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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

    BACKGROUND: Enhanced lysosomal trafficking is associated with metastatic cancer. In an attempt to discover cancer relevant lysosomal motor proteins, we compared the lysosomal proteomes from parental MCF-7 breast cancer cells with those from highly invasive MCF-7 cells that express an active form of...... the ErbB2 (DeltaN-ErbB2). METHODOLOGY/PRINCIPAL FINDINGS: Mass spectrometry analysis identified kinesin heavy chain protein KIF5B as the only microtubule motor associated with the lysosomes in MCF-7 cells, and ectopic DeltaN-ErbB2 enhanced its lysosomal association. KIF5B associated with lysosomes...... also in HeLa cervix carcinoma cells as analyzed by subcellular fractionation. The depletion of KIF5B triggered peripheral aggregations of lysosomes followed by lysosomal destabilization, and cell death in HeLa cells. Lysosomal exocytosis in response to plasma membrane damage as well as fluid phase...

  2. Virulent Brucella abortus Prevents Lysosome Fusion and Is Distributed within Autophagosome-Like Compartments

    OpenAIRE

    Pizarro-Cerdá, Javier; Moreno, Edgardo; Sanguedolce, Veronique; Mege, Jean-Louis; Gorvel, Jean-Pierre

    1998-01-01

    Virulent and attenuated Brucella abortus strains attach to and penetrate nonprofessional phagocytic HeLa cells. Compared to pathogenic Brucella, the attenuated strain 19 hardly replicates within cells. The majority of the strain 19 bacteria colocalized with the lysosome marker cathepsin D, suggesting that Brucella-containing phagosomes had fused with lysosomes, in which they may have degraded. The virulent bacteria prevented lysosome-phagosome fusion and were found distributed in the perinucl...

  3. Polarized secretion of lysosomal enzymes: co-distribution of cation- independent mannose-6-phosphate receptors and lysosomal enzymes along the osteoclast exocytic pathway

    OpenAIRE

    1988-01-01

    The osteoclast is a polarized cell which secretes large amounts of newly synthesized lysosomal enzymes into an apical extracellular lacuna where bone resorption takes place. Using immunocytochemical techniques, we have localized the cation-independent mannose-6-phosphate (Man6P) receptor and lysosomal enzymes in this cell type in order to determine the expression and distribution of this receptor and its ligands. The results demonstrate that the osteoclast expresses large amounts of immunorea...

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

    Directory of Open Access Journals (Sweden)

    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.

  5. Impaired lysosomal cobalamin transport in Alzheimer's disease.

    Science.gov (United States)

    Zhao, Hua; Li, Hongyun; Ruberu, Kalani; Garner, Brett

    2015-01-01

    Cobalamin (vitamin B12) is required for erythrocyte formation and DNA synthesis and it plays a crucial role in maintaining neurological function. As a coenzyme for methionine synthase and methylmalonyl-CoA mutase, cobalamin utilization depends on its efficient transit through the intracellular lysosomal compartment and subsequent delivery to the cytosol and mitochondria. Lysosomal function deteriorates in Alzheimer's disease (AD). Lysosomal acidification is defective in AD and lysosomal proteolysis is disrupted by AD-related presenilin 1 mutation. In this study, we propose that AD related lysosomal dysfunction may impair lysosomal cobalamin transport. The experiments use in vitro and in vivo models of AD to define how lysosomal dysfunction directly affects cobalamin utilization. SH-SY5Y-AβPP mutant cells were treated with a proteasome inhibitor to induce lysosomal amyloid-β accumulation. We metabolically labeled these cells with [57Co] cobalamin and isolated purified lysosomes, mitochondria, and cytosol fractions. The results indicated that proteasome inhibition was associated with lysosomal amyloid-β accumulation and a doubling of lysosomal [57Co] cobalamin levels. We also used AβPPxPS1 transgenic AD mice that were intraperitoneally injected with [57Co] cobalamin. The amount of [57Co] cobalamin in the major organs of these mice was measured and the subcellular [57Co] cobalamin distribution in the brain was assessed. The results demonstrated that lysosomal [57Co] cobalamin level was significantly increased by 56% in the AβPPxPS1 AD mouse brains as compared to wild type control mice. Together these data provide evidence that lysosomal cobalamin may be impaired in AD in association with amyloid-β accumulation. PMID:25125476

  6. Demographic characteristics and distribution of lysosomal storage disorder subtypes in Eastern China.

    Science.gov (United States)

    Chen, Xueru; Qiu, Wenjuan; Ye, Jun; Han, Lianshu; Gu, Xuefan; Zhang, Huiwen

    2016-04-01

    Lysosomal storage disorders (LSDs) are a group of >50 different types of inherited metabolic disorders that result from defects in the lysosome. The aim of this study was to investigate the distribution and demographic characteristics of the different subtypes of LSDs in Eastern China. From 2006 to 2012, 376 out of 1331 clinically suspected patients were diagnosed with 17 different subtypes of LSDs at our hospital. Mucopolysaccharidoses (MPS) were the most common group of LSDs (50.5%), followed by sphingolipidoses (25.4%) and Pompe disease (19.8%). Mucolipidosis type II/III accounted for the remaining 4% of diagnosed LSDs. MPS II was the most common form of MPS, comprising 47.4% of all MPS cases diagnosed, followed by MPS IVA (26.8%) and MPS I (16.3%). Gaucher disease and Niemann-Pick disease type A/B were the two most common forms of sphingolipidoses. There was a large variation in the time between disease onset and eventual diagnosis, from 0.3 years in infantile-onset Pompe disease to 30 years in Fabry disease, highlighting timely and accurate diagnosis of LSDs as the main challenge in China. PMID:26740238

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

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

    International Nuclear Information System (INIS)

    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

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

    Directory of Open Access Journals (Sweden)

    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.

  10. Alteration of Dynein Function Affects α-Synuclein Degradation via the Autophagosome-Lysosome Pathway

    OpenAIRE

    Da Li; Ji-Jun Shi; Cheng-Jie Mao; Sha Liu; Jian-Da Wang; Jing Chen; Fen Wang; Ya-Ping Yang; Wei-Dong Hu; Li-Fang Hu; Chun-Feng Liu

    2013-01-01

    Growing evidence suggests that dynein dysfunction may be implicated in the pathogenesis of neurodegeneration. It plays a central role in aggresome formation, the delivery of autophagosome to lysosome for fusion and degradation, which is a pro-survival mechanism essential for the bulk degradation of misfolded proteins and damaged organells. Previous studies reported that dynein dysfuntion was associated with aberrant aggregation of α-synuclein, which is a major component of inclusion bodies in...

  11. Tubular lysosome morphology and distribution within macrophages depend on the integrity of cytoplasmic microtubules

    Energy Technology Data Exchange (ETDEWEB)

    Swanson, J.; Bushnell, A.; Silverstein, S.C.

    1987-04-01

    Pinocytosis of the fluorescent dye lucifer yellow labels elongated, membrane-bound tubular organelles in several cell types, including cultured human monocytes, thioglycolate-elicited mouse peritoneal macrophages, and the macrophage-like cell line J774.2. These tubular structures can be identified as lysosomes by acid phosphatase histochemistry and immunofluorescence localization of cathepsin L. The abundance of tubular lysosomes is markedly increased by treatment with phorbol 12-myristate 13-acetate. When labeled by pinocytosis of microperoxidase and examined by electron microscopic histochemistry, the tubular lysosomes have an outside diameter of approx. = 75 nm and a length of several micrometers; they radiate from the cell's centrosphere in alignment with cytoplasmic microtubules and intermediate filaments. Incubation of phorbol myristate acetate-treated macrophages at 4/sup 0/C or in medium containing 5 ..mu..M colchicine or nocodazole at 37/sup 0/C leads to disassembly of microtubules and fragmentation of the tubular lysosomes. Return of the cultures to 37/sup 0/C or removal of nocodazole from the medium leads to reassembly of microtubules and the reappearance of tubular lysosomes within 10-20 min. The authors conclude that microtubules are essential for the maintenance of tubular lysosome morphology and that, in macrophages, a significant proportion of the lysosomal compartment is contained within these tubular structures.

  12. Tubular lysosome morphology and distribution within macrophages depend on the integrity of cytoplasmic microtubules

    International Nuclear Information System (INIS)

    Pinocytosis of the fluorescent dye lucifer yellow labels elongated, membrane-bound tubular organelles in several cell types, including cultured human monocytes, thioglycolate-elicited mouse peritoneal macrophages, and the macrophage-like cell line J774.2. These tubular structures can be identified as lysosomes by acid phosphatase histochemistry and immunofluorescence localization of cathepsin L. The abundance of tubular lysosomes is markedly increased by treatment with phorbol 12-myristate 13-acetate. When labeled by pinocytosis of microperoxidase and examined by electron microscopic histochemistry, the tubular lysosomes have an outside diameter of ≅ 75 nm and a length of several micrometers; they radiate from the cell's centrosphere in alignment with cytoplasmic microtubules and intermediate filaments. Incubation of phorbol myristate acetate-treated macrophages at 40C or in medium containing 5 μM colchicine or nocodazole at 370C leads to disassembly of microtubules and fragmentation of the tubular lysosomes. Return of the cultures to 370C or removal of nocodazole from the medium leads to reassembly of microtubules and the reappearance of tubular lysosomes within 10-20 min. The authors conclude that microtubules are essential for the maintenance of tubular lysosome morphology and that, in macrophages, a significant proportion of the lysosomal compartment is contained within these tubular structures

  13. Burden of lysosomal storage disorders in India: experience of 387 affected children from a single diagnostic facility.

    Science.gov (United States)

    Sheth, Jayesh; Mistri, Mehul; Sheth, Frenny; Shah, Raju; Bavdekar, Ashish; Godbole, Koumudi; Nanavaty, Nidhish; Datar, Chaitanya; Kamate, Mahesh; Oza, Nrupesh; Ankleshwaria, Chitra; Mehta, Sanjeev; Jackson, Marie

    2014-01-01

    Lysosomal storage disorders (LSDs) are considered to be a rare metabolic disease for the national health forum, clinicians, and scientists. This study aimed to know the prevalence of different LSDs, their geographical variation, and burden on the society. It included 1,110 children from January 2002 to December 2012, having coarse facial features, hepatomegaly or hepatosplenomegaly, skeletal dysplasia, neuroregression, leukodystrophy, developmental delay, cerebral-cerebellar atrophy, and abnormal ophthalmic findings. All subjects were screened for I-cell disease, glycolipid storage disorders (Niemann-Pick disease A/B, Gaucher), and mucopolysaccharide disorders followed by confirmatory lysosomal enzymes study from leucocytes and/or fibroblasts. Niemann-Pick disease-C (NPC) was confirmed by fibroblasts study using filipin stain. Various storage disorders were detected in 387 children (34.8 %) with highest prevalence of glycolipid storage disorders in 48 %, followed by mucopolysaccharide disorders in 22 % and defective sulfatide degradation in 14 % of the children. Less common defects were glycogen degradation defect and protein degradation defect in 5 % each, lysosomal trafficking protein defect in 4 %, and transport defect in 3 % of the patients. This study demonstrates higher incidence of Gaucher disease (16 %) followed by GM2 gangliosidosis that includes Tay-Sachs disease (10 %) and Sandhoff disease (7.8 %) and mucopolysaccharide disorders among all LSDs. Nearly 30 % of the affected children were born to consanguineous parents and this was higher (72 %) in children with Batten disease. Our study also demonstrates two common mutations c.1277_1278insTATC in 14.28 % (4/28) and c.964G>T (p.D322Y) in 10.7 % (3/28) for Tay-Sachs disease in addition to the earlier reported c.1385A>T (p.E462V) mutation in 21.42 % (6/28). PMID:23852624

  14. Subcellular distributions of lipids in cultured BHK cells: evidence for the enrichment of lysobisphosphatidic acid and neutral lipids in lysosomes.

    Science.gov (United States)

    Brotherus, J; Renkonen, O

    1977-03-01

    Homogenates of cultured hamster fibroblasts (BHK 21 cells) were fractionated by differential centrifugation into six main fractions: nuclear, mitochondrial, light mitochondrial, microsomal, soluble, and floating. The contents of several lipids and some marker enzymes were measured. According to the enzyme distributions, lysosomes were enriched both in the floating fraction and in the light mitochondrial fraction. Lysobisphosphatidic acid was enriched in the floating fraction more than tenfold relative to phospholipid. Cholesteryl esters and triglycerides were the main constituents of the fraction (70% of total lipids). Lysobisphosphatidic acid, triglycerides, and cholesteryl esters were enriched also in the light mitochondrial fraction. Their distribution patterns were different from those of the other lipids. Electron microscopy showed that the floating fraction contained numerous lipofuscin-like particles with darkly stained peripheries and with core regions staining like droplets of neutral lipids. Similar particles, frequently containing prominent multilamellar formations, were also common in intact cells. They contained cytochemically identified acid phosphatase. We conclude that lysobisphosphatidic acid was enriched in the lysosomes of the BHK cells and that the lysosomes also contained variable amounts of neutral lipids in the form of intralysosomal droplets. PMID:845501

  15. Invariant Natural Killer T cells are not affected by lysosomal storage in patients with Niemann-Pick disease type C

    OpenAIRE

    Speak, Anneliese O; Platt, Nicholas; Salio, Mariolina; te Vruchte, Danielle Taylor; Smith, David A.; Shepherd, Dawn; Veerapen, Natacha; Besra, Gurdyal; Yanjanin, Nicole M.; Simmons, Louise; Imrie, Jackie; Wraith, James E.; Lachmann, Robin; Hartung, Ralf; Runz, Heiko

    2012-01-01

    Invariant Natural Killer T (iNKT) cells are a specialised subset of T cells that are restricted to the MHC class I like molecule, CD1d. The ligands for iNKT cells are lipids, with the canonical superagonist being α-galactosylceramide, a non-mammalian glycosphingolipid. Trafficking of CD1d through the lysosome is required for the development of murine iNKT cells. Niemann-Pick type C (NPC) disease is a lysosomal storage disorder caused by dysfunction in either of two lysosomal proteins, NPC1 or...

  16. Lysosomal Trafficking Regulator (LYST).

    Science.gov (United States)

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

    2016-01-01

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

  17. Cancer-associated lysosomal changes

    DEFF Research Database (Denmark)

    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......-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...

  18. The enlarged lysosomes in beigej cells result from decreased lysosome fission and not increased lysosome fusion

    OpenAIRE

    Durchfort, Nina; Verhoef, Shane; Vaughn, Michael B.; Shrestha, Rishna; Adam, Dieter; Kaplan, Jerry; Ward, Diane McVey

    2011-01-01

    Chediak-Higashi Syndrome is an autosomal recessive disorder that affects vesicle morphology. The Chs1/Lyst protein is a member of the BEACH family of proteins. The absence of Chs1/Lyst gives rise to enlarged lysosomes. Lysosome size is regulated by a balance between vesicle fusion and fission and can be reversibly altered by acidifying the cytoplasm using Acetate Ringer’s or by incubating with the drug vacuolin-1. We took advantage of these procedures to determine rates of lysosome fusion and...

  19. The awesome lysosome

    OpenAIRE

    Ballabio, Andrea

    2016-01-01

    In the early 50s, Christian De Duve identified a new cellular structure, the lysosome, defined as the cell's “suicide bag” (de Duve, 2005). Sixty years later, it is clear that the lysosome greatly exceeded the expectations of its discoverer. Over 50 different types of lysosomal storage diseases have been identified, each due to the deficiency or malfunction of a specific lysosomal protein. In addition, an important role of the lysosome has been unveiled in several common human diseases, such ...

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

    Science.gov (United States)

    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

    2016-01-01

    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. PMID

  1. Proteomics of the Lysosome

    OpenAIRE

    Lübke, Torben; Lobel, Peter; Sleat, David

    2008-01-01

    Defects in lysosomal function have been associated with numerous monogenic human diseases typically classified as lysosomal storage diseases. However, there is increasing evidence that lysosomal proteins are also involved in more widespread human diseases including cancer and Alzheimer disease. Thus, there is a continuing interest in understanding the cellular functions of the lysosome and an emerging approach to this is the identification of its constituent proteins by proteomic analyses. To...

  2. Lysosomal Signaling Molecules Regulate Longevity in Caenorhabditis elegans

    OpenAIRE

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

    2015-01-01

    Lysosomes are crucial cellular organelles for human health that function in digestion and recycling of extracellular and intracellular macromolecules. We describe a signaling role for lysosomes that affects aging. In the worm, Caenorhabditis elegans, the lysosomal acid lipase LIPL-4 triggered nuclear translocalization of a lysosomal lipid chaperone LBP-8, consequently promoting longevity by activating the nuclear hormone receptors NHR-49 and NHR-80. We used high-throughput metabolomic analysi...

  3. Lysosome Transport as a Function of Lysosome Diameter

    OpenAIRE

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

    2014-01-01

    Lysosomes are membrane-bound organelles responsible for the transport and degradation of intracellular and extracellular cargo. The intracellular motion of lysosomes is both diffusive and active, mediated by motor proteins moving lysosomes along microtubules. We sought to determine how lysosome diameter influences lysosome transport. We used osmotic swelling to double the diameter of lysosomes, creating a population of enlarged lysosomes. This allowed us to directly examine the intracellular ...

  4. 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

  5. Lysosomes, cholesterol and atherosclerosis

    OpenAIRE

    Jerome, W. Gray

    2010-01-01

    Cholesterol-engorged macrophage foam cells are a critical component of the atherosclerotic lesion. Reducing the sterol deposits in lesions reduces clinical events. Sterol accumulations within lysosomes have proven to be particularly hard to mobilize out of foam cells. Moreover, excess sterol accumulation in lysosomes has untoward effects, including a complete disruption of lysosome function. Recently, we demonstrated that treatment of sterol-engorged macrophages in culture with triglyceride-c...

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

    Science.gov (United States)

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

    2015-01-01

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

  7. 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

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

    Directory of Open Access Journals (Sweden)

    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.

  9. Targeting the lysosome in cancer

    OpenAIRE

    Piao, Shengfu; Amaravadi, Ravi K.

    2015-01-01

    Lysosomes are membrane-bound intracellular organelles that receive macromolecules delivered by endocytosis, phagocytosis, and autophagy for degradation and recycling. Over the last decade, advances in lysosome research have established a broad role for the lysosome in the pathophysiology of disease. In this review, we highlight the recent discoveries in lysosome biology, with an emphasis on their implications for cancer therapy. We focus on targeting the lysosome in cancer by exploring lysoso...

  10. TFEB regulates lysosomal proteostasis.

    Science.gov (United States)

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

    2013-05-15

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

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

    Science.gov (United States)

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

    2015-01-01

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

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

    International Nuclear Information System (INIS)

    Highlights: ► p18 is a membrane adaptor that anchors mTORC1 to late endosomes/lysosomes. ► We examine the role of the p18-mTORC1 pathway in lysosome biogenesis. ► The loss of p18 causes accumulation of intact late endosomes by arresting lysosome maturation. ► Inhibition of mTORC1 activity with rapamycin phenocopies the defects of p18 loss. ► 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.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  14. 溶酶体贮积病%Lysosomal storage disorders

    Institute of Scientific and Technical Information of China (English)

    Yong QU

    2006-01-01

    Lysosomal storage disorders (LSDs) are genetic defects caused by lysosomal hydrolase deficiencies. These deficiencies lead to substrate accumulation affecting cells, tissues and organs. Detecting abnormal compound excretion and deficient enzymes assist diagnosis of these disorders for treatment and prevention. This mini review summarizes clinical presentations and diagnostic workup of LSDs and updates the new development in the area.

  15. Mutations in HYAL1, a member of a tandemly distributed multigene family encoding disparate hyaluronidase activities, cause a newly described lysosomal disorder, mucopolysaccharidosis IX

    OpenAIRE

    Triggs-Raine, Barbara; Salo, Timothy J.; Zhang, Hong; Wicklow, Brandy A; Natowicz, Marvin R.

    1999-01-01

    Hyaluronan (HA), a large glycosaminoglycan abundant in the extracellular matrix, is important in cell migration during embryonic development, cellular proliferation, and differentiation and has a structural role in connective tissues. The turnover of HA requires endoglycosidic breakdown by lysosomal hyaluronidase, and a congenital deficiency of hyaluronidase has been thought to be incompatible with life. However, a patient with a deficiency of serum hyaluronidase, now designated as mucopolysa...

  16. 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.

  17. The Biogenesis of Lysosomes and Lysosome-Related Organelles

    Science.gov (United States)

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

    2014-01-01

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

  18. 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.

  19. 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.

  20. Astrocytes and lysosomal storage diseases.

    Science.gov (United States)

    Rama Rao, K V; Kielian, T

    2016-05-26

    Lysosomal storage diseases (LSDs) encompass a wide range of disorders characterized by inborn errors of lysosomal function. The majority of LSDs result from genetic defects in lysosomal enzymes, although some arise from mutations in lysosomal proteins that lack known enzymatic activity. Neuropathological abnormalities are a feature of several LSDs and when severe, represent an important determinant in disease outcome. Glial dysfunction, particularly in astrocytes, is also observed in numerous LSDs and has been suggested to impact neurodegeneration. This review will discuss the potential role of astrocytes in LSDs and highlight the possibility of targeting glia as a beneficial strategy to counteract the neuropathology associated with LSDs. PMID:26037807

  1. Transmembrane protein 106B, a risk factor in frontotemporal lobar degeneration, is a lysosomal type II transmembrane protein and affects autophagy

    OpenAIRE

    Lang, Christina

    2016-01-01

    Frontotemporal lobar degeneration (FTLD) is a fatal neurodegenerative disease with presenile onset. Clinically, it mainly presents with language disorders or personality and behavioural changes whereas pathologically patients show atrophy of the frontal and temporal lobes of the brain. Like in other neurodegenerative disorders, abnormal protein deposition can be detected in the affected areas of the brain nervous system. However, several different proteins have been identified ...

  2. Stereotyped distribution of proliferating keratinocytes in disorders affecting the epidermis

    International Nuclear Information System (INIS)

    We used the technique of autoradiography after incorporation of tritiated thymidine (3H-TdR) to evaluate keratinocyte proliferation in basal, epibasal, and other epidermal layers in 30 diseases affecting the epidermis. The number and proportion of 3H-TdR-labeled keratinocytes were counted in the different layers of the epidermis. Significant correlations were found between the proliferative indices of the different epidermal layers. Such links indicate that the epidermis responds in a rather stereotyped way to various pathological conditions. There exists some regulation in the distribution, number, and proportion of 3H-TdR-labeled keratinocytes in the various layers of the epidermis

  3. Gene therapy for lysosomal storage disorders: a good start.

    Science.gov (United States)

    Biffi, Alessandra

    2016-04-15

    Lysosomal storage disorders (LSDs) are a heterogeneous group of inherited diseases with a collective frequency of ∼1 in 7000 births, resulting from the deficiency in one or more enzymes or transporters that normally reside within the lysosomes. Pathology results from the progressive accumulation of uncleaved lipids, glycoproteins and/or glycosaminoglycans in the lysosomes and secondary damages that affect the brain, viscera, bones and connective tissues. Most treatment modalities developed for LSD, including gene therapy (GT), are based on the lysosome-specific cross-correction mechanism, by which close proximity of normal cells leads to the correction of the biochemical consequences of enzymatic deficiency within the neighboring cells. Here, GT efforts addressing these disorders are reviewed with an up-to-date discussion of their impact on the LSD disease phenotype in animal models and patients. PMID:26604151

  4. 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.

  5. Lysosomal Adaptation: How the Lysosome Responds to External Cues

    OpenAIRE

    Settembre, C.; Ballabio, A

    2014-01-01

    Recent evidence indicates that the importance of the lysosome in cell metabolism and organism physiology goes far beyond the simple disposal of cellular garbage. This dynamic organelle is situated at the crossroad of the most important cellular pathways and is involved in sensing, signaling, and transcriptional mechanisms that respond to environmental cues, such as nutrients. Two main mediators of these lysosomal adaptation mechanisms are the mTORC1 kinase complex and the transcription factor...

  6. Factors affecting daughters distribution among progeny testing Holstein bulls

    Directory of Open Access Journals (Sweden)

    Martino Cassandro

    2012-01-01

    Full Text Available The aim of this study was to investigate factors influencing the number of daughters of Holstein bulls during the progeny testing using data provided by the Italian Holstein Friesian Cattle Breeders Association. The hypothesis is that there are no differences among artificial insemination studs (AIS on the daughters distribution among progeny testing bulls. For each bull and beginning from 21 months of age, the distribution of daughters over the progeny testing period was calculated. Data were available on 1973 bulls born between 1986 and 2004, progeny tested in Italy and with at least 4 paternal half-sibs. On average, bulls exited the genetic centre at 11.3±1.1 months and reached their first official genetic proof at 58.0±3.1 months of age. An analysis of variance was performed on the cumulative frequency of daughters at 24, 36, 48, and 60 months. The generalized linear model included the fixed effects of year of birth of the bull (18 levels, artificial insemination stud (4 levels and sire of bull (137 levels. All effects significantly affected the variability of studied traits. Artificial insemination stud was the most important source of variation, followed by year of birth and sire of bull. Significant differences among AI studs exist, probably reflecting different strategies adopted during progeny testing.

  7. Lysosomal Dysfunction and α-Synuclein Aggregation in Parkinson's Disease: Diagnostic Links.

    Science.gov (United States)

    Moors, Tim; Paciotti, Silvia; Chiasserini, Davide; Calabresi, Paolo; Parnetti, Lucilla; Beccari, Tommaso; van de Berg, Wilma D J

    2016-06-01

    Lysosomal impairment is increasingly recognized as a central event in the pathophysiology of PD. Genetic associations between lysosomal storage disorders, including Gaucher disease and PD, highlight common risk factors and pathological mechanisms. Because the autophagy-lysosomal system is involved in the intralysosomal hydrolysis of dysfunctional proteins, lysosomal impairment may contribute to α-synuclein aggregation in PD. The degradation of α-synuclein is a complex process involving different proteolytic mechanisms depending on protein burden, folding, posttranslational modifications, and yet unknown factors. In this review, evidence for lysosomal dysfunction in PD and its intimate relationship with α-synuclein aggregation are discussed, after which the question of whether lysosomal proteins may serve as diagnostic biomarkers for PD is addressed. Changes in lysosomal enzymes, such as reduced glucocerebrosidase and cathepsin levels, have been observed in affected brain regions in PD patients. The detection of lysosomal proteins in CSF may provide a read-out of lysosomal dysfunction in PD and holds promise for the development of diagnostic PD biomarkers. Initial PD biomarker studies demonstrated altered lysosomal enzyme activities in CSF of PD patients when compared with controls. However, CSF lysosomal enzyme activities alone could not discriminate between PD patients and controls. The combination of CSF lysosomal markers with α-synuclein species and indicators of mitochondrial dysfunction, inflammation, and other pathological proteins in PD may be able to facilitate a more accurate diagnosis of PD. Further CSF biomarker studies are needed to investigate the utility of CSF lysosomal proteins as measures of disease state and disease progression in PD. © 2016 International Parkinson and Movement Disorder Society. PMID:26923732

  8. Lysosomal cell death at a glance

    DEFF Research Database (Denmark)

    Aits, Sonja; Jaattela, Marja

    2013-01-01

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

  9. The FTLD risk factor TMEM106B and MAP6 control dendritic trafficking of lysosomes

    NARCIS (Netherlands)

    Schwenk, B.M.; Lang, C.M.; Hogl, S.; Tahirovic, S.; Orozco, D.; Rentzsch, K.; Lichtenthaler, S.F.; Hoogenraad, Casper; Capell, A.; Haass, C.; Edbauer, D.

    2014-01-01

    TMEM106B is a major risk factor for frontotemporal lobar degeneration with TDP‐43 pathology. TMEM106B localizes to lysosomes, but its function remains unclear. We show that TMEM106B knockdown in primary neurons affects lysosomal trafficking and blunts dendritic arborization. We identify microtubule‐

  10. 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.

  11. 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,…

  12. Human recombinant lysosomal enzymes produced in microorganisms.

    Science.gov (United States)

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

    2015-01-01

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

  13. 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...... evolution of relative humidity based on statistical rate theory and computational fluid dynamics is developed. In our approach we use short-term steady-state steps to simulate the slowly evolving evaporation in the system. Experiments demonstrate considerably good agreement with the computer modeling and...

  14. Factors and pharmaceuticals that affect the radiopharmaceuticals bio distributions

    International Nuclear Information System (INIS)

    The pattern of biodistribution of radiopharmaceuticals may be affected by various agents and therapeutical procedures, chemotherapy agents, thyroid hormones, metals, radiotherapy, surgery, anesthetic agents, dialysis other radiopharmaceutical interactions. Recommendations for the detection of altered biodistribution in patients by causes not directly related with the pathology itself was given. pathology itself was given

  15. 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.

  16. Inhibitors of lysosomal cysteine proteases

    OpenAIRE

    Lyanna O. L.; Chorna V. I.

    2011-01-01

    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 anal...

  17. 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.

  18. Intracellular targeting of peroxiredoxin 6 to lysosomal organelles requires MAPK activity and binding to 14-3-3ε

    OpenAIRE

    Sorokina, Elena M.; Feinstein, Sheldon I.; Zhou, Suiping; Fisher, Aron B.

    2011-01-01

    Peroxiredoxin 6 (Prdx6), a bifunctional protein with GSH peroxidase and lysosomal-type phospholipase A2 activities, has been localized to both cytosolic and acidic compartments (lamellar bodies and lysosomes) in lung alveolar epithelium. We postulate that Prdx6 subcellular localization affects the balance between the two activities. Immunostaining localized Prdx6 to lysosome-related organelles in the MLE12 and A549 alveolar epithelial cell lines. Inhibition of trafficking by brefeldin A indic...

  19. 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

  20. Potential pitfalls and solutions for use of fluorescent fusion proteins to study the lysosome.

    Directory of Open Access Journals (Sweden)

    Ling Huang

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

  1. 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. PMID:27241615

  2. Role of lysosomes in cancer therapy

    Directory of Open Access Journals (Sweden)

    Halaby R

    2015-09-01

    Full Text Available Reginald Halaby Department of Biology, Montclair State University, Montclair, NJ, USA Abstract: Lysosomes are acidic organelles that are involved in cellular digestion by endocytosis, phagocytosis, and autophagy. They contain more than 50 hydrolases that are capable of degrading all macromolecules. There is accumulating evidence that lysosomal enzymes can provoke apoptotic cell death. This has important implications for cancer, where proapoptotic genes are mutated and antiapoptotic genes are often overexpressed leading to chemoresistance. Lysosomes play a dual role in cancer development depending on their subcellular localization. When they are located extracellularly they can promote invasion, angiogenesis, and metastasis. However, when they are located intracellularly they can trigger apoptosis by leaking into the cytosol. In this review, we examine the pathways by which lysosomes can evoke both apoptosis and tumorigenesis. Although cancer cells have defects in their apoptotic machinery, they can still undergo lysosomal cell death. We offer several strategies to explain how targeting lysosomes can serve as a putative model for the development of novel anticancer agents. Furthermore, we propose that lysosomal cell death is an effective treatment against apoptosis-resistant cancer cells and thus holds great potential as a therapeutic strategy for circumventing apoptosis deficiency in tumors. Keywords: cathepsins, lysosomal membrane permeability, apoptosis, chemoresistance 

  3. 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)P2, a lysosome-localized phosphoinositide, acts upstream of TRPML1. Furthermore, the PtdIns(3,5)P2-TRPML1-ALG-2-dynein signalling is necessary for lysosome tubulation and reformation. In contrast, the TRPML1 pathway is not required for the perinuclear accumulation of lysosomes observed in many LSDs, which is instead likely to be caused by secondary cholesterol accumulation that constitutively activates Rab7-RILP-dependent retrograde transport. Ca(2+) release from lysosomes thus provides an on-demand mechanism regulating lysosome motility, positioning and tubulation. PMID:26950892

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

    International Nuclear Information System (INIS)

    Radioactive galactose, covalently bound to cell surface glycoconjugates on mouse macrophage cells, P388D1, was used as a membrane marker to study the composition, and the kinetics of exchange, of plasma membrane-derived constituents in the membrane of secondary lysosomes. Secondary lysosomes were separated from endosomes and plasma membrane by self-forming Percoll density gradients. Horseradish peroxidase, taken up by fluid-phase pinocytosis, served as a vesicle contents marker to monitor transfer of endosomal contents into secondary lysosomes. Concurrently, the fraction of plasma membrane-derived label of secondary lysosomes increased by first order kinetics from 4 PAGE, labeled molecules of M/sub r/ 160-190 kD were depleted and of the M/sub r/ 100-120 kD were enriched in lysosome membrane compared with the relative composition of label on the cell surface. No corresponding selectivity was observed for the degradation of label, with all M/sub r/ classes being affected to the same relative extent. The results indicate that endocytosis-derived transfer of plasma membrane constitutents to secondary lysosomes is a limited and selective process, and that only ∼1% of internalized membrane is recycled via a membrane pool of secondary lysosomes

  5. Podocytes degrade endocytosed albumin primarily in lysosomes.

    Science.gov (United States)

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

    2014-01-01

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

  6. 77 FR 32717 - Distribution of Continued Dumping and Subsidy Offset to Affected Domestic Producers

    Science.gov (United States)

    2012-06-01

    ... FR 48546) on September 21, 2001, which was effective as of that date, in order to implement the CDSOA... Continued Dumping and Subsidy Offset to Affected Domestic Producers; Notice #0;#0;Federal Register / Vol. 77... Border Protection Distribution of Continued Dumping and Subsidy Offset to Affected Domestic...

  7. 76 FR 31019 - Distribution of Continued Dumping and Subsidy Offset to Affected Domestic Producers

    Science.gov (United States)

    2011-05-27

    ... the Federal Register (66 FR 48546) on September 21, 2001, which was effective as of that date, in... of Continued Dumping and Subsidy Offset to Affected Domestic Producers; Notice #0;#0;Federal Register... and Border Protection Distribution of Continued Dumping and Subsidy Offset to Affected...

  8. 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...

  9. 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.

  10. Nanoparticles restore lysosomal acidification defects: Implications for Parkinson and other lysosomal-related diseases.

    Science.gov (United States)

    Bourdenx, Mathieu; Daniel, Jonathan; Genin, Emilie; Soria, Federico N; Blanchard-Desce, Mireille; Bezard, Erwan; Dehay, Benjamin

    2016-03-01

    Lysosomal impairment causes lysosomal storage disorders (LSD) and is involved in pathogenesis of neurodegenerative diseases, notably Parkinson disease (PD). Strategies enhancing or restoring lysosomal-mediated degradation thus appear as tantalizing disease-modifying therapeutics. Here we demonstrate that poly(DL-lactide-co-glycolide) (PLGA) acidic nanoparticles (aNP) restore impaired lysosomal function in a series of toxin and genetic cellular models of PD, i.e. ATP13A2-mutant or depleted cells or glucocerebrosidase (GBA)-mutant cells, as well as in a genetic model of lysosomal-related myopathy. We show that PLGA-aNP are transported to the lysosome within 24 h, lower lysosomal pH and rescue chloroquine (CQ)-induced toxicity. Re-acidification of defective lysosomes following PLGA-aNP treatment restores lysosomal function in different pathological contexts. Finally, our results show that PLGA-aNP may be detected after intracerebral injection in neurons and attenuate PD-related neurodegeneration in vivo by mechanisms involving a rescue of compromised lysosomes. PMID:26761717

  11. Lysosomes and autophagy in aquatic animals.

    Science.gov (United States)

    Moore, Michael N; Kohler, Angela; Lowe, David; Viarengo, Aldo

    2008-01-01

    The lysosomal-autophagic system appears to be a common target for many environmental pollutants, as lysosomes accumulate many toxic metals and organic xenobiotics, which perturb normal function and damage the lysosomal membrane. In fact, autophagic reactions frequently involving reduced lysosomal membrane integrity or stability appear to be effective generic indicators of cellular well-being in eukaryotes: in social amoebae (slime mold), mollusks and fish, autophagy/membrane destabilization is correlated with many stress and toxicological responses and pathological reactions. Prognostic use of adverse lysosomal and autophagic reactions to environmental pollutants can be used for predicting cellular dysfunction and health in aquatic animals, such as shellfish and fish, which are extensively used as sensitive bioindicators in monitoring ecosystem health; and also represent a significant food resource for at least 20% of the global human population. Explanatory frameworks for prediction of pollutant impact on health have been derived encompassing a conceptual mechanistic model linking lysosomal damage and autophagic dysfunction with injury to cells and tissues. Methods are described for tracking in vivo autophagy of fluorescently labeled cytoplasmic proteins, measuring degradation of radiolabeled intracellular proteins and morphometric measurement of lysosomal/cytoplasmic volume ratio. Additional methods for the determination of lysosomal membrane stability in lower animals are also described, which can be applied to frozen tissue sections, protozoans and isolated cells in vivo. Experimental and simulated results have also indicated that nutritional deprivation (analogous in marine mussels to caloric restriction)-induced autophagy has a protective function against toxic effects mediated by reactive oxygen species (ROS). Finally, coupled measurement of lysosomal-autophagic reactions and simulation modelling is proposed as a practical toolbox for predicting toxic

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

    International Nuclear Information System (INIS)

    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.

  13. The distribution of attention in RSVP tasks and its sensitivity to affect, personality traits and estradiol

    OpenAIRE

    Dhinakaran, Janani

    2014-01-01

    In this thesis, the influences of positive and negative affect state, the personality trait neuroticism, and the hormone estradiol on selective attention are explored in three studies. In Study 1 a double-stream RSVP paradigm was used to explore if attention can be spatially diffused and focused by affect. A spatial interpretation of the diffuse mental state was not found. Study 2 studied the distribution of attention as influenced by neuroticism and cognitive control. Results confirmed that ...

  14. The transport and distribution of 3H-ABA affected by al sress on soybean seedig

    International Nuclear Information System (INIS)

    A hydroponic experiment combining radioisotope techniques was carried out to understand the effect of Al stress on the transport and the distribution of 3H-ABA by using Jilin70, a soybean variety of Al resistance. The transport and distribution of ABA affected by Al stress on soybean seedling were studied with radioisotope technique. The results showed that ABA could be transported up or down in soybean seedling. The stress of Al accelerated the transport of ABA and enhanced the distribution of ABA in the roots by Al stress. The paper present the foundation for the mechanisms of ABA under Al stress in plant. (authors)

  15. 75 FR 30529 - Distribution of Continued Dumping and Subsidy Offset to Affected Domestic Producers

    Science.gov (United States)

    2010-06-01

    ... the Federal Register (66 FR 48546) on September 21, 2001, which was effective as of that date, in... Homeland Security Customs and Border Protection Distribution of Continued Dumping and Subsidy Offset to... Dumping and Subsidy Offset to Affected Domestic Producers AGENCY: U.S. Customs and Border...

  16. ErbB2-associated changes in the lysosomal proteome

    DEFF Research Database (Denmark)

    Nylandsted, Jesper; Becker, Andrea C; Bunkenborg, Jakob;

    2011-01-01

    Late endosomes and lysosomes (hereafter referred to as lysosomes) play an essential role in the turnover of cellular macromolecules and organelles. Their biochemical characterization has so far depended on purification methods based on either density gradient centrifugations or magnetic...... purification of iron-loaded organelles. Owing to dramatic changes in lysosomal density and stability associated with lysosomal diseases and cancer, these methods are not optimal for the comparison of normal and pathological lysosomes. Here, we introduce an efficient method for the purification of intact...... lysosomes by magnetic immunoprecipitation with antibodies against the vacuolar-type H(+) -ATPase. Quantitative MS-based proteomics analysis of the obtained lysosomal membranes identified 60 proteins, most of which have previously been associated with the lysosomal compartment. Interestingly, the lysosomal...

  17. Lysosome-associated membrane proteins (LAMPs) regulate intracellular positioning of mitochondria in MC3T3-E1 cells.

    Science.gov (United States)

    Rajapakshe, Anupama R; Podyma-Inoue, Katarzyna A; Terasawa, Kazue; Hasegawa, Katsuya; Namba, Toshimitsu; Kumei, Yasuhiro; Yanagishita, Masaki; Hara-Yokoyama, Miki

    2015-02-01

    The intracellular positioning of both lysosomes and mitochondria meets the requirements of degradation and energy supply, which are respectively the two major functions for cellular maintenance. The positioning of both lysosomes and mitochondria is apparently affected by the nutrient status of the cells. However, the mechanism coordinating the positioning of the organelles has not been sufficiently elucidated. Lysosome-associated membrane proteins-1 and -2 (LAMP-1 and LAMP-2) are highly glycosylated proteins that are abundant in lysosomal membranes. In the present study, we demonstrated that the siRNA-mediated downregulation of LAMP-1, LAMP-2 or their combination enhanced the perinuclear localization of mitochondria, in the pre-osteoblastic cell line MC3T3-E1. On the other hand, in the osteocytic cell line MLO-Y4, in which both the lysosomes and mitochondria originally accumulate in the perinuclear region and mitochondria also fill dendrites, the effect of siRNA of LAMP-1 or LAMP-2 was barely observed. LAMPs are not directly associated with mitochondria, and there do not seem to be any accessory molecules commonly required to recruit the motor proteins to lysosomes and mitochondria. Our results suggest that LAMPs may regulate the positioning of lysosomes and mitochondria. A possible mechanism involving the indirect and context-dependent action of LAMPs is discussed. PMID:25246127

  18. Effect of subsurface drainage on salt movement and distribution in salt-affected soils

    International Nuclear Information System (INIS)

    This study was carried out to evaluate different subsurface drainage treatments (combinations of depth and spacing) on salt movement and distribution. The soil is clay and the drainage was designed according to the steady-state condition (Hooghoudt's equation). Three spacings and two depths resulted in six drainage treatments. Soil samples represented the initial state of every treatment and after 14 months they (cotton followed by wheat) were analysed. The data show that drain depth has its effective role in salt leaching, while drain spacing has its effect on salt distribution in the soil profile. The leaching rate of each specific ion is also affected by the different drainage treatments. In general, the salt movement and distribution should be taken into consideration when evaluating the design of drainage systems. (author)

  19. 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.

  20. Screening and Optimization of Ligand Conjugates for Lysosomal Targeting

    OpenAIRE

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

    2011-01-01

    The use of lysosome-targeted liposomes may significantly improve the delivery of therapeutic enzymes and chaperones into lysosomes for the treatment of lysosomal storage disorders. The aim of this research was to synthesize new potentially lysosomotropic ligands on a base of Neutral Red and rhodamine B and to study their ability to enhance specific lysosomal delivery of surface-modified liposomes loaded with a model compound, fluorescein isothiocyanate-dextran (FD). The delivery of these lipo...

  1. Purification of Lysosomes Using Supraparamagnetic Iron Oxide Nanoparticles (SPIONs).

    Science.gov (United States)

    Rofe, Adam P; Pryor, Paul R

    2016-04-01

    Lysosomes can be rapidly isolated from tissue culture cells using supraparamagnetic iron oxide particles (SPIONs). In this protocol, colloidal iron dextran (FeDex) particles, a type of SPION, are taken up by cultured mouse macrophage cells via the endocytic pathway. The SPIONs accumulate in lysosomes, the end point of the endocytic pathway, permitting the lysosomes to be isolated magnetically. The purified lysosomes are suitable for in vitro fusion assays or for proteomic analysis. PMID:27037068

  2. Promotion of Proapoptotic Signals by Lysosomal Photodamage.

    Science.gov (United States)

    Kessel, David; Reiners, John J

    2015-01-01

    We previously reported that low-level lysosomal photodamage enhanced the efficacy of subsequent mitochondrial photodamage, resulting in a substantial promotion of apoptotic cell death. We now extend our analysis of the sequential PDT protocol to include two additional lysosomal-targeting photosensitizers. These agents, because of enhanced permeability, are more potent than the agent (N-aspartyl chlorin E6, NPe6) used in the initial study. Addition of the cell-permeable cysteine protease inhibitor E-64d and calcium chelator BAPTA-AM almost completely suppressed sequential PDT-induced loss of mitochondrial membrane potential and activation of procaspases-3 and -7. These inhibitors did not, however, suppress the proapoptotic effect of a BH3 mimetic or mitochondrial photodamage. Knockdowns of ATG7 or ATG5, proteins normally associated with autophagy, suppressed photodamage induced by the sequential PDT protocol. These effects appear to be independent of the autophagic process as pharmacological inhibition of autophagy offered no such protection. Effects of ATG7 and ATG5 knockdowns may reflect the role that ATG7 plays in regulating lysosome permeability, and the likelihood that a proteolytic fragment of ATG5 amplifies mitochondrial proapoptotic processes. Our results suggest that low-dose photodamage that sequentially targets lysosomes and mitochondria may offer significant advantages over the use of single photosensitizers. PMID:25873082

  3. 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

  4. Loss of Niemann-Pick C1 or C2 protein results in similar biochemical changes suggesting that these proteins function in a common lysosomal pathway.

    Directory of Open Access Journals (Sweden)

    Sayali S Dixit

    Full Text Available Niemann-Pick Type C (NPC disease is a lysosomal storage disorder characterized by accumulation of unesterified cholesterol and other lipids in the endolysosomal system. NPC disease results from a defect in either of two distinct cholesterol-binding proteins: a transmembrane protein, NPC1, and a small soluble protein, NPC2. NPC1 and NPC2 are thought to function closely in the export of lysosomal cholesterol with both proteins binding cholesterol in vitro but they may have unrelated lysosomal roles. To investigate this possibility, we compared biochemical consequences of the loss of either protein. Analyses of lysosome-enriched subcellular fractions from brain and liver revealed similar decreases in buoyant densities of lysosomes from NPC1 or NPC2 deficient mice compared to controls. The subcellular distribution of both proteins was similar and paralleled a lysosomal marker. In liver, absence of either NPC1 or NPC2 resulted in similar alterations in the carbohydrate processing of the lysosomal protease, tripeptidyl peptidase I. These results highlight biochemical alterations in the lysosomal system of the NPC-mutant mice that appear secondary to lipid storage. In addition, the similarity in biochemical phenotypes resulting from either NPC1 or NPC2 deficiency supports models in which the function of these two proteins within lysosomes are linked closely.

  5. Close encounters of the lysosome/peroxisome kind

    OpenAIRE

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

    2015-01-01

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

  6. Lysosome-targeted stress reveals increased stability of lipofuscin-containing lysosomes

    OpenAIRE

    Stroikin, Yuri; Mild, Hanna; Johansson, Uno; Roberg, Karin; Öllinger, Karin

    2008-01-01

    Cellular ageing is associated with accumulation of undegradable intralysosomal material, called lipofuscin. In order to accelerate the lipofuscin-accumulation, confluent, growth arrested human fibroblasts were cultured under hyperoxic conditions. To provide a better insight into the effects of lipofuscin on cellular functions, we compared lysosomal stability in control and lipofuscin-loaded human fibroblasts under conditions of lysosome-targeted stress induced by exposure to either the lysoso...

