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Sample records for lysosomal membrane stability

  1. Gastroprotection and lysosomal membrane stabilization by sulglicotide.

    PubMed

    Porta, R; Niada, R; Pescador, R; Mantovani, M; Prino, G

    1986-07-01

    Well-known agents that induce gastric ulcers cause a decrease in lysosomal stability, with release of lytic enzymes. Some antiulcer and cytoprotective agents have lysosomal membrane stabilizing activity when tested in vitro and ex vivo. Sulglicotide (Gliptide), a polysulfated glycopeptide with antiulcer and cytoprotective activities, was able to stabilize lysosomal membranes in vitro at concentrations between 9 and 36 micrograms/ml. The ratio of potency of sulglicotide to that of carbenoxolone was 12.2. In ex vivo experiments in rats, it was found that sulglicotide stabilized lysosomes after oral treatment. The effect was dose-dependent after intravenous treatment. Carbenoxolone, injected i.v. under the same experimental conditions, was less active (potency ratio 0.65). 16,16-dimethyl-PGE2, administered at a dose of 10 micrograms/kg orally or intravenously, had an activity equivalent to that of sulglicotide at a dose of 12.5 mg/kg i.v. or 200 mg/kg p.o. Sulglicotide (200-400 mg/kg p.o.) was also able to prevent the release of acid phosphatase from stomachs challenged for 10 min or 3 h with absolute ethanol. The same result was obtained with 200 mg/kg p.o. of carbenoxolone. These data show that sulglicotide is a potent lysosomal membrane stabilizer in vitro and ex vivo, and could explain the cytoprotective activity of this compound in different experimental models of ulcer.

  2. Enantioselective effects of methamidophos on the coelomocytes lysosomal membrane stability of Eisenia fetida.

    PubMed

    Chen, Linhua; Lu, Xianting; Ma, Yun

    2012-12-01

    Many of organophosphorous insecticides are chiral compounds. In this study, the enantioselective effects of organophosphate insecticide methamidophos on the coelomocytes lysosomal membrane stability of earthworm Eisenia fetida were studied: (1) The enantiomers of methamidophos were absolutely separated by high-performance liquid chromatography with a commercial chiral column; (2) The neutral red retention assay was used to judge the lysosomal membrane stability. The results showed that with the concentration increasing, lysosomal membranes have been significantly destroyed by individual stereoisomers and racemate of methamidophos. The neutral red retention times were significantly descended from 76.88 to 29.78 min. Both (+)- and (-)-methamidophos showed more prone to destroy the integrity of the lysosomal membrane than the racemate. However, the different effect between stereoisomers is slight.

  3. Hepatocyte Lysosomal Membrane Stabilization by Olive Leaves against Chemically Induced Hepatocellular Neoplasia in Rats

    PubMed Central

    Abdel-Hamid, N. M.; El-Moselhy, M. A.; El-Baz, A.

    2011-01-01

    Extensive efforts are exerted looking for safe and effective chemotherapy for hepatocellular carcinoma (HCC). Specific and sensitive early biomarkers for HCC still in query. Present work to study proteolytic activity and lysosomal membrane integrity by hepatocarcinogen, trichloroacetic acid (TCA), in Wistar rats against aqueous olive leaf extract (AOLE).TCA showed neoplastic changes as oval- or irregular-shaped hepatocytes and transformed, vesiculated, and binucleated liver cells. The nuclei were pleomorphic and hyperchromatic. These changes were considerably reduced by AOLE. The results added, probably for the first time, that TCA-induced HCC through disruption of hepatocellular proteolytic enzymes as upregulation of papain, free cathepsin-D and nonsignificant destabilization of lysosomal membrane integrity, a prerequisite for cancer invasion and metastasis. AOLE introduced a promising therapeutic value in liver cancer, mostly through elevating lysosomal membrane integrity. The study substantiated four main points: (1) the usefulness of proteolysis and lysosomalmembrane integrity in early prediction of HCC. (2) TCA carcinogenesis is possibly mediated by lysosomal membrane destabilization, through cathepsin-D disruption, which could be reversed by AOLE administration. (3) A new strategy for management of HCC, using dietary olive leaf system may be a helpful phytotherapeutic trend. (4) A prospective study on serum proteolytic enzyme activity may introduce novel diagnostic tools. PMID:21994869

  4. Membrane stabilizer

    DOEpatents

    Mingenbach, William A.

    1988-01-01

    A device is provided for stabilizing a flexible membrane secured within a frame, wherein a plurality of elongated arms are disposed radially from a central hub which penetrates the membrane, said arms imposing alternately against opposite sides of the membrane, thus warping and tensioning the membrane into a condition of improved stability. The membrane may be an opaque or translucent sheet or other material.

  5. DNA adducts, benzo(a)pyrene monooxygenase activity, and lysosomal membrane stability in Mytilus galloprovincialis from different areas in Taranto coastal waters (Italy).

    PubMed

    Pisoni, M; Cogotzi, L; Frigeri, A; Corsi, I; Bonacci, S; Iacocca, A; Lancini, L; Mastrototaro, F; Focardi, S; Svelto, M

    2004-10-01

    The aim of this study was to investigate the impact of environmental pollution at different stations along the Taranto coastline (Ionian Sea, Puglia, Italy) using several biomarkers of exposure and the effect on mussels, Mytilus galloprovincialis, collected in October 2001 and October 2002. Five sampling sites were compared with a "cleaner" reference site in the Aeronautics Area. In this study we also investigated the differences between adduct levels in gills and digestive gland. This Taranto area is the most significant industrial settlement on the Ionian Sea known to be contaminated by polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls, heavy metals, etc. Exposure to PAHs was evaluated by measuring DNA adduct levels and benzo(a)pyrene monooxygenase activity (B(a)PMO); DNA adducts were analyzed by 32P-postlabeling with nuclease P1 enhancement in both gills and digestive glands to evaluate differences between DNA adduct levels in the two tissues. B(a)PMO was assayed in the microsomal fraction of the digestive glands as a result of the high expression of P450-metabolizing enzymes in this tissue. Lysosomal membrane stability, a potential biomarker of anthropogenic stress, was also evaluated in the digestive glands of mussels, by measuring the latent activity of beta-N-acetylhexosaminidase. Induction of DNA adducts was evident in both tissues, although the results revealed large tissue differences in DNA adduct formation. In fact, gills showed higher DNA adduct levels than did digestive gland. No significant differences were found in DNA adduct levels over time, with both tissues providing similar results in both years. DNA adduct levels were correlated with B(a)PMO activity in digestive gland in both years (r = 0.60 in 2001; r = 0.73 in 2002). Increases were observed in B(a)PMO activity and DNA adduct levels at different stations; no statistical difference was observed in B(a)PMO activity over the two monitoring campaigns. The membrane labilization

  6. PIG7 promotes leukemia cell chemosensitivity via lysosomal membrane permeabilization

    PubMed Central

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

    2016-01-01

    PIG7 localizes to lysosomal membrane in leukemia cells. Our previous work has shown that transduction of pig7 into a series of leukemia cell lines did not result in either apoptosis or differentiation of most tested cell lines. Interestingly, it did significantly sensitize these cell lines to chemotherapeutic drugs. Here, we further investigated the mechanism underlying pig7-induced improved sensitivity of acute leukemia cells to chemotherapy. Our results demonstrated that the sensitization effect driven by exogenous pig7 was more effective in drug-resistant leukemia cell lines which had lower endogenous pig7 expression. Overexpression of pig7 did not directly activate the caspase apoptotic pathway, but decreased the lysosomal stability. The expression of pig7 resulted in lysosomal membrane permeabilization (LMP) and lysosomal protease (e.g. cathepsin B, D, L) release. Moreover, we also observed increased reactive oxygen species (ROS) and decreased mitochondrial membrane potential (ΔΨm) induced by pig7. Some autophagy markers such as LC3I/II, ATG5 and Beclin-1, and necroptosis maker MLKL were also stimulated. However, intrinsic antagonism such as serine/cysteine protease inhibitors Spi2A and Cystatin C prevented downstream effectors from triggering leukemia cells, which were only on the “verge of apoptosis”. When combined with chemotherapy, LMP increased and more proteases were released. Once this process was beyond the limit of intrinsic antagonism, it induced programmed cell death cooperatively via caspase-independent and caspase-dependent pathways. PMID:26716897

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

    PubMed

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

    2016-01-26

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

  8. Membrane stabilizer

    DOEpatents

    Mingenbach, W.A.

    1988-02-09

    A device is provided for stabilizing a flexible membrane secured within a frame, wherein a plurality of elongated arms are disposed radially from a central hub which penetrates the membrane, said arms imposing alternately against opposite sides of the membrane, thus warping and tensioning the membrane into a condition of improved stability. The membrane may be an opaque or translucent sheet or other material. 10 figs.

  9. 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).

    PubMed

    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 (p<0.001) reduced the ability of Dp44mT to inhibit cancer cell proliferation (i.e., increased the IC(50)) by 140-fold. On the other hand, cholesterol extraction using methyl-β-cyclodextrin enhanced Dp44mT-induced LMP and significantly (p<0.01) increased its anti-proliferative activity. The protective effect of U18666A in increasing lysosomal cholesterol and preventing the cytotoxic activity of Dp44mT was not due to induced autophagy. Instead, U18666A was found to decrease lysosomal turnover, resulting in autophagosome accumulation. Moreover, preincubation with U18666A did not prevent the ability of Dp44mT to induce autophagosome synthesis, indicating that autophagic initiation via Dp44mT occurs independently of LMP. These studies demonstrate the significance of lysosomal membrane stability in relation to the ability of Dp44mT to execute tumor cell death and overcome pro-survival autophagy. Hence, lysosomal-dependent cell death induced by Dp44mT serves as an important anti-tumor strategy. These results are important for comprehensively understanding the mechanism of action of Dp44mT.

  10. Using lysosomal membrane stability of haemocytes in Ruditapes philippinarum as a biomarker of cellular stress to assess contamination by caffeine, ibuprofen, carbamazepine and novobiocin.

    PubMed

    Aguirre-Martínez, Gabriela V; Buratti, Sara; Fabbr, Elena; DelValls, Angel T; Martín-Díaz, M Laura

    2013-07-01

    Although pharmaceuticals have been detected in the environment only in the range from ng/L to microg/L, it has been demonstrated that they can adversely affect the health status of aquatic organisms. Lysosomal membrane stability (LMS) has previously been applied as an indicator of cellular well-being to determine health status in bivalve mussels. The objective of this study is to evaluate LMS in Ruditapes philippinarum haemolymph using the neutral red retention assay (NRRA). Clams were exposed in laboratory conditions to caffeine (0.1, 5, 15, 50 microg/L), ibuprofen (0.1, 5, 10, 50 microg/L), carbamazepine and novobiocin (both at 0.1, 1, 10, 50 microg/L) for 35 days. Results show a dose-dependent effect of the pharmaceuticals. The neutral red retention time measured at the end of the bioassay was significantly reduced by 50% after exposure to environmental concentrations (p < 0.05) (caffeine = 15 microg/L; ibuprofen = 10 microg/L; carbamazepine = 1 microg/L and novobiocin = 1 microg/L), compared to controls. Clams exposed to these pharmaceuticals were considered to present a diminished health status (retention time < 45 min), significantly worse than controls (96 min) (p < 0.05). The predicted no environmental effect concentration (PNEC) results showed that these pharmaceuticals are very toxic at the environmental concentrations tested. Measurement of the alteration of LMS has been found to be a sensitive technique that enables evaluation of the health status of clams after exposure to pharmaceuticals under laboratory conditions, thus representing a robust Tier-1 screening biomarker.

  11. Methods for the quantification of lysosomal membrane permeabilization: a hallmark of lysosomal cell death.

    PubMed

    Aits, Sonja; Jäättelä, Marja; Nylandsted, Jesper

    2015-01-01

    Lysosomal cell death is triggered by lysosomal membrane permeabilization (LMP) and subsequent release of lysosomal hydrolases from the lysosomal lumen into the cytosol. Once released into the cytosol, the lysosomal cathepsin proteases act as executioner proteases for the subsequent cell death-either autonomously without caspase activation or in concert with the classical apoptotic machinery. Lysosomal cell death usually remains functional in apoptosis-resistant cancer cells and thus holds great potential as a therapeutic strategy for circumventing apoptosis deficiency in cancers. Notably, lysosomal cell death also plays an important role in normal physiology, e.g., during the regression of the mammary gland. Here we present four complementary methods for the quantification and visualization of LMP during the onset of death: (1) enzymatic activity measurements of released lysosomal hydrolases in the cytosol after digitonin extraction, (2) direct visualization of LMP by monitoring the release of fluorescent dextran from lysosomes into the cytosol, (3) immunocytochemistry to detect cathepsins released into the cytosol, and (4) detection of the translocation of galectins to damaged lysosomes. The methods presented here can ideally be combined as needed to provide solid evidence for LMP after a given cytotoxic stimuli.

