photoreactive arylazide derivative of sialic acid, is shown to be a powerful competitive inhibitor of lysosomal neuraminidase from bovine testis (Ki ≈ 21 μM). Photoaffinity labeling and partial purification of preparations containing this lysosomal neuramin

  12. Lysosome/lipid droplet interplay in metabolic diseases.

    Science.gov (United States)

    Dugail, Isabelle

    2014-01-01

    Lysosomes and lipid droplets are generally considered as intracellular compartments with divergent roles in cell metabolism, lipid droplets serving as lipid reservoirs in anabolic pathways, whereas lysosomes are specialized in the catabolism of intracellular components. During the last few years, new insights in the biology of lysosomes has challenged this view by providing evidence for the importance of lysosome recycling as a sparing mechanism to maintain cellular fitness. On the other hand the understanding of lipid droplets has evolved from an inert intracellular deposit toward the status of an intracellular organelle with dynamic roles in cellular homeostasis beyond storage. These unrelated aspects have also recently converged in the finding of unexpected lipid droplet/lysosome communication through autophagy, and the discovery of lysosome-mediated lipid droplet degradation called lipopagy. Furthermore, adipocytes which are professional cells for lipid droplet formation were also shown to be active in peptide antigen presentation a pathway requiring lysosomal activity. The potential importance of lipid droplet/lysosome interplay is discussed in the context of metabolic diseases and the setting of chronic inflammation.

  13. Artesunate induces cell death in human cancer cells via enhancing lysosomal function and lysosomal degradation of ferritin.

    Science.gov (United States)

    Yang, Nai-Di; Tan, Shi-Hao; Ng, Shukie; Shi, Yin; Zhou, Jing; Tan, Kevin Shyong Wei; Wong, Wai-Shiu Fred; Shen, Han-Ming

    2014-11-28

    Artesunate (ART) is an anti-malaria drug that has been shown to exhibit anti-tumor activity, and functional lysosomes are reported to be required for ART-induced cancer cell death, whereas the underlying molecular mechanisms remain largely elusive. In this study, we aimed to elucidate the molecular mechanisms underlying ART-induced cell death. We first confirmed that ART induces apoptotic cell death in cancer cells. Interestingly, we found that ART preferably accumulates in the lysosomes and is able to activate lysosomal function via promotion of lysosomal V-ATPase assembly. Furthermore, we found that lysosomes function upstream of mitochondria in reactive oxygen species production. Importantly, we provided evidence showing that lysosomal iron is required for the lysosomal activation and mitochondrial reactive oxygen species production induced by ART. Finally, we showed that ART-induced cell death is mediated by the release of iron in the lysosomes, which results from the lysosomal degradation of ferritin, an iron storage protein. Meanwhile, overexpression of ferritin heavy chain significantly protected cells from ART-induced cell death. In addition, knockdown of nuclear receptor coactivator 4, the adaptor protein for ferritin degradation, was able to block ART-mediated ferritin degradation and rescue the ART-induced cell death. In summary, our study demonstrates that ART treatment activates lysosomal function and then promotes ferritin degradation, subsequently leading to the increase of lysosomal iron that is utilized by ART for its cytotoxic effect on cancer cells. Thus, our data reveal a new mechanistic action underlying ART-induced cell death in cancer cells.

  14. Protective Role of Endogenous Gangliosides for Lysosomal Pathology in a Cellular Model of Synucleinopathies

    OpenAIRE

    Wei, Jianshe; Fujita, Masayo; Nakai, Masaaki; Waragai, Masaaki; Sekigawa, Akio; Sugama, Shuei; Takenouchi, Takato; Masliah, Eliezer; Hashimoto,Makoto

    2009-01-01

    Gangliosides may be involved in the pathogenesis of Parkinson’s disease and related disorders, although the precise mechanisms governing this involvement remain unknown. In this study, we determined whether changes in endogenous ganglioside levels affect lysosomal pathology in a cellular model of synucleinopathy. For this purpose, dementia with Lewy body-linked P123H β-synuclein (β-syn) neuroblastoma cells transfected with α-synuclein were used as a model system because these cells were chara...

  15. Mitochondrial respiration controls lysosomal function during inflammatory T cell responses

    Science.gov (United States)

    Baixauli, Francesc; Acín-Pérez, Rebeca; Villarroya-Beltrí, Carolina; Mazzeo, Carla; Nuñez-Andrade, Norman; Gabandé-Rodriguez, Enrique; Dolores Ledesma, Maria; Blázquez, Alberto; Martin, Miguel Angel; Falcón-Pérez, Juan Manuel; Redondo, Juan Miguel; Enríquez, Jose Antonio; Mittelbrunn, Maria

    2016-01-01

    Summary The endolysosomal system is critical for the maintenance of cellular homeostasis. However, how endolysosomal compartment is regulated by mitochondrial function is largely unknown. We have generated a mouse model with defective mitochondrial function in CD4+ T lymphocytes by genetic deletion of the mitochondrial transcription factor A (Tfam). Mitochondrial respiration-deficiency impairs lysosome function, promotes p62 and sphingomyelin accumulation and disrupts endolysosomal trafficking pathways and autophagy, thus linking a primary mitochondrial dysfunction to a lysosomal storage disorder. The impaired lysosome function in Tfam-deficient cells subverts T cell differentiation toward pro-inflammatory subsets and exacerbates the in vivo inflammatory response. Restoration of NAD+ levels improves lysosome function and corrects the inflammatory defects in Tfam-deficient T cells. Our results uncover a mechanism by which mitochondria regulate lysosome function to preserve T cell differentiation and effector functions, and identify novel strategies for intervention in mitochondrial-related diseases. PMID:26299452

  16. Hormonal and cholinergic influences on pancreatic lysosomal and digestive enzymes in rats.

    Science.gov (United States)

    Evander, A; Ihse, I; Lundquist, I

    1983-01-01

    Hormonal and cholinergic influences on lysosomal and digestive enzyme activities in pancreatic tissue were studied in normal adult rats. Hormonal stimulation by the cholecystokinin analogue, caerulein, induced a marked enhancement of the activities of cathepsin D and N-acetyl-beta-D-glucosaminidase in pancreatic tissue, whereas the activities of amylase and lipase tended to decrease. Acid phosphatase activity was not affected. Further, caerulein was found to induce a significant increase of cathepsin D output in bile-pancreatic juice. This output largely parallelled that of amylase. Cholinergic stimulation by the muscarinic agonist carbachol, at a dose level giving the same output of amylase as caerulein, did not affect pancreatic activities of cathepsin D and N-acetyl-beta-D-glucosaminidase. Further, cholinergic stimulation induced an increase of amylase activity and a slight decrease of acid phosphatase activity in pancreatic tissue. Lipase activity was not affected. No apparent effect on cathepsin D output in bile-pancreatic juice was encountered after cholinergic stimulation. The activities of neither the digestive nor the lysosomal enzymes were influenced by the administration of secretin. The results suggest a possible lysosomal involvement in caerulein-induced secretion and/or inactivation of pancreatic digestive enzymes, whereas cholinergic stimulation seems to act through different mechanisms.

  17. Lung perfusion and emphysema distribution affect the outcome of endobronchial valve therapy

    Directory of Open Access Journals (Sweden)

    Thomsen C

    2016-06-01

    Full Text Available Christian Thomsen,1 Dorothea Theilig,2 Dominik Herzog,1 Alexander Poellinger,2 Felix Doellinger,2 Nils Schreiter,3 Vera Schreiter,2 Dirk Schürmann,1 Bettina Temmesfeld-Wollbrueck,1 Stefan Hippenstiel,1 Norbert Suttorp,1 Ralf-Harto Hubner1 1Department of Internal Medicine/Infectious Diseases and Respiratory Medicine, 2Institute of Radiology, 3Institute of Nuclear Medicine, Charité – Universitätsmedizin Berlin, Berlin, Germany Abstract: The exclusion of collateral ventilation (CV and other factors affect the clinical success of endoscopic lung volume reduction (ELVR. However, despite its benefits, the outcome of ELVR remains difficult to predict. We investigated whether clinical success could be predicted by emphysema distribution assessed by computed tomography scan and baseline perfusion assessed by perfusion scintigraphy. Data from 57 patients with no CV in the target lobe (TL were retrospectively analyzed after ELVR with valves. Pulmonary function tests (PFT, St George’s Respiratory Questionnaire (SGRQ, and 6-minute walk tests (6MWT were performed on patients at baseline. The sample was grouped into high and low levels at the median of TL perfusion, ipsilateral nontarget lobe (INL perfusion, and heterogeneity index (HI. These groups were analyzed for association with changes in outcome parameters from baseline to 3 months follow-up. Compared to baseline, patients showed significant improvements in PFT, SGRQ, and 6MWT (all P≤0.001. TL perfusion was not associated with changes in the outcome. High INL perfusion was significantly associated with increases in 6MWT (P=0.014, and high HI was associated with increases in forced expiratory volume in 1 second (FEV1, (P=0.012. Likewise, there were significant correlations for INL perfusion and improvement of 6MWT (r=0.35, P=0.03 and for HI and improvement in FEV1 (r=0.45, P=0.001. This study reveals new attributes that associate with positive outcomes for patient selection prior to ELVR

  18. Activation of peroxisome proliferator-activated receptor α induces lysosomal biogenesis in brain cells: implications for lysosomal storage disorders.

    Science.gov (United States)

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

    2015-04-17

    Lysosomes are ubiquitous membrane-enclosed organelles filled with an acidic interior and are central to the autophagic, endocytic, or phagocytic pathway. In contrast to its classical function as the waste management machinery, lysosomes are now considered to be an integral part of various cellular signaling processes. The diverse functionality of this single organelle requires a very complex and coordinated regulation of its activity with transcription factor EB (TFEB), a master regulator of lysosomal biogenesis, at its core. However, mechanisms by which TFEB is regulated are poorly understood. This study demonstrates that gemfibrozil, an agonist of peroxisome proliferator-activated receptor (PPAR) α, alone and in conjunction with all-trans-retinoic acid is capable of enhancing TFEB in brain cells. We also observed that PPARα, but not PPARβ and PPARγ, is involved in gemfibrozil-mediated up-regulation of TFEB. Reporter assay and chromatin immunoprecipitation studies confirmed the recruitment of retinoid X receptor α, PPARα, and PGC1α on the PPAR-binding site on the Tfeb promoter as well. Subsequently, the drug-mediated induction of TFEB caused an increase in lysosomal protein and the lysosomal abundance in cell. Collectively, this study reinforces the link between lysosomal biogenesis and lipid metabolism with TFEB at the crossroads. Furthermore, gemfibrozil may be of therapeutic value in the treatment of lysosomal storage disorders in which autophagy-lysosome pathway plays an important role.

  19. Analysis of geological structure and anthropological factors affecting arsenic distribution in the Lahore aquifer, Pakistan

    Science.gov (United States)

    Muhammad, Akhtar Malik; Zhonghua, Tang; Sissou, Zakari; Mohamadi, Bahaa; Ehsan, Muhsan

    2016-11-01

    This study investigated the potential factors affecting arsenic concentration in the groundwater system of Lahore, Pakistan. The effects of several factors such as population density (PD), pumping rate (PR), impermeable land use (LU), surface elevation (SE), and water-table elevation (WL) on arsenic concentration were studied in 101 union councils of Lahore. Forty single and multi-factor models were established using geographic information system (GIS) techniques to develop an arsenic contamination map and to investigate the most effective combinations among factors. Additionally, statistical tests were used to evaluate arsenic concentration between classes of the same single factor. The arsenic concentration in the Lahore aquifer varied from 0.001 to 0.143 mg L-1. The highest arsenic concentrations were detected in the Walled City and the town of Shahdara. Among the 40 raster models, groundwater arsenic concentration showed the best matching frequency with single-factor models for PD (50.70 %) and SE (47 %). Thus, PD and SE were used to develop an arsenic distribution raster map, and they were also used to study the effect of aquifer depth on arsenic concentration. PD was found to have hidden latent variables such as PR and LU. The shallow aquifer depth was negatively correlated with arsenic concentration ( r = -0.23) and positively with PR ( r = 0.15). Therefore, when there was high PR in wells with smaller aquifer depth, the arsenic concentration was high. The existing water treatment and alternative water resources are good options, which should be developed to deal with Lahore wells contaminated with arsenic at high concentrations.

  20. Effects of contaminant exposure and food restriction on hepatic autophagic lysosomal parameters in Herring Gull (Larus argentatus) chicks.

    Science.gov (United States)

    Hegseth, Marit Nøst; Gorbi, Stephania; Bocchetti, Raffaella; Camus, Lionel; Gabrielsen, Geir Wing; Regoli, Francesco

    2014-08-01

    Lysosomal autophagic responses, such as lysosomal membrane stability, neutral lipids (NL), lipofuscin (LF), and malondialdehyde (MDA) levels, are valuable measures of cellular early-onset effects induced by environmental stress factors, such as contaminant exposure and fasting. In this study, these parameters were analysed and related to levels of halogenated organic contaminants (HOCs) in 40 Herring Gull (Larus argentatus) chicks. Chicks were experimentally exposed to HOCs through diet and went through a period of nutrient deprivation at the end of the experiment. HOC exposure and fasting were conducted separately and in combination. NL storages were depleted, and lysosomal membranes were destabilised after HOC exposure and nutrient deprivation. These responses were not related specifically to one type of stress or the extent of the treatment. No synergistic or additive effects from the combination of HOC exposure and fasting were observed. LF accumulated, and MDA levels increased as a result of fasting, but were unaffected by HOC exposure. LF accumulation was strongly associated with the percent weight change in the chicks. Large weight loss was associated with high LF levels, and slight weight gain was associated with low LF levels. Hence, food deprivation affected all the measured parameters, and HOC exposure decreased NL levels and lysosomal membrane stability in HG chick liver. Furthermore, autophagic lysosomal parameters have frequently been applied as biomarkers of cellular health status in previous studies of marine and terrestrial invertebrates, and this study suggests that these parameters may be good candidates for biomarkers of cellular health status in seabirds as well.

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

    Science.gov (United States)

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

    2015-01-30

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

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

    Directory of Open Access Journals (Sweden)

    Stern Stephan T

    2012-06-01

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

  3. Does vertebroplasty affect radiation dose distribution?: comparison of spatial dose distributions in a cement-injected vertebra as calculated by treatment planning system and actual spatial dose distribution.

    Science.gov (United States)

    Komemushi, Atsushi; Tanigawa, Noboru; Kariya, Shuji; Yagi, Rie; Nakatani, Miyuki; Suzuki, Satoshi; Sano, Akira; Ikeda, Koshi; Utsunomiya, Keita; Harima, Yoko; Sawada, Satoshi

    2012-01-01

    Purpose. To assess differences in dose distribution of a vertebral body injected with bone cement as calculated by radiation treatment planning system (RTPS) and actual dose distribution. Methods. We prepared two water-equivalent phantoms with cement, and the other two phantoms without cement. The bulk density of the bone cement was imported into RTPS to reduce error from high CT values. A dose distribution map for the phantoms with and without cement was calculated using RTPS with clinical setting and with the bulk density importing. Actual dose distribution was measured by the film density. Dose distribution as calculated by RTPS was compared to the dose distribution measured by the film dosimetry. Results. For the phantom with cement, dose distribution was distorted for the areas corresponding to inside the cement and on the ventral side of the cement. However, dose distribution based on film dosimetry was undistorted behind the cement and dose increases were seen inside cement and around the cement. With the equivalent phantom with bone cement, differences were seen between dose distribution calculated by RTPS and that measured by the film dosimetry. Conclusion. The dose distribution of an area containing bone cement calculated using RTPS differs from actual dose distribution.

  4. Distribution and abundance of predators that affect duck production--prairie pothole region

    Science.gov (United States)

    Sargeant, A.B.; Greenwood, R.J.; Sovada, M.A.; Shaffer, T.L.

    1993-01-01

    During 1983-88, the relative abundance of 18 species and species-groups of mammalian and avian predators affecting duck production in the prairie pothole region was determined in 33 widely scattered study areas ranging in size from 23-26 km2. Accounts of each studied species and species-group include habitat and history, population structure and reported densities, and information on distribution and abundance from the present study. Index values of undetected, scarce, uncommon, common, or numerous were used to rate abundance of nearly all species in each study area. Principal survey methods were livetrapping of striped skunks (Mephitis mephitis) and Franklin's ground squirrels (Spermophilus franklinii), systematic searches for carnivore tracks in quarter sections (0.65 km2), daily records of sightings of individual predator species, and systematic searches for occupied nests of tree-nesting avian predators. Abundances of predators in individual areas were studied 1-3 years.The distribution and abundance of predator species throughout the prairie pothole region have undergone continual change since settlement of the region by Europeans in the late 1800's. Predator populations in areas we studied differed markedly from those of pristine times. The changes occurred from habitat alterations, human-inflicted mortality of predators, and interspecific relations among predator species. Indices from surveys of tracks revealed a decline in the abundance of red foxes (Vulpes vulpes) and an albeit less consistent decline in the abundance of raccoons (Procyon lotor) with an increase in the abundance of coyotes (Canis latrans). Records of locations of occupied nests revealed great horned owls (Bubo virginianus) and red-tailed hawks (Buteo jamaicensis) tended to nest 0.5 km apart, and American crows (Corvus brachyrhynchos) tended to avoid nesting 0.5 km of nests of red-tailed hawks. Excluding large gulls, for which no measurements of abundance were obtained, the number of

  5. Factors affecting the occurrence and distribution of entomopathogenic fungi in natural and cultivated soils.

    Science.gov (United States)

    Quesada-Moraga, Enrique; Navas-Cortés, Juan A; Maranhao, Elizabeth A A; Ortiz-Urquiza, Almudena; Santiago-Alvarez, Cándido

    2007-08-01

    Factors affecting the occurrence and distribution of entomopathogenic fungi in 244 soil samples collected from natural and cultivated areas in Spain were studied using an integrated approach based on univariate and multivariate analyses. Entomopathogenic fungi were isolated from 175 of the 244 (71.7%) soil samples, with only two species found, Beauveria bassiana and Metarhizium anisopliae. Of the 244 soil samples, 104 yielded B. bassiana (42.6%), 18 yielded M. anisopliae (7.3%), and 53 soil samples (21.7%) harboured both fungi. Log-linear models indicated no significant effect of habitat on the occurrence of B. bassiana, but a strong association between M. anisopliae and soils from cultivated habitats, particularly field crops. Also, irrespective of habitat type, B. bassiana predominated over M. anisopliae in soils with a higher clay content, higher pH, and lower organic matter content. Logistic regression analyses showed that pH and clay content were predictive variables for the occurrence of B. bassiana, whereas organic matter content was the predictive variable for M. anisopliae. Also, latitude and longitude predicted the occurrence of these same species, but in opposite directions. Altitude was found to be predictive for the occurrence of B. bassiana. Using principal component analysis, four factors (1 to 4) accounted for 86% of the total variance; 32.8, 22.9, 19.6 and 10.4% of the cumulative variance explained, respectively. Factor 1 was associated with high positive weights for soil clay and silt content and high negative weights for soil sand content. Factor 2 was associated with high positive weights for soil organic matter content and high negative weights for soil pH. Factor 3 was associated with high positive weights for latitude and longitude of the sampled localities and factor 4, had high positive weights only for the altitude. Bi-plot displays representing soil samples were developed for different factor combinations and indicated that, irrespective

  6. Morphology, chemistry and distribution of neoformed spherulites in agricultural land affected by metallurgical point-source pollution

    NARCIS (Netherlands)

    Leguedois, S.; Oort, van F.; Jongmans, A.G.; Chevalier, P.

    2004-01-01

    Metal distribution patterns in superficial soil horizons of agricultural land affected by metallurgical point-source pollution were studied using optical and electron microscopy, synchrotron radiation and spectroscopy analyses. The site is located in northern France, at the center of a former entry

  7. Lysosomal membrane stability in laboratory- and field-exposed terrestrial isopods Porcellio scaber (Isopoda, Crustacea).

    Science.gov (United States)

    Nolde, Natasa; Drobne, Damjana; Valant, Janez; Padovan, Ingrid; Horvat, Milena

    2006-08-01

    Two established methods for assessment of the cytotoxicity of contaminants, the lysosomal latency (LL) assay and the neutral red retention (NRR) assay, were successfully applied to in toto digestive gland tubes (hepatopancreas) of the terrestrial isopod Porcellio scaber (Isopoda, Crustacea). In vitro exposure of isolated gland tubes to copper was used as a positive control to determine the performance of the two methods. Lysosomal latency and the NRR assay were then used on in vivo (via food) laboratory-exposed animals and on field populations. Arbitrarily selected criteria for determination of the fitness of P. scaber were set on the basis of lysosomal membrane stability (LMS) as assessed with in toto digestive gland tubes. Decreased LMS was detected in animals from all polluted sites, but cytotoxicity data were not in agreement with concentrations of pollutants. Lysosomal membrane stability in the digestive gland tubes of animals from an environment in Idrija, Slovenia that was highly polluted with mercury (260 microg/g dry wt food and 1,600 microg/g dry wt soil) was less affected than LMS in laboratory animals fed with 5 and 50 microg Hg/g dry weight for 3 d. This probably indicates tolerance of P. scaber to mercury in the mercury-polluted environment and/or lower bioavailability of environmental mercury. In animals from the vicinity of a thermal power plant with environmental mercury concentrations three to four orders of magnitude lower than those in Idrija, LMS was severely affected. In general, the LL assay was more sensitive than the NRR assay. The LMS assay conducted on digestive gland tubes of terrestrial isopods is highly recommended for integrated biomarker studies.

  8. A Maximum-Entropy Compound Distribution Model for Extreme Wave Heights of Typhoon-Affected Sea Areas

    Institute of Scientific and Technical Information of China (English)

    WANG Li-ping; SUN Xiao-guang; LU Ke-bo; XU De-lun

    2012-01-01

    A new compound distribution model for extreme wave heights of typhoon-affected sea areas is proposed on the basis of the maximum-entropy principle.The new model is formed by nesting a discrete distribution in a continuous one,having eight parameters which can be determined in terms of observed data of typhoon occurrence-frequency and extreme wave heights by numerically solving two sets of equations derived in this paper.The model is examined by using it to predict the N-year return-periodwave height at two hydrology stations in the Yellow Sea,and the predicted results are compared with those predicted by use of some other compound distribution models.Examinations and comparisons show that the model has some advantages for predicting the N-year return-period wave height in typhoon-affected sea areas.

  9. Two pore channel 2 (TPC2) inhibits autophagosomal-lysosomal fusion by alkalinizing lysosomal pH.

    Science.gov (United States)

    Lu, Yingying; Hao, Bai-Xia; Graeff, Richard; Wong, Connie W M; Wu, Wu-Tian; Yue, Jianbo

    2013-08-16

    Autophagy is an evolutionarily conserved lysosomal degradation pathway, yet the underlying mechanisms remain poorly understood. Nicotinic acid adenine dinucleotide phosphate (NAADP), one of the most potent Ca(2+) mobilizing messengers, elicits Ca(2+) release from lysosomes via the two pore channel 2 (TPC2) in many cell types. Here we found that overexpression of TPC2 in HeLa or mouse embryonic stem cells inhibited autophagosomal-lysosomal fusion, thereby resulting in the accumulation of autophagosomes. Treatment of TPC2 expressing cells with a cell permeant-NAADP agonist, NAADP-AM, further induced autophagosome accumulation. On the other hand, TPC2 knockdown or treatment of cells with Ned-19, a NAADP antagonist, markedly decreased the accumulation of autophagosomes. TPC2-induced accumulation of autophagosomes was also markedly blocked by ATG5 knockdown. Interestingly, inhibiting mTOR activity failed to increase TPC2-induced autophagosome accumulation. Instead, we found that overexpression of TPC2 alkalinized lysosomal pH, and lysosomal re-acidification abolished TPC2-induced autophagosome accumulation. In addition, TPC2 overexpression had no effect on general endosomal-lysosomal degradation but prevented the recruitment of Rab-7 to autophagosomes. Taken together, our data demonstrate that TPC2/NAADP/Ca(2+) signaling alkalinizes lysosomal pH to specifically inhibit the later stage of basal autophagy progression.

  10. Lysosomal localization of TRPML3 depends on TRPML2 and the mucolipidosis-associated protein TRPML1.

    Science.gov (United States)

    Venkatachalam, Kartik; Hofmann, Thomas; Montell, Craig

    2006-06-23

    Mucolipidosis type IV is an autosomal recessive lysosomal storage disorder characterized by severe neurodegeneration, achlorhydria, and visual impairments such as corneal opacity and strabismus. The disease arises due to mutations in a group 2 transient receptor potential (TRP)-related cation channel, TRPML1. Mammals encode two additional TRPML proteins named TRPML2 and TRPML3. Information regarding the propensity of these proteins to multimerize, their subcellular distribution and mechanisms that regulate their trafficking are limited. Here we demonstrate that TRPMLs interact to form homo- and heteromultimers. Moreover, the presence of either TRPML1 or TRPML2 specifically influences the spatial distribution of TRPML3. TRPML1 and TRPML2 homomultimers are lysosomal proteins, whereas TRPML3 homomultimers are in the endoplasmic reticulum. However, TRPML3 localizes to lysosomes when coexpressed with either TRPML1 or TRPML2 and is comparably mislocalized when lysosomal targeting of TRPML1 and TRPML2 is disrupted. Conversely, TRPML3 does not cause retention of TRPML1 or TRPML2 in the endoplasmic reticulum. These data demonstrate that there is a hierarchy controlling the subcellular distributions of the TRPMLs such that TRPML1 and TRPML2 dictate the localization of TRPML3 and not vice versa.

  11. Secondary Lysosomal Changes in Liver in Preclinical Drug Development

    Institute of Scientific and Technical Information of China (English)

    Vincent P. Meador; D. V. M.; Ph. D.; Diplomate ACVP

    2005-01-01

    @@ Lysosomes are intracytoplasmic membrane-bound organelles that function to degrade intracellular substances by enzymatic digestion. They occur normally in all cells, being especially prominent in phagocytic cells of the reticuloendothelial system.

  12. Endosome-lysosomes, ubiquitin and neurodegeneration.

    Science.gov (United States)

    Mayer, R J; Tipler, C; Arnold, J; Laszlo, L; Al-Khedhairy, A; Lowe, J; Landon, M

    1996-01-01

    Before the advent of ubiquitin immunochemistry and immunogold electron microscopy, there was no known intracellular molecular commonality between neurodegenerative diseases. The application of antibodies which primarily detect ubiquitin protein conjugates has shown that all of the human and animal idiopathic and transmissible chronic neurodegenerative diseases, (including Alzheimer's disease (AD), Lewy body disease (LBD), amyotrophic lateral sclerosis (ALS), Creutzfeldt-Jakob disease (CJD) and scrapie) are related by some form of intraneuronal inclusion which contains ubiquitin protein conjugates. In addition, disorders such as Alzheimer's disease, CJD and sheep scrapie, are characterised by deposits of amyloid, arising through incomplete breakdown of membrane proteins which may be associated with cytoskeletal reorganisation. Although our knowledge about these diseases is increasing, they remain largely untreatable. Recently, attention has focused on the mechanisms of production of different types of amyloid and the likely involvement within cells of the endosome-lysosome system, organelles which are immuno-positive for ubiquitin protein conjugates. These organelles may be 'bioreactor' sites for the unfolding and partial degradation of membrane proteins to generate the amyloid materials or their precursors which subsequently become expelled from the cell, or are released from dead cells, and accumulate as pathological entities. Such common features of the disease processes give new direction to therapeutic intervention.

  13. Magnesium Modulates Doxorubicin Activity through Drug Lysosomal Sequestration and Trafficking.

    Science.gov (United States)

    Trapani, Valentina; Luongo, Francesca; Arduini, Daniela; Wolf, Federica I

    2016-03-21

    Magnesium is directly involved in the control of cell growth and survival, but its role in cancer biology and therapy is multifaceted; in particular, it is highly controversial whether magnesium levels can affect therapy outcomes. Here we investigated whether magnesium availability can modulate cellular responses to the widely used chemotherapeutic doxorubicin. We used an in vitro model consisting of mammary epithelial HC11 cells and found that high magnesium availability was correlated with diminished sensitivity both in cells chronically adapted to high magnesium concentrations and in acutely magnesium-supplemented cells. This decrease in sensitivity resulted from reduced intracellular doxorubicin accumulation in the face of a similar drug uptake rate. We observed that high-magnesium conditions caused a decrease in intracellular drug retention by altering drug lysosomal sequestration and trafficking. In our model, magnesium supplementation correspondingly modulated expression of the TRPM7 channel, which is known to control cytoskeletal organization and dynamics and may be involved in the proposed mechanism. Our findings suggest that magnesium supplementation in hypomagnesemic cancer patients may hinder response to therapy.

  14. Lysosomal trafficking functions of mucolipin-1 in murine macrophages

    Directory of Open Access Journals (Sweden)

    Dang Hope

    2007-12-01

    Full Text Available Abstract 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.

  15. Lysosomal enzymes and their receptors in invertebrates: an evolutionary perspective.

    Science.gov (United States)

    Kumar, Nadimpalli Siva; Bhamidimarri, Poorna M

    2015-01-01

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

  16. Revealing the fate of cell surface human P-glycoprotein (ABCB1): The Lysosomal Degradation Pathway

    Science.gov (United States)

    Katayama, Kazuhiro; Kapoor, Khyati; Ohnuma, Shinobu; Patel, Atish; Swaim, William; Ambudkar, Indu S.; Ambudkar, Suresh V.

    2015-01-01

    P-glycoprotein (P-gp) transports a variety of chemically dissimilar amphipathic compounds including anticancer drugs. Although mechanisms of P-gp drug transport are widely studied, the pathways involving its internalization are poorly understood. The present study is aimed at elucidating the pathways involved in degradation of cell surface P-gp. The fate of P-gp at the cell surface was determined by biotinylating cell surface proteins followed by flow cytometry and Western blotting. Our data shows that the half-life of endogenously expressed P-gp is 26.7 ± 1.1 h in human colorectal cancer HCT-15 cells. Treatment of cells with Bafilomycin A1 (BafA1) a vacuolar H+ ATPase inhibitor increased the half-life of P-gp at the cell surface to 36.1± 0.5 h. Interestingly, treatment with the proteasomal inhibitors MG132, MG115 or lactacystin alone did not alter the half-life of the protein. When cells were treated with both lysosomal and proteasomal inhibitors (BafA1 and MG132), the half-life was further prolonged to 39-50 h. Functional assays done with rhodamine 123 or calcein-AM, fluorescent substrates of P-gp, indicated that the transport function of P-gp was not affected by either biotinylation or treatment with BafA1 or proteasomal inhibitors. Immunofluorescence studies done with the antibody against lysosomal marker LAMP1 and the P-gp-specific antibody UIC2 in permeabilized cells indicated that intracellular P-gp is primarily localized in the lysosomal compartment. Our results suggest that the lysosomal degradation system could be targeted to increase the sensitivity of P-gp expressing cancer cells towards chemotherapeutic drugs. PMID:26057472

  17. Revealing the fate of cell surface human P-glycoprotein (ABCB1): The lysosomal degradation pathway.

    Science.gov (United States)

    Katayama, Kazuhiro; Kapoor, Khyati; Ohnuma, Shinobu; Patel, Atish; Swaim, William; Ambudkar, Indu S; Ambudkar, Suresh V

    2015-10-01

    P-glycoprotein (P-gp) transports a variety of chemically dissimilar amphipathic compounds including anticancer drugs. Although mechanisms of P-gp drug transport are widely studied, the pathways involving its internalization are poorly understood. The present study is aimed at elucidating the pathways involved in degradation of cell surface P-gp. The fate of P-gp at the cell surface was determined by biotinylating cell surface proteins followed by flow cytometry and Western blotting. Our data shows that the half-life of endogenously expressed P-gp is 26.7±1.1 h in human colorectal cancer HCT-15 cells. Treatment of cells with Bafilomycin A1 (BafA1) a vacuolar H+ ATPase inhibitor increased the half-life of P-gp at the cell surface to 36.1±0.5 h. Interestingly, treatment with the proteasomal inhibitors MG132, MG115 or lactacystin alone did not alter the half-life of the protein. When cells were treated with both lysosomal and proteasomal inhibitors (BafA1 and MG132), the half-life was further prolonged to 39-50 h. Functional assays done with rhodamine 123 or calcein-AM, fluorescent substrates of P-gp, indicated that the transport function of P-gp was not affected by either biotinylation or treatment with BafA1 or proteasomal inhibitors. Immunofluorescence studies done with the antibody against lysosomal marker LAMP1 and the P-gp-specific antibody UIC2 in permeabilized cells indicated that intracellular P-gp is primarily localized in the lysosomal compartment. Our results suggest that the lysosomal degradation system could be targeted to increase the sensitivity of P-gp- expressing cancer cells towards chemotherapeutic drugs.

  18. Factors influencing the measurement of lysosomal enzymes activity in human cerebrospinal fluid.

    Directory of Open Access Journals (Sweden)

    Emanuele Persichetti

    Full Text Available Measurements of the activities of lysosomal enzymes in cerebrospinal fluid have recently been proposed as putative biomarkers for Parkinson's disease and other synucleinopathies. To define the operating procedures useful for ensuring the reliability of these measurements, we analyzed several pre-analytical factors that may influence the activity of β-glucocerebrosidase, α-mannosidase, β-mannosidase, β-galactosidase, α-fucosidase, β-hexosaminidase, cathepsin D and cathepsin E in cerebrospinal fluid. Lysosomal enzyme activities were measured by well-established fluorimetric assays in a consecutive series of patients (n = 28 with different neurological conditions, including Parkinson's disease. The precision, pre-storage and storage conditions, and freeze/thaw cycles were evaluated. All of the assays showed within- and between-run variabilities below 10%. At -20°C, only cathepsin D was stable up to 40 weeks. At -80°C, the cathepsin D, cathepsin E, and β-mannosidase activities did not change significantly up to 40 weeks, while β-glucocerebrosidase activity was stable up to 32 weeks. The β-galactosidase and α-fucosidase activities significantly increased (+54.9±38.08% after 4 weeks and +88.94±36.19% after 16 weeks, respectively. Up to four freeze/thaw cycles did not significantly affect the activities of cathepsins D and E. The β-glucocerebrosidase activity showed a slight decrease (-14.6% after two freeze/thaw cycles. The measurement of lysosomal enzyme activities in cerebrospinal fluid is reliable and reproducible if pre-analytical factors are accurately taken into consideration. Therefore, the analytical recommendations that ensue from this study may contribute to the establishment of actual values for the activities of cerebrospinal fluid lysosomal enzymes as putative biomarkers for Parkinson's disease and other neurodegenerative disorders.

  19. Serial Spike Time Correlations Affect Probability Distribution of Joint Spike Events.

    Science.gov (United States)

    Shahi, Mina; van Vreeswijk, Carl; Pipa, Gordon

    2016-01-01

    Detecting the existence of temporally coordinated spiking activity, and its role in information processing in the cortex, has remained a major challenge for neuroscience research. Different methods and approaches have been suggested to test whether the observed synchronized events are significantly different from those expected by chance. To analyze the simultaneous spike trains for precise spike correlation, these methods typically model the spike trains as a Poisson process implying that the generation of each spike is independent of all the other spikes. However, studies have shown that neural spike trains exhibit dependence among spike sequences, such as the absolute and relative refractory periods which govern the spike probability of the oncoming action potential based on the time of the last spike, or the bursting behavior, which is characterized by short epochs of rapid action potentials, followed by longer episodes of silence. Here we investigate non-renewal processes with the inter-spike interval distribution model that incorporates spike-history dependence of individual neurons. For that, we use the Monte Carlo method to estimate the full shape of the coincidence count distribution and to generate false positives for coincidence detection. The results show that compared to the distributions based on homogeneous Poisson processes, and also non-Poisson processes, the width of the distribution of joint spike events changes. Non-renewal processes can lead to both heavy tailed or narrow coincidence distribution. We conclude that small differences in the exact autostructure of the point process can cause large differences in the width of a coincidence distribution. Therefore, manipulations of the autostructure for the estimation of significance of joint spike events seem to be inadequate.

  20. Contact analysis and load distribution of double-envelop hourglassworm gearing affected by errors and load

    Institute of Scientific and Technical Information of China (English)

    XU Wujiao; QIN Datong; SHI Wankai

    2003-01-01

    An approach for the contact analysis and load distribution of double-envelop hourglass worm gearing is presented,which is based on a 3-D elastic contact finite element method (FEM) model that 3ccommodates the influence of errors and load.As compared with existing tooth contact analysis model that assumes rigidity for the contacting surfaces, the proposed model provides a more realistic analysis on the contact patterns, the distribution of contact load and transmission errors. It is also capable of exploring the influence of different errors on meshing performances, the contact deformation, the shift of the contact zone and load share among the meshing tooth-pairs under different load.

  1. 26 CFR 1.332-5 - Distributions in liquidation as affecting minority interests.

    Science.gov (United States)

    2010-04-01

    ... liquidation as affecting minority interests. Upon the liquidation of a corporation in pursuance of a plan of complete liquidation, the gain or loss of minority shareholders shall be determined without regard to... minority interests. 1.332-5 Section 1.332-5 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF...

  2. 78 FR 32713 - Distribution of Continued Dumping and Subsidy Offset to Affected Domestic Producers

    Science.gov (United States)

    2013-05-31

    ... 504 of the Tax Relief, Unemployment Insurance Reauthorization, and Job Creation Act of 2010... money available for distribution can be expected to continue diminishing over time. It should also be...-337-804......... 731-TA-776........ Preserved LK Bowman. Mushrooms/ Chile. Modern Mushroom...

  3. 76 FR 31019 - Distribution of Continued Dumping and Subsidy Offset to Affected Domestic Producers

    Science.gov (United States)

    2011-05-27

    ..., Unemployment Insurance Reauthorization, and Job Creation Act of 2010. Consequently, the full impact of the... will be continued for an undetermined period; however, the amount of money available for distribution... Preserved Mushrooms/ LK Bowman Chile. Modern Mushroom Farms Monterey Mushrooms Mount Laurel Canning...

  4. Low temperature treatment affects concentration and distribution of chrysanthemum stunt viroid in Argyranthemum

    Directory of Open Access Journals (Sweden)

    Zhibo eZhang

    2016-03-01

    Full Text Available Chrysanthemum stunt viroid (CSVd can infect Argyranthemum and cause serious economic loss. Low temperature treatment combined with meristem culture has been applied to eradicate viroids from their hosts, but without success in eliminating CSVd from diseased Argyranthemum. The objectives of this work were to investigate 1 the effect of low temperature treatment combined with meristem culture on elimination of CSVd, 2 the effect of low temperature treatment on CSVd distribution pattern in shoot apical meristem (SAM, and 3 CSVd distribution in flowers and stems of two infected Argyranthemum cultivars. After treatment with low temperature combined with meristem tip culture, two CSVd-free plants were found in ‘Border Dark Red’, but none in ‘Yellow Empire’. With the help of in situ hybridization, we found that CSVd distribution patterns in the SAM showed no changes in diseased ‘Yellow Empire’ following 5oC treatment, compared with non-treated plants. However, the CSVd-free area in SAM was enlarged in diseased ‘Border Dark Red’ following prolonged 5oC treatment. Localization of CSVd in the flowers and stems of infected ‘Border Dark Red’ and ‘Yellow Empire’ indicated that seeds could not transmit CSVd in these two cultivars, and CSVd existed in phloem. Results obtained in the study contributed to better understanding of the distribution of CSVd in systemically infected plants and the combination of low temperature treatment and meristem tip culture for production of viroid-free plants.

  5. Distributed Modeling of soil erosion and deposition affected by buffer strips

    DEFF Research Database (Denmark)

    Khademalrasoul, Ataalah; Heckrath, Goswin Johann; Iversen, Bo Vangsø

    and dimension of buffer zones in the landscape can be optimized by means of spatially distributed erosion and deposition modeling. During the period from 1998 to 2000 field campaigns were done on a range of agricultural land in Denmark. On 21 slope units and adjacent buffer zones, rill erosion and deposition...

  6. Soil organic matter distribution and microaggregate characteristics as affected by agricultural management and earthworm activity

    NARCIS (Netherlands)

    Pulleman, M.M.; Six, J.; Breemen, van N.; Jongmans, A.G.

    2005-01-01

    Stable microaggregates can physically protect occluded soil organic matter (SOM) against decomposition. We studied the effects of agricultural management on the amount and characteristics of microaggregates and on SOM distribution in a marine loam soil in the Netherlands. Three long-term farming sys

  7. Patchy Distributions of Competitors Affect the Growth of a Clonal Plant When the Competitor Density Is High

    OpenAIRE

    Wei Xue; Lin Huang; Bi-Cheng Dong; Ming-Xiang Zhang; Fei-Hai Yu

    2013-01-01

    Environments are patchy in not only abiotic factors but also biotic ones. Many studies have examined effects of spatial heterogeneity in abiotic factors such as light, water and nutrients on the growth of clonal plants, but few have tested those in biotic factors. We conducted a greenhouse experiment to examine how patchy distributions of competitors affect the growth of a rhizomatous wetland plant Bolboschoenus planiculmis and whether such effects depend on the density of the competitors. We...

  8. Numerical Simulation for Flow Distribution in ACE7 Fuel Assemblies affected by a Spacer Grid Deformation

    Energy Technology Data Exchange (ETDEWEB)

    Park, Jongpil; Jeong, Ji Hwan [Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of)

    2014-05-15

    In spite of various efforts to understand hydraulic phenomena in a rod bundle containing deformed rods due to swelling and/or ballooning of clad, the studies for flow blockage due to spacer grid deformation have been limited. In the present work, 3D CFD analysis for flow blockage was performed to evaluate coolant flow within ACE7 fuel assemblies (FAs) containing a FA affected by a spacer grid deformation. The real geometry except for inner grids was used in the simulation and the region including inner grid was replaced by porous media. In the present work, the numerical simulation was performed to predict coolant flow within ACE7 FAs affected by a Mid grid deformation. The 3D CFD result shows that approximately 60 subchannel hydraulic diameter is required to fully recover coolant flow under normal operating condition.

  9. Proteasome Activity Is Affected by Fluctuations in Insulin-Degrading Enzyme Distribution.

    Science.gov (United States)

    Sbardella, Diego; Tundo, Grazia Raffaella; Sciandra, Francesca; Bozzi, Manuela; Gioia, Magda; Ciaccio, Chiara; Tarantino, Umberto; Brancaccio, Andrea; Coletta, Massimo; Marini, Stefano

    2015-01-01

    Insulin-Degrading-Enzyme (IDE) is a Zn2+-dependent peptidase highly conserved throughout evolution and ubiquitously distributed in mammalian tissues wherein it displays a prevalent cytosolic localization. We have recently demonstrated a novel Heat Shock Protein-like behaviour of IDE and its association with the 26S proteasome. In the present study, we examine the mechanistic and molecular features of IDE-26S proteasome interaction in a cell experimental model, extending the investigation also to the effect of IDE on the enzymatic activities of the 26S proteasome. Further, kinetic investigations indicate that the 26S proteasome activity undergoes a functional modulation by IDE through an extra-catalytic mechanism. The IDE-26S proteasome interaction was analyzed during the Heat Shock Response and we report novel findings on IDE intracellular distribution that might be of critical relevance for cell metabolism.

  10. Proteasome Activity Is Affected by Fluctuations in Insulin-Degrading Enzyme Distribution.

    Directory of Open Access Journals (Sweden)

    Diego Sbardella

    Full Text Available Insulin-Degrading-Enzyme (IDE is a Zn2+-dependent peptidase highly conserved throughout evolution and ubiquitously distributed in mammalian tissues wherein it displays a prevalent cytosolic localization. We have recently demonstrated a novel Heat Shock Protein-like behaviour of IDE and its association with the 26S proteasome. In the present study, we examine the mechanistic and molecular features of IDE-26S proteasome interaction in a cell experimental model, extending the investigation also to the effect of IDE on the enzymatic activities of the 26S proteasome. Further, kinetic investigations indicate that the 26S proteasome activity undergoes a functional modulation by IDE through an extra-catalytic mechanism. The IDE-26S proteasome interaction was analyzed during the Heat Shock Response and we report novel findings on IDE intracellular distribution that might be of critical relevance for cell metabolism.

  11. Distribution of elements in needles of Pinus massoniana (Lamb.) was uneven and affected by needle age.

    Science.gov (United States)

    Kuang, Yuan Wen; Wen, Da Zhi; Zhou, Guoyi; Liu, Shi Zhong

    2007-02-01

    Macronutrients (P, S, K, Na, Mg, Ca), heavy metals (Fe, Zn, Mn, Cu, Pb, Cr, Ni, Cd,) and Al concentrations as well as values of Ca/Al in the tip, middle and base sections, and sheaths of current year and previous year needles of Pinus massoniana from Xiqiao Mountain were analyzed and the distribution patterns of those elements were compared. The results indicated that many elements were unevenly distributed among the different components of needles. Possible deficiency of P, K, Ca, Mn and Al toxicity occurred in needles under air pollution. Heavy metals may threaten the health of Masson pine. Needle sheaths were good places to look for particulate pollutants, in this case including Fe, Cu, Zn, Pb, Cr, Cd and Al.

  12. Distribution of elements in needles of Pinus massoniana (Lamb.) was uneven and affected by needle age

    Energy Technology Data Exchange (ETDEWEB)

    Kuang Yuanwen [South China Botanical Garden, Chinese Academy of Sciences, 510650 Guangzhou (China)]. E-mail: kuangyw@scbg.ac.cn; Wen Dazhi [South China Botanical Garden, Chinese Academy of Sciences, 510650 Guangzhou (China)]. E-mail: dzwen@scbg.ac.cn; Zhou Guoyi [South China Botanical Garden, Chinese Academy of Sciences, 510650 Guangzhou (China)]. E-mail: gyzhou@scbg.ac.cn; Liu Shizhong [South China Botanical Garden, Chinese Academy of Sciences, 510650 Guangzhou (China)]. E-mail: lsz@scbg.ac.cn

    2007-02-15

    Macronutrients (P, S, K, Na, Mg, Ca), heavy metals (Fe, Zn, Mn, Cu, Pb, Cr, Ni, Cd,) and Al concentrations as well as values of Ca/Al in the tip, middle and base sections, and sheaths of current year and previous year needles of Pinus massoniana from Xiqiao Mountain were analyzed and the distribution patterns of those elements were compared. The results indicated that many elements were unevenly distributed among the different components of needles. Possible deficiency of P, K, Ca, Mn and Al toxicity occurred in needles under air pollution. Heavy metals may threaten the health of Masson pine. Needle sheaths were good places to look for particulate pollutants, in this case including Fe, Cu, Zn, Pb, Cr, Cd and Al. - Pine needle sections as bioindicator for heavy metals and nutrient deficiency particularly needle sheath for particle pollutants.

  13. Distribution of elements in needles of Pinus massoniana (Lamb.) was uneven and affected by needle age

    Energy Technology Data Exchange (ETDEWEB)

    Kuang Yuanwen [Institute of Ecology, South China Botanical Garden, Chinese Academy of Sciences, 510650 Guangzhou (China)]. E-mail: kuangyw@scbg.ac.cn; Wen Dazhi [Institute of Ecology, South China Botanical Garden, Chinese Academy of Sciences, 510650 Guangzhou (China)]. E-mail: dzwen@scbg.ac.cn; Zhou Guoyi [Institute of Ecology, South China Botanical Garden, Chinese Academy of Sciences, 510650 Guangzhou (China)]. E-mail: gyzhou@scbg.ac.cn; Liu Shizhong [Institute of Ecology, South China Botanical Garden, Chinese Academy of Sciences, 510650 Guangzhou (China)]. E-mail: lsz@scbg.ac.cn

    2007-01-15

    Macronutrients (P, S, K, Na, Mg, Ca), heavy metals (Fe, Zn, Mn, Cu, Pb, Cr, Ni, Cd) and Al concentrations as well as values of Ca/Al in the tip, middle, base sections and sheaths of current year and previous year needles of Pinus massoniana from Xiqiao Mountain were analyzed and the distribution patterns of those elements were compared. The results indicated that many elements were unevenly distributed among the different components of needles. Possible deficiency of P, K, Ca, Mn and Al toxicity occurred in needles under air pollution. Heavy metals may threaten the health of Masson pine. Needle sheaths were good places to look for particulate pollutants, in this case including Fe, Cu, Zn, Pb, Cr, Cd and Al. - Pine needle sections as bioindicator for heavy metals and nutrient deficiency particularly needle sheath for particle pollutants.

  14. Lysosomal {beta}-mannosidase: cDNA cloning and characterization

    Energy Technology Data Exchange (ETDEWEB)

    Chen, H.; Leipprandt, J.R.; Traviss, C.E. [Michigan State Univ., East Lansing, MI (United States)] [and others

    1994-09-01

    Lysosomal {beta}-mannosidase is an exoglycosidase that cleaves the single {beta}-linked mannose residue from the non-reducing end of all N-linked glycoprotein oligosaccharides. Deficiency of this enzyme results in {beta}-mannosidosis, a severe neurodegenerative disease in goats and cattle. The human cases described have a milder, highly variable presentation. Study of the molecular pathology of this disease in ruminants and humans and development of the animal model for gene therapy studies required cloning of the gene for {beta}-mannosidase has been cloned. {beta}-Mannosidase cDNA were obtained from a bovine thyroid cDNA library by screening with mixed oligonucleotides derived from peptide sequences resulting from microsequencing of bovine {beta}-mannosidase peptides. A total of six independent positive clones were identified from 5 x 10{sup 5} plaques, covering about 80% of the C-terminal region. The missing 5{prime} region was obtained using 5{prime} RACE. The full-length construct contains 3852-bp nucleotides, encoding 879 amino acids. The initiation codon is followed by 17 amino acids containing the characteristics of a typical signal peptide sequence. The deduced amino acid sequence is colinear with all peptide sequences determined by protein microsequencing. Northern blot analysis demonstrated a 4.2 kb single transcript in various tissues from both normal and affected goats and calves. The mRNA level was decreased in affected {beta}-mannosidosis animals. The gene encoding {beta}-mannosidase was localized on human chromosome 4 by Southern analysis of rodent/human somatic cell hybrids. The mutation in bovine {beta}-mannosidosis has been identified. This is the first report of cloning of the {beta}-mannosidase gene.

  15. Human and environmental factors affecting Aedes aegypti distribution in an arid urban environment.

    Science.gov (United States)

    Walker, Kathleen R; Joy, Teresa K; Ellers-Kirk, Christa; Ramberg, Frank B

    2011-06-01

    Aedes aegypti has reappeared in urban communities in the southwestern U.S.A. in the 1990s after a 40-year absence. In 2003 and 2004, a systematic survey was conducted throughout metropolitan Tucson, AZ, to identify human and environmental factors associated with Ae. aegypti distribution within an arid urban area. Aedes aegypti presence and abundance were measured monthly using the Centers for Disease Control and Prevention enhanced oviposition traps at sampling sites established in a grid at 3- to 4-km intervals across the city. Sampling occurred in the summer rainy season (July through September), the peak of mosquito activity in the region. Multiple regression analyses were conducted to determine relationships between mosquito density and factors that could influence mosquito distribution. House age was the only factor that showed a consistent significant association with Ae. aegypti abundance in both years: older houses had more mosquito eggs. This is the 1st study of Ae. aegypti distribution at a local level to identify house age as an explanatory factor independent of other human demographic factors. Further research into the reasons why mosquitoes were more abundant around older homes may help inform and refine future vector surveillance and control efforts in the event of a dengue outbreak in the region.

  16. Lysosomal storage disorders: Molecular basis and laboratory testing

    Directory of Open Access Journals (Sweden)

    Filocamo Mirella

    2011-03-01

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

  17. How the Assumed Size Distribution of Dust Minerals Affects the Predicted Ice Forming Nuclei

    Science.gov (United States)

    Perlwitz, Jan P.; Fridlind, Ann M.; Garcia-Pando, Carlos Perez; Miller, Ron L.; Knopf, Daniel A.

    2015-01-01

    The formation of ice in clouds depends on the availability of ice forming nuclei (IFN). Dust aerosol particles are considered the most important source of IFN at a global scale. Recent laboratory studies have demonstrated that the mineral feldspar provides the most efficient dust IFN for immersion freezing and together with kaolinite for deposition ice nucleation, and that the phyllosilicates illite and montmorillonite (a member of the smectite group) are of secondary importance.A few studies have applied global models that simulate mineral specific dust to predict the number and geographical distribution of IFN. These studies have been based on the simple assumption that the mineral composition of soil as provided in data sets from the literature translates directly into the mineral composition of the dust aerosols. However, these tables are based on measurements of wet-sieved soil where dust aggregates are destroyed to a large degree. In consequence, the size distribution of dust is shifted to smaller sizes, and phyllosilicates like illite, kaolinite, and smectite are only found in the size range 2 m. In contrast, in measurements of the mineral composition of dust aerosols, the largest mass fraction of these phyllosilicates is found in the size range 2 m as part of dust aggregates. Conversely, the mass fraction of feldspar is smaller in this size range, varying with the geographical location. This may have a significant effect on the predicted IFN number and its geographical distribution.An improved mineral specific dust aerosol module has been recently implemented in the NASA GISS Earth System ModelE2. The dust module takes into consideration the disaggregated state of wet-sieved soil, on which the tables of soil mineral fractions are based. To simulate the atmospheric cycle of the minerals, the mass size distribution of each mineral in aggregates that are emitted from undispersed parent soil is reconstructed. In the current study, we test the null

  18. Stability and heavy metal distribution of soil aggregates affected by application of apatite, lime, and charcoal.

    Science.gov (United States)

    Cui, Hongbiao; Ma, Kaiqiang; Fan, Yuchao; Peng, Xinhua; Mao, Jingdong; Zhou, Dongmei; Zhang, Zhongbin; Zhou, Jing

    2016-06-01

    Only a few studies have been reported on the stability and heavy metal distribution of soil aggregates after soil treatments to reduce the availability of heavy metals. In this study, apatite (22.3 t ha(-1)), lime (4.45 t ha(-1)), and charcoal (66.8 t ha(-1)) were applied to a heavy metal-contaminated soil for 4 years. The stability and heavy metal distribution of soil aggregates were investigated by dry and wet sieving. No significant change in the dry mean weight diameter was observed in any treatments. Compared with the control, three-amendment treatments significantly increased the wet mean weight diameter, but only charcoal treatment significantly increased the wet aggregate stability. The soil treatments increased the content of soil organic carbon, and the fraction 0.25-2 mm contained the highest content of soil organic carbon. Amendments' application slightly increased soil total Cu and Cd, but decreased the concentrations of CaCl2 -extractable Cu and Cd except for the fraction 2 and 0.25-2 mm contained the highest concentrations of CaCl2-extractable Cu and Cd, accounted for about 74.5-86.8 % of CaCl2-extractable Cu and Cd in soil. The results indicated that amendments' application increased the wet soil aggregate stability and decreased the available Cu and Cd. The distribution of available heavy metals in wet soil aggregates was not controlled by soil aggregate stability, but possibly by soil organic carbon.

  19. Distribution and prevalence of crown rot pathogens affecting wheat crops in southern Chile

    Directory of Open Access Journals (Sweden)

    Ernesto Moya-Elizondo

    2015-03-01

    Full Text Available Crown rot pathogens are associated with higher losses for wheat crop farmers, but information about the distribution and prevalence of these pathogens in Chile is inadequate. Distribution and prevalence of wheat (Triticum aestivum L. crown rot pathogens were examined in a survey of 48 commercial fields from December 2011 to February 2012 in southern Chile. These fields were located between Collipulli (37°56'00" S; 72°26'39" W and Purranque (40°50'30" S; 73°22'03" W. Severity of crown rot disease was determined through visual assessment of the first internode of 20 tillers obtained from each field. Incidence of crown rot pathogens per field was determined by plating the 20 tillers on Petri plates with 20% potato dextrose agar amended with lactic acid (aPDA medium. Resulting fungal colonies from monoxenic culture were identified by morphological or molecular-assisted identification. Severity of crown rot varied between 11.3% and 80% for individual fields. Culture plate analysis showed 72.2% of stems were infected with some fungus. Fusarium avenaceum, F. graminearum, and F. culmorum, pathogens associated with Fusarium crown rot disease were isolated from 13.5% of tillers. Gaeumannomyces graminis, causal agent of take-all disease in cereals, was isolated from 11.1% of culms. Phaeosphaeria sp., an endophyte and possibly a non-pathogenic fungus, was isolated from 13.9% of tillers. Pathogenic fungi such as Rhizoctonia spp. and Microdochium nivale, other saprophyte, and several unidentified non-sporulating fungi were isolated at frequencies lower than 3% of the total. Fusarium crown rot and take-all were the most prevalent and distributed crown rot diseases present in wheat crops in southern Chile.

  20. Is the distribution of Prochlorococcus and Synechococcus ecotypes in the Mediterranean Sea affected by global warming?

    Science.gov (United States)

    Mella-Flores, D.; Mazard, S.; Humily, F.; Partensky, F.; Mahé, F.; Bariat, L.; Courties, C.; Marie, D.; Ras, J.; Mauriac, R.; Jeanthon, C.; Mahdi Bendif, E.; Ostrowski, M.; Scanlan, D. J.; Garczarek, L.

    2011-09-01

    Biological communities populating the Mediterranean Sea, which is situated at the northern boundary of the subtropics, are often claimed to be particularly affected by global warming. This is indicated, for instance, by the introduction of (sub)tropical species of fish or invertebrates that can displace local species. This raises the question of whether microbial communities are similarly affected, especially in the Levantine basin where sea surface temperatures have significantly risen over the last 25 years (0.50 ± 0.11 °C in average per decade, P libraries of the 16S-23S ribosomal DNA Internal Transcribed Spacer (ITS) region, with a focus on the abundance of clades that may constitute bioindicators of warm waters. During both cruises, the dominant Prochlorococcus clade in the upper mixed layer at all stations was HLI, a clade typical of temperate waters, whereas the HLII clade, the dominant group in (sub)tropical waters, was only present at very low concentrations. The Synechococcus community was dominated by clades I, III and IV in the northwestern waters of the Gulf of Lions and by clade III and groups genetically related to clades WPC1 and VI in the rest of the Mediterranean Sea. In contrast, only a few sequences of clade II, a group typical of warm waters, were observed. These data indicate that local cyanobacterial populations have not yet been displaced by their (sub)tropical counterparts.

  1. Planting densities and bird and rodent absence affect size distributions of four dicots in synthetic tallgrass communities.

