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Sample records for 5b affects lysosomal

  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. 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...

  3. 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.

  4. 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

  5. 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.

  6. 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

  7. 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...

  8. 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...

  9. 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 ...

  10. 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

    2016-05-02

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

  11. 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

  12. 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

  13. 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...

  14. 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 ...

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

    Directory of Open Access Journals (Sweden)

    Aleksandra Glogowska

    2012-05-01

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

  16. Lysosomal Storage Diseases

    Directory of Open Access Journals (Sweden)

    Joseph Alroy DVM, DACVP

    2014-03-01

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

  17. 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...

  18. 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

  19. 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.

  20. Mitochondrial Dysfunction in Lysosomal Storage Disorders

    Directory of Open Access Journals (Sweden)

    Mario de la Mata

    2016-10-01

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

  1. 34 CFR 5b.13 - Fees.

    Science.gov (United States)

    2010-07-01

    ... 34 Education 1 2010-07-01 2010-07-01 false Fees. 5b.13 Section 5b.13 Education Office of the Secretary, Department of Education PRIVACY ACT REGULATIONS § 5b.13 Fees. (a) Policy. Where applicable, fees for copying records will be charged in accordance with the schedule set forth in this section....

  2. 34 CFR 5b.11 - Exempt systems.

    Science.gov (United States)

    2010-07-01

    ... Chief Information Officer, Regulatory Information Management Group, Washington, DC 20202-4651. (f... 34 Education 1 2010-07-01 2010-07-01 false Exempt systems. 5b.11 Section 5b.11 Education Office of the Secretary, Department of Education PRIVACY ACT REGULATIONS § 5b.11 Exempt systems. (a)...

  3. 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

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

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    Leen Mathys

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

  5. The lysosome and neurodegenerative diseases

    Institute of Scientific and Technical Information of China (English)

    Lisha Zhang; Rui Sheng; Zhenghong Qin

    2009-01-01

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

  6. Neuroinflammatory paradigms in lysosomal storage diseases

    Directory of Open Access Journals (Sweden)

    Megan Elizabeth Bosch

    2015-10-01

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

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

    Science.gov (United States)

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

    2000-07-01

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

  8. 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...

  9. 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...

  10. 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...

  11. 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‐

  12. Conventional kinesin KIF5B mediates adiponectin secretion in 3T3-L1 adipocytes.

    Science.gov (United States)

    Cui, Ju; Pang, Jing; Lin, Ya-Jun; Jiang, Ping; Gong, Huan; Wang, Zai; Li, Jian; Cai, Jian-Ping; Huang, Jian-Dong; Zhang, Tie-Mei

    2016-08-01

    Insulin stimulates adiponectin secretion and glucose transporter type 4 (GLUT4) translocation in adipocyte to regulate metabolism homeostasis. Similar to GLUT4 translocation, intracellular trafficking and release of adiponectin in adipocytes relies on the trans-Golgi network and endosomal system. Recent studies show that the heavy chain of conventional kinesin (KIF5B) mediates GLUT4 translocation in murine 3T3-L1 adipocytes, however, the motor machinery involved in mediating intracellular trafficking and release of adiponectin is unknown. Here, we examined the role of KIF5B in the regulation of adiponectin secretion. The KIF5B level was up-regulated during 3T3-L1 adipogenesis. This increase in cytosolic KIF5B was synchronized with the induction of adiponectin. Endogenous KIF5B and adiponectin were partially colocalized at the peri-nuclear and cytosolic regions. In addition, adiponectin-containing vesicles were co-immunoprecipitated with KIF5B. Knockdown of KIF5B resulted in a marked inhibition of adiponectin secretion and overexpression of KIF5B enhanced adiponectin release, whereas leptin secretion was not affected by changes in KIF5B expression. These data suggest that the secretion of adiponectin, but not leptin, is dependent on functional KIF5B. PMID:27264953

  13. 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,…

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

    Science.gov (United States)

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

    2000-01-01

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

  15. 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.

  16. 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...

  17. 45 CFR 5b.4 - Maintenance of records.

    Science.gov (United States)

    2010-10-01

    ... 45 Public Welfare 1 2010-10-01 2010-10-01 false Maintenance of records. 5b.4 Section 5b.4 Public Welfare DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL ADMINISTRATION PRIVACY ACT REGULATIONS § 5b.4 Maintenance of records. (a) No record will be maintained by the Department unless: (1) It is relevant and necessary to accomplish a...

  18. 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.

  19. 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 

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

    Science.gov (United States)

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

    2016-04-01

    To mediate the degradation of biomacromolecules, lysosomes must traffic towards cargo-carrying vesicles for subsequent membrane fusion or fission. Mutations of the lysosomal Ca(2+) channel TRPML1 cause lysosomal storage disease (LSD) characterized by disordered lysosomal membrane trafficking in cells. Here we show that TRPML1 activity is required to promote Ca(2+)-dependent centripetal movement of lysosomes towards the perinuclear region (where autophagosomes accumulate) following autophagy induction. ALG-2, an EF-hand-containing protein, serves as a lysosomal Ca(2+) sensor that associates physically with the minus-end-directed dynactin-dynein motor, while PtdIns(3,5)P(2), a lysosome-localized phosphoinositide, acts upstream of TRPML1. Furthermore, the PtdIns(3,5)P(2)-TRPML1-ALG-2-dynein signalling is necessary for lysosome tubulation and reformation. In contrast, the TRPML1 pathway is not required for the perinuclear accumulation of lysosomes observed in many LSDs, which is instead likely to be caused by secondary cholesterol accumulation that constitutively activates Rab7-RILP-dependent retrograde transport. Ca(2+) release from lysosomes thus provides an on-demand mechanism regulating lysosome motility, positioning and tubulation. PMID:26950892

  1. Analysis list: Stat5b [Chip-atlas[Archive

    Lifescience Database Archive (English)

    Full Text Available Stat5b Blood,Breast + mm9 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/target/Sta...t5b.1.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/target/Stat5b.5.tsv http://dbarchive.biosciencedbc....jp/kyushu-u/mm9/target/Stat5b.10.tsv http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/colo/Stat5b.Blood.tsv,http://dbarchive.bioscien...cedbc.jp/kyushu-u/mm9/colo/Stat5b.Breast.tsv http://dbarchive.bioscien...cedbc.jp/kyushu-u/mm9/colo/Blood.gml,http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/colo/Breast.gml ...

  2. 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

    2016-01-01

    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

  3. 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.

  4. STAT5B deficiency: Impacts on human growth and immunity.

    Science.gov (United States)

    Hwa, Vivian

    2016-06-01

    Growth hormone (GH) promotes postnatal human growth primarily by regulating insulin-like growth factor (IGF)-I production through activation of the GH receptor (GHR)-signal transducer and activator of transcription (STAT)-5B signaling cascade. The critical importance of STAT5B in human IGF-I production was confirmed with the identification of the first homozygous, autosomal recessive, STAT5B mutation in a young female patient who phenotypically resembled patients with classical growth hormone insensitivity (GHI) syndrome (Laron syndrome) due to mutations in the GHR gene, presenting with severe postnatal growth failure and marked IGF-I deficiency. Of note, the closely related STAT5A, which shares >95% amino acid identity with STAT5B, could not compensate for loss of functional STAT5B. To date, 7 homozygous, inactivating, STAT5B mutations in 10 patients have been reported. STAT5B deficient patients, unlike patients deficient in GHR, can also present with a novel, potentially fatal, primary immunodeficiency, which can manifest as chronic pulmonary disease. STAT5B deficiency may be underestimated in endocrine, immunology and pulmonary clinics. PMID:26703237

  5. Variants of MUC5B minisatellites and the susceptibility of bladder cancer.

    Science.gov (United States)

    Ahn, Eun-Kyung; Kim, Wun-Jae; Kwon, Jeong-Ah; Choi, Phil-Jo; Kim, Woo Jin; Sunwoo, Yangil; Heo, Jeonghoon; Leem, Sun-Hee

    2009-04-01

    The human MUC5B gene, which is primarily expressed in the tracheobronchial tract, is clustered to chromosome 11p15.5 with three other secreted gel-forming mucins, MUC6, MUC2, and MUC5AC. In this study, we identified seven variable number of tandem repeats (VNTRs; minisatellites) from the entire MUC5B region. Six (MUC5B-MS1, -MS2, -MS3, -MS4, -MS5, and -MS7) of the seven minisatellites evaluated in this study were novel minisatellites, but the MUC5B-MS6 minisatellite was described in a previous study. These minisatellites of MUC5B were analyzed in genomic DNA extracted from controls, cancer patients, and multigenerational families. Three (MUC5B-MS3, -MS6, and -MS7) of the seven minisatellites were found to be polymorphic and transmitted through meiosis following Mendelian inheritance in seven families; therefore, these minisatellite polymorphisms could be useful as markers for paternity mapping and DNA fingerprinting. In addition, we evaluated allelic variation in these minisatellites to determine if such variation affected the susceptibility to various carcinomas. To accomplish this, we conducted a case-control study in which the genomic DNA of 789 cancer-free controls and cancer patients with five types of cancer were compared. A statistically significant association between the long rare MUC5B-MS6 alleles and the occurrence of bladder cancer was identified in the younger group (<60; odds ratio, 4.54; 95% confidence interval, 1.0-20.7; p=0.03). This observation suggests that the long rare MUC5B-MS6 alleles evaluated in this study could be used to identify the risk of bladder cancer. PMID:19191526

  6. Kinesin 5B (KIF5B is required for progression through female meiosis and proper chromosomal segregation in mitotic cells.

    Directory of Open Access Journals (Sweden)

    Dawit Kidane

    Full Text Available The fidelity of chromosomal segregation during cell division is important to maintain chromosomal stability in order to prevent cancer and birth defects. Although several spindle-associated molecular motors have been shown to be essential for cell division, only a few chromosome arm-associated motors have been described. Here, we investigated the role of Kinesin 5b (Kif5b during female mouse meiotic cell development and mitotic cell division. RNA interference (RNAi-mediated silencing of Kif5b in mouse oocytes induced significant delay in germinal vesicle breakdown (GVBD and failure in extrusion of the first polar body (PBE. In mitotic cells, knockdown of Kif5b leads to centrosome amplification and a chromosomal segregation defect. These data suggest that KIF5B is critical in suppressing chromosomal instability at the early stages of female meiotic cell development and mitotic cell division.

  7. Kinesin 5B (KIF5B) is required for progression through female meiosis and proper chromosomal segregation in mitotic cells.

    Science.gov (United States)

    Kidane, Dawit; Sakkas, Denny; Nottoli, Timothy; McGrath, James; Sweasy, Joann B

    2013-01-01

    The fidelity of chromosomal segregation during cell division is important to maintain chromosomal stability in order to prevent cancer and birth defects. Although several spindle-associated molecular motors have been shown to be essential for cell division, only a few chromosome arm-associated motors have been described. Here, we investigated the role of Kinesin 5b (Kif5b) during female mouse meiotic cell development and mitotic cell division. RNA interference (RNAi)-mediated silencing of Kif5b in mouse oocytes induced significant delay in germinal vesicle breakdown (GVBD) and failure in extrusion of the first polar body (PBE). In mitotic cells, knockdown of Kif5b leads to centrosome amplification and a chromosomal segregation defect. These data suggest that KIF5B is critical in suppressing chromosomal instability at the early stages of female meiotic cell development and mitotic cell division.

  8. Muc5b is required for airway defence

    Science.gov (United States)

    Roy, Michelle G.; Livraghi-Butrico, Alessandra; Fletcher, Ashley A.; McElwee, Melissa M.; Evans, Scott E.; Boerner, Ryan M.; Alexander, Samantha N.; Bellinghausen, Lindsey K.; Song, Alfred S.; Petrova, Youlia M.; Tuvim, Michael J.; Adachi, Roberto; Romo, Irlanda; Bordt, Andrea S.; Bowden, M. Gabriela; Sisson, Joseph H.; Woodruff, Prescott G.; Thornton, David J.; Rousseau, Karine; de La Garza, Maria M.; Moghaddam, Seyed J.; Karmouty-Quintana, Harry; Blackburn, Michael R.; Drouin, Scott M.; Davis, C. William; Terrell, Kristy A.; Grubb, Barbara R.; O'Neal, Wanda K.; Flores, Sonia C.; Cota-Gomez, Adela; Lozupone, Catherine A.; Donnelly, Jody M.; Watson, Alan M.; Hennessy, Corinne E.; Keith, Rebecca C.; Yang, Ivana V.; Barthel, Lea; Henson, Peter M.; Janssen, William J.; Schwartz, David A.; Boucher, Richard C.; Dickey, Burton F.; Evans, Christopher M.

    2014-01-01

    Respiratory surfaces are exposed to billions of particulates and pathogens daily. A protective mucus barrier traps and eliminates them through mucociliary clearance (MCC). However, excessive mucus contributes to transient respiratory infections and to the pathogenesis of numerous respiratory diseases. MUC5AC and MUC5B are evolutionarily conserved genes that encode structurally related mucin glycoproteins, the principal macromolecules in airway mucus. Genetic variants are linked to diverse lung diseases, but specific roles for MUC5AC and MUC5B in MCC, and the lasting effects of their inhibition, are unknown. Here we show that mouse Muc5b (but not Muc5ac) is required for MCC, for controlling infections in the airways and middle ear, and for maintaining immune homeostasis in mouse lungs, whereas Muc5ac is dispensable. Muc5b deficiency caused materials to accumulate in upper and lower airways. This defect led to chronic infection by multiple bacterial species, including Staphylococcus aureus, and to inflammation that failed to resolve normally. Apoptotic macrophages accumulated, phagocytosis was impaired, and interleukin-23 (IL-23) production was reduced in Muc5b-/- mice. By contrast, in mice that transgenically overexpress Muc5b, macrophage functions improved. Existing dogma defines mucous phenotypes in asthma and chronic obstructive pulmonary disease (COPD) as driven by increased MUC5AC, with MUC5B levels either unaffected or increased in expectorated sputum. However, in many patients, MUC5B production at airway surfaces decreases by as much as 90%. By distinguishing a specific role for Muc5b in MCC, and by determining its impact on bacterial infections and inflammation in mice, our results provide a refined framework for designing targeted therapies to control mucin secretion and restore MCC.

  9. Muc5b is required for airway defence.

    Science.gov (United States)

    Roy, Michelle G; Livraghi-Butrico, Alessandra; Fletcher, Ashley A; McElwee, Melissa M; Evans, Scott E; Boerner, Ryan M; Alexander, Samantha N; Bellinghausen, Lindsey K; Song, Alfred S; Petrova, Youlia M; Tuvim, Michael J; Adachi, Roberto; Romo, Irlanda; Bordt, Andrea S; Bowden, M Gabriela; Sisson, Joseph H; Woodruff, Prescott G; Thornton, David J; Rousseau, Karine; De la Garza, Maria M; Moghaddam, Seyed J; Karmouty-Quintana, Harry; Blackburn, Michael R; Drouin, Scott M; Davis, C William; Terrell, Kristy A; Grubb, Barbara R; O'Neal, Wanda K; Flores, Sonia C; Cota-Gomez, Adela; Lozupone, Catherine A; Donnelly, Jody M; Watson, Alan M; Hennessy, Corinne E; Keith, Rebecca C; Yang, Ivana V; Barthel, Lea; Henson, Peter M; Janssen, William J; Schwartz, David A; Boucher, Richard C; Dickey, Burton F; Evans, Christopher M

    2014-01-16

    Respiratory surfaces are exposed to billions of particulates and pathogens daily. A protective mucus barrier traps and eliminates them through mucociliary clearance (MCC). However, excessive mucus contributes to transient respiratory infections and to the pathogenesis of numerous respiratory diseases. MUC5AC and MUC5B are evolutionarily conserved genes that encode structurally related mucin glycoproteins, the principal macromolecules in airway mucus. Genetic variants are linked to diverse lung diseases, but specific roles for MUC5AC and MUC5B in MCC, and the lasting effects of their inhibition, are unknown. Here we show that mouse Muc5b (but not Muc5ac) is required for MCC, for controlling infections in the airways and middle ear, and for maintaining immune homeostasis in mouse lungs, whereas Muc5ac is dispensable. Muc5b deficiency caused materials to accumulate in upper and lower airways. This defect led to chronic infection by multiple bacterial species, including Staphylococcus aureus, and to inflammation that failed to resolve normally. Apoptotic macrophages accumulated, phagocytosis was impaired, and interleukin-23 (IL-23) production was reduced in Muc5b(-/-) mice. By contrast, in mice that transgenically overexpress Muc5b, macrophage functions improved. Existing dogma defines mucous phenotypes in asthma and chronic obstructive pulmonary disease (COPD) as driven by increased MUC5AC, with MUC5B levels either unaffected or increased in expectorated sputum. However, in many patients, MUC5B production at airway surfaces decreases by as much as 90%. By distinguishing a specific role for Muc5b in MCC, and by determining its impact on bacterial infections and inflammation in mice, our results provide a refined framework for designing targeted therapies to control mucin secretion and restore MCC.

  10. Disruption of STAT5b-Regulated Sexual Dimorphism of the Liver Transcriptome by Diverse Factors Is a Common Event.

    Science.gov (United States)

    Oshida, Keiyu; Vasani, Naresh; Waxman, David J; Corton, J Christopher

    2016-01-01

    Signal transducer and activator of transcription 5b (STAT5b) is a growth hormone (GH)-activated transcription factor and a master regulator of sexually dimorphic gene expression in the liver. Disruption of the GH hypothalamo-pituitary-liver axis controlling STAT5b activation can lead to metabolic dysregulation, steatosis, and liver cancer. Computational approaches were developed to identify factors that disrupt STAT5b function in a mouse liver gene expression compendium. A biomarker comprised of 144 STAT5b-dependent genes was derived using comparisons between wild-type male and wild-type female mice and between STAT5b-null and wild-type mice. Correlations between the STAT5b biomarker gene set and a test set comprised of expression datasets (biosets) with known effects on STAT5b function were evaluated using a rank-based test (the Running Fisher algorithm). Using a similarity p-value ≤ 10(-4), the test achieved a balanced accuracy of 99% and 97% for detection of STAT5b activation or STAT5b suppression, respectively. The STAT5b biomarker gene set was then used to identify factors that activate (masculinize) or suppress (feminize) STAT5b function in an annotated mouse liver and primary hepatocyte gene expression compendium of ~1,850 datasets. Disruption of GH-regulated STAT5b is a common phenomenon in liver in vivo, with 5% and 29% of the male datasets, and 11% and 13% of the female datasets, associated with masculinization or feminization, respectively. As expected, liver STAT5b activation/masculinization occurred at puberty and suppression/feminization occurred during aging and in mutant mice with defects in GH signaling. A total of 70 genes were identified that have effects on STAT5b activation in genetic models in which the gene was inactivated or overexpressed. Other factors that affected liver STAT5b function were shown to include fasting, caloric restriction and infections. Together, these findings identify diverse factors that perturb the hypothalamo

  11. Assembly of the Respiratory Mucin MUC5B

    OpenAIRE

    Ridley, C.; Kouvatsos, Nikos; Thornton, David J.; Raynal, Bertrand D; Howard, Marj; Collins, Richard F.; Desseyn, Jean-Luc; Jowitt, Thomas A.; Baldock, Clair; Davis, C. William; Timothy E. Hardingham

    2014-01-01

    Mucins are essential components in mucus gels that form protective barriers at all epithelial surfaces, but much remains unknown about their assembly, intragranular organization, and post-secretion unfurling to form mucus. MUC5B is a major polymeric mucin expressed by respiratory epithelia, and we investigated the molecular mechanisms involved during its assembly. Studies of intact polymeric MUC5B revealed a single high affinity calcium-binding site, distinct from multiple low affinity sites ...

  12. 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...

  13. On the structure of Lattice code WIMSD-5B

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Won Young; Min, Byung Joo

    2004-03-15

    The WIMS-D code is a freely available thermal reactor physics lattice code used widely for thermal research and power reactor calculation. Now the code WIMS-AECL, developed on the basis of WIMS-D, has been used as one of lattice codes for the cell calculation in Canada and also, in 1998, the latest version WIMSD-5B is released for OECD/NEA Data Bank. While WIMS-KAERI was developed and has been used, originated from WIMS-D, in Korea, it was adjusted for the cell calculation of research reactor HANARO and so it has no confirmaty to CANDU reactor. Therefore, the code development applicable to cell calculation of CANDU reactor is necessary not only for technological independence and but also for the establishment of CANDU safety analysis system. A lattice code WIMSD-5B was analyzed in order to set the system of reactor physics computer codes, to be used in the assessment of void reactivity effect. In order to improve and validate WIMSD-5B code, the analysis of the structure of WIMSD-5B lattice code was made and so its structure, algorithm and the subroutines of WIMSD-5B were presented for the cluster type and the pij method modelling the CANDU-6 fuel

  14. On the structure of Lattice code WIMSD-5B

    International Nuclear Information System (INIS)

    The WIMS-D code is a freely available thermal reactor physics lattice code used widely for thermal research and power reactor calculation. Now the code WIMS-AECL, developed on the basis of WIMS-D, has been used as one of lattice codes for the cell calculation in Canada and also, in 1998, the latest version WIMSD-5B is released for OECD/NEA Data Bank. While WIMS-KAERI was developed and has been used, originated from WIMS-D, in Korea, it was adjusted for the cell calculation of research reactor HANARO and so it has no confirmaty to CANDU reactor. Therefore, the code development applicable to cell calculation of CANDU reactor is necessary not only for technological independence and but also for the establishment of CANDU safety analysis system. A lattice code WIMSD-5B was analyzed in order to set the system of reactor physics computer codes, to be used in the assessment of void reactivity effect. In order to improve and validate WIMSD-5B code, the analysis of the structure of WIMSD-5B lattice code was made and so its structure, algorithm and the subroutines of WIMSD-5B were presented for the cluster type and the pij method modelling the CANDU-6 fuel

  15. Affectivity

    OpenAIRE

    Stenner, Paul; Greco, Monica

    2013-01-01

    The concept of affectivity has assumed central importance in much recent scholarship, and many in the social sciences and humanities now talk of an ‘affective turn’. The concept of affectivity at play in this ‘turn’ remains, however, somewhat vague and slippery. Starting with Silvan Tomkins’ influential theory of affect, this paper will explore the relevance of the general assumptions (or ‘utmost abstractions’) that inform thinking about affectivity. The technological and instrumentalist char...

  16. Analysis of mutant NS5B proteins encoded by isolates from chimpanzees chronically infected following clonal HCV RNA inoculation

    International Nuclear Information System (INIS)

    We hypothesized that mutations in the HCV NS5B polymerase, which occur during infection, may affect RNA-dependent RNA polymerase (RdRp) activity. NS5B proteins corresponding to a genotype 1a infectious clone and mutants identified in chimpanzees following inoculation with the clone were expressed and purified and their in vitro RdRp activity was compared to a NS5B genotype 1b control. A Gln-65-to-His mutation increased RdRp activity by 1.8-fold as compared to the infectious clone. Moreover, this NS5B1a protein had RdRp activity similar to the NS5B1b control. Three NS5B proteins representing mutations found in another animal had no in vitro RdRp activity. All mutations were maintained in the majority circulating virus for at least 216 weeks. The results demonstrate that some in vivo mutations of NS5B directly enhance in vitro RdRp activity. In addition, they suggest that the in vitro RdRp activity of NS5B may not always reflect in vivo activity within replication complexes

  17. 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.

  18. 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

  19. 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...

  20. 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

  1. 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

  2. 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.

  3. 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

  4. 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.

  5. 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...

  6. 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...

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

    DEFF Research Database (Denmark)

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

    2014-01-01

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

  8. 45 CFR Appendix C to Part 5b... - [Reserved

    Science.gov (United States)

    2010-10-01

    ... 45 Public Welfare 1 2010-10-01 2010-10-01 false C Appendix C to Part 5b-Delegations of Authority Public Welfare DEPARTMENT OF HEALTH AND HUMAN SERVICES GENERAL ADMINISTRATION PRIVACY ACT REGULATIONS Appendix C to Part 5b—Delegations of Authority...

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

    Science.gov (United States)

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

    2011-10-01

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

  12. 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.

  13. 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.

  14. Discovery of an irreversible HCV NS5B polymerase inhibitor.

    Science.gov (United States)

    Zeng, Qingbei; Nair, Anilkumar G; Rosenblum, Stuart B; Huang, Hsueh-Cheng; Lesburg, Charles A; Jiang, Yueheng; Selyutin, Oleg; Chan, Tin-Yau; Bennett, Frank; Chen, Kevin X; Venkatraman, Srikanth; Sannigrahi, Mousumi; Velazquez, Francisco; Duca, Jose S; Gavalas, Stephen; Huang, Yuhua; Pu, Haiyan; Wang, Li; Pinto, Patrick; Vibulbhan, Bancha; Agrawal, Sony; Ferrari, Eric; Jiang, Chuan-kui; Li, Cheng; Hesk, David; Gesell, Jennifer; Sorota, Steve; Shih, Neng-Yang; Njoroge, F George; Kozlowski, Joseph A

    2013-12-15

    The discovery of lead compound 2e was described. Its covalent binding to HCV NS5B polymerase enzyme was investigated by X-ray analysis. The results of distribution, metabolism and pharmacokinetics were reported. Compound 2e was demonstrated to be potent (replicon GT-1b EC50 = 0.003 μM), highly selective, and safe in in vitro and in vivo assays.

  15. An Assessment of Resonance Treatment in WIMSD-5B

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Won Young; You, Guk Jong; Min, Byung Joo; Park, Joo Hwan [Korea Atomic Energy Research Institute, Taejon (Korea, Republic of)

    2005-07-01

    WIMSD-5B is a lattice code with a depletion capability for the analysis of reactor physics problems related to a design and safety. It is released from the OECD/NEA Data Bank in 1998 and is now being used widely for thermal research and power reactor calculations. The purpose of this study is to assess and improve the resonance treatment method in WIMSD- 5B, through the introduction of a new method with a high accuracy in treating the resonance, as one of the development works for WIMS/CANDU, which is being developed for replacing WIMS-AECL, for the physics analysis of CANDU reactors. In this article, we specifically describe the recent improvements in the resonance integral method using the Carlvik's approximation. As a result, a comparison for the resonance calculation on the CANDU-6 fuel lattice was performed between the WIMSD-5B code and the WIMS/CANDU code with the 69-energy groups of the ENDF/B-VI nuclear data library and the WIMS-AECL code with the 89-energy group of the ENDF/B-VI nuclear data library.

  16. Pharmacoinformatics approach for investigation of alternative potential hepatitis C virus nonstructural protein 5B inhibitors

    Directory of Open Access Journals (Sweden)

    Mirza MU

    2015-03-01

    Full Text Available Muhammad Usman Mirza,1 Noor-Ul-Huda Ghori,2 Nazia Ikram,3 Abdur Rehman Adil,4 Sadia Manzoor3 1Centre for Research in Molecular Medicine (CRiMM, The University of Lahore, Lahore, 2Atta-ur-Rehman School of Applied Biosciences (ASAB, National University of Science and Technology, Islamabad, 3Institute of Molecular Biology and Biotechnology (IMBB, The University of Lahore, Lahore, Pakistan; 4Centre for Excellence in Molecular Biology (CEMB, The University of Punjab, Lahore, Pakistan Abstract: Hepatitis C virus (HCV is one of the major viruses affecting the world today. It is a highly variable virus, having a rapid reproduction and evolution rate. The variability of genomes is due to hasty replication catalyzed by nonstructural protein 5B (NS5B which is also a potential target site for the development of anti-HCV agents. Recently, the US Food and Drug Administration approved sofosbuvir as a novel oral NS5B inhibitor for the treatment of HCV. Unfortunately, it is much highlighted for its pricing issues. Hence, there is an urgent need to scrutinize alternate therapies against HCV that are available at affordable price and do not have associated side effects. Such a need is crucial especially in underdeveloped countries. The search for various new bioactive compounds from plants is a key part of pharmaceutical research. In the current study, we applied a pharmacoinformatics-based approach for the identification of active plant-derived compounds against NS5B. The results were compared to docking results of sofosbuvir. The lead compounds with high-binding ligands were further analyzed for pharmacokinetic and pharmacodynamic parameters based on in silico absorption, distribution, metabolism, excretion, and toxicity (ADMET profile. The results showed the potential alternative lead compounds that can be developed into commercial drugs having high binding energy and promising ADMET properties. Keywords: hepatitis C, NS5B inhibitors, molecular docking, Auto

  17. 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...

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

    Directory of Open Access Journals (Sweden)

    Cristina I López Sanjurjo

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

  19. 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.

  20. Screening and Optimization of Ligand Conjugates for Lysosomal Targeting

    Science.gov (United States)

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

    2011-01-01

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

  1. 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

  2. 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.

  3. 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

  4. 46 CFR Appendix A to Subpart A of... - Example of Escrow Agreement for Use Under 46 CFR 540.5(b)

    Science.gov (United States)

    2010-10-01

    ... 46 Shipping 9 2010-10-01 2010-10-01 false Example of Escrow Agreement for Use Under 46 CFR 540.5(b) A Appendix A to Subpart A of Part 540 Shipping FEDERAL MARITIME COMMISSION REGULATIONS AFFECTING... Agreement for Use Under 46 CFR 540.5(b) Escrow Agreement 1. Legal name(s), state(s) of...

  5. 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...

  6. 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...

  7. A lysosome-centered view of nutrient homeostasis.

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

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

    2012-01-01

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

  9. 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 (...

  10. Combination Therapies for Lysosomal Storage Diseases: A Complex Answer to a Simple Problem.

    Science.gov (United States)

    Macauley, Shannon L

    2016-06-01

    Abstract Lysosomal storage diseases (LSDs) are a group of 40-50 rare monogenic disorders that result in disrupted lysosomal function and subsequent lysosomal pathology. Depending on the protein or enzyme deficiency associated with each disease, LSDs affect an array of organ systems and elicit a complex set of secondary disease mechanisms that make many of these disorders difficult to fully treat. The etiology of most LSDs is known and the innate biology of lysosomal enzymes favors therapeutic intervention, yet most attempts at treating LSDs with enzyme replacement strategies fall short of being curative. Even with the advent of more sophisticated approaches, like substrate reduction therapy, pharmacologic chaperones, gene therapy or stem cell therapy, comprehensive treatments for LSDs have yet to be achieved. Given the limitations with individual therapies, recent research has focused on using a combination approach to treat LSDs. By coupling protein-, cell-, and gene- based therapies with small molecule drugs, researchers have found greater success in eradicating the clinical features of disease. This review seeks to discuss the positive and negatives of singular therapies used to treat LSDs, and discuss how, in combination, studies have demonstrated a more holistic benefit on pathological and functional parameters. By optimizing routes of delivery, therapeutic timing, and targeting secondary disease mechanisms, combination therapy represents the future for LSD treatment. PMID:27491211

  11. 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

  12. Data of evolutionary structure change: 1AKMC-3GD5B [Confc[Archive

    Lifescience Database Archive (English)

    Full Text Available 1AKMC-3GD5B 1AKM 3GD5 C B SGFYHKHFLKLLDFTPAELNSLLQLAAKLKADKKSGKEE...AKLTGKNIALIFEKDSTRTRCSFEVAAYDQGARVTYLGPSGSQIGHKESIKDTARVLGRMYDGIQYRGYGQEIVETLAEYASVPVWNGLTNEFHPTQLLADLLTMQEHLPGKAFNEMTLVYAGD...FAQTELEEYAHYAGIPVINALTDHEHPCQVVADLLTIRENFG--RLAGLKLAYVGDG-NNVAHSLLLGCAKVGMSIAVATPEGFTPDPAVSARASEIAGRTGAEVQIL...hain> 3GD5 B 3GD5B...ndel> 1 3GD5 B 3GD

  13. 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...

  14. Photoaffinity labeling of the lysosomal neuraminidase from bovine testis

    NARCIS (Netherlands)

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

    1990-01-01

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

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

    Science.gov (United States)

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

    2016-02-01

    Many cellular activities and pharmaceutical interventions involve endocytosis and delivery to lysosomes for processing. Hence, lysosomal processing defects can cause cell and tissue damage, as in lysosomal storage diseases (LSDs) characterized by lysosomal accumulation of undegraded materials. This storage causes endocytic and trafficking alterations, which exacerbate disease and hinder treatment. However, there have been no systematic studies comparing different endocytic routes in LSDs. Here, we used genetic and pharmacological models of four LSDs (type A Niemann-Pick, type C Niemann-Pick, Fabry, and Gaucher diseases) and evaluated the pinocytic and receptor-mediated activity of the clathrin-, caveolae-, and macropinocytic routes. Bulk pinocytosis was diminished in all diseases, suggesting a generic endocytic alteration linked to lysosomal storage. Fluid-phase (dextran) and ligand (transferrin) uptake via the clathrin route were lower for all LSDs. Fluid-phase and ligand (cholera toxin B) uptake via the caveolar route were both affected but less acutely in Fabry or Gaucher diseases. Epidermal growth factor-induced macropinocytosis was altered in Niemann-Pick cells but not other LSDs. Intracellular trafficking of ligands was also distorted in LSD versus wild-type cells. The extent of these endocytic alterations paralleled the level of cholesterol storage in disease cell lines. Confirming this, pharmacological induction of cholesterol storage in wild-type cells disrupted endocytosis, and model therapeutics restored uptake in proportion to their efficacy in attenuating storage. This suggests a proportional and reversible relationship between endocytosis and lipid (cholesterol) storage. By analogy, the accumulation of biological material in other diseases, or foreign material from drugs or their carriers, may cause similar deficits, warranting further investigation. PMID:26702793

  16. 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

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

    Science.gov (United States)

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

    2016-01-01

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

  18. 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...

  19. 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

  20. 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.

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

    Science.gov (United States)

    Evander, A; Ihse, I; Lundquist, I

    1983-01-01

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

  2. Diabetes alters KIF1A and KIF5B motor proteins in the hippocampus.

    Directory of Open Access Journals (Sweden)

    Filipa I Baptista

    Full Text Available Diabetes mellitus is the most common metabolic disorder in humans. Diabetic encephalopathy is characterized by cognitive and memory impairments, which have been associated with changes in the hippocampus, but the mechanisms underlying those impairments triggered by diabetes, are far from being elucidated. The disruption of axonal transport is associated with several neurodegenerative diseases and might also play a role in diabetes-associated disorders affecting nervous system. We investigated the effect of diabetes (2 and 8 weeks duration on KIF1A, KIF5B and dynein motor proteins, which are important for axonal transport, in the hippocampus. The mRNA expression of motor proteins was assessed by qRT-PCR, and also their protein levels by immunohistochemistry in hippocampal slices and immunoblotting in total extracts of hippocampus from streptozotocin-induced diabetic and age-matched control animals. Diabetes increased the expression and immunoreactivity of KIF1A and KIF5B in the hippocampus, but no alterations in dynein were detected. Since hyperglycemia is considered a major player in diabetic complications, the effect of a prolonged exposure to high glucose on motor proteins, mitochondria and synaptic proteins in hippocampal neurons was also studied, giving particular attention to changes in axons. Hippocampal cell cultures were exposed to high glucose (50 mM or mannitol (osmotic control; 25 mM plus 25 mM glucose for 7 days. In hippocampal cultures incubated with high glucose no changes were detected in the fluorescence intensity or number of accumulations related with mitochondria in the axons of hippocampal neurons. Nevertheless, high glucose increased the number of fluorescent accumulations of KIF1A and synaptotagmin-1 and decreased KIF5B, SNAP-25 and synaptophysin immunoreactivity specifically in axons of hippocampal neurons. These changes suggest that anterograde axonal transport mediated by these kinesins may be impaired in hippocampal

  3. 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

  4. 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.

  5. 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.

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

    Directory of Open Access Journals (Sweden)

    Anne-Claire Jacomin

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

  7. Crystal structure of translation initiation factor 5B from the crenarchaeon Aeropyrum pernix.

    Science.gov (United States)

    Murakami, Ryo; Miyoshi, Tomohiro; Uchiumi, Toshio; Ito, Kosuke

    2016-05-01

    Initiation factor 5B (IF5B) is a universally conserved translational GTPase that catalyzes ribosomal subunit joining. In eukaryotes, IF5B directly interacts via a groove in its domain IV with initiation factor 1A (IF1A), another universally conserved initiation factor, to accomplish efficient subunit joining. Here, we have determined the first structure of a crenarchaeal IF5B, which revealed that the archaea-specific region of IF5B (helix α15) binds and occludes the groove of domain IV. Therefore, archaeal IF5B cannot access IF1A in the same manner as eukaryotic IF5B. This fact suggests that different relationships between IF5B and IF1A exist in archaea and eukaryotes. Proteins 2016; 84:712-717. © 2016 Wiley Periodicals, Inc. PMID:26868175

  8. Obesity-resistant S5B rats showed great cocaine conditioned place preference than the obesity-prone OM rats

    Energy Technology Data Exchange (ETDEWEB)

    Thanos, P.K.; Wang, G.; Thanos, P.K..; Kim, R.; Cho, J.; Michaelides, M.; Anderson, B.J.; Primeaux, S.D.; Bray, G.A.; Wang, G.-J.; Robinson, J.K.; Volkow, N.D.

    2010-12-01

    Dopamine (DA) and the DA D2 receptor (D2R) are involved in the rewarding and conditioned responses to food and drug rewards. Osborne-Mendel (OM) rats are genetically prone and S5B/P rats are genetically resistant to obesity when fed a high-fat diet. We hypothesized that the differential sensitivity of these two rat strains to natural rewards may also be reflected in sensitivity to drugs of abuse. Therefore, we tested whether OM and S5B/P rats showed a differential preference to cocaine using conditioned place preference (CPP). To also evaluate whether there is specific involvement of the D2R in this differential conditioning sensitivity, we then tested whether the D2R agonist bromocriptine (BC) would differentially affect the effects of cocaine in the two strains. OM and S5B/P rats were conditioned with cocaine (5 or 10 mg/kg) in one chamber and saline in another for 8 days. Rats were then tested for cocaine preference. The effects of BC (0.5, 1, 5, 10, 20 mg/kg) on cocaine preference were then assessed in subsequent test sessions. OM rats did not show a significant preference for the cocaine-paired chamber on test day. Only the S5B/P rats showed cocaine CPP. Later treatment with only the highest dose of BC resulted in reduced cocaine CPP in S5B/P rats when treated with 5 mg/kg cocaine and in OM rats treated with 10 mg/kg cocaine. Our results indicated that obesity-resistant S5B rats showed greater cocaine CPP than the obesity-prone OM rats. These findings do not support a theory of common vulnerability for reinforcer preferences (food and cocaine). However, they show that BC reduced cocaine conditioning effects supporting at least a partial regulatory role of D2R in conditioned responses to drugs.

  9. Chemical and Hormonal Effects on STAT5b-Dependent Sexual Dimorphism of the Liver Transcriptome.

    Science.gov (United States)

    The growth hormone (GH)-activated transcription factor signal transducer and activator of transcription 5b (STAT5b) is a key regulator of sexually dimorphic gene expression in the liver. Suppression of hepatic STAT5b signaling is associated with lipid metabolic dysfunction leadi...

  10. 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.

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

    Science.gov (United States)

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

    1996-10-14

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

  12. 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

  13. 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.