  7. A Proteolytic Cascade Controls Lysosome Rupture and Necrotic Cell Death Mediated by Lysosome-Destabilizing Adjuvants

    OpenAIRE

    Jürgen Brojatsch; Heriberto Lima; Alak K Kar; Jacobson, Lee S.; Stefan M Muehlbauer; Kartik Chandran; Felipe Diaz-Griffero

    2014-01-01

    Recent studies have linked necrotic cell death and proteolysis of inflammatory proteins to the adaptive immune response mediated by the lysosome-destabilizing adjuvants, alum and Leu-Leu-OMe (LLOMe). However, the mechanism by which lysosome-destabilizing agents trigger necrosis and proteolysis of inflammatory proteins is poorly understood. The proteasome is a cellular complex that has been shown to regulate both necrotic cell death and proteolysis of inflammatory proteins. We found that the p...

  8. Pigeon monocyte/macrophage lysosomes during beta VLDL uptake. Induction of acid phosphatase activity. A model for complex arterial lysosomes.

    OpenAIRE

    Jones, N L; Jerome, W. G.; Lewis, J. C.

    1991-01-01

    Lysosomes have long been implicated as a factor contributing to the progression and complication of atherosclerosis. The authors' laboratory previously has shown that lysosomal ultrastructure in arterial macrophage foam cells is altered as primary lysosomes give rise to large pleiomorphic organelles on lipid accumulation during lesion progression. To further explore the subcellular alterations in lysosomes and associated organelles during foam cell formation, three-dimensional (3D) intermedia...

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

    OpenAIRE

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

    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 Torin1), but not by an allosteric inhibitor (rapamycin), leads to activation of lysosomal function. Second, we provided evidence that activation of lysosomal f...

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

    OpenAIRE

    Settembre C; Zoncu R; Medina DL; Vetrini F; Erdin S; Huynh T; Ferron M; Karsenty G; Vellard MC; Facchinetti V; Sabatini DM; Ballabio A.

    2012-01-01

    The lysosome plays a key role in cellular homeostasis by controlling both cellular clearance and energy production to respond to environmental cues. However, the mechanisms mediating lysosomal adaptation are largely unknown. Here, we show that the Transcription Factor EB (TFEB), a master regulator of lysosomal biogenesis, colocalizes with master growth regulator mTOR complex 1 (mTORC1) on the lysosomal membrane. When nutrients are present, phosphorylation of TFEB by mTORC1 inhibits TFEB activ...

  11. Changes in plantar load distribution and gait pattern following foot drop correction in leprosy affected patients.

    Science.gov (United States)

    Karmakar, Mrinmoy; Joshua, Jerry; Mahato, Nidhu

    2015-09-01

    This study was done to compare the changes in plantar load (weight distribution) and gait patterns before and after tibialis posterior transfer surgery in people affected by leprosy. Changes in gait patterns were observed and proportionate changes in plantar load were quantified using data captured by a baropodometer. All the eight patients who underwent tibialis posterior transfer surgery in 2013 in our hospital were included in the study. In addition to the regular pre-operative and post-operative assessments, the patients also underwent baropodometric evaluation. There was a significant change in plantar load at the heel, lateral border and forefoot. Using the foot pressure scan, it was noted that the progression of the centre of mass (displayed graphically as 'the gait line') was also affected by the altered pattern of weight distribution. This study reiterates the importance of tibialis posterior transfer because: it restores the normal gait pattern of 1, 2, 3 (where 1 is heel strike, 2 is mid foot contact and 3 is forefoot contact) and provides a more uniform distribution of planter load. PMID:26665356

  12. 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

    The endosomal-lysosomal system (ELS) has been suggested to play a role in the pathogenesis of prion diseases. The purpose of this study was to examine how experimental observations can be translated to human neuropathology and whether alterations of the ELS relate to neuropathologic changes....... Combined with stereologic techniques, we examined components of the ELS in human sporadic Creutzfeldt-Jakob disease brains. We immunostained for the early endosomal marker Rab5 and lysosomal enzymes cathepsin D and B. We determined neuron-specific changes in their expression and correlated these with the......-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...

  13. 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

    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......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...

  14. Diagnosing lysosomal storage diseases in a Brazilian non-newborn population by tandem mass spectrometry

    Directory of Open Access Journals (Sweden)

    Guilherme Dotto Brand

    2013-11-01

    Full Text Available OBJECTIVES: High-throughput mass spectrometry methods have been developed to screen newborns for lysosomal storage disorders, allowing the implementation of newborn screening pilot studies in North America and Europe. It is currently feasible to diagnose Pompe, Fabry, Gaucher, Krabbe, and Niemann-Pick A/B diseases, as well as mucopolysaccharidosis I, by tandem mass spectrometry in dried blood spots, which offers considerable technical advantages compared with standard methodologies. We aimed to investigate whether the mass spectrometry methodology for lysosomal storage disease screening, originally developed for newborns, can also discriminate between affected patients and controls of various ages. METHODS: A total of 205 control individuals were grouped according to age and subjected to mass spectrometry quantification of lysosomal α-glucosidase, β-glucocerebrosidase, α-galactosidase, acid sphingomyelinase, galactocerebrosidase, and α−L-iduronidase activities. Additionally, 13 affected patients were analyzed. RESULTS: The median activities for each enzyme and each age group were determined. Enzyme activities were significantly lower in individuals aged older than 18 years compared with those in newborns. Affected patients presented enzymatic activities corresponding to less than 20% of the age-matched controls. CONCLUSIONS: Our data indicate that the mass spectrometry methodology can be used for the screening of lysosomal storage diseases in non-newborn patients. However, for some diseases, such as Fabry and mucopolysaccharidosis I, a combination of biochemical and clinical data may be necessary to achieve accurate diagnoses.

  15. Mechanisms of Dendritic Cell Lysosomal Killing of Cryptococcus

    Science.gov (United States)

    Hole, Camaron R.; Bui, Hoang; Wormley, Floyd L.; Wozniak, Karen L.

    2012-10-01

    Cryptococcus neoformans is an opportunistic pulmonary fungal pathogen that disseminates to the CNS causing fatal meningitis in immunocompromised patients. Dendritic cells (DCs) phagocytose C. neoformans following inhalation. Following uptake, cryptococci translocate to the DC lysosomal compartment and are killed by oxidative and non-oxidative mechanisms. DC lysosomal extracts kill cryptococci in vitro; however, the means of antifungal activity remain unknown. Our studies determined non-oxidative antifungal activity by DC lysosomal extract. We examined DC lysosomal killing of cryptococcal strains, anti-fungal activity of purified lysosomal enzymes, and mechanisms of killing against C. neoformans. Results confirmed DC lysosome fungicidal activity against all cryptococcal serotypes. Purified lysosomal enzymes, specifically cathepsin B, inhibited cryptococcal growth. Interestingly, cathepsin B combined with its enzymatic inhibitors led to enhanced cryptococcal killing. Electron microscopy revealed structural changes and ruptured cryptococcal cell walls following treatment. Finally, additional studies demonstrated that osmotic lysis was responsible for cryptococcal death.

  16. Lysosome: regulator of lipid degradation pathways

    OpenAIRE

    Settembre, Carmine; Ballabio, Andrea

    2014-01-01

    Autophagy is a catabolic pathway that has a fundamental role in the adaptation to fasting and primarily relies on the activity of the endolysosomal system, to which the autophagosome targets substrates for degradation. Recent studies have revealed that the lysosomal–autophagic pathway plays an important part in the early steps of lipid degradation. In this review, we discuss the transcriptional mechanisms underlying co-regulation between lysosome, autophagy, and other steps of lipid catabolis...

  17. Lichen-Associated Fungal Community in Hypogymnia hypotrypa (Parmeliaceae, Ascomycota) Affected by Geographic Distribution and Altitude

    Science.gov (United States)

    Wang, Yanyan; Zheng, Yong; Wang, Xinyu; Wei, Xinli; Wei, Jiangchun

    2016-01-01

    Lichen-associated fungal species have already been investigated in almost all the main growth forms of lichens, however, whether or not they are homogeneous and constant within each lichen species are still inconclusive. Moreover, the related ecological factors to affect and structure the fungal composition have been poorly studied. In order to answer these questions, we took Hypogymnia hypotrypa as a model to study the relationship between the lichen-associated fungal composition and two ecological factors, i.e., site and altitude, using the method of IlluminaMiSeq sequencing. Four different sites and two levels of altitude were included in this study, and the effects of site and altitude on fungal community composition were assessed at three levels, i.e., operational taxonomic unit (OTU), class and phylum. The results showed that a total of 50 OTUs were identified and distributed in 4 phyla, 13 classes, and 20 orders. The lichen-associated fungal composition within H. hypotrypa were significantly affected by both site and altitude at OTU and class levels, while at the phylum level, it was only affected by altitude. While the lichen associated fungal communities were reported to be similar with endophytic fungi of the moss, our results indicated the opposite results in some degree. But whether there exist specific OTUs within this lichen species corresponding to different sites and altitudes is still open. More lichen species and ecological factors would be taken into the integrated analyses to address these knowledge gaps in the near future. PMID:27547204

  18. Lichen-Associated Fungal Community in Hypogymnia hypotrypa (Parmeliaceae, Ascomycota) Affected by Geographic Distribution and Altitude.

    Science.gov (United States)

    Wang, Yanyan; Zheng, Yong; Wang, Xinyu; Wei, Xinli; Wei, Jiangchun

    2016-01-01

    Lichen-associated fungal species have already been investigated in almost all the main growth forms of lichens, however, whether or not they are homogeneous and constant within each lichen species are still inconclusive. Moreover, the related ecological factors to affect and structure the fungal composition have been poorly studied. In order to answer these questions, we took Hypogymnia hypotrypa as a model to study the relationship between the lichen-associated fungal composition and two ecological factors, i.e., site and altitude, using the method of IlluminaMiSeq sequencing. Four different sites and two levels of altitude were included in this study, and the effects of site and altitude on fungal community composition were assessed at three levels, i.e., operational taxonomic unit (OTU), class and phylum. The results showed that a total of 50 OTUs were identified and distributed in 4 phyla, 13 classes, and 20 orders. The lichen-associated fungal composition within H. hypotrypa were significantly affected by both site and altitude at OTU and class levels, while at the phylum level, it was only affected by altitude. While the lichen associated fungal communities were reported to be similar with endophytic fungi of the moss, our results indicated the opposite results in some degree. But whether there exist specific OTUs within this lichen species corresponding to different sites and altitudes is still open. More lichen species and ecological factors would be taken into the integrated analyses to address these knowledge gaps in the near future. PMID:27547204

  19. Ultraviolet induced lysosome activity in corneal epithelium

    International Nuclear Information System (INIS)

    A 5.000 W Xe-Hg high pressure lamp and a double monochromator were used to produce a 3.3 nm half-bandpass ultraviolet radiation at 295 nm. Pigmented rabbit eyes were irradiated with radiant exposures from 140 Jm-2 to 10.000 Jm-2 and evaluated by slit-lamp biomicroscopy, light and electron microscopy. Corneal threshold (Hsub(c) was 200 Jm-2 and lens threshold (Hsub(L)) was 7.500 Jm-2. The most repeatable and reliable corneal response to these levels of UV was the development of corneal epithelial granules. Histological changes included a loss of superficial epithelial cells and selective UV induced autolysis of the wing cells. It is suggested that the biomicroscopically observed granules are the clinical manifestation of the secondary lysosomes revealed by light and electron microscopy. It is proposed that UV breaks down the primary lysosome membranes to release hydrolytic enzymes which in turn form the secondary lysosomes during autolysis. Extreme levels of radiant exposure at 295 nm result in indiscriminate destruction of all layers of the corneal epithelium, but the posterior cornea was spared. (orig.)

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

    International Nuclear Information System (INIS)

    In the present study, measurements of naturally occurring radioactive materials and 137Cs 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 226Ra and 232Th 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 137Cs and 40K. The results suggest that these two radionuclides are attached to different mobile particulates. Increasing salinity tends to strengthen the adsorption of 137Cs and solubilization of 40K in sediment. On the other hand, sediment with high organic matter content traps 137Cs and 40K associated particulates to bottom sediment. - Highlights: • 226Ra and 232Th are uniformly distributed in Burullus Lake. • Increasing salinity increases the adsorption of 137Cs and solubilization of 40K in sediment. • Sediment with high organic matter content traps 137Cs and 40K. • 137Cs and 40K are not associated with the same particulate material

  1. Nearest Neighbor Interactions Affect the Conformational Distribution in the Unfolded State of Peptides

    Science.gov (United States)

    Toal, Siobhan; Schweitzer-Stenner, Reinhard; Rybka, Karin; Schwalbe, Hardol

    2013-03-01

    In order to enable structural predictions of intrinsically disordered proteins (IDPs) the intrinsic conformational propensities of amino acids must be complimented by information on nearest-neighbor interactions. To explore the influence of nearest-neighbors on conformational distributions, we preformed a joint vibrational (Infrared, Vibrational Circular Dichroism (VCD), polarized Raman) and 2D-NMR study of selected GxyG host-guest peptides: GDyG, GSyG, GxLG, GxVG, where x/y ={A,K,LV}. D and S (L and V) were chosen at the x (y) position due to their observance to drastically change the distribution of alanine in xAy tripeptide sequences in truncated coil libraries. The conformationally sensitive amide' profiles of the respective spectra were analyzed in terms of a statistical ensemble described as a superposition of 2D-Gaussian functions in Ramachandran space representing sub-ensembles of pPII-, β-strand-, helical-, and turn-like conformations. Our analysis and simulation of the amide I' band profiles exploits excitonic coupling between the local amide I' vibrational modes in the tetra-peptides. The resulting distributions reveal that D and S, which themselves have high propensities for turn-structures, strongly affect the conformational distribution of their downstream neighbor. Taken together, our results indicate that Dx and Sx motifs might act as conformational randomizers in proteins, attenuating intrinsic propensities of neighboring residues. Overall, our results show that nearest neighbor interactions contribute significantly to the Gibbs energy landscape of disordered peptides and proteins.

  2. The Role of Microscopy in Understanding Atherosclerotic Lysosomal Lipid Metabolism

    Science.gov (United States)

    Gray Jerome, W.; Yancey, Patricia G.

    2003-02-01

    Microscopy has played a critical role in first identifying and then defining the role of lysosomes in formation of atherosclerotic foam cells. We review the evidence implicating lysosomal lipid accumulation as a factor in the pathogenesis of atherosclerosis with reference to the role of microscopy. In addition, we explore mechanisms by which lysosomal lipid engorgement occurs. Low density lipoproteins which have become modified are the major source of lipid for foam cell formation. These altered lipoproteins are taken into the cell via receptor-mediated endocytosis and delivered to lysosomes. Under normal conditions, lipids from these lipoproteins are metabolized and do not accumulate in lysosomes. In the atherosclerotic foam cell, this normal metabolism is inhibited so that cholesterol and cholesteryl esters accumulate in lysosomes. Studies of cultured cells incubated with modified lipoproteins suggests this abnormal metabolism occurs in two steps. Initially, hydrolysis of lipoprotein cholesteryl esters occurs normally, but the resultant free cholesterol cannot exit the lysosome. Further lysosomal cholesterol accumulation inhibits hydrolysis, producing a mixture of cholesterol and cholesteryl esters within swollen lysosomes. Various lipoprotein modifications can produce this lysosomal engorgement in vitro and it remains to be seen which modifications are most important in vivo.

  3. Starch Binding Domain-containing Protein 1 Plays a Dominant Role in Glycogen Transport to Lysosomes in Liver.

    Science.gov (United States)

    Sun, Tao; Yi, Haiqing; Yang, Chunyu; Kishnani, Priya S; Sun, Baodong

    2016-08-01

    A small portion of cellular glycogen is transported to and degraded in lysosomes by acid α-glucosidase (GAA) in mammals, but it is unclear why and how glycogen is transported to the lysosomes. Stbd1 has recently been proposed to participate in glycogen trafficking to lysosomes. However, our previous study demonstrated that knockdown of Stbd1 in GAA knock-out mice did not alter lysosomal glycogen storage in skeletal muscles. To further determine whether Stbd1 participates in glycogen transport to lysosomes, we generated GAA/Stbd1 double knock-out mice. In fasted double knock-out mice, glycogen accumulation in skeletal and cardiac muscles was not affected, but glycogen content in liver was reduced by nearly 73% at 3 months of age and by 60% at 13 months as compared with GAA knock-out mice, indicating that the transport of glycogen to lysosomes was suppressed in liver by the loss of Stbd1. Exogenous expression of human Stbd1 in double knock-out mice restored the liver lysosomal glycogen content to the level of GAA knock-out mice, as did a mutant lacking the Atg8 family interacting motif (AIM) and another mutant that contains only the N-terminal 24 hydrophobic segment and the C-terminal starch binding domain (CBM20) interlinked by an HA tag. Our results demonstrate that Stbd1 plays a dominant role in glycogen transport to lysosomes in liver and that the N-terminal transmembrane region and the C-terminal CBM20 domain are critical for this function. PMID:27358407

  4. 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.

  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. Diagnosing Lysosomal Storage Disorders: The GM2 Gangliosidoses.

    Science.gov (United States)

    Hall, Patricia; Minnich, Sara; Teigen, Claire; Raymond, Kimiyo

    2014-01-01

    The GM2 gangliosidoses are a group of autosomal recessive lysosomal storage disorders caused by defective β-hexosaminidase. There are three clinical conditions in this group: Tay-Sachs disease (TSD), Sandhoff disease (SD), and hexosaminidase activator deficiency. The three conditions are clinically indistinguishable. TSD and SD have been identified with infantile, juvenile, and adult onset forms. The activator deficiency is only known to present with infantile onset. Diagnosis of TSD and SD is based on decreased hexosaminidase activity and a change in the percentage of activity between isoforms. There are no biochemical tests currently available for activator deficiency. This unit provides a detailed procedure for identifying TSD and SD in affected individuals and carriers from leukocyte samples, the most robust sample type available. PMID:25271840

  7. 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...

  8. 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

  9. 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

  10. Chinese hamster ovary cell lysosomes rapidly exchange contents

    OpenAIRE

    1987-01-01

    We have used cell fusion to address the question of whether macromolecules are rapidly exchanged between lysosomes. Donor cell lysosomes were labeled by the long-term internalization of the fluid- phase pinocytic markers, invertase (sucrase), Lucifer Yellow, FITC- conjugated dextran, or Texas red-conjugated dextran. Recipient cells contained lysosomes swollen by long-term internalization of dilute sucrose or marked by an overnight FITC-dextran uptake. Cells were incubated for 1 or 2 h in mark...

  11. Inhibition of macrophage phagosome-lysosome fusion by Salmonella typhimurium.

    OpenAIRE

    Buchmeier, N A; Heffron, F

    1991-01-01

    Salmonella typhimurium-infected macrophages were examined by electron microscopy to determine whether intracellular survival of S. typhimurium is associated with failure of bacteria containing phagosomes to fuse with secondary lysosomes. S. typhimurium 14028 actively inhibited phagosome-lysosome fusion and appeared to preferentially divide within unfused phagocytic vesicles. In comparison with Escherichia coli, S. typhimurium inhibited phagosome-lysosome fusion in peritoneal macrophages, J774...

  12. A lysosome-centered view of nutrient homeostasis.

    Science.gov (United States)

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

    2016-04-01

    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. PMID:27050453

  13. Decreased T2 signal in the thalami may be a sign of lysosomal storage disease

    International Nuclear Information System (INIS)

    Lysosomal disorders are rare and are caused by genetically transmitted lysosomal enzyme deficiencies. A decreased T2 signal in the thalamus has occasionally been reported. Because the finding of bilateral abnormal signal intensity of the thalamus on T2-weighted images has not been systematically reviewed, and its value as a diagnostic tool critically evaluated, we carried out a systematic review of the literature. Articles in English with 30 trios of keywords were collected from PubMed. Exclusion criteria were lack of conventional T2-weighted images in the protocol and not being a human study. Finally, 111 articles were included. The thalamus was considered affected only if mentioned in the text or in the figure legends. Some 117 patients with various lysosomal diseases and five patients with ceruloplasmin deficiency were reported to have a bilateral decrease in T2 signal intensity. At least one article reported a bilateral decrease in signal intensity of the thalami on T2-weighted images in association with GM1 and GM2 gangliosidosis and with Krabbe's disease, aspartylglucosaminuria, mannosidosis, fucosidosis, and mucolipidosis IV. Furthermore, thalamic alteration was a consistent finding in several types of neuronal ceroid lipofuscinosis (NCL) including CLN1 (infantile NCL), CLN2 (classic late infantile NCL), CLN3 (juvenile NCL), CLN5 (Finnish variant late infantile NCL), and CLN7 (Turkish variant late infantile NCL). A decrease in T2 signal intensity in the thalami seems to be a sign of lysosomal disease. (orig.)

  14. Current molecular genetics strategies for the diagnosis of lysosomal storage disorders.

    Science.gov (United States)

    Giugliani, Roberto; Brusius-Facchin, Ana-Carolina; Pasqualim, Gabriela; Leistner-Segal, Sandra; Riegel, Mariluce; Matte, Ursula

    2016-01-01

    Lysosomal storage disorders (LSDs) are a group of almost 50 monogenic diseases characterized by mutations causing deficiency of lysosomal enzymes or non-enzyme proteins involved in transport across the lysosomal membrane, protein maturation or lysosomal biogenesis. Usually, affected patients are normal at birth and have a progressive and severe disease with high morbidity and reduced life expectancy. The overall incidence of LSDs is usually estimated as 1:5000, but newborn screening studies are indicating that it could be much higher. Specific therapies were already developed for selected LSDs, making the timely and correct diagnosis very important for successful treatment and also for genetic counseling. In most LSD cases the biochemical techniques provide a reliable diagnosis. However, the identification of pathogenic mutations by genetic analysis is being increasingly recommended to provide additional information. In this paper we discuss the conventional methods for genetic analysis used in the LSDs [restriction fragment length polymorphism (RFLP), amplification-refractory mutation system (ARMS), single strand conformation polymorphism (SSCP), denaturing high performance liquid chromatography (dHPLC), real-time polymerase chain reaction, high resolution melting (HRM), multiplex ligation-dependent probe amplification (MLPA), Sanger sequencing] and also the newer approaches [massive parallel sequencing, array comparative genomic hybridization (CGH)]. PMID:26567866

  15. Septins promote macropinosome maturation and traffic to the lysosome by facilitating membrane fusion.

    Science.gov (United States)

    Dolat, Lee; Spiliotis, Elias T

    2016-08-29

    Macropinocytosis, the internalization of extracellular fluid and material by plasma membrane ruffles, is critical for antigen presentation, cell metabolism, and signaling. Macropinosomes mature through homotypic and heterotypic fusion with endosomes and ultimately merge with lysosomes. The molecular underpinnings of this clathrin-independent endocytic pathway are largely unknown. Here, we show that the filamentous septin GTPases associate preferentially with maturing macropinosomes in a phosphatidylinositol 3,5-bisphosphate-dependent manner and localize to their contact/fusion sites with macropinosomes/endosomes. Septin knockdown results in large clusters of docked macropinosomes, which persist longer and exhibit fewer fusion events. Septin depletion and overexpression down-regulates and enhances, respectively, the delivery of fluid-phase cargo to lysosomes, without affecting Rab5 and Rab7 recruitment to macropinosomes/endosomes. In vitro reconstitution assays show that fusion of macropinosomes/endosomes is abrogated by septin immunodepletion and function-blocking antibodies and is induced by recombinant septins in the absence of cytosol and polymerized actin. Thus, septins regulate fluid-phase cargo traffic to lysosomes by promoting macropinosome maturation and fusion with endosomes/lysosomes. PMID:27551056

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

    OpenAIRE

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

    2015-01-01

    Intracellular pH plays an important role in the response to cancer invasion. We have designed and synthesized a series of new fluorescent probes (Superior LysoProbes) with the capacity to label acidic organelles and monitor lysosomal pH. Unlike commercially available fluorescent dyes, Superior LysoProbes are lysosome-specific and are highly stable. The use of Superior LysoProbes facilitates the direct visualization of the lysosomal response to lobaplatin elicited in human chloangiocarcinoma (...

  17. 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.

  18. A Review of Gene Therapy in Canine and Feline Models of Lysosomal Storage Disorders

    OpenAIRE

    Bradbury, Allison M.; Gurda, Brittney L.; Casal, Margret L.; Ponder, Katherine P; Vite, Charles H.; Haskins, Mark E.

    2015-01-01

    Lysosomal storage disorders (LSDs) are inherited diseases that result from the intracellular accumulation of incompletely degraded macromolecules. The majority of LSDs affect both the peripheral and central nervous systems and are not effectively treated by enzyme replacement therapy, substrate reduction therapy, or bone marrow transplantation. Advances in adeno-associated virus and retroviral vector development over the past decade have resurged gene therapy as a promising therapeutic interv...

  19. From Consumer Incomes to Car Ages: How the Distribution of Income Affects the Distribution of Vehicle Vintages

    OpenAIRE

    Yurko, Anna

    2008-01-01

    This paper studies the relationship between consumer incomes and ages of the durable goods consumed. At the household level, it presents evidence from the Consumer Expenditure Survey of a negative correlation between incomes and ages of the vehicles owned. At the aggregate level, it constructs a dynamic, heterogeneous agents, discrete choice model, to study the relationship between the distribution of consumer incomes and the distribution of vehicle vintages. The model's parameters are cal...

  20. Engaging the lysosomal compartment to combat B cell malignancies

    DEFF Research Database (Denmark)

    Gronbaek, K.; Jaattela, M.

    2009-01-01

    generation of therapeutic anti-CD20 mAbs. In this issue of the JCI, Ivanov and colleagues identify the lysosomal compartment as a target for type II mAbs (see the related article beginning on page 2143). These data encourage the further clinical development of type II mAbs as well as other lysosome...

  1. Lysosomal phospholipids from rat liver after treatment with different drugs.

    Science.gov (United States)

    Tjiong, H B; Lepthin, J; Debuch, H

    1978-01-01

    Rats were treated with 5 different drugs p-ethoxyacetanilide (I), indometacin (II) and nor-amidopyrine-methanesulfonate (III), O,O'-bis(diethylaminoethyl)hexestrol(IV) and choloroquine (V) for 3 - 4 weeks. Liver cell fractions were isolated by discontinuous gradient centrifugation and the specific activity of acid phosphatase was determined in each. Lysosomal fractions contained widely varying amounts of this marker enzyme, indicating that the concentration of lysosomes within these fractions differed. The amounts and patterns of phospholipids reflected this fact. Since we assumed bis(monoacylglycero)phosphate [(MAG)2-P; synonym:lysobisphosphatidic acid] is a marker lipid for secondary lysosomes, we expected and found significant quantities of this acidic phospholipid only in those lysosomal fractions which were also rich in acid phosphatase activity. 12% of the lysosomal phospholipids from animals receiving the hexestrol derivative (IV), and 19% of those from the chloroquine (V) experiment were present as (MAG)2P. The fatty acid compositions of this lysosomal phospholipid were not the same in all lysosome fractions. The more (MAG)2P present in the lysosomes, the more unsaturated are the fatty acids. Thus, after treatment with chloroquine, more than 90% of the fatty acids from (MAG)2P are unsaturated; C22:6 represents about 70% of the total. PMID:627402

  2. Homotypic Lysosome Fusion in Macrophages: Analysis Using an In Vitro Assay

    OpenAIRE

    Diane M Ward; Jonathan D Leslie; Kaplan, Jerry

    1997-01-01

    Lysosomes are dynamic structures capable of fusing with endosomes as well as other lysosomes. We examined the biochemical requirements for homotypic lysosome fusion in vitro using lysosomes obtained from rabbit alveolar macrophages or the cultured macrophage-like cell line, J774E. The in vitro assay measures the formation of a biotinylated HRP–avidin conjugate, in which biotinylated HRP and avidin were accumulated in lysosomes by receptor-mediated endocytosis. We determined that lysosome fusi...

  3. Lysosomal Dysfunction Caused by Cellular Accumulation of Silica Nanoparticles.

    Science.gov (United States)

    Schütz, Irene; Lopez-Hernandez, Tania; Gao, Qi; Puchkov, Dmytro; Jabs, Sabrina; Nordmeyer, Daniel; Schmudde, Madlen; Rühl, Eckart; Graf, Christina M; Haucke, Volker

    2016-07-01

    Nanoparticles (NPs) are widely used as components of drugs or cosmetics and hold great promise for biomedicine, yet their effects on cell physiology remain poorly understood. Here we demonstrate that clathrin-independent dynamin 2-mediated caveolar uptake of surface-functionalized silica nanoparticles (SiNPs) impairs cell viability due to lysosomal dysfunction. We show that internalized SiNPs accumulate in lysosomes resulting in inhibition of autophagy-mediated protein turnover and impaired degradation of internalized epidermal growth factor, whereas endosomal recycling proceeds unperturbed. This phenotype is caused by perturbed delivery of cargo via autophagosomes and late endosomes to SiNP-filled cathepsin B/L-containing lysosomes rather than elevated lysosomal pH or altered mTOR activity. Given the importance of autophagy and lysosomal protein degradation for cellular proteostasis and clearance of aggregated proteins, these results raise the question of beneficial use of NPs in biomedicine and beyond. PMID:27226546

  4. 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

  5. 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...

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

    Science.gov (United States)

    Thomsen, Christian; Theilig, Dorothea; Herzog, Dominik; Poellinger, Alexander; Doellinger, Felix; Schreiter, Nils; Schreiter, Vera; Schürmann, Dirk; Temmesfeld-Wollbrueck, Bettina; Hippenstiel, Stefan; Suttorp, Norbert; Hubner, Ralf-Harto

    2016-01-01

    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. Patients with high perfusions in INL demonstrated greater improvements in 6MWT, while patients with high HI were more likely to respond in FEV1. PMID:27354783

  7. Lysosomal Storage Causes Cellular Dysfunction in Mucolipidosis II Skin Fibroblasts*

    Science.gov (United States)

    Otomo, Takanobu; Higaki, Katsumi; Nanba, Eiji; Ozono, Keiichi; Sakai, Norio

    2011-01-01

    Mucolipidosis II (ML-II) is a fatal inherited metabolic disease caused by deficiency of GlcNAc-phosphotransferase, which plays a role in generating the mannose 6-phosphate recognition marker on lysosomal enzymes. In ML-II, many lysosomal acid hydrolases are mistargeted out of cells, and lysosomes become filled with undigested substrates, which explains inclusion cell disease as an alternative name for this disease. In this study, we revealed various cellular phenotypes in ML-II skin fibroblasts. We quantitated phospholipid and cholesterol within cells and showed ∼2-fold accumulation in ML-II as compared with normal cells. Lysosomal pH of ML-II cells was higher than that of normal cells (5.29 ± 0.08 versus 4.79 ± 0.10, p < 0.001). The proliferated lysosomes in ML-II cells were accumulated ∼3-fold in amount as compared with normal cells. Intracellular logistics including endocytosis and mannose 6-phosphate receptor recycling were impaired in ML-II cells. To confirm whether these ML-II cellular phenotypes derive from deficient lysosomal acid hydrolases within lysosomes, we performed supplementation of lysosomal enzymes using a partially purified total enzyme mixture, which was derived from the conditioned culture medium of normal skin fibroblasts after NH4Cl treatment. This supplementation corrected all of the previously described ML-II phenotypes. In addition, the autophagic and mitochondrial impairment that we have previously reported improved, and inclusion bodies disappeared on electron micrography following total lysosomal enzyme supplementation. Our results indicate that various cellular phenotypes in ML-II are caused by the deficiency of many lysosomal enzymes and massive accumulation of undigested substrates. PMID:21846724

  8. Diagnosing lysosomal storage disorders: mucopolysaccharidosis type I.

    Science.gov (United States)

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

    2015-01-01

    Mucopolysaccharidosis type I (MPS I) is a lysosomal storage disorder due to deficiency of alpha iduronidase (IDUA). Progressive storage of dermatan and heparan sulfate throughout the body lead to a multiorgan presentation including short stature, dysostosis multiplex, corneal clouding, hearing loss, coarse facies, hepatosplenomegaly, and intellectual disability. Diagnosis of MPS I is based on IDUA enzyme analysis in leukocytes or dried blood spots (DBS) followed by molecular confirmation of the IDUA gene mutations in individuals with low enzyme activity. The advent of mass spectrometry methods for enzyme analysis in DBS has enabled high-throughput screening for MPS I in symptomatic individuals and newborn infants. The following unit provides the detailed analytical protocol for measurement of IDUA activity in DBS using tandem mass spectrometry. PMID:25599668

  9. Cyclodextrin induces calcium-dependent lysosomal exocytosis.

    Directory of Open Access Journals (Sweden)

    Fannie W Chen

    Full Text Available Cyclodextrins (CDs have long been used to manipulate cellular cholesterol levels both in vitro and in vivo, but their direct effects at a cellular level are not well characterized. Recently, CDs have garnered much interest because of their ability to clear stored cholesterol from Niemann Pick Type C (NPC cells and markedly prolong the life of NPC1 disease mice. Here, we investigate the hypothesis that treatment with 2-hydroxypropyl- β-cyclodextrin (HPB-CD stimulates lysosomal exocytosis in a calcium-enhanced manner. We propose that this exocytosis is the mechanism by which HPB-CD ameliorates the endolysosomal cholesterol storage phenotype in NPC cells. These findings have significant implications for the use of HPB-CD in biochemical assays and data interpretation as well as for their use for the treatment for NPC and other disorders.

  10. Building ensemble representations: How the shape of preceding distractor distributions affects visual search.

    Science.gov (United States)

    Chetverikov, Andrey; Campana, Gianluca; Kristjánsson, Árni

    2016-08-01

    Perception allows us to extract information about regularities in the environment. Observers can quickly determine summary statistics of a group of objects and detect outliers. The existing body of research has, however, not revealed how such ensemble representations develop over time. Moreover, the correspondence between the physical distribution of features in the external world and their potential internal representation as a probability density function (PDF) by the visual system is still unknown. Here, for the first time we demonstrate that such internal PDFs are built during visual search and show how they can be assessed with repetition and role-reversal effects. Using singleton search for an oddly oriented target line among differently oriented distractors (a priming of pop-out paradigm), we test how different properties of previously observed distractor distributions (mean, variability, and shape) influence search times. Our results indicate that observers learn properties of distractor distributions over and above mean and variance; in fact, response times also depend on the shape of the preceding distractor distribution. Response times decrease as a function of target distance from the mean of preceding Gaussian distractor distributions, and the decrease is steeper when preceding distributions have small standard deviations. When preceding distributions are uniform, however, this decrease in response times can be described by a two-piece function corresponding to the uniform distribution PDF. Moreover, following skewed distributions response times function is skewed in accordance with the skew in distributions. Indeed, internal PDFs seem to be specifically tuned to the observed feature distribution. PMID:27232163

  11. Distribution of radiocesium and radiostrontium in undisturbed soil affected by Fukushima nuclear power plant accident

    International Nuclear Information System (INIS)

    contaminated with 90Sr. To understand the geochemical behavior of Cs and Sr, distribution coefficient (Kd) and soil parameters have been determined experimentally and the important parameters affecting Cs and Sr sorption have been identified. The present study will be useful for understanding the migration processes and for long-term prediction of activity depth profiles in soil. (author)

  12. Rotenone Upregulates Alpha-Synuclein and Myocyte Enhancer Factor 2D Independently from Lysosomal Degradation Inhibition

    Directory of Open Access Journals (Sweden)

    Gessica Sala

    2013-01-01

    Full Text Available Dysfunctions of chaperone-mediated autophagy (CMA, the main catabolic pathway for alpha-synuclein, have been linked to the pathogenesis of Parkinson’s disease (PD. Since till now there is limited information on how PD-related toxins may affect CMA, in this study we explored the effect of mitochondrial complex I inhibitor rotenone on CMA substrates, alpha-synuclein and MEF2D, and effectors, lamp2A and hsc70, in a human dopaminergic neuroblastoma SH-SY5Y cell line. Rotenone induced an upregulation of alpha-synuclein and MEF2D protein levels through the stimulation of their de novo synthesis rather than through a reduction of their CMA-mediated degradation. Moreover, increased MEF2D transcription resulted in higher nuclear protein levels that exert a protective role against mitochondrial dysfunction and oxidative stress. These results were compared with those obtained after lysosome inhibition with ammonium chloride. As expected, this toxin induced the cytosolic accumulation of both alpha-synuclein and MEF2D proteins, as the result of the inhibition of their lysosome-mediated degradation, while, differently from rotenone, ammonium chloride decreased MEF2D nuclear levels through the downregulation of its transcription, thus reducing its protective function. These results highlight that rotenone affects alpha-synuclein and MEF2D protein levels through a mechanism independent from lysosomal degradation inhibition.

  13. 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.

  14. Presence of detergent-resistant microdomains in lysosomal membranes.

    Science.gov (United States)

    Taute, Antje; Wätzig, Kristin; Simons, Brigitte; Lohaus, Christiane; Meyer, Helmut; Hasilik, Andrej

    2002-10-18

    We examined the association of acetyl-CoA:alpha-glucosaminide N-acetyltransferase, a lysosomal enzyme participating in the degradation of heparan sulfate with other components of the lysosomal membrane. We prepared lysosomal membranes from human placenta and treated them with zwitterionic and non-ionic detergents. Membrane proteins were solubilized either in the presence of CHAPS at room temperature or of Triton X-100 at 4 degrees C. The CHAPS-containing extract was subjected to gel filtration in a column with the nominal size exclusion of 0.6 MDa. Under these conditions the enzyme fractionated near the void volume. To examine the association of the enzyme with detergent-resistant lipid microdomains, the extract that had been prepared with Triton X-100 was subjected to flotation in a density gradient medium. After centrifugation, a major portion of the activity of the acetyltransferase was found at the top of the gradient along with the bulk of alkaline phosphatase. Alkaline phosphatase is a glycosylphosphatidylinositol-anchored protein; possibly a contaminant in the lysosomal fraction originating from the plasma membrane and adventitiously an internal control for the flotation in the gradient. In contrast, acetyltransferase is a genuine lysosomal protein that obligatorily spans the membrane since it transfers acetyl residues from acetyl-CoA in cytosol to glucosaminyl residues in heparan sulfate fragments in the lysosomal matrix. To our knowledge this is the first report on association of a lysosomal membrane protein with detergent-resistant membrane microdomains or rafts. PMID:12379211

  15. Oral small molecule therapy for lysosomal storage diseases.

    Science.gov (United States)

    Weinreb, Neal J

    2013-11-01

    For more than 20 years, "enzyme replacement therapy" (ERT) has been the prevalent treatment approach for lysosomal storage disorders (LSDs). Unfortunately, ERT, as currently administered, is ineffective for primary neuronopathic LSDs. For LSDs whose major disease burden is non-neurological, ERT efficacy is limited by uneven tissue distribution and penetration, immunological intolerance, and disturbed intracellular homeostasis associated with persistent mutant enzymes that are not "replaced" by ERT. Many of these limitations might be circumvented by oral, low molecular weight pharmaceuticals that address relevant LSD pathophysiology and distribute widely in steady state concentrations in all cells and body tissues including the CNS. Two oral small molecule drugs (miglustat and cysteamine) are currently approved for clinical use and two (eliglustat and migalastat) are in advanced stage clinical trials. Several others are in early stages of clinical or pre-clinical investigation. This article reviews current knowledge of small molecule treatment for LSDs including approaches such as substrate synthesis inhibition, pharmacological chaperones, and proteostasis modification. PMID:24380126

  16. 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

    Directory of Open Access Journals (Sweden)

    Atsushi Komemushi

    2012-01-01

    Full Text Available 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.

  17. 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

    International Nuclear Information System (INIS)

    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

  18. Lamellar granule biogenesis: a role for ceramide glucosyltransferase, lysosomal enzyme transport, and the Golgi.

    Science.gov (United States)

    Madison, K C; Sando, G N; Howard, E J; True, C A; Gilbert, D; Swartzendruber, D C; Wertz, P W

    1998-08-01

    Although lamellar granules are critical to the formation of the epidermal permeability barrier and are a known marker of late keratinocyte differentiation, very little is known about the physiologic regulators of lamellar granule assembly and extrusion. Ceramide glucosyltransferase (CGT), the enzyme responsible for the synthesis of lamellar granule glucosylceramides (GlcCer; the precursors of the stratum corneum ceramides), is localized to the Golgi apparatus in other cell types. We have found that CGT is induced during keratinocyte culture differentiation coincident with increased GlcCer content and the appearance of lamellar granules. In this study we show that the differentiation-related CGT induction is likely mediated at the transcriptional level. In addition, all-trans retinoic acid, a well-known inhibitor of keratinocyte differentiation, prevents the appearance of lamellar granules and decreases culture CGT activity and GlcCer content without affecting sphingomyelin or total lipid content, indicating a specific inhibition of this enzymatic pathway. These data show a direct relationship between CGT activity and epidermal differentiation, suggesting that regulation of CGT expression is a critical part of epidermal barrier generation. The differentiation dependence of CGT activity, the key role of this Golgi-localized enzyme in epidermal GlcCer synthesis, and our previous finding that ceramides are converted to GlcCer in the Golgi apparatus in keratinocyte cultures, strongly suggest a Golgi origin for lamellar granules. In contrast to CGT, the activity of the lysosomal enzymes acid lipase and glucocerebrosidase is less clearly related to epidermal differentiation and the appearance of lamellar granules, although both enzymes show striking colocalization and enrichment in a subcellular lamellar granule fraction derived from pig epidermis. Acid lipase activity in the lamellar granule fraction was found to contain primarily a small lysosomal form of the enzyme

  19. 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.

  20. 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...... the diffusion of neutral and ionic molecules across biomembranes, protonation to mono- or bivalent ions, adsorption to lipids, and electrical attraction or repulsion. Based on simulation results, high and selective accumulation in lysosomes was found for weak mono- and bivalent bases with intermediate...... to high log K ow. These findings were validated with experimental results and by a comparison to the properties of antimalarial drugs in clinical use. For ten active compounds, nine were predicted to accumulate to a greater extent in lysosomes than in other organelles, six of these were in the...

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

    Directory of Open Access Journals (Sweden)

    Gabriel C Baltazar

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

  2. Importance of lysosomal cysteine proteases in lung disease

    OpenAIRE

    Chapman Harold A; Wolters Paul J

    2000-01-01

    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 ...

  3. A Lysosome-Targeting AIEgen for Autophagy Visualization.

    Science.gov (United States)

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

    2016-02-18

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

  4. Extracellular Acidification Alters Lysosomal Trafficking in Human Breast Cancer Cells

    OpenAIRE

    Kristine Glunde; Sandra E. Guggino; Meiyappan Solaiyappan; Pathak, Arvind P.; Yoshitaka Ichikawa; Bhujwalla, Zaver M.

    2003-01-01

    Cancer cells invade by secreting degradative enzymes, which are sequestered in lysosomal vesicles. In this study, the impact of an acidic extracellular environment on lysosome size, number, and distance from the nucleus in human mammary epithelial cells (HMECs) and breast cancer cells of different degrees of malignancy was characterized because the physiological microenvironment of tumors is frequently characterized by extracellular acidity. An acidic extracellular pH (pHe) resulted in a dist...

  5. Unequally distributed psychological assets: are there social disparities in optimism, life satisfaction, and positive affect?

    Directory of Open Access Journals (Sweden)

    Julia K Boehm

    Full Text Available Socioeconomic status is associated with health disparities, but underlying psychosocial mechanisms have not been fully identified. Dispositional optimism may be a psychosocial process linking socioeconomic status with health. We hypothesized that lower optimism would be associated with greater social disadvantage and poorer social mobility. We also investigated whether life satisfaction and positive affect showed similar patterns. Participants from the Midlife in the United States study self-reported their optimism, satisfaction, positive affect, and socioeconomic status (gender, race/ethnicity, education, occupational class and prestige, income. Social disparities in optimism were evident. Optimistic individuals tended to be white and highly educated, had an educated parent, belonged to higher occupational classes with more prestige, and had higher incomes. Findings were generally similar for satisfaction, but not positive affect. Greater optimism and satisfaction were also associated with educational achievement across generations. Optimism and life satisfaction are consistently linked with socioeconomic advantage and may be one conduit by which social disparities influence health.

  6. Unequally Distributed Psychological Assets: Are There Social Disparities in Optimism, Life Satisfaction, and Positive Affect?

    Science.gov (United States)

    Boehm, Julia K.; Chen, Ying; Williams, David R.; Ryff, Carol; Kubzansky, Laura D.

    2015-01-01

    Socioeconomic status is associated with health disparities, but underlying psychosocial mechanisms have not been fully identified. Dispositional optimism may be a psychosocial process linking socioeconomic status with health. We hypothesized that lower optimism would be associated with greater social disadvantage and poorer social mobility. We also investigated whether life satisfaction and positive affect showed similar patterns. Participants from the Midlife in the United States study self-reported their optimism, satisfaction, positive affect, and socioeconomic status (gender, race/ethnicity, education, occupational class and prestige, income). Social disparities in optimism were evident. Optimistic individuals tended to be white and highly educated, had an educated parent, belonged to higher occupational classes with more prestige, and had higher incomes. Findings were generally similar for satisfaction, but not positive affect. Greater optimism and satisfaction were also associated with educational achievement across generations. Optimism and life satisfaction are consistently linked with socioeconomic advantage and may be one conduit by which social disparities influence health. PMID:25671665

  7. Role of Nanotechnology for Enzyme Replacement Therapy in Lysosomal Diseases. A Focus on Gaucher's Disease.

    Science.gov (United States)

    Martín-Banderas, L; Holgado, M A; Durán-Lobato, M; Infante, J J; Álvarez-Fuentes, J; Fernández-Arévalo, M

    2016-01-01

    Lysosomal storage diseases (LSDs) comprise a group of rare inherited chronic syndromes that cause deficiency of specific native enzymes within the lysosomes. The macromolecular compounds that are usually catabolized by lysosomal enzymes are accumulated within these organelles, causing progressive damage to tissues, skeleton and organs and, in several cases, the central nervous system (CNS). The damage caused by substrate accumulation finally results in physical deterioration, functional impairment and potential death. Up to date, the most promising therapy for most LSDs is enzyme-replacement therapy (ERT), which provides patients with the corresponding active enzyme. However, these enzymes do not have enough stability in blood, the treatment must be therefore periodically administrated by i.v. infusion under medical supervision, and immunogenicity issues are frequent. In addition, affected areas within the CNS, where the blood-brain barrier (BBB) is a major obstacle, cannot be reached by the enzymes. Nanotechnology can provide useful carriers to successfully protect and preserve enzymes, and transport them through the BBB towards brain locations. Several strategies based on targeting specific receptors on the BBB have led to nanoparticles that successfully carry sensitive molecules to the brain. Then, the main LSDs are described and a thorough review of nanotechnology strategies for brain delivery studied up to date is presented. PMID:26860997

  8. Prevention of lysosomal storage diseases and derivation of mutant stem cell lines by preimplantation genetic diagnosis.