    Science.gov (United States)

    Martínez-Garza, Cristina; Saha, Sonali; Torres, Veronica; Brown, Joel S; Howe, Henry F

    2004-05-01

    Variability in the size distributions of populations is usually studied in monocultures or in mixed plantings of two species. Variability of size distributions of populations in more complex communities has been neglected. The effects of seeding density (35 or 350 seeds/species/m2) and presence of small vertebrates on the variability of size distributions were studied for a total of 1,920 individuals of 4 species in replicated synthetic communities of 18 species in northern Illinois. End-of season height and above-ground biomass were measured for prairie perennials Dalea purpurea (purple prairie clover), Echinacea purpurea (purple coneflower), Desmanthus illinoensis (Illinois bundleflower) and Heliopsis helianthoides (early sunflower). Variability in biomass distribution of the four target species was twice as great at low than at high densities when small vertebrates were excluded. Our results suggest that inter- and intraspecific competition may affect all individuals more under high-density conditions, thereby reducing the variability in their biomass distributions within this community. This result, a consequence of plant-plant interaction, is obscured when small birds or mammals are present, presumably because either or both add variance that overwhelms the pattern.

  2. From daily movements to population distributions: weather affects competitive ability in a guild of soaring birds.

    Science.gov (United States)

    Shepard, Emily L C; Lambertucci, Sergio A

    2013-11-06

    The ability of many animals to access and exploit food is dependent on the ability to move. In the case of scavenging birds, which use soaring flight to locate and exploit ephemeral resources, the cost and speed of movement vary with meteorological factors. These factors are likely to modify the nature of interspecific interactions, as well as individual movement capacity, although the former are less well understood. We used aeronautical models to examine how soaring performance varies with weather within a guild of scavenging birds and the consequences this has for access to a common resource. Birds could be divided broadly into those with low wing loading that are more competitive in conditions with weak updraughts and low winds (black vultures and caracaras), and those with high wing loading that are well adapted for soaring in strong updraughts and moderate to high winds (Andean condors). Spatial trends in meteorological factors seem to confine scavengers with high wing loading to the mountains where they out-compete other birds; a trend that is borne out in worldwide distributions of the largest species. However, model predictions and carcass observations suggest that the competitive ability of these and other birds varies with meteorological conditions in areas where distributions overlap. This challenges the view that scavenging guilds are structured by fixed patterns of dominance and suggests that competitive ability varies across spatial and temporal scales, which may ultimately be a mechanism promoting diversity among aerial scavengers.

  3. Elevation and stream-size thresholds affect distributions of native and exotic warmwater fishes in Wyoming

    Science.gov (United States)

    Quist, M.C.; Hubert, W.A.; Rahel, F.J.

    2004-01-01

    This study was conducted to assess the influence of elevation and stream width on the occurrence of 28 native and six exotic fish species using data collected (1954-2003) from 1,114 stream reaches in Wyoming. Medians and ranges of elevation and stream width were used to assess how elevation and stream width influenced the occurrence of individual species and to indicate which species had large and small ranges of distribution. Twenty-four species were common at elevations below 1,550 m and 31 species occurred in streams less than 20 m wide. The six exotic species had the potential to overlap all of the native species with regard to both elevation and stream width. In general, species that were collected over a wide range of elevations were also collected over a wide range of stream widths. Red shiner (Cyprinella lutrensis) and river carpsucker (Carpiodes carpio) occurred over the smallest elevation ranges ( 2,500 m). Longnose sucker and white sucker (Catostomus commersoni) occurred over the greatest ranges in stream widths (> 90 m), and brook stickleback (Culaea inconstans), black bullhead (Ameiurus melas), and quillback (Carpiodes cyprinus) were found over the lowest ranges in stream widths (< 12 m). The distributions of native and exotic species in streams that transition from the Rocky Mountains to the Great Plains were largely explained by elevation and stream width.

  4. TRPML1: an ion channel in the lysosome.

    Science.gov (United States)

    Wang, Wuyang; Zhang, Xiaoli; Gao, Qiong; Xu, Haoxing

    2014-01-01

    The first member of the mammalian mucolipin TRP channel subfamily (TRPML1) is a cation-permeable channel that is predominantly localized on the membranes of late endosomes and lysosomes (LELs) in all mammalian cell types. In response to the regulatory changes of LEL-specific phosphoinositides or other cellular cues, TRPML1 may mediate the release of Ca(2+) and heavy metal Fe(2+)/Zn(2+)ions into the cytosol from the LEL lumen, which in turn may regulate membrane trafficking events (fission and fusion), signal transduction, and ionic homeostasis in LELs. Human mutations in TRPML1 result in type IV mucolipidosis (ML-IV), a childhood neurodegenerative lysosome storage disease. At the cellular level, loss-of-function mutations of mammalian TRPML1 or its C. elegans or Drosophila homolog gene results in lysosomal trafficking defects and lysosome storage. In this chapter, we summarize recent advances in our understandings of the cell biological and channel functions of TRPML1. Studies on TRPML1's channel properties and its regulation by cellular activities may provide clues for developing new therapeutic strategies to delay neurodegeneration in ML-IV and other lysosome-related pediatric diseases.

  5. Eucommia ulmoides Oliver extract, aucubin, and geniposide enhance lysosomal activity to regulate ER stress and hepatic lipid accumulation.

    Directory of Open Access Journals (Sweden)

    Hwa-Young Lee

    Full Text Available Eucommia ulmoides Oliver is a natural product widely used as a dietary supplement and medicinal plant. Here, we examined the potential regulatory effects of Eucommia ulmoides Oliver extracts (EUE on hepatic dyslipidemia and its related mechanisms by in vitro and in vivo studies. EUE and its two active constituents, aucubin and geniposide, inhibited palmitate-induced endoplasmic reticulum (ER stress, reducing hepatic lipid accumulation through secretion of apolipoprotein B and associated triglycerides and cholesterol in human HepG2 hepatocytes. To determine how EUE diminishes the ER stress response, lysosomal and proteasomal protein degradation activities were analyzed. Although proteasomal activity was not affected, lysosomal enzyme activities including V-ATPase were significantly increased by EUE as well as aucubin and geniposide in HepG2 cells. Treatment with the V-ATPase inhibitor, bafilomycin, reversed the inhibition of ER stress, secretion of apolipoprotein B, and hepatic lipid accumulation induced by EUE or its component, aucubin or geniposide. In addition, EUE was determined to regulate hepatic dyslipidemia by enhancing lysosomal activity and to regulate ER stress in rats fed a high-fat diet. Together, these results suggest that EUE and its active components enhance lysosomal activity, resulting in decreased ER stress and hepatic dyslipidemia.

  6. Actin-binding protein coronin 1A controls osteoclastic bone resorption by regulating lysosomal secretion of cathepsin K

    Science.gov (United States)

    Ohmae, Saori; Noma, Naruto; Toyomoto, Masayasu; Shinohara, Masahiro; Takeiri, Masatoshi; Fuji, Hiroaki; Takemoto, Kenji; Iwaisako, Keiko; Fujita, Tomoko; Takeda, Norihiko; Kawatani, Makoto; Aoyama, Mineyoshi; Hagiwara, Masatoshi; Ishihama, Yasushi; Asagiri, Masataka

    2017-01-01

    Osteoclasts degrade bone matrix proteins via the secretion of lysosomal enzymes. However, the precise mechanisms by which lysosomal components are transported and fused to the bone-apposed plasma membrane, termed ruffled border membrane, remain elusive. Here, we identified coronin 1A as a negative regulator of exocytotic release of cathepsin K, one of the most important bone-degrading enzymes in osteoclasts. The modulation of coronin 1A expression did not alter osteoclast differentiation and extracellular acidification, but strongly affected the secretion of cathepsin K and osteoclast bone-resorption activity, suggesting the coronin 1A-mediated regulation of lysosomal trafficking and protease exocytosis. Further analyses suggested that coronin 1A prevented the lipidation-mediated sorting of the autophagy-related protein LC3 to the ruffled border and attenuated lysosome–plasma membrane fusion. In this process, the interactions between coronin 1A and actin were crucial. Collectively, our findings indicate that coronin 1A is a pivotal component that regulates lysosomal fusion and the secretion pathway in osteoclast-lineage cells and may provide a novel therapeutic target for bone diseases. PMID:28300073

  7. Mangrove forest distributions and dynamics (19752005) of the tsunami-affected region of Asia

    Science.gov (United States)

    Giri, C.; Zhu, Z.; Tieszen, L.L.; Singh, A.; Gillette, S.; Kelmelis, J.A.

    2008-01-01

    Aim: We aimed to estimate the present extent of tsunami-affected mangrove forests and determine the rates and causes of deforestation from 1975 to 2005. Location: Our study region covers the tsunami-affected coastal areas of Indonesia, Malaysia, Thailand, Burma (Myanmar), Bangladesh, India and Sri Lanka in Asia. Methods: We interpreted time-series Landsat data using a hybrid supervised and unsupervised classification approach. Landsat data were geometrically corrected to an accuracy of plus-or-minus half a pixel, an accuracy necessary for change analysis. Each image was normalized for solar irradiance by converting digital number values to the top-of-the atmosphere reflectance. Ground truth data and existing maps and data bases were used to select training samples and also for iterative labelling. We used a post-classification change detection approach. Results: were validated with the help of local experts and/or high-resolution commercial satellite data. Results The region lost 12% of its mangrove forests from 1975 to 2005, to a present extent of c. 1,670,000 ha. Rates and causes of deforestation varied both spatially and temporally. Annual deforestation was highest in Burma (c. 1%) and lowest in Sri Lanka (0.1%). In contrast, mangrove forests in India and Bangladesh remained unchanged or gained a small percentage. Net deforestation peaked at 137,000 ha during 1990-2000, increasing from 97,000 ha during 1975-90, and declining to 14,000 ha during 2000-05. The major causes of deforestation were agricultural expansion (81%), aquaculture (12%) and urban development (2%). Main conclusions: We assessed and monitored mangrove forests in the tsunami-affected region of Asia using the historical archive of Landsat data. We also measured the rates of change and determined possible causes. The results of our study can be used to better understand the role of mangrove forests in saving lives and property from natural disasters such as the Indian Ocean tsunami, and to identify

  8. Interrill erosion, runoff and sediment size distribution as affected by slope steepness and antecedent moisture content

    Directory of Open Access Journals (Sweden)

    M. B. Defersha

    2010-08-01

    Full Text Available Soil erosion is a two-phase process consisting of the detachment of individual particles and their transport by erosive agents such as flowing water. The rate at which erosion occurs depends upon the individual as well as interactive effects of different parameters responsible for soil erosion. The study discusses results of a laboratory analysis and evaluates the effect of slope steepness and antecedent moisture content on sediment yield (wash and runoff rate. Interrill sediment yield, splash detachment, runoff, and sediment size distribution were measured in laboratory erosion pans under simulated total duration of 90 min. Rainfall intensity at 120 mm/hr, 70 mm/hr, and 55 mm/hr were applied sequentially at 9, 25, and 45% slope steepness for three soils (Alemaya Black soil, Regosols, and Cambisols varied from clay to sandy clay loam in texture with wet and dry antecedent water contents. As slope steepness increased from 9 to 25% splash increased for five treatments and decreased for the remaining treatment; washed sediment increased for all treatments. As slope increased from 25 to 45% splash decreased for five treatments but increased for one treatment, and washed sediment increased for three treatments but decreased for the other three treatments. Pre-wetting decreased splash detachment for all soil treatments and rate of reduction was high for the highly aggregated soil, Alemaya Black soil and low for the less aggregated soil Regosols. Splash sediment and sediment yield was not correlated. Change in splash with increase in slope steepness was also not correlated with change in sediment yield. Change in runoff rate with increase in slope steepness was correlated (r=0.66 with change in sediment yield. For Alemaya Black soil and Regosols, splashed sediment size distribution was correlated with washed sediment size distribution. Interrill erosion models that include runoff and rainfall intensity parameters were a better fit for these data

  9. Photoperiod affects distribution of dynorphin A in the brain of Siberian hamster.

    Science.gov (United States)

    Meyza, Ksenia Z; Sotowska-Brochocka, Jolanta

    2006-01-01

    Dynorphin A1-77 (DYN A1-17) acting in the CNS is known to affect thermoregulation, water and energy balance in the short time scale. In this study a long-term alteration of these functions induced by changes of day length in the highly photoperiodic species, the Siberian hamster (Phodopus sungorus) was studied using immunohistochemistry for DYN A1-17. We found that in the long day (LD, L:D 16 h:8 h) more brain areas express DYN A1-17 peptide than in the short day (SD, L:D 8 h:16 h) conditions. Structures of the hypothalamo-pituitary axis as well as cells of the ependyma, subcomissural organ and choroid plexus of the lateral and third brain ventricles are immunoreactive to anti-dynorphin IgG only in the LD. This might indicate a seasonal regulatory role of DYN A1-17 in physiological adaptations to severe climate changes.

  10. Alters Intratumoral Drug Distribution and Affects Therapeutic Synergy of Antiangiogenic Organoselenium Compound

    Directory of Open Access Journals (Sweden)

    Youcef M. Rustum

    2010-01-01

    Full Text Available Tumor differentiation enhances morphologic and microvascular heterogeneity fostering hypoxia that retards intratumoral drug delivery, distribution, and compromise therapeutic efficacy. In this study, the influence of tumor biologic heterogeneity on the interaction between cytotoxic chemotherapy and selenium was examined using a panel of human tumor xenografts representing cancers of the head and neck and lung along with tissue microarray analysis of human surgical samples. Tumor differentiation status, microvessel density, interstitial fluid pressure, vascular phenotype, and drug delivery were correlated with the degree of enhancement of chemotherapeutic efficacy by selenium. Marked potentiation of antitumor activity was observed in H69 tumors that exhibited a well-vascularized, poorly differentiated phenotype. In comparison, modulation of chemotherapeutic efficacy by antiangiogenic selenium was generally lower or absent in well-differentiated tumors with multiple avascular hypoxic, differentiated regions. Tumor histomorphologic heterogeneity was found prevalent in the clinical samples studied and represents a primary and critical physiological barrier to chemotherapy.

  11. Is the distribution of Prochlorococcus and Synechococcus ecotypes in the Mediterranean Sea affected by global warming?

    Directory of Open Access Journals (Sweden)

    M. Ostrowski

    2011-05-01

    Full Text Available Biological communities populating the Mediterranean Sea, which is situated at the northern boundary of the subtropics, are often claimed to be particularly affected by global warming. This is indicated, for instance, by the introduction of (subtropical species of fish or invertebrates that can displace local species. This raises the question of whether microbial communities are similarly affected, especially in the Levantine basin where sea surface temperatures have risen in recent years. In this paper, the genetic diversity of the two most abundant members of the phytoplankton community, the picocyanobacteria Prochlorococcus and Synechococcus, was examined on a transect from the South coast of France to Cyprus in the summer of 2008 (BOUM cruise. Diversity was studied using dot blot hybridization with clade-specific 16S rRNA oligonucleotide probes and clone libraries of the 16S–23S ribosomal DNA Internal Transcribed Spacer (ITS region. Data were compared with those obtained during the PROSOPE cruise held almost a decade earlier, with a focus on the abundance of clades that may constitute bioindicators of warm waters. During both cruises, the dominant Prochlorococcus clade in the upper mixed layer at all stations was HLI, a clade typical of temperate waters, whereas the HLII clade, the dominant group in (subtropical waters, was only present at very low concentrations. The Synechococcus community was dominated by clades I, III and IV in the northwestern waters of the Gulf of Lions and by clade III and groups genetically related to clades WPC1 and VI in the rest of the Mediterranean Sea. In contrast, only a few sequences of clade II, a group typical of warm waters, were observed. These data indicate that local cyanobacterial populations have not yet been displaced by their (subtropical counterparts. This is discussed in the context of the low phosphorus concentrations found in surface waters in the eastern Mediterranean basin, as this may

  12. Do arbuscular mycorrhizal fungi affect cadmium uptake kinetics, subcellular distribution and chemical forms in rice?

    Science.gov (United States)

    Li, Hui; Luo, Na; Zhang, Li Jun; Zhao, Hai Ming; Li, Yan Wen; Cai, Quan Ying; Wong, Ming Hung; Mo, Ce Hui

    2016-11-15

    Rice (Oryza sativa L.) plants were inoculated with two species of arbuscular mycorrhizal fungi (AMF) - Rhizophagus intraradices (RI) and Funneliformis mosseae (FM) and grown for 60days to ensure strong colonization. Subsequently, a short-term hydroponic experiment was carried out to investigate the effects of AMF on cadmium (Cd) uptake kinetics, subcellular distribution and chemical forms in rice exposed to six Cd levels (0, 0.005, 0.01, 0.025, 0.05, 0.1mM) for three days. The results showed that the uptake kinetics of Cd fitted the Michaelis-Menten model well (R(2)>0.89). AMF significantly decreased the Cd concentrations both in shoots and roots in Cd solutions. Furthermore, the decrement of Cd concentrations by FM was significantly higher than RI treatment in roots. AMF reduced the Cd concentrations markedly in the cell wall fractions at high Cd substrate (≥0.025mM). The main subcellular fraction contributed to Cd detoxification was cell wall at low Cd substrate (<0.05mM), while vacuoles at high Cd substrate (≥0.05mM). Moreover, the concentrations and proportions of Cd in inorganic and water-soluble form also reduced by AMF colonization at high Cd substrate (≥0.05mM), both in shoots and roots. This suggested that AMF could convert Cd into inactive forms which were less toxic. Therefore, AMF could enhance rice resistance to Cd through altering subcellular distribution and chemical forms of Cd in rice.

  13. Involvement of the endosomal-lysosomal system correlates with regional pathology in Creutzfeldt-Jakob disease

    DEFF Research Database (Denmark)

    Kovács, Gábor G; Gelpi, Ellen; Ströbel, Thomas;

    2007-01-01

    these with the severity of neuropathologic changes. In regions with mild pathology and scant abnormal prion protein (PrP) deposition, neurons showed an increased volume of Rab5-immunopositive early endosomes. In contrast, neurons in regions with prominent pathology had an increased volume of cathepsin D- or B......-immunoreactive lysosomes. The intraneuronal distribution of cathepsin D and B diverges between Purkinje cells and frontal cortical neurons in sporadic Creutzfeldt-Jakob disease brains. We demonstrated focal intra- and perineuronal colocalization of cathepsin D and PrP. Our results indicate that effects in the ELS...

  14. Circulating TFH subset distribution is strongly affected in lupus patients with an active disease.

    Directory of Open Access Journals (Sweden)

    Carole Le Coz

    Full Text Available Follicular helper T cells (TFH represent a distinct subset of CD4(+ T cells specialized in providing help to B lymphocytes, which may play a central role in autoimmune diseases having a major B cell component such as systemic lupus erythematosus. Recently, TFH subsets that share common phenotypic and functional characteristics with TFH cells from germinal centers, have been described in the peripheral blood from healthy individuals. The aim of this study was to analyze the distribution of such populations in lupus patients. Circulating TFH cell subsets were defined by multicolor flow cytometry as TFH17 (CXCR3(-CCR6(+, TFH1 (CXCR3 (+ CCR6(- or TFH2 (CXCR3(-CCR6(- cells among CXCR5 (+ CD45RA(-CD4(+ T cells in the peripheral blood of 23 SLE patients and 23 sex and age-matched healthy controls. IL-21 receptor expression by B cells was analyzed by flow cytometry and the serum levels of IL-21 and Igs were determined by ELISA tests. We found that the TFH2 cell subset frequency is strongly and significantly increased in lupus patients with an active disease (SLEDAI score>8, while the TFH1 cell subset percentage is greatly decreased. The TFH2 and TFH1 cell subset frequency alteration is associated with the presence of high Ig levels and autoantibodies in patient's sera. Moreover, the TFH2 cell subset enhancement correlates with an increased frequency of double negative memory B cells (CD27(-IgD(-CD19(+ cells expressing the IL-21R. Finally, we found that IgE levels in lupus patients' sera correlate with disease activity and seem to be associated with high TFH2 cell subset frequency. In conclusion, our study describes for the first time the distribution of circulating TFH cell subsets in lupus patients. Interestingly, we found an increased frequency of TFH2 cells, which correlates with disease activity. Our results suggest that this subset might play a key role in lupus pathogenesis.

  15. Distribution of organic carbon in physical fractions of soils as affected by agricultural management

    Energy Technology Data Exchange (ETDEWEB)

    Sindhu, Jagadamma [Oak Ridge National Laboratory (ORNL); Lal, Dr. Rattan [Ohio State University, The, Columbus

    2010-08-01

    Soil organic carbon (SOC) is distributed heterogeneously among different-sized primary particles and aggregates. Further, the SOC associated with different physical fractions respond differently to managements. Therefore, this study was conducted with the objective to quantify the SOC associated with all the three structural levels of SOC (particulate organic matter, soil separates and aggregate-size fractions) as influenced by long-term change in management. The study also aims at reevaluating the concept that the SOC sink capacity of individual size-fractions is limited. Long-term tillage and crop rotation effects on distribution of SOC among fractions were compared with soil from adjacent undisturbed area under native vegetation for the mixed, mesic, Typic Fragiudalf of Wooster, OH. Forty five years of no-till (NT) management resulted in more SOC accumulation in soil surface (0 7.5 cm) than in chisel tillage and plow tillage (PT) treatments. However, PT at this site resulted in a redistribution of SOC from surface to deeper soil layers. The soils under continuous corn accumulated significantly more SOC than those under corn soybean rotation at 7.5 45 cm depth. Although soil texture was dominated by the silt-sized particles, most of the SOC pool was associated with the clay fraction. Compared to PT, the NT treatment resulted in (i) significantly higher proportion of large macroaggregates (>2,000 m) and (ii) 1.5 2.8 times higher SOC concentrations in all aggregate-size classes. A comparative evaluation using radar graphs indicated that among the physical fractions, the SOC associated with sand and silt fractions quickly changed with a land use conversion from native vegetation to agricultural crops. A key finding of this study is the assessment of SOC sink capacity of individual fractions, which revealed that the clay fraction of agricultural soils continues to accumulate more SOC, albeit at a slower rate, with progressive increase in total SOC concentration

  16. Changing head model extent affects finite element predictions of transcranial direct current stimulation distributions

    Science.gov (United States)

    Indahlastari, Aprinda; Chauhan, Munish; Schwartz, Benjamin; Sadleir, Rosalind J.

    2016-12-01

    Objective. In this study, we determined efficient head model sizes relative to predicted current densities in transcranial direct current stimulation (tDCS). Approach. Efficiency measures were defined based on a finite element (FE) simulations performed using nine human head models derived from a single MRI data set, having extents varying from 60%-100% of the original axial range. Eleven tissue types, including anisotropic white matter, and three electrode montages (T7-T8, F3-right supraorbital, Cz-Oz) were used in the models. Main results. Reducing head volume extent from 100% to 60%, that is, varying the model’s axial range from between the apex and C3 vertebra to one encompassing only apex to the superior cerebellum, was found to decrease the total modeling time by up to half. Differences between current density predictions in each model were quantified by using a relative difference measure (RDM). Our simulation results showed that {RDM} was the least affected (a maximum of 10% error) for head volumes modeled from the apex to the base of the skull (60%-75% volume). Significance. This finding suggested that the bone could act as a bioelectricity boundary and thus performing FE simulations of tDCS on the human head with models extending beyond the inferior skull may not be necessary in most cases to obtain reasonable precision in current density results.

  17. Carbon amendment and soil depth affect the distribution and abundance of denitrifiers in agricultural soils.

    Science.gov (United States)

    Barrett, M; Khalil, M I; Jahangir, M M R; Lee, C; Cardenas, L M; Collins, G; Richards, K G; O'Flaherty, V

    2016-04-01

    The nitrite reductase (nirS and nirK) and nitrous oxide reductase-encoding (nosZ) genes of denitrifying populations present in an agricultural grassland soil were quantified using real-time polymerase chain reaction (PCR) assays. Samples from three separate pedological depths at the chosen site were investigated: horizon A (0-10 cm), horizon B (45-55 cm), and horizon C (120-130 cm). The effect of carbon addition (treatment 1, control; treatment 2, glucose-C; treatment 3, dissolved organic carbon (DOC)) on denitrifier gene abundance and N2O and N2 fluxes was determined. In general, denitrifier abundance correlated well with flux measurements; nirS was positively correlated with N2O, and nosZ was positively correlated with N2 (P soil (GCC) varied in response to carbon type amendment (P soil depth directly affected bacterial, archaeal, and denitrifier abundance, possibly due to changes in soil carbon availability with depth.

  18. How does the preparation of rye porridge affect molecular weight distribution of extractable dietary fibers?

    Science.gov (United States)

    Rakha, Allah; Aman, Per; Andersson, Roger

    2011-01-01

    Extractable dietary fiber (DF) plays an important role in nutrition. This study on porridge making with whole grain rye investigated the effect of rest time of flour slurries at room temperature before cooking and amount of flour and salt in the recipe on the content of DF components and molecular weight distribution of extractable fructan, mixed linkage (1→3)(1→4)-β-d-glucan (β-glucan) and arabinoxylan (AX) in the porridge. The content of total DF was increased (from about 20% to 23% of dry matter) during porridge making due to formation of insoluble resistant starch. A small but significant increase in the extractability of β-glucan (P = 0.016) and AX (P = 0.002) due to rest time was also noted. The molecular weight of extractable fructan and AX remained stable during porridge making. However, incubation of the rye flour slurries at increased temperature resulted in a significant decrease in extractable AX molecular weight. The molecular weight of extractable β-glucan decreased greatly during a rest time before cooking, most likely by the action of endogenous enzymes. The amount of salt and flour used in the recipe had small but significant effects on the molecular weight of β-glucan. These results show that whole grain rye porridge made without a rest time before cooking contains extractable DF components maintaining high molecular weights. High molecular weight is most likely of nutritional importance.

  19. How Does the Preparation of Rye Porridge Affect Molecular Weight Distribution of Extractable Dietary Fibers?

    Directory of Open Access Journals (Sweden)

    Roger Andersson

    2011-05-01

    Full Text Available Extractable dietary fiber (DF plays an important role in nutrition. This study on porridge making with whole grain rye investigated the effect of rest time of flour slurries at room temperature before cooking and amount of flour and salt in the recipe on the content of DF components and molecular weight distribution of extractable fructan, mixed linkage (1→3(1→4-β-D-glucan (β-glucan and arabinoxylan (AX in the porridge. The content of total DF was increased (from about 20% to 23% of dry matter during porridge making due to formation of insoluble resistant starch. A small but significant increase in the extractability of β-glucan (P = 0.016 and AX (P = 0.002 due to rest time was also noted. The molecular weight of extractable fructan and AX remained stable during porridge making. However, incubation of the rye flour slurries at increased temperature resulted in a significant decrease in extractable AX molecular weight. The molecular weight of extractable β-glucan decreased greatly during a rest time before cooking, most likely by the action of endogenous enzymes. The amount of salt and flour used in the recipe had small but significant effects on the molecular weight of β-glucan. These results show that whole grain rye porridge made without a rest time before cooking contains extractable DF components maintaining high molecular weights. High molecular weight is most likely of nutritional importance.

  20. Distribution of Pseudomonas Species in a Dairy Plant Affected by Occasional Blue Discoloration

    Science.gov (United States)

    Lomonaco, Sara; Nucera, Daniele; Garoglio, Davide; Dalmasso, Alessandra; Civera, Tiziana

    2014-01-01

    During 2010 many cases of discoloration in mozzarella, popularly termed as blue mozzarella, have been reported to the attention of public opinion. Causes of the alteration were bacteria belonging to the genus Pseudomonas. The strong media impact of such cases has created confusion, not only among consumers, but also among experts. In order to help improving the knowledge on microbial ecology of this microorganism a study has been set up with the collaboration of a medium-sized dairy plant producing fresh mozzarella cheese, with occasional blue discoloration, conducting surveys and sampling in the pre-operational, operational and post-operational process phase, milk before and after pasteurization, water (n=12), environmental surfaces (n=22) and the air (n=27). A shelf life test was conducted on finished products stored at different temperatures (4-8°C). Among the isolates obtained from the microbiological analysis of the samples, 60 were subjected to biomolecular tests in order to confirm the belonging to Pseudomonas genus and to get an identification at species level by the amplification and sequencing of the gyrB gene. The results of microbiological tests demonstrated the presence of microorganisms belonging to the genus Pseudomonas along the entire production lane; molecular tests showed 7 different species among the 40 isolates identified. One particular species (Pseudomonas koreensis) was isolated from blue discolored mozzarella cheese and was indicated as the most relevant for the production plant, both for the distribution along the processing chain and for the consequences on the finished product.

  1. Vertical Distribution and Seasonal Fluctuation of Nematode Trophic Groups as Affected by Land Use

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    A field investigation was conducted at the Shenyang Experimental Station of Ecology, Chinese Academy of Sciences,in an aquic brown soil of Northeast China under three land use types (cropland, abandoned cropland, and woodland) in order to evaluate whether the vertical distribution and seasonal fluctuation for the number of total nematodes and trophic groups could reflect soil ecosystem differences and to determine the relationships between soil chemical properties and soil nematodes. The majority of soil nematodes were present in the 0-20 cm soil layers, and for these land use types plant parasites were the most abundant trophic group. In the abandoned cropland the numbers of plant parasites reached a peak on the August sampling date, whereas the cropland and woodland peaked on the October sampling date. Meanwhile,in all land use types the number of total nematodes, bacterivores, plant parasites, and omnivores-predators was negatively(P < 0.05, except for bacterivores in cropland, which was not significant) correlated with bulk density, and positively(P < 0.05, except for fungivores in abandoned cropland, which was not significant) correlated with total organic carbon and total nitrogen.

  2. Environmental stability affects phenotypic evolution in a globally distributed marine picoplankton

    Science.gov (United States)

    Schaum, C-Elisa; Rost, Björn; Collins, Sinéad

    2016-01-01

    Marine phytoplankton can evolve rapidly when confronted with aspects of climate change because of their large population sizes and fast generation times. Despite this, the importance of environment fluctuations, a key feature of climate change, has received little attention—selection experiments with marine phytoplankton are usually carried out in stable environments and use single or few representatives of a species, genus or functional group. Here we investigate whether and by how much environmental fluctuations contribute to changes in ecologically important phytoplankton traits such as C:N ratios and cell size, and test the variability of changes in these traits within the globally distributed species Ostreococcus. We have evolved 16 physiologically distinct lineages of Ostreococcus at stable high CO2 (1031±87 μatm CO2, SH) and fluctuating high CO2 (1012±244 μatm CO2, FH) for 400 generations. We find that although both fluctuation and high CO2 drive evolution, FH-evolved lineages are smaller, have reduced C:N ratios and respond more strongly to further increases in CO2 than do SH-evolved lineages. This indicates that environmental fluctuations are an important factor to consider when predicting how the characteristics of future phytoplankton populations will have an impact on biogeochemical cycles and higher trophic levels in marine food webs. PMID:26125683

  3. Patchy distributions of competitors affect the growth of a clonal plant when the competitor density is high.

    Directory of Open Access Journals (Sweden)

    Wei Xue

    Full Text Available Environments are patchy in not only abiotic factors but also biotic ones. Many studies have examined effects of spatial heterogeneity in abiotic factors such as light, water and nutrients on the growth of clonal plants, but few have tested those in biotic factors. We conducted a greenhouse experiment to examine how patchy distributions of competitors affect the growth of a rhizomatous wetland plant Bolboschoenus planiculmis and whether such effects depend on the density of the competitors. We grew one ramet of B. planiculmis in the center of each of the experimental boxes without competitors (Schoenoplectus triqueter, with a homogeneous distribution of the competitors of low or high density, and with a patchy distribution of the competitors of low or high density. The presence of competitors markedly decreased the growth (biomass, number of ramets, number of tubers and rhizome length of the B. planiculmis clones. When the density of the competitors was low, the growth of B. planiculmis did not differ significantly between the competitor patches and competitor-free patches. However, when the density of the competitors was high, the growth of B. planiculmis was significantly higher in the competitor-free patches than in the competitor patches. Therefore, B. planiculmis can respond to patchy distributions of competitors by placing more ramets in competition-free patches when the density of competitors is high, but cannot do so when the density of competitors is low.

  4. Patchy distributions of competitors affect the growth of a clonal plant when the competitor density is high.

    Science.gov (United States)

    Xue, Wei; Huang, Lin; Dong, Bi-Cheng; Zhang, Ming-Xiang; Yu, Fei-Hai

    2013-01-01

    Environments are patchy in not only abiotic factors but also biotic ones. Many studies have examined effects of spatial heterogeneity in abiotic factors such as light, water and nutrients on the growth of clonal plants, but few have tested those in biotic factors. We conducted a greenhouse experiment to examine how patchy distributions of competitors affect the growth of a rhizomatous wetland plant Bolboschoenus planiculmis and whether such effects depend on the density of the competitors. We grew one ramet of B. planiculmis in the center of each of the experimental boxes without competitors (Schoenoplectus triqueter), with a homogeneous distribution of the competitors of low or high density, and with a patchy distribution of the competitors of low or high density. The presence of competitors markedly decreased the growth (biomass, number of ramets, number of tubers and rhizome length) of the B. planiculmis clones. When the density of the competitors was low, the growth of B. planiculmis did not differ significantly between the competitor patches and competitor-free patches. However, when the density of the competitors was high, the growth of B. planiculmis was significantly higher in the competitor-free patches than in the competitor patches. Therefore, B. planiculmis can respond to patchy distributions of competitors by placing more ramets in competition-free patches when the density of competitors is high, but cannot do so when the density of competitors is low.

  5. Distribution of Pseudomonas species in a dairy plant affected by occasional blue discoloration

    Directory of Open Access Journals (Sweden)

    Francesco Chiesa

    2014-12-01

    Full Text Available During 2010 many cases of discoloration in mozzarella, popularly termed as blue mozzarella, have been reported to the attention of public opinion. Causes of the alteration were bacteria belonging to the genus Pseudomonas. The strong media impact of such cases has created confusion, not only among consumers, but also among experts. In order to help improving the knowledge on microbial ecology of this microorganism a study has been set up with the collaboration of a medium-sized dairy plant producing fresh mozzarella cheese, with occasional blue discoloration, conducting surveys and sampling in the pre-operational, operational and post-operational process phase, milk before and after pasteurization, water (n=12, environmental surfaces (n=22 and the air (n=27. A shelf life test was conducted on finished products stored at different temperatures (4-8°C. Among the isolates obtained from the microbiological analysis of the samples, 60 were subjected to biomolecular tests in order to confirm the belonging to Pseudomonas genus and to get an identification at species level by the amplification and sequencing of the gyrB gene. The results of microbiological tests demonstrated the presence of microorganisms belonging to the genus Pseudomonas along the entire production lane; molecular tests showed 7 different species among the 40 isolates identified. One particular species (Pseudomonas koreensis was isolated from blue discolored mozzarella cheese and was indicated as the most relevant for the production plant, both for the distribution along the processing chain and for the consequences on the finished product.

  6. PDT: loss of autophagic cytoprotection after lysosomal photodamage

    Science.gov (United States)

    Kessel, David; Price, Michael

    2012-02-01

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

  7. Lysosome stability during lytic infection by simian virus 40.

    Science.gov (United States)

    Einck, K H; Norkin, L C

    1979-01-01

    By 48 h postinfection, 40--80% of SV40-infected CV-1 cells have undergone irreversible injury as indicated by trypan blue staining. Nevertheless, at this time the lysosomes of these cells appear as discrete structures after vital staining with either acridine orange or neutral red. Lysosomes, vitally stained with neutral red at 24 h postinfection, were still intact in cells stained with trypan blue at 48 h. Acid phosphatase activity is localized in discrete cytoplasmic particles at 48 h, as indicated by histochemical staining of both fixed and unfixed cells.

  8. Importance of lysosomal cysteine proteases in lung disease

    Directory of Open Access Journals (Sweden)

    Chapman Harold A

    2000-11-01

    Full Text Available Abstract The human lysosomal cysteine proteases are a family of 11 proteases whose members include cathepsins B, C, H, L, and S. The biology of these proteases was largely ignored for decades because of their lysosomal location and the belief that their function was limited to the terminal degradation of proteins. In the past 10 years, this view has changed as these proteases have been found to have specific functions within cells. This review highlights some of these functions, specifically their roles in matrix remodeling and in regulating the immune response, and their relationship to lung diseases.

  9. The endoplasmic reticulum, not the pH gradient, drives calcium refilling of lysosomes.

    Science.gov (United States)

    Garrity, Abigail G; Wang, Wuyang; Collier, Crystal Md; Levey, Sara A; Gao, Qiong; Xu, Haoxing

    2016-05-23

    Impaired homeostasis of lysosomal Ca(2+) causes lysosome dysfunction and lysosomal storage diseases (LSDs), but the mechanisms by which lysosomes acquire and refill Ca(2+) are not known. We developed a physiological assay to monitor lysosomal Ca(2+) store refilling using specific activators of lysosomal Ca(2+) channels to repeatedly induce lysosomal Ca(2+) release. In contrast to the prevailing view that lysosomal acidification drives Ca(2+) into the lysosome, inhibiting the V-ATPase H(+) pump did not prevent Ca(2+) refilling. Instead, pharmacological depletion or chelation of Endoplasmic Reticulum (ER) Ca(2+) prevented lysosomal Ca(2+) stores from refilling. More specifically, antagonists of ER IP3 receptors (IP3Rs) rapidly and completely blocked Ca(2+) refilling of lysosomes, but not in cells lacking IP3Rs. Furthermore, reducing ER Ca(2+) or blocking IP3Rs caused a dramatic LSD-like lysosome storage phenotype. By closely apposing each other, the ER may serve as a direct and primary source of Ca(2+)for the lysosome.

  10. Methods for monitoring Ca(2+) and ion channels in the lysosome.

    Science.gov (United States)

    Zhong, Xi Zoë; Yang, Yiming; Sun, Xue; Dong, Xian-Ping

    2016-12-09

    Lysosomes and lysosome-related organelles are emerging as intracellular Ca(2+) stores and play important roles in a variety of membrane trafficking processes, including endocytosis, exocytosis, phagocytosis and autophagy. Impairment of lysosomal Ca(2+) homeostasis and membrane trafficking has been implicated in many human diseases such as lysosomal storage diseases (LSDs), neurodegeneration, myopathy and cancer. Lysosomal membrane proteins, in particular ion channels, are crucial for lysosomal Ca(2+) signaling. Compared with ion channels in the plasma membrane, lysosomal ion channels and their roles in lysosomal Ca(2+) signaling are less understood, largely due to their intracellular localization and the lack of feasible functional assays directly applied to the native environment. Recent advances in biomedical methodology have made it possible to directly investigate ion channels in the lysosomal membrane. In this review, we provide a summary of the newly developed methods for monitoring lysosomal Ca(2+) and ion channels, as well as the recent discovery of lysosomal ion channels and their significances in intracellular Ca(2+) signaling. These new techniques will expand our research scope and our understanding of the nature of lysosomes and lysosome-related diseases.

  11. Processes affecting the spatial distribution of seagrass meadow sedimentary material on Yao Yai Island, Thailand

    Science.gov (United States)

    Quak, Michelle S. Y.; Ziegler, Alan D.; Benner, Shawn G.; Evans, Sam; Todd, Peter A.; Gillis, Lucy G.; Vongtanaboon, Sukanya; Jachowski, Nick; Bouma, Tjeerd J.

    2016-12-01

    Many islands throughout SE Asia are experiencing rapid development and land-cover conversion that potentially threaten sensitive coastal ecosystems, such as seagrasses, through increased loading of sediment and nutrients originating from disturbed catchments draining to the sea. To evaluate this threat for one such island in Southern Thailand (Yao Yai), we perform sediment source tracing via end-member mixing analysis using stable isotopes δ13C and δ15N in organic matter to explore sediment loading in a seagrass meadow. The analysis indicates that sedimentary material in the meadow originates mostly from ocean-associated sources (∼62% from seagrass detritus, seston, and ocean sediments). Terrestrial material comprises ∼19% of the organic material found in the seagrass meadow, with another 20% originating from an adjacent mangrove forest. Approximately one-fourth of the seagrass meadow material (24%) is detritus that has been (re)deposited internally. The high contribution of terrestrial-derived organic matter deposited near the river mouth demonstrates that substantial quantities of sediment are being transferred from upslope erosion sources into the seagrass meadow. However, only a small amount of this material is deposited throughout the entire bay because much of the terrestrial- and mangrove-derived sediment is transferred to the open ocean via channels that are periodically dredged to allow boat access to two small inland harbours. This positive affect of dredging has not received very much attention in existing literature. River water flowing to the channels during falling tide delivers sediment to these efficient pathways, where much of it bypasses the seagrass meadow at periods of time when sediment deposition would normally be the greatest. There is growing concern that ongoing land-cover changes and planned urbanization related to tourism and agriculture on the island may boost sediment/nutrients above a critical threshold, beyond that revealed in

  12. Distribution of Exchangeable Calcium,Magnesium and Potassium as Affected by Fertilizer Application to Red Soil

    Institute of Scientific and Technical Information of China (English)

    SHENREN-FANG; ZHAOQI-GUO

    1995-01-01

    A leaching experiment was Carried out with repacked soil columns in laboratory to study the leaching process of a red soil derived from sandstone as affected by various fertilization practices.The treatments were CK(as a control),CaCO3,CaSO4,MgCO3,Ca(H2PO4)2,Urea,KCl,Multiple(a mixture of the above mentioned fertilizers) and KNO3,The fertilizers were added to the bare surface of the soil columns,and then the columns were leached with 120 mL deionized water daily through perstaltic pumps over a period of 92 days,At the end of leaching process,soils were sampled from different depths of the soil profiles ,i.o.,of 92 days,At the end of leaching process,soils were sampled from different depths of the soil profiles,I.e.0-5cm,5-10cm,10-20cm,20-40cm,and 40-60cm,The results showed when applying Ca,Mg,and K to the bare surface of the soil columns,exchangeable Ca2+,Mg2+,and K+ in the upper layer of the soil profile increased correspondingly,with an extent depending mainly on the application rates of Ca,Mg,and K and showing a downward trend,CaCO3,CaSO4,MgCO3,and Ca(H2PO4)2 treatments had scarcely and effect on movement of exchangeable K+,while CaCO3,and CaSO4 treatments singnificantly promoted the downward movement of exchangealble Mg2+ although these two treatments had no obvious effect on leaching losses of Mg,The fact that under Urea treatment,exchangeable Ca2+ and Mg2+,were higher as compared to CK treatment showed urea could prevent leaching of exchangeable Ca2+and Mg2+,the obvious downward movement of exchangeable Ca2+and Mg2+ was noticed in KCl treatment ,In Multiple treatment,the downward movement of exchangeable Ca2+ and Mg2+ was evident,while that of K+ was less evident,Application of KNO3 strongly promoted the downward movement of exchangeable Ca2+and Mg2+ in the soil profile.

  13. Study of distribution and factors affecting syphilis epidemic among inner-city minorities of Baltimore.

    Science.gov (United States)

    Williams, P B; Ekundayo, O

    2001-11-01

    Disparities in health and medical conditions among ethnic and racial groups have been repeatedly documented. These inequalities, which have been noted in the recent past, include health outcomes such as quality of life and mortality, process, accessibility and appropriateness of care, and the prevalence of certain degenerative conditions and infectious diseases. Syphilis, a sexually transmitted disease (STD) which seemed to have disappeared or had been controlled over the years, has now re-emerged as a major public health problem in many rural, urban and suburban communities. Progression of the current rate of syphilis, which erupted in Baltimore during the later part of 1994, has continued unabated, most especially among the ethnic minorities, despite efforts of the Baltimore City Health Department and Maryland Department of Health and Mental Hygiene to control the epidemic. With the current incidence rates of 270 per 100 000 live births for congenital syphilis and 99.3 per 100 000 population for primary, secondary and latent syphilis (96% of the cases being in the non-white population), Baltimore becomes the city with the highest number of syphilis cases in the nation, surpassing the national average of 2.6 cases per 100 000 population. This study, which utilizes a combination of retrospective and questionnaire-oriented approach, was designed to assess factors that influenced the high incidence of syphilis among Baltimore inner-city dwellers between 1994 and 1998. Data for the study included syphilis reports from private physicians, the Baltimore City Health Department, STD clinics, the Center for Disease Control (CDC), and ethnographic interviews. Factors favoring the distribution and infectivity of the disease among the inner-city dwellers include greater poverty, high level of communication gaps between providers and a cross-section of minority inner-city dwellers, exchange of sex for crack cocaine, lower educational background, and inadequate and

  14. Soil Tillage Management Affects Maize Grain Yield by Regulating Spatial Distribution Coordination of Roots, Soil Moisture and Nitrogen Status.

    Science.gov (United States)

    Wang, Xinbing; Zhou, Baoyuan; Sun, Xuefang; Yue, Yang; Ma, Wei; Zhao, Ming

    2015-01-01

    The spatial distribution of the root system through the soil profile has an impact on moisture and nutrient uptake by plants, affecting growth and productivity. The spatial distribution of the roots, soil moisture, and fertility are affected by tillage practices. The combination of high soil density and the presence of a soil plow pan typically impede the growth of maize (Zea mays L.).We investigated the spatial distribution coordination of the root system, soil moisture, and N status in response to different soil tillage treatments (NT: no-tillage, RT: rotary-tillage, SS: subsoiling) and the subsequent impact on maize yield, and identify yield-increasing mechanisms and optimal soil tillage management practices. Field experiments were conducted on the Huang-Huai-Hai plain in China during 2011 and 2012. The SS and RT treatments significantly reduced soil bulk density in the top 0-20 cm layer of the soil profile, while SS significantly decreased soil bulk density in the 20-30 cm layer. Soil moisture in the 20-50 cm profile layer was significantly higher for the SS treatment compared to the RT and NT treatment. In the 0-20 cm topsoil layer, the NT treatment had higher soil moisture than the SS and RT treatments. Root length density of the SS treatment was significantly greater than density of the RT and NT treatments, as soil depth increased. Soil moisture was reduced in the soil profile where root concentration was high. SS had greater soil moisture depletion and a more concentration root system than RT and NT in deep soil. Our results suggest that the SS treatment improved the spatial distribution of root density, soil moisture and N states, thereby promoting the absorption of soil moisture and reducing N leaching via the root system in the 20-50 cm layer of the profile. Within the context of the SS treatment, a root architecture densely distributed deep into the soil profile, played a pivotal role in plants' ability to access nutrients and water. An optimal

  15. Lysosomal Localization of TRPML3 Depends on TRPML2 and the Mucolipidosis-associated Protein TRPML1*S

    OpenAIRE

    2006-01-01

    Mucolipidosis type IV is an autosomal recessive lysosomal storage disorder characterized by severe neurodegeneration, achlorhydria, and visual impairments such as corneal opacity and strabismus. The disease arises due to mutations in a group 2 transient receptor potential (TRP)-related cation channel, TRPML1. Mammals encode two additional TRPML proteins named TRPML2 and TRPML3. Information regarding the propensity of these proteins to multimerize, their subcellular distribution and mechanisms...

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

    Directory of Open Access Journals (Sweden)

    Md Suhail Alam

    Full Text Available Niemann-Pick Type C (NPC disease is a rare, genetic, lysosomal disorder with progressive neurodegeneration. Poor understanding of the pathophysiology and a lack of blood-based diagnostic markers are major hurdles in the treatment and management of NPC and several additional, neurological lysosomal disorders. To identify disease severity correlates, we undertook whole genome expression profiling of sentinel organs, brain, liver, and spleen of Balb/c Npc1(-/- mice relative to Npc1(+/- at an asymptomatic stage, as well as early- and late-symptomatic stages. Unexpectedly, we found prominent up regulation of innate immunity genes with age-dependent change in their expression, in all three organs. We shortlisted a set of 12 secretory genes whose expression steadily increased with age in both brain and liver, as potential plasma correlates of neurological and/or liver disease. Ten were innate immune genes with eight ascribed to lysosomes. Several are known to be elevated in diseased organs of murine models of other lysosomal diseases including Gaucher's disease, Sandhoff disease and MPSIIIB. We validated the top candidate lysozyme, in the plasma of Npc1(-/- as well as Balb/c Npc1(nmf164 mice (bearing a point mutation closer to human disease mutants and show its reduction in response to an emerging therapeutic. We further established elevation of innate immunity in Npc1(-/- mice through multiple functional assays including inhibition of bacterial infection as well as cellular analysis and immunohistochemistry. These data revealed neutrophil elevation in the Npc1(-/- spleen and liver (where large foci were detected proximal to damaged tissue. Together our results yield a set of lysosomal, secretory innate immunity genes that have potential to be developed as pan or specific plasma markers for neurological diseases associated with lysosomal storage and where diagnosis is a major problem. Further, the accumulation of neutrophils in diseased organs

  17. Factors Affecting Distribution of Vegetation Types on Abandoned Cropland in the Hilly-Gullied Loess Plateau Region of China

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    A study was conducted in the forest-steppe region of the Loess Plateau to provide insight into the factors affecting the process of vegetation establishment,and to provide recommendations for the selection of indigenous species in order to speed up the succession process and to allow the establishment of vegetation more resistant to soil erosion.Four distinctive vegetation types were identified,and their distribution was affected not only by the time since abandonment but also by other environmental factors,mainly soil water and total P in the upper soil layers.One of the vegetation types,dominated by Artemisia scoparia,formed the early successional stage after abandonment while the other three types formed later successional stages with their distribution determined by the soil water content and total P.It can be concluded that the selection of appropriate species for introduction to accelerate succession should be determined by the local conditions and especially the total P concentration and soil water content.

  18. The critical factors that affected the distribution of aboveground biomass in the alpine steppe and meadow, Tibetan Plateau

    Directory of Open Access Journals (Sweden)

    J. Sun

    2012-10-01

    Full Text Available Tibetan Plateau – the third pole of the world, with its extremly harsh and fragile ecological environment, is so sensitive to global change that it attracts many scientists' attention. Alpine grassland here is an important component of the global carbon cycle. Many studies have examined links between environmental factors and distribution of biomass, but little showed the critical environmental factors affecting the distribution of biomass. To document the general relationships between the habitat factors and aboveground biomass (AGB in Tibetan Plateau, and to identify the critical factors for the distribution of AGB in the alpine steppe and meadow, the data of AGB and habitat factors from 110 field sites across the widely distributed alpine steppe and meadow of the plateau were compiled and analyzed with the classification and regression tree (CART model, and the generalized additive model (GAM. The results showed that (1 the spatial pattern of AGB in alpine steppe was determined by six major environmental factors: soil organic carbon density of soil 0–30 cm depth (SOC1, longitude, mean annual precipitation (MAP, latitude, clay and soil moisture. As to the alpine meadow, the major factors were altitude, soil moisture, nitrogen, MAP and mean annual temperature (MAT. (2 As to the alpine steppe, increased SOC1, MAP and latitude were associated with increased AGB abundance, but increased longitude resulted in lower abundance of AGB. As to the alpine meadow, the distribution of AGB had strong negative relationships with altitude and soil moisture, but a positive correlation with soil nitrogen content across sites. The results suggested that the combined effects of meteorological factors, topographic factors, and soil factors were more significant for the spatial pattern of AGB in Tibetan Plateau. In addition, our work highlights the importance of further studies to seek effects of slope and aspect in alpine grassland.

  19. Accuracy of travel time distribution (TTD) models as affected by TTD complexity, observation errors, and model and tracer selection

    Science.gov (United States)

    Green, Christopher T.; Zhang, Yong; Jurgens, Bryant C.; Starn, J. Jeffrey; Landon, Matthew K.

    2014-01-01

    Analytical models of the travel time distribution (TTD) from a source area to a sample location are often used to estimate groundwater ages and solute concentration trends. The accuracies of these models are not well known for geologically complex aquifers. In this study, synthetic datasets were used to quantify the accuracy of four analytical TTD models as affected by TTD complexity, observation errors, model selection, and tracer selection. Synthetic TTDs and tracer data were generated from existing numerical models with complex hydrofacies distributions for one public-supply well and 14 monitoring wells in the Central Valley, California. Analytical TTD models were calibrated to synthetic tracer data, and prediction errors were determined for estimates of TTDs and conservative tracer (NO3−) concentrations. Analytical models included a new, scale-dependent dispersivity model (SDM) for two-dimensional transport from the watertable to a well, and three other established analytical models. The relative influence of the error sources (TTD complexity, observation error, model selection, and tracer selection) depended on the type of prediction. Geological complexity gave rise to complex TTDs in monitoring wells that strongly affected errors of the estimated TTDs. However, prediction errors for NO3− and median age depended more on tracer concentration errors. The SDM tended to give the most accurate estimates of the vertical velocity and other predictions, although TTD model selection had minor effects overall. Adding tracers improved predictions if the new tracers had different input histories. Studies using TTD models should focus on the factors that most strongly affect the desired predictions.

  20. Ghost of habitat past: historic habitat affects the contemporary distribution of giant garter snakes in a modified landscape.

    Science.gov (United States)

    Halstead, Brian J.; Wylie, Glenn D.; Casazza, Michael L.

    2014-01-01

    Historic habitat conditions can affect contemporary communities and populations, but most studies of historic habitat are based on the reduction in habitat extent or connectivity. Little is known about the effects of historic habitat on contemporary species distributions when historic habitat has been nearly completely removed, but species persist in a highly altered landscape. More than 93% of the historic wetlands in the Central Valley of California, USA, have been drained and converted to agricultural and other uses, but agricultural wetlands, such as rice and its supporting infrastructure of canals, allow some species to persist. Little is known about the distribution of giant garter snakes Thamnophis gigas, a rare aquatic snake species inhabiting this predominantly agricultural landscape, or the variables that affect where this species occurs. We used occupancy modeling to examine the distribution of giant garter snakes at the landscape scale in the Sacramento Valley (northern portion of the Central Valley) of California, with an emphasis on the relative strength of historic and contemporary variables (landscape-scale habitat, local microhabitat, vegetation composition and relative prey counts) for predicting giant garter snake occurrence. Proximity to historic marsh best explained variation in the probability of occurrence of giant garter snakes at the landscape scale, with greater probability of occurrence near historic marsh. We suspect that the importance of distance to historic marsh represents dispersal limitations of giant garter snakes. These results suggest that preserving and restoring areas near historic marsh, and minimizing activities that reduce the extent of marsh or marsh-like (e.g. rice agriculture, canal) habitats near historic marsh may be advantageous to giant garter snakes.

  1. Variations in Concentration and Distribution of Health-Related Elements Affected by Environmental and Genotypic Differences in Rice Grains

    Institute of Scientific and Technical Information of China (English)

    REN Xue-liang; LIU Qing-long; WU Dian-xing; SHU Qing-yao

    2006-01-01

    A research work was conducted to investigate the variations in concentration and distribution of health-related elements affected by environmental and genotypic differences in rice grains. The grain of Xieqingzao B (indica rice variety) and Xiushui 110 (japonica rice variety) were divided into: hull, bran and milled rice, based on the conventional rice consumption and process. Xieqingzao B was grown at four different locations, and at one location, it was planted in the same field and season as Xiushui 110. In addition, another four indica and four japonica varieties were cultivated in the same field and time to analyze the elements in milled rice. The average concentrations of total P and phytic acid P were the highest in the bran, followed by milled rice and hull; Zn, K, Mg, and As concentrations were the highest in bran, followed by hull and milled rice, while Fe, Ca, and Cu concentrations were the highest in the hull, but similar in bran and milled rice. The result indicated that genotype and environment significantly affected the concentrations of all the tested elements, while the distribution of the above elements in grains was not in the same order as concentration. Moreover, all the elements except 97.7% of Cu and 93.2% of Fe was deposited in the hull on average, were mostly distributed either in the bran (37.3% and 57.7% for K and phytic acid P) or in milled rice (41.7%, 42.6%, 40.3%, 49.8% for Zn, Mg, As, total P, respectively).

  2. The governance of natural resources: Issues affecting better management of revenues and distribution of benefits within Papua New Guinea

    Directory of Open Access Journals (Sweden)

    Hitelai Polume-Kiele

    2014-09-01

    Full Text Available Papua New Guinea is rich in natural resources, including minerals, oil, gas, timber and fish, and cash crops such as coffee, palm oil, cocoa, copra, rubber, tea and spices which contribute significantly to Papua New Guinea’s overall development. Several mining, oil and gas companies are currently operating in Porgera, Ok Tedi, Lihir, Hidden Valley, Sinivit, Simberi, Tolukuma, Kutubu and Gobe. The operations of these companies have generated an estimated K13.42 billion to Papua New Guinea’s economy. Landowners affected by these developments also receive royalties from those operations. However this wealth has not been translated into tangible human development across the country, as shown in persistently poorly performing social indicators. Instead income from the exploitation of natural resources is being used in unplanned projects and not focused on the delivery of core social functions, such as the provision of a stable and non-distorting policy aimed at building and sustaining the development of a modern market, and legislative and regulatory frameworks, social services, social security and social infrastructure which would lead to the improvement in the delivery of essential services to all Papua New Guineans. There is widespread evidence of benefits not being distributed to all landowners. Landowners are yet to fulfil their aspirations regarding these developments and to see improvements in their living standards. This paper discusses two case studies: the Porgera and Lihir mines, outlining the landowners associations’ experiences, which illustrate issues of governance and management of the distribution of benefit flows from the exploitation of Papua New Guinea’s natural resource wealth.The focus of the article’s discussion is on governance and management issues that affect the distribution of benefits, delivery of essential services to rural areas of PNG, stability within government, and the expectations of landowners.

  3. Porcine islet isolation outcome is not affected by the amount and distribution of collagen in the pancreas.

    Science.gov (United States)

    Hilling, Denise E; Rijkelijkhuizen, Josephine K R A; Töns, H Annemiek M; Terpstra, Onno T; Bouwman, Eelco

    2010-01-01

    Variable islet yields in porcine islet isolation may be caused by the collagen substrate within the pancreas. The aim of the present study was to determine the total amount and distribution of collagen within porcine pancreata and their relationship to islet isolation outcome. A total of 64 juvenile and 76 adult porcine pancreata of eight purebred breeds were histologically examined. The amount of collagen was quantitatively assessed in tissue samples stained with Sirius Red. Collagen distribution was semi-quantitatively determined by assessing the presence of collagen in the endocrine-exocrine interface and within the islet, in tissue samples stained with Sirius Red and anti-insulin. Islet isolation was performed in 58 pancreata of the adult group. Total collagen content and islet encapsulation ranged widely in both adult and juvenile pigs. However, the majority of islets in adult and juvenile pigs had no or only a limited collagen capsule. The difference in collagen content between adult and juvenile pigs could not be explained by age. Furthermore, no differences between adult and juvenile pigs were found in islet encapsulation or the amount of intra-islet collagen. In adult pigs, no significant relationships were found between obtained islet yield and total collagen content, islet encapsulation or amount of collagen within the islet. Considering the limitations in experimental design (staining method) and study material, isolation outcome does not seem to be affected by the total collagen content or collagen distribution. The influence of other matrix elements and collagen subtypes should be investigated.