  14. 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

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

    Science.gov (United States)

    Kumar, Nadimpalli Siva; Bhamidimarri, Poorna M

    2015-01-01

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

  16. Coffee Polyphenols Change the Expression of STAT5B and ATF-2 Modifying Cyclin D1 Levels in Cancer Cells

    Directory of Open Access Journals (Sweden)

    Carlota Oleaga

    2012-01-01

    Full Text Available Background. Epidemiological studies suggest that coffee consumption reduces the risk of cancer, but the molecular mechanisms of its chemopreventive effects remain unknown. Objective. To identify differentially expressed genes upon incubation of HT29 colon cancer cells with instant caffeinated coffee (ICC or caffeic acid (CA using whole-genome microarrays. Results. ICC incubation of HT29 cells caused the overexpression of 57 genes and the underexpression of 161, while CA incubation induced the overexpression of 12 genes and the underexpression of 32. Using Venn-Diagrams, we built a list of five overexpressed genes and twelve underexpressed genes in common between the two experimental conditions. This list was used to generate a biological association network in which STAT5B and ATF-2 appeared as highly interconnected nodes. STAT5B overexpression was confirmed at the mRNA and protein levels. For ATF-2, the changes in mRNA levels were confirmed for both ICC and CA, whereas the decrease in protein levels was only observed in CA-treated cells. The levels of cyclin D1, a target gene for both STAT5B and ATF-2, were downregulated by CA in colon cancer cells and by ICC and CA in breast cancer cells. Conclusions. Coffee polyphenols are able to affect cyclin D1 expression in cancer cells through the modulation of STAT5B and ATF-2.

  17. 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

  18. 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.

  19. 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

  20. 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...

  1. 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...

  2. 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...

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

    Science.gov (United States)

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

    2015-10-01

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

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

    Directory of Open Access Journals (Sweden)

    Emanuele Persichetti

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

  5. Stable knockdown of Kif5b in MDCK cells leads to epithelial–mesenchymal transition

    Energy Technology Data Exchange (ETDEWEB)

    Cui, Ju, E-mail: juzi.cui@gmail.com [The Key Laboratory of Geriatrics, Beijing Hospital & Beijing Institute of Geriatrics, Ministry of Health, Beijing (China); Department of Biochemistry, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong SAR (China); Jin, Guoxiang; Yu, Bin [Department of Biochemistry, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong SAR (China); Wang, Zai [Department of Biochemistry, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong SAR (China); Institute of Clinical Medical Sciences, China-Japan Friendship Hospital, Beijing (China); Lin, Raozhou [Department of Biochemistry, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong SAR (China); Huang, Jian-Dong, E-mail: jdhuang@hku.hk [Department of Biochemistry, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong SAR (China); The Centre for Synthetic Biology Engineering Research, Shenzhen Institutes of Advanced Technology, Shenzhen (China)

    2015-07-17

    Polarization of epithelial cells requires vectorial sorting and transport of polarity proteins to apical or basolateral domains. Kif5b is the mouse homologue of the human ubiquitous Kinesin Heavy Chain (uKHC). To investigate the function of Kif5b in epithelial cells, we examined the phenotypes of Kif5b-deficient MDCK cells. Stable knockdown of Kif5b in MDCK cells resulted in reduced cell proliferation rate, profound changes in cell morphology, loss of epithelial cell marker, and gain of mesenchymal marker, as well as increased cell migration, invasion, and tumorigenesis abilities. E-cadherin and NMMIIA could interact with Kif5b in polarized MDCK cells, and their expression levels were decreased in Kif5b-deficient MDCK cells. Overexpression of E-cadherin and NMMIIA in Kif5b depleted MDCK cells could decrease mesenchymal marker expression and cell migration ability. These results indicate that stable knockdown of Kif5b in MDCK cells can lead to epithelial–mesenchymal transition, which is mediated by defective E-cadherin and NMMIIA expression. - Highlights: • Knockdown of Kif5b in MDCK cells resulted in reduced cell proliferation rate. • Kif5b deficient MDCK cells underwent epithelial–mesenchymal transition. • E-cadherin and NMMIIA could interact with Kif5b in polarized MDCK cells. • Decreased E-cadherin and NMMIIA levels mediate EMT in Kif5b deficient MDCK cells. • Overexpression of E-cadherin and NMMIIA reverse the effects of Kif5b knockdown.

  6. Histone demethylase KDM5B is a key regulator of genome stability

    OpenAIRE

    LI, XIN; Liu, Ling; Yang, Shangda; Song, Nan; Zhou, Xing; Gao, Jie; Yu, Na; Shan, Lin; Wang, Qian; Liang, Jing; Xuan, Chenghao; Wang, Yan; Shang, Yongfeng; Shi, Lei

    2014-01-01

    DNA double-strand breaks are generally repaired in the context of highly organized chromatin. However, how epigenetic mechanisms are involved in the maintenance of the genetic fidelity remains poorly understood. Here we report that lysine-specific histone demethylase 5B (KDM5B), a well-defined transcriptional repressor, promotes double-strand break signaling and is required for efficient DNA repairs. We demonstrated that KDM5B, in doing so, functions to orchestrate checkpoint activation and c...

  7. 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

  8. Unprecedentedly mild direct Pd-catalyzed arylation of oxazolo[4,5-b]pyridine

    DEFF Research Database (Denmark)

    Zhuravlev, Fedor

    2006-01-01

    Pd-catalyzed C-2 arylation of oxazolo[4,5-b]pyridine proceeds efficiently at 30 degrees C and tolerates a variety of aryl halides, including derivatized amino acids for which no racemization was observed during the reaction. Experimental evidence for facile deprotonation of oxazolo[4,5-b]pyridine......Pd-catalyzed C-2 arylation of oxazolo[4,5-b]pyridine proceeds efficiently at 30 degrees C and tolerates a variety of aryl halides, including derivatized amino acids for which no racemization was observed during the reaction. Experimental evidence for facile deprotonation of oxazolo[4,5-b...

  9. 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.

  10. Assembly and Regulation of the Membrane Attack Complex Based on Structures of C5b6 and sC5b9

    Directory of Open Access Journals (Sweden)

    Michael A. Hadders

    2012-03-01

    Full Text Available Activation of the complement system results in formation of membrane attack complexes (MACs, pores that disrupt lipid bilayers and lyse bacteria and other pathogens. Here, we present the crystal structure of the first assembly intermediate, C5b6, together with a cryo-electron microscopy reconstruction of a soluble, regulated form of the pore, sC5b9. Cleavage of C5 to C5b results in marked conformational changes, distinct from those observed in the homologous C3-to-C3b transition. C6 captures this conformation, which is preserved in the larger sC5b9 assembly. Together with antibody labeling, these structures reveal that complement components associate through sideways alignment of the central MAC-perforin (MACPF domains, resulting in a C5b6-C7-C8β-C8α-C9 arc. Soluble regulatory proteins below the arc indicate a potential dual mechanism in protection from pore formation. These results provide a structural framework for understanding MAC pore formation and regulation, processes important for fighting infections and preventing complement-mediated tissue damage.

  11. 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

  12. Quantitative modeling of selective lysosomal targeting for drug design

    DEFF Research Database (Denmark)

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

    2008-01-01

    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...... predicted by the model and three were close. Five of the antimalarial drugs were lipophilic weak dibasic compounds. The predicted optimum properties for a selective accumulation of weak bivalent bases in lysosomes are consistent with experimental values and are more accurate than any prior calculation...

  13. 49 CFR 173.5b - Portable and mobile refrigeration systems.

    Science.gov (United States)

    2010-10-01

    ... 49 Transportation 2 2010-10-01 2010-10-01 false Portable and mobile refrigeration systems. 173.5b...-GENERAL REQUIREMENTS FOR SHIPMENTS AND PACKAGINGS General § 173.5b Portable and mobile refrigeration... refrigeration systems, which may or may not be permanently mounted to a transport vehicle, used for...

  14. Corticothalamic Spike Transfer via the L5B-POm Pathway in vivo.

    Science.gov (United States)

    Mease, Rebecca A; Sumser, Anton; Sakmann, Bert; Groh, Alexander

    2016-08-01

    The cortex connects to the thalamus via extensive corticothalamic (CT) pathways, but their function in vivo is not well understood. We investigated "top-down" signaling from cortex to thalamus via the cortical layer 5B (L5B) to posterior medial nucleus (POm) pathway in the whisker system of the anesthetized mouse. While L5B CT inputs to POm are extremely strong in vitro, ongoing activity of L5 neurons in vivo might tonically depress these inputs and thereby block CT spike transfer. We find robust transfer of spikes from the cortex to the thalamus, mediated by few L5B-POm synapses. However, the gain of this pathway is not constant but instead is controlled by global cortical Up and Down states. We characterized in vivo CT spike transfer by analyzing unitary PSPs and found that a minority of PSPs drove POm spikes when CT gain peaked at the beginning of Up states. CT gain declined sharply during Up states due to frequency-dependent adaptation, resulting in periodic high gain-low gain oscillations. We estimate that POm neurons receive few (2-3) active L5B inputs. Thus, the L5B-POm pathway strongly amplifies the output of a few L5B neurons and locks thalamic POm sub-and suprathreshold activity to cortical L5B spiking. PMID:27178196

  15. Data of evolutionary structure change: 1JDZC-2IQ5B [Confc[Archive

    Lifescience Database Archive (English)

    Full Text Available > 2IQ5B VRLDG-ASLHF >EEE - ...SYNQGGLFYQY e>E HHHHHHture> 2IQ5B QTESH----AVKIV > HH----HHHHH...ain>1JDZC EELEKSVMDGAKAV re...> HHHHHHHHHHHH> ATOM 5061 CA GLU C 217 95.716 121.480 11.777 1.

  16. Loss of COX5B inhibits proliferation and promotes senescence via mitochondrial dysfunction in breast cancer.

    Science.gov (United States)

    Gao, Shui-Ping; Sun, He-Fen; Jiang, Hong-Lin; Li, Liang-Dong; Hu, Xin; Xu, Xiao-En; Jin, Wei

    2015-12-22

    COX5B, a peripheral subunit of the cytochrome c oxidase complex, has previously been reported to maintain the stability of this complex. However, its functions and mechanisms involved in breast cancer progression remain unclear. Here, by performing SILAC assays in breast cancer cell models and detecting COX5B expression in tissues, we found that COX5B expression was elevated in breast cancer. Down-regulation of COX5B in breast cancer cell lines can suppress cell proliferation and induced cell senescence which was accompanied by elevating production of IL-8 and other cytokines. Interestingly, conditioned medium from COX5B knockdown cells could promote breast cancer cell migration. Mechanistic studies reveal that COX5B silence induces an increase in production of ROS, depolarization of MMP and a decrease in ATP. What's more, silence of COX5B leads to metabolic disorders, such as increased glucose uptake and decreased lactate secretion. Collectively, our study shows that loss of COX5B induces mitochondrial dysfunction and subsequently leads to cell growth suppression and cell senescence. Cytokines such as IL-8 secreted by senescent cells may in turn alter the microenvironment which could enhance cell migration. These findings may provide a novel paradigm for the treatment which combined anti-cancer drugs with particular cytokine inhibitors such as IL-8 blockers.

  17. 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

  18. 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

  19. A single amino acid change within the R2 domain of the VvMYB5b transcription factor modulates affinity for protein partners and target promoters selectivity

    Directory of Open Access Journals (Sweden)

    Granier Thierry

    2011-08-01

    Full Text Available Abstract Background Flavonoid pathway is spatially and temporally controlled during plant development and the transcriptional regulation of the structural genes is mostly orchestrated by a ternary protein complex that involves three classes of transcription factors (R2-R3-MYB, bHLH and WDR. In grapevine (Vitis vinifera L., several MYB transcription factors have been identified but the interactions with their putative bHLH partners to regulate specific branches of the flavonoid pathway are still poorly understood. Results In this work, we describe the effects of a single amino acid substitution (R69L located in the R2 domain of VvMYB5b and predicted to affect the formation of a salt bridge within the protein. The activity of the mutated protein (name VvMYB5bL, the native protein being referred as VvMYB5bR was assessed in different in vivo systems: yeast, grape cell suspensions, and tobacco. In the first two systems, VvMYB5bL exhibited a modified trans-activation capability. Moreover, using yeast two-hybrid assay, we demonstrated that modification of VvMYB5b transcriptional properties impaired its ability to correctly interact with VvMYC1, a grape bHLH protein. These results were further substantiated by overexpression of VvMYB5bR and VvMYB5bL genes in tobacco. Flowers from 35S::VvMYB5bL transgenic plants showed a distinct phenotype in comparison with 35S::VvMYB5bR and the control plants. Finally, significant differences in transcript abundance of flavonoid metabolism genes were observed along with variations in pigments accumulation. Conclusions Taken together, our findings indicate that VvMYB5bL is still able to bind DNA but the structural consequences linked to the mutation affect the capacity of the protein to activate the transcription of some flavonoid genes by modifying the interaction with its co-partner(s. In addition, this study underlines the importance of an internal salt bridge for protein conformation and thus for the establishment

  20. 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

  1. 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

  2. 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.

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

    NARCIS (Netherlands)

    D.J.J. Halley (Dicky)

    1980-01-01

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

  4. 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 ...

  5. 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.

  6. Identification of STAT5A and STAT5B target genes in human T cells.

    Directory of Open Access Journals (Sweden)

    Takahiro Kanai

    Full Text Available Signal transducer and activator of transcription (STAT comprises a family of universal transcription factors that help cells sense and respond to environmental signals. STAT5 refers to two highly related proteins, STAT5A and STAT5B, with critical function: their complete deficiency is lethal in mice; in humans, STAT5B deficiency alone leads to endocrine and immunological problems, while STAT5A deficiency has not been reported. STAT5A and STAT5B show peptide sequence similarities greater than 90%, but subtle structural differences suggest possible non-redundant roles in gene regulation. However, these roles remain unclear in humans. We applied chromatin immunoprecipitation followed by DNA sequencing using human CD4(+ T cells to detect candidate genes regulated by STAT5A and/or STAT5B, and quantitative-PCR in STAT5A or STAT5B knock-down (KD human CD4(+ T cells to validate the findings. Our data show STAT5A and STAT5B play redundant roles in cell proliferation and apoptosis via SGK1 interaction. Interestingly, we found a novel, unique role for STAT5A in binding to genes involved in neural development and function (NDRG1, DNAJC6, and SSH2, while STAT5B appears to play a distinct role in T cell development and function via DOCK8, SNX9, FOXP3 and IL2RA binding. Our results also suggest that one or more co-activators for STAT5A and/or STAT5B may play important roles in establishing different binding abilities and gene regulation behaviors. The new identification of these genes regulated by STAT5A and/or STAT5B has major implications for understanding the pathophysiology of cancer progression, neural disorders, and immune abnormalities.

  7. 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

  8. 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 i...

  9. 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.

  10. Main design elements of the superconducting magnet for the LIN-5B baseball trap

    International Nuclear Information System (INIS)

    A design of superconducting magnet system (SMS) is described for the LIN-5B baseball trap elaborated for the OGRA-3B experimental installation. The general layout of the trap, cross sections of the superconducting coil and load-bearing frame, the current leads arrangement and main LIN-5B SMS parameters are presented. A three-year experience in operation of the baseball LIN-5B SMS and successful tests followed by achieving critical conditions four times permit to make the conclusion that the design and SMS assembly meet the requirements

  11. 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

  12. Myo5b knockout mice as a model of microvillus inclusion disease

    NARCIS (Netherlands)

    Carton-Garcia, Fernando; Overeem, Arend W.; Nieto, Rocio; Bazzocco, Sarah; Dopeso, Higinio; Macaya, Irati; Bilic, Josipa; Landolfi, Stefania; Hernandez-Losa, Javier; Schwartz, Simo; Ramon y Cajal, Santiago; van Ijzendoorn, Sven C. D.; Arango, Diego

    2015-01-01

    Inherited MYO5B mutations have recently been associated with microvillus inclusion disease (MVID), an autosomal recessive syndrome characterized by intractable, life-threatening, watery diarrhea appearing shortly after birth. Characterization of the molecular mechanisms underlying this disease and d

  13. LOFT CIS analysis S-5B penetration 3'' IA-296-AB

    International Nuclear Information System (INIS)

    The 3'' IA-296-AB line from the containment penetration S-5B was analyzed to ASME Code, Subsection NC (Class 2) criteria. This section of piping is part of the Containment Isolation System; the model considered the line from penetration S-5B outward through a series of elbows and through the third isolation valve. Results of this analysis show that the section of line described will meet Class 2 requirements if additional supports are installed at three locations

  14. Chemical and Hormonal Effects on STAT5b-Dependent Sexual Dimorphism of the Liver Transcriptome.

    Directory of Open Access Journals (Sweden)

    Keiyu Oshida

    Full Text Available The growth hormone (GH-activated transcription factor signal transducer and activator of transcription 5b (STAT5b is a key regulator of sexually dimorphic gene expression in the liver. Suppression of hepatic STAT5b signaling is associated with lipid metabolic dysfunction leading to steatosis and liver cancer. In the companion publication, a STAT5b biomarker gene set was identified and used in a rank-based test to predict both increases and decreases in liver STAT5b activation status/function with high (≥ 97% accuracy. Here, this computational approach was used to identify chemicals and hormones that activate (masculinize or suppress (feminize STAT5b function in a large, annotated mouse liver and primary hepatocyte gene expression compendium. Exposure to dihydrotestosterone and thyroid hormone caused liver masculinization, whereas glucocorticoids, fibroblast growth factor 15, and angiotensin II caused liver feminization. In mouse models of diabetes and obesity, liver feminization was consistently observed and was at least partially reversed by leptin or resveratrol exposure. Chemical-induced feminization of male mouse liver gene expression profiles was a relatively frequent phenomenon: of 156 gene expression biosets from chemically-treated male mice, 29% showed feminization of liver STAT5b function, while <1% showed masculinization. Most (93% of the biosets that exhibited feminization of male liver were also associated with activation of one or more xenobiotic-responsive receptors, most commonly constitutive activated receptor (CAR or peroxisome proliferator-activated receptor alpha (PPARα. Feminization was consistently associated with increased expression of peroxisome proliferator-activated receptor gamma (Pparg but not other lipogenic transcription factors linked to steatosis. GH-activated STAT5b signaling in mouse liver is thus commonly altered by diverse chemicals, and provides a linkage between chemical exposure and dysregulated gene

  15. Screening of Rice Genes Interacting with p5b of Rice Black-Streaked Dwarf Virus

    Institute of Scientific and Technical Information of China (English)

    LU Ying; YANG Jian; ZHANG Heng-mu; CHEN Jian-ping

    2013-01-01

    Rice black-streaked dwarf virus (RBSDV) is a recognized member of the genus Fijivirus,family Reoviridae.Its genome has ten double-stranded RNA (dsRNA) segments (S1-S10),in which the fifth genome segment (S5) contains two open reading frames (ORFs) with a partially overlapping region.The second ORF of RBSDV S5 encodes a viral nonstructural protein named p5b with unknown function.To reveal the function of p5b,its gene was ligated into the bait plasmid pGBKT7 and an expression library containing rice cDNAs was constructed using plasmid pGADT7 for yeast two-hybrid assay.The bait protein p5b was detected in yeast by western blot,and the result of an auto-activation test showed that p5b could not autonomously activate the expression of reporter genes in yeast.Then the bait protein p5b was used for screening the cDNA expression libraries of rice.Gene fragments of some pivotal enzymes involved in photosynthesis,respiration and other important metabolic processes,were identified to interact with p5b in yeast,suggesting that these interactions may play roles in symptom development in infected plants.

  16. 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

  17. 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

  18. Reduced mucociliary clearance in old mice is associated with a decrease in Muc5b mucin.

    Science.gov (United States)

    Grubb, Barbara R; Livraghi-Butrico, Alessandra; Rogers, Troy D; Yin, Weining; Button, Brian; Ostrowski, Lawrence E

    2016-05-01

    Respiratory infections are a major cause of morbidity and mortality in the elderly. Previous reports have suggested that mucociliary clearance (MCC) is impaired in older individuals, but the cause is unclear. To unravel the mechanisms responsible for the age-associated decline in MCC, we investigated the MCC system in young (3 mo) and old (2 yr) C57BL/6 mice. We found that old mice had significantly reduced MCC function in both the upper and lower airways compared with young mice. Measurement of bioelectric properties of isolated tracheal and bronchial tissue revealed a significant decrease in Cl(-) secretion, suggesting that the older mice may have a reduced ability to maintain a sufficiently hydrated airway surface for efficient MCC. Ciliary beat frequency was also observed to be reduced in the older animals; however, this reduction was small relative to the reduction in MCC. Interestingly, the level of the major secreted mucin, Muc5b, was found to be reduced in both bronchioalveolar lavage and isolated tracheal tissue. Our previous studies of Muc5b(-/-) mice have demonstrated that Muc5b is essential for normal MCC in the mouse. Furthermore, examination of Muc5b(+/-) and wild-type animals revealed that heterozygous animals, which secrete ∼50% of the wild-type level of Muc5b, also demonstrate a markedly reduced level of MCC, confirming the importance of Muc5b levels to MCC. These results demonstrate that aged mice exhibit a decrease in MCC and suggest that a reduced level of secretion of both Cl(-) and Muc5b may be responsible. PMID:26968767

  19. 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...

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

    DEFF Research Database (Denmark)

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

    2013-01-01

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

  1. Role of alveolar macrophage lysosomes in metal detoxification.

    Science.gov (United States)

    Berry, J P; Zhang, L; Galle, P; Ansoborlo, E; Hengé-Napoli, M H; Donnadieu-Claraz, M

    1997-02-15

    The intracellular behaviour of different toxic mineral elements inhaled as soluble aerosols or as insoluble particles was studied in the rat by electron microscopy, electron probe microanalysis, and electron microdiffraction. This study showed that, after inhalation, aerosols of soluble elements like cerous chloride, chromic chloride, uranyl nitrate, and aluminium chloride, are concentrated in the lysosomes of alveolar macrophages and are precipitated in the lysosomes in the form of insoluble phosphate, probably due to the activity of acid phosphatase (intralysosomial enzyme). Also, after inhalation of crystalline particles that are insoluble or poorly soluble in water such as the illites (phyllosilicates), ceric oxides (opaline), and industrial uranium oxides (U3O8), the small crystals are captured by the alveolar macrophage lysosomes and transformed over time into an amorphous form. This structural transformation is associated with changes in the chemical nature of particles inhaled in the oxide form. Microanalysis of amorphous deposits observed after inhalation of uranium or ceric oxides has shown that they contain high concentrations of phosphorus associated with the initial elements cerium and uranium. These different processes tend to limit the diffusion of these toxic elements within the organism, whether they are inhaled in soluble form or not. PMID:9140931

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

    Science.gov (United States)

    Tripathi, Vijay; Gupta, Dwijendra Kumar

    2014-01-01

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

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

    Science.gov (United States)

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

    1999-01-01

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

  4. 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

  5. A NEW CELL CLONE DERIVED FROM TRICHOPLUSIA NI TN5B1-4 CELLS

    Institute of Scientific and Technical Information of China (English)

    Jian-xiaoTian; Chang-youLi; Gui-lingZheng; Guo-xunLi; PingWang; Granados

    2004-01-01

    The characteristics of a cultured cell line do not always remain stable and may change upon continuous passage. Most continuous cell lines, even after cloning, possess several genotypes that are constantly changing. There are numerous selective and adaptive culture processes, in addition to genetic instability, that may improve phenotypic change in cell growth, virus susceptibility, gene expression, and production of virus. Similar detrimental effects of long term passaging of insect cells have also been reported for continuous cell lines, for example, Tn5B 1-4 cells, which are the most widely used for the baculovirus expression vector system (BEVS), provide superior production of recombinant proteins,however, this high productivity may be more evident in low passage cells. In this paper, we describe the isolation of a cell clone, Tn5B-40, from low passage Tn5B 1-4 cells. The growth characteristics,productions of virus, and high level of recombinant protein productions were determined. The results showed the susceptibility of both clone and Tn5B 1-4 cells to wild-type AcNPV was approximately the same rate with over 95% of infection; when the cloned cells were infected with recombinant baculoviruses expressing β-galactosidase and secreted alkaline phosphatase (SEAP), expression of the recombinant proteins from the cloned cells exceeded that from the parental Tn5B 1-4 cells.

  6. Overexpressed KDM5B is associated with the progression of glioma and promotes glioma cell growth via downregulating p21

    International Nuclear Information System (INIS)

    Highlights: • KDM5B is overexpressed in glioma samples. • KDM5B stimulated proliferation of glioma cells. • Inhibition of p21contributes to KDM5B-induced proliferation. - Abstract: Epigenetic alterations such as aberrant expression of histone-modifying enzymes have been implicated in tumorigenesis. Upregulation of lysine (K)-specific demethylase 5B (KDM5B) has been reported in a variety of malignant tumors. However, the impact of KDM5B in glioma remains unclear. The objective of this study was to investigate the expression and prognostic value of KDM5B in glioma. In clinical glioma samples, we found that KDM5B expression was significantly upregulated in cancer lesions compared with normal brain tissues. Kaplan–Meier analysis showed that patients with glioma and higher KDM5B expression tend to have shorter overall survival time. By silencing or overexpressing KDM5B in glioma cells, we found that KDM5B could promote cell growth both in vitro and in vivo. Moreover, we demonstrated that KDM5B promoted glioma proliferation partly via regulation of the expression of p21. Our study provided evidence that KDM5B functions as a novel tumor oncogene in glioma and may be a potential therapeutic target for glioma management

  7. Overexpressed KDM5B is associated with the progression of glioma and promotes glioma cell growth via downregulating p21

    Energy Technology Data Exchange (ETDEWEB)

    Dai, Bin [Department of Neurosurgery, Beijing Shijitan Hospital, Capital Medical University, Beijing 100038 (China); Hu, Zhiqiang, E-mail: zhiqhutg@126.com [Department of Neurosurgery, Beijing Shijitan Hospital, Capital Medical University, Beijing 100038 (China); Huang, Hui; Zhu, Guangtong; Xiao, Zhiyong [Department of Neurosurgery, Beijing Shijitan Hospital, Capital Medical University, Beijing 100038 (China); Wan, Weiqing; Zhang, Peng; Jia, Wang; Zhang, Liwei [Department of Neurosurgery, Beijing Tian Tan Hospital, Capital Medical University, Beijing 100050 (China)

    2014-11-07

    Highlights: • KDM5B is overexpressed in glioma samples. • KDM5B stimulated proliferation of glioma cells. • Inhibition of p21contributes to KDM5B-induced proliferation. - Abstract: Epigenetic alterations such as aberrant expression of histone-modifying enzymes have been implicated in tumorigenesis. Upregulation of lysine (K)-specific demethylase 5B (KDM5B) has been reported in a variety of malignant tumors. However, the impact of KDM5B in glioma remains unclear. The objective of this study was to investigate the expression and prognostic value of KDM5B in glioma. In clinical glioma samples, we found that KDM5B expression was significantly upregulated in cancer lesions compared with normal brain tissues. Kaplan–Meier analysis showed that patients with glioma and higher KDM5B expression tend to have shorter overall survival time. By silencing or overexpressing KDM5B in glioma cells, we found that KDM5B could promote cell growth both in vitro and in vivo. Moreover, we demonstrated that KDM5B promoted glioma proliferation partly via regulation of the expression of p21. Our study provided evidence that KDM5B functions as a novel tumor oncogene in glioma and may be a potential therapeutic target for glioma management.

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

    Science.gov (United States)

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

    2014-01-01

    The use of lysosome-targeted liposomes may significantly improve a delivery of therapeutic enzymes into lysosomes for the treatment of lysosome-associated diseases. The aim of this research was to achieve a specific intracellular targeting of lysosomes, by using liposomes modified with the lysosomotropic octadecyl-rhodamine B (RhB) and loaded with a model compound, fluorescein isothiocyanate (FITC)–dextran (FD). Plain and RhB-modified liposomes were prepared by hydration of lipid films and loaded with FD or with 5-dodecanoylaminofluorescein di-β-D-galactopyranoside (C12FDG), a specific substrate for the intralysosomal β-galactosidase. The delivery of these liposomes and their content to lysosomes in HeLa cells was investigated by confocal microscopy, flow cytometry, and subcellular fractionation. Confocal microscopy demonstrated that RhB-liposomes co-localize well with the specific lysosomal markers, unlike plain liposomes. The comparison of the FITC fluorescence of the lysosomes isolated by subcellular fractionation also showed that the efficiency of FD delivery into lysosomes by RhB-modified liposomes was significantly higher compared with plain liposomes. These results were additionally confirmed by the flow cytometry of the intact cells treated with C12FDG-loaded liposomes that also demonstrated increased lysosomal targeting by RhB-modified liposomes. The modification of the liposomal surface with a lysosomotropic ligand, such as octadecyl-RhB, can significantly increase the delivery of liposomal loads to lysosomes. PMID:21275828

  9. 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

  10. The chromosomal passenger protein birc5b organizes microfilaments and germ plasm in the zebrafish embryo.

    Directory of Open Access Journals (Sweden)

    Sreelaja Nair

    2013-04-01

    Full Text Available Microtubule-microfilament interactions are important for cytokinesis and subcellular localization of proteins and mRNAs. In the early zebrafish embryo, astral microtubule-microfilament interactions also facilitate a stereotypic segregation pattern of germ plasm ribonucleoparticles (GP RNPs, which is critical for their eventual selective inheritance by germ cells. The precise mechanisms and molecular mediators for both cytoskeletal interactions and GP RNPs segregation are the focus of intense research. Here, we report the molecular identification of a zebrafish maternal-effect mutation motley as Birc5b, a homolog of the mammalian Chromosomal Passenger Complex (CPC component Survivin. The meiosis and mitosis defects in motley/birc5b mutant embryos are consistent with failed CPC function, and additional defects in astral microtubule remodeling contribute to failures in the initiation of cytokinesis furrow ingression. Unexpectedly, the motley/birc5b mutation also disrupts cortical microfilaments and GP RNP aggregation during early cell divisions. Birc5b localizes to the tips of astral microtubules along with polymerizing cortical F-actin and the GP RNPs. Mutant Birc5b co-localizes with cortical F-actin and GP RNPs, but fails to associate with astral microtubule tips, leading to disorganized microfilaments and GP RNP aggregation defects. Thus, maternal Birc5b localizes to astral microtubule tips and associates with cortical F-actin and GP RNPs, potentially linking the two cytoskeletons to mediate microtubule-microfilament reorganization and GP RNP aggregation during early embryonic cell cycles in zebrafish. In addition to the known mitotic function of CPC components, our analyses reveal a non-canonical role for an evolutionarily conserved CPC protein in microfilament reorganization and germ plasm aggregation.

  11. Fragment-based discovery of hepatitis C virus NS5b RNA polymerase inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Antonysamy, Stephen S.; Aubol, Brandon; Blaney, Jeff; Browner, Michelle F.; Giannetti, Anthony M.; Harris, Seth F.; Hébert, Normand; Hendle, Jörg; Hopkins, Stephanie; Jefferson, Elizabeth; Kissinger, Charles; Leveque, Vincent; Marciano, David; McGee, Ethel; Nájera, Isabel; Nolan, Brian; Tomimoto, Masaki; Torres, Eduardo; Wright, Tobi (SGX); (Roche)

    2009-07-22

    Non-nucleoside inhibitors of HCV NS5b RNA polymerase were discovered by a fragment-based lead discovery approach, beginning with crystallographic fragment screening. The NS5b binding affinity and biochemical activity of fragment hits and inhibitors was determined by surface plasmon resonance (Biacore) and an enzyme inhibition assay, respectively. Crystallographic fragment screening hits with {approx}1-10 mM binding affinity (K{sub D}) were iteratively optimized to give leads with {approx}200 nM biochemical activity and low {micro}M cellular activity in a Replicon assay.

  12. Methyl-CpG binding protein 2 (Mecp2 Regulates Sensory Function through Sema5b and Robo2

    Directory of Open Access Journals (Sweden)

    Wan Ying eLeong

    2015-12-01

    Full Text Available Mutations in the gene encoding the MECP2 underlies Rett syndrome, a neurodevelopmental disorder in young females. Although reduced pain sensitivity in Rett syndrome patients and in partial MeCP2 deficient mice had been reported, these previous studies focused predominantly on motor impairments. Therefore, it is still unknown how MeCP2 is involved in these sensory defects. In addition, the human disease manifestations where males with mutations in MECP2 gene normally do not survive and females show typical neurological symptoms only after 18 months of age, is profoundly different in MeCP2-deficient mouse where all animals survived, and males but not females displayed Rett syndrome phenotypes at an early age. Thus, the mecp2-deficient zebrafish serves as an additional animal model to aid in deciphering the role and mechanisms of Mecp2 in neurodevelopment. Here, we used 2 independent methods of silencing expression of Mecp2 in zebrafish to uncover a novel role of Mecp2 in trigeminal ganglion sensory neurons during the embryonic development. mecp2-null mutation and morpholino-mediated silencing of Mecp2 in the zebrafish embryos resulted in defects in peripheral innervation of trigeminal sensory neurons and consequently affecting the sensory function. These defects were demonstrated to be dependent on the expression of Sema5b and Robo2. The expression of both proteins together could better overcome the defects caused by Mecp2 deficiency as compared to the expression of either Sema5b or Robo2 alone. Sema5b and Robo2 were downregulated upon Mecp2 silencing or in mecp2-null embryos, and Chromatin immunoprecipitation (ChIP assay using antibody against Mecp2 was able to pull down specific regions of both Sema5b and Robo2 promoters, showing interaction between Mecp2 and the promoters of both genes. In addition, cell-specific expression of Mecp2 can overcome the innervation and sensory response defects in Mecp2 morphants indicating that these MeCP2-mediated

  13. Identification of a major human high molecular weight salivary mucin (MG1) as tracheobronchial mucin MUC5B

    DEFF Research Database (Denmark)

    Nielsen, P A; Bennett, E P; Wandall, H H;

    1997-01-01

    . Northern analysis of salivary gland RNA probed with SAL1 suggested that MUC5B was highly expressed in salivary glands. In situ hybridization was performed with a SAL1/MUC5B probe and a MUC7 probe. All mucous cells from the submandibular, sublingual, palatine, and labial glands labeled with the MUC5B probe...

  14. 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.)

  15. 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...

  16. HATS-5b: A Transiting hot-Saturn from the HATSouth Survey

    CERN Document Server

    Zhou, G; Penev, K; Bakos, G Á; Hartman, J D; Jordán, A; Mancini, L; Mohler, M; Csubry, Z; Ciceri, S; Brahm, R; Rabus, M; Buchhave, L; Henning, T; Suc, V; Espinoza, N; Béky, B; Noyes, R W; Schmidt, B; Butler, R P; Shectman, S; Thompson, I; Crane, J; Sato, B; Csák, B; Lázár, J; Papp, I; Sári, P; Nikolov, N

    2014-01-01

    We report the discovery of HATS-5b, a transiting hot-Saturn orbiting a G type star, by the HAT-South survey. HATS-5b has a mass of Mp=0.24 Mj, radius of Rp=0.91 Rj, and transits its host star with a period of P=4.7634d. The radius of HATS-5b is consistent with both theoretical and empirical models. The host star has a V band magnitude of 12.6, mass of 0.94 Msun, and radius of 0.87 Rsun. The relatively high scale height of HATS-5b, and the bright, photometrically quiet host star, make this planet a favourable target for future transmission spectroscopy follow-up observations. We reexamine the correlations in radius, equilibrium temperature, and metallicity of the close-in gas-giants, and find hot Jupiter-mass planets to exhibit the strongest dependence between radius and equilibrium temperature. We find no significant dependence in radius and metallicity for the close-in gas-giant population.

  17. Phospholipase C-related catalytically inactive protein (PRIP controls KIF5B-mediated insulin secretion

    Directory of Open Access Journals (Sweden)

    Satoshi Asano

    2014-05-01

    Full Text Available We previously reported that phospholipase C-related catalytically inactive protein (PRIP-knockout mice exhibited hyperinsulinemia. Here, we investigated the role of PRIP in insulin granule exocytosis using Prip-knockdown mouse insulinoma (MIN6 cells. Insulin release from Prip-knockdown MIN6 cells was higher than that from control cells, and Prip knockdown facilitated movement of GFP-phogrin-labeled insulin secretory vesicles. Double-immunofluorescent staining and density step-gradient analyses showed that the KIF5B motor protein co-localized with insulin vesicles in Prip-knockdown MIN6 cells. Knockdown of GABAA-receptor-associated protein (GABARAP, a microtubule-associated PRIP-binding partner, by Gabarap silencing in MIN6 cells reduced the co-localization of insulin vesicles with KIF5B and the movement of vesicles, resulting in decreased insulin secretion. However, the co-localization of KIF5B with microtubules was not altered in Prip- and Gabarap-knockdown cells. The presence of unbound GABARAP, freed either by an interference peptide or by Prip silencing, in MIN6 cells enhanced the co-localization of insulin vesicles with microtubules and promoted vesicle mobility. Taken together, these data demonstrate that PRIP and GABARAP function in a complex to regulate KIF5B-mediated insulin secretion, providing new insights into insulin exocytic mechanisms.

  18. Data of evolutionary structure change: 1CF5B-2JJRA [Confc[Archive

    Lifescience Database Archive (English)

    Full Text Available 1CF5B-2JJRA 1CF5 2JJR B A DVNFDLSTATAKTYTKFIEDFRATLPFSHKVYDIPLLYS...ID> A 2JJRA SYFFNEASATE LEU CA 336 2JJR A 2JJRA...Chain> 2JJR A 2JJRA...bChain>A 2JJRA GKVTS-DIALL

  19. WNT-5A and WNT-5B modulate calcium homeostasis in airway smooth muscle

    NARCIS (Netherlands)

    Koopmans, Tim; Kumawat, Kudleer; Van Den Berge, Maarten; Hoffmann, Roland; Halayko, Andrew J.; Gosens, Reinoud

    2014-01-01

    Rationale Airway hyperresponsiveness is a common feature of asthma explained in part by an excessive contractile response of the airway smooth muscle (ASM). The underlying mechanisms are complex and in need of study. WNT-5A and WNT-5B, two members of the WNT signaling pathway, may be of significance

  20. Genome Sequence Analysis of the Biogenic Amine-Degrading Strain Lactobacillus casei 5b

    OpenAIRE

    Ladero Losada, Víctor Manuel; Herrero, Ana; Martínez Álvarez, Noelia; Río Lagar, Beatriz del; Linares, Daniel M.; Fernández García, María; Martín, M. Cruz; Álvarez González, Miguel Ángel

    2014-01-01

    We here report a 3.02-Mbp annotated draft assembly of the Lactobacillus casei 5b genome. The sequence of this biogenic amine-degrading dairy isolate may help identify the mechanisms involved in the catabolism of biogenic amines and perhaps shed light on ways to reduce the presence of these toxic compounds in food.

  1. Data of evolutionary structure change: 1AO5B-1ELCA [Confc[Archive

    Lifescience Database Archive (English)

    Full Text Available 1AO5B-1ELCA 1AO5 1ELC B A VVGGFNCEKNSQPWQVAVYYQ----KEHICGGVLLDRNW...TPTWQKPDDLQCVFITLLPNENCAKV--YLQKVTDVMLCAGEMGGGKDTCRDDSGGPLICD----GILQGTTSYGPV-PCGKPGVPAIYTNLIKFNSWIKDTMMKNA TRP CA 491 1ELC A 1ELCA...hain> 1ELC A 1ELCA PLHCLVNGQYAVHG... PRO CA 225 1ELC A 1ELCA

  2. Progress on Developing an Interface Program between WIMSD-5B and RFSP

    Energy Technology Data Exchange (ETDEWEB)

    You, Guk Jong; Kim, Won Young; Park, Joo Hwan [Korea Atomic Energy Research Institute, Taejon (Korea, Republic of)

    2005-07-01

    WIMS (Winfrith Improved Multigroup Scheme) code is a multi-group transport code for the reactor lattice calculations which includes a fuel depletion or burn-up routine. The code, created at the United Kingdom Atomic Energy Authority Establishment, Winfrith (AEEW), was intended to perform the lattice calculations with an acceptable accuracy for the analysis of the experiments in a wide range of geometries. As one of its branches, WIMSD-5B is a code which was released from OECD/NEA Data Bank in 1998 and now has been used widely for thermal research and power reactor calculation. Also one of WIMS codes, WIMS-AECL, has been developed by AECL in Canada as an independent version of the original AEEW code. While WIMS-AECL produces a data file which can generate the information required by other code such as RFSP, WIMSD-5B does not. The data file is used for the reactor analysis by WIMSAECL in connection with RFSP. This study is to develop an interface data file (Tape 16) of WIMSD-5B with RFSP and to develop a process utility to provide the group collapsing and cell average cross-section generation for a CANDU-6 core analysis on the WINDOW system. With this utility, the physics analysis of a CANDU-6 reactor will be performed by RFSP code using the lattice parameters generated by WIMSD-5B.