    Science.gov (United States)

    Altarescu, Gheona; Beeri, Rachel; Eiges, Rachel; Epsztejn-Litman, Silvina; Eldar-Geva, Talia; Elstein, Deborah; Zimran, Ari; Margalioth, Ehud J; Levy-Lahad, Ephrat; Renbaum, Paul

    2012-01-01

    Preimplantation genetic diagnosis (PGD) allows birth of unaffected children for couples at risk for a genetic disorder. We present the strategy and outcome of PGD for four lysosomal storage disorders (LSD): Tay-Sachs disease (TSD), Gaucher disease (GD), Fabry disease (FD), and Hunter syndrome (HS), and subsequent development of stem cell lines. For each disease, we developed a family-specific fluorescent multiplex single-cell PCR protocol that included the familial mutation and informative markers surrounding the mutation. Embryo biopsy and PGD analysis were performed on either oocytes (polar bodies one and two) or on single blastomeres from a six-cell embryo. We treated twenty families carrying mutations in these lysosomal storage disorders, including 3 couples requiring simultaneous analysis for two disorders (TSD/GD, TSD/balanced Robertsonian translocation 45XYder(21;14), and HS/oculocutaneus albinism). These analyses led to an overall pregnancy rate/embryo transfer of 38% and the birth of 20 unaffected children from 17 families. We have found that PGD for lysosomal disorders is a safe and effective method to prevent birth of affected children. In addition, by using mutant embryos for the derivation of stem cell lines, we have successfully established GD and HS hESC lines for use as valuable models in LSD research. PMID:23320174

  9. Prevention of Lysosomal Storage Diseases and Derivation of Mutant Stem Cell Lines by Preimplantation Genetic Diagnosis

    Directory of Open Access Journals (Sweden)

    Gheona Altarescu

    2012-01-01

    Full Text Available Preimplantation genetic diagnosis (PGD allows birth of unaffected children for couples at risk for a genetic disorder. We present the strategy and outcome of PGD for four lysosomal storage disorders (LSD: Tay-Sachs disease (TSD, Gaucher disease (GD, Fabry disease (FD, and Hunter syndrome (HS, and subsequent development of stem cell lines. For each disease, we developed a family-specific fluorescent multiplex single-cell PCR protocol that included the familial mutation and informative markers surrounding the mutation. Embryo biopsy and PGD analysis were performed on either oocytes (polar bodies one and two or on single blastomeres from a six-cell embryo. We treated twenty families carrying mutations in these lysosomal storage disorders, including 3 couples requiring simultaneous analysis for two disorders (TSD/GD, TSD/balanced Robertsonian translocation 45XYder(21;14, and HS/oculocutaneus albinism. These analyses led to an overall pregnancy rate/embryo transfer of 38% and the birth of 20 unaffected children from 17 families. We have found that PGD for lysosomal disorders is a safe and effective method to prevent birth of affected children. In addition, by using mutant embryos for the derivation of stem cell lines, we have successfully established GD and HS hESC lines for use as valuable models in LSD research.

  10. Lysosomal function in macromolecular homeostasis and bioenergetics in Parkinson's disease

    Directory of Open Access Journals (Sweden)

    Zhang Jianhua

    2010-04-01

    Full Text Available Abstract The pathological changes occurring in Parkinson's and several other neurodegenerative diseases are complex and poorly understood, but all clearly involve protein aggregation. Also frequently appearing in neurodegeneration is mitochondrial dysfunction which may precede, coincide or follow protein aggregation. These observations led to the concept that protein aggregation and mitochondrial dysfunction either arise from the same etiological factors or are interactive. Understanding the mechanisms and regulation of processes that lead to protein aggregation or mitochondrial dysfunction may therefore contribute to the design of better therapeutics. Clearance of protein aggregates and dysfunctional organelles is dependent on macroautophagy which is the process through which aged or damaged proteins and organelles are first degraded by the lysosome and then recycled. The macroautophagy-lysosomal pathway is essential for maintaining protein and energy homeostasis. Not surprisingly, failure of the lysosomal system has been implicated in diseases that have features of protein aggregation and mitochondrial dysfunction. This review summarizes 3 major topics: 1 the current understanding of Parkinson's disease pathogenesis in terms of accumulation of damaged proteins and reduction of cellular bioenergetics; 2 evolving insights into lysosomal function and biogenesis and the accumulating evidence that lysosomal dysfunction may cause or exacerbate Parkinsonian pathology and finally 3 the possibility that enhancing lysosomal function may provide a disease modifying therapy.

  11. 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.

  12. Lysosomes from rabbit type II cells catabolize surfactant lipids.

    Science.gov (United States)

    Rider, E D; Ikegami, M; Pinkerton, K E; Peake, J L; Jobe, A H

    2000-01-01

    The role of a lysosome fraction from rabbit type II cells in surfactant dipalmitoylphosphatidylcholine (DPPC) catabolism was investigated in vivo using radiolabeled DPPC and dihexadecylphosphatidylcholine (1, 2-dihexadecyl-sn-glycero-3-phosphocholine; DEPC), a phospholipase A(1)- and A(2)-resistant analog of DPPC. Freshly isolated type II cells were gently disrupted by shearing, and lysosomes were isolated with Percoll density gradients (density range 1.0591-1.1457 g/ml). The lysosome fractions were relatively free of contaminating organelles as determined by electron microscopy and organelle marker enzymes. After intratracheal injection of rabbits with [(3)H]DPPC and [(14)C]DEPC associated with a trace amount of natural rabbit surfactant, the degradation-resistant DEPC accumulated 16-fold compared with DPPC in lysosome fractions at 15 h. Lysosomes can be isolated from freshly isolated type II cells, and lysosomes from type II cells are the primary catabolic organelle for alveolar surfactant DPPC following reuptake by type II cells in vivo. PMID:10645892

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

    OpenAIRE

    Ernesto Moya-Elizondo; Nolberto Arismendi; María Paz Castro; Herman Doussoulin

    2015-01-01

    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 d...

  14. 78 FR 32713 - Distribution of Continued Dumping and Subsidy Offset to Affected Domestic Producers

    Science.gov (United States)

    2013-05-31

    ... Offset to Affected Domestic Producers) in the Federal Register (66 FR 48546) on September 21, 2001, which.... Revere Copper Products. United Steelworkers of America. A-122-605......... 731-TA-367........ Color.... Stockham Valves & Fittings. U-Brand. Ward Manufacturing. A-351-602......... 731-TA-308........ Carbon...

  15. Glucosamine-Bound Near-Infrared Fluorescent Probes with Lysosomal Specificity for Breast Tumor Imaging1

    OpenAIRE

    Li, Cong; Greenwood, Tiffany R; Glunde, Kristine

    2008-01-01

    Noninvasive imaging of lysosomes will be useful 1) to elucidate the role of lysosomal parameters in cancer, 2) to diagnose malignant lesions, and 3) to evaluate future lysosome-targeted anticancer therapies. Lysosome-specific labeling of glucosamine-bound near-infrared (NIR) fluorescent probes, IR-1 and IR-2, but not control probe IR-15 without the glucosamine moiety, was observed by fluorescence microscopy in human breast epithelial cell lines. Lysosome labeling and tumor specificity of thes...

  16. Spinster is required for autophagic lysosome reformation and mTOR reactivation following starvation

    OpenAIRE

    Rong, Yueguang; McPhee, Christina K; Deng, Shuangshen; Huang, Lei; Chen, Lilian; Liu, Mei; Tracy, Kirsten; Baehrecke, Eric H.; Yu, Li; Lenardo, Michael J.

    2011-01-01

    Autophagy is a conserved cellular process to degrade and recycle cytoplasmic components. During autophagy, lysosomes fuse with an autophagosome to form an autolysosome. Sequestered components are degraded by lysosomal hydrolases and presumably released into the cytosol by lysosomal efflux permeases. Following starvation-induced autophagy, lysosome homeostasis is restored by autophagic lysosome reformation (ALR) requiring activation of the “target of rapamycin” (TOR) kinase. Spinster (Spin) en...

  17. Glucosamine-Bound Near-Infrared Fluorescent Probes with Lysosomal Specificity for Breast Tumor Imaging

    OpenAIRE

    Cong Li; Greenwood, Tiffany R; Kristine Glunde

    2008-01-01

    Noninvasive imaging of lysosomes will be useful 1) to elucidate the role of lysosomal parameters in cancer, 2) to diagnose malignant lesions, and 3) to evaluate future lysosome-targeted anticancer therapies. Lysosome-specific labeling of glucosamine-bound near-infrared (NIR) fluorescent probes, IR-1 and IR-2, but not control probe IR-15 without the glucosamine moiety, was observed by fluorescence microscopy in human breast epithelial cell lines. Lysosome labeling and tumor specificity of thes...

  18. Gene Therapy for Lysosomal Storage Diseases (LSDs) in Large Animal Models

    OpenAIRE

    Haskins, Mark

    2009-01-01

    Lysosomal storage diseases (LSDs) are inherited metabolic disorders caused by deficient activity of a single lysosomal enzyme or other defects resulting in deficient catabolism of large substrates in lysosomes. There are more than 40 forms of inherited LSDs known to occur in humans, with an aggregate incidence estimated at 1 in 7,000 live births. Clinical signs result from the inability of lysosomes to degrade large substrates; because most lysosomal enzymes are ubiquitously expressed, a defi...

  19. Body mass index distribution affects discrepancies in weight classifications in children

    Science.gov (United States)

    The aim of the present study was to investigate the effect of body mass index (BMI) distribution, ethnicity, and age at menarche on the consistency in the prevalence of underweight and overweight as defined by the Centers for Disease Control and Prevention (CDC) and the International Obesity Task Fo...

  20. Low Temperature Treatment Affects Concentration and Distribution of Chrysanthemum Stunt Viroid in Argyranthemum.

    Science.gov (United States)

    Zhang, Zhibo; Lee, YeonKyeong; Sivertsen, Astrid; Skjeseth, Gry; Haugslien, Sissel; Clarke, Jihong Liu; Wang, Qiao-Chun; Blystad, Dag-Ragnar

    2016-01-01

    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 5°C treatment, compared with non-treated plants. However, the CSVd-free area in SAM was enlarged in diseased 'Border Dark Red' following prolonged 5°C 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. PMID:26973607

  1. Non-gaussian distributions affect identification of expression patterns, functional annotation, and prospective classification in human cancer genomes.

    Directory of Open Access Journals (Sweden)

    Nicholas F Marko

    Full Text Available INTRODUCTION: Gene expression data is often assumed to be normally-distributed, but this assumption has not been tested rigorously. We investigate the distribution of expression data in human cancer genomes and study the implications of deviations from the normal distribution for translational molecular oncology research. METHODS: We conducted a central moments analysis of five cancer genomes and performed empiric distribution fitting to examine the true distribution of expression data both on the complete-experiment and on the individual-gene levels. We used a variety of parametric and nonparametric methods to test the effects of deviations from normality on gene calling, functional annotation, and prospective molecular classification using a sixth cancer genome. RESULTS: Central moments analyses reveal statistically-significant deviations from normality in all of the analyzed cancer genomes. We observe as much as 37% variability in gene calling, 39% variability in functional annotation, and 30% variability in prospective, molecular tumor subclassification associated with this effect. CONCLUSIONS: Cancer gene expression profiles are not normally-distributed, either on the complete-experiment or on the individual-gene level. Instead, they exhibit complex, heavy-tailed distributions characterized by statistically-significant skewness and kurtosis. The non-Gaussian distribution of this data affects identification of differentially-expressed genes, functional annotation, and prospective molecular classification. These effects may be reduced in some circumstances, although not completely eliminated, by using nonparametric analytics. This analysis highlights two unreliable assumptions of translational cancer gene expression analysis: that "small" departures from normality in the expression data distributions are analytically-insignificant and that "robust" gene-calling algorithms can fully compensate for these effects.

  2. Targeted disruption of the M(r) 46,000 mannose 6-phosphate receptor gene in mice results in misrouting of lysosomal proteins.

    Science.gov (United States)

    Köster, A; Saftig, P; Matzner, U; von Figura, K; Peters, C; Pohlmann, R

    1993-01-01

    Lysosomal enzymes containing mannose 6-phosphate recognition markers are sorted to lysosomes by mannose 6-phosphate receptors (MPRs). The physiological importance of this targeting mechanism is illustrated by I-cell disease, a fatal lysosomal storage disorder caused by the absence of mannose 6-phosphate residues in lysosomal enzymes. Most mammalian cells express two MPRs. Although the binding specificities, subcellular distribution and expression pattern of the two receptors can be differentiated, their coexpression is not understood. The larger of the two receptors with an M(r) of approximately 300,000 (MPR300), which also binds IGFII, appears to have a dominant role in lysosomal enzyme targeting, while the function of the smaller receptor with an M(r) of 46,000 (MPR46) is less clear. To investigate the in vivo function of the MPR46, we generated MPR46-deficient mice using gene targeting in embryonic stem cells. Reduced intracellular retention of newly synthesized lysosomal proteins in cells from MPR46 -/- mice demonstrated an essential sorting function of MPR46. The phenotype of MPR46 -/- mice was normal, indicating mechanisms that compensate the MPR46 deficiency in vivo. Images PMID:8262064

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

    OpenAIRE

    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...

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

    International Nuclear Information System (INIS)

    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

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

    International Nuclear Information System (INIS)

    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

  6. Testing an agent-based model of bacterial cell motility: How nutrient concentration affects speed distribution

    OpenAIRE

    Garcia, Victor; Birbaumer, Mirko; Schweitzer, Frank

    2011-01-01

    We revisit a recently proposed agent-based model of active biological motion and compare its predictions with own experimental findings for the speed distribution of bacterial cells, \\emph{Salmonella typhimurium}. Agents move according to a stochastic dynamics and use energy stored in an internal depot for metabolism and active motion. We discuss different assumptions of how the conversion from internal to kinetic energy $d(v)$ may depend on the actual speed, to conclude that $d_{2}v^{\\xi}$ w...

  7. Testing an agent-based model of bacterial cell motility: How nutrient concentration affects speed distribution

    Science.gov (United States)

    Garcia, V.; Birbaumer, M.; Schweitzer, F.

    2011-08-01

    We revisit a recently proposed agent-based model of active biological motion and compare its predictions with own experimental findings for the speed distribution of bacterial cells, Salmonella typhimurium. Agents move according to a stochastic dynamics and use energy stored in an internal depot for metabolism and active motion. We discuss different assumptions of how the conversion from internal to kinetic energy d( v) may depend on the actual speed, to conclude that d 2 v ξ with either ξ = 2 or 1 speed distribution of bacteria which were obtained in media of different nutrient concentration and at different times. We find that both hypotheses are in line with the experimental observations, with ξ between 1.67 and 2.0. Regarding the influence of a higher nutrient concentration, we conclude that the take-up of energy by bacterial cells is indeed increased. But this energy is not used to increase the speed, with 40 μm/s as the most probable value of the speed distribution, but is rather spend on metabolism and growth.

  8. Mercury distribution in a mangrove tidal creek affected by intensive shrimp farming.

    Science.gov (United States)

    Costa, B G B; Soares, T M; Torres, R F; Lacerda, L D

    2013-05-01

    In this study, the Hg distributions in water and sediments from a mangrove creek that receives intensive shrimp farming effluents were determined. The average dissolved and particulate Hg concentrations in the water varied from 3.1 to 9.2 ng L(-1) and from 4.4 to 9.4 ng L(-1), respectively. However, the Hg concentrations in the suspended particulate matter and the bottom sediments varied from 95.4 to 115.7 ng g(-1) and from 1.6 to 10.3 ng g(-1), respectively. A Ward quadratic distance cluster analysis based on the Hg concentrations and hydro- and geochemical parameters (oxygen, salinity, temperature, pH, and organic matter and aluminum content) showed the effects of shrimp farming effluents on the Hg distribution pattern. Furthermore, these results were supported by the Hg distribution in the sediment cores. This study emphasizes the necessity of including Hg as a potential pollutant when monitoring the environmental impacts of intensive shrimp farming. PMID:23370694

  9. Decision factors affecting transmission and distribution efficiency improvements by Northwest electric utilities

    Energy Technology Data Exchange (ETDEWEB)

    Hendrickson, P.L.; Darwin, R.F.

    1986-01-01

    The principal objective of this report was to assess and document the attitude of Northwest electric utilities toward possible BPA conservation acquisition programs that may provide incentive(s) to reduce losses on T and D lines. Secondary objectives were to examine existing incentives for making such improvements, to categorize prior T and D efficiency improvements, and to examine factors affecting the decision-making process for system improvements. Much of the information presented in the report is derived from a survey administered during personal interviews at 29 Northwest electric utilities between November 1984 and January 1985.

  10. 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. PMID:26893180

  11. 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.

  12. Investigations on temperature distribution of satellite surfaces affected by solar absorptivity

    Science.gov (United States)

    Liu, Y.; Pan, X. X.; Li, G. H.; Liu, X.; Jiang, L. X.

    2010-09-01

    Based on the Monte Carlo ray-tracing method, the network coefficients of thermal network model describing the radiation heat transfer among satellite surfaces is solved by considering the surface material optical characters. It is superiority to the conventional Gebhart's method in view of the grey body and the diffuse reflection assumptions. The zone leveling method is used to discrete the governing equations and the solar absorpivity is separated and considered to be an important correction parameter. Effects of the solar incidence round angle, the zenith angle and the ratio of absorpivity to emissivity (RAE) on temperature distribution are numerically simulated and discussed in detail. The higher or the lower the RAE may be lead to the alternative heating and cooling tend with a larger heating or cooling velocity of main body surfaces than the solar array surfaces. Furthermore, maximum temperature of main body is almost larger than solar arrays. Under the same RAE, solar incidence angle make a great effect on the uniform character of temperature distribution.

  13. Handle Shape Affects the Grip Force Distribution and the Muscle Loadings During Power Grip Tasks.

    Science.gov (United States)

    Rossi, Jérémy; Goislard De Monsabert, Benjamin; Berton, Eric; Vigouroux, Laurent

    2015-12-01

    The objectives of this study were to investigate the effect of handle shape on the grip force distribution in the hand and on the muscle forces during maximal power grip tasks. Eleven subjects maximally grasped 3 handles with different external shapes (circular, elliptic, and double-frustum). A handle dynamometer, equipped with both a force sensor and a pressure map, was used to record the forces exerted at the hand/handle interface. The finger and wrist joint postures were also computed from synchronized kinematic measurement. These processed data were then used as input of a biomechanical hand model to estimate muscle forces. The results showed that handle shape influences the maximal grip force, the grip force distribution, and the finger joint postures. Particularly, we observed that the elliptical shape resulted in a 6.6% lower maximal grip force compared with the circular and double-frustum handle. Concomitantly, the estimated muscle forces also varied significantly according to the handle shape, with up to 48% differences for the flexor digitorum superficialis muscle for example. Interestingly, different muscle coordination strategies were observed depending on the handle shape, therefore suggesting a potential influence of these geometrical characteristics on pathological risks such as tendonitis. PMID:26214057

  14. Distribution of 137Cs in surface soils as affected by forest clear-cutting

    International Nuclear Information System (INIS)

    The distribution of 137Cs was studied in podzol soil profiles from a 5 year old forest clear-cut area and an adjacent mature spruce forest in central Norway in order to assess the effects of clear-cutting on the distribution and mobility of radiocaesium in surface soils. A distinctly higher radiocaesium activity observed in the humus layer from the clear-cut compared to the forest area strongly indicates an increase in organic surface soil 137Cs activity within the first 5 years following forest clear-cutting. Such an increase, previously observed for Ca, Mg, Mn and Zn, is explained by increased supply of radiocaesium from decomposing logging residue, such as lichens and needles. Roughly 25% of the activity leached from decomposing residue had been transported into the A-E layer 5 years after clear-cutting. High 137Cs activity in the eluvial (E) horizon and a distinct decrease in deeper horizons indicates a certain leaching of 137Cs from the humus layer into the E horizon, which may act as an effective barrier against further leaching of radiocaesium. (Copyright (c) 1999 Elsevier Science B.V., Amsterdam. All rights reserved.)

  15. 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

  16. 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

  17. Numerical analyses of flow distributions in nuclear fuel assemblies affected by grid deformations

    International Nuclear Information System (INIS)

    Highlights: • Deformed spacer grid of a fuel assembly restricts coolant flow. • CFD analyses are conducted to assess flow redistribution and recovery. • Flow field is analyzed for normal operation, blowdown and reflood phases. • Forty-five times hydraulic diameter is required to recover 95% of flow rate. - Abstract: In the event of a safety shutdown earthquake (SSE) in a nuclear power plant, the spacer grid of the fuel assembly will be deformed as a result of the vibrations. If the flow area in a subchannel is reduced due to the grid deformation, the coolant flow will be restricted and consequently a loss of flow occurs in the affected fuel assembly during the accident. In this study, computational fluid dynamics (CFD) analyses are conducted in order to assess the flow redistribution and flow recovery in fuel assemblies. The real geometries of an outer grid and mixing vane are used in the simulation, and the region including the inner grid is modeled as a porous media zone. The resistance coefficients of the porous media model are determined using CFD analyses. The Reynolds-averaged Navier–Stokes equation with a non-linear turbulence model was used to solve the three-dimensional anisotropic turbulence flow in the rod bundles during normal operation, blowdown, and reflood phases following a loss-of-coolant accident (LOCA). In these analyses, it is assumed that forty percent of the flow area is blocked by grid deformations. The results demonstrate that a downstream distance of 45 times the hydraulic diameter is required for the coolant flow to recover to 95% of the original flow rate in the affected fuel assembly

  18. 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.

  19. N-acetylglucosamine-1-Phosphate Transferase Suppresses Lysosomal Hydrolases in Dysfunctional Osteoclasts: A Potential Mechanism for Vascular Calcification

    Directory of Open Access Journals (Sweden)

    Yang Lei

    2015-04-01

    Full Text Available In addition to increased differentiation of vascular smooth muscle cells into osteoblast-like phenotypes, the limited accumulation of osteoclasts in atherosclerotic plaques or their dysfunction may participate in potential mechanisms for vascular calcification. N-acetylglucosamine-1-phosphate transferase containing alpha and beta subunits (GNPTAB is a transmembrane enzyme complex that mediates the vesicular transport of lysosomal hydrolases. GNPTAB may also regulate the biogenesis of lysosomal hydrolases from bone-marrow derived osteoclasts. In this study, the areas surrounding calcification in human atherosclerotic plaques contained high levels of GNPTAB and low levels of lysosomal hydrolases such as cathepsin K (CTSK and tartrate-resistant acid phosphatase (TRAP, as demonstrated by immunohistochemistry and laser-capture microdissection-assisted mRNA expression analysis. We therefore hypothesized that GNPTAB secretion may suppress the release of CTSK and TRAP by vascular osteoclast-like cells, thus causing their dysfunction and reducing the resorption of calcification. We used human primary macrophages derived from peripheral blood mononuclear cells, an established osteoclast differentiation model. GNPTAB siRNA silencing accelerated the formation of functional osteoclasts as detected by increased secretion of CTSK and TRAP and increased their bone resorption activity as gauged by resorption pits assay. We concluded that high levels of GNPTAB inhibit secretion of lysosomal hydrolases in dysfunctional osteoclasts, thereby affecting their resorption potential in cardiovascular calcification.

  20. Do changes in the azimuthal distribution of maize leaves over time affect canopy light absorption?

    International Nuclear Information System (INIS)

    In maize canopies, when modelling the architecture and light regime one usually assumes leaf azimuths are distributed uniformly. Once we had demonstrated azimuthal re-orientation of maize leaves during the vegetative phase, we tested the weight of this hypothesis on the light absorbed daily by the canopy. We thus modelled the three-dimensional (3D) geometry of maize canopies with various plant densities and at different developmental stages using plant digitizing under field conditions. We simulated radiative transfer using a volume-based approach within actual and hypothetical canopies, obtained by simply rearranging leaf azimuths. Simulations indicated that changes to horizontal heterogeneity have little effect on daily light absorption efficiency. It is concluded that changes in leaf azimuths do not have to be taken into account in crop-functioning models. (author)

  1. Exchangeable basic cations and nitrogen distribution in soil as affected by crop residues and nitrogen

    Directory of Open Access Journals (Sweden)

    Ciro Antonio Rosolem

    2011-06-01

    Full Text Available In this work, a greenhouse experiment was conducted to study the effects of N fertilization and residues of pearl millet, black oats and oilseed radish on pH and Ca, Mg, K, NO3-, and NH4+ distribution within the profile of a Distroferric Red Latosol. The equivalent of 8 t ha-1 of plant residues were placed on soil surface. Lime was applied on the soil surface and nitrogen was applied over the straw at 0, 50, 100, and 150 mg kg-1, as ammonium nitrate. Corn was grown for 57 days. Calcium contents and pH in the soil profile were decreased by Pearl millet residue, while black oat and oilseed radish increased Ca contents and these effects are not related with Ca contents in residue tissue. However, the presence of plant residues increased nitrate, ammonium, and potassium contents in the deeper layers of the pots.

  2. 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.

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

    DEFF Research Database (Denmark)

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

    bodies. Buffer zones can be efficient in terms of retaining sediment and phosphorus transported by water erosion. This study aimed at parameterizing a spatial distributed erosion model to evaluate the effect of different buffer zone properties and dimension. It was our hypothesis that the placement and......Soil degradation and environmental impacts due to water erosion are a growing concern globally. Large parts of Denmark are covered by gently rolling moraine landscape with moderately to locally highly erodible soils where water erosion causes off-site problems in the form of eutrophication of water...... was surveyed during the runoff season. In addition, organic carbon and phosphorous contents as well as bulk density were determined in soils of eroding and depositional sites. General buffer zone properties were recorded. Here we present results from scenario analyses comparing measured sediment...

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

    International Nuclear Information System (INIS)

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

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

    Science.gov (United States)

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

    2007-01-24

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

  6. Zinc(II)-Thiosemicarbazone Complexes Are Localized to the Lysosomal Compartment Where They Transmetallate with Copper Ions to Induce Cytotoxicity.

    Science.gov (United States)

    Stacy, Alexandra E; Palanimuthu, Duraippandi; Bernhardt, Paul V; Kalinowski, Danuta S; Jansson, Patric J; Richardson, Des R

    2016-05-26

    As the di-2-pyridylketone thiosemicarbazone (DpT) and 2-acetylpyridine thiosemicarbazone (ApT) series show potent antitumor activity in vitro and in vivo, we synthesized their fluorescent zinc(II) complexes to assess their intracellular distribution. The Zn(II) complexes generally showed significantly greater cytotoxicity than the thiosemicarbazones alone in several tumor cell-types. Notably, specific structure-activity relationships demonstrated the importance of the di-2-pyridyl pharmacophore in their activity. Confocal fluorescence imaging and live cell microscopy showed that the Zn(II) complex of our lead compound, di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC), which is scheduled to enter clinical trials, was localized to lysosomes. Under lysosomal conditions, the Zn(II) complexes were shown to transmetallate with copper ions, leading to redox-active copper complexes that induced lysosomal membrane permeabilization (LMP) and cytotoxicity. This is the first study to demonstrate direct lysosomal targeting of our novel Zn(II)-thiosemicarbazone complexes that mediate their activity via transmetalation with copper ions and LMP. PMID:27023111

  7. 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.

  8. Distribution and retention of Cs radioisotopes in soil affected by Fukushima nuclear plant accident

    International Nuclear Information System (INIS)

    There was a large release of radio cesium (134Cs and 137Cs) to the atmosphere during Fukushima Daiichi Nuclear Power Plant (FDNPP) accident and contaminated soil over a vast area, due to fallout activity. Therefore, studies on the behavior of radio cesium especially migration in soil and its retention on soil particles is very important for external dose assessment and root uptake. We have determined the sorption coefficient (Kd) for Cs using laboratory batch method in soil samples collected from a contaminated area affected by FDNPP accident and the effect of various soil parameters on the Kd value has been studied. We have noticed that Cs sorption is mostly influenced by cation exchange process and sorbed on the surface of clay particles. From vertical depth profile of Cs in soil shows most of it is retained on the top layer within 5cm thickness. Sequential extraction of soil using various reagents may be helpful to understand better on the mechanism of Cs retention. (author)

  9. 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.

  10. Factors Affecting the Distribution of Wild Rice (Zizania palustris) and the Surrounding Macrophyte Community.

    Science.gov (United States)

    Pillsbury, R. W.; McGuire, M.

    2005-05-01

    A recent decline in wild rice wetlands is cause for concern due to its importance as a food source, refuge for wildlife, and cultural significance. Sixty wetlands in Wisconsin and Minnesota (USA) were sampled, with approximately equal numbers displaying dense, moderate and sparse wild rice production. Chemical, physical, and watershed parameters were measured as well as macrophyte densities. Data were analyzed using multivariate statistics (CCA). Moderate levels of phosphorus appear beneficial to the overall success of wild rice, while free-floating macrophytes show an overwhelming positive response to higher levels of P. The distribution of macrophytes bordering wild rice beds is correlated to pH,with Potamogeton robbinsii and filamentous green algae responding most strongly to its increase. Healthy stands of wild rice exhibit a narrow circum-neutral range of pH (6.1-8.0)which is significantly different from the greater range exhibited by sparse wild rice wetlands (6.5-8.5). This pattern was paralleled when considering depth which suggests that deeper wetlands may be more susceptible to wild rice loss. Management of existing wild rice wetlands should focus monitoring on pH, depth, phosphorus concentrations and shore development. We are currently using this data base to locate the best reintroduction sites for wild rice.

  11. The distribution of parasite strains among hosts affects disease spread in a social insect.

    Science.gov (United States)

    Ulrich, Yuko; Schmid-Hempel, Paul

    2015-06-01

    Social insects present highly interesting and experimentally amenable systems for the study of disease transmission because they naturally live in dense groups of frequently interacting individuals. Using experimental inoculations of five trypanosomatid strains into groups of its natural host, the bumblebee Bombus terrestris, we investigate the effects of the initial parasite strain distribution across group members on the establishment and transmission success of the different strains to new hosts. For a given number of parasite strains circulating within a host group, transmission to new hosts was increased when the strains were initially inoculated as mixed infections (as opposed to separate single infections), presumably because mixed infections generally favored fast replicating strains. In contrast, separate single infections reduced transmission at least in part through a precedence effect, whereby weak strains appeared to persist by making their host unavailable to superinfection. These results suggest that host groups could benefit from 'compartmentalizing' infections by different parasite strains across different group members, which might be achieved in social insects, for example, by division of labor. PMID:25858120

  12. Element distribution and morphology of spotted golden goatfish fish scales as affected by demineralisation.

    Science.gov (United States)

    Chuaychan, Sira; Benjakul, Soottawat; Nuthong, Pornpot

    2016-04-15

    Scales of spotted golden goatfish were subjected to non-collagenous protein removal followed by demineralisation with hydrochloric acid at different concentrations (0.25, 0.5, 0.75 and 1 M) for various times (30, 60 and 90 min). The morphology and element composition/distribution of scales from spotted golden goatfish as influenced by demineralisation conditions were determined. The appropriate demineralisation was pertained using 0.75 M hydrochloric acid for 30 min, in which the ash content was 0.62% (dry weight basis). The scales having non-collagenous protein removal with, and without, subsequent demineralisation were analysed for element content using X-ray fluorescence spectrometer. Images of different scales were determined using scanning electron microscopy (SEM) and scanning electron microscopy with energy dispersive X-ray spectroscopy (SEM-EDX). Based on the images, an external layer rich in inorganic elements was removed. Most of Ca and P were eliminated with the coincidental increases in organic substances (C, N and O) after demineralisation. Demineralisation therefore mainly removed the external layer of scales, which facilitated the further extraction of collagen or gelatin. PMID:26617021

  13. Factors Affecting the Distribution of the Amphipod Corophium volutatorin Two Estuaries in South-east England

    Science.gov (United States)

    Hughes, R. G.; Gerdol, V.

    1997-05-01

    The distribution of Corophium volutator(Pallas) in the estuaries of the Rivers Blackwater and Crouch in South-east England was examined by taking samples of mud from 137 sites at approximately 0·5 m below mean high water of neap tidal level. Corophium volutatorwere approximately twice as abundant in creeks and semi-enclosed bays than on the open mud flats, a difference that was significant statistically. There was no correlation between the abundance of C. volutatorand the median particle size of the sediment nor the mud content. There was a significant but weak negative correlation between the abundance of C. volutatorand the polychaete Nereis diversicolor. The aggregation of C. volutatorin the creeks and bays was attributed to their dispersal behaviour of swimming on the flood tide, which would sweep the amphipods into such areas where the tide rises but does not flow laterally. On the open mud flats, displacement of swimming amphipods by the flood tide further upstream and into semi-enclosed areas would occur. Their dispersal behaviour places C. volutatorin the creeks and bays within the saltmarsh vegetation, where their bioturbatory feeding habits may be responsible, in part, for the significant loss of pioneer zone vegetation that occurs there.

  14. Genetic Coding Variant in GPR65 Alters Lysosomal pH and Links Lysosomal Dysfunction with Colitis Risk.

    Science.gov (United States)

    Lassen, Kara G; McKenzie, Craig I; Mari, Muriel; Murano, Tatsuro; Begun, Jakob; Baxt, Leigh A; Goel, Gautam; Villablanca, Eduardo J; Kuo, Szu-Yu; Huang, Hailiang; Macia, Laurence; Bhan, Atul K; Batten, Marcel; Daly, Mark J; Reggiori, Fulvio; Mackay, Charles R; Xavier, Ramnik J

    2016-06-21

    Although numerous polymorphisms have been associated with inflammatory bowel disease (IBD), identifying the function of these genetic factors has proved challenging. Here we identified a role for nine genes in IBD susceptibility loci in antibacterial autophagy and characterized a role for one of these genes, GPR65, in maintaining lysosome function. Mice lacking Gpr65, a proton-sensing G protein-coupled receptor, showed increased susceptibly to bacteria-induced colitis. Epithelial cells and macrophages lacking GPR65 exhibited impaired clearance of intracellular bacteria and accumulation of aberrant lysosomes. Similarly, IBD patient cells and epithelial cells expressing an IBD-associated missense variant, GPR65 I231L, displayed aberrant lysosomal pH resulting in lysosomal dysfunction, impaired bacterial restriction, and altered lipid droplet formation. The GPR65 I231L polymorphism was sufficient to confer decreased GPR65 signaling. Collectively, these data establish a role for GPR65 in IBD susceptibility and identify lysosomal dysfunction as a potentially causative element in IBD pathogenesis with effects on cellular homeostasis and defense. PMID:27287411

  15. 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.

  16. Sink-source proximity affecting photosynthate distributive pattern in barley (Hordeum Vulgare L.)

    International Nuclear Information System (INIS)

    Yielding ability of cereals is an interplay of source-sink capacities and is controlled presumably by physiological events at different growth stages. The translocation of 14C-sucrose from the flag leaf to the ears and other plant parts of the main tiller of barley (H. vulgare L. cv 292) were studied at its appearance (I), maturation (II) and senescence initiation (III) stages. At every stage, there was preferentially higher 14C-translocation from the flag leaf to the ears in comparison to other plant parts, indicating thereby the crucial role played by sink-source proximity in PHS'ate(photosynthate) Partg(partitioning). In order to ascertain such Partg behaviour of the different leaves on the main tiller at stage-II, the leaves starting basipetally, from flag to the fifth leaf, were labelled with 14C-sucrose and their relative translocation to the ears and roots were determined. There was a differential 14C-translocation to the sinks due to the change in the sink-source proximity, viz. >90% 14C were translocated to the ears from flag leaf, which declined to ≤5% only from the fifth leaf, the reverse being true for the roots. Further, spikelet's central grains preferentially accumulated more 14C-assimilates than the peripheral ones, probably due to being in close proximity to the unloading points. Thus, the distributive pattern of photoassimilates showed stage-specific quantitative changes and that the sink-source proximity played a crucial role in the PHS'ate Partg. (author). 3 figs., 29 refs

  17. Distribution pattern of pelagic amphipods and its affecting facors in the East China Sea

    Institute of Scientific and Technical Information of China (English)

    XU Zhaoli; JIANG Mei

    2006-01-01

    On the basis of data from four seasonal cruises in the East China Sea (23°30'~33°N,118°30'~128°E) from 1997 to 2000, horizontal distribution of amphipods, seasonal variations of abundance and their dynamic mechanisms were approached with quantitative and qualitative methods. Results showed that the average value of amphipod abundance in autumn was 115×10-2 ind./m3, the highest in four seasons. The second occurred in summer with a mean value of 44×10-2 ind./m3 and the lowest occurred in spring with a mean of 10×10-2 ind./m3. Amphipods were hardly observed in the north nearshore (29°30'~33°N and 122°30'~125°E) in winter and spring.In all seasons, except for the north of the sea in autumn and the south in summer, the abundance in the offshore area was higher than that nearshore.The major species influencing the amphipod abundance were Lestrigonus schizogeneios in spring, L. macrophthalmus and L. schizogeneios in summer, L. bengalensis and Hyperioides sibaginis in autumn and Gammarus sp. in winter. The amphipod abundance showed a prominent linear correlation with the surface water temperature, but no significant correlation with both other water temperatures and salinity in spring. It was found that water temperature and salinity were not significantly correlated with the variation of amphipod abundance in summer, autumn and winter. In addition, the amphipod abundance in all four seasons can be linearly correlated with the bottom water temperature. The highest abundance area was located in the mixture waters near the side of warm water current in summer. Peak of amphipod abundance in autumn, not in summer told a temporal process for amphipods to develop their populations. The continuance of warm current was beneficial to the development of amphipod abundance from summer to autumn.Amphipods are kinds of very important diets for fishes in the offshore East China Sea.

  18. 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

    2016-01-01

    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. PMID:27213518

  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. The membrane composition of coated pits, microvilli, endosomes, and lysosomes is distinctive in the rat kidney proximal tubule cell

    OpenAIRE

    1986-01-01

    The distribution of a number of membrane proteins on plasmalemmal microdomains (microvilli, coated pits) and in endosomes and lysosomes of the proximal tubule epithelial cell was determined in normal rat kidneys by immunofluorescence and immunoelectron microscopy. Two major brush border proteins, 130 and 94 kD, and gamma-glutamyl transpeptidase were detected on the membranes of the microvilli but were not found on membranes of coated pits. Gp330, the Heymann nephritis antigen, and clathrin we...

  1. 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.

  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. 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).

  4. A Rab3a-dependent complex essential for lysosome positioning and plasma membrane repair.

    Science.gov (United States)

    Encarnação, Marisa; Espada, Lília; Escrevente, Cristina; Mateus, Denisa; Ramalho, José; Michelet, Xavier; Santarino, Inês; Hsu, Victor W; Brenner, Michael B; Barral, Duarte; Vieira, Otília V

    2016-06-20

    Lysosome exocytosis plays a major role in resealing plasma membrane (PM) disruptions. This process involves two sequential steps. First, lysosomes are recruited to the periphery of the cell and then fuse with the damaged PM. However, the trafficking molecular machinery involved in lysosome exocytosis and PM repair (PMR) is poorly understood. We performed a systematic screen of the human Rab family to identify Rabs required for lysosome exocytosis and PMR. Rab3a, which partially localizes to peripheral lysosomes, was one of the most robust hits. Silencing of Rab3a or its effector, synaptotagmin-like protein 4a (Slp4-a), leads to the collapse of lysosomes to the perinuclear region and inhibition of PMR. Importantly, we have also identified a new Rab3 effector, nonmuscle myosin heavy chain IIA, as part of the complex formed by Rab3a and Slp4-a that is responsible for lysosome positioning at the cell periphery and lysosome exocytosis. PMID:27325790

  5. 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 deficient due to mutations in the sulfatase-modifying factor 1 (SUMF1) gene, encoding the essential activator of all sulfatases. We identify a novel regulatory layer of sulfate metabolism mediated by a microRNA. miR-95 depletes SUMF1 protein levels and suppresses sulfatase activity, causing the disruption...

  6. Plasma Membrane Repair Is Regulated Extracellularly by Proteases Released from Lysosomes

    OpenAIRE

    Castro-Gomes, Thiago; Corrotte, Matthias; Tam, Christina; Andrews, Norma W.

    2016-01-01

    Eukaryotic cells rapidly repair wounds on their plasma membrane. Resealing is Ca2+-dependent, and involves exocytosis of lysosomes followed by massive endocytosis. Extracellular activity of the lysosomal enzyme acid sphingomyelinase was previously shown to promote endocytosis and wound removal. However, whether lysosomal proteases released during cell injury participate in resealing is unknown. Here we show that lysosomal proteases regulate plasma membrane repair. Extracellular proteolysis is...

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

    OpenAIRE

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

    2015-01-01

    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 ...

  8. Host cell invasion by trypanosomes requires lysosomes and microtubule/kinesin-mediated transport

    OpenAIRE

    1996-01-01

    Invasion of mammalian cells by the protozoan parasite Trypanosoma cruzi occurs by an actin-independent mechanism distinct from phagocytosis. Clusters of host lysosomes are observed at the site of parasite attachment, and lysosomal markers are detected in the vacuolar membrane at early stages of the entry process. These observations led to the hypothesis that the trypanosomes recruit host lysosomes to their attachment site, and that lysosomal fusion serves as a source of membrane to form the p...

  9. Structure of human saposin A at lysosomal pH

    Energy Technology Data Exchange (ETDEWEB)

    Hill, Chris H.; Read, Randy J.; Deane, Janet E., E-mail: jed55@cam.ac.uk [University of Cambridge, Wellcome Trust/MRC Building, Cambridge Biomedical Campus, Hills Road, Cambridge CB2 0XY (United Kingdom)

    2015-06-27

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

  10. 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

  11. Clinical, biochemical and genetic heterogeneity in lysosomal storage diseases

    NARCIS (Netherlands)

    A.J.J. Reuser (Arnold)

    1977-01-01

    textabstractThe history of lysosomal storage diseases dates back to the end of the last century when the first clinical reports appeared of patients suffering from these genetic, metabolic disorders (Tay, 1881; Gaucher, 1882; Sachs, 1887; Fabry, 1898). About seventy years wouid pass before the term

  12. Lysosomal Exocytosis in Schwann Cells Contributes to Axon Remyelination

    Institute of Scientific and Technical Information of China (English)

    GANG CHEN; ZHIJUN ZHANG; ZHONGYA WEI; QIONG CHENG; XIA LI; WEI LI; SHUMIN DUAN; XIAOSONG GU

    2012-01-01

    Myelin biogenesis is a complex process involving coordinated exocytosis, endocytosis, mRNA transport, and cytoskeletal dynamics. Although abnormalities of myelin are common in lysosomal storage diseases, our understanding of the role of lysosomes in the formation and maintenance of myelin is still limited. Here, we show that late endosomes/lysosomes in Schwann cells contain abundant myelin protein P0, which accounts for over half the total protein of compact myelin in the peripheral nervous system and exhibit Ca2+-dependent exocytosis in response to various stimuli. Downregulation of Rab27a, a small GTPase required for the trafficking of the secretory lysosomes to the plasma membrane, largely blocked lysosomal exocytosis in Schwann cells and reduced the remyelination of regenerated sciatic nerve. These findings highlight a novel role for lysosomes in Schwann cells and suggest that the regulated lysosome exocytosis in Schwann cells may have important physiological and pathological significance in the peripheral nervous%髓鞘形成是一个包括协同性的胞吐、内吞、mRNA转运和细胞骨架的动态变化的复杂过程.尽管髓鞘的异常在溶酶体贮积症中很常见,但对溶酶体在髓鞘形成和维持中所扮演的角色仍不清楚.本文发现Schwann细胞中的晚期内涵体/溶酶体包含大量的髓鞘蛋白P0,含量占超过一半的外周神经系统中的致密髓鞘的总蛋白并且在不同的刺激下表现出Ca2+依赖性的胞吐作用.Rab27a(一种将分泌溶酶体运输至细胞膜的小GTP酶)下调,极大地阻碍了Schwann细胞中的溶酶体胞吐作用,减少了再生坐骨神经的髓鞘形成.这些发现强调了Schwann细胞中溶酶体的新角色,提示调节Schwann细胞中的溶酶体胞吐作用在外周神经系统中有很重要的生理和病理意义.

  13. Protective effect of enterosgel on rat liver lysosomes during cytostatic treatment.

    Science.gov (United States)

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

    2002-10-01

    Polychemotherapy with a complex of cytostatics (cyclophosphamide, doxorubicin, vincristine, prednisolone) induces progressive damage to hepatocyte membranes, which manifested in labilization of lysosomes and activation of lysosomal enzymes and serum transaminases. Enterosgel stabilized liver lysosomes and reduced manifestation of hepatocyte cytolysis. PMID:12533758

  14. Lysosome size, motility and stress response regulated by fronto-temporal dementia modifier TMEM106B.

    Science.gov (United States)

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

    2014-07-01

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

  15. 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

  16. Enrichment and analysis of secretory lysosomes from lymphocyte populations

    Directory of Open Access Journals (Sweden)

    Leippe Matthias

    2009-07-01

    Full Text Available Abstract Background In specialized cells, such as mast cells, macrophages, T lymphocytes and Natural Killer cells in the immune system and for instance melanocytes in the skin, secretory lysosomes (SL have evolved as bifunctional organelles that combine degradative and secretory properties. Mutations in lysosomal storage, transport or sorting molecules are associated with severe immunodeficiencies, autoimmunity and (partial albinism. In order to analyze the function and content of secretory lysosomes in different cell populations, an efficient enrichment of these organelles is mandatory. Results Based on a combination of differential and density gradient centrifugation steps, we provide a protocol to enrich intact SL from expanded hematopoietic cells, here T lymphocytes and Natural Killer cells. Individual fractions were initially characterized by Western blotting using antibodies against an array of marker proteins for intracellular compartments. As indicated by the presence of LAMP-3 (CD63 and FasL (CD178, we obtained a selective enrichment of SL in one of the resulting organelle fractions. The robustness and reproducibility of the applied separation protocol was examined by a high-resolution proteome analysis of individual SL preparations of different donors by 2D difference gel electrophoresis (2D-DIGE. Conclusion The provided protocol is readily applicable to enrich and isolate intact secretory vesicles from individual cell populations. It can be used to compare SL of normal and transformed cell lines or primary cell populations from healthy donors and patients with lysosomal storage or transport diseases, or from corresponding mutant mice. A subsequent proteome analysis allows the characterization of molecules involved in lysosomal maturation and cytotoxic effector function at high-resolution.

  17. Leaky lysosomes in lung transplant macrophages: azithromycin prevents oxidative damage

    Directory of Open Access Journals (Sweden)

    Persson H L

    2012-09-01

    Full Text Available Abstract Background Lung allografts contain large amounts of iron (Fe, which inside lung macrophages may promote oxidative lysosomal membrane permeabilization (LMP, cell death and inflammation. The macrolide antibiotic azithromycin (AZM accumulates 1000-fold inside the acidic lysosomes and may interfere with the lysosomal pool of Fe. Objective Oxidative lysosomal leakage was assessed in lung macrophages from lung transplant recipients without or with AZM treatment and from healthy subjects. The efficiency of AZM to protect lysosomes and cells against oxidants was further assessed employing murine J774 macrophages. Methods Macrophages harvested from 8 transplant recipients (5 without and 3 with ongoing AZM treatment and 7 healthy subjects, and J774 cells pre-treated with AZM, a high-molecular-weight derivative of the Fe chelator desferrioxamine or ammonium chloride were oxidatively stressed. LMP, cell death, Fe, reduced glutathione (GSH and H-ferritin were assessed. Results Oxidant challenged macrophages from transplants recipients without AZM exhibited significantly more LMP and cell death than macrophages from healthy subjects. Those macrophages contained significantly more Fe, while GSH and H-ferritin did not differ significantly. Although macrophages from transplant recipients treated with AZM contained both significantly more Fe and less GSH, which would sensitize cells to oxidants, these macrophages resisted oxidant challenge well. The preventive effect of AZM on oxidative LMP and J774 cell death was 60 to 300 times greater than the other drugs tested. Conclusions AZM makes lung transplant macrophages and their lysososomes more resistant to oxidant challenge. Possibly, prevention of obliterative bronchiolitis in lung transplants by AZM is partly due to this action.