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

    NASA Astrophysics Data System (ADS)

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

    1995-05-01

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

  13. Sensitive detection of lysosomal membrane permeabilization by lysosomal galectin puncta assay

    PubMed Central

    Aits, Sonja; Kricker, Jennifer; Liu, Bin; Ellegaard, Anne-Marie; Hämälistö, Saara; Tvingsholm, Siri; Corcelle-Termeau, Elisabeth; Høgh, Søren; Farkas, Thomas; Holm Jonassen, Anna; Gromova, Irina; Mortensen, Monika; Jäättelä, Marja

    2015-01-01

    Lysosomal membrane permeabilization (LMP) contributes to tissue involution, degenerative diseases, and cancer therapy. Its investigation has, however, been hindered by the lack of sensitive methods. Here, we characterize and validate the detection of galectin puncta at leaky lysosomes as a highly sensitive and easily manageable assay for LMP. LGALS1/galectin-1 and LGALS3/galectin-3 are best suited for this purpose due to their widespread expression, rapid translocation to leaky lysosomes and availability of high-affinity antibodies. Galectin staining marks individual leaky lysosomes early during lysosomal cell death and is useful when defining whether LMP is a primary or secondary cause of cell death. This sensitive method also reveals that cells can survive limited LMP and confirms a rapid formation of autophagic structures at the site of galectin puncta. Importantly, galectin staining detects individual leaky lysosomes also in paraffin-embedded tissues allowing us to demonstrate LMP in tumor xenografts in mice treated with cationic amphiphilic drugs and to identify a subpopulation of lysosomes that initiates LMP in involuting mouse mammary gland. The use of ectopic fluorescent galectins renders the galectin puncta assay suitable for automated screening and visualization of LMP in live cells and animals. Thus, the lysosomal galectin puncta assay opens up new possibilities to study LMP in cell death and its role in other cellular processes such as autophagy, senescence, aging, and inflammation. PMID:26114578

  14. Lysosomal membrane stability, phagocytosis and tolerance to emersion in the mussel Perna viridis (Bivalvia: Mytilidae) following exposure to acute, sublethal, copper.

    PubMed

    Nicholson, S

    2003-08-01

    The mytilid mussel Perna viridis is distributed throughout the Indo-Pacific region and is potentially a suitable candidate for biological effects (biomarker) monitoring in the subtropics. A suite of cytological and physiological responses to acute (48-72 h) copper exposures of 50-200 microgl(-1) were assessed in order to determine the suitability of P. viridis for marine pollution monitoring. Copper elicited significant destabilisation of the haemocyte lysosomal membranes and also impaired phagocytosis. Survival during emersion following exposure to copper was not related to the experimental copper exposures suggesting that higher metal concentrations may be required to interfere with anaerobic enzymes responsible for suppression of metabolism. Based on this preliminary study, cytological biomarkers evaluated in the haemocytes extracted from P. viridis should prove an effective non-destructive means of assessing metal pollution throughout the mussels subtropical range.

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

    SciTech Connect

    Chance, S.C.

    1987-01-01

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

  16. FIG4 regulates lysosome membrane homeostasis independent of phosphatase function.

    PubMed

    Bharadwaj, Rajnish; Cunningham, Kathleen M; Zhang, Ke; Lloyd, Thomas E

    2016-02-15

    FIG4 is a phosphoinositide phosphatase that is mutated in several diseases including Charcot-Marie-Tooth Disease 4J (CMT4J) and Yunis-Varon syndrome (YVS). To investigate the mechanism of disease pathogenesis, we generated Drosophila models of FIG4-related diseases. Fig4 null mutant animals are viable but exhibit marked enlargement of the lysosomal compartment in muscle cells and neurons, accompanied by an age-related decline in flight ability. Transgenic animals expressing Drosophila Fig4 missense mutations corresponding to human pathogenic mutations can partially rescue lysosomal expansion phenotypes, consistent with these mutations causing decreased FIG4 function. Interestingly, Fig4 mutations predicted to inactivate FIG4 phosphatase activity rescue lysosome expansion phenotypes, and mutations in the phosphoinositide (3) phosphate kinase Fab1 that performs the reverse enzymatic reaction also causes a lysosome expansion phenotype. Since FIG4 and FAB1 are present together in the same biochemical complex, these data are consistent with a model in which FIG4 serves a phosphatase-independent biosynthetic function that is essential for lysosomal membrane homeostasis. Lysosomal phenotypes are suppressed by genetic inhibition of Rab7 or the HOPS complex, demonstrating that FIG4 functions after endosome-to-lysosome fusion. Furthermore, disruption of the retromer complex, implicated in recycling from the lysosome to Golgi, does not lead to similar phenotypes as Fig4, suggesting that the lysosomal defects are not due to compromised retromer-mediated recycling of endolysosomal membranes. These data show that FIG4 plays a critical noncatalytic function in maintaining lysosomal membrane homeostasis, and that this function is disrupted by mutations that cause CMT4J and YVS.

  17. FIG4 regulates lysosome membrane homeostasis independent of phosphatase function

    PubMed Central

    Bharadwaj, Rajnish; Cunningham, Kathleen M.; Zhang, Ke; Lloyd, Thomas E.

    2016-01-01

    FIG4 is a phosphoinositide phosphatase that is mutated in several diseases including Charcot-Marie-Tooth Disease 4J (CMT4J) and Yunis-Varon syndrome (YVS). To investigate the mechanism of disease pathogenesis, we generated Drosophila models of FIG4-related diseases. Fig4 null mutant animals are viable but exhibit marked enlargement of the lysosomal compartment in muscle cells and neurons, accompanied by an age-related decline in flight ability. Transgenic animals expressing Drosophila Fig4 missense mutations corresponding to human pathogenic mutations can partially rescue lysosomal expansion phenotypes, consistent with these mutations causing decreased FIG4 function. Interestingly, Fig4 mutations predicted to inactivate FIG4 phosphatase activity rescue lysosome expansion phenotypes, and mutations in the phosphoinositide (3) phosphate kinase Fab1 that performs the reverse enzymatic reaction also causes a lysosome expansion phenotype. Since FIG4 and FAB1 are present together in the same biochemical complex, these data are consistent with a model in which FIG4 serves a phosphatase-independent biosynthetic function that is essential for lysosomal membrane homeostasis. Lysosomal phenotypes are suppressed by genetic inhibition of Rab7 or the HOPS complex, demonstrating that FIG4 functions after endosome-to-lysosome fusion. Furthermore, disruption of the retromer complex, implicated in recycling from the lysosome to Golgi, does not lead to similar phenotypes as Fig4, suggesting that the lysosomal defects are not due to compromised retromer-mediated recycling of endolysosomal membranes. These data show that FIG4 plays a critical noncatalytic function in maintaining lysosomal membrane homeostasis, and that this function is disrupted by mutations that cause CMT4J and YVS. PMID:26662798

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

    PubMed

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

    2012-01-01

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

  19. Lysosomal membrane glycoproteins bind cholesterol and contribute to lysosomal cholesterol export

    PubMed Central

    Li, Jian; Pfeffer, Suzanne R

    2016-01-01

    LAMP1 and LAMP2 proteins are highly abundant, ubiquitous, mammalian proteins that line the lysosome limiting membrane, and protect it from lysosomal hydrolase action. LAMP2 deficiency causes Danon’s disease, an X-linked hypertrophic cardiomyopathy. LAMP2 is needed for chaperone-mediated autophagy, and its expression improves tissue function in models of aging. We show here that human LAMP1 and LAMP2 bind cholesterol in a manner that buries the cholesterol 3β-hydroxyl group; they also bind tightly to NPC1 and NPC2 proteins that export cholesterol from lysosomes. Quantitation of cellular LAMP2 and NPC1 protein levels suggest that LAMP proteins represent a significant cholesterol binding site at the lysosome limiting membrane, and may signal cholesterol availability. Functional rescue experiments show that the ability of human LAMP2 to facilitate cholesterol export from lysosomes relies on its ability to bind cholesterol directly. DOI: http://dx.doi.org/10.7554/eLife.21635.001 PMID:27664420

  20. Passive diffusion of non-electrolytes across the lysosome membrane.

    PubMed Central

    Iveson, G P; Bird, S J; Lloyd, J B

    1989-01-01

    An osmotic-protection method has been used to study the permeability of rat liver lysosomes to 43 organic non-electrolytes of formula weights ranging from 62 to 1000. A lysosome-rich centrifugal fraction of rat liver homogenate was resuspended in an unbuffered 0.25 M solution of test solute, pH 7.0, and incubated at 25 degrees C for 60 min. The free and total activities of 4-methylumbelliferyl N-acetyl-beta-D-glucosaminidase were measured after incubation for 0, 30 and 60 min. Three patterns of results were seen. In pattern A the percentage free activity remained low throughout the 60 min incubation, indicating little or no solute entry into the lysosomes. In pattern B, the percentage free activity was initially low, but rose substantially during the incubation, indicating solute entry. In pattern C there was not even initial osmotic protection, indicating very rapid solute entry. The rapidity of solute entry into the lysosomes showed no correlation with the formula weight, but a perfect inverse correlation with the hydrogen-bonding capacity of the solutes. The results, which can be used to predict the ability of further compounds to cross the lysosome membrane by unassisted diffusion, are discussed in the context of metabolite and drug release from lysosomes in vivo. PMID:2775227

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

    PubMed

    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.

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

    PubMed Central

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

    2001-01-01

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

  3. The Chaperone-Mediated Autophagy Receptor Organizes in Dynamic Protein Complexes at the Lysosomal Membrane ▿ †

    PubMed Central

    Bandyopadhyay, Urmi; Kaushik, Susmita; Varticovski, Lyuba; Cuervo, Ana Maria

    2008-01-01

    Chaperone-mediated autophagy (CMA) is a selective type of autophagy by which specific cytosolic proteins are sent to lysosomes for degradation. Substrate proteins bind to the lysosomal membrane through the lysosome-associated membrane protein type 2A (LAMP-2A), one of the three splice variants of the lamp2 gene, and this binding is limiting for their degradation via CMA. However, the mechanisms of substrate binding and uptake remain unknown. We report here that LAMP-2A organizes at the lysosomal membrane into protein complexes of different sizes. The assembly and disassembly of these complexes are a very dynamic process directly related to CMA activity. Substrate proteins only bind to monomeric LAMP-2A, while the efficient translocation of substrates requires the formation of a particular high-molecular-weight LAMP-2A complex. The two major chaperones related to CMA, hsc70 and hsp90, play critical roles in the functional dynamics of the LAMP-2A complexes at the lysosomal membrane. Thus, we have identified a novel function for hsc70 in the disassembly of LAMP-2A from these complexes, whereas the presence of lysosome-associated hsp90 is essential to preserve the stability of LAMP-2A at the lysosomal membrane. PMID:18644871

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

    PubMed

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

    2015-11-01

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

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

    PubMed

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

    2012-09-01

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

  6. Endolyn-78, a membrane glycoprotein present in morphologically diverse components of the endosomal and lysosomal compartments: implications for lysosome biogenesis

    PubMed Central

    1989-01-01

    A monoclonal antibody (2C5) raised against rat liver lysosomal membranes was used to identify a 78-kD glycoprotein that is present in the membranes of both endosomes and lysosomes and, therefore, is designated endolyn-78. In cultures of rat hepatoma (Fu5C8) and kidney cells (NRK), this glycoprotein could not be labeled with [35S]methionine or with [32P]inorganic phosphate but was easily labeled with [35S]cysteine and [3H]mannose. Pulse-chase experiments and determinations of endoglycosidase H (endo H) sensitivity showed that endolyn-78 is derived from a precursor of Mr 58-62 kD that is processed to the mature form with a t1/2 of 15-30 min. The protein has a 22-kD polypeptide backbone that is detected after a brief pulse in tunicamycin-treated cells. During a chase in the presence of the drug, this is converted into an O-glycosylated product of 46 kD that despite the absence of N-linked oligosaccharides is effectively transferred to lysosomes. This demonstrates that the delivery of endolyn-78 to this organelle is not mediated by the mannose-6-phosphate receptor (MPR). Immunocytochemical experiments showed that endolyn-78 is present in the limiting membranes and the interior membranous structures of morphologically identifiable secondary lysosomes that contain the lysosomal hydrolase beta-glucuronidase, lack the MPR, and could not be labeled with alpha-2-macroglobulin at 18.5 degrees C, a temperature which prevents appearance of endocytosed markers in lysosomes. Endolyn- 78 was present at low levels in the plasma membrane and in peripheral tubular endosomes, but was prominent in morphologically diverse components of the endosomal compartment (vacuolar endosomes and various types of multivesicular bodies) which acquired alpha-2-macroglobulin at 18.5 degrees C, and frequently contained substantial levels of the MPR and variable levels of beta-glucuronidase. On the other hand, the MPR was very rarely found in endolyn-containing structures that were not labeled with

  7. Attenuation of the lysosomal death pathway by lysosomal cholesterol accumulation.

    PubMed

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

    2011-02-01

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

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

    PubMed Central

    Encarnação, Marisa; Mateus, Denisa; Michelet, Xavier; Santarino, Inês; Hsu, Victor W.; Brenner, Michael B.

    2016-01-01

    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

  9. Quantification of Lysosomal Membrane Permeabilization by Cytosolic Cathepsin and β-N-Acetyl-Glucosaminidase Activity Measurements.