  3. MYO5B mutations in patients with microvillus inclusion disease presenting with transient renal Fanconi syndrome

    NARCIS (Netherlands)

    Golachowska, Magdalena R.; van Dael, Carin M. L.; Keuning, Hilda; Karrenbeld, Arend; Hoekstra, Dick; Gijsbers, Carolien F. M.; Benninga, Marc A.; Rings, Edmond H. H. M.; van IJzendoorn, Sven C. D.

    2012-01-01

    Background and Objective: Microvillus inclusion disease (MVID) is a rare congenital enteropathy associated with brush border atrophy and reduced expression of enzymes at the enterocytes' apical surface. MVID is associated with mutations in the MYO5B gene, which is expressed in all epithelial tissues

  4. HATS-5b: A TRANSITING HOT SATURN FROM THE HATSouth SURVEY

    Energy Technology Data Exchange (ETDEWEB)

    Zhou, G.; Bayliss, D.; Schmidt, B. [Research School of Astronomy and Astrophysics, Australian National University, Canberra, ACT 2611 (Australia); Penev, K.; Bakos, G. Á.; Hartman, J. D.; Csubry, Z. [Department of Astrophysical Sciences, Princeton University, NJ 08544 (United States); Jordán, A.; Brahm, R.; Rabus, M.; Suc, V.; Espinoza, N. [Departamento de Astronomía y Astrofísica, Pontificia Universidad Católica de Chile, Av. Vicuña Mackenna 4860, 7820436 Macul, Santiago (Chile); Mancini, L.; Mohler, M.; Ciceri, S.; Henning, T. [Max Planck Institute for Astronomy, Heidelberg (Germany); Buchhave, L. [Niels Bohr Institute, Copenhagen University (Denmark); Béky, B.; Noyes, R. W. [Harvard-Smithsonian Center for Astrophysics, Cambridge, MA 02138 (United States); Butler, R. P., E-mail: george.zhou@anu.edu.au [Department of Terrestrial Magnetism, Carnegie Institution of Washington, 5241 Broad Branch Road NW, Washington, DC 20015-1305 (United States); and others

    2014-06-01

    We report the discovery of HATS-5b, a transiting hot Saturn orbiting a G-type star, by the HATSouth survey. HATS-5b has a mass of M{sub p} ≈ 0.24 M {sub J}, radius of R{sub p} ≈ 0.91 R {sub J}, and transits its host star with a period of P ≈ 4.7634 days. The radius of HATS-5b is consistent with both theoretical and empirical models. The host star has a V-band magnitude of 12.6, mass of 0.94 M {sub ☉}, and radius of 0.87 R {sub ☉}. The relatively high scale height of HATS-5b and the bright, photometrically quiet host star make this planet a favorable target for future transmission spectroscopy follow-up observations. We reexamine the correlations in radius, equilibrium temperature, and metallicity of the close-in gas giants and find hot Jupiter-mass planets to exhibit the strongest dependence between radius and equilibrium temperature. We find no significant dependence in radius and metallicity for the close-in gas giant population.

  5. 45 CFR Appendix A to Part 5b - Employee Standards of Conduct

    Science.gov (United States)

    2010-10-01

    ... implementing the Act are set forth in 45 CFR 5b. Instruction on the requirements of the Act and regulation... subject to criminal liability as set forth below and in 5 U.S.C. 552a (i): (a) Any officer or employee of... not in compliance with the Act and regulation; (c) Make a disclosure of records within the...

  6. 45 CFR 5b.7 - Procedures for correction or amendment of records.

    Science.gov (United States)

    2010-10-01

    ... corrected or amended, the subject individual will be informed in writing of the refusal to correct or amend... 45 Public Welfare 1 2010-10-01 2010-10-01 false Procedures for correction or amendment of records... PRIVACY ACT REGULATIONS § 5b.7 Procedures for correction or amendment of records. (a) Any...

  7. 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...

  8. 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...

  9. 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 ...

  10. 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....

  11. 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...

  12. 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...

  13. 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...

  14. 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...

  15. 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...

  16. 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 ...

  17. 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...

  18. Age-related changes of serum tartrate-resistant acid phosphatase 5b and the relationship with bone mineral density in Chinese women

    Institute of Scientific and Technical Information of China (English)

    Yue-juan QIN; Zhen-lin ZHANG; Hao ZHANG; Wei-wei HU; Yu-juan LIU; Yun-qiu HU; Miao LI; Jie-mei GU; Jin-wei HE

    2008-01-01

    Aim: Ostcoclastic activity is mainly assessed by measurement of urinary markers (eg C-terminal cross-linked telopeptides of type I collagen, N-terminal cross-linked telopeptides of type I collagen etc), the levels of which could often be affected by renal clearance. Recently, serum tartrate-resistant acid phosphatase 5b (TRACP5b) has been used as an alternative serum marker to evaluate osteoclastic activity. We investigated the age-related changes of TRACP5b level and its association with bone mineral density (BMD) in Chinese women. Methods: Seven-hundred and twenty-two Chinese mainland women aged 20-79 years were recruited in the study. Serum TRACP5b level was measured using immunoassay to evaluate the state of bone resorption. Bone mineral density (BMD) (g/cm2) at lumbar spine 1-4 and proximal femur were measured by duel-energy X-ray absorptiometry. Results: The serum TRACP5b level reached a bottom value in premenopausal women aged 30-39, gradually increased in women aged 40-49, rapidly rose in women aged 50-59, and culminated with a maximum value in women aged 60-69 before a slow drop in women aged 70-79. The average level of TRACPSb was significantly higher in postmenopausal women [(3.29±1.07) U/L] than in premenopausal women ([1.70±0.59] U/L). The levels of TRACP5b were inversely correlated with BMD at all measured sites (P<0.001). Furthermore, the level of TRACP5b was obviously higher in women with osteoporosis and osteopenia than those with normal bone mass (P<0.001). Conclusion: We have established the reference values of serum TRACPSb in Chinese mainland women, and found that postmenopausal women had higher TRACP5b concentration than younger women. The results showed that serum TRACPSb was a sensitive and useful parameter for the evaluation of age-related changes of bone absorption.

  19. Protection and Delivery of Anthelmintic Protein Cry5B to Nematodes Using Mesoporous Silicon Particles.

    Science.gov (United States)

    Wu, Chia-Chen; Hu, Yan; Miller, Melanie; Aroian, Raffi V; Sailor, Michael J

    2015-06-23

    The ability of nano- and microparticles of partially oxidized mesoporous silicon (pSi) to sequester, protect, and deliver the anthelmintic pore-forming protein Cry5B to nematodes is assessed in vitro and in vivo. Thermally oxidized pSi particles are stable under gastric conditions and show relatively low toxicity to nematodes. Fluorescence images of rhodamine-labeled pSi particles within the nematodes Caenorhabditis elegans and Ancylostoma ceylanicum show that ingestion is dependent on particle size: particles of a 0.4 ± 0.2 μm size are noticeably ingested by both species within 2 h of introduction in vitro, whereas 5 ± 2 μm particles are excluded from C. elegans but enter the pharynx region of A. ceylanicum after 24 h. The anthelmintic protein Cry5B, a pore-forming crystal (Cry) protein derived from Bacillus thuringiensis, is incorporated into the pSi particles by aqueous infiltration. Feeding of Cry5B-loaded pSi particles to C. elegans leads to significant intoxication of the nematode. Protein-loaded particles of size 0.4 μm display the highest level of in vitro toxicity toward C. elegans on a drug-mass basis. The porous nanostructure protects Cry5B from hydrolytic and enzymatic (pepsin) degradation in simulated gastric fluid (pH 1.2) for time periods up to 2 h. In vivo experiments with hookworm-infected hamsters show no significant reduction in worm burden with the Cry5B-loaded particles, which is attributed to slow release of the protein from the particles and/or short residence time of the particles in the duodenum of the animal.

  20. Protection and Delivery of Anthelmintic Protein Cry5B to Nematodes Using Mesoporous Silicon Particles.

    Science.gov (United States)

    Wu, Chia-Chen; Hu, Yan; Miller, Melanie; Aroian, Raffi V; Sailor, Michael J

    2015-06-23

    The ability of nano- and microparticles of partially oxidized mesoporous silicon (pSi) to sequester, protect, and deliver the anthelmintic pore-forming protein Cry5B to nematodes is assessed in vitro and in vivo. Thermally oxidized pSi particles are stable under gastric conditions and show relatively low toxicity to nematodes. Fluorescence images of rhodamine-labeled pSi particles within the nematodes Caenorhabditis elegans and Ancylostoma ceylanicum show that ingestion is dependent on particle size: particles of a 0.4 ± 0.2 μm size are noticeably ingested by both species within 2 h of introduction in vitro, whereas 5 ± 2 μm particles are excluded from C. elegans but enter the pharynx region of A. ceylanicum after 24 h. The anthelmintic protein Cry5B, a pore-forming crystal (Cry) protein derived from Bacillus thuringiensis, is incorporated into the pSi particles by aqueous infiltration. Feeding of Cry5B-loaded pSi particles to C. elegans leads to significant intoxication of the nematode. Protein-loaded particles of size 0.4 μm display the highest level of in vitro toxicity toward C. elegans on a drug-mass basis. The porous nanostructure protects Cry5B from hydrolytic and enzymatic (pepsin) degradation in simulated gastric fluid (pH 1.2) for time periods up to 2 h. In vivo experiments with hookworm-infected hamsters show no significant reduction in worm burden with the Cry5B-loaded particles, which is attributed to slow release of the protein from the particles and/or short residence time of the particles in the duodenum of the animal. PMID:25950754

  1. 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.

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

    Science.gov (United States)

    Seggewiß, Nicole; Paulmann, Dajana; Dotzauer, Andreas

    2016-01-01

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

  3. Reduced sulfation of muc5b is linked to xerostomia in patients with Sjögren syndrome

    DEFF Research Database (Denmark)

    Alliende, C; Kwon, Y-J; Brito, M;

    2008-01-01

    the amount of MUC5B (mRNA and protein) as well as sulfation levels in salivary glands of patients with normal or altered unstimulated salivary flow. Localisation of MUC5B and sulfated MUC5B, as well as total levels sulfated groups were determined and compared with acini basal lamina disorganisation. PATIENTS...... AND METHODS: In all, 18 patients with normal or altered unstimulated salivary flow and 16 controls were studied. MUC5B mRNA and protein were evaluated in salivary glands by semiquantitative RT-PCR and Western blot analysis. MUC5B sulfation was determined by Western blotting. MUC5B and sulfo-Lewis(a) antigen...... disorganised basal lamina. CONCLUSION: Disorganisation of the basal lamina observed in patients with Sjögren syndrome may lead to dedifferentiation of acinar mucous cells and, as a consequence, alter sulfation of MUC5B. These changes are suggested to represent a novel mechanism that may explain xerostomia...

  4. 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

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

    Directory of Open Access Journals (Sweden)

    Arnaud M. Labrousse

    2011-10-01

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

  6. Comparative Investigation of Normal Modes and Molecular Dynamics of Hepatitis C NS5B Protein

    Science.gov (United States)

    Asafi, M. S.; Yildirim, A.; Tekpinar, M.

    2016-04-01

    Understanding dynamics of proteins has many practical implications in terms of finding a cure for many protein related diseases. Normal mode analysis and molecular dynamics methods are widely used physics-based computational methods for investigating dynamics of proteins. In this work, we studied dynamics of Hepatitis C NS5B protein with molecular dynamics and normal mode analysis. Principal components obtained from a 100 nanoseconds molecular dynamics simulation show good overlaps with normal modes calculated with a coarse-grained elastic network model. Coarse-grained normal mode analysis takes at least an order of magnitude shorter time. Encouraged by this good overlaps and short computation times, we analyzed further low frequency normal modes of Hepatitis C NS5B. Motion directions and average spatial fluctuations have been analyzed in detail. Finally, biological implications of these motions in drug design efforts against Hepatitis C infections have been elaborated.

  7. The influence of alloying elements in aluminium on the grain refinement with ALTI5B1

    Directory of Open Access Journals (Sweden)

    Naglič I.

    2009-07-01

    Full Text Available This work deals with the influence of alloying elements in aluminium on the grain refinement with various additions of AlTi5B1. Grain-refinement tests were made at a cooling rate of 15 °C/s. The results revealed that in both aluminium and an Al-Fe alloy the grain size decreases with increasing additions of the AlTi5B1 grain refiner. We found that for the same boron content the grain size was smaller in the case of the Al-Fe alloy. The difference in the grain sizes for the same content of boron was approximately 15 μm; this is considerably smaller than the difference between the grain sizes in samples with the same difference of growth-restricting factor made at slower cooling rates.

  8. 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.

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

    Directory of Open Access Journals (Sweden)

    Marika Salvalaio

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

  10. Unit 3 Hobbies《牛津小学英语》(译林版)5B

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    @@ 一、教材分析 本课是(译林版)5B Unit 3 Hobbies的第一课时.教学内容是四个动词词组go shopping,take photos,collect stamps和grow flowers,以及句型"Do you have any hobbies?Yes.I do.I like...He/She likes...too.".

  11. Qatar Exoplanet Survey : Qatar-3b, Qatar-4b and Qatar-5b

    CERN Document Server

    Alsubai, Khalid A; Tsvetanov, Zlatan I; Latham, David W; Bieryla, Allyson; Buchhave, Lars A; Esquerdo, Gilbert A; Bramich, D M; Pyrzas, Stylianos; Vilchez, Nicolas P E; Mancini, Luigi; Southworth, John; Evans, Daniel F; Henning, Thomas; Ciceri, Simona

    2016-01-01

    We report the discovery of Qatar-3b, Qatar-4b, and Qatar-5b, three new transiting planets identified by the Qatar Exoplanet Survey (QES). The three planets belong to the hot Jupiter family, with orbital periods of $P_{Q3b}$=2.5079204 days, $P_{Q4b}$=1.8053949 days, and $P_{Q5b}$=2.8792319 days. Follow-up spectroscopic observations reveal the masses of the planets to be $M_{Q3b}$=4.31$M_{\\rm J}$, $M_{Q4b}$=5.85$M_{\\rm J}$, and $M_{Q5b}$=4.32$M_{\\rm J}$, while model fits to the transit light curves yield radii of $R_{Q3b}$=1.096$R_{\\rm J}$, $R_{Q4b}$=1.552$R_{\\rm J}$, and $R_{Q5b}$=1.107$R_{\\rm J}$. No evidence of eccentric orbit is seen in the radial velocity curve of any of the planets. The host stars are typical main sequence stars with masses and radii $M_{Q3}$=1.145$M_{\\odot}$, $M_{Q4}$=0.954$M_{\\odot}$, $M_{Q5}$=1.128$M_{\\odot}$ and $R_{Q3}$=1.272$R_{\\odot}$, $R_{Q4}$=1.115$R_{\\odot}$ and $R_{Q5}$=1.076$R_{\\odot}$ for the Qatar-3, 4 and 5 respectively. All three new planets can be classified as heavy hot ...

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

    Science.gov (United States)

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

    2013-01-01

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

  13. Structural analysis of human KDM5B guides histone demethylase inhibitor development.

    Science.gov (United States)

    Johansson, Catrine; Velupillai, Srikannathasan; Tumber, Anthony; Szykowska, Aleksandra; Hookway, Edward S; Nowak, Radoslaw P; Strain-Damerell, Claire; Gileadi, Carina; Philpott, Martin; Burgess-Brown, Nicola; Wu, Na; Kopec, Jola; Nuzzi, Andrea; Steuber, Holger; Egner, Ursula; Badock, Volker; Munro, Shonagh; LaThangue, Nicholas B; Westaway, Sue; Brown, Jack; Athanasou, Nick; Prinjha, Rab; Brennan, Paul E; Oppermann, Udo

    2016-07-01

    Members of the KDM5 (also known as JARID1) family are 2-oxoglutarate- and Fe(2+)-dependent oxygenases that act as histone H3K4 demethylases, thereby regulating cell proliferation and stem cell self-renewal and differentiation. Here we report crystal structures of the catalytic core of the human KDM5B enzyme in complex with three inhibitor chemotypes. These scaffolds exploit several aspects of the KDM5 active site, and their selectivity profiles reflect their hybrid features with respect to the KDM4 and KDM6 families. Whereas GSK-J1, a previously identified KDM6 inhibitor, showed about sevenfold less inhibitory activity toward KDM5B than toward KDM6 proteins, KDM5-C49 displayed 25-100-fold selectivity between KDM5B and KDM6B. The cell-permeable derivative KDM5-C70 had an antiproliferative effect in myeloma cells, leading to genome-wide elevation of H3K4me3 levels. The selective inhibitor GSK467 exploited unique binding modes, but it lacked cellular potency in the myeloma system. Taken together, these structural leads deliver multiple starting points for further rational and selective inhibitor design. PMID:27214403

  14. 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...

  15. 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...

  16. 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.

  17. 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

  18. GR-127935-sensitive mechanism mediating hypotension in anesthetized rats: are 5-HT5B receptors involved?

    Science.gov (United States)

    Sánchez-Maldonado, Carolina; López-Sánchez, Pedro; Anguiano-Robledo, Liliana; Leopoldo, Marcello; Lacivita, Enza; Terrón, José A

    2015-04-01

    The 5-HT1B/1D receptor antagonist, GR-127935, inhibits hypotensive responses produced by the 5-HT1A, 5-HT1B/1D and 5-HT7 receptor agonist, and 5-HT5A/5B receptor ligand, 5-carboxamidotryptamine (5-CT), in rats. This work further characterized the above mechanism using more selective 5-HT1B and 5-HT1D receptor antagonists. Also, expression of 5-HT5A and 5-HT5B receptor mRNAs in blood vessels was searched by reverse transcription polymerase chain reaction. Decreases in diastolic blood pressure induced by 5-CT (0.001-10 μg/kg, intravenously) were analyzed in anesthetized rats that had received intravenous vehicle (1 mL/kg), SB-224289 (5-HT1B antagonist; 0.3 and 1.0 mg/kg), BRL15572 (5-HT1D antagonist; 0.3 and 1.0 mg/kg), SB-224289 + BRL15572 (0.3 mg/kg, each), or SB-224289 + BRL15572 (0.3 mg/kg, each) + GR-127935 (1 mg/kg). Because only the latter treatment inhibited 5-CT-induced hypotension, suggestive of a mechanism unrelated to 5-HT1B/1D receptors, the effects of antagonists/ligands at 5-HT5A (SB-699551, 1 mg/kg), 5-HT6 (SB-399885, 1 mg/kg), and 5-HT1B/1D/5A/5B/7 receptors (ergotamine, 0.1 mg/kg) on 5-CT-induced hypotension were tested. Interestingly, only ergotamine blocked 5-CT-induced responses; this effect closely paralleled that of SB-224289 + BRL-15572 + GR-127935. Neither did ergotamine nor GR-127935 inhibit hypotensive responses induced by the 5-HT7 receptor agonist, LP-44. Faint but clear bands corresponding to 5-HT5A and 5-HT5B receptor mRNAs in aorta and mesenteric arteries were detected. Results suggest that the GR-127935-sensitive mechanism mediating hypotension in rats is unrelated to 5-HT1B, 5-HT1D, 5-HT5A, 5-HT6, and 5-HT7 receptors. This mechanism, however, resembles putative 5-HT5B receptors. PMID:25502305

  19. Over-expression of Stat5b confers protection against diabetes in the non-obese diabetic (NOD) mice via up-regulation of CD4+CD25+ regulatory T cells

    International Nuclear Information System (INIS)

    Highlights: ► This is the first study to provide direct evidence of the role of Stat5b in NOD mice. ► Over-expression of wild type Stat5b transgene protects NOD mice against diabetes. ► This protection may be mediated by the up-regulation of CD4+CD25+ Tregs. -- Abstract: The signal transducers and activators of transcription (STAT) family of proteins play a critical role in cytokine signaling required for fine tuning of immune regulation. Previous reports showed that a mutation (L327M) in the Stat5b protein leads to aberrant cytokine signaling in the NOD mice. To further elaborate the role of Stat5b in diabetes, we established a NOD transgenic mouse that over-expresses the wild type Stat5b gene. The incidences of spontaneous diabetes as well as cyclophosphamide-induced diabetes were significantly reduced and delayed in the Stat5b transgenic NOD mice compared to their littermate controls. The total cell numbers of CD4+ T cells and especially CD8+ T cells in the spleen and pancreatic lymph node were increased in the Stat5b transgenic NOD mice. Consistent with these findings, CD4+ and CD8+ T cells from the Stat5b transgenic NOD mice showed a higher proliferation capacity and up-regulation of multiple cytokines including IL-2, IFN-γ, TNF-α and IL-10 as well as anti-apoptotic gene Bcl-xl. Furthermore, the number and proportion of CD4+CD25+ regulatory T cells were significantly increased in transgenic mice although in vitro suppression ability of the regulatory T-cells was not affected by the transgene. Our results suggest that Stat5b confers protection against diabetes in the NOD mice by regulating the numbers and function of multiple immune cell types, especially by up-regulating CD4+CD25+ regulatory T cells.

  20. The lysosome as a command-and-control center for cellular metabolism.

    Science.gov (United States)

    Lim, Chun-Yan; Zoncu, Roberto

    2016-09-12

    Lysosomes are membrane-bound organelles found in every eukaryotic cell. They are widely known as terminal catabolic stations that rid cells of waste products and scavenge metabolic building blocks that sustain essential biosynthetic reactions during starvation. In recent years, this classical view has been dramatically expanded by the discovery of new roles of the lysosome in nutrient sensing, transcriptional regulation, and metabolic homeostasis. These discoveries have elevated the lysosome to a decision-making center involved in the control of cellular growth and survival. Here we review these recently discovered properties of the lysosome, with a focus on how lysosomal signaling pathways respond to external and internal cues and how they ultimately enable metabolic homeostasis and cellular adaptation. PMID:27621362

  1. 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

  2. MUC5B silencing reduces chemo-resistance of MCF-7 breast tumor cells and impairs maturation of dendritic cells.

    Science.gov (United States)

    García, Enrique P; Tiscornia, Inés; Libisch, Gabriela; Trajtenberg, Felipe; Bollati-Fogolín, Mariela; Rodríguez, Ernesto; Noya, Verónica; Chiale, Carolina; Brossard, Natalie; Robello, Carlos; Santiñaque, Federico; Folle, Gustavo; Osinaga, Eduardo; Freire, Teresa

    2016-05-01

    Mucins participate in cancer progression by regulating cell growth, adhesion, signaling, apoptosis or chemo-resistance to drugs. The secreted mucin MUC5B, the major component of the respiratory tract mucus, is aberrantly expressed in breast cancer, where it could constitute a cancer biomarker. In this study we evaluated the role of MUC5B in breast cancer by gene silencing the MUC5B expression with short hairpin RNA on MCF-7 cells. We found that MUC5B-silenced MCF-7 cells have a reduced capacity to grow, adhere and form cell colonies. Interestingly, MUC5B knock-down increased the sensitivity to death induced by chemotherapeutic drugs. We also show that MUC5B silencing impaired LPS-maturation of DCs, and production of cytokines. Furthermore, MUC5B knock-down also influenced DC-differentiation and activation since it resulted in an upregulation of IL-1β, IL-6 and IL-10, cytokines that might be involved in cancer progression. Thus, MUC5B could enhance the production of LPS-induced cytokines, suggesting that the use of MUC5B-based cancer vaccines combined with DC-maturation stimuli, could favor the induction of an antitumor immune response.

  3. Physico-chemical Properties and Bioactivities of a Glycoconjugate LbGp5B from Lycium barbarum L.

    Institute of Scientific and Technical Information of China (English)

    PENG,Xue-Mei(彭雪梅); PENG,Xue-Mei; QI,Chun-Hui(齐春会); QI,Chun-Hui; TIAN,Geng-Yuan (田庚元); TIAN,Geng-Yuan; ZHANG,Yong-Xiang(张永详); ZHANG,Yong-Xiang

    2001-01-01

    A glycoconjugatedesignated as LbGp5B was isolated from the fruit of Lyciun barbarum L. and purified to homogeneity by gel filtration .LbGp5B is composed of rhamnose (Rha), arabinose (Ara), galactose (Gal), glucose (Glc), galacturonic acid (GalA) and seveateen amino acids. The molecular weight of LbGp5B was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and by matrix-asisted laser desorption/ionization (MALDI) time of fight (T OF) mass spectrometry (MS). The preliminary experiments showed that LbGp5B promoted splenocyte proliferation in mice and inhihited the peroxidation of low density lipoprotein (LDL).

  4. A quantitative assay for lysosomal acidification rates in human osteoclasts

    DEFF Research Database (Denmark)

    Jensen, Vicki Kaiser; Nosjean, Olivier; Dziegiel, Morten Hanefeld;

    2011-01-01

    The osteoclast initiates resorption by creating a resorption lacuna. The ruffled border surrounding the lacunae arises from exocytosis of lysosomes. To dissolve the inorganic phase of the bone, the vacuolar adenosine triphosphatase, located in the ruffled border, pumps protons into the resorption...... lacunae. The electroneutrality of the lacunae is maintained by chloride transport through the chloride-proton antiporter chloride channel 7. Inhibition of either proton or chloride transport prevents bone resorption. The aims of this study were to validate the human osteoclastic microsome- based influx......, the effect of valinomycin, inhibitor sensitivity, and the ion profile of the human osteoclast microsomes. The expression level of chloride channel 7 was increased in the human osteoclastic microsomes compared with whole osteoclasts. Acid influx was induced by 1.25 mM adenosine triphosphate. Further 1.1 μ...

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

    Science.gov (United States)

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

    1998-01-01

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

  6. KIF5B and Nup358 Cooperatively Mediate the Nuclear Import of HIV-1 during Infection.

    Science.gov (United States)

    Dharan, Adarsh; Talley, Sarah; Tripathi, Abhishek; Mamede, João I; Majetschak, Matthias; Hope, Thomas J; Campbell, Edward M

    2016-06-01

    Following envelope mediated fusion, the HIV-1 core is released into the cytoplasm of the target cell and undergoes a series of trafficking and replicative steps that result in the nuclear import of the viral genome, which ultimately leads to the integration of the proviral DNA into the host cell genome. Previous studies have found that disruption of microtubules, or depletion of dynein or kinesin motors, perturb the normal uncoating and trafficking of the viral genome. Here, we show that the Kinesin-1 motor, KIF5B, induces a relocalization of the nuclear pore component Nup358 into the cytoplasm during HIV-1 infection. This relocalization of NUP358 is dependent on HIV-1 capsid, and NUP358 directly associates with viral cores following cytoplasmic translocation. This interaction between NUP358 and the HIV-1 core is dependent on multiple capsid binding surfaces, as this association is not observed following infection with capsid mutants in which a conserved hydrophobic binding pocket (N74D) or the cyclophilin A binding loop (P90A) is disrupted. KIF5B knockdown also prevents the nuclear entry and infection by HIV-1, but does not exert a similar effect on the N74D or P90A capsid mutants which do not rely on Nup358 for nuclear import. Finally, we observe that the relocalization of Nup358 in response to CA is dependent on cleavage protein and polyadenylation factor 6 (CPSF6), but independent of cyclophilin A. Collectively, these observations identify a previously unappreciated role for KIF5B in mediating the Nup358 dependent nuclear import of the viral genome during infection. PMID:27327622

  7. KIF5B and Nup358 Cooperatively Mediate the Nuclear Import of HIV-1 during Infection.

    Directory of Open Access Journals (Sweden)

    Adarsh Dharan

    2016-06-01

    Full Text Available Following envelope mediated fusion, the HIV-1 core is released into the cytoplasm of the target cell and undergoes a series of trafficking and replicative steps that result in the nuclear import of the viral genome, which ultimately leads to the integration of the proviral DNA into the host cell genome. Previous studies have found that disruption of microtubules, or depletion of dynein or kinesin motors, perturb the normal uncoating and trafficking of the viral genome. Here, we show that the Kinesin-1 motor, KIF5B, induces a relocalization of the nuclear pore component Nup358 into the cytoplasm during HIV-1 infection. This relocalization of NUP358 is dependent on HIV-1 capsid, and NUP358 directly associates with viral cores following cytoplasmic translocation. This interaction between NUP358 and the HIV-1 core is dependent on multiple capsid binding surfaces, as this association is not observed following infection with capsid mutants in which a conserved hydrophobic binding pocket (N74D or the cyclophilin A binding loop (P90A is disrupted. KIF5B knockdown also prevents the nuclear entry and infection by HIV-1, but does not exert a similar effect on the N74D or P90A capsid mutants which do not rely on Nup358 for nuclear import. Finally, we observe that the relocalization of Nup358 in response to CA is dependent on cleavage protein and polyadenylation factor 6 (CPSF6, but independent of cyclophilin A. Collectively, these observations identify a previously unappreciated role for KIF5B in mediating the Nup358 dependent nuclear import of the viral genome during infection.

  8. Pds5B is required for cohesion establishment and Aurora B accumulation at centromeres

    OpenAIRE

    Carretero, María; Ruiz-Torres, Miguel; Rodríguez-Corsino, Miriam; Barthelemy, Isabel; Losada, Ana

    2013-01-01

    Cohesin mediates sister chromatid cohesion and contributes to the organization of interphase chromatin through DNA looping. In vertebrate somatic cells, cohesin consists of Smc1, Smc3, Rad21, and either SA1 or SA2. Three additional factors Pds5, Wapl, and Sororin bind to cohesin and modulate its dynamic association with chromatin. There are two Pds5 proteins in vertebrates, Pds5A and Pds5B, but their functional specificity remains unclear. Here, we demonstrate that Pds5 proteins are essential...

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

    Science.gov (United States)

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

    2010-12-01

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

  10. 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

  11. 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

  12. Compensatory Role of Inositol 5-Phosphatase INPP5B to OCRL in Primary Cilia Formation in Oculocerebrorenal Syndrome of Lowe.

    Directory of Open Access Journals (Sweden)

    Na Luo

    Full Text Available Inositol phosphatases are important regulators of cell signaling, polarity, and vesicular trafficking. Mutations in OCRL, an inositol polyphosphate 5-phosphatase, result in Oculocerebrorenal syndrome of Lowe, an X-linked recessive disorder that presents with congenital cataracts, glaucoma, renal dysfunction and mental retardation. INPP5B is a paralog of OCRL and shares similar structural domains. The roles of OCRL and INPP5B in the development of cataracts and glaucoma are not understood. Using ocular tissues, this study finds low levels of INPP5B present in human trabecular meshwork but high levels in murine trabecular meshwork. In contrast, OCRL is localized in the trabecular meshwork and Schlemm's canal endothelial cells in both human and murine eyes. In cultured human retinal pigmented epithelial cells, INPP5B was observed in the primary cilia. A functional role for INPP5B is revealed by defects in cilia formation in cells with silenced expression of INPP5B. This is further supported by the defective cilia formation in zebrafish Kupffer's vesicles and in cilia-dependent melanosome transport assays in inpp5b morphants. Taken together, this study indicates that OCRL and INPP5B are differentially expressed in the human and murine eyes, and play compensatory roles in cilia development.

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

    Science.gov (United States)

    Opperdoes, F R; Van Roy, J

    1982-09-01

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

  14. 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.

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

    Directory of Open Access Journals (Sweden)

    Geum-Hwa Lee

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

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

    Science.gov (United States)

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

    2016-01-01

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

  17. A mouse model of KIF5B-RET fusion-dependent lung tumorigenesis.

    Science.gov (United States)

    Saito, Motonobu; Ishigame, Teruhide; Tsuta, Koji; Kumamoto, Kensuke; Imai, Toshio; Kohno, Takashi

    2014-11-01

    Oncogenic fusion of the RET (rearranged during transfection) gene was recently identified as a novel driver gene aberration not only for the development of thyroid carcinoma but also of lung adenocarcinoma, the most frequent histological type of lung cancer. This study constructed and analyzed transgenic mice expressing KIF5B-RET, the predominant form of RET fusion gene specific for lung adenocarcinoma, under the control of the SPC (surfactant protein C) gene promoter. The mice expressed the KIF5B-RET fusion gene specifically in lung alveolar epithelial cells, and developed multiple tumors in the lungs. Treatment of the transgenic mice with vandetanib, which is a RET tyrosine kinase inhibitor approved by the U.S. Food and Drug Administration for the treatment of thyroid carcinoma, for 8 or 20 weeks led to a marked reduction in the number of lung tumors (3.3 versus 0 and 6.5 versus 0.2 per tissue section, respectively; P < 0.01, t-test). The results suggest that the RET fusion functions as a driver for the development of lung tumors, whose growth is inhibited by RET tyrosine kinase inhibitors. PMID:25064355

  18. Classification of HCV NS5B Polymerase Inhibitors Using Support Vector Machine

    Directory of Open Access Journals (Sweden)

    Changyuan Yu

    2012-03-01

    Full Text Available Using a support vector machine (SVM, three classification models were built to predict whether a compound is an active or weakly active inhibitor based on a dataset of 386 hepatitis C virus (HCV NS5B polymerase NNIs (non-nucleoside analogue inhibitors fitting into the pocket of the NNI III binding site. For each molecule, global descriptors, 2D and 3D property autocorrelation descriptors were calculated from the program ADRIANA.Code. Three models were developed with the combination of different types of descriptors. Model 2 based on 16 global and 2D autocorrelation descriptors gave the highest prediction accuracy of 88.24% and MCC (Matthews correlation coefficient of 0.789 on test set. Model 1 based on 13 global descriptors showed the highest prediction accuracy of 86.25% and MCC of 0.732 on external test set (including 80 compounds. Some molecular properties such as molecular shape descriptors (InertiaZ, InertiaX and Span, number of rotatable bonds (NRotBond, water solubility (LogS, and hydrogen bonding related descriptors performed important roles in the interactions between the ligand and NS5B polymerase.

  19. Comparative analysis of reactor cycle neutron characteristics using different WIMSD5B nuclear data libraries

    International Nuclear Information System (INIS)

    The accuracy and quality of steady-state and transient calculations with 3D core kinetics codes depend heavily on the quality of neutronic constants used in calculations. Current paper presents comparative analysis of VVER-1000 core calculations by means of DYN3D code with neutronic constants prepared using WIMSD5B code with implementation of different nuclear data libraries. The quality of results obtained using WIMSD5B can be improved by means of updating its nuclear data library. Libraries under consideration have been developed as a part of WIMSD Library Update Project (WLUP) held by IAEA and have the following names: ENDFB-6, ENDFB-7, IAEA, JEF2.2, JEFF3.1 and JENDL3. Libraries performance was tested by simulating the 8th fuel cycle of KhNPP-2 and comparing simulation results with real operational parameters. Libraries listed above were used for preparation of neutronic constants for VVER-1000 fuel, operated at KhNPP-2 during 1-7 fuel cycles and, as a result, separate neutronic constants' sets and initial burnup distribution for the 8th fuel cycle were obtained for each library. Simulation and operational data were compared based on such criteria as cycle duration, boron concentration and power peaking factors over core for four different moments of cycle. Results verify that simulation data is in good agreement with operational data, and results obtained using JEF2.2 library are of good quality in terms of criteria listed above

  20. 水稻小G蛋白OsRab5b的亚细胞定位研究%Subcellular Localization of Rice Small G Protein OsRab5b in BY-2 Cells

    Institute of Scientific and Technical Information of China (English)

    邵军丽; 龙跃生; 徐增富

    2015-01-01

    observed in the nuclei and cytoplasm of the transgenic cells, which were not affected by the wortmannin or BFA treatment. OsRab5b is localized to the prevacuolar compartment of BY-2 cells and the Gly2 site is essential for the correct subcellular localization of OsRab5b.

  1. 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

  2. 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...

  3. 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.

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

    Science.gov (United States)

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

    2016-03-01

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

  5. Mechanistic and single-dose in vivo therapeutic studies of Cry5B anthelmintic action against hookworms.

    Directory of Open Access Journals (Sweden)

    Yan Hu

    Full Text Available BACKGROUND: Hookworm infections are one of the most important parasitic infections of humans worldwide, considered by some second only to malaria in associated disease burden. Single-dose mass drug administration for soil-transmitted helminths, including hookworms, relies primarily on albendazole, which has variable efficacy. New and better hookworm therapies are urgently needed. Bacillus thuringiensis crystal protein Cry5B has potential as a novel anthelmintic and has been extensively studied in the roundworm Caenorhabditis elegans. Here, we ask whether single-dose Cry5B can provide therapy against a hookworm infection and whether C. elegans mechanism-of-action studies are relevant to hookworms. METHODOLOGY/PRINCIPAL FINDINGS: To test whether the C. elegans invertebrate-specific glycolipid receptor for Cry5B is relevant in hookworms, we fed Ancylostoma ceylanicum hookworm adults Cry5B with and without galactose, an inhibitor of Cry5B-C. elegans glycolipid interactions. As with C. elegans, galactose inhibits Cry5B toxicity in A. ceylanicum. Furthermore, p38 mitogen-activated protein kinase (MAPK, which controls one of the most important Cry5B signal transduction responses in C. elegans, is functionally operational in hookworms. A. ceylanicum hookworms treated with Cry5B up-regulate p38 MAPK and knock down of p38 MAPK activity in hookworms results in hypersensitivity of A. ceylanicum adults to Cry5B attack. Single-dose Cry5B is able to reduce by >90% A. ceylanicum hookworm burdens from infected hamsters, in the process eliminating hookworm egg shedding in feces and protecting infected hamsters from blood loss. Anthelmintic activity is increased about 3-fold, eliminating >97% of the parasites with a single 3 mg dose (∼30 mg/kg, by incorporating a simple formulation to help prevent digestion in the acidic stomach of the host mammal. CONCLUSIONS/SIGNIFICANCE: These studies advance the development of Cry5B protein as a potent, safe single

  6. Bupivacaine can enhance lysosomal activity in mouse muscle myoblasts%布比卡因增强小鼠成肌细胞溶酶体的活性

    Institute of Scientific and Technical Information of China (English)

    熊静薇; 毛雨; 李荣荣; 丁正年

    2015-01-01

    Objective To investigate the effects of bupivacaine on lysosomal abundance and activity in mouse muscle myoblasts.Methods Mouse myoblasts C2C12 was randomly divided into control group (without any treatment) and bupivacaine group (treated with bupivacaine 600 μ mol/L for 6 h).After then,the changes of lysosomal pH was assessed by LysoSensor pH indicator.The content of lysosomes was detected by LysoTracker probe.The expression of lysosomal-associated membrane protein-1 (LAMP-1) and Cathepsin B was detected by Western blot analysis.The activity of lysosomal proteolytic enzymes Cathepsin B was determined by MagicRed assay kit.Results Bupivacaine did not affect lysosomal pH.However,compared with the controls,lysosomal abundance was significantly increased 15.15% following bupivacaine treatment(P<0.01).Moreover,protein expression levels of LAMP-1 and Cathepsin B were significantly upregulated 36.41% and 35.29% respetctively by bupivacaine (P<0.01).Furthermore,the activity of Cathepsin B was significantly increased 23.74% by bupivacaine(P<0.01).Conclusions Bupivacaine increased lysosomal content and enhance lysosomal activity in mouse muscle myoblasts.%目的 探讨局部麻醉药布比卡因对小鼠成肌细胞溶酶体的影响. 方法 将体外培养的小鼠成肌细胞C2C12分为2组.对照组:不加任何药物;布比卡因组:以600μmol/L布比卡因刺激细胞6h.实验结束后,用LysoSensor探针评价溶酶体腔pH,用LysoTrackor探针检测溶酶体含量,用蛋白免疫印迹法检测溶酶体相关膜蛋白-1(LAMP-1)和溶酶体蛋白水解酶Cathepsin B的表达水平,并以MagicRed染色法测定Cathepsin B的活性.结果 布比卡因对溶酶体腔pH没有影响.但是,与对照组相比,布比卡因组溶酶体含量增加15.15% (P<0.01),LAMP-1与Cathepsin B表达量分别增加36.41%、35.29% (P<0.01),Cathepsin B活性增加23.74%(P<0.01).结论 布比卡因能增加小鼠成肌细胞溶酶体含量,增强溶酶体活性.