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

    Science.gov (United States)

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

    2014-08-01

    Development of higher rates of nondiabetic glomerulosclerosis (GS) in African Americans has been attributed to two coding sequence variants (G1 and G2) in the APOL1 gene. To date, the cellular function and the role of APOL1 variants (Vs) in GS are still unknown. In this study, we examined the effects of overexpressing wild-type (G0) and kidney disease risk variants (G1 and G2) of APOL1 in human podocytes using a lentivirus expression system. Interestingly, G0 inflicted podocyte injury only at a higher concentration; however, G1 and G2 promoted moderate podocyte injury at lower and higher concentrations. APOL1Vs expressing podocytes displayed diffuse distribution of both Lucifer yellow dye and cathepsin L as manifestations of enhanced lysosomal membrane permeability (LMP). Chloroquine attenuated the APOL1Vs-induced increase in podocyte injury, consistent with targeting lysosomes. The chloride channel blocker DIDS prevented APOL1Vs- induced injury, indicating a role for chloride influx in osmotic swelling of lysosomes. Direct exposure of noninfected podocytes with conditioned media from G1- and G2-expressing podocytes also induced injury, suggesting a contributory role of the secreted component of G1 and G2 as well. Adverse host factors (AHFs) such as hydrogen peroxide, hypoxia, TNF-α, and puromycin aminonucleoside augmented APOL1- and APOL1Vs-induced podocyte injury, while the effect of human immunodeficiency virus (HIV) on podocyte injury was overwhelming under conditions of APOLVs expression. We conclude that G0 and G1 and G2 APOL1 variants have the potential to induce podocyte injury in a manner which is further augmented by AHFs, with HIV infection being especially prominent. PMID:24899058

  19. 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. PMID:26682800

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

    International Nuclear Information System (INIS)

    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 lane to a bunker of World War II, temporarily paved with coarse industrial waste fragments and removed at the end of the war. Thin sections made from undisturbed soil samples from A and B horizons were studied. Optical microscopy revealed the occurrence of yellow micrometer-sized (Ap horizon) and red decamicrometer-sized spherulites (AB, B1g horizons) as well as distinct distribution patterns. The chemical composition of the spherulites was dominated by Fe, Mn, Zn, Pb, Ca, and P. Comparison of calculated Zn stocks, both in the groundmass and in spherulites, showed a quasi-exclusive Zn accumulation in these neoformed features. Their formation was related to several factors: (i) liberation of metal elements due to weathering of waste products, (ii) Ca and P supply from fertilizing practices, (iii) co-precipitation of metal elements and Ca and P in a porous soil environment, after slow exudation of a supersaturated soil solution in more confined mineral media. - Metal spherulites may act as high metal-trapping mineral phases in polluted agricultural soils

  1. 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

  2. Research on influence of temporary distribution of power of laser radiation with a hybrid processing on a depth heat affected zone

    International Nuclear Information System (INIS)

    Investigations on the influence of the temporal distribution of the laser radiation power per pulse of hybrid processing on a depth heat affected zone. Recommendations on use of specific distribution of the laser radiation power in the combined laser and plasma processing for decrease in energy consumption

  3. Lysosomal and endosomal heterogeneity in the liver: A comparison of the intracellular pathways of endocytosis in rat liver cells

    International Nuclear Information System (INIS)

    Air-filled albumin microspheres, asialoorosomucoid and formaldehyde-treated serum albumin are selectively taken up by endocytosis in rat liver Kupffer cells, parenchymal cells and endothelial cells, respectively. Intracellular transport and degradation of endocytosed material were studied by subcellular fractionation in sucrose and Nycodenz gradients after intravenous injection of the ligand. By using ligands labeled with 125I-tyramine-cellobiose, the subcellular distribution of labeled degradation products can be studied because they are trapped at the site of formation. The results show that the kinetics of intracellular transport are different in hepatic parenchymal, endothelial and Kupffer cells. In endothelial cells, the ligand is associated with two types of endosomes during the first minutes after internalization and then is transferred rapidly to the lysosomes. In parenchymal cells, 125I-tyramine-cellobiose-asialoorosomucoid was located in a relatively slowly sedimenting vesicle during the first minute after internalization and subsequently in denser endosomes. Degradation of 125I-tyramine-cellobiose-asialoorosomucoid in parenchymal cells started later than that of 125I-tyramine-cellobiose-formaldehyde-treated serum albumin in endothelial cells. Furthermore, the ligand seemed to be transferred relatively slowly from endosomes to lysosomes, and most of the undegraded ligand was in the endosomes. The rate-limiting step of proteolysis in parenchymal cells is probably the transport from endosomes to lysosomes. In Kupffer cells, most 125I-tyramine-cellobiose-microspheres are found as undegraded material in very dense endosomes up to 3 hr after injection. After 20 hr, most of the ligand is degraded in lysosomes distributed at a lower density than the endosomes in Nycodenz and sucrose gradients

  4. Rab7 and Arl8 GTPases are necessary for lysosome tubulation in macrophages.

    Science.gov (United States)

    Mrakovic, Amra; Kay, Jason G; Furuya, Wendy; Brumell, John H; Botelho, Roberto J

    2012-12-01

    Lysosomes provide a niche for molecular digestion and are a convergence point for endocytic trafficking, phagosome maturation and autophagy. Typically, lysosomes are small, globular organelles that appear punctate under the fluorescence microscope. However, activating agents like phorbol esters transform macrophage lysosomes into tubular lysosomes (TLs), which have been implicated in retention of pinocytic uptake and phagosome maturation. Moreover, dendritic cells exposed to lipopolysaccharides (LPSs) convert their punctate class II major histocompatibility complex compartment, a lysosome-related organelle, into a tubular network that is thought to be involved in antigen presentation. Other than a requirement for microtubules and kinesin, little is known about the molecular mechanisms that drive lysosome tubulation. Here, we show that macrophage cell lines readily form TLs after LPS exposure, with a requirement for the Rab7 GTPase and its effectors RILP (Rab7-interacting lysosomal protein) and FYCO1 (coiled-coil domain-containing protein 1), which respectively modulate the dynein and kinesin microtubule motor proteins. We also show that Arl8B, a recently identified lysosomal GTPase, and its effector SKIP, are also important for TL biogenesis. Finally, we reveal that TLs are significantly more motile than punctate lysosomes within the same LPS-treated cells. Therefore, we identify the first molecular regulators of lysosome tubulation and we show that TLs represent a more dynamic lysosome population. PMID:22909026

  5. 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. PMID:25976368

  6. Disease models for the development of therapies for lysosomal storage diseases.

    Science.gov (United States)

    Xu, Miao; Motabar, Omid; Ferrer, Marc; Marugan, Juan J; Zheng, Wei; Ottinger, Elizabeth A

    2016-05-01

    Lysosomal storage diseases (LSDs) are a group of rare diseases in which the function of the lysosome is disrupted by the accumulation of macromolecules. The complexity underlying the pathogenesis of LSDs and the small, often pediatric, population of patients make the development of therapies for these diseases challenging. Current treatments are only available for a small subset of LSDs and have not been effective at treating neurological symptoms. Disease-relevant cellular and animal models with high clinical predictability are critical for the discovery and development of new treatments for LSDs. In this paper, we review how LSD patient primary cells and induced pluripotent stem cell-derived cellular models are providing novel assay systems in which phenotypes are more similar to those of the human LSD physiology. Furthermore, larger animal disease models are providing additional tools for evaluation of the efficacy of drug candidates. Early predictors of efficacy and better understanding of disease biology can significantly affect the translational process by focusing efforts on those therapies with the higher probability of success, thus decreasing overall time and cost spent in clinical development and increasing the overall positive outcomes in clinical trials. PMID:27144735

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

    OpenAIRE

    Tessitore, Alessandra; Pirozzi, Marinella; Auricchio, Alberto

    2009-01-01

    Background Lysosomal storage diseases are characterized by intracellular accumulation of metabolites within lysosomes. Recent evidence suggests that lysosomal storage impairs autophagy resulting in accumulation of polyubiquitinated proteins and dysfunctional mitochondria, ultimately leading to apoptosis. We studied the relationship between lysosome storage and impairment of different intracellular pathways and organelle function in mucopolysaccharidosis VI, which is characterized by accumulat...

  8. Zinc Chelation Mediates the Lysosomal Disruption without Intracellular ROS Generation

    Science.gov (United States)

    Matias, Andreza Cândido; Manieri, Tânia Maria; Cerchiaro, Giselle

    2016-01-01

    We report the molecular mechanism for zinc depletion caused by TPEN (N,N,N′,N′-Tetrakis(2-pyridylmethyl)ethylenediamine) in neuroblastoma cells. The activation of p38 MAP kinase and subsequently caspase 3 is not due to or followed by redox imbalance or ROS generation, though these are commonly observed in literature. We found that TPEN is not responsible for ROS generation and the mechanism involves essentially lysosomal disruption caused by intracellular zinc depletion. We also observed a modest activation of Bax and no changes in the Bcl-2 proteins. As a result, we suggest that TPEN causes intracellular zinc depletion which can influence the breakdown of lysosomes and cell death without ROS generation. PMID:27123155

  9. 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

  10. 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.

  11. A genetic model with specifically impaired autophagosome–lysosome fusion

    OpenAIRE

    Takáts, Szabolcs; Juhász, Gábor

    2013-01-01

    Yeast studies identified the evolutionarily conserved core ATG genes responsible for autophagosome formation. However, the SNARE-dependent machinery involved in autophagosome fusion with the vacuole in yeast is not conserved. We recently reported that the SNARE complex consisting of Syx17 (Syntaxin 17), ubisnap (SNAP-29) and Vamp7 is required for the fusion of autophagosomes with late endosomes and lysosomes in Drosophila. Syx17 mutant flies are viable but exhibit neuronal dysfunction, locomo...

  12. The Use of Lysosomotropic Dyes to Exclude Lysosomal Membrane Permeabilization.

    Science.gov (United States)

    Repnik, Urška; Česen, Maruša Hafner; Turk, Boris

    2016-01-01

    Progressive lowering of pH is characteristic for the endocytic pathway and enables efficient degradation of molecules by hydrolytic enzymes at its distal end. The existence of the proton gradient over the endosomal/lysosomal membranes depends on the action of the vacuolar ATPase (v-ATPase). During lysosomal membrane permeabilization (LMP), protons leak through the destabilized membrane, resulting in loss of the pH gradient. Here, we present a protocol showing how this effect can be detected by staining cells with lysosomotropic dyes, which accumulate in acidic organelles after protonation. During LMP, cells lose the ability to retain these dyes and therefore appear pale. Among the most commonly used lysosomotropic dyes are LysoTracker reagents and acridine orange. Cells can be analyzed with a fluorescence microscope; however, flow-cytometric analysis enables fast, objective, and reliable evaluation of differences between samples. Advantages of the technique include the fact that sample preparation is relatively simple and can be scaled-up to test several different compounds or conditions. However, as we will discuss, cells treated with v-ATPase inhibitors also lose the pH gradient across lysosomal membranes and cannot be stained with lysosomotropic dyes, although this is not accompanied by LMP. Therefore, merely observing loss of staining is not in itself a proof of LMP. PMID:27140914

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

    International Nuclear Information System (INIS)

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

  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

    2016-01-01

    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 Ca2+-dependent mechanism independent of mTOR. Exogenous oxidants or increasing mitochondrial ROS levels directly and specifically activate lysosomal TRPML1 channels, inducing lysosomal Ca2+ 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. PMID:27357649

  16. Principles of lysosomal membrane digestion: stimulation of sphingolipid degradation by sphingolipid activator proteins and anionic lysosomal lipids.

    Science.gov (United States)

    Kolter, Thomas; Sandhoff, Konrad

    2005-01-01

    Sphingolipids and glycosphingolipids are membrane components of eukaryotic cell surfaces. Their constitutive degradation takes place on the surface of intra-endosomal and intra-lysosomal membrane structures. During endocytosis, these intra-lysosomal membranes are formed and prepared for digestion by a lipid-sorting process during which their cholesterol content decreases and the concentration of the negatively charged bis(monoacylglycero)phosphate (BMP)--erroneously also called lysobisphosphatidic acid (LBPA)--increases. Glycosphingolipid degradation requires the presence of water-soluble acid exohydrolases, sphingolipid activator proteins, and anionic phospholipids like BMP. The lysosomal degradation of sphingolipids with short hydrophilic head groups requires the presence of sphingolipid activator proteins (SAPs). These are the saposins (Saps) and the GM2 activator protein. Sphingolipid activator proteins are membrane-perturbing and lipid-binding proteins with different specificities for the bound lipid and the activated enzyme-catalyzed reaction. Their inherited deficiency leads to sphingolipid- and membrane-storage diseases. Sphingolipid activator proteins not only facilitate glycolipid digestion but also act as glycolipid transfer proteins facilitating the association of lipid antigens with immunoreceptors of the CD1 family. PMID:16212488

  17. 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.)

  18. Simulation study for lifetime distribution of middle-of-line time-dependent dielectric breakdown affected by global and local spacing variations

    Science.gov (United States)

    Yokogawa, Shinji

    2016-06-01

    The impact of process fluctuations for the lifetime distribution of middle-of-line time-dependent dielectric breakdown was investigated by Monte-Carlo simulation. Global and local variations were simulated using a doubly truncated normal distribution. A goodness of fit to the generated data was determined statistically in terms of the Weibull distribution and clustering model. A change in the standard deviation of the global variation shows a large contribution to lifetime variation. However, it does not affect the average lifetime. A change in the standard deviation of the local variation contributes to both the average lifetime and the distribution variation. The upper limit of the process variation induces the convex upward shape of the lifetime distribution on the Weibull plot. The clustering model well explains the distribution shape.

  19. Expression of the lysosomal-associated membrane protein-1 (LAMP-1) in astrocytomas

    DEFF Research Database (Denmark)

    Jensen, Stine S; Aaberg-Jessen, Charlotte; Christensen, Karina G;

    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...... 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...

  20. Effect of glutamate on lysosomal membrane permeabilization in primary cultured cortical neurons

    OpenAIRE

    Yan, Min; Zhu, Wenbo; Zheng, Xiaoke; Li, Yuan; TANG, LIPENG; LU, BINGZHENG; Chen, WenLi; Qiu, Pengxin; Leng, Tiandong; Lin, Suizhen; Yan, Guangmei; Yin, Wei

    2016-01-01

    Glutamate is the principal neurotransmitter in the central nervous system. Glutamate-mediated excitotoxicity is the predominant cause of cerebral damage. Recent studies have shown that lysosomal membrane permeabilization (LMP) is involved in ischemia-associated neuronal death in non-human primates. This study was designed to investigate the effect of glutamate on lysosomal stability in primary cultured cortical neurons. Glutamate treatment for 30 min induced the permeabilization of lysosomal ...

  1. Lysosomal-specific Cholesterol Reduction by Biocleavable Polyrotaxanes for Ameliorating Niemann-Pick Type C Disease

    OpenAIRE

    Atsushi Tamura; Nobuhiko Yui

    2014-01-01

    Niemann-Pick type C (NPC) disease is an autosomal recessive lysosomal trafficking disorder, in which the cholesterols are abnormally accumulated in lysosomes. Recently, the β-cyclodextrin (CD) derivatives are revealed to show therapeutic effect for NPC disease through the removal of accumulated cholesterols in lysosomes. Herein, to enhance the therapeutic effect and reduce the toxicity of β-CD derivatives, biocleavable Pluronic/β-CD-based polyrotaxanes (PRXs) bearing terminal disulfide linkag...

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

    OpenAIRE

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

    2012-01-01

    Multidrug resistance (MDR) remains a primary hindrance to curative cancer therapy. Thus, introduction of novel strategies to overcome MDR is of paramount therapeutic significance. Sequestration of chemotherapeutics in lysosomes is an established mechanism of drug resistance. Here, we show that MDR cells display a marked increase in lysosome number. We further demonstrate that imidazoacridinones (IAs), which are cytotoxic fluorochromes, undergo a dramatic compartmentalization in lysosomes beca...

  3. Phagosome-lysosome fusion is a calcium-independent event in macrophages

    OpenAIRE

    1996-01-01

    Phagosome-lysosome membrane fusion is a highly regulated event that is essential for intracellular killing of microorganisms. Functionally, it represents a form of polarized regulated secretion, which is classically dependent on increases in intracellular ionized calcium ([Ca2+]i). Indeed, increases in [Ca2+]i are essential for phagosome- granule (lysosome) fusion in neutrophils and for lysosomal fusion events that mediate host cell invasion by Trypanosoma cruzi trypomastigotes. Since several...

  4. FLCN Maintains the Leucine Level in Lysosome to Stimulate mTORC1

    OpenAIRE

    Wu, Xiaochun; Zhao, Lingling; Chen, Zhi; Ji, Xin; Qiao, Xianfeng; Jin, Yaping; Liu, Wei

    2016-01-01

    The intracellular amino acid pool within lysosome is a signal that stimulates the nutrient-sensing mTORC1 signalling pathway. The signal transduction cascade has garnered much attention, but little is known about the sequestration of the signalling molecules within the lysosome. Using human HEK293 cells as a model, we found that suppression of the BHD syndrome gene FLCN reduced the leucine level in lysosome, which correlated with decreased mTORC1 activity. Both consequences could be reversed ...

  5. 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....

  6. Deviant Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP)-mediated Ca2+ Signaling upon Lysosome Proliferation*

    OpenAIRE

    Dickinson, G. D.; Churchill, G. C.; Brailoiu, E; Patel, S.

    2010-01-01

    Accumulating evidence suggests that the endolysosomal system is a novel intracellular Ca2+ pool mobilized by the second messenger, nicotinic acid adenine dinucleotide phosphate (NAADP). Although lysosomes in neurons are known to proliferate in numerous neurodegenerative diseases and during the normal course of aging, little is known concerning the effect of lysosomal proliferation on Ca2+ homeostasis. Here, we induce proliferation of lysosomes in primary cultures of rat hippocampal neurons an...

  7. Role of lysosome rupture in controlling Nlrp3 signaling and necrotic cell death

    OpenAIRE

    Lima, Jr., Heriberto; Jacobson, Lee S.; Goldberg, Michael F.; Chandran, Kartik; Diaz-Griffero, Felipe; Lisanti, Michael P; Brojatsch, Jürgen

    2013-01-01

    The Nod-like receptor, Nlrp3, has been linked to inflammatory diseases and adjuvant-mediated immune responses. A wide array of structurally diverse agents does not interact directly with Nlrp3, but is thought to activate the Nlrp3 inflammasome by inducing a common upstream signal, such as lysosome rupture. To test the connection between lysosome integrity and Nlrp3 signaling, we analyzed inflammasome activation following stimulation of murine macrophages with lysosome-destabilizing agents and...

  8. Coordinated host responses during pyroptosis: caspase-1-dependent lysosome exocytosis and inflammatory cytokine maturation

    OpenAIRE

    Bergsbaken, Tessa; Fink, Susan L.; den Hartigh, Andreas B.; Loomis, Wendy P.; Cookson, Brad T.

    2011-01-01

    Activation of caspase-1 leads to pyroptosis, a program of cell death characterized by cell lysis and inflammatory cytokine release. Caspase-1 activation triggered by multiple NLRs (NLRC4, NLRP1b, or NLRP3) leads to loss of lysosomes via their fusion with the cell surface, or lysosome exocytosis. Active caspase-1 increased cellular membrane permeability and intracellular calcium levels, which facilitated lysosome exocytosis and release of host antimicrobial factors and microbial products. Lyso...

  9. Signals for the lysosome: a control center for cellular clearance and energy metabolism

    OpenAIRE

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

    2013-01-01

    For a long time lysosomes were considered merely to be cellular “incinerators” involved in the degradation and recycling of cellular waste. However, there is now compelling evidence indicating that lysosomes have a much broader function and that they are involved in fundamental processes such as secretion, plasma membrane repair, signaling and energy metabolism. Furthermore, the essential role of lysosomes in the autophagic pathway puts these organelles at the crossroads of several cellular p...

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

    Directory of Open Access Journals (Sweden)

    Minjeong Jung

    Full Text Available 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.

  11. Glucosamine-Bound Near-Infrared Fluorescent Probes with Lysosomal Specificity for Breast Tumor Imaging

    Directory of Open Access Journals (Sweden)

    Cong Li

    2008-04-01

    Full Text Available Noninvasive imaging of lysosomes will be useful 1 to elucidate the role of lysosomal parameters in cancer, 2 to diagnose malignant lesions, and 3 to evaluate future lysosome-targeted anticancer therapies. Lysosome-specific labeling of glucosamine-bound near-infrared (NIR fluorescent probes, IR-1 and IR-2, but not control probe IR-15 without the glucosamine moiety, was observed by fluorescence microscopy in human breast epithelial cell lines. Lysosome labeling and tumor specificity of these NIR probes were investigated by dynamic optical imaging and immunofluorescence staining in human breast tumor xenografts. IR-1 and IR-2 demonstrated faster lysosome labeling rates in highly aggressive MDA-MB-231 and MDA-MB-435 cells compared with less aggressive MCF-7 and nontumorigenic MCF-12A cells. IR-1 and IR-2, but not IR-15, accumulated in human MDA-MB-231, MDA-MB-435, and MCF-7 breast tumor xenografts in vivo. IR-2 demonstrated the highest maximum fluorescence and tumor/normal tissue ratios in all tumor models. Specific lysosome labeling from IR-2 in vivo was validated by colocalization of the NIR fluorescence with CD63 immunofluorescence in tumor sections. IR-1 and IR-2 demonstrated high lysosome-labeling ability and breast tumor-targeting specificity in vitro and in vivo. They are promising for diagnosing malignant lesions and may provide a means for evaluating and monitoring future lysosome-targeted anticancer therapies.

  12. 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.

  13. Receiving enzyme replacement therapy for a lysosomal storage disorder: a preliminary exploration of the experiences of young patients and their families.

    Science.gov (United States)

    Freedman, R; Sahhar, M; Curnow, L; Lee, J; Peters, H

    2013-08-01

    Medical intervention for lysosomal storage disorders becomes part of life, shaping the reality of the condition for affected individuals and families. Enzyme replacement therapy (ERT) is available to treat some lysosomal storage disorders. ERT is costly and time consuming, requiring frequent hospital visits to receive intravenous infusions. This qualitative study sought to explore the impact of receiving ERT for a lysosomal storage disorder on the health related quality of life (HRQoL) of young patients and their families. Fifteen semi-structured interviews were conducted with young people and parents and siblings of young people accessing ERT for Pompe disease, Gaucher disease or mucopolysaccharidosis types I or II living in Victoria, Australia. Interviews were transcribed then analyzed thematically. The biopsychosocial model assisted in interpreting themes. Findings revealed positive attitudes towards ERT, with noticed improvements in physical and psychosocial well-being. Participants prioritised intervention over other activities and provided suggestions for improving current service delivery. Communication with family members and professionals was deemed important, especially in respect to information provision. Participants described challenges associated with living with a lysosomal storage disorder and receiving ERT and coping strategies, such as positive thinking and ways to manage uncertainty. These findings provide valuable insights into the impact of living with a chronic genetic condition and receiving intensive treatment on HRQoL. PMID:23536258

  14. Factors affecting stress distribution and displacements in crystals III-V grown by Czochralski method with liquid encapsulation

    International Nuclear Information System (INIS)

    A mathematical model based on the finite element method for calculating temperature and shear stress distributions in III-V crystals grown by LEC technique was developed. The calculated temperature are in good agreements with the experimental measurements. The shear stress distribution was calculated for several environmental conditions. The results showed that the magnitude and the distribution of shear stresses are highly sensitive to the crystal environment, including thickness and temperature distribution in boron oxides and the gas. The shear stress is also strongly influenced by interface curvature and cystals radius. (author)

  15. Lysosomal sphingomyelinase is not solicited for apoptosis signaling.

    Science.gov (United States)

    Bezombes, C; Ségui, B; Cuvillier, O; Bruno, A P; Uro-Coste, E; Gouazé, V; Andrieu-Abadie, N; Carpentier, S; Laurent, G; Salvayre, R; Jaffrézou, J P; Levade, T

    2001-02-01

    Stress-induced activation of an acidic sphingomyelinase leading to generation of ceramide, an important lipid mediator, has been associated with apoptosis; however, the implication of this hydrolase has been questioned. The present study aimed at re-evaluating the role of this lysosomal enzyme in apoptosis initiated by different apoptotic inducers. The sensitivity of a series of acid sphingomyelinase-deficient cell lines derived from Niemann-Pick disease patients to stress-induced apoptosis was investigated. We have now shown that stress stimuli, such as anthracyclines, ionizing radiation, and Fas ligation trigger similar apoptotic hallmarks in normal and acid sphingomyelinase-deficient cell lines. Retrovirus-mediated gene correction of enzyme deficiency in Niemann-Pick cells does not modify response to apoptosis. Ceramide production is comparable in normal and Niemann-Pick cells, and increased activity of neutral sphingomyelinase is observed. Thus, our findings cast serious doubts that lysosomal sphingomyelinase activation is responsible for stress-induced apoptosis of cultured cells. PMID:11156942

  16. 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.

  17. NEW EMBO MEMBER’S REVIEW: Lysosomal cysteine proteases: facts and opportunities

    OpenAIRE

    Turk, Vito; Turk, Boris; Turk, Dušan

    2001-01-01

    From their discovery in the first half of the 20th century, lysosomal cysteine proteases have come a long way: from being the enzymes non-selectively degrading proteins in lysosomes to being those responsible for a number of important cellular processes. Some of the features and roles of their structures, specificity, regulation and physiology are discussed.

  18. Plasma Membrane Repair Is Regulated Extracellularly by Proteases Released from Lysosomes.

    Directory of Open Access Journals (Sweden)

    Thiago Castro-Gomes

    Full Text Available Eukaryotic cells rapidly repair wounds on their plasma membrane. Resealing is Ca(2+-dependent, and involves exocytosis of lysosomes followed by massive endocytosis. Extracellular activity of the lysosomal enzyme acid sphingomyelinase was previously shown to promote endocytosis and wound removal. However, whether lysosomal proteases released during cell injury participate in resealing is unknown. Here we show that lysosomal proteases regulate plasma membrane repair. Extracellular proteolysis is detected shortly after cell wounding, and inhibition of this process blocks repair. Conversely, surface protein degradation facilitates plasma membrane resealing. The abundant lysosomal cysteine proteases cathepsin B and L, known to proteolytically remodel the extracellular matrix, are rapidly released upon cell injury and are required for efficient plasma membrane repair. In contrast, inhibition of aspartyl proteases or RNAi-mediated silencing of the lysosomal aspartyl protease cathepsin D enhances resealing, an effect associated with the accumulation of active acid sphingomyelinase on the cell surface. Thus, secreted lysosomal cysteine proteases may promote repair by facilitating membrane access of lysosomal acid sphingomyelinase, which promotes wound removal and is subsequently downregulated extracellularly by a process involving cathepsin D.

  19. Plasma Membrane Repair Is Regulated Extracellularly by Proteases Released from Lysosomes.

    Science.gov (United States)

    Castro-Gomes, Thiago; Corrotte, Matthias; Tam, Christina; Andrews, Norma W

    2016-01-01

    Eukaryotic cells rapidly repair wounds on their plasma membrane. Resealing is Ca(2+)-dependent, and involves exocytosis of lysosomes followed by massive endocytosis. Extracellular activity of the lysosomal enzyme acid sphingomyelinase was previously shown to promote endocytosis and wound removal. However, whether lysosomal proteases released during cell injury participate in resealing is unknown. Here we show that lysosomal proteases regulate plasma membrane repair. Extracellular proteolysis is detected shortly after cell wounding, and inhibition of this process blocks repair. Conversely, surface protein degradation facilitates plasma membrane resealing. The abundant lysosomal cysteine proteases cathepsin B and L, known to proteolytically remodel the extracellular matrix, are rapidly released upon cell injury and are required for efficient plasma membrane repair. In contrast, inhibition of aspartyl proteases or RNAi-mediated silencing of the lysosomal aspartyl protease cathepsin D enhances resealing, an effect associated with the accumulation of active acid sphingomyelinase on the cell surface. Thus, secreted lysosomal cysteine proteases may promote repair by facilitating membrane access of lysosomal acid sphingomyelinase, which promotes wound removal and is subsequently downregulated extracellularly by a process involving cathepsin D. PMID:27028538

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

    Directory of Open Access Journals (Sweden)

    Fang Chen

    2016-04-01

    Full Text Available 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 < 0.05 in 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.

  1. 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

    2016-01-01

    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 < 0.05) in 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. PMID:27120618

  2. Lysosomal and autophagic reactions as predictive indicators of environmental impact in aquatic animals.

    Science.gov (United States)

    Moore, Michael N; Allen, J Icarus; McVeigh, Allan; Shaw, Jenny

    2006-01-01

    The lysosomal-autophagic system appears to be a common target for many environmental pollutants as lysosomes accumulate many toxic metals and organic xenobiotics, which perturb normal function and damage the lysosomal membrane. In fact, lysosomal membrane integrity or stability appears to be an effective generic indicator of cellular well-being in eukaryotes: in bivalve molluscs and fish, stability is correlated with many toxicological responses and pathological reactions. Prognostic use of adverse lysosomal and autophagic reactions to environmental pollutants has been explored in relation to predicting cellular dysfunction and health in marine mussels, which are extensively used as sensitive bioindicators in monitoring ecosystem health. Derivation of explanatory frameworks for prediction of pollutant impact on health is a major goal; and we have developed a conceptual mechanistic model linking lysosomal damage and autophagic dysfunction with injury to cells and tissues. This model has also complemented the creation of a cell-based computational model for molluscan hepatopancreatic cells that simulates lysosomal, autophagic and other cellular reactions to pollutants. Experimental and simulated results have also indicated that nutritional deprivation-induced autophagy has a protective function against toxic effects mediated by reactive oxygen species (ROS). Finally, coupled measurement of lysosomal-autophagic reactions and modelling is proposed as a practical toolbox for predicting toxic environmental risk. PMID:16874099

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

    Science.gov (United States)

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

    2012-01-01

    Multidrug resistance (MDR) remains a primary hindrance to curative cancer therapy. Thus, introduction of novel strategies to overcome MDR is of paramount therapeutic significance. Sequestration of chemotherapeutics in lysosomes is an established mechanism of drug resistance. Here, we show that MDR cells display a marked increase in lysosome number. We further demonstrate that imidazoacridinones (IAs), which are cytotoxic fluorochromes, undergo a dramatic compartmentalization in lysosomes because of their hydrophobic weak base nature. We hence developed a novel photoactivation-based pharmacological Trojan horse approach to target and eradicate MDR cancer cells based on photo-rupture of IA-loaded lysosomes and tumor cell lysis via formation of reactive oxygen species. Illumination of IA-loaded cells resulted in lysosomal photodestruction and restoration of parental cell drug sensitivity. Lysosomal photodestruction of MDR cells overexpressing the key MDR efflux transporters ABCG2, ABCB1 or ABCC1 resulted in 10- to 52-fold lower IC(50) values of various IAs, thereby restoring parental cell sensitivity. Finally, in vivo application of this photodynamic therapy strategy after i.v. injection of IAs in human ovarian tumor xenografts in the chorioallantoic membrane model revealed selective destruction of tumors and their associated vasculature. These findings identify lysosomal sequestration of IAs as an Achilles heel of MDR cells that can be harnessed to eradicate MDR tumor cells via lysosomal photodestruction. PMID:22476101

  4. Taking Out TB–Lysosomal Trafficking and Mycobactericidal Ubiquitin-Derived Peptides

    OpenAIRE

    Purdy, Georgiana E.

    2011-01-01

    Tuberculosis remains a significant global health concern. The hallmark of Mycobacterium tuberculosis pathogenicity is its ability to infect resting macrophages and establish an intracellular niche. Activated and autophagic macrophages control mycobacterial infections through bactericidal mechanisms ranging from reactive oxygen and nitrogen intermediates to the delivery of the bacterium to the acidified, hydrolytically active lysosome. The mycobactericidal activity of the lysosome is due in pa...

  5. Taking out TB – A role for lysosomal ubiquitin-derived peptides

    OpenAIRE

    Georgiana ePurdy

    2011-01-01

    Tuberculosis remains a significant global health concern. The hallmark of Mycobacterium tuberculosis pathogenicity is its ability to infect resting macrophages and establish an intracellular niche. Activated and autophagic macrophages control mycobacterial infections through bactericidal mechanisms ranging from reactive oxygen and nitrogen intermediates to the delivery of the bacterium to the acidified, hydrolytically active lysosome. The mycobactericidal activity of the lysosome is due in pa...

  6. Lysosome stabilization in slices of rat liver when incubated with vitamin A excess

    International Nuclear Information System (INIS)

    An organ culture of slices of livers from adult rats was used to study effect of vitamin A (all-trans retinol) on lysosome stability. Lysosomes were purified by centrifugation in Percoll gradients. Preparations were monitored by electron microscopy and evaluated by morphometry and assays of marker enzymes. Enrichments relative to homogenates and crude pellets were estimated from latent (triton X-100) acid p-nitrophenylphosphatase specific activities. Lysosomes prepared from unincubated slices were enriched 50-fold in latent acid phosphatase relative to homogenates. In contrast, lysosomes prepared from slices incubated for 30 min in PBS alone were enriched only 20-fold. When 25 μg/ml retinol was included in the incubation medium, enrichments of 40-fold were obtained. The integrity of the slices was monitored by electron microscopy and their viability was confirmed by a sustained uptake and incorporation of [3H]leucine into protein (up to 2 h in culture). The loss of lysosomes from homogenates of slices incubated in the absence of retinol was accompanied by a loss of acid phosphatase from the lysosomal pellet to the supernatant during purification. Addition of retinol to slices just prior to homogenization was without effect. The results demonstrate a stabilizing influence of vitamin A on lysosomes during incubation of licer slices. The findings contrast earlier reports of retinol-induced lysosome fragility in other in vitro systems

  7. The stereochemical configuration of lysosomal phosphatidylcholine and phosphatidylethanolamine: comparison with lysobisphosphatidic acid.

    Science.gov (United States)

    Joutti, A; Renkonen, O

    1979-02-01

    Lysosomal phosphatidylcholine and phosphatidylethanolamine were isolated from liver of rats treated with Triton WR 1339 and from cultured BHK-cells. Stereochemical analysis proved that these lipids, in contrast to the lysosomal lysobisphosphatidic acid, were derivatives of sn-glycero-3-phosphate. PMID:438662

  8. Non-inhibitory antibodies impede lysosomal storage reduction during enzyme replacement therapy of a lysosomal storage disease.

    Science.gov (United States)

    Matzner, Ulrich; Matthes, Frank; Weigelt, Cecilia; Andersson, Claes; Eistrup, Carl; Fogh, Jens; Gieselmann, Volkmar

    2008-04-01

    Enzyme replacement therapy is a treatment option for several lysosomal storage disorders. We reported previously that treatment of a knockout mouse model of the sphingolipid storage disease metachromatic leukodystrophy (MLD) by intravenous injection of recombinant human arylsulfatase A (rhASA) reduces sulfatide storage and improves nervous system pathology and function. Here, we show that treated mice can develop anti-rhASA antibodies, which impede sulfatide clearance without inhibiting enzyme activity. The neutralizing effect of antibodies was reproduced in cell culture models of MLD by demonstrating that mouse immune serum reduces the ability of rhASA to clear sulfatide from cultured ASA-deficient Schwann and kidney cells. We show that reduced clearance is due to an antibody-mediated blockade of mannose 6-phosphate receptor-dependent enzyme uptake, retargeting of rhASA from sulfatide-storing cells to macrophages, intracellular misrouting of rhASA, and reduction of enzyme stability. Induction of immunotolerance to rhASA by transgenic expression of an active site mutant of human ASA restores sulfatide clearance in mice. The data indicate that the influence of non-inhibitory antibodies must be more intensively considered in evaluating the therapeutic efficacy of enzyme replacement in lysosomal storage disorders in general and in patients without cross-reacting material specifically. PMID:18360747

  9. Lysosomal ATP imaging in living cells by a water-soluble cationic polythiophene derivative.

    Science.gov (United States)

    Huang, Bing-Huan; Geng, Zhi-Rong; Ma, Xiao-Yan; Zhang, Cui; Zhang, Zhi-Yang; Wang, Zhi-Lin

    2016-09-15

    Lysosomes in astrocytes and microglia can release ATP as the signaling molecule for the cells through ca(2+)-dependent exocytosis in response to various stimuli. At present, fluorescent probes that can detect ATP in lysosomes have not been reported. In this work, we have developed a new water-soluble cationic polythiophene derivative that can be specifically localized in lysosomes and can be utilized as a fluorescent probe to sense ATP in cells. PEMTEI exhibits high selectivity and sensitivity to ATP at physiological pH values and the detection limit of ATP is as low as 10(-11)M. The probe has low cytotoxicity, good permeability and high photostability in living cells and has been applied successfully to real-time monitoring of the change in concentrations of ATP in lysosomes though fluorescence microscopy. We also demonstrated that lysosomes in Hela cells can release ATP through Ca(2+)-dependent exocytosis in response to drug stimuli. PMID:27131993

  10. Changes in the distribution of multispecies pest assemblages affect levels of crop damage in warming tropical Andes.

    Science.gov (United States)

    Crespo-Pérez, Verónica; Régnière, Jacques; Chuine, Isabelle; Rebaudo, François; Dangles, Olivier

    2015-01-01

    Climate induced species range shifts might create novel interactions among species that may outweigh direct climatic effects. In an agricultural context, climate change might alter the intensity of competition or facilitation interactions among pests with, potentially, negative consequences on the levels of damage to crop. This could threaten the productivity of agricultural systems and have negative impacts on food security, but has yet been poorly considered in studies. In this contribution, we constructed and evaluated process-based species distribution models for three invasive potato pests in the Tropical Andean Region. These three species have been found to co-occur and interact within the same potato tuber, causing different levels of damage to crop. Our models allowed us to predict the current and future distribution of the species and therefore, to assess how damage to crop might change in the future due to novel interactions. In general, our study revealed the main challenges related to distribution modeling of invasive pests in highly heterogeneous regions. It yielded different results for the three species, both in terms of accuracy and distribution, with one species surviving best at lower altitudes and the other two performing better at higher altitudes. As to future distributions our results suggested that the three species will show different responses to climate change, with one of them expanding to higher altitudes, another contracting its range and the other shifting its distribution to higher altitudes. These changes will result in novel areas of co-occurrence and hence, interactions of the pests, which will cause different levels of damage to crop. Combining population dynamics and species distribution models that incorporate interspecific trade-off relationships in different environments revealed a powerful approach to provide predictions about the response of an assemblage of interacting species to future environmental changes and their

  11. 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. PMID:23071517

  12. 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].

  13. The probability distribution of maintenance cost of a system affected by the gamma process of degradation: Finite time solution

    International Nuclear Information System (INIS)

    The stochastic gamma process has been widely used to model uncertain degradation in engineering systems and structures. The optimization of the condition-based maintenance (CBM) policy is typically based on the minimization of the asymptotic cost rate. In the financial planning of a maintenance program, however, a more accurate prediction interval for the cost is needed for prudent decision making. The prediction interval cannot be estimated unless the probability distribution of cost is known. In this context, the asymptotic cost rate has a limited utility. This paper presents the derivation of the probability distribution of maintenance cost, when the system degradation is modelled as a stochastic gamma process. A renewal equation is formulated to derive the characteristic function, then the discrete Fourier transform of the characteristic function leads to the complete probability distribution of cost in a finite time setting. The proposed approach is useful for a precise estimation of prediction limits and optimization of the maintenance cost.

  14. A novel melano-lysosome in the retinal epithelium of rhesus monkeys.

    Science.gov (United States)

    Gouras, Peter; Brown, Kristy; Ivert, Lena; Neuringer, Martha

    2011-12-01

    The large phagocytic load that confronts the retinal pigment epithelium (RPE) is thought to play a possible role in the pathogenesis of age related macular degeneration (AMD) that afflicts both humans and monkeys. Our knowledge of how RPE degrades phagosomes and other intra-cellular material by lysosomal action is still rudimentary. In this paper we examine organelles that play a role in this process, melanosome, lysosomes and phagosomes, in the RPE of young and old rhesus monkeys in order to better understand lysosomal autophagy and heterophagy in the RPE and its possible role in AMD. We used electron microscopy to detect and describe the characteristics of melanosomes and lysosome-like organelles in the macular RPE of rhesus monkeys (Macaca mulatta) that were 1, 6, 24, 24, 26 and 35 years of age. The measurements include the number, shape and size of these organelles located in the basal, middle and apical regions of RPE cells. Phaagosomes were also examined but not counted or measured for size or shape because of their rarity. Melanosomes were homogeneously dark with a circular or elliptical shape and decreased in number with age. Smaller melanosomes were more common at the basal side of the RPE. Among the small melanosomes, we found an organelle that was losing melanin in varying degrees; in some cases was nearly devoid of melanin. Because of the melanin loss, we considered this organelle to be a unique type of autophagic melano-lysosome, which we called a Type 1 lysosome. We found another organelle, more canonically lysosomal, which we called a Type 2 lysosome. This organelle was composed of a light matrix containing melanosomes in various stages of degradation. Type 2 lysosomes without melanosomes were rare. Type 2 lysosomes increased while Type 1 decreased in number with age. Phagosomes were rare in both young and old monkeys. They made close contact with Type 2 lysosomes which we considered responsible for their degradation. Melanosomes are being lost from

  15. Availability and Temporal Heterogeneity of Water Supply Affect the Vertical Distribution and Mortality of a Belowground Herbivore and Consequently Plant Growth

    OpenAIRE

    Tsunoda, Tomonori; Kachi, Naoki; Suzuki, Jun-Ichirou

    2014-01-01

    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 belowgr...

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

    OpenAIRE

    He, Hongqing; Wan, Weixing

    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 e...

  17. 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 (pstrategy. These results are important for comprehensively understanding the mechanism of action of Dp44mT. PMID:27102538

  18. The UL24 protein of herpes simplex virus 1 affects the sub-cellular distribution of viral glycoproteins involved in fusion

    International Nuclear Information System (INIS)

    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. - Highlights: • UL24 affects the sub-cellular distribution of viral glycoproteins required for fusion. • Sub-cellular distribution of viral glycoproteins varies in cell-type dependent manner. • Drugs targeting actin microfilaments affect formation of UL24-related syncytia in HFFs

  19. 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

    biochemical markers pregnancy-associated plasma protein-A (PAPP-A) and free β-human chorionic gonadotropin (free β-hCG) in twin pregnancies relative to singleton pregnancies and establish improved screening procedure for chromosomal anomalies such as trisomy 21 in twin pregnancies. METHODS A total of 4843...... 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 8.0% to...

  20. The Biogeochemistry of Pu and U: Distribution of Radionuclides Affected by Micro-Organisms and Their Siderophores, Reductants, and Exopolymers

    International Nuclear Information System (INIS)

    Investigations to date focused on studying the dissolution of oxides and desorption of metals by the siderophore, Desferrioxamine B (DFB), with different metal ions adsorbed onto the solids. X-ray absorption spectroscopy (XAS) was used to probe the surface structural environment of sorbed metal ions. Results indicated that while DFB effectively dissolved iron oxides with different adsorbed metals, this process was also affected by the type of the metal adsorbed. For pure hematite, samples with adsorbed metals had less dissolved Fe by DFB than the one without. Different type of metal ion seemed to have no significant effects on Fe dissolution under these experimental conditions. This result suggested that while adsorbed metals blocked available surface sites on hematite surfaces for DFB causing less Fe release, Fe dissolution by DFB from the well crystalline structure of hematite was not affected by the adsorbed metal ions

  1. 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...

  2. Trees in Towns: Factors Affecting the Distribution of Trees in High Density Residential Areas of Greater Manchester

    OpenAIRE

    Hall, Justine Michelle

    2010-01-01

    The distribution of trees across urban areas of the UK has been shown to be uneven, with lower density residential areas containing many more trees and much higher tree cover than areas of higher density housing. However, in Greater Manchester, tree number within high density housing areas also varies substantially. This thesis sought to explore the reasons for this variation in tree cover, whether tree cover should be increased and if so, how. The research investigated a potential cause for ...

  3. 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.

  4. 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. PMID:26603631

  5. Proteolytic activity within Lysosomes and turnover of pinocytic vesicles: a kinetic analysis

    International Nuclear Information System (INIS)

    Degradation of exogenous [125I] ribonuclease by renal lysosomes follows first-order kinetics in ribonuclease concentration. To demonstrate this, it was necessary to apply corrections for the presence of labeled but digestively inactive particles, either pinocytic vesicles or lysosomes damaged during preparation. Such kinetics were not observed under conditions favoring lysosmal breakdown, i.e., in isotonic KCl, or in the absence of EDTA. The kinetic analysis allows determination of half-times for lysosomal protein digestion. This facilitates comparison of different lysosome preparations, or of in vitro degradation rates with results of in vivo metabolism studies. Degradation of [125I] ribonuclease showed a half-time of about 11 and one-half minutes in isotonic sucrose or saline media. This is less than the half-time for decrease of kidney radioactivity in vivo after uptake of [125I] ribonuclease. The proportion of exogenous labeled protein contained within secondary lysosomes was determined as a function of time after injection of ribonuclease to monitor transfer of the protein from pinocytic vesicles to lysosomes. Ribonuclease molecules remained in pinocytic vesicles for approximately three minutes after uptake, before passage into the lysosomes

  6. 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.