    PubMed

    Jäättelä, Marja; Nylandsted, Jesper

    2015-11-02

    Programmed cell death involving lysosomal membrane permeabilization (LMP) is an alternative cell death pathway induced under various cellular conditions and by numerous cytotoxic stimuli. The method presented here to quantify LMP takes advantage of the detergent digitonin, which creates pores in cellular membranes by replacing cholesterol. The difference in cholesterol content between the plasma membrane (high) and lysosomal membrane (low) allows titration of digitonin to a concentration that permeabilizes the plasma membrane but leaves lysosomal membranes intact. The extent of LMP is determined by measuring the cytosolic activity of lysosomal hydrolases (e.g., cysteine cathepsins) and/or β-N-acetyl-glucosaminidase in the digitonin-extracted cytoplasm and comparing it to the total cellular enzyme activity. Digitonin extraction of the cytosol can be combined with precipitation of protein and/or western blot analysis for detection of lysosomal proteins (e.g., cathepsins).

  10. Membrane Cholesterol Regulates Lysosome-Plasma Membrane Fusion Events and Modulates Trypanosoma cruzi Invasion of Host Cells

    PubMed Central

    Hissa, Bárbara; Duarte, Jacqueline G.; Kelles, Ludmila F.; Santos, Fabio P.; del Puerto, Helen L.; Gazzinelli-Guimarães, Pedro H.; de Paula, Ana M.; Agero, Ubirajara; Mesquita, Oscar N.; Guatimosim, Cristina; Chiari, Egler; Andrade, Luciana O.

    2012-01-01

    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 lysosomes are

  11. Alterations in membrane trafficking and pathophysiological implications in lysosomal storage disorders.

    PubMed

    Kuech, Eva-Maria; Brogden, Graham; Naim, Hassan Y

    2016-11-01

    Lysosomal storage disorders are a heterogeneous group of more than 50 distinct inborn metabolic diseases affecting about 1 in 5000 to 7000 live births. The diseases often result from mutations followed by functional deficiencies of enzymes or transporters within the acidic environment of the lysosome, which mediate the degradation of a wide subset of substrates, including glycosphingolipids, glycosaminoglycans, cholesterol, glycogen, oligosaccharides, peptides and glycoproteins, or the export of the respective degradation products from the lysosomes. The progressive accumulation of uncleaved substrates occurs in multiple organs and finally causes a broad spectrum of different pathologies including visceral, neurological, skeletal and hematologic manifestations. Besides deficient lysosomal enzymes and transporters other defects may lead to lysosomal storage disorders, including activator defects, membrane defects or defects in modifier proteins. In this review we concentrate on four different lysosomal storage disorders: Niemann-Pick type C, Fabry disease, Gaucher disease and Pompe disease. While the last three are caused by defective lysosomal hydrolases, Niemann-Pick type C is caused by the inability to export LDL-derived cholesterol out of the lysosome. We want to emphasise potential implications of membrane trafficking defects on the pathology of these diseases, as many mutations interfere with correct lysosomal protein trafficking and alter cellular lipid homeostasis. Current therapeutic strategies are summarised, including substrate reduction therapy as well as pharmacological chaperone therapy which directly aim to improve folding and lysosomal transport of misfolded mutant proteins.

  12. Approaches for plasma membrane wounding and assessment of lysosome-mediated repair responses

    PubMed Central

    Corrotte, M.; Castro-Gomes, T.; Koushik, A.B.; Andrews, N.W.

    2016-01-01

    Rapid plasma membrane repair is essential to restore cellular homeostasis and improve cell survival after injury. Several mechanisms for plasma membrane repair have been proposed, including formation of an intracellular vesicle patch, reduction of plasma membrane tension, lesion removal by endocytosis, and/or shedding of the wounded membrane. Under all conditions studied to date, plasma membrane repair is strictly dependent on the entry of calcium into cells, from the extracellular medium. Calcium-dependent exocytosis of lysosomes is an important early step in the plasma membrane repair process, and defects in plasma membrane repair have been observed in cells carrying mutations responsible for serious lysosomal diseases, such as Chediak–Higashi (Huynh, Roth, Ward, Kaplan, & Andrews, 2004) and Niemann–Pick Disease type A (Tam et al., 2010). A functional role for release of the lysosomal enzyme acid sphingomyelinase, which generates ceramide on the cell surface and triggers endocytosis, has been described (Corrotte et al., 2013; Tam et al., 2010). Therefore, procedures for measuring the extent of lysosomal fusion with the plasma membrane of wounded cells are important indicators of the cellular repair response. The importance of carefully selecting the methodology for experimental plasma membrane injury, in order not to adversely impact the membrane repair machinery, is becoming increasingly apparent. Here, we describe physiologically relevant methods to induce different types of cellular wounds, and sensitive assays to measure the ability of cells to secrete lysosomes and reseal their plasma membrane. PMID:25665445

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

    NASA Astrophysics Data System (ADS)

    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) are created in the plasma membrane in contrast to larger diameter pores (> 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.

  14. Lipophilic cationic drugs increase the permeability of lysosomal membranes in a cell culture system.

    PubMed

    Kornhuber, Johannes; Henkel, Andreas W; Groemer, Teja W; Städtler, Sven; Welzel, Oliver; Tripal, Philipp; Rotter, Andrea; Bleich, Stefan; Trapp, Stefan

    2010-07-01

    Lysosomes accumulate many drugs several fold higher compared to their extracellular concentration. This mechanism is believed to be responsible for many pharmacological effects. So far, uptake and release kinetics are largely unknown and interactions between concomitantly administered drugs often provoke mutual interference. In this study, we addressed these questions in a cell culture model. The molecular mechanism for lysosomal uptake kinetics was analyzed by live cell fluorescence microscopy in SY5Y cells using four drugs (amantadine, amitriptyline, cinnarizine, flavoxate) with different physicochemical properties. Drugs with higher lipophilicity accumulated more extensively within lysosomes, whereas a higher pK(a) value was associated with a more rapid uptake. The drug-induced displacement of LysoTracker was neither caused by elevation of intra-lysosomal pH, nor by increased lysosomal volume. We extended our previously developed numerical single cell model by introducing a dynamic feedback mechanism. The empirical data were in good agreement with the results obtained from the numerical model. The experimental data and results from the numerical model lead to the conclusion that intra-lysosomal accumulation of lipophilic xenobiotics enhances lysosomal membrane permeability. Manipulation of lysosomal membrane permeability might be useful to overcome, for example, multi-drug resistance by altering subcellular drug distribution.

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

    PubMed Central

    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-fold) and 13 down-regulated (2.0-fold) spots in relation to melanin exposure. Conclusions It has been found that lysosomal membrane proteins are associated with melanin to decolorize and quantity through cellular activation of lysosome. PMID:27158002

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

    PubMed

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

    2016-04-25

    Ginsenoside Rh2 is a potential pharmacologically active metabolite of ginseng. Previously, we have reported that an octyl ester derivative of ginsenoside Rh2 (Rh2-O), has been confirmed to possess higher bioavailability and anticancer effect than Rh2 in vitro. In order to better assess the possibility that Rh2-O could be used as an anticancer compound, the underlying mechanism was investigated in this study. The present results revealed that lysosomal destabilization was involved in the early stage of cell apoptosis in HepG2 cells induced by Rh2-O. Rh2-O could induce an early lysosomal membrane permeabilization with the release of lysosomal protease cathepsins to the cytosol in HepG2 cells. The Cat B inhibitor (leu) and Cat D inhibitor (pepA) inhibited Rh2-O-induced HepG2 apoptosis as well as tBid production and Δφm depolarization, indicating that lysosomal permeabilization occurred upstream of mitochondrial dysfunction. In addition, Rh2-O induced a significant increase in the protein levels of DRAM1 and Bax (p < 0.05) in lysosomes of HepG2 cells. Knockdown of Bax partially inhibited Rh2-O-induced Cat D release from lysosomes. Thus it was concluded that Rh2-O induced apoptosis of HepG2 cells through activation of the lysosomal-mitochondrial apoptotic pathway involving the translocation of Bax to the lysosome.

  17. Transport of the lysosomal membrane glycoprotein lgp120 (lgp-A) to lysosomes does not require appearance on the plasma membrane

    PubMed Central

    1992-01-01

    We have used stably transfected CHO cell lines to characterize the pathway of intracellular transport of the lgp120 (lgp-A) to lysosomes. Using several surface labeling and internalization assays, our results suggest that lgp120 can reach its final destination with or without prior appearance on the plasma membrane. The extent to which lgp120 was transported via the cell surface was determined by two factors: expression level and the presence of a conserved glycine-tyrosine motif in the cytoplasmic tail. In cells expressing low levels of wild-type lgp120, the majority of newly synthesized molecules reached lysosomes without becoming accessible to antibody or biotinylation reagents added extracellularly at 4 degrees C. With increased expression levels, however, an increased fraction of transfected lgp120, as well as some endogenous lgp-B, appeared on the plasma membrane. The fraction of newly synthesized lgp120 reaching the cell surface was also increased by mutations affecting the cytoplasmic domain tyrosine or glycine residues. A substantial fraction of both mutants reached the surface even at low expression levels. However, only the lgp120G----A7 mutant was rapidly internalized and delivered from the plasma membrane to lysosomes. Taken together, our results show that the majority of newly synthesized wild-type lgp120 does not appear to pass through the cell surface en route to lysosomes. Instead, it is likely that lysosomal targeting involves a saturable intracellular sorting site whose affinity for lgp's is dependent on a glycine-tyrosine motif in the lgp120 cytoplasmic tail. PMID:1560028

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

    PubMed

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

    1993-05-01

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

  19. [Cestode lysosomes].

    PubMed

    Smirnov, L P; Bogdan, V V

    1989-01-01

    By differential centrifugation method a lysosomal fraction was obtained from five species of cestodes, which possesses the highest specific activity of acidic phosphatases as compared to other subcellular fractions. By isopyknic centrifugation in the density gradient of saccharose the lysosomal fraction is divided into primary and secondary lysosomes. Lysosomes of cestodes are similar to those of vertebrate animals in the character of fractional distribution of acidic phosphatase, sedimentation abilities and sensitivity of membranes to triton X-100.

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

    SciTech Connect

    Magini, Alessandro; Polchi, Alice; Urbanelli, Lorena; Cesselli, Daniela; Beltrami, Antonio; Tancini, Brunella; Emiliani, Carla

    2013-10-18

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

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

    PubMed

    Einck, K H; Norkin, L C

    1979-01-01

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

  2. Increased expression of lysosome membrane protein 2 in glomeruli of patients with idiopathic membranous nephropathy.

    PubMed

    Rood, Ilse M; Merchant, Michael L; Wilkey, Daniel W; Zhang, Terry; Zabrouskov, Vlad; van der Vlag, Johan; Dijkman, Henry B; Willemsen, Brigith K; Wetzels, Jack F; Klein, Jon B; Deegens, Jeroen K

    2015-11-01

    Urinary microvesicles constitute a rich source of membrane-bound and intracellular proteins that may provide important clues of pathophysiological mechanisms in renal disease. In the current study, we analyzed and compared the proteome of urinary microvesicles from patients with idiopathic membranous nephropathy (iMN), idiopathic focal segmental glomerulosclerosis (iFSGS), and normal controls using an approach that combined both proteomics and pathology analysis. Lysosome membrane protein-2 (LIMP-2) was increased greater than twofold in urinary microvesicles obtained from patients with iMN compared to microvesicles of patients with iFSGS and normal controls. Immunofluorescence analysis of renal biopsies confirmed our proteomics findings that LIMP-2 was upregulated in glomeruli from patients with iMN but not in glomeruli of diseased patients (iFSGS, minimal change nephropathy, IgA nephropathy, membranoproliferative glomerulonephritis) and normal controls. Confocal laser microscopy showed co-localization of LIMP-2 with IgG along the glomerular basement membrane. Serum antibodies against LIMP-2 could not be detected. In conclusion, our data show the value of urinary microvesicles in biomarker discovery and provide evidence for de novo expression of LIMP-2 in glomeruli of patients with iMN.

  3. Mechanism of Aloe Vera extract protection against UVA: shelter of lysosomal membrane avoids photodamage.

    PubMed

    Rodrigues, Daniela; Viotto, Ana Cláudia; Checchia, Robert; Gomide, Andreza; Severino, Divinomar; Itri, Rosangela; Baptista, Maurício S; Martins, Waleska Kerllen

    2016-03-01

    The premature aging (photoaging) of skin characterized by wrinkles, a leathery texture and mottled pigmentation is a well-documented consequence of exposure to sunlight. UVA is an important risk factor for human cancer also associated with induction of inflammation, immunosuppression, photoaging and melanogenesis. Although herbal compounds are commonly used as photoprotectants against the harmful effects of UVA, the mechanisms involved in the photodamage are not precisely known. In this study, we investigated the effects of Aloe Vera (Aloe barbadensis mil) on the protection against UVA-modulated cell killing of HaCaT keratinocytes. Aloe Vera exhibited the remarkable ability of reducing both in vitro and in vivo photodamage, even though it does not have anti-radical properties. Interestingly, the protection conferred by Aloe Vera was associated with the maintenance of membrane integrity in both mimetic membranes and intracellular organelles. The increased lysosomal stability led to a decrease in lipofuscinogenesis and cell death. This study explains why Aloe Vera extracts offer protection against photodamage at a cellular level in both the UV and visible spectra, leading to its beneficial use as a supplement in protective dermatological formulations.