  7. STAT5b as Molecular Target in Pancreatic Cancer—Inhibition of Tumor Growth, Angiogenesis, and Metastases

    Directory of Open Access Journals (Sweden)

    Christian Moser

    2012-10-01

    Full Text Available The prognosis of patients suffering from pancreatic cancer is still poor and novel therapeutic options are urgently needed. Recently, the transcription factor signal transducer and activator of transcription 5b (STAT5b was associated with tumor progression in human solid cancer. Hence, we assessed whether STAT5b might serve as an anticancer target in ductal pancreatic adenocarcinoma (DPAC. We found that nuclear expression of STAT5b can be detected in approximately 50% of DPAC. Blockade of STAT5b by stable shRNA-mediated knockdown showed no effects on tumor cell growth in vitro. However, inhibition of tumor cell motility was found even in response to stimulation with epidermal growth factor or interleukin-6. These findings were paralleled by a reduction of prometastatic and proangiogenic factors in vitro. Subsequent in vivo experiments revealed a strong growth inhibition on STAT5b blockade in subcutaneous and orthotopic models. These findings were paralleled by impaired tumor angiogenesis in vivo. In contrast to the subcutaneous model, the orthotopic model revealed a strong reduction of tumor cell proliferation that emphasizes the meaning of assessing targets in an appropriate microenvironment. Taken together, our results suggest that STAT5b might be a potential novel target for human DPAC.

  8. 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.

  9. UNC5B receptor deletion exacerbates DSS-induced colitis in mice by increasing epithelial cell apoptosis.

    Science.gov (United States)

    Ranganathan, Punithavathi; Jayakumar, Calpurnia; Li, Dean Y; Ramesh, Ganesan

    2014-07-01

    The netrin-1 administration or overexpression is known to protect colon from acute colitis. However, the receptor that mediates netrin-1 protective activities in the colon during colitis remains unknown. We tested the hypothesis that UNC5B receptor is a critical mediator of protective function of netrin-1 in dextran sodium sulfate (DSS)-induced colitis using mice with partial deletion of UNC5B receptor. DSS colitis was performed in mice with partial genetic UNC5B deficiency (UNC5B(+/-) mice) or wild-type mice to examine the role of endogenous UNC5B. These studies were supported by in vitro models of DSS-induced apoptosis in human colon epithelial cells. WT mice developed colitis in response to DSS feeding as indicated by reduction in bw, reduction in colon length and increase in colon weight. These changes were exacerbated in heterozygous UNC5B knockout mice treated with DSS. Periodic Acid-Schiff stained section shows damages in colon epithelium and mononuclear cell infiltration in WT mice, which was further increased in UNC5B heterozygous knockout mice. This was associated with large increase in inflammatory mediators such as cytokine and chemokine expression and extensive apoptosis of epithelial cells in heterozygous knockout mice as compared to WT mice. Overexpression of UNC5B human colon epithelial cells suppressed DSS-induced apoptosis and caspase-3 activity. Moreover, DSS induced large amount of netrin-1 and shRNA mediated knockdown of netrin-1 induction exacerbated DSS-induced epithelial cell apoptosis. Our results suggest that UNC5B is a critical mediator of cell survival in response to stress in colon.

  10. The HCV non-nucleoside inhibitor Tegobuvir utilizes a novel mechanism of action to inhibit NS5B polymerase function.

    Directory of Open Access Journals (Sweden)

    Christy M Hebner

    Full Text Available Tegobuvir (TGV is a novel non-nucleoside inhibitor (NNI of HCV RNA replication with demonstrated antiviral activity in patients with genotype 1 chronic HCV infection. The mechanism of action of TGV has not been clearly defined despite the identification of resistance mutations mapping to the NS5B polymerase region. TGV does not inhibit NS5B enzymatic activity in biochemical assays in vitro, suggesting a more complex antiviral mechanism with cellular components. Here, we demonstrate that TGV exerts anti-HCV activity utilizing a unique chemical activation and subsequent direct interaction with the NS5B protein. Treatment of HCV subgenomic replicon cells with TGV results in a modified form of NS5B with a distinctly altered mobility on a SDS-PAGE gel. Further analysis reveals that the aberrantly migrating NS5B species contains the inhibitor molecule. Formation of this complex does not require the presence of any other HCV proteins. The intensity of the aberrantly migrating NS5B species is strongly dependent on cellular glutathione levels as well as CYP 1A activity. Furthermore analysis of NS5B protein purified from a heterologous expression system treated with TGV by mass spectrometry suggests that TGV undergoes a CYP- mediated intracellular activation step and the resulting metabolite, after forming a glutathione conjugate, directly and specifically interacts with NS5B. Taken together, these data demonstrate that upon metabolic activation TGV is a specific, covalent inhibitor of the HCV NS5B polymerase and is mechanistically distinct from other classes of the non-nucleoside inhibitors (NNI of the viral polymerase.

  11. 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

  12. 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.

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

    Science.gov (United States)

    Coutinho, Maria Francisca; Santos, Juliana Inês; Alves, Sandra

    2016-01-01

    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. PMID:27384562

  14. 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

  15. 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.

  16. 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

  17. Activating mutations of STAT5B and STAT3 in lymphomas derived from ??-T or NK cells.

    OpenAIRE

    Lack, Nathan A.; Şen, Emel; Kucuk, Can; Jiang, Bei; Hu, Xiaozhou; Zhang, Wenyan; Chan, John K. C.; Xiao, Wenming; Alkan, Can; Williams, John C.; Avery, Kendra N.; Kavak, Pinar; Scuto, Anna; Gaulard, Philippe; Staudt, Lou; Iqbal, Javeed; Zhang, Weiwei; Cornish, Adam; Gong, Qiang; Yang, Qunpei; Sun, Hong; d'Amore, Francesco; Leppa, Sirpa; Liu, Weiping; Fu, Kai; de Leval, Laurence; McKeithan, Timothy; Chan, Wing C.

    2015-01-01

    Lymphomas arising from NK or gamma delta-T cells are very aggressive diseases and little is known regarding their pathogenesis. Here we report frequent activating mutations of STAT3 and STAT5B in NK/T-cell lymphomas (n - 51), gamma delta-T-cell lymphomas (n - 43) and their cell lines (n = 9) through next generation and/or Sanger sequencing. STAT5B N642H is particularly frequent in all forms of gamma delta-T-cell lymphomas. STAT3 and STAT5B mutations are associated with increased phosphorylate...

  18. 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.

  19. 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 ...

  20. 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.

  1. 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...

  2. 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 ...

  3. 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...

  4. 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...

  5. 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...

  6. The BH3 Mimetic Obatoclax Accumulates in Lysosomes and Causes Their Alkalinization.

    Directory of Open Access Journals (Sweden)

    Vasileios A Stamelos

    Full Text Available Obatoclax belongs to a class of compounds known as BH3 mimetics which function as antagonists of Bcl-2 family apoptosis regulators. It has undergone extensive preclinical and clinical evaluation as a cancer therapeutic. Despite this, it is clear that obatoclax has additional pharmacological effects that contribute to its cytotoxic activity. It has been claimed that obatoclax, either alone or in combination with other molecularly targeted therapeutics, induces an autophagic form of cell death. In addition, obatoclax has been shown to inhibit lysosomal function, but the mechanism of this has not been elucidated. We have evaluated the mechanism of action of obatoclax in eight ovarian cancer cell lines. Consistent with its function as a BH3 mimetic, obatoclax induced apoptosis in three cell lines. However, in the remaining cell lines another form of cell death was evident because caspase activation and PARP cleavage were not observed. Obatoclax also failed to show synergy with carboplatin and paclitaxel, chemotherapeutic agents which we have previously shown to be synergistic with authentic Bcl-2 family antagonists. Obatoclax induced a profound accumulation of LC-3 but knockdown of Atg-5 or beclin had only minor effects on the activity of obatoclax in cell growth assays suggesting that the inhibition of lysosomal function rather than stimulation of autophagy may play a more prominent role in these cells. To evaluate how obatoclax inhibits lysosomal function, confocal microscopy studies were conducted which demonstrated that obatoclax, which contains two basic pyrrole groups, accumulates in lysosomes. Studies using pH sensitive dyes demonstrated that obatoclax induced lysosomal alkalinization. Furthermore, obatoclax was synergistic in cell growth/survival assays with bafilomycin and chloroquine, two other drugs which cause lysosomal alkalinization. These studies explain, for the first time, how obatoclax inhibits lysosomal function and suggest that

  7. 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...

  8. 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

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

    DEFF Research Database (Denmark)

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

    2007-01-01

    these with the severity of neuropathologic changes. In regions with mild pathology and scant abnormal prion protein (PrP) deposition, neurons showed an increased volume of Rab5-immunopositive early endosomes. In contrast, neurons in regions with prominent pathology had an increased volume of cathepsin D- or B...... correlate with regional pathology. Overloading of this system might impair the function of lysosomal enzymes and thus may mimic some features of lysosomal storage disorders. Udgivelsesdato: 2007-Jul...

  10. 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.

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

    Science.gov (United States)

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

    2010-07-15

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

  12. 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

  13. 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

  14. Expansion of CD5 - B cells in multiple sclerosis correlates with CD80 (B7-1) expression

    DEFF Research Database (Denmark)

    Sellebjerg, F; Jensen, J; Jensen, C.V.;

    2002-01-01

    ) and a panel of serum autoantibodies in patients with clinically isolated syndromes (CIS), suggestive of MS and patients with clinically definite MS (CDMS). Patients with CDMS had a higher percentage of CD5- B cells in CSF than did control subjects (P = 0.02). CIS patients with immunoglobulin G (Ig......The pathogenetic role of autoantibodies in multiple sclerosis (MS) is uncertain. CD5+ B cells commonly produce autoantibodies, but CD5 expression has also been implicated in B-cell tolerance. We studied B-cell subsets, anti-myelin protein antibody-secreting cells in cerebrospinal fluid (CSF......G) oligoclonal bands in CSF or multiple lesions on magnetic resonance imaging (MRI) had a higher percentage of CD5- B cells in CSF than did the remaining CIS patients (P = 0.03). The percentage of CD5- and CD80+ B cells correlated positively and the percentage of CD5+ B cells correlated negatively...

  15. I. Novel HCV NS5B polymerase inhibitors: Discovery of indole 2-carboxylic acids with C3-heterocycles

    Energy Technology Data Exchange (ETDEWEB)

    Anilkumar, Gopinadhan N.; Lesburg, Charles A.; Selyutin, Oleg; Rosenblum, Stuart B.; Zeng, Qingbei; Jiang, Yueheng; Chan, Tin-Yau; Pu, Haiyan; Vaccaro, Henry; Wang, Li; Bennett, Frank; Chen, Kevin X.; Duca, Jose; Gavalas, Stephen; Huang, Yuhua; Pinto, Patrick; Sannigrahi, Mousumi; Velazquez, Francisco; Venkatraman, Srikanth; Vibulbhan, Bancha; Agrawal, Sony; Butkiewicz, Nancy; Feld, Boris; Ferrari, Eric; He, Zhiqing; Jiang, Chuan-kui; Palermo, Robert E.; Mcmonagle, Patricia; Huang, H.-C.; Shih, Neng-Yang; Njoroge, George; Kozlowski, Joseph A. (Merck)

    2012-05-03

    SAR development of indole-based palm site inhibitors of HCV NS5B polymerase exemplified by initial indole lead 1 (NS5B IC{sub 50} = 0.9 {micro}M, replicon EC{sub 50} > 100 {micro}M) is described. Structure-based drug design led to the incorporation of novel heterocyclic moieties at the indole C3-position which formed a bidentate interaction with the protein backbone. SAR development resulted in leads 7q (NS5B IC{sub 50} = 0.032 {micro}M, replicon EC{sub 50} = 1.4 {micro}M) and 7r (NS5B IC{sub 50} = 0.017 {micro}M, replicon EC{sub 50} = 0.3 {micro}M) with improved enzyme and replicon activity.

  16. Visual detection of STAT5B gene expression in living cell using the hairpin DNA modified gold nanoparticle beacon.

    Science.gov (United States)

    Xue, Jianpeng; Shan, Lingling; Chen, Haiyan; Li, Yang; Zhu, Hongyan; Deng, Dawei; Qian, Zhiyu; Achilefu, Samuel; Gu, Yueqing

    2013-03-15

    Signal transducer and activator of transcription 5B (STAT5B) is an important protein in JAK-STAT signaling pathway that is responsible for the metastasis and proliferation of tumor cells. Determination of the STAT5B messenger Ribonucleic Acid (mRNA) relating to the STAT5B expression provides insight into the mechanism of tumor progression. In this study, we designed and used a special hairpin deoxyribonucleic acid (DNA) for human STAT5B mRNA to functionalize gold nanoparticles, which served as a beacon for detecting human STAT5B expression. Up to 90% quenching efficiency was achieved. Upon hybridizing with the target mRNA, the hairpin DNA modified gold nanoparticle beacons (hDAuNP beacons) release the fluorophores attached at 5' end of the oligonucleotide sequence. The fluorescence properties of the beacon before and after the hybridization with the complementary DNA were confirmed in vitro. The stability of hDAuNP beacons against degradation by DNase I and GSH indicated that the prepared beacon is stable inside cells. The detected fluorescence in MCF-7 cancer cells correlates with the specific STAT5B mRNA expression, which is consistent with the result from PCR measurement. Fluorescence microscopy showed that the hDAuNP beacons internalized in cells without using transfection agents, with intracellular distribution in the cytoplasm rather than the nucleus. The results demonstrated that this beacon could directly provide quantitative measurement of the intracellular STAT5B mRNA in living cells. Compared to the previous approaches, this beacon has advantages of higher target to background ratio of detection and an increased resistance to nuclease degradation. The strategy reported in this study is a promising approach for the intracellular measurement of RNA or protein expression in living cells, and has great potential in the study of drug screening and discovery.

  17. STAT5B mutations in heterozygous state have negative impact on height: another clue in human stature heritability

    Science.gov (United States)

    Scalco, Renata C; Hwa, Vivian; Domené, Horacio M.; Jasper, Héctor G.; Belgorosky, Alicia; Marino, Roxana; Pereira, Alberto M.; Tonelli, Carlos A.; Wit, Jan M.; Rosenfeld, Ron G.; Jorge, Alexander A.L.

    2016-01-01

    Context and objective Growth hormone insensitivity with immune dysfunction caused by signal transducer and activator of transcription 5B (STAT5B) mutations is an autosomal recessive condition. Heterozygous mutations in other genes involved in growth regulation were previously associated with a mild height reduction. Our objective was to assess for the first time the phenotype of heterozygous STAT5B mutations. Methods We genotyped and performed clinical and laboratorial evaluations in 52 relatives of 2 previously described Brazilian brothers with homozygous STAT5B c.424_427del mutation (21 heterozygous). Additionally, we obtained height data and genotype from 1,104 adult control individuals from the same region in Brazil and identified 5 additional families harboring the same mutation (18 individuals, 11 heterozygous). Furthermore, we gathered the available height data from first-degree relatives of patients with homozygous STAT5B mutations (17 individuals from 7 families). Data from heterozygous individuals and non-carriers were compared. Results Individuals carrying heterozygous STAT5B c.424_427del mutation were 0.6 SDS shorter than their non-carrier relatives (p= 0.009). Heterozygous subjects also had significantly lower SDS for serum concentrations of IGF-1 (p=0.028) and IGFBP-3 (p=0.02) than their non-carrier relatives. The 17 heterozygous first-degree relatives of patients carrying homozygous STAT5B mutations had an average height SDS of −1.4 ± 0.8 when compared with population-matched controls (p < 0.001). Conclusions STAT5B mutations in heterozygous state have a significant negative impact on height (approximately 3.9 cm). This effect is milder than the effect seen in the homozygous state, with height usually within the normal range. Our results support the hypothesis that heterozygosity of rare pathogenic variants contributes to normal height heritability. PMID:26034074

  18. Hepatitis C virus non-structural 5B protein interacts with cyclin A2 and regulates viral propagation

    DEFF Research Database (Denmark)

    Pham, Long; Ngo, HT; Lim, YS;

    2012-01-01

    ) specifically interacted with CycA2 in vitro and in vivo. Protein interaction was mediated through the cyclin box of CycA2 and the palm domain of NS5B. We further showed that R/HxL motif in the palm domain of HCV NS5B mediated protein interaction with CycA2 and this interaction was necessary for HCV replication...

  19. 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.

  20. 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

  1. 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

  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. The evaluation of Tracp5b as a marker for monitoring treatment results of bone metastasis in breast cancer patients

    Institute of Scientific and Technical Information of China (English)

    Xiaoyun Huang; Yan Si; Jia Zhao; Qiang Ding

    2008-01-01

    Objective:To evaluate the sensitivity of serum tartrate-resistant acid phosphatase 5b(Tracp5b) activity in monitoring bisphosphonate treatment results of bone metastasis in breast cancer(BC) patients. Methods:The serum activities of Tracp5b, CEA, CA153 were measured in 58 BC patients, including 26 without bone metastasis, 32 with bone metastasis. The serum activities of Tracp5b, CEA, CA153 were also measured in 19 patients with bone metastasis after 3 months of bisphosphonate treatment. Eighteen healthy women with age from 34 to 70 served as control. Results:Serum Tracp5b was significantly elevated in patients with bone metastasis compared with that in all any other groups(P< 0.05). The sensitivity of TracpSb was 78.13% and the specificity was 86.36%. The sensitivity of CA153 was 37.50% and the specificity was 77.27%. The sensitivity of CEA was 21.88% and the specificity was 84.09%. The serum activity of Tracp5b decreased significantly(P < 0.05) after 3 months of bisphosphonate treatment, while the levels of CA153 and CEA were unchanged. Conclusion:Serum TracpSb activity is a useful diagnostic marker for bone metastasis in BC patients and can be used to evaluate the treatment results of bisphosphonate.

  4. Complete genome sequence of Shigella flexneri 5b and comparison with Shigella flexneri 2a

    Directory of Open Access Journals (Sweden)

    Xue Ying

    2006-07-01

    Full Text Available Abstract Background Shigella bacteria cause dysentery, which remains a significant threat to public health. Shigella flexneri is the most common species in both developing and developed countries. Five Shigella genomes have been sequenced, revealing dynamic and diverse features. To investigate the intra-species diversity of S. flexneri genomes further, we have sequenced the complete genome of S. flexneri 5b strain 8401 (abbreviated Sf8401 and compared it with S. flexneri 2a (Sf301. Results The Sf8401 chromosome is 4.5-Mb in size, a little smaller than that of Sf301, mainly because the former lacks the SHI-1 pathogenicity island (PAI. Compared with Sf301, there are 6 inversions and one translocation in Sf8401, which are probably mediated by insertion sequences (IS. There are clear differences in the known PAIs between these two genomes. The bacteriophage SfV segment remaining in SHI-O of Sf8401 is clearly larger than the remnants of bacteriophage SfII in Sf301. SHI-1 is absent from Sf8401 but a specific related protein is found next to the pheV locus. SHI-2 is involved in one intra-replichore inversion near the origin of replication, which may change the expression of iut/iuc genes. Moreover, genes related to the glycine-betaine biosynthesis pathway are present only in Sf8401 among the known Shigella genomes. Conclusion Our data show that the two S. flexneri genomes are very similar, which suggests a high level of structural and functional conservation between the two serotypes. The differences reflect different selection pressures during evolution. The ancestor of S. flexneri probably acquired SHI-1 and SHI-2 before SHI-O was integrated and the serotypes diverged. SHI-1 was subsequently deleted from the S. flexneri 5b genome by recombination, but stabilized in the S. flexneri 2a genome. These events may have contributed to the differences in pathogenicity and epidemicity between the two serotypes of S. flexneri.

  5. Crystallization kinetics of an amorphous Co77Si11.5B11.5 alloy

    Directory of Open Access Journals (Sweden)

    R. Nowosielski

    2006-04-01

    Full Text Available Purpose: This paper describes crystallization kinetics and changes magnetic properties involved by process of crystallization Co-Si-B amorphous alloy.Design/methodology/approach: The following experimental techniques were used: X-ray diffraction (XRD, electrical resistivity in situ measurements (four-point probe static and dynamic measurements of magnetic properties (magnetic balance, fluxmeter, Maxwell-Wien bridge.Findings: In this work has been performed influence of thermal annealing on crystallization kinetics and magnetic properties amorphous Co77Si11.5B11.5 alloy.Practical implications: The attractive properties of Co-Si-B alloy are of special interest for basic research on the materials as well as for their potential applications, like magnetic sensors. The Co soft magnetic material is used in noise filters, saturable reactors, miniature inductance elements for abating spike noise, mains transformers, choke coils, zero-phase current transformers, and magnetic heads etc., i.e., devices which are expected to exhibit high levels of permeability at high frequencies.Originality/value: It has been shown that thermal annealing at temperature close to the crystallization temperature leads to a significant increase of the initial magnetic permeability.

  6. Episodic adaptive diversification of classical swine fever virus RNA-dependent RNA polymerase NS5B.

    Science.gov (United States)

    Li, Yan; Yang, Zexiao

    2015-12-01

    Classical swine fever virus (CSFV) is the pathogen that causes a highly infectious disease of pigs and has led to disastrous losses to pig farms and related industries. The RNA-dependent RNA polymerase (RdRp) NS5B is a central component of the replicase complex (RC) in some single-stranded RNA viruses, including CSFV. On the basis of genetic variation, the CSFV RdRps could be clearly divided into 2 major groups and a minor group, which is consistent with the phylogenetic relationships and virulence diversification of the CSFV isolates. However, the adaptive signature underlying such an evolutionary profile of the polymerase and the virus is still an interesting open question. We analyzed the evolutionary trajectory of the CSFV RdRps over different timescales to evaluate the potential adaptation. We found that adaptive selection has driven the diversification of the RdRps between, but not within, CSFV major groups. Further, the major adaptive divergence-related sites are located in the surfaces relevant to the interaction with other component(s) of RC and the entrance and exit of the template-binding channel. These results might shed some light on the nature of the RdRp in virulence diversification of CSFV groups.

  7. A study of microstructure and properties of cast Fe-10Cr-1.5B alloy

    Directory of Open Access Journals (Sweden)

    Zhang Haibin

    2014-05-01

    Full Text Available In the present study, the microstructure and mechanical properties of cast Fe-10Cr-1.5B (FCB alloy after different heat treatments were studied. The results showed that the as-cast microstructure of FCB alloy consists of ?Fe, M(M=Cr, Fe, Mn2(B, C and M(M=Cr, Fe, Mn7(C, B3 type borocarbides, and small amounts of pearlite and austenite. After oil quenching treatment, metal matrix transformed into the martensite from the mixture of martensite, pearlite and austenite. There are many M(M=Cr,Fe,Mn23(C,B6 type borocarbide precipitates in the metal matrix, and eutectic borocarbide appears with an apparent disconnection and isolated phenomenon. When the quenching temperature reaches 1,050 oC, the hardness of FCB alloy is the highest, but the change of quenching temperature has no obvious effect on impact toughness of FCB alloy. After tempering, the eutectic microstructure of FCB alloy appears with a "two links" trend. With the increase of tempering temperature, the hardness of FCB alloy decreases gradually and impact toughness increases gradually. Cast FCB alloy oil-quenched from 1,050 oC and tempered from 200 oC has excellent combined properties; its hardness and impact toughness are 61.5 HRC and 8.8 J.m-2 respectively.

  8. Episodic adaptive diversification of classical swine fever virus RNA-dependent RNA polymerase NS5B.

    Science.gov (United States)

    Li, Yan; Yang, Zexiao

    2015-12-01

    Classical swine fever virus (CSFV) is the pathogen that causes a highly infectious disease of pigs and has led to disastrous losses to pig farms and related industries. The RNA-dependent RNA polymerase (RdRp) NS5B is a central component of the replicase complex (RC) in some single-stranded RNA viruses, including CSFV. On the basis of genetic variation, the CSFV RdRps could be clearly divided into 2 major groups and a minor group, which is consistent with the phylogenetic relationships and virulence diversification of the CSFV isolates. However, the adaptive signature underlying such an evolutionary profile of the polymerase and the virus is still an interesting open question. We analyzed the evolutionary trajectory of the CSFV RdRps over different timescales to evaluate the potential adaptation. We found that adaptive selection has driven the diversification of the RdRps between, but not within, CSFV major groups. Further, the major adaptive divergence-related sites are located in the surfaces relevant to the interaction with other component(s) of RC and the entrance and exit of the template-binding channel. These results might shed some light on the nature of the RdRp in virulence diversification of CSFV groups. PMID:26485449

  9. ARID5B Genetic Polymorphisms Contribute to Racial Disparities in the Incidence and Treatment Outcome of Childhood Acute Lymphoblastic Leukemia

    Science.gov (United States)

    Xu, Heng; Cheng, Cheng; Devidas, Meenakshi; Pei, Deqing; Fan, Yiping; Yang, Wenjian; Neale, Geoff; Scheet, Paul; Burchard, Esteban G.; Torgerson, Dara G.; Eng, Celeste; Dean, Michael; Antillon, Frederico; Winick, Naomi J.; Martin, Paul L.; Willman, Cheryl L.; Camitta, Bruce M.; Reaman, Gregory H.; Carroll, William L.; Loh, Mignon; Evans, William E.; Pui, Ching-Hon; Hunger, Stephen P.; Relling, Mary V.; Yang, Jun J.

    2012-01-01

    Purpose Recent genome-wide screens have identified genetic variations in ARID5B associated with susceptibility to childhood acute lymphoblastic leukemia (ALL). We sought to determine the contribution of ARID5B single nucleotide polymorphisms (SNPs) to racial disparities in ALL susceptibility and treatment outcome. Patients and Methods We compared the association between ARID5B SNP genotype and ALL susceptibility in whites (> 95% European genetic ancestry; 978 cases and 1,046 controls) versus in Hispanics (> 10% Native American ancestry; 330 cases and 541 controls). We determined the relationships between ARID5B SNP genotype and ALL relapse risk in 1,605 children treated on the Children's Oncology Group (COG) P9904/9905 clinical trials. Results Among 49 ARID5B SNPs interrogated, 10 were significantly associated with ALL susceptibility in both whites and Hispanics (P < .05), with risk alleles consistently more frequent in Hispanics than in whites. rs10821936 exhibited the most significant association in both races (P = 8.4 × 10−20 in whites; P = 1 × 10−6 in Hispanics), and genotype at this SNP was highly correlated with local Native American genetic ancestry (P = 1.8 × 10−8). Multivariate analyses in Hispanics identified an additional SNP associated with ALL susceptibility independent of rs10821936. Eight ARID5B SNPs were associated with both ALL susceptibility and relapse hazard; the alleles related to higher ALL incidence were always linked to poorer treatment outcome and were more frequent in Hispanics. Conclusion ARID5B polymorphisms are important determinants of childhood ALL susceptibility and treatment outcome, and they contribute to racial disparities in this disease. PMID:22291082

  10. 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 ...

  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. Reconstitution of Bacillus cereus 5/B/6 metallo-[beta]-lactamase activity with copper

    Energy Technology Data Exchange (ETDEWEB)

    Hilliard, N.P.; Shaw, R.W. (Texas Tech Univ., Lubbock (United States))

    1992-01-01

    Pathogenic bacteria become resistant to [beta]-lactam antibiotics such as penicillins and cephalosporins through the production of enzymes called [beta]-lactamases. The authors have successfully reconstituted the enzymatic activity of the metallo-[beta]-lactamase of Bacillus cereus 5/B/6 purified from an E. coli expression vector system by the addition of Cu(II) to the apoenzyme. This is the first report that copper supports catalytic activity in this enzyme. Maximal activity of the copper-reconstituted enzyme was achieved by a careful addition of a stoichiometric amount of CuSO[sub 4] to 200 [mu]M apoenzyme. Using either benzylpenicillin or cephalosporin C as the substrate, reconstitution of the activity by addition of copper to the apoenzyme resulted in the recovery of approximately 35% of the control activity of the native Zn(II) enzyme. In agreement with previous reports, in the presence of excess Cu(II), the preparation did not possess measurable catalytic activity. Electronic spectra of the copper-reconstituted enzyme displayed adsorption maxima at 394, 698 and 1,022 nm with extinction coefficients of 2,656, 55 and < 3 M[sup [minus]1]cm[sup [minus]1] respectively. Circular dichorism spectra in the ultraviolent region (UVCD) of the copper-reconstituted enzyme were identical with those of the native Zn(II) enzyme. Addition of excess cephalosporin C to the copper-reconstituted enzyme caused a decrease of about 50% of the absorbance of the 394 nm band and the formation of a new feature at 350 nm.

  14. Tartrate-resistant acid phosphatase 5b is a potential biomarker for rheumatoid arthritis: a pilot study in Han Chinese

    Institute of Scientific and Technical Information of China (English)

    Cheng Tao; Wang Mingjun; Chen Zhiwei; Robert A Eisenberg; Zhang Yu; Zou Yaohong; Deng Yingsu

    2014-01-01

    Background Bone damage around the joints is one of the major pathophysiological mechanisms that leads to rheumatoid arthritis (RA) chronic disability.Serum tartrate-resistant acid phosphatase 5b (TRACP-5b) is secreted by osteoclasts,its activity can be used as a clinically relevant bone resorption marker.The aim of this study was to test whether the measurement of serum levels of TRACP-5b in patients with RA would correlate with measures of disease activity and with responses to therapy.Methods Fifty-six patients were randomly assigned to receive recombinant human cytotoxic tlymphocyte-associated antigen-4 immunoglobulin (RhCTLA4-lg),infliximab or methotrexate (MTX).The clinical and serologic indicators of RA activity were evaluated at baseline and at 24 weeks.Serum TRACP-5b was measured by Enzyme-linked Immunosorbent Assay (ELISA) at 0,12 and 24 weeks.Hand X-rays were obtained at baseline.Results At baseline,the levels of TRACP-5b correlated with the severity of X-ray damage,disease duration (r=0.332,P=0.012),and tender joint count (r=0.408,P=0.002).The 24 weeks values of TRACP-5b for RhCTLA4-lg group and infliximab group differed significantly from the baseline values in each group (P <0.05; P <0.05),whereas only the value for RhCTLA4-lg group differed significantly from the 24 weeks value for the MTX group (P <0.01).Considering the two biologics-treated groups together,the TRACP-5b levels at 24 weeks differed significantly from the baseline values only in those patients who reached an ACR70 level (P <0.05).Conclusions Measurement of serum TRACP-5b in RA patients reflects clinical and radiological measures of disease activity,treatment with certain biologics,and degree of response to therapy.TRACP-5b should be investigated further as a potential biomarker to predict response to therapy,including slowing of radiographic progression.

  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. The salivary mucin MUC5B and lactoperoxidase can be used for layer-by-layer film formation.

    Science.gov (United States)

    Lindh, Liselott; Svendsen, Ida E; Svensson, Olof; Cárdenas, Marité; Arnebrant, Thomas

    2007-06-01

    In situ ellipsometry was used to study layer-by-layer film formation on hydrophilic and hydrophobized silica surfaces by alternating sequential adsorption of human mucin MUC5B and cationic proteins lysozyme, lactoferrin, lactoperoxidase or histatin 5, respectively. The stability of the multilayers was investigated by addition of sodium dodecyl sulfate solution (SDS). Atomic force microscopy was employed to investigate morphological structures on the surfaces during the layer-by-layer film build-up. It was clearly shown that, on both hydrophilic and hydrophobized silica, only MUC5B and lactoperoxidase showed the ability for multilayer formation, resulting in an approximately linear increase in adsorbed amount and film thickness with each deposition cycle. The net increase in amounts per cycle was larger on the hydrophilic silica. Further, MUC5B needs to be adsorbed first on the hydrophilic substrates to obtain this fast build-up behavior. Generally, addition of SDS solution showed that a large fraction of the adsorbed film could be desorbed. However, films on the hydrophobized silica were more resistant to surfactant elution. In conclusion, MUC5B-cationic protein multilayers can be formed on hydrophilic and hydrophobized silica, depending on the choice of the cationic protein as well as in which order the build-up is started on hydrophilic silica. Additionally, SDS disrupts the layer-by-layer film formed by MUC5B and lactoperoxidase.

  17. Over-expression of Stat5b confers protection against diabetes in the non-obese diabetic (NOD) mice via up-regulation of CD4{sup +}CD25{sup +} regulatory T cells

    Energy Technology Data Exchange (ETDEWEB)

    Jin, Yulan; Purohit, Sharad [Center for Biotechnology and Genomic Medicine, Georgia Health Sciences University, GA (United States); Department of Pathology, Medical College of Georgia, Georgia Health Sciences University, GA (United States); Chen, Xueqin; Yi, Bing [Center for Biotechnology and Genomic Medicine, Georgia Health Sciences University, GA (United States); She, Jin-Xiong, E-mail: jshe@georgiahealth.edu [Center for Biotechnology and Genomic Medicine, Georgia Health Sciences University, GA (United States); Department of Pathology, Medical College of Georgia, Georgia Health Sciences University, GA (United States)

    2012-08-10

    Highlights: Black-Right-Pointing-Pointer This is the first study to provide direct evidence of the role of Stat5b in NOD mice. Black-Right-Pointing-Pointer Over-expression of wild type Stat5b transgene protects NOD mice against diabetes. Black-Right-Pointing-Pointer This protection may be mediated by the up-regulation of CD4{sup +}CD25{sup +} Tregs. -- Abstract: The signal transducers and activators of transcription (STAT) family of proteins play a critical role in cytokine signaling required for fine tuning of immune regulation. Previous reports showed that a mutation (L327M) in the Stat5b protein leads to aberrant cytokine signaling in the NOD mice. To further elaborate the role of Stat5b in diabetes, we established a NOD transgenic mouse that over-expresses the wild type Stat5b gene. The incidences of spontaneous diabetes as well as cyclophosphamide-induced diabetes were significantly reduced and delayed in the Stat5b transgenic NOD mice compared to their littermate controls. The total cell numbers of CD4{sup +} T cells and especially CD8{sup +} T cells in the spleen and pancreatic lymph node were increased in the Stat5b transgenic NOD mice. Consistent with these findings, CD4{sup +} and CD8{sup +} T cells from the Stat5b transgenic NOD mice showed a higher proliferation capacity and up-regulation of multiple cytokines including IL-2, IFN-{gamma}, TNF-{alpha} and IL-10 as well as anti-apoptotic gene Bcl-xl. Furthermore, the number and proportion of CD4{sup +}CD25{sup +} regulatory T cells were significantly increased in transgenic mice although in vitro suppression ability of the regulatory T-cells was not affected by the transgene. Our results suggest that Stat5b confers protection against diabetes in the NOD mice by regulating the numbers and function of multiple immune cell types, especially by up-regulating CD4{sup +}CD25{sup +} regulatory T cells.

  18. 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

  19. 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.

  20. 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.

  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. 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

  3. 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

  4. Giant Lysosomes as a Chemotherapy Resistance Mechanism in Hepatocellular Carcinoma Cells.

    Directory of Open Access Journals (Sweden)

    Federico Colombo

    Full Text Available Despite continuous improvements in therapeutic protocols, cancer-related mortality is still one of the main problems facing public health. The main cause of treatment failure is multi-drug resistance (MDR: simultaneous insensitivity to different anti-cancer agents, the underlying molecular and biological mechanisms of which include the activity of ATP binding cassette (ABC proteins and drug compartmentalisation in cell organelles. We investigated the expression of the main ABC proteins and the role of cytoplasmic vacuoles in the MDR of six hepatocellular carcinoma (HCC cell lines, and confirmed the accumulation of the yellow anti-cancer drug sunitinib in giant (four lines and small cytoplasmic vacuoles of lysosomal origin (two lines. ABC expression analyses showed that the main ABC protein harboured by all of the cell lines was PGP, whose expression was not limited to the cell membrane but was also found on lysosomes. MTT assays showed that the cell lines with giant lysosomes were more resistant to sorafenib treatment than those with small lysosomes (p<0.01, and that verapamil incubation can revert this resistance, especially if it is administered after drug pre-incubation. The findings of this study demonstrate the involvement of PGP-positive lysosomes in drug sequestration and MDR in HCC cell lines. The possibility of modulating this mechanism using PGP inhibitors could lead to the development of new targeted strategies to enhance HCC treatment.

  5. 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

  6. Interakcija modifikacijske zlitine AlTi5B1 s talino zlitine AlCu6PbBi:

    OpenAIRE

    Križman, Alojz; Spaić, Savo; Zupanič, Franc

    1996-01-01

    The course of melting and dissolution of the AlTi5B1 grain-refining alloy in the melt of AlCu6PbBi alloy has been investigated. The kind of possible nucleating agents has also been determined. The melting and the dissolution process begin preferentially in the regions of good contact between AlTi5B1 and AlCu6PbBi as well as in the regions where low-melting microstructural constituents are prsent in the grain refining alloy. During the dissolution titanium and boron are difusing from the grain...

  7. Characterization of two-pore channel 2 (TPCN2)-mediated Ca2+ currents in isolated lysosomes.

    Science.gov (United States)

    Schieder, Michael; Rötzer, Katrin; Brüggemann, Andrea; Biel, Martin; Wahl-Schott, Christian A

    2010-07-01

    Two-pore channels (TPCNs) have been proposed to form lysosomal Ca(2+) release channels that are activated by nicotinic acid adenine dinucleotide phosphate. Here, we employ a glass chip-based method to record for the first time nicotinic acid adenine dinucleotide phosphate -dependent currents through a two-pore channel (TPCN2) from intact lysosomes. We show that TPCN2 is a highly selective Ca(2+) channel that is regulated by intralysosomal pH. Using site-directed mutagenesis, we identify an amino acid residue in the putative pore region that is crucial for conferring high Ca(2+) selectivity. Our glass chip-based method will provide electrophysiological access not only to lysosomal TPCN channels but also to a broad range of other intracellular ion channels.

  8. Characterization of Two-pore Channel 2 (TPCN2)-mediated Ca2+ Currents in Isolated Lysosomes*

    Science.gov (United States)

    Schieder, Michael; Rötzer, Katrin; Brüggemann, Andrea; Biel, Martin; Wahl-Schott, Christian A.