  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. Triptolide induces lysosomal-mediated programmed cell death in MCF-7 breast cancer cells

    Directory of Open Access Journals (Sweden)

    Owa C

    2013-09-01

    Full Text Available Chie Owa, Michael E Messina Jr, Reginald HalabyDepartment of Biology, Montclair State University, Montclair, NJ, USABackground: Breast cancer is a major cause of death; in fact, it is the most common type, in order of the number of global deaths, of cancer in women worldwide. This research seeks to investigate how triptolide, an extract from the Chinese herb Tripterygium wilfordii Hook F, induces apoptosis in MCF-7 human breast cancer cells. Accumulating evidence suggests a role for lysosomal proteases in the activation of apoptosis. However, there is also some controversy regarding the direct participation of lysosomal proteases in activation of key apoptosis-related caspases and release of mitochondrial cytochrome c. In the present study, we demonstrate that triptolide induces an atypical, lysosomal-mediated apoptotic cell death in MCF-7 cells because they lack caspase-3.Methods: MCF-7 cell death was characterized via cellular morphology, chromatin condensation, 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide colorimetric cell growth inhibition assay and the expression levels of proapoptotic proteins. Acridine orange and LysoTracker® staining were performed to visualize lysosomes. Lysosomal enzymatic activity was monitored using an acid phosphatase assay and western blotting of cathepsin B protein levels in the cytosolic fraction, which showed increased enzymatic activity in drug-treated cells.Results: These experiments suggest that triptolide-treated MCF-7 cells undergo atypical apoptosis and that, during the early stages, lysosomal enzymes leak into the cytosol, indicating lysosomal membrane permeability.Conclusion: Our results suggest that further studies are warranted to investigate triptolide's potential as an anticancer therapeutic agent.Keywords: triptolide, MCF-7 breast cancer cells, apoptosis, lysosomes, lysosomal membrane permeabilization (LMP

  9. Use of triton-WR-1339 (TWR) for a quantitative determination of the lysosomal binding of transuranium elements in the rat liver

    International Nuclear Information System (INIS)

    The subcellular localisation of 239Pu and 241Am in the rat liver was investigated. A biochemical separation method with modified lysosomes was applied for the first time; the method permitted unique statements on the mitochondrial or lysosomal association of transuranium elements. Lysosomal association of 239Pu and 241Am between days 4 and 6 after radionuclide injection was most easily detected by follow-up treatment with TWR, i.e. the nonionic detergent was applied 2 days after injection of the radionuclide and 4 days before the onset of the analytical procedure. Extracellular transuranium depots could not be proved to exist neither by comparing absolute retention figures in perfused and nonperfused livers nor by subcellular distribution studies. A quantitative relation between the DTPA mobilisation efficiency in the total liver and the subcellular distribution patterns obtained for americium could be estimated for the period between the first and 18th day after radionuclide injection. It suggests on intracellular effect of DTPA. (orig./MG)

  10. 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.

  11. Physico-chemical characteristics affect the spatial distribution of pesticide and transformation product loss to an agricultural brook.

    Science.gov (United States)

    Gassmann, M; Olsson, O; Stamm, C; Weiler, M; Kümmerer, K

    2015-11-01

    Diffuse entry of pesticide residues from agriculture into rivers is spatially unevenly distributed. Therefore, the identification of critical source areas (CSAs) may support water quality management in agricultural catchments. In contrast to former studies, we followed the hypothesis that not only hydrological and topographical characteristics but also physico-chemical properties of pesticide residues have a major influence on their loss to rivers and on corresponding formation of CSAs. We designed a virtual experiment, i.e. a numerical experiment as close as possible to environmental conditions, in a headwater catchment where pronounced spatial differences in hydrological transport processes were identified in the past. 144 scenarios with different combinations of adsorption coefficients (KOC = 10-1000 ml/g) and transformation half-lives (DT50 = 3-60 days) for pesticide parent compounds (PCs) and their transformation products (TPs) were simulated using the catchment-scale spatially distributed reactive transport model ZIN-AgriTra. Export fractions of substances in the virtual experiment ranged from 0.001-15% for pesticides and 0.001-1.8% for TPs. The results of the scenario investigations suggest that more of the calculated export mass variability could be attributed to KOC than to DT50 for both PCs and TPs. CSAs for TPs were spatially more equally distributed in the catchment than for PC export which was likely an effect of changing physico-chemical properties during transformation. The ranking of highest export fields was different between PCs and TPs for most of the investigated scenarios but six fields appeared among the top ten export fields in 95% of the scenarios, which shows the influence of site characteristics such as tile drains or soil properties in the catchment. Thus, the highest export fields were determined by a combination of site characteristics and substance characteristics. Therefore, despite the challenge of widely differing physico

  12. 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

  13. Distribution Of 15N Fertilizer Added To Sandy Soil Under Drip Irrigation System As Affected By Irrigation Frequencies

    International Nuclear Information System (INIS)

    Neutron moisture meter and stable nitrogen isotope (15N) were used to follow horizontal and vertical water movement and N-fertilizer added to soil before and after irrigation. The data indicated that soil moisture distribution and values of total hydraulic potential depend on soil moisture content. Characterization of nitrogen in soil for all sites around the emitter indicated spatial variability with different soil depths due to leaching and volatilization processes. Moreover, water movement and flow direction greatly were characterized by active evaporation depth which was 30 cm.

  14. Oxycodone Induces Overexpression of P-Glycoprotein (ABCB1) and Affects Paclitaxel’s Tissue Distribution in Sprague Dawley Rats

    OpenAIRE

    Hassan, Hazem E.; MYERS, ALAN L.; LEE, INSONG J.; Coop, Andrew; Eddington, Natalie D.

    2007-01-01

    Previous studies suggest that P-glycoprotein (P-gp) modulates the PK/PD of many compounds including opioid agonists and chemotherapeutic agents. The objective of this study was to assess the P-gp affinity status of oxycodone, the P-gp expression, and the paclitaxel’s tissue distribution in oxycodone-treated rats. P-gp ATPase assay, Caco-2 transepithelial permeability studies, and mdr1a/b (−/−) mice were used to assess the P-gp affinity status of oxycodone. P-gp expression was determined by We...

  15. Gradient moduli lens models: how material properties and application of forces can affect deformation and distributions of stress

    Science.gov (United States)

    Wang, Kehao; Venetsanos, Demetrios; Wang, Jian; Pierscionek, Barbara K.

    2016-01-01

    The human lens provides one-third of the ocular focussing power and is responsible for altering focus over a range of distances. This ability, termed accommodation, defines the process by which the lens alters shape to increase or decrease ocular refractive power; this is mediated by the ciliary muscle through the zonule. This ability decreases with age such that around the sixth decade of life it is lost rendering the eye unable to focus on near objects. There are two opponent theories that provide an explanation for the mechanism of accommodation; definitive support for either of these requires investigation. This work aims to elucidate how material properties can affect accommodation using Finite Element models based on interferometric measurements of refractive index. Gradients of moduli are created in three models from representative lenses, aged 16, 35 and 48 years. Different forms of zonular attachments are studied to determine which may most closely mimic the physiological form by comparing stress and displacement fields with simulated shape changes to accommodation in living lenses. The results indicate that for models to mimic accommodation in living eyes, the anterior and posterior parts of the zonule need independent force directions. Choice of material properties affects which theory of accommodation is supported. PMID:27507665

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

    Science.gov (United States)

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

    2016-01-01

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

  17. Biliary copper excretion by hepatocyte lysosomes in the rat. Major excretory pathway in experimental copper overload.

    OpenAIRE

    Gross, J B; Myers, B M; Kost, L J; Kuntz, S M; LaRusso, N F

    1989-01-01

    We investigated the hypothesis that lysosomes are the main source of biliary copper in conditions of hepatic copper overload. We used a rat model of oral copper loading and studied the relationship between the biliary output of copper and lysosomal hydrolases. Male Sprague-Dawley rats were given tap water with or without 0.125% copper acetate for up to 36 wk. Copper loading produced a 23-fold increase in the hepatic copper concentration and a 30-65% increase in hepatic lysosomal enzyme activi...

  18. Expression of the lysosomal-associated membrane protein-1 (LAMP-1) in astrocytomas

    DEFF Research Database (Denmark)

    Jensen, Stine Skov; Christensen, Karina; Aaberg-Jessen, Charlotte;

    aim of this study was to investigate the immunohistochemical expression of LAMP-1, a membrane bound protein in lysosomes, in formalin fixed paraffin embedded tumor tissue from 23 diffuse astrocytomas, 17 anaplastic astrocytomas and 72 glioblastomas. The LAMP-1 expression was scored and compared with......Targeting lysosomes is a novel approach in cancer therapy providing a possible way of killing the otherwise apoptosis-resistant cancer cells. Recent research has thus shown that lysosome targeting compounds induce cell death in a cervix cancer cell line. Tumor stem cells in glioblastomas have...

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

    Science.gov (United States)

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

    2016-01-01

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

  20. [Analysis on factors affecting net primary productivity distribution in Changbai Mountain based on process model for landscape scale].

    Science.gov (United States)

    Zhang, Na; Yu, Guirui; Yu, Zhenliang; Zhao, Shidong

    2003-05-01

    Based on the data received from remote sensing images, the spatial distribution of annual net primary productivity (NPP) was simulated by the process model (EPPML), and the relationships between annual NPP and environmental conditions were analyzed. The results showed that NPP in 1995 was 0.680 kg C.m-2.yr.-1, mostly ranging from 0.105 to 1.241 kg C.m-2.yr.-1, accounting for 82.1%. The highest NPP (1.084 kg C.m-2.yr.-1) appears in mixed broad-leaved and Korean pine forest. Environmental conditions decide the main trend of the spatial distribution of annual NPP (Carbon) in Changbai Mountain. Soil water content had a negative correlativity with NPP, and the correlation coefficient (R) was -0.65. Therefore, water was sufficient for the growth of plants in Changbai Mountain. NPP was highly correlated with LAI (R = 0.81). When LAI was greater than 4-5 m2.m-2, NPP became saturated. NPP was also highly correlated with canopy transpiration (R = 0.77). The response of NPP on environmental conditions, LAI and canopy transpiration in Betula ermanii and broad-leaved forests were different from those in other vegetation. PMID:12924113

  1. A preliminary investigation of the variables affecting the distribution of giant gartersnakes (Thamnophis gigas) in the Sacramento Valley, California

    Science.gov (United States)

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

    2015-01-01

    Giant gartersnakes (Thamnophis gigas) comprise a species of rare, semi-aquatic snake precinctive to the Central Valley of California. Because of the loss of more than 90% of their natural habitat, giant gartersnakes are listed as Threatened by the United States and California endangered species acts. Little is known, however, about the distribution of giant gartersnakes in the Sacramento Valley, which is where most extant populations occur. We conducted detection-nondetection surveys for giant gartersnakes throughout the rice-growing regions of the Sacramento Valley, and used occupancy models to examine evidence for the effects of landscape-scale GIS-derived variables, local habitat and vegetation composition, and prey communities on patterns of giant gartersnake occurrence. Although our results are based on a relatively small sample of sites, we found that distance to historic marsh, relative fish count, and an interaction of distance to historic marsh with proportion of habitat composed of submerged vegetation were important variables for explaining occupancy of giant gartersnakes. In particular, giant gartersnakes were more likely to occur closer to historic marsh and where relatively fewer fish were captured in traps. At locations in or near historic marsh, giant gartersnakes were more likely to occur in areas with less submerged vegetation, but this relationship was reversed (and more uncertain) at sites distant from historic marsh. Additional research with a larger sample of sites would further elucidate the distribution of giant gartersnakes in the Sacramento Valley.

  2. Native defects affecting the Li atom distribution tune the optical emission of ZnO:Li epitaxial thin film

    International Nuclear Information System (INIS)

    It is found that the oxygen vacancy (VO) defect concentration affecting the separation between individual species in LiZn-Lii complex influences the optical emission property of Li0.06Zn0.94O epitaxial thin film grown by pulsed laser deposition. The film grown under low oxygen partial pressure (n-type conductivity)/higher partial pressure (resistive-type) has broad emission at ∼2.99 eV/∼2.1 eV and a narrower emission at 3.63 eV/3.56 eV, respectively. First principle based mBJLDA electronic structure calculation suggests that the emission at 2.99 eV is due to the LiZn-Lii pair complex and the emission at 2.1 eV is when the component species are away from each other

  3. Native defects affecting the Li atom distribution tune the optical emission of ZnO:Li epitaxial thin film

    Science.gov (United States)

    Sahu, R.; Dileep, K.; Loukya, B.; Datta, R.

    2014-02-01

    It is found that the oxygen vacancy (VO) defect concentration affecting the separation between individual species in LiZn-Lii complex influences the optical emission property of Li0.06Zn0.94O epitaxial thin film grown by pulsed laser deposition. The film grown under low oxygen partial pressure (n-type conductivity)/higher partial pressure (resistive-type) has broad emission at ˜2.99 eV/˜2.1 eV and a narrower emission at 3.63 eV/3.56 eV, respectively. First principle based mBJLDA electronic structure calculation suggests that the emission at 2.99 eV is due to the LiZn-Lii pair complex and the emission at 2.1 eV is when the component species are away from each other.

  4. Storage temperature affects distribution of carbon, VFA, ammonia, phosphorus, copper and zinc in raw pig slurry and its separated liquid fraction.

    Science.gov (United States)

    Popovic, Olga; Jensen, Lars Stoumann

    2012-08-01

    Chemical-mechanical separation of pig slurry into a solid fraction rich in dry matter, P, Cu and Zn and a liquid fraction rich in inorganic N but poor in dry matter may allow farmers to manage surplus slurry by exporting the solid fraction to regions with no nutrient surplus. Pig slurry can be applied to arable land only in certain periods during the year, so it is commonly stored prior to field application. This study investigated the effect of storage duration and temperature on chemical characteristics and P, Cu and Zn distribution between particle size classes of raw slurry and its liquid separation fraction. Dry matter, VFA, total N and ammonium content of both slurry products decreased during storage and were affected by temperature, showing higher losses at higher storage temperatures. In both products, total P, Cu and Zn concentrations were not significantly affected by storage duration or temperature. Particle size distribution was affected by slurry separation, storage duration and temperature. In raw slurry, particles larger than 1 mm decreased, whereas particles 250 μm-1 mm increased. The liquid fraction produced was free of particles >500 μm, with the highest proportions of P, Cu and Zn in the smallest particle size class (particles particle size classes followed a similar pattern to dry matter. PMID:22591817

  5. 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.

  6. The Coupled Mars Dust and Water Cycles: Understanding How Clouds Affect the Vertical Distribution and Meridional Transport of Dust and Water.

    Science.gov (United States)

    Kahre, M. A.

    2015-01-01

    The dust and water cycles are crucial to the current Martian climate, and they are coupled through cloud formation. Dust strongly impacts the thermal structure of the atmosphere and thus greatly affects atmospheric circulation, while clouds provide radiative forcing and control the hemispheric exchange of water through the modification of the vertical distributions of water and dust. Recent improvements in the quality and sophistication of both observations and climate models allow for a more comprehensive understanding of how the interaction between the dust and water cycles (through cloud formation) affects the dust and water cycles individually. We focus here on the effects of clouds on the vertical distribution of dust and water, and how those vertical distributions control the net meridional transport of water. For this study, we utilize observations of temperature, dust and water ice from the Mars Climate Sounder (MCS) on the Mars Reconnaissance Orbiter (MRO) combined with the NASA ARC Mars Global Climate Model (MGCM). We demonstrate that the magnitude and nature of the net meridional transport of water between the northern and southern hemispheres during NH summer is sensitive to the vertical structure of the simulated aphelion cloud belt. We further examine how clouds influence the atmospheric thermal structure and thus the vertical structure of the cloud belt. Our goal is to identify and understand the importance of radiative/dynamic feedbacks due to the physical processes involved with cloud formation and evolution on the current climate of Mars.

  7. 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.

  8. Quantum efficiency affected by localized carrier distribution near the V-defect in GaN based quantum well

    International Nuclear Information System (INIS)

    It is known that due to the formation of in-plane local energy barrier, V-defects can screen the carriers which non-radiatively recombine in threading dislocations (TDs) and hence, enhance the internal quantum efficiency in GaN based light-emitting diodes. By a theoretical modeling capable of describing the inhomogeneous carrier distribution near the V-defect in GaN based quantum wells, we show that the efficient suppression of non-radiative (NR) recombination via TD requires the local energy barrier height of V-defect larger than ∼80 meV. The NR process in TD combined with V-defect influences the quantum efficiency mainly in the low injection current density regime suitably described by the linear dependence of carrier density. We provide a simple phenomenological expression for the NR recombination rate based on the model result

  9. Fertilizer 15N Accumulation, Recovery and Distribution in Cotton Plant as Affected by N Rate and Split

    Institute of Scientific and Technical Information of China (English)

    YANG Guo-zheng; CHU Kun-yan; TANG Hao-yue; NIE Yi-chun; ZHANG Xian-long

    2013-01-01

    N fertilization of 300 kg N ha-1 is normally applied to cotton crops in three splits:pre-plant application (PPA, 30%), first bloom application (FBA, 40%) and peak bloom application (PBA, 30%) in the Yangtze River Valley China. However, low fertilizer N plant recovery (NPR) (30-35%) causes problems such as cotton yield stagnation even in higher N rate, low profit margin of cotton production and fertilizer release to the environment. Therefore, it is questioned:Are these three splits the same significance to cotton N uptake and distribution? An outdoor pot trial was conducted with five N rates and 15N labeled urea to determine the recovery and distribution of 15N from different splits in cotton (Gossypium hirsutum L. cv. Huazamian H318) plant. The results showed that, cotton plant absorbed fertilizer 15N during the whole growing period, the majority during flowering for 18-20 d regardless of N rates (150-600 kg ha-1). Fertilizer 15N proportion to the total N accumulated in cotton plant increased with N rates, and it was the highest in reproductive organs (88%averaged across N rates) among all the plant parts. FBA had the highest NPR (70%), the lowest fertilizer N lose (FNL, 19%), and the highest contribution to the fertilizer 15N proportion to the total N (46%) in cotton plant, whereas PPA had the reverse effect. It suggests that FBA should be the most important split for N absorption and yield formation comparatively and allocating more fertilizer N for late application from PPA should improve the benefit from fertilizer.

  10. Roles of CUP-5, the Caenorhabditis elegans orthologue of human TRPML1, in lysosome and gut granule biogenesis

    Directory of Open Access Journals (Sweden)

    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.

  11. Sowing Density: A Neglected Factor Fundamentally Affecting Root Distribution and Biomass Allocation of Field Grown Spring Barley (Hordeum Vulgare L.

    Directory of Open Access Journals (Sweden)

    Vera L Hecht

    2016-06-01

    Full Text Available Studies after 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 top soil 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 two 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 top soil (0-10 cm. Greater sowing density increased specific root length partly through greater production of fine roots in the top soil. Rooting depth 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 SRL and increased 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

  12. 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.

  13. Purification of the lysosomal sialic acid transporter. Functional characteristics of a monocarboxylate transporter

    NARCIS (Netherlands)

    A.C. Havelaar (Adrie); G.M.S. Mancini (Grazia); C.E.M.T. Beerens (Cecile); R.M. Souren; F.W. Verheijen (Frans)

    1998-01-01

    textabstractSialic acid and glucuronic acid are monocarboxylated monosaccharides, which are normally present in sugar side chains of glycoproteins, glycolipids, and glycosaminoglycans. After degradation of these compounds in lysosomes, the free monosaccharides are relea

  14. 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...... 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...... fibroblasts. Blocking lysosomal cysteine proteases with the inhibitor E64d resulted in strong accumulation of collagen IV in lysosomes in wild-type cells, but only very weak intracellular fluorescence accumulation in uPARAP/endo180-deficient fibroblasts. We conclude that uPARAP/endo180 is critical for...

  15. 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...... human CHMP2B mutation brain than in neurodegenerative disease or age-matched control brains. These data suggest that lysosomal storage pathology is the major neuronal pathology in FTD caused by CHMP2B mutation. Recent evidence suggests that two other genes associated with FTD, GRN and TMEM106B are...... important for lysosomal function. Our identification of lysosomal storage pathology in FTD caused by CHMP2B mutation now provides evidence that endolysosomal dysfunction is a major degenerative pathway in FTD....

  16. 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

  17. Lysosomal acid lipase: At the crossroads of normal and atherogenic cholesterol metabolism

    Directory of Open Access Journals (Sweden)

    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.

  18. Updated distribution of Osmoderma eremita in Abruzzo (Italy and agro-pastoral practices affecting its conservation (Coleoptera: Scarabaeidae

    Directory of Open Access Journals (Sweden)

    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.

  19. Less Is More: Substrate Reduction Therapy for Lysosomal Storage Disorders

    Directory of Open Access Journals (Sweden)

    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.

  20. Saposin C-LBPA interaction in late-endosomes/lysosomes

    International Nuclear Information System (INIS)

    Acidic phospholipids and saposins associations are involved in the degradation process of glycosphingolipids/sphingolipids in late endosomes/lysosomes. In this report, we showed the colocalization of saposin C and lysobisphosphatidic acid (LBPA) in human fibroblasts by using cytoimmunofluorescence analysis. This colocalization pattern was not seen with other saposins. Large numbers of saposins A, B, and D illustrated the staining patterns that differ from LBPA. In addition, ingested anti-LBPA antibody altered the location of saposin C in human wild-type fibroblasts. In vitro assays demonstrated that saposin C at nM concentrations induced membrane fusion of LBPA containing phospholipid vesicles. Under the same condition, other saposins had no fusion induction on these vesicles. These results suggested a specific interaction between saposin C and LBPA. Total saposin-deficient fibroblasts showed a massive accumulation of multivesicular bodies (MVBs) by electron microscopic analysis. No significant increase of MVBs was found in saposins A and B deficient cells. Interestingly, the accumulated MVBs were significantly reduced by loading saposin C alone into the total saposin-deficient cells. Therefore, we propose that saposin C-LBPA interaction plays a role in the regulation of MVB formation in cells

  1. Saposin C-LBPA interaction in late-endosomes/lysosomes.

    Science.gov (United States)

    Chu, Zhengtao; Witte, David P; Qi, Xiaoyang

    2005-02-15

    Acidic phospholipids and saposins associations are involved in the degradation process of glycosphingolipids/sphingolipids in late endosomes/lysosomes. In this report, we showed the colocalization of saposin C and lysobisphosphatidic acid (LBPA) in human fibroblasts by using cytoimmunofluorescence analysis. This colocalization pattern was not seen with other saposins. Large numbers of saposins A, B, and D illustrated the staining patterns that differ from LBPA. In addition, ingested anti-LBPA antibody altered the location of saposin C in human wild-type fibroblasts. In vitro assays demonstrated that saposin C at nM concentrations induced membrane fusion of LBPA containing phospholipid vesicles. Under the same condition, other saposins had no fusion induction on these vesicles. These results suggested a specific interaction between saposin C and LBPA. Total saposin-deficient fibroblasts showed a massive accumulation of multivesicular bodies (MVBs) by electron microscopic analysis. No significant increase of MVBs was found in saposins A and B deficient cells. Interestingly, the accumulated MVBs were significantly reduced by loading saposin C alone into the total saposin-deficient cells. Therefore, we propose that saposin C-LBPA interaction plays a role in the regulation of MVB formation in cells. PMID:15652344

  2. Transferrin coupled azanthraquinone enhances the killing effect on trypanosomes. The role of lysosomal mannosidase

    Directory of Open Access Journals (Sweden)

    Nok A.J.

    2002-12-01

    Full Text Available Partially purified azanthraquinone (AQ extract from Mitracarpus scaber was coupled to bovine transferrin (Tf using azidophenyl glyoxal (APG. The AQ-APG-Tf conjugate was found to possess an enhanced in vitro trypanocidal activity against Trypanosoma congolense and T. brucei brucei. At low concentrations of 0.39-90 mg/ml, the conjugate diminished the growth of T. congolense and T. b. brucei dose dependently at the logarithmic phase. Both parasites were more sensitive to AQ-APG-Tf than to the free (AQ extract. Growth inhibition on the parasites by the free extract was observed at 20-200 mg/ml. The total activity of the lysosomal enzyme a-mannosidase was reduced in the T. congolense cells treated with AQ-APG-Tf in a dose related pattern. However, the activity of the mannosidase in the T. b. brucei treated cells is less affected. The AQ-APG-Tf is more effective on a mannosidase than free AQ, eight and four fold for T. congolense and T. b. brucei respectively. The results are discussed as regards the potency of using transferrin as suitable drug carrier in the chemotherapy of Human sleeping sickness.

  3. Lysosomal exocytosis in response to subtle membrane damage following nanosecond pulse exposure

    Science.gov (United States)

    Dalzell, Danielle R.; Roth, Caleb C.; Bernhard, Joshua A.; Payne, Jason A.; Wilmink, Gerald J.; Ibey, Bennett L.

    2011-03-01

    The cellular response to subtle membrane damage following exposure to nanosecond electric pulses (nsEP) is not well understood. Recent work has shown that when cells are exposed to nsEP, ion permeable nanopores ( 2nm) created by longer micro and millisecond duration pulses. Macroscopic damage to a plasma membrane by a micropipette has been shown to cause internal vesicles (lysosomes) to undergo exocytosis to repair membrane damage, a calcium mediated process called lysosomal exocytosis. Formation of large pores in the plasma membrane by electrical pulses has been shown to elicit lysosomal exocytosis in a variety of cell types. Our research objective is to determine whether lysosomal exocytosis will occur in response to nanopores formed by exposure to nsEP. In this paper we used propidium iodide (PI) and Calcium Green-1 AM ester (CaGr) to differentiate between large and small pores formed in CHO-K1 cells following exposure to either 1 or 20, 600-ns duration electrical pulses at 16.2 kV/cm. This information was compared to changes in membrane organization observed by increases in FM1-43 fluorescence, both in the presence and absence of calcium ions in the outside buffer. In addition, we monitored the real time migration of lysosomes within the cell using Cellular Lights assay to tag LAMP-1, a lysosomal membrane protein. Both 1 and 20 pulses elicited a large influx of extracellular calcium, while little PI uptake was observed following a single pulse exposure. Statistically significant increases in FM1-43 fluorescence were seen in samples containing calcium suggesting that calcium-triggered membrane repair may be occurring. Lastly, density of lysosomes within cells, specifically around the nucleus, appeared to change rapidly upon nsEP stimulation suggesting lysosomal migration.

  4. Exosome Secretion Ameliorates Lysosomal Storage of Cholesterol in Niemann-Pick Type C Disease*

    OpenAIRE

    STRAUSS, K; C. GOEBEL; Runz, H.; Mobius, W.; Weiss, S; Feussner, I.; M. Simons; A. Schneider

    2010-01-01

    Niemann-Pick type C1 disease is an autosomal-recessive lysosomal storage disorder. Loss of function of the npc1 gene leads to abnormal accumulation of free cholesterol and sphingolipids within the late endosomal and lysosomal compartments resulting in progressive neurodegeneration and dysmyelination. Here, we show that oligodendroglial cells secrete cholesterol by exosomes when challenged with cholesterol or U18666A, which induces late endosomal cholesterol accumulation. Up-regulation of exos...

  5. Vitamin A-deficiency and its effects on the lysosomal enzymes of fish.

    Science.gov (United States)

    Harikumar, P; Kakati, R; Goswami, U C

    1996-01-01

    The effect of vitamin A-deficiency on the structural integrity of lysosomes in the skeletal muscle and skin of Heteropneustes fossilis, a dehydroretinol-rich freshwater siluroid used in pisciculture, has been evaluated. Dietary stress was found to cause enhanced release of acid hydrolases from both skeletal muscle and skin tissues. The results indicate that the regulation of lysosomal membrane stability in these tissues is a function of vitamin A. PMID:8843982

  6. Analysis of lysosomal membrane proteins exposed to melanin in HeLa cells

    OpenAIRE

    Bang, Seung Hyuck; Park, Dong Jun; Kim, Yang-Hoon; Min, Jiho

    2016-01-01

    Objectives There have been developed to use targeting ability for antimicrobial, anticancerous, gene therapy and cosmetics through analysis of various membrane proteins isolated from cell organelles. Methods It was examined about the lysosomal membrane protein extracted from lysosome isolated from HeLa cell treated by 100 ppm melanin for 24 hours in order to find associated with targeting ability to melanin using by 2-dimensional electrophoresis. Results The result showed 14 up-regulated (1.5...

  7. 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. PMID:27113357

  8. Oxidative Stress and Autophagy in the Regulation of Lysosome-Dependent Neuron Death

    OpenAIRE

    Pivtoraiko, Violetta N.; Stone, Sara L; Roth, Kevin A.; Shacka, John J

    2009-01-01

    Lysosomes critically regulate the pH-dependent catabolism of extracellular and intracellular macromolecules delivered from the endocytic/heterophagy and autophagy pathways, respectively. The importance of lysosomes to cell survival is underscored not only by their unique ability effectively to degrade metalloproteins and oxidatively damaged macromolecules, but also by the distinct potential for induction of both caspase-dependent and -independent cell death with a compromise in the integrity ...

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

    Science.gov (United States)

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

    2005-07-01

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

  10. The HOPS complex mediates autophagosome–lysosome fusion through interaction with syntaxin 17

    OpenAIRE

    Jiang, Peidu; Nishimura, Taki; Sakamaki, Yuriko; Itakura, Eisuke; Hatta, Tomohisa; Natsume, Tohru; Mizushima, Noboru

    2014-01-01

    Membrane fusion is generally controlled by Rabs, soluble N-ethylmaleimide–sensitive factor attachment protein receptors (SNAREs), and tethering complexes. Syntaxin 17 (STX17) was recently identified as the autophagosomal SNARE required for autophagosome–lysosome fusion in mammals and Drosophila. In this study, to better understand the mechanism of autophagosome–lysosome fusion, we searched for STX17-interacting proteins. Immunoprecipitation and mass spectrometry analysis identified vacuolar p...

  11. Activation of macrophages by lymphokines: enhancement of phagosome-lysosome fusion and killing of Coccidioides immitis.

    OpenAIRE

    Beaman, L; Benjamini, E; Pappagianis, D

    1983-01-01

    Previously, it was shown that arthroconidia of Coccidioides immitis appear to inhibit phagosome-lysosome fusion and survive within normal mouse peritoneal macrophages. However, when these macrophages are exposed to antigen-stimulated T lymphocytes from immune mice, activation occurs, leading to enhanced phagosome-lysosome fusion and killing of C. immitis. Results indicate that the activation of macrophages can be effected after incubation with soluble lymphocyte product(s) (lymphokines). The ...

  12. Cryptococcus neoformans-induced macrophage lysosome damage crucially contributes to fungal virulence1

    OpenAIRE

    Davis, Michael J.; Eastman, Alison J.; Qiu, Yafeng; Gregorka, Brian; Kozel, Thomas R.; Osterholzer, John J.; Curtis, Jeffrey L; Swanson, Joel A.; Michal A Olszewski

    2015-01-01

    Upon ingestion by macrophages, Cryptococcus neoformans (Cn) can survive and replicate intracellularly unless the macrophages become classically activated. The mechanism enabling intracellular replication is not fully understood; neither are the mechanisms which allow classical activation to counteract replication. Cn-induced lysosome damage was observed in infected murine bone marrow-derived macrophages, increased with time and required yeast viability. To demonstrate lysosome damage in the i...

  13. EGFRvIII escapes down-regulation due to impaired internalization and sorting to lysosomes

    DEFF Research Database (Denmark)

    Grandal, Michael V; Zandi, Roza; Pedersen, Mikkel W;

    2007-01-01

    . Moreover, internalized EGFRvIII is recycled rather than delivered to lysosomes. EGFRvIII binds the ubiquitin ligase c-Cbl via Grb2, whereas binding via phosphorylated tyrosine residue 1045 seems to be limited. Despite c-Cbl binding, the receptor fails to become effectively ubiquitinylated. Thus, our...... results suggest that the long lifetime of EGFRvIII is caused by inefficient internalization and impaired sorting to lysosomes due to lack of effective ubiquitinylation....

  14. Distinct Lysosome Phenotypes Influence Inflammatory Function in Peritoneal and Bone Marrow-Derived Macrophages

    OpenAIRE

    Kassandra Weber; Schilling, Joel D.

    2014-01-01

    Lysosomes play a critical role in the degradation of both extracellular and intracellular material. These dynamic organelles also contribute to nutrient sensing and cell signaling pathways. Macrophages represent a heterogeneous group of phagocytic cells that contribute to tissue homeostasis and inflammation. Recently, there has been a renewed interest in understanding the role of macrophage autophagy and lysosome function in health and disease. Thioglycollate-elicited peritoneal and bone marr...

  15. Effects of ethanol and protein deficiency on pancreatic digestive and lysosomal enzymes.

    OpenAIRE

    Apte, M V; Wilson, J. S.; Korsten, M A; McCaughan, G W; Haber, P S; Pirola, R. C.

    1995-01-01

    The pathogenesis of alcoholic pancreatitis is not fully understood. An increase in pancreatic digestive and lysosomal enzyme synthesis because of ethanol consumption could contribute to the development of pancreatic injury in alcoholics. This study aimed, firstly, to determine the effect of ethanol on the content and messenger RNA levels of pancreatic digestive enzymes and on the messenger RNA level of the lysosomal enzyme cathepsin B, and secondly, to examine the influence of concomitant pro...

  16. Comparative study on lysosomal accumulation of 67Ga and 111In in Morris hepatoma 7316A

    International Nuclear Information System (INIS)

    Intracellular localization of 67Ga and 111In was investigated in Morris hepatoma 7316A and in normal Buffalo rat liver cells by a cell fractionation method at 48 hr after an intraperitoneal injection of the nuclides. Lysosomal fractions of the tumor and normal liver cells had the highest relative specific radioactivities of the nuclides (p 67Ga (p 67Ga seemed to indicate that 67Ga determines lysosomal functions of tumor cells more precisely than 111In

  17. Extracellular Acidification Alters Lysosomal Trafficking in Human Breast Cancer Cells1

    OpenAIRE

    Glunde, Kristine; Sandra E. Guggino; Solaiyappan, Meiyappan; Pathak, Arvind P.; Ichikawa, Yoshitaka; Bhujwalla, Zaver M.

    2003-01-01

    Cancer cells invade by secreting degradative enzymes, which are sequestered in lysosomal vesicles. In this study, the impact of an acidic extracellular environment on lysosome size, number, and distance from the nucleus in human mammary epithelial cells (HMECs) and breast cancer cells of different degrees of malignancy was characterized because the physiological microenvironment of tumors is frequently characterized by extracellular acidity. An acidic extracellular pH (pHe) resulted in a dist...

  18. Situation of classical swine fever and the epidemiologic and ecologic aspects affecting its distribution in the American continent.

    Science.gov (United States)

    Vargas Terán, Moisés; Calcagno Ferrat, Nelson; Lubroth, Juan

    2004-10-01

    Classical swine fever (CSF) is a viral transboundary animal disease that is highly contagious among domestic and wild pigs, such as boars and peccaries. Today, far from being what was classically described historically, the disease is characterized as having a varied clinical picture, and its diagnosis depends on resorting to proper sample collection and prompt dispatch to a laboratory that can employ several techniques to obtain a definitive diagnosis. Laboratory findings should be complemented with a field analysis of the occurrence of disease to have a better understanding of its epidemiology. The disease is still present in various regions and countries of Latin America and the Caribbean, thus hindering production, trade, and the livestock economy in the region. Consequently, it is among the diseases included in List A of the Office International des Epizooties (OIE). Currently, there are epidemiologic and ecologic aspects that characterize its geographical distribution in the region such as: continued trends in the demand for pork and pork products; an increase in swine investment with low production costs which are able to compete advantageously in international markets; the convention of associating CSF in the syndrome of "swine hemorrhagic diseases" owing to the historical description of its acute presentation and not to the new and more frequent subacute presentations or the diseases with which it may be confused (notably, porcine reproductive and respiratory syndrome and porcine dermopathic nephropathy syndrome, among others); dissemination of the virus through asymptomatic hosts such as piglets infected in utero; frequent lack of quality control and registration of vaccines and vaccinations; feeding of swine with contaminated food waste (swill); the common practice of smuggling animals and by-products across borders; the backyard family production system or extensive open field methods of swine rearing with minimal input in care and feeding; poor

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

    Science.gov (United States)

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

    2014-01-01

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

  20. An efficient ratiometric fluorescent probe for tracking dynamic changes in lysosomal pH.

    Science.gov (United States)

    Wang, Qianqian; Zhou, Liyi; Qiu, Liping; Lu, Danqing; Wu, Yongxiang; Zhang, Xiao-Bing

    2015-08-21

    Lysosomes are acidic organelles (approximately pH 4.5-5.5) and tracking the changes in lysosomal pH is of great biological importance. To address this issue, quite a few of fluorescent probes have been developed. However, few of these probes can realize the tracking of dynamic changes in lysosomal pH. Herein, we report a new lysosome-targeted ratiometric fluorescent probe (FR-Lys) by hybridizing morpholine with a xanthane derivative and an o-hydroxy benzoxazole group. In this probe, the morpholine group serves as a targeting unit for lysosome, the xanthane derivative exhibits a pH-modulated open/close reaction of the spirocycle, while the o-hydroxy benzoxazole moiety shows a pH modulated excited-state intramolecular proton transfer (ESIPT) process. Such a design affords the probe a ratiometric fluorescence response towards pH with pH values ranging from 4.0 to 6.3. The response of the probe to pH was fast and reversible with high selectivity. Moreover, this probe possesses further advantages such as easy synthesis, high photostability and low cytotoxicity. These features are favorable for tracking dynamic pH changes in biosystems. It was then applied for dynamic imaging pH changes in lysosomes with satisfactory results. PMID:26107774

  1. N-Glycan Branching Affects the Subcellular Distribution of and Inhibition of Matriptase by HAI-2/Placental Bikunin.

    Directory of Open Access Journals (Sweden)

    Ying-Jung J Lai

    Full Text Available The gene product of SPINT 2, that encodes a transmembrane, Kunitz-type serine protease inhibitor independently designated as HAI-2 or placenta bikunin (PB, is involved in regulation of sodium absorption in human gastrointestinal track. Here, we show that SPINT 2 is expressed as two species of different size (30-40- versus 25-kDa due to different N-glycans on Asn-57. The N-glycan on 25-kDa HAI-2 appears to be of the oligomannose type and that on 30-40-kDa HAI-2 to be of complex type with extensive terminal N-acetylglucosamine branching. The two different types of N-glycan differentially mask two epitopes on HAI-2 polypeptide, recognized by two different HAI-2 mAbs. The 30-40-kDa form may be mature HAI-2, and is primarily localized in vesicles/granules. The 25-kDa form is likely immature HAI-2, that remains in the endoplasmic reticulum (ER in the perinuclear regions of mammary epithelial cells. The two different N-glycans could, therefore, represent different maturation stages of N-glycosylation with the 25-kDa likely a precursor of the 30-40-kDa HAI-2, with the ratio of their levels roughly similar among a variety of cells. In breast cancer cells, a significant amount of the 30-40-kDa HAI-2 can translocate to and inhibit matriptase on the cell surface, followed by shedding of the matriptase-HAI-2 complex. The 25-kDa HAI-2 appears to have also exited the ER/Golgi, being localized at the cytoplasmic face of the plasma membrane of breast cancer cells. While the 25-kDa HAI-2 was also detected at the extracellular face of plasma membrane at very low levels it appears to have no role in matriptase inhibition probably due to its paucity on the cell surface. Our study reveals that N-glycan branching regulates HAI-2 through different subcellular distribution and subsequently access to different target proteases.

  2. The Abnormality of Lysosomal Iron Metabolism and Kidney Injury%溶酶体铁代谢异常与肾脏损伤

    Institute of Scientific and Technical Information of China (English)

    许碧华; 许琛; 王丽姝; 刘华锋; 刘伟敬; 梁东

    2013-01-01

    The lysosomal compartment is essential for a variety of cellular functions. Degradation of iron-containing macromolecules results in a large amount of low mass iron which exists in redox-active Fe(II) form in lysosomes. Fe(II) is known to catalyze Fenton reactions that may jeopardize lysosomal membrane integrity during oxidative stress. This results in the release of hydrolases and redox-active iron into the cytosol with ensuing damage or cell death. Besides,autophagy act as a crucial role in iron metabolism. Adversely affecting lysosomal iron handling can contribute to a variety of kidney diseases.%  溶酶体的完整性对维持细胞正常的代谢非常重要。溶酶体降解含铁的物质会使其内存在大量低质量铁。这些铁主要以还原型Fe(Ⅱ)形式存在并能诱导芬顿反应的发生,导致溶酶体膜完整性破坏。溶酶体膜透性增加会使一系列水解酶和还原铁进入胞浆进而诱导细胞损伤或死亡。此外,自噬在溶酶体铁代谢中也起着至关重要的作用。溶酶体铁处理的异常在一系列肾脏疾病中发挥着重要作用。

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

    International Nuclear Information System (INIS)

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

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

    Energy Technology Data Exchange (ETDEWEB)

    Magini, Alessandro [Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Perugia (Italy); Department of Medical and Biological Sciences (DSMB), University of Udine, Udine (Italy); Polchi, Alice; Urbanelli, Lorena [Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Perugia (Italy); Cesselli, Daniela; Beltrami, Antonio [Department of Medical and Biological Sciences (DSMB), University of Udine, Udine (Italy); Tancini, Brunella [Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Perugia (Italy); Emiliani, Carla, E-mail: carla.emiliani@unipg.it [Department of Experimental Medicine and Biochemical Sciences, University of Perugia, Perugia (Italy)

    2013-10-18

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

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

    Science.gov (United States)

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

    2016-02-19

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

  6. Nuclear distribution and chromatin association of DNA polymerase α-primase is affected by TEV protease cleavage of Cdc23 (Mcm10 in fission yeast

    Directory of Open Access Journals (Sweden)

    Gregan Juraj

    2005-06-01

    Full Text Available Abstract Background Cdc23/Mcm10 is required for the initiation and elongation steps of DNA replication but its biochemical function is unclear. Here, we probe its function using a novel approach in fission yeast, involving Cdc23 cleavage by the TEV protease. Results Insertion of a TEV protease cleavage site into Cdc23 allows in vivo removal of the C-terminal 170 aa of the protein by TEV protease induction, resulting in an S phase arrest. This C-terminal fragment of Cdc23 is not retained in the nucleus after cleavage, showing that it lacks a nuclear localization signal and ability to bind to chromatin. Using an in situ chromatin binding procedure we have determined how the S phase chromatin association of DNA polymerase α-primase and the GINS (Sld5-Psf1-Psf2-Psf3 complex is affected by Cdc23 inactivation. The chromatin binding and sub-nuclear distribution of DNA primase catalytic subunit (Spp1 is affected by Cdc23 cleavage and also by inactivation of Cdc23 using a degron allele, implying that DNA polymerase α-primase function is dependent on Cdc23. In contrast to the effect on Spp1, the chromatin association of the Psf2 subunit of the GINS complex is not affected by Cdc23 inactivation. Conclusion An important function of Cdc23 in the elongation step of DNA replication may be to assist in the docking of DNA polymerase α-primase to chromatin.

  7. Er3+ infrared fluorescence affected by spatial distribution synchronicity of Ba2+ and Er3+ in Er3+-doped BaO–SiO2 glasses

    Directory of Open Access Journals (Sweden)

    Atsunobu Masuno

    2016-02-01

    Full Text Available Glasses with the composition xBaO–(99.9 − xSiO2–0.1ErO3/2 (0 ≤x ≤ 34.9 were fabricated by a levitation technique. The glasses in the immiscibility region were opaque due to chemical inhomogeneity, while the other glasses were colorless and transparent. The scanning electron microscope observations and electron probe microanalysis scan profiles revealed that more Er3+ ions were preferentially distributed in the regions where more Ba2+ ions existed in the chemically inhomogeneous glasses. The synchronicity of the spatial distributions of the two ions initially increased with increasing x and then decreased when the Ba2+ concentration exceeded a certain value. The peak shape and lifetime of the fluorescence at 1.55 μm depended on x as well as the spatial distribution of both ions. These results indicate that although ErOn polyhedra are preferentially coordinated with Ba2+ ions and their local structure is affected by the coordination of Ba2+, there is a maximum in the amount of Ba2+ ions that can coordinate ErOn polyhedra since the available space for Ba2+ ions is limited. These findings provide us with efficient ways to design the chemical composition of glasses with superior Er3+ fluorescence properties for optical communication network systems.

  8. 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. PMID:26401639

  9. Evaluation of Geostatistical Techniques for Mapping Spatial Distribution of Soil PH, Salinity and Plant Cover Affected by Environmental Factors in Southern Iran

    Directory of Open Access Journals (Sweden)

    Mohammad ZARE-MEHRJARDI

    2010-12-01

    Full Text Available The study presented in this paper attempts to evaluate some interpolation techniques for mapping spatial distribution of soil pH, salinity and plant cover in Hormozgan province, Iran. The relationships among environmental factors and distribution of vegetation types were also investigated. Plot sampling was applied in the study area. Landform parameters of each plot were recorded and canopy cover percentages of each species were measured while stoniness and browsing damage were estimated. Results indicated that there was a significant difference in vegetation cover for high and low slope steepness. Also, vegetation cover was greater than other cases in the mountains with calcareous lithology. In general, there were no significant relationships among vegetation cover and soil properties such as pH, EC, and texture. Other soil properties, such as soil depth and gravel percentage were significantly affected by vegetation cover. Moreover, the geostatistical results showed that kriging and cokriging methods were better than inverse distance weighting (IDW method for prediction of the spatial distribution of soil properties. Also, the results indicated that all the concerned soil and plant parameters were better determined by means of a cokriging method. Land elevation, which was highly correlated with studied parameters, was used as an auxiliary parameter.

  10. 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

    2016-01-01

    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

  11. Lack of lysosomal fusion with phagosomes containing Ehrlichia risticii in P388D1 cells: abrogation of inhibition with oxytetracycline.

    OpenAIRE

    Wells, M Y; Rikihisa, Y

    1988-01-01

    Fusion of lysosomes with phagosomes containing Ehrlichia risticii, an obligate intracellular parasite, was evaluated in P388D1 murine macrophagelike cells. Lysosomes in cells ranging in infectivity from 30 to 70% were labeled cytochemically with acid phosphatase or via endocytosis of thorium dioxide or cationized ferritin to document phagosome-lysosome (P-L) fusion in untreated cells and cells treated with oxytetracycline. Regardless of the marker used, P-L fusion was generally not observed i...

  12. Protective effect of squalene on certain lysosomal hydrolases and free amino acids in isoprenaline-induced myocardial infarction in rats

    DEFF Research Database (Denmark)

    Farvin, Sabeena; Surendraraj, A.; Anandan, R.

    2010-01-01

    This study was aimed to evaluate the preventive role of squalene on free amino acids and lysosomal alterations in experimentally induced myocardial infarction in rats. The levels of lysosomal enzymes (beta-glucuronidase, beta-galactosidase, beta-glucosidase, acid phosphatase and cathepsin D) in...... plasma and lysosomal fractions, hydroxyproline content and free amino acids in heart tissue were determined. Isoprenaline administration to rats resulted in decreased stability of the membranes which was reflected by significantly (p...

  13. Lysosome fusion to the cell membrane is mediated by the dysferlin C2A domain in coronary arterial endothelial cells

    OpenAIRE

    Han, Wei-Qing; Xia, Min; Xu, Ming; Krishna M Boini; Ritter, Joseph K.; Li, Ning-Jun; Li, Pin-Lan

    2012-01-01

    Dysferlin has recently been reported to participate in cell membrane repair in muscle and other cells through lysosome fusion. Given that lysosome fusion is a crucial mechanism that leads to membrane raft clustering, the present study attempted to determine whether dysferlin is involved in this process and its related signalling, and explores the mechanism underlying dysferlin-mediated lysosome fusion in bovine coronary arterial endothelial cells (CAECs). We found that dysferlin is clustered ...