  4. Ethambutol-induced toxicity is mediated by zinc and lysosomal membrane permeabilization in cultured retinal cells

    SciTech Connect

    Chung, Hyewon; Yoon, Young Hee; Hwang, Jung Jin; Cho, Kyung Sook; Koh, Jae Young; Kim, June-Gone

    2009-03-01

    Ethambutol, an efficacious antituberculosis agent, can cause irreversible visual loss in a small but significant fraction of patients. However, the mechanism of ocular toxicity remains to be established. We previously reported that ethambutol caused severe vacuole formation in cultured retinal cells, and that the addition of zinc along with ethambutol aggravated vacuole formation whereas addition of the cell-permeable zinc chelator, N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN), reduced vacuole formation. To investigate the origin of vacuoles and to obtain an understanding of drug toxicity, we used cultured primary retinal cells from newborn Sprague-Dawley rats and imaged ethambutol-treated cells stained with FluoZin-3, zinc-specific fluorescent dye, under a confocal microscope. Almost all ethambutol-induced vacuoles contained high levels of labile zinc. Double staining with LysoTracker or MitoTracker revealed that almost all zinc-containing vacuoles were lysosomes and not mitochondria. Intracellular zinc chelation with TPEN markedly blocked both vacuole formation and zinc accumulation in the vacuole. Immunocytochemistry with antibodies to lysosomal-associated membrane protein-2 (LAMP-2) and cathepsin D, an acid lysosomal hydrolase, disclosed lysosomal activation after exposure to ethambutol. Immunoblotting after 12 h exposure to ethambutol showed that cathepsin D was released into the cytosol. In addition, cathepsin inhibitors attenuated retinal cell toxicity induced by ethambutol. This is consistent with characteristics of lysosomal membrane permeabilization (LMP). TPEN also inhibited both lysosomal activation and LMP. Thus, accumulation of zinc in lysosomes, and eventual LMP, may be a key mechanism of ethambutol-induced retinal cell death.

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

    PubMed

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

    2016-12-18

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

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

    PubMed

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

    2011-02-18

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

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

    PubMed

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

    2013-01-01

    Targeting of lysosomes is a novel therapeutic anti-cancer strategy for killing the otherwise apoptosis-resistant cancer cells. Such strategies are urgently needed for treatment of brain tumors, especially the glioblastoma, which is the most frequent and most malignant type. The aim of the present study was to investigate the presence of lysosomes in astrocytic brain tumors focussing also on the therapy resistant tumor stem cells. Expression of the lysosomal marker LAMP-1 (lysosomal-associated membrane protein-1) was investigated by immunohistochemistry in 112 formalin fixed paraffin embedded astrocytomas and compared with tumor grade and overall patient survival. Moreover, double immunofluorescence stainings were performed with LAMP-1 and the astrocytic marker GFAP and the putative stem cell marker CD133 on ten glioblastomas. Most tumors expressed the LAMP-1 protein in the cytoplasm of the tumor cells, while the blood vessels were positive in all tumors. The percentage of LAMP-1 positive tumor cells and staining intensities increased with tumor grade but variations in tumors of the same grade were also found. No association was found between LAMP-1 expression and patient overall survival in the individual tumor grades. LAMP-1/GFAP showed pronounced co-expression and LAMP-1/CD133 was co-expressed as well suggesting that tumor cells including the proposed tumor stem cells contain lysosomes. The results suggest that high amounts of lysosomes are present in glioblastomas and in the proposed tumor stem cells. Targeting of lysosomes may be a promising novel therapeutic strategy against this highly malignant neoplasm.

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

    SciTech Connect

    Bame, K.J.

    1986-01-01

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

  9. Brucella suis-Impaired Specific Recognition of Phagosomes by Lysosomes due to Phagosomal Membrane Modifications

    PubMed Central

    Naroeni, Aroem; Jouy, Nicolas; Ouahrani-Bettache, Safia; Liautard, Jean-Pierre; Porte, Françoise

    2001-01-01

    Brucella species are gram-negative, facultatively intracellular bacteria that infect humans and animals. These organisms can survive and replicate within a membrane-bound compartment in phagocytic and nonprofessional phagocytic cells. Inhibition of phagosome-lysosome fusion has been proposed as a mechanism for intracellular survival in both types of cells. However, the biochemical mechanisms and microbial factors implicated in Brucella maturation are still completely unknown. We developed two different approaches in an attempt to gain further insight into these mechanisms: (i) a fluorescence microscopy analysis of general intracellular trafficking on whole cells in the presence of Brucella and (ii) a flow cytometry analysis of in vitro reconstitution assays showing the interaction between Brucella suis-containing phagosomes and lysosomes. The fluorescence microscopy results revealed that fusion properties of latex bead-containing phagosomes with lysosomes were not modified in the presence of live Brucella suis in the cells. We concluded that fusion inhibition was restricted to the pathogen phagosome and that the host cell fusion machinery was not altered by the presence of live Brucella in the cell. By in vitro reconstitution experiments, we observed a specific association between killed B. suis-containing phagosomes and lysosomes, which was dependent on exogenously supplied cytosol, energy, and temperature. This association was observed with killed bacteria but not with live bacteria. Hence, this specific recognition inhibition seemed to be restricted to the pathogen phagosomal membrane, as noted in the in vivo experiments. PMID:11119541

  10. How and why intralumenal membrane fragments form during vacuolar lysosome fusion

    PubMed Central

    Mattie, Sevan; McNally, Erin K.; Karim, Mahmoud A.; Vali, Hojatollah; Brett, Christopher L.

    2017-01-01

    Lysosomal membrane fusion mediates the last step of the autophagy and endocytosis pathways and supports organelle remodeling and biogenesis. Because fusogenic proteins and lipids concentrate in a ring at the vertex between apposing organelle membranes, the encircled area of membrane can be severed and internalized within the lumen as a fragment upon lipid bilayer fusion. How or why this intralumenal fragment forms during fusion, however, is not entirely clear. To better understand this process, we studied fragment formation during homotypic vacuolar lysosome membrane fusion in Saccharomyces cerevisiae. Using cell-free fusion assays and light microscopy, we find that GTPase activation and trans-SNARE complex zippering have opposing effects on fragment formation and verify that this affects the morphology of the fusion product and regulates transporter protein degradation. We show that fragment formwation is limited by stalk expansion, a key intermediate of the lipid bilayer fusion reaction. Using electron microscopy, we present images of hemifusion diaphragms that form as stalks expand and propose a model describing how the fusion machinery regulates fragment formation during lysosome fusion to control morphology and protein lifetimes. PMID:27881666

  11. Phase coexistence in a triolein-phosphatidylcholine system. Implications for lysosomal membrane properties.

    PubMed

    Pakkanen, Kirsi I; Duelund, Lars; Vuento, Matti; Ipsen, John Hjort

    2010-02-01

    The effects of tri- and monoglycerides on phospholipid (POPC) membranes were studied using spectroscopical methods. Triolein was found to form two types of POPC-rich membranes, both with POPC or as a three-component system with monopalmitin. These two membrane types were determined as co-existing phases based on their spontaneous and stable separation and named heavy and light phase according to their sedimentation behaviour. Marked differences were seen in the physical properties of these phases, even though only minor compositional variation was detected. The light, less polar phase was found to be less ordered and more fluid and seemed to allow significantly lower amount of water penetration into the membrane-water interface than pure POPC membrane. The heavy phase, apart from their slightly altered water penetration, resembled more a pure POPC membrane. As triglycerides are present in lysosomal membranes, the present results can be seen as an implication for polarity-based water permeability barrier possibly contributing to the integrity of lysosomes.

  12. Lysosomal Membrane Permeabilization is an Early Event in Sigma-2 Receptor Ligand Mediated Cell Death in Pancreatic Cancer

    PubMed Central

    2012-01-01

    Background Sigma-2 receptor ligands have been studied for treatment of pancreatic cancer because they are preferentially internalized by proliferating cells and induce apoptosis. This mechanism of apoptosis is poorly understood, with varying reports of caspase-3 dependence. We evaluated multiple sigma-2 receptor ligands in this study, each shown to decrease tumor burden in preclinical models of human pancreatic cancer. Results Fluorescently labeled sigma-2 receptor ligands of two classes (derivatives of SW43 and PB282) localize to cell membrane components in Bxpc3 and Aspc1 pancreatic cancer cells and accumulate in lysosomes. We found that interactions in the lysosome are critical for cell death following sigma-2 ligand treatment because selective inhibition of a protective lysosomal membrane glycoprotein, LAMP1, with shRNA greatly reduced the viability of cells following treatment. Sigma-2 ligands induced lysosomal membrane permeabilization (LMP) and protease translocation triggering downstream effectors of apoptosis. Subsequently, cellular oxidative stress was greatly increased following treatment with SW43, and the hydrophilic antioxidant N-acetylcysteine (NAC) gave greater protection against this than a lipophilic antioxidant, α-tocopherol (α-toco). Conversely, PB282-mediated cytotoxicity relied less on cellular oxidation, even though α-toco did provide protection from this ligand. In addition, we found that caspase-3 induction was not as significantly inhibited by cathepsin inhibitors as by antioxidants. Both NAC and α-toco protected against caspase-3 induction following PB282 treatment, while only NAC offered protection following SW43 treatment. The caspase-3 inhibitor DEVD-FMK offered significant protection from PB282, but not SW43. Conclusions Sigma-2 ligand SW43 commits pancreatic cancer cells to death by a caspase-independent process involving LMP and oxidative stress which is protected from by NAC. PB282 however undergoes a caspase-dependent death

  13. 58-F, a flavanone from Ophiopogon japonicus, prevents hepatocyte death by decreasing lysosomal membrane permeability

    PubMed Central

    Yan, Xiaofeng; Ye, Tingjie; Hu, Xudong; Zhao, Pei; Wang, Xiaoling

    2016-01-01

    Lysosome membrane permeabilization (LMP) has been implicated in cell death. In the present study, we investigated the relationship between cell death and H2O2-/CCl4-induced LMP in hepatocytes in vitro and following acute liver injury in vivo. The key finding was that H2O2 triggered LMP by oxidative stress, as evidenced by a suppression of LAMP1 expression, a reduction in LysoTracker Green and AO staining, and the leakage of proton and cathepsin B/D from the lysosome to the cytoplasm, resulting in cell death. CCl4 also triggered hepatocyte death by decreasing lysosome LAMP1 expression and by inducing the accumulation of products of peroxidative lipids and oxidized proteins. Furthermore, a novel compound 5,8-dimethoxy-6-methyl-7-hydroxy-3-3(2-hydroxy-4-methoxybenzyl) chroman-4-one (58-F) was extracted from Ophiopogon japonicus and served as a potential therapeutic drug. In vivo and in vitro results showed that 58-F effectively rescued hepatocytes by decreasing LMP and by inducing lysosomal enzyme translocation to the cytosol. PMID:27306715

  14. Kinetic evidence that newly-synthesized endogenous lysosome-associated membrane protein-1 (LAMP-1) first transits early endosomes before it is delivered to lysosomes.

    PubMed

    Ebrahim, Roshan; Thilo, Lutz

    2011-05-01

    After de novo synthesis of lysosome-associated membrane proteins (LAMPs), they are sorted in the trans-Golgi network (TGN) for delivery to lysosomes. Opposing views prevail on whether LAMPs are targeted to lysosomes directly, or indirectly via prelysosomal stages of the endocytic pathway, in particular early endosomes. Conflicting evidence is based on kinetic measurements with too limited quantitative data for sufficient temporal and organellar resolution. Using cells of the mouse macrophage cell line, P338D(1), this study presents detailed kinetic data that describe the extent of, and time course for, the appearance of newly-synthesized LAMP-1 in organelles of the endocytic pathway, which had been loaded selectively with horse-radish peroxidase (HRP) by appropriate periods of endocytosis. After a 5-min pulse of metabolic labelling, LAMP-1 was trapped in the respective organelles by HRP-catalyzed crosslinking with membrane-permeable diaminobenzidine (DAB). These kinetic observations provide sufficient quantitative evidence that in P338D(1) cells the bulk of newly-synthesized endogenous LAMP-1 first appeared in early endosomes, before it was delivered to late endosomes and lysosomes about 25 min later.