    2010-01-01

    Two-pore channels (TPCNs) have been proposed to form lysosomal Ca2+ release channels that are activated by nicotinic acid adenine dinucleotide phosphate. Here, we employ a glass chip-based method to record for the first time nicotinic acid adenine dinucleotide phosphate -dependent currents through a two-pore channel (TPCN2) from intact lysosomes. We show that TPCN2 is a highly selective Ca2+ channel that is regulated by intralysosomal pH. Using site-directed mutagenesis, we identify an amino acid residue in the putative pore region that is crucial for conferring high Ca2+ selectivity. Our glass chip-based method will provide electrophysiological access not only to lysosomal TPCN channels but also to a broad range of other intracellular ion channels. PMID:20495006

  9. Guanidinylated neomycin mediates heparan sulfate-dependent transport of active enzymes to lysosomes.

    Science.gov (United States)

    Sarrazin, Stéphane; Wilson, Beth; Sly, William S; Tor, Yitzhak; Esko, Jeffrey D

    2010-07-01

    Guanidinylated neomycin (GNeo) can transport bioactive, high molecular weight cargo into the interior of cells in a process that depends on cell surface heparan sulfate proteoglycans. In this report, we show that GNeo-modified quantum dots bind to cell surface heparan sulfate, undergo endocytosis and eventually reach the lysosomal compartment. An N-hydroxysuccinimide activated ester of GNeo (GNeo-NHS) was prepared and conjugated to two lysosomal enzymes, beta-D-glucuronidase (GUS) and alpha-L-iduronidase. Conjugation did not interfere with enzyme activity and enabled binding of the enzymes to heparin-Sepharose and heparan sulfate on primary human fibroblasts. Cells lacking the corresponding lysosomal enzyme took up sufficient amounts of the conjugated enzymes to restore normal turnover of glycosaminoglycans. The high capacity of proteoglycan-mediated uptake suggests that this method of delivery might be used for enzyme replacement or introduction of foreign enzymes into cells.

  10. 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.

  11. 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

  12. Changes in lysosomal morphology and enzyme activities during the development of adriamycin-induced cardiomyopathy.

    Science.gov (United States)

    Singal, P K; Segstro, R J; Singh, R P; Kutryk, M J

    1985-03-01

    Morphologic and enzymic changes in heart lysosomes were studied following a chronic treatment of animals with a cumulative dose of 15 mg/kg of adriamycin. Myocardial cell damage due to adriamycin included lysosomal changes, sarcotubular swelling, vacuolization and myofibrillar drop-out. These structural changes were more pronounced in the 6-week treated group as opposed to the 3-week treated group. The number of lysosomes per unit area increased from a control value of 3.6 +/- 1.7 to 17.8 +/- 4.0 in the 3-week treated group and 35.9 +/- 9.2 in the 6-week treated groups, respectively. The scatter in the size distribution of lysosomes was much wider in treated animals. Lysosomal hydrolases in the 3-week and 6-week adriamycin-treated group changed as follows: N acetyl beta-glucosaminidase activity fell in the homogenate (H) and nonsedimentable (NS) and rose in the serum (Ser) fractions; a drop in alpha-mannosidase was seen in the sedimentable (S) and Ser fractions; an increase in beta-galactosidase was noted in the H, S and Ser fractions; acid phosphatase was increased in H, S, NS and Ser fractions. Lanthanum staining, used as a cytochemical probe for normal membrane permeability, revealed intracytoplasmic localization of the tracer only in the 6-week group. Malondialdehyde content was increased significantly in the 3-week and 6-weed treated groups. These results show lysosomal changes in adriamycin-treated hearts which precede as well as accompany nonspecific permeability changes in the sarcolemma.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:3931886

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

    Science.gov (United States)

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

    2012-11-01

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

  14. Lysosomal exoglycosidases in serum and urine of patients with pancreatic adenocarcinoma.

    Directory of Open Access Journals (Sweden)

    Anna Stypułkowska

    2010-11-01

    Full Text Available Lysosomal exoglycosidases: N-acetyl-β-D-hexosaminidase (HEX, β-D-galactosidase (GAL, ι-L-fucosidase (FUC and ι-D-mannosidase (MAN modify oligosaccharide chains of glycoconjugates in endoplasmatic reticulum and/or Golgi apparatus and degrade them in lysosomes. In acid environment of lysosome, exoglycosidases degrade oligosaccharide chains of glycoproteins, glycolipids and glycosaminoglycans by eliminating single sugars from the edges of oligosaccharide chains. Neoplasms change biochemical processes in tissues and may significantly change the activity of many enzymes including the activity of lysosomal exoglycosidasses in serum and urine of persons with neoplasmatic diseases. The aim of the present paper was evaluation the activity of HEX, GAL, FUC and MAN in serum and urine of patients with pancreatic adenocarcinoma. Serum and urine samples were collected from 15 patients with adenocarcinoma of the pancreas and 15 healthy persons. The activity of lysosomal exoglycosidases was determined by the method of Marciniak et al. adapted to serum and urine of patients with adenocarcinoma of the pancreas. Our results indicate significant decrease in activity of GAL (p=0.037 in serum of patients with pancreatic adenocarcinoma, significant increase in activity of HEX (p<0.001 and FUC (p=0.027 in serum, and HEX (p=0.003 in urine, as well as significant decrease of FUC (p=0.016 and MAN (p=0.029 in urine o patients with adenocarcinoma of the pancreas, in comparison to the control group. Increase in activity of some lysosomal enzymes in serum and urine of pancreatic adenocarcinoma patients, may indicate on destruction of pancreatic tissue by pancreatic adenocarcinoma. Determination of the HEX, GAL, FUC and MAN in serum and urine may be useful in diagnostics of pancreatic adenocarcinoma.

  15. 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 ...

  16. 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...

  17. Lysosomal membrane stability of the mussel, Mytilus galloprovincialis (L.), as a biomarker of tributyltin exposure.

    Science.gov (United States)

    Okoro, Hussein K; Snyman, Reinette G; Fatoki, Olalekan S; Adekola, Folahan A; Ximba, Bhekumusa J; Slabber, Michelle Y

    2015-05-01

    The effect of tributyltin (TBT) on the stability of hemocytic lysosome membranes of the mussel, Mytilus galloprovincialis, and the use thereof as a biomarker of TBT-induced stress, was investigated. Mussels were exposed to 0.1 and 1.0 µg/L tributyltin respectively for 4 weeks. Lysosomal membrane stability of hemocytes was tested weekly by means of the neutral red retention time (NRRT) assay, after which the mussel samples were analyzed for TBT content. The two exposed groups exhibited significantly increased (p galloprovincialis.

  18. Reduced sulfation of muc5b is linked to xerostomia in patients with Sjögren syndrome

    NARCIS (Netherlands)

    C. Alliende; Y.-J. Kwon; M. Brito; C. Molina; S. Aguilera; P. Pérez; L. Leyton; A.F.G. Quest; U. Mandel; E. Veerman; M. Espinosa; H. Clausen; C. Leyton; R. Romo; S. González

    2008-01-01

    Objectives: MUC5B contains sulfated and sialylated oligosaccharides that sequester water required for moisturising the oral mucosa. Xerostomia, in patients with Sjögren syndrome, is generally associated with reduced quantities, rather than altered properties, of saliva. Here, we determined the amoun

  19. Anomalous grain growth in nanocrystalline Fe73.5Cu1Nb3Su13.5B9 alloys

    DEFF Research Database (Denmark)

    Jiang, Jianzhong

    1997-01-01

    The grain growth of the FeSi phase during the crystallization process of the amorphous Fe73.5Cu1Nb3Si13.5B9 alloy was studied using transmission electron microscopy and x-ray diffractometry. An anomalous grain growth behaviour of the FeSi phase in the samples annealed in temperature range from 74...

  20. Mossbauer studies of amorphous Fe73.5Cu1Nb3Si13.5B9 alloy

    DEFF Research Database (Denmark)

    Jiang, Jianzhong

    1996-01-01

    This paper reports a Mossbauer study of amorphous Fe73.5Cu1Nb3Si13.5B9 alloy between 10 and 673 K. The Curie temperature Tc is found to be 620-+ 1 K. The temperature dependence of the reduced average hyperfine field can be explained on the basis of Handrich's model of amorphous ferromagnetism...

  1. Iron content and acid phosphatase activity in hepatic parenchymal lysosomes of patients with hemochromatosis before and after phlebotomy treatment

    Energy Technology Data Exchange (ETDEWEB)

    Cleton, M.I.; de Bruijn, W.C.; van Blokland, W.T.; Marx, J.J.; Roelofs, J.M.; Rademakers, L.H.

    1988-03-01

    Lysosomal structures in liver parenchymal cells of 3 patients with iron overload and of 3 subjects without iron-storage disorders were investigated. A combination of enzyme cytochemistry--with cerium as a captive ion to demonstrate lysosomal acid phosphatase activity--and electron probe X-ray microanalysis (EPMA) was used. We were able (1) to define and quantify lysosomal structures as lysosomes, siderosomes, or residual bodies, (2) to quantify the amount of iron and cerium simultaneously in these structures, and (3) to evaluate a possible relation between iron storage and enzyme activity. With histopathologically increased iron storage, the number of siderosomes had increased at the cost of lysosomes, with a corresponding increase in acid phosphatase activity in both organelles. In histopahtologically severe iron overload, however, acid phosphatase activity was low or not detectable and most of the iron was stored in residual bodies. After phlebotomy treatment, the number of siderosomes had decreased in favor of the lysosomes, approaching values obtained in control subjects, and acid phosphatase activity was present in all iron-containing structures. In this way a relationship between iron storage and enzyme activity was established. The iron content of the individual lysosomal structures per unit area had increased with histopathologically increased iron storage and had decreased after phlebotomy treatment. From this observation, it is concluded that the iron status of the patient is not only reflected by the amount of iron-containing hepatocytes but, as well, by the iron content lysosomal unit area.

  2. A specific box switches the cell fate determining activity of XOTX2 and XOTX5b in the Xenopus retina

    Directory of Open Access Journals (Sweden)

    He Rong-Qiao

    2007-06-01

    Full Text Available Abstract Background Otx genes, orthologues of the Drosophila orthodenticle gene (otd, play crucial roles in vertebrate brain development. In the Xenopus eye, Xotx2 and Xotx5b promote bipolar and photoreceptor cell fates, respectively. The molecular basis of their differential action is not completely understood, though the carboxyl termini of the two proteins seem to be crucial. To define the molecular domains that make the action of these proteins so different, and to determine whether their retinal abilities are shared by Drosophila OTD, we performed an in vivo molecular dissection of their activity by transfecting retinal progenitors with several wild-type, deletion and chimeric constructs of Xotx2, Xotx5b and otd. Results We identified a small 8–10 amino acid divergent region, directly downstream of the homeodomain, that is crucial for the respective activities of XOTX2 and XOTX5b. In lipofection experiments, the exchange of this 'specificity box' completely switches the retinal activity of XOTX5b into that of XOTX2 and vice versa. Moreover, the insertion of this box into Drosophila OTD, which has no effect on retinal cell fate, endows it with the specific activity of either XOTX protein. Significantly, in cell transfection experiments, the diverse ability of XOTX2 and XOTX5b to synergize with NRL, a cofactor essential for vertebrate rod development, to transactivate the rhodopsin promoter is also switched depending on the box. We also show by GST-pull down that XOTX2 and XOTX5b differentially interact with NRL, though this property is not strictly dependent on the box. Conclusion Our data provide molecular evidence on how closely related homeodomain gene products can differentiate their functions to regulate distinct cell fates. A small 'specificity box' is both necessary and sufficient to confer on XOTX2 and XOTX5b their distinct activities in the developing frog retina and to convert the neutral orthologous OTD protein of Drosophila

  3. Diabetes Alters KIF1A and KIF5B Motor Proteins in the Hippocampus

    OpenAIRE

    Baptista, Filipa I.; Pinto, Maria J.; Filipe Elvas; Ramiro D Almeida; Ambrósio, António F.

    2013-01-01

    Diabetes mellitus is the most common metabolic disorder in humans. Diabetic encephalopathy is characterized by cognitive and memory impairments, which have been associated with changes in the hippocampus, but the mechanisms underlying those impairments triggered by diabetes, are far from being elucidated. The disruption of axonal transport is associated with several neurodegenerative diseases and might also play a role in diabetes-associated disorders affecting nervous system. We investigated...

  4. 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.

  5. The Possible "Proton Sponge " Effect of Polyethylenimine (PEI) Does Not Include Change in Lysosomal pH

    DEFF Research Database (Denmark)

    Søndergaard, Rikke Vicki; Mattebjerg, Maria Ahlm; Henriksen, Jonas Rosager;

    2013-01-01

    is still elusive. The "proton sponge " hypothesis remains the most generally accepted mechanism, although it is heavily debated. This hypothesis is associated with the large buffering capacity of PEI and other polycations, which has been interpreted to cause an increase in lysosomal pH even though...... no conclusive proof has been provided. In the present study, we have used a nanoparticle pH sensor that was developed for pH measurements in the endosomal/lysosomal pathway. We have carried out quantitative measurements of lysosomal pH as a function of PEI content and correlate the results to the "proton sponge...... " hypothesis. Our measurements show that PEI does not induce change in lysosomal pH as previously suggested and quantification of PEI concentrations in lysosomes makes it uncertain that the "proton sponge " effect is the dominant mechanism of polyplex escape.Molecular Therapy (2012); doi:10.1038/mt.2012.185....

  6. 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.

  7. 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.

  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. 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.

  10. 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.

  11. 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.

  12. Transcriptional control of the autophagy-lysosome system in pancreatic cancer

    Science.gov (United States)

    Perera, Rushika M.; Stoykova, Svetlana; Nicolay, Brandon N.; Ross, Kenneth N.; Fitamant, Julien; Boukhali, Myriam; Lengrand, Justine; Deshpande, Vikram; Selig, Martin K.; Ferrone, Cristina R.; Settleman, Jeff; Stephanopoulos, Gregory; Dyson, Nicholas J.; Zoncu, Roberto; Ramaswamy, Sridhar; Haas, Wilhelm; Bardeesy, Nabeel

    2016-01-01

    Activation of cellular stress response pathways to maintain metabolic homeostasis is emerging as a critical growth and survival mechanism in many cancers1. The pathogenesis of pancreatic ductal adenocarcinoma (PDA) requires high levels of autophagy2–4, a conserved self-degradative process5. However, the regulatory circuits that activate autophagy and reprogram PDA cell metabolism are unknown. We now show that autophagy induction in PDA occurs as part of a broader transcriptional program that coordinates activation of lysosome biogenesis and function, and nutrient scavenging, mediated by the MiT/TFE family transcription factors. In PDA cells, the MiT/TFE proteins6 – MITF, TFE3 and TFEB – are decoupled from regulatory mechanisms that control their cytoplasmic retention. Increased nuclear import in turn drives the expression of a coherent network of genes that induce high levels of lysosomal catabolic function essential for PDA growth. Unbiased global metabolite profiling reveals that MiT/TFE-dependent autophagy-lysosomal activation is specifically required to maintain intracellular amino acid (AA) pools. These results identify the MiT/TFE transcription factors as master regulators of metabolic reprogramming in pancreatic cancer and demonstrate activation of clearance pathways converging on the lysosome as a novel hallmark of aggressive malignancy. PMID:26168401

  13. (-)-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

  14. Lysosomal Enzyme Glucocerebrosidase Protects against Aβ1-42 Oligomer-Induced Neurotoxicity.

    Directory of Open Access Journals (Sweden)

    Seulah Choi

    Full Text Available Glucocerebrosidase (GCase functions as a lysosomal enzyme and its mutations are known to be related to many neurodegenerative diseases, including Gaucher's disease (GD, Parkinson's disease (PD, and Dementia with Lewy Bodies (DLB. However, there is little information about the role of GCase in the pathogenesis of Alzheimer's disease (AD. Here we demonstrate that GCase protein levels and enzyme activity are significantly decreased in sporadic AD. Moreover, Aβ1-42 oligomer treatment results in neuronal cell death that is concomitant with decreased GCase protein levels and enzyme activity, as well as impairment in lysosomal biogenesis and acidification. Importantly, overexpression of GCase promotes the lysosomal degradation of Aβ1-42 oligomers, restores the lysosomal impairment, and protects against the toxicity in neurons treated with Aβ1-42 oligomers. Our findings indicate that a deficiency of GCase could be involved in progression of AD pathology and suggest that augmentation of GCase activity may be a potential therapeutic option for the treatment of AD.

  15. 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

  16. 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

  17. 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

  18. Neural stem cell transplantation as a therapeutic approach for treating lysosomal storage diseases.

    Science.gov (United States)

    Shihabuddin, Lamya S; Cheng, Seng H

    2011-10-01

    Treating the central nervous system manifestations of subjects with neuropathic lysosomal storage diseases remains a major technical challenge. This is because of the low efficiency by which lysosomal enzymes in systemic circulation are able to traverse the blood brain barrier into the central nervous system. Intracranial transplantation of neural stems cells genetically modified to overexpress the respective deficient enzymes represents a potential approach to addressing this group of diseases. The unique properties of neural stem cells and progenitor cells, such as their ability to migrate to distal sites, differentiate into various cell types and integrate within the host brain without disrupting normal function, making them particularly attractive therapeutic agents. In addition, neural stem cells are amenable to ex vivo propagation and modification by gene transfer vectors. In this regard, transplanted cells can serve not only as a source of lysosomal enzymes but also as a means to potentially repair the injured brain by replenishing the organ with healthy cells and effecting the release of neuroprotective factors. This review discusses some of the well-characterized neural stem cell types and their possible use in treating neuropathic lysosomal storage diseases such as the Niemann Pick A disease.

  19. 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...

  20. KIF5B-RET fusion gene and non-small cell lung cancer%KIF5B-RET融合基因与非小细胞肺癌

    Institute of Scientific and Technical Information of China (English)

    韩英; 成志勇

    2013-01-01

    Lung cancer is the leading cause of mortality in cancer worldwide. Molecular targeted therapy is the hotpot of lung cancer study in recent years. In 2012, a novel fusion gene KIF5B-RET was identified in non-small cell lung cancer. This fusion gene is more frequently detected in the lung adenocarcinoma, with no or little history of cigarette smoking. The mutually exclusive nature of the RET fusions and other oncogenic alterations such as EGFR,K-Ras,ALK,etc. .suggests that the KIF5B-RET fusion is a new driver mutation. It could be a promising molecular target for the personalized diagnosis and treatment of non-small cell lung cancer.%肺癌是全世界范围死亡率最高的肿瘤.近年来,靶向治疗成为肺癌研究的热点.2012年研究发现肺癌中存在一种新的融合基因KIF5B-RET,其阳性患者多为不吸烟或很少吸烟的腺癌患者.其存在与其他已知的基因改变如EGFR、K-Ras、ALK等相互排斥,提示KIF5B-RET是一种新的致癌驱动突变,有可能成为非小细胞肺癌个体化诊断与治疗的一个分子靶点.

  1. 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.

  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. Inhibitor candidates's identification of HCV's RNA polymerase NS5B using virtual screening against iPPI-library

    Science.gov (United States)

    Sulistyawati, Indah; Sulistyo Dwi K., P.; Ichsan, Mochammad

    2016-03-01

    Hepatitis C is one of the major causes of chronic liver failure that caused by Hepatitis C Virus (HCV). Preventing the progression of HCV's replication through the inhibition of The RNA polymerase NS5B of Hepatitis C virus (NS5B) can be achieved via 4 binding regions: Site I (Thumb I), Site II (Thumb II), Site III (Palm I), and Site IV (Palm II). The aim of this research is to identify a candidate of NS5B inhibitor as an alternative for Hepatitis C treatment. An NS5B's 3D structure (PDB ID = 3D5M) used in this study has met some criteria of a good model to be used in virtual screening againts iPPI-lib using MTiOpenScreen webserver. The top two natural compounds resulted here then docked using Pyrix 0.8 and discovered trans-6-Benzamido-2-methyldecahydroisoquinoline (-9,1kcal/mol) and 2,4-dichloro-5-[4-(2 methoxyphenyl) piperazine-1-carbonyl]-N-[3-(trifluoromethyl)phenyl] benzenesulfonamide (9,4 kcal/mol) can bind to Tyr448 similar with all three established inhibitors, such as setrobuvir (-11,4 kcal/mol; site 3 inhibitor), CHEMBL379677 (-9,1 kcal/mol; site 1 inhibitor), and nesbuvir (-7,7 kcal/mol; site 4 inhibitor). The results of this study are relatively still needs to be tested, both in vitro and in vivo, in order to obtain more comprehensive knowledges as a follow-up of this predictive study.

  4. Models of neocortical layer 5b pyramidal cells capturing a wide range of dendritic and perisomatic active properties.

    Directory of Open Access Journals (Sweden)

    Etay Hay

    2011-07-01

    Full Text Available The thick-tufted layer 5b pyramidal cell extends its dendritic tree to all six layers of the mammalian neocortex and serves as a major building block for the cortical column. L5b pyramidal cells have been the subject of extensive experimental and modeling studies, yet conductance-based models of these cells that faithfully reproduce both their perisomatic Na(+-spiking behavior as well as key dendritic active properties, including Ca(2+ spikes and back-propagating action potentials, are still lacking. Based on a large body of experimental recordings from both the soma and dendrites of L5b pyramidal cells in adult rats, we characterized key features of the somatic and dendritic firing and quantified their statistics. We used these features to constrain the density of a set of ion channels over the soma and dendritic surface via multi-objective optimization with an evolutionary algorithm, thus generating a set of detailed conductance-based models that faithfully replicate the back-propagating action potential activated Ca(2+ spike firing and the perisomatic firing response to current steps, as well as the experimental variability of the properties. Furthermore, we show a useful way to analyze model parameters with our sets of models, which enabled us to identify some of the mechanisms responsible for the dynamic properties of L5b pyramidal cells as well as mechanisms that are sensitive to morphological changes. This automated framework can be used to develop a database of faithful models for other neuron types. The models we present provide several experimentally-testable predictions and can serve as a powerful tool for theoretical investigations of the contribution of single-cell dynamics to network activity and its computational capabilities.

  5. 3D-QSAR and molecular docking studies on designing inhibitors of the hepatitis C virus NS5B polymerase

    Science.gov (United States)

    Li, Wenlian; Si, Hongzong; Li, Yang; Ge, Cuizhu; Song, Fucheng; Ma, Xiuting; Duan, Yunbo; Zhai, Honglin

    2016-08-01

    Viral hepatitis C infection is one of the main causes of the hepatitis after blood transfusion and hepatitis C virus (HCV) infection is a global health threat. The HCV NS5B polymerase, an RNA dependent RNA polymerase (RdRp) and an essential role in the replication of the virus, has no functional equivalent in mammalian cells. So the research and development of efficient NS5B polymerase inhibitors provides a great strategy for antiviral therapy against HCV. A combined three-dimensional quantitative structure-activity relationship (QSAR) modeling was accomplished to profoundly understand the structure-activity correlation of a train of indole-based inhibitors of the HCV NS5B polymerase to against HCV. A comparative molecular similarity indices analysis (COMSIA) model as the foundation of the maximum common substructure alignment was developed. The optimum model exhibited statistically significant results: the cross-validated correlation coefficient q2 was 0.627 and non-cross-validated r2 value was 0.943. In addition, the results of internal validations of bootstrapping and Y-randomization confirmed the rationality and good predictive ability of the model, as well as external validation (the external predictive correlation coefficient rext2 = 0.629). The information obtained from the COMSIA contour maps enables the interpretation of their structure-activity relationship. Furthermore, the molecular docking study of the compounds for 3TYV as the protein target revealed important interactions between active compounds and amino acids, and several new potential inhibitors with higher activity predicted were designed basis on our analyses and supported by the simulation of molecular docking. Meanwhile, the OSIRIS Property Explorer was introduced to help select more satisfactory compounds. The satisfactory results from this study may lay a reliable theoretical base for drug development of hepatitis C virus NS5B polymerase inhibitors.

  6. Ground-based detection of the near-infrared emission from the dayside of WASP-5b

    CERN Document Server

    Chen, Guo; Madhusudhan, Nikku; Wang, Hongchi; Nikolov, Nikolay; Seemann, Ulf; Henning, Thomas

    2014-01-01

    (Abridged) WASP-5b is a highly irradiated dense hot Jupiter orbiting a G4V star every 1.6 days. We observed two secondary eclipses of WASP-5b in the J, H and K bands simultaneously. Thermal emission of WASP-5b is detected in the J and K bands. The retrieved planet-to-star flux ratios in the J and K bands are 0.168 +0.050/-0.052% and 0.269+/-0.062%, corresponding to brightness temperatures of 2996 +212/-261K and 2890 +246/-269K, respectively. No thermal emission is detected in the H band, with a 3-sigma upper limit of 0.166%, corresponding to a maximum temperature of 2779K. On the whole, our J, H, K results can be explained by a roughly isothermal temperature profile of ~2700K in the deep layers of the planetary dayside atmosphere that are probed at these wavelengths. Together with Spitzer observations, which probe higher layers that are found to be at ~1900K, a temperature inversion is ruled out in the range of pressures probed by the combined data set. While an oxygen-rich model is unable to explain all the ...

  7. Paulus als Schriftuitlegger Paulus’ vertolking van Genesis 15:5b-6 in Romeinen 4:3

    Directory of Open Access Journals (Sweden)

    Pieter K. Baaij

    2005-07-01

    Full Text Available Paul as expositor of Scripture. Paul’s interpretation of Genesis 15:5b-6 in Romans 4:3 The author of this article has since 1986 been working exclusively on the translation and exegesis of the Epistle to the Romans. In the course of this work, he discovered that Paul first carefully considered his text in Biblical Hebrew before writing it accurately in Greek. It then transpires that “the difficult Paul” proclaimed the gospel of the crowning of the Law (10:4 with great clarity. The real Paul is pre-eminently the expositor of Scripture. Provided we use the instruments provided to the reader by Paul, we shall thus not only understand the proclamation of Paul better but also Scripture itself.  In this article the author illustrates that Paul in Romans 4:3 accurately renders what is written in Genesis 15:5b and 6. Paul teaches us to understand the Hebrew text better by indicating where the emphasis lies in the Hebrew text and how we must interpret the Hebrew terms used. For this reason Paul’s interpretation of Genesis 15:5b and 6 in Romans 4:3 is of great importance in the continually more topical debate on how we must translate the Bible.

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

    Science.gov (United States)

    Oh, Doo-Byoung

    2015-08-01

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

  9. 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

  10. A new role for an old drug: Ambroxol triggers lysosomal exocytosis via pH-dependent Ca²⁺ release from acidic Ca²⁺ stores.

    Science.gov (United States)

    Fois, Giorgio; Hobi, Nina; Felder, Edward; Ziegler, Andreas; Miklavc, Pika; Walther, Paul; Radermacher, Peter; Haller, Thomas; Dietl, Paul

    2015-12-01

    Ambroxol (Ax) is a frequently prescribed drug used to facilitate mucociliary clearance, but its mode of action is yet poorly understood. Here we show by X-ray spectroscopy that Ax accumulates in lamellar bodies (LBs), the surfactant storing, secretory lysosomes of type II pneumocytes. Using lyso- and acidotropic substances in combination with fluorescence imaging we confirm that these vesicles belong to the class of acidic Ca(2+) stores. Ax lead to a significant neutralization of LB pH, followed by intracellular Ca(2+) release, and to a dose-dependent surfactant exocytosis. Ax-induced Ca(2+) release was significantly reduced and slowed down by pretreatment of the cells with bafilomycin A1 (Baf A1), an inhibitor of the vesicular H(+) ATPase. These results could be nearly reproduced with NH3/NH4(+). The findings suggest that Ax accumulates within LBs and severely affects their H(+) and Ca(2+) homeostasis. This is further supported by an Ax-induced change of nanostructural assembly of surfactant layers. We conclude that Ax profoundly affects LBs presumably by disordering lipid bilayers and by acting as a weak base. The pH change triggers - at least in part - Ca(2+) release from stores and secretion of surfactant from type II cells. This novel mechanism of Ax as a lysosomal secretagogue may also play a role for its recently discussed use for lysosomal storage and other degenerative diseases.

  11. OCRL-mutated fibroblasts from patients with Dent-2 disease exhibit INPP5B-independent phenotypic variability relatively to Lowe syndrome cells.

    Science.gov (United States)

    Montjean, Rodrick; Aoidi, Rifdat; Desbois, Pierrette; Rucci, Julien; Trichet, Michaël; Salomon, Rémi; Rendu, John; Fauré, Julien; Lunardi, Joël; Gacon, Gérard; Billuart, Pierre; Dorseuil, Olivier

    2015-02-15

    OCRL mutations are associated with both Lowe syndrome and Dent-2 disease, two rare X-linked conditions. Lowe syndrome is an oculo-cerebro-renal disorder, whereas Dent-2 patients mainly present renal proximal tubulopathy. Loss of OCRL-1, a phosphoinositide-5-phosphatase, leads in Lowe patients' fibroblasts to phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) accumulation, with defects in F-actin network, α-actinin distribution and ciliogenesis, whereas fibroblasts of Dent-2 patients are still uncharacterized. To search for mechanisms linked to clinical variability observed between these two OCRL mutation-associated pathologies, we compared dermal fibroblasts from independent patients, four affected by Dent-2 disease and six with Lowe syndrome. For the first time, we describe that Dent-2 fibroblasts with OCRL loss-of-function (LOF) mutations exhibit decrease in actin stress fibers, appearance of punctate α-actinin signals and alteration in primary cilia formation. Interestingly, we quantified these phenotypes as clearly intermediate between Lowe and control fibroblasts, thus suggesting that levels of these defects correlate with clinical variations observed between patients with OCRL mutations. In addition, we show that Lowe and Dent-2 fibroblasts display similar PI(4,5)P2 accumulation levels. Finally, we analyzed INPP5B, a paralogous gene already reported to exhibit functional redundancy with OCRL, and report neither differences in its expression at RNA or protein levels, nor specific allelic variations between fibroblasts of patients. Altogether, we describe here differential phenotypes between fibroblasts from Lowe and Dent-2 patients, both associated with OCRL LOF mutations, we exclude direct roles of PI(4,5)P2 and INPP5B in this phenotypic variability and we underline potential key alterations leading to ocular and neurological clinical features in Lowe syndrome. PMID:25305077

  12. 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...

  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. Inhibition of the histone demethylase Kdm5b promotes neurogenesis and derepresses Reln (reelin) in neural stem cells from the adult subventricular zone of mice.

    Science.gov (United States)

    Zhou, Qiong; Obana, Edwin A; Radomski, Kryslaine L; Sukumar, Gauthaman; Wynder, Christopher; Dalgard, Clifton L; Doughty, Martin L

    2016-02-15

    The role of epigenetic regulators in the control of adult neurogenesis is largely undefined. We show that the histone demethylase enzyme Kdm5b (Jarid1b) negatively regulates neurogenesis from adult subventricular zone (SVZ) neural stem cells (NSCs) in culture. shRNA-mediated depletion of Kdm5b in proliferating adult NSCs decreased proliferation rates and reduced neurosphere formation in culture. When transferred to differentiation culture conditions, Kdm5b-depleted adult NSCs migrated from neurospheres with increased velocity. Whole-genome expression screening revealed widespread transcriptional changes with Kdm5b depletion, notably the up-regulation of reelin (Reln), the inhibition of steroid biosynthetic pathway component genes and the activation of genes with intracellular transport functions in cultured adult NSCs. Kdm5b depletion increased extracellular reelin concentration in the culture medium and increased phosphorylation of the downstream reelin signaling target Disabled-1 (Dab1). Sequestration of extracellular reelin with CR-50 reelin-blocking antibodies suppressed the increase in migratory velocity of Kdm5b-depleted adult NSCs. Chromatin immunoprecipitation revealed that Kdm5b is present at the proximal promoter of Reln, and H3K4me3 methylation was increased at this locus with Kdm5b depletion in differentiating adult NSCs. Combined the data suggest Kdm5b negatively regulates neurogenesis and represses Reln in neural stem cells from the adult SVZ. PMID:26739753

  15. Hepatitis C Genotype Prevalence in Monastir Region, Tunisia: Correlation between 5' Untranslated Region (5'UTR), Non-structural 5B (NS5B), and Core Sequences in HCV Subtyping.

    Science.gov (United States)

    Souii, Amira; Elargoubi, Aida; Fallecker, Catherine; Mastouri, Maha; Drouet, Emmanuel

    2016-09-01

    Hepatitis C virus (HCV) is a causative agent of chronic liver disease, cirrhosis, and hepatocellular carcinoma. It constitutes a major public health around the world. There is no vaccine available against HCV, and current therapies are effective in only small percentage of patients. HCV has wide population-specific genotype variability. Genotype knowledge and viral load assessment are equally important for designing therapeutic strategies. Taking into account that the molecular epidemiology of HCV variants circulating in Tunisia is not yet well elucidated, and that, at present, little is known about the distribution pattern of HCV in Monastir region (Tunisia), we aimed, herein, to evaluate the prevalence of HCV genotypes in Monastir and to identify risk-related factors. For this purpose, 50 anti-HCV antibody-positive cases were diagnosed and subjected to viral RNA extraction, amplification, genotyping, and viral load quantification. Molecular epidemiology was studied by 5' untranslated region (5' UTR) sequencing as compared with the non-structural 5B (NS5B) and core region sequences. Overall concordance between 5' UTR, core, and NS5B sequencing was 100 %. The highest prevalent genotype was 1b (50 %) followed by genotypes 1a (16 %), 4a (12 %), 2a (10 %), 2c (8 %), and 3a (4 %). Interestingly, the subtype 1b had a statistically significant higher viral load than the other genotypes followed by subtype 1a. Based on these data, this study revealed a high prevalence of HCV genotype 1 (subtypes 1b and 1a) compared to other genotypes. A continued monitoring of HCV and knowledge of circulating genotypes could impact on future vaccine formulations. PMID:27189386

  16. 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.)

  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. 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...... 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...

  20. 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

    Collagen turnover is crucial for tissue homeostasis and remodeling and pathological processes such as cancer invasion, but the underlying molecular mechanisms are poorly understood. A major pathway appears to be internalization and degradation by fibroblasts. We now show that the endocytic...... 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...

  1. 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 aggregates are an early and progressive pathology that occur at 3 months of age and increase in both size and number over time. These autofluorescent aggregates are not observed in mice expressing wild-type CHMP2B, or in non-transgenic controls, indicating that they are a specific pathology caused by mutant...... in 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...

  2. Role of phospholipids in destabilization of lysosomal membranes in chronic alcohol poisoning

    Energy Technology Data Exchange (ETDEWEB)

    Tadevosyan, Y.V.; Batikyan, T.B.; Gevorkyan, G.A.; Karagezyan, K.G.

    1986-04-01

    The aim of this investigation was to study changes in the phospholipids (PL) spectrum and possible activity of membrane-bound phospholipase A/sub 2/ in lysosomal membranes from albino rat liver under conditions of the normally metabolizing tissue and during long-term alcohol poisoning. Changes in stability of the lysosomal membranes were determined by measuring nonsedimented acid phosphatase (AP) activity. The substance 1-acyl-2-(1-/sup 14/C)-oleoyl-phosphatidyl-choline (/sup 14/C-PCh) was synthesized by an enzymic method. Phospholipase A/sub 2/ activity was determined in an incubation medium of Tris-Maleate buffer containing 20 nanomoles (/sup 14/C)-PCH, 8 mM CaC1/sub 2/, and about 100 micrograms protein.

  3. 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 ...

  4. 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...

  5. Arsenic induces apoptosis by the lysosomal-mitochondrial pathway in INS-1 cells.

    Science.gov (United States)

    Pan, Xiao; Jiang, Liping; Zhong, Laifu; Geng, Chengyan; Jia, Li; Liu, Shuang; Guan, Huai; Yang, Guang; Yao, Xiaofeng; Piao, Fengyuan; Sun, Xiance

    2016-02-01

    Recently, long term arsenic exposure was considered to be associated with an increased risk of diabetes mellitus. While a relation of cause-and-effect between apoptosis of pancreatic β-cells and arsenic exposure, the precise mechanisms of these events remains unclear. The aim of this study was to explore arsenic-induced pancreatic β-cell apoptosis and the mechanisms of through the possible link between lysosomal and the mitochondrial apoptotic pathway. After exposure to 10 μM of arsenic, the reactive oxygen species (ROS) level was significantly increased at 12 h, while the mitochondrial membrane potential was reduced at 24 h and the lysosomal membrane integrity was disrupted at 48 h. A significant increase in protein expression for cytochrome c was also observed using Western blot analysis after exposure to arsenic for 48 h. To further demonstrate that arsenic reduced the lysosomal membrane integrity, cells pretreated with NH4 Cl and exposed to arsenic harbored a lower fluorescence increase than cells that were only exposed to arsenic. In addition, apoptosis was mesured using Hoechst 33342/PI dual staining by microscopy and annexin V-FITC/propidium iodide dual staining by flow cytometry. The results show an increased uptake of the arsenic dose and the cells changed from dark blue to light blue, karyopyknosis, nuclear chromatin condensation, side set or fracture, and a correlation was found between the number of apoptotic cells and arsenic dose. The result of present study suggest that arsenic may induce pancreatic β-cell apoptosis through activation of the lysosome-mitochondrial pathway.

  6. 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 ...

  7. 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, ...

  8. 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...

  9. 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...

  10. 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...

  11. 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...

  12. Lysosomal trafficking of {beta}-catenin induced by the tea polyphenol epigallocatechin-3-gallate

    Energy Technology Data Exchange (ETDEWEB)

    Dashwood, Wan-Mohaiza [Linus Pauling Institute, 571 Weniger Hall, Oregon State University, Corvallis, OR 97331-6512 (United States); Carter, Orianna [Linus Pauling Institute, 571 Weniger Hall, Oregon State University, Corvallis, OR 97331-6512 (United States); Al-Fageeh, Mohamed [Linus Pauling Institute, 571 Weniger Hall, Oregon State University, Corvallis, OR 97331-6512 (United States); Li, Qingjie [Linus Pauling Institute, 571 Weniger Hall, Oregon State University, Corvallis, OR 97331-6512 (United States); Dashwood, Roderick H. [Linus Pauling Institute, 571 Weniger Hall, Oregon State University, Corvallis, OR 97331-6512 (United States)]. E-mail: Rod.Dashwood@oregonstate.edu

    2005-12-11

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

  13. 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...

  14. Glutathione transferase mu 2 protects glioblastoma cells against aminochrome toxicity by preventing autophagy and lysosome dysfunction

    Science.gov (United States)

    Huenchuguala, Sandro; Muñoz, Patricia; Zavala, Patricio; Villa, Mónica; Cuevas, Carlos; Ahumada, Ulises; Graumann, Rebecca; Nore, Beston F; Couve, Eduardo; Mannervik, Bengt; Paris, Irmgard; Segura-Aguilar, Juan

    2014-01-01

    U373MG cells constitutively express glutathione S-transferase mu 2 (GSTM2) and exhibit 3H-dopamine uptake, which is inhibited by 2 µM of nomifensine and 15 µM of estradiol. We generated a stable cell line (U373MGsiGST6) expressing an siRNA against GSTM2 that resulted in low GSTM2 expression (26% of wild-type U373MG cells). A significant increase in cell death was observed when U373MGsiGST6 cells were incubated with 50 µM purified aminochrome (18-fold increase) compared with wild-type cells. The incubation of U373MGsiGST6 cells with 75 µM aminochrome resulted in the formation of autophagic vacuoles containing undigested cellular components, as determined using transmission electron microscopy. A significant increase in autophagosomes was determined by measuring endogenous LC3-II, a significant decrease in cell death was observed in the presence of bafilomycin A1, and a significant increase in cell death was observed in the presence of trehalose. A significant increase in LAMP2 immunostaining was observed, a significant decrease in bright red fluorescence of lysosomes with acridine orange was observed, and bafilomycin A1 pretreatment reduced the loss of lysosome acidity. A significant increase in cell death was observed in the presence of lysosomal protease inhibitors. Aggregation of TUBA/α-tubulin (tubulin, α) and SQSTM1 protein accumulation were also observed. Moreover, a significant increase in the number of lipids droplets was observed compared with U373MG cells with normal expression of GSTM2. These results support the notion that GSTM2 is a protective enzyme against aminochrome toxicity in astrocytes and that aminochrome cell death in U373MGsiGST6 cells involves autophagic-lysosomal dysfunction. PMID:24434817

  15. 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

  16. 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

  17. Endo-lysosomal TRP mucolipin-1 channels trigger global ER Ca2+ release and Ca2+ influx

    Science.gov (United States)

    Kilpatrick, Bethan S.; Yates, Elizabeth; Grimm, Christian; Schapira, Anthony H.