  14. Study of Se subcellular distribution in human tissues by neutron activation analysis

    International Nuclear Information System (INIS)

    Subcellular distribution of selenium in certain important human tissues is studied by neutron activation analysis combined with differential centrifugation method. The standard reference materials, bovine liver NIST 1577a and horse kidney IAEA H8, are used to validate the analytical quality. The experimental results show that the slenium levels in various individuals are significantly different. Body selenium is highly enriched in human kidney. Different subcellular selenium distribution patterns are also found in different tissues. The selenium levels in various organelles are sequenced as mitochondria >nuclei>cytosol>lysosome, microsome in liver; nuclei>mitochondria>cytosol>lysosome, microsome in kidney; and mitochondria, nuclei>cytosol, lysosome, microsome in heart

  15. 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.

  16. Burn-induced stimulation of lysosomal enzyme synthesis in skeletal muscle

    International Nuclear Information System (INIS)

    A localized burn injury to a rat hindlimb results in atrophy of soleus muscle (in the absence of cellular damage) which is attributable to an increase in muscle protein breakdown. Previous work has shown that lysosomal enzyme activities (cathepsins B, H, L, and D) are elevated in muscle from the burned leg by 50% to 100%. There is no change in endogenous neutral protease activity (+/- Ca++). The increase in protease activity can not be attributed to changes in endogenous protease inhibitors. The latency [(Triton X100 treated - control)/triton treated] of lysosomal enzymes is approximately 50% and is not altered by burn injury. The rate of sucrose uptake is also not altered by burn. These experiments suggest that the rate of substrate supply to the lysosomal apparatus via endocytosis or autophagocytosis is not altered by burn. When muscles are preincubated with 3H-phenylalanine or 3H-mannose burn increased incorporation into protein of the fraction containing lysosomes by 100%. Preincubation in the presence of tunicamycin (an inhibitor of glycoprotein synthesis) inhibited incorporation of both labels into a microsomal fraction of the muscle from the burned leg, but has little effect on incorporation in the control muscle. These findings are consistent with the hypothesis that the burn-induced increase in protein breakdown is caused by an increase in lysosomal protease synthesis

  17. TNFα Post-Translationally Targets ZnT2 to Accumulate Zinc in Lysosomes.

    Science.gov (United States)

    Hennigar, Stephen R; Kelleher, Shannon L

    2015-10-01

    Mammary epithelial cells undergo widespread lysosomal-mediated cell death (LCD) during early mammary gland involution. Recently, we demonstrated that tumor necrosis factor-α (TNFα), a cytokine released during early involution, redistributes the zinc (Zn) transporter ZnT2 to accumulate Zn in lysosomes and activate LCD and involution. The objective of this study is to determine how TNFα retargets ZnT2 to lysosomes. We tested the hypothesis that TNFα signaling dephosphorylates ZnT2 to uncover a highly conserved dileucine motif (L294L) in the C-terminus of ZnT2, allowing adaptor protein complex-3 (AP-3) to bind and traffic ZnT2 to lysosomes. Confocal micrographs showed that TNFα redistributed wild-type (WT) ZnT2 from late endosomes (Pearson's coefficient = 0.202 ± 0.05 and 0.097 ± 0.03; Pwomen with variation in the C-terminus of ZnT2 may be at risk for inadequate involution and breast disease due the inability to traffic ZnT2 to lysosomes. PMID:25808614

  18. Lysosomes and apoptosis%溶酶体与细胞凋亡

    Institute of Scientific and Technical Information of China (English)

    赵凯; 卫涛涛

    2011-01-01

    在特定条件下,包括活性氧、鞘氨醇、细胞凋亡效应因子Bax等在内的多种刺激因子均可诱发溶酶体膜通透,之后溶酶体内含的蛋白酶(如组织蛋白酶等)及其他水解酶从溶酶体释放至胞浆中,通过剪切效应分子、激活包括凋亡酶在内的其他水解酶而启动细胞凋亡程序的执行.简要概括了引发溶酶体膜通透的可能机制及溶酶体参与细胞凋亡的主要途径.%In certain conditions, lysosomal membrane permeabilization could be induced by a broad array of stimuli including reactive oxygen species (ROS), sphingosine, and some endogenous cell death effector proteins such as Bax. As a consequence of LMP, lysosomal proteases (such as cathepsins) and other hydrolases were released from the lysosomal lumen to the cytosol, where they lead to apoptosis by the activation apoptotic cascades. This review describes the possible molecular mechanisms underlying the occurrence of lysosomal membrane permeabilization and the consequent lysosome-mediated apoptosis.

  19. Cathepsin B launches an apoptotic exit effort upon cell death-associated disruption of lysosomes.

    Science.gov (United States)

    de Castro, M A G; Bunt, G; Wouters, F S

    2016-01-01

    The release of cathepsin proteases from disrupted lysosomes results in lethal cellular autodigestion. Lysosomal disruption-related cell death is highly variable, showing both apoptotic and necrotic outcomes. As the substrate spectrum of lysosomal proteases encompasses the apoptosis-regulating proteins of the Bcl-2 family, their degradation could influence the cell death outcome upon lysosomal disruption. We used Förster resonance energy transfer (FRET)-based biosensors to image the real-time degradation of the Bcl-2-family members, Bcl-xl, Bax and Bid, in living cells undergoing lysosomal lysis and identified an early chain of proteolytic events, initiated by the release of cathepsin B, which directs cells toward apoptosis. In this apoptotic exit strategy, cathepsin B's proteolytic activity results in apoptosis-inducing Bid and removes apoptosis-preventing Bcl-xl. Cathepsin B furthermore appears to degrade a cystein protease that would otherwise have eliminated apoptosis-supporting Bax, indirectly keeping cellular levels of the Bax protein up. The concerted effort of these three early events shifts the balance of cell fate away from necrosis and toward apoptosis. PMID:27551506

  20. 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.

  1. Cytochemical localisation of lysosomal enzymes and acidic mucopolysaccharides in the salivary glands of Aplysia depilans (Opisthobranchia).

    Science.gov (United States)

    Lobo-da-Cunha, A

    2002-04-01

    Three types of secretory cells were reported in the salivary glands of Aplysia depilans: granular cells, vacuolated cells and mucocytes. To improve the characterisation of these cells, cytochemical methods for the detection of lysosomal enzymes and acidic mucopolysaccharides were applied. In granular cells, acid phosphatase and arylsulphatase were present in small lysosomes and in some secretory granules. The secretory granules could have received these enzymes after fusion with the small lysosomes that were frequently found very close to them. These cells were not stained with colloidal iron because they do not contain acidic mucopolysaccharides. In vacuolated cells, acid phosphatase and arylsulphatase were detected in lysosomes but not in the secretory vacuoles. Colloidal iron staining revealed the presence of acidic mucopolysaccharides in the vacuoles and in the Golgi apparatus of these cells. In mucocytes, lysosomes were very rare, but the secretion of these cells was very rich in acidic mucopolysaccharides. The filamentous network within the secretory vesicles was completely covered with iron particles, but practically no particles were observed over the granular masses attached to the membrane of the vesicles. Iron particles were also found in the trans-face cisternae of the U-shaped Golgi stacks, but were not seen in the cis-face cisternae or in the rough endoplasmic reticulum. PMID:12117284

  2. Low-dose, continuous enzyme replacement therapy ameliorates brain pathology in the neurodegenerative lysosomal disorder mucopolysaccharidosis type IIIA.

    Science.gov (United States)

    King, Barbara; Hassiotis, Sofia; Rozaklis, Tina; Beard, Helen; Trim, Paul J; Snel, Marten F; Hopwood, John J; Hemsley, Kim M

    2016-05-01

    Repeated replacement of sulphamidase via cerebrospinal fluid injection is an effective treatment for pathological changes in the brain in mice and dogs with the lysosomal storage disorder, mucopolysaccharidosis type IIIA (MPS IIIA). Investigational trials of this approach are underway in children with this condition, however, infusions require attendance at a specialist medical facility. We sought to comprehensively evaluate the effectiveness of sustained-release (osmotic pump-delivered) enzyme replacement therapy in murine MPS IIIA as this method, if applied to humans, would require only subcutaneous administration of enzyme once the pump was installed. Six-week-old MPS IIIA and unaffected mice were implanted with subcutaneous mini-osmotic pumps connected to an infusion cannula directed at the right lateral ventricle. Either recombinant human sulphamidase or vehicle were infused over the course of 7 weeks, with pumps replaced part-way through the experimental period. We observed near-normalisation of primarily stored substrate (heparan sulphate) in both hemispheres of the MPS IIIA brain and cervical spinal cord, as determined using tandem mass spectrometry. Immunohistochemistry indicated a reduction in secondarily stored GM 3 ganglioside and neuroinflammatory markers. A bias towards the infusion side was seen in some, but not all outcomes. The recombinant enzyme appears stable under pump-like conditions for at least 1 month. Given that infusion pumps are in clinical use in other nervous system disorders, e.g. for treatment of spasticity or brain tumours, this treatment method warrants consideration for testing in large animal models of MPS IIIA and other lysosomal storage disorders that affect the brain. Clinical trials of repeated injection of replacement enzyme into CSF are underway in patients with the inherited neurodegenerative disorder mucopolysaccharidosis type IIIA. In this pre-clinical study, we examined an alternative approach - slow, continual

  3. 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.

  4. 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. PMID:20504381

  5. Elimination of paternal mitochondria through the lysosomal degradation pathway in C. elegans.

    Science.gov (United States)

    Zhou, Qinghua; Li, Haimin; Xue, Ding

    2011-12-01

    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 mitochondria 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. PMID:22105480

  6. 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.

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

    International Nuclear Information System (INIS)

    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 GM2AP, and the soluble hydrolases cathepsin D, cathepsin H, and acid sphingomyelinase. Sortilin belongs to a growing family of receptors with homology to the yeast Vps10 protein, which acts as a lysosomal sorting receptor for carboxypeptidase Y. In this study we examined the effects of the sortilin gene inactivation in mice. The inactivation of this gene did not yield any noticeable lysosomal pathology. To determine the existence of an alternative receptor complementing the sorting function of sortilin, we quantified the concentration of prosaposin in the lysosomes of the nonciliated epithelial cells lining the efferent ducts. These cells were chosen because they express sortilin and have a large number of lysosomes containing prosaposin. In addition, the nonciliated cells are known to endocytose luminal prosaposin that is synthesized and secreted by Sertoli cells into the seminiferous luminal fluids. Consequently, the nonciliated cells are capable of targeting both exogenous and endogenous prosaposin to the lysosomes. Using electron microscope immunogold labeling and quantitative analysis, our results demonstrate that inactivation of the sortilin gene produces a significant decrease of prosaposin in the lysosomes. When luminal prosaposin was excluded from the efferent ducts, the level of prosaposin in lysosomes was even lower in the mutant mice. Nonetheless, a significant amount of prosaposin continues to reach the lysosomal compartment. These results strongly suggest the existence of an alternative receptor that complements the function of sortilin and explains the lack of lysosomal storage disorders in the sortilin-deficient mice.

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

    Science.gov (United States)

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

    2014-02-26

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

  9. 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

  10. A dual-site two-photon fluorescent probe for visualizing lysosomes and tracking lysosomal hydrogen sulfide with two different sets of fluorescence signals in the living cells and mouse liver tissues.

    Science.gov (United States)

    Liu, Yong; Meng, Fangfang; He, Longwei; Liu, Keyin; Lin, Weiying

    2016-05-19

    Herein, we have developed a novel dual-site two-photon fluorescent probe as the first paradigm of the probes, which can concurrently report lysosomes and lysosomal H2S with two different sets of fluorescence signals in the living cells and tissues. PMID:27159054

  11. An adenosine triphosphate-dependent calcium uptake pump in human neutrophil lysosomes.

    OpenAIRE

    Klemper, M S

    1985-01-01

    Regulation of the cytosolic free calcium concentration is important to neutrophil function. In these studies, an ATP-dependent calcium uptake pump has been identified in human neutrophil lysosomes. This energy-dependent Ca++ uptake pump has a high affinity for Ca++ (Michaelis constant [Km] Ca++ = 107 nM) and a maximum velocity (Vmax) of 5.3 pmol/mg of protein per min. ATP was the only nucleotide that supported Ca++ uptake by lysosomes. The Km for ATP was 177 microM. ATP-dependent Ca++ uptake ...

  12. Three-layer poly(methyl methacrylate) microsystem for analysis of lysosomal enzymes for diagnostic purposes

    DEFF Research Database (Denmark)

    Kwapiszewski, Radoslaw; Kwapiszewska, Karina; Kutter, Jörg P;

    2015-01-01

    Lysosomal storage diseases are chronic, progressive and typically have a devastating impact on the patient and the family. The diagnosis of these diseases is still a challenge, however, even for trained specialists. Accurate diagnostic methods and high-throughput tools that could be readily...... incorporated into existing screening laboratories are urgently required. We propose a new method for measuring the activity of lysosomal enzymes using a microfluidic device. The principle of the method is the fluorometric determination of a protonated form of 4-methylumbelliferone directly in the enzymatic...

  13. Inhibition of Endosome-Lysosome System Acidification Enhances Porcine Circovirus 2 Infection of Porcine Epithelial Cells▿

    OpenAIRE

    Misinzo, Gerald; Delputte, Peter; Nauwynck, Hans

    2007-01-01

    Recently, Misinzo et al. (G. Misinzo, P. Meerts, M. Bublot, J. Mast, H. M. Weingartl, and H. J. Nauwynck, J. Gen. Virol. 86:2057-2068, 2005) reported that inhibiting endosome-lysosome system acidification reduced porcine circovirus 2 (PCV2) infection of monocytic 3D4/31 cells. The present study examined the effect of inhibiting endosome-lysosome system acidification in epithelial cells, since epithelial cells support PCV2 infection in vivo and are used in culturing PCV2 in vitro. Ammonium chl...

  14. Cocaine induces a mixed lysosomal lipidosis in cultured fibroblasts, by inactivation of acid sphingomyelinase and inhibition of phospholipase A1

    International Nuclear Information System (INIS)

    This paper reports that cocaine may induce a lysosomal storage disorder. Indeed, culture of Rat-1 fibroblasts with 250-500 μM cocaine induced after 2-3 days a major accumulation in lysosomes of electron-dense lamellar structures. By subcellular fractionation, this was reflected by a selective decrease of the buoyant density of several lysosomal enzymes, indicating lysosomal lipid overload. Biochemical analysis confirmed an increased cellular content of major phospholipids and sphingomyelin, but not of cholesterol. Cocaine, a membrane-permeant weak base, is concentrated by acidotropic sequestration, because its accumulation was abrogated by the proton ionophore, monensin and the vacuolar ATPase inhibitor, bafilomycin A1. At its estimated lysosomal concentration, cocaine almost completely inhibited phospholipase A1 activity on liposomes. Cell incubation with cocaine, but not with its inactive metabolite, benzoylecgonine, rapidly inactivated acid sphingomyelinase, as reflected by a 10-fold decrease in Vmax with identical Km. Acid sphingomyelinase inactivation was fully prevented by the thiol proteinases inhibitors, leupeptin and E64, indicating that cocaine induces selective sphingomyelinase proteolysis. Upon cocaine removal, acid sphingomyelinase activity was rapidly restored, pointing to its fast turnover. In contrast, the cellular content of several other lysosomal hydrolases was increased up to 2-fold. Together, these data show that acidotropic accumulation of cocaine in lysosomes rapidly inhibits acid phospholipase A1 and inactivates acid sphingomyelinase, which can explain induction of a mixed lysosomal lipidosis

  15. The influence of the type of sulphate bond and degree of sulphation of glycosaminoglycans on their interaction with lysosomal enzymes.

    Science.gov (United States)

    Avila, J L

    1978-01-01

    Significant differences occur between the interaction of several sulphated glycosaminoglycans with a particular lysosomal protein, leading to inhibition in the case of lysosomal enzymes. The order of strength of inhibition at pH4 was: heparin greater than chondroitin 4-sulphate = chondroitin 6-sulphate greater than dermatan sulphate. PMID:656058

  16. Distribution of polychlorinated biphenyls in an urban riparian zone affected by wastewater treatment plant effluent and the transfer to terrestrial compartment by invertebrates

    International Nuclear Information System (INIS)

    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 δ13C 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−2⋅y−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 terrestrial

  17. 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.

  18. 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

  19. Lysosomally cleavable peptide-containing polymersomes modified with anti-EGFR antibody for systemic cancer chemotherapy.

    Science.gov (United States)

    Lee, Jung S; Groothuis, Tom; Cusan, Claudia; Mink, Daniel; Feijen, Jan

    2011-12-01

    Polymersomes (Ps) based on a biodegradable and biocompatible block copolymer of methoxy poly(ethylene glycol) (mPEG) and poly(D,L-lactide) (PDLLA) in which apeptide sequence, Gly-Phe-Leu-Gly-Phe (GFLGF), was introduced in between the two blocks(mPEG-pep-PDLLA) were developed. The peptide linker is cleavable by the lysosomal enzymecathepsin B (Cath B). Ps containing the peptide linker (Ps(pep)) with an average diameter of about 124 nm were prepared by injecting a THF solution of the block copolymer into DI water. The Ps had a membrane thickness of about 15 nm as determined by transmission electron microscopy (TEM). In order to investigate the enzymatic degradation of the Ps (pep), dynamic light scattering (DLS) measurements of Ps(pep) dispersions with different concentrations of Cath B at pH 5.5 and 7.4 were performed as a function of time. A gradual decrease in kilo counts per second (Kcps) of the Ps (pep) over 7 d was observed after incubation of the Ps (pep) dispersions with 5 units/ml of Cath B at pH 5.5 at 37 °C. The size distribution became also bimodal, indicating that aggregation and precipitation of Ps (pep) occurred by disintegration of the Ps (pep) as a result of cleavage of the peptide. The rate of disintegration of the Ps (pep) was depending on the concentration of Cath Band the pH. No changes by DLS were seen when the dispersions were incubated with the enzyme at pH 7.4. Acridine orange (AO) was encapsulated in Ps (pep)as a model drug and rapid release of AO triggered by Cath B degradation of Ps (pep) was observed at pH 5.5. Anti-epidermal growth factor receptor (anti-EGFR) antibody (abEGFR) was immobilized on the surface of Ps(pep)in order to enhance the cellular uptake of Ps (pep). Fluorescein isothiocyanate labeled dextran (40,000 g/mol) (FD40) was incorporated in the Ps (pep) for the cell study and Ps either without peptide or antibody or without both peptide and antibody were used as negative controls. After 3 d exposure to SKBR3 cells, ab

  20. 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.

  1. Rotenone causing dysfunctional mitochondria and lysosomes in cerebral ganglions of Lumbricus terrestris degenerate giant fibers and neuromuscular junctions.

    Science.gov (United States)

    Subaraja, Mamangam; Vanisree, Arambakkam Janardhanam

    2016-06-01

    Rotenone is well-documented to cause neurodegenerative condition such as Parkinson's, in the exposed systems. However, its detrimental effect on particular sites of neuronal pathway is still under investigation. We aimed at elucidating the impact of rotenone on cerebral ganglions (CG) of Lumbricus terrestris which control movement and behaviour of the worms. Worms were exposed to 0-0.4 ppm/mL of rotenone. Mitochondrial and lysosomal integrities were found to be affected beyond 0.2 ppm/mL of rotenone. Activities of cholinergic enzymes and the expression of tyrosine hydroxylase showed an impaired neuronal transmission in CGs at the dose of 0.2 ppm/mL of rotenone. Histopathological and immunoflourescent analyses showed neuronal apoptosis, reduced nucleic acid content and inhibited of neurosecretion at 0.3 ppm/mL. Electron microscopy showed that the neurons and neuromuscular junctions were affected at 0.2 ppm/mL. Dose-dependent changes were also observed in the motor function such as burrowing behaviours and locomotion. Conduction velocity (CV) and locomotion assessment showed that the CV of lateral giant fiber (LGF) was reduced while that of MGF remains unaffected at 0.2 ppm, the dose at which the burrowing behaviour was also not affected. LGF, cholinergic enzymes and tyrosine hydroxylase are primarily targeted by rotenone affecting locomotion at 0.2 ppm/mL while MGF, neuropile and the burrowing behaviour were affected at 0.3 ppm/mL. We demonstrate, in addition to dose-dependent effects, that the bioaccumulation factors range 0.28-0.32 ppm/μg of rotenone cause degenerative impact on giant fibers affecting neuronal behaviors/locomotion of worms. We also propose worms for studying mechanisms of neuronal pathology caused by chemicals prevailing in earth's atmosphere. PMID:27003369

  2. Environmental factors affecting the distribution of land snails in the Atlantic Rain Forest of Ilha Grande, Angra dos Reis, RJ, Brazil.

    Science.gov (United States)

    Nunes, G K M; Santos, S B

    2012-02-01

    The distribution and abundance of terrestrial molluscs are affected by environmental factors, but data are lacking for Brazilian land snails. The aim of this study was to understand the relationship between measured environmental factors and the land-snail species composition of two hillsides covered with Atlantic Rain Forest on Ilha Grande. On each hillside, five plots located at 100 m intervals between 100 to 500 m asl were chosen. Each plot was sampled by carrying out timed searches and collecting and sorting litter samples from ten quadrats of 25 × 75 cm. A range of environmental data was measured for each of the quadrats in a plot. A Cluster Analysis was carried out for the richness and abundance data. The environmental variables were analysed using a Pearson Correlation Matrix and Discriminant Analysis. Our results show that the two mountains are similar in species richness, but species composition and abundance are different, probably reflecting observed differences in environmental conditions. The environmental factors associated with compositional variation between the two mountains were: atmospheric temperature, soil temperature, litter depth, and relative air humidity. Distinct luminosity and canopy closure conditions were related to the composition of the land-snail community of one hillside. PMID:22437388

  3. Distribution of polychlorinated biphenyls in an urban riparian zone affected by wastewater treatment plant effluent and the transfer to terrestrial compartment by invertebrates.

    Science.gov (United States)

    Yu, Junchao; Wang, Thanh; Han, Shanlong; Wang, Pu; Zhang, Qinghua; Jiang, Guibin

    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(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(-2)⋅y(-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. PMID:23811358

  4. 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.

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

    OpenAIRE

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

    2010-01-01

    Photodynamic therapy (PDT) with lysosome-targeted photosensitizers induces the intrinsic pathway of apoptosis via the cleavage and activation of the BH3-only protein Bid by proteolytic enzymes released from photo-disrupted lysosomes. To investigate the role of Bid in apoptosis induction and the role of damaged lysosomes on cell killing by lysosome-targeted PDT, we compared the responses of wild type and Bid-knock-out murine embryonic fibroblasts toward a mitochondrion/endoplasmic reticulum-bi...

  6. uPARAP/endo180 directs lysosomal delivery and degradation of collagen IV

    DEFF Research Database (Denmark)

    Kjøller, Lars; Engelholm, Lars H; Høyer-Hansen, Maria; Danø, Keld; Bugge, Thomas H; Behrendt, Niels

    2004-01-01

    fibroblasts. Blocking lysosomal cysteine proteases with the inhibitor E64d resulted in strong accumulation of collagen IV inlysosomes in wild-type cells, but only very weak intracellular fluorescence accumulation in uPARAP/endo180-deficient fibroblasts. We conclude that uPARAP/endo180 is critical for targeted...

  7. Gallium and Functionalized-Porphyrins Combine to Form Potential Lysosome-Specific Multimodal Bioprobes.

    Science.gov (United States)

    Pan, Jie; Harriss, Bethany I; Chan, Chi-Fai; Jiang, Lijun; Tsoi, Tik-Hung; Long, Nicholas J; Wong, Wing-Tak; Wong, Wai-Kwok; Wong, Ka-Leung

    2016-07-18

    A water-soluble bimetallic normal ("cold") and radiochemical ("hot") gallium-porphyrin-ruthenium-bipyridine complex (GaporRu-1) has been synthesized by microwave methodology in short reaction times with good (>85%) yields. (68)GaporRu-1 is demonstrated to be a potential multimodal and functional bioprobe for positron emission tomography (PET), lysosome specific optical imaging, and photodynamic therapy. PMID:27355871

  8. Prion infection impairs lysosomal degradation capacity by interfering with rab7 membrane attachment in neuronal cells.

    Science.gov (United States)

    Shim, Su Yeon; Karri, Srinivasarao; Law, Sampson; Schatzl, Hermann M; Gilch, Sabine

    2016-01-01

    Prions are proteinaceous infectious particles which cause fatal neurodegenerative disorders in humans and animals. They consist of a mostly β-sheeted aggregated isoform (PrP(Sc)) of the cellular prion protein (PrP(c)). Prions replicate autocatalytically in neurons and other cell types by inducing conformational conversion of PrP(c) into PrP(Sc). Within neurons, PrP(Sc) accumulates at the plasma membrane and in vesicles of the endocytic pathway. To better understand the mechanisms underlying neuronal dysfunction and death it is critical to know the impact of PrP(Sc) accumulation on cellular pathways. We have investigated the effects of prion infection on endo-lysosomal transport. Our study demonstrates that prion infection interferes with rab7 membrane association. Consequently, lysosomal maturation and degradation are impaired. Our findings indicate a mechanism induced by prion infection that supports stable prion replication. We suggest modulation of endo-lysosomal vesicle trafficking and enhancement of lysosomal maturation as novel targets for the treatment of prion diseases. PMID:26865414

  9. 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.

  10. (-)-Oleocanthal rapidly and selectively induces cancer cell death via lysosomal membrane permeabilization

    Science.gov (United States)

    LeGendre, Onica; Breslin, Paul AS; Foster, David A

    2015-01-01

    (-)-Oleocanthal (OC), a phenolic compound present in extra-virgin olive oil (EVOO), has been implicated in the health benefits associated with diets rich in EVOO. We investigated the effect of OC on human cancer cell lines in culture and found that OC induced cell death in all cancer cells examined as rapidly as 30 minutes after treatment in the absence of serum. OC treatment of non-transformed cells suppressed their proliferation but did not cause cell death. OC induced both primary necrotic and apoptotic cell death via induction of lysosomal membrane permeabilization (LMP). We provide evidence that OC promotes LMP by inhibiting acid sphingomyelinase (ASM) activity, which destabilizes the interaction between proteins required for lysosomal membrane stability. The data presented here indicate that cancer cells, which tend to have fragile lysosomal membranes compared to non-cancerous cells, are susceptible to cell death induced by lysosomotropic agents. Therefore, targeting lysosomal membrane stability represents a novel approach for the induction of cancer-specific cell death. PMID:26380379

  11. 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......, 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...

  12. Lysosome triggered near-infrared fluorescence imaging of cellular trafficking processes in real time.

    Science.gov (United States)

    Grossi, Marco; Morgunova, Marina; Cheung, Shane; Scholz, Dimitri; Conroy, Emer; Terrile, Marta; Panarella, Angela; Simpson, Jeremy C; Gallagher, William M; O'Shea, Donal F

    2016-01-01

    Bioresponsive NIR-fluorophores offer the possibility for continual visualization of dynamic cellular processes with added potential for direct translation to in vivo imaging. Here we show the design, synthesis and lysosome-responsive emission properties of a new NIR fluorophore. The NIR fluorescent probe design differs from typical amine functionalized lysosomotropic stains with off/on fluorescence switching controlled by a reversible phenol/phenolate interconversion. Emission from the probe is shown to be highly selective for the lysosomes in co-imaging experiments using a HeLa cell line expressing the lysosomal-associated membrane protein 1 fused to green fluorescent protein. The responsive probe is capable of real-time continuous imaging of fundamental cellular processes such as endocytosis, lysosomal trafficking and efflux in 3D and 4D. The advantage of the NIR emission allows for direct translation to in vivo tumour imaging, which is successfully demonstrated using an MDA-MB-231 subcutaneous tumour model. This bioresponsive NIR fluorophore offers significant potential for use in live cellular and in vivo imaging, for which currently there is a deficit of suitable molecular fluorescent tools. PMID:26927507

  13. Osteoclasts derive from hematopoietic stem cells according to marker, giant lysosomes of beige mice

    International Nuclear Information System (INIS)

    To ascertain the origin of multinucleated osteoclasts from hematopoietic stem cells, giant lysosomes peculiar to cells of beige mice (bg bg) were used as marker cells of that provenance. Radiation chimeras were established reciprocally between bg bg mice and osteopetrotic mi mi mice with defective osteoclasts. As a result, all the derivative cells of the hematopoietic stem cell would depend on the donor's cell line, whereas osteogenesis would remain the province of the host. It was affirmed in the chimeras mi mi/bg bg that the osteopetrosis was cured within six weeks. Thereafter the definitive osteoclasts of the chimeras contained giant lysosomes attributable to the beige cell line. However, the cure was well advanced before donor osteoclasts were prominent, for which several reasons are offered. In the mouse chimeras, bg bg/mi mi, there was a delay of some six weeks before osteopetrosis became evident, histologically before radiologically, at the major metaphyseal growth centers. During the period one to two months after establishment, osteoclasts appeared to be a mixture of two cell lines according to quantitative assessments for giant lysosomes. Assessments consisted of measurements of the percentage area of osteoclasts occupied by lysosomes over 1 micrometer diameter. The means were 0.018% +/- 0.008% for nonbeige stock and 2.09% +/- 0.58% for beige stock

  14. Inhibition of lysosomal protease cathepsin D reduces renal fibrosis in murine chronic kidney disease.

    Science.gov (United States)

    Fox, Christopher; Cocchiaro, Pasquale; Oakley, Fiona; Howarth, Rachel; Callaghan, Krystena; Leslie, Jack; Luli, Saimir; Wood, Katrina M; Genovese, Federica; Sheerin, Neil S; Moles, Anna

    2016-01-01

    During chronic kidney disease (CKD) there is a dysregulation of extracellular matrix (ECM) homeostasis leading to renal fibrosis. Lysosomal proteases such as cathepsins (Cts) regulate this process in other organs, however, their role in CKD is still unknown. Here we describe a novel role for cathepsins in CKD. CtsD and B were located in distal and proximal tubular cells respectively in human disease. Administration of CtsD (Pepstatin A) but not B inhibitor (Ca074-Me), in two mouse CKD models, UUO and chronic ischemia reperfusion injury, led to a reduction in fibrosis. No changes in collagen transcription or myofibroblasts numbers were observed. Pepstatin A administration resulted in increased extracellular urokinase and collagen degradation. In vitro and in vivo administration of chloroquine, an endo/lysosomal inhibitor, mimicked Pepstatin A effect on renal fibrosis. Therefore, we propose a mechanism by which CtsD inhibition leads to increased collagenolytic activity due to an impairment in lysosomal recycling. This results in increased extracellular activity of enzymes such as urokinase, triggering a proteolytic cascade, which culminates in more ECM degradation. Taken together these results suggest that inhibition of lysosomal proteases, such as CtsD, could be a new therapeutic approach to reduce renal fibrosis and slow progression of CKD. PMID:26831567

  15. Dietary protein deficiency reduces lysosomal and nonlysosomal ATP-dependent proteolysis in muscle

    Science.gov (United States)

    Tawa, N. E. Jr; Kettelhut, I. C.; Goldberg, A. L.

    1992-01-01

    When rats are fed a protein deficient (PD) diet for 7 days, rates of proteolysis in skeletal muscle decrease by 40-50% (N. E. Tawa, Jr., and A. L. Goldberg. Am. J. Physiol. 263 (Endocrinol. Metab. 26): E317-325, 1992). To identify the underlying biochemical adaptations, we measured different proteolytic processes in incubated muscles. The capacity for intralysosomal proteolysis, as shown by sensitivity to methylamine or lysosomal protease inhibitors, fell 55-75% in muscles from PD rats. Furthermore, extracts of muscles of PD rats showed 30-70% lower activity of many lysosomal proteases, including cathepsins B, H, and C, and carboxypeptidases A and C, as well as other lysosomal hydrolases. The fall in cathepsin B and proteolysis was evident by 3 days on the PD diet, and both returned to control levels 3 days after refeeding of the normal diet. In muscles maintained under optimal conditions, 80-90% of protein breakdown occurs by nonlysosomal pathways. In muscles of PD rats, this ATP-dependent process was also 40-60% slower. Even though overall proteolysis decreased in muscles of PD rats, their capacity for Ca(2+)-dependent proteolysis increased (by 66%), as did the activity of the calpains (+150-250%). Thus the lysosomal and the ATP-dependent processes decrease coordinately and contribute to the fall in muscle proteolysis in PD animals.

  16. Matrix protein 2 of influenza A virus blocks autophagosome fusion with lysosomes

    DEFF Research Database (Denmark)

    Gannagé, Monique; Dormann, Dorothee; Albrecht, Randy;

    2009-01-01

    demonstrate that influenza A virus inhibits macroautophagy, a cellular process known to be manipulated by diverse pathogens. Influenza A virus infection causes accumulation of autophagosomes by blocking their fusion with lysosomes, and one viral protein, matrix protein 2, is necessary and sufficient for this...

  17. 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.

  18. Lysosome vs. mitochondrion as photosensitizer binding site: how does the tortoise overtake the hare?

    Science.gov (United States)

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

    2008-02-01

    Pc 4, a photosensitizer first synthesized at Case Western Reserve University and now in clinical trial at University Hospitals Case Medical Center, has been shown to bind preferentially and with high affinity to mitochondrial and endoplasmic reticulum membranes. Upon photoirradiation of Pc 4-loaded cells, membrane components, especially the anti-apoptotic protein Bcl-2, are photodamaged. Apoptosis, as indicated by activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase, is triggered by the initial photodamage. A series of analogues of Pc 4 has been synthesized containing two axial ligands, one identical to the single ligand of Pc 4 and the other either the same as the Pc 4 ligand or bearing one or more hydroxyl groups. The hydroxyl-bearing axial ligands reduce the aggregation of the Pc in polar environments and direct the Pc's to lysosomes. Photoirradiation of cells that have taken up these Pc's into their lysosomes is 4-6 times more efficient at killing cells, as defined by loss of clonogenicity, than with Pc 4. Whereas PDT with Pc 4 photodamages Bcl-2 and Bcl-xL over the same dose response range as for cell killing, PDT with Pc 181 or the other lysosome-localizing Pc's causes much less photodamage to Bcl-2 relative to cell killing. Furthermore, in the case of the lysosome-bound Pc's, little or no caspase-3-dependent apoptosis is observed.

  19. Lysosome triggered near-infrared fluorescence imaging of cellular trafficking processes in real time

    Science.gov (United States)

    Grossi, Marco; Morgunova, Marina; Cheung, Shane; Scholz, Dimitri; Conroy, Emer; Terrile, Marta; Panarella, Angela; Simpson, Jeremy C.; Gallagher, William M.; O'Shea, Donal F.

    2016-01-01

    Bioresponsive NIR-fluorophores offer the possibility for continual visualization of dynamic cellular processes with added potential for direct translation to in vivo imaging. Here we show the design, synthesis and lysosome-responsive emission properties of a new NIR fluorophore. The NIR fluorescent probe design differs from typical amine functionalized lysosomotropic stains with off/on fluorescence switching controlled by a reversible phenol/phenolate interconversion. Emission from the probe is shown to be highly selective for the lysosomes in co-imaging experiments using a HeLa cell line expressing the lysosomal-associated membrane protein 1 fused to green fluorescent protein. The responsive probe is capable of real-time continuous imaging of fundamental cellular processes such as endocytosis, lysosomal trafficking and efflux in 3D and 4D. The advantage of the NIR emission allows for direct translation to in vivo tumour imaging, which is successfully demonstrated using an MDA-MB-231 subcutaneous tumour model. This bioresponsive NIR fluorophore offers significant potential for use in live cellular and in vivo imaging, for which currently there is a deficit of suitable molecular fluorescent tools. PMID:26927507

  20. Sorting Nexin 11 Regulates Lysosomal Degradation of Plasma Membrane TRPV3.

    Science.gov (United States)

    Li, Caiyue; Ma, Wenbo; Yin, Shikui; Liang, Xin; Shu, Xiaodong; Pei, Duanqing; Egan, Terrance M; Huang, Jufang; Pan, Aihua; Li, Zhiyuan

    2016-05-01

    The trafficking of ion channels to/from the plasma membrane is considered an important mechanism for cellular activity and an interesting approach for disease therapies. The transient receptor potential vanilloid 3 (TRPV3) ion channel is widely expressed in skin keratinocytes, and its trafficking mechanism to/from the plasma membrane is unknown. Here, we report that the vesicular trafficking protein sorting nexin 11 (SNX11) downregulates the level of the TRPV3 plasma membrane protein. Overexpression of SNX11 causes a decrease in the level of TRPV3 current and TRPV3 plasma membrane protein in TRPV3-transfected HEK293T cells. Subcellular localizations and western blots indicate that SNX11 interacts with TRPV3 and targets it to lysosomes for degradation, which is blocked by the lysosomal inhibitors chloroquine and leupeptin. Both TRPV3 and SNX11 are highly expressed in HaCaT cells. We show that TRPV3 agonists-activated Ca(2+) influxes and the level of native TRPV3 total protein in HaCaT cells are decreased by overexpression of SNX11 and increased by knockdown of SNX11. Our findings reveal that SNX11 promotes the trafficking of TRPV3 from the plasma membrane to lysosomes for degradation via protein-protein interactions, which demonstrates a previously unknown function of SNX11 as a regulator of TRPV3 trafficking from the plasma membrane to lysosomes. PMID:26818531

  1. Melanoma cell lysosome secretory burst neutralizes the CTL-mediated cytotoxicity at the lytic synapse

    Science.gov (United States)

    Khazen, Roxana; Müller, Sabina; Gaudenzio, Nicolas; Espinosa, Eric; Puissegur, Marie-Pierre; Valitutti, Salvatore

    2016-01-01

    Human melanoma cells express various tumour antigens that are recognized by CD8+ cytotoxic T lymphocytes (CTLs) and elicit tumour-specific responses in vivo. However, natural and therapeutically enhanced CTL responses in melanoma patients are of limited efficacy. The mechanisms underlying CTL effector phase failure when facing melanomas are still largely elusive. Here we show that, on conjugation with CTL, human melanoma cells undergo an active late endosome/lysosome trafficking, which is intensified at the lytic synapse and is paralleled by cathepsin-mediated perforin degradation and deficient granzyme B penetration. Abortion of SNAP-23-dependent lysosomal trafficking, pH perturbation or impairment of lysosomal proteolytic activity restores susceptibility to CTL attack. Inside the arsenal of melanoma cell strategies to escape immune surveillance, we identify a self-defence mechanism based on exacerbated lysosome secretion and perforin degradation at the lytic synapse. Interfering with this synaptic self-defence mechanism might be useful in potentiating CTL-mediated therapies in melanoma patients. PMID:26940455

  2. 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. PMID:26907692

  3. Digestion of thyroglobulin with purified thyroid lysosomes: preferential release of iodoamino acids

    International Nuclear Information System (INIS)

    [131I]Thyroglobulin [( 131I]Tg), prepared by either enzymatic iodination of human goiter Tg in vitro or isolation from the thyroids of rats previously injected with 131I, was digested with a solubilized enzyme mixture prepared from purified hog thyroid lysosomes. The digestion was performed at 37 C for 24 h under nitrogen at pH 5.0 in the presence of 4 mM dithiothreitol. Under these conditions the release of free [131I] iodoamino acids (MIT, DIT, T4, and T3) was quantitatively very similar to that observed with a standard pronase digestion procedure. To determine whether other amino acids in Tg were released as quantitatively as the iodoamino acids, free amino acids in the lysosomal digest were measured, and total free amino acid release was compared with a similar analysis performed after digestion of [131I]Tg with 6 N HCl. Total amino acid release was much less complete than iodoamino acid release, indicating preferential release of iodoamino acids from Tg by lysosomal digestion. Analysis of the lysosomal digest by HPLC on a size exclusion column indicated that Tg was degraded to peptides with a mol wt less than 4000. Assuming that the in vitro lysosomal digestion system represents a valid model for the physiological proteolytic system that degrades Tg, the results of the present study suggest that a substantial portion of the Tg in the thyroid is not degraded to free amino acids and that peptide fragments of Tg are normally present in the thyroid. In such a case, the fate and possible physiological activity of these fragments require further elucidation

  4. 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. PMID:24880782

  5. The GATOR2 Component Wdr24 Regulates TORC1 Activity and Lysosome Function

    Science.gov (United States)

    Cai, Weili; Wei, Youheng; Jarnik, Michal; Reich, John; Lilly, Mary A.

    2016-01-01

    TORC1 is a master regulator of metabolism in eukaryotes that responds to multiple upstream signaling pathways. The GATOR complex is a newly defined upstream regulator of TORC1 that contains two sub-complexes, GATOR1, which inhibits TORC1 activity in response to amino acid starvation and GATOR2, which opposes the activity of GATOR1. While the GATOR1 complex has been implicated in a wide array of human pathologies including cancer and hereditary forms of epilepsy, the in vivo relevance of the GATOR2 complex remains poorly understood in metazoans. Here we define the in vivo role of the GATOR2 component Wdr24 in Drosophila. Using a combination of genetic, biochemical, and cell biological techniques we demonstrate that Wdr24 has both TORC1 dependent and independent functions in the regulation of cellular metabolism. Through the characterization of a null allele, we show that Wdr24 is a critical effector of the GATOR2 complex that promotes the robust activation of TORC1 and cellular growth in a broad array of Drosophila tissues. Additionally, epistasis analysis between wdr24 and genes that encode components of the GATOR1 complex revealed that Wdr24 has a second critical function, the TORC1 independent regulation of lysosome dynamics and autophagic flux. Notably, we find that two additional members of the GATOR2 complex, Mio and Seh1, also have a TORC1 independent role in the regulation of lysosome function. These findings represent a surprising and previously unrecognized function of GATOR2 complex components in the regulation of lysosomes. Consistent with our findings in Drosophila, through the characterization of a wdr24-/- knockout HeLa cell line we determined that Wdr24 promotes lysosome acidification and autophagic flux in mammalian cells. Taken together our data support the model that Wdr24 is a key effector of the GATOR2 complex, required for both TORC1 activation and the TORC1 independent regulation of lysosomes. PMID:27166823

  6. Secretory Lysosomes and Diseases%分泌型溶酶体与疾病

    Institute of Scientific and Technical Information of China (English)

    王俊伟; 周逸蒋; 竺可青

    2011-01-01

    The classical lysosomes contain a variety of hydrolytic enzymes and lipase to decompose proteins and membrane structures and considered as the cellular terminus digestive organelle. The secretory lysosomes are dicovered in certain cell types with the functions of both intracellular digestion and regulatory exocytosis. Rab27a plays a central role in the regulation of the exocytosis of lysosomal proteins. The exocytosis-related gene mutations, particularly in the regulatory proteins, can cause a variety of immune deficiency phenotypes. The ATP released from the astrocytes lysosomes can be used as the transmission signal between neurons and glial cells. The secretory lysosomes also get involved in the invasion and metastasis of cancer cells.%传统意义的溶酶体被认为是细胞内消化途径的终点站,是含有多种水解酶和脂肪酶的细胞器,可以消化蛋白以及膜结构等.某些特殊类型的细胞中存在分泌型溶酶体,它既有胞内消化的功能,又有调节分泌功能.在调节溶酶体胞吐的蛋白质中,Rab27a蛋白起到了核心作用.相关基因特别是控制胞吐的基因突变,可造成各种免疫缺陷综合症.星型胶质细胞溶酶体胞吐释放ATP,在神经元和胶质细胞之间传递信息.分泌型溶酶体还参与了肿瘤细胞浸润、转移的过程.

  7. 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.

  8. Occurrence, distribution, and potential affecting factors of organophosphate flame retardants in sewage sludge of wastewater treatment plants in Henan Province, Central China.

    Science.gov (United States)

    Pang, Long; Yuan, Yiting; He, Han; Liang, Kang; Zhang, Hongzhong; Zhao, Jihong

    2016-06-01

    Organophosphate esters (OPEs) are widely used as flame retardants. In this study, the occurrence and distribution of six OPEs were investigated in sewage sludge from 24 wastewater treatment plants (WWTPs) in 18 cities of Henan province, Central China. The results indicated that all target OPEs were detected in the sludge samples with the detection rate of 95.8%, except tris(dichloropropyl)phosphate (TDCP). The total concentration of the six OPEs ranged from 38.6 to 508 μg kg(-1). Tris(2-chloroethyl)phosphate (TCEP), tris(2-butoxyethyl)phosphate (TBEP), and tris(2-chloroiso-propyl)phosphate (TCPP) were found to be predominant, with concentrations ranging from 2.50 to 203, 1.60 to 383, and 6.70-161 μg kg(-1), respectively. The potential factors affecting OPE levels in sewage sludge, such as wastewater source, sludge characteristics, operational conditions, treatment techniques, and total organic carbon (TOC) of sludge in WWTPs were investigated. The results indicated that the total concentration of OPEs in sewage sludge has no significant relationship with the individual parameters (p > 0.05). However, significant correlations were found between triphenyl phosphate (TPhP) level and treatment capacity (R = 0.484, p < 0.05), processing volume (R = 0.495, p < 0.05), and serving population (R = 0.591, p < 0.05). Furthermore, the relationship between treatment techniques and the total concentration of OPEs in sewage sludge was also investigated in this study, and the results illustrated that the levels of OPEs in sludge were independent of the solid retention time (SRT). PMID:26974479

  9. Spatial Distribution of Magnetic Properties and Selected Heavy Metals in Calcareous Soils as Affected by Land Use in the Isfahan Region, Central Iran

    Institute of Scientific and Technical Information of China (English)

    Z. DANKOUB; S. AYOUBI; H. KHADEMI; LU Sheng-Gao

    2012-01-01

    Anthropogenic activities have caused the accumulation of heavy metals in the soil environment.Pollution of the soils significantly reduces environmental quality and affects human health. In many recent studies,magnetic susceptibility measurements have been used for pollution monitoring.The objective of this research was to determine the spatial variability of magnetic properties and selected heavy metals and the effect of land use on their variability in the surface soils of the Isfahan region,Central Iran.A total of 158 composite surface (0-5 cm) samples of calcareous soils were collected from an area of about 700 km2,located along a cross-border transect from Isfahan City to a steel plant,covering urban,industrial,agricultural,and uncultivated land uses. Concentrations of copper (Cu),zinc (Zn),lead (Pb),manganese (Mn),iron (Fe),nickel (Ni),chromium (Cr),and cobalt (Co) and magnetic parameters,magnetic susceptibility at low frequency (xlf),natural remanent magnetization (NRM),saturation isothermal remanent magnetization (SIRM),and isothermal remanent magnetization at the field of 100 mT (IRM100mT) and the backfield of 100 mT (IRM-100mT),were measured in all the soil samples.Results showed that magnetic susceptibility in the urban and industrial land topsoils (0-5 cm) samples was significantly higher than that in the agricultural and uncultivated land soils in the study area.Concentrations of Cu,Zn,Pb,Mn,and Fe were positively correlated with magnetic properties (xlf,IRM100mT,SIRM,IRM-100mT,and NRM),which could be attributed to their inputs from traffic emissions and industrial activities at the study sites.Ni and Cr concentrations showed significant negative correlations with magnetic properties.No significant correlation was found between Co concentration and magnetic parameters.The Tomlinson pollution load index (PLI) showed significant correlation with the magnetic properties (xlf,IRM100mT,SIRM,IRM-100mT,and NRM).The spatial distribution of the selected heavy

  10. NLRP3 inflammasome signaling is activated by low-level lysosome disruption but inhibited by extensive lysosome disruption: roles for K+ efflux and Ca2+ influx.