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

    PubMed

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

    2013-11-01

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

  16. Characterization and cloning of lgp110, a lysosomal membrane glycoprotein from mouse and rat cells.

    PubMed

    Granger, B L; Green, S A; Gabel, C A; Howe, C L; Mellman, I; Helenius, A

    1990-07-15

    lgp110 is a heavily glycosylated intrinsic protein of lysosomal membranes. Initially defined by monoclonal antibodies against mouse liver lysosomes, it consists of a 45-kilodalton core polypeptide with O-linked and 17 asparagine-linked oligosaccharide side chains in mouse cells. Sialic acid residues make the mature protein extremely acidic, with an isoelectric point of between 2 and 4 in both normal tissues and most cultured cell lines. Partial sequencing of mouse lgp110 allowed oligonucleotide probes to be constructed for the screening of several mouse cDNA libraries. A partial cDNA clone for mouse lgp110 was found and used for additional library screening, generating a cDNA clone covering all of the coding sequence of mature rat lgp110 as well as genomic clones covering most of the mouse gene. These new clones bring to seven the number of lysosomal membrane proteins whose amino acid sequences can be deduced, and two distinct but highly similar groups (designated lgp-A and lgp-B) can now be defined. Sequence comparisons suggest that differences within each group reflect species variations of the same protein and that lgp-A and lgp-B probably diverged from a common ancestor prior to the evolup4f1ary divergence of birds and mammals. Individual cells and individual lysosomes possess both lgp-A and lgp-B, suggesting that these two proteins have different functions. Mouse lgp110 is encoded by at least seven exons; intron positions suggest that the two homologous ectodomains of each lgp arose through gene duplication.

  17. Pneumolysin activates macrophage lysosomal membrane permeabilization and executes apoptosis by distinct mechanisms without membrane pore formation.

    PubMed

    Bewley, Martin A; Naughton, Michael; Preston, Julie; Mitchell, Andrea; Holmes, Ashleigh; Marriott, Helen M; Read, Robert C; Mitchell, Timothy J; Whyte, Moira K B; Dockrell, David H

    2014-10-07

    Intracellular killing of Streptococcus pneumoniae is complemented by induction of macrophage apoptosis. Here, we show that the toxin pneumolysin (PLY) contributes both to lysosomal/phagolysosomal membrane permeabilization (LMP), an upstream event programing susceptibility to apoptosis, and to apoptosis execution via a mitochondrial pathway, through distinct mechanisms. PLY is necessary but not sufficient for the maximal induction of LMP and apoptosis. PLY's ability to induce both LMP and apoptosis is independent of its ability to form cytolytic pores and requires only the first three domains of PLY. LMP involves TLR (Toll-like receptor) but not NLRP3/ASC (nucleotide-binding oligomerization domain [Nod]-like receptor family, pyrin domain-containing protein 3/apoptosis-associated speck-like protein containing a caspase recruitment domain) signaling and is part of a PLY-dependent but phagocytosis-independent host response that includes the production of cytokines, including interleukin-1 beta (IL-1β). LMP involves progressive and selective permeability to 40-kDa but not to 250-kDa fluorescein isothiocyanate (FITC)-labeled dextran, as PLY accumulates in the cytoplasm. In contrast, the PLY-dependent execution of apoptosis requires phagocytosis and is part of a host response to intracellular bacteria that also includes NO generation. In cells challenged with PLY-deficient bacteria, reconstitution of LMP using the lysomotrophic detergent LeuLeuOMe favored cell necrosis whereas PLY reconstituted apoptosis. The results suggest that PLY contributes to macrophage activation and cytokine production but also engages LMP. Following bacterial phagocytosis, PLY triggers apoptosis and prevents macrophage necrosis as a component of a broad-based antimicrobial strategy. This illustrates how a key virulence factor can become the focus of a multilayered and coordinated innate response by macrophages, optimizing pathogen clearance and limiting inflammation. Importance: Streptococcus

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

    PubMed

    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.

  19. LAMP-3 (Lysosome-Associated Membrane Protein 3) Promotes the Intracellular Proliferation of Salmonella typhimurium

    PubMed Central

    Lee, Eun-Ju; Park, Kwan-Sik; Jeon, In-Sook; Choi, Jae-Woon; Lee, Sang-Jeon; Choy, Hyun E.; Song, Ki-Duk; Lee, Hak-Kyo; Choi, Joong-Kook

    2016-01-01

    Lysosomes are cellular organelles containing diverse classes of catabolic enzymes that are implicated in diverse cellular processes including phagocytosis, autophagy, lipid transport, and aging. Lysosome-associated membrane proteins (LAMP-1 and LAMP-2) are major glycoproteins important for maintaining lysosomal integrity, pH, and catabolism. LAMP-1 and LAMP-2 are constitutively expressed in Salmonella-infected cells and are recruited to Salmonella-containing vacuoles (SCVs) as well as Salmonella-induced filaments (Sifs) that promote the survival and proliferation of the Salmonella. LAMP-3, also known as DC-LAMP/CD208, is a member of the LAMP family of proteins, but its role during Salmonella infection remains unclear. DNA microarray analysis identified LAMP-3 as one of the genes responding to LPS stimulation in THP-1 macrophage cells. Subsequent analyses reveal that LPS and Salmonella induced the expression of LAMP-3 at both the transcriptional and translational levels. Confocal Super resolution N-SIM imaging revealed that LAMP-3, like LAMP-2, shifts its localization from the cell surface to alongside Salmonella. Knockdown of LAMP-3 by specific siRNAs decreased the number of Salmonella recovered from the infected cells. Therefore, we conclude that LAMP-3 is induced by Salmonella infection and recruited to the Salmonella pathogen for intracellular proliferation. PMID:27329040

  20. LAMP-3 (Lysosome-Associated Membrane Protein 3) Promotes the Intracellular Proliferation of Salmonella typhimurium.

    PubMed

    Lee, Eun-Ju; Park, Kwan-Sik; Jeon, In-Sook; Choi, Jae-Woon; Lee, Sang-Jeon; Choy, Hyun E; Song, Ki-Duk; Lee, Hak-Kyo; Choi, Joong-Kook

    2016-07-01

    Lysosomes are cellular organelles containing diverse classes of catabolic enzymes that are implicated in diverse cellular processes including phagocytosis, autophagy, lipid transport, and aging. Lysosome-associated membrane proteins (LAMP-1 and LAMP-2) are major glycoproteins important for maintaining lysosomal integrity, pH, and catabolism. LAMP-1 and LAMP-2 are constitutively expressed in Salmonella-infected cells and are recruited to Salmonella-containing vacuoles (SCVs) as well as Salmonella-induced filaments (Sifs) that promote the survival and proliferation of the Salmonella. LAMP-3, also known as DC-LAMP/CD208, is a member of the LAMP family of proteins, but its role during Salmonella infection remains unclear. DNA microarray analysis identified LAMP-3 as one of the genes responding to LPS stimulation in THP-1 macrophage cells. Subsequent analyses reveal that LPS and Salmonella induced the expression of LAMP-3 at both the transcriptional and translational levels. Confocal Super resolution N-SIM imaging revealed that LAMP-3, like LAMP-2, shifts its localization from the cell surface to alongside Salmonella. Knockdown of LAMP-3 by specific siRNAs decreased the number of Salmonella recovered from the infected cells. Therefore, we conclude that LAMP-3 is induced by Salmonella infection and recruited to the Salmonella pathogen for intracellular proliferation.

  1. Membrane Cholesterol Removal Changes Mechanical Properties of Cells and Induces Secretion of a Specific Pool of Lysosomes

    PubMed Central

    Roma, Paula Magda S.; Alves, Ana Paula; Rocha, Carolina D.; Valverde, Thalita M.; Aguiar, Pedro Henrique N.; Almeida, Fernando P.; Guimarães, Allan J.; Guatimosim, Cristina; Silva, Aristóbolo M.; Fernandes, Maria C.; Andrews, Norma W.; Viana, Nathan B.; Mesquita, Oscar N.; Agero, Ubirajara; Andrade, Luciana O.

    2013-01-01

    In a previous study we had shown that membrane cholesterol removal induced unregulated lysosomal exocytosis events leading to the depletion of lysosomes located at cell periphery. However, the mechanism by which cholesterol triggered these exocytic events had not been uncovered. In this study we investigated the importance of cholesterol in controlling mechanical properties of cells and its connection with lysosomal exocytosis. Tether extraction with optical tweezers and defocusing microscopy were used to assess cell dynamics in mouse fibroblasts. These assays showed that bending modulus and surface tension increased when cholesterol was extracted from fibroblasts plasma membrane upon incubation with MβCD, and that the membrane-cytoskeleton relaxation time increased at the beginning of MβCD treatment and decreased at the end. We also showed for the first time that the amplitude of membrane-cytoskeleton fluctuation decreased during cholesterol sequestration, showing that these cells become stiffer. These changes in membrane dynamics involved not only rearrangement of the actin cytoskeleton, but also de novo actin polymerization and stress fiber formation through Rho activation. We found that these mechanical changes observed after cholesterol sequestration were involved in triggering lysosomal exocytosis. Exocytosis occurred even in the absence of the lysosomal calcium sensor synaptotagmin VII, and was associated with actin polymerization induced by MβCD. Notably, exocytosis triggered by cholesterol removal led to the secretion of a unique population of lysosomes, different from the pool mobilized by actin depolymerizing drugs such as Latrunculin-A. These data support the existence of at least two different pools of lysosomes with different exocytosis dynamics, one of which is directly mobilized for plasma membrane fusion after cholesterol removal. PMID:24376622

  2. Effect of aloe vera leaf gel extract on membrane bound phosphatases and lysosomal hydrolases in rats with streptozotocin diabetes.

    PubMed

    Rajasekaran, S; Sriram, N; Arulselvan, P; Subramanian, S

    2007-03-01

    Diabetes mellitus is known to promote deterioration of membrane function and impair intra cellular metabolism in the organism. The aim of the present study was to examine the effect of the ethanolic extract from Aloe vera leaf gel on membrane bound phosphatases and lysosomal hydrolases in the liver and kidney of streptozotocin (STZ)-induced diabetic rats. The rats treated with STZ showed significant alterations in the activities of membrane bound phosphatases and lysosomal hydrolases in the liver and kidney. Oral administration of Aloe vera gel extract at a dose of 300 mg/kg body weight/day to STZ-induced diabetic rats for a period of 21 days significantly restored the alterations in enzymes activity to near normalcy. These results were compared with glibenclamide, a reference drug. Thus, the present study confirms that Aloe vera gel extract possesses a significant beneficial effect on membrane bound phosphatases and lysosomal hydrolases.

  3. Effects of pH and Iminosugar Pharmacological Chaperones on Lysosomal Glycosidase Structure and Stability

    SciTech Connect

    Lieberman, Raquel L.; D’aquino, J. Alejandro; Ringe, Dagmar; Petsko, Gregory A.

    2009-06-05

    Human lysosomal enzymes acid-{beta}-glucosidase (GCase) and acid-{alpha}-galactosidase ({alpha}-Gal A) hydrolyze the sphingolipids glucosyl- and globotriaosylceramide, respectively, and mutations in these enzymes lead to the lipid metabolism disorders Gaucher and Fabry disease, respectively. We have investigated the structure and stability of GCase and {alpha}-Gal A in a neutral-pH environment reflective of the endoplasmic reticulum and an acidic-pH environment reflective of the lysosome. These details are important for the development of pharmacological chaperone therapy for Gaucher and Fabry disease, in which small molecules bind mutant enzymes in the ER to enable the mutant enzyme to meet quality control requirements for lysosomal trafficking. We report crystal structures of apo GCase at pH 4.5, at pH 5.5, and in complex with the pharmacological chaperone isofagomine (IFG) at pH 7.5. We also present thermostability analysis of GCase at pH 7.4 and 5.2 using differential scanning calorimetry. We compare our results with analogous experiments using {alpha}-Gal A and the chaperone 1-deoxygalactonijirimycin (DGJ), including the first structure of {alpha}-Gal A with DGJ. Both GCase and {alpha}-Gal A are more stable at lysosomal pH with and without their respective iminosugars bound, and notably, the stability of the GCase-IFG complex is pH sensitive. We show that the conformations of the active site loops in GCase are sensitive to ligand binding but not pH, whereas analogous galactose- or DGJ-dependent conformational changes in {alpha}-Gal A are not seen. Thermodynamic parameters obtained from {alpha}-Gal A unfolding indicate two-state, van't Hoff unfolding in the absence of the iminosugar at neutral and lysosomal pH, and non-two-state unfolding in the presence of DGJ. Taken together, these results provide insight into how GCase and {alpha}-Gal A are thermodynamically stabilized by iminosugars and suggest strategies for the development of new pharmacological

  4. Membrane Stability Testing

    SciTech Connect

    Hobbs, D.T.

    1997-09-30

    The Electrosynthesis Co. Inc. (ESC) was contracted by the Westinghouse Savannah River Company to investigate the long term performance and durability of cell components (anode, membrane, cathode) in an electrochemical caustic recovery process using a simulated SRC liquid waste as anolyte solution. This report details the results of two long-term studies conducted using an ICI FM01 flow cell. This cell is designed and has previously been demonstrated to scale up directly into the commercial scale ICI FM21 cell.

  5. Lysosomal integral membrane protein 2 (LIMP-2) restricts the invasion of Trypanosoma cruzi extracellular amastigotes through the activity of the lysosomal enzyme β-glucocerebrosidase.