    2016-01-01

    ABSTRACT Transient receptor potential (TRP) mucolipins (TRPMLs), encoded by the MCOLN genes, are patho-physiologically relevant endo-lysosomal ion channels crucial for membrane trafficking. Several lines of evidence suggest that TRPMLs mediate localised Ca2+ release but their role in Ca2+ signalling is not clear. Here, we show that activation of endogenous and recombinant TRPMLs with synthetic agonists evoked global Ca2+ signals in human cells. These signals were blocked by a dominant-negative TRPML1 construct and a TRPML antagonist. We further show that, despite a predominant lysosomal localisation, TRPML1 supports both Ca2+ release and Ca2+ entry. Ca2+ release required lysosomal and ER Ca2+ stores suggesting that TRPMLs, like other endo-lysosomal Ca2+ channels, are capable of ‘chatter’ with ER Ca2+ channels. Our data identify new modalities for TRPML1 action. PMID:27577094

  18. The pore-forming protein Cry5B elicits the pathogenicity of Bacillus sp. against Caenorhabditis elegans.

    Directory of Open Access Journals (Sweden)

    Melanie F Kho

    Full Text Available The soil bacterium Bacillus thuringiensis is a pathogen of insects and nematodes and is very closely related to, if not the same species as, Bacillus cereus and Bacillus anthracis. The defining characteristic of B. thuringiensis that sets it apart from B. cereus and B. anthracis is the production of crystal (Cry proteins, which are pore-forming toxins or pore-forming proteins (PFPs. Although it is known that PFPs are important virulence factors since their elimination results in reduced virulence of many pathogenic bacteria, the functions by which PFPs promote virulence are incompletely understood. Here we study the effect of Cry proteins in B. thuringiensis pathogenesis of the nematode Caenorhabditis elegans. We find that whereas B. thuringiensis on its own is not able to infect C. elegans, the addition of the PFP Cry protein, Cry5B, results in a robust lethal infection that consumes the nematode host in 1-2 days, leading to a "Bob" or bag-of-bacteria phenotype. Unlike other infections of C. elegans characterized to date, the infection by B. thuringiensis shows dose-dependency based on bacterial inoculum size and based on PFP concentration. Although the infection process takes 1-2 days, the PFP-instigated infection process is irreversibly established within 15 minutes of initial exposure. Remarkably, treatment of C. elegans with Cry5B PFP is able to instigate many other Bacillus species, including B. anthracis and even "non-pathogenic" Bacillus subtilis, to become lethal and infectious agents to C. elegans. Co-culturing of Cry5B-expressing B. thuringiensis with B. anthracis can result in lethal infection of C. elegans by B. anthracis. Our data demonstrate that one potential property of PFPs is to sensitize the host to bacterial infection and further that C. elegans and probably other roundworms can be common hosts for B. cereus-group bacteria, findings with important ecological and research implications.

  19. Confirmation of childhood acute lymphoblastic leukemia variants, ARID5B and IKZF1, and interaction with parental environmental exposures.

    Directory of Open Access Journals (Sweden)

    Tiffany-Jane Evans

    Full Text Available Genome wide association studies (GWAS have established association of ARID5B and IKZF1 variants with childhood acute lymphoblastic leukemia (ALL. Epidemiological studies suggest that environmental factors alone appear to make a relatively minor contribution to disease risk. The polygenic nature of childhood ALL predisposition together with the timing of environmental triggers may hold vital clues for disease etiology. This study presents results from an Australian GWAS of childhood ALL cases (n = 358 and population controls (n = 1192. Furthermore, we utilised family trio (n = 204 genotypes to extend our investigation to gene-environment interaction of significant loci with parental exposures before conception, and child's sex and age. Thirteen SNPs achieved genome wide significance in the population based case/control analysis; ten annotated to ARID5B and three to IKZF1. The most significant SNPs in these regions were ARID5B rs4245595 (OR 1.63, CI 1.38-1.93, P = 2.13×10(-9, and IKZF1 rs1110701 (OR 1.69, CI 1.42-2.02, p = 7.26×10(-9. There was evidence of gene-environment interaction for risk genotype at IKZF1, whereby an apparently stronger genetic effect was observed if the mother took folic acid or if the father did not smoke prior to pregnancy (respective interaction P-values: 0.04, 0.05. There were no interactions of risk genotypes with age or sex (P-values >0.2. Our results evidence that interaction of genetic variants and environmental exposures may further alter risk of childhood ALL however, investigation in a larger population is required. If interaction of folic acid supplementation and IKZF1 variants holds, it may be useful to quantify folate levels prior to initiating use of folic acid supplements.

  20. Non-covalent DNA groove-binding by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine.

    OpenAIRE

    Marsch, G A; Ward, R. L.; Colvin, M.; Turteltaub, K W

    1994-01-01

    The cooked meat mutagen 2-amino-1-methyl-6-phenyl-imidazo[4,5-b]pyridine (PhIP) is metabolized in vivo to electrophilic intermediates that covalently bind to DNA guanines. Here we address the mechanism of PhIP's non-covalent interaction with DNA by using spectroscopic and computational methodologies. NMR methodologies indicated that upon addition of DNA, PhIP aromatic protons underwent a small, 0.11-0.12 p.p.m. upfield shift. DNA phosphorus resonances of non-covalent PhIP-DNA complexes broade...

  1. Biomonitoring the Cooked Meat Carcinogen 2-Amino-1-methy-6-phenylimidazo[4,5-b]pyridine in Canine Fur

    OpenAIRE

    Gu, Dan; Neuman, Zachary L.; Modiano, Jaime F; Turesky, Robert J.

    2012-01-01

    2-Amino-1-methyl-6-phenylimidazo[4, 5-b]pyridine (PhIP) is a heterocyclic aromatic amine (HAA) that is formed during the cooking of meat, poultry, and fish. PhIP is a rodent carcinogen and thought to contribute to several diet-related cancers in humans. PhIP is present in the hair of human omnivores but not in the hair of vegetarians. We have now identified PhIP in the fur of fourteen out of sixteen healthy dogs consuming different brands of commercial pet food. The levels of PhIP in canine f...

  2. Planar patch clamp approach to characterize ionic currents from intact lysosomes.

    Science.gov (United States)

    Schieder, Michael; Rötzer, Katrin; Brüggemann, Andrea; Biel, Martin; Wahl-Schott, Christian

    2010-01-01

    Since its launch in the early 1980s, the patch clamp method has been extensively used to study ion channels in the plasma membrane, but its application to the study of intracellular ion channels has been limited. Unlike the plasma membrane, intracellular membranes are usually not stable enough to withstand mechanical manipulation by glass electrodes during seal formation and rupturing of the membrane. To circumvent these problems, we developed a method involving the immobilization of isolated organelles on a solid matrix planar glass chip. This glass chip contains a microstructured hole that supports the formation of gigaseals and subsequent electrophysiological recordings despite the high fragility of intracellular membranes. Here, we report the experimental details of this method using lysosomes, which are the smallest cellular organelles, as a model system. We demonstrate that we can record endogenous ionic currents from wild-type lysosomes, as well as from lysosomes overexpressing ion channels, and expect that this method will provide electrophysiological access to a broad range of intracellular ion channels.

  3. 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.

  4. A lysosome-targeted drug delivery system based on sorbitol backbone towards efficient cancer therapy.

    Science.gov (United States)

    Maniganda, Santhi; Sankar, Vandana; Nair, Jyothi B; Raghu, K G; Maiti, Kaustabh K

    2014-09-14

    A straightforward synthetic approach was adopted for the construction of a lysosome-targeted drug delivery system (TDDS) using sorbitol scaffold (Sor) linked to octa-guanidine and tetrapeptide GLPG, a peptide substrate of lysosomal cysteine protease, cathepsin B. The main objective was to efficiently deliver the potential anticancer drug, doxorubicin to the target sites, thereby minimizing dose-limiting toxicity. Three TDDS vectors were synthesized viz., DDS1: Sor-GLPG-Fl, DDS2: Sor-Fl (control) and DDS3: Sor-GLPGC-SMCC-Dox. Dox release from DDS3 in the presence of cathepsin B was studied by kinetics measurement based on the fluorescent property of Dox. The cytotoxicity of DDS1 was assessed and found to be non-toxic. Cellular internalization and colocalization studies of all the 3 systems were carried out by flow cytometry and confocal microscopy utilizing cathepsin B-expressing HeLa cells. DDS1 and DDS3 revealed significant localization within the lysosomes, in contrast to DDS2 (control). The doxorubicin-conjugated carrier, DDS3, demonstrated significant cytotoxic effect when compared to free Dox by MTT assay and also by flow cytometric analysis. The targeted approach with DDS3 is expected to be promising, because it is indicated to be advantageous over free Dox, which possesses dose-limiting toxicity, posing risk of injury to normal tissues.

  5. Quantitative Differences in the Urinary Proteome of Siblings Discordant for Type 1 Diabetes Include Lysosomal Enzymes.

    Science.gov (United States)

    Suh, Moo-Jin; Tovchigrechko, Andrey; Thovarai, Vishal; Rolfe, Melanie A; Torralba, Manolito G; Wang, Junmin; Adkins, Joshua N; Webb-Robertson, Bobbie-Jo M; Osborne, Whitney; Cogen, Fran R; Kaplowitz, Paul B; Metz, Thomas O; Nelson, Karen E; Madupu, Ramana; Pieper, Rembert

    2015-08-01

    Individuals with type 1 diabetes (T1D) often have higher than normal blood glucose levels, causing advanced glycation end product formation and inflammation and increasing the risk of vascular complications years or decades later. To examine the urinary proteome in juveniles with T1D for signatures indicative of inflammatory consequences of hyperglycemia, we profiled the proteome of 40 T1D patients with an average of 6.3 years after disease onset and normal or elevated HbA1C levels, in comparison with a cohort of 41 healthy siblings. Using shotgun proteomics, 1036 proteins were identified, on average, per experiment, and 50 proteins showed significant abundance differences using a Wilcoxon signed-rank test (FDR q-value ≤ 0.05). Thirteen lysosomal proteins were increased in abundance in the T1D versus control cohort. Fifteen proteins with functional roles in vascular permeability and adhesion were quantitatively changed, including CD166 antigen and angiotensin-converting enzyme 2. α-N-Acetyl-galactosaminidase and α-fucosidase 2, two differentially abundant lysosomal enzymes, were detected in western blots with often elevated quantities in the T1D versus control cohort. Increased release of proteins derived from lysosomes and vascular epithelium into urine may result from hyperglycemia-associated inflammation in the kidney vasculature.

  6. [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

  7. 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

  8. 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

  9. 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.

  10. Alpha-synuclein induces lysosomal rupture and cathepsin dependent reactive oxygen species following endocytosis.

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    David Freeman

    Full Text Available α-synuclein dysregulation is a critical aspect of Parkinson's disease pathology. Recent studies have observed that α-synuclein aggregates are cytotoxic to cells in culture and that this toxicity can be spread between cells. However, the molecular mechanisms governing this cytotoxicity and spread are poorly characterized. Recent studies of viruses and bacteria, which achieve their cytoplasmic entry by rupturing intracellular vesicles, have utilized the redistribution of galectin proteins as a tool to measure vesicle rupture by these organisms. Using this approach, we demonstrate that α-synuclein aggregates can induce the rupture of lysosomes following their endocytosis in neuronal cell lines. This rupture can be induced by the addition of α-synuclein aggregates directly into cells as well as by cell-to-cell transfer of α-synuclein. We also observe that lysosomal rupture by α-synuclein induces a cathepsin B dependent increase in reactive oxygen species (ROS in target cells. Finally, we observe that α-synuclein aggregates can induce inflammasome activation in THP-1 cells. Lysosomal rupture is known to induce mitochondrial dysfunction and inflammation, both of which are well established aspects of Parkinson's disease, thus connecting these aspects of Parkinson's disease to the propagation of α-synuclein pathology in cells.

  11. Alpha Adrenergic Induction of Transport of Lysosomal Enzyme across the Blood-Brain Barrier.

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    Akihiko Urayama

    Full Text Available The impermeability of the adult blood-brain barrier (BBB to lysosomal enzymes impedes the ability to treat the central nervous system manifestations of lysosomal storage diseases. Here, we found that simultaneous stimulation of the alpha1 and alpha2 adrenoreceptor restores in adult mice the high rate of transport for the lysosomal enzyme P-GUS that is seen in neonates but lost with development. Beta adrenergics, other monoamines, and acetylcholine did not restore this transport. A high dose (500 microg/mouse of clonidine, a strong alpha2 and weak alpha1 agonist, was able to act as monotherapy in the stimulation of P-GUS transport. Neither use of alpha1 plus alpha2 agonists nor the high dose clonidine disrupted the BBB to albumin. In situ brain perfusion and immunohistochemistry studies indicated that adrengerics act on transporters already at the luminal surface of brain endothelial cells. These results show that adrenergic stimulation, including monotherapy with clonidine, could be key for CNS enzyme replacement therapy.

  12. Characterization of lysosome-destabilizing DOPE/PLGA nanoparticles designed for cytoplasmic drug release.

    Science.gov (United States)

    Chhabra, Resham; Grabrucker, Andreas M; Veratti, Patrizia; Belletti, Daniela; Boeckers, Tobias M; Vandelli, Maria Angela; Forni, Flavio; Tosi, Giovanni; Ruozi, Barbara

    2014-08-25

    Polymeric nanoparticles (NPs) offer a promising approach for therapeutic intracellular delivery of proteins, conventionally hampered by short half-lives, instability and immunogenicity. Remarkably, NPs uptake occurs via endocytic internalization leading to NPs content's release within lysosomes. To overcome lysosomal degradation and achieve NPs and/or loaded proteins release into cytosol, we propose the formulation of hybrid NPs by adding 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) as pH sensitive component in the formulation of poly-lactide-co-glycolide (PLGA) NPs. Hybrid NPs, featured by different DOPE/PLGA ratios, were characterized in terms of structure, stability and lipid organization within the polymeric matrix. Experiments on NIH cells and rat primary neuronal cultures highlighted the safety profile of hybrid NPs. Moreover, after internalization, NPs are able to transiently destabilize the integrity of lysosomes in which they are taken up, speeding their escape and favoring cytoplasmatic localization. Thus, these DOPE/PLGA-NPs configure themselves as promising carriers for intracellular protein delivery.

  13. 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

  14. 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

  15. 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...

  16. 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...

  17. 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...

  18. Endocytic pathway rapidly delivers internalized molecules to lysosomes: an analysis of vesicle trafficking, clustering and mass transfer.

    Science.gov (United States)

    Pangarkar, Chinmay; Dinh, Anh-Tuan; Mitragotri, Samir

    2012-08-20

    Lysosomes play a critical role in intracellular drug delivery. For enzyme-based therapies, they represent a potential target site whereas for nucleic acid or many protein drugs, they represent the potential degradation site. Either way, understanding the mechanisms and processes involved in routing of materials to lysosomes after cellular entry is of high interest to the field of drug delivery. Most therapeutic cargoes other than small hydrophobic molecules enter the cells through endocytosis. Endocytosed cargoes are routed to lysosomes via microtubule-based transport and are ultimately shared by various lysosomes via tethering and clustering of endocytic vesicles followed by exchange of their contents. Using a combined experimental and numerical approach, here we studied the rates of mass transfer into and among the endocytic vesicles in a model cell line, 3T3 fibroblasts. In order to understand the relationship of mass transfer with microtubular transport and vesicle clustering, we varied both properties through various pharmacological agents. At the same time, microtubular transport and vesicle clustering were modeled through diffusion-advection equations and the Smoluchowski equations, respectively. Our analysis revealed that the rate of mass transfer is optimally related to microtubular transport and clustering properties of vesicles. Further, the rate of mass transfer is highest in the innate state of the cell. Any perturbation to either microtubular transport or vesicle aggregation led to reduced mass transfer to lysosome. These results suggest that in the absence of an external intervention the endocytic pathway appears to maximize molecular delivery to lysosomes. Strategies are discussed to reduce mass transfer to lysosomes so as to extend the residence time of molecules in endosomes or late endosomes, thus potentially increasing the likelihood of their escape before disposition in the lysosomes.

  19. 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...

  20. 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.

  1. Cardenolide-Induced Lysosomal Membrane Permeabilization Demonstrates Therapeutic Benefits in Experimental Human Non-Small Cell Lung Cancers

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    Tatjana Mijatovic

    2006-05-01

    Full Text Available Non-small cell lung cancers (NSCLCs are the leading cause of cancer deaths in most developed countries. Targeting heat shock protein 70 (Hsp70 expression and function, together with the induction of lysosomal membrane permeabilization (LMP, could overcome the multiple anti-cell death mechanisms evidenced in NSCLCs that are responsible for the failure of currently used chemotherapeutic drugs. Because cardenolides bind to the sodium pump, they affect multiple signaling pathways and thus have a number of marked effects on tumor cell behavior. The aim of the present study was to characterize in vitro and in vivo the antitumor effects of a new cardenolide (UNBS1450 on experimental human NSCLCs. UNBS1450 is a potent source of in vivo antitumor activity in the case of paclitaxeland oxaliplatin-resistant subcutaneous human NCIH727 and orthotopic A549 xenografts in nude mice. In vitro UNBS1450-mediated antitumor activity results from the induction of nonapoptotic cell death. UNBS1450 mediates the decrease of Hsp70 at both mRNA and protein levels, and this is at least partly due to UNBS1450-induced downregulation of NFAT5/ TonEBP (a factor responsible for the transcriptional control of Hsp70. These effects were paralleled by the induction of LMP, as evidenced by acridine orange staining and immunofluorescence analysis for cathepsin B accumulation.

  2. 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

  3. Ca2+ -regulated lysosome fusion mediates angiotensin II-induced lipid raft clustering in mesenteric endothelial cells.

    Science.gov (United States)

    Han, Wei-Qing; Chen, Wen-Dong; Zhang, Ke; Liu, Jian-Jun; Wu, Yong-Jie; Gao, Ping-Jin

    2016-04-01

    It has been reported that intracellular Ca2+ is involved in lysosome fusion and membrane repair in skeletal cells. Given that angiotensin II (Ang II) elicits an increase in intracellular Ca2+ and that lysosome fusion is a crucial mediator of lipid raft (LR) clustering, we hypothesized that Ang II induces lysosome fusion and activates LR formation in rat mesenteric endothelial cells (MECs). We found that Ang II acutely increased intracellular Ca2+ content, an effect that was inhibited by the extracellular Ca2+ chelator ethylene glycol tetraacetic acid (EGTA) and the inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release inhibitor 2-aminoethoxydiphenyl borate (2-APB). Further study showed that EGTA almost completely blocked Ang II-induced lysosome fusion, the translocation of acid sphingomyelinase (ASMase) to LR clusters, ASMase activation and NADPH (nicotinamide adenine dinucleotide phosphate) oxidase activation. In contrast, 2-APB had a slight inhibitory effect. Functionally, both the lysosome inhibitor bafilomycin A1 and the ASMase inhibitor amitriptyline reversed Ang II-induced impairment of vasodilation. We conclude that Ca2+ -regulated lysosome fusion mediates the Ang II-induced regulation of the LR-redox signaling pathway and mesenteric endothelial dysfunction.

  4. 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.

  5. 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

  6. Contribution of mitochondria and lysosomes to photodynamic therapy-induced death in cancer cells

    Science.gov (United States)

    Nieminen, Anna-Liisa; Azizuddin, Kashif; Zhang, Ping; Kenney, Malcolm E.; Pediaditakis, Peter; Lemasters, John J.; Oleinick, Nancy L.

    2008-02-01

    In photodynamic therapy (PDT), visible light activates a photosensitizing drug added to a tissue, resulting in singlet oxygen formation and cell death. Employing confocal microscopy, we previously found that the phthalocyanine Pc 4 localized primarily to mitochondrial membranes in various cancer cell lines, resulting in mitochondrial reactive oxygen species (ROS) production, followed by inner membrane permeabilization (mitochondrial permeability transition) with mitochondrial depolarization and swelling, which in turn led to cytochrome c release and apoptotic death. Recently, derivatives of Pc 4 with OH groups added to one of the axial ligands were synthesized. These derivatives appeared to be taken up more avidly by cells and caused more cytotoxicity than the parent compound Pc 4. Using organelle-specific fluorophores, we found that one of these derivatives, Pc 181, accumulated into lysosomes and that PDT with Pc 181 caused rapid disintegration of lysosomes. We hypothesized that chelatable iron released from lysosomes during PDT contributes to mitochondrial damage and subsequent cell death. We monitored cytosolic Fe2+ concentrations after PDT with calcein. Fe2+ binds to calcein causing quenching of calcein fluorescence. After bafilomycin, an inhibitor of the vacuolar proton-translocating ATPase, calcein fluorescence became quenched, an effect prevented by starch desferal s-DFO, an iron chelator that enters cells by endocytosis. After Pc 181-PDT, cytosolic calcein fluorescence also decreased, indicating increased chelatable Fe2+ in the cytosol, and apoptosis occurred. s-DFO decreased Pc 181-PDT-induced apoptosis as measured by a decrease of caspase-3 activation. In isolated mitochondria preparations, Fe2+ induced mitochondrial swelling, which was prevented by Ru360, an inhibitor of the mitochondrial Ca2+ uniporter. The data support a hypothesis of oxidative injury in which Pc 181-PDT disintegrates lysosomes and releases constituents that synergistically promote

  7. Protective effects of positive lysosomal modulation in Alzheimer's disease transgenic mouse models.

    Directory of Open Access Journals (Sweden)

    David Butler

    Full Text Available Alzheimer's disease (AD is an age-related neurodegenerative pathology in which defects in proteolytic clearance of amyloid β peptide (Aβ likely contribute to the progressive nature of the disorder. Lysosomal proteases of the cathepsin family exhibit up-regulation in response to accumulating proteins including Aβ(1-42. Here, the lysosomal modulator Z-Phe-Ala-diazomethylketone (PADK was used to test whether proteolytic activity can be enhanced to reduce the accumulation events in AD mouse models expressing different levels of Aβ pathology. Systemic PADK injections in APP(SwInd and APPswe/PS1ΔE9 mice caused 3- to 8-fold increases in cathepsin B protein levels and 3- to 10-fold increases in the enzyme's activity in lysosomal fractions, while neprilysin and insulin-degrading enzyme remained unchanged. Biochemical analyses indicated the modulation predominantly targeted the active mature forms of cathepsin B and markedly changed Rab proteins but not LAMP1, suggesting the involvement of enhanced trafficking. The modulated lysosomal system led to reductions in both Aβ immunostaining as well as Aβ(x-42 sandwich ELISA measures in APP(SwInd mice of 10-11 months. More extensive Aβ deposition in 20-22-month APPswe/PS1ΔE9 mice was also reduced by PADK. Selective ELISAs found that a corresponding production of the less pathogenic Aβ(1-38 occurs as Aβ(1-42 levels decrease in the mouse models, indicating that PADK treatment leads to Aβ truncation. Associated with Aβ clearance was the elimination of behavioral and synaptic protein deficits evident in the two transgenic models. These findings indicate that pharmacologically-controlled lysosomal modulation reduces Aβ(1-42 accumulation, possibly through intracellular truncation that also influences extracellular deposition, and in turn offsets the defects in synaptic composition and cognitive functions. The selective modulation promotes clearance at different levels of Aβ pathology and provides proof

  8. 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

  9. 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

  10. Synthesis of 2-mercapto-5-methoxyimidazo[4,5-b] pyridine%2-巯基-5-甲氧基咪唑并[4,5-b]吡啶的合成工艺

    Institute of Scientific and Technical Information of China (English)

    戴立言; 程国林; 王晓钟; 陈英奇

    2007-01-01

    泰妥拉唑(tenatoprazole,TU-199),化学名称是5-甲氧基-2-(4-甲氧基-3,5-二甲基吡啶-2-甲亚磺酰基)-咪唑并[4,5-b]吡啶,是由日本东京田边、日本三菱公司和日本北陆制药公司联合研制开发的一种新型胃H+/K+_ATP酶质子泵抑制剂,用于治疗消化性溃疡。结构如下

  11. Effect of sugarcane molasses extract on the formation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in a model system.

    Science.gov (United States)

    Yu, Di; Chen, Ming-Shun; Yu, Shu-Juan

    2016-04-15

    Molasses, the main by-product of sugar production, is a well-known source of antioxidants. In this study, sugarcane molasses extract was investigated for its total phenolic content and in vitro antioxidant capacity. The experimental total phenolic content was 101.3 mg of gallic acid equivalent (GAE) in 1 g of extract, IC50 of Trolox and sugarcane molasses extract were 125.33 μg/ml and 126.0 μg/ml, respectively. A chemical model system showed that the sugarcane molasses extract effectively reduced the formation of phenylacetaldehyde and the aldol condensation product, meanwhile, the amount of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) also decreased. This could be due to the reaction between the phenolic compounds of sugarcane molasses extract and the carbonyl group of phenylacetaldehyde inhibiting the aldol condensation product formation, and this would suppress the formation of PhIP. A pathway that phenolic compounds inhibited the formation of PhIP is proposed. This pathway also suggested a mechanism for how the sugarcane affects the formation of PHIP. PMID:26617035

  12. Identification of an Allosteric Binding Site on Human Lysosomal Alpha-Galactosidase Opens the Way to New Pharmacological Chaperones for Fabry Disease

    Science.gov (United States)

    den-Haan, Helena; Pérez-Sánchez, Horacio; Del Prete, Rosita; Liguori, Ludovica; Cimmaruta, Chiara; Lukas, Jan; Andreotti, Giuseppina

    2016-01-01

    Personalized therapies are required for Fabry disease due to its large phenotypic spectrum and numerous different genotypes. In principle, missense mutations that do not affect the active site could be rescued with pharmacological chaperones. At present pharmacological chaperones for Fabry disease bind the active site and couple a stabilizing effect, which is required, to an inhibitory effect, which is deleterious. By in silico docking we identified an allosteric hot-spot for ligand binding where a drug-like compound, 2,6-dithiopurine, binds preferentially. 2,6-dithiopurine stabilizes lysosomal alpha-galactosidase in vitro and rescues a mutant that is not responsive to a mono-therapy with previously described pharmacological chaperones, 1-deoxygalactonojirimycin and galactose in a cell based assay. PMID:27788225

  13. 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.

  14. 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

  15. 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.

  16. Repetitive stimulation of autophagy-lysosome machinery by intermittent fasting preconditions the myocardium to ischemia-reperfusion injury.

    Science.gov (United States)

    Godar, Rebecca J; Ma, Xiucui; Liu, Haiyan; Murphy, John T; Weinheimer, Carla J; Kovacs, Attila; Crosby, Seth D; Saftig, Paul; Diwan, Abhinav

    2015-01-01

    Autophagy, a lysosomal degradative pathway, is potently stimulated in the myocardium by fasting and is essential for maintaining cardiac function during prolonged starvation. We tested the hypothesis that intermittent fasting protects against myocardial ischemia-reperfusion injury via transcriptional stimulation of the autophagy-lysosome machinery. Adult C57BL/6 mice subjected to 24-h periods of fasting, every other day, for 6 wk were protected from in-vivo ischemia-reperfusion injury on a fed day, with marked reduction in infarct size in both sexes as compared with nonfasted controls. This protection was lost in mice heterozygous null for Lamp2 (coding for lysosomal-associated membrane protein 2), which demonstrate impaired autophagy in response to fasting with accumulation of autophagosomes and SQSTM1, an autophagy substrate, in the heart. In lamp2 null mice, intermittent fasting provoked progressive left ventricular dilation, systolic dysfunction and hypertrophy; worsening cardiomyocyte autophagosome accumulation and lack of protection to ischemia-reperfusion injury, suggesting that intact autophagy-lysosome machinery is essential for myocardial homeostasis during intermittent fasting and consequent ischemic cardioprotection. Fasting and refeeding cycles resulted in transcriptional induction followed by downregulation of autophagy-lysosome genes in the myocardium. This was coupled with fasting-induced nuclear translocation of TFEB (transcription factor EB), a master regulator of autophagy-lysosome machinery; followed by rapid decline in nuclear TFEB levels with refeeding. Endogenous TFEB was essential for attenuation of hypoxia-reoxygenation-induced cell death by repetitive starvation, in neonatal rat cardiomyocytes, in-vitro. Taken together, these data suggest that TFEB-mediated transcriptional priming of the autophagy-lysosome machinery mediates the beneficial effects of fasting-induced autophagy in myocardial ischemia-reperfusion injury. PMID:26103523

  17. Novel synthesis of the hexahydroimidazo[1,5b]isoquinoline scaffold: application to the synthesis of glucocorticoid receptor modulators.

    Science.gov (United States)

    Xiao, Hai-Yun; Wu, Dauh-Rurng; Malley, Mary F; Gougoutas, Jack Z; Habte, Sium F; Cunningham, Mark D; Somerville, John E; Dodd, John H; Barrish, Joel C; Nadler, Steven G; Dhar, T G Murali

    2010-02-11

    The first stereoselective synthesis of the hexahydroimidazo[1,5b]isoquinoline (HHII) scaffold as a surrogate for the steroidal A-B ring system is described. The structure-activity relationships of the analogs derived from this scaffold show that the basic imidazole moiety is tolerated by the glucocorticoid receptor (GR) in terms of binding affinity, although the partial agonist activity in the transrepressive assays depends on the substitution pattern on the B-ring. More importantly, most compounds in the HHII series bearing a tertiary alcohol moiety on the B-ring are either inactive or significantly less active in inducing GR-mediated transactivation, thus displaying a "dissociated" pharmacology in vitro. PMID:20047280

  18. Cell penetrable humanized-VH/V(H)H that inhibit RNA dependent RNA polymerase (NS5B) of HCV.

    Science.gov (United States)

    Thueng-in, Kanyarat; Thanongsaksrikul, Jeeraphong; Srimanote, Potjanee; Bangphoomi, Kunan; Poungpair, Ornnuthchar; Maneewatch, Santi; Choowongkomon, Kiattawee; Chaicumpa, Wanpen

    2012-01-01

    NS5B is pivotal RNA dependent RNA polymerase (RdRp) of HCV and NS5B function interfering halts the virus infective cycle. This work aimed to produce cell penetrable humanized single domain antibodies (SdAb; VH/V(H)H) that interfere with the RdRp activity. Recombinant NS5BΔ55 of genotype 3a HCV with de novo RNA synthetic activity was produced and used in phage biopanning for selecting phage clones that displayed NS5BΔ55 bound VH/V(H)H from a humanized-camel VH/V(H)H display library. VH/V(H)H from E. coli transfected with four selected phage clones inhibited RdRp activity when tested by ELISA inhibition using 3'di-cytidylate 25 nucleotide directed in vitro RNA synthesis. Deduced amino acid sequences of two clones showed V(H)H hallmark and were designated V(H)H6 and V(H)H24; other clones were conventional VH, designated VH9 and VH13. All VH/V(H)H were linked molecularly to a cell penetrating peptide, penetratin. The cell penetrable VH9, VH13, V(H)H6 and V(H)H24 added to culture of Huh7 cells transfected with JHF-1 RNA of genotype 2a HCV reduced the amounts of RNA intracellularly and in culture medium implying that they inhibited the virus replication. VH/V(H)H mimotopes matched with residues scattered on the polymerase fingers, palm and thumb which were likely juxtaposed to form conformational epitopes. Molecular docking revealed that the antibodies covered the RdRp catalytic groove. The transbodies await further studies for in vivo role in inhibiting HCV replication.

  19. 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

  20. 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

  1. 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

  2. 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.

  3. 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.

  4. 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...

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1978-07-01

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

  6. 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

    signaling pathways. Failure to attenuate signaling by receptor down-regulation could be one of the major mechanisms by which EGFRvIII becomes oncogenic. Using a cell system expressing either EGFR or EGFRvIII with no expression of other EGFR family members and with endogenous levels of key degradation....... 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...

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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

  12. Discovery of Novel Thiophene-Based, Thumb Pocket 2 Allosteric Inhibitors of the Hepatitis C NS5B Polymerase with Improved Potency and Physicochemical Profiles.

    Science.gov (United States)

    Court, John J; Poisson, Carl; Ardzinski, Andrzej; Bilimoria, Darius; Chan, Laval; Chandupatla, Kishan; Chauret, Nathalie; Collier, Philip N; Das, Sanjoy Kumar; Denis, Francois; Dorsch, Warren; Iyer, Ganesh; Lauffer, David; L'Heureux, Lucille; Li, Pan; Luisi, Brian S; Mani, Nagraj; Nanthakumar, Suganthi; Nicolas, Olivier; Rao, B Govinda; Ronkin, Steven; Selliah, Subajini; Shawgo, Rebecca S; Tang, Qing; Waal, Nathan D; Yannopoulos, Constantin G; Green, Jeremy

    2016-07-14

    The hepatitis C viral proteins NS3/4A protease, NS5B polymerase, and NS5A are clinically validated targets for direct-acting antiviral therapies. The NS5B polymerase may be inhibited directly through the action of nucleosides or nucleotide analogues or allosterically at a number of well-defined sites. Herein we describe the further development of a series of thiophene carboxylate allosteric inhibitors of NS5B polymerase that act at the thumb pocket 2 site. Lomibuvir (1) is an allosteric HCV NS5B inhibitor that has demonstrated excellent antiviral activity and potential clinical utility in combination with other direct acting antiviral agents. Efforts to further explore and develop this series led to compound 23, a compound with comparable potency and improved physicochemical properties.

  13. Natural transformation of chlordecone into 5b-hydrochlordecone in French West Indies soils: statistical evidence for investigating long-term persistence of organic pollutants.

    Science.gov (United States)

    Devault, Damien A; Laplanche, Christophe; Pascaline, Hélène; Bristeau, Sébastien; Mouvet, Christophe; Macarie, Hervé

    2016-01-01

    Chlordecone (CLD) was an organochlorine insecticide whose previous use resulted in an extensive pollution of the environment with severe health effects and social consequences. A closely related compound, 5b-hydrochlordecone (5b-hydroCLD), has been searched for and often detected in environmental matrices from the geographical area where CLD was applied. The current consensus considered that its presence was not the result of a biotic or abiotic dechlorination of CLD in these matrices but rather the consequence of its presence as impurity (synthesis by-product) in the CLD released into the environment. The aim of the present study was to determine if and to what extent degradation of CLD into 5b-hydroCLD occurred in the field. To test this hypothesis, the ratios of 5b-hydroCLD and CLD concentrations in a dataset of 810 soils collected between 2006 and 2012 in Martinique were compared to the ratios measured in 3 samples of the CLD dust commercial formulations applied in the banana fields of French West Indies (FWI) and 1 sample of the technical-grade CLD corresponding to the active ingredient used in such formulations. Soil data were processed with a hierarchical Bayesian model to account for random measurement errors and data censoring. Any pathway of CLD transformation into 5b-hydroCLD occurring over the long term in FWI soils would indeed change the ratio of 5b-hydroCLD/CLD compared to what it was in the initially applied formulations. Results showed a significant increase of the 5b-hydroCLD/CLD ratio in the soils-25 times greater in soil than in commercial formulations-which suggested that natural CLD transformation into 5b-hydroCLD over the long term occurred in these soils. Results from this study may impact future decisions for the remediation of the polluted areas. PMID:26122571

  14. 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

  15. Microglial migration mediated by ATP-induced ATP release from lysosomes

    Institute of Scientific and Technical Information of China (English)

    Ying Dou; Qing-ming Luo; Shumin Duan; Hang-jun Wu; Hui-quan Li; Song Qin; Yin-er Wang; Jing Li; Hui-fang Lou; Zhong Chen; Xiao-ming Li

    2012-01-01

    Microglia are highly motile cells that act as the main form of active immune defense in the central nervous system.Attracted by factors released from damaged cells,microglia are recruited towards the damaged or infected site,where they are involved in degenerative and regenerative responses and phagocytotic clearance of cell debris.ATP release from damaged neural tissues has been suggested to mediate the rapid extension of microglial process towards the site of injury.However,the mechanisms of the long-range migration of microglia remain to be clarified.Here,we found that lysosomes in microglia contain abundant ATP and exhibit Ca2+-dependent exocytosis in response to various stimuli.By establishing an efficient in vitro chemotaxis assay,we demonstrated that endogenously-released ATP from microglia triggered by local microinjection of ATPγS is critical for the long-range chemotaxis of microglia,a response that was significantly inhibited in microglia treated with an agent inducing iysosome osmodialysis or in cells derived from mice deficient in Rab 27a (ashen mice),a small GTPase required for the trafficking and exocytosis of secretory iysosomes.These results suggest that microglia respond to extracellular ATP by releasing ATP themselves through lysosomal exocytosis,thereby providing a positive feedback mechanism to generate a long-range extracellular signal for attracting distant microglia to migrate towards and accumulate at the site of injury.

  16. 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

  17. 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.

  18. 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

  19. 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

  20. [High cost drugs for rare diseases in Brazil: the case of lysosomal storage disorders].

    Science.gov (United States)

    de Souza, Mônica Vinhas; Krug, Bárbara Corrêa; Picon, Paulo Dornelles; Schwartz, Ida Vanessa Doederlein

    2010-11-01

    This paper approaches in a critical way aspects of Brazilian public policies for drugs, emphasizing those classified as high cost and for rare diseases. The lysosomal storage diseases was taken as an example because of their rarity and the international trend for the development of new drugs for their treatment, all at high costs. Three lysosomal storage diseases were approached: Gaucher disease, Fabry disease and mucopolysaccharidosis type I. Gaucher disease has its treatment drug licensed in Brazil and guidelines for its use are established through a clinical protocol by the Ministry of Health. The others have their drug treatments registered in Brazil; however, no treatment guidelines for them have been developed by the government. The objective of the paper was to foster the discussion on the role of health technology assessment for high-cost drugs for rare diseases in Brazil, emphasizing the need for establishing health policies with legitimacy towards these diseases. Despite the difficulties in establishing a health policy for each rare disease, it is possible to create rational models to deal with this growing challenge.

  1. 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.

  2. 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.

  3. LINGO-1 promotes lysosomal degradation of amyloid-β protein precursor

    Directory of Open Access Journals (Sweden)

    Rian de Laat

    2015-03-01

    Full Text Available Sequential proteolytic cleavages of amyloid-β protein precursor (AβPP by β-secretase and γ-secretase generate amyloid β (Aβ peptides, which are thought to contribute to Alzheimer's disease (AD. Much of this processing occurs in endosomes following endocytosis of AβPP from the plasma membrane. However, this pathogenic mode of processing AβPP may occur in competition with lysosomal degradation of AβPP, a common fate of membrane proteins trafficking through the endosomal system. Following up on published reports that LINGO-1 binds and promotes the amyloidogenic processing of AβPP we have examined the consequences of LINGO-1/AβPP interactions. We report that LINGO-1 and its paralogs, LINGO-2 and LINGO-3, decrease processing of AβPP in the amyloidogenic pathway by promoting lysosomal degradation of AβPP. We also report that LINGO-1 levels are reduced in AD brain, representing a possible pathogenic mechanism stimulating the generation of Aβ peptides in AD.