  4. The relationship between Cd-induced autophagy and lysosomal activation in WRL-68 cells.

    Science.gov (United States)

    Meng, Su-Fang; Mao, Wei-Ping; Wang, Fang; Liu, Xiao-Qian; Shao, Luan-Luan

    2015-11-01

    This study shows that Cd induces autophagy in the human's embryonic normal liver cell line (WRL-68). The expression of LC3B-II and the mature cathepsin L were analyzed by Western blotting. The autophagosomes and lysosomes were directly visualized by electron microscopy and confocal microscopy analysis in Cd-exposed WRL-68 cells. In this study, we first found that autophagy induced the activation of lysosomal function in WRL-68 cells. The lysosomal activation was markedly decreased when the cells were co-treated with 3-MA (an inhibitor of autophagy). Secondly, we provided the evidence that the activation of lysosomal function depended on autophagosome-lysosome fusion. The colocalization of lysosome-associated membrane protein-2 (LAMP2) and GFP-LC3 was significantly reduced, when they were treated with thapsigargin (an inhibitor of autophagosome-lysosome fusion). We demonstrated that deletion or blockage of the autophagosome-lysosome fusion process effectively diminished lysosomal activation, which suggests that lysosomal activation occurring in the course of autophagy is dependent on autophagosome-lysosome fusion. Thirdly, we provided evidence that the activation of lysosomal function was associated with lysosomal acid. We investigated the relationship between autophagosome-lysosome fusion and pH in acidic compartments by visualizing fusion process in WRL-68 cells. This suggests that increasing pH in acidic compartments in WRL-68 cells inhibits the autophagosome-lysosome fusion. Finally, we found that the activation of lysosomal function was associated with Ca(2+) stores and the intracellular Ca(2+) channels or pumps were possibly pH-dependent.

  5. PIG7 promotes leukemia cell chemosensitivity via lysosomal membrane permeabilization.

    Science.gov (United States)

    Liu, Jiazhuo; Peng, Leiwen; Niu, Ting; Wu, Yu; Li, Jianjun; Wang, Fangfang; Zheng, Yuhuan; Liu, Ting

    2016-01-26

    PIG7 localizes to lysosomal membrane in leukemia cells. Our previous work has shown that transduction of pig7 into a series of leukemia cell lines did not result in either apoptosis or differentiation of most tested cell lines. Interestingly, it did significantly sensitize these cell lines to chemotherapeutic drugs. Here, we further investigated the mechanism underlying pig7-induced improved sensitivity of acute leukemia cells to chemotherapy. Our results demonstrated that the sensitization effect driven by exogenous pig7 was more effective in drug-resistant leukemia cell lines which had lower endogenous pig7 expression. Overexpression of pig7 did not directly activate the caspase apoptotic pathway, but decreased the lysosomal stability. The expression of pig7 resulted in lysosomal membrane permeabilization (LMP) and lysosomal protease (e.g. cathepsin B, D, L) release. Moreover, we also observed increased reactive oxygen species (ROS) and decreased mitochondrial membrane potential (ΔΨm) induced by pig7. Some autophagy markers such as LC3I/II, ATG5 and Beclin-1, and necroptosis maker MLKL were also stimulated. However, intrinsic antagonism such as serine/cysteine protease inhibitors Spi2A and Cystatin C prevented downstream effectors from triggering leukemia cells, which were only on the "verge of apoptosis". When combined with chemotherapy, LMP increased and more proteases were released. Once this process was beyond the limit of intrinsic antagonism, it induced programmed cell death cooperatively via caspase-independent and caspase-dependent pathways.

  6. Recent advances in gene therapy for lysosomal storage disorders

    Directory of Open Access Journals (Sweden)

    Rastall DP

    2015-06-01

    Full Text Available David PW Rastall,1 Andrea Amalfitano1,2 1Department of Microbiology and Molecular Genetics, 2Department of Pediatrics, College of Osteopathic Medicine, Michigan State University, East Lansing, MI, USA Abstract: Lysosomal storage disorders (LSDs are a group of genetic diseases that result in metabolic derangements of the lysosome. Most LSDs are due to the genetic absence of a single catabolic enzyme, causing accumulation of the enzyme's substrate within the lysosome. Over time, tissue-specific substrate accumulations result in a spectrum of symptoms and disabilities that vary by LSD. LSDs are promising targets for gene therapy because delivery of a single gene into a small percentage of the appropriate target cells may be sufficient to impact the clinical course of the disease. Recently, there have been several significant advancements in the potential for gene therapy of these disorders, including the first human trials. Future clinical trials will build upon these initial attempts, with an improved understanding of immune system responses to gene therapy, the obstacle that the blood–brain barrier poses for neuropathic LSDs, as well other biological barriers that, when overcome, may facilitate gene therapy for LSDs. In this manuscript, we will highlight the recent innovations in gene therapy for LSDs and discuss the clinical limitations that remain to be overcome, with the goal of fostering an understanding and further development of this important field. Keywords: human trials, clinical trials, gene therapy, lysosomal storage disease, blood-brain barrier, adeno-associated virus, lentivirus, adenovirus 

  7. The frequency of lysosomal storage diseases in The Netherlands

    NARCIS (Netherlands)

    Poorthuis, BJHM; Wevers, RA; Kleijer, WJ; Groener, JEM; de Jong, JGN; van Weely, S; Niezen-Koning, KE; van Diggelen, OP

    1999-01-01

    We have calculated the relative frequency and the birth prevalence of lysosomal storage diseases (LSDs) in The Netherlands based on all 963 enzymatically confirmed cases diagnosed during the period 1970-1996. The combined birth prevalence for all LSDs is 14 per 100,000 live births. Glycogenosis type

  8. Lysosomal acid phosphatase is internalized via clathrin-coated pits

    NARCIS (Netherlands)

    Klumperman, J.; Hille, A.; Geuze, H.J.; Peters, C.; Brodsky, F.M.; Figura, K. von

    1992-01-01

    The presence of lysosomal acid phosphatase (LAP) in coated pits at the plasma membrane was investigated by immunocytochemistry in thymidine kinase negative mouse L-cells (Ltk-) and baby hamster kidney (BHK) cells overexpressing human LAP (Ltk-LAP and BHK-LAP cells). Double immunogold labeling showed

  9. Release and uptake of lysosomal enzymes : studied in cultured cells

    NARCIS (Netherlands)

    D.J.J. Halley (Dicky)

    1980-01-01

    textabstractThe purpose of the experimental work described in this thesiswas to investigate some aspects of the release and uptake of lysosomal enzymes. The experiments involved the use of normal human and animal fibroblasts and some other cell types such as hepatocytes and hepatoma cells as sources

  10. Glycolipid-dependent sorting of melanosomal from lysosomal membrane proteins by lumenal determinants

    NARCIS (Netherlands)

    Groux-Degroote, S.; Dijk, S.M. van; Wolthoorn, J.; Neumann, S.; Theos, A.C.; Mazière, A.M. de; Klumperman, J.; Meer, G. van; Sprong, H.

    2008-01-01

    Melanosomes are lysosome-related organelles that coexist with lysosomes in mammalian pigment cells. Melanosomal and lysosomal membrane proteins share similar sorting signals in their cytoplasmic tail, raising the question how they are segregated. We show that in control melanocytes, the melanosomal

  11. Sulindac metabolites induce proteosomal and lysosomal degradation of the epidermal growth factor receptor.

    Science.gov (United States)

    Pangburn, Heather A; Ahnen, Dennis J; Rice, Pamela L

    2010-04-01

    The epidermal growth factor receptor (EGFR) is a member of the ErbB family of receptor tyrosine kinases. In response to ligand, EGFR is internalized and degraded by the ubiquitin-proteasome/lysosome pathway. We previously reported that metabolites of the nonsteroidal anti-inflammatory drug sulindac downregulate the expression of EGFR and inhibit basal and EGF-induced EGFR signaling through extracellular signal-regulated kinase 1/2. We now have evaluated the mechanisms of sulindac metabolite-induced downregulation of EGFR. EGF-induced downregulation of EGFR occurs within 10 minutes and lasts for 24 hours. By contrast, downregulation of EGFR by sulindac sulfide and sulindac sulfone was first evident at 4 and 24 hours, respectively, with maximal downregulation at 72 hours. Pretreatment with either the lysosomal inhibitor chloroquine or the proteosomal inhibitor MG132 blocked sulindac metabolite-induced downregulation of EGFR. Sulindac metabolites also increased the ubiquitination of EGFR. Whereas sulindac metabolites inhibited phosphorylation of EGFR pY1068, they increased phosphorylation of EGFR pY1045, the docking site where c-Cbl binds, thereby enabling receptor ubiquitination and degradation. Immunofluorescence analysis of EGF and EGFR distribution confirmed the biochemical observations that sulindac metabolites alter EGFR localization and EGFR internalization in a manner similar to that seen with EGF treatment. Expression of ErbB family members HER2 and HER3 was also downregulated by sulindac metabolites. We conclude that downregulation of EGFR expression by sulindac metabolites is mediated via lysosomal and proteosomal degradation that may be due to drug-induced phosphorylation at pY1045 with resultant ubiquitination of EGFR. Thus, sulindac metabolite-induced downregulation of EGFR seems to be mediated through mechanism(s) similar, at least in part, to those involved in EGF-induced downregulation of EGFR.

  12. Lysosomal membrane stability plays a major role in the cytotoxic activity of the anti-proliferative agent, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT).

    Science.gov (United States)

    Gutierrez, Elaine M; Seebacher, Nicole A; Arzuman, Laila; Kovacevic, Zaklina; Lane, Darius J R; Richardson, Vera; Merlot, Angelica M; Lok, Hiu; Kalinowski, Danuta S; Sahni, Sumit; Jansson, Patric J; Richardson, Des R

    2016-07-01

    The potent and selective anti-tumor agent, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT), localizes in lysosomes and forms cytotoxic copper complexes that generate reactive oxygen species (ROS), resulting in lysosomal membrane permeabilization (LMP) and cell death. Herein, the role of lysosomal membrane stability in the anti-tumor activity of Dp44mT was investigated. Studies were performed using molecules that protect lysosomal membranes against Dp44mT-induced LMP, namely heat shock protein 70 (HSP70) and cholesterol. Up-regulation or silencing of HSP70 expression did not affect Dp44mT-induced LMP in MCF7 cells. In contrast, cholesterol accumulation in lysosomes induced by the well characterized cholesterol transport inhibitor, 3-β-[2-(diethyl-amino)ethoxy]androst-5-en-17-one (U18666A), inhibited Dp44mT-induced LMP and markedly and significantly (peffect of U18666A in increasing lysosomal cholesterol and preventing the cytotoxic activity of Dp44mT was not due to induced autophagy. Instead, U18666A was found to decrease lysosomal turnover, resulting in autophagosome accumulation. Moreover, preincubation with U18666A did not prevent the ability of Dp44mT to induce autophagosome synthesis, indicating that autophagic initiation via Dp44mT occurs independently of LMP. These studies demonstrate the significance of lysosomal membrane stability in relation to the ability of Dp44mT to execute tumor cell death and overcome pro-survival autophagy. Hence, lysosomal-dependent cell death induced by Dp44mT serves as an important anti-tumor strategy. These results are important for comprehensively understanding the mechanism of action of Dp44mT.

  13. Fusion of lysosomes with secretory organelles leads to uncontrolled exocytosis in the lysosomal storage disease mucolipidosis type IV.

    Science.gov (United States)

    Park, Soonhong; Ahuja, Malini; Kim, Min Seuk; Brailoiu, G Cristina; Jha, Archana; Zeng, Mei; Baydyuk, Maryna; Wu, Ling-Gang; Wassif, Christopher A; Porter, Forbes D; Zerfas, Patricia M; Eckhaus, Michael A; Brailoiu, Eugen; Shin, Dong Min; Muallem, Shmuel

    2016-02-01

    Mutations in TRPML1 cause the lysosomal storage disease mucolipidosis type IV (MLIV). The role of TRPML1 in cell function and how the mutations cause the disease are not well understood. Most studies focus on the role of TRPML1 in constitutive membrane trafficking to and from the lysosomes. However, this cannot explain impaired neuromuscular and secretory cells' functions that mediate regulated exocytosis. Here, we analyzed several forms of regulated exocytosis in a mouse model of MLIV and, opposite to expectations, we found enhanced exocytosis in secretory glands due to enlargement of secretory granules in part due to fusion with lysosomes. Preliminary exploration of synaptic vesicle size, spontaneous mEPSCs, and glutamate secretion in neurons provided further evidence for enhanced exocytosis that was rescued by re-expression of TRPML1 in neurons. These features were not observed in Niemann-Pick type C1. These findings suggest that TRPML1 may guard against pathological fusion of lysosomes with secretory organelles and suggest a new approach toward developing treatment for MLIV.

  14. Acute effects of the sigma-2 receptor agonist siramesine on lysosomal and extra-lysosomal proteolytic systems in lens epithelial cells

    OpenAIRE

    Jonhede, S.; Petersen, A; Zetterberg, M.; Karlsson, J-O

    2010-01-01

    Purpose The aim of the present study was to examine the effects of the sigma-2 receptor agonist, siramesine, on morphology, growth, cell death, lysosomal function, and effects on extra-lysosomal proteolytic systems in human lens epithelial cells. Methods Human lens epithelial cells in culture were exposed to siramesine and examined for morphological changes using Nomarski optics or calcein. Lysosomes were evaluated using acridine orange and Magic Red (RR-cresyl violet). Nuclear morphology was...

  15. The use of dried blood spot samples in the diagnosis of lysosomal storage disorders--current status and perspectives.

    Science.gov (United States)

    Reuser, Arnold J; Verheijen, Frans W; Bali, Deeksha; van Diggelen, Otto P; Germain, Dominique P; Hwu, Wuh-Liang; Lukacs, Zoltan; Mühl, Adolf; Olivova, Petra; Piraud, Monique; Wuyts, Birgit; Zhang, Kate; Keutzer, Joan

    2011-01-01

    Dried blood spot (DBS) methods are currently available for identification of a range of lysosomal storage disorders (LSDs). These disorders are generally characterized by a deficiency of activity of a lysosomal enzyme and by a broad spectrum of phenotypes. Diagnosis of LSD patients is often delayed, which is of particular concern as therapeutic outcomes (e.g. enzyme replacement therapy) are generally more favorable in early disease stages. Experts in the field of LSDs diagnostics and screening programs convened and reviewed experiences with the use of DBS methods, and discuss the diagnostic challenges, possible applications and quality programs in this paper. Given the easy sampling and shipping and stability of samples, DBS has evident advantages over other laboratory methods and can be particularly helpful in the early identification of affected LSD patients through neonatal screening, high-risk population screening or family screening.

  16. Trace metal distribution in pristine permafrost-affected soils of the Lena River Delta and its Hinterland, Northern Siberia, Russia

    Directory of Open Access Journals (Sweden)

    I. Antcibor

    2013-02-01

    Full Text Available Soils are an important compartment of ecosystems and have the ability to immobilize chemicals preventing their movement to other environment compartments. Predicted climatic changes together with other anthropogenic influences on Arctic terrestrial environments may affect biogeochemical processes enhancing leaching and migration of trace elements in permafrost-affected soils. This is especially important since the Arctic ecosystems are considered to be very sensitive to climatic changes as well as to chemical contamination. This study characterizes background levels of trace metals in permafrost-affected soils of the Lena River Delta and its hinterland in northern Siberia (73.5° N–69.5° N representing a remote region far from evident anthropogenic trace metal sources. Investigations on total element contents of iron (Fe, arsenic (As, manganese (Mn, zinc (Zn, nickel (Ni, copper (Cu, lead (Pb, cadmium (Cd, cobalt (Co and mercury (Hg in different soil types developed in different geological parent materials have been carried out. The highest concentrations of the majority of the measured elements were observed in soils belonging to ice-rich permafrost sediments formed during the Pleistocene (ice-complex in the Lena River Delta region. Correlation analyses of trace metal concentrations and soil chemical and physical properties at a Holocene estuarine terrace and two modern floodplain levels in the southern-central Lena River Delta (Samoylov Island showed that the main factors controlling the trace metal distribution in these soils are organic matter content, soil texture and contents of iron and manganese-oxides. Principal Component Analysis (PCA revealed that soil oxides play a significant role in trace metal distribution in both top and bottom horizons. Occurrence of organic matter contributes to Cd binding in top soils and Cu binding in bottom horizons. Observed ranges of the background concentrations of the majority of trace elements were

  17. Autophagic flux promotes cisplatin resistance in human ovarian carcinoma cells through ATP-mediated lysosomal function.

    Science.gov (United States)

    Ma, Liwei; Xu, Ye; Su, Jing; Yu, Huimei; Kang, Jinsong; Li, Hongyan; Li, Xiaoning; Xie, Qi; Yu, Chunyan; Sun, Liankun; Li, Yang

    2015-11-01

    Lysosomes are involved in promoting resistance of cancer cells to chemotherapeutic agents. However, the mechanisms underlying lysosomal influence of cisplatin resistance in ovarian cancer remain incompletely understood. We report that, compared with cisplatin-sensitive SKOV3 cells, autophagy increases in cisplatin-resistant SKOV3/DDP cells treated with cisplatin. Inhibition of early-stage autophagy enhanced cisplatin-mediated cytotoxicity in SKOV3/DDP cells, but autophagy inhibition at a later stage by disturbing autophagosome-lysosome fusion is more effective. Notably, SKOV3/DDP cells contained more lysosomes than cisplatin-sensitive SKOV3 cells. Abundant lysosomes and lysosomal cathepsin D activity were required for continued autolysosomal degradation and maintenance of autophagic flux in SKOV3/DDP cells. Furthermore, SKOV3/DDP cells contain abundant lysosomal ATP required for lysosomal function, and inhibition of lysosomal ATP accumulation impaired lysosomal function and blocked autophagic flux. Therefore, our findings suggest that lysosomes at least partially contribute to cisplatin resistance in ovarian cancer cells through their role in cisplatin-induced autophagic processes, and provide insight into the mechanism of cisplatin resistance in tumors.

  18. The Role of Oxidized Cholesterol in Diabetes-Induced Lysosomal Dysfunction in the Brain.

    Science.gov (United States)

    Sims-Robinson, Catrina; Bakeman, Anna; Rosko, Andrew; Glasser, Rebecca; Feldman, Eva L

    2016-05-01

    Abnormalities in lysosomal function have been reported in diabetes, aging, and age-related degenerative diseases. These lysosomal abnormalities are an early manifestation of neurodegenerative diseases and often precede the onset of clinical symptoms such as learning and memory deficits; however, the mechanism underlying lysosomal dysfunction is not known. In the current study, we investigated the mechanism underlying lysosomal dysfunction in the cortex and hippocampi, key structures involved in learning and memory, of a type 2 diabetes (T2D) mouse model, the leptin receptor deficient db/db mouse. We demonstrate for the first time that diabetes leads to destabilization of lysosomes as well as alterations in the protein expression, activity, and/or trafficking of two lysosomal enzymes, hexosaminidase A and cathepsin D, in the hippocampus of db/db mice. Pioglitazone, a thiazolidinedione (TZD) commonly used in the treatment of diabetes due to its ability to improve insulin sensitivity and reverse hyperglycemia, was ineffective in reversing the diabetes-induced changes on lysosomal enzymes. Our previous work revealed that pioglitazone does not reverse hypercholesterolemia; thus, we investigated whether cholesterol plays a role in diabetes-induced lysosomal changes. In vitro, cholesterol promoted the destabilization of lysosomes, suggesting that lysosomal-related changes associated with diabetes are due to elevated levels of cholesterol. Since lysosome dysfunction precedes neurodegeneration, cognitive deficits, and Alzheimer's disease neuropathology, our results may provide a potential mechanism that links diabetes with complications of the central nervous system.

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

    Science.gov (United States)

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

    2015-02-01

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

  20. Myelin lesions associated with lysosomal and peroxisomal disorders.

    Science.gov (United States)

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

    2010-09-01

    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.

  1. Targeting Androgen Receptor by Lysosomal Degradation in Prostate Cancer

    Science.gov (United States)

    2014-09-01

    were done as described.13 Protein Sample Preparation and Mass Spectrometry Tandem Affinity Purification of FLAG-His-EWS-Fli-1- Interacting Proteins . Forty...incubated with Ni-NTA agarose (Qiagen), FLAG-His-EWS-Fli-1 and its interacting proteins were collected by centrifugation, washed three times with TN buffer...the lysosome fraction was loaded at 100x compared to the input. ■ RESULTS AND DISCUSSION Proteomic Analysis of the EWS-Fli-1- Interacting Proteins To

  2. Recent advances in gene therapy for lysosomal storage disorders.

    Science.gov (United States)

    Rastall, David Pw; Amalfitano, Andrea

    2015-01-01

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

  3. Discriminating lysosomal membrane protein types using dynamic neural network.

    Science.gov (United States)

    Tripathi, Vijay; Gupta, Dwijendra Kumar

    2014-01-01

    This work presents a dynamic artificial neural network methodology, which classifies the proteins into their classes from their sequences alone: the lysosomal membrane protein classes and the various other membranes protein classes. In this paper, neural networks-based lysosomal-associated membrane protein type prediction system is proposed. Different protein sequence representations are fused to extract the features of a protein sequence, which includes seven feature sets; amino acid (AA) composition, sequence length, hydrophobic group, electronic group, sum of hydrophobicity, R-group, and dipeptide composition. To reduce the dimensionality of the large feature vector, we applied the principal component analysis. The probabilistic neural network, generalized regression neural network, and Elman regression neural network (RNN) are used as classifiers and compared with layer recurrent network (LRN), a dynamic network. The dynamic networks have memory, i.e. its output depends not only on the input but the previous outputs also. Thus, the accuracy of LRN classifier among all other artificial neural networks comes out to be the highest. The overall accuracy of jackknife cross-validation is 93.2% for the data-set. These predicted results suggest that the method can be effectively applied to discriminate lysosomal associated membrane proteins from other membrane proteins (Type-I, Outer membrane proteins, GPI-Anchored) and Globular proteins, and it also indicates that the protein sequence representation can better reflect the core feature of membrane proteins than the classical AA composition.

  4. Vamp-7 Mediates Vesicular Transport from Endosomes to Lysosomes

    Science.gov (United States)

    Advani, Raj J.; Yang, Bin; Prekeris, Rytis; Lee, Kelly C.; Klumperman, Judith; Scheller, Richard H.

    1999-01-01

    A more complete picture of the molecules that are critical for the organization of membrane compartments is beginning to emerge through the characterization of proteins in the vesicle-associated membrane protein (also called synaptobrevin) family of membrane trafficking proteins. To better understand the mechanisms of membrane trafficking within the endocytic pathway, we generated a series of monoclonal and polyclonal antibodies against the cytoplasmic domain of vesicle-associated membrane protein 7 (VAMP-7). The antibodies recognize a 25-kD membrane-associated protein in multiple tissues and cell lines. Immunohistochemical analysis reveals colocalization with a marker of late endosomes and lysosomes, lysosome-associated membrane protein 1 (LAMP-1), but not with other membrane markers, including p115 and transferrin receptor. Treatment with nocodozole or brefeldin A does not disrupt the colocalization of VAMP-7 and LAMP-1. Immunoelectron microscopy analysis shows that VAMP-7 is most concentrated in the trans-Golgi network region of the cell as well as late endosomes and transport vesicles that do not contain the mannose-6 phosphate receptor. In streptolysin- O–permeabilized cells, antibodies against VAMP-7 inhibit the breakdown of epidermal growth factor but not the recycling of transferrin. These data are consistent with a role for VAMP-7 in the vesicular transport of proteins from the early endosome to the lysosome. PMID:10459012

  5. Dynamics of aggregate stability and soil organic C distribution as affected by climatic aggressiveness: a mesocosm approach

    Science.gov (United States)

    Pellegrini, Sergio; Elio Agnelli, Alessandro; Costanza Andrenelli, Maria; Barbetti, Roberto; Castelli, Fabio; Costantini, Edoardo A. C.; Lagomarsino, Alessandra; Pasqui, Massimiliano; Tomozeiu, Rodica; Razzaghi, Somayyeh; Vignozzi, Nadia

    2014-05-01

    In the framework of a research project aimed at evaluating the adaptation scenarios of the Italian agriculture to the current climate change, a mesocosm experiment under controlled conditions was set up for studying the dynamics of soil aggregate stability and organic C in different size fractions. Three alluvial loamy soils (BOV - Typic Haplustalfs coarse-loamy; CAS - Typic Haplustalfs fine-loamy; MED - Typic Hapludalfs fine-loamy) along a climatic gradient (from dryer to moister pedoclimatic conditions) in the river Po valley (northern Italy), under crop rotation for animal husbandry from more than 40 years, were selected. The Ap horizons (0-30cm) were taken and placed in 9 climatic chambers under controlled temperature and rainfall. Each soil was subjected to three different climate scenarios in terms of erosivity index obtained by combining Modified Fournier and Bagnouls-Gaussen indexes: i) typical (TYP), the median year of each site related to the 1961-1990 reference period; ii) maximum aggressive year (MAX) observed in the same period, and iii) the simulated climate (SIM), obtained by projections of climate change precipitation and temperature for the period 2021-2050 as provided by the IPCC-A1B emission scenario. In the climatic chambers the year climate was reduced to six months. The soils were analyzed for particle size distribution, aggregate stability by wet and dry sieving, and organic C content at the beginning and at the end of the trial. The soils showed different behaviour in terms of aggregate stability and dynamics of organic C in the diverse size fractions. The soils significantly differed in terms of initial mean weight diameter (MWD) (CAS>MED>BOV). A general reduction of MWD in all sites was observed at the end of the experiment, with the increase of the smallest aggregate fractions (0.250-0.05 mm). In particular, BOV showed the maximum decrease of the aggregate stability and MED the lowest. C distribution in aggregate fractions significantly

  6. Subtle changes in bone mineralization density distribution in most severely affected patients with chronic obstructive pulmonary disease.

    Science.gov (United States)

    Misof, B M; Roschger, P; Jorgetti, V; Klaushofer, K; Borba, V Z C; Boguszewski, C L; Cohen, A; Shane, E; Zhou, H; Dempster, D W; Moreira, C A

    2015-10-01

    Chronic obstructive pulmonary disease (COPD) is associated with low aBMD as measured by DXA and altered microstructure as assessed by bone histomorphometry and microcomputed tomography. Knowledge of bone matrix mineralization is lacking in COPD. Using quantitative backscatter electron imaging (qBEI), we assessed cancellous (Cn.) and cortical (Ct.) bone mineralization density distribution (BMDD) in 19 postmenopausal women (62.1 ± 7.3 years of age) with COPD. Eight had sustained fragility fractures, and 13 had received treatment with inhaled glucocorticoids. The BMDD outcomes from the patients were compared with healthy reference data and were correlated with previous clinical and histomorphometric findings. In general, the BMDD outcomes for the patients were not significantly different from the reference data. Neither the subgroups of with or without fragility fractures or of who did or did not receive inhaled glucocorticoid treatment, showed differences in BMDD. However, subgroup comparison according to severity revealed 10% decreased cancellous mineralization heterogeneity (Cn.CaWidth) for the most severely affected compared with less affected patients (p=0.042) and compared with healthy premenopausal controls (p=0.021). BMDD parameters were highly correlated with histomorphometric cancellous bone volume (BV/TV) and formation indices: mean degree of mineralization (Cn.CaMean) versus BV/TV (r=0.58, p=0.009), and Cn.CaMean and Ct.CaMean versus bone formation rate (BFR/BS) (r=-0.71, p50th percentile) BV/TV. The normality in most of the BMDD parameters and bone formation rates as well as the significant correlations between them suggests unaffected mineralization processes in COPD. Our findings also indicate no significant negative effect of treatment with inhaled glucocorticoids on the bone mineralization pattern. However, the observed concomitant occurrence of relatively lower bone volumes with lower bone matrix mineralization will both contribute to the reduced a

  7. Vertical distribution of radiocesium in soils of the area affected by the Fukushima Dai-ichi nuclear power plant accident

    Science.gov (United States)

    Konoplev, A. V.; Golosov, V. N.; Yoschenko, V. I.; Nanba, K.; Onda, Y.; Takase, T.; Wakiyama, Y.

    2016-05-01

    Presented are results of the study of radiocesium vertical distribution in the soils of the irrigation pond catchments in the near field 0.25 to 8 km from the Fukushima Dai-ichi NPP, on sections of the Niida River floodplain, and in a forest ecosystem typical of the territory contaminated after the accident. It is shown that the vertical migration of radiocesium in undisturbed forest and grassland soils in the zone affected by the Fukushima accident is faster than it was in the soils of the 30-km zone of the Chernobyl NPP for a similar time interval after the accident. The effective dispersion coefficients in the Fukushima soils are several times higher than those for the Chernobyl soils. This may be associated with higher annual precipitation (by about 2.5 times) in Fukushima as compared to the Chernobyl zone. In the forest soils the radiocesium dispersion is faster as compared to grassland soils, both in the Fukushima and Chernobyl zones. The study and analysis of the vertical distribution of the Fukushima origin radiocesium in the Niida gawa floodplain soils has made it possible to identify areas of contaminated sediment accumulation on the floodplain. The average accumulation rate for sediments at the study locations on the Niida gawa floodplain varied from 0.3 to 3.3 cm/year. Taking into account the sediments accumulation leading to an increase in the radiocesium inventory in alluvial soils is key for predicting redistribution of radioactive contamination after the Fukushima accident on the river catchments, as well as for decision-making on contaminated territories remediation and clean-up. Clean-up of alluvial soils does not seem to be worthwhile because of the following accumulation of contaminated sediments originating from more contaminated areas, including the exclusion zone.

  8. Simvastatin promotes NPC1-mediated free cholesterol efflux from lysosomes through CYP7A1/LXRα signalling pathway in oxLDL-loaded macrophages.

    Science.gov (United States)

    Xu, Xiaoyang; Zhang, Aolin; Halquist, Matthew S; Yuan, Xinxu; Henderson, Scott C; Dewey, William L; Li, Pin-Lan; Li, Ningjun; Zhang, Fan

    2017-02-01

    Statins, 3-hydroxyl-3-methylglutaryl coenzyme A reductase inhibitors, are the first-line medications prescribed for the prevention and treatment of coronary artery diseases. The efficacy of statins has been attributed not only to their systemic cholesterol-lowering actions but also to their pleiotropic effects that are unrelated to cholesterol reduction. These pleiotropic effects have been increasingly recognized as essential in statins therapy. This study was designed to investigate the pleiotropic actions of simvastatin, one of the most commonly prescribed statins, on macrophage cholesterol homeostasis with a focus on lysosomal free cholesterol egression. With simultaneous nile red and filipin staining, analysis of confocal/multi-photon imaging demonstrated that simvastatin markedly attenuated unesterified (free) cholesterol buildup in macrophages loaded with oxidized low-density lipoprotein but had little effect in reducing the sizes of cholesteryl ester-containing lipid droplets; the reduction in free cholesterol was mainly attributed to decreases in lysosome-compartmentalized cholesterol. Functionally, the egression of free cholesterol from lysosomes attenuated pro-inflammatory cytokine secretion. It was determined that the reduction of lysosomal free cholesterol buildup by simvastatin was due to the up-regulation of Niemann-Pick C1 (NPC1), a lysosomal residing cholesterol transporter. Moreover, the enhanced enzymatic production of 7-hydroxycholesterol by cytochrome P450 7A1 and the subsequent activation of liver X receptor α underscored the up-regulation of NPC1. These findings reveal a novel pleiotropic effect of simvastatin in affecting lysosomal cholesterol efflux in macrophages and the associated significance in the treatment of atherosclerosis.

  9. The tumor suppressor p53 regulates autophagosomal and lysosomal biogenesis in lung cancer cells by targeting transcription factor EB.

    Science.gov (United States)

    Zhang, Zengli; Wang, Hongfeng; Ding, Qifeng; Xing, Yufei; Xu, Delai; Xu, Zhonghua; Zhou, Tong; Qian, Bin; Ji, Chenghong; Pan, Xue; Zhong, Anyuan; Ying, Zheng; Zhou, Caicun; Shi, Minhua

    2017-03-10

    The cellular protein degradation system, such as proteasomal or autophagy-lysosomal system plays an important role in the pathogenesis of a variety of human diseases including cancer. Transcription factor EB (TFEB) is a master transcriptional factor in the regulation of autophagy-lysosome pathway (ALP), and it has multiple biological functions including protein degradation, cell homeostasis and cell survival. In the present study we show that the tumor suppressor p53 can regulate TFEB nuclear translocation and activity in lung cancer cells. We found p53 deletion or chemical inhibition of p53 using pifithrin-α could promote the translocation of TFEB from cytoplasm to the nucleus, thus increased the TFEB-mediated lysosomal and autophagosomal biogenesis in lung cancer cells. Moreover, re-expression of p53 could decrease the expression levels of TFEB-targeting genes involved in ALP, and knockdown of TFEB could abolish the effect of p53 on the regulation of ALP gene expression. Taken together, our data indicate that p53 affects ALP through regulating TFEB nuclear translocation in lung cancer cells. Importantly, our study reveals a critical link between two keys factors in tumourigenesis and autophagy, and suggests a potential important role of p53-TFEB signaling axis in lung cancer.

  10. Andrographolide sensitizes cisplatin-induced apoptosis via suppression of autophagosome-lysosome fusion in human cancer cells.

    Science.gov (United States)

    Zhou, Jing; Hu, Shuai-Er; Tan, Shi-Hao; Cao, Ruoxi; Chen, Yiyang; Xia, Dajing; Zhu, Xinqiang; Yang, Xing-Fen; Ong, Choon-Nam; Shen, Han-Ming

    2012-03-01

    Suppression of autophagy has been increasingly recognized as a novel cancer therapeutic approach. Andrographolide (Andro), a diterpenoid lactone isolated from an herbal plant Andrographis paniculata, is known to possess anti-inflammatory and anticancer activity. In this study, we sought to examine the effect of Andro on autophagy, and to evaluate whether such effect is relevant to the sensitization effect of Andro on apoptosis induced by DNA damage agents in cancer cells. First, we found that Andro is able to significantly enhance autophagic markers in various cancer cell lines, including GFP-LC3 puncta and LC3-II level. Interestingly, Andro treatment also led to marked increase of p62 protein level and addition of chloroquine (CQ) failed to further enhance either LC3-II or p62 level, indicating that Andro is likely to suppress autophagic flux at the maturation and degradation stage. Next, we provided evidence that Andro inhibits autophagosome maturation not by affecting the lysosomal function, but by impairing autophagosome-lysosome fusion. Lastly, we demonstrated that treatment with cisplatin, a DNA damage agent, induces autophagy in cancer cells. Importantly, Andro is capable of sensitizing cisplatin-induced cell killing determined with both short-term apoptosis assays and long-term clonogenic test, via suppression of autophagy, a process independent of p53. In summary, these observations collectively suggest that Andro could be a promising anti-cancer agent in combination therapy via its potent inhibitory effect on autophagy by disrupting autophagosome-lysosome fusion.

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

    Energy Technology Data Exchange (ETDEWEB)

    Li, Hongmei [Ministry of Education Key Laboratory of Synthetic and Natural Functional Molecule Chemistry, College of Chemistry & Materials Science, Northwest University, Xi' an 710069 (China); Wang, Cuiling [Key Laboratory of Resource Biology and Biotechnology in Western China, Ministry of Education, College of Life Science, Northwest University, Xi' an 710069 (China); She, Mengyao; Zhu, Yuelu; Zhang, Jidong; Yang, Zheng [Ministry of Education Key Laboratory of Synthetic and Natural Functional Molecule Chemistry, College of Chemistry & Materials Science, Northwest University, Xi' an 710069 (China); Liu, Ping, E-mail: liuping@nwu.edu.cn [Ministry of Education Key Laboratory of Synthetic and Natural Functional Molecule Chemistry, College of Chemistry & Materials Science, Northwest University, Xi' an 710069 (China); Wang, Yaoyu [Ministry of Education Key Laboratory of Synthetic and Natural Functional Molecule Chemistry, College of Chemistry & Materials Science, Northwest University, Xi' an 710069 (China); Li, Jianli, E-mail: lijianli@nwu.edu.cn [Ministry of Education Key Laboratory of Synthetic and Natural Functional Molecule Chemistry, College of Chemistry & Materials Science, Northwest University, Xi' an 710069 (China)

    2015-11-05

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

  12. Profile Distributions of Dissolved and Colloidal Phosphorus as Affected by Degree of Phosphorus Saturation in Paddy Soil

    Institute of Scientific and Technical Information of China (English)

    ZANG Ling; TIAN Guang-Ming; LIANG Xin-Qing; HE Miao-Miao; BAO Qi-Bei; YAO Jin-Hua

    2013-01-01

    Soil dissolved phosphorus (P) and colloidal P mobilization could be closely related to the degree of phosphorus saturation (DPS).Effects of a wide range of DPS on the distributions of dissolved P and colloidal P in a paddy soil profile were investigated in this study.Dissolved P and colloidal P in water-dispersible soil colloid suspension increased obviously with increasing DPS.The change point of DPS was at 0.12 by using a split-line model.Above the value,dissolved P (3.1 mg P kg-1) in soil profile would increase sharply and then transfer downward.Compared with dissolved P,colloidal P was the dominant fraction (78%-91%) of P in soil colloid suspension,and positively related to DPS without a significant change point.The high release of colloids in subsoils with low DPS was attributed to the low ionic strength and high pH value in subsoils.The DPS also had a significant and positive correlation with electrical conductivity (EC),but it showed a negative correlation with pH value.However,the concentration of colloidal P was not greatly correlated to the pH value,EC and optical density of the soil colloid suspension.The results indicated that DPS was an important factor that may affect the accumulation and mobilization of water-extractable colloidal P and dissolved P.

  13. Vertical Distribution of Cadmium and Lead on Soils Affected by Metropolitan Refuse Disposal in Owerri, Southeastern Nigeria

    Directory of Open Access Journals (Sweden)

    E.U. Onweremadu

    2011-01-01

    Full Text Available The authors investigated distribution of cadmium (Cd and lead (Pb in soil profile pits affected by municipal solid wastes in Avu dumpsite in Owerri, Southeastern Nigeria in 2010. Transect soil survey technique was used in aligning profile pits for field studies and sampling. Standard procedures were used in digging, describing and sampling from profile pits. Sieved soil samples were subjected to laboratory analyses and data were analyzed statistically using coefficient of variation measured in percentages. Results showed higher values of % CV in silt and clay contents. Variability of clay increased from dumpsite (CV=43.77 % to moderately dumped site (CV=62.73% decreased in slightly dumped side (20.98%. Highest mean values of organic matter (26.8 g/kg and pH water (5.7 were reported in heavily dumped site. Organic Matter showed very significant positive relationship with Cd (r = 0.92; p = 0.01 and Pb (r=0.97; P = 0.97. There is need to include more soil attributes; results of which should be subjected to multi-variate techniques for more reliability and confidence especially in field applications.

  14. The Hydrological Regimes Brought by the Three Gorges Project Affected Riparian Vegetation Distribution and Diversity in 2009 and 2010

    Science.gov (United States)

    Miao, Ling-Feng; Liu, Wei-Wei; Yang, Fan

    2017-01-01

    Post-dam riparian vegetations affected by the new hydrological regimes in the Three Gorges Reservoir (TGR) were investigated in 2009 and 2010, respectively. The investigation in 2009 showed that about 231 vascular plant species belonging to 169 genera of 61 families were distributed in the water-level-fluctuation zone (WLFZ) of the (TGR). Three vegetation types, including Chuanjiang, Gorge, and other vegetation types, were classified efficiently via cluster analysis. Alpha diversity analysis indicated that species richness gradually decreased with decreasing elevation. Beta diversity analysis indicated that high environment heterogeneity was existed between the lower section and the other two sections, and environment homogeneity was also existed between middle section and upper section. Using the analysis of the field growth in the 2009 and 2010 field surveys as bases, we proposed a list of perennial herb species and woody species that may potentially occurred in the WLFZ of the TGR. In addition, we predicted plant community structural changes in the different altitude sections of WLFZ in the future.

  15. MCOLN1 is a ROS sensor in lysosomes that regulates autophagy.

    Science.gov (United States)

    Zhang, Xiaoli; Cheng, Xiping; Yu, Lu; Yang, Junsheng; Calvo, Raul; Patnaik, Samarjit; Hu, Xin; Gao, Qiong; Yang, Meimei; Lawas, Maria; Delling, Markus; Marugan, Juan; Ferrer, Marc; Xu, Haoxing

    2016-06-30

    Cellular stresses trigger autophagy to remove damaged macromolecules and organelles. Lysosomes 'host' multiple stress-sensing mechanisms that trigger the coordinated biogenesis of autophagosomes and lysosomes. For example, transcription factor (TF)EB, which regulates autophagy and lysosome biogenesis, is activated following the inhibition of mTOR, a lysosome-localized nutrient sensor. Here we show that reactive oxygen species (ROS) activate TFEB via a lysosomal Ca(2+)-dependent mechanism independent of mTOR. Exogenous oxidants or increasing mitochondrial ROS levels directly and specifically activate lysosomal TRPML1 channels, inducing lysosomal Ca(2+) release. This activation triggers calcineurin-dependent TFEB-nuclear translocation, autophagy induction and lysosome biogenesis. When TRPML1 is genetically inactivated or pharmacologically inhibited, clearance of damaged mitochondria and removal of excess ROS are blocked. Furthermore, TRPML1's ROS sensitivity is specifically required for lysosome adaptation to mitochondrial damage. Hence, TRPML1 is a ROS sensor localized on the lysosomal membrane that orchestrates an autophagy-dependent negative-feedback programme to mitigate oxidative stress in the cell.

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

    Directory of Open Access Journals (Sweden)

    Seydoux E

    2014-08-01

    of BMDCs to degrade soluble antigen, without affecting their ability to induce antigen-specific CD4+ T-cell proliferation. Co-localization studies between PS particles and lysosomes using laser scanning confocal microscopy detected a significantly higher frequency of co-localized 20 nm particles as compared with their 1,000 nm counterparts. Neither size of PS particle caused lysosomal leakage, expression of endoplasmic reticulum stress gene markers, or changes in cytokines profiles. Conclusion: These data indicate that although supposedly inert PS nanoparticles did not induce DC activation or alteration in CD4+ T-cell stimulating capacity, 20 nm (but not 1,000 nm PS particles may reduce antigen degradation through interference in the lysosomal compartment. These findings emphasize the importance of performing in-depth analysis of DC function when developing novel approaches for immune modulation with nanoparticles. Keywords: polystyrene particles, nanoparticles, immune modulation, mouse dendritic cells, CD4+ T-cells

  17. The clinical spectrum and pathophysiology of skeletal complications in lysosomal storage disorders.

    Science.gov (United States)

    Clarke, Lorne A; Hollak, Carla E M

    2015-03-01

    Lysosomal storage disorders affect multiple organs including the skeleton. Disorders with prominent skeletal symptoms are type 1 and 3 Gaucher disease, the mucopolysaccharidoses, the glycoproteinoses and pycnodysostosis. Clinical manifestations range from asymptomatic radiographical evidence of bone pathology to overt bone crises (Gaucher), short stature with typical imaging features known as dysostosis multiplex (MPS), with spine and joint deformities (mucopolysaccharidoses, mucolipidosis), or osteopetrosis with pathological fractures (pynodysostosis). The pathophysiology of skeletal disease is only partially understood and involves direct substrate storage, inflammation and other complex alterations of cartilage and bone metabolism. Current treatments are enzyme replacement therapy, substrate reduction therapy and hematopoietic stem cell transplantation. However, effects of these interventions on skeletal disease manifestations are less well established and outcomes are highly dependent on disease burden at treatment initiation. It is now clear that adjunctive treatments that target skeletal disease are needed and should be part of future research agenda.

  18. Nuclear morphology and lysosomal stability of molluskan hemocytes as possible biomarkers of arsenic toxicity

    Energy Technology Data Exchange (ETDEWEB)

    Chakraborty, Sudipta [Post Graduate Department of Zoology, Parasitology and Medical Entomology Laboratory, Darjeeling Government College, Darjeeling (India); Ray, Sajal [Department of Zoology, Aquatic Toxicology Laboratory, University of Calcutta, Kolkata (India)

    2009-10-15

    The frequency of nuclear aberrations and neutral red retention time of hemocytes in the mollusk Lamellidens marginalis were recorded under exposure to sublethal concentrations of sodium arsenite in order to examine the sensitivity and effectiveness of these inexpensive assays for screening the toxicity of As{sup 3+}in a freshwater ecosystem. A dose and time dependent increase in the density of micronucleated and binucleated hemocytes and gill cells was indicative of the pronounced genotoxic effect of arsenic on this animal. The disruption of intrahemocyte homeostasis imposed by this natural toxicant was evident from a dose and time dependent reduction in the lysosomal stability of the hemocytes of the animal. The tested parameters are indicative of arsenic toxicity in L. marginalis in the freshwater systems of the arsenic affected geographical areas of West Bengal, India. (Abstract Copyright [2009], Wiley Periodicals, Inc.)

  19. Cathepsin B modulates lysosomal biogenesis and host defense against Francisella novicida infection.

    Science.gov (United States)

    Qi, Xiaopeng; Man, Si Ming; Malireddi, R K Subbarao; Karki, Rajendra; Lupfer, Christopher; Gurung, Prajwal; Neale, Geoffrey; Guy, Clifford S; Lamkanfi, Mohamed; Kanneganti, Thirumala-Devi

    2016-09-19

    Lysosomal cathepsins regulate an exquisite range of biological functions, and their deregulation is associated with inflammatory, metabolic, and degenerative diseases in humans. In this study, we identified a key cell-intrinsic role for cathepsin B as a negative feedback regulator of lysosomal biogenesis and autophagy. Mice and macrophages lacking cathepsin B activity had increased resistance to the cytosolic bacterial pathogen Francisella novicida Genetic deletion or pharmacological inhibition of cathepsin B down-regulated mechanistic target of rapamycin activity and prevented cleavage of the lysosomal calcium channel TRPML1. These events drove transcription of lysosomal and autophagy genes via transcription factor EB, which increased lysosomal biogenesis and activation of autophagy initiation kinase ULK1 for clearance of the bacteria. Our results identified a fundamental biological function of cathepsin B in providing a checkpoint for homeostatic maintenance of lysosome populations and basic recycling functions in the cell.

  20. TFEB and TFE3: Linking Lysosomes to Cellular Adaptation to Stress.

    Science.gov (United States)

    Raben, Nina; Puertollano, Rosa

    2016-10-06

    In recent years, our vision of lysosomes has drastically changed. Formerly considered to be mere degradative compartments, they are now recognized as key players in many cellular processes. The ability of lysosomes to respond to different stimuli revealed a complex and coordinated regulation of lysosomal gene expression. This review discusses the participation of the transcription factors TFEB and TFE3 in the regulation of lysosomal function and biogenesis, as well as the role of the lysosomal pathway in cellular adaptation to a variety of stress conditions, including nutrient deprivation, mitochondrial dysfunction, protein misfolding, and pathogen infection. We also describe how cancer cells make use of TFEB and TFE3 to promote their own survival and highlight the potential of these transcription factors as therapeutic targets for the treatment of neurological and lysosomal diseases.

  1. A TRP Channel in the Lysosome Regulates Large Particle Phagocytosis via Focal Exocytosis

    OpenAIRE

    2013-01-01

    Phagocytosis of large extracellular particles such as apoptotic bodies requires delivery of the intracellular endosomal and lysosomal membranes to form plasmalemmal pseudopods. Here we identified Mucolipin TRP channel 1 (TRPML1) as the key lysosomal Ca2+ channel regulating focal exocytosis and phagosome biogenesis. Both particle ingestion and lysosomal exocytosis are inhibited by synthetic TRPML1 blockers, and are defective in macrophages isolated from TRPML1 knockout mice. Furthermore, TRPML...

  2. Disruption of Lysosome Function Promotes Tumor Growth and Metastasis in Drosophila *

    OpenAIRE

    Chi, Congwu; Zhu, Huanhu; Han,Min; Zhuang, Yuan; Wu, Xiaohui; Xu, Tian

    2010-01-01

    Lysosome function is essential to many physiological processes. It has been suggested that deregulation of lysosome function could contribute to cancer. Through a genetic screen in Drosophila, we have discovered that mutations disrupting lysosomal degradation pathway components contribute to tumor development and progression. Loss-of-function mutations in the Class C vacuolar protein sorting (VPS) gene, deep orange (dor), dramatically promote tumor overgrowth and invasion of the RasV12 cells....

  3. Sub-lethal oxidative stress induces lysosome biogenesis via a lysosomal membrane permeabilization-cathepsin-caspase 3-transcription factor EB-dependent pathway.

    Science.gov (United States)

    Leow, San Min; Chua, Shu Xian Serene; Venkatachalam, Gireedhar; Shen, Liang; Luo, Le; Clement, Marie-Veronique

    2016-12-18

    Here we provide evidence to link sub-lethal oxidative stress to lysosomal biogenesis. Exposure of cells to sub-lethal concentrations of exogenously added hydrogen peroxide resulted in cytosol to nuclear translocation of the Transcription Factor EB (TFEB), the master controller of lysosome biogenesis and function. Nuclear translocation of TFEB was dependent upon the activation of a cathepsin-caspase 3 signaling pathway, downstream of a lysosomal membrane permeabilization and accompanied by a significant increase in lysosome numbers as well as induction of TFEB dependent lysosome-associated genes expression such as Ctsl, Lamp2 and its spliced variant Lamp2a, Neu1and Ctsb and Sqstm1 and Atg9b. The effects of sub-lethal oxidative stress on lysosomal gene expression and biogenesis were rescued upon gene silencing of caspase 3 and TFEB. Notably, caspase 3 activation was not associated with phenotypic hallmarks of apoptosis, evidenced by the absence of caspase 3 substrate cleavage, such as PARP, Lamin A/C or gelsolin. Taken together, these data demonstrate for the first time an unexpected and non-canonical role of a cathepsin-caspase 3 axis in the nuclear translocation of TFEB leading to lysosomes biogenesis under conditions of sub-lethal oxidative stress.

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

    Science.gov (United States)

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

    2014-05-22

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

  5. Cathepsin inhibition-induced lysosomal dysfunction enhances pancreatic beta-cell apoptosis in high glucose.

    Science.gov (United States)

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

    2015-01-01

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

  6. hLGDB: a database of human lysosomal genes and their regulation.

    Science.gov (United States)

    Brozzi, Alessandro; Urbanelli, Lorena; Germain, Pierre Luc; Magini, Alessandro; Emiliani, Carla

    2013-01-01

    Lysosomes are cytoplasmic organelles present in almost all eukaryotic cells, which play a fundamental role in key aspects of cellular homeostasis such as membrane repair, autophagy, endocitosis and protein metabolism. The characterization of the genes and enzymes constituting the lysosome represents a central issue to be addressed toward a better understanding of the biology of this organelle. In humans, mutations that cause lysosomal enzyme deficiencies result in >50 different disorders and severe pathologies. So far, many experimental efforts using different methodologies have been carried out to identity lysosomal genes. The Human Lysosome Gene Database (hLGDB) is the first resource that provides a comprehensive and accessible census of the human genes belonging to the lysosomal system. This database was developed by collecting and annotating gene lists from many different sources. References to the studies that have identified each gene are provided together with cross databases gene related information. Special attention has been given to the regulation of the genes through microRNAs and the transcription factor EB. The hLGDB can be easily queried to retrieve, combine and analyze information on different lists of lysosomal genes and their regulation by microRNA (binding sites predicted by five different algorithms). The hLGDB is an open access dynamic project that will permit in the future to collapse in a unique publicly accessible resource all the available biological information about lysosome genes and their regulation. Database URL: http://lysosome.unipg.it/.

  7. Lysosomes serve as a platform for hepatitis A virus particle maturation and nonlytic release.

    Science.gov (United States)

    Seggewiß, Nicole; Paulmann, Dajana; Dotzauer, Andreas

    2016-01-01

    Early studies on hepatitis A virus (HAV) in cell culture demonstrated the inclusion of several viral particles in an intracellular lipid-bilayer membrane. However, the origin of these virus-associated membranes and the mechanism for the non-lytic release of HAV into bile are still unknown. Analyzing the association of this virus with cell organelles, we found that newly synthesized HAV particles accumulate in lysosomal organelles and that lysosomal enzymes are involved in the maturation cleavage of the virion. Furthermore, by inhibiting the processes of fusion of lysosomes with the plasma membrane, we found that the nonlytic release of HAV from infected cells occurs via lysosome-related organelles.

  8. Lipid Storage Disorders Block Lysosomal Trafficking By Inhibiting TRP Channel and Calcium Release

    OpenAIRE

    2012-01-01

    Lysosomal lipid accumulation, defects in membrane trafficking, and altered Ca2+ homeostasis are common features in many lysosomal storage diseases. Mucolipin TRP channel 1 (TRPML1) is the principle Ca2+ channel in the lysosome. Here we show that TRPML1-mediated lysosomal Ca2+ release, measured using a genetically-encoded Ca2+ indicator (GCaMP3) attached directly to TRPML1 and elicited by a potent membrane-permeable synthetic agonist, is dramatically reduced in Niemann-Pick (NP) disease cells....

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

    Directory of Open Access Journals (Sweden)

    Roger Gilabert-Oriol

    2014-05-01

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

  10. Impact of high glucose and AGEs on cultured kidney-derived cells. Effects on cell viability, lysosomal enzymes and effectors of cell signaling pathways.

    Science.gov (United States)

    Peres, Giovani B; Schor, Nestor; Michelacci, Yara M

    2017-04-01

    We have previously reported decreased expression and activities of lysosomal cathepsins B and L in diabetic kidney. Relevant morphological changes were observed in proximal tubules, suggesting that these cells are implicated in the early stages of the disease. The aim of the present study was to investigate the mechanisms that lead to these changes. The effects of high glucose (HG) and advanced glycation end products (AGEs) on cell viability, lysosomal enzymes and other effectors of cell signaling of cultured kidney cells were studied. HG increased viable mesangial cells (ihMC) in 48 h, while epithelial tubular cells were not affected (LLC-PK1 and MDCK). In contrast, the number of viable cells was markedly decreased, for all cell lines, by AGE-BSA. Concerning lysosomal enzymes, the main cysteine-protease expressed by these cells was cathepsin B, and its concentration was much higher in epithelial than in mesangial cells. Exposure to HG had no effect on the cathepsin B activity, but AGE-BSA caused a marked decrease in LLC-PK1, and increased the enzyme activities in the other cell lines. The levels of nitric oxide (NO) was increased by AGE-BSA in all cell lines, suggesting oxidative stress, and Western blotting has shown that, among the investigated proteins, cathepsin B, mTOR and transcription factor EB (TFEB) were the most significantly affected by exposure to AGE-BSA. As mTOR induces anabolism and inhibits autophagy, and TFEB is a master transcription factor for lysosomal enzymes, it is possible that this pathway plays a role in the inhibition of lysosomal enzymes in proximal tubule cells.

  11. Megalin/Cubulin-Lysosome-mediated Albumin Reabsorption Is Involved in the Tubular Cell Activation of NLRP3 Inflammasome and Tubulointerstitial Inflammation.

    Science.gov (United States)

    Liu, Dan; Wen, Yi; Tang, Tao-Tao; Lv, Lin-Li; Tang, Ri-Ning; Liu, Hong; Ma, Kun-Ling; Crowley, Steve D; Liu, Bi-Cheng

    2015-07-17

    Albuminuria contributes to the development and progression of chronic kidney disease by inducing tubulointerstitial inflammation (TI) and fibrosis. However, the exact mechanisms of TI in response to albuminuria are unresolved. We previously demonstrated that NLRP3 and inflammasomes mediate albumin-induced lesions in tubular cells. Here, we further investigated the role of endocytic receptors and lysosome rupture in NLRP3 inflammasome activation. A murine proteinuric nephropathy model was induced by albumin overload as described previously. The priming and activation signals for inflammasome complex formation were evoked simultaneously by albumin excess in tubular epithelial cells. The former signal was dependent on a albumin-triggered NF-κB pathway activation. This process is mediated by the endocytic receptor, megalin and cubilin. However, the silencing of megalin or cubilin inhibited the albumin-induced NLRP3 signal. Notably, subsequent lysosome rupture and the corresponding release of lysosomal hydrolases, especially cathepsin B, were observed in tubular epithelial cells exposed to albumin. Cathepsin B release and distribution are essential for NLRP3 signal activation, and inhibitors of cathepsin B suppressed the NLRP3 signal in tubular epithelial cells. Taken together, our findings suggest that megalin/cubilin and lysosome rupture are involved in albumin-triggered tubular injury and TI. This study provides novel insights into albuminuria-induced TI and implicates the active control of albuminuria as a critical strategy to halt the progression of chronic kidney disease.

  12. Urothelial endocytic vesicle recycling and lysosomal degradative pathway regulated by lipid membrane composition.

    Science.gov (United States)

    Grasso, E J; Calderón, R O

    2013-02-01

    The urothelium, a specialized epithelium that covers the mucosa cell surface of the urinary bladder, undergoes dramatic morphological changes during the micturition cycle that involve a membrane apical traffic. This traffic was first described as a lysosomal pathway, in addition to the known endocytosis/exocytosis membrane recycling. In an attempt to understand the role of membrane lipid composition in those effects, we previously described the lipid-dependent leakage of the endocytosed vesicle content. In this work, we demonstrated clear differences in the traffic of both the fluid probe and the membrane-bound probe in urothelial umbrella cells by using spectrofluorometry and/or confocal and epifluorescence microscopy. Different membrane lipid compositions were established by using three diet formulae enriched in oleic acid, linoleic acid and a commercial formula. Between three and five animals for each dietary treatment were used for each analysis. The decreased endocytosis of both fluid and membrane-bound probes (approximately 32 and 49 % lower, respectively) in oleic acid-derived umbrella cells was concomitant with an increased recycling (approximately 4.0 and 3.7 times, respectively) and diminished sorting to the lysosome (approximately 23 and 37 %, respectively) when compared with the control umbrella cells. The higher intravesicular pH and the impairment of the lysosomal pathway of oleic acid diet-derived vesicles compared to linoleic acid diet-derived vesicles and control diet-derived vesicles correlate with our findings of a lower V-ATPase activity previously reported. We integrated the results obtained in the present and previous work to determine the sorting of endocytosed material (fluid and membrane-bound probes) into the different cell compartments. Finally, the weighted average effect of the individual alterations on the intracellular distribution was evaluated. The results shown in this work add evidences for the modulatory role of the membrane

  13. Crystal structure of the conserved domain of the DC lysosomal associated membrane protein: implications for the lysosomal glycocalyx

    OpenAIRE

    Wilke Sonja; Krausze Joern; Büssow Konrad

    2012-01-01

    Abstract Background The family of lysosome-associated membrane proteins (LAMP) comprises the multifunctional, ubiquitous LAMP-1 and LAMP-2, and the cell type-specific proteins DC-LAMP (LAMP-3), BAD-LAMP (UNC-46, C20orf103) and macrosialin (CD68). LAMPs have been implicated in a multitude of cellular processes, including phagocytosis, autophagy, lipid transport and aging. LAMP-2 isoform A acts as a receptor in chaperone-mediated autophagy. LAMP-2 deficiency causes the fatal Danon disease. The ...