    PubMed

    Gonçalves, Viviane Martinelli; D'Almeida, Vânia; Müller, Karen Barbosa; Real, Fernando; Mortara, Renato Arruda

    2014-03-01

    Lysosomal integral membrane protein 2 (LIMP-2, SCARB2) is directly linked to β-glucocerebrosidase enzyme (βGC) and mediates the transport of this enzyme from the Golgi complex to lysosomes. Active βGC cleaves the β-glycosidic linkages of glucosylceramide, an intermediate in the metabolism of sphingoglycolipids, generating ceramide. In this study we used mouse embryonic fibroblasts (MEFs) deficient for LIMP-2 and observed that these cells were more susceptible to infection by extracellular amastigotes of the protozoan parasite Trypanosoma cruzi when compared to wild-type (WT) fibroblasts. The absence of LIMP-2 decreases the activity of βGC measured in fibroblast extracts. Replacement of βGC enzyme in LIMP-2 deficient fibroblasts restores the infectivity indices to those of WT cells in T. cruzi invasion assays. Considering the participation of βGC in the production of host cell ceramide, we propose that T. cruzi extracellular amastigotes are more invasive to cells deficient in this membrane component. These results contribute to our understanding of the role of host cell lysosomal components in T. cruzi invasion.

  6. Membrane-active antimicrobial peptides and human placental lysosomal extracts are highly active against mycobacteria.

    PubMed

    Jena, Prajna; Mishra, Bibhuti; Leippe, Matthias; Hasilik, Andrej; Griffiths, Gareth; Sonawane, Avinash

    2011-05-01

    Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, manifests discreet strategies to subvert host immune responses, which enable the pathogen to survive and multiply inside the macrophages. This problem is further worsened by the emergence of multidrug resistant mycobacterial strains, which make most of the anti-tuberculous drugs ineffective. It is thus imperative to search for and design better therapeutic strategies, including employment of new antibiotics. Recently, naturally produced antimicrobial molecules such as enzymes, peptides and their synthetic analogs have emerged as compounds with potentially significant therapeutical applications. Although, many antimicrobial peptides have been identified only very few of them have been tested against mycobacteria. A major limitation in using peptides as therapeutics is their sensitivity to enzymatic degradation or inactivity under certain physiological conditions such as relatively high salt concentration. Here, we show that NK-2, a peptide representing the cationic core region of the lymphocytic effector protein NK-lysin, and Ci-MAM-A24, a synthetic salt-tolerant peptide derived from immune cells of Ciona intestinalis, efficiently kill Mycobacterium smegmatis and Mycobacterium bovis-BCG. In addition, NK-2 and Ci-MAM-A24 showed a synergistic killing effect against M. smegmatis, no cytotoxic effect on mouse macrophages at bactericidal concentrations, and were even found to kill mycobacteria residing inside the macrophages. We also show that human placental lysosomal contents exert potent killing effect against mycobacteria under acidic and reducing growth conditions. Electron microscopic studies demonstrate that the lysosomal extract disintegrate bacterial cell membrane resulting in killing of mycobacteria.

  7. Azadirachtin-induced apoptosis involves lysosomal membrane permeabilization and cathepsin L release in Spodoptera frugiperda Sf9 cells.

    PubMed

    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.

  8. Iron-Mediated Lysosomal Membrane Permeabilization in Ethanol-Induced Hepatic Oxidative Damage and Apoptosis: Protective Effects of Quercetin.

    PubMed

    Li, Yanyan; Chen, Man; Xu, Yanyan; Yu, Xiao; Xiong, Ting; Du, Min; Sun, Jian; Liu, Liegang; Tang, Yuhan; Yao, Ping

    2016-01-01

    Iron, in its free ferrous states, can catalyze Fenton reaction to produce OH∙, which is recognized as a crucial role in the pathogenesis of alcoholic liver diseases (ALD). As a result of continuous decomposition of iron-containing compounds, lysosomes contain a pool of redox-active iron. To investigate the important role of intralysosomal iron in alcoholic liver injury and the potential protection of quercetin, male C57BL/6J mice fed by Lieber De Carli diets containing ethanol (30% of total calories) were cotreated by quercetin or deferoxamine (DFO) for 15 weeks and ethanol-incubated mice primary hepatocytes were pretreated with FeCl3, DFO, and bafilomycin A1 at their optimal concentrations and exposure times. Chronic ethanol consumption caused an evident increase in lysosomal redox-active iron accompanying sustained oxidative damage. Iron-mediated ROS could trigger lysosomal membrane permeabilization (LMP) and subsequent mitochondria apoptosis. The hepatotoxicity was attenuated by reducing lysosomal iron while being exacerbated by escalating lysosomal iron. Quercetin substantially alleviated the alcoholic liver oxidative damage and apoptosis by decreasing lysosome iron and ameliorating iron-mediated LMP, which provided a new prospective of the use of quercetin against ALD.

  9. Relationship between lysosomal membrane destabilization and chemical body burden in eastern oysters (Crassostrea virginica) from Galveston Bay, Texas, USA.

    PubMed

    Hwang, Hyun-Min; Wade, Terry L; Sericano, Jose L

    2002-06-01

    Lysosomal destabilization was measured by using hemocytes of eastern oysters (Crassostrea virginica) collected along a chemical concentration gradient in Galveston Bay, Texas, USA. Results of the lysosomal response were compared to concentrations of organic compounds and trace elements in oyster tissue. Concentrations (on a dry-wt basis) ranged from 288 to 2,390 ng/g for polycyclic aromatic hydrocarbons (PAHs), 38 to 877 ng Sn/g for tri-n-butyltin (TBT), 60 to 562 ng/g for polyclorinated biphenyls (PCBs), and 7 to 71 ng/g for total DDT. Trace element concentrations (on a dry-wt basis) ranged from 1.1 to 4.0 microg/g for Cd, 105 to 229 microg/g for Cu, 212 to 868 microg/g for Al, and 1,200 to 8,180 microg/g for Zn. The percentage of destabilized lysosomes ranged from 34 to 81%. A significant positive correlation (p < 0.05) was observed between lysosomal destabilization and body burden of organic compounds (PAHs, PCBs, TBT, and chlorinated pesticides). No significant correlation was found between metal concentrations and lysosomal destabilization. Based on lysosomal destabilization, the study sites in Galveston Bay can be placed in one of three groups: healthy (Hanna Reef and Confederate Bay), moderately damaged (Offats Bayou and Todd's Dump), and highly damaged (Yacht Club and Ship Channel). Lysosomal destabilization that is consistent with toxic chemical body burdens supports previous observations that lysosomal membranes are damaged by toxic chemicals and indicates that this method can serve as an early screening tool to assess overall ecosystem health by using oysters.

  10. The Arabidopsis tonoplast is almost devoid of glycoproteins with complex N-glycans, unlike the rat lysosomal membrane

    PubMed Central

    Pedrazzini, Emanuela; Caprera, Andrea; Fojadelli, Ilaria; Stella, Alessandra; Rocchetti, Alessandra; Bassin, Barbara; Martinoia, Enrico; Vitale, Alessandro

    2016-01-01

    The distribution of the N-glycoproteome in integral membrane proteins of the vacuolar membrane (tonoplast) or the plasma membrane of Arabidopsis thaliana and, for further comparison, of the Rattus norvegicus lysosomal and plasma membranes, was analyzed. In silico analysis showed that potential N-glycosylation sites are much less frequent in tonoplast proteins. Biochemical analysis of Arabidopsis subcellular fractions with the lectin concanavalin A, which recognizes mainly unmodified N-glycans, or with antiserum against Golgi-modified N-glycans confirmed the in silico results and showed that, unlike the plant plasma membrane, the tonoplast is almost or totally devoid of N-glycoproteins with Golgi-modified glycans. Lysosomes share with vacuoles the hydrolytic functions and the position along the secretory pathway; however, our results indicate that their membranes had a divergent evolution. We propose that protection against the luminal hydrolases that are abundant in inner hydrolytic compartments, which seems to have been achieved in many lysosomal membrane proteins by extensive N-glycosylation of the luminal domains, has instead been obtained in the vast majority of tonoplast proteins by limiting the length of such domains. PMID:26748395

  11. An approach to the assessment of membrane stability of cultured cells.

    PubMed

    Thaw, H H; Lukinius, A; Brunk, U T; Collins, V P

    1983-01-01

    A simple method for assessing the combined stability of the plasma and lysosomal membranes of cultured cells is described. Monolayers of normal, human glial cells were incubated in situ in an isotonic, buffered sucrose solution (pH 5.0) containing the acid phosphatase (AP) enzyme substrate p-nitrophenyl phosphate (PNPP). The rate of appearance, in the solution, of the reaction product p-nitrophenol (PNP) was measured spectrophotometrically, curves then plotted, and fitted by computer. "Lag time" (LT) was calculated, and an index of membrane lability constructed, termed "fragility index" (FI). Transmission electron microscopy (TEM), "vital" staining of the cells with fluorescein diacetate (FDA) and Evans Blue (EB), and use of a Gomori-type cytochemical technique, indicate that the data reflects the combined stability of lysosomal and plasma membranes. The latter playing the more critical role. Cell cultures pre-incubated with various membrane labilizing or stabilizing agents were compared. Control, 0.3 M sucrose, and normal saline treated cells demonstrated similar stability. Distilled water decreased AP latency (increased fragility), and the magnitude of this effect was time dependent. Cells fixed in glutaraldehyde (GA) retained much of their osmotic reactivity, as confirmed by distilled water treatment. Oxygen derived free radicals caused pronounced fragility, while dexamethasone, a membrane stabilizing agent, decreased membrane fragility. Triton X-100 abolished latency completely, and total AP activity was very rapidly recovered outside the cells in the surrounding incubation medium. These results suggest this technique yields a measure of membrane stability which is sensitive enough to differentiate between known stabilizers and labilizers of membranes. Hence, this may prove an easy and useful aid for the assessment of how various substances and environments modulate the lysosomal and plasma membrane stability of cultured cells.

  12. Targeting of Salmonella typhimurium to vesicles containing lysosomal membrane glycoproteins bypasses compartments with mannose 6-phosphate receptors

    PubMed Central

    1995-01-01

    Salmonella typhimurium is an intracellular bacterial pathogen that remains enclosed in vacuoles (SCV) upon entry into the host cell. In this study we have examined the intracellular trafficking route of S. typhimurium within epithelial cells. Indirect immunofluorescence analysis showed that bacteria initiated fusion with lysosomal membrane glycoprotein (lgp)-containing compartments approximately 15 min after bacterial internalization. This process was completed approximately 75 min later and did not require microtubules. Cation-independent (CI)- or cation-dependent (CD)-mannose 6-phosphate receptors (M6PRs) were not observed at detectable levels in SCV. Lysosomal enzymes showed a different distribution in SCV: lysosomal-acid phosphatase (LAP) was incorporated into these vacuoles with the same kinetics as lgps, while cathepsin D was present in a low proportion (approximately 30%) of SCV. Uptake experiments with fluid endocytic tracers such as fluorescein- dextran sulphate (F-DX) or horseradish-peroxidase (HRP) showed that after 2 h of uptake, F-DX was present in approximately 75% of lgp- containing vesicles in uninfected cells, while only approximately 15% of SCV contained small amounts of the tracer during the same uptake period. SCV also showed only partial fusion with HRP-preloaded secondary lysosomes, with approximately 30% of SCV having detectable amounts of HRP at 6 h after infection. These results indicate that SCV show limited accessibility to fluid endocytic tracers and mature lysosomes, and are therefore functionally separated from the endocytic route. Moreover, the unusual intracellular trafficking route of S. typhimurium inside epithelial cells has allowed us to establish the existence of two different lgp-containing vesicles in Salmonella- infected cells: one population is separated from the endocytic route, fusogenic with incoming SCV and may arise from a secretory pathway, while the second involves the classical secondary or mature lysosomes. PMID

  13. Lysosomal integral membrane protein Sidt2 plays a vital role in insulin secretion.

    PubMed

    Gao, Jialin; Yu, Cui; Xiong, Qianyin; Zhang, Yao; Wang, Lizhuo

    2015-01-01

    Abnormal insulin secretion results in impaired glucose tolerance and is one of the causal factors in the etiology of type 2 diabetes mellitus. Sidt2, a lysosomal integral membrane protein, plays a critical role in insulin secretion. Here, we further investigate its regulation in insulin secretion. We show that Sidt2(-/-) mice exhibit weight loss, decreased postnatal survival rate with aging, increased fasting glucose and impaired glucose tolerance. After loading high levels of glucose in their diet, Sidt2(-/-) mice produce notably lower insulin levels at the first-phase secretion compared with Sidt2(+/+) mice. Consistent with the in vivo study, INS-1 cells treated with Sidt2 siRNA produced less insulin when loaded with 16.7 mM of glucose. Only 2 of the 13 genes, synap1 and synap3 which encode soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins, showed significantly decreased expression in Sidt2(-/-) mice. In conclusion, Sdit2 may play a vital role in the regulation of insulin secretion via two SNARE proteins synap1 and syanp3.

  14. Niemann-Pick C1 functions independently of Niemann-Pick C2 in the initial stage of retrograde transport of membrane-impermeable lysosomal cargo.