  4. Actin cytoskeleton-dependent Rab GTPase-regulated angiotensin type I receptor lysosomal degradation studied by fluorescence lifetime imaging microscopy

    Science.gov (United States)

    Li, Hewang; Yu, Peiying; Sun, Yuansheng; Felder, Robin A.; Periasamy, Ammasi; Jose, Pedro A.

    2010-09-01

    The dynamic regulation of the cellular trafficking of human angiotensin (Ang) type 1 receptor (AT1R) is not well understood. Therefore, we investigated the cellular trafficking of AT1R-enhanced green fluorescent protein (EGFP) (AT1R-EGFP) heterologously expressed in HEK293 cells by determining the change in donor lifetime (AT1R-EGFP) in the presence or absence of acceptor(s) using fluorescence lifetime imaging-fluorescence resonance energy transfer (FRET) microscopy. The average lifetime of AT1R-EGFP in our donor-alone samples was ~2.33 ns. The basal state lifetime was shortened slightly in the presence of Rab5 (2.01+/-0.10 ns) or Rab7 (2.11+/-0.11 ns) labeled with Alexa 555, as the acceptor fluorophore. A 5-min Ang II treatment markedly shortened the lifetime of AT1R-EGFP in the presence of Rab5-Alexa 555 (1.78+/-0.31 ns) but was affected minimally in the presence of Rab7-Alexa 555 (2.09+/-0.37 ns). A 30-min Ang II treatment further decreased the AT1R-EGFP lifetime in the presence of both Rab5- and Rab7-Alexa 555. Latrunculin A but not nocodazole pretreatment blocked the ability of Ang II to shorten the AT1R-EGFP lifetime. The occurrence of FRET between AT1R-EGFP (donor) and LAMP1-Alexa 555 (acceptor) with Ang II stimulation was impaired by photobleaching the acceptor. These studies demonstrate that Ang II-induced AT1R lysosomal degradation through its association with LAMP1 is regulated by Rab5/7 via mechanisms that are dependent on intact actin cytoskeletons.

  5. 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

  6. Label Free Inhibitor Screening of Hepatitis C Virus (HCV) NS5B Viral Protein Using RNA Oligonucleotide

    Science.gov (United States)

    Roh, Changhyun; Kim, Sang Eun; Jo, Sung-Kee

    2011-01-01

    Globally, over 170 million people (ca. 3% of the World’s population) are infected with the hepatitis C virus (HCV), which can cause serious liver diseases such as chronic hepatitis, evolving into subsequent health problems. Driven by the need to detect the presence of HCV, as an essential factor in diagnostic medicine, the monitoring of viral protein has been of great interest in developing simple and reliable HCV detection methods. Despite considerable advances in viral protein detection as an HCV disease marker, the current enzyme linked immunosorbent assay (ELISA) based detection methods using antibody treatment have several drawbacks. To overcome this bottleneck, an RNA aptamer become to be emerged as an antibody substitute in the application of biosensor for detection of viral protein. In this study, we demonstrated a streptavidin-biotin conjugation method, namely, the RNA aptamer sensor system that can quantify viral protein with detection level of 700 pg mL−1 using a biotinylated RNA oligonucleotide on an Octet optical biosensor. Also, we showed this method can be used to screen inhibitors of viral protein rapidly and simply on a biotinylated RNA oligonucleotide biosensor. Among the inhibitors screened, (−)-Epigallocatechin gallate showed high binding inhibition effect on HCV NS5B viral protein. The proposed method can be considered a real-time monitoring method for inhibitor screening of HCV viral protein and is expected to be applicable to other types of diseases. PMID:22163979

  7. Biomonitoring the cooked meat carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in canine fur.

    Science.gov (United States)

    Gu, Dan; Neuman, Zachary L; Modiano, Jaime F; Turesky, Robert J

    2012-09-12

    2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a heterocyclic aromatic amine (HAA) that is formed during the cooking of meat, poultry, and fish. PhIP is a rodent carcinogen and is thought to contribute to several diet-related cancers in humans. PhIP is present in the hair of human omnivores but not in the hair of vegetarians. We have now identified PhIP in the fur of 14 out of 16 healthy dogs consuming different brands of commercial pet food. The levels of PhIP in canine fur varied by over 85-fold and were comparable to the levels of PhIP present in human hair. However, high density fur containing PhIP covers a very high proportion of the body surface area of dogs, whereas high density terminal hair primarily covers the scalp and pubis body surface area of humans. These findings signify that the exposure and bioavailability of PhIP are high in canines. A potential role for PhIP in the etiology of canine cancer should be considered. PMID:22906298

  8. ANGRA-1 neutron kinetics model at BOL using WIMSD-5B and PARCS V2.7 codes

    Energy Technology Data Exchange (ETDEWEB)

    Hamers, Adolfo R.; Reis, Patricia A.L.; Rodrigues, Thiago D.A.; Pereira, Claubia; Costa, Antonella L., E-mail: adolforomerohamers@hotmail.com, E-mail: patricialire@yahoo.com.br, E-mail: claubia@nuclear.ufmg.br, E-mail: antonella@nuclear.ufmg.br [Universidade Federal de Minas Gerais (UFMG), Belo Horizonte (Brazil). Departamento de Engenheria Nuclear; Miro, R.; Verdu, G., E-mail: rmiro@iqn.upv.es, E-mail: gverdu@iqn.upv.es [Universitat Politecnica de Valencia (Spain)

    2015-07-01

    A steady-state neutron kinetics model of the Angra-1 NPP at BOL (Beginning Of Life) has been developed with the PARCS V2.7 neutron diffusion code. The information of the burnable poison rods, fuel enrichments and control rod banks distributions within the core have been taken from the Angra-1 FSAR (Final Safety Analysis Report) and implemented in the model. The macroscopic cross sections for the fast and thermal neutron groups have been calculated with the WIMSD-5B lattice cell code. The cross sections were obtained for the rodded and unrodded cases for each composition in the core. In order to establish the initial steady-state, an eigenvalue was made with the PARCS V2.7 code for three steady-state scenario cases reported at the FSAR; a K{sub eff} of 1.0733 was obtained for the unrodded case, K{sub eff} of 1.0718 for a 24% of bank D inserted case and K{sub eff} of 0.8512 for the full rodded case. The normalized core power density distributions were obtained and compared with the corresponding FSAR case. (author)

  9. Magnetoelastic effects in Nd4Fe77.5B18.5 nano-composite magnet

    Directory of Open Access Journals (Sweden)

    M. R. Alinejad

    2005-03-01

    Full Text Available   In this research, magnetostriction and thermal expansion of polymer-bonded Nd4Fe77.5B18.5 nano-composites are studied using strain gage method. The effect of sintering on samples is also studied. X-ray diffraction patterns and SEM pictures of the original and annealed samples show that the grain size of a -Fe and Fe3B soft magnetic phases in amorphous matrix increases and mechanical hardness of the samples improves after annealing at 700 ° C for 20 min.. Overall behavior of the thermal expansion is similar for samples in both cases, with about 20% larger coefficient for the annealed one. The magnetostriction measurements show huge magnitudes in order of 10-4 at the presence of relatively weak external magnetic fields. The magnitude of magnetostriction noticeably decreases after annealing which can originate from two different sources. Magnetostriction of the original sample mainly originates from single particle interactions of Nd-sublattice in Nd2Fe14B phase, but the situation is very different for the annealed sample. The complicate variations of the magnetostrictive isotherms of the annealed sample are discussed based on the following three factors,internal stresses, magnetocrystalline anisotropy and mechanisms of coercivity.

  10. Microwave assisted synthesis of some novel Flurbiprofen hydrazide- hydrazones as anti-HCV NS5B and anticancer agents

    Directory of Open Access Journals (Sweden)

    Sevil Aydın

    2013-01-01

    Full Text Available The synthesis of a new series of flurbiprofen hydrazide-hydrazones using microwave assisted reactions is described. Substituted aldehydes were condensed with flurbiprofen hydrazide by microwave irradiation to corresponding hydrazones. Synthesis of N’-[(4-bromothiophen-2-ylmethylidene]-2-(2-fluorobiphenyl-4-yl propanehydrazide (3o employing microwave assisted process resulted in higher yields, in faster time and with less chemical waste compared to traditional techniques. (2-fluorobiphenyl-4-yl-N’-(phenylmethylidenepropanehydrazide (3p andN’-[(2-chloro-6-fluorophenyl methylidene]-2-(2-fluorobiphenyl-4-ylpropanehydrazide (3s inhibited the growth of a leukemia cancercell line HL-60 (TB by 66.37% and an ovarian cancer cell line OVCAR-4 by 77.34% (singledose, 10μM, respectively at the National Cancer Institute (NCI, but had no significant ef-fect on a panel of sixty human tumor cell lines. Flurbiprofen hydrazide-hydrazones were weak inhibitors of hepatitis C virus NS5B polymerase activity with N’-[(5-ethylfuran-2-ylmethylidene]-2-(2-fluorobiphenyl-4-ylpropanehydrazide (3m being the most active of this series. Binding mode investigations of compound 3m suggested that allosteric pocket (AP-B may be the potential binding site for flurbiprofen hydrazones and these results will alsoassist in further derivatization of 3m using the green chemistry approach and improve the potency of S-flurbiprofen hydrazide hydrazones

  11. Multivariate regression modelling of antifungal activity of some benzoxazole and oxazolo[4,5-b]pyridine derivatives.

    Science.gov (United States)

    Kovačević, Strahinja Z; Podunavac Kuzmanović, Sanja O; Jevrić, Lidija R

    2013-01-01

    In the present study, principal component analysis (PCA) followed by principal component regression (PCR) and partial least squares (PLS) method was applied in order to identify the most important in silico molecular descriptors and quantify their influence on antifungal activity (expressed as minimal inhibitory concentration) of selected benzoxazole and oxazolo[4,5-b]pyridine derivatives against Candida albicans. PLS regression showed the best statistical performance, according to the lowest value of the standard error (root mean square errors of calibration of 0.02526 and cross-validation of 0.04533), while PCR model was characterized by root mean square errors of calibration of 0.03176 and cross-validation of 0.05661. The most important descriptors in both PLS and PCR model are solubility in water, expressed as AClogS and ABlogS, and lipophilicity, expressed as XlogP2 and ABlogP. Very good predictive ability of the established models, confirmed by corresponding statistical parameters, allows us to estimate antifungal activity of structurally similar compounds.

  12. Label Free Inhibitor Screening of Hepatitis C Virus (HCV NS5B Viral Protein Using RNA Oligonucleotide

    Directory of Open Access Journals (Sweden)

    Sang Eun Kim

    2011-06-01

    Full Text Available Globally, over 170 million people (ca. 3% of the World’s population are infected with the hepatitis C virus (HCV, which can cause serious liver diseases such as chronic hepatitis, evolving into subsequent health problems. Driven by the need to detect the presence of HCV, as an essential factor in diagnostic medicine, the monitoring of viral protein has been of great interest in developing simple and reliable HCV detection methods. Despite considerable advances in viral protein detection as an HCV disease marker, the current enzyme linked immunosorbent assay (ELISA based detection methods using antibody treatment have several drawbacks. To overcome this bottleneck, an RNA aptamer become to be emerged as an antibody substitute in the application of biosensor for detection of viral protein. In this study, we demonstrated a streptavidin-biotin conjugation method, namely, the RNA aptamer sensor system that can quantify viral protein with detection level of 700 pg mL−1 using a biotinylated RNA oligonucleotide on an Octet optical biosensor. Also, we showed this method can be used to screen inhibitors of viral protein rapidly and simply on a biotinylated RNA oligonucleotide biosensor. Among the inhibitors screened, (−-Epigallocatechin gallate showed high binding inhibition effect on HCV NS5B viral protein. The proposed method can be considered a real-time monitoring method for inhibitor screening of HCV viral protein and is expected to be applicable to other types of diseases.

  13. Impact of the loss of caveolin-1 on lung mass and cholesterol metabolism in mice with and without the lysosomal cholesterol transporter, Niemann-Pick type C1.

    Science.gov (United States)

    Mundy, Dorothy I; Lopez, Adam M; Posey, Kenneth S; Chuang, Jen-Chieh; Ramirez, Charina M; Scherer, Philipp E; Turley, Stephen D

    2014-07-01

    Caveolin-1 (Cav-1) is a major structural protein in caveolae in the plasma membranes of many cell types, particularly endothelial cells and adipocytes. Loss of Cav-1 function has been implicated in multiple diseases affecting the cardiopulmonary and central nervous systems, as well as in specific aspects of sterol and lipid metabolism in the liver and intestine. Lungs contain an exceptionally high level of Cav-1. Parameters of cholesterol metabolism in the lung were measured, initially in Cav-1-deficient mice (Cav-1(-/-)), and subsequently in Cav-1(-/-) mice that also lacked the lysosomal cholesterol transporter Niemann-Pick C1 (Npc1) (Cav-1(-/-):Npc1(-/-)). In 50-day-old Cav-1(-/-) mice fed a low- or high-cholesterol chow diet, the total cholesterol concentration (mg/g) in the lungs was marginally lower than in the Cav-1(+/+) controls, but due to an expansion in their lung mass exceeding 30%, whole-lung cholesterol content (mg/organ) was moderately elevated. Lung mass (g) in the Cav-1(-/-):Npc1(-/-) mice (0.356±0.022) markedly exceeded that in their Cav-1(+/+):Npc1(+/+) controls (0.137±0.009), as well as in their Cav-1(-/-):Npc1(+/+) (0.191±0.013) and Cav-1(+/+):Npc1(-/-) (0.213±0.022) littermates. The corresponding lung total cholesterol contents (mg/organ) in mice of these genotypes were 6.74±0.17, 0.71±0.05, 0.96±0.05 and 3.12±0.43, respectively, with the extra cholesterol in the Cav-1(-/-):Npc1(-/-) and Cav-1(+/+):Npc1(-/-) mice being nearly all unesterified (UC). The exacerbation of the Npc1 lung phenotype and increase in the UC level in the Cav-1(-/-):Npc1(-/-) mice imply a regulatory role of Cav-1 in pulmonary cholesterol metabolism when lysosomal sterol transport is disrupted.

  14. Expression of HIV-1 Vpu leads to loss of the viral restriction factor CD317/Tetherin from lipid rafts and its enhanced lysosomal degradation.

    Directory of Open Access Journals (Sweden)

    Ruth Rollason

    Full Text Available CD317/tetherin (aka BST2 or HM1.24 antigen is an interferon inducible membrane protein present in regions of the lipid bilayer enriched in sphingolipids and cholesterol (often termed lipid rafts. It has been implicated in an eclectic mix of cellular processes including, most notably, the retention of fully formed viral particles at the surface of cells infected with HIV and other enveloped viruses. Expression of the HIV viral accessory protein Vpu has been shown to lead to intracellular sequestration and degradation of tetherin, thereby counteracting the inhibition of viral release. There is evidence that tetherin interacts directly with Vpu, but it remains unclear where in the cell this interaction occurs or if Vpu expression affects the lipid raft localisation of tetherin. We have addressed these points using biochemical and cell imaging approaches focused on endogenous rather than ectopically over-expressed tetherin. We find i no evidence for an interaction between Vpu and endogenous tetherin at the cell surface, ii the vast majority of endogenous tetherin that is at the cell surface in control cells is in lipid rafts, iii internalised tetherin is present in non-raft fractions, iv expression of Vpu in cells expressing endogenous tetherin leads to the loss of tetherin from lipid rafts, v internalised tetherin enters early endosomes, and late endosomes, in both control cells and cells expressing Vpu, but the proportion of tetherin molecules destined for degradation rather than recycling is increased in cells expressing Vpu vi lysosomes are the primary site for degradation of endogenous tetherin in cells expressing Vpu. Our studies underlie the importance of studying endogenous tetherin and let us propose a model in which Vpu intercepts newly internalised tetherin and diverts it for lysosomal destruction rather than recycling to the cell surface.

  15. Differential Roles of Arabidopsis Dynamin-Related Proteins DRP3A,DRP3B,and DRP5B in Organelle Division

    Institute of Scientific and Technical Information of China (English)

    Kyaw Aung; Jianping Hu

    2012-01-01

    Dynamin-related proteins (DRPs) are key components of the organelle division machineries,functioning as molecular scissors during the fission process.In Arabidopsis,DRP3A and DRP3B are shared by peroxisomal and mitochondrial division,whereas the structurally-distinct DRP5B (ARC5) protein is involved in the division of chloroplasts and peroxisomes.Here,we further investigated the roles of DRP3A,DRP3B,and DRP5B in organelle division and plant development.Despite DRP5B's lack of stable association with mitochondria,drp5B mutants show defects in mitochondrial division.The drp3A-2 drp3B-2 drp5B-2 triple mutant exhibits enhanced mitochondrial division phenotypes over drp3A-2 drp3B-2,but its peroxisomal morphology and plant growth phenotypes resemble those of the double mutant.We further demonstrated that DRP3A and DRP3B form a supercomplex in vivo,in which DRP3A is the major component,yet DRP5B is not a constituent of this complex.We thus conclude that DRP5B participates in the division of three types of organelles in Arabidopsis,acting independently of the DRP3 complex.Our findings will help elucidate the precise composition of the DRP3 complex at organelle division sites,and will be instrumental to studies aimed at understanding how the same protein mediates the morphogenesis of distinct organelles that are linked by metabolism.

  16. CD1d(hi)CD5+ B cells expanded by GM-CSF in vivo suppress experimental autoimmune myasthenia gravis.

    Science.gov (United States)

    Sheng, Jian Rong; Quan, Songhua; Soliven, Betty

    2014-09-15

    IL-10-competent subset within CD1d(hi)CD5(+) B cells, also known as B10 cells, has been shown to regulate autoimmune diseases. Whether B10 cells can prevent or suppress the development of experimental autoimmune myasthenia gravis (EAMG) has not been studied. In this study, we investigated whether low-dose GM-CSF, which suppresses EAMG, can expand B10 cells in vivo, and whether adoptive transfer of CD1d(hi)CD5(+) B cells would prevent or suppress EAMG. We found that treatment of EAMG mice with low-dose GM-CSF increased the proportion of CD1d(hi)CD5(+) B cells and B10 cells. In vitro coculture studies revealed that CD1d(hi)CD5(+) B cells altered T cell cytokine profile but did not directly inhibit T cell proliferation. In contrast, CD1d(hi)CD5(+) B cells inhibited B cell proliferation and its autoantibody production in an IL-10-dependent manner. Adoptive transfer of CD1d(hi)CD5(+) B cells to mice could prevent disease, as well as suppress EAMG after disease onset. This was associated with downregulation of mature dendritic cell markers and expansion of regulatory T cells resulting in the suppression of acetylcholine receptor-specific T cell and B cell responses. Thus, our data have provided significant insight into the mechanisms underlying the tolerogenic effects of B10 cells in EAMG. These observations suggest that in vivo or in vitro expansion of CD1d(hi)CD5(+) B cells or B10 cells may represent an effective strategy in the treatment of human myasthenia gravis.

  17. Resistance Analyses of HCV NS3/4A Protease and NS5B Polymerase from Clinical Studies of Deleobuvir and Faldaprevir

    Science.gov (United States)

    Berger, Kristi L.; Sarrazin, Christoph; Nelson, David R.; Scherer, Joseph; Sha, Nanshi; Marquis, Martin; Côté-Martin, Alexandra; Vinisko, Richard; Stern, Jerry O.; Mensa, Federico J.; Kukolj, George

    2016-01-01

    Background & Aim The resistance profile of anti-hepatitis C virus (HCV) agents used in combination is important to guide optimal treatment regimens. We evaluated baseline and treatment-emergent NS3/4A and NS5B amino-acid variants among HCV genotype (GT)-1a and -1b-infected patients treated with faldaprevir (HCV protease inhibitor), deleobuvir (HCV polymerase non-nucleoside inhibitor), and ribavirin in multiple clinical studies. Methods HCV NS3/4A and NS5B population sequencing (Sanger method) was performed on all baseline plasma samples (n = 1425 NS3; n = 1556 NS5B) and on post-baseline plasma samples from patients with virologic failure (n = 113 GT-1a; n = 221 GT-1b). Persistence and time to loss of resistance-associated variants (RAVs) was estimated using Kaplan–Meier analysis. Results Faldaprevir RAVs (NS3 R155 and D168) and deleobuvir RAVs (NS5B 495 and 496) were rare (90%). Virologic relapse was associated with RAVs in both NS3 and NS5B (53% GT-1b; 52% GT-1b); some virologic relapses had NS3 RAVs only (47% GT-1a; 17% GT-1b). Median time to loss of GT-1b NS5B P495 RAVs post-treatment (5 months) was less than that of GT-1b NS3 D168 (8.5 months) and GT-1a R155 RAVs (11.5 months). Conclusion Faldaprevir and deleobuvir RAVs are more prevalent among virologic failures than at baseline. Treatment response was not compromised by common NS3 polymorphisms; however, alanine at NS5B amino acid 499 at baseline (wild-type in GT-1a, polymorphism in GT-1b) may reduce response to this deleobuvir-based regimen. PMID:27494410

  18. N-acetyltransferase-dependent activation of 2-hydroxyamino-1-methyl-6-phenylimidazo[4,5-b]pyridine: formation of 2-amino-1-methyl-6-(5-hydroxy)phenylimidazo [4,5-b]pyridine, a possible biomarker for the reactive dose of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine

    DEFF Research Database (Denmark)

    Frandsen, Henrik Lauritz; Alexander, J.

    2000-01-01

    2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is a mutagenic and carcinogenic heterocyclic amine formed during ordinary cooking. PhIP is metabolically activated to the ultimate mutagenic metabolite by CYP P450-mediated N-hydroxylation followed by phase II esterification, Incubation of N......-hydroxy-PhIP (N-OH-PhIP) with cytosol, acetyl coenzyme A (AcCoA) and 2'-deoxyguanosine for 24 h resulted in the formation of three different adducts: N-2-(deoxyguanosin-8-yl)-PhIP, N-2-(guanosin-8-yl) -PhIP and PhIP-xanthine. One additional product, 5-hydroxy-PhIP (5-OH-PhIP), was also identified......M) to the incubation mixture resulted in a 50% reduction in both adducts and 5-hydroxy-PhIP formation in liver cytosol, The main product detected was PhIP, suggesting glutathione-dependent reduction of the N-acetoxy-PhIP, Addition of glutathione to incubation mixtures from the other cytosolic preparations had less...

  19. Pharmacokinetic, Pharmacodynamic, and Drug-Interaction Profile of the Hepatitis C Virus NS5B Polymerase Inhibitor Sofosbuvir.

    Science.gov (United States)

    Kirby, Brian J; Symonds, William T; Kearney, Brian P; Mathias, Anita A

    2015-07-01

    Sofosbuvir (SOVALDI(®)), a potent, once-daily, orally administered nucleotide analog prodrug inhibitor of the hepatitis C virus (HCV) NS5B polymerase is approved in the USA, EU, Canada, and other regions for the treatment of HCV infection as a component of an antiviral treatment regimen. Sofosbuvir undergoes intracellular activation to form GS-461203 (active triphosphate, not detected in plasma), and ultimately the inactive, renally eliminated metabolite GS-331007. GS-331007 was identified as the primary analyte of interest for clinical pharmacology studies as it accounted for >90 % of systemic drug-related material exposure, and provided comparable exposure-response relationships for viral kinetics as observed for sofosbuvir. GS-331007 and sofosbuvir exhibit linear pharmacokinetics with minimal accumulation upon multiple dosing. Compared to healthy subjects, HCV-infected patients had modestly lower (39 %) GS-331007 area under the plasma concentration-time curve (AUC) and higher sofosbuvir AUC (60 %). Sofosbuvir can be administered without dose modification in HCV-infected patients with any degree of hepatic impairment or mild to moderate renal impairment. Sofosbuvir has a low propensity for clinically significant drug interactions with common concomitant medications used by HCV-infected patients. Clinically significant alterations in GS-331007 or sofosbuvir exposures are limited to potent inducers of intestinal P-glycoprotein that may lower exposure. In HCV-infected patients, demographic variables do not significantly influence GS-331007 and sofosbuvir exposures and no consistent exposure-response relationships were observed for efficacy or safety. This review focuses on the clinical pharmacokinetics, pharmacodynamics, and pharmacokinetic-pharmacodynamic relationships of sofosbuvir, and summarizes a number of drug interaction studies with important concomitant medications commonly used by HCV-infected patients.

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

    Science.gov (United States)

    Grasso, E J; Calderón, R O

    2013-02-01

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

  1. Comparison of the effects of eldecalcitol with either raloxifene or bisphosphonate on serum tartrate resistant acid phosphatase-5b, a bone resorption marker, in postmenopausal osteoporosis

    Science.gov (United States)

    Takada, Junichi; Ikeda, Satoshi; Kusanagi, Tetsuya; Mizuno, Satoshi; Wada, Hiroshi; Iba, Kousuke; Yoshizaki, Takashi; Yamashita, Toshihiko

    2016-01-01

    Summary Objective This study analyzes whether concomitant raloxifene (RLX) or bisphosphonates (BP) plus eldecalcitol (ELD) has excessive suppressive effects on a bone resorption marker during the first 6 months of treatment in postmenopausal women in real-world setting. Methods 285 postmenopausal osteoporotic patients who had been treated with RLX or BP plus ELD were evaluated the bone resorption marker, serum tartrate resistant acid phosphatase-5b (TRACP-5b), during the first 6 months of treatment. Results In drug-naïve group (not received osteoporosis medications before the administration, n=70), the concomitant RLX or BP with ELD significantly decreased levels of TRACP-5b without severe suppression. In vitamin D switch group [RLX or BP plus alfacalcidol (ALF) and then switched to RLX or BP plus ELD, n=215], the replacing ALF with ELD further and significantly decreased TRACP-5b and tertile analyses based on baseline values were significantly decreased far more in the highest, compared with the lowest tertile in the ELD+RLX and ELD+BP groups. Conclusion ELD combined with RLX or BP administered for 6 months to postmenopausal women with osteoporosis who were drug-naïve or who had switched medications significantly reduced and maintained TRACP-5b values within the reference range. PMID:27252739

  2. 2,2',4,4'-Tetrabromodiphenyl ether injures cell viability and mitochondrial function of mouse spermatocytes by decreasing mitochondrial proteins Atp5b and Uqcrc1.

    Science.gov (United States)

    Huang, Shaoping; Wang, Jing; Cui, Yiqiang

    2016-09-01

    Our object was to explore direct effects and mechanism of BDE47 on GC2 (immortalized mouse spermatocyte). GC2 were exposed to DMSO, 0.1, 1, 10, 100μM BDE47 for 48h. Cell viability was detected by trypan-blue exclusion; ultrastructure by electron-microscopy; cell cycle, mitochondrial membrane motential (MMP), reactive oxygen species (ROS) by flow-cytometry; ATP production by luminometer; Atp5b, Uqcrc1, Bcl-2 level by WB. To explore whether the decreased mitochondrial proteins play an important role in apoptosis, MMP and apoptosis were detected after Atp5b or Uqcrc1 knockdown in GC2. Results showed BDE47 reduced cell viability, caused condensation of nuclear and vacuolated mitochondria, decreased MMP and ATP, induced ROS, cell cycle arrest at S and G2/M phase, reduced Atp5b, Uqcrc1, Bcl-2 in GC2. Knockdown of Atp5b or Uqcrc1 decreased MMP, induced apoptosis in GC2. Results suggested that BDE47 reduced cell viability, injured mitochondria in spermatocytes probably by decreasing mitochondrial protein Atp5b and Uqcrc1. PMID:27525561

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

    Science.gov (United States)

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

    2014-07-01

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

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

    Institute of Scientific and Technical Information of China (English)

    Tian-Fang Jiang; Sheng-Di Chen

    2012-01-01

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

  5. [Lysosomal proteinasen and peptidasen in serum of children with inflammatory diseases (author's transl)].

    Science.gov (United States)

    Appel, W; Huth, E; Herrmann, H

    1976-08-01

    In the serum of 43 children the activities of proteinases and peptidases by mean of 41 substrates have been determined in order to get knowledge of overall activities and differentiation of lysosomal proteolytic enzymes. Proteinases, cathepsins A, B, C and D, aminopeptidases, carboxypeptidases, dipeptidases, tripeptidases and aminoacidarylamidases have been checked. The enzyme pattern of the serum of a collective of 15 healthy children or those without serious clinical signs is demonstrated, also the alterations and differentiations in the serum of children with leucemia, pneumonia, inflammatory diseases of the respiratory tract, other inflammatory diseases and common diseases. Leucyl-glycyl-glycyltripeptidase, glycyl-glycyl-glycyltripeptidase, a proteosterase, carboxypeptidase A, a neutrale proteinase and basic proteinase (cathepsin B) and cathepsin C are increased. A distinct elevation has been found only in children with leucemia and pneumonia.

  6. The Underexploited Role of Non-Coding RNAs in Lysosomal Storage Diseases

    Science.gov (United States)

    de Queiroz, Matheus Trovão; Pereira, Vanessa Gonçalves; do Nascimento, Cinthia Castro; D’Almeida, Vânia

    2016-01-01

    Non-coding RNAs (ncRNAs) are a functional class of RNA involved in the regulation of several cellular processes which may modulate disease onset, progression, and prognosis. Lysosomal storage diseases (LSD) are a group of rare disorders caused by mutations of genes encoding specific hydrolases or non-enzymatic proteins, characterized by a wide spectrum of manifestations. The alteration of ncRNA levels is well established in several human diseases such as cancer and auto-immune disorders; however, there is a lack of information focused on the role of ncRNA in rare diseases. Recent reports related to changes in ncRNA expression and its consequences on LSD physiopathology show us the importance to keep advancing in this field. This article will summarize recent findings and provide key points for further studies on LSD and ncRNA association.

  7. Concentration measurement of lysosome enzymes in blood by fluorimetric analysis method

    Science.gov (United States)

    Strinadko, Marina M.; Strinadko, Elena M.

    2002-02-01

    The diagnostics of heritable disease series and sugar diabetes, myocardial infarction, collagenosis and kidney diseases widely uses the measurement of lysosomic enzymes in blood. In the present research work the definition procedure of concentration (beta) -glucuronidase with the help of fluorimetric analysis is offered, which allows using microamounts of biological fluids and samples with low enzyme activity which is especially important in paediatric practice. Due to the sharp sensibility of fluorimetric analysis and high speed of luminescent reactions the procedure gives an opportunity to obtain the result in the minimum terms as well as the use of small amounts of reaction mixture. The incubation in large dilution leads thereby to the elimination of influence of endogenic inhibitors and activators.

  8. Structure and function of lysosomal phospholipase A2 and lecithin:cholesterol acyltransferase

    Science.gov (United States)

    Glukhova, Alisa; Hinkovska-Galcheva, Vania; Kelly, Robert; Abe, Akira; Shayman, James A.; Tesmer, John J. G.

    2015-03-01

    Lysosomal phospholipase A2 (LPLA2) and lecithin:cholesterol acyltransferase (LCAT) belong to a structurally uncharacterized family of key lipid-metabolizing enzymes responsible for lung surfactant catabolism and for reverse cholesterol transport, respectively. Whereas LPLA2 is predicted to underlie the development of drug-induced phospholipidosis, somatic mutations in LCAT cause fish eye disease and familial LCAT deficiency. Here we describe several high-resolution crystal structures of human LPLA2 and a low-resolution structure of LCAT that confirms its close structural relationship to LPLA2. Insertions in the α/β hydrolase core of LPLA2 form domains that are responsible for membrane interaction and binding the acyl chains and head groups of phospholipid substrates. The LCAT structure suggests the molecular basis underlying human disease for most of the known LCAT missense mutations, and paves the way for rational development of new therapeutics to treat LCAT deficiency, atherosclerosis and acute coronary syndrome.

  9. The two-pore channel TPCN2 mediates NAADP-dependent Ca(2+)-release from lysosomal stores.

    Science.gov (United States)

    Zong, Xiangang; Schieder, Michael; Cuny, Hartmut; Fenske, Stefanie; Gruner, Christian; Rötzer, Katrin; Griesbeck, Oliver; Harz, Hartmann; Biel, Martin; Wahl-Schott, Christian

    2009-09-01

    Second messenger-induced Ca(2+)-release from intracellular stores plays a key role in a multitude of physiological processes. In addition to 1,4,5-inositol trisphosphate (IP(3)), Ca(2+), and cyclic ADP ribose (cADPR) that trigger Ca(2+)-release from the endoplasmatic reticulum (ER), nicotinic acid adenine dinucleotide phosphate (NAADP) has been identified as a cellular metabolite that mediates Ca(2+)-release from lysosomal stores. While NAADP-induced Ca(2+)-release has been found in many tissues and cell types, the molecular identity of the channel(s) conferring this release remained elusive so far. Here, we show that TPCN2, a novel member of the two-pore cation channel family, displays the basic properties of native NAADP-dependent Ca(2+)-release channels. TPCN2 transcripts are widely expressed in the body and encode a lysosomal protein forming homomers. TPCN2 mediates intracellular Ca(2+)-release after activation with low-nanomolar concentrations of NAADP while it is desensitized by micromolar concentrations of this second messenger and is insensitive to the NAADP analog nicotinamide adenine dinucleotide phosphate (NADP). Furthermore, TPCN2-mediated Ca(2+)-release is almost completely abolished when the capacity of lysosomes for storing Ca(2+) is pharmacologically blocked. By contrast, TPCN2-specific Ca(2+)-release is unaffected by emptying ER-based Ca(2+) stores. In conclusion, these findings indicate that TPCN2 is a major component of the long-sought lysosomal NAADP-dependent Ca(2+)-release channel.

  10. Partial restoration of mutant enzyme homeostasis in three distinct lysosomal storage disease cell lines by altering calcium homeostasis.

    Directory of Open Access Journals (Sweden)

    Ting-Wei Mu

    2008-02-01

    Full Text Available A lysosomal storage disease (LSD results from deficient lysosomal enzyme activity, thus the substrate of the mutant enzyme accumulates in the lysosome, leading to pathology. In many but not all LSDs, the clinically most important mutations compromise the cellular folding of the enzyme, subjecting it to endoplasmic reticulum-associated degradation instead of proper folding and lysosomal trafficking. A small molecule that restores partial mutant enzyme folding, trafficking, and activity would be highly desirable, particularly if one molecule could ameliorate multiple distinct LSDs by virtue of its mechanism of action. Inhibition of L-type Ca2+ channels, using either diltiazem or verapamil-both US Food and Drug Administration-approved hypertension drugs-partially restores N370S and L444P glucocerebrosidase homeostasis in Gaucher patient-derived fibroblasts; the latter mutation is associated with refractory neuropathic disease. Diltiazem structure-activity studies suggest that it is its Ca2+ channel blocker activity that enhances the capacity of the endoplasmic reticulum to fold misfolding-prone proteins, likely by modest up-regulation of a subset of molecular chaperones, including BiP and Hsp40. Importantly, diltiazem and verapamil also partially restore mutant enzyme homeostasis in two other distinct LSDs involving enzymes essential for glycoprotein and heparan sulfate degradation, namely alpha-mannosidosis and type IIIA mucopolysaccharidosis, respectively. Manipulation of calcium homeostasis may represent a general strategy to restore protein homeostasis in multiple LSDs. However, further efforts are required to demonstrate clinical utility and safety.

  11. Intraneuronal aggregation of the β-CTF fragment of APP (C99) induces Aβ-independent lysosomal-autophagic pathology.

    Science.gov (United States)

    Lauritzen, Inger; Pardossi-Piquard, Raphaëlle; Bourgeois, Alexandre; Pagnotta, Sophie; Biferi, Maria-Grazia; Barkats, Martine; Lacor, Pascale; Klein, William; Bauer, Charlotte; Checler, Frederic

    2016-08-01

    Endosomal-autophagic-lysosomal (EAL) dysfunction is an early and prominent neuropathological feature of Alzheimers's disease, yet the exact molecular mechanisms contributing to this pathology remain undefined. By combined biochemical, immunohistochemical and ultrastructural approaches, we demonstrate a link between EAL pathology and the intraneuronal accumulation of the β-secretase-derived βAPP fragment (C99) in two in vivo models, 3xTgAD mice and adeno-associated viral-mediated C99-infected mice. We present a pathological loop in which the accumulation of C99 is both the effect and causality of impaired lysosomal-autophagic function. The deleterious effect of C99 was found to be linked to its aggregation within EAL-vesicle membranes leading to disrupted lysosomal proteolysis and autophagic impairment. This effect was Aβ independent and was even exacerbated when γ-secretase was pharmacologically inhibited. No effect was observed in inhibitor-treated wild-type animals suggesting that lysosomal dysfunction was indeed directly linked to C99 accumulation. In some brain areas, strong C99 expression also led to inflammatory responses and synaptic dysfunction. Taken together, this work demonstrates a toxic effect of C99 which could underlie some of the early-stage anatomical hallmarks of Alzheimer's disease pathology. Our work also proposes molecular mechanisms likely explaining some of the unfavorable side-effects associated with γ-secretase inhibitor-directed therapies. PMID:27138984

  12. PEG-lipid micelles enable cholesterol efflux in Niemann-Pick Type C1 disease-based lysosomal storage disorder.

    Science.gov (United States)

    Brown, Anna; Patel, Siddharth; Ward, Carl; Lorenz, Anna; Ortiz, Mauren; DuRoss, Allison; Wieghardt, Fabian; Esch, Amanda; Otten, Elsje G; Heiser, Laura M; Korolchuk, Viktor I; Sun, Conroy; Sarkar, Sovan; Sahay, Gaurav

    2016-01-01

    2-Hydroxy-propyl-β-cyclodextrin (HPβCD), a cholesterol scavenger, is currently undergoing Phase 2b/3 clinical trial for treatment of Niemann Pick Type C-1 (NPC1), a fatal neurodegenerative disorder that stems from abnormal cholesterol accumulation in the endo/lysosomes. Unfortunately, the extremely high doses of HPβCD required to prevent progressive neurodegeneration exacerbates ototoxicity, pulmonary toxicity and autophagy-based cellular defects. We present unexpected evidence that a poly (ethylene glycol) (PEG)-lipid conjugate enables cholesterol clearance from endo/lysosomes of Npc1 mutant (Npc1(-/-)) cells. Herein, we show that distearyl-phosphatidylethanolamine-PEG (DSPE-PEG), which forms 12-nm micelles above the critical micelle concentration, accumulates heavily inside cholesterol-rich late endosomes in Npc1(-/-) cells. This potentially results in cholesterol solubilization and leakage from lysosomes. High-throughput screening revealed that DSPE-PEG, in combination with HPβCD, acts synergistically to efflux cholesterol without significantly aggravating autophagy defects. These well-known excipients can be used as admixtures to treat NPC1 disorder. Increasing PEG chain lengths from 350 Da-30 kDa in DSPE-PEG micelles, or increasing DSPE-PEG content in an array of liposomes packaged with HPβCD, improved cholesterol egress, while Pluronic block copolymers capable of micelle formation showed slight effects at high concentrations. We postulate that PEG-lipid based nanocarriers can serve as bioactive drug delivery systems for effective treatment of lysosomal storage disorders. PMID:27572704

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

    NARCIS (Netherlands)

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

    2014-01-01

    There is still no treatment for polyglutamine disorders, but clearance of mutant proteins might represent a potential therapeutic strategy. Autophagy, the major pathway for organelle and protein turnover, has been implicated in these diseases. To determine whether the autophagy/lysosome system contr

  14. 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

  15. Concanavalin A/IFN-gamma triggers autophagy-related necrotic hepatocyte death through IRGM1-mediated lysosomal membrane disruption.