  14. Noxa couples lysosomal membrane permeabilization and apoptosis during oxidative stress.

    Science.gov (United States)

    Eno, Colins O; Zhao, Guoping; Venkatanarayan, Avinashnarayan; Wang, Bing; Flores, Elsa R; Li, Chi

    2013-12-01

    The exact roles of lysosomal membrane permeabilization (LMP) in oxidative stress-triggered apoptosis are not completely understood. Here, we first studied the temporal relation between LMP and mitochondrial outer membrane permeabilization (MOMP) during the initial stage of apoptosis caused by the oxidative stress inducer H2O2. Despite its essential role in mediating apoptosis, the expression of the BH3-only Bcl-2 protein Noxa was dispensable for LMP. In contrast, MOMP was dependent on Noxa expression and occurred downstream of LMP. When lysosomal membranes were stabilized by the iron-chelating agent desferrioxamine, H2O2-induced increase in DNA damage, Noxa expression, and subsequent apoptosis were abolished by the inhibition of LMP. Importantly, LMP-induced Noxa expression increase was mediated by p53 and seems to be a unique feature of apoptosis caused by oxidative stress. Finally, exogenous iron loading recapitulated the effects of H2O2 on the expression of BH3-only Bcl-2 proteins. Overall, these data reveal a Noxa-mediated signaling pathway that couples LMP with MOMP and ultimate apoptosis during oxidative stress.

  15. Klebsiella pneumoniae survives within macrophages by avoiding delivery to lysosomes.

    Science.gov (United States)

    Cano, Victoria; March, Catalina; Insua, Jose Luis; Aguiló, Nacho; Llobet, Enrique; Moranta, David; Regueiro, Verónica; Brennan, Gerard P; Millán-Lou, Maria Isabel; Martín, Carlos; Garmendia, Junkal; Bengoechea, José A

    2015-11-01

    Klebsiella pneumoniae is an important cause of community-acquired and nosocomial pneumonia. Evidence indicates that Klebsiella might be able to persist intracellularly within a vacuolar compartment. This study was designed to investigate the interaction between Klebsiella and macrophages. Engulfment of K. pneumoniae was dependent on host cytoskeleton, cell plasma membrane lipid rafts and the activation of phosphoinositide 3-kinase (PI3K). Microscopy studies revealed that K. pneumoniae resides within a vacuolar compartment, the Klebsiella-containing vacuole (KCV), which traffics within vacuoles associated with the endocytic pathway. In contrast to UV-killed bacteria, the majority of live bacteria did not co-localize with markers of the lysosomal compartment. Our data suggest that K. pneumoniae triggers a programmed cell death in macrophages displaying features of apoptosis. Our efforts to identify the mechanism(s) whereby K. pneumoniae prevents the fusion of the lysosomes to the KCV uncovered the central role of the PI3K-Akt-Rab14 axis to control the phagosome maturation. Our data revealed that the capsule is dispensable for Klebsiella intracellular survival if bacteria were not opsonized. Furthermore, the environment found by Klebsiella within the KCV triggered the down-regulation of the expression of cps. Altogether, this study proves evidence that K. pneumoniae survives killing by macrophages by manipulating phagosome maturation that may contribute to Klebsiella pathogenesis.

  16. From Lysosomal Storage Diseases to NKT Cell Activation and Back

    Science.gov (United States)

    Pereira, Cátia S.; Ribeiro, Helena; Macedo, M. Fatima

    2017-01-01

    Lysosomal storage diseases (LSDs) are inherited metabolic disorders characterized by the accumulation of different types of substrates in the lysosome. With a multisystemic involvement, LSDs often present a very broad clinical spectrum. In many LSDs, alterations of the immune system were described. Special emphasis was given to Natural Killer T (NKT) cells, a population of lipid-specific T cells that is activated by lipid antigens bound to CD1d (cluster of differentiation 1 d) molecules at the surface of antigen-presenting cells. These cells have important functions in cancer, infection, and autoimmunity and were altered in a variety of LSDs’ mouse models. In some cases, the observed decrease was attributed to defects in either lipid antigen availability, trafficking, processing, or loading in CD1d. Here, we review the current knowledge about NKT cells in the context of LSDs, including the alterations detected, the proposed mechanisms to explain these defects, and the relevance of these findings for disease pathology. Furthermore, the effect of enzyme replacement therapy on NKT cells is also discussed. PMID:28245613

  17. Frustrated phagocytosis on micro-patterned immune complexes to characterize lysosome movements in live macrophages.

    Directory of Open Access Journals (Sweden)

    Arnaud M. Labrousse

    2011-10-01

    Full Text Available Lysosome mobilization is a key cellular process in phagocytes for bactericidal activities and trans-matrix migration. The molecular mechanisms that regulate lysosome mobilization are still poorly known. Lysosomes are hard to track as they move towards phagosomes throughout the cell volume. In order to anticipate cell regions where lysosomes are recruited to, human and RAW264.7 macrophages were seeded on surfaces that were micro-patterned with immune complexes (ICs as 4 µm-side squares. Distances between IC patterns were adapted to optimize cell spreading in order to constrain lysosome movements mostly in 2 dimensions. Fc receptors triggered local frustrated phagocytosis, frustrated phagosomes appeared as rings of F-actin dots around the IC patterns as early as 5 minutes after cells made contact with the substratum. Frustrated phagosomes recruited actin-associated proteins (vinculin, paxillin and gelsolin. The fusion of lysosomes with frustrated phagosomes was shown by the release of beta-hexosaminidase and the recruitment of Lamp-1 to frustrated phagosomes. Lysosomes of RAW264.7 macrophages were labeled with cathepsinD-mCherry to visualize their movements towards frustrated phagosomes. Lysosomes saltatory movements were markedly slowed down compared to cells layered on non-opsonized patterns. In addition, the linearity of the trajectories and the frequency and duration of contacts of lysosomes with frustrated phagosomes were measured.¬¬¬¬¬¬¬¬ Using PP2 we showed that instant velocity, pauses and frequency of lysosome/phagosome contacts were at least in part dependent on Src tyrosine kinases. This experimental set-up is the first step towards deciphering molecular mechanisms which are involved in lysosome movements in the cytoplasm (directionality, docking and fusion using RNA interference, pharmacological inhibition or mutant expression.

  18. Preubiquitinated chimeric ErbB2 is constitutively endocytosed and subsequently degraded in lysosomes

    Energy Technology Data Exchange (ETDEWEB)

    Vuong, Tram Thu [Institute of Clinical Medicine, University of Oslo, Rikshospitalet, 0027 Oslo (Norway); Berger, Christian [Department of Pathology, Oslo University Hospital, Rikshospitalet, P.O. Box 4950 Nydalen, 0424 Oslo (Norway); Bertelsen, Vibeke; Rødland, Marianne Skeie [Institute of Clinical Medicine, University of Oslo, Rikshospitalet, 0027 Oslo (Norway); Stang, Espen [Department of Pathology, Oslo University Hospital, Rikshospitalet, P.O. Box 4950 Nydalen, 0424 Oslo (Norway); Madshus, Inger Helene, E-mail: i.h.madshus@medisin.uio.no [Institute of Clinical Medicine, University of Oslo, Rikshospitalet, 0027 Oslo (Norway); Department of Pathology, Oslo University Hospital, Rikshospitalet, P.O. Box 4950 Nydalen, 0424 Oslo (Norway)

    2013-02-01

    The oncoprotein ErbB2 is endocytosis-deficient, probably due to its interaction with Heat shock protein 90. We previously demonstrated that clathrin-dependent endocytosis of ErbB2 is induced upon incubation of cells with Ansamycin derivatives, such as geldanamycin and its derivative 17-AAG. Furthermore, we have previously demonstrated that a preubiquitinated chimeric EGFR (EGFR-Ub{sub 4}) is constitutively endocytosed in a clathrin-dependent manner. We now demonstrate that also an ErbB2-Ub{sub 4} chimera is endocytosed constitutively and clathrin-dependently. Upon expression, the ErbB2-Ub{sub 4} was further ubiquitinated, and by Western blotting, we demonstrated the formation of both Lys48-linked and Lys63-linked polyubiquitin chains. ErbB2-Ub{sub 4} was constitutively internalized and eventually sorted to late endosomes and lysosomes where the fusion protein was degraded. ErbB2-Ub{sub 4} was not cleaved prior to internalization. Interestingly, over-expression of Ubiquitin Interaction Motif-containing dominant negative fragments of the clathrin adaptor proteins epsin1 and Eps15 negatively affected endocytosis of ErbB2. Altogether, this argues that ubiquitination is sufficient to induce clathrin-mediated endocytosis and lysosomal degradation of the otherwise plasma membrane localized ErbB2. Also, it appears that C-terminal cleavage is not required for endocytosis. -- Highlights: ► A chimera containing ErbB2 and a tetra-Ubiquitin chain internalizes constitutively. ► Receptor fragmentation is not required for endocytosis of ErbB2. ► Ubiquitination is sufficient to induce endocytosis and degradation of ErbB2. ► ErbB2-Ub4 is internalized clathrin-dependently.

  19. Targeted Polymeric Nanoparticles for Brain Delivery of High Molecular Weight Molecules in Lysosomal Storage Disorders.

    Directory of Open Access Journals (Sweden)

    Marika Salvalaio

    Full Text Available Lysosomal Storage Disorders (LSDs are a group of metabolic syndromes, each one due to the deficit of one lysosomal enzyme. Many LSDs affect most of the organ systems and overall about 75% of the patients present neurological impairment. Enzyme Replacement Therapy, although determining some systemic clinical improvements, is ineffective on the CNS disease, due to enzymes' inability to cross the blood-brain barrier (BBB. With the aim to deliver the therapeutic enzymes across the BBB, we here assayed biodegradable and biocompatible PLGA-nanoparticles (NPs in two murine models for LSDs, Mucopolysaccharidosis type I and II (MPS I and MPS II. PLGA-NPs were modified with a 7-aminoacid glycopeptide (g7, yet demonstrated to be able to deliver low molecular weight (MW molecules across the BBB in rodents. We specifically investigated, for the first time, the g7-NPs ability to transfer a model drug (FITC-albumin with a high MW, comparable to the enzymes to be delivered for LSDs brain therapy. In vivo experiments, conducted on wild-type mice and knockout mouse models for MPS I and II, also included a whole series of control injections to obtain a broad preliminary view of the procedure efficiency. Results clearly showed efficient BBB crossing of albumin in all injected mice, underlying the ability of NPs to deliver high MW molecules to the brain. These results encourage successful experiments with enzyme-loaded g7-NPs to deliver sufficient amounts of the drug to the brain district on LSDs, where exerting a corrective effect on the pathological phenotype.

  20. Presenilin 1 regulates epidermal growth factor receptor turnover and signaling in the endosomal-lysosomal pathway.

    Science.gov (United States)

    Repetto, Emanuela; Yoon, Il-Sang; Zheng, Hui; Kang, David E

    2007-10-26

    Mutations in the gene encoding presenilin 1 (PS1) cause the most aggressive form of early-onset familial Alzheimer disease. In addition to its well established role in Abeta production and Notch proteolysis, PS1 has been shown to mediate other physiological activities, such as regulation of the Wnt/beta-catenin signaling pathway, modulation of phosphatidylinositol 3-kinase/Akt and MEK/ERK signaling, and trafficking of select membrane proteins and/or intracellular vesicles. In this study, we present evidence that PS1 is a critical regulator of a key signaling receptor tyrosine kinase, epidermal growth factor receptor (EGFR). Specifically, EGFR levels were robustly increased in fibroblasts deficient in both PS1 and PS2 (PS(-/-)) due to delayed turnover of EGFR protein. Stable transfection of wild-type PS1 but not PS2 corrected EGFR to levels comparable to PS(+/+) cells, while FAD PS1 mutations showed partial loss of activity. The C-terminal fragment of PS1 was sufficient to fully reduce EGFR levels. In addition, the rapid ligand-induced degradation of EGFR was markedly delayed in PS(-/-) cells, resulting in prolonged signal activation. Despite the defective turnover of EGFR, ligand-induced autophosphorylation, ubiquitination, and endocytosis of EGFR were not affected by the lack of PS1. Instead, the trafficking of EGFR from early endosomes to lysosomes was severely delayed by PS1 deficiency. Elevation of EGFR was also seen in brains of adult mice conditionally ablated in PS1 and in skin tumors associated with the loss of PS1. These findings demonstrate a critical role of PS1 in the trafficking and turnover of EGFR and suggest potential pathogenic effects of elevated EGFR as well as perturbed endosomal-lysosomal trafficking in cell cycle control and Alzheimer disease.

  1. Ouabain-induced internalization and lysosomal degradation of the Na+/K+-ATPase.

    Science.gov (United States)

    Cherniavsky-Lev, Marina; Golani, Ofra; Karlish, Steven J D; Garty, Haim

    2014-01-10

    Internalization of the Na(+)/K(+)-ATPase (the Na(+) pump) has been studied in the human lung carcinoma cell line H1299 that expresses YFP-tagged α1 from its normal genomic localization. Both real-time imaging and surface biotinylation have demonstrated internalization of α1 induced by ≥100 nm ouabain which occurs in a time scale of hours. Unlike previous studies in other systems, the ouabain-induced internalization was insensitive to Src or PI3K inhibitors. Accumulation of α1 in the cells could be augmented by inhibition of lysosomal degradation but not by proteosomal inhibitors. In agreement, the internalized α1 could be colocalized with the lysosomal marker LAMP1 but not with Golgi or nuclear markers. In principle, internalization could be triggered by a conformational change of the ouabain-bound Na(+)/K(+)-ATPase molecule or more generally by the disruption of cation homeostasis (Na(+), K(+), Ca(2+)) due to the partial inhibition of active Na(+) and K(+) transport. Overexpression of ouabain-insensitive rat α1 failed to inhibit internalization of human α1 expressed in the same cells. In addition, incubating cells in a K(+)-free medium did not induce internalization of the pump or affect the response to ouabain. Thus, internalization is not the result of changes in the cellular cation balance but is likely to be triggered by a conformational change of the protein itself. In physiological conditions, internalization may serve to eliminate pumps that have been blocked by endogenous ouabain or other cardiac glycosides. This mechanism may be required due to the very slow dissociation of the ouabain·Na(+)/K(+)-ATPase complex.

  2. Autophagy-lysosomal pathway is involved in lipid degradation in rat liver.

    Science.gov (United States)

    Skop, V; Cahová, M; Papáčková, Z; Páleníčková, E; Daňková, H; Baranowski, M; Zabielski, P; Zdychová, J; Zídková, J; Kazdová, L

    2012-01-01

    We present data supporting the hypothesis that the lysosomal-autophagy pathway is involved in the degradation of intracellular triacylglycerols in the liver. In primary hepatocytes cultivated in the absence of exogenous fatty acids (FFA), both inhibition of autophagy flux (asparagine) or lysosomal activity (chloroquine) decreased secretion of VLDL (very low density lipoproteins) and formation of FFA oxidative products while the stimulation of autophagy by rapamycine increased some of these parameters. Effect of rapamycine was completely abolished by inactivation of lysosomes. Similarly, when autophagic activity was influenced by cultivating the hepatocytes in "starving" (amino-acid poor medium) or "fed" (serum-supplemented medium) conditions, VLDL secretion and FFA oxidation mirrored the changes in autophagy being higher in starvation and lower in fed state. Autophagy inhibition as well as lysosomal inactivation depressed FFA and DAG (diacylglycerol) formation in liver slices in vitro. In vivo, intensity of lysosomal lipid degradation depends on the formation of autophagolysosomes, i.e. structures bringing the substrate for degradation and lysosomal enzymes into contact. We demonstrated that lysosomal lipase (LAL) activity in liver autophagolysosomal fraction was up-regulated in fasting and down-regulated in fed state together with the increased translocation of LAL and LAMP2 proteins from lysosomal pool to this fraction. Changes in autophagy intensity (LC3-II/LC3-I ratio) followed a similar pattern.

  3. The phytoestrogen genistein modulates lysosomal metabolism and transcription factor EB (TFEB) activation.

    Science.gov (United States)

    Moskot, Marta; Montefusco, Sandro; Jakóbkiewicz-Banecka, Joanna; Mozolewski, Paweł; Węgrzyn, Alicja; Di Bernardo, Diego; Węgrzyn, Grzegorz; Medina, Diego L; Ballabio, Andrea; Gabig-Cimińska, Magdalena

    2014-06-13

    Genistein (5,7-dihydroxy-3-(4-hydroxyphenyl)-4H-1-benzopyran-4-one) has been previously proposed as a potential drug for use in substrate reduction therapy for mucopolysaccharidoses, a group of inherited metabolic diseases caused by mutations leading to inefficient degradation of glycosaminoglycans (GAGs) in lysosomes. It was demonstrated that this isoflavone can cross the blood-brain barrier, making it an especially desirable potential drug for the treatment of neurological symptoms present in most lysosomal storage diseases. So far, no comprehensive genomic analyses have been performed to elucidate the molecular mechanisms underlying the effect elicited by genistein. Therefore, the aim of this work was to identify the genistein-modulated gene network regulating GAG biosynthesis and degradation, taking into consideration the entire lysosomal metabolism. Our analyses identified over 60 genes with known roles in lysosomal biogenesis and/or function whose expression was enhanced by genistein. Moreover, 19 genes whose products are involved in both GAG synthesis and degradation pathways were found to be remarkably differentially regulated by genistein treatment. We found a regulatory network linking genistein-mediated control of transcription factor EB (TFEB) gene expression, TFEB nuclear translocation, and activation of TFEB-dependent lysosome biogenesis to lysosomal metabolism. Our data indicate that the molecular mechanism of genistein action involves not only impairment of GAG synthesis but more importantly lysosomal enhancement via TFEB. These findings contribute to explaining the beneficial effects of genistein in lysosomal storage diseases as well as envisage new therapeutic approaches to treat these devastating diseases.

  4. Protective effects of sinapic acid on lysosomal dysfunction in isoproterenol induced myocardial infarcted rats.

    Science.gov (United States)

    Roy, Subhro Jyoti; Stanely Mainzen Prince, Ponnian

    2012-11-01

    In the pathology of myocardial infarction, lysosomal lipid peroxidation and resulting enzyme release play an important role. We evaluated the protective effects of sinapic acid on lysosomal dysfunction in isoproterenol induced myocardial infarcted rats. Male Wistar rats were treated with sinapic acid (12 mg/kg body weight) orally daily for 10 days and isoproterenol (100 mg/kg body weight) was injected twice at an interval of 24 h (9th and 10th day). Then, lysosomal lipid peroxidation, lysosomal enzymes in serum, heart homogenate, lysosomal fraction and myocardial infarct size were measured. Isoproterenol induced myocardial infarcted rats showed a significant increase in serum creatine kinase-MB and lysosomal lipid peroxidation. The activities of β-glucuronidase, β-galactosidase, cathepsin-B and D were significantly increased in serum, heart and the activities of β-glucuronidase and cathepsin-D were significantly decreased in lysosomal fraction of myocardial infarcted rats. Pre-and-co-treatment with sinapic acid normalized all the biochemical parameters and reduced myocardial infarct size in myocardial infarcted rats. In vitro studies confirmed the free radical scavenging effects of sinapic acid. The possible mechanisms for the observed effects are attributed to sinapic acid's free radical scavenging and membrane stabilizing properties. Thus, sinapic acid has protective effects on lysosomal dysfunction in isoproterenol induced myocardial infarcted rats.

  5. The Octyl Ester of Ginsenoside Rh2 Induces Lysosomal Membrane Permeabilization via Bax Translocation.

    Science.gov (United States)

    Chen, Fang; Zhang, Bing; Sun, Yong; Xiong, Zeng-Xing; Peng, Han; Deng, Ze-Yuan; Hu, Jiang-Ning

    2016-04-25

    Ginsenoside Rh2 is a potential pharmacologically active metabolite of ginseng. Previously, we have reported that an octyl ester derivative of ginsenoside Rh2 (Rh2-O), has been confirmed to possess higher bioavailability and anticancer effect than Rh2 in vitro. In order to better assess the possibility that Rh2-O could be used as an anticancer compound, the underlying mechanism was investigated in this study. The present results revealed that lysosomal destabilization was involved in the early stage of cell apoptosis in HepG2 cells induced by Rh2-O. Rh2-O could induce an early lysosomal membrane permeabilization with the release of lysosomal protease cathepsins to the cytosol in HepG2 cells. The Cat B inhibitor (leu) and Cat D inhibitor (pepA) inhibited Rh2-O-induced HepG2 apoptosis as well as tBid production and Δφm depolarization, indicating that lysosomal permeabilization occurred upstream of mitochondrial dysfunction. In addition, Rh2-O induced a significant increase in the protein levels of DRAM1 and Bax (p lysosomes of HepG2 cells. Knockdown of Bax partially inhibited Rh2-O-induced Cat D release from lysosomes. Thus it was concluded that Rh2-O induced apoptosis of HepG2 cells through activation of the lysosomal-mitochondrial apoptotic pathway involving the translocation of Bax to the lysosome.

  6. Expression Pattern of Lysosomal Protective Protein/Cathepsin A: Implications for the analysis of hnman galactosialidosis

    NARCIS (Netherlands)

    R.J. Rottier (Robbert)

    1998-01-01

    textabstractThe lysosome represents a well characterized, membrane-contained intracellular digestive system. Iu this important organelle a battery of lysosomal hydro lases and accessory proteins work in concert on the step-wise conversion of macromolecular substrates into small biological building b

  7. Calpains mediate epithelial-cell death during mammary gland involution: mitochondria and lysosomal destabilization.

    Science.gov (United States)

    Arnandis, T; Ferrer-Vicens, I; García-Trevijano, E R; Miralles, V J; García, C; Torres, L; Viña, J R; Zaragozá, R

    2012-09-01

    Our aim was to elucidate the physiological role of calpains (CAPN) in mammary gland involution. Both CAPN-1 and -2 were induced after weaning and its activity increased in isolated mitochondria and lysosomes. CAPN activation within the mitochondria could trigger the release of cytochrome c and other pro-apoptotic factors, whereas in lysosomes it might be essential for tissue remodeling by releasing cathepsins into the cytosol. Immunohistochemical analysis localized CAPNs mainly at the luminal side of alveoli. During weaning, CAPNs translocate to the lysosomes processing membrane proteins. To identify these substrates, lysosomal fractions were treated with recombinant CAPN and cleaved products were identified by 2D-DIGE. The subunit b(2) of the v-type H(+) ATPase is proteolyzed and so is the lysosomal-associated membrane protein 2a (LAMP2a). Both proteins are also cleaved in vivo. Furthermore, LAMP2a cleavage was confirmed in vitro by addition of CAPNs to isolated lysosomes and several CAPN inhibitors prevented it. Finally, in vivo inhibition of CAPN1 in 72-h-weaned mice decreased LAMP2a cleavage. Indeed, calpeptin-treated mice showed a substantial delay in tissue remodeling and involution of the mammary gland. These results suggest that CAPNs are responsible for mitochondrial and lysosomal membrane permeabilization, supporting the idea that lysosomal-mediated cell death is a new hallmark of mammary gland involution.

  8. Lysosomal cholesterol accumulation : driver on the road to inflammation during atherosclerosis and non-alcoholic steatohepatitis

    NARCIS (Netherlands)

    Hendrikx, T.; Walenbergh, S. M. A.; Hofker, M. H.; Shiri-Sverdlov, R.

    2014-01-01

    Many studies show an association between the accumulation of cholesterol inside lysosomes and the progression towards inflammatory disease states that are closely related to obesity. While in the past, the knowledge regarding lysosomal cholesterol accumulation was limited to its association with pla

  9. A TRP channel in the lysosome regulates large particle phagocytosis via focal exocytosis.

    Science.gov (United States)

    Samie, Mohammad; Wang, Xiang; Zhang, Xiaoli; Goschka, Andrew; Li, Xinran; Cheng, Xiping; Gregg, Evan; Azar, Marlene; Zhuo, Yue; Garrity, Abigail G; Gao, Qiong; Slaugenhaupt, Susan; Pickel, Jim; Zolov, Sergey N; Weisman, Lois S; Lenk, Guy M; Titus, Steve; Bryant-Genevier, Marthe; Southall, Noel; Juan, Marugan; Ferrer, Marc; Xu, Haoxing

    2013-09-16

    Phagocytosis of large extracellular particles such as apoptotic bodies requires delivery of the intracellular endosomal and lysosomal membranes to form plasmalemmal pseudopods. Here, we identified mucolipin TRP channel 1 (TRPML1) as the key lysosomal Ca2+ channel regulating focal exocytosis and phagosome biogenesis. Both particle ingestion and lysosomal exocytosis are inhibited by synthetic TRPML1 blockers and are defective in macrophages isolated from TRPML1 knockout mice. Furthermore, TRPML1 overexpression and TRPML1 agonists facilitate both lysosomal exocytosis and particle uptake. Using time-lapse confocal imaging and direct patch clamping of phagosomal membranes, we found that particle binding induces lysosomal PI(3,5)P2 elevation to trigger TRPML1-mediated lysosomal Ca2+ release specifically at the site of uptake, rapidly delivering TRPML1-resident lysosomal membranes to nascent phagosomes via lysosomal exocytosis. Thus phagocytic ingestion of large particles activates a phosphoinositide- and Ca2+-dependent exocytosis pathway to provide membranes necessary for pseudopod extension, leading to clearance of senescent and apoptotic cells in vivo.

  10. 1,25-Dihydroxyvitamin D3-mediated intestinal calcium transport. Biochemical identification of lysosomes containing calcium and calcium-binding protein (calbindin-D28K).

    Science.gov (United States)

    Nemere, I; Leathers, V; Norman, A W

    1986-12-05

    A variety of intestinal cell organelles and proteins have been proposed to mediate 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3)-stimulated calcium absorption. In the present study biochemical analyses were undertaken to determine the subcellular localization of 45Ca after calcium transport in vivo in ligated duodenal loops of vitamin D-deficient chicks injected with 1.3 nmol of 1,25-(OH)2D3 or vehicle 15 h prior to experimentation. Separation of Golgi, mitochondria, basal lateral membrane, and lysosome fractions in the epithelial homogenates was achieved by differential sedimentation followed by centrifugation in Percoll gradients and evaluation of appropriate marker enzyme activities. Both vitamin D-deficient and 1,25-(OH)2D3-treated chicks had the highest levels of 45Ca-specific activity in lysosomal fractions. The lysosomes were also the only organelles to exhibit a 1,25-(OH)2D3-mediated difference in calcium content, increasing to 138% of controls. Lysosomes prepared from 1,25-(OH)2D3-treated chicks also contained the greatest levels of immunoreactive calbindin-D28k (calcium-binding protein). Chloroquine, a drug known to interfere with lysosomal function, was tested and found to inhibit 1,25-(OH)2D3-stimulated intestinal calcium absorption. Neither 1,25-(OH)2D3 nor chloroquine affected [3H]2O transport. In additional experiments, microsomal membranes (105,000 X g pellets) were subjected to gradient centrifugation. The highest levels of 45Ca-specific activity and calcium-binding protein in material from 1,25-(OH)2D3-treated chicks were found in fractions denser than endoplasmic reticulum and may represent endocytic vesicles. In studies on intestinal mucosa of 1,25-(OH)2D3-treated birds fractionated after 30 min of exposure to lumenal Ca2+ or Ca2+ plus chloroquine, 45Ca was found to accumulate in lysosomes and putative endocytic vesicles, relative to controls. A mechanism involving vesicular flow is proposed for 1,25-(OH)2D3-mediated intestinal calcium transport

  11. Ubiquitin trafficking to the lysosome: keeping the house tidy and getting rid of unwanted guests.

    Science.gov (United States)

    Purdy, Georgiana E; Russell, David G

    2007-01-01

    Bacterial killing by autophagic delivery to the lysosomal compartment has been shown for Mycobacteria, Streptococcus, Shigella, Legionella and Salmonella, indicating an important role for this conserved trafficking pathway for the control of intracellular bacterial pathogens.(1-5) In a recent study we found that solubilized lysosomes isolated from bone marrow-derived macrophages had potent antibacterial properties against M. tuberculosis and M. smegmatis that were associated with ubiquitin and ubiquitin-derived peptides. We propose that ubiquitinated proteins are delivered to the lysosomal compartment, where degradation by lysosomal proteinases generates ubiquitin-derived peptides with antimycobacterial properties. This surprising finding provokes a number of questions regarding the nature and trafficking of ubiquitin and ubiquitin-modified proteins in mammalian cells. We discuss the possible role(s) that the multivesicular body (MVB), the late endosome and the autophagosome may play in trafficking of ubiquitinated proteins to the lysosome.

  12. Metallothionein-3 regulates lysosomal function in cultured astrocytes under both normal and oxidative conditions.

    Science.gov (United States)

    Lee, Sook-Jeong; Park, Mi-Ha; Kim, Hyun-Jae; Koh, Jae-Young

    2010-08-01

    Cellular zinc plays a key role in lysosomal change and cell death in neurons and astrocytes under oxidative stress. Here, using astrocytes lacking metallothionein-3 (MT3), a potential source of labile zinc in the brain, we studied the role of MT3 in oxidative stress responses. H(2)O(2) induced a large increase in labile zinc in wild-type (WT) astrocytes, but stimulated only a modest rise in MT3-null astrocytes. In addition, H(2)O(2)-induced lysosomal membrane permeabilization (LMP) and cell death were comparably attenuated in MT3-null astrocytes. Expression and glycosylation of Lamp1 (lysosome-associated membrane protein 1) and Lamp2 were increased in MT3-null astrocytes, and the activities of several lysosomal enzymes were significantly reduced, indicating an effect of MT3 on lysosomal components. Consistent with lysosomal dysfunction in MT3-null cells, the level of LC3-II (microtubule-associated protein 1 light chain 3), a marker of early autophagy, was increased by oxidative stress in WT astrocytes, but not in MT3-null cells. Similar changes in Lamp1, LC3, and cathepsin-D were induced by the lysosomal inhibitors bafilomycin A1, chloroquine, and monensin, indicating that lysosomal dysfunction may lie upstream of changes observed in MT3-null astrocytes. Consistent with this idea, lysosomal accumulation of cholesterol and lipofuscin were augmented in MT3-null astrocytes. Similar to the results seen in MT3-null cells, MT3 knockdown by siRNA inhibited oxidative stress-induced increases in zinc and LMP. These results indicate that MT3 may play a key role in normal lysosomal function in cultured astrocytes.

  13. Mild MPP(+) exposure impairs autophagic degradation through a novel lysosomal acidity-independent mechanism.

    Science.gov (United States)

    Miyara, Masatsugu; Kotake, Yaichiro; Tokunaga, Wataru; Sanoh, Seigo; Ohta, Shigeru

    2016-10-01

    Parkinson's disease (PD) is the second most common neurodegenerative disorder, but its underlying cause remains unknown. Although recent studies using PD-related neurotoxin MPP(+) suggest autophagy involvement in the pathogenesis of PD, the effect of MPP(+) on autophagic processes under mild exposure, which mimics the slow progressive nature of PD, remains largely unclear. We examined the effect of mild MPP(+) exposure (10 and 200 μM for 48 h), which induces a more slowly developing cell death, on autophagic processes and the mechanistic differences with acute MPP(+) toxicity (2.5 and 5 mM for 24 h). In SH-SY5Y cells, mild MPP(+) exposure predominantly inhibited autophagosome degradation, whereas acute MPP(+) exposure inhibited both autophagosome degradation and basal autophagy. Mild MPP(+) exposure reduced lysosomal hydrolase cathepsin D activity without changing lysosomal acidity, whereas acute exposure decreased lysosomal density. Lysosome biogenesis enhancers trehalose and rapamycin partially alleviated mild MPP(+) exposure induced impaired autophagosome degradation and cell death, but did not prevent the pathogenic response to acute MPP(+) exposure, suggesting irreversible lysosomal damage. We demonstrated impaired autophagic degradation by MPP(+) exposure and mechanistic differences between mild and acute MPP(+) toxicities. Mild MPP(+) toxicity impaired autophagosome degradation through novel lysosomal acidity-independent mechanisms. Sustained mild lysosomal damage may contribute to PD. We examined the effects of MPP(+) on autophagic processes under mild exposure, which mimics the slow progressive nature of Parkinson's disease, in SH-SY5Y cells. This study demonstrated impaired autophagic degradation through a reduction in lysosomal cathepsin D activity without altering lysosomal acidity by mild MPP(+) exposure. Mechanistic differences between acute and mild MPP(+) toxicity were also observed. Sustained mild damage of lysosome may be an underlying cause

  14. Identification of cytoskeleton-associated proteins essential for lysosomal stability and survival of human cancer cells.

    Science.gov (United States)

    Groth-Pedersen, Line; Aits, Sonja; Corcelle-Termeau, Elisabeth; Petersen, Nikolaj H T; Nylandsted, Jesper; Jäättelä, Marja

    2012-01-01

    Microtubule-disturbing drugs inhibit lysosomal trafficking and induce lysosomal membrane permeabilization followed by cathepsin-dependent cell death. To identify specific trafficking-related proteins that control cell survival and lysosomal stability, we screened a molecular motor siRNA library in human MCF7 breast cancer cells. SiRNAs targeting four kinesins (KIF11/Eg5, KIF20A, KIF21A, KIF25), myosin 1G (MYO1G), myosin heavy chain 1 (MYH1) and tropomyosin 2 (TPM2) were identified as effective inducers of non-apoptotic cell death. The cell death induced by KIF11, KIF21A, KIF25, MYH1 or TPM2 siRNAs was preceded by lysosomal membrane permeabilization, and all identified siRNAs induced several changes in the endo-lysosomal compartment, i.e. increased lysosomal volume (KIF11, KIF20A, KIF25, MYO1G, MYH1), increased cysteine cathepsin activity (KIF20A, KIF25), altered lysosomal localization (KIF25, MYH1, TPM2), increased dextran accumulation (KIF20A), or reduced autophagic flux (MYO1G, MYH1). Importantly, all seven siRNAs also killed human cervix cancer (HeLa) and osteosarcoma (U-2-OS) cells and sensitized cancer cells to other lysosome-destabilizing treatments, i.e. photo-oxidation, siramesine, etoposide or cisplatin. Similarly to KIF11 siRNA, the KIF11 inhibitor monastrol induced lysosomal membrane permeabilization and sensitized several cancer cell lines to siramesine. While KIF11 inhibitors are under clinical development as mitotic blockers, our data reveal a new function for KIF11 in controlling lysosomal stability and introduce six other molecular motors as putative cancer drug targets.

  15. Breeding resource distribution affects selection gradients on male phenotypic traits: experimental study on lifetime reproductive success in the bitterling fish (Rhodeus amarus).

    Science.gov (United States)

    Reichard, Martin; Ondracková, Markéta; Bryjová, Anna; Smith, Carl; Bryja, Josef

    2009-02-01

    The spatial distribution of breeding resources can have pronounced demographic and evolutionary consequences. We used 20 experimental groups of the bitterling (Rhodeus amarus), an annual fish with a promiscuous, resource-based mating system, and extended breeding season to investigate how the spatial distribution (clumped or regular) of bitterling oviposition sites (live freshwater mussels) affected offspring production, variation in reproductive success, and directional selection on phenotypic traits over their entire reproductive lifetime. We did not detect any effect of resource distribution on offspring production or variation in reproductive success among individual fish, although variation between replicates was higher with a clumped distribution. This finding is discussed with regard to the incidence of alternative mating behaviors (sneaking) within the limitations imposed by our experimental design. Breeding resource distribution had a significant effect on selection on male phenotypic traits. Stronger directional selection on traits associated with intrasexual competition for fertilizations, gonad mass (an indicator of sperm competition), and the extent of red, carotenoid-based pigment in the iris (an index of dominance status), was detected with a clumped resource distribution. With a regular resource distribution, a stronger positive selection on male body size was detected. We discuss the implications of our results for natural populations.

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

    DEFF Research Database (Denmark)

    Petersen, Nikolaj H T; Olsen, Ole D; Groth-Pedersen, Line

    2013-01-01

    Lysosomal membrane permeabilization and subsequent cell death may prove useful in cancer treatment, provided that cancer cell lysosomes can be specifically targeted. Here, we identify acid sphingomyelinase (ASM) inhibition as a selective means to destabilize cancer cell lysosomes. Lysosome-destab...... 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....

  17. Septins as modulators of endo-lysosomal membrane traffic

    Directory of Open Access Journals (Sweden)

    Kyungyeun Song

    2016-11-01

    Full Text Available Septins constitute a family of GTP-binding proteins, which assemble into non-polar filaments in a nucleotide-dependent manner. These filaments can be recruited to negatively charged membrane surfaces. When associated with membranes septin filaments can act as diffusion barriers, which confine subdomains of distinct biological functions. In addition, they serve scaffolding roles by recruiting cytosolic proteins and other cytoskeletal elements. Septins have been implicated in a large variety of membrane-dependent processes, including cytokinesis, signaling, cell migration, and membrane traffic, and several family members have been implicated in disease. However, surprisingly little is known about the molecular mechanisms underlying their biological functions. This review summarizes evidence in support of regulatory roles of septins during endo-lysosomal sorting, with a particular focus on phosphoinositides, which serve as spatial landmarks guiding septin recruitment to distinct subcellular localizations.

  18. How will climate change affect the potential distribution of Eurasian tree sparrows Passer montanus in North America?

    Directory of Open Access Journals (Sweden)

    Jim GRAHAM, Catherine JARNEVICH, Nick YOUNG, Greg NEWMAN, Thomas STOHLGREN

    2011-10-01

    Full Text Available Habitat suitability models have been used to predict the present and future potential distribution of a variety of species. Eurasian tree sparrows Passer montanus, native to Eurasia, have established populations in other parts of the world. In North America, their current distribution is limited to a relatively small region around its original introduction to St. Louis, Missouri. We combined data from the Global Biodiversity Information Facility with current and future climate data to create habitat suitability models using Maxent for this species. Under projected climate change scenarios, our models show that the distribution and range of the Eurasian tree sparrow could increase as far as the Pacific Northwest and Newfoundland. This is potentially important information for prioritizing the management and control of this non-native species [Current Zoology 57 (5: 648–654, 2011].

  19. How will climate change affect the potential distribution of Eurasian tree sparrows Passer montanus in North America?

    Institute of Scientific and Technical Information of China (English)

    Jim GRAHAM; Catherine JARNEVICH; Nick YOUNG; Greg NEWMAN; Thomas STOHLGREN

    2011-01-01

    Habitat suitability models have been used to predict the present and future potential distribution of a variety of species.Eurasian tree sparrows Passer montanus,native to Eurasia,have established populations in other parts of the world.In North America,their current distribution is limited to a relatively small region around its original introduction to St.Louis,Missouri.We combined data from the Global Biodiversity Information Facility with current and future climate data to create habitat suitability models using Maxent for this species.Under projected climate change scenarios,our models show that the distribution and range of the Eurasian tree sparrow could increase as far as the Pacific Northwest and Newfoundland.This is potentially important information for prioritizing the management and control of this non-native species [Current Zoology 57 (5):648-654,2011].

  20. Antioxidant, genotoxic and lysosomal biomarkers in the freshwater bivalve (Unio pictorum) transplanted in a metal polluted river basin.

    Science.gov (United States)

    Guidi, Patrizia; Frenzilli, Giada; Benedetti, Maura; Bernardeschi, Margherita; Falleni, Alessandra; Fattorini, Daniele; Regoli, Francesco; Scarcelli, Vittoria; Nigro, Marco

    2010-10-01

    The freshwater painter's mussel (Unio pictorum) was used as sentinel species to assess the chemical disturbance in an Italian river (the river Cecina) characterized by elevated levels of trace metals of both natural and anthropogenic origin. Organisms were transplanted for 4 weeks in different locations of the river basin and the bioaccumulation of metals was integrated with a wide battery of biomarkers consisting of oxidative, genotoxic and lysosomal responses. Such parameters included the levels of individual antioxidants (catalase, glutathione-S-transferases, glutathione reductase, Se-dependent and Se-independent glutathione peroxidases, total glutathione), the total oxyradical scavenging capacity (TOSC), metallothionein-like proteins, the assessment of DNA integrity, chromosomal damages and lysosomal membrane stability. Elevated levels of several metals were measured in sediments, but the relatively low tissue concentrations suggested a moderate bioaccumulation, possibly due to a high excretion efficiency, of U. pictorum and/or to a limited bioavailability of these elements, partly deriving from erosion of bedrocks. Among antioxidant responses, those based on glutathione metabolism and the activity of catalase were mostly affected in bivalves showing a significant accumulation of arsenic, mercury and/or nickel. In these specimens, the content of glutathione and the activities of glutathione reductase and glutathione peroxidases (H2O2) were respectively 9-, 6- and 4-fold lower than in controls, while a 3-fold increase was observed for catalase. Despite some differences in the response of individual antioxidants, a significant reduction of the capability to neutralize peroxyl radicals was observed in bivalves caged in all the impacted sites of the river basin; these organisms also exhibited a significant impairment at the DNA, chromosomal and lysosomal levels. Considering the mild contamination gradient in the investigated area, the overall results suggested that

  1. Finite element lumbar spine facet contact parameter predictions are affected by the cartilage thickness distribution and initial joint gap size.

    Science.gov (United States)

    Woldtvedt, Daniel J; Womack, Wesley; Gadomski, Benjamin C; Schuldt, Dieter; Puttlitz, Christian M

    2011-06-01

    Current finite element modeling techniques utilize geometrically inaccurate cartilage distribution representations in the lumbar spine. We hypothesize that this shortcoming severely limits the predictive fidelity of these simulations. Specifically, it is unclear how these anatomically inaccurate cartilage representations alter range of motion and facet contact predictions. In the current study, cadaveric vertebrae were serially sectioned, and images were taken of each slice in order to identify the osteochondral interface and the articulating surface. A series of custom-written algorithms were utilized in order to quantify each facet joint's three-dimensional cartilage distribution using a previously developed methodology. These vertebrae-dependent thickness cartilage distributions were implemented on an L1 through L5 lumbar spine finite element model. Moments were applied in three principal planes of motion, and range of motion and facet contact predictions from the variable thickness and constant thickness distribution models were determined. Initial facet gap thickness dimensions were also parameterized. The data indicate that the mean and maximum cartilage thickness increased inferiorly from L1 to L5, with an overall mean thickness value of 0.57 mm. Cartilage distribution and initial facet joint gap thickness had little influence on the lumbar range of motion in any direction, whereas the mean contact pressure, total contact force, and total contact area predictions were altered considerably. The data indicate that range of motion predictions alone are insufficient to establish model validation intended to predict mechanical contact parameters. These data also emphasize the need for the careful consideration of the initial facet joint gap thickness with respect to the spinal condition being studied.

  2. Combined effects of thermal stress and Cd on lysosomal biomarkers and transcription of genes encoding lysosomal enzymes and HSP70 in mussels, Mytilus galloprovincialis.

    Science.gov (United States)

    Izagirre, Urtzi; Errasti, Aitzpea; Bilbao, Eider; Múgica, María; Marigómez, Ionan

    2014-04-01

    In estuaries and coastal areas, intertidal organisms may be subject to thermal stress resulting from global warming, together with pollution. In the present study, the combined effects of thermal stress and exposure to Cd were investigated in the endo-lysosomal system of digestive cells in mussels, Mytilus galloprovincialis. Mussels were maintained for 24h at 18°C and 26°C seawater temperature in absence and presence of 50 μg Cd/L seawater. Cadmium accumulation in digestive gland tissue, lysosomal structural changes and membrane stability were determined. Semi-quantitative PCR was applied to reveal the changes elicited by the different experimental conditions in hexosaminidase (hex), β-glucuronidase (gusb), cathepsin L (ctsl) and heat shock protein 70 (hsp70) gene transcription levels. Thermal stress provoked lysosomal enlargement whilst Cd-exposure led to fusion of lysosomes. Both thermal stress and Cd-exposure caused lysosomal membrane destabilisation. hex, gusb and ctsl genes but not hsp70 gene were transcriptionally up-regulated as a result of thermal stress. In contrast, all the studied genes were transcriptionally down-regulated in response to Cd-exposure. Cd bioaccumulation was comparable at 18°C and 26°C seawater temperatures but interactions between thermal stress and Cd-exposure were remarkable both in lysosomal biomarkers and in gene transcription. hex, gusb and ctsl genes, reacted to elevated temperature in absence of Cd but not in Cd-exposed mussels. Therefore, thermal stress resulting from global warming might influence the use and interpretation of lysosomal biomarkers in marine pollution monitoring programmes and, vice versa, the presence of pollutants may condition the capacity of mussels to respond against thermal stress in a climate change scenario.

  3. Knockout of Lysosomal Enzyme-Targeting Gene Causes Abnormalities in Mouse Pup Isolation Calls

    Science.gov (United States)

    Barnes, Terra D.; Holy, Timothy E.

    2017-01-01

    Humans lacking a working copy of the GNPTAB gene suffer from the metabolic disease Mucolipidosis type II (MLII). MLII symptoms include mental retardation, skeletal deformities and cartilage defects as well as a speech delay with most subjects unable to utter single words (Otomo et al., 2009; Cathey et al., 2010; Leroy et al., 2012). Here we asked whether mice lacking a copy of Gnptab gene exhibited vocal abnormities. We recorded ultrasonic vocalizations from 5 to 8 day old mice separated from their mother and littermates. Although Gnptab−/− pups emitted a similar number of calls, several features of the calls were different from their wild type littermates. Gnptab−/− mice showed a decrease in the length of calls, an increase in the intra-bout pause duration, significantly fewer pitch jumps with smaller mean size, and an increase in the number of isolated calls. In addition, Gnptab−/− mice vocalizations had less power, particularly in the higher frequencies. Gnptab+/− mouse vocalizations did not appear to be affected. We then attempted to classify these recordings using these features to determine the genotype of the animal. We were able to correctly identify 87% of the recordings as either Gnptab−/− or Gnptab+/+ pup, significantly better than chance, demonstrating that genotype is a strong predictor of vocalization phenotype. These data show that deletion of genes in the lysosomal enzyme targeting pathway affect mouse pup isolation calls.

  4. A re-assessment of biochemical marker distributions in T21 affected and unaffected twin pregnancies in the first trimester

    DEFF Research Database (Denmark)

    Madsen, Helen Nordahl; Tørring, Niels

    unaffected and 47 trisomy 21 affected twin pregnancies were included in the study. Chorionicity specific medians were generated for PAPP-A and free β-hCG from gestational age 8 to 14 weeks. Multiple of the median for each of the markers were calculated. Detection rates (DR) and false-positive rates (FPR....... Allowing for gestation and chorionicity, twin pregnancies affected with trisomy 21 had higher levels of free β-hCG and lower levels of PAPP-A. Adding biochemistry into the risk assessment increased the DR for fetal trisomy 21 in dizygotic twin pregnancies from 78% to 90%, and decreased the FPR from 8......INTRODUCTION Serum biochemical marker concentrations in twin pregnancies reflect the presence of two fetuses rather than one. In general, the concentrations are approximately double those found in singleton pregnancies. The objective was to estimate the difference between levels of the two...

  5. The Statistical and Numerical Study of the Longitudinally Asymmetric Distribution of Solar Proton Events Affecting the Earth Environment of 1996-2011

    CERN Document Server

    He, Hongqing

    2015-01-01

    Large solar proton events (SPEs) affect the solar-terrestrial space environment and become a very important aspect in space weather research. In this work, we statistically investigate 78 solar proton events of 1996-2011 and find that there exists a longitudinally asymmetric distribution of flare sources of the solar proton events observed near 1 AU, namely, with the same longitude separation between magnetic field line footpoint of observer and flare sources, the number of the solar proton events originating from sources located at eastern side of the nominal magnetic footpoint of observer is much larger than that of the solar proton events originating from sources located at western side. A complete model calculation of solar energetic particle (SEP) propagation in the three-dimensional Parker interplanetary magnetic field is presented to give a numerical explanation for this longitudinally asymmetric distribution phenomenon. We find that the longitudinally asymmetric distribution of solar proton events res...

  6. THE CHARACTERISTICS OF TEMPORAL AND SPATIAL DISTRIBUTION OF TROPICAL CYCLONE FREQUENCIES OVER THE SOUTH CHINA SEA AND ITS AFFECTING OCEANIC FACTORS IN THE PAST 50 YEARS

    Institute of Scientific and Technical Information of China (English)

    LI Chun-hui; LIU Chun-xia; CHENG Zheng-quan

    2007-01-01

    The characteristics of temporal and spatial distribution of tropical cyclone frequencies over the South China Sea areas and its affecting factors in the past 50yrs are analyzed based on typhoon data that provided by CMA and Simple Ocean Data Assimilation (SODA). The results show that the tropical cyclone frequencies from June to October show concentrated geographic distribution, for they mainly distribute over the SCS area from 15 - 20 °N. The characteristics present significant interdecadal changes. The impact of oceanic factors on the tropical cyclone frequencies in the SCS area is mainly realized by La Ni(n)a and La Ni(n)a-like events before 1975 but mainly by El Ni(n)o and El Nifo-like events after 1975.

  7. Action of low-energy monochromatic coherent light on the stability of retinal lysosomes

    Science.gov (United States)

    Metelitsina, Irina P.; Leus, N. F.

    1995-05-01

    The data had been obtained during the experiment in vitro by irradiation of solubilized lysosomal enzymes, retinal homogenates and native lysosomes enabled us to conclude that the laser beam ((lambda) equals 632.8 nm, power density from 0.1 to 15.0 mWt/cm2) acts on the level of membranous structures of lysosomes. During irradiation of rabbits eyes in vitro with an unfocused laser beam (power density on the cornea aur face from 0.01 to 15.0 mWt/cm2 was shown, that low-energy, ranged from 0.01 to 1.0 mWt/cm2 promotes stabilization of lysosomal membranes. Irradiation with laser beam of 8.0 mWt/cm2 and more power induces destabilization of lysosomal membranes. We have also shown that vitamins A and E effecting membranotropic on lysosomes may be corrected by low-energy radiation of helium-neon laser. It is substantiated experimentally that the stabilizing effect of vitamin E may be intensified in case of the combined action of laser radiation on lysosomes. The labilizing effect of vitamin A on membranes of organelles, as was studied, may be weakened by application of laser radiation of low intensities.

  8. Chlamydia species-dependent differences in the growth requirement for lysosomes.

    Directory of Open Access Journals (Sweden)

    Scot P Ouellette

    Full Text Available Genome reduction is a hallmark of obligate intracellular pathogens such as Chlamydia, where adaptation to intracellular growth has resulted in the elimination of genes encoding biosynthetic enzymes. Accordingly, chlamydiae rely heavily on the host cell for nutrients yet their specific source is unclear. Interestingly, chlamydiae grow within a pathogen-defined vacuole that is in close apposition to lysosomes. Metabolically-labeled uninfected host cell proteins were provided as an exogenous nutrient source to chlamydiae-infected cells, and uptake and subsequent labeling of chlamydiae suggested lysosomal degradation as a source of amino acids for the pathogen. Indeed, Bafilomycin A1 (BafA1, an inhibitor of the vacuolar H(+/ATPase that blocks lysosomal acidification and functions, impairs the growth of C. trachomatis and C. pneumoniae, and these effects are especially profound in C. pneumoniae. BafA1 induced the marked accumulation of material within the lysosomal lumen, which was due to the inhibition of proteolytic activities, and this response inhibits chlamydiae rather than changes in lysosomal acidification per se, as cathepsin inhibitors also inhibit the growth of chlamydiae. Finally, the addition of cycloheximide, an inhibitor of eukaryotic protein synthesis, compromises the ability of lysosomal inhibitors to block chlamydial growth, suggesting chlamydiae directly access free amino acids in the host cytosol as a preferred source of these nutrients. Thus, chlamydiae co-opt the functions of lysosomes to acquire essential amino acids.

  9. Eucommia ulmoides cortex, geniposide and aucubin regulate lipotoxicity through the inhibition of lysosomal BAX.

    Science.gov (United States)

    Lee, Geum-Hwa; Lee, Mi-Rin; Lee, Hwa-Young; Kim, Seung Hyun; Kim, Hye-Kyung; Kim, Hyung-Ryong; Chae, Han-Jung

    2014-01-01

    In this study we examined the inhibition of hepatic dyslipidemia by Eucommia ulmoides extract (EUE). Using a screening assay for BAX inhibition we determined that EUE regulates BAX-induced cell death. Among various cell death stimuli tested EUE regulated palmitate-induced cell death, which involves lysosomal BAX translocation. EUE rescued palmitate-induced inhibition of lysosomal V-ATPase, α-galactosidase, α-mannosidase, and acid phosphatase, and this effect was reversed by bafilomycin, a lysosomal V-ATPase inhibitor. The active components of EUE, aucubin and geniposide, showed similar inhibition of palmitate-induced cell death to that of EUE through enhancement of lysosome activity. Consistent with these in vitro findings, EUE inhibited the dyslipidemic condition in a high-fat diet animal model by regulating the lysosomal localization of BAX. This study demonstrates that EUE regulates lipotoxicity through a novel mechanism of enhanced lysosomal activity leading to the regulation of lysosomal BAX activation and cell death. Our findings further indicate that geniposide and aucubin, active components of EUE, may be therapeutic candidates for non-alcoholic fatty liver disease.

  10. Involvement of lysosomes in the uptake of macromolecular material by bloodstream forms of Trypanosoma brucei.

    Science.gov (United States)

    Opperdoes, F R; Van Roy, J

    1982-09-01

    To investigate whether the lysosomes of Trypanosoma brucei are capable of uptake of macromolecules after internalization by the cell, we used Triton WR-1339, a non-digestible macromolecular compound, which is known to cause a marked decrease in the density of hepatic lysosomes due to massive intralysosomal storage. Intraperitoneal administration of 0.4 g/kg Triton WR-1339 to rats infected with T. brucei led to the development of a large vacuole in the trypanosomes between nucleus and kinetoplast within 22 h. Higher doses (2 g/kg) led to the disappearance of the trypanosomes from the blood and resulted in permanent cures (greater than 100 days). Lysosomes isolated from the trypanosomes of animals treated with a sub-curative dose showed a decrease in equilibrium density of 0.03 g/cm3 in sucrose gradients. These lysosomes were partly damaged as evidenced by a reduction in latency and an increase in the non-sedimentable part of lysosomal enzymes. We conclude that acid proteinase and alpha-mannosidase-containing organelles of T. brucei take up exogenous macromolecules and must therefore be considered as true lysosomes and that Triton WR-1339 acts in T. brucei as a true lysosomotropic drug. Its trypanocidal action probably results from an interference with lysosomal function.

  11. Lysosomal cholesterol accumulation: driver on the road to inflammation during atherosclerosis and non-alcoholic steatohepatitis.

    Science.gov (United States)

    Hendrikx, T; Walenbergh, S M A; Hofker, M H; Shiri-Sverdlov, R

    2014-05-01

    Many studies show an association between the accumulation of cholesterol inside lysosomes and the progression towards inflammatory disease states that are closely related to obesity. While in the past, the knowledge regarding lysosomal cholesterol accumulation was limited to its association with plaque severity during atherosclerosis, recently, a growing body of evidence indicates a causal link between lysosomal cholesterol accumulation and inflammation. These findings make lysosomal cholesterol accumulation an important target for intervention in metabolic diseases that are characterized by the presence of an inflammatory response. In this review, we aim to show the importance of cholesterol trapping inside lysosomes to the development of inflammation by focusing upon cardiovascular disease and non-alcoholic steatohepatitis (NASH) in particular. We summarize current data supporting the hypothesis that lysosomal cholesterol accumulation plays a key role in the development of inflammation during atherosclerosis and NASH. In addition, potential mechanisms by which disturbed lysosomal function can trigger the inflammatory response, the challenges in improving cholesterol trafficking in macrophages and recent successful research directions will be discussed.

  12. Antimicrobial Properties of Lysosomal Enzymes Immobilized on NH₂Functionalized Silica-Encapsulated Magnetite Nanoparticles.

    Science.gov (United States)

    Bang, Seung Hyuck; Sekhon, Simranjeet Singh; Cho, Sung-Jin; Kim, So Jeong; Le, Thai-Hoang; Kim, Pil; Ahn, Ji-Young; Kim, Yang-Hoon; Min, Jiho

    2016-01-01

    The immobilization efficiency, antimicrobial activity and recovery of lysosomal enzymes on NH2 functionalized magnetite nanoparticles have been studied under various conditions. The immobi- lization efficiency depends upon the ratio of the amount of enzyme and magnetite and it shows an increase with magnetite concentration which is due to the presence of amine group at the magnetite surface that leads to a strong attraction. The optimized reaction time to immobilize the lysosomal enzymes on magnetite was determined by using a rolling method. The immobilization efficiency increases with reaction time and reached a plateau after 5 minutes and then remained constant for 10 minutes. However, after 30 minutes the immobilization efficiency decreased to 85%, which is due to the weaker electrostatic interactions between magnetite and detached lysosomal enzymes. The recovery and stability of immobilized lysosomal enzymes has also been studied. The antimicrobial activity was almost 100% but it decreased upon reuse and no activity was observed after its reuse for seven times. The storage stability of lysosomal enzymes as an antimicrobial agent was about 88%, which decreased to 53% after one day and all activity of immobilized lysosomal enzymes was maintained after five days. Thus, the lysosomal enzymes immobilized on magnetite nanoparticles could potentially be used as antimicrobial agents to remove bacteria.

  13. Ecological and social factors affecting the local habitat distribution of western sandpipers wintering at Bah?Santa Mar? Northwest Mexico

    OpenAIRE

    2005-01-01

    The process of habitat selection often requires individuals to choose among habitats that differ in foraging profitability and predation danger. The local habitat distribution of Western Sandpipers (Calidris maur~] was studied at Bahia Santa Maria, northwest Mexico, for three non-breeding seasons (1 999-2002). Western Sandpipers are highly sexually dimorphic, males being lighter and smaller-billed than females, and thus males may use visual foraging more often, be more susceptible to interfer...