    PubMed

    Goldman, Stephen D B; Krise, Jeffrey P

    2010-02-12

    The rare neurodegenerative disease Niemann-Pick Type C (NPC) results from mutations in either NPC1 or NPC2, which are membrane-bound and soluble lysosomal proteins, respectively. Previous studies have shown that mutations in either protein result in biochemically indistinguishable phenotypes, most notably the hyper-accumulation of cholesterol and other cargo in lysosomes. We comparatively evaluated the kinetics of [(3)H]dextran release from lysosomes of wild type, NPC1, NPC2, and NPC1/NPC2 pseudo-double mutant cells and found significant differences between all cell types examined. Specifically, NPC1 or NPC2 mutant fibroblasts treated with NPC1 or NPC2 siRNA (to create NPC1/NPC2 pseudo-double mutants) secreted dextran less efficiently than did either NPC1 or NPC2 single mutant cell lines, suggesting that the two proteins may work independently of one another in the egress of membrane-impermeable lysosomal cargo. To investigate the basis for these differences, we examined the role of NPC1 and NPC2 in the retrograde fusion of lysosomes with late endosomes to create so-called hybrid organelles, which is believed to be the initial step in the egress of cargo from lysosomes. We show here that cells with mutated NPC1 have significantly reduced rates of late endosome/lysosome fusion relative to wild type cells, whereas cells with mutations in NPC2 have rates that are similar to those observed in wild type cells. Instead of being involved in hybrid organelle formation, we show that NPC2 is required for efficient membrane fission events from nascent hybrid organelles, which is thought to be required for the reformation of lysosomes and the release of lysosomal cargo-containing membrane vesicles. Collectively, these results suggest that NPC1 and NPC2 can function independently of one another in the egress of certain membrane-impermeable lysosomal cargo.

  15. Lysosome-associated membrane proteins-1 and -2 (LAMP-1 and LAMP-2) assemble via distinct modes.

    PubMed

    Terasawa, Kazue; Tomabechi, Yuri; Ikeda, Mariko; Ehara, Haruhiko; Kukimoto-Niino, Mutsuko; Wakiyama, Motoaki; Podyma-Inoue, Katarzyna A; Rajapakshe, Anupama R; Watabe, Tetsuro; Shirouzu, Mikako; Hara-Yokoyama, Miki

    2016-10-21

    Lysosome-associated membrane proteins 1 and 2 (LAMP-1 and LAMP-2) have a large, heavily glycosylated luminal domain composed of two subdomains, and are the most abundant protein components in lysosome membranes. LAMP-1 and LAMP-2 have distinct functions, and the presence of both proteins together is required for the essential regulation of autophagy to avoid embryonic lethality. However, the structural aspects of LAMP-1 and LAMP-2 have not been elucidated. In the present study, we demonstrated that the subdomains of LAMP-1 and LAMP-2 adopt the unique β-prism fold, similar to the domain structure of the dendritic cell-specific-LAMP (DC-LAMP, LAMP-3), confirming the conserved aspect of this family of lysosome-associated membrane proteins. Furthermore, we evaluated the effects of the N-domain truncation of LAMP-1 or LAMP-2 on the assembly of LAMPs, based on immunoprecipitation experiments. We found that the N-domain of LAMP-1 is necessary, whereas that of LAMP-2 is repressive, for the organization of a multimeric assembly of LAMPs. Accordingly, the present study suggests for the first time that the assembly modes of LAMP-1 and LAMP-2 are different, which may underlie their distinct functions.

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

    SciTech Connect

    Yang, Mei; Zhang, Minfang; Tahara, Yoshio; Chechetka, Svetlana; Miyako, Eijiro; Iijima, Sumio; Yudasaka, Masako

    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.

  17. The potential role of lysosome-associated membrane protein 3 (LAMP3) on cardiac remodelling

    PubMed Central

    Jiang, Ding-Sheng; Yi, Xin; Huo, Bo; Liu, Xin-Xin; Li, Rui; Zhu, Xue-Hai; Wei, Xiang

    2016-01-01

    Lysosome-associated membrane protein 3 (LAMP3) was first identified as a cell surface marker of mature dendritic cells and specifically expressed in lung tissues. Recently studies demonstrated that LAMP3 plays a critical role in several cancers, and regulated by hypoxia. However, whether LAMP3 expressed in the heart and cardiomyocytes and changed its expression level in the hearts with cardiac remodelling was largely unknown. In this study, we first cultured H9C2 (a clonal muscle cell line from rat heart) and stimulated with 1 μM angiotensin II (Ang II), or 100 μM isoproterenol (ISO), or 100 μM phenylephrine (PE) for indicated times. We found that LAMP3 expression level was significantly increased after these stimulation. Next, the pressure overload-induced cardiac remodelling mouse model was performed in the wild type C57BL/6J mice. After 4 and 8 weeks of transverse aortic constriction (TAC), obvious cardiac remodelling was observed in the wild type mice compared with sham group. Importantly, LAMP3 expression level was gradually elevated from 2 weeks to 8 weeks after TAC surgery. Furthermore, in human dilated cardiomyopathy (DCM) hearts, severe cardiac remodelling was observed, as evidenced by remarkably increased cardiomyocytes cross sectional area and collagen deposition. Notably, the mRNA and protein level of LAMP3 were significantly increased in the DCM hearts compared with donor hearts. Immunohistochemistry assay showed that LAMP3 was expression in the cardiomyocytes and responsible for its increased expression in the hearts. Our data indicated that LAMP3 might have a potential role in the process of cardiac remodelling. PMID:27069538

  18. Lysosomal integral membrane protein type-2 (LIMP-2/SCARB2) is a substrate of cathepsin-F, a cysteine protease mutated in type-B-Kufs-disease.

    PubMed

    Peters, Judith; Rittger, Andrea; Weisner, Rebecca; Knabbe, Johannes; Zunke, Friederike; Rothaug, Michelle; Damme, Markus; Berkovic, Samuel F; Blanz, Judith; Saftig, Paul; Schwake, Michael

    2015-02-13

    The lysosomal integral membrane protein type-2 (LIMP-2/SCARB2) has been identified as a receptor for enterovirus 71 uptake and mannose-6-phosphate-independent lysosomal trafficking of the acid hydrolase β-glucocerebrosidase. Here we show that LIMP-2 undergoes proteolytic cleavage mediated by lysosomal cysteine proteases. Heterologous expression and in vitro studies suggest that cathepsin-F is mainly responsible for the lysosomal processing of wild-type LIMP-2. Furthermore, examination of purified lysosomes revealed that LIMP-2 undergoes proteolysis in vivo. Mutations in the gene encoding cathepsin-F (CTSF) have recently been associated with type-B-Kufs-disease, an adult form of neuronal ceroid-lipofuscinosis. In this study we show that disease-causing cathepsin-F mutants fail to cleave LIMP-2. Our findings provide evidence that LIMP-2 represents an in vivo substrate of cathepsin-F with relevance for understanding the pathophysiology of type-B-Kufs-disease.

  19. Application and evaluation of the neutral red retention (NRR) assay for lysosomal stability in mussel populations along the Iberian Mediterranean coast.

    PubMed

    Martínez-Gómez, Concepción; Benedicto, José; Campillo, Juan Antonio; Moore, Michael

    2008-04-01

    The neutral red retention (NRR) assay to determine lysosomal membrane stability (LMS) was applied to wild mussels (Mytilus galloprovincialis) sampled from fourteen sites with different degrees of chemical pollution along the Iberian Mediterranean coast in 2002 and 2003. Total body burdens for a range of contaminants were measured in pooled samples (n = 50) of whole soft tissues in order to explore possible causality. Mean LMS values (n = 15) were significantly greater in mussels from one of the selected two reference sites (San Diego) than in chemically well-characterized hotspot sites. Mean LMS values were inversely correlated with contaminant concentrations in mussel tissues (except for Zn). The multidimensional scaling ordination technique classified the sampling sites into three groups according to their chemical-physical-biological similarities and the results were statistically confirmed using ANOSIM analysis. The results show that lysosomal stability is an effective indicator of health status in mussels along the Iberian Mediterranean coast and that it is related to body burden of contaminants, particularly polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs).

  20. Point mutation of adenosine triphosphate-binding motif generated rigor kinesin that selectively blocks anterograde lysosome membrane transport

    PubMed Central

    1995-01-01

    In the study of motor proteins, the molecular mechanism of mechanochemical coupling, as well as the cellular role of these proteins, is an important issue. To assess these questions we introduced cDNA of wild-type and site-directed mutant kinesin heavy chains into fibroblasts, and analyzed the behavior of the recombinant proteins and the mechanisms involved in organelle transports. Overexpression of wild-type kinesin significantly promoted elongation of cellular processes. Wild-type kinesin accumulated at the tips of the long processes, whereas the kinesin mutants, which contained either a T93N- or T93I mutation in the ATP-binding motif, tightly bound to microtubules in the center of the cells. These mutant kinesins could bind to microtubules in vitro, but could not dissociate from them even in the presence of ATP, and did not support microtubule motility in vitro, thereby indicating rigor-type mutations. Retrograde transport from the Golgi apparatus to the endoplasmic reticulum, as well as lysosome dispersion, was shown to be a microtubule-dependent, plus-end- directed movement. The latter was selectively blocked in the rigor- mutant cells, although the microtubule minus-end-directed motion of lysosomes was not affected. We found the point mutations that make kinesin motor in strong binding state with microtubules in vitro and showed that this mutant causes a dominant effect that selectively blocks anterograde lysosome membrane transports in vivo. PMID:7490281

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

    PubMed Central

    García-Del Portillo, Francisco; Jungnitz, Heidrun; Rohde, Manfred; Guzmán, Carlos A.

    2000-01-01

    Dendritic cells (DCs) play a central role in the generation of acquired immunity to infections by pathogenic microorganisms. Salmonella enterica serotype Typhimurium is known to survive and proliferate intracellularly within macrophages and nonphagocytic cells, but no data exist on how this pathogen interacts with DCs. In this report, we show the capacity of serotype Typhimurium to survive within the established mouse DC line CB1. In contrast to the case for the macrophage model, the compartments of DCs containing serotype Typhimurium are devoid of lysosomal membrane glycoproteins and the PhoPQ two-component regulatory system is not essential for pathogen intracellular survival. PMID:10768999

  2. Characterization of the complex formed by β-glucocerebrosidase and the lysosomal integral membrane protein type-2.

    PubMed

    Zunke, Friederike; Andresen, Lisa; Wesseler, Sophia; Groth, Johann; Arnold, Philipp; Rothaug, Michelle; Mazzulli, Joseph R; Krainc, Dimitri; Blanz, Judith; Saftig, Paul; Schwake, Michael

    2016-04-05

    The lysosomal integral membrane protein type-2 (LIMP-2) plays a pivotal role in the delivery of β-glucocerebrosidase (GC) to lysosomes. Mutations in GC result in Gaucher's disease (GD) and are the major genetic risk factor for the development of Parkinson's disease (PD). Variants in the LIMP-2 gene cause action myoclonus-renal failure syndrome and also have been linked to PD. Given the importance of GC and LIMP-2 in disease pathogenesis, we studied their interaction sites in more detail. Our previous data demonstrated that the crystal structure of LIMP-2 displays a hydrophobic three-helix bundle composed of helices 4, 5, and 7, of which helix 5 and 7 are important for ligand binding. Here, we identified a similar helical motif in GC through surface potential analysis. Coimmunoprecipitation and immunofluorescence studies revealed a triple-helical interface region within GC as critical for LIMP-2 binding and lysosomal transport. Based on these findings, we generated a LIMP-2 helix 5-derived peptide that precipitated and activated recombinant wild-type and GD-associated N370S mutant GC in vitro. The helix 5 peptide fused to a cell-penetrating peptide also activated endogenous lysosomal GC and reduced α-synuclein levels, suggesting that LIMP-2-derived peptides can be used to activate endogenous as well as recombinant wild-type or mutant GC efficiently. Our data also provide a structural model of the LIMP-2/GC complex that will facilitate the development of GC chaperones and activators as potential therapeutics for GD, PD, and related synucleinopathies.