    Science.gov (United States)

    Katsnelson, Michael A; Lozada-Soto, Kristen M; Russo, Hana M; Miller, Barbara A; Dubyak, George R

    2016-07-01

    Nucleotide-binding domain, leucine-rich-repeat-containing family, pyrin domain-containing 3 (NLRP3) is a cytosolic protein that nucleates assembly of inflammasome signaling platforms, which facilitate caspase-1-mediated IL-1β release and other inflammatory responses in myeloid leukocytes. NLRP3 inflammasomes are assembled in response to multiple pathogen- or environmental stress-induced changes in basic cell physiology, including the destabilization of lysosome integrity and activation of K(+)-permeable channels/transporters in the plasma membrane (PM). However, the quantitative relationships between lysosome membrane permeabilization (LMP), induction of increased PM K(+) permeability, and activation of NLRP3 signaling are incompletely characterized. We used Leu-Leu-O-methyl ester (LLME), a soluble lysosomotropic agent, to quantitatively track the kinetics and extent of LMP in relation to NLRP3 inflammasome signaling responses (ASC oligomerization, caspase-1 activation, IL-1β release) and PM cation fluxes in murine bone marrow-derived dendritic cells (BMDCs). Treatment of BMDCs with submillimolar (≤1 mM) LLME induced slower and partial increases in LMP that correlated with robust NLRP3 inflammasome activation and K(+) efflux. In contrast, supramillimolar (≥2 mM) LLME elicited extremely rapid and complete collapse of lysosome integrity that was correlated with suppression of inflammasome signaling. Supramillimolar LLME also induced dominant negative effects on inflammasome activation by the canonical NLRP3 agonist nigericin; this inhibition correlated with an increase in NLRP3 ubiquitination. LMP elicited rapid BMDC death by both inflammasome-dependent pyroptosis and inflammasome-independent necrosis. LMP also triggered Ca(2+) influx, which attenuated LLME-stimulated NLRP3 inflammasome signaling but potentiated LLME-induced necrosis. Taken together, these studies reveal a previously unappreciated signaling network that defines the coupling between LMP, changes

  11. Mice Doubly-Deficient in Lysosomal Hexosaminidase A and Neuraminidase 4 Show Epileptic Crises and Rapid Neuronal Loss

    OpenAIRE

    Volkan Seyrantepe; Pablo Lema; Aurore Caqueret; Larbi Dridi; Samar Bel Hadj; Stephane Carpentier; Francine Boucher; Thierry Levade; Lionel Carmant; Gravel, Roy A; Edith Hamel; Pascal Vachon; Graziella Di Cristo; 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 α-subunit of lysosomal β-hexosaminidase A, which converts G(M2) to G(M3) ganglioside. Hexa(-/-) mice, depleted of β-hexosaminidase A, remain asymptomatic to 1 year of age, because they catabolise G(M2) ganglioside via a lysosomal sialidase into glycolipid G(A2), which is further processed by β-hexosaminidase B to lactosyl-ceramide, thereby bypassing the β-hexosaminidase A defect. Since this by...

  12. Silymarin and vitamin E reduce amiodarone-induced lysosomal phospholipidosis in rats

    International Nuclear Information System (INIS)

    Several antioxidants have been shown to reduce lysosomal phospholipidosis, which is a potential mechanism of amiodarone toxicity, and prevent amiodarone toxicity by antioxidant and/or non-antioxidant mechanisms. The aim of this study was to test whether the co-administration of two structurally different antioxidants vitamin E and silymarin with amiodarone can reduce amiodarone-induced lysosomal phospholipidosis, and if yes, by reducing the tissue concentration of amiodarone and desethylamiodarone or by their antioxidant action. To this end, male Fischer 344 rats were treated by gavage once a day for 3 weeks and randomly assigned to the following four experimental groups: 1, control; 2, amiodarone (150 mg/(kg per day)); 3, amiodarone (150 mg/(kg per day)) plus vitamin E (100 mg/(kg per day)); 4, amiodarone (150 mg/(kg per day)) plus silymarin (60 mg/(kg per day)) treated groups. Total plasma phospholipid (PL), liver-conjugated diene, thiobarbituric acid reactive substances (TBARSs), amiodarone and desethylamiodarone concentrations were determined and the extent of lysosomal phospholipidosis in the liver was estimated by a semi-quantitative electron microscopic method. Amiodarone treatment increased significantly the liver-conjugated diene (P<0.001), TBARS (P=0.012), plasma total PL (P<0.001) concentrations compared with control. Antioxidants combined with amiodarone significantly decreased the liver-conjugated diene (P<0.001 for both), TBARS (P=0.016 for vitamin E, P=0.053 borderline for silymarin) and plasma total PL (P=0.058 borderline for vitamin E, P<0.01 for silymarin) concentrations compared with amiodarone treatment alone. Silymarin significantly (P=0.021) reduced liver amiodarone, but only tended to decrease desethylamiodarone concentration; however, vitamin E failed to do so. Amiodarone treatment increased lysosomal phospholipidosis (P<0.001) estimated by semi-quantitative electron microscopic method and both antioxidants combined with amiodarone reduced

  13. 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

  14. Transport of radiolabelled glycoprotein to cell surface and lysosome-like bodies of absorptive cells in cultured small-intestinal tissue from normal subjects and patients with a lysosomal storage disease

    International Nuclear Information System (INIS)

    The transport of 3H-fucose and 3H-glucosamine-labelled glycoproteins in the absorptive cells of cultured human small-intestinal tissue was investigated with light- and electron-microscopical autoradiography. The findings showed that these glycoproteins were completed in the Golgi apparatus and transported in small vesicular structures to the apical cytoplasm of these cells. Since this material arrived in the cell coat on the microvilli and in the lysosome-like bodies simultaneously, a crinophagic function of these organelles in the regulation of the transport or secretion of cell-coat material was supported. In the absorptive cells of patients with fucosidosis or Hunter's type of lysosomal storage disease, a similar transport of cell-coat material to the lysosome-like bodies and a congenital defect of a lysosomal hydrolase normally involved in the degradation of cell-coat material, can explain the accumulation of this material in the dense bodies. (orig.)

  15. 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

  16. 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.

  17. 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.

  18. Lysosome-associated miniSOG as a photosensitizer for mammalian cells.

    Science.gov (United States)

    Ryumina, Alina P; Serebrovskaya, Ekaterina O; Staroverov, Dmitry B; Zlobovskaya, Olga A; Shcheglov, Alexander S; Lukyanov, Sergey A; Lukyanov, Konstantin A

    2016-01-01

    Genetically encoded photosensitizers represent a promising optogenetic tool for the induction of light-controlled oxidative stress strictly localized to a selected intracellular compartment. Here we tested the phototoxic effects of the flavin-containing phototoxic protein miniSOG targeted to the cytoplasmic surfaces of late endosomes and lysosomes by fusion with Rab7. In HeLa Kyoto cells stably expressing miniSOG-Rab7, we demonstrated a high level of cell death upon blue-light illumination. Pepstatin A completely abolished phototoxicity of miniSOG-Rab7, showing a key role for cathepsin D in this model. Using a far-red fluorescence sensor for caspase-3, we observed caspase-3 activation during miniSOG-Rab7-mediated cell death. We conclude that upon illumination, miniSOG-Rab7 induces lysosomal membrane permeabilization (LMP) and leakage of cathepsins into the cytosol, resulting in caspase-dependent apoptosis. PMID:27528074

  19. Activity of lysosomal and mitochondrial ferments in serum and liver tissue at controlled and treated by leukotitin animals

    International Nuclear Information System (INIS)

    In this chapter author describes the experiments on rats and gives the information on activity of lysosomal and mitochondrial ferments in serum and liver tissue at controlled and treated by leukotitin animals

  20. 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

    Polycations such as polyethylenimine (PEI) are used in many novel nonviral vector designs and there are continuous efforts to increase our mechanistic understanding of their interactions with cells. Even so, the mechanism of polyplex escape from the endosomal/lysosomal pathway after internalization...... 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...

  1. Mucopolysaccharidosis IIIB, a lysosomal storage disease, triggers a pathogenic CNS autoimmune response

    OpenAIRE

    Popovich Phillip G; Divers Erin; DiRosario Julianne; Killedar Smruti; McCarty Douglas M; Fu Haiyan

    2010-01-01

    Abstract Background Recently, using a mouse model of mucopolysaccharidosis (MPS) IIIB, a lysosomal storage disease with severe neurological deterioration, we showed that MPS IIIB neuropathology is accompanied by a robust neuroinflammatory response of unknown consequence. This study was to assess whether MPS IIIB lymphocytes are pathogenic. Methods Lymphocytes from MPS IIIB mice were adoptively transferred to naïve wild-type mice. The recipient animals were then evaluated for signs of disease ...

  2. WASH is required for lysosomal recycling and efficient autophagic and phagocytic digestion

    OpenAIRE

    King, Jason S.; Gueho, Aurélie; Hagedorn, Monica; Gopaldass, Navin Andréw; Leuba, Florence; Soldati, Thierry; Insall, Robert H.

    2013-01-01

    Wiskott-Aldrich syndrome protein and SCAR homologue (WASH) is an important regulator of vesicle trafficking. By generating actin on the surface of intracellular vesicles, WASH is able to directly regulate endosomal sorting and maturation. We report that, in Dictyostelium, WASH is also required for the lysosomal digestion of both phagocytic and autophagic cargo. Consequently, Dictyostelium cells lacking WASH are unable to grow on many bacteria or to digest their own cytoplasm to survive starva...

  3. Relative acidic compartment volume as a lysosomal storage disorder–associated biomarker

    OpenAIRE

    te Vruchte, Danielle; Speak, Anneliese O; Wallom, Kerri L.; Al Eisa, Nada; Smith, David A.; Hendriksz, Christian J.; Simmons, Louise; Lachmann, Robin H.; Cousins, Alison; Hartung, Ralf; Mengel, Eugen; Runz, Heiko; Beck, Michael; Amraoui, Yasmina; Imrie, Jackie

    2014-01-01

    Lysosomal storage disorders (LSDs) occur at a frequency of 1 in every 5,000 live births and are a common cause of pediatric neurodegenerative disease. The relatively small number of patients with LSDs and lack of validated biomarkers are substantial challenges for clinical trial design. Here, we evaluated the use of a commercially available fluorescent probe, Lysotracker, that can be used to measure the relative acidic compartment volume of circulating B cells as a potentially universal bioma...

  4. Mechanism of the lysosomal membrane enzyme acetyl coenzyme A: alpha-glucosaminide N-acetyltransferase

    International Nuclear Information System (INIS)

    Acetyl-CoA:α-glucosaminide N-acetyltransferase is a lysosomal membrane enzyme, deficient in the genetic disease Sanfilippo C syndrome. The enzyme catalyzes the transfer of an acetyl group from cytoplasmic acetyl-CoA to terminal α-glucosamine residues of heparan sulfate within the organelle. The reaction mechanism was examined using high purified lysosomal membranes from rat liver and human fibroblasts. The N-acetyltransferase reaction is optimal above pH 5.5 and a 2-3 fold stimulation of activity is observed in the presence of 0.1% taurodeoxycholate. Double reciprocal analysis and product inhibition studies indicate that the enzyme works by a Di-Iso Ping Pong Bi Bi mechanism. The binding of acetyl-CoA to the enzyme is measured by exchange label from [3H]CoA to acetyl-CoA, and is optimal at pH's above 7.0. The acetyl-enzyme intermediate is formed by incubating membranes with [3H]acetyl-CoA. The acetyl group can be transferred to glucosamine, forming [3H]N-acetylglucosamine; the transfer is optimal between pH 4 and 5. Lysosomal membranes from Sanfilippo C fibroblasts confirm that these half reactions carried out by the N-acetyltransferase. The enzyme is inactivated by N-bromosuccinimide and diethylpyrocarbonate, indicating that a histidine is involved in the reaction. These results suggest that the histidine residue is at the active site of the enzyme. The properties of the N-acetyltransferase in the membrane, the characterization of the enzyme kinetics, the chemistry of a histidine mediated acetylation and the pH difference across the lysosomal membrane all support a transmembrane acetylation mechanism

  5. Phagosome-lysosome fusion inhibited by algal symbionts of Hydra viridis

    OpenAIRE

    1982-01-01

    Certain species of Chlorella live within the digestive cells of the fresh water cnidarian Hydra viridis. When introduced into the hydra gut, these symbiotic algae are phagocytized by digestive cells but avoid host digestion and persist at relatively constant numbers within host cells. In contrast, heat-killed symbionts are rapidly degraded after phagocytosis. Live symbionts appear to persist because host lysosomes fail to fuse with phagosomes containing live symbionts. Neither acid phosphatas...

  6. C. elegans BLOC-1 functions in trafficking to lysosome-related gut granules.

    Directory of Open Access Journals (Sweden)

    Greg J Hermann

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

  7. Elimination of paternal mitochondria through the lysosomal degradation pathway in C. elegans

    OpenAIRE

    Zhou, Qinghua; Li, Haimin; Xue, Ding

    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 ...

  8. Autophagy Induction Protects Against 7-Oxysterol-induced Cell Death via Lysosomal Pathway and Oxidative Stress

    OpenAIRE

    Xi-Ming Yuan; Nargis Sultana; Nabeel Siraj; Ward, Liam J.; Bijar Ghafouri; Wei Li

    2016-01-01

    7-Oxysterols are major toxic components in oxidized low-density lipoprotein and human atheroma lesions, which cause lysosomal membrane permeabilization (LMP) and cell death. Autophagy may function as a survival mechanism in this process. Here, we investigated whether 7-oxysterols mixed in an atheroma-relevant proportion induce autophagy, whether autophagy induction influences 7-oxysterol-mediated cell death, and the underlying mechanisms, by focusing on cellular lipid levels, oxidative stress...

  9. Lysosomal trafficking of β-catenin induced by the tea polyphenol epigallocatechin-3-gallate

    International Nuclear Information System (INIS)

    β-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 β-catenin, since this provides an avenue for the prevention of colorectal and other cancers in which β-catenin is frequently over-expressed. We show here that physiologically relevant concentrations of the tea polyphenol epigallocatechin-3-gallate (EGCG) inhibited β-catenin/TCF-dependent reporter activity in human embryonic kidney 293 cells transfected with wild type or mutant β-catenins, and there was a corresponding decrease in β-catenin protein levels in the nuclear, cytosolic and membrane-associated fractions. However, β-catenin accumulated as punctate aggregates in response to EGCG treatment, including in human colon cancer cells over-expressing β-catenin endogenously. Confocal microscopy studies revealed that the aggregated β-catenin in HEK293 cells was extra-nuclear and co-localized with lysosomes, suggesting that EGCG activated a pathway involving lysosomal trafficking of β-catenin. Lysosomal inhibitors leupeptin and transepoxysuccinyl-L-leucylamido(4-guanido)butane produced an increase in β-catenin protein in total cell lysates, without a concomitant increase in β-catenin transcriptional activity. These data provide the first evidence that EGCG facilitates the trafficking of β-catenin into lysosomes, presumably as a mechanism for sequestering β-catenin and circumventing further nuclear transport and activation of β-catenin/TCF/LEF signaling

  10. Defective lysosomal targeting of activated fibroblast growth factor receptor 3 in achondroplasia

    OpenAIRE

    Cho, Jay Y.; Guo, Changsheng; Torello, Monica; Lunstrum, Gregory P.; Iwata, Tomoko; Deng, Chuxia; Horton, William A.

    2003-01-01

    Mutations of fibroblast growth factor receptor 3 (FGFR3) are responsible for achondroplasia (ACH) and related dwarfing conditions in humans. The pathogenesis involves constitutive activation of FGFR3, which inhibits proliferation and differentiation of growth plate chondrocytes. Here we report that activating mutations in FGFR3 increase the stability of the receptor. Our results suggest that the mutations disrupt c-Cbl-mediated ubiquitination that serves as a targeting signal for lysosomal de...

  11. New biotechnological and nanomedicine strategies for treatment of lysosomal storage disorders

    OpenAIRE

    Muro, Silvia

    2010-01-01

    This review discusses the multiple bio- and nano-technological strategies developed in the last few decades for treatment of a group of fatal genetic diseases, termed lysosomal storage disorders. Some basic foundation on the biomedical causes and social and clinical relevance of these diseases is provided. Several treatment modalities, from those currently available to novel therapeutic approaches under development, are also discussed; these include gene and cell therapies, substrate reductio...

  12. PHOSPHATIDYLINOSITOL 3,5-BISPHOSPHATE IS AN ESSENTIAL REGULATOR OF LYSOSOME MORPHOLOGY

    OpenAIRE

    Asanuma, Ken; Takasuga, Shunsuke; Sasaki, Junko; Sasaki, Takehiko

    2013-01-01

    Phosphoinositides are lipid second messengers that act as key players in endosomal membrane trafficking, and mutations in several phosphatases that metabolize these lipids cause severe genetic diseases. We previously reported that type III phosphatidylinositol phosphate kinase (PIPKIII) is a critical regulator of lysosome size. However, the lipid products that mediate PIPKIII function have not been well characterized. Using a series of phosphoinositide phosphatase expression vectors, we show ...

  13. OXIDATIVE STRESS TRIGGERS CA2+-DEPENDENT LYSOSOME TRAFFICKING AND ACTIVATION OF ACID SPHINGOMYELINASE

    OpenAIRE

    Li, Xiang; Gulbins, Erich; Zhang, Yang

    2012-01-01

    Recent studies demonstrate that rapid translocation of the acid sphingomyelinase (ASM), a lysosomal hydrolase, to the outer leaflet of the cell membrane and concomitant release of ceramide constitute a common cellular signaling cascade to various stimuli including CD95 ligation, UV-irradiation, bacterial and viral infections. Reactive oxygen species (ROS) were shown to play a crucial role in regulating this signaling cascade at least for some bacterial infections and UV-irradiation. However, ...

  14. Possible Existence of Lysosome-Like Organella within Mitochondria and Its Role in Mitochondrial Quality Control

    OpenAIRE

    Yuji Miyamoto; Noriaki Kitamura; Yasuyuki Nakamura; Manabu Futamura; Takafumi Miyamoto; Masaki Yoshida; Masaya Ono; Shizuko Ichinose; Hirofumi Arakawa

    2011-01-01

    The accumulation of unhealthy mitochondria results in mitochondrial dysfunction, which has been implicated in aging, cancer, and a variety of degenerative diseases. However, the mechanism by which mitochondrial quality is regulated remains unclear. Here, we show that Mieap, a novel p53-inducible protein, induces intramitochondrial lysosome-like organella that plays a critical role in mitochondrial quality control. Mieap expression is directly regulated by p53 and is frequently lost in human c...

  15. Protein breakdown in muscle wasting: Role of autophagy-lysosome and ubiquitin-proteasome

    OpenAIRE

    Sandri, Marco

    2013-01-01

    Skeletal muscle adapts its mass as consequence of physical activity, metabolism and hormones. Catabolic conditions or inactivity induce signaling pathways that regulate the process of muscle loss. Muscle atrophy in adult tissue occurs when protein degradation rates exceed protein synthesis. Two major protein degradation pathways, the ubiquitin-proteasome and the autophagy-lysosome systems, are activated during muscle atrophy and variably contribute to the loss of muscle mass. These degradatio...

  16. Vacuolin-1 potently and reversibly inhibits autophagosome-lysosome fusion by activating RAB5A

    OpenAIRE

    Lu, Yingying; Dong, Shichen; Hao, Baixia; Li, Chang; Zhu, Kaiyuan; Guo, Wenjing; Wang, Qian; Cheung, King-Ho; Wong, Connie WM; Wu, Wu-Tian; Markus, Huss; Yue, Jianbo

    2014-01-01

    Autophagy is a catabolic lysosomal degradation process essential for cellular homeostasis and cell survival. Dysfunctional autophagy has been associated with a wide range of human diseases, e.g., cancer and neurodegenerative diseases. A large number of small molecules that modulate autophagy have been widely used to dissect this process and some of them, e.g., chloroquine (CQ), might be ultimately applied to treat a variety of autophagy-associated human diseases. Here we found that vacuolin-1...

  17. Pancreatic duct obstruction in rabbits causes digestive zymogen and lysosomal enzyme colocalization.

    OpenAIRE

    Saluja, A; Saluja, M; Villa, A.; Leli, U; Rutledge, P.; Meldolesi, J.; Steer, M.

    1989-01-01

    The pancreatic duct of anesthetized rabbits was cannulated and, in some animals, flow of pancreatic exocrine secretions was blocked by raising the cannula to a vertical position. Blockage for 3-7 h caused a rapid and significant rise in serum amylase activity and an increase in amylase activity within the pancreas. The concentration of lysosomal enzymes in the pancreas was not altered but they became redistributed among subcellular fractions and, as a result, an increased amount was recovered...

  18. The role of lysosomal proteolytic enzymes in invasion and dissemination of malignant melanoma

    International Nuclear Information System (INIS)

    Preoperative chemo- and radiation therapy was followed by a decrease in lysosomal cathepsins activity in metastatic lymph nodes which, however, did not reach the level established for intact lymph nodes. The pathogenetic role of proteolytic endopeptidases in invasion and sissemination of malignant melanoma is discussed as well as the value of their level measurement for assessing metastatic potential of tumor and prognosis of disease of disease on the basis of tumor site, degree of invasion regional lymph node status

  19. Y682 mutation of amyloid precursor protein promotes endo-lysosomal dysfunction by disrupting APP-SorLA interaction

    Directory of Open Access Journals (Sweden)

    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.

  20. LYST controls the biogenesis of the endosomal compartment required for secretory lysosome function.

    Science.gov (United States)

    Sepulveda, Fernando E; Burgess, Agathe; Heiligenstein, Xavier; Goudin, Nicolas; Ménager, Mickaël M; Romao, Maryse; Côte, Marjorie; Mahlaoui, Nizar; Fischer, Alain; Raposo, Graça; Ménasché, Gaël; de Saint Basile, Geneviève

    2015-02-01

    Chediak-Higashi syndrome (CHS) is caused by mutations in the gene encoding LYST protein, the function of which remains poorly understood. Prominent features of CHS include defective secretory lysosome exocytosis and the presence of enlarged, lysosome-like organelles in several cell types. In order to get further insight into the role of LYST in the biogenesis and exocytosis of cytotoxic granules, we analyzed cytotoxic T lymphocytes (CTLs) from patients with CHS. Using confocal microscopy and correlative light electron microscopy, we showed that the enlarged organelle in CTLs is a hybrid compartment that contains proteins components from recycling-late endosomes and lysosomes. Enlargement of cytotoxic granules results from the progressive clustering and then fusion of normal-sized endolysosomal organelles. At the immunological synapse (IS) in CHS CTLs, cytotoxic granules have limited motility and appear docked while nevertheless unable to degranulate. By increasing the expression of effectors of lytic granule exocytosis, such as Munc13-4, Rab27a and Slp3, in CHS CTLs, we were able to restore the dynamics and the secretory ability of cytotoxic granules at the IS. Our results indicate that LYST is involved in the trafficking of the effectors involved in exocytosis required for the terminal maturation of perforin-containing vesicles into secretory cytotoxic granules. PMID:25425525

  1. A Contiguous Compartment Functions as Endoplasmic Reticulum and Endosome/Lysosome in Giardia lamblia▿ †

    Science.gov (United States)

    Abodeely, Marla; DuBois, Kelly N.; Hehl, Adrian; Stefanic, Sasa; Sajid, Mohammed; deSouza, Wanderley; Attias, Marcia; Engel, Juan C.; Hsieh, Ivy; Fetter, Richard D.; McKerrow, James H.

    2009-01-01

    The dynamic evolution of organelle compartmentalization in eukaryotes and how strictly compartmentalization is maintained are matters of ongoing debate. While the endoplasmic reticulum (ER) is classically envisioned as the site of protein cotranslational translocation, it has recently been proposed to have pluripotent functions. Using transfected reporter constructs, organelle-specific markers, and functional enzyme assays, we now show that in an early-diverging protozoan, Giardia lamblia, endocytosis and subsequent degradation of exogenous proteins occur in the ER or in an adjacent and communicating compartment. The Giardia endomembrane system is simple compared to those of typical eukaryotes. It lacks peroxisomes, a classical Golgi apparatus, and canonical lysosomes. Giardia orthologues of mammalian lysosomal proteases function within an ER-like tubulovesicular compartment, which itself can dynamically communicate with clathrin-containing vacuoles at the periphery of the cell to receive endocytosed proteins. These primitive characteristics support Giardia's proposed early branching and could serve as a model to study the compartmentalization of endocytic and lysosomal functions into organelles distinct from the ER. This system also may have functional similarity to the retrograde transport of toxins and major histocompatibility complex class I function in the ER of mammals. PMID:19749174

  2. A contiguous compartment functions as endoplasmic reticulum and endosome/lysosome in Giardia lamblia.

    Science.gov (United States)

    Abodeely, Marla; DuBois, Kelly N; Hehl, Adrian; Stefanic, Sasa; Sajid, Mohammed; DeSouza, Wanderley; Attias, Marcia; Engel, Juan C; Hsieh, Ivy; Fetter, Richard D; McKerrow, James H

    2009-11-01

    The dynamic evolution of organelle compartmentalization in eukaryotes and how strictly compartmentalization is maintained are matters of ongoing debate. While the endoplasmic reticulum (ER) is classically envisioned as the site of protein cotranslational translocation, it has recently been proposed to have pluripotent functions. Using transfected reporter constructs, organelle-specific markers, and functional enzyme assays, we now show that in an early-diverging protozoan, Giardia lamblia, endocytosis and subsequent degradation of exogenous proteins occur in the ER or in an adjacent and communicating compartment. The Giardia endomembrane system is simple compared to those of typical eukaryotes. It lacks peroxisomes, a classical Golgi apparatus, and canonical lysosomes. Giardia orthologues of mammalian lysosomal proteases function within an ER-like tubulovesicular compartment, which itself can dynamically communicate with clathrin-containing vacuoles at the periphery of the cell to receive endocytosed proteins. These primitive characteristics support Giardia's proposed early branching and could serve as a model to study the compartmentalization of endocytic and lysosomal functions into organelles distinct from the ER. This system also may have functional similarity to the retrograde transport of toxins and major histocompatibility complex class I function in the ER of mammals. PMID:19749174

  3. 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.

  4. Inhibitor screening of pharmacological chaperones for lysosomal β-glucocerebrosidase by capillary electrophoresis.

    Science.gov (United States)

    Shanmuganathan, Meera; Britz-McKibbin, Philip

    2011-03-01

    Pharmacological chaperones (PCs) represent a promising therapeutic strategy for treatment of lysosomal storage disorders based on enhanced stabilization and trafficking of mutant protein upon orthosteric and/or allosteric binding. Herein, we introduce a simple yet reliable enzyme assay using capillary electrophoresis (CE) for inhibitor screening of PCs that target the lysosomal enzyme, β-glucocerebrosidase (GCase). The rate of GCase-catalyzed hydrolysis of the synthetic substrate, 4-methylumbelliferyl-β-D: -glucopyranoside was performed using different classes of PCs by CE with UV detection under standardized conditions. The pH and surfactant dependence of inhibitor binding on recombinant GCase activity was also examined. Enzyme inhibition studies were investigated for five putative PCs including isofagomine (IFG), ambroxol, bromhexine, diltiazem, and fluphenazine. IFG was confirmed as a potent competitive inhibitor of recombinant GCase with half-maximal inhibitory concentration (IC(50)) of 47.5 ± 0.1 and 4.6 ± 1.4 nM at pH 5.2 and pH 7.2, respectively. In contrast, the four other non-carbohydrate amines were demonstrated to function as mixed-type inhibitors with high micromolar activity at neutral pH relative to acidic pH conditions reflective of the lysosome. CE offers a convenient platform for characterization of PCs as a way to accelerate the clinical translation of previously approved drugs for oral treatment of rare genetic disorders, such as Gaucher disease. PMID:21286689

  5. Chylomicron remnant model emulsions induce intracellular cholesterol accumulation and cell death due to lysosomal destabilization.

    Science.gov (United States)

    Wakita, Kyoko; Morita, Shin-ya; Okamoto, Naoko; Takata, Eriko; Handa, Tetsurou; Nakano, Minoru

    2015-05-01

    Chylomicron remnants, which carry dietary fats and cholesterol, play a role in promoting atherosclerosis. Chylomicron remnants are characterized by high cholesterol content at the surface, different from low-density lipoproteins (LDLs) containing high amounts of esterified cholesterol (CE) in the core. We prepared cholesterol-rich emulsions (TO-PC/cholesterol emulsions) as models for chylomicron remnants and compared their effects on J774 macrophages with acetylated-LDL (ac-LDL). Internalization of TO-PC/cholesterol emulsions into macrophages reduced cell viability, whereas ac-LDL did not. Surprisingly, there was no difference in intracellular free cholesterol content between cells incubated with TO-PC/cholesterol emulsions and with ac-LDL. Furthermore, cholesterol in TO-PC/cholesterol emulsions and ac-LDL both were internalized into J774 macrophages; however, incubation with TO-PC/cholesterol emulsions induced leakage of lysosomal protease, cathepsin-L, to cytosol, which was not observed for incubation with ac-LDL. Inhibition of the activity of cathepsin-L recovered the viability of macrophages that ingested TO-PC/cholesterol emulsions. We suggest an alternative fate of cholesterol-rich emulsions taken up by macrophages, which is different from other atherogenic lipoproteins rich in CE; internalization of TO-PC/cholesterol emulsions into macrophages induces rapid free cholesterol accumulation in lysosomes and cell death due to lysosomal destabilization. PMID:25661161

  6. Cerebrospinal Fluid from Sporadic Amyotrophic Lateral Sclerosis Patients Induces Mitochondrial and Lysosomal Dysfunction.

    Science.gov (United States)

    Sharma, Aparna; Varghese, Anu Mary; Vijaylakshmi, Kalyan; Sumitha, Rajendrarao; Prasanna, V K; Shruthi, S; Chandrasekhar Sagar, B K; Datta, Keshava K; Gowda, Harsha; Nalini, Atchayaram; Alladi, Phalguni Anand; Christopher, Rita; Sathyaprabha, Talakad N; Raju, Trichur R; Srinivas Bharath, M M

    2016-05-01

    In our laboratory, we have developed (1) an in vitro model of sporadic Amyotrophic Lateral Sclerosis (sALS) involving exposure of motor neurons to cerebrospinal fluid (CSF) from sALS patients and (2) an in vivo model involving intrathecal injection of sALS-CSF into rat pups. In the current study, we observed that spinal cord extract from the in vivo sALS model displayed elevated reactive oxygen species (ROS) and mitochondrial dysfunction. Quantitative proteomic analysis of sub-cellular fractions from spinal cord of the in vivo sALS model revealed down-regulation of 35 mitochondrial proteins and 4 lysosomal proteins. Many of the down-regulated mitochondrial proteins contribute to alterations in respiratory chain complexes and organellar morphology. Down-regulated lysosomal proteins Hexosaminidase, Sialidase and Aryl sulfatase also displayed lowered enzyme activity, thus validating the mass spectrometry data. Proteomic analysis and validation by western blot indicated that sALS-CSF induced the over-expression of the pro-apoptotic mitochondrial protein BNIP3L. In the in vitro model, sALS-CSF induced neurotoxicity and elevated ROS, while it lowered the mitochondrial membrane potential in rat spinal cord mitochondria in the in vivo model. Ultra structural alterations were evident in mitochondria of cultured motor neurons exposed to ALS-CSF. These observations indicate the first line evidence that sALS-CSF mediated mitochondrial and lysosomal defects collectively contribute to the pathogenesis underlying sALS. PMID:26646005

  7. [Structuro-functional changes in dog liver and regional lymph node lysosomes in toxic hepatitis].

    Science.gov (United States)

    Borodin, Iu I; Korolenko, T A; Malygin, A E; Pupyshev, A B; Sharaĭkina, E O

    1978-10-01

    Structural and functional changes in the dog liver and regional lymph nodes lysosomes were studied during toxic hepatitis induced by CCl4 administration (single and repeated). Total activity of lysosomal enzymes (acid RNA-ase and beta-galactosidase) was higher in the regional lymph nodes than in the liver, reflecting the barrier, protective function of the organ. During acute toxic hepatitis the specific activities of acid RNA-ase and cathepsin D displayed a sharp rise. No normalization of the indices under study occurred during the observation period (from 8 to 30 days). At the same time there was a rise of the regional lymph node weight and an elevation of the relative macrophage and neutrophil content in the sinuses. The increased activity of the lysosome enzymes in the regional lymph nodes in injury of the liver was connected with greater functional load on the lymph nodes effecting hydrolysis of biopolymeres which penetrated into the regional lymphatic node with the lymph. PMID:708870

  8. BNIP3 and NIX mediate Mieap-induced accumulation of lysosomal proteins within mitochondria.

    Directory of Open Access Journals (Sweden)

    Yasuyuki Nakamura

    Full Text Available Mieap, a p53-inducible protein, controls mitochondrial quality by repairing unhealthy mitochondria. During repair, Mieap induces the accumulation of intramitochondrial lysosomal proteins (designated MALM for Mieap-induced accumulation of lysosome-like organelles within mitochondria by interacting with NIX, leading to the elimination of oxidized mitochondrial proteins. Here, we report that an additional mitochondrial outer membrane protein, BNIP3, is also involved in MALM. BNIP3 interacts with Mieap in a reactive oxygen species (ROS-dependent manner via the BH3 domain of BNIP3 and the coiled-coil domains of Mieap. The knockdown of endogenous BNIP3 expression severely inhibited MALM. Although the overexpression of either BNIP3 or NIX did not cause a remarkable change in the mitochondrial membrane potential (MMP, the co-expression of all three exogenous proteins, Mieap, BNIP3 and NIX, caused a dramatic reduction in MMP, implying that the physical interaction of Mieap, BNIP3 and NIX at the mitochondrial outer membrane may regulate the opening of a pore in the mitochondrial double membrane. This effect was not related to cell death. These results suggest that two mitochondrial outer membrane proteins, BNIP3 and NIX, mediate MALM in order to maintain mitochondrial integrity. The physical interaction of Mieap, BNIP3 and NIX at the mitochondrial outer membrane may play a critical role in the translocation of lysosomal proteins from the cytoplasm to the mitochondrial matrix.

  9. Lysosomal storage of oligosaccharide and glycosphingolipid in imino sugar treated cells.

    Science.gov (United States)

    Boomkamp, Stephanie D; Rountree, J S Shane; Neville, David C A; Dwek, Raymond A; Fleet, George W J; Butters, Terry D

    2010-04-01

    Sandhoff and Tay-Sachs disease are autosomal recessive GM2 gangliosidoses where a deficiency of lysosomal beta-hexosaminidase results in storage of glycoconjugates. Imino sugar (2-acetamido-1,4-imino-1,2,4-trideoxy-L-arabinitol) inhibition of beta-hexosaminidase in murine RAW264.7 macrophage-like cells led to lysosomal storage of glycoconjugates that were characterised structurally using fluorescence labelling of the free or glycolipid-derived oligosaccharides followed by HPLC and mass spectrometry. Stored glycoconjugates were confirmed as containing non-reducing GlcNAc or GalNAc residues resulting from the incomplete degradation of N-linked glycoprotein oligosaccharide and glycolipids, respectively. When substrate reduction therapeutics N-butyl-deoxynojirimycin (NB-DNJ) or N-butyldeoxygalactonojirimycin (NB-DGJ) were applied to the storage phenotype cells, an increase in glucosylated and galactosylated oligosaccharide species was observed due to endoplasmic reticulum alpha-glucosidases and lysosomal beta-galactosidase inhibition, respectively. Hexosaminidase inhibition triggered a tightly regulated cytokine-mediated inflammatory response that was normalised using imino sugars NB-DNJ and NB-DGJ, which restored the GM2 ganglioside storage burden but failed to reduce the levels of GA2 glycolipid or glycoprotein-derived N-linked oligosaccharides. Using a chemically induced gangliosidosis phenotype that can be modulated with substrate lowering drugs, the critical role of GM2 ganglioside in the progression of inflammatory disease is also demonstrated. PMID:20186478

  10. Inflammatory cytokine response to Bacillus anthracis peptidoglycan requires phagocytosis and lysosomal trafficking.

    Science.gov (United States)

    Iyer, Janaki K; Khurana, Taruna; Langer, Marybeth; West, Christopher M; Ballard, Jimmy D; Metcalf, Jordan P; Merkel, Tod J; Coggeshall, K Mark

    2010-06-01

    During advanced stages of inhalation anthrax, Bacillus anthracis accumulates at high levels in the bloodstream of the infected host. This bacteremia leads to sepsis during late-stage anthrax; however, the mechanisms through which B. anthracis-derived factors contribute to the pathology of infected hosts are poorly defined. Peptidoglycan, a major component of the cell wall of Gram-positive bacteria, can provoke symptoms of sepsis in animal models. We have previously shown that peptidoglycan of B. anthracis can induce the production of proinflammatory cytokines by cells in human blood. Here, we show that biologically active peptidoglycan is shed from an active culture of encapsulated B. anthracis strain Ames in blood. Peptidoglycan is able to bind to surfaces of responding cells, and internalization of peptidoglycan is required for the production of inflammatory cytokines. We also show that the peptidoglycan traffics to lysosomes, and lysosomal function is required for cytokine production. We conclude that peptidoglycan of B. anthracis is initially bound by an unknown extracellular receptor, is phagocytosed, and traffics to lysosomes, where it is degraded to a product recognized by an intracellular receptor. Binding of the peptidoglycan product to the intracellular receptor causes a proinflammatory response. These findings provide new insight into the mechanism by which B. anthracis triggers sepsis during a critical stage of anthrax disease. PMID:20308305

  11. Cellular uptake of saposin (SAP) precursor and lysosomal delivery by the low density lipoprotein receptor-related protein (LRP).

    OpenAIRE

    Hiesberger, T; Hüttler, S; Rohlmann, A; Schneider, W; Sandhoff, K.; Herz, J.

    1998-01-01

    Sphingolipid activator proteins SAP-A, -B, -C and -D (also called saposins) are generated by proteolytic processing from a 73 kDa precursor and function as obligatory activators of lysosomal enzymes involved in glycosphingolipid metabolism. Although the SAP precursor can be recognized by the mannose-6-phosphate (M-6-P) receptor and shuttled directly from the secretory pathway to the lysosome, a substantial fraction of newly synthesized precursor is secreted from the cell where it may particip...

  12. Crystal structures of native and inhibited forms of human cathepsin D: implications for lysosomal targeting and drug design.

    OpenAIRE

    Baldwin, E. T.; Bhat, T N; Gulnik, S; Hosur, M. V.; Sowder, R C; Cachau, R.E.; Collins, J.; A. M. Silva; Erickson, J. W.

    1993-01-01

    Cathepsin D (EC 3.4.23.5) is a lysosomal protease suspected to play important roles in protein catabolism, antigen processing, degenerative diseases, and breast cancer progression. Determination of the crystal structures of cathepsin D and a complex with pepstatin at 2.5 A resolution provides insights into inhibitor binding and lysosomal targeting for this two-chain, N-glycosylated aspartic protease. Comparison with the structures of a complex of pepstatin bound to rhizopuspepsin and with a h...

  13. Distinct Mechanisms of Ferritin Delivery to Lysosomes in Iron-Depleted and Iron-Replete Cells ▿

    OpenAIRE

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

    2011-01-01

    Ferritin is a cytosolic protein that stores excess iron, thereby protecting cells from iron toxicity. Ferritin-stored iron is believed to be utilized when cells become iron deficient; however, the mechanisms underlying the extraction of iron from ferritin have yet to be fully elucidated. Here, we demonstrate that ferritin is degraded in the lysosome under iron-depleted conditions and that the acidic environment of the lysosome is crucial for iron extraction from ferritin and utilization by ce...

  14. Repetitive stimulation of autophagy-lysosome machinery by intermittent fasting preconditions the myocardium to ischemia-reperfusion injury

    OpenAIRE

    Godar, Rebecca J.; Ma, Xiucui; Liu, Haiyan; Murphy, John T.; Carla J Weinheimer; Kovacs, Attila; Seth D Crosby; 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 fe...

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

    OpenAIRE

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

    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...

  16. Visualization of cholesterol deposits in lysosomes of Niemann-Pick type C fibroblasts using recombinant perfringolysin O

    OpenAIRE

    Kwiatkowska, Katarzyna; Marszałek–Sadowska, Ewelina; Traczyk, Gabriela; Koprowski, Piotr; Musielak, Małgorzata; Ługowska, Agnieszka; Kulma, Magdalena; Grzelczyk, Anna; Sobota, Andrzej

    2014-01-01

    Background Niemann-Pick disease type C (NPC) is caused by defects in cholesterol efflux from lysosomes due to mutations of genes coding for NPC1 and NPC2 proteins. As a result, massive accumulation of unesterified cholesterol in late endosomes/lysosomes is observed. At the level of the organism these cholesterol metabolism disorders are manifested by progressive neurodegeneration and hepatosplenomegaly. Until now filipin staining of cholesterol deposits in cells has been widely used for NPC d...

  17. ESCRT-Dependent Cell Death in a Caenorhabditis elegans Model of the Lysosomal Storage Disorder Mucolipidosis Type IV.

    Science.gov (United States)

    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. PMID:26596346

  18. Azadirachtin-induced apoptosis involves lysosomal membrane permeabilization and cathepsin L release in Spodoptera frugiperda Sf9 cells.

    Science.gov (United States)

    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. PMID:25849458

  19. Iron-Mediated Lysosomal Membrane Permeabilization in Ethanol-Induced Hepatic Oxidative Damage and Apoptosis: Protective Effects of Quercetin

    Directory of Open Access Journals (Sweden)

    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.

  20. A six-membered-ring incorporated Si-rhodamine for imaging of copper(ii) in lysosomes.

    Science.gov (United States)

    Wang, Baogang; Cui, Xiaoyan; Zhang, Zhiqiang; Chai, Xiaoyun; Ding, Hao; Wu, Qiuye; Guo, Zhongwu; Wang, Ting

    2016-07-12

    The regulation of copper homeostasis in lysosomes of living cells is closely related to various physiological and pathological processes. Thus, it is of urgent need to develop a fluorescent probe for selectively and sensitively monitoring the location and concentration of lysosomal Cu(2+). Herein, a six-membered ring, thiosemicarbazide, was incorporated into a Si-rhodamine (SiR) scaffold for the first time, affording a SiR-based fluorescent probe SiRB-Cu. Through the effective Cu(2+)-triggered ring-opening process, the probe exhibits fast NIR chromogenic and fluorogenic responses to Cu(2+) within 2 min as the result of formation of a highly fluorescent product SiR-NCS. Compared with a five-membered ring, the expanded ring retains great tolerance to H(+), ensuring the superior sensitivity with a detection limit as low as 7.7 nM and 200-fold enhancement of relative fluorescence in the presence of 1.0 equiv. of Cu(2+) in pH = 5.0 solution, the physiological pH of lysosome. Moreover, the thiosemicarbazide moiety acts not only as the chelating and reactive site, but also as an efficient lysosome-targeting group, leading to the proactive accumulation of the probe into lysosomes. Taking advantage of these distinct properties, SiRB-Cu provides a functional probe suitable for imaging exogenous and endogenous lysosomal Cu(2+) with high imaging contrast and fidelity. PMID:27314426

  1. The anticancer agent di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT) overcomes prosurvival autophagy by two mechanisms: persistent induction of autophagosome synthesis and impairment of lysosomal integrity.

    Science.gov (United States)

    Gutierrez, Elaine; Richardson, Des R; Jansson, Patric J

    2014-11-28

    Autophagy functions as a survival mechanism during cellular stress and contributes to resistance against anticancer agents. The selective antitumor and antimetastatic chelator di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT) causes lysosomal membrane permeabilization and cell death. Considering the integral role of lysosomes in autophagy and cell death, it was important to assess the effect of Dp44mT on autophagy to further understand its mechanism of action. Notably, Dp44mT affected autophagy by two mechanisms. First, concurrent with its antiproliferative activity, Dp44mT increased the expression of the classical autophagic marker LC3-II as a result of induced autophagosome synthesis. Second, this effect was supplemented by a reduction in autophagosome degradation as shown by the accumulation of the autophagic substrate and receptor p62. Conversely, the classical iron chelator desferrioxamine induced autophagosome accumulation only by inhibiting autophagosome degradation. The formation of redox-active iron or copper Dp44mT complexes was critical for its dual effect on autophagy. The cytoprotective antioxidant N-acetylcysteine inhibited Dp44mT-induced autophagosome synthesis and p62 accumulation. Importantly, Dp44mT inhibited autophagosome degradation via lysosomal disruption. This effect prevented the fusion of lysosomes with autophagosomes to form autolysosomes, which is crucial for the completion of the autophagic process. The antiproliferative activity of Dp44mT was suppressed by Beclin1 and ATG5 silencing, indicating the role of persistent autophagosome synthesis in Dp44mT-induced cell death. These studies demonstrate that Dp44mT can overcome the prosurvival activity of autophagy in cancer cells by utilizing this process to potentiate cell death. PMID:25301941

  2. Inhibitory effect of mTOR activator MHY1485 on autophagy: suppression of lysosomal fusion.

    Directory of Open Access Journals (Sweden)

    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

  3. Evaluation of Geostatistical Techniques for Mapping Spatial Distribution of Soil PH, Salinity and Plant Cover Affected by Environmental Factors in Southern Iran

    OpenAIRE

    Mohammad ZARE-MEHRJARDI; Ruhollah TAGHIZADEH-MEHRJARDI; Ali AKBARZADEH

    2010-01-01

    The study presented in this paper attempts to evaluate some interpolation techniques for mapping spatial distribution of soil pH, salinity and plant cover in Hormozgan province, Iran. The relationships among environmental factors and distribution of vegetation types were also investigated. Plot sampling was applied in the study area. Landform parameters of each plot were recorded and canopy cover percentages of each species were measured while stoniness and browsing damage were estimated. Res...

  4. A theory of local and global processes which affect solar wind electrons. I - The origin of typical 1 AU velocity distribution functions - Steady state theory

    Science.gov (United States)

    Scudder, J. D.; Olbert, S.

    1979-01-01

    A kinetic theory for the velocity distribution of solar wind electrons which illustrates the global and local properties of the solar wind expansion is proposed. By means of the Boltzmann equation with the Krook collision operator accounting for Coulomb collisions, it is found that Coulomb collisions determine the population and shape of the electron distribution function in both the thermal and suprathermal energy regimes. For suprathermal electrons, the cumulative effects of Coulomb interactions are shown to take place on the scale of the heliosphere itself, whereas the Coulomb interactions of thermal electrons occur on a local scale near the point of observation (1 AU). The bifurcation of the electron distribution between thermal and suprathermal electrons is localized to the deep solar corona (1 to 10 solar radii).