  14. Predicting probability of occurrence and factors affecting distribution and abundance of three Ozark endemic crayfish species at multiple spatial scales

    Science.gov (United States)

    Nolen, Matthew S.; Magoulick, Daniel D.; DiStefano, Robert J.; Imhoff, Emily M.; Wagner, Brian K.

    2014-01-01

    Crayfishes and other freshwater aquatic fauna are particularly at risk globally due to anthropogenic demand, manipulation and exploitation of freshwater resources and yet are often understudied. The Ozark faunal region of Missouri and Arkansas harbours a high level of aquatic biological diversity, especially in regard to endemic crayfishes. Three such endemics, Orconectes eupunctus,Orconectes marchandi and Cambarus hubbsi, are threatened by limited natural distribution and the invasions of Orconectes neglectus.

  15. Diversity and Distribution of Arsenic-Related Genes Along a Pollution Gradient in a River Affected by Acid Mine Drainage.

    Science.gov (United States)

    Desoeuvre, Angélique; Casiot, Corinne; Héry, Marina

    2016-04-01

    Some microorganisms have the capacity to interact with arsenic through resistance or metabolic processes. Their activities contribute to the fate of arsenic in contaminated ecosystems. To investigate the genetic potential involved in these interactions in a zone of confluence between a pristine river and an arsenic-rich acid mine drainage, we explored the diversity of marker genes for arsenic resistance (arsB, acr3.1, acr3.2), methylation (arsM), and respiration (arrA) in waters characterized by contrasted concentrations of metallic elements (including arsenic) and pH. While arsB-carrying bacteria were representative of pristine waters, Acr3 proteins may confer to generalist bacteria the capacity to cope with an increase of contamination. arsM showed an unexpected wide distribution, suggesting biomethylation may impact arsenic fate in contaminated aquatic ecosystems. arrA gene survey suggested that only specialist microorganisms (adapted to moderately or extremely contaminated environments) have the capacity to respire arsenate. Their distribution, modulated by water chemistry, attested the specialist nature of the arsenate respirers. This is the first report of the impact of an acid mine drainage on the diversity and distribution of arsenic (As)-related genes in river waters. The fate of arsenic in this ecosystem is probably under the influence of the abundance and activity of specific microbial populations involved in different As biotransformations.

  16. SVD identifies transcript length distribution functions from DNA microarray data and reveals evolutionary forces globally affecting GBM metabolism.

    Science.gov (United States)

    Bertagnolli, Nicolas M; Drake, Justin A; Tennessen, Jason M; Alter, Orly

    2013-01-01

    To search for evolutionary forces that might act upon transcript length, we use the singular value decomposition (SVD) to identify the length distribution functions of sets and subsets of human and yeast transcripts from profiles of mRNA abundance levels across gel electrophoresis migration distances that were previously measured by DNA microarrays. We show that the SVD identifies the transcript length distribution functions as "asymmetric generalized coherent states" from the DNA microarray data and with no a-priori assumptions. Comparing subsets of human and yeast transcripts of the same gene ontology annotations, we find that in both disparate eukaryotes, transcripts involved in protein synthesis or mitochondrial metabolism are significantly shorter than typical, and in particular, significantly shorter than those involved in glucose metabolism. Comparing the subsets of human transcripts that are overexpressed in glioblastoma multiforme (GBM) or normal brain tissue samples from The Cancer Genome Atlas, we find that GBM maintains normal brain overexpression of significantly short transcripts, enriched in transcripts that are involved in protein synthesis or mitochondrial metabolism, but suppresses normal overexpression of significantly longer transcripts, enriched in transcripts that are involved in glucose metabolism and brain activity. These global relations among transcript length, cellular metabolism and tumor development suggest a previously unrecognized physical mode for tumor and normal cells to differentially regulate metabolism in a transcript length-dependent manner. The identified distribution functions support a previous hypothesis from mathematical modeling of evolutionary forces that act upon transcript length in the manner of the restoring force of the harmonic oscillator.

  17. Combined effects of thermal stress and Cd on lysosomal biomarkers and transcription of genes encoding lysosomal enzymes and HSP70 in mussels, Mytilus galloprovincialis

    Energy Technology Data Exchange (ETDEWEB)

    Izagirre, Urtzi; Errasti, Aitzpea; Bilbao, Eider; Múgica, María; Marigómez, Ionan, E-mail: ionan.marigomez@ehu.es

    2014-04-01

    Highlights: • Thermal stress and Cd caused lysosomal enlargement and membrane destabilisation. • hex, gusb and ctsl but not hsp70 were up-regulated at elevated temperature but down-regulated by Cd. • Thermal stress influenced lysosomal responses to Cd exposure. • The presence of Cd jeopardised responsiveness against thermal stress. - Abstract: In estuaries and coastal areas, intertidal organisms may be subject to thermal stress resulting from global warming, together with pollution. In the present study, the combined effects of thermal stress and exposure to Cd were investigated in the endo-lysosomal system of digestive cells in mussels, Mytilus galloprovincialis. Mussels were maintained for 24 h at 18 °C and 26 °C seawater temperature in absence and presence of 50 μg Cd/L seawater. Cadmium accumulation in digestive gland tissue, lysosomal structural changes and membrane stability were determined. Semi-quantitative PCR was applied to reveal the changes elicited by the different experimental conditions in hexosaminidase (hex), β-glucuronidase (gusb), cathepsin L (ctsl) and heat shock protein 70 (hsp70) gene transcription levels. Thermal stress provoked lysosomal enlargement whilst Cd-exposure led to fusion of lysosomes. Both thermal stress and Cd-exposure caused lysosomal membrane destabilisation. hex, gusb and ctsl genes but not hsp70 gene were transcriptionally up-regulated as a result of thermal stress. In contrast, all the studied genes were transcriptionally down-regulated in response to Cd-exposure. Cd bioaccumulation was comparable at 18 °C and 26 °C seawater temperatures but interactions between thermal stress and Cd-exposure were remarkable both in lysosomal biomarkers and in gene transcription. hex, gusb and ctsl genes, reacted to elevated temperature in absence of Cd but not in Cd-exposed mussels. Therefore, thermal stress resulting from global warming might influence the use and interpretation of lysosomal biomarkers in marine pollution

  18. Lysosomal glycosphingolipid catabolism by acid ceramidase: formation of glycosphingoid bases during deficiency of glycosidases.

    Science.gov (United States)

    Ferraz, Maria J; Marques, André R A; Appelman, Monique D; Verhoek, Marri; Strijland, Anneke; Mirzaian, Mina; Scheij, Saskia; Ouairy, Cécile M; Lahav, Daniel; Wisse, Patrick; Overkleeft, Herman S; Boot, Rolf G; Aerts, Johannes M

    2016-03-01

    Glycosphingoid bases are elevated in inherited lysosomal storage disorders with deficient activity of glycosphingolipid catabolizing glycosidases. We investigated the molecular basis of the formation of glucosylsphingosine and globotriaosylsphingosine during deficiency of glucocerebrosidase (Gaucher disease) and α-galactosidase A (Fabry disease). Independent genetic and pharmacological evidence is presented pointing to an active role of acid ceramidase in both processes through deacylation of lysosomal glycosphingolipids. The potential pathophysiological relevance of elevated glycosphingoid bases generated through this alternative metabolism in patients suffering from lysosomal glycosidase defects is discussed.

  19. Neither folic acid supplementation nor pregnancy affects the distribution of folate forms in the red blood cells of women.

    Science.gov (United States)

    Hartman, Brenda A; Fazili, Zia; Pfeiffer, Christine M; O'Connor, Deborah L

    2014-09-01

    It is not known whether folate metabolism is altered during pregnancy to support increased DNA and RNA biosynthesis. By using a state-of-the-art LC tandem mass spectrometry technique, the aim of this study was to investigate differences in RBC folate forms between pregnant and nonpregnant women and between nonpregnant women consuming different concentrations of supplemental folic acid. Forms of folate in RBCs were used to explore potential shifts in folate metabolism during early erythropoiesis. Total RBC folate and folate forms [tetrahydrofolate; 5-methyltetrahydrofolate (5-methyl-THF); 4α-hydroxy-5-methyl-tetrahydrofolate (an oxidation product of 5-methyl-THF); 5-formyl-tetrahydrofolate; and 5,10-methenyl-tetrahydrofolate] were measured in 4 groups of women (n = 26): pregnant women (PW) (30-36 wk of gestation) consuming 1 mg/d of folic acid, and nonpregnant women consuming 0 mg/d (NPW-0), 1 mg/d (NPW-1), and 5 mg/d (NPW-5) folic acid. The mean ± SD RBC folate concentration of the NPW-0 group (890 ± 530 nmol/L) was lower than the NPW-1 (1660 ± 350 nmol/L) and NPW-5 (1980 ± 570 nmol/L) groups as assessed by microbiologic assay (n = 26, P folic acid supplements had detectable concentrations of 5,10-methenyl-tetrahydrofolate (LOD = 0.31 nmol/L). However, there was no difference in the relative distribution of 5-methyl-THF (83-84%), sum of non-methyl folates (0.6-3%), or individual non-methyl folate forms in RBCs across groups. We conclude that although folic acid supplementation in nonpregnant women increases RBC total folate and the concentration of individual folate forms, it does not alter the relative distribution of folate forms. Similarly, distribution of RBC folate forms did not differ between pregnant and nonpregnant women. This trial was registered at clinicaltrials.gov as NCT01741077.

  20. SVD identifies transcript length distribution functions from DNA microarray data and reveals evolutionary forces globally affecting GBM metabolism.

    Directory of Open Access Journals (Sweden)

    Nicolas M Bertagnolli

    Full Text Available To search for evolutionary forces that might act upon transcript length, we use the singular value decomposition (SVD to identify the length distribution functions of sets and subsets of human and yeast transcripts from profiles of mRNA abundance levels across gel electrophoresis migration distances that were previously measured by DNA microarrays. We show that the SVD identifies the transcript length distribution functions as "asymmetric generalized coherent states" from the DNA microarray data and with no a-priori assumptions. Comparing subsets of human and yeast transcripts of the same gene ontology annotations, we find that in both disparate eukaryotes, transcripts involved in protein synthesis or mitochondrial metabolism are significantly shorter than typical, and in particular, significantly shorter than those involved in glucose metabolism. Comparing the subsets of human transcripts that are overexpressed in glioblastoma multiforme (GBM or normal brain tissue samples from The Cancer Genome Atlas, we find that GBM maintains normal brain overexpression of significantly short transcripts, enriched in transcripts that are involved in protein synthesis or mitochondrial metabolism, but suppresses normal overexpression of significantly longer transcripts, enriched in transcripts that are involved in glucose metabolism and brain activity. These global relations among transcript length, cellular metabolism and tumor development suggest a previously unrecognized physical mode for tumor and normal cells to differentially regulate metabolism in a transcript length-dependent manner. The identified distribution functions support a previous hypothesis from mathematical modeling of evolutionary forces that act upon transcript length in the manner of the restoring force of the harmonic oscillator.

  1. Disturbance frequency and vertical distribution of seeds affect long-term population dynamics: a mechanistic seed bank model.

    Science.gov (United States)

    Eager, Eric Alan; Haridas, Chirakkal V; Pilson, Diana; Rebarber, Richard; Tenhumberg, Brigitte

    2013-08-01

    Seed banks are critically important for disturbance specialist plants because seeds of these species germinate only in disturbed soil. Disturbance and seed depth affect the survival and germination probability of seeds in the seed bank, which in turn affect population dynamics. We develop a density-dependent stochastic integral projection model to evaluate the effect of stochastic soil disturbances on plant population dynamics with an emphasis on mimicking how disturbances vertically redistribute seeds within the seed bank. We perform a simulation analysis of the effect of the frequency and mean depth of disturbances on the population's quasi-extinction probability, as well as the long-term mean and variance of the total density of seeds in the seed bank. We show that increasing the frequency of disturbances increases the long-term viability of the population, but the relationship between the mean depth of disturbance and the long-term viability of the population are not necessarily monotonic for all parameter combinations. Specifically, an increase in the probability of disturbance increases the long-term viability of the total seed bank population. However, if the probability of disturbance is too low, a shallower mean depth of disturbance can increase long-term viability, a relationship that switches as the probability of disturbance increases. However, a shallow disturbance depth is beneficial only in scenarios with low survival in the seed bank.

  2. Epigallocatechin-3-gallate affects the growth of LNCaP cells via membrane fluidity and distribution of cellular zinc

    Institute of Scientific and Technical Information of China (English)

    Jun-guo YANG; Hai-ning YU; Shi-li SUN; Lan-cui ZHANG; Guo-qing HE; Undurti N. DAS; Hui RUAN; Sheng-rong SHEN

    2009-01-01

    Objective: To evaluate effects of epigallocatechin-3-gallate (EGCG) on the viability, membrane properties, and zinc distribution, with and without the presence of Zn2+, in human prostate carcinoma LNCaP cells. Methods: We examined changes in cellular morphology and membrane fluidity of LNCaP cells, distribution of cellular zinc, and the incorporated portion of EGCG after treatments with EGCG, Zn2+, and EGCG+Zn2+. Results: We observed an alteration in cellular morphology and a decrease in membrane fluidity of LNCaP cells after treatment with EGCG or Zn2+. The proportion of EGCG incorporated into liposomes treated with the mixture of EGCG and Zn2+ at the ratio of 1:l was 90.57%, which was significantly higher than that treated with EGCG alone (30.33%). Electron spin resonance (ESR) studies and determination of fatty acids showed that the effects of EGCG on the membrane fluidity of LNCaP were decreased by Zn2+. EGCG accelerated the accumulation of zinc in the mitochondria and cytosol as observed by atomic absorption spectrometer. Conclusion: These results show that EGCG interacted with cell membrane,decreased the membrane fluidity of LNCaP cells, and accelerated zinc accumulation in the mitochondria and cytosol, which could be the mechanism by which EGCG inhibits proliferation of LNCaP cells. In addition, high concentrations of Zn2+ could attenuate the actions elicited by EGCG.

  3. Addition of pectin and soy soluble polysaccharide affects the particle size distribution of casein suspensions prepared from acidified skim milk.

    Science.gov (United States)

    Liu, JinRu; Nakamura, Akihiro; Corredig, Milena

    2006-08-23

    Pectins are negatively charged polysaccharides employed as stabilizers in acidified milk dispersions, where caseins aggregate because of the low pH and serum separation needs to be prevented. The objective of this research was to study the effect of charge on the stabilizing functionality of the polysaccharide in acid milk drinks. Unstandardized pectins with various charges (as degree of esterification, DE) as well as soybean soluble polysaccharide (SSPS) were tested for their stabilizing behavior as a function of pH and concentration. Skim milk was acidified by glucono-delta-lactone and then homogenized in the presence of polysaccharide at different pH values (in the range from 4.2 to 3.0). Measurements of particle size distribution demonstrated that pectins with a DE of 71.4, 68.6, and 67.4 stabilized milk at pH > 4.0. Pectins with a lower DE (63.9%) needed a higher concentration (0.4%) at the same pH to show a monomodal distribution of particle sizes. Pectins with lower DE (milk, at pH milk dispersion was pH-dependent, and results were similar to those for samples containing pectin alone. This suggested that in the mixture, pectin dominated the behavior over SSPS, even when an excess of SSPS was added to the dispersions before homogenization.

  4. A post-implementation evaluation of ceramic water filters distributed to tsunami-affected communities in Sri Lanka.

    Science.gov (United States)

    Casanova, Lisa M; Walters, Adam; Naghawatte, Ajith; Sobsey, Mark D

    2012-06-01

    Sri Lanka was devastated by the 2004 Indian Ocean tsunami. During recovery, the Red Cross distributed approximately 12,000 free ceramic water filters. This cross-sectional study was an independent post-implementation assessment of 452 households that received filters, to determine the proportion still using filters, household characteristics associated with use, and quality of household drinking water. The proportion of continued users was high (76%). The most common household water sources were taps or shallow wells. The majority (82%) of users used filtered water for drinking only. Mean filter flow rate was 1.12 L/hr (0.80 L/hr for households with taps and 0.71 for those with wells). Water quality varied by source; households using tap water had source water of high microbial quality. Filters improved water quality, reducing Escherichia coli for households (largely well users) with high levels in their source water. Households were satisfied with filters and are potentially long-term users. To promote sustained use, recovery filter distribution efforts should try to identify households at greatest long-term risk, particularly those who have not moved to safer water sources during recovery. They should be joined with long-term commitment to building supply chains and local production capacity to ensure safe water access.

  5. Distribution of rare earth elements in an alluvial aquifer affected by acid mine drainage: the Guadiamar aquifer (SW Spain)

    Energy Technology Data Exchange (ETDEWEB)

    Olias, M. [Departamento de Geodinamica y Paleontologia, Universidad de Huelva, Avda. de las Fuerza Armadas s/n, 21071 Huelva (Spain)]. E-mail: manuel.olias@dgyp.uhu.es; Ceron, J.C. [Departamento de Geodinamica y Paleontologia, Universidad de Huelva, Avda. de las Fuerza Armadas s/n, 21071 Huelva (Spain); Fernandez, I. [Departamento de Geodinamica y Paleontologia, Universidad de Huelva, Avda. de las Fuerza Armadas s/n, 21071 Huelva (Spain); Rosa, J. de la [Departamento de Geologia, Universidad de Huelva, Avda. de las Fuerza Armadas s/n, 21071 Huelva (Spain)

    2005-05-01

    This work analyses the spatial distribution, the origin, and the shale-normalised fractionation patterns of the rare earth elements (REE) in the alluvial aquifer of the Guadiamar River (south-western Spain). This river received notoriety in April 1998 for a spill that spread a great amount of slurry (mainly pyrites) and acid waters in a narrow strip along the river course. Groundwaters and surface waters were sampled to analyse, among other elements, the REEs. Their spatial distribution shows a peak close to the mining region, in an area with low values of pH and high concentrations of sulphates and other metals such as Zn, Cu, Co, Ni, Pb, and Cd. The patterns of shale-normalised fractionation at the most-contaminated points show an enrichment in the middle rare earth elements (MREE) with respect to the light (LREE) and heavy (HREE) ones, typical of acid waters. The Ce-anomaly becomes more negative as pH increases, due to the preferential fractionation of Ce in oxyhydroxides of Fe. - Pollution of the aquifer with rare earth elements is documented at a site of a major spill from a mining operation.

  6. Bupivacaine can enhance lysosomal activity in mouse muscle myoblasts%布比卡因增强小鼠成肌细胞溶酶体的活性

    Institute of Scientific and Technical Information of China (English)

    熊静薇; 毛雨; 李荣荣; 丁正年

    2015-01-01

    Objective To investigate the effects of bupivacaine on lysosomal abundance and activity in mouse muscle myoblasts.Methods Mouse myoblasts C2C12 was randomly divided into control group (without any treatment) and bupivacaine group (treated with bupivacaine 600 μ mol/L for 6 h).After then,the changes of lysosomal pH was assessed by LysoSensor pH indicator.The content of lysosomes was detected by LysoTracker probe.The expression of lysosomal-associated membrane protein-1 (LAMP-1) and Cathepsin B was detected by Western blot analysis.The activity of lysosomal proteolytic enzymes Cathepsin B was determined by MagicRed assay kit.Results Bupivacaine did not affect lysosomal pH.However,compared with the controls,lysosomal abundance was significantly increased 15.15% following bupivacaine treatment(P<0.01).Moreover,protein expression levels of LAMP-1 and Cathepsin B were significantly upregulated 36.41% and 35.29% respetctively by bupivacaine (P<0.01).Furthermore,the activity of Cathepsin B was significantly increased 23.74% by bupivacaine(P<0.01).Conclusions Bupivacaine increased lysosomal content and enhance lysosomal activity in mouse muscle myoblasts.%目的 探讨局部麻醉药布比卡因对小鼠成肌细胞溶酶体的影响. 方法 将体外培养的小鼠成肌细胞C2C12分为2组.对照组:不加任何药物;布比卡因组:以600μmol/L布比卡因刺激细胞6h.实验结束后,用LysoSensor探针评价溶酶体腔pH,用LysoTrackor探针检测溶酶体含量,用蛋白免疫印迹法检测溶酶体相关膜蛋白-1(LAMP-1)和溶酶体蛋白水解酶Cathepsin B的表达水平,并以MagicRed染色法测定Cathepsin B的活性.结果 布比卡因对溶酶体腔pH没有影响.但是,与对照组相比,布比卡因组溶酶体含量增加15.15% (P<0.01),LAMP-1与Cathepsin B表达量分别增加36.41%、35.29% (P<0.01),Cathepsin B活性增加23.74%(P<0.01).结论 布比卡因能增加小鼠成肌细胞溶酶体含量,增强溶酶体活性.

  7. Roles of CUP-5, the Caenorhabditis elegans orthologue of human TRPML1, in lysosome and gut granule biogenesis

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    Fares Hanna

    2010-06-01

    Full Text Available Abstract Background CUP-5 is a Transient Receptor Potential protein in C. elegans that is the orthologue of mammalian TRPML1. Loss of TRPML1 results in the lysosomal storage disorder Mucolipidosis type IV. Loss of CUP-5 results in embryonic lethality and the accumulation of enlarged yolk granules in developing intestinal cells. The embryonic lethality of cup-5 mutants is rescued by mutations in mrp-4, which is required for gut granule differentiation. Gut granules are intestine-specific lysosome-related organelles that accumulate birefringent material. This link between CUP-5 and gut granules led us to determine the roles of CUP-5 in lysosome and gut granule biogenesis in developing intestinal cells. Results We show that CUP-5 protein localizes to lysosomes, but not to gut granules, in developing intestinal cells. Loss of CUP-5 results in defects in endo-lysosomal transport in developing intestinal cells of C. elegans embryos. This ultimately leads to the appearance of enlarged terminal vacuoles that show defective lysosomal degradation and that have lysosomal and endosomal markers. In contrast, gut granule biogenesis is normal in the absence of CUP-5. Furthermore, loss of CUP-5 does not result in inappropriate fusion or mixing of content between lysosomes and gut granules. Conclusions Using an in vivo model of MLIV, we show that there is a defect in lysosomal transport/biogenesis that is earlier than the presumed function of TRPML1 in terminal lysosomes. Our results indicate that CUP-5 is required for the biogenesis of lysosomes but not of gut granules. Thus, cellular phenotypes in Mucolipidosis type IV are likely not due to defects in lysosome-related organelle biogenesis, but due to progressive defects in lysosomal transport that lead to severe lysosomal dysfunction.

  8. Factors affecting distribution of microbiotic crusts in the grain-for-green land of the loess region,northern Shaanxi,China

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    A field survey was conducted in the grain-for-green land of the loess region,northern Shaanxi,China,from July to August of 2005 to provide a scientific evaluation of the grain-for-green project,including its soil and water conservation and other ecological benefits for the region.The distribution of microbiotic crusts were studied,while human disturbance,aspect,topography,vegetation structure and other factors affecting it were obtained from the analysis of survey data from 78 sample plots.Results show that crust coverage is larger on lessdisturbed plots than on highly-disturbed ones,on northfacing plots than on south-facing ones and on gully-slopes than on ridge-slopes.Coverage increases with herbal coverage and trees can provide better conditions for distribution of crusts than shrubs.Therefore,crust coverage is larger in herb-dominated plots than in tree-dominated ones and crusts in shrub-dominated plots are smaller.However,we made no progress in our study on deciding how slope degrees and herb species affect the distribution of crusts.We believe that more studies are necessary for a further exploration of the relationship between them.

  9. Distribution and factors affecting adsorption of sterols in the surface sediments of Bosten Lake and Manas Lake, Xinjiang.

    Science.gov (United States)

    Liu, Jiang; Yao, Xiaorui; Lu, Jianjiang; Qiao, Xiuwen; Liu, Zilong; Li, Shanman

    2016-03-01

    This study investigated the concentrations and distribution of eight sterol compounds in the surface sediments of Bosten Lake and Manas Lake, Xinjiang, China. The ratios of sterols as diagnostic indices were used to identify pollution sources. The sediment of the two lakes was selected as an adsorbent to investigate the adsorption behaviour of sterols. Results showed that the sterols were widely distributed in the sediments of the lakes in the study areas. The total concentrations of the detected sterols in Bosten Lake and in Manas Lake were 1.584-27.897 and 2.048-18.373 μg g(-1)∙dw, respectively. In all of the sampling sites, the amount of faecal sterols was less than that of plant sterols. β-sitosterol was the dominant plant sterol with a mean concentration of 2.378 ± 2.234 μg g(-1)∙dw; cholesterol was the most abundant faecal sterol with a mean concentration of 1.060 ± 1.402 μg g(-1)∙dw. The pollution level was higher in Bosten Lake than in Manas Lake. Majority of the ratios clearly demonstrated that the contamination by human faecal sources was occurring at stations which are adjacent to residential areas and water inlets. The adsorption behaviour of sterols to sediment suggested that the sterol adsorption coefficients were reduced as temperature increased. As salinity increased, the adsorption quantity also increased. As pH increased, the sediment adsorption of sterol slightly increased because the strong alkaline solution is not conducive to the adsorption of sterols. The ratios between sterols did not change largely with the change in external factors.

  10. Enantioselective effects of methamidophos on the coelomocytes lysosomal membrane stability of Eisenia fetida.

    Science.gov (United States)

    Chen, Linhua; Lu, Xianting; Ma, Yun

    2012-12-01

    Many of organophosphorous insecticides are chiral compounds. In this study, the enantioselective effects of organophosphate insecticide methamidophos on the coelomocytes lysosomal membrane stability of earthworm Eisenia fetida were studied: (1) The enantiomers of methamidophos were absolutely separated by high-performance liquid chromatography with a commercial chiral column; (2) The neutral red retention assay was used to judge the lysosomal membrane stability. The results showed that with the concentration increasing, lysosomal membranes have been significantly destroyed by individual stereoisomers and racemate of methamidophos. The neutral red retention times were significantly descended from 76.88 to 29.78 min. Both (+)- and (-)-methamidophos showed more prone to destroy the integrity of the lysosomal membrane than the racemate. However, the different effect between stereoisomers is slight.

  11. Lysosome associated membrane proteins maintain pancreatic acinar cell homeostasis : LAMP-2 deficient mice develop pancreatitis

    NARCIS (Netherlands)

    Mareninova, Olga A; Sendler, Matthias; Malla, Sudarshan Ravi; Yakubov, Iskandar; French, Samuel W; Tokhtaeva, Elmira; Vagin, Olga; Oorschot, Viola; Lüllmann-Rauch, Renate; Blanz, Judith; Dawson, David; Klumperman, Judith; Lerch, Markus M; Mayerle, Julia; Gukovsky, Ilya; Gukovskaya, Anna S

    2015-01-01

    BACKGROUND & AIMS: The pathogenic mechanism of pancreatitis is poorly understood. Recent evidence implicates defective autophagy in pancreatitis responses; however, the pathways mediating impaired autophagy in pancreas remain largely unknown. Here, we investigate the role of lysosome associated memb

  12. Lysosomal acid lipase: At the crossroads of normal and atherogenic cholesterol metabolism

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    Joshua A Dubland

    2015-02-01

    Full Text Available Unregulated cellular uptake of apolipoprotein B-containing lipoproteins in the arterial intima leads to the formation of foam cells in atherosclerosis. Lysosomal acid lipase (LAL plays a crucial role in both lipoprotein lipid catabolism and excess lipid accumulation as it is the primary enzyme that hydrolyzes cholesteryl esters derived from both low density lipoprotein (LDL and modified forms of LDL. Evidence suggests that as atherosclerosis progresses, accumulation of excess free cholesterol in lysosomes leads to impairment of LAL activity, resulting in accumulation of cholesteryl esters in the lysosome as well as the cytosol in foam cells. Impaired metabolism and release of cholesterol from lysosomes can lead to downstream defects in ATP-binding cassette transporter A1 regulation, needed to offload excess cholesterol from plaque foam cells. This review focuses on the role LAL plays in normal cholesterol metabolism and how the associated changes in its enzymatic activity may ultimately contribute to atherosclerosis progression.

  13. Lysosome dysfunction enhances oxidative stress-induced apoptosis through ubiquitinated protein accumulation in Hela cells.

    Science.gov (United States)

    Yu, Chunyan; Huang, Xiaowei; Xu, Ye; Li, Hongyan; Su, Jing; Zhong, Jiateng; Kang, Jinsong; Liu, Yuhe; Sun, Liankun

    2013-01-01

    The role of lysosomal system in oxidative stress-induced apoptosis in cancer cells is not fully understood. Menadione is frequently used as oxidative stress model. It is indicated that menadione could induce autophagy in Hela cells. In the present study, we examined whether the lysosomal inhibitor, ammonium chloride (NH(4)Cl) could prevent the autophagy flux by inhibiting the fusion of autophagosomes with lysosomes and enhance apoptosis induced by menadione via mitochondrial pathway. The results demonstrated generation and accumulation of reactive oxygen species and increased levels of ubiquitinated proteins and GRP78 in cells treated with both menadione and NH(4)Cl. Our data indicates that lysosomal system through autophagy plays an important role in preventing menadione-induced apoptosis in Hela cells by clearing misfolded proteins, which alleviates endoplasmic reticulum stress.

  14. Less Is More: Substrate Reduction Therapy for Lysosomal Storage Disorders

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    Maria Francisca Coutinho

    2016-07-01

    Full Text Available Lysosomal storage diseases (LSDs are a group of rare, life-threatening genetic disorders, usually caused by a dysfunction in one of the many enzymes responsible for intralysosomal digestion. Even though no cure is available for any LSD, a few treatment strategies do exist. Traditionally, efforts have been mainly targeting the functional loss of the enzyme, by injection of a recombinant formulation, in a process called enzyme replacement therapy (ERT, with no impact on neuropathology. This ineffectiveness, together with its high cost and lifelong dependence is amongst the main reasons why additional therapeutic approaches are being (and have to be investigated: chaperone therapy; gene enhancement; gene therapy; and, alternatively, substrate reduction therapy (SRT, whose aim is to prevent storage not by correcting the original enzymatic defect but, instead, by decreasing the levels of biosynthesis of the accumulating substrate(s. Here we review the concept of substrate reduction, highlighting the major breakthroughs in the field and discussing the future of SRT, not only as a monotherapy but also, especially, as complementary approach for LSDs.

  15. Sowing Density: A Neglected Factor Fundamentally Affecting Root Distribution and Biomass Allocation of Field Grown Spring Barley (Hordeum Vulgare L.).

    Science.gov (United States)

    Hecht, Vera L; Temperton, Vicky M; Nagel, Kerstin A; Rascher, Uwe; Postma, Johannes A

    2016-01-01

    Studies on the function of root traits and the genetic variation in these traits are often conducted under controlled conditions using individual potted plants. Little is known about root growth under field conditions and how root traits are affected by agronomic practices in particular sowing density. We hypothesized that with increasing sowing density, root length density (root length per soil volume, cm cm(-3)) increases in the topsoil as well as specific root length (root length per root dry weight, cm g(-1)) due to greater investment in fine roots. Therefore, we studied two spring barley cultivars at ten different sowing densities (24-340 seeds m(-2)) in 2 consecutive years in a clay loam field in Germany and established sowing density dose-response curves for several root and shoot traits. We took soil cores for measuring roots up to a depth of 60 cm in and between plant rows (inter-row distance 21 cm). Root length density increased with increasing sowing density and was greatest in the plant row in the topsoil (0-10 cm). Greater sowing density increased specific root length partly through greater production of fine roots in the topsoil. Rooting depth (D50) of the major root axes (root diameter class 0.4-1.0 mm) was not affected. Root mass fraction decreased, while stem mass fraction increased with sowing density and over time. Leaf mass fraction was constant over sowing density but greater leaf area was realized through increased specific leaf area. Considering fertilization, we assume that light competition caused plants to grow more shoot mass at the cost of investment into roots, which is partly compensated by increased specific root length and shallow rooting. Increased biomass per area with greater densities suggest that density increases the efficiency of the cropping system, however, declines in harvest index at densities over 230 plants m(-2) suggest that this efficiency did not translate into greater yield. We conclude that plant density is a

  16. Reduction of mutant huntingtin accumulation and toxicity by lysosomal cathepsins D and B in neurons

    OpenAIRE

    Ouyang Xiaosen; Liang Qiuli; Schneider Lonnie; Zhang Jianhua

    2011-01-01

    Abstract Background Huntington's disease is caused by aggregation of mutant huntingtin (mHtt) protein containing more than a 36 polyQ repeat. Upregulation of macroautophagy was suggested as a neuroprotective strategy to degrade mutant huntingtin. However, macroautophagy initiation has been shown to be highly efficient in neurons whereas lysosomal activities are rate limiting. The role of the lysosomal and other proteases in Huntington is not clear. Some studies suggest that certain protease a...

  17. Action of polystyrene nanoparticles of different sizes on lysosomal function and integrity

    OpenAIRE

    Fröhlich Eleonore; Meindl Claudia; Roblegg Eva; Ebner Birgit; Absenger Markus; Pieber Thomas R

    2012-01-01

    Abstract Background Data from environmental exposure to nanoparticles (NPs) suggest that chronic exposure may increase the incidence of lung, cardiovascular and neurodegenerative diseases. Impairment of cell function by intracellular accumulation of NPs is also suspected. Many types of NPs have been detected in the endosomal-lysosomal system and, upon repeated exposure, alterations of the endosomal-lysosomal system may occur. To identify such effects we compared the effect of carboxyl polysty...

  18. Lysosomal responses to heat-shock of seasonal temperature extremes in Cd-exposed mussels.

    Science.gov (United States)

    Múgica, M; Izagirre, U; Marigómez, I

    2015-07-01

    The present study was aimed at determining the effect of temperature extremes on lysosomal biomarkers in mussels exposed to a model toxic pollutant (Cd) at different seasons. For this purpose, temperature was elevated 10°C (from 12°C to 22°C in winter and from 18°C to 28°C in summer) for a period of 6h (heat-shock) in control and Cd-exposed mussels, and then returned back to initial one. Lysosomal membrane stability and lysosomal structural changes in digestive gland were investigated. In winter, heat-shock reduced the labilisation period (LP) of the lysosomal membrane, especially in Cd-exposed mussels, and provoked transient lysosomal enlargement. LP values recovered after the heat-shock cessation but lysosomal enlargement prevailed in both experimental groups. In summer, heat-shock induced remarkable reduction in LP and lysosomal enlargement (more markedly in Cd-exposed mussels), which recovered within 3 days. Besides, whilst heat-shock effects on LP were practically identical for Cd-exposed mussels in winter and summer, the effects were longer-lasting in summer than in winter for control mussels. Thus, lysosomal responsiveness after heat-shock was higher in summer than in winter but recovery was faster as well, and therefore the consequences of the heat shock seem to be more decisive in winter. In contrast, inter-season differences were attenuated in the presence of Cd. Consequently, mussels seem to be better prepared in summer than in winter to stand short periods of abrupt temperature change; this is, however, compromised when mussels are exposed to pollutants such as Cd.

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

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    Vasileios A Stamelos

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

  20. Observation of intracellular interactions between DNA origami and lysosomes by the fluorescence localization method.

    Science.gov (United States)

    Fu, Meifang; Dai, Luru; Jiang, Qiao; Tang, Yunqing; Zhang, Xiaoming; Ding, Baoquan; Li, Junbai

    2016-07-28

    We obtained the fluorescence localization images of tube DNA origami nanostructures in NIH 3T3 cells for the first time. The fluorescence localization images of tube DNA origami nanostructures and TIRF images of lysosomes were combined and they revealed the detailed interactions between the two structures. Quantitative analysis illustrated that the tube origami can be captured as well as degraded by lysosomes with time.

  1. Streptococcus oralis Induces Lysosomal Impairment of Macrophages via Bacterial Hydrogen Peroxide.

    Science.gov (United States)

    Okahashi, Nobuo; Nakata, Masanobu; Kuwata, Hirotaka; Kawabata, Shigetada

    2016-07-01

    Streptococcus oralis, an oral commensal, belongs to the mitis group of streptococci and occasionally causes opportunistic infections, such as bacterial endocarditis and bacteremia. Recently, we found that the hydrogen peroxide (H2O2) produced by S. oralis is sufficient to kill human monocytes and epithelial cells, implying that streptococcal H2O2 is a cytotoxin. In the present study, we investigated whether streptococcal H2O2 impacts lysosomes, organelles of the intracellular digestive system, in relation to cell death. S. oralis infection induced the death of RAW 264 macrophages in an H2O2-dependent manner, which was exemplified by the fact that exogenous H2O2 also induced cell death. Infection with either a mutant lacking spxB, which encodes pyruvate oxidase responsible for H2O2 production, or Streptococcus mutans, which does not produce H2O2, showed less cytotoxicity. Visualization of lysosomes with LysoTracker revealed lysosome deacidification after infection with S. oralis or exposure to H2O2, which was corroborated by acridine orange staining. Similarly, fluorescent labeling of lysosome-associated membrane protein-1 gradually disappeared during infection with S. oralis or exposure to H2O2 The deacidification and the following induction of cell death were inhibited by chelating iron in lysosomes. Moreover, fluorescent staining of cathepsin B indicated lysosomal destruction. However, treatment of infected cells with a specific inhibitor of cathepsin B had negligible effects on cell death; instead, it suppressed the detachment of dead cells from the culture plates. These results suggest that streptococcal H2O2 induces cell death with lysosomal destruction and then the released lysosomal cathepsins contribute to the detachment of the dead cells.

  2. Availability and temporal heterogeneity of water supply affect the vertical distribution and mortality of a belowground herbivore and consequently plant growth.

    Directory of Open Access Journals (Sweden)

    Tomonori Tsunoda

    Full Text Available We examined how the volume and temporal heterogeneity of water supply changed the vertical distribution and mortality of a belowground herbivore, and consequently affected plant biomass. Plantago lanceolata (Plantaginaceae seedlings were grown at one per pot under different combinations of water volume (large or small volume and heterogeneity (homogeneous water conditions, watered every day; heterogeneous conditions, watered every 4 days in the presence or absence of a larva of the belowground herbivorous insect, Anomala cuprea (Coleoptera: Scarabaeidae. The larva was confined in different vertical distributions to top feeding zone (top treatment, middle feeding zone (middle treatment, or bottom feeding zone (bottom treatment; alternatively no larva was introduced (control treatment or larval movement was not confined (free treatment. Three-way interaction between water volume, heterogeneity, and the herbivore significantly affected plant biomass. With a large water volume, plant biomass was lower in free treatment than in control treatment regardless of heterogeneity. Plant biomass in free treatment was as low as in top treatment. With a small water volume and in free treatment, plant biomass was low (similar to that under top treatment under homogeneous water conditions but high under heterogeneous ones (similar to that under middle or bottom treatment. Therefore, there was little effect of belowground herbivory on plant growth under heterogeneous water conditions. In other watering regimes, herbivores would be distributed in the shallow soil and reduced root biomass. Herbivore mortality was high with homogeneous application of a large volume or heterogeneous application of a small water volume. Under the large water volume, plant biomass was high in pots in which the herbivore had died. Thus, the combinations of water volume and heterogeneity affected plant growth via the change of a belowground herbivore.

  3. Distribution of hydroxyanthracene derivatives in Cassia alata and the factors affecting the quality of the raw material

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    Niwan Intaraksa

    2003-07-01

    Full Text Available Analyses have been carried out on the content of hydroxyanthracene derivatives of the leaves, flowers and pods of Cassia alata, which had been collected at different harvesting times and different leaf-positions. It was found that when the leaves had been harvested in March, June or September, the hydroxyanthracene derivatives were accumulated more in the leaf-positions 1-3 (1.82, 1.25, 1.63 %w/w, respectively and 4-6 (1.39, 1.58, 1.09 %w/w, respectively. In December (the flowering and fruiting season, hydroxyanthracene derivatives were accumulated more in the flowers (2.21%w/w and the pods (1.82 %w/w, respectively. The method and temperature of drying markedly affected the hydroxyanthracene derivative content. Drying of the leaves in a hot air oven at 50ºC gave a higher hydroxyanthracene derivative content (1.43 %w/w than drying in a hot air oven at 80ºC (0.44 %w/w or drying in the sun (0.95 %w/w. Study on the stability of hydroxyanthracene derivatives in C. alata leaf powder, which was kept in tight container at room temperature, found that the hydroxyanthracene derivative content did not decrease within 9 months.

  4. Updated distribution of Osmoderma eremita in Abruzzo (Italy and agro-pastoral practices affecting its conservation (Coleoptera: Scarabaeidae

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    Patrizia Giangregorio

    2015-12-01

    Full Text Available New records of Osmoderma eremita (Scopoli, 1763 (Coleoptera: Scarabaeidae: Cetoniinae are reported for Abruzzo (Italy, together with a review of its distribution in this region. O. eremita is a saproxylic beetle dependent on the presence of hollow deciduous trees with abundant wood mould in their cavities. The major threats for the species are habitat loss and fragmentation. EU Habitats Directive requests to the member States its protection and the monitoring of its conservation status. Detection of its occurrence is the first step to protect the species. The surveys have been carried out in ten sites of Abruzzo by using black cross-windows traps baited with specific pheromone. The species has been recorded for the first time in the Sant’Antonio forest and its presence is confirmed in the Peligna Valley, after a decade. The populations seem to be confined to small patches of suitable habitats. At local level, the abandonment of the pollarding practice (willow and beech forests and the use of pollarded trees as biomass for fuel are the major threats for this species. Indeed some key actions, such as the protection of old hollow trees and the continuation of pollarding practice in rural landscape, could be key factors for the conservation strategies of the species in the study area.

  5. The Role of Next-Generation Sequencing in the Diagnosis of Lysosomal Storage Disorders

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    Katalin Komlosi MD, PhD

    2016-10-01

    Full Text Available Next-generation sequencing (NGS panels are used widely in clinical diagnostics to identify genetic causes of various monogenic disease groups including neurometabolic disorders and, more recently, lysosomal storage disorders (LSDs. Many new challenges have been introduced through these new technologies, both at the laboratory level and at the bioinformatics level, with consequences including new requirements for interpretation of results, and for genetic counseling. We review some recent examples of the application of NGS technologies, with purely diagnostic and with both diagnostic and research aims, for establishing a rapid genetic diagnosis in LSDs. Given that NGS can be applied in a way that takes into account the many issues raised by international consensus guidelines, it can have a significant role even early in the course of the diagnostic process, in combination with biochemical and clinical data. Besides decreasing the delay in diagnosis for many patients, a precise molecular diagnosis is extremely important as new therapies are becoming available within the LSD spectrum for patients who share specific types of mutations. A genetic diagnosis is also the prerequisite for genetic counseling, family planning, and the individual choice of reproductive options in affected families.

  6. Reduction of mutant huntingtin accumulation and toxicity by lysosomal cathepsins D and B in neurons

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    Ouyang Xiaosen

    2011-06-01

    Full Text Available Abstract Background Huntington's disease is caused by aggregation of mutant huntingtin (mHtt protein containing more than a 36 polyQ repeat. Upregulation of macroautophagy was suggested as a neuroprotective strategy to degrade mutant huntingtin. However, macroautophagy initiation has been shown to be highly efficient in neurons whereas lysosomal activities are rate limiting. The role of the lysosomal and other proteases in Huntington is not clear. Some studies suggest that certain protease activities may contribute to toxicity whereas others are consistent with protection. These discrepancies may be due to a number of mechanisms including distinct effects of the specific intermediate digestion products of mutant huntingtin generated by different proteases. These observations suggested a critical need to investigate the consequence of upregulation of individual lysosomal enzyme in mutant huntingtin accumulation and toxicity. Results In this study, we used molecular approaches to enhance lysosomal protease activities and examined their effects on mutant huntingtin level and toxicity. We found that enhanced expression of lysosomal cathepsins D and B resulted in their increased enzymatic activities and reduced both full-length and fragmented huntingtin in transfected HEK cells. Furthermore, enhanced expression of cathepsin D or B protected against mutant huntingtin toxicity in primary neurons, and their neuroprotection is dependent on macroautophagy. Conclusions These observations demonstrate a neuroprotective effect of enhancing lysosomal cathepsins in reducing mutant huntingtin level and toxicity in transfected cells. They highlight the potential importance of neuroprotection mediated by cathepsin D or B through macroautophagy.

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

    Science.gov (United States)

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

    2015-11-01

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

  8. TMEM175 deficiency impairs lysosomal and mitochondrial function and increases α-synuclein aggregation

    Science.gov (United States)

    Jinn, Sarah; Drolet, Robert E.; Cramer, Paige E.; Wong, Andus Hon-Kit; Toolan, Dawn M.; Gretzula, Cheryl A.; Voleti, Bhavya; Vassileva, Galya; Disa, Jyoti; Tadin-Strapps, Marija; Stone, David J.

    2017-01-01

    Parkinson disease (PD) is a neurodegenerative disorder pathologically characterized by nigrostriatal dopamine neuron loss and the postmortem presence of Lewy bodies, depositions of insoluble α-synuclein, and other proteins that likely contribute to cellular toxicity and death during the disease. Genetic and biochemical studies have implicated impaired lysosomal and mitochondrial function in the pathogenesis of PD. Transmembrane protein 175 (TMEM175), the lysosomal K+ channel, is centered under a major genome-wide association studies peak for PD, making it a potential candidate risk factor for the disease. To address the possibility that variation in TMEM175 could play a role in PD pathogenesis, TMEM175 function was investigated in a neuronal model system. Studies confirmed that TMEM175 deficiency results in unstable lysosomal pH, which led to decreased lysosomal catalytic activity, decreased glucocerebrosidase activity, impaired autophagosome clearance by the lysosome, and decreased mitochondrial respiration. Moreover, TMEM175 deficiency in rat primary neurons resulted in increased susceptibility to exogenous α-synuclein fibrils. Following α-synuclein fibril treatment, neurons deficient in TMEM175 were found to have increased phosphorylated and detergent-insoluble α-synuclein deposits. Taken together, data from these studies suggest that TMEM175 plays a direct and critical role in lysosomal and mitochondrial function and PD pathogenesis and highlight this ion channel as a potential therapeutic target for treating PD. PMID:28193887

  9. Cross-talk between TRPML1 channel, lipids and lysosomal storage diseases.

    Science.gov (United States)

    Weiss, Norbert

    2012-03-01

    Described by the Belgian cytologist Christian De Duve in 1949,(1) lysosomes (from the Greek "digestive bodies") are ubiquitous specialized intracellular organelles that ensure the degradation/recycling of macromolecules (proteins, lipids, membranes) through the activity of specific enzymes (i.e., acid hydrolases). They receive their substrates through different internalization pathways (i.e., endocytosis, phagocytosis and autophagy) and are involved in a wide range of physiological functions from cell death and signaling to cholesterol homeostasis and plasma membrane repair.(2) In Mammals, 50 soluble lysosomal hydrolases have been described, each targeting specific substrates. They are confined in the lumen of the lysosome and require an optimum pH (i.e., pH 4.5) to work. This acidic pH compared with the slightly alkaline pH of the cytosol (i.e., ~pH 7.2) is maintained by the activity of integral lysosomal membrane proteins (LMPs, that represent the second class of lysosomal proteins), including the V-type proton (H(+))-ATPase(3) and the chloride ion channel CLC7(4) that pumps protons from the cytosol across the lysosomal membrane.

  10. IL-10 conditioning of human skin affects the distribution of migratory dendritic cell subsets and functional T cell differentiation.

    Science.gov (United States)

    Lindenberg, Jelle J; Oosterhoff, Dinja; Sombroek, Claudia C; Lougheed, Sinéad M; Hooijberg, Erik; Stam, Anita G M; Santegoets, Saskia J A M; Tijssen, Henk J; Buter, Jan; Pinedo, Herbert M; van den Eertwegh, Alfons J M; Scheper, Rik J; Koenen, Hans J P M; van de Ven, Rieneke; de Gruijl, Tanja D

    2013-01-01

    In cancer patients pervasive systemic suppression of Dendritic Cell (DC) differentiation and maturation can hinder vaccination efficacy. In this study we have extensively characterized migratory DC subsets from human skin and studied how their migration and T cell-stimulatory abilities were affected by conditioning of the dermal microenvironment through cancer-related suppressive cytokines. To assess effects in the context of a complex tissue structure, we made use of a near-physiological skin explant model. By 4-color flow cytometry, we identified migrated Langerhans Cells (LC) and five dermis-derived DC populations in differential states of maturation. From a panel of known tumor-associated suppressive cytokines, IL-10 showed a unique ability to induce predominant migration of an immature CD14(+)CD141(+)DC-SIGN(+) DC subset with low levels of co-stimulatory molecules, up-regulated expression of the co-inhibitory molecule PD-L1 and the M2-associated macrophage marker CD163. A similarly immature subset composition was observed for DC migrating from explants taken from skin overlying breast tumors. Whereas predominant migration of mature CD1a(+) subsets was associated with release of IL-12p70, efficient Th cell expansion with a Th1 profile, and expansion of functional MART-1-specific CD8(+) T cells, migration of immature CD14(+) DDC was accompanied by increased release of IL-10, poor expansion of CD4(+) and CD8(+) T cells, and skewing of Th responses to favor coordinated FoxP3 and IL-10 expression and regulatory T cell differentiation and outgrowth. Thus, high levels of IL-10 impact the composition of skin-emigrated DC subsets and appear to favor migration of M2-like immature DC with functional qualities conducive to T cell tolerance.

  11. In Absence of the Cellular Prion Protein, Alterations in Copper Metabolism and Copper-Dependent Oxidase Activity Affect Iron Distribution.

    Science.gov (United States)

    Gasperini, Lisa; Meneghetti, Elisa; Legname, Giuseppe; Benetti, Federico

    2016-01-01

    Essential elements as copper and iron modulate a wide range of physiological functions. Their metabolism is strictly regulated by cellular pathways, since dysregulation of metal homeostasis is responsible for many detrimental effects. Neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease and prion diseases are characterized by alterations of metal ions. These neurodegenerative maladies involve proteins that bind metals and mediate their metabolism through not well-defined mechanisms. Prion protein, for instance, interacts with divalent cations via multiple metal-binding sites and it modulates several metal-dependent physiological functions, such as S-nitrosylation of NMDA receptors. In this work we focused on the effect of prion protein absence on copper and iron metabolism during development and adulthood. In particular, we investigated copper and iron functional values in serum and several organs such as liver, spleen, total brain and isolated hippocampus. Our results show that iron content is diminished in prion protein-null mouse serum, while it accumulates in liver and spleen. Our data suggest that these alterations can be due to impairments in copper-dependent cerulopalsmin activity which is known to affect iron mobilization. In prion protein-null mouse total brain and hippocampus, metal ion content shows a fluctuating trend, suggesting the presence of homeostatic compensatory mechanisms. However, copper and iron functional values are likely altered also in these two organs, as indicated by the modulation of metal-binding protein expression levels. Altogether, these results reveal that the absence of the cellular prion protein impairs copper metabolism and copper-dependent oxidase activity, with ensuing alteration of iron mobilization from cellular storage compartments.

  12. IL-10 conditioning of human skin affects the distribution of migratory dendritic cell subsets and functional T cell differentiation.

    Directory of Open Access Journals (Sweden)

    Jelle J Lindenberg

    Full Text Available In cancer patients pervasive systemic suppression of Dendritic Cell (DC differentiation and maturation can hinder vaccination efficacy. In this study we have extensively characterized migratory DC subsets from human skin and studied how their migration and T cell-stimulatory abilities were affected by conditioning of the dermal microenvironment through cancer-related suppressive cytokines. To assess effects in the context of a complex tissue structure, we made use of a near-physiological skin explant model. By 4-color flow cytometry, we identified migrated Langerhans Cells (LC and five dermis-derived DC populations in differential states of maturation. From a panel of known tumor-associated suppressive cytokines, IL-10 showed a unique ability to induce predominant migration of an immature CD14(+CD141(+DC-SIGN(+ DC subset with low levels of co-stimulatory molecules, up-regulated expression of the co-inhibitory molecule PD-L1 and the M2-associated macrophage marker CD163. A similarly immature subset composition was observed for DC migrating from explants taken from skin overlying breast tumors. Whereas predominant migration of mature CD1a(+ subsets was associated with release of IL-12p70, efficient Th cell expansion with a Th1 profile, and expansion of functional MART-1-specific CD8(+ T cells, migration of immature CD14(+ DDC was accompanied by increased release of IL-10, poor expansion of CD4(+ and CD8(+ T cells, and skewing of Th responses to favor coordinated FoxP3 and IL-10 expression and regulatory T cell differentiation and outgrowth. Thus, high levels of IL-10 impact the composition of skin-emigrated DC subsets and appear to favor migration of M2-like immature DC with functional qualities conducive to T cell tolerance.

  13. In Absence of the Cellular Prion Protein, Alterations in Copper Metabolism and Copper-Dependent Oxidase Activity Affect Iron Distribution

    Science.gov (United States)

    Gasperini, Lisa; Meneghetti, Elisa; Legname, Giuseppe; Benetti, Federico

    2016-01-01

    Essential elements as copper and iron modulate a wide range of physiological functions. Their metabolism is strictly regulated by cellular pathways, since dysregulation of metal homeostasis is responsible for many detrimental effects. Neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease and prion diseases are characterized by alterations of metal ions. These neurodegenerative maladies involve proteins that bind metals and mediate their metabolism through not well-defined mechanisms. Prion protein, for instance, interacts with divalent cations via multiple metal-binding sites and it modulates several metal-dependent physiological functions, such as S-nitrosylation of NMDA receptors. In this work we focused on the effect of prion protein absence on copper and iron metabolism during development and adulthood. In particular, we investigated copper and iron functional values in serum and several organs such as liver, spleen, total brain and isolated hippocampus. Our results show that iron content is diminished in prion protein-null mouse serum, while it accumulates in liver and spleen. Our data suggest that these alterations can be due to impairments in copper-dependent cerulopalsmin activity which is known to affect iron mobilization. In prion protein-null mouse total brain and hippocampus, metal ion content shows a fluctuating trend, suggesting the presence of homeostatic compensatory mechanisms. However, copper and iron functional values are likely altered also in these two organs, as indicated by the modulation of metal-binding protein expression levels. Altogether, these results reveal that the absence of the cellular prion protein impairs copper metabolism and copper-dependent oxidase activity, with ensuing alteration of iron mobilization from cellular storage compartments. PMID:27729845

  14. Investigation of factors affecting the accumulation of vinyl chloride in polyvinyl chloride piping used in drinking water distribution systems.

    Science.gov (United States)

    Walter, Ryan K; Lin, Po-Hsun; Edwards, Marc; Richardson, Ruth E

    2011-04-01

    Plastic piping made of polyvinyl chloride (PVC), and chlorinated PVC (CPVC), is being increasingly used for drinking water distribution lines. Given the formulation of the material from vinyl chloride (VC), there has been concern that the VC (a confirmed human carcinogen) can leach from the plastic piping into drinking water. PVC/CPVC pipe reactors in the laboratory and tap samples collected from consumers homes (n = 15) revealed vinyl chloride accumulation in the tens of ng/L range after a few days and hundreds of ng/L after two years. While these levels did not exceed the EPA's maximum contaminant level (MCL) of 2 μg/L, many readings that simulated stagnation times in homes (overnight) exceeded the MCL-Goal of 0 μg/L. Considerable differences in VC levels were seen across different manufacturers, while aging and biofilm effects were generally small. Preliminary evidence suggests that VC may accumulate not only via chemical leaching from the plastic piping, but also as a disinfection byproduct (DBP) via a chlorine-dependent reaction. This is supported from studies with CPVC pipe reactors where chlorinated reactors accumulated more VC than dechlorinated reactors, copper pipe reactors that accumulated VC in chlorinated reactors and not in dechlorinated reactors, and field samples where VC levels were the same before and after flushing the lines where PVC/CPVC fittings were contributing. Free chlorine residual tests suggest that VC may be formed as a secondary, rather than primary, DBP. Further research and additional studies need to be conducted in order to elucidate reaction mechanisms and tease apart relative contributions of VC accumulation from PVC/CPVC piping and chlorine-dependent reactions.

  15. Cuspy no more: how outflows affect the central dark matter and baryon distribution in Λ cold dark matter galaxies

    Science.gov (United States)

    Governato, F.; Zolotov, A.; Pontzen, A.; Christensen, C.; Oh, S. H.; Brooks, A. M.; Quinn, T.; Shen, S.; Wadsley, J.

    2012-05-01

    We examine the evolution of the inner dark matter (DM) and baryonic density profile of a new sample of simulated field galaxies using fully cosmological, Λ cold dark matter (ΛCDM), high-resolution SPH+N-Body simulations. These simulations include explicit H2 and metal cooling, star formation (SF) and supernovae-driven gas outflows. Starting at high redshift, rapid, repeated gas outflows following bursty SF transfer energy to the DM component and significantly flatten the originally 'cuspy' central DM mass profile of galaxies with present-day stellar masses in the 104.5-109.8 M⊙ range. At z= 0, the central slope of the DM density profile of our galaxies (measured between 0.3 and 0.7 kpc from their centre) is well fitted by ρDM ∝ rα with α≃-0.5 + 0.35 log10(M★/108 M⊙), where M★ is the stellar mass of the galaxy and 4 < log M★ < 9.4. These values imply DM profiles flatter than those obtained in DM-only simulations and in close agreement with those inferred in galaxies from the THINGS and LITTLE THINGS surveys. Only in very small haloes, where by z= 0 SF has converted less than ˜0.03 per cent of the original baryon abundance into stars, outflows do not flatten the original cuspy DM profile out to radii resolved by our simulations. The mass (DM and baryonic) measured within the inner 500 pc of each simulated galaxy remains nearly constant over 4 orders of magnitudes in stellar mass for M★ < 109 M⊙. This finding is consistent with estimates for faint Local Group dwarfs and field galaxies. These results address one of the outstanding problems faced by the CDM model, namely the strong discrepancy between the original predictions of cuspy DM profiles and the shallower central DM distribution observed in galaxies.

  16. The second report of a new hypomyelinating disease due to a defect in the VPS11 gene discloses a massive lysosomal involvement.