    Directory of Open Access Journals (Sweden)

    Chih-Peng Chang

    Full Text Available Interferon-gamma (IFN-γ, a potent Th1 cytokine with multiple biological functions, can induce autophagy to enhance the clearance of the invading microorganism or cause cell death. We have reported that Concanavalin A (Con A can cause autophagic cell death in hepatocytes and induce both T cell-dependent and -independent acute hepatitis in immunocompetent and immunodeficient mice, respectively. Although IFN-γ is known to enhance liver injury in Con A-induced hepatitis, its role in autophagy-related hepatocyte death is not clear. In this study we report that IFN-γ can enhance Con A-induced autophagic flux and cell death in hepatoma cell lines. A necrotic cell death with increased lysosomal membrane permeabilization (LMP is observed in Con A-treated hepatoma cells in the presence of IFN-γ. Cathepsin B and L were released from lysosomes to cause cell death. Furthermore, IFN-γ induces immunity related GTPase family M member 1(IRGM1 translocation to lysosomes and prolongs its activity in Con A-treated hepatoma cells. Knockdown of IRGM1 inhibits the IFN-γ/Con A-induced LMP change and cell death. Furthermore, IFN-γ(-/- mice are resistant to Con A-induced autophagy-associated necrotic hepatocyte death. We conclude that IFN-γ enhances Con A-induced autophagic flux and causes an IRGM1-dependent lysosome-mediated necrotic cell death in hepatocytes.

  16. Sorting of ligand-activated epidermal growth factor receptor to lysosomes requires its actin-binding domain

    NARCIS (Netherlands)

    Stoorvogel, W; Kerstens, S; Fritzsche, I; den Hartigh, JC; Oud, R; van der Heyden, MAG; Henegouwen, PMPVE

    2004-01-01

    Ligand-induced down-regulation of the epidermal growth factor receptor (EGFR) comprises activation of two sequential transport steps. The first involves endocytic uptake by clathrin-coated vesicles, the second transfer of endocytosed EGFR from endosomes to lysosomes. Here we demonstrate that the sec

  17. LRRK2 and RAB7L1 coordinately regulate axonal morphology and lysosome integrity in diverse cellular contexts.

    Science.gov (United States)

    Kuwahara, Tomoki; Inoue, Keiichi; D'Agati, Vivette D; Fujimoto, Tetta; Eguchi, Tomoya; Saha, Shamol; Wolozin, Benjamin; Iwatsubo, Takeshi; Abeliovich, Asa

    2016-01-01

    Leucine-rich repeat kinase 2 (LRRK2) has been linked to several clinical disorders including Parkinson's disease (PD), Crohn's disease, and leprosy. Furthermore in rodents, LRRK2 deficiency or inhibition leads to lysosomal pathology in kidney and lung. Here we provide evidence that LRRK2 functions together with a second PD-associated gene, RAB7L1, within an evolutionarily conserved genetic module in diverse cellular contexts. In C. elegans neurons, orthologues of LRRK2 and RAB7L1 act coordinately in an ordered genetic pathway to regulate axonal elongation. Further genetic studies implicated the AP-3 complex, which is a known regulator of axonal morphology as well as of intracellular protein trafficking to the lysosome compartment, as a physiological downstream effector of LRRK2 and RAB7L1. Additional cell-based studies implicated LRRK2 in the AP-3 complex-related intracellular trafficking of lysosomal membrane proteins. In mice, deficiency of either RAB7L1 or LRRK2 leads to prominent age-associated lysosomal defects in kidney proximal tubule cells, in the absence of frank CNS pathology. We hypothesize that defects in this evolutionarily conserved genetic pathway underlie the diverse pathologies associated with LRRK2 in humans and in animal models. PMID:27424887

  18. Singlet oxygen mediated apoptosis by anthrone involving lysosomes and mitochondria at ambient UV exposure

    Energy Technology Data Exchange (ETDEWEB)

    Mujtaba, Syed Faiz [Photobiology Division, (CSIR)-Indian Institute of Toxicology Research, Post Box No. 80, M.G. Marg, Lucknow 226001, Uttar Pradesh (India); College of Pharmacy, Faculty of Pharmaceutical Sciences, Pt. B.D.S University of Health Sciences, Rohtak, Haryana (India); Dwivedi, Ashish; Yadav, Neera [Photobiology Division, (CSIR)-Indian Institute of Toxicology Research, Post Box No. 80, M.G. Marg, Lucknow 226001, Uttar Pradesh (India); Ray, R.S., E-mail: ratanray.2011@rediffmail.com [Photobiology Division, (CSIR)-Indian Institute of Toxicology Research, Post Box No. 80, M.G. Marg, Lucknow 226001, Uttar Pradesh (India); Singh, Gajendra [College of Pharmacy, Faculty of Pharmaceutical Sciences, Pt. B.D.S University of Health Sciences, Rohtak, Haryana (India)

    2013-05-15

    Highlights: ► Photomodification of anthrone at ambient environmental intensities of UV-radiation. ► Phototoxicity of anthrone through type-II photodynamic reaction by generating {sup 1}O{sub 2}. ► Role of DNA damage and lipid peroxidation in anthrone phototoxicity. ► Apototic cell death and involvement of lysosomes and mitochondria. ► Up-regulation of p21 and bax concomitantly down regulation of bcl2 genes expression. -- Abstract: Anthrone a tricyclic aromatic hydrocarbon which is toxic environmental pollutant comes in the environment through photooxidation of anthracene. We have studied the photomodification of anthrone under environmental conditions. Anthrone generates reactive oxygen species (ROS) like {sup 1}O{sub 2} through Type-II photodynamic reaction. Significant intracellular ROS generation was measured through dichlorohydrofluorescein fluorescence intensity. The generation of {sup 1}O{sub 2} was further substantiated by using specific quencher like sodium azide. UV induced photodegradation of 2-deoxyguanosine and photoperoxidation of linoleic acid accorded the involvement of {sup 1}O{sub 2} in the manifestation of anthrone phototoxicity. Phototoxicity of anthrone was done on human keratinocytes (HaCaT) through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide and neutral red uptake assays. Anthrone induced cell cycle arrest (G2/M-phase) and DNA damage in a concentration dependent manner. We found apoptosis as a pattern of cell death which was confirmed through sub-G1 fraction, morphological changes, caspase-3 activation, acridine orange/ethidium bromide staining and phosphatidylserine translocation. Mitochondrial depolarization and lysosomal destabilization was parallel to apoptotic process. Our RT-PCR results strongly supports our view point of apoptotic cell death through up-regulation of pro-apoptotic genes p21 and Bax, and down regulation of anti-apoptotic gene Bcl{sub 2}. Therefore, much attention should be paid to concomitant

  19. Singlet oxygen mediated apoptosis by anthrone involving lysosomes and mitochondria at ambient UV exposure

    International Nuclear Information System (INIS)

    Highlights: ► Photomodification of anthrone at ambient environmental intensities of UV-radiation. ► Phototoxicity of anthrone through type-II photodynamic reaction by generating 1O2. ► Role of DNA damage and lipid peroxidation in anthrone phototoxicity. ► Apototic cell death and involvement of lysosomes and mitochondria. ► Up-regulation of p21 and bax concomitantly down regulation of bcl2 genes expression. -- Abstract: Anthrone a tricyclic aromatic hydrocarbon which is toxic environmental pollutant comes in the environment through photooxidation of anthracene. We have studied the photomodification of anthrone under environmental conditions. Anthrone generates reactive oxygen species (ROS) like 1O2 through Type-II photodynamic reaction. Significant intracellular ROS generation was measured through dichlorohydrofluorescein fluorescence intensity. The generation of 1O2 was further substantiated by using specific quencher like sodium azide. UV induced photodegradation of 2-deoxyguanosine and photoperoxidation of linoleic acid accorded the involvement of 1O2 in the manifestation of anthrone phototoxicity. Phototoxicity of anthrone was done on human keratinocytes (HaCaT) through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide and neutral red uptake assays. Anthrone induced cell cycle arrest (G2/M-phase) and DNA damage in a concentration dependent manner. We found apoptosis as a pattern of cell death which was confirmed through sub-G1 fraction, morphological changes, caspase-3 activation, acridine orange/ethidium bromide staining and phosphatidylserine translocation. Mitochondrial depolarization and lysosomal destabilization was parallel to apoptotic process. Our RT-PCR results strongly supports our view point of apoptotic cell death through up-regulation of pro-apoptotic genes p21 and Bax, and down regulation of anti-apoptotic gene Bcl2. Therefore, much attention should be paid to concomitant exposure of anthrone and UV-R for its total

  20. New Palladium-Catalyzed Domino Reaction with Intramolecular Ring Closure of an N-(2-Chloro-3-heteroaryl) arylamide: First Synthesis of Oxazolo[4,5-b] pyrazines

    DEFF Research Database (Denmark)

    Demmer, Charles S.; Hansen, Jacob C.; Kehler, Jan;

    2014-01-01

    lipophilicity and target interaction points are often desired. In this respect, the oxazolo[4,5-b]pyrazine is an attractive heterocyclic scaffold as it possesses increased water solubility as well as two additional hydrogen bonding acceptors. We here report a new Pd(II)-catalyzed domino reaction comprising...... is required for the domino reaction to proceed. The robustness of the methodology is confirmed by the synthesis of 23 2-substituted oxazolo[4,5-b]pyrazine analogues in good-to-high yields and containing both electron-withdrawing as well as electron-donating substituents on the reacting arylamide....

  1. Structure and magnetic properties of nanocomposites with polyethylene matrix including Fe73.5Cu1Nb3Si13.5B9 powder

    International Nuclear Information System (INIS)

    The paper presents research results concerning the influence of thermal and mechanical nanocrystallization on the magnetic properties and the structure of powders made of the Fe73.5Cu1Nb3Si13.5B9 alloy. Furthermore, the structure and magnetic properties of nanocomposites were examined. The nanocomposites were formed in result of connecting powders of Fe73.5Cu1Nb3Si13.5B9 alloy and polyethylene (PHED) - 10% wt. and were obtained in an uniaxial, one-sided pressing process at the temperature of 170 oC and pressure of 200 MPa/ (author)

  2. Lysosomal membrane permeabilization: Carbon nanohorn-induced reactive oxygen species generation and toxicity by this neglected mechanism

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Mei, E-mail: happy_deercn@163.com [Nanotube Research Center, National Institute of Advanced Industrial Science and Technology 5-2, 1-1-1 Higashi, Tsukuba 305-8565 (Japan); Zhang, Minfang; Tahara, Yoshio; Chechetka, Svetlana; Miyako, Eijiro [Nanotube Research Center, National Institute of Advanced Industrial Science and Technology 5-2, 1-1-1 Higashi, Tsukuba 305-8565 (Japan); Iijima, Sumio [Nanotube Research Center, National Institute of Advanced Industrial Science and Technology 5-2, 1-1-1 Higashi, Tsukuba 305-8565 (Japan); Faculty of Science and Technology, Meijo University, 1-501 Shiogamaguchi, Tenpaku, Nagoya 468-8502 (Japan); Yudasaka, Masako, E-mail: m-yudasaka@aist.go.jp [Nanotube Research Center, National Institute of Advanced Industrial Science and Technology 5-2, 1-1-1 Higashi, Tsukuba 305-8565 (Japan)

    2014-10-01

    Understanding the molecular mechanisms responsible for the cytotoxic effects of carbon nanomaterials is important for their future biomedical applications. Carbon nanotubular materials induce the generation of reactive oxygen species (ROS), which causes cell death; however, the exact details of this process are still unclear. Here, we identify a mechanism of ROS generation that is involved in the apoptosis of RAW264.7 macrophages caused by excess uptake of carbon nanohorns (CNHs), a typical type of carbon nanotubule. CNH accumulated in the lysosomes, where they induced lysosomal membrane permeabilization (LMP) and the subsequent release of lysosomal proteases, such as cathepsins, which in turn caused mitochondrial dysfunction and triggered the generation of ROS in the mitochondria. The nicotinamide adenine dinucleotide phosphate oxidase was not directly involved in CNH-related ROS production, and the ROS generation cannot be regulated by mitochondrial electron transport chain. ROS fed back to amplify the mitochondrial dysfunction, leading to the subsequent activation of caspases and cell apoptosis. Carbon nanotubules commonly accumulate in the lysosomes after internalization in cells; however, lysosomal dysfunction has not attracted much attention in toxicity studies of these materials. These results suggest that LMP, a neglected mechanism, may be the primary reason for carbon nanotubule toxicity. - Highlights: • We clarify an apoptotic mechanism of RAW264.7 cells caused by carbon nanohorns. • In the meantime, the mechanism of CNH-induced ROS generation is identified. • LMP is the initial factor of CNH-induced ROS generation and cell death. • Cathepsins work as mediators that connect LMP and mitochondrial dysfunction.

  3. Toll-like receptor 4 is not targeted to the lysosome in cystic fibrosis airway epithelial cells.

    Science.gov (United States)

    Kelly, Catriona; Canning, Paul; Buchanan, Paul J; Williams, Mark T; Brown, Vanessa; Gruenert, Dieter C; Elborn, J Stuart; Ennis, Madeleine; Schock, Bettina C

    2013-03-01

    The innate immune response to bacterial infection is mediated through Toll-like receptors (TLRs), which trigger tightly regulated signaling cascades through transcription factors including NF-κB. LPS activation of TLR4 triggers internalization of the receptor-ligand complex which is directed toward lysosomal degradation or endocytic recycling. Cystic fibrosis (CF) patients display a robust and uncontrolled inflammatory response to bacterial infection, suggesting a defect in regulation. This study examined the intracellular trafficking of TLR4 in CF and non-CF airway epithelial cells following stimulation with LPS. We employed cells lines [16hBE14o-, CFBE41o- (CF), and CFTR-complemented CFBE41o-] and confirmed selected experiments in primary nasal epithelial cells from non-CF controls and CF patients (F508del homozygous). In control cells, TLR4 expression (surface and cytoplasmic) was reduced after LPS stimulation but remained unchanged in CF cells and was accompanied by a heightened inflammatory response 24 h after stimulation. All cells expressed markers of the early (EEA1) and late (Rab7b) endosomes at basal levels. However, only CF cells displayed persistent expression of Rab7b following LPS stimulation. Rab7 variants may directly internalize bacteria to the Golgi for recycling or to the lysosome for degradation. TLR4 colocalized with the lysosomal marker LAMP1 in 16 hBE14o- cells, suggesting that TLR4 is targeted for lysosomal degradation in these cells. However, this colocalization was not observed in CFBE41o- cells, where persistent expression of Rab7 and release of proinflammatory cytokines was detected. Consistent with the apparent inability of CF cells to target TLR4 toward the lysosome for degradation, we observed persistent surface and cytoplasmic expression of this pathogen recognition receptor. This defect may account for the prolonged cycle of chronic inflammation associated with CF. PMID:23316065

  4. A Novel High Content Imaging-Based Screen Identifies the Anti-Helminthic Niclosamide as an Inhibitor of Lysosome Anterograde Trafficking and Prostate Cancer Cell Invasion.

    Directory of Open Access Journals (Sweden)

    Magdalena L Circu

    Full Text Available Lysosome trafficking plays a significant role in tumor invasion, a key event for the development of metastasis. Previous studies from our laboratory have demonstrated that the anterograde (outward movement of lysosomes to the cell surface in response to certain tumor microenvironment stimulus, such as hepatocyte growth factor (HGF or acidic extracellular pH (pHe, increases cathepsin B secretion and tumor cell invasion. Anterograde lysosome trafficking depends on sodium-proton exchanger activity and can be reversed by blocking these ion pumps with Troglitazone or EIPA. Since these drugs cannot be advanced into the clinic due to toxicity, we have designed a high-content assay to discover drugs that block peripheral lysosome trafficking with the goal of identifying novel drugs that inhibit tumor cell invasion. An automated high-content imaging system (Cellomics was used to measure the position of lysosomes relative to the nucleus. Among a total of 2210 repurposed and natural product drugs screened, 18 "hits" were identified. One of the compounds identified as an anterograde lysosome trafficking inhibitor was niclosamide, a marketed human anti-helminthic drug. Further studies revealed that niclosamide blocked acidic pHe, HGF, and epidermal growth factor (EGF-induced anterograde lysosome redistribution, protease secretion, motility, and invasion of DU145 castrate resistant prostate cancer cells at clinically relevant concentrations. In an effort to identify the mechanism by which niclosamide prevented anterograde lysosome movement, we found that this drug exhibited no significant effect on the level of ATP, microtubules or actin filaments, and had minimal effect on the PI3K and MAPK pathways. Niclosamide collapsed intralysosomal pH without disruption of the lysosome membrane, while bafilomycin, an agent that impairs lysosome acidification, was also found to induce JLA in our model. Taken together, these data suggest that niclosamide promotes

  5. Insights into Ubiquitin Transfer Cascades from a Structure of a UbcH5B[is equivalent to]Ubiquitin-HECT[superscript NEDD4L] Complex

    Energy Technology Data Exchange (ETDEWEB)

    Kamadurai, Hari B.; Souphron, Judith; Scott, Daniel C.; Duda, David M.; Miller, Darcie J.; Stringer, Daniel; Piper, Robert C.; Schulman, Brenda A.; (SJCH)

    2010-02-23

    In E1-E2-E3 ubiquitin (Ub) conjugation cascades, the E2 first forms a transient E2 {approx} Ub covalent complex and then interacts with an E3 for Ub transfer. For cascades involving E3s in the HECT class, Ub is transferred from an associated E2 to the acceptor cysteine in the HECT domain C lobe. To gain insights into this process, we determined the crystal structure of a complex between the HECT domain of NEDD4L and the E2 UbcH5B bearing a covalently linked Ub at its active site (UbcH5B {approx} Ub). Noncovalent interactions between UbcH5B and the HECT N lobe and between Ub and the HECT domain C lobe lead to an overall compact structure, with the Ub C terminus sandwiched between UbcH5B and HECT domain active sites. The structure suggests a model for E2-to-HECT Ub transfer, in which interactions between a donor Ub and an acceptor domain constrain upstream and downstream enzymes for conjugation.

  6. The breast cancer resistance protein (Bcrp1/Abcg2) restricts exposure to the dietary carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine

    NARCIS (Netherlands)

    van Herwaarden, AE; Jonker, JW; Wagenaar, E; Brinkhuis, RF; Schellens, JHM; Beijnen, JH; Schinkel, AH

    2003-01-01

    The food carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is the most abundant heterocyclic amine found in various protein containing foods. PhIP is mutagenic and carcinogenic in rodents, inducing lymphomas in mice and colon, mammary and prostate carcinomas in rats. It has also been

  7. 17 CFR 230.153b - Definition of “preceded by a prospectus”, as used in section 5(b)(2), in connection with certain...

    Science.gov (United States)

    2010-04-01

    ... 17 Commodity and Securities Exchanges 2 2010-04-01 2010-04-01 false Definition of âpreceded by a prospectusâ, as used in section 5(b)(2), in connection with certain transactions in standardized options. 230... RULES AND REGULATIONS, SECURITIES ACT OF 1933 General § 230.153b Definition of “preceded by a...

  8. Hepatitis C Virus (HCV) NS5B Nonnucleoside Inhibitors Specifically Block Single-Stranded Viral RNA Synthesis Catalyzed by HCV Replication Complexes In Vitro▿

    OpenAIRE

    Yang, Wengang; Sun, Yongnian; Phadke, Avinash; Deshpande, Milind; Huang, Mingjun

    2006-01-01

    Replication complexes of hepatitis C virus synthesized two major species of viral RNA in vitro, double stranded and single stranded. NS5B nonnucleoside inhibitors inhibited dose dependently the synthesis of single-stranded RNA but not double-stranded RNA. Moreover, replication complexes carrying a mutation resistant to a nonnucleoside inhibitor lost their susceptibilities to the inhibitor.

  9. Validation study of the reactor physics lattice transport code WIMSD-5B by TRX and BAPL critical experiments of light water reactors

    International Nuclear Information System (INIS)

    Highlights: • To validate the reactor physics lattice code WIMSD-5B by this analysis. • To model TRX and BAPL critical experiments using WIMSD-5B. • To compare the calculated results with experiment and MCNP results. • To rely on WIMSD-5B code for TRIGA calculations. - Abstract: The aim of this analysis is to validate the reactor physics lattice transport code WIMSD-5B by TRX (thermal reactor-one region lattice) and BAPL (Bettis Atomic Power Laboratory-one region lattice) critical experiments of light water reactors for neutronics analysis of 3 MW TRIGA Mark-II research reactor at AERE, Dhaka, Bangladesh. This analysis is achieved through the analysis of integral parameters of five light water reactor critical experiments TRX-1, TRX-2, BAPL-UO2-1, BAPL-UO2-2 and BAPL-UO2-3 based on evaluated nuclear data libraries JEFF-3.1 and ENDF/B-VII.1. In integral measurements, these experiments are considered as standard benchmark lattices for validating the reactor physics lattice transport code WIMSD-5B as well as evaluated nuclear data libraries. The integral parameters of the said critical experiments are calculated using the reactor physics lattice transport code WIMSD-5B. The calculated integral parameters are compared to the measured values as well as the earlier published MCNP results based on the Chinese evaluated nuclear data library CENDL-3.0 for assessment of deterministic calculation. It was found that the calculated integral parameters give mostly reasonable and globally consistent results with the experiment and the MCNP results. Besides, the group constants in WIMS format for the isotopes U-235 and U-238 between two data files have been compared using WIMS library utility code WILLIE and it was found that the group constants are well consistent with each other. Therefore, this analysis reveals the validation study of the reactor physics lattice transport code WIMSD-5B based on JEFF-3.1 and ENDF/B-VII.1 libraries and can also be essential to execute

  10. Netrin-1 induces the migration of Schwann cells via p38 MAPK and PI3K-Akt signaling pathway mediated by the UNC5B receptor

    International Nuclear Information System (INIS)

    Schwann cells (SCs) play an essentially supportive role in the regeneration of injured peripheral nerve system (PNS). As Netrin-1 is crucial for the normal development of nervous system (NS) and can direct the process of damaged PNS regeneration, our study was designed to determine the role of Netrin-1 in RSC96 Schwann cells (an immortalized rat Schwann cell line) proliferation and migration. Our studies demonstrated that Netrin-1 had no effect on RSC96 cells proliferation, while significantly promoted RSC96 cells migration. The Netrin-1-induced RSC96 cells migration was significantly attenuated by inhibition of p38 and PI3K through pretreatment with SB203580 and LY294002 respectively, but not inhibition of MEK1/2 and JNK by U0126-EtOH and SP600125 individually. Treatment with Netrin-1 enhanced the phosphorylation of p38 and Akt. QRT-PCR indicated that Netrin-1 and only its receptors Unc5a, Unc5b and Neogenin were expressed in RSC96 cells, among which Unc5b expressed the most. And UNC5B protein was significantly increased after stimulated by Netrin-1. In conclusion, we show here that Netrin-1-enhanced SCs migration is mediated by activating p38 MAPK and PI3K-Akt signal cascades via receptor UNC5B, which suggests that Netrin-1 could serve as a new therapeutic strategy and has potential application value for PNS regeneration. - Highlights: • Netrin-1 attracts RSC96 Schwann cells migration in a dose dependent manner. • Netrin-1 induced Schwann cells migration is p38 and PI3K-Akt signaling dependent. • UNC5B may be dominant receptor mediating Netrin-1′ effect on RSC96 cells motility. • Netrin-1 may promote peripheral nerve repair by enhancing Schwann cells motility

  11. Association study of single nucleotide polymorphisms in JAK2 and STAT5B genes and their differential mRNA expression with mastitis susceptibility in Chinese Holstein cattle.

    Science.gov (United States)

    Usman, T; Wang, Y; Liu, C; Wang, X; Zhang, Y; Yu, Y

    2015-08-01

    The JAK-STAT pathway plays a key role in mediating immune responses. The genetic effects of single nucleotide polymorphisms (SNPs) in JAK2 and STAT5B were investigated for serum cytokines, mastitis indicators and productions traits in a population of 468 Chinese Holstein cattle. Pooled DNA sequencing revealed one SNP (BTA8:g.39645396A>G) in JAK2 and two SNPs (BTA19:g.43673888A>G and BTA19:g.43660093T>C) in STAT5B. A fixed effect model considering the effects of SNPs, parity, herd, season and year of calving was used by way of the general linear model procedure of sas. Genotype frequencies of these SNPs in the population were in Hardy-Weinberg equilibrium (P > 0.05). A novel SNP (g.39645396A>G) in JAK2 was predicted to change the amino acid from lysine to asparagine and was significantly associated with the somatic cell count (SCC) and somatic cell score (SCS), whereas g.43673888A>G in STAT5B was significantly associated with SCC, SCS and interleukin-4 (IL-4) (P G in JAK2 was significant for SCS, and its additive effect was significant for SCC, whereas the dominant effect of g.43673888A>G in STAT5B was significant for SCS and IL-4 (P G in JAK2 and g.43673888A>G in STAT5B showed a significant effect on SCC, SCS, IL-4 and TNF-α (P G and GG genotype g.43673888A>G indicated higher mRNA expression level and were significantly different from other genotypes (P cattle against mastitis development.

  12. Anti-HIV-1 activity of salivary MUC5B and MUC7 mucins from HIV patients with different CD4 counts

    Directory of Open Access Journals (Sweden)

    Roux Paul

    2010-10-01

    Full Text Available Abstract Background We have previously shown that MUC5B and MUC7 mucins from saliva of HIV negative individuals inhibit HIV-1 activity by 100% in an in vitro assay. The purpose of this subsequent study was to investigate whether MUC5B and MUC7 from saliva of HIV patients or with full blown AIDS had a similar inhibitory activity against the virus. Methods Salivary MUC5B and MUC7 from HIV patients with different CD4 counts ( 400 were incubated with HIV-1 prior to infection of the human T lymphoblastoid cell line (CEM SS cells. Cells were then cultured and viral replication was measured by a qualitative p24 antigen assay. The size, charge and immunoreactivity of mucins from HIV negative and positive individuals was also analysed by SDS-PAGE, Western blot and ELISA respectively. Results It was shown that irrespective of their CD4 counts both MUC5B and MUC7 from HIV patients, unlike the MUC5B and MUC7 from HIV negative individuals, did not inhibit HIV-1 activity. Size, charge and immunoreactivity differences between the mucins from HIV negative and positive individuals and among the mucins from HIV patients of different CD4 count was observed by SDS-PAGE, Western blot and ELISA. Conclusions Purified salivary mucins from HIV positive patients do not inhibit the AIDS virus in an in vitro assay. Although the reason for the inability of mucins from infected individuals to inhibit the virus is not known, it is likely that there is an alteration of the glycosylation pattern, and therefore of charge of mucin, in HIV positive patients. The ability to inhibit the virus by aggregation by sugar chains is thus diminished.

  13. Netrin-1 induces the migration of Schwann cells via p38 MAPK and PI3K-Akt signaling pathway mediated by the UNC5B receptor

    Energy Technology Data Exchange (ETDEWEB)

    Lv, Jianwei [General Hospital of Tianjin Medical University, No. 154, Anshan Road, Heping District, Tianjin 300052 (China); Tianjin Institute of Orthopedics in Traditional Chinese and Western Medicine, No. 155, Munan Road, Tianjin 300050 (China); Sun, Xiaolei; Ma, Jianxiong [Tianjin Institute of Orthopedics in Traditional Chinese and Western Medicine, No. 155, Munan Road, Tianjin 300050 (China); Ma, Xinlong, E-mail: gengxiao502@163.com [General Hospital of Tianjin Medical University, No. 154, Anshan Road, Heping District, Tianjin 300052 (China); Tianjin Institute of Orthopedics in Traditional Chinese and Western Medicine, No. 155, Munan Road, Tianjin 300050 (China); Zhang, Yang; Li, Fengbo; Li, Yanjun; Zhao, Zhihu [Tianjin Institute of Orthopedics in Traditional Chinese and Western Medicine, No. 155, Munan Road, Tianjin 300050 (China)

    2015-08-14

    Schwann cells (SCs) play an essentially supportive role in the regeneration of injured peripheral nerve system (PNS). As Netrin-1 is crucial for the normal development of nervous system (NS) and can direct the process of damaged PNS regeneration, our study was designed to determine the role of Netrin-1 in RSC96 Schwann cells (an immortalized rat Schwann cell line) proliferation and migration. Our studies demonstrated that Netrin-1 had no effect on RSC96 cells proliferation, while significantly promoted RSC96 cells migration. The Netrin-1-induced RSC96 cells migration was significantly attenuated by inhibition of p38 and PI3K through pretreatment with SB203580 and LY294002 respectively, but not inhibition of MEK1/2 and JNK by U0126-EtOH and SP600125 individually. Treatment with Netrin-1 enhanced the phosphorylation of p38 and Akt. QRT-PCR indicated that Netrin-1 and only its receptors Unc5a, Unc5b and Neogenin were expressed in RSC96 cells, among which Unc5b expressed the most. And UNC5B protein was significantly increased after stimulated by Netrin-1. In conclusion, we show here that Netrin-1-enhanced SCs migration is mediated by activating p38 MAPK and PI3K-Akt signal cascades via receptor UNC5B, which suggests that Netrin-1 could serve as a new therapeutic strategy and has potential application value for PNS regeneration. - Highlights: • Netrin-1 attracts RSC96 Schwann cells migration in a dose dependent manner. • Netrin-1 induced Schwann cells migration is p38 and PI3K-Akt signaling dependent. • UNC5B may be dominant receptor mediating Netrin-1′ effect on RSC96 cells motility. • Netrin-1 may promote peripheral nerve repair by enhancing Schwann cells motility.

  14. Bacillus thuringiensis Cry5B protein is highly efficacious as a single-dose therapy against an intestinal roundworm infection in mice.

    Directory of Open Access Journals (Sweden)

    Yan Hu

    Full Text Available Intestinal parasitic nematode diseases are one of the great diseases of our time. Intestinal roundworm parasites, including hookworms, whipworms, and Ascaris, infect well over 1 billion people and cause significant morbidity, especially in children and pregnant women. To date, there is only one drug, albendazole, with adequate efficacy against these parasites to be used in mass drug administration, although tribendimidine may emerge as a second. Given the hundreds of millions of people to be treated, the threat of parasite resistance, and the inadequacy of current treatments, new anthelmintics are urgently needed. Bacillus thuringiensis (Bt crystal (Cry proteins are the most common used biologically produced insecticides in the world and are considered non-toxic to vertebrates.Here we study the ability of a nematicidal Cry protein, Cry5B, to effect a cure in mice of a chronic roundworm infection caused by the natural intestinal parasite, Heligmosomoides bakeri (formerly polygyrus. We show that Cry5B produced from either of two Bt strains can act as an anthelmintic in vivo when administered as a single dose, achieving a approximately 98% reduction in parasite egg production and approximately 70% reduction in worm burdens when delivered per os at approximately 700 nmoles/kg (90-100 mg/kg. Furthermore, our data, combined with the findings of others, suggest that the relative efficacy of Cry5B is either comparable or superior to current anthelmintics. We also demonstrate that Cry5B is likely to be degraded quite rapidly in the stomach, suggesting that the actual dose reaching the parasites is very small.This study indicates that Bt Cry proteins such as Cry5B have excellent anthelmintic properties in vivo and that proper formulation of the protein is likely to reveal a superior anthelmintic.

  15. Inspired by nonenveloped viruses escaping from endo-lysosomes: a pH-sensitive polyurethane micelle for effective intracellular trafficking

    Science.gov (United States)

    Song, Nijia; Zhou, Lijuan; Li, Jiehua; Pan, Zhicheng; He, Xueling; Tan, Hong; Wan, Xinyuan; Li, Jianshu; Ran, Rong; Fu, Qiang

    2016-03-01

    A multifunctional drug delivery system (DDS) for cancer therapy still faces great challenges due to multiple physiological barriers encountered in vivo. To increase the efficacy of current cancer treatment a new anticancer DDS mimicking the response of nonenveloped viruses, triggered by acidic pH to escape endo-lysosomes, is developed. Such a smart DDS is self-assembled from biodegradable pH-sensitive polyurethane containing hydrazone bonds in the backbone, named pHPM. The pHPM exhibits excellent micellization characteristics and high loading capacity for hydrophobic chemotherapeutic drugs. The responses of the pHPM in acidic media, undergoing charge conversion and hydrophobic core exposure, resulting from the detachment of the hydrophilic polyethylene glycol (PEG) shell, are similar to the behavior of a nonenveloped virus when trapped in acidic endo-lysosomes. Moreover, the degradation mechanism was verified by gel permeation chromatography (GPC). The endo-lysosomal membrane rupture induced by these transformed micelles is clearly observed by transmission electron microscopy. Consequently, excellent antitumor activity is confirmed both in vitro and in vivo. The results verify that the pHPM could be a promising new drug delivery tool for the treatment of cancer and other diseases.A multifunctional drug delivery system (DDS) for cancer therapy still faces great challenges due to multiple physiological barriers encountered in vivo. To increase the efficacy of current cancer treatment a new anticancer DDS mimicking the response of nonenveloped viruses, triggered by acidic pH to escape endo-lysosomes, is developed. Such a smart DDS is self-assembled from biodegradable pH-sensitive polyurethane containing hydrazone bonds in the backbone, named pHPM. The pHPM exhibits excellent micellization characteristics and high loading capacity for hydrophobic chemotherapeutic drugs. The responses of the pHPM in acidic media, undergoing charge conversion and hydrophobic core

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

    Science.gov (United States)

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

    2015-04-01

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

  17. Affective Urbanism

    DEFF Research Database (Denmark)

    Samson, Kristine

    . Under these circumstances affective aesthetics operate strategically within the urban field of interests, capital flows and desires of the social. This ‘affective urbanism’ (Anderson & Holden 2008) is linked to a society influenced by new kinds of information flows, where culture is mediated and enacted...... and cultural festivals, both practices indicate that design is implemented as means of creating affective spaces in the city. Both cases show how immaterial production of affects and emotions in the city can be seen in relation to economic potential and urban development. Finally, I will discuss whether urban......Urban design and architecture are increasingly used as material and affective strategies for setting the scene, for manipulation and the production of urban life: The orchestration of atmospheres, the framing and staging of urban actions, the programming for contemplation, involvement, play...

  18. Effects of TiO2 coating dosage and operational parameters on a TiO2/Ag photocatalysis system for decolorizing Procion red MX-5B.

    Science.gov (United States)

    Lin, Yu-Chih; Lee, Ho-Shan

    2010-07-15

    In this study, titanium dioxide (TiO(2)) powder was coated onto the surface of a dendritic silver (Ag) carrier to synthesize TiO(2)/Ag for decolorizing Procion red MX-5B (MX-5B), and related operation factors were also studied. The results showed that even without ultraviolet-A (UVA) irradiation, the Ag carrier from the TiO(2)/Ag catalyst had oxidizing ability, which could effectively degrade MX-5B color, but TiO(2) was ineffective. In addition, TiO(2) from TiO(2)/Ag demonstrated photocatalysis performance when irradiated, and the Ag carrier further showed an electron-scavenging ability to mitigate electron-hole pair recombination, which can improve the photocatalytic efficacy. With the oxidization and electron-scavenging ability of Ag and the photocatalysis ability of TiO(2), TiO(2)/Ag can decolor MX-5B more efficiently than TiO(2). The heavier Ag carrier also improves the solid-liquid separation of nano-TiO(2), making TiO(2)/Ag more suitable for application in slurry systems of photocatalytic water treatment. When the TiO(2)/Ag coating ratio was 50% by weight, there was a sufficient amount of TiO(2) on Ag's surface with a good distribution, and it exhibited a good photocatalysis decolorizing effect. In a study of how operational factors impact the decolorizing of MX-5B in the TiO(2)/Ag photocatalysis system with UVA irradiation (UVA-TiO(2)/Ag), the decolorization efficiency was optimal when the solution was maintained at pH 6.35. The addition of 0.01 M hydrogen peroxide (H(2)O(2)) aided the photocatalysis decolorization efficiency, although excessive H(2)O(2) reacted with hydroxyl free radicals and decreased the active groups in the system, thereby reducing the photocatalysis activity. An operating temperature of 40 degrees C was conducive to MX-5B decolorization, which was better than operating at room temperature.

  19. Accounting for target flexibility and water molecules by docking to ensembles of target structures: the HCV NS5B palm site I inhibitors case study.

    Science.gov (United States)

    Barreca, Maria Letizia; Iraci, Nunzio; Manfroni, Giuseppe; Gaetani, Rosy; Guercini, Chiara; Sabatini, Stefano; Tabarrini, Oriana; Cecchetti, Violetta

    2014-02-24

    The introduction of new anti-HCV drugs in therapy is an imperative need and is necessary with a view to develop an interferon-free therapy. Thus, the discovery and development of novel small molecule inhibitors of the viral NS5B polymerase represent an exciting area of research for many pharmaceutical companies and academic groups. This study represents a contribution to this field and relies on the identification of the best NS5B model(s) to be used in structure-based computational approaches aimed at identifying novel non-nucleoside inhibitors of one of the protein allosteric sites, namely, palm site I. First, the NS5B inhibitors at palm site I were classified as water-mediated or nonwater-mediated ligands depending on their ability to interact with or displace a specific water molecule. Then, we took advantage of the available X-ray structures of the NS5B/ligand complexes to build different models of protein/water combinations, which were used to investigate the influence on docking studies of solvent sites as well as of the influence of the protein conformations. As the overall trend, we observed improved performance in the docking results of the water-mediated inhibitors by inclusion of explicit water molecules, with an opposite behavior generally happening for the nonwater-mediated inhibitors. The best performing target structures for the two ligand sets were then used for virtual screening simulations of a library containing the known NS5B inhibitors along with related decoys to assess the best performing targets ensembles on the basis of their ability to discriminate active and inactive compounds as well as to generate the correct binding modes. The parallel use of different protein structures/water sets outperformed the use of a single target structure, with the two-protein 3H98/2W-2FVC/7W and 3HKY/NoW-3SKE/NoW models resulting in the best performing ensembles for water-mediated inhibitors and nonwater-mediated inhibitors, respectively. The information

  20. High proportion of mannosidosis and fucosidosis among lysosomal storage diseases in Cuba.

    Science.gov (United States)

    Menéndez-Sainz, C; González-Quevedo, A; González-García, S; Peña-Sánchez, M; Giugliani, R

    2012-08-13

    Although lysosomal storage disorders (LSDs) are considered individually rare, as a group they present a non-negligible frequency. Few studies have been made of populational occurrence of LSDs; they have been conducted predominantly on Caucasian populations. We studied the occurrence of LSDs in Cuba. Data from individuals who had been referred to the Institute of Neurology and Neurosurgery in Havana from hospitals all over the country between January 1990 and December 2005 were analyzed. This institute was the only laboratory to provide enzyme-based diagnostic testing for 19 LSDs in Cuba during this period. Occurrence rates were calculated by dividing the number of postnatal diagnoses by the number of births during the study period. The combined occurrence of LSDs in Cuba was 5.6 per 100,000, lower than that reported in other studies conducted on Caucasian populations. The most frequent individual LSDs were: mucopolysaccharidosis type I (1.01 per 100,000) and, surprisingly, alpha-mannosidosis (0.72 per 100,000) and fucosidosis (0.62 per 100,000). These findings may be related to specific genetic characteristics and admixture of the Cuban population. This is the first comprehensive study of the occurrence of LSDs in Cuba. We conclude that the epidemiology of these diseases can vary regionally, and we stress the need for similar surveys in other Latin American countries.