  5. A theory of local and global processes which affect solar wind electrons. 1: The origin of typical 1 AU velocity distribution functions: Steady state theory

    Science.gov (United States)

    Scudder, J. D.

    1978-01-01

    A detailed first principle kinetic theory for electrons which is neither a classical fluid treatment nor an exospheric calculation is presented. This theory illustrates the global and local properties of the solar wind expansion that shape the observed features of the electron distribution function, such as its bifurcation, its skewness and the differential temperatures of the thermal and suprathermal subpopulations. Coulomb collisions are substantial mediators of the interplanetary electron velocity distribution function and they place a zone for a bifurcation of the electron distribution function deep in the corona. The local cause and effect precept which permeates the physics of denser media is modified for electrons in the solar wind. The local form of transport laws and equations of state which apply to collision dominated plasmas are replaced with global relations that explicitly depend on the relative position of the observer to the boundaries of the system.

  6. 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. PMID:26103523

  7. 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. PMID:27154979

  8. Apical secretion of lysosomal enzymes in rabbit pancreas occurs via a secretagogue regulated pathway and is increased after pancreatic duct obstruction.

    OpenAIRE

    Hirano, T; Saluja, A; Ramarao, P; Lerch, M M; Saluja, M; Steer, M L

    1991-01-01

    Lysosomal hydrolases such as cathepsin B are apically secreted from rabbit pancreatic acinar cells via a regulated as opposed to a constitutive pathway. Intravenous infusion of the cholecystokinin analogue caerulein results in highly correlated apical secretion of digestive and lysosomal enzymes, suggesting that they are discharged from the same presecretory compartment (zymogen granules). Lysosomal enzymes appear to enter that compartment as a result of missorting. After 7 h of duct obstruct...

  9. Understanding interannual variability in the distribution of, and transport processes affecting, the early life stages of Todarodes pacificus using behavioral-hydrodynamic modeling approaches

    Science.gov (United States)

    Kim, Jung Jin; Stockhausen, William; Kim, Suam; Cho, Yang-Ki; Seo, Gwang-Ho; Lee, Joon-Soo

    2015-11-01

    To understand interannual variability in the distribution of the early life stages of Todarodes pacificus summer spawning population, and to identify the key transport processes influencing this variability, we used a coupled bio-physical model that combines an individual-based model (IBM) incorporating ontogenetic vertical migration for paralarval behavior and temperature-dependent survival process with a ROMS oceanographic model. Using the distribution of paralarvae observed in the northern East China Sea (ECS) during several field cruises as an end point, the spawning ground for the summer-spawning population was estimated to extend from southeast Jeju Island to the central ECS near 29°N by running the model backwards in time. Running the model forward, interannual variability in the distribution of paralarvae predicted by the model was consistent with that observed in several field surveys; surviving individuals in the northern ECS were substantially more abundant in late July 2006 than in 2007, in agreement with observed paralarval distributions. The total number of surviving individuals at 60 days after release based on the simulation throughout summer spawning period (June-August) was 20,329 for 2006, compared with 13,816 for 2007. The surviving individuals were mainly distributed in the East/Japan Sea (EJS), corresponding to a pathway following the nearshore branch of the Tsushima Warm Current flowing along the Japanese coast during both years. In contrast, the abundance of surviving individuals was extremely low in 2007 compared to 2006 on the Pacific side of Japan. Interannual variability in transport and survival processes made a substantial impact on not only the abundance of surviving paralarvae, but also on the flux of paralarvae to adjacent waters. Our simulation results for between-year variation in paralarval abundance coincide with recruitment (year n + 1) variability of T. pacificus in the field. The agreement between the simulation and field

  10. Three-layer poly(methyl methacrylate) microsystem for analysis of lysosomal enzymes for diagnostic purposes

    International Nuclear Information System (INIS)

    Highlights: • New approach for measuring the activity of lysosomal enzymes. • Determination of a protonated form of 4-MU directly in the enzymatic mixture. • Elimination of a long incubation step. • Significant reduction of the processing time and simplification of the procedure. - Abstract: Lysosomal storage diseases are chronic, progressive and typically have a devastating impact on the patient and the family. The diagnosis of these diseases is still a challenge, however, even for trained specialists. Accurate diagnostic methods and high-throughput tools that could be readily incorporated into existing screening laboratories are urgently required. We propose a new method for measuring the activity of lysosomal enzymes using a microfluidic device. The principle of the method is the fluorometric determination of a protonated form of 4-methylumbelliferone directly in the enzymatic mixture. Compared to the standard diagnostic protocols, the method eliminates the necessity to add alkaline buffer to stop the enzymatic reaction, and thus, the number of analytical steps is reduced. The system allows for on-chip short-term incubation of the enzymatic reagents, leading to a much simplified analytical procedure and a significantly shortened processing time. We measured the activity of β-galactosidase in RPMI-1788 human B lymphocytes and in isolated leukocytes from healthy adults. The method shows a good agreement with the standard diagnostic method. The agreement was confirmed by statistical analysis including construction of a Bland–Altman plot. The approach presented can be an alternative for the currently used diagnostic procedures. The method developed has a potential for the implementation into complex microfluidic devices thus becoming a powerful tool for a high-throughput and multiplex screening of newborns

  11. Three-layer poly(methyl methacrylate) microsystem for analysis of lysosomal enzymes for diagnostic purposes

    Energy Technology Data Exchange (ETDEWEB)

    Kwapiszewski, Radoslaw, E-mail: r.kwapiszewski@gmail.com [Department of Microbioanalytics, Faculty of Chemistry, Warsaw University of Technology, Noakowskiego 3, 00-664 Warsaw (Poland); Kwapiszewska, Karina [Department of Microbioanalytics, Faculty of Chemistry, Warsaw University of Technology, Noakowskiego 3, 00-664 Warsaw (Poland); Institute of Physical Chemistry, Polish Academy of Sciences, Kasprzaka 44/52, 01-224 Warsaw (Poland); Kutter, Jörg P. [Department of Pharmacy, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, 2100 Copenhagen (Denmark); Brzozka, Zbigniew [Department of Microbioanalytics, Faculty of Chemistry, Warsaw University of Technology, Noakowskiego 3, 00-664 Warsaw (Poland)

    2015-01-01

    Highlights: • New approach for measuring the activity of lysosomal enzymes. • Determination of a protonated form of 4-MU directly in the enzymatic mixture. • Elimination of a long incubation step. • Significant reduction of the processing time and simplification of the procedure. - Abstract: Lysosomal storage diseases are chronic, progressive and typically have a devastating impact on the patient and the family. The diagnosis of these diseases is still a challenge, however, even for trained specialists. Accurate diagnostic methods and high-throughput tools that could be readily incorporated into existing screening laboratories are urgently required. We propose a new method for measuring the activity of lysosomal enzymes using a microfluidic device. The principle of the method is the fluorometric determination of a protonated form of 4-methylumbelliferone directly in the enzymatic mixture. Compared to the standard diagnostic protocols, the method eliminates the necessity to add alkaline buffer to stop the enzymatic reaction, and thus, the number of analytical steps is reduced. The system allows for on-chip short-term incubation of the enzymatic reagents, leading to a much simplified analytical procedure and a significantly shortened processing time. We measured the activity of β-galactosidase in RPMI-1788 human B lymphocytes and in isolated leukocytes from healthy adults. The method shows a good agreement with the standard diagnostic method. The agreement was confirmed by statistical analysis including construction of a Bland–Altman plot. The approach presented can be an alternative for the currently used diagnostic procedures. The method developed has a potential for the implementation into complex microfluidic devices thus becoming a powerful tool for a high-throughput and multiplex screening of newborns.

  12. Diagnostic and clinical relevance of the autophago-lysosomal network in human gliomas

    Science.gov (United States)

    Jennewein, Lukas; Ronellenfitsch, Michael W.; Antonietti, Patrick; Ilina, Elena I.; Jung, Jennifer; Stadel, Daniela; Flohr, Lisa-Marie; Zinke, Jenny; von Renesse, Janusz; Drott, Ulrich; Baumgarten, Peter; Braczynski, Anne K.; Penski, Cornelia; Burger, Michael C.; Theurillat, Jean-Philippe; Steinbach, Joachim P.; Plate, Karl-Heinz; Dikic, Ivan; Fulda, Simone; Brandts, Christian; Kögel, Donat; Behrends, Christian; Harter, Patrick N.; Mittelbronn, Michel

    2016-01-01

    Recently, the conserved intracellular digestion mechanism ‘autophagy’ has been considered to be involved in early tumorigenesis and its blockade proposed as an alternative treatment approach. However, there is an ongoing debate about whether blocking autophagy has positive or negative effects in tumor cells. Since there is only poor data about the clinico-pathological relevance of autophagy in gliomas in vivo, we first established a cell culture based platform for the in vivo detection of the autophago-lysosomal components. We then investigated key autophagosomal (LC3B, p62, BAG3, Beclin1) and lysosomal (CTSB, LAMP2) molecules in 350 gliomas using immunohistochemistry, immunofluorescence, immunoblotting and qPCR. Autophagy was induced pharmacologically or by altering oxygen and nutrient levels. Our results show that autophagy is enhanced in astrocytomas as compared to normal CNS tissue, but largely independent from the WHO grade and patient survival. A strong upregulation of LC3B, p62, LAMP2 and CTSB was detected in perinecrotic areas in glioblastomas suggesting micro-environmental changes as a driver of autophagy induction in gliomas. Furthermore, glucose restriction induced autophagy in a concentration-dependent manner while hypoxia or amino acid starvation had considerably lesser effects. Apoptosis and autophagy were separately induced in glioma cells both in vitro and in vivo. In conclusion, our findings indicate that autophagy in gliomas is rather driven by micro-environmental changes than by primary glioma-intrinsic features thus challenging the concept of exploitation of the autophago-lysosomal network (ALN) as a treatment approach in gliomas. PMID:26956048

  13. The Serotonin Transporter Undergoes Constitutive Internalization and Is Primarily Sorted to Late Endosomes and Lysosomal Degradation*

    Science.gov (United States)

    Rahbek-Clemmensen, Troels; Bay, Tina; Eriksen, Jacob; Gether, Ulrik; Jørgensen, Trine Nygaard

    2014-01-01

    The serotonin transporter (SERT) plays a critical role in regulating serotonin signaling by mediating reuptake of serotonin from the extracellular space. The molecular and cellular mechanisms controlling SERT levels in the membrane remain poorly understood. To study trafficking of the surface resident SERT, two functional epitope-tagged variants were generated. Fusion of a FLAG-tagged one-transmembrane segment protein Tac to the SERT N terminus generated a transporter with an extracellular epitope suited for trafficking studies (TacSERT). Likewise, a construct with an extracellular antibody epitope was generated by introducing an HA (hemagglutinin) tag in the extracellular loop 2 of SERT (HA-SERT). By using TacSERT and HA-SERT in antibody-based internalization assays, we show that SERT undergoes constitutive internalization in a dynamin-dependent manner. Confocal images of constitutively internalized SERT demonstrated that SERT primarily co-localized with the late endosomal/lysosomal marker Rab7, whereas little co-localization was observed with the Rab11, a marker of the “long loop” recycling pathway. This sorting pattern was distinct from that of a prototypical recycling membrane protein, the β2-adrenergic receptor. Furthermore, internalized SERT co-localized with the lysosomal marker LysoTracker and not with transferrin. The sorting pattern was further confirmed by visualizing internalization of SERT using the fluorescent cocaine analog JHC1-64 and by reversible and pulse-chase biotinylation assays showing evidence for lysosomal degradation of the internalized transporter. Finally, we found that SERT internalized in response to stimulation with 12-myristate 13-acetate co-localized primarily with Rab7- and LysoTracker-positive compartments. We conclude that SERT is constitutively internalized and that the internalized transporter is sorted mainly to degradation. PMID:24973209

  14. Lysosome biogenesis/scattering increases host cell susceptibility to invasion by Trypanosoma cruzi metacyclic forms and resistance to tissue culture trypomastigotes.

    Science.gov (United States)

    Cortez, Cristian; Real, Fernando; Yoshida, Nobuko

    2016-05-01

    A fundamental question to be clarified concerning the host cell invasion by Trypanosoma cruzi is whether the insect-borne and mammalian-stage parasites use similar mechanisms for invasion. To address that question, we analysed the cell invasion capacity of metacyclic trypomastigotes (MT) and tissue culture trypomastigotes (TCT) under diverse conditions. Incubation of parasites for 1 h with HeLa cells in nutrient-deprived medium, a condition that triggered lysosome biogenesis and scattering, increased MT invasion and reduced TCT entry into cells. Sucrose-induced lysosome biogenesis increased HeLa cell susceptibility to MT and resistance to TCT. Treatment of cells with rapamycin, which inhibits mammalian target of rapamycin (mTOR), induced perinuclear lysosome accumulation and reduced MT invasion while augmenting TCT invasion. Metacylic trypomastigotes, but not TCT, induced mTOR dephosphorylation and the nuclear translocation of transcription factor EB (TFEB), a mTOR-associated lysosome biogenesis regulator. Lysosome biogenesis/scattering was stimulated upon HeLa cell interaction with MT but not with TCT. Recently, internalized MT, but not TCT, were surrounded by colocalized lysosome marker LAMP2 and mTOR. The recombinant gp82 protein, the MT-specific surface molecule that mediates invasion, induced mTOR dephosphorylation, nuclear TFEB translocation and lysosome biogenesis/scattering. Taken together, our data clearly indicate that MT invasion is mainly lysosome-dependent, whereas TCT entry is predominantly lysosome-independent. PMID:26572924

  15. Adaptor protein complexes AP-1 and AP-3 are required by the HHV-7 Immunoevasin U21 for rerouting of class I MHC molecules to the lysosomal compartment.

    Directory of Open Access Journals (Sweden)

    Lisa A Kimpler

    Full Text Available The human herpesvirus-7 (HHV-7 U21 gene product binds to class I major histocompatibility complex (MHC molecules and reroutes them to a lysosomal compartment. Trafficking of integral membrane proteins to lysosomes is mediated through cytoplasmic sorting signals that recruit heterotetrameric clathrin adaptor protein (AP complexes, which in turn mediate protein sorting in post-Golgi vesicular transport. Since U21 can mediate rerouting of class I molecules to lysosomes even when lacking its cytoplasmic tail, we hypothesize the existence of a cellular protein that contains the lysosomal sorting information required to escort class I molecules to the lysosomal compartment. If such a protein exists, we expect that it might recruit clathrin adaptor protein complexes as a means of lysosomal sorting. Here we describe experiments demonstrating that the μ adaptins from AP-1 and AP-3 are involved in U21-mediated trafficking of class I molecules to lysosomes. These experiments support the idea that a cellular protein(s is necessary for U21-mediated lysosomal sorting of class I molecules. We also examine the impact of transient versus chronic knockdown of these adaptor protein complexes, and show that the few remaining μ subunits in the cells are eventually able to reroute class I molecules to lysosomes.

  16. Historical Cohort Study on the Factors Affecting Blood Pressure in Workers of Polyacryl Iran Corporation Using Bayesian Multilevel Modeling with Skew T Distribution

    OpenAIRE

    Gholami Fesharaki, Mohammad; Kazemnejad, Anoshirvan; Zayeri, Farid; Sanati, Javad; Akbari, Hamed

    2013-01-01

    Background Hypertension is considered as a major public health problem in most countries due to its association with ischemic heart disease which causes cerebrovascular disease and death. Objectives The purpose of the present study was to study factors affecting Blood Pressure (BP). Patients and Methods The data were extracted from annual observation of the workers who worked in Polyacryl Iran Corporation (PIC) between 1998 and 2010. In this research, we assessed the effect of Body Mass Index...

  17. Lysosomal-Associated Transmembrane Protein 5 (LAPTM5) Is a Molecular Partner of CD1e

    OpenAIRE

    Angénieux, Catherine; Waharte, François; Gidon, Alexandre; Signorino-Gelo, François; Wurtz, Virginie; Hojeij, Rim; Proamer, Fabienne; Gachet, Christian; Van Dorsselaer, Alain; Hanau, Daniel; Salamero, Jean; de la Salle, Henri

    2012-01-01

    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 compar...

  18. Phorbol myristate acetate stimulates phagosome-lysosome fusion in mouse macrophages

    OpenAIRE

    1981-01-01

    The effect of the tumor promoter phorbol myristate acetate (PMA) on phagosome-lysosome (P-L) fusion in mouse macrophages has been studied using a previously described (10) fluorescence assay. Treatment with 0.1--1.0 microgram PMA/ml caused a striking increase in the rate and extent of P-L fusion. Exposure of cells to phorbol, free myristate, or the monoesters of PMA did not reproduce this effect. Macrophages required from 2 to 3 h of pretreatment to express maximal P-L fusion, and this was ma...

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

    Science.gov (United States)

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

    2015-02-01

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

  20. Lysosomal Fusion Dysfunction as a Unifying Hypothesis for Alzheimer's Disease Pathology

    Directory of Open Access Journals (Sweden)

    Kristen E. Funk

    2012-01-01

    Full Text Available Alzheimer's disease is characterized pathologically by extracellular senile plaques, intracellular neurofibrillary tangles, and granulovacuolar degeneration. It has been debated whether these hallmark lesions are markers or mediators of disease progression, and numerous paradigms have been proposed to explain the appearance of each lesion individually. However, the unfaltering predictability of these lesions suggests a single pathological nidus central to disease onset and progression. One of the earliest pathologies observed in Alzheimer's disease is endocytic dysfunction. Here we review the recent literature of endocytic dysfunction with particular focus on disrupted lysosomal fusion and propose it as a unifying hypothesis for the three most-studied lesions of Alzheimer's disease.

  1. 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

    occurrence on light-demanding species via size-asymmetric competition for light, but a facilitative effect on shade-tolerant species. In order to compare the relative importance of tree cover, four models with different combinations of variables (climate, soil and tree cover) were run for each species. Then......, we simulated a removal experiment by comparing the elevational distribution of each species under high and low tree cover. Tree cover improved model performances and species’ response curves to a tree cover gradient varied depending on their shade tolerance, supporting the hypothesized antagonistic...... role. Results indicated that high tree cover causes range contraction, especially at the upper limit, for light-demanding species, whereas it causes shade-tolerant species to extend their range upwards and downwards. Tree cover thus drives plant-plant interactions to shape plant species distribution...

  2. Autophagy regulation revealed by SapM-induced block of autophagosome-lysosome fusion via binding RAB7

    Energy Technology Data Exchange (ETDEWEB)

    Hu, Dong, E-mail: austhudong@126.com [Institute of Infection and Immunology, Department of Medical Immunology, Medical School, Anhui University of Science and Technology, Huainan (China); Wu, Jing, E-mail: wujing8008@126.com [Institute of Infection and Immunology, Department of Medical Immunology, Medical School, Anhui University of Science and Technology, Huainan (China); Wang, Wan; Mu, Min; Zhao, Runpeng; Xu, Xuewei; Chen, Zhaoquan [Institute of Infection and Immunology, Department of Medical Immunology, Medical School, Anhui University of Science and Technology, Huainan (China); Xiao, Jian [School of Pharmacy, Wenzhou Medical College, Wenzhou (China); Hu, Fengyu; Yang, Yabo [Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou (China); Zhang, Rongbo, E-mail: lory456@126.com [Institute of Infection and Immunology, Department of Medical Immunology, Medical School, Anhui University of Science and Technology, Huainan (China)

    2015-05-29

    The mechanism underlying autophagy alteration by mycobacterium tuberculosis remains unclear. Our previous study shows LpqH, a lipoprotein of mycobacterium tuberculosis, can cause autophagosomes accumulation in murine macrophages. It is well known that SapM, another virulence factor, plays an important role in blocking phagosome-endosome fusion. However, the mechanism that SapM interferes with autophagy remains poorly defined. In this study, we report that SapM suppresses the autophagy flux by blocking autophagosome fusion with lysosome. Exposure to SapM results in accumulations of autophagosomes and decreased co-localization of autophagosome with lysosome. Molecularly, Rab7, a small GTPase, is blocked by SapM through its CT domain and is prevented from involvement of autophagosome-lysosome fusion. In conclusion, our study reveals that SapM takes Rab7 as a previously unknown target to govern a distinct molecular mechanism underlying autophagosome-lysosome fusion, which may bring light to a new thought about developing potential drugs or vaccines against tuberculosis. - Highlights: • A mechanism for disrupting autophagosome-lysosome fusion induced by SapM. • Rab7 is involved in SapM-inhibited autophagy. • SapM interacts with Rab7 by CT-domain. • CT-domain is indispensable to SapM-inhibited autophagy.

  3. Autophagy regulation revealed by SapM-induced block of autophagosome-lysosome fusion via binding RAB7

    International Nuclear Information System (INIS)

    The mechanism underlying autophagy alteration by mycobacterium tuberculosis remains unclear. Our previous study shows LpqH, a lipoprotein of mycobacterium tuberculosis, can cause autophagosomes accumulation in murine macrophages. It is well known that SapM, another virulence factor, plays an important role in blocking phagosome-endosome fusion. However, the mechanism that SapM interferes with autophagy remains poorly defined. In this study, we report that SapM suppresses the autophagy flux by blocking autophagosome fusion with lysosome. Exposure to SapM results in accumulations of autophagosomes and decreased co-localization of autophagosome with lysosome. Molecularly, Rab7, a small GTPase, is blocked by SapM through its CT domain and is prevented from involvement of autophagosome-lysosome fusion. In conclusion, our study reveals that SapM takes Rab7 as a previously unknown target to govern a distinct molecular mechanism underlying autophagosome-lysosome fusion, which may bring light to a new thought about developing potential drugs or vaccines against tuberculosis. - Highlights: • A mechanism for disrupting autophagosome-lysosome fusion induced by SapM. • Rab7 is involved in SapM-inhibited autophagy. • SapM interacts with Rab7 by CT-domain. • CT-domain is indispensable to SapM-inhibited autophagy

  4. How inhomogeneous salt distribution can affect the sensory properties of salt-reduced multi-component food: contribution of a mixture experimental design approach applied to pizza

    OpenAIRE

    Guilloux, Marion; PROST, Carole; Courcoux, Philippe; Le Bail, Alain

    2015-01-01

    Salt reduction in food is a major concern for public health authorities but remains a challenge for the food industry. Aims of this study are to modulate salt distribution between the ingredients of salt-reduced pizza (-30%) by modifying the salt content of each ingredient without changing the total salt content of pizza using mixture experimental design, to demonstrate its impact on sensory properties, and to determine the formulation with sensory properties, evaluated by Quantitative Descri...

  5. Time course of export of 14C-assimilates and their distribution pattern as affected by feeding time and night temperature in cucumber plants

    International Nuclear Information System (INIS)

    The export and distribution pattern of 14C fed intermittently to the single leaves of cucumber plants were investigated and compared with the results obtained on tomato plants in the previous report. The percentage of the total export of 14C 4 hours after feeding was about 20% irrespective of the feeding time, and amounted to 30 to 35% by the next morning. The ratio of the 14C exported in the daytime to that during the following night was about 1 : 1. The comparatively slow rate of increase of 14C export from 4 to 8 hours after the earlier feeding seemed to be due to the incorporation of 14C into starch and the resultant reduction of the pool size of 14C-sugars as they translocate. The export of 14C fed in the afternoon was retarded at low temperature at night, and especially in the plants at the 20-leaf stage. The percentage distribution to the lower parts including the roots 16 hours after feeding was comparatively high at the 7-leaf stage, and it decreased with rising night temperature. At the 20-leaf stage, it was highest at 12 deg C and decreased at both lower and higher temperatures. It was considered that the low values at low temperature was due to the delay of export. There was little difference between cucumber and tomato plants not only in the time course of the export but also in the effects of night temperature on the distribution pattern. (Kaihara, S.)

  6. Chemical and physical properties affecting strontium distribution coefficients of surficial-sediment samples at the Idaho National Engineering and Environmental Laboratory, Idaho

    International Nuclear Information System (INIS)

    The U.S. Geological Survey and Idaho State University, in cooperation with the U.S. Department of Energy, conducted a study to determine strontium distribution coefficients (Kds) of surficial sediments at the Idaho National Engineering and Environmental Laboratory (INEEL). Batch experiments using synthesized aqueous solutions were used to determine Kds, which describe the distribution of a solute between the solution and solid phase, of 20 surficial-sediment samples from the INEEL. The Kds for the 20 surficial-sediment samples ranged from 36 to 275 ml/g. Many properties of both the synthesized aqueous solutions and sediments used in the experiments also were determined. Solution properties determined were initial and equilibrium concentrations of calcium, magnesium, and strontium, pH and specific conductance, and initial concentrations of potassium and sodium. Sediment properties determined were grainsize distribution, bulk mineralogy, whole-rock major-oxide and strontium and barium concentrations, and Brunauer-Emmett-Teller (BET) surface area. Solution and sediment properties were correlated with strontium Kds of the 20 surficial sediments using Pearson correlation coefficients. Solution properties with the strongest correlations with strontium Kds were equilibrium pH and equilibrium calcium concentration correlation coefficients, 0.6598 and -0.6518, respectively. Sediment properties with the strongest correlations with strontium Kds were manganese oxide (MnO), BET surface area, and the >4.75-mm-grain-size fraction correlation coefficients, 0.7054, 0.7022, and -0.6660, respectively. Effects of solution properties on strontium Kds were interpreted as being due to competition among similarly charged and sized cations in solution for strontium-sorption sites; effects of sediment properties on strontium Kds were interpreted as being surface-area related. Multivariate analyses of these solution and sediment properties resulted in r2 values of 0.8071 when all five

  7. Factors affecting the potential of direct load control for non-generating utilities. Final report. [Distribution and wholesale power supply interaction

    Energy Technology Data Exchange (ETDEWEB)

    None

    1979-04-01

    Several alternatives are available for achieving load management, including direct or voluntary control of customer loads, customer or utility energy storage systems for diurnal load shifting, and expanded interconnection and operation of electric power systems. All of these alternatives are available to the fully integrated (generating, transmitting and distributing) electric utility and the analysis of their effects encompasses the power supply and delivery system. However, the costs and benefits of the alternatives to the fully integrated electric utility are perhaps not so obvious. Therefore, by considering a non-generating utility, this analysis focuses upon the distribution system and wholesale power supply interaction as a step toward an analysis including the power supply and delivery system. This report develops an analysis procedure and discusses some of the relevant factors to be consdered in the application of direct load control for a non-generating utility system. The analysis concentrates on the distribution system only to determine the effect of rates and payback as a result of direct load control. Thus, the study is responsive to the specific needs of the non-generating utility. This analysis of direct load control encompasses the determination of those loads amenable to control, the selection of a suitable one-way communications system to rend control and the estimation of expected benefits and costs. The complementary functions to the application of direct load control such as automatic meter reading via the addition of a bi-directional communications system and voltage control are not included in the analysis but are detailed for future consideration.

  8. Estimation of uranium isotopes in soil affected by Fukushima nuclear power plant accident and its mobility based on distribution coefficient and soil properties

    Energy Technology Data Exchange (ETDEWEB)

    Sahoo, S.K. [National Institute of Radiological Sciences (Japan); Mishra, S. [Bhabha Atomic Research Centre (India); Sorimachi, A.; Hosoda, M.; Tokonami, S. [Hirosaki University (Japan); Kritsananuwat, R. [Tokyo Metropolitan University (Japan); Ishikawa, T. [Fukushima Medical University (Japan)

    2014-07-01

    An extraordinary earthquake of magnitude 9.0 followed by Tsunami on 11 March 2011 caused serious nuclear accident at the Fukushima Daiichi Nuclear Power Plant (FDNPP) about 250 km north to Tokyo, capital of Japan. This resulted in radioactive contamination due to deposition of long-lived radionuclides. Contaminated soil can cause an enhanced radiation exposure even after many years. Depending upon environmental conditions radionuclides can be mobilized to aquatic systems. Therefore, the fate and transfer of these radionuclides in the soil water system is very important for radiation protection and dose assessment. In the present study, emphasis has been given on isotope ratio measurement of uranium that may give some idea about its contamination during accident. Soil and water samples were collected from contaminated areas around FDNPP. Inductively coupled plasma mass spectrometry (ICPMS) is used for total uranium concentration and thermal ionization mass spectrometry (TIMS) has been used for uranium isotopes measurement. Extraction chromatography has been used for the separation of uranium. We have observed, isotope ratio {sup 235}U/{sup 238}U is of natural origin, however in a few soil samples {sup 236}U has been detected. For the migration behavior, its distribution coefficient (K{sub d}) has been determined using laboratory batch method. Depleted uranium is used as tracer for uranium K{sub d} estimation. Chemical characterization of soil with respect to different parameters has been carried out. The effect of these soil parameters on distribution coefficient of uranium has been studied in order to explain the radionuclide mobility in this particular area. The distribution coefficient values for uranium are found to vary from 30-35679 L/Kg. A large variation in the distribution coefficient values shows the retention or mobility of uranium is highly dependent on soil characteristics in the particular area. This variation is explained with respect to pH, Fe, Mn

  9. Pathogenic LRRK2 mutations, through increased kinase activity, produce enlarged lysosomes with reduced degradative capacity and increase ATP13A2 expression.

    Science.gov (United States)

    Henry, Anastasia G; Aghamohammadzadeh, Soheil; Samaroo, Harry; Chen, Yi; Mou, Kewa; Needle, Elie; Hirst, Warren D

    2015-11-01

    Lysosomal dysfunction plays a central role in the pathogenesis of several neurodegenerative disorders, including Parkinson's disease (PD). Several genes linked to genetic forms of PD, including leucine-rich repeat kinase 2 (LRRK2), functionally converge on the lysosomal system. While mutations in LRRK2 are commonly associated with autosomal-dominant PD, the physiological and pathological functions of this kinase remain poorly understood. Here, we demonstrate that LRRK2 regulates lysosome size, number and function in astrocytes, which endogenously express high levels of LRRK2. Expression of LRRK2 G2019S, the most common pathological mutation, produces enlarged lysosomes and diminishes the lysosomal capacity of these cells. Enlarged lysosomes appears to be a common phenotype associated with pathogenic LRRK2 mutations, as we also observed this effect in cells expressing other LRRK2 mutations; R1441C or Y1699C. The lysosomal defects associated with these mutations are dependent on both the catalytic activity of the kinase and autophosphorylation of LRRK2 at serine 1292. Further, we demonstrate that blocking LRRK2's kinase activity, with the potent and selective inhibitor PF-06447475, rescues the observed defects in lysosomal morphology and function. The present study also establishes that G2019S mutation leads to a reduction in lysosomal pH and increased expression of the lysosomal ATPase ATP13A2, a gene linked to a parkinsonian syndrome (Kufor-Rakeb syndrome), in brain samples from mouse and human LRRK2 G2019S carriers. Together, these results demonstrate that PD-associated LRRK2 mutations perturb lysosome function in a kinase-dependent manner, highlighting the therapeutic promise of LRRK2 kinase inhibitors in the treatment of PD. PMID:26251043

  10. Glucocerebrosidase deficiency and lysosomal storage of glucocerebroside induced in cultured macrophages

    International Nuclear Information System (INIS)

    A cell culture model simulating the genetic deficiency of glucocerebrosidase has been developed, utilizing macrophages and conduritol B epoxide (CBE), the specific irreversible inhibitor of the enzyme. Rat peritoneal macrophage glucocerebrosidase was completely inhibited when cells were treated with 10 +M CBE for 16 h or 100 μM CBE for 2 h. The t/sub 1/2/ of inactivation was 30 min at 10+M concentration. When cells were washed free of CBE, the enzyme activity reappeared linearly with time, reaching 50% of control activity 48 h after removal of the inhibitor. CBE-treated macrophages have normal phagocytic activity toward [3H]glycine-coupled latex beads and a normal number of mannose receptors. CBE was found to have no effect on other lysosomal enzymes. When [14C]glucocerebroside, encapsulated in multilamellar liposomes with α-D-mannopyranoside covalently coupled to the surface, was fed to glucocerebrosidase-depleted macrophages, the radiolabelled glycolipid accumulated and was undegraded. Subcellular fractionation on a Percol density gradient demonstrated that the stored glucocerebroside in the CBE-treated macrophages was localized in lysosomes

  11. Influence of cucumariosides upon intracellular [Ca2+]i and lysosomal activity of macrophages.

    Science.gov (United States)

    Agafonova, Irina G; Aminin, Dmitry L; Avilov, Sergey A; Stonik, Valentin A

    2003-11-19

    Biological effects of the triterpene glycosides, cucumariosides A(2)-2 and A(7)-1 from the edible sea cucumber Cucumaria japonica and their aglycones were investigated using embryos of the sea urchin Strongylocentrotus nudus and the BALB/C line mouse peritoneal macrophages. Cucumariosides were highly cytotoxic in a sea urchin embryo development test with EC(50) values of 0.3 and 1.98 microg/mL, respectively. The aglycone was completely lacking in cytotoxicity. In subtoxic concentrations (0.001-0.1 microg/mL), cucumarioside A(2)-2 showed more then 2-fold stimulation of lysosomal activity and induced a rapid short-term increase in cytosolic Ca(2+) content in mouse macrophages. The maximal stimulatory effect was detected after 1-2 h of cultivation of cells with this glycoside. Cucumarioside A(7)-1 demonstrated more weak effects and even slightly inhibited lysosomal activity, while the aglycone was completely ineffective. At the toxic concentration (1 microg/mL), cucumarioside A(2)-2 induced the sharp irreversable increase of intracellular Ca(2+) concentration. We suggested that cucumariosides, especially A(2)-2, may act as Ca(2+) agonists due to their membranolytic properties. PMID:14611158

  12. Lyso-glycosphingolipid abnormalities in different murine models of lysosomal storage disorders.

    Science.gov (United States)

    Ferraz, Maria J; Marques, André R A; Gaspar, Paulo; Mirzaian, Mina; van Roomen, Cindy; Ottenhoff, Roelof; Alfonso, Pilar; Irún, Pilar; Giraldo, Pilar; Wisse, Patrick; Sá Miranda, Clara; Overkleeft, Herman S; Aerts, Johannes M

    2016-02-01

    In lysosomal glycosphingolipid storage disorders, marked elevations in corresponding glycosphingoid bases (lyso-glycosphingolipids) have been reported, such as galactosylsphingosine in Krabbe disease, glucosylsphingosine in Gaucher disease and globotriaosylsphingosine in Fabry disease. Using LC–MS/MS, we comparatively investigated the occurrence of abnormal lyso-glycosphingolipids in tissues and plasma of mice with deficiencies in lysosomal α-galactosidase A, glucocerebrosidase and galactocerebrosidase. The nature and specificity of lyso-glycosphingolipid abnormalities are reported and compared to that in correspondingly more abundant N-acylated glycosphingolipids. Specific elevations in tissue and plasma globotriaosylsphingosine were detected in α-galactosidase A-deficient mice; glucosylsphingosine in glucocerebrosidase-deficient mice and galactosylsphingosine in galactocerebrosidase-deficient animals. A similar investigation was conducted for two mouse models of Niemann Pick type C (Npc1nih and Npc1nmf164), revealing significant tissue elevation of several neutral glycosphingolipids and concomitant increased plasma glucosylsphingosine. This latter finding was recapitulated by analysis of plasma of NPC patients. The value of plasma glucosylsphingosine in biochemical confirmation of the diagnosis of NPC is discussed. PMID:26750750

  13. WO3/Pt nanoparticles promote light-induced lipid peroxidation and lysosomal instability within tumor cells

    Science.gov (United States)

    Clark, Andrea J.; Petty, Howard R.

    2016-02-01

    Although metal-metal oxide nanoparticles have attracted considerable interest as catalysts, they have attracted little interest in nanomedicine. This is likely due to the fact that metal oxide semiconductors generally require biologically harmful ultraviolet excitation. In contrast, this study focuses upon WO3/Pt nanoparticles, which can be excited by visible light. To optimize the nanoparticles’ catalytic performance, platinization was performed at alkaline pH. These nanoparticles destroyed organic dyes, consumed dissolved oxygen and produced hydroxyl radicals. 4T1 breast cancer cells internalized WO3/Pt nanoparticles within the membrane-bound endo-lysosomal compartment as shown by electron and fluorescence microscopy. During visible light exposure, but not in darkness, WO3/Pt nanoparticles manufacture reactive oxygen species, promote lipid peroxidation, and trigger lysosomal membrane disruption. As cells of the immune system degrade organic molecules, produce reactive oxygen species, and activate the lipid peroxidation pathway within target cells, these nanoparticles mimic the chemical attributes of immune effector cells. These biomimetic nanoparticles should become useful in managing certain cancers, especially ocular cancer.

  14. Retromer Ensures the Degradation of Autophagic Cargo by Maintaining Lysosome Function in Drosophila.

    Science.gov (United States)

    Maruzs, Tamás; Lőrincz, Péter; Szatmári, Zsuzsanna; Széplaki, Szilvia; Sándor, Zoltán; Lakatos, Zsolt; Puska, Gina; Juhász, Gábor; Sass, Miklós

    2015-10-01

    The retromer is an evolutionarily conserved coat complex that consists of Vps26, Vps29, Vps35 and a heterodimer of sorting nexin (Snx) proteins in yeast. Retromer mediates the recycling of transmembrane proteins from endosomes to the trans-Golgi network, including receptors that are essential for the delivery of hydrolytic enzymes to lysosomes. Besides its function in lysosomal enzyme receptor recycling, involvement of retromer has also been proposed in a variety of vesicular trafficking events, including early steps of autophagy and endocytosis. Here we show that the late stages of autophagy and endocytosis are impaired in Vps26 and Vps35 deficient Drosophila larval fat body cells, but formation of autophagosomes and endosomes is not compromised. Accumulation of aberrant autolysosomes and amphisomes in the absence of retromer function appears to be the consequence of decreased degradative capacity, as they contain undigested cytoplasmic material. Accordingly, we show that retromer is required for proper cathepsin L trafficking mainly independent of LERP, the Drosophila homolog of the cation-independent mannose 6-phosphate receptor. Finally, we find that Snx3 and Snx6 are also required for proper autolysosomal degradation in Drosophila larval fat body cells. PMID:26172538

  15. Newborn Screening for Lysosomal Storage Disorders: Views of Genetic Healthcare Providers.

    Science.gov (United States)

    Lisi, Emily C; McCandless, Shawn E

    2016-04-01

    Lysosomal storage diseases (LSDs), lysosomal enzyme deficiencies causing multi-system organ damage, have come to the forefront in newborn screening (NBS) initiatives due to new screening technologies and emerging treatments. We developed a qualitative discussion tool to explore opinions of genetic healthcare providers (HCPs) regarding population-based NBS for MPS types 1 and 2, Pompe, Gaucher, Fabry, and Krabbe diseases. Thirty-eight telephone interviews conducted by a single researcher were analyzed and coded for thematic trends. Six major themes emerged: 1) treatment availability and efficacy is crucial; 2) early age of disease onset is important; 3) ambiguity regarding prognosis is undesirable; 4) parents' ability to make reproductive decisions is seen by some as a benefit of NBS; 5) paucity of resources for follow-up exists; and 6) the decision-making process for adding conditions to mandated NBS is concerning to HCPs. Among the LSDs discussed, Pompe was considered most appropriate, and Krabbe least appropriate, for NBS. MPS1 and MPS2 were overall considered favorably for screening, but MPS1 ranked higher, due to a perception of better efficacy of therapeutic options. Fabry and Gaucher diseases were viewed less favorably due to later age of onset. The themes identified in this study must be addressed by decision-makers in expanding NBS for LSDs and may be applied to many diseases being considered for NBS in the future. PMID:26315880

  16. The role of autophagic and lysosomal pathways in ischemic brain injury******

    Institute of Scientific and Technical Information of China (English)

    Zhaohua Gu; Nan Shi; Qian Zhang; Wei Zhang; Meizhen Zhao; Xiaojiang Sun; Yinyi Sun; Kangyong Liu; Fen Wang; Ting Zhang; Qiang Li; Liwei Shen; Ling Zhou; Liang Dong

    2013-01-01

    Autophagy is involved in neural cel death after cerebral ischemia. Our previous studies showed that rapamycin-induced autophagy decreased the rate of apoptosis, but the rate of apoptosis was creased after the autophagy inhibitor, 3-methyladenine, was used. In this study, a suture-occluded method was performed to generate a rat model of brain ischemia. Under a transmission electron microscope, autophagic bodies and autophagy lysosomes were markedly accumulated in neurons at 4 hours post brain ischemic injury, with their numbers gradual y reducing over time. Western blotting demonstrated that protein levels of light chain 3-II and cathepsin B were significantly in-creased within 4 hours of ischemic injury, but these levels were not persistently upregulated over time. Confocal microscopy showed that autophagy was mainly found in neurons with positive light chain 3 signal. Injection of rapamycin via tail vein promoted the occurrence of autophagy in rat brain tissue after cerebral ischemia and elevated light chain 3 and cathepsin B expression. However, in-jection of 3-methyladenine significantly diminished light chain 3-II and cathepsin B expression. Results verified that autophagic and lysosomal activity is increased in ischemic neurons. Abnormal components in cel s can be eliminated through upregulating cel autophagy or inhibiting autophagy after ischemic brain injury, resulting in a dynamic balance of substances in cel s. Moreover, drugs that interfere with autophagy may be potential therapies for the treatment of brain injury.

  17. Mucopolysaccharidosis IIIB, a lysosomal storage disease, triggers a pathogenic CNS autoimmune response

    Directory of Open Access Journals (Sweden)

    Popovich Phillip G

    2010-07-01

    Full Text Available Abstract Background Recently, using a mouse model of mucopolysaccharidosis (MPS IIIB, a lysosomal storage disease with severe neurological deterioration, we showed that MPS IIIB neuropathology is accompanied by a robust neuroinflammatory response of unknown consequence. This study was to assess whether MPS IIIB lymphocytes are pathogenic. Methods Lymphocytes from MPS IIIB mice were adoptively transferred to naïve wild-type mice. The recipient animals were then evaluated for signs of disease and inflammation in the central nervous system. Results Our results show for the first time, that lymphocytes isolated from MPS IIIB mice caused a mild paralytic disease when they were injected systemically into naïve wild-type mice. This disease is characterized by mild tail and lower trunk weakness with delayed weight gain. The MPS IIIB lymphocytes also trigger neuroinflammation within the CNS of recipient mice characterized by an increase in transcripts of IL2, IL4, IL5, IL17, TNFα, IFNα and Ifi30, and intraparenchymal lymphocyte infiltration. Conclusions Our data suggest that an autoimmune response directed at CNS components contributes to MPS IIIB neuropathology independent of lysosomal storage pathology. Adoptive transfer of purified T-cells will be needed in future studies to identify specific effector T-cells in MPS IIIB neuroimmune pathogenesis.

  18. Interconversion of the specificities of human lysosomal enzymes associated with Fabry and Schindler diseases.

    Science.gov (United States)

    Tomasic, Ivan B; Metcalf, Matthew C; Guce, Abigail I; Clark, Nathaniel E; Garman, Scott C

    2010-07-01

    The human lysosomal enzymes alpha-galactosidase (alpha-GAL, EC 3.2.1.22) and alpha-N-acetylgalactosaminidase (alpha-NAGAL, EC 3.2.1.49) share 46% amino acid sequence identity and have similar folds. The active sites of the two enzymes share 11 of 13 amino acids, differing only where they interact with the 2-position of the substrates. Using a rational protein engineering approach, we interconverted the enzymatic specificity of alpha- GAL and alpha-NAGAL. The engineered alpha-GAL (which we call alpha-GAL(SA)) retains the antigenicity of alpha-GAL but has acquired the enzymatic specificity of alpha-NAGAL. Conversely, the engineered alpha-NAGAL (which we call alpha-NAGAL(EL)) retains the antigenicity of alpha-NAGAL but has acquired the enzymatic specificity of the alpha-GAL enzyme. Comparison of the crystal structures of the designed enzyme alpha-GAL(SA) to the wild-type enzymes shows that active sites of alpha-GAL(SA) and alpha-NAGAL superimpose well, indicating success of the rational design. The designed enzymes might be useful as non-immunogenic alternatives in enzyme replacement therapy for treatment of lysosomal storage disorders such as Fabry disease. PMID:20444686

  19. Interconversion of the Specificities of Human Lysosomal Enzymes Associated with Fabry and Schindler Diseases

    Energy Technology Data Exchange (ETDEWEB)

    Tomasic, Ivan B.; Metcalf, Matthew C.; Guce, Abigail I.; Clark, Nathaniel E.; Garman, Scott C. (UMASS, Amherst)

    2010-09-03

    The human lysosomal enzymes {alpha}-galactosidase ({alpha}-GAL, EC 3.2.1.22) and {alpha}-N-acetylgalactosaminidase ({alpha}-NAGAL, EC 3.2.1.49) share 46% amino acid sequence identity and have similar folds. The active sites of the two enzymes share 11 of 13 amino acids, differing only where they interact with the 2-position of the substrates. Using a rational protein engineering approach, we interconverted the enzymatic specificity of {alpha}-GAL and {alpha}-NAGAL. The engineered {alpha}-GAL (which we call {alpha}-GALSA) retains the antigenicity of {alpha}-GAL but has acquired the enzymatic specificity of {alpha}-NAGAL. Conversely, the engineered {alpha}-NAGAL (which we call {alpha}-NAGAL{sup EL}) retains the antigenicity of {alpha}-NAGAL but has acquired the enzymatic specificity of the {alpha}-GAL enzyme. Comparison of the crystal structures of the designed enzyme {alpha}-GAL{sup SA} to the wild-type enzymes shows that active sites of {alpha}-GAL{sup SA} and {alpha}-NAGAL superimpose well, indicating success of the rational design. The designed enzymes might be useful as non-immunogenic alternatives in enzyme replacement therapy for treatment of lysosomal storage disorders such as Fabry disease.

  20. Low Serum Lysosomal Acid Lipase Activity Correlates with Advanced Liver Disease

    Directory of Open Access Journals (Sweden)

    Eyal Shteyer

    2016-02-01

    Full Text Available Fatty liver has become the most common liver disorder and is recognized as a major health burden in the Western world. The causes for disease progression are not fully elucidated but lysosomal impairment is suggested. Here we evaluate a possible role for lysosomal acid lipase (LAL activity in liver disease. To study LAL levels in patients with microvesicular, idiopathic cirrhosis and nonalcoholic fatty liver disease (NAFLD. Medical records of patients with microvesicular steatosis, cryptogenic cirrhosis and NAFLD, diagnosed on the basis of liver biopsies, were included in the study. Measured serum LAL activity was correlated to clinical, laboratory, imaging and pathological data. No patient exhibited LAL activity compatible with genetic LAL deficiency. However, serum LAL activity inversely predicted liver disease severity. A LAL level of 0.5 was the most sensitive for detecting both histologic and noninvasive markers for disease severity, including lower white blood cell count and calcium, and elevated γ-glutamyltransferase, creatinine, glucose, glycated hemoglobin, uric acid and coagulation function. Serum LAL activity <0.5 indicates severe liver injury in patients with fatty liver and cirrhosis. Further studies should define the direct role of LAL in liver disease severity and consider the possibility of replacement therapy.