    Science.gov (United States)

    Hörtnagel, Konstanze; Krägeloh-Mann, Inge; Bornemann, Antje; Döcker, Miriam; Biskup, Saskia; Mayrhofer, Heidi; Battke, Florian; du Bois, Gabriele; Harzer, Klaus

    2016-11-01

    Vesicular protein sorting-associated proteins (VPS, including VPS11) are indispensable in the endocytic network, in particular the endosome-lysosome biogenesis. Exome sequencing revealed the homozygous variant p.Leu387_ Gly395del in the VPS11 gene in two siblings. On immunoblotting, the mutant VPS11 protein showed a distinctly reduced immunostaining intensity. The children presented with primary and severe developmental delay associated with myoclonic seizures, spastic tetraplegia, trunk and neck hypotonia, blindness, hearing loss, and microcephaly. Neuro-imaging showed severe hypomyelination affecting cerebral and cerebellar white matter and corpus callosum, in the absence of a peripheral neuropathy. Electron microscopy of a skin biopsy revealed clusters of membranous cytoplasmic bodies in dermal unmyelinated nerve axons, and numbers of vacuoles in eccrine sweat glands, similar to what is seen in a classic lysosomal storage disease (LSD). Bone marrow cytology showed a high number of storage macrophages with a micro-vacuolated cytoplasm. Biochemically, changes in urinary glycosphingolipids were reminiscent of those in prosaposin deficiency (another LSD). The clinical and neuro-imaged features in our patients were almost identical to those in some recently reported patients with another variant in the VPS11 gene, p.Cys846Gly; underlining the presumed pathogenic potential of VPS11 defects. A new feature was the morphological evidence for lysosomal storage in VPS11 deficiency: This newly characterised disease can be viewed as belonging to the complex field of LSD.

  17. Enhancing lysosomal biogenesis and autophagic flux by activating the transcription factor EB protects against cadmium-induced neurotoxicity

    Science.gov (United States)

    Pi, Huifeng; Li, Min; Tian, Li; Yang, Zhiqi; Yu, Zhengping; Zhou, Zhou

    2017-01-01

    Cadmium (Cd), a highly ubiquitous heavy metal, is a well-known inducer of neurotoxicity. However, the mechanism underlying cadmium-induced neurotoxicity remains unclear. In this study, we found that Cd inhibits autophagosome-lysosome fusion and impairs lysosomal function by reducing the levels of lysosomal-associated membrane proteins, inhibiting lysosomal proteolysis and altering lysosomal pH, contributing to defects in autophagic clearance and subsequently leading to nerve cell death. In addition, Cd decreases transcription factor EB (TFEB) expression at both the mRNA and protein levels. Furthermore, Cd induces the nuclear translocation of TFEB and TFEB target-gene expression, associated with compromised lysosomal function or a compensatory effect after the impairment of the autophagic flux. Notably, restoration of the levels of lysosomal-associated membrane protein, lysosomal proteolysis, lysosomal pH and autophagic flux through Tfeb overexpression protects against Cd-induced neurotoxicity, and this protective effect is incompletely dependent on TFEB nuclear translocation. Moreover, gene transfer of the master autophagy regulator TFEB results in the clearance of toxic proteins and the correction of Cd-induced neurotoxicity in vivo. Our study is the first to demonstrate that Cd disrupts lysosomal function and autophagic flux and manipulation of TFEB signalling may be a therapeutic approach for antagonizing Cd-induced neurotoxicity. PMID:28240313

  18. LITAF mutations associated with Charcot-Marie-Tooth disease 1C show mislocalization from the late endosome/lysosome to the mitochondria.

    Directory of Open Access Journals (Sweden)

    Andressa Ferreira Lacerda

    Full Text Available Charcot-Marie-Tooth (CMT disease is one of the most common heritable neuromuscular disorders, affecting 1 in every 2500 people. Mutations in LITAF have been shown to be causative for CMT type 1C disease. In this paper we explore the subcellular localization of wild type LITAF and mutant forms of LITAF known to cause CMT1C (T49M, A111G, G112S, T115N, W116G, L122V and P135T. The results show that LITAF mutants A111G, G112S, W116G, and T115N mislocalize from the late endosome/lysosome to the mitochondria while the mutants T49M, L122V, and P135T show partial mislocalization with a portion of the total protein present in the late endosome/lysosome and the remainder of the protein localized to the mitochondria. This suggests that different mutants of LITAF will produce differing severity of disease. We also explored the effect of the presence of mutant LITAF on wild-type LITAF localization. We showed that in cells heterozygous for LITAF, CMT1C mutants T49M and G112S are dominant since wild-type LITAF localized to the mitochondria when co-transfected with a LITAF mutant. Finally, we demonstrated how LITAF transits to the endosome and mitochondria compartments of the cell. Using Brefeldin A to block ER to Golgi transport we demonstrated that wild type LITAF traffics through the secretory pathway to the late endosome/lysosome while the LITAF mutants transit to the mitochondria independent of the secretory pathway. In addition, we demonstrated that the C-terminus of LITAF is necessary and sufficient for targeting of wild-type LITAF to the late endosome/lysosome and the mutants to the mitochondria. Together these data provide insight into how mutations in LITAF cause CMT1C disease.

  19. Fucosylation of LAMP-1 and LAMP-2 by FUT1 correlates with lysosomal positioning and autophagic flux of breast cancer cells.

    Science.gov (United States)

    Tan, Keng-Poo; Ho, Ming-Yi; Cho, Huan-Chieh; Yu, John; Hung, Jung-Tung; Yu, Alice Lin-Tsing

    2016-08-25

    Alpha1,2-fucosyltransferases, FUT1 and FUT2, which transfer fucoses onto the terminal galactose of N-acetyl-lactosamine via α1,2-linkage have been shown to be highly expressed in various types of cancers. A few studies have shown the involvement of FUT1 substrates in tumor cell proliferation and migration. Lysosome-associated membrane protein 1, LAMP-1, has been reported to carry alpha1,2-fucosylated Lewis Y (LeY) antigens in breast cancer cells, however, the biological functions of LeY on LAMP-1 remain largely unknown. Whether or not its family member, LAMP-2, displays similar modifications and functions as LAMP-1 has not yet been addressed. In this study, we have presented evidence supporting that both LAMP-1 and 2 are substrates for FUT1, but not FUT2. We have also demonstrated the presence of H2 and LeY antigens on LAMP-1 by a targeted nanoLC-MS(3) and the decreased levels of fucosylation on LAMP-2 by MALDI-TOF analysis upon FUT1 knockdown. In addition, we found that the expression of LeY was substantial in less invasive ER+/PR+/HER- breast cancer cells (MCF-7 and T47D) but negligible in highly invasive triple-negative MDA-MB-231 cells, of which LeY levels were correlated with the levels of LeY carried by LAMP-1 and 2. Intriguingly, we also observed a striking change in the subcellular localization of lysosomes upon FUT1 knockdown from peripheral distribution of LAMP-1 and 2 to a preferential perinuclear accumulation. Besides that, knockdown of FUT1 led to an increased rate of autophagic flux along with diminished activity of mammalian target of rapamycin complex 1 (mTORC1) and enhanced autophagosome-lysosome fusion. This may be associated with the predominantly perinuclear distribution of lysosomes mediated by FUT1 knockdown as lysosomal positioning has been reported to regulate mTOR activity and autophagy. Taken together, our results suggest that downregulation of FUT1, which leads to the perinuclear localization of LAMP-1 and 2, is correlated with increased

  20. The Farmers Perception on Effectiveness of Private Forest Revolving Fund Distribution and Factors Affecting its Repayment: Case in South Lampung District, Lampung Province

    Directory of Open Access Journals (Sweden)

    Sanudin

    2016-04-01

    Full Text Available Commercial s are ed providing for forest plantation development bank not interest in fund community based . - Therefore, in this case, non bank institutions such Forest Development Funding Center (pusat pembiayaan pembangunan hutan, PPPH private forest are highly required. This paper is aimed to find out the effectiveness of revolving fund and factors affecting its repayment distribution . The research was conducted during September–December 2014 in Private Forest Farmer Groups in Katibung Sub-District South Lampung Dist 3 , , rict Lampung Province. The data was collected through household surveys and in-depth interviews. The household surveys were done using structured questionnaires that included questions related to: characteristics of the borrowers, characteristics of private forest, characteristics of loan, and household perceptions on private forest revolving fund Household perceptions on private forest revolving fund are loan . pre requirement, loan procedure, realization, interest rate, , and repayment procedure The effectiveness of private forest length of repayment periode . revolving fund d t and factors affecting repayment of loan was analyzed by istribution was analyzed by liker scale logistic regression. ult private forest revolving fund in The res showed that: 1 three private forest farmer groups in Katibung Sub-District, South Lampung effective District was 2 income from non-private forest and amount of loan , are factors affecting repayment of private forest revolving fund, 3 faced private forest revolving f the problem in und distribution PPPH private could be overcame by maximizing the role of field officers in assisting and facilitating forest revolving fund ors debit candidate.

  1. Distribution of free radicals and intermediates during the photodegradation of polychlorinated biphenyls strongly affected by cosolvents and TiO₂ catalyst.

    Science.gov (United States)

    Zhu, Xiangdong; Wang, Yujun; Qin, Wenxiu; Zhang, Shicheng; Zhou, Dongmei

    2016-02-01

    Polychlorinated biphenyls (PCBs) pose potential ecological risk because of their high toxicity and carcinogenicity. Photodegradation, which is an important process for the removal of PCBs, is greatly influenced by the cosolvent and catalyst. Hence, it is important to explore their effects on the photodegradation behavior of PCBs. In this study, 2,4,4'-trichlorobiphenyl (PCB28) was selected as a model compound, and the effects of two typical cosolvents, namely acetone and ethanol, and TiO2 catalyst on the distributions of free radicals and intermediates were investigated. Interestingly, the TiO2 catalyst did not promote PCB28 photodegradation. Moreover, the free radical distribution was greatly influenced in the presence of the TiO2 catalyst, while was only slightly affected in its absence by the cosolvent kinds. The main photodegradation pathways are proposed on the basis of the distribution of detected intermediates, which were significantly regulated by both the cosolvent and TiO2 catalyst. The results provide novel insights into the photodegradation of PCBs and may have important implications for choosing cosolvent in desorbing soil PCBs and consequently enhancing PCBs degradation.

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

    Science.gov (United States)

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

    2013-05-01

    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.

  3. The Phosphoinositide-Gated Lysosomal Ca(2+) Channel, TRPML1, Is Required for Phagosome Maturation.

    Science.gov (United States)

    Dayam, Roya M; Saric, Amra; Shilliday, Ryan E; Botelho, Roberto J

    2015-09-01

    Macrophages internalize and sequester pathogens into a phagosome. Phagosomes then sequentially fuse with endosomes and lysosomes, converting into degradative phagolysosomes. Phagosome maturation is a complex process that requires regulators of the endosomal pathway including the phosphoinositide lipids. Phosphatidylinositol-3-phosphate and phosphatidylinositol-3,5-bisphosphate (PtdIns(3,5)P2 ), which respectively control early endosomes and late endolysosomes, are both required for phagosome maturation. Inhibition of PIKfyve, which synthesizes PtdIns(3,5)P2 , blocked phagosome-lysosome fusion and abated the degradative capacity of phagosomes. However, it is not known how PIKfyve and PtdIns(3,5)P2 participate in phagosome maturation. TRPML1 is a PtdIns(3,5)P2 -gated lysosomal Ca(2+) channel. Because Ca(2+) triggers membrane fusion, we postulated that TRPML1 helps mediate phagosome-lysosome fusion. Using Fcγ receptor-mediated phagocytosis as a model, we describe our research showing that silencing of TRPML1 hindered phagosome acquisition of lysosomal markers and reduced the bactericidal properties of phagosomes. Specifically, phagosomes isolated from TRPML1-silenced cells were decorated with lysosomes that docked but did not fuse. We could rescue phagosome maturation in TRPML1-silenced and PIKfyve-inhibited cells by forcible Ca(2+) release with ionomycin. We also provide evidence that cytosolic Ca(2+) concentration increases upon phagocytosis in a manner dependent on TRPML1 and PIKfyve. Overall, we propose a model where PIKfyve and PtdIns(3,5)P2 activate TRPML1 to induce phagosome-lysosome fusion.

  4. Expression of the lysosomal-associated membrane protein-1 (LAMP-1) in astrocytomas.

    Science.gov (United States)

    Jensen, Stine S; Aaberg-Jessen, Charlotte; Christensen, Karina G; Kristensen, Bjarne

    2013-01-01

    Targeting of lysosomes is a novel therapeutic anti-cancer strategy for killing the otherwise apoptosis-resistant cancer cells. Such strategies are urgently needed for treatment of brain tumors, especially the glioblastoma, which is the most frequent and most malignant type. The aim of the present study was to investigate the presence of lysosomes in astrocytic brain tumors focussing also on the therapy resistant tumor stem cells. Expression of the lysosomal marker LAMP-1 (lysosomal-associated membrane protein-1) was investigated by immunohistochemistry in 112 formalin fixed paraffin embedded astrocytomas and compared with tumor grade and overall patient survival. Moreover, double immunofluorescence stainings were performed with LAMP-1 and the astrocytic marker GFAP and the putative stem cell marker CD133 on ten glioblastomas. Most tumors expressed the LAMP-1 protein in the cytoplasm of the tumor cells, while the blood vessels were positive in all tumors. The percentage of LAMP-1 positive tumor cells and staining intensities increased with tumor grade but variations in tumors of the same grade were also found. No association was found between LAMP-1 expression and patient overall survival in the individual tumor grades. LAMP-1/GFAP showed pronounced co-expression and LAMP-1/CD133 was co-expressed as well suggesting that tumor cells including the proposed tumor stem cells contain lysosomes. The results suggest that high amounts of lysosomes are present in glioblastomas and in the proposed tumor stem cells. Targeting of lysosomes may be a promising novel therapeutic strategy against this highly malignant neoplasm.

  5. Glucagon-like Peptide-1 Protects Pancreatic Beta-cells from Death by Increasing Autophagic Flux and Restoring Lysosomal Function.

    Science.gov (United States)

    Zummo, Francesco P; Cullen, Kirsty S; Honkanen-Scott, Minna; Shaw, James Am; Lovat, Penny E; Arden, Catherine

    2017-02-23

    Studies in animal models of type 2 diabetes have shown that glucagon-like peptide-1 (GLP-1) receptor agonists prevent β-cell loss. Whether GLP-1 mediates β-cell survival via the key lysosomal-mediated process of autophagy is unknown.Here we report that treatment of INS-1E β-cells and primary islets with glucolipotoxicity (0.5mmol/l palmitate, 25mmol/l glucose) increases LC3 II, a marker of autophagy. Further analysis indicates a blockage in autophagic flux associated with lysosomal dysfunction. Accumulation of defective lysosomes leads to lysosomal membrane permeabilisation (LMP) and release of Cathepsin D, which contributes to cell death. Our data further demonstrated defects in autophagic flux and lysosomal staining in human samples of type 2 diabetes. Co-treatment with the GLP-1 receptor agonist exendin-4 reversed the lysosomal dysfunction, relieving the impairment in autophagic flux and further stimulated autophagy. siRNA knockdown showed the restoration of autophagic flux is also essential for the protective effects of exendin-4.Collectively, our data highlights lysosomal dysfunction as a critical mediator of β-cell loss and shows that exendin-4 improves cell survival via restoration of lysosomal function and autophagic flux. Modulation of autophagy / lysosomal homeostasis may thus define a novel therapeutic strategy for type 2 diabetes, with the GLP-1 signalling pathway as a potential focus.

  6. Evidence for lysosomal exocytosis and release of aggrecan-degrading hydrolases from hypertrophic chondrocytes, in vitro and in vivo

    Directory of Open Access Journals (Sweden)

    Edward R. Bastow

    2012-02-01

    The abundant proteoglycan, aggrecan, is resorbed from growth plate cartilage during endochondral bone ossification, yet mice with genetically-ablated aggrecan-degrading activity have no defects in bone formation. To account for this apparent anomaly, we propose that lysosomal hydrolases degrade extracellular, hyaluronan-bound aggrecan aggregates in growth plate cartilage, and that lysosomal hydrolases are released from hypertrophic chondrocytes into growth plate cartilage via Ca2+-dependent lysosomal exocytosis. In this study we confirm that hypertrophic chondrocytes release hydrolases via lysosomal exocytosis in vitro and we show in vivo evidence for lysosomal exocytosis in hypertrophic chondrocytes during skeletal development. We show that lysosome-associated membrane protein 1 (LAMP1 is detected at the cell surface following in vitro treatment of epiphyseal chondrocytes with the calcium ionophore, ionomycin. Furthermore, we show that in addition to the lysosomal exocytosis markers, cathepsin D and β-hexosaminidase, ionomycin induces release of aggrecan- and hyaluronan-degrading activity from cultured epiphyseal chondrocytes. We identify VAMP-8 and VAMP7 as v-SNARE proteins with potential roles in lysosomal exocytosis in hypertrophic chondrocytes, based on their colocalisation with LAMP1 at the cell surface in secondary ossification centers in mouse tibiae. We propose that resorbing growth plate cartilage involves release of destructive hydrolases from hypertrophic chondrocytes, via lysosomal exocytosis.

  7. Not nanocarbon but dispersant induced abnormality in lysosome in macrophages in vivo

    Science.gov (United States)

    Yudasaka, Masako; Zhang, Minfang; Matsumura, Sachiko; Yuge, Ryota; Ichihashi, Toshinari; Irie, Hiroshi; Shiba, Kiyotaka; Iijima, Sumio

    2015-05-01

    The properties of nanocarbons change from hydrophobic to hydrophilic as a result of coating them with dispersants, typically phospholipid polyethylene glycols, for biological studies. It has been shown that the dispersants remain attached to the nanocarbons when they are injected in mice and influence the nanocarbons’ biodistribution in vivo. We show in this report that the effects of dispersants also appear at the subcellular level in vivo. Carbon nanohorns (CNHs), a type of nanocarbon, were dispersed with ceramide polyethylene glycol (CPEG) and intravenously injected in mice. Histological observations and electron microscopy with energy dispersive x-ray analysis revealed that, in liver and spleen, the lysosome membranes were damaged, and the nanohorns formed a complex with hemosiderin in the lysosomes of the macrophages. It is inferred that the lysosomal membrane was damaged by sphigosine generated as a result of CPEG decomposition, which changed the intra lysosomal conditions, inducing the formation of the CPEG-CNH and hemosiderin complex. For comparison, when glucose was used instead of CPEG, neither the nanohorn-hemosiderin complex nor lysosomal membrane damage was found. Our results suggest that surface functionalization can control the behavior of nancarbons in cells in vivo and thereby improve their suitability for medical applications.

  8. Color reduction of melanin by lysosomal and peroxisomal enzymes isolated from mammalian cells.

    Science.gov (United States)

    Park, Dong Jun; Sekhon, Simranjeet Singh; Yoon, Jihee; Kim, Yang-Hoon; Min, Jiho

    2016-02-01

    Lysosomes and peroxisomes are organelles with many functions in all eukaryotic cells. Lysosomes contain hydrolytic enzymes (lysozyme) that degrade molecules, whereas peroxisomes contain enzymes such as catalase that convert hydrogen peroxide (H2O2) to water and oxygen and neutralize toxicity. In contrast, melanin is known as a helpful element to protect the skin against harmful ultraviolet rays. However, a high quantity of melanin leads to hyperpigmentation or skin cancer in human. New materials have already been discovered to inhibit tyrosinase in melanogenesis; however, melanin reduction does not suggest their preparation. In this study, we report that the color intensity because of melanin decreased by the cellular activation of lysosomes and peroxisomes. An increase in the superficial intensity of lysosome and peroxisome activities of HeLa cells was observed. In addition, a decrease in the amount of melanin has also been observed in mammalian cells without using any other chemical, showing that the process can work in vivo for treating melanin. Therefore, the results of this study indicate that the amount of melanin decreases by the lysosome and peroxisome activity after entering the cells, and functional organelles are effective in color reduction. This mechanism can be used in vivo for treating melanin.

  9. LAMP-2 is required for incorporating syntaxin-17 into autophagosomes and for their fusion with lysosomes.

    Science.gov (United States)

    Hubert, Virginie; Peschel, Andrea; Langer, Brigitte; Gröger, Marion; Rees, Andrew; Kain, Renate

    2016-10-15

    Autophagy is an evolutionarily conserved process used for removing surplus and damaged proteins and organelles from the cytoplasm. The unwanted material is incorporated into autophagosomes that eventually fuse with lysosomes, leading to the degradation of their cargo. The fusion event is mediated by the interaction between the Qa-SNARE syntaxin-17 (STX17) on autophagosomes and the R-SNARE VAMP8 on lysosomes. Cells deficient in lysosome membrane-associated protein-2 (LAMP-2) have increased numbers of autophagosomes but the underlying mechanism is poorly understood. By transfecting LAMP-2-deficient and LAMP-1/2--double-deficient mouse embryonic fibroblasts (MEFs) with a tandem fluorescent-tagged LC3 we observed a failure of fusion between the autophagosomes and the lysosomes that could be rescued by complementation with LAMP-2A. Although we observed no change in expression and localization of VAMP8, its interacting partner STX17 was absent from autophagosomes of LAMP-2-deficient cells. Thus, LAMP-2 is essential for STX17 expression by the autophagosomes and this absence is sufficient to explain their failure to fuse with lysosomes. The results have clear implications for situations associated with a reduction of LAMP-2 expression.

  10. Disruption of lysosome function promotes tumor growth and metastasis in Drosophila.

    Science.gov (United States)

    Chi, Congwu; Zhu, Huanhu; Han, Min; Zhuang, Yuan; Wu, Xiaohui; Xu, Tian

    2010-07-09

    Lysosome function is essential to many physiological processes. It has been suggested that deregulation of lysosome function could contribute to cancer. Through a genetic screen in Drosophila, we have discovered that mutations disrupting lysosomal degradation pathway components contribute to tumor development and progression. Loss-of-function mutations in the Class C vacuolar protein sorting (VPS) gene, deep orange (dor), dramatically promote tumor overgrowth and invasion of the Ras(V12) cells. Knocking down either of the two other components of the Class C VPS complex, carnation (car) and vps16A, also renders Ras(V12) cells capable for uncontrolled growth and metastatic behavior. Finally, chemical disruption of the lysosomal function by feeding animals with antimalarial drugs, chloroquine or monensin, leads to malignant tumor growth of the Ras(V12) cells. Taken together, our data provide evidence for a causative role of lysosome dysfunction in tumor growth and invasion and indicate that members of the Class C VPS complex behave as tumor suppressors.

  11. The movement and distribution of Helicoverpa armigera (Hübner) larvae on pea plants is affected by egg placement and flowering.

    Science.gov (United States)

    Perkins, L E; Cribb, B W; Hanan, J; Zalucki, M P

    2010-10-01

    The distribution and movement of 1st instar Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) larvae on whole garden pea (Pisum sativum L.) plants were determined in glasshouse trials. This economically-important herbivore attacks a wide variety of agricultural, horticultural and indigenous plants. To investigate the mechanisms underlying larval intra-plant movement, we used early-flowering and wild-type plant genotypes and placed eggs at different vertical heights within the plants, one egg per plant. Leaf water and nitrogen content and cuticle hardness were measured at the different plant heights. Of 92 individual larvae, 41% did not move from the node of eclosion, 49% moved upwards and 10% moved downwards with the distance moved being between zero and ten plant nodes. Larvae from eggs placed on the lower third of the plant left the natal leaf more often and moved further than larvae from eggs placed in the middle or upper thirds. The low nutritive value of leaves was the most likely explanation for more movement away from lower plant regions. Although larvae on flowering plants did not move further up or down than larvae on non-flowering plants, they more often departed the leaflet (within a leaf) where they eclosed. The final distribution of larvae was affected by plant genotype, with larvae on flowering plants found less often on leaflets and more often on stipules, tendrils and reproductive structures. Understanding intra-plant movement by herbivorous insects under natural conditions is important because such movement determines the value of economic loss to host crops. Knowing the behaviour underlying the spatial distribution of herbivores on plants will assist us to interpret field data and should lead to better informed pest management decisions.

  12. Analysis of the factors that affect the distribution and abundance of three Neobuxbaumia species (Cactaceae) that differ in their degree of rarity

    Science.gov (United States)

    Ruedas, Marcela; Valverde, Teresa; Zavala-Hurtado, José Alejandro

    2006-03-01

    We studied three species of columnar cacti in the genus Neobuxbaumia which differ in their degree of rarity: Neobuxbaumia macrocephala (the rarest), Neobuxbaumia tetetzo (intermediate), and Neobuxbaumia mezcalaensis (the most common). To investigate the ecological factors that limit their distribution and abundance, we surveyed 80 localities within the region of Tehuacan-Cuicatlán, in Central Mexico. At each locality we measured several environmental variables, and the density of the Neobuxbaumia populations present. We used a principal component analysis (PCA) to identify the factors that are associated to the presence/absence of each species. Additionally, we carried out multiple regressions between environmental variables and population density to test whether the variation in these variables was related to changes in abundance. The results show that factors significantly affecting the distribution of these species are mean annual temperature, altitude, rainfall, and soil properties such as texture and organic matter content. N. mezcalaensis reaches maximum population densities of 14,740 plants per ha (average density = 3943 plants per ha) and is associated with localities with relatively abundant rainfall. N. tetetzo shows maximum population densities of 14,060 plants per ha (average = 3070 plants per ha), and is associated with sites located at high latitudes and with high phosphorous content in the soil. The rarest species, N. macrocephala, shows maximum densities of 1180 plants per ha (average = 607 plants per ha) and is associated with localities with high soil calcium content. The distribution of this species is limited to sites with specific values of the environmental variables recorded, conferring it a high habitat specificity which accounts for its rarity.

  13. Characterization of two-pore channel 2 (TPCN2)-mediated Ca2+ currents in isolated lysosomes.

    Science.gov (United States)

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

    2010-07-01

    Two-pore channels (TPCNs) have been proposed to form lysosomal Ca(2+) release channels that are activated by nicotinic acid adenine dinucleotide phosphate. Here, we employ a glass chip-based method to record for the first time nicotinic acid adenine dinucleotide phosphate -dependent currents through a two-pore channel (TPCN2) from intact lysosomes. We show that TPCN2 is a highly selective Ca(2+) channel that is regulated by intralysosomal pH. Using site-directed mutagenesis, we identify an amino acid residue in the putative pore region that is crucial for conferring high Ca(2+) selectivity. Our glass chip-based method will provide electrophysiological access not only to lysosomal TPCN channels but also to a broad range of other intracellular ion channels.

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

    Science.gov (United States)

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

    2010-01-01

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

  15. Guanidinylated neomycin mediates heparan sulfate-dependent transport of active enzymes to lysosomes.

    Science.gov (United States)

    Sarrazin, Stéphane; Wilson, Beth; Sly, William S; Tor, Yitzhak; Esko, Jeffrey D

    2010-07-01

    Guanidinylated neomycin (GNeo) can transport bioactive, high molecular weight cargo into the interior of cells in a process that depends on cell surface heparan sulfate proteoglycans. In this report, we show that GNeo-modified quantum dots bind to cell surface heparan sulfate, undergo endocytosis and eventually reach the lysosomal compartment. An N-hydroxysuccinimide activated ester of GNeo (GNeo-NHS) was prepared and conjugated to two lysosomal enzymes, beta-D-glucuronidase (GUS) and alpha-L-iduronidase. Conjugation did not interfere with enzyme activity and enabled binding of the enzymes to heparin-Sepharose and heparan sulfate on primary human fibroblasts. Cells lacking the corresponding lysosomal enzyme took up sufficient amounts of the conjugated enzymes to restore normal turnover of glycosaminoglycans. The high capacity of proteoglycan-mediated uptake suggests that this method of delivery might be used for enzyme replacement or introduction of foreign enzymes into cells.

  16. Elimination of paternal mitochondria through the lysosomal degradation pathway in C.elegans

    Institute of Scientific and Technical Information of China (English)

    Qinghua Zhou; Haimin Li; Ding Xue

    2011-01-01

    In mammals,the inheritance of mitochondrion and its DNA (mtDNA) is strictly maternal,despite the fact that a sperm can inject up to 100 functional mitochondria into the oocyte during fertilization.The mechanisms responsible for the elimination of the paternal mitochondria remain largely unknown.We report here that this paternal mitochondrial elimination process is conserved in Caenorhabditis elegans,and that the lysosomal pathway actively participates in this process.Molecular and cell biological analyses indicate that in wild-type animals paternal mitoehondria and mtDNA are destroyed within two hours after fertilization.In animals with compromised lysosomes,paternal mitochondria persist until late embryonic stages.Therefore,the lysosomal pathway plays an important role in degrading paternal mitochondria introduced into the oocyte during fertilization.Our study indicates that C.elegans is an excellent animal model for understanding and dissecting this conserved biological process critical for animal development and reproduction.

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

    Energy Technology Data Exchange (ETDEWEB)

    Zeng, Jibin; Racicott, Jesse [Department of Anatomy and Cell Biology, McGill University, Montreal (Canada); Morales, Carlos R., E-mail: carlos.morales@mcgill.ca [Department of Anatomy and Cell Biology, McGill University, Montreal (Canada)

    2009-11-01

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

  18. Lysosomal and mitochondrial permeabilization mediates zinc(II) cationic phthalocyanine phototoxicity.

    Science.gov (United States)

    Marino, Julieta; García Vior, María C; Furmento, Verónica A; Blank, Viviana C; Awruch, Josefina; Roguin, Leonor P

    2013-11-01

    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

  19. Lysosomal exoglycosidases in serum and urine of patients with pancreatic adenocarcinoma.

    Directory of Open Access Journals (Sweden)

    Anna Stypułkowska

    2010-11-01

    Full Text Available Lysosomal exoglycosidases: N-acetyl-β-D-hexosaminidase (HEX, β-D-galactosidase (GAL, ι-L-fucosidase (FUC and ι-D-mannosidase (MAN modify oligosaccharide chains of glycoconjugates in endoplasmatic reticulum and/or Golgi apparatus and degrade them in lysosomes. In acid environment of lysosome, exoglycosidases degrade oligosaccharide chains of glycoproteins, glycolipids and glycosaminoglycans by eliminating single sugars from the edges of oligosaccharide chains. Neoplasms change biochemical processes in tissues and may significantly change the activity of many enzymes including the activity of lysosomal exoglycosidasses in serum and urine of persons with neoplasmatic diseases. The aim of the present paper was evaluation the activity of HEX, GAL, FUC and MAN in serum and urine of patients with pancreatic adenocarcinoma. Serum and urine samples were collected from 15 patients with adenocarcinoma of the pancreas and 15 healthy persons. The activity of lysosomal exoglycosidases was determined by the method of Marciniak et al. adapted to serum and urine of patients with adenocarcinoma of the pancreas. Our results indicate significant decrease in activity of GAL (p=0.037 in serum of patients with pancreatic adenocarcinoma, significant increase in activity of HEX (p<0.001 and FUC (p=0.027 in serum, and HEX (p=0.003 in urine, as well as significant decrease of FUC (p=0.016 and MAN (p=0.029 in urine o patients with adenocarcinoma of the pancreas, in comparison to the control group. Increase in activity of some lysosomal enzymes in serum and urine of pancreatic adenocarcinoma patients, may indicate on destruction of pancreatic tissue by pancreatic adenocarcinoma. Determination of the HEX, GAL, FUC and MAN in serum and urine may be useful in diagnostics of pancreatic adenocarcinoma.

  20. Cytosolic peroxidases protect the lysosome of bloodstream African trypanosomes from iron-mediated membrane damage.

    Directory of Open Access Journals (Sweden)

    Corinna Hiller

    2014-04-01

    Full Text Available African trypanosomes express three virtually identical non-selenium glutathione peroxidase (Px-type enzymes which preferably detoxify lipid-derived hydroperoxides. As shown previously, bloodstream Trypanosoma brucei lacking the mitochondrial Px III display only a weak and transient proliferation defect whereas parasites that lack the cytosolic Px I and Px II undergo extremely fast lipid peroxidation and cell lysis. The phenotype can completely be rescued by supplementing the medium with the α-tocopherol derivative Trolox. The mechanism underlying the rapid cell death remained however elusive. Here we show that the lysosome is the origin of the cellular injury. Feeding the px I-II knockout parasites with Alexa Fluor-conjugated dextran or LysoTracker in the presence of Trolox yielded a discrete lysosomal staining. Yet upon withdrawal of the antioxidant, the signal became progressively spread over the whole cell body and was completely lost, respectively. T. brucei acquire iron by endocytosis of host transferrin. Supplementing the medium with iron or transferrin induced, whereas the iron chelator deferoxamine and apo-transferrin attenuated lysis of the px I-II knockout cells. Immunofluorescence microscopy with MitoTracker and antibodies against the lysosomal marker protein p67 revealed that disintegration of the lysosome precedes mitochondrial damage. In vivo experiments confirmed the negligible role of the mitochondrial peroxidase: Mice infected with px III knockout cells displayed only a slightly delayed disease development compared to wild-type parasites. Our data demonstrate that in bloodstream African trypanosomes, the lysosome, not the mitochondrion, is the primary site of oxidative damage and cytosolic trypanothione/tryparedoxin-dependent peroxidases protect the lysosome from iron-induced membrane peroxidation. This process appears to be closely linked to the high endocytic rate and distinct iron acquisition mechanisms of the infective

  1. Lysosomal membrane stability of the mussel, Mytilus galloprovincialis (L.), as a biomarker of tributyltin exposure.

    Science.gov (United States)

    Okoro, Hussein K; Snyman, Reinette G; Fatoki, Olalekan S; Adekola, Folahan A; Ximba, Bhekumusa J; Slabber, Michelle Y

    2015-05-01

    The effect of tributyltin (TBT) on the stability of hemocytic lysosome membranes of the mussel, Mytilus galloprovincialis, and the use thereof as a biomarker of TBT-induced stress, was investigated. Mussels were exposed to 0.1 and 1.0 µg/L tributyltin respectively for 4 weeks. Lysosomal membrane stability of hemocytes was tested weekly by means of the neutral red retention time (NRRT) assay, after which the mussel samples were analyzed for TBT content. The two exposed groups exhibited significantly increased (p galloprovincialis.

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

    OpenAIRE

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

    2010-01-01

    Two-pore channels (TPCNs) have been proposed to form lysosomal Ca2+ release channels that are activated by nicotinic acid adenine dinucleotide phosphate. Here, we employ a glass chip-based method to record for the first time nicotinic acid adenine dinucleotide phosphate -dependent currents through a two-pore channel (TPCN2) from intact lysosomes. We show that TPCN2 is a highly selective Ca2+ channel that is regulated by intralysosomal pH. Using site-directed mutagenesis, we identify an amino ...

  3. Impact of lysosomal storage disorders on biology of mesenchymal stem cells: Evidences from in vitro silencing of glucocerebrosidase (GBA) and alpha-galactosidase A (GLA) enzymes.

    Science.gov (United States)

    Squillaro, Tiziana; Antonucci, Ivana; Alessio, Nicola; Esposito, Anna; Cipollaro, Marilena; Melone, Mariarosa Anna Beatrice; Peluso, Gianfranco; Stuppia, Liborio; Galderisi, Umberto

    2017-01-18

    Lysosomal storage disorders (LDS) comprise a group of rare multisystemic diseases resulting from inherited gene mutations that impair lysosomal homeostasis. The most common LSDs, Gaucher disease (GD), and Fabry disease (FD) are caused by deficiencies in the lysosomal glucocerebrosidase (GBA) and alpha-galactosidase A (GLA) enzymes, respectively. Given the systemic nature of enzyme deficiency, we hypothesized that the stem cell compartment of GD and FD patients might be also affected. Among stem cells, mesenchymal stem cells (MSCs) are a commonly investigated population given their role in hematopoiesis and the homeostatic maintenance of many organs and tissues. Since the impairment of MSC functions could pose profound consequences on body physiology, we evaluated whether GBA and GLA silencing could affect the biology of MSCs isolated from bone marrow and amniotic fluid. Those cell populations were chosen given the former's key role in organ physiology and the latter's intriguing potential as an alternative stem cell model for human genetic disease. Our results revealed that GBA and GLA deficiencies prompted cell cycle arrest along with the impairment of autophagic flux and an increase of apoptotic and senescent cell percentages. Moreover, an increase in ataxia-telangiectasia-mutated staining 1 hr after oxidative stress induction and a return to basal level at 48 hr, along with persistent gamma-H2AX staining, indicated that MSCs properly activated DNA repair signaling, though some damages remained unrepaired. Our data therefore suggest that MSCs with reduced GBA or GLA activity are prone to apoptosis and senescence due to impaired autophagy and DNA repair capacity.

  4. Therapeutic effects of remediating autophagy failure in a mouse model of Alzheimer disease by enhancing lysosomal proteolysis.

    Science.gov (United States)

    Yang, Dun-Sheng; Stavrides, Philip; Mohan, Panaiyur S; Kaushik, Susmita; Kumar, Asok; Ohno, Masuo; Schmidt, Stephen D; Wesson, Daniel W; Bandyopadhyay, Urmi; Jiang, Ying; Pawlik, Monika; Peterhoff, Corrinne M; Yang, Austin J; Wilson, Donald A; St George-Hyslop, Peter; Westaway, David; Mathews, Paul M; Levy, Efrat; Cuervo, Ana M; Nixon, Ralph A

    2011-07-01

    The extensive autophagic-lysosomal pathology in Alzheimer disease (AD) brain has revealed a major defect: in the proteolytic clearance of autophagy substrates. Autophagy failure contributes on several levels to AD pathogenesis and has become an important therapeutic target for AD and other neurodegenerative diseases. We recently observed broad therapeutic effects of stimulating autophagic-lysosomal proteolysis in the TgCRND8 mouse model of AD that exhibits defective proteolytic clearance of autophagic substrates, robust intralysosomal amyloid-β peptide (Aβ) accumulation, extracellular β-amyloid deposition and cognitive deficits. By genetically deleting the lysosomal cysteine protease inhibitor, cystatin B (CstB), to selectively restore depressed cathepsin activities, we substantially cleared Aβ, ubiquitinated proteins and other autophagic substrates from autolysosomes/lysosomes and rescued autophagic-lysosomal pathology, as well as reduced total Aβ40/42 levels and extracellular amyloid deposition, highlighting the underappreciated importance of the lysosomal system for Aβ clearance. Most importantly, lysosomal remediation prevented the marked learning and memory deficits in TgCRND8 mice. Our findings underscore the pathogenic significance of autophagic-lysosomal dysfunction in AD and demonstrate the value of reversing this dysfunction as an innovative therapeautic strategy for AD.

  5. Iron content and acid phosphatase activity in hepatic parenchymal lysosomes of patients with hemochromatosis before and after phlebotomy treatment

    Energy Technology Data Exchange (ETDEWEB)

    Cleton, M.I.; de Bruijn, W.C.; van Blokland, W.T.; Marx, J.J.; Roelofs, J.M.; Rademakers, L.H.

    1988-03-01

    Lysosomal structures in liver parenchymal cells of 3 patients with iron overload and of 3 subjects without iron-storage disorders were investigated. A combination of enzyme cytochemistry--with cerium as a captive ion to demonstrate lysosomal acid phosphatase activity--and electron probe X-ray microanalysis (EPMA) was used. We were able (1) to define and quantify lysosomal structures as lysosomes, siderosomes, or residual bodies, (2) to quantify the amount of iron and cerium simultaneously in these structures, and (3) to evaluate a possible relation between iron storage and enzyme activity. With histopathologically increased iron storage, the number of siderosomes had increased at the cost of lysosomes, with a corresponding increase in acid phosphatase activity in both organelles. In histopahtologically severe iron overload, however, acid phosphatase activity was low or not detectable and most of the iron was stored in residual bodies. After phlebotomy treatment, the number of siderosomes had decreased in favor of the lysosomes, approaching values obtained in control subjects, and acid phosphatase activity was present in all iron-containing structures. In this way a relationship between iron storage and enzyme activity was established. The iron content of the individual lysosomal structures per unit area had increased with histopathologically increased iron storage and had decreased after phlebotomy treatment. From this observation, it is concluded that the iron status of the patient is not only reflected by the amount of iron-containing hepatocytes but, as well, by the iron content lysosomal unit area.

  6. Partial correction of the CNS lysosomal storage defect in a mouse model of juvenile neuronal ceroid lipofuscinosis by neonatal CNS administration of an adeno-associated virus serotype rh.10 vector expressing the human CLN3 gene.

    Science.gov (United States)

    Sondhi, Dolan; Scott, Emma C; Chen, Alvin; Hackett, Neil R; Wong, Andrew M S; Kubiak, Agnieszka; Nelvagal, Hemanth R; Pearse, Yewande; Cotman, Susan L; Cooper, Jonathan D; Crystal, Ronald G

    2014-03-01

    Juvenile neuronal ceroid lipofuscinosis (JNCL or CLN3 disease) is an autosomal recessive lysosomal storage disease resulting from mutations in the CLN3 gene that encodes a lysosomal membrane protein. The disease primarily affects the brain with widespread intralysosomal accumulation of autofluorescent material and fibrillary gliosis, as well as the loss of specific neuronal populations. As an experimental treatment for the CNS manifestations of JNCL, we have developed a serotype rh.10 adeno-associated virus vector expressing the human CLN3 cDNA (AAVrh.10hCLN3). We hypothesized that administration of AAVrh.10hCLN3 to the Cln3(Δex7/8) knock-in mouse model of JNCL would reverse the lysosomal storage defect, as well as have a therapeutic effect on gliosis and neuron loss. Newborn Cln3(Δex7/8) mice were administered 3 × 10(10) genome copies of AAVrh.10hCLN3 to the brain, with control groups including untreated Cln3(Δex7/8) mice and wild-type littermate mice. After 18 months, CLN3 transgene expression was detected in various locations throughout the brain, particularly in the hippocampus and deep anterior cortical regions. Changes in the CNS neuronal lysosomal accumulation of storage material were assessed by immunodetection of subunit C of ATP synthase, luxol fast blue staining, and periodic acid-Schiff staining. For all parameters, Cln3(Δex7/8) mice exhibited abnormal lysosomal accumulation, but AAVrh.10hCLN3 administration resulted in significant reductions in storage material burden. There was also a significant decrease in gliosis in AAVrh.10hCLN3-treated Cln3(Δex7/8) mice, and a trend toward improved neuron counts, compared with their untreated counterparts. These data demonstrate that AAVrh.10 delivery of a wild-type cDNA to the CNS is not harmful and instead provides a partial correction of the neurological lysosomal storage defect of a disease caused by a lysosomal membrane protein, indicating that this may be an effective therapeutic strategy for JNCL and

  7. Low pH affects survival, growth, size distribution, and carapace quality of the postlarvae and early juveniles of the freshwater prawn Macrobrachium rosenbergii de Man

    Science.gov (United States)

    Kawamura, Gunzo; Bagarinao, Teodora; Yong, Annita Seok Kian; Chen, Chiau Yu; Noor, Siti Norasidah Mat; Lim, Leong Seng

    2015-06-01

    Acidification of rain water caused by air pollutants is now recognized as a serious threat to aquatic ecosystems. We examined the effects of low pH (control pH 7.5, pH 6, pH 5, pH 4) on the survival, growth, and shell quality of Macrobrachium rosenbergii postlarvae and early juveniles in the laboratory. Hatcheryproduced postlarvae (PL 5) were stocked at 250 PL per aquarium, acclimated over 7 d to experimental pH adjusted with hydrochloric acid, and reared for 30 d. Dead specimens were removed and counted twice a day. After 27 d rearing, all specimens were measured for total length and body weight. Carapace quality was assessed by spectrophotometry. Survival of juveniles was highest at pH 6 (binomial 95% confidence interval 79 - 89%) followed by control pH 7.5 (56 - 68%) and pH 5 (50 - 60%) and was lowest for unmetamorphosed postlarvae and juveniles at pH 4 (43 - 49%). The final median total length and body weight of juveniles were similar at control pH 7.5 (18.2 TL, 50.2 mg BW) and pH 6 (17.7 mm TL, 45.0 mg BW) but significantly less at pH 5 (16.7 mm TL, 38.2 mg BW); at pH 4, the postlarvae did not metamorphose and measured only 9.8 mm TL, 29.3 mg BW. Length frequency distribution showed homogeneous growth at pH 6, positive skew at control pH 7.5 and pH 5, and extreme heterogeneity at pH 4. The carapace showed different transmittance spectra and lower total transmittance (i.e. thicker carapace) in juveniles at pH 7.5, pH 6, and pH 5 than in unmetamorphosed postlarvae and juveniles with thinner carapace at pH 4. Thus, survival, growth, size distribution, and carapace quality of M. rosenbergii postlarvae and early juveniles were negatively affected by pH 5 and especially pH 4. The thinner carapace of the survivors at pH 4 was mostly due to their small size and failure to metamorphose. Natural waters affected by acid rain could decimate M. rosenbergii populations in the wild.

  8. Distribution of polychlorinated biphenyls in an urban riparian zone affected by wastewater treatment plant effluent and the transfer to terrestrial compartment by invertebrates

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Junchao [State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing, 100085 (China); Environment Research Institute, Shandong University, Jinan, 250100 (China); Wang, Thanh, E-mail: bswang@rcees.ac.cn [State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing, 100085 (China); Han, Shanlong [Environment Research Institute, Shandong University, Jinan, 250100 (China); Wang, Pu; Zhang, Qinghua; Jiang, Guibin [State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing, 100085 (China)

    2013-10-01

    In this study, we investigated the distribution of polychlorinated biphenyls (PCBs) in a riparian zone affected by the effluent from a wastewater treatment plant (WWTP). River water, sediment, aquatic invertebrates and samples from the surrounding terrestrial compartment such as soil, reed plants and several land based invertebrates were collected. A relatively narrow range of δ{sup 13}C values was found among most invertebrates (except butterflies, grasshoppers), indicating a similar energy source. The highest concentration of total PCBs was observed in zooplankton (151.1 ng/g lipid weight), and soil dwelling invertebrates showed higher concentrations than phytophagous insects at the riparian zone. The endobenthic oligochaete Tubifex tubifex (54.28 ng/g lw) might be a useful bioindicator of WWTP derived PCBs contamination. High bioaccumulation factors (BAFs) were observed in collected aquatic invertebrates, although the biota-sediment/soil accumulation factors (BSAF) remained relatively low. Emerging aquatic insects such as chironomids could carry waterborne PCBs to the terrestrial compartment via their lifecycles. The estimated annual flux of PCBs for chironomids ranged from 0.66 to 265 ng⋅m{sup −2}⋅y{sup −1}. Although a high prevalence of PCB-11 and PCB-28 was found for most aquatic based samples in this riparian zone, the mid-chlorinated congeners (e.g. PCB-153 and PCB-138) became predominant among chironomids and dragonflies as well as soil dwelling invertebrates, which might suggest a selective biodriven transfer of different PCB congeners. Highlights: • The distribution of PCBs in an urban riparian zone around a wastewater effluent affected river was investigated. • Relatively high abundances of PCB-11 and PCB-28 were found for most samples. • Mid-chlorinated congeners (PCB-153 and PCB-138) were more accumulated in chironomids and dragonflies as well as soil dwelling invertebrates. • Emerging invertebrates can carry waterborne PCBs to the

  9. The Possible "Proton Sponge " Effect of Polyethylenimine (PEI) Does Not Include Change in Lysosomal pH

    DEFF Research Database (Denmark)

    Søndergaard, Rikke Vicki; Mattebjerg, Maria Ahlm; Henriksen, Jonas Rosager;

    2013-01-01

    is still elusive. The "proton sponge " hypothesis remains the most generally accepted mechanism, although it is heavily debated. This hypothesis is associated with the large buffering capacity of PEI and other polycations, which has been interpreted to cause an increase in lysosomal pH even though...... no conclusive proof has been provided. In the present study, we have used a nanoparticle pH sensor that was developed for pH measurements in the endosomal/lysosomal pathway. We have carried out quantitative measurements of lysosomal pH as a function of PEI content and correlate the results to the "proton sponge...... " hypothesis. Our measurements show that PEI does not induce change in lysosomal pH as previously suggested and quantification of PEI concentrations in lysosomes makes it uncertain that the "proton sponge " effect is the dominant mechanism of polyplex escape.Molecular Therapy (2012); doi:10.1038/mt.2012.185....

  10. Role of lysosomal enzymes released by alveolar macrophages in the pathogenesis of the acute phase of hypersensitivity pneumonitis

    Directory of Open Access Journals (Sweden)

    J. L. Pérez-Arellano

    1995-01-01

    Full Text Available Hydrolytic enzymes are the major constituents of alveolar macrophages (AM and have been shown to be involved in many aspects of the inflammatory pulmonary response. The aim of this study was to evaluate the role of lysosomal enzymes in the acute phase of hypersensitivity pneumonitis (HPs. An experimental study on AM lysosomal enzymes of an HP-guinea-pig model was performed. The results obtained both in vivo and in vitro suggest that intracellular enzymatic activity decrease is, at least partly, due to release of lysosomal enzymes into the medium. A positive but slight correlation was found between extracellular lysosomal activity and four parameters of lung lesion (lung index, bronchoalveolar fluid total (BALF protein concentration, BALF LDH and BALF alkaline phosphatase activities. All the above findings suggest that the AM release of lysosomal enzymes during HP is a factor involved, although possibly not the only one, in the pulmonary lesions appearing in this disease.

  11. In Vivo Evidence for Lysosome Depletion and Impaired Autophagic Clearance in Hereditary Spastic Paraplegia Type SPG11.

    Directory of Open Access Journals (Sweden)

    Rita-Eva Varga

    2015-08-01

    Full Text Available Hereditary spastic paraplegia (HSP is characterized by a dying back degeneration of corticospinal axons which leads to progressive weakness and spasticity of the legs. SPG11 is the most common autosomal-recessive form of HSPs and is caused by mutations in SPG11. A recent in vitro study suggested that Spatacsin, the respective gene product, is needed for the recycling of lysosomes from autolysosomes, a process known as autophagic lysosome reformation. The relevance of this observation for hereditary spastic paraplegia, however, has remained unclear. Here, we report that disruption of Spatacsin in mice indeed causes hereditary spastic paraplegia-like phenotypes with loss of cortical neurons and Purkinje cells. Degenerating neurons accumulate autofluorescent material, which stains for the lysosomal protein Lamp1 and for p62, a marker of substrate destined to be degraded by autophagy, and hence appears to be related to autolysosomes. Supporting a more generalized defect of autophagy, levels of lipidated LC3 are increased in Spatacsin knockout mouse embryonic fibrobasts (MEFs. Though distinct parameters of lysosomal function like processing of cathepsin D and lysosomal pH are preserved, lysosome numbers are reduced in knockout MEFs and the recovery of lysosomes during sustained starvation impaired consistent with a defect of autophagic lysosome reformation. Because lysosomes are reduced in cortical neurons and Purkinje cells in vivo, we propose that the decreased number of lysosomes available for fusion with autophagosomes impairs autolysosomal clearance, results in the accumulation of undegraded material and finally causes death of particularly sensitive neurons like cortical motoneurons and Purkinje cells in knockout mice.

  12. In Vivo Evidence for Lysosome Depletion and Impaired Autophagic Clearance in Hereditary Spastic Paraplegia Type SPG11.

    Science.gov (United States)

    Varga, Rita-Eva; Khundadze, Mukhran; Damme, Markus; Nietzsche, Sandor; Hoffmann, Birgit; Stauber, Tobias; Koch, Nicole; Hennings, J Christopher; Franzka, Patricia; Huebner, Antje K; Kessels, Michael M; Biskup, Christoph; Jentsch, Thomas J; Qualmann, Britta; Braulke, Thomas; Kurth, Ingo; Beetz, Christian; Hübner, Christian A

    2015-08-01

    Hereditary spastic paraplegia (HSP) is characterized by a dying back degeneration of corticospinal axons which leads to progressive weakness and spasticity of the legs. SPG11 is the most common autosomal-recessive form of HSPs and is caused by mutations in SPG11. A recent in vitro study suggested that Spatacsin, the respective gene product, is needed for the recycling of lysosomes from autolysosomes, a process known as autophagic lysosome reformation. The relevance of this observation for hereditary spastic paraplegia, however, has remained unclear. Here, we report that disruption of Spatacsin in mice indeed causes hereditary spastic paraplegia-like phenotypes with loss of cortical neurons and Purkinje cells. Degenerating neurons accumulate autofluorescent material, which stains for the lysosomal protein Lamp1 and for p62, a marker of substrate destined to be degraded by autophagy, and hence appears to be related to autolysosomes. Supporting a more generalized defect of autophagy, levels of lipidated LC3 are increased in Spatacsin knockout mouse embryonic fibrobasts (MEFs). Though distinct parameters of lysosomal function like processing of cathepsin D and lysosomal pH are preserved, lysosome numbers are reduced in knockout MEFs and the recovery of lysosomes during sustained starvation impaired consistent with a defect of autophagic lysosome reformation. Because lysosomes are reduced in cortical neurons and Purkinje cells in vivo, we propose that the decreased number of lysosomes available for fusion with autophagosomes impairs autolysosomal clearance, results in the accumulation of undegraded material and finally causes death of particularly sensitive neurons like cortical motoneurons and Purkinje cells in knockout mice.

  13. In Vivo Evidence for Lysosome Depletion and Impaired Autophagic Clearance in Hereditary Spastic Paraplegia Type SPG11

    Science.gov (United States)

    Varga, Rita-Eva; Khundadze, Mukhran; Damme, Markus; Nietzsche, Sandor; Hoffmann, Birgit; Stauber, Tobias; Koch, Nicole; Hennings, J. Christopher; Franzka, Patricia; Huebner, Antje K.; Kessels, Michael M.; Biskup, Christoph; Jentsch, Thomas J.; Qualmann, Britta; Braulke, Thomas; Kurth, Ingo; Beetz, Christian; Hübner, Christian A.

    2015-01-01

    Hereditary spastic paraplegia (HSP) is characterized by a dying back degeneration of corticospinal axons which leads to progressive weakness and spasticity of the legs. SPG11 is the most common autosomal-recessive form of HSPs and is caused by mutations in SPG11. A recent in vitro study suggested that Spatacsin, the respective gene product, is needed for the recycling of lysosomes from autolysosomes, a process known as autophagic lysosome reformation. The relevance of this observation for hereditary spastic paraplegia, however, has remained unclear. Here, we report that disruption of Spatacsin in mice indeed causes hereditary spastic paraplegia-like phenotypes with loss of cortical neurons and Purkinje cells. Degenerating neurons accumulate autofluorescent material, which stains for the lysosomal protein Lamp1 and for p62, a marker of substrate destined to be degraded by autophagy, and hence appears to be related to autolysosomes. Supporting a more generalized defect of autophagy, levels of lipidated LC3 are increased in Spatacsin knockout mouse embryonic fibrobasts (MEFs). Though distinct parameters of lysosomal function like processing of cathepsin D and lysosomal pH are preserved, lysosome numbers are reduced in knockout MEFs and the recovery of lysosomes during sustained starvation impaired consistent with a defect of autophagic lysosome reformation. Because lysosomes are reduced in cortical neurons and Purkinje cells in vivo, we propose that the decreased number of lysosomes available for fusion with autophagosomes impairs autolysosomal clearance, results in the accumulation of undegraded material and finally causes death of particularly sensitive neurons like cortical motoneurons and Purkinje cells in knockout mice. PMID:26284655

  14. TPC1 Has Two Variant Isoforms, and Their Removal Has Different Effects on Endo-Lysosomal Functions Compared to Loss of TPC2

    OpenAIRE

    Ruas, Margarida; Chuang, Kai-Ting; Davis, Lianne C.; Al-Douri, Areej; Tynan, Patricia W.; Tunn, Ruth; Teboul, Lydia; Galione, Antony; Parrington, John

    2014-01-01

    Organelle ion homeostasis within the endo-lysosomal system is critical for physiological functions. Two-pore channels (TPCs) are cation channels that reside in endo-lysosomal organelles, and overexpression results in endo-lysosomal trafficking defects. However, the impact of a lack of TPC expression on endo-lysosomal trafficking is unknown. Here, we characterize Tpcn1 expression in two transgenic mouse lines (Tpcn1 XG716 and Tpcn1 T159) and show expression of a novel evolutionarily conserved ...

  15. Phototoxic effects of lysosome-associated genetically encoded photosensitizer KillerRed

    Science.gov (United States)

    Serebrovskaya, Ekaterina O.; Ryumina, Alina P.; Boulina, Maria E.; Shirmanova, Marina V.; Zagaynova, Elena V.; Bogdanova, Ekaterina A.; Lukyanov, Sergey A.; Lukyanov, Konstantin A.

    2014-07-01

    KillerRed is a unique phototoxic red fluorescent protein that can be used to induce local oxidative stress by green-orange light illumination. Here we studied phototoxicity of KillerRed targeted to cytoplasmic surface of lysosomes via fusion with Rab7, a small GTPase that is known to be attached to membranes of late endosomes and lysosomes. It was found that lysosome-associated KillerRed ensures efficient light-induced cell death similar to previously reported mitochondria- and plasma membrane-localized KillerRed. Inhibitory analysis demonstrated that lysosomal cathepsins play an important role in the manifestation of KillerRed-Rab7 phototoxicity. Time-lapse monitoring of cell morphology, membrane integrity, and nuclei shape allowed us to conclude that KillerRed-Rab7-mediated cell death occurs via necrosis at high light intensity or via apoptosis at lower light intensity. Potentially, KillerRed-Rab7 can be used as an optogenetic tool to direct target cell populations to either apoptosis or necrosis.

  16. Lysosomal-associated transmembrane protein 5 (LAPTM5 is a molecular partner of CD1e.

    Directory of Open Access Journals (Sweden)

    Catherine Angénieux

    Full Text Available The CD1e protein participates in the presentation of lipid antigens in dendritic cells. Its transmembrane precursor is transported to lysosomes where it is cleaved into an active soluble form. In the presence of bafilomycin, which inhibits vacuolar ATPase and consequently the acidification of endosomal compartments, CD1e associates with a 27 kD protein. In this work, we identified this molecular partner as LAPTM5. The latter protein and CD1e colocalize in trans-Golgi and late endosomal compartments. The quantity of LAPTM5/CD1e complexes increases when the cells are treated with bafilomycin, probably due to the protection of LAPTM5 from lysosomal proteases. Moreover, we could demonstrate that LAPTM5/CD1e association occurs under physiological conditions. Although LAPTM5 was previously shown to act as a platform recruiting ubiquitin ligases and facilitating the transport of receptors to lysosomes, we found no evidence that LATPM5 controls either CD1e ubiquitination or the generation of soluble lysosomal CD1e proteins. Notwithstanding these last observations, the interaction of LAPTM5 with CD1e and their colocalization in antigen processing compartments both suggest that LAPTM5 might influence the role of CD1e in the presentation of lipid antigens.

  17. Lysosomal Enzyme Glucocerebrosidase Protects against Aβ1-42 Oligomer-Induced Neurotoxicity

    Science.gov (United States)

    Kam, Tae-In; Yun, Seungpil; Kim, Sangjune; Park, Hyejin; Hwang, Heehong; Pletnikova, Olga; Troncoso, Juan C.; Dawson, Valina L.; Dawson, Ted M.; Ko, Han Seok

    2015-01-01

    Glucocerebrosidase (GCase) functions as a lysosomal enzyme and its mutations are known to be related to many neurodegenerative diseases, including Gaucher’s disease (GD), Parkinson’s disease (PD), and Dementia with Lewy Bodies (DLB). However, there is little information about the role of GCase in the pathogenesis of Alzheimer’s disease (AD). Here we demonstrate that GCase protein levels and enzyme activity are significantly decreased in sporadic AD. Moreover, Aβ1–42 oligomer treatment results in neuronal cell death that is concomitant with decreased GCase protein levels and enzyme activity, as well as impairment in lysosomal biogenesis and acidification. Importantly, overexpression of GCase promotes the lysosomal degradation of Aβ1–42 oligomers, restores the lysosomal impairment, and protects against the toxicity in neurons treated with Aβ1–42 oligomers. Our findings indicate that a deficiency of GCase could be involved in progression of AD pathology and suggest that augmentation of GCase activity may be a potential therapeutic option for the treatment of AD. PMID:26629917

  18. Lysosomal Enzyme Glucocerebrosidase Protects against Aβ1-42 Oligomer-Induced Neurotoxicity.

    Directory of Open Access Journals (Sweden)

    Seulah Choi

    Full Text Available Glucocerebrosidase (GCase functions as a lysosomal enzyme and its mutations are known to be related to many neurodegenerative diseases, including Gaucher's disease (GD, Parkinson's disease (PD, and Dementia with Lewy Bodies (DLB. However, there is little information about the role of GCase in the pathogenesis of Alzheimer's disease (AD. Here we demonstrate that GCase protein levels and enzyme activity are significantly decreased in sporadic AD. Moreover, Aβ1-42 oligomer treatment results in neuronal cell death that is concomitant with decreased GCase protein levels and enzyme activity, as well as impairment in lysosomal biogenesis and acidification. Importantly, overexpression of GCase promotes the lysosomal degradation of Aβ1-42 oligomers, restores the lysosomal impairment, and protects against the toxicity in neurons treated with Aβ1-42 oligomers. Our findings indicate that a deficiency of GCase could be involved in progression of AD pathology and suggest that augmentation of GCase activity may be a potential therapeutic option for the treatment of AD.

  19. Transcriptional control of the autophagy-lysosome system in pancreatic cancer

    Science.gov (United States)

    Perera, Rushika M.; Stoykova, Svetlana; Nicolay, Brandon N.; Ross, Kenneth N.; Fitamant, Julien; Boukhali, Myriam; Lengrand, Justine; Deshpande, Vikram; Selig, Martin K.; Ferrone, Cristina R.; Settleman, Jeff; Stephanopoulos, Gregory; Dyson, Nicholas J.; Zoncu, Roberto; Ramaswamy, Sridhar; Haas, Wilhelm; Bardeesy, Nabeel

    2016-01-01

    Activation of cellular stress response pathways to maintain metabolic homeostasis is emerging as a critical growth and survival mechanism in many cancers1. The pathogenesis of pancreatic ductal adenocarcinoma (PDA) requires high levels of autophagy2–4, a conserved self-degradative process5. However, the regulatory circuits that activate autophagy and reprogram PDA cell metabolism are unknown. We now show that autophagy induction in PDA occurs as part of a broader transcriptional program that coordinates activation of lysosome biogenesis and function, and nutrient scavenging, mediated by the MiT/TFE family transcription factors. In PDA cells, the MiT/TFE proteins6 – MITF, TFE3 and TFEB – are decoupled from regulatory mechanisms that control their cytoplasmic retention. Increased nuclear import in turn drives the expression of a coherent network of genes that induce high levels of lysosomal catabolic function essential for PDA growth. Unbiased global metabolite profiling reveals that MiT/TFE-dependent autophagy-lysosomal activation is specifically required to maintain intracellular amino acid (AA) pools. These results identify the MiT/TFE transcription factors as master regulators of metabolic reprogramming in pancreatic cancer and demonstrate activation of clearance pathways converging on the lysosome as a novel hallmark of aggressive malignancy. PMID:26168401

  20. Two-photon fluorescence probes for imaging of mitochondria and lysosomes.

    Science.gov (United States)

    Yang, Wanggui; Chan, Pui Shan; Chan, Miu Shan; Li, King Fai; Lo, Pik Kwan; Mak, Nai Ki; Cheah, Kok Wai; Wong, Man Shing

    2013-04-28

    Novel biocompatible cyanines show not only a very large two-photon cross-section of up to 5130 GM at 910 nm in aqueous medium for high-contrast and -brightness two-photon fluorescence live cell imaging but also highly selective subcellular localization properties including localization of mitochondria and lysosomes.

  1. Histopathologic correlates of radial stripes on MR images in lysosomal storage disorders.

    NARCIS (Netherlands)

    Voorn, J.P. van der; Pouwels, P.J.; Kamphorst, W.; Powers, J.M.; Lammens, M.M.Y.; Barkhof, F.; Knaap, M.S. van der

    2005-01-01

    BACKGROUND AND PURPOSE: Radially oriented hypointense stripes in hyperintense cerebral white matter are recognized on T2-weighted images of certain lysosomal storage disorders. We compared in vivo and postmortem MR imaging with histopathologic findings in three patients with metachromatic leukodystr

  2. Brucella suis-Impaired Specific Recognition of Phagosomes by Lysosomes due to Phagosomal Membrane Modifications

    Science.gov (United States)

    Naroeni, Aroem; Jouy, Nicolas; Ouahrani-Bettache, Safia; Liautard, Jean-Pierre; Porte, Françoise

    2001-01-01

    Brucella species are gram-negative, facultatively intracellular bacteria that infect humans and animals. These organisms can survive and replicate within a membrane-bound compartment in phagocytic and nonprofessional phagocytic cells. Inhibition of phagosome-lysosome fusion has been proposed as a mechanism for intracellular survival in both types of cells. However, the biochemical mechanisms and microbial factors implicated in Brucella maturation are still completely unknown. We developed two different approaches in an attempt to gain further insight into these mechanisms: (i) a fluorescence microscopy analysis of general intracellular trafficking on whole cells in the presence of Brucella and (ii) a flow cytometry analysis of in vitro reconstitution assays showing the interaction between Brucella suis-containing phagosomes and lysosomes. The fluorescence microscopy results revealed that fusion properties of latex bead-containing phagosomes with lysosomes were not modified in the presence of live Brucella suis in the cells. We concluded that fusion inhibition was restricted to the pathogen phagosome and that the host cell fusion machinery was not altered by the presence of live Brucella in the cell. By in vitro reconstitution experiments, we observed a specific association between killed B. suis-containing phagosomes and lysosomes, which was dependent on exogenously supplied cytosol, energy, and temperature. This association was observed with killed bacteria but not with live bacteria. Hence, this specific recognition inhibition seemed to be restricted to the pathogen phagosomal membrane, as noted in the in vivo experiments. PMID:11119541

  3. Cancer cell injury by cytotoxins from cobra venom is mediated through lysosomal damage.

    Science.gov (United States)

    Feofanov, Alexei V; Sharonov, George V; Astapova, Maria V; Rodionov, Dmitriy I; Utkin, Yuriy N; Arseniev, Alexander S

    2005-08-15

    Cytotoxins from cobra venom are known to manifest cytotoxicity in various cell types. It is widely accepted that the plasma membrane is a target of cytotoxins, but the mechanism of their action remains obscure. Using the confocal spectral imaging technique, we show for the first time that cytotoxins from cobra venom penetrate readily into living cancer cells and accumulate markedly in lysosomes. Cytotoxins CT1 and CT2 from Naja oxiana, CT3 from Naja kaouthia and CT1 from Naja haje are demonstrated to possess this property with respect to human lung adenocarcinoma A549 and promyelocytic leukaemia HL60 cells. Immobilized plasma membrane binding accompanies the internalization of CT3 from Naja kaouthia in the HL60 cells, but it is very weak for other cytotoxins. Detectable membrane binding is not a property of any of the cytotoxins tested in A549 cells. The kinetics and concentration-dependence of cytotoxin accumulation in lysosomes correlate well with their cytotoxic effects. On the basis of the results obtained, we propose that lysosomes are a primary target of the lytic action of cytotoxins. Plasma membrane permeabilization seems to be a downstream event relative to lysosome rupture. Direct damage to the plasma membrane may be a complementary mechanism, but its relative contribution to the cytotoxic action depends on the cytotoxin structure and cell type.

  4. Frontotemporal dementia caused by CHMP2B mutation is characterised by neuronal lysosomal storage pathology

    DEFF Research Database (Denmark)

    Clayton, Emma L.; Mizielinska, Sarah; Edgar, James R.;

    2015-01-01

    Mutations in the charged multivesicular body protein 2B (CHMP2B) cause frontotemporal dementia (FTD). We report that mice which express FTD-causative mutant CHMP2B at physiological levels develop a novel lysosomal storage pathology characterised by large neuronal autofluorescent aggregates. The a...

  5. Lysosomal acid lipase deficiency--an under-recognized cause of dyslipidaemia and liver dysfunction.

    Science.gov (United States)

    Reiner, Željko; Guardamagna, Ornella; Nair, Devaki; Soran, Handrean; Hovingh, Kees; Bertolini, Stefano; Jones, Simon; Ćorić, Marijana; Calandra, Sebastiano; Hamilton, John; Eagleton, Terence; Ros, Emilio

    2014-07-01

    Lysosomal acid lipase deficiency (LAL-D) is a rare autosomal recessive lysosomal storage disease caused by deleterious mutations in the LIPA gene. The age at onset and rate of progression vary greatly and this may relate to the nature of the underlying mutations. Patients presenting in infancy have the most rapidly progressive disease, developing signs and symptoms in the first weeks of life and rarely surviving beyond 6 months of age. Children and adults typically present with some combination of dyslipidaemia, hepatomegaly, elevated transaminases, and microvesicular hepatosteatosis on biopsy. Liver damage with progression to fibrosis, cirrhosis and liver failure occurs in a large proportion of patients. Elevated low-density lipoprotein cholesterol levels and decreased high-density lipoprotein cholesterol levels are common features, and cardiovascular disease may manifest as early as childhood. Given that these clinical manifestations are shared with other cardiovascular, liver and metabolic diseases, it is not surprising that LAL-D is under-recognized in clinical practice. This article provides practical guidance to lipidologists, endocrinologists, cardiologists and hepatologists on how to recognize individuals with this life-limiting disease. A diagnostic algorithm is proposed with a view to achieving definitive diagnosis using a recently developed blood test for lysosomal acid lipase. Finally, current management options are reviewed in light of the ongoing development of enzyme replacement therapy with sebelipase alfa (Synageva BioPharma Corp., Lexington, MA, USA), a recombinant human lysosomal acid lipase enzyme.

  6. Identification of cytoskeleton-associated proteins essential for lysosomal stability and survival of human cancer cells

    DEFF Research Database (Denmark)

    Groth-Pedersen, Line; Aits, Sonja; Corcelle-Termeau, Elisabeth

    2012-01-01

    Microtubule-disturbing drugs inhibit lysosomal trafficking and induce lysosomal membrane permeabilization followed by cathepsin-dependent cell death. To identify specific trafficking-related proteins that control cell survival and lysosomal stability, we screened a molecular motor siRNA library...... in human MCF7 breast cancer cells. SiRNAs targeting four kinesins (KIF11/Eg5, KIF20A, KIF21A, KIF25), myosin 1G (MYO1G), myosin heavy chain 1 (MYH1) and tropomyosin 2 (TPM2) were identified as effective inducers of non-apoptotic cell death. The cell death induced by KIF11, KIF21A, KIF25, MYH1 or TPM2 si......), increased dextran accumulation (KIF20A), or reduced autophagic flux (MYO1G, MYH1). Importantly, all seven siRNAs also killed human cervix cancer (HeLa) and osteosarcoma (U-2-OS) cells and sensitized cancer cells to other lysosome-destabilizing treatments, i.e. photo-oxidation, siramesine, etoposide...

  7. Drosophila Vps16A is required for trafficking to lysosomes and biogenesis of pigment granules.

    Science.gov (United States)

    Pulipparacharuvil, Suprabha; Akbar, Mohammed Ali; Ray, Sanchali; Sevrioukov, Evgueny A; Haberman, Adam S; Rohrer, Jack; Krämer, Helmut

    2005-08-15

    Mutations that disrupt trafficking to lysosomes and lysosome-related organelles cause multiple diseases, including Hermansky-Pudlak syndrome. The Drosophila eye is a model system for analyzing such mutations. The eye-color genes carnation and deep orange encode two subunits of the Vps-C protein complex required for endosomal trafficking and pigment-granule biogenesis. Here we demonstrate that dVps16A (CG8454) encodes another Vps-C subunit. Biochemical experiments revealed a specific interaction between the dVps16A C-terminus and the Sec1/Munc18 homolog Carnation but not its closest homolog, dVps33B. Instead, dVps33B interacted with a related protein, dVps16B (CG18112). Deep orange bound both Vps16 homologs. Like a deep orange null mutation, eye-specific RNAi-induced knockdown of dVps16A inhibited lysosomal delivery of internalized ligands and interfered with biogenesis of pigment granules. Ubiquitous knockdown of dVps16A was lethal. Together, these findings demonstrate that Drosophila Vps16A is essential for lysosomal trafficking. Furthermore, metazoans have two types of Vps-C complexes with non-redundant functions.

  8. The UL24 protein of herpes simplex virus 1 affects the sub-cellular distribution of viral glycoproteins involved in fusion.

    Science.gov (United States)

    Ben Abdeljelil, Nawel; Rochette, Pierre-Alexandre; Pearson, Angela

    2013-09-01

    Mutations in UL24 of herpes simplex virus type 1 can lead to a syncytial phenotype. We hypothesized that UL24 affects the sub-cellular distribution of viral glycoproteins involved in fusion. In non-immortalized human foreskin fibroblasts (HFFs) we detected viral glycoproteins B (gB), gD, gH and gL present in extended blotches throughout the cytoplasm with limited nuclear membrane staining; however, in HFFs infected with a UL24-deficient virus (UL24X), staining for the viral glycoproteins appeared as long, thin streaks running across the cell. Interestingly, there was a decrease in co-localized staining of gB and gD with F-actin at late times in UL24X-infected HFFs. Treatment with chemical agents that perturbed the actin cytoskeleton hindered the formation of UL24X-induced syncytia in these cells. These data support a model whereby the UL24 syncytial phenotype results from a mislocalization of viral glycoproteins late in infection.

  9. Muscle fiber-type distribution, fiber-type-specific damage, and the Pompe disease phenotype.

    Science.gov (United States)

    van den Berg, L E M; Drost, M R; Schaart, G; de Laat, J; van Doorn, P A; van der Ploeg, A T; Reuser, A J J

    2013-09-01

    Pompe disease is a lysosomal storage disorder caused by acid α-glucosidase deficiency and characterized by progressive muscle weakness. Enzyme replacement therapy (ERT) has ameliorated patients' perspectives, but reversal of skeletal muscle pathology remains a challenge. We studied pretreatment biopsies of 22 patients with different phenotypes to investigate to what extent fiber-type distribution and fiber-type-specific damage contribute to clinical diversity. Pompe patients have the same fiber-type distribution as healthy persons, but among nonclassic patients with the same GAA mutation (c.-32-13T>G), those with early onset of symptoms tend to have more type 2 muscle fibers than those with late-onset disease. Further, it seemed that the older, more severely affected classic infantile patients and the wheelchair-bound and ventilated nonclassic patients had a greater proportion of type 2x muscle fibers. However, as in other diseases, this may be caused by physical inactivity of those patients.

  10. Lysine suppresses protein degradation through autophagic-lysosomal system in C2C12 myotubes.

    Science.gov (United States)

    Sato, Tomonori; Ito, Yoshiaki; Nedachi, Taku; Nagasawa, Takashi

    2014-06-01

    Muscle mass is determined between protein synthesis and protein degradation. Reduction of muscle mass leads to bedridden condition and attenuation of resistance to diseases. Moreover, bedridden condition leads to additional muscle loss due to disuse muscle atrophy. In our previous study (Sato et al. 2013), we showed that administered lysine (Lys), one of essential amino acid, suppressed protein degradation in skeletal muscle. In this study, we investigated that the mechanism of the suppressive effects of Lys on skeletal muscle proteolysis in C2C12 cell line. C2C12 myotubes were incubated in the serum-free medium containing 10 mM Lys or 20 mM Lys, and myofibrillar protein degradation was determined by the rates of 3-methylhistidine (MeHis) release from the cells. The mammalian target of rapamycin (mTOR) activity from the phosphorylation levels of p70-ribosormal protein S6 kinase 1 and eIF4E-binding protein 1 and the autophagic-lysosomal system activity from the ratio of LC3-II/I in C2C12 myotubes stimulated by 10 mM Lys for 0-3 h were measured. The rates of MeHis release were markedly reduced by addition of Lys. The autophagic-lysosomal system activity was inhibited upon 30 min of Lys supplementation. The activity of mTOR was significantly increased upon 30 min of Lys supplementation. The suppressive effect of Lys on the proteolysis by the autophagic-lysosomal system was maintained partially when mTOR activity was inhibited by 100 nM rapamycin, suggesting that some regulator other than mTOR signaling, for example, Akt, might also suppress the autophagic-lysosomal system. From these results, we suggested that Lys suppressed the activity of the autophagic-lysosomal system in part through activation of mTOR and reduced myofibrillar protein degradation in C2C12 myotubes.

  11. Glucolipotoxicity diminishes cardiomyocyte TFEB and inhibits lysosomal autophagy during obesity and diabetes.

    Science.gov (United States)

    Trivedi, Purvi C; Bartlett, Jordan J; Perez, Lester J; Brunt, Keith R; Legare, Jean Francois; Hassan, Ansar; Kienesberger, Petra C; Pulinilkunnil, Thomas

    2016-12-01

    Impaired cardiac metabolism in the obese and diabetic heart leads to glucolipotoxicity and ensuing cardiomyopathy. Glucolipotoxicity causes cardiomyocyte injury by increasing energy insufficiency, impairing proteasomal-mediated protein degradation and inducing apoptosis. Proteasome-evading proteins are degraded by autophagy in the lysosome, whose metabolism and function are regulated by master regulator transcription factor EB (TFEB). Limited studies have examined the impact of glucolipotoxicity on intra-lysosomal signaling proteins and their regulators. By utilizing a mouse model of diet-induced obesity, type-1 diabetes (Akita) and ex-vivo model of glucolipotoxicity (H9C2 cells and NRCM, neonatal rat cardiomyocyte), we examined whether glucolipotoxicity negatively targets TFEB and lysosomal proteins to dysregulate autophagy and cause cardiac injury. Despite differential effects of obesity and diabetes on LC3B-II, expression of proteins facilitating autophagosomal clearance such as TFEB, LAMP-2A, Hsc70 and Hsp90 were decreased in the obese and diabetic heart. In-vivo data was recapitulated in H9C2 and NRCM cells, which exhibited impaired autophagic flux and reduced TFEB content when exposed to a glucolipotoxic milieu. Notably, overloading myocytes with a saturated fatty acid (palmitate) but not an unsaturated fatty acid (oleate) depleted cellular TFEB and suppressed autophagy, suggesting a fatty acid specific regulation of TFEB and autophagy in the cardiomyocyte. The effect of glucolipotoxicity to reduce TFEB content was also confirmed in heart tissue from patients with Class-I obesity. Therefore, during glucolipotoxicity, suppression of lysosomal autophagy was associated with reduced lysosomal content, decreased cathepsin-B activity and diminished cellular TFEB content likely rendering myocytes susceptible to cardiac injury.

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

    Science.gov (United States)

    Lamore, Sarah D; Wondrak, Georg T

    2013-06-05

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

  13. Endothelial Nlrp3 inflammasome activation associated with lysosomal destabilization during coronary arteritis.

    Science.gov (United States)

    Chen, Yang; Li, Xiang; Boini, Krishna M; Pitzer, Ashley L; Gulbins, Erich; Zhang, Yang; Li, Pin-Lan

    2015-02-01

    Inflammasomes play a critical role in the development of vascular diseases. However, the molecular mechanisms activating the inflammasome in endothelial cells and the relevance of this inflammasome activation is far from clear. Here, we investigated the mechanisms by which an Nlrp3 inflammasome is activated to result in endothelial dysfunction during coronary arteritis by Lactobacillus casei (L. casei) cell wall fragments (LCWE) in a mouse model for Kawasaki disease. Endothelial dysfunction associated with increased vascular cell adhesion protein 1 (VCAM-1) expression and endothelial-leukocyte adhesion was observed during coronary arteritis in mice treated with LCWE. Accompanied with these changes, the inflammasome activation was also shown in coronary arterial endothelium, which was characterized by a marked increase in caspase-1 activity and IL-1β production. In cultured endothelial cells, LCWE induced Nlrp3 inflammasome formation, caspase-1 activation and IL-1β production, which were blocked by Nlrp3 gene silencing or lysosome membrane stabilizing agents such as colchicine, dexamethasone, and ceramide. However, a potassium channel blocker glibenclamide or an oxygen free radical scavenger N-acetyl-l-cysteine had no effects on LCWE-induced inflammasome activation. LCWE also increased endothelial cell lysosomal membrane permeability and triggered lysosomal cathepsin B release into cytosol. Silencing cathepsin B blocked LCWE-induced Nlrp3 inflammasome formation and activation in endothelial cells. In vivo, treatment of mice with cathepsin B inhibitor also abolished LCWE-induced inflammasome activation in coronary arterial endothelium. It is concluded that LCWE enhanced lysosomal membrane permeabilization and consequent release of lysosomal cathepsin B, resulting in activation of the endothelial Nlrp3 inflammasome, which may contribute to the development of coronary arteritis.

  14. Neurologic abnormalities in mouse models of the lysosomal storage disorders mucolipidosis II and mucolipidosis III γ.

    Directory of Open Access Journals (Sweden)

    Rachel A Idol

    Full Text Available UDP-GlcNAc:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase is an α2β2γ2 hexameric enzyme that catalyzes the synthesis of the mannose 6-phosphate targeting signal on lysosomal hydrolases. Mutations in the α/β subunit precursor gene cause the severe lysosomal storage disorder mucolipidosis II (ML II or the more moderate mucolipidosis III alpha/beta (ML III α/β, while mutations in the γ subunit gene cause the mildest disorder, mucolipidosis III gamma (ML III γ. Here we report neurologic consequences of mouse models of ML II and ML III γ. The ML II mice have a total loss of acid hydrolase phosphorylation, which results in depletion of acid hydrolases in mesenchymal-derived cells. The ML III γ mice retain partial phosphorylation. However, in both cases, total brain extracts have normal or near normal activity of many acid hydrolases reflecting mannose 6-phosphate-independent lysosomal targeting pathways. While behavioral deficits occur in both models, the onset of these changes occurs sooner and the severity is greater in the ML II mice. The ML II mice undergo progressive neurodegeneration with neuronal loss, astrocytosis, microgliosis and Purkinje cell depletion which was evident at 4 months whereas ML III γ mice have only mild to moderate astrocytosis and microgliosis at 12 months. Both models accumulate the ganglioside GM2, but only ML II mice accumulate fucosylated glycans. We conclude that in spite of active mannose 6-phosphate-independent targeting pathways in the brain, there are cell types that require at least partial phosphorylation function to avoid lysosomal dysfunction and the associated neurodegeneration and behavioral impairments.

  15. Campylobacter jejuni cell lysates differently target mitochondria and lysosomes on HeLa cells.

    Science.gov (United States)

    Canonico, B; Campana, R; Luchetti, F; Arcangeletti, M; Betti, M; Cesarini, E; Ciacci, C; Vittoria, E; Galli, L; Papa, S; Baffone, W

    2014-08-01

    Campylobacter jejuni is the most common cause of bacterial gastroenteritis in humans. The synthesis of cytolethal distending toxin appears essential in the infection process. In this work we evaluated the sequence of lethal events in HeLa cells exposed to cell lysates of two distinct strains, C. jejuni ATCC 33291 and C. jejuni ISS3. C. jejuni cell lysates (CCLys) were added to HeLa cell monolayers which were analysed to detect DNA content, death features, bcl-2 and p53 status, mitochondria/lysosomes network and finally, CD54 and CD59 alterations, compared to cell lysates of C. jejuni 11168H cdtA mutant. We found mitochondria and lysosomes differently targeted by these bacterial lysates. Death, consistent with apoptosis for C. jejuni ATCC 33291 lysate, occurred in a slow way (>48 h); concomitantly HeLa cells increase their endolysosomal compartment, as a consequence of toxin internalization besides a simultaneous and partial lysosomal destabilization. C. jejuni CCLys induces death in HeLa cells mainly via a caspase-dependent mechanism although a p53 lysosomal pathway (also caspase-independent) seems to appear in addition. In C. jejuni ISS3-treated cells, the p53-mediated oxidative degradation of mitochondrial components seems to be lost, inducing the deepest lysosomal alterations. Furthermore, CD59 considerably decreases, suggesting both a degradation or internalisation pathway. CCLys-treated HeLa cells increase CD54 expression on their surface, because of the action of lysate as its double feature of toxin and bacterial peptide. In conclusion, we revealed that C. jejuni CCLys-treated HeLa cells displayed different features, depending on the particular strain.

  16. Impaired Lysosomal Integral Membrane Protein 2-dependent Peroxiredoxin 6 Delivery to Lamellar Bodies Accounts for Altered Alveolar Phospholipid Content in Adaptor Protein-3-deficient pearl Mice.

    Science.gov (United States)

    Kook, Seunghyi; Wang, Ping; Young, Lisa R; Schwake, Michael; Saftig, Paul; Weng, Xialian; Meng, Ying; Neculai, Dante; Marks, Michael S; Gonzales, Linda; Beers, Michael F; Guttentag, Susan

    2016-04-15

    The Hermansky Pudlak syndromes (HPS) constitute a family of disorders characterized by oculocutaneous albinism and bleeding diathesis, often associated with lethal lung fibrosis. HPS results from mutations in genes of membrane trafficking complexes that facilitate delivery of cargo to lysosome-related organelles. Among the affected lysosome-related organelles are lamellar bodies (LB) within alveolar type 2 cells (AT2) in which surfactant components are assembled, modified, and stored. AT2 from HPS patients and mouse models of HPS exhibit enlarged LB with increased phospholipid content, but the mechanism underlying these defects is unknown. We now show that AT2 in the pearl mouse model of HPS type 2 lacking the adaptor protein 3 complex (AP-3) fails to accumulate the soluble enzyme peroxiredoxin 6 (PRDX6) in LB. This defect reflects impaired AP-3-dependent trafficking of PRDX6 to LB, because pearl mouse AT2 cells harbor a normal total PRDX6 content. AP-3-dependent targeting of PRDX6 to LB requires the transmembrane protein LIMP-2/SCARB2, a known AP-3-dependent cargo protein that functions as a carrier for lysosomal proteins in other cell types. Depletion of LB PRDX6 in AP-3- or LIMP-2/SCARB2-deficient mice correlates with phospholipid accumulation in lamellar bodies and with defective intraluminal degradation of LB disaturated phosphatidylcholine. Furthermore, AP-3-dependent LB targeting is facilitated by protein/protein interaction between LIMP-2/SCARB2 and PRDX6 in vitro and in vivo Our data provide the first evidence for an AP-3-dependent cargo protein required for the maturation of LB in AT2 and suggest that the loss of PRDX6 activity contributes to the pathogenic changes in LB phospholipid homeostasis found HPS2 patients.

  17. Cytosolic chloride ion is a key factor in lysosomal acidification and function of autophagy in human gastric cancer cell.

    Science.gov (United States)

    Hosogi, Shigekuni; Kusuzaki, Katsuyuki; Inui, Toshio; Wang, Xiangdong; Marunaka, Yoshinori

    2014-06-01

    The purpose of the present study was to clarify roles of cytosolic chloride ion (Cl(-) ) in regulation of lysosomal acidification [intra-lysosomal pH (pHlys )] and autophagy function in human gastric cancer cell line (MKN28). The MKN28 cells cultured under a low Cl(-) condition elevated pHlys and reduced the intra-lysosomal Cl(-) concentration ([Cl(-) ]lys ) via reduction of cytosolic Cl(-) concentration ([Cl(-) ]c ), showing abnormal accumulation of LC3II and p62 participating in autophagy function (dysfunction of autophagy) accompanied by inhibition of cell proliferation via G0 /G1 arrest without induction of apoptosis. We also studied effects of direct modification of H(+) transport on lysosomal acidification and autophagy. Application of bafilomycin A1 (an inhibitor of V-type H(+) -ATPase) or ethyl isopropyl amiloride [EIPA; an inhibitor of Na(+) /H(+) exchanger (NHE)] elevated pHlys and decreased [Cl(-) ]lys associated with inhibition of cell proliferation via induction of G0 /G1 arrest similar to the culture under a low Cl(-) condition. However, unlike low Cl(-) condition, application of the compound, bafilomycin A1 or EIPA, induced apoptosis associated with increases in caspase 3 and 9 without large reduction in [Cl(-) ]c compared with low Cl(-) condition. These observations suggest that the lowered [Cl(-) ]c primarily causes dysfunction of autophagy without apoptosis via dysfunction of lysosome induced by disturbance of intra-lysosomal acidification. This is the first study showing that cytosolic Cl(-) is a key factor of lysosome acidification and autophagy.

  18. A new role for an old drug: Ambroxol triggers lysosomal exocytosis via pH-dependent Ca²⁺ release from acidic Ca²⁺ stores.

    Science.gov (United States)

    Fois, Giorgio; Hobi, Nina; Felder, Edward; Ziegler, Andreas; Miklavc, Pika; Walther, Paul; Radermacher, Peter; Haller, Thomas; Dietl, Paul

    2015-12-01

    Ambroxol (Ax) is a frequently prescribed drug used to facilitate mucociliary clearance, but its mode of action is yet poorly understood. Here we show by X-ray spectroscopy that Ax accumulates in lamellar bodies (LBs), the surfactant storing, secretory lysosomes of type II pneumocytes. Using lyso- and acidotropic substances in combination with fluorescence imaging we confirm that these vesicles belong to the class of acidic Ca(2+) stores. Ax lead to a significant neutralization of LB pH, followed by intracellular Ca(2+) release, and to a dose-dependent surfactant exocytosis. Ax-induced Ca(2+) release was significantly reduced and slowed down by pretreatment of the cells with bafilomycin A1 (Baf A1), an inhibitor of the vesicular H(+) ATPase. These results could be nearly reproduced with NH3/NH4(+). The findings suggest that Ax accumulates within LBs and severely affects their H(+) and Ca(2+) homeostasis. This is further supported by an Ax-induced change of nanostructural assembly of surfactant layers. We conclude that Ax profoundly affects LBs presumably by disordering lipid bilayers and by acting as a weak base. The pH change triggers - at least in part - Ca(2+) release from stores and secretion of surfactant from type II cells. This novel mechanism of Ax as a lysosomal secretagogue may also play a role for its recently discussed use for lysosomal storage and other degenerative diseases.

  19. Excessive ammonia inhibited transcription of MsU2 gene and furthermore affected accumulation distribution of allantoin and amino acids in alfalfa Medicago sativa

    Institute of Scientific and Technical Information of China (English)

    WANG Li; JIANG Lin-lin; Nomura Mika; Tajima Shigeyuki; CHENG Xian-guo

    2015-01-01

    In legume plants, uricase gene (Nodulin-35) plays a positive role in metabolism of ureide and amide compounds in symbiotic nitrogen-ifxing in the nodules. In this study, a pot experiment was performed to examine the effects of ammonium application on the transcription of MsU2 gene and distribution of major nitrogen compounds in alfalfa Medicago sativa. Data showed that alfalfa plant has a signiifcant difference in contents of nitrogen compounds in xylem saps compared with soybean plant, and belongs to typical amide type legume plants with little ureide accumulation, and the accumulation of asparagines and ureide in the tissues of alfalfa is mainly gathered in the nodules. Northern blotting showed that excessive ammonium signiifcantly inhibited the transcription of MsU2 gene in the nodules and roots, and mRNA accumulation of MsU2 gene in the plants exposed to excessive ammonium decreased gradual y with culture time extension, indicating that application of ammonium signiifcantly inhibited the transcription of MsU2 gene in the alfalfa plants. Although the application of exces-sive ammonium increased the contents of amino acids in various tissues of alfalfa, the accumulation of al antoin relfecting the strength of uricase activity is remarkably reduced in the xylem saps, stems and nodules when alfalfa plants exposed to excessive ammonium, suggesting that application of excessive ammonium generated a negative effect on symbiosis ifxing-nitrogen system due to inhibition of ammonium ion on uricase activity in the nodules of alfalfa. This result seems to imply that application of excessive ammonium in legume plants should not be proposed to avoid affecting the ability of ifxing nitrogen in the nodules of legume plants, and reasonable dose of ammonium should be recommended to effectively utilize the ifxed N from atmosphere in legume plant production.

  20. Characterization of the egg vesicular components in the seaweed, Fucus serratus L. (Fucales, Phaeophyta), using enzyme histochemistry and vital staining: the search for a lysosome-like body.

    Science.gov (United States)

    Holland, R D; Pitt, D; Moore, M N; Brownlee, C

    1997-03-01

    Fucus serratus eggs were examined for evidence of the existence of a lysosome-like body using enzyme histochemical and vital staining techniques. Simultaneous coupling azo-dye techniques for lysosomal acid phosphatase proved inappropriate owing to endogenous phenolic binding artefacts. The large number of alginate polysaccharide and polyphenolic egg vesicles interfered with vital staining techniques for lysosomes. Lysosomal esterase activity was detected in the abundant egg lipid bodies. The role of the egg lipid body as an equivalent lysosome-like body of higher plants, the spherosome, is discussed in relation to egg fertilization and early zygote development.

  1. Mice Doubly-deficient in lysosomal hexosaminidase a and neuraminidase 4 show epileptic crises and rapid neuronal loss

    OpenAIRE

    Seyrantepe, Volkan; Lema, Pablo; Caqueret, Aurore; Dridi, Larbi; Hadj, Samar Bel; Carpentier, Stephane; Boucher, Francine; Levade, Thierry; Carmant, Lionel; Gravel, Roy A.; Hamel, Edith; Vachon, Pascal; Di Cristo, Graziella; Michaud, Jacques L.; Morales, Carlos R.

    2010-01-01

    Tay-Sachs disease is a severe lysosomal disorder caused by mutations in the HexA gene coding for the a-subunit of lysosomal β-hexosaminidase A, which converts GM2 to GM3 ganglioside. Hexa-/- mice, depleted of b-hexosaminidase A, remain asymptomatic to 1 year of age, because they catabolise GM2 ganglioside via a lysosomal sialidase into glycolipid GA2, which is further processed by β-hexosaminidase B to lactosyl-ceramide, thereby bypassing the β-hexosaminidase A defect. Since this bypass is no...

  2. Physical-chemical requirements for the catalysis of substrates by lysosomal phospholipase A1.

    Science.gov (United States)

    Robinson, M; Waite, M

    1983-12-10

    The catalytic properties of a 1440-fold purified preparation of lysosomal phospholipase A1 were examined. The preparation was at least 95% specific for the sn-1 position of neat phosphatidylethanolamine (PE). The apparent specificity of the enzyme toward substrates was affected by three factors: the physical arrangement of molecules in the substrate aggregate, the charge on the lipid-water interface and the chemical structure of the substrate as it relates to the active site of the enzyme. Of various phospholipids tested in the absence of detergent PE was the preferred substrate, phosphatidylcholine (PC) was hydrolyzed at one-fifth the rate of PE, while phosphatidylinositol (PI), phosphatidylserine (PS), and phosphatidylglycerol (PG) were degraded very slowly. Triton WR1339 stimulated the hydrolysis of PC, PI, PS, and PG but inhibited the hydrolysis of PE, with PG the preferred substrate at a 6:1 Triton/phospholipid ratio. The preference for PC over PE in detergent mixtures was attributed to the active site fit of the chemical structures of the substrate molecules. The enzyme preferentially hydrolyzed neat PE containing palmitic and oleic acids at position 1. A negative surface charge was required for the hydrolysis of PC and PE. Ca2+ stimulated the hydrolysis of PI, PS, and PG but inhibited the hydrolysis of PE. The inhibition of PE hydrolysis by Ca2+ was the result of an alteration in the surface charge of the PE vesicle. Chromatography of phospholipase A1 on concanavalin A-Sepharose resulted in a loss of activity toward acidic phospholipids which could be restored with Ca2+. Plasmalogen PE was found to inhibit the hydrolysis of diacyl-PE at the level of interfacial binding but not by competition for the active site of the enzyme. These results suggest that the hexagonal structure of PE represents a preferred physical form for catalysis by phospholipase A1, while the bilayer form is less readily attacked. Dispersion of the substrate in the inert detergent

  3. Effects of pH and Iminosugar Pharmacological Chaperones on Lysosomal Glycosidase Structure and Stability

    Energy Technology Data Exchange (ETDEWEB)

    Lieberman, Raquel L.; D’aquino, J. Alejandro; Ringe, Dagmar; Petsko, Gregory A.; (Harvard-Med); (Brandeis)

    2009-06-05

    Human lysosomal enzymes acid-{beta}-glucosidase (GCase) and acid-{alpha}-galactosidase ({alpha}-Gal A) hydrolyze the sphingolipids glucosyl- and globotriaosylceramide, respectively, and mutations in these enzymes lead to the lipid metabolism disorders Gaucher and Fabry disease, respectively. We have investigated the structure and stability of GCase and {alpha}-Gal A in a neutral-pH environment reflective of the endoplasmic reticulum and an acidic-pH environment reflective of the lysosome. These details are important for the development of pharmacological chaperone therapy for Gaucher and Fabry disease, in which small molecules bind mutant enzymes in the ER to enable the mutant enzyme to meet quality control requirements for lysosomal trafficking. We report crystal structures of apo GCase at pH 4.5, at pH 5.5, and in complex with the pharmacological chaperone isofagomine (IFG) at pH 7.5. We also present thermostability analysis of GCase at pH 7.4 and 5.2 using differential scanning calorimetry. We compare our results with analogous experiments using {alpha}-Gal A and the chaperone 1-deoxygalactonijirimycin (DGJ), including the first structure of {alpha}-Gal A with DGJ. Both GCase and {alpha}-Gal A are more stable at lysosomal pH with and without their respective iminosugars bound, and notably, the stability of the GCase-IFG complex is pH sensitive. We show that the conformations of the active site loops in GCase are sensitive to ligand binding but not pH, whereas analogous galactose- or DGJ-dependent conformational changes in {alpha}-Gal A are not seen. Thermodynamic parameters obtained from {alpha}-Gal A unfolding indicate two-state, van't Hoff unfolding in the absence of the iminosugar at neutral and lysosomal pH, and non-two-state unfolding in the presence of DGJ. Taken together, these results provide insight into how GCase and {alpha}-Gal A are thermodynamically stabilized by iminosugars and suggest strategies for the development of new pharmacological

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

    Science.gov (United States)

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

    2015-11-01

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

  5. The expression changes of Nicastrin in neuronal cells after lysosomal inhibitors treatment%溶酶体酶抑制剂对神经细胞内Nicastrin表达水平的影响

    Institute of Scientific and Technical Information of China (English)

    彭雪华; 龙志敏; 骆世芳; 贺桂琼

    2012-01-01

    Objective To explore whether the lysosomal pathway was involved in the degradation of Alzheimer' s disease ( AD ) -related protein Nicastrin( NCT). Methods Following generation of NCT stable cell lines, various methods such as Western blotting, double immunofluorescent staining and cell fractionation, combined with lysosomal inhibition were used to check NCT expression level in NCT stable cell line. Results Cell fractionation experiment showed that NCT distributed primarily in ER and Golgi apparatus, few NCT located in lyso-some. Treatment of cells with lysosomal inhibitors significantly increased both endogenous and exogenous mature NCT ( mNCT) in NCT stable cells or non-transfected neuronal cells, and the effect of lysosomal inhibitor on mNCT was time- and dose-dependent; however, lysosomal inhibitor had no effect on immature NCT (imNCT). Immunofluorescent microscopic analysis showed that lysosomal inhibition leaded to the accumulation of NCT in lysosomal apparatus. Conclusions The lysosomal pathway is involved in the degradation of mNCT in neuronal cells%目的 探讨溶酶体酶抑制剂对阿尔茨海默病(AD)相关蛋白Nicastrin(NCT)表达的影响,以期明确NCT的蛋白降解是否与溶酶体途径有关.方法 在用人神经母细胞瘤细胞SH-SY5Y建立稳定表达NCT细胞株的基础上,应用溶酶体酶抑制剂处理NCT细胞株,并结合Western印迹、亚细胞器的分级分离、免疫荧光双标等技术,检测溶酶体酶抑制剂处理后神经细胞内NCT的表达变化.结果 亚细胞器分级分离实验显示,正常情况下神经细胞内的NCT主要分布于内质网和高尔基复合体,少量NCT分布于溶酶体.Western印迹结果显示,溶酶体酶抑制剂处理后,神经细胞内源性和外源性成熟NCT(mNCT)的表达显著增强,且溶酶体酶抑制剂氯喹对mNCT蛋白表达的增强效应呈剂量依赖性和时间依赖性,但溶酶体抑制剂对非成熟NCT(imNCT)的表达无影响;免疫荧光双标结果显

  6. CNS penetration of intrathecal-lumbar idursulfase in the monkey, dog and mouse: implications for neurological outcomes of lysosomal storage disorder.

    Directory of Open Access Journals (Sweden)

    Pericles Calias

    Full Text Available A major challenge for the treatment of many central nervous system (CNS disorders is the lack of convenient and effective methods for delivering biological agents to the brain. Mucopolysaccharidosis II (Hunter syndrome is a rare inherited lysosomal storage disorder resulting from a deficiency of iduronate-2-sulfatase (I2S. I2S is a large, highly glycosylated enzyme. Intravenous administration is not likely to be an effective therapy for disease-related neurological outcomes that require enzyme access to the brain cells, in particular neurons and oligodendrocytes. We demonstrate that intracerebroventricular and lumbar intrathecal administration of recombinant I2S in dogs and nonhuman primates resulted in widespread enzyme distribution in the brain parenchyma, including remarkable deposition in the lysosomes of both neurons and oligodendrocytes. Lumbar intrathecal administration also resulted in enzyme delivery to the spinal cord, whereas little enzyme was detected there after intraventricular administration. Mucopolysaccharidosis II model is available in mice. Lumbar administration of recombinant I2S to enzyme deficient animals reduced the storage of glycosaminoglycans in both superficial and deep brain tissues, with concurrent morphological improvements. The observed patterns of enzyme transport from cerebrospinal fluid to the CNS tissues and the resultant biological activity (a warrant further investigation of intrathecal delivery of I2S via lumbar catheter as an experimental treatment for the neurological symptoms of Hunter syndrome and (b may have broader implications for CNS treatment with biopharmaceuticals.

  7. CNS penetration of intrathecal-lumbar idursulfase in the monkey, dog and mouse: implications for neurological outcomes of lysosomal storage disorder.

    Science.gov (United States)

    Calias, Pericles; Papisov, Mikhail; Pan, Jing; Savioli, Nancy; Belov, Vasily; Huang, Yan; Lotterhand, Jason; Alessandrini, Mary; Liu, Nan; Fischman, Alan J; Powell, Jan L; Heartlein, Michael W

    2012-01-01

    A major challenge for the treatment of many central nervous system (CNS) disorders is the lack of convenient and effective methods for delivering biological agents to the brain. Mucopolysaccharidosis II (Hunter syndrome) is a rare inherited lysosomal storage disorder resulting from a deficiency of iduronate-2-sulfatase (I2S). I2S is a large, highly glycosylated enzyme. Intravenous administration is not likely to be an effective therapy for disease-related neurological outcomes that require enzyme access to the brain cells, in particular neurons and oligodendrocytes. We demonstrate that intracerebroventricular and lumbar intrathecal administration of recombinant I2S in dogs and nonhuman primates resulted in widespread enzyme distribution in the brain parenchyma, including remarkable deposition in the lysosomes of both neurons and oligodendrocytes. Lumbar intrathecal administration also resulted in enzyme delivery to the spinal cord, whereas little enzyme was detected there after intraventricular administration. Mucopolysaccharidosis II model is available in mice. Lumbar administration of recombinant I2S to enzyme deficient animals reduced the storage of glycosaminoglycans in both superficial and deep brain tissues, with concurrent morphological improvements. The observed patterns of enzyme transport from cerebrospinal fluid to the CNS tissues and the resultant biological activity (a) warrant further investigation of intrathecal delivery of I2S via lumbar catheter as an experimental treatment for the neurological symptoms of Hunter syndrome and (b) may have broader implications for CNS treatment with biopharmaceuticals.

  8. The role of lysosomal cysteine proteases in crustacean immune response

    Directory of Open Access Journals (Sweden)

    FL Garcia-Carreño

    2014-04-01

    Full Text Available Over the long course of evolution and under the selective pressure exerted by pathogens and parasites, animals have selectively fixed a number of defense mechanisms against the constant attack of intruders. The immune response represents a key component to optimize the biological fitness of individuals. Two decades ago, prevention and control of diseases in crustacean aquaculture systems were considered priorities in most shrimp-producing countries, but knowledge was scarce and various pathogens have severely affected aquaculture development around the world. Scientific contributions have improved our understanding of the crustacean immune response. Several studies confirm the central role played by proteases in the immune response of animals, and the cooperative interaction of these enzymes in a wide variety of organisms is well known. This review summarizes the current information regarding the role of cysteine proteases in the immune system of Crustacea and points to aspects that are needed to provide a better integration of our knowledge.

  9. Localization of acid hydrolases in protoplasts. Examination of the proposed lysosomal function of the mature vacuole

    Energy Technology Data Exchange (ETDEWEB)

    Butcher, H.C.; Wagner, G.J.; Siegelman, H.W.

    1977-06-01

    The development of techniques to isolate and purify relatively large quantities of intact vacuoles from mature tissues permits direct biochemical analysis of this ubiquitous mature plant cell organelle. Vacuoles and a fraction enriched in soluble cytoplasmic constituents were quantitatively prepared from Hippeastrum flower petal protoplasts. Vacuolar lysate and soluble cytoplasmic fractions were examined for acid hydrolase activities commonly associated with animal lysosomes, and pH optima were determined. Esterase, protease, carboxypeptidase, ..beta..-galactosidase, ..cap alpha..-glycosidase and ..beta..-glycosidase, not found in the vacuole lysate fraction, were components of the soluble cytoplasmic fraction. Acid phosphatase, RNase and DNase were present in both fractions. Vacuolar enzyme activities were also examined as a function of flower development from bud through senescent stages. The data obtained are not consistent with the concept that the mature plant cell vacuole functions as a generalized lysosome.

  10. Campylobacter jejuni survives within epithelial cells by avoiding delivery to lysosomes.

    Directory of Open Access Journals (Sweden)

    Robert O Watson

    2008-01-01

    Full Text Available Campylobacter jejuni is one of the major causes of infectious diarrhea world-wide, although relatively little is know about its mechanisms of pathogenicity. This bacterium can gain entry into intestinal epithelial cells, which is thought to be important for its ability to persistently infect and cause disease. We found that C. jejuni is able to survive within intestinal epithelial cells. However, recovery of intracellular bacteria required pre-culturing under oxygen-limiting conditions, suggesting that C. jejuni undergoes significant physiological changes within the intracellular environment. We also found that in epithelial cells the C. jejuni-containing vacuole deviates from the canonical endocytic pathway immediately after a unique caveolae-dependent entry pathway, thus avoiding delivery into lysosomes. In contrast, in macrophages, C. jejuni is delivered to lysosomes and consequently is rapidly killed. Taken together, these studies indicate that C. jejuni has evolved specific adaptations to survive within host cells.

  11. uPARAP/endo180 directs lysosomal delivery and degradation of collagen IV

    DEFF Research Database (Denmark)

    Kjøller, Lars; Engelholm, Lars H; Høyer-Hansen, Maria

    2004-01-01

    transmembrane glycoprotein urokinase plasminogen activator receptor-associated protein (uPARAP/endo180) directs collagen IV for lysosomal delivery and degradation. In wild-type fibroblasts, fluorescently labeled collagen IV was first internalized into vesicular structures with diffuse fluorescence eventually......Collagen turnover is crucial for tissue homeostasis and remodeling and pathological processes such as cancer invasion, but the underlying molecular mechanisms are poorly understood. A major pathway appears to be internalization and degradation by fibroblasts. We now show that the endocytic...... appearing uniformly within the wild-type cells after longer incubation times. In these cells, some collagen-containing vesicles were identified as lysosomes by staining for LAMP-1. In contrast, collagen IV remained extracellular and associated with fiber-like structures on uPARAP/endo180-deficient...

  12. Preventive effect of phytic acid on lysosomal hydrolases in normal and isoproterenol-induced myocardial infarction in Wistar rats.

    Science.gov (United States)

    Brindha, E; Rajasekapandiyan, M

    2015-02-01

    This study was aimed to evaluate the preventive role of phytic acid on lysosomal enzymes in isoproterenol (ISO)-induced myocardial infarction (MI) in male Wistar rats. Rats subcutaneously injected with ISO (85 mg/kg) at an interval of 24 h for two days showed a significant increase in the activities of lysosomal enzymes (glucuronidase, N-acetyl glucosaminidase, galactosidase, cathepsin-B and cathepsin-D) were increased significantly in serum and the heart of ISO-induced rats, but the activities of glucuronidase and cathepsin-D were decreased significantly in the lysosomal fraction of the heart. Pretreatment with phytic acid (25 and 50 mg/kg) daily for a period of 56 d positively altered activities of lysosomal hydrolases in ISO-induced rats. Thus, phytic acid possesses a cardioprotective effect in ISO-induced MI in rats.

  13. Constitutively internalized dopamine transporter is targeted to late endosomes and lysosomal degradation in heterologous cell lines and dopaminergic neurons

    DEFF Research Database (Denmark)

    Eriksen, Jacob; Madsen, Kenneth; Vægter, Christian Bjerggaard;

    (leupeptin, chloroquine, or ammonium chloride) increased the amount of transporter accumulated intracellularly over time, suggesting that constitutively endocytosed transporter was targeted to lysosomal degradation. This was further supported by expression of Tac-DAT in the immortalized dopaminergic cell...

  14. Endo-lysosomal TRP mucolipin-1 channels trigger global ER Ca2+ release and Ca2+ influx

    Science.gov (United States)

    Kilpatrick, Bethan S.; Yates, Elizabeth; Grimm, Christian; Schapira, Anthony H.

    2016-01-01

    ABSTRACT Transient receptor potential (TRP) mucolipins (TRPMLs), encoded by the MCOLN genes, are patho-physiologically relevant endo-lysosomal ion channels crucial for membrane trafficking. Several lines of evidence suggest that TRPMLs mediate localised Ca2+ release but their role in Ca2+ signalling is not clear. Here, we show that activation of endogenous and recombinant TRPMLs with synthetic agonists evoked global Ca2+ signals in human cells. These signals were blocked by a dominant-negative TRPML1 construct and a TRPML antagonist. We further show that, despite a predominant lysosomal localisation, TRPML1 supports both Ca2+ release and Ca2+ entry. Ca2+ release required lysosomal and ER Ca2+ stores suggesting that TRPMLs, like other endo-lysosomal Ca2+ channels, are capable of ‘chatter’ with ER Ca2+ channels. Our data identify new modalities for TRPML1 action. PMID:27577094

  15. A Genome-wide RNAi Screen for Microtubule Bundle Formation and Lysosome Motility Regulation in Drosophila S2 Cells

    Directory of Open Access Journals (Sweden)

    Amber L. Jolly

    2016-01-01

    Full Text Available Long-distance intracellular transport of organelles, mRNA, and proteins (“cargo” occurs along the microtubule cytoskeleton by the action of kinesin and dynein motor proteins, but the vast network of factors involved in regulating intracellular cargo transport are still unknown. We capitalize on the Drosophila melanogaster S2 model cell system to monitor lysosome transport along microtubule bundles, which require enzymatically active kinesin-1 motor protein for their formation. We use an automated tracking program and a naive Bayesian classifier for the multivariate motility data to analyze 15,683 gene phenotypes and find 98 proteins involved in regulating lysosome motility along microtubules and 48 involved in the formation of microtubule filled processes in S2 cells. We identify innate immunity genes, ion channels, and signaling proteins having a role in lysosome motility regulation and find an unexpected relationship between the dynein motor, Rab7a, and lysosome motility regulation.

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

    OpenAIRE

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

    2015-01-01

    Para-toluenesulfonamide (PTS) has been implicated with anticancer effects against a variety of tumors. In the present study, we investigated the inhibitory effects of PTS on tongue squamous cell carcinoma (Tca-8113) and explored the lysosomal and mitochondrial changes after PTS treatment in vitro. High-performance liquid chromatography showed that PTS selectively accumulated in Tca-8113 cells with a relatively low concentration in normal fibroblasts. Next, the effects of PTS on cell viability...

  17. Lysosomal trafficking of {beta}-catenin induced by the tea polyphenol epigallocatechin-3-gallate

    Energy Technology Data Exchange (ETDEWEB)

    Dashwood, Wan-Mohaiza [Linus Pauling Institute, 571 Weniger Hall, Oregon State University, Corvallis, OR 97331-6512 (United States); Carter, Orianna [Linus Pauling Institute, 571 Weniger Hall, Oregon State University, Corvallis, OR 97331-6512 (United States); Al-Fageeh, Mohamed [Linus Pauling Institute, 571 Weniger Hall, Oregon State University, Corvallis, OR 97331-6512 (United States); Li, Qingjie [Linus Pauling Institute, 571 Weniger Hall, Oregon State University, Corvallis, OR 97331-6512 (United States); Dashwood, Roderick H. [Linus Pauling Institute, 571 Weniger Hall, Oregon State University, Corvallis, OR 97331-6512 (United States)]. E-mail: Rod.Dashwood@oregonstate.edu

    2005-12-11

    {beta}-Catenin is a cadherin-binding protein involved in cell-cell adhesion, which also functions as a transcriptional activator when complexed in the nucleus with members of the T-cell factor (TCF)/lymphoid enhancer factor (LEF) family of proteins. There is considerable interest in mechanisms that down-regulate {beta}-catenin, since this provides an avenue for the prevention of colorectal and other cancers in which {beta}-catenin is frequently over-expressed. We show here that physiologically relevant concentrations of the tea polyphenol epigallocatechin-3-gallate (EGCG) inhibited {beta}-catenin/TCF-dependent reporter activity in human embryonic kidney 293 cells transfected with wild type or mutant {beta}-catenins, and there was a corresponding decrease in {beta}-catenin protein levels in the nuclear, cytosolic and membrane-associated fractions. However, {beta}-catenin accumulated as punctate aggregates in response to EGCG treatment, including in human colon cancer cells over-expressing {beta}-catenin endogenously. Confocal microscopy studies revealed that the aggregated {beta}-catenin in HEK293 cells was extra-nuclear and co-localized with lysosomes, suggesting that EGCG activated a pathway involving lysosomal trafficking of {beta}-catenin. Lysosomal inhibitors leupeptin and transepoxysuccinyl-L-leucylamido(4-guanido)butane produced an increase in {beta}-catenin protein in total cell lysates, without a concomitant increase in {beta}-catenin transcriptional activity. These data provide the first evidence that EGCG facilitates the trafficking of {beta}-catenin into lysosomes, presumably as a mechanism for sequestering {beta}-catenin and circumventing further nuclear transport and activation of {beta}-catenin/TCF/LEF signaling.

  18. Characterization and cloning of lgp110, a lysosomal membrane glycoprotein from mouse and rat cells.

    Science.gov (United States)

    Granger, B L; Green, S A; Gabel, C A; Howe, C L; Mellman, I; Helenius, A

    1990-07-15

    lgp110 is a heavily glycosylated intrinsic protein of lysosomal membranes. Initially defined by monoclonal antibodies against mouse liver lysosomes, it consists of a 45-kilodalton core polypeptide with O-linked and 17 asparagine-linked oligosaccharide side chains in mouse cells. Sialic acid residues make the mature protein extremely acidic, with an isoelectric point of between 2 and 4 in both normal tissues and most cultured cell lines. Partial sequencing of mouse lgp110 allowed oligonucleotide probes to be constructed for the screening of several mouse cDNA libraries. A partial cDNA clone for mouse lgp110 was found and used for additional library screening, generating a cDNA clone covering all of the coding sequence of mature rat lgp110 as well as genomic clones covering most of the mouse gene. These new clones bring to seven the number of lysosomal membrane proteins whose amino acid sequences can be deduced, and two distinct but highly similar groups (designated lgp-A and lgp-B) can now be defined. Sequence comparisons suggest that differences within each group reflect species variations of the same protein and that lgp-A and lgp-B probably diverged from a common ancestor prior to the evolup4f1ary divergence of birds and mammals. Individual cells and individual lysosomes possess both lgp-A and lgp-B, suggesting that these two proteins have different functions. Mouse lgp110 is encoded by at least seven exons; intron positions suggest that the two homologous ectodomains of each lgp arose through gene duplication.

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

    Science.gov (United States)

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

    2014-05-01

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

  20. Arsenic induces apoptosis by the lysosomal-mitochondrial pathway in INS-1 cells.

    Science.gov (United States)

    Pan, Xiao; Jiang, Liping; Zhong, Laifu; Geng, Chengyan; Jia, Li; Liu, Shuang; Guan, Huai; Yang, Guang; Yao, Xiaofeng; Piao, Fengyuan; Sun, Xiance

    2016-02-01

    Recently, long term arsenic exposure was considered to be associated with an increased risk of diabetes mellitus. While a relation of cause-and-effect between apoptosis of pancreatic β-cells and arsenic exposure, the precise mechanisms of these events remains unclear. The aim of this study was to explore arsenic-induced pancreatic β-cell apoptosis and the mechanisms of through the possible link between lysosomal and the mitochondrial apoptotic pathway. After exposure to 10 μM of arsenic, the reactive oxygen species (ROS) level was significantly increased at 12 h, while the mitochondrial membrane potential was reduced at 24 h and the lysosomal membrane integrity was disrupted at 48 h. A significant increase in protein expression for cytochrome c was also observed using Western blot analysis after exposure to arsenic for 48 h. To further demonstrate that arsenic reduced the lysosomal membrane integrity, cells pretreated with NH4 Cl and exposed to arsenic harbored a lower fluorescence increase than cells that were only exposed to arsenic. In addition, apoptosis was mesured using Hoechst 33342/PI dual staining by microscopy and annexin V-FITC/propidium iodide dual staining by flow cytometry. The results show an increased uptake of the arsenic dose and the cells changed from dark blue to light blue, karyopyknosis, nuclear chromatin condensation, side set or fracture, and a correlation was found between the number of apoptotic cells and arsenic dose. The result of present study suggest that arsenic may induce pancreatic β-cell apoptosis through activation of the lysosome-mitochondrial pathway.

  1. A genetic screen in Drosophila reveals novel cytoprotective functions of the autophagy-lysosome pathway.

    Directory of Open Access Journals (Sweden)

    Andrew M Arsham

    Full Text Available The highly conserved autophagy-lysosome pathway is the primary mechanism for breakdown and recycling of macromolecular and organellar cargo in the eukaryotic cell. Autophagy has recently been implicated in protection against cancer, neurodegeneration, and infection, and interest is increasing in additional roles of autophagy in human health, disease, and aging. To search for novel cytoprotective features of this pathway, we carried out a genetic mosaic screen for mutations causing increased lysosomal and/or autophagic activity in the Drosophila melanogaster larval fat body. By combining Drosophila genetics with live-cell imaging of the fluorescent dye LysoTracker Red and fixed-cell imaging of autophagy-specific fluorescent protein markers, the screen was designed to identify essential metazoan genes whose disruption causes increased flux through the autophagy-lysosome pathway. The screen identified a large number of genes associated with the protein synthesis and ER-secretory pathways (e.g. aminoacyl tRNA synthetases, Oligosaccharyl transferase, Sec61alpha, and with mitochondrial function and dynamics (e.g. Rieske iron-sulfur protein, Dynamin-related protein 1. We also observed that increased lysosomal and autophagic activity were consistently associated with decreased cell size. Our work demonstrates that disruption of the synthesis, transport, folding, or glycosylation of ER-targeted proteins at any of multiple steps leads to autophagy induction. In addition to illuminating cytoprotective features of autophagy in response to cellular damage, this screen establishes a genetic methodology for investigating cell biological phenotypes in live cells, in the context of viable wild type organisms.

  2. SIRT1 regulates accumulation of oxidized LDL in HUVEC via the autophagy-lysosomal pathway.

    Science.gov (United States)

    Zhang, Yanlin; Sun, Juanjuan; Yu, Xiaoyan; Shi, Luyao; Du, Wenxiu; Hu, Lifang; Liu, Chunfeng; Cao, Yongjun

    2016-01-01

    Autophagy is involved in the degradation of oxidized low-density lipoprotein (ox-LDL) in human umbilical vein endothelial cells (HUVECs). Sirtuin1 (SIRT1), a new anti-atherosclerotic factor, can induce autophagy in cardiac myocytes. In the present study, we observed the effect of SIRT1 on the accumulation of ox-LDL in HUVECs, and elucidated whether its effect is relative with the autophagy-lysosomal pathway. The results showed that treatment with either SIRT1 siRNA or SIRT1 inhibitor nicotinamide (NAM) increased Dil-labelled-ox-LDL (Dil-ox-LDL) accumulation in HUVECs, and the SIRT1 inducer resveratrol (RSV) decreased it. Knockdown of autophagy-related protein 5 or inhibit the lysosomal degradation by chloroquine (CQ) decreased the effect of RSV. In HUVECs with ox-LDL, expression of LC3II and LC3 puncta was decreased by treatment with SIRT1 siRNA or NAM, but increased by RSV treatment; sequestosome 1 p62 expression showed the opposite effects. Moreover, Dil-ox-LDL combined with SIRT1 siRNA or NAM showed a much smaller degree of overlap of Lamp1 or Cathepsin D with Dil-ox-LDL than in cells with Dil-ox-LDL alone, and RSV treatment resulted in a greater degree of overlap. These results suggest that SIRT1 can decrease the accumulation of ox-LDL in HUVECs, and this effect is related to the autophagy-lysosomal pathway.

  3. Planar patch clamp approach to characterize ionic currents from intact lysosomes.

    Science.gov (United States)

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

    2010-01-01

    Since its launch in the early 1980s, the patch clamp method has been extensively used to study ion channels in the plasma membrane, but its application to the study of intracellular ion channels has been limited. Unlike the plasma membrane, intracellular membranes are usually not stable enough to withstand mechanical manipulation by glass electrodes during seal formation and rupturing of the membrane. To circumvent these problems, we developed a method involving the immobilization of isolated organelles on a solid matrix planar glass chip. This glass chip contains a microstructured hole that supports the formation of gigaseals and subsequent electrophysiological recordings despite the high fragility of intracellular membranes. Here, we report the experimental details of this method using lysosomes, which are the smallest cellular organelles, as a model system. We demonstrate that we can record endogenous ionic currents from wild-type lysosomes, as well as from lysosomes overexpressing ion channels, and expect that this method will provide electrophysiological access to a broad range of intracellular ion channels.

  4. Lack of the Lysosomal Membrane Protein, GLMP, in Mice Results in Metabolic Dysregulation in Liver.

    Directory of Open Access Journals (Sweden)

    Xiang Yi Kong

    Full Text Available Ablation of glycosylated lysosomal membrane protein (GLMP, formerly known as NCU-G1 has been shown to cause chronic liver injury which progresses into liver fibrosis in mice. Both lysosomal dysfunction and chronic liver injury can cause metabolic dysregulation. Glmp gt/gt mice (formerly known as Ncu-g1gt/gt mice were studied between 3 weeks and 9 months of age. Body weight gain and feed efficiency of Glmp gt/gt mice were comparable to wild type siblings, only at the age of 9 months the Glmp gt/gt siblings had significantly reduced body weight. Reduced size of epididymal fat pads was accompanied by hepatosplenomegaly in Glmp gt/gt mice. Blood analysis revealed reduced levels of blood glucose, circulating triacylglycerol and non-esterified fatty acids in Glmp gt/gt mice. Increased flux of glucose, increased de novo lipogenesis and lipid accumulation were detected in Glmp gt/gt primary hepatocytes, as well as elevated triacylglycerol levels in Glmp gt/gt liver homogenates, compared to hepatocytes and liver from wild type mice. Gene expression analysis showed an increased expression of genes involved in fatty acid uptake and lipogenesis in Glmp gt/gt liver compared to wild type. Our findings are in agreement with the metabolic alterations observed in other mouse models lacking lysosomal proteins, and with alterations characteristic for advanced chronic liver injury.

  5. Alpha Adrenergic Induction of Transport of Lysosomal Enzyme across the Blood-Brain Barrier.

    Directory of Open Access Journals (Sweden)

    Akihiko Urayama

    Full Text Available The impermeability of the adult blood-brain barrier (BBB to lysosomal enzymes impedes the ability to treat the central nervous system manifestations of lysosomal storage diseases. Here, we found that simultaneous stimulation of the alpha1 and alpha2 adrenoreceptor restores in adult mice the high rate of transport for the lysosomal enzyme P-GUS that is seen in neonates but lost with development. Beta adrenergics, other monoamines, and acetylcholine did not restore this transport. A high dose (500 microg/mouse of clonidine, a strong alpha2 and weak alpha1 agonist, was able to act as monotherapy in the stimulation of P-GUS transport. Neither use of alpha1 plus alpha2 agonists nor the high dose clonidine disrupted the BBB to albumin. In situ brain perfusion and immunohistochemistry studies indicated that adrengerics act on transporters already at the luminal surface of brain endothelial cells. These results show that adrenergic stimulation, including monotherapy with clonidine, could be key for CNS enzyme replacement therapy.

  6. Quantitative Differences in the Urinary Proteome of Siblings Discordant for Type 1 Diabetes Include Lysosomal Enzymes.

    Science.gov (United States)

    Suh, Moo-Jin; Tovchigrechko, Andrey; Thovarai, Vishal; Rolfe, Melanie A; Torralba, Manolito G; Wang, Junmin; Adkins, Joshua N; Webb-Robertson, Bobbie-Jo M; Osborne, Whitney; Cogen, Fran R; Kaplowitz, Paul B; Metz, Thomas O; Nelson, Karen E; Madupu, Ramana; Pieper, Rembert

    2015-08-01

    Individuals with type 1 diabetes (T1D) often have higher than normal blood glucose levels, causing advanced glycation end product formation and inflammation and increasing the risk of vascular complications years or decades later. To examine the urinary proteome in juveniles with T1D for signatures indicative of inflammatory consequences of hyperglycemia, we profiled the proteome of 40 T1D patients with an average of 6.3 years after disease onset and normal or elevated HbA1C levels, in comparison with a cohort of 41 healthy siblings. Using shotgun proteomics, 1036 proteins were identified, on average, per experiment, and 50 proteins showed significant abundance differences using a Wilcoxon signed-rank test (FDR q-value ≤ 0.05). Thirteen lysosomal proteins were increased in abundance in the T1D versus control cohort. Fifteen proteins with functional roles in vascular permeability and adhesion were quantitatively changed, including CD166 antigen and angiotensin-converting enzyme 2. α-N-Acetyl-galactosaminidase and α-fucosidase 2, two differentially abundant lysosomal enzymes, were detected in western blots with often elevated quantities in the T1D versus control cohort. Increased release of proteins derived from lysosomes and vascular epithelium into urine may result from hyperglycemia-associated inflammation in the kidney vasculature.

  7. Unfolded protein response activates glycogen synthase kinase-3 via selective lysosomal degradation.

    Science.gov (United States)

    Nijholt, Diana A T; Nölle, Anna; van Haastert, Elise S; Edelijn, Hessel; Toonen, Ruud F; Hoozemans, Jeroen J M; Scheper, Wiep

    2013-07-01

    The unfolded protein response (UPR) is a stress response that is activated upon disturbed homeostasis in the endoplasmic reticulum. In Alzheimer's disease, as well as in other tauopathies, the UPR is activated in neurons that contain early tau pathology. A recent genome-wide association study identified genetic variation in a UPR transducer as a risk factor for tauopathy, supporting a functional connection between UPR activation and tau pathology. Here we show that UPR activation increases the activity of the major tau kinase glycogen synthase kinase (GSK)-3 in vitro via a selective removal of inactive GSK-3 phosphorylated at Ser(21/9). We demonstrate that this is mediated by the autophagy/lysosomal pathway. In brain tissue from patients with different tauopathies, lysosomal accumulations of pSer(21/9) GSK-3 are found in neurons with markers for UPR activation. Our data indicate that UPR activation increases the activity of GSK-3 by a novel mechanism, the lysosomal degradation of the inactive pSer(21/9) GSK-3. This may provide a functional explanation for the close association between UPR activation and early tau pathology in neurodegenerative diseases.

  8. Characterization of lysosome-destabilizing DOPE/PLGA nanoparticles designed for cytoplasmic drug release.

    Science.gov (United States)

    Chhabra, Resham; Grabrucker, Andreas M; Veratti, Patrizia; Belletti, Daniela; Boeckers, Tobias M; Vandelli, Maria Angela; Forni, Flavio; Tosi, Giovanni; Ruozi, Barbara

    2014-08-25

    Polymeric nanoparticles (NPs) offer a promising approach for therapeutic intracellular delivery of proteins, conventionally hampered by short half-lives, instability and immunogenicity. Remarkably, NPs uptake occurs via endocytic internalization leading to NPs content's release within lysosomes. To overcome lysosomal degradation and achieve NPs and/or loaded proteins release into cytosol, we propose the formulation of hybrid NPs by adding 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) as pH sensitive component in the formulation of poly-lactide-co-glycolide (PLGA) NPs. Hybrid NPs, featured by different DOPE/PLGA ratios, were characterized in terms of structure, stability and lipid organization within the polymeric matrix. Experiments on NIH cells and rat primary neuronal cultures highlighted the safety profile of hybrid NPs. Moreover, after internalization, NPs are able to transiently destabilize the integrity of lysosomes in which they are taken up, speeding their escape and favoring cytoplasmatic localization. Thus, these DOPE/PLGA-NPs configure themselves as promising carriers for intracellular protein delivery.

  9. A lysosome-targeted drug delivery system based on sorbitol backbone towards efficient cancer therapy.

    Science.gov (United States)

    Maniganda, Santhi; Sankar, Vandana; Nair, Jyothi B; Raghu, K G; Maiti, Kaustabh K

    2014-09-14

    A straightforward synthetic approach was adopted for the construction of a lysosome-targeted drug delivery system (TDDS) using sorbitol scaffold (Sor) linked to octa-guanidine and tetrapeptide GLPG, a peptide substrate of lysosomal cysteine protease, cathepsin B. The main objective was to efficiently deliver the potential anticancer drug, doxorubicin to the target sites, thereby minimizing dose-limiting toxicity. Three TDDS vectors were synthesized viz., DDS1: Sor-GLPG-Fl, DDS2: Sor-Fl (control) and DDS3: Sor-GLPGC-SMCC-Dox. Dox release from DDS3 in the presence of cathepsin B was studied by kinetics measurement based on the fluorescent property of Dox. The cytotoxicity of DDS1 was assessed and found to be non-toxic. Cellular internalization and colocalization studies of all the 3 systems were carried out by flow cytometry and confocal microscopy utilizing cathepsin B-expressing HeLa cells. DDS1 and DDS3 revealed significant localization within the lysosomes, in contrast to DDS2 (control). The doxorubicin-conjugated carrier, DDS3, demonstrated significant cytotoxic effect when compared to free Dox by MTT assay and also by flow cytometric analysis. The targeted approach with DDS3 is expected to be promising, because it is indicated to be advantageous over free Dox, which possesses dose-limiting toxicity, posing risk of injury to normal tissues.

  10. LAMP-3 (Lysosome-Associated Membrane Protein 3) Promotes the Intracellular Proliferation of Salmonella typhimurium.

    Science.gov (United States)

    Lee, Eun-Ju; Park, Kwan-Sik; Jeon, In-Sook; Choi, Jae-Woon; Lee, Sang-Jeon; Choy, Hyun E; Song, Ki-Duk; Lee, Hak-Kyo; Choi, Joong-Kook

    2016-07-01

    Lysosomes are cellular organelles containing diverse classes of catabolic enzymes that are implicated in diverse cellular processes including phagocytosis, autophagy, lipid transport, and aging. Lysosome-associated membrane proteins (LAMP-1 and LAMP-2) are major glycoproteins important for maintaining lysosomal integrity, pH, and catabolism. LAMP-1 and LAMP-2 are constitutively expressed in Salmonella-infected cells and are recruited to Salmonella-containing vacuoles (SCVs) as well as Salmonella-induced filaments (Sifs) that promote the survival and proliferation of the Salmonella. LAMP-3, also known as DC-LAMP/CD208, is a member of the LAMP family of proteins, but its role during Salmonella infection remains unclear. DNA microarray analysis identified LAMP-3 as one of the genes responding to LPS stimulation in THP-1 macrophage cells. Subsequent analyses reveal that LPS and Salmonella induced the expression of LAMP-3 at both the transcriptional and translational levels. Confocal Super resolution N-SIM imaging revealed that LAMP-3, like LAMP-2, shifts its localization from the cell surface to alongside Salmonella. Knockdown of LAMP-3 by specific siRNAs decreased the number of Salmonella recovered from the infected cells. Therefore, we conclude that LAMP-3 is induced by Salmonella infection and recruited to the Salmonella pathogen for intracellular proliferation.

  11. Disintegration of lysosomes mediated by GTPgammaS-treated cytosol: possible involvement of phospholipases.

    Science.gov (United States)

    Sai, Y; Matsuda, T; Arai, K; Ohkuma, S

    1998-04-01

    We showed previously that cytosol treated with guanosine 5'-O-(3-thiotriphosphate) (GTP-gammaS) disintegrated lysosomes in vitro [Sai, Y. et al. (1994) Biochem. Biophys. Res. Commun. 198, 869-877] in time-, temperature-, and dose-dependent manners. This also requires ATP, however, the latter can be substituted with deoxy-ATP, ADP, or ATPgammaS, suggesting no requirement of ATP hydrolysis. The lysis was inhibited by several chemical modifiers, including N-ethylmaleimide, 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole, and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, and by various phospholipase inhibitors (trifluoperazine, p-bromophenacyl bromide, nordihydroguaiaretic acid, W-7, primaquine, compound 48/80, neomycin, and gentamicin), but not by ONO-RS-082, an inhibitor of phospholipase A2. The reaction was also inhibited by phospholipids (phosphatidylinositol, phosphatidylserine, phosphatidic acid, and phosphatidylcholine) and diacylglycerol. Among the phospholipase A2 hydrolysis products of phospholipids, unsaturated fatty acids (oleate, linoleate, and arachidonate) and lysophospholipid (lysophosphatidylcholine) by themselves broke lysosomes down directly, whereas saturated fatty acids (palmitate and stearate) had little effect. We found that GTPgammaS-stimulated cytosolic phospholipase A2 activity was highly sensitive to ONO-RS-082. These results suggest the participation of phospholipase(s), though not cytosolic phospholipase A2, in the GTPgammaS-dependent lysis of lysosomes.

  12. Investigation of endosome and lysosome biology by ultra pH-sensitive nanoprobes.

    Science.gov (United States)

    Wang, Chensu; Zhao, Tian; Li, Yang; Huang, Gang; White, Michael A; Gao, Jinming

    2016-09-06

    Endosomes and lysosomes play a critical role in various aspects of cell physiology such as nutrient sensing, receptor recycling, protein/lipid catabolism, and cell death. In drug delivery, endosomal release of therapeutic payloads from nanocarriers is also important in achieving efficient delivery of drugs to reach their intracellular targets. Recently, we invented a library of ultra pH-sensitive (UPS) nanoprobes with exquisite fluorescence response to subtle pH changes. The UPS nanoprobes also displayed strong pH-specific buffer effect over small molecular bases with broad pH responses (e.g., chloroquine and NH4Cl). Tunable pH transitions from 7.4 to 4.0 of UPS nanoprobes cover the entire physiological pH of endocytic organelles (e.g., early and late endosomes) and lysosomes. These unique physico-chemical properties of UPS nanoprobes allowed a 'detection and perturbation' strategy for the investigation of luminal pH in cell signaling and metabolism, which introduces a nanotechnology-enabled paradigm for the biological studies of endosomes and lysosomes.

  13. Endocytic pathway rapidly delivers internalized molecules to lysosomes: an analysis of vesicle trafficking, clustering and mass transfer.

    Science.gov (United States)

    Pangarkar, Chinmay; Dinh, Anh-Tuan; Mitragotri, Samir

    2012-08-20

    Lysosomes play a critical role in intracellular drug delivery. For enzyme-based therapies, they represent a potential target site whereas for nucleic acid or many protein drugs, they represent the potential degradation site. Either way, understanding the mechanisms and processes involved in routing of materials to lysosomes after cellular entry is of high interest to the field of drug delivery. Most therapeutic cargoes other than small hydrophobic molecules enter the cells through endocytosis. Endocytosed cargoes are routed to lysosomes via microtubule-based transport and are ultimately shared by various lysosomes via tethering and clustering of endocytic vesicles followed by exchange of their contents. Using a combined experimental and numerical approach, here we studied the rates of mass transfer into and among the endocytic vesicles in a model cell line, 3T3 fibroblasts. In order to understand the relationship of mass transfer with microtubular transport and vesicle clustering, we varied both properties through various pharmacological agents. At the same time, microtubular transport and vesicle clustering were modeled through diffusion-advection equations and the Smoluchowski equations, respectively. Our analysis revealed that the rate of mass transfer is optimally related to microtubular transport and clustering properties of vesicles. Further, the rate of mass transfer is highest in the innate state of the cell. Any perturbation to either microtubular transport or vesicle aggregation led to reduced mass transfer to lysosome. These results suggest that in the absence of an external intervention the endocytic pathway appears to maximize molecular delivery to lysosomes. Strategies are discussed to reduce mass transfer to lysosomes so as to extend the residence time of molecules in endosomes or late endosomes, thus potentially increasing the likelihood of their escape before disposition in the lysosomes.

  14. Effect of collection, transport, processing and storage of blood specimens on the activity of lysosomal enzymes in plasma and leukocytes

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    Burin M.

    2000-01-01

    Full Text Available This study was designed to evaluate the effect of different conditions of collection, transport and storage on the quality of blood samples from normal individuals in terms of the activity of the enzymes ß-glucuronidase, total hexosaminidase, hexosaminidase A, arylsulfatase A and ß-galactosidase. The enzyme activities were not affected by the different materials used for collection (plastic syringes or vacuum glass tubes. In the evaluation of different heparin concentrations (10% heparin, 5% heparin, and heparinized syringe in the syringes, it was observed that higher doses resulted in an increase of at least 1-fold in the activities of ß-galactosidase, total hexosaminidase and hexosaminidase A in leukocytes, and ß-glucuronidase in plasma. When the effects of time and means of transportation were studied, samples that had been kept at room temperature showed higher deterioration with time (72 and 96 h before processing, and in this case it was impossible to isolate leukocytes from most samples. Comparison of heparin and acid citrate-dextrose (ACD as anticoagulants revealed that ß-glucuronidase and hexosaminidase activities in plasma reached levels near the lower normal limits when ACD was used. In conclusion, we observed that heparin should be used as the preferable anticoagulant when measuring these lysosomal enzyme activities, and we recommend that, when transport time is more than 24 h, samples should be shipped by air in a styrofoam box containing wet ice.

  15. Y682 mutation of amyloid precursor protein promotes endo-lysosomal dysfunction by disrupting APP-SorLA interaction

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    Luca Rosario La Rosa

    2015-04-01

    Full Text Available The intracellular transport and localization of amyloid precursor protein (APP are critical determinants of APP processing and β-amyloid peptide production, thus crucially important for the pathophysiology of Alzheimer’s disease (AD. Notably, the C-terminal Y682ENPTY687 domain of APP binds to specific adaptors controlling APP trafficking and sorting in neurons. Mutation on the Y682 residue to glycine (Y682G leads to altered APP sorting in hippocampal neurons that favors its accumulation in intracellular compartments and the release of soluble APPα. Such alterations induce premature aging and learning and cognitive deficits in APP Y682G mutant mice (APPYG/YG. Here, we report that Y682G mutation affects formation of the APP complex with sortilin-related receptor (SorLA, resulting in endo-lysosomal dysfunctions and neuronal degeneration. Moreover, disruption of the APP/SorLA complex changes the trafficking pathway of SorLA, with its consequent increase in secretion outside neurons. Mutations in the SorLA gene are a prognostic factor in AD, and increases in SorLA levels in cerebrospinal fluid are predictive of AD in humans. These results might open new possibilities in comprehending the role played by SorLA in its interaction with APP and in the progression of neuronal degeneration. In addition, they further underline the crucial role played by Y682 residue in controlling APP trafficking in neurons.

  16. Cardenolide-Induced Lysosomal Membrane Permeabilization Demonstrates Therapeutic Benefits in Experimental Human Non-Small Cell Lung Cancers

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    Tatjana Mijatovic

    2006-05-01

    Full Text Available Non-small cell lung cancers (NSCLCs are the leading cause of cancer deaths in most developed countries. Targeting heat shock protein 70 (Hsp70 expression and function, together with the induction of lysosomal membrane permeabilization (LMP, could overcome the multiple anti-cell death mechanisms evidenced in NSCLCs that are responsible for the failure of currently used chemotherapeutic drugs. Because cardenolides bind to the sodium pump, they affect multiple signaling pathways and thus have a number of marked effects on tumor cell behavior. The aim of the present study was to characterize in vitro and in vivo the antitumor effects of a new cardenolide (UNBS1450 on experimental human NSCLCs. UNBS1450 is a potent source of in vivo antitumor activity in the case of paclitaxeland oxaliplatin-resistant subcutaneous human NCIH727 and orthotopic A549 xenografts in nude mice. In vitro UNBS1450-mediated antitumor activity results from the induction of nonapoptotic cell death. UNBS1450 mediates the decrease of Hsp70 at both mRNA and protein levels, and this is at least partly due to UNBS1450-induced downregulation of NFAT5/ TonEBP (a factor responsible for the transcriptional control of Hsp70. These effects were paralleled by the induction of LMP, as evidenced by acridine orange staining and immunofluorescence analysis for cathepsin B accumulation.

  17. Iron-Mediated Lysosomal Membrane Permeabilization in Ethanol-Induced Hepatic Oxidative Damage and Apoptosis: Protective Effects of Quercetin.

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    Li, Yanyan; Chen, Man; Xu, Yanyan; Yu, Xiao; Xiong, Ting; Du, Min; Sun, Jian; Liu, Liegang; Tang, Yuhan; Yao, Ping

    2016-01-01

    Iron, in its free ferrous states, can catalyze Fenton reaction to produce OH∙, which is recognized as a crucial role in the pathogenesis of alcoholic liver diseases (ALD). As a result of continuous decomposition of iron-containing compounds, lysosomes contain a pool of redox-active iron. To investigate the important role of intralysosomal iron in alcoholic liver injury and the potential protection of quercetin, male C57BL/6J mice fed by Lieber De Carli diets containing ethanol (30% of total calories) were cotreated by quercetin or deferoxamine (DFO) for 15 weeks and ethanol-incubated mice primary hepatocytes were pretreated with FeCl3, DFO, and bafilomycin A1 at their optimal concentrations and exposure times. Chronic ethanol consumption caused an evident increase in lysosomal redox-active iron accompanying sustained oxidative damage. Iron-mediated ROS could trigger lysosomal membrane permeabilization (LMP) and subsequent mitochondria apoptosis. The hepatotoxicity was attenuated by reducing lysosomal iron while being exacerbated by escalating lysosomal iron. Quercetin substantially alleviated the alcoholic liver oxidative damage and apoptosis by decreasing lysosome iron and ameliorating iron-mediated LMP, which provided a new prospective of the use of quercetin against ALD.

  18. Preventive effects of p-coumaric acid on lysosomal dysfunction and myocardial infarct size in experimentally induced myocardial infarction.

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    Jyoti Roy, Abhro; Stanely Mainzen Prince, P

    2013-01-15

    The present study was designed to evaluate the preventive effects of p-coumaric acid on lysosomal dysfunction and myocardial infarct size in isoproterenol induced myocardial infarcted rats. Male albino Wistar rats were pretreated with p-coumaric acid (8 mg/kg body weight) daily for a period of 7 days after which isoproterenol (100mg/kg body weight) was injected subcutaneously into rats twice at an interval of 24h (8th and 9th day).The activity/levels of serum cardiac diagnostic markers, heart lysosomal lipid peroxidation products and the activities of lysosomal enzymes (β-glucuronidase, β-galactosidase, cathepsin-B and cathepsin-D) were significantly (Plysosomal fraction. The pretreatment with p-coumaric acid significantly (Plysosomal lipid peroxidation products and the activities of lysosomal enzymes. In addition, p-coumaric acid greatly reduced myocardial infarct size. p-Coumaric acid pretreatment (8 mg/kg body weight) to normal rats did not show any significant effect. Thus, this study showed that p-coumaric acid prevents lysosomal dysfunction against cardiac damage induced by isoproterenol and brings back the levels of lipid peroxidation products and activities of lysosomal enzymes to near normal levels. The in vitro study also revealed the free radical scavenging activity of p-coumaric acid. Thus, the observed effects are due to p-coumaric acid's free radical scavenging and membrane stabilizing properties.

  19. Polyketide synthase (PKS) reduces fusion of Legionella pneumophila-containing vacuoles with lysosomes and contributes to bacterial competitiveness during infection.

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    Shevchuk, Olga; Pägelow, Dennis; Rasch, Janine; Döhrmann, Simon; Günther, Gabriele; Hoppe, Julia; Ünal, Can Murat; Bronietzki, Marc; Gutierrez, Maximiliano Gabriel; Steinert, Michael

    2014-11-01

    L. pneumophila-containing vacuoles (LCVs) exclude endocytic and lysosomal markers in human macrophages and protozoa. We screened a L. pneumophila mini-Tn10 transposon library for mutants, which fail to inhibit the fusion of LCVs with lysosomes by loading of the lysosomal compartment with colloidal iron dextran, mechanical lysis of infected host cells, and magnetic isolation of LCVs that have fused with lysosomes. In silico analysis of the mutated genes, D. discoideum plaque assays and infection assays in protozoa and U937 macrophage-like cells identified well established as well as novel putative L. pneumophila virulence factors. Promising candidates were further analyzed for their co-localization with lysosomes in host cells using fluorescence microscopy. This approach corroborated that the O-methyltransferase, PilY1, TPR-containing protein and polyketide synthase (PKS) of L. pneumophila interfere with lysosomal degradation. Competitive infections in protozoa and macrophages revealed that the identified PKS contributes to the biological fitness of pneumophila strains and may explain their prevalence in the epidemiology of Legionnaires' disease.

  20. Ca2+ -regulated lysosome fusion mediates angiotensin II-induced lipid raft clustering in mesenteric endothelial cells.

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    Han, Wei-Qing; Chen, Wen-Dong; Zhang, Ke; Liu, Jian-Jun; Wu, Yong-Jie; Gao, Ping-Jin

    2016-04-01

    It has been reported that intracellular Ca2+ is involved in lysosome fusion and membrane repair in skeletal cells. Given that angiotensin II (Ang II) elicits an increase in intracellular Ca2+ and that lysosome fusion is a crucial mediator of lipid raft (LR) clustering, we hypothesized that Ang II induces lysosome fusion and activates LR formation in rat mesenteric endothelial cells (MECs). We found that Ang II acutely increased intracellular Ca2+ content, an effect that was inhibited by the extracellular Ca2+ chelator ethylene glycol tetraacetic acid (EGTA) and the inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release inhibitor 2-aminoethoxydiphenyl borate (2-APB). Further study showed that EGTA almost completely blocked Ang II-induced lysosome fusion, the translocation of acid sphingomyelinase (ASMase) to LR clusters, ASMase activation and NADPH (nicotinamide adenine dinucleotide phosphate) oxidase activation. In contrast, 2-APB had a slight inhibitory effect. Functionally, both the lysosome inhibitor bafilomycin A1 and the ASMase inhibitor amitriptyline reversed Ang II-induced impairment of vasodilation. We conclude that Ca2+ -regulated lysosome fusion mediates the Ang II-induced regulation of the LR-redox signaling pathway and mesenteric endothelial dysfunction.

  1. ESCRT-Dependent Cell Death in a Caenorhabditis elegans Model of the Lysosomal Storage Disorder Mucolipidosis Type IV.

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    Huynh, Julie M; Dang, Hope; Munoz-Tucker, Isabel A; O'Ketch, Marvin; Liu, Ian T; Perno, Savannah; Bhuyan, Natasha; Crain, Allison; Borbon, Ivan; Fares, Hanna

    2016-02-01

    Mutations in MCOLN1, which encodes the cation channel protein TRPML1, result in the neurodegenerative lysosomal storage disorder Mucolipidosis type IV. Mucolipidosis type IV patients show lysosomal dysfunction in many tissues and neuronal cell death. The ortholog of TRPML1 in Caenorhabditis elegans is CUP-5; loss of CUP-5 results in lysosomal dysfunction in many tissues and death of developing intestinal cells that results in embryonic lethality. We previously showed that a null mutation in the ATP-Binding Cassette transporter MRP-4 rescues the lysosomal defect and embryonic lethality of cup-5(null) worms. Here we show that reducing levels of the Endosomal Sorting Complex Required for Transport (ESCRT)-associated proteins DID-2, USP-50, and ALX-1/EGO-2, which mediate the final de-ubiquitination step of integral membrane proteins being sequestered into late endosomes, also almost fully suppresses cup-5(null) mutant lysosomal defects and embryonic lethality. Indeed, we show that MRP-4 protein is hypo-ubiquitinated in the absence of CUP-5 and that reducing levels of ESCRT-associated proteins suppresses this hypo-ubiquitination. Thus, increased ESCRT-associated de-ubiquitinating activity mediates the lysosomal defects and corresponding cell death phenotypes in the absence of CUP-5.

  2. Azadirachtin-induced apoptosis involves lysosomal membrane permeabilization and cathepsin L release in Spodoptera frugiperda Sf9 cells.

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    Wang, Zheng; Cheng, Xingan; Meng, Qianqian; Wang, Peidan; Shu, Benshui; Hu, Qiongbo; Hu, Meiying; Zhong, Guohua

    2015-07-01

    Azadirachtin as a kind of botanical insecticide has been widely used in pest control. We previously reported that azadirachtin could induce apoptosis of Spodoptera litura cultured cell line Sl-1, which involves in the up-regulation of P53 protein. However, the detailed mechanism of azadirachtin-induced apoptosis is not clearly understood in insect cultured cells. The aim of the present study was to address the involvement of lysosome and lysosomal protease in azadirachtin-induced apoptosis in Sf9 cells. The result confirmed that azadirachtin indeed inhibited proliferation and induced apoptosis. The lysosomes were divided into different types as time-dependent manner, which suggested that changes of lysosomes were necessarily physiological processes in azadirachtin-induced apoptosis in Sf9 cells. Interestingly, we noticed that azadirachtin could trigger lysosomal membrane permeabilization and cathepsin L releasing to cytosol. Z-FF-FMK (a cathepsin L inhibitor), but not CA-074me (a cathepsin B inhibitor), could effectively hinder the apoptosis induced by azadirachtin in Sf9 cells. Meanwhile, the activity of caspase-3 could also be inactivated by the inhibition of cathepsin L enzymatic activity induced by Z-FF-FMK. Taken together, our findings suggest that azadirachtin could induce apoptosis in Sf9 cells in a lysosomal pathway, and cathepsin L plays a pro-apoptosis role in this process through releasing to cytosol and activating caspase-3.

  3. Iron-Mediated Lysosomal Membrane Permeabilization in Ethanol-Induced Hepatic Oxidative Damage and Apoptosis: Protective Effects of Quercetin

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    Yanyan Li

    2016-01-01

    Full Text Available Iron, in its free ferrous states, can catalyze Fenton reaction to produce OH∙, which is recognized as a crucial role in the pathogenesis of alcoholic liver diseases (ALD. As a result of continuous decomposition of iron-containing compounds, lysosomes contain a pool of redox-active iron. To investigate the important role of intralysosomal iron in alcoholic liver injury and the potential protection of quercetin, male C57BL/6J mice fed by Lieber De Carli diets containing ethanol (30% of total calories were cotreated by quercetin or deferoxamine (DFO for 15 weeks and ethanol-incubated mice primary hepatocytes were pretreated with FeCl3, DFO, and bafilomycin A1 at their optimal concentrations and exposure times. Chronic ethanol consumption caused an evident increase in lysosomal redox-active iron accompanying sustained oxidative damage. Iron-mediated ROS could trigger lysosomal membrane permeabilization (LMP and subsequent mitochondria apoptosis. The hepatotoxicity was attenuated by reducing lysosomal iron while being exacerbated by escalating lysosomal iron. Quercetin substantially alleviated the alcoholic liver oxidative damage and apoptosis by decreasing lysosome iron and ameliorating iron-mediated LMP, which provided a new prospective of the use of quercetin against ALD.

  4. Contribution of mitochondria and lysosomes to photodynamic therapy-induced death in cancer cells

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    Nieminen, Anna-Liisa; Azizuddin, Kashif; Zhang, Ping; Kenney, Malcolm E.; Pediaditakis, Peter; Lemasters, John J.; Oleinick, Nancy L.

    2008-02-01

    In photodynamic therapy (PDT), visible light activates a photosensitizing drug added to a tissue, resulting in singlet oxygen formation and cell death. Employing confocal microscopy, we previously found that the phthalocyanine Pc 4 localized primarily to mitochondrial membranes in various cancer cell lines, resulting in mitochondrial reactive oxygen species (ROS) production, followed by inner membrane permeabilization (mitochondrial permeability transition) with mitochondrial depolarization and swelling, which in turn led to cytochrome c release and apoptotic death. Recently, derivatives of Pc 4 with OH groups added to one of the axial ligands were synthesized. These derivatives appeared to be taken up more avidly by cells and caused more cytotoxicity than the parent compound Pc 4. Using organelle-specific fluorophores, we found that one of these derivatives, Pc 181, accumulated into lysosomes and that PDT with Pc 181 caused rapid disintegration of lysosomes. We hypothesized that chelatable iron released from lysosomes during PDT contributes to mitochondrial damage and subsequent cell death. We monitored cytosolic Fe2+ concentrations after PDT with calcein. Fe2+ binds to calcein causing quenching of calcein fluorescence. After bafilomycin, an inhibitor of the vacuolar proton-translocating ATPase, calcein fluorescence became quenched, an effect prevented by starch desferal s-DFO, an iron chelator that enters cells by endocytosis. After Pc 181-PDT, cytosolic calcein fluorescence also decreased, indicating increased chelatable Fe2+ in the cytosol, and apoptosis occurred. s-DFO decreased Pc 181-PDT-induced apoptosis as measured by a decrease of caspase-3 activation. In isolated mitochondria preparations, Fe2+ induced mitochondrial swelling, which was prevented by Ru360, an inhibitor of the mitochondrial Ca2+ uniporter. The data support a hypothesis of oxidative injury in which Pc 181-PDT disintegrates lysosomes and releases constituents that synergistically promote

  5. Protective effects of positive lysosomal modulation in Alzheimer's disease transgenic mouse models.

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

    2011-01-01

    Alzheimer's disease (AD) is an age-related neurodegenerative pathology in which defects in proteolytic clearance of amyloid β peptide (Aβ) likely contribute to the progressive nature of the disorder. Lysosomal proteases of the cathepsin family exhibit up-regulation in response to accumulating proteins including Aβ(1-42). Here, the lysosomal modulator Z-Phe-Ala-diazomethylketone (PADK) was used to test whether proteolytic activity can be enhanced to reduce the accumulation events in AD mouse models expressing different levels of Aβ pathology. Systemic PADK injections in APP(SwInd) and APPswe/PS1ΔE9 mice caused 3- to 8-fold increases in cathepsin B protein levels and 3- to 10-fold increases in the enzyme's activity in lysosomal fractions, while neprilysin and insulin-degrading enzyme remained unchanged. Biochemical analyses indicated the modulation predominantly targeted the active mature forms of cathepsin B and markedly changed Rab proteins but not LAMP1, suggesting the involvement of enhanced trafficking. The modulated lysosomal system led to reductions in both Aβ immunostaining as well as Aβ(x-42) sandwich ELISA measures in APP(SwInd) mice of 10-11 months. More extensive Aβ deposition in 20-22-month APPswe/PS1ΔE9 mice was also reduced by PADK. Selective ELISAs found that a corresponding production of the less pathogenic Aβ(1-38) occurs as Aβ(1-42) levels decrease in the mouse models, indicating that PADK treatment leads to Aβ truncation. Associated with Aβ clearance was the elimination of behavioral and synaptic protein deficits evident in the two transgenic models. These findings indicate that pharmacologically-controlled lysosomal modulation reduces Aβ(1-42) accumulation, possibly through intracellular truncation that also influences extracellular deposition, and in turn offsets the defects in synaptic composition and cognitive functions. The selective modulation promotes clearance at different levels of Aβ pathology and provides proof

  6. Inhibitory effect of mTOR activator MHY1485 on autophagy: suppression of lysosomal fusion.

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    Yeon Ja Choi

    Full Text Available Autophagy is a major degradative process responsible for the disposal of cytoplasmic proteins and dysfunctional organelles via the lysosomal pathway. During the autophagic process, cells form double-membraned vesicles called autophagosomes that sequester disposable materials in the cytoplasm and finally fuse with lysosomes. In the present study, we investigated the inhibition of autophagy by a synthesized compound, MHY1485, in a culture system by using Ac2F rat hepatocytes. Autophagic flux was measured to evaluate the autophagic activity. Autophagosomes were visualized in Ac2F cells transfected with AdGFP-LC3 by live-cell confocal microscopy. In addition, activity of mTOR, a major regulatory protein of autophagy, was assessed by western blot and docking simulation using AutoDock 4.2. In the result, treatment with MHY1485 suppressed the basal autophagic flux, and this inhibitory effect was clearly confirmed in cells under starvation, a strong physiological inducer of autophagy. The levels of p62 and beclin-1 did not show significant change after treatment with MHY1485. Decreased co-localization of autophagosomes and lysosomes in confocal microscopic images revealed the inhibitory effect of MHY1485 on lysosomal fusion during starvation-induced autophagy. These effects of MHY1485 led to the accumulation of LC3II and enlargement of the autophagosomes in a dose- and time-dependent manner. Furthermore, MHY1485 induced mTOR activation and correspondingly showed a higher docking score than PP242, a well-known ATP-competitive mTOR inhibitor, in docking simulation. In conclusion, MHY1485 has an inhibitory effect on the autophagic process by inhibition of fusion between autophagosomes and lysosomes leading to the accumulation of LC3II protein and enlarged autophagosomes. MHY1485 also induces mTOR activity, providing a possibility for another regulatory mechanism of autophagy by the MHY compound. The significance of this study is the finding of a novel

  7. Protective effects of positive lysosomal modulation in Alzheimer's disease transgenic mouse models.

    Directory of Open Access Journals (Sweden)

    David Butler

    Full Text Available Alzheimer's disease (AD is an age-related neurodegenerative pathology in which defects in proteolytic clearance of amyloid β peptide (Aβ likely contribute to the progressive nature of the disorder. Lysosomal proteases of the cathepsin family exhibit up-regulation in response to accumulating proteins including Aβ(1-42. Here, the lysosomal modulator Z-Phe-Ala-diazomethylketone (PADK was used to test whether proteolytic activity can be enhanced to reduce the accumulation events in AD mouse models expressing different levels of Aβ pathology. Systemic PADK injections in APP(SwInd and APPswe/PS1ΔE9 mice caused 3- to 8-fold increases in cathepsin B protein levels and 3- to 10-fold increases in the enzyme's activity in lysosomal fractions, while neprilysin and insulin-degrading enzyme remained unchanged. Biochemical analyses indicated the modulation predominantly targeted the active mature forms of cathepsin B and markedly changed Rab proteins but not LAMP1, suggesting the involvement of enhanced trafficking. The modulated lysosomal system led to reductions in both Aβ immunostaining as well as Aβ(x-42 sandwich ELISA measures in APP(SwInd mice of 10-11 months. More extensive Aβ deposition in 20-22-month APPswe/PS1ΔE9 mice was also reduced by PADK. Selective ELISAs found that a corresponding production of the less pathogenic Aβ(1-38 occurs as Aβ(1-42 levels decrease in the mouse models, indicating that PADK treatment leads to Aβ truncation. Associated with Aβ clearance was the elimination of behavioral and synaptic protein deficits evident in the two transgenic models. These findings indicate that pharmacologically-controlled lysosomal modulation reduces Aβ(1-42 accumulation, possibly through intracellular truncation that also influences extracellular deposition, and in turn offsets the defects in synaptic composition and cognitive functions. The selective modulation promotes clearance at different levels of Aβ pathology and provides proof

  8. Groundwater arsenic contamination in one of the 107 arsenic-affected blocks in West Bengal, India: Status, distribution, health effects and factors responsible for arsenic poisoning.

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    Roychowdhury, Tarit

    2010-11-01

    A somewhat detailed study was carried out in Gaighata, one of the 107 arsenic-affected blocks in West Bengal, India, to determine the degree of groundwater contamination with arsenic, its depth wise distribution, correlation with iron, arsenical health effects to the inhabitants and the factors responsible for arsenic poisoning. Groundwater in all the 107 mouzas over 13 gram-panchayets in Gaighata block contains arsenic above 0.01mgl(-1) and in 91 mouzas, arsenic concentration has been found above 0.05mgl(-1). About 59.2 and 40.3% of the tubewell water samples contain arsenic above 0.01 and 0.05mgl(-1), respectively. The approximate population drinking arsenic-contaminated water above 0.01 and 0.05mgl(-1) are 106,560 and 72,540, respectively. The tubewells that were installed within the depth range of 15.4-30.3m are mostly arsenic-contaminated. Even the shallow groundwater level (7.87-15.1m) is arsenic-contaminated. Both arsenic and iron concentrations in groundwater gradually increase from lower depth to higher depth up to 39.4m, and then decrease with increasing depth. About 58% of the deep tubewell water samples (depth range 122-182m, n=31) contain arsenic ≥0.05mgl(-1). About 72% of the arsenic-contaminated deep tubewells (n=18) were safe when surveyed first time. But within a span of 2-5 years, they became contaminated with arsenic. The linear regression shows direct correlation between arsenic and iron concentrations in groundwater (r(2)=0.8114, p<0.0001, n=912). Intakes of inorganic arsenic from water by an adult male and female in the surveyed areas are 11.7 and 13.1μg/kg body wt./day, respectively and these values are higher than the WHO recommended PTDI value of inorganic arsenic (2.1μg/kg body wt./day). Mean arsenic concentrations in urine, hair and nail samples, collected from the inhabitants of Gutri mouza are higher than their normal level and the values are 292μgl(-1) (range: 8.35-1024μg l(-1), n=193), 2.50mgkg(-1) (range: 0.17-5.99mgkg(-1), n

  9. Neuronal transport of acid hydrolases and peroxidase within the lysosomal system or organelles: involvement of agranular reticulum-like cisterns.

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    Broadwell, R D; Oliver, C; Brightman, M W

    1980-04-01

    Neurosecretory neurons of the hyperosmotically stressed hypothalamo-neurohypophysial system have been a useful model with which to demonstrate interrelationships among perikaryal lysosomes, agranular reticulum-like cisterns, endocytotic vacuoles, and the axoplasmic transport of acid hydrolases and horseradish peroxidase. Supraoptic neurons from normal mice and mice given 2% salt water to drink for 5--8 days have been studied using enzyme cytochemical techniques for peroxidase and lysosomal acid hydrolases. Peroxidase-labeling of these neurons was accomplished by intravenous injection or cerebral ventriculocisternal perfusion of the protein as previously reported (Broadwell and Brightman, '79). Compared to normal controls, supraoptic cell bodies from hyperosmotically stimulated mice contained elevated concentrations of peroxidase-labeled dense bodies demonstrated to be secondary lysosomes and acid hydrolase-positive and peroxidase-positive cisterns either attached or unattached to secondary lysosomes. These cisterns were smooth-surfaced and 400--1,000 A wide. Their morphology was similar to that of the agranular reticulum. Some of the cisterns contained both peroxidase and acid hydrolase activities. The cisterns probably represent an elongated form of lysosome and, therefore, are not elements of the agranular reticulum per se. By virtue of their direct connections with perikaryal secondary lysosomes, these cisterns may provide the route by which acid hydrolases and exogenous macromolecules can leave perikaryal secondary lysosomes for anterograde flow down the axon. Very few smooth-surfaced cisterns were involved in the retrograde transport of peroxidase within pituitary stalk axons from normal and salt-treated mice injected intravenously with peroxidase. Peroxidase undergoing retrograde transport was predominantly in endocytotic structures such as vacuoles and cup-shaped organelles, which deliver this exogenous macromolecule directly to secondary lysosomes for

  10. Lysosome-associated protein 1 (LAMP-1) and lysosome-associated protein 2 (LAMP-2) in a larger family carrier of Fabry disease.

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    Pereira, Ester M; do Monte, Semiramis J H; do Nascimento, Fernando F; de Castro, Jose A F; Sousa, Jackeline L M; Filho, Henrique C S A L C; da Silva, Raimundo N; Labilloy, Anatália; Monte Neto, José T; da Silva, Adalberto S

    2014-02-15

    This study investigated the potential relationship between the expression levels of lysosome-associated membrane proteins (LAMP) 1 and 2 and responses to enzyme replacement therapy (ERT) in the members of a single family with Fabry disease (FD). LAMP levels were assessed by flow cytometry in leukocytes from 17 FD patients who received an eight-month course of ERT course and 101 healthy individuals. We found that phagocytic cells from the FD patients had higher expression levels of both LAMP-1 and LAMP-2, relative to the levels in phagocytes from the healthy controls (p=0.001). Furthermore, the LAMP-1 and LAMP-2 levels in phagocytes from the FD carriers continuously decreased with ERT administration to reach levels similar to those in healthy controls. We suggest that LAMP-1 and LAMP-2 could be used as additional markers with which to assess ERT effectiveness in FD.

  11. Bone cement distribution in the vertebral body affects chances of recompression after percutaneous vertebroplasty treatment in elderly patients with osteoporotic vertebral compression fractures

    Science.gov (United States)

    Zhang, Liang; Wang, Qiang; Wang, Lin; Shen, Jian; Zhang, Qiwei; Sun, Changtai

    2017-01-01

    Objective Percutaneous vertebroplasty (PVP) is a surgical procedure that has been widely used to treat patients suffering from osteoporotic vertebral compression fractures (OVCFs). The procedure involves injection of bone cement into a fractured vertebra. In this study, we investigated whether the distribution of the cement in the vertebral body is related to the occurrence of recompression after surgery. Patients and methods A total of 172 patients diagnosed with OVCF, from January 2008 to June 2013, were retrospectively reviewed. Fifty of these patients experienced recompression after surgery during the follow-up period (recompression group), and 122 patients had no recompression observed during the follow-up period (control group). Statistical analysis was performed to compare clinical and operative parameters between these two groups. Results Differences were found in bone cement distribution between the recompression group and control group (P=0.001). Patients with bone cement distributed around both upper and lower endplates had a significantly less incidence of recompression (4/50 patients), when compared to other patterns of cement distribution (eg, below upper endplate, above lower endplate, and in the middle of vertebral body). The logistic multiple regression analysis also indicated that patients with bone cement distributed around both the upper and lower endplates had a lower risk of recompression when compared to patients with bone cement distributed in the middle of vertebral body (odds ratio =0.223, P=0.003). Conclusion We herein suggest that the control of bone cement distribution during surgery provides beneficial effects on reducing the risks of recompression after PVP treatment in patients with OVCF. PMID:28260871

  12. Identification of an Allosteric Binding Site on Human Lysosomal Alpha-Galactosidase Opens the Way to New Pharmacological Chaperones for Fabry Disease

    Science.gov (United States)

    den-Haan, Helena; Pérez-Sánchez, Horacio; Del Prete, Rosita; Liguori, Ludovica; Cimmaruta, Chiara; Lukas, Jan; Andreotti, Giuseppina

    2016-01-01

    Personalized therapies are required for Fabry disease due to its large phenotypic spectrum and numerous different genotypes. In principle, missense mutations that do not affect the active site could be rescued with pharmacological chaperones. At present pharmacological chaperones for Fabry disease bind the active site and couple a stabilizing effect, which is required, to an inhibitory effect, which is deleterious. By in silico docking we identified an allosteric hot-spot for ligand binding where a drug-like compound, 2,6-dithiopurine, binds preferentially. 2,6-dithiopurine stabilizes lysosomal alpha-galactosidase in vitro and rescues a mutant that is not responsive to a mono-therapy with previously described pharmacological chaperones, 1-deoxygalactonojirimycin and galactose in a cell based assay. PMID:27788225

  13. Chronic high glucose inhibits albumin reabsorption by lysosomal alkalinization in cultured porcine proximal tubular epithelial cells (LLC-PK1).

    Science.gov (United States)

    Ishibashi, Fukashi

    2006-06-01

    Lysosomal acidification is a key step of albumin reabsorption in proximal tubular epithelial cells (PTECs). This study was performed to examine the influence of chronic high glucose on lysosomal acidification in cultured PTECs. Porcine PTECs (LLC-PK(1) cells) were cultured in 16.7 mM (300 mg/dl) glucose (HG) alone or with 0.5 mM phlorizin for 24 weeks and subsequently for 12 weeks in 5.5 mM (100 mg/dl) glucose (NG). Chronic HG inhibited the fluorescein isothiocyanate (FITC)-albumin (A) uptake progressively, while phlorizin reversed the inhibition. NG for 12 weeks after HG normalized the uptake. The time-dependent uptake of FITC-A was inhibited by HG and bafilomycin A(1) (BafA(1)) after 15 min and by 4,4'-diisothiocyanato-2,2'-disulfonic acid (DIDS) and N-ethyl-N-isopropyl-amiloride (EIPA) after 3 min. Cellular ATP was depleted by HG and restored by NG. Lysosomal pH, assessed by an acidotropic fluorescent probe, was alkalinized (pH 4.5-7.8) with 5.5-27.8 mM glucose and normalized by subsequent NG. BafA(1) alkalinized lysosomes, and the concentration required to 50% change for the pH and 50% inhibition of FITC-A uptake was similar. EIPA inhibited FITC-A uptake, but did not influence lysosomal pH. DIDS inhibited FITC-A uptake, and unexpectedly lowered lysosomal pH. Real time PCR showed that HG reduced the mRNA level for vacuolar H(+)-ATPase, but did not alter those of chloride channel-5 and Na(+)-H(+)-exchanger-3. In conclusion, the chronic HG inhibits albumin reabsorption by lysosomal alkalinization in PTECs, probably due to ATP depletion and down-regulation of vacuolar H(+)-ATPase.

  14. Ceria nanoparticles stabilized by organic surface coatings activate the lysosome-autophagy system and enhance autophagic clearance.

    Science.gov (United States)

    Song, Wensi; Soo Lee, Seung; Savini, Marzia; Popp, Lauren; Colvin, Vicki L; Segatori, Laura

    2014-10-28

    Cerium oxide nanoparticles (nanoceria) are widely used in a variety of industrial applications including UV filters and catalysts. The expanding commercial scale production and use of ceria nanoparticles have inevitably increased the risk of release of nanoceria into the environment as well as the risk of human exposure. The use of nanoceria in biomedical applications is also being currently investigated because of its recently characterized antioxidative properties. In this study, we investigated the impact of ceria nanoparticles on the lysosome-autophagy system, the main catabolic pathway that is activated in mammalian cells upon internalization of exogenous material. We tested a battery of ceria nanoparticles functionalized with different types of biocompatible coatings (N-acetylglucosamine, polyethylene glycol and polyvinylpyrrolidone) expected to have minimal effect on lysosomal integrity and function. We found that ceria nanoparticles promote activation of the transcription factor EB, a master regulator of lysosomal function and autophagy, and induce upregulation of genes of the lysosome-autophagy system. We further show that the array of differently functionalized ceria nanoparticles tested in this study enhance autophagic clearance of proteolipid aggregates that accumulate as a result of inefficient function of the lysosome-autophagy system. This study provides a mechanistic understanding of the interaction of ceria nanoparticles with the lysosome-autophagy system and demonstrates that ceria nanoparticles are activators of autophagy and promote clearance of autophagic cargo. These results provide insights for the use of nanoceria in biomedical applications, including drug delivery. These findings will also inform the design of engineered nanoparticles with safe and precisely controlled impact on the environment and the design of nanotherapeutics for the treatment of diseases with defective autophagic function and accumulation of lysosomal storage material.

  15. Turning the gun on cancer: Utilizing lysosomal P-glycoprotein as a new strategy to overcome multi-drug resistance.

    Science.gov (United States)

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

    2016-07-01

    Oxidative stress plays a role in the development of drug resistance in cancer cells. Cancer cells must constantly and rapidly adapt to changes in the tumor microenvironment, due to alterations in the availability of nutrients, such as glucose, oxygen and key transition metals (e.g., iron and copper). This nutrient flux is typically a consequence of rapid growth, poor vascularization and necrosis. It has been demonstrated that stress factors, such as hypoxia and glucose deprivation up-regulate master transcription factors, namely hypoxia inducible factor-1α (HIF-1α), which transcriptionally regulate the multi-drug resistance (MDR), transmembrane drug efflux transporter, P-glycoprotein (Pgp). Interestingly, in addition to the established role of plasma membrane Pgp in MDR, a new paradigm of intracellular resistance has emerged that is premised on the ability of lysosomal Pgp to transport cytotoxic agents into this organelle. This mechanism is enabled by the topological inversion of Pgp via endocytosis resulting in the transporter actively pumping agents into the lysosome. In this way, classical Pgp substrates, such as doxorubicin (DOX), can be actively transported into this organelle. Within the lysosome, DOX becomes protonated upon acidification of the lysosomal lumen, causing its accumulation. This mechanism efficiently traps DOX, preventing its cytotoxic interaction with nuclear DNA. This review discusses these effects and highlights a novel mechanism by which redox-active and protonatable Pgp substrates can utilize lysosomal Pgp to gain access to this compartment, resulting in catastrophic lysosomal membrane permeabilization and cell death. Hence, a key MDR mechanism that utilizes Pgp (the "gun") to sequester protonatable drug substrates safely within lysosomes can be "turned on" MDR cancer cells to destroy them from within.

  16. Repetitive stimulation of autophagy-lysosome machinery by intermittent fasting preconditions the myocardium to ischemia-reperfusion injury.

    Science.gov (United States)

    Godar, Rebecca J; Ma, Xiucui; Liu, Haiyan; Murphy, John T; Weinheimer, Carla J; Kovacs, Attila; Crosby, Seth D; Saftig, Paul; Diwan, Abhinav

    2015-01-01

    Autophagy, a lysosomal degradative pathway, is potently stimulated in the myocardium by fasting and is essential for maintaining cardiac function during prolonged starvation. We tested the hypothesis that intermittent fasting protects against myocardial ischemia-reperfusion injury via transcriptional stimulation of the autophagy-lysosome machinery. Adult C57BL/6 mice subjected to 24-h periods of fasting, every other day, for 6 wk were protected from in-vivo ischemia-reperfusion injury on a fed day, with marked reduction in infarct size in both sexes as compared with nonfasted controls. This protection was lost in mice heterozygous null for Lamp2 (coding for lysosomal-associated membrane protein 2), which demonstrate impaired autophagy in response to fasting with accumulation of autophagosomes and SQSTM1, an autophagy substrate, in the heart. In lamp2 null mice, intermittent fasting provoked progressive left ventricular dilation, systolic dysfunction and hypertrophy; worsening cardiomyocyte autophagosome accumulation and lack of protection to ischemia-reperfusion injury, suggesting that intact autophagy-lysosome machinery is essential for myocardial homeostasis during intermittent fasting and consequent ischemic cardioprotection. Fasting and refeeding cycles resulted in transcriptional induction followed by downregulation of autophagy-lysosome genes in the myocardium. This was coupled with fasting-induced nuclear translocation of TFEB (transcription factor EB), a master regulator of autophagy-lysosome machinery; followed by rapid decline in nuclear TFEB levels with refeeding. Endogenous TFEB was essential for attenuation of hypoxia-reoxygenation-induced cell death by repetitive starvation, in neonatal rat cardiomyocytes, in-vitro. Taken together, these data suggest that TFEB-mediated transcriptional priming of the autophagy-lysosome machinery mediates the beneficial effects of fasting-induced autophagy in myocardial ischemia-reperfusion injury.

  17. Large-scale biotic interaction effects - tree cover interacts with shade toler-ance to affect distribution patterns of herb and shrub species across the Alps

    DEFF Research Database (Denmark)

    Nieto-Lugilde, Diego; Lenoir, Jonathan; Abdulhak, Sylvain;

    2012-01-01

    distributions has scarcely been investigated. Here, we used species distribution modeling (SDM) to assess the effect of tree cover on the elevational range limits of 1,378 herb and shrub species across the Alps, based on 18,798 vegetation plots. We hypothesize that tree cover will have a negative effect...... on the occurrence on light-demanding species via size-asymmetr