  3. Characterization of the complex formed by β-glucocerebrosidase and the lysosomal integral membrane protein type-2

    PubMed Central

    Zunke, Friederike; Andresen, Lisa; Wesseler, Sophia; Groth, Johann; Arnold, Philipp; Rothaug, Michelle; Mazzulli, Joseph R.; Krainc, Dimitri; Blanz, Judith; Saftig, Paul; Schwake, Michael

    2016-01-01

    The lysosomal integral membrane protein type-2 (LIMP-2) plays a pivotal role in the delivery of β-glucocerebrosidase (GC) to lysosomes. Mutations in GC result in Gaucher's disease (GD) and are the major genetic risk factor for the development of Parkinson's disease (PD). Variants in the LIMP-2 gene cause action myoclonus-renal failure syndrome and also have been linked to PD. Given the importance of GC and LIMP-2 in disease pathogenesis, we studied their interaction sites in more detail. Our previous data demonstrated that the crystal structure of LIMP-2 displays a hydrophobic three-helix bundle composed of helices 4, 5, and 7, of which helix 5 and 7 are important for ligand binding. Here, we identified a similar helical motif in GC through surface potential analysis. Coimmunoprecipitation and immunofluorescence studies revealed a triple-helical interface region within GC as critical for LIMP-2 binding and lysosomal transport. Based on these findings, we generated a LIMP-2 helix 5-derived peptide that precipitated and activated recombinant wild-type and GD-associated N370S mutant GC in vitro. The helix 5 peptide fused to a cell-penetrating peptide also activated endogenous lysosomal GC and reduced α-synuclein levels, suggesting that LIMP-2–derived peptides can be used to activate endogenous as well as recombinant wild-type or mutant GC efficiently. Our data also provide a structural model of the LIMP-2/GC complex that will facilitate the development of GC chaperones and activators as potential therapeutics for GD, PD, and related synucleinopathies. PMID:27001828

  4. Ubiquitination and dynactin regulate TMEPAI lysosomal trafficking

    PubMed Central

    Luo, Shenheng; Jing, Lei; Zhao, Tian; Li, Yuyin; Liu, Zhenxing; Diao, Aipo

    2017-01-01

    The transmembrane prostate androgen-induced protein (TMEPAI) has been reported to be elevated in various tumor cells, is localized to the lysosome and promotes lysosome stability. The molecular mechanism of TMEPAI trafficking however to the lysosome is unknown. Here we report that clathrin and CI-M6PR mediate TMEPAI transport from the Golgi directly into the endo-lysosomal pathway. TMEPAI is ubiquitinated at its C-terminal region and ubiquitination modification of TMEPAI is a signal for its lysosomal trafficking. Moreover, TMEPAI binds the ubiquitin binding proteins Hrs and STAM which is required for its lysosomal transport. In addition, TMEPAI interacts with the dynactin pointed-end complex subunits dynactin 5 and dynactin 6. The aa 132–155 domain is essential for specific TMEPAI binding and deletion of this binding site leads to mis-trafficking of TMEPAI to the plasma membrane. These results reveal the pathway and mechanism regulating transport of TMEPAI to the lysosome, which helps to further understand the role of TMEPAI in tumorigenesis. PMID:28218281

  5. Morphological alteration, lysosomal membrane fragility and apoptosis of the cells of Indian freshwater sponge exposed to washing soda (sodium carbonate).

    PubMed

    Mukherjee, Soumalya; Ray, Mitali; Dutta, Manab Kumar; Acharya, Avanti; Mukhopadhyay, Sandip Kumar; Ray, Sajal

    2015-12-01

    Washing soda is chemically known as sodium carbonate and is a component of laundry detergent. Domestic effluent, drain water and various anthropogenic activities have been identified as major routes of sodium carbonate contamination of the freshwater ecosystem. The freshwater sponge, Eunapius carteri, bears ecological and evolutionary significance and is considered as a bioresource in aquatic ecosystems. The present study involves estimation of morphological damage, lysosomal membrane integrity, activity of phosphatases and apoptosis in the cells of E. carteri under the environmentally realistic concentrations of washing soda. Exposure to washing soda resulted in severe morphological alterations and damages in cells of E. carteri. Fragility and destabilization of lysosomal membranes of E. carteri under the sublethal exposure was indicative to toxin induced physiological stress in sponge. Prolonged exposure to sodium carbonate resulted a reduction in the activity of acid and alkaline phosphatases in the cells of E. carteri. Experimental concentration of 8 mg/l of washing soda for 192 h yielded an increase in the physiological level of cellular apoptosis among the semigranulocytes and granulocytes of E. carteri, which was suggestive to possible shift in apoptosis mediated immunoprotection. The results were indicative of an undesirable shift in the immune status of sponge. Contamination of the freshwater aquifers by washing soda thus poses an alarming ecotoxicological threat to sponges.

  6. The AP-3 adaptor complex mediates sorting of yeast and mammalian PQ-loop-family basic amino acid transporters to the vacuolar/lysosomal membrane

    PubMed Central

    Llinares, Elisa; Barry, Abdoulaye Oury; André, Bruno

    2015-01-01

    The limiting membrane of lysosomes in animal cells and that of the vacuole in yeast include a wide variety of transporters, but little is known about how these proteins reach their destination membrane. The mammalian PQLC2 protein catalyzes efflux of basic amino acids from the lysosome, and the similar Ypq1, −2, and −3 proteins of yeast perform an equivalent function at the vacuole. We here show that the Ypq proteins are delivered to the vacuolar membrane via the alkaline phosphatase (ALP) trafficking pathway, which requires the AP-3 adaptor complex. When traffic via this pathway is deficient, the Ypq proteins pass through endosomes from where Ypq1 and Ypq2 properly reach the vacuolar membrane whereas Ypq3 is missorted to the vacuolar lumen via the multivesicular body pathway. When produced in yeast, PQLC2 also reaches the vacuolar membrane via the ALP pathway, but tends to sort to the vacuolar lumen if AP-3 is defective. Finally, in HeLa cells, inhibiting the synthesis of an AP-3 subunit also impairs sorting of PQLC2 to lysosomes. Our results suggest the existence of a conserved AP-3-dependent trafficking pathway for proper delivery of basic amino acid exporters to the yeast vacuole and to lysosomes of human cells. PMID:26577948

  7. Membrane Stability during Biopreservation of Blood Cells

    PubMed Central

    Stoll, Christoph; Wolkers, Willem F.

    2011-01-01

    Summary Storage methods, which can be taken into consideration for red blood cells and platelets, include liquid storage, cryopreservation and freeze-drying. Red blood cells can be hypothermically stored at refrigerated temperatures, whereas platelets are chilling sensitive and therefore cannot be stored at temperatures below 20 °C. Here we give an overview of available cryopreservation and freeze-drying procedures for blood cells and discuss the effects of these procedures on cells, particularly on cellular membranes. Cryopreservation and freeze-drying may result in chemical and structural modifications of cellular membranes. Membranes undergo phase and permeability changes during freezing and drying. Cryo- and lyoprotective agents prevent membrane damage by different mechanisms. Cryoprotective agents are preferentially excluded from membrane surfaces. They decrease the activation energy for water transport during freezing and control the rate of cellular dehydration. Lyoprotectants are thought to stabilize membranes during drying by forming direct hydrogen bonding interactions with phospholipid head groups. In addition, lyoprotectants can form a glassy state at room temperature. Recently liposomes have been investigated to stabilize blood cells during freezing and freeze-drying. Liposomes modify the composition of cellular membranes by lipid and cholesterol transfer, which can stabilize or destabilize the low temperature response of cells. PMID:21566710

  8. Stabilization of Erythrocyte Membranes by Polyamines

    NASA Astrophysics Data System (ADS)

    Ballas, Samir K.; Mohandas, Narla; Marton, Laurence J.; Shohet, Stephen B.

    1983-04-01

    Using a laser diffraction technique, we have studied the effects of putrescine, spermidine, and spermine, the three physiologic polyamines, on the deformability and mechanical stability of human erythrocyte membranes. Ghosts resealed with polyamines were subjected to high fluid shear stress in an ektacytometer. All polyamines increased the membrane shear modulus (decreased deformability) in a concentration- and time-dependent manner. The order of effectiveness was spermine > spermidine > putrescine. At 10 μ M, spermine appreciably decreased membrane deformability. For the measurement of membrane mechanical stability, resealed ghosts were subjected to constant high shear stress in the ektacytometer and deformability was continuously recorded as the deformable ghosts fragmented into rigid spherical vesicles. Polyamines, especially spermine, caused a noticeable increase in the t1/2 for fragmentation. These effects could not be ascribed to proteolysis or Ca2+-induced transglutamination. That the effects of polyamines were specific and not simply due to their positive charge was demonstrated by the finding that Ca2+ and Mg2+ destabilized the erythrocyte membrane as evidenced by decreasing the t1/2 for fragmentation. Extracellular polyamines were not effective except under conditions that caused significant accumulation inside the cell. The data indicate that intracellular physiologic polyamines, especially spermine, decrease erythrocyte membrane deformability and stabilize the membrane skeleton, making it more resistant to fragmentation.

  9. The proteome of lysosomes.

    PubMed

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

    2010-11-01

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

  10. A LAPF/phafin1-like protein regulates TORC1 and lysosomal membrane permeabilization in response to endoplasmic reticulum membrane stress

    PubMed Central

    Kim, Adam; Cunningham, Kyle W.

    2015-01-01

    Lysosomal membrane permeabilization (LMP) is a poorly understood regulator of programmed cell death that involves leakage of luminal lysosomal or vacuolar hydrolases into the cytoplasm. In Saccharomyces cerevisiae, LMP can be induced by antifungals and endoplasmic reticulum stressors when calcineurin also has been inactivated. A genome-wide screen revealed Pib2, a relative of LAPF/phafin1 that regulates LMP in mammals, as a pro-LMP protein in yeast. Pib2 associated with vacuolar and endosomal limiting membranes in unstressed cells in a manner that depended on its FYVE domain and on phosphatidylinositol 3-phosphate (PI(3)P) biosynthesis. Genetic experiments suggest that Pib2 stimulates the activity of TORC1, a vacuole-associated protein kinase that is sensitive to rapamycin, in a pathway parallel to the Ragulator/EGO complex containing the GTPases Gtr1 and Gtr2. A hyperactivating mutation in the catalytic subunit of TORC1 restored LMP to the gtr1∆ and pib2∆ mutants and also prevented the synthetic lethality of the double mutants. These findings show novel roles of PI(3)P and Pib2 in the regulation of TORC1, which in turn promoted LMP and nonapoptotic death of stressed cells. Rapamycin prevented the death of the pathogenic yeast Candida albicans during exposure to fluconazole plus a calcineurin inhibitor, suggesting that TORC1 broadly promotes sensitivity to fungistats in yeasts. PMID:26510498

  11. Ceramic membranes with enhanced thermal stability

    DOEpatents

    Anderson, Marc A.; Xu, Qunyin; Bischoff, Brian L.

    1993-01-01

    A method of creating a ceramic membrane with enhanced thermal stability is disclosed. The method involves combining quantities of a first metal alkoxide with a second metal, the quantities selected to give a preselected metal ratio in the resultant membrane. A limited amount of water and acid is added to the combination and stirred until a colloidal suspension is formed. The colloid is dried to a gel, and the gel is fired at a temperature greater than approximately 400.degree. C. The porosity and surface area of ceramic membranes formed by this method are not adversely affected by this high temperature firing.

  12. Regulators of Lysosome Function and Dynamics in Caenorhabditis elegans

    PubMed Central

    Gee, Kevin; Zamora, Danniel; Horm, Teresa; George, Laeth; Upchurch, Cameron; Randall, Justin; Weaver, Colby; Sanford, Caitlin; Miller, Austin; Hernandez, Sebastian; Dang, Hope; Fares, Hanna

    2017-01-01

    Lysosomes, the major membrane-bound degradative organelles, have a multitude of functions in eukaryotic cells. Lysosomes are the terminal compartments in the endocytic pathway, though they display highly dynamic behaviors, fusing with each other and with late endosomes in the endocytic pathway, and with the plasma membrane during regulated exocytosis and for wound repair. After fusing with late endosomes, lysosomes are reformed from the resulting hybrid organelles through a process that involves budding of a nascent lysosome, extension of the nascent lysosome from the hybrid organelle, while remaining connected by a membrane bridge, and scission of the membrane bridge to release the newly formed lysosome. The newly formed lysosomes undergo cycles of homotypic fusion and fission reactions to form mature lysosomes. In this study, we used a forward genetic screen in Caenorhabditis elegans to identify six regulators of lysosome biology. We show that these proteins function in different steps of lysosome biology, regulating lysosome formation, lysosome fusion, and lysosome degradation. PMID:28122949

  13. Membrane chemical stability and seed longevity.

    PubMed

    Golovina, Elena A; Van As, Henk; Hoekstra, Folkert A

    2010-03-01

    Here, we investigate the relationships between the chemical stability of the membrane surface and seed longevity. Dry embryos of long-lived tomato and short-lived onion seeds were labeled with 5-doxyl-stearic acid (5-DS). Temperature-induced loss of the electron spin resonance signal caused by chemical conversion of 5-DS to nonparamagnetic species was used to characterize the membrane surface chemical stability. No difference was found between temperature plots of 5-DS signal intensity in dry onion and tomato below 345 K. Above this temperature, the 5-DS signal remained unchanged in tomato embryos and irreversibly disappeared in onion seeds. The role of the physical state and chemical status of the membrane environment in the chemical stability of membrane surfaces was estimated for model systems containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) dried alone or in the presence of trehalose or glucose. Fourier transform infrared spectroscopy was used to follow temperature-induced structural changes in dry POPC. Spin-label technique was used to relate the chemical stability of 5-DS with the dynamic properties of the bilayer and 5-DS motion behavior. In all the models, the decrease in 5-DS signal intensity was always observed above T(m) for the membrane surface. The 5-DS signal was irreversibly lost at high temperature when dry POPC was embedded in a glucose matrix. The loss of 5-DS signal was moderate when POPC was dried alone or in the presence of trehalose. Comparison of model and in vivo data shows that the differences in longevity between onion and tomato seeds are caused by differences in the chemical status of the membrane surface rather than the degree of its immobilization.

  14. α-Defensin HD5 Inhibits Human Papillomavirus 16 Infection via Capsid Stabilization and Redirection to the Lysosome

    PubMed Central

    Wiens, Mayim E.

    2017-01-01

    ABSTRACT α-Defensins are an important class of abundant innate immune effectors that are potently antiviral against a number of nonenveloped viral pathogens; however, a common mechanism to explain their ability to block infection by these unrelated viruses is lacking. We previously found that human defensin 5 (HD5) blocks a critical host-mediated proteolytic processing step required for human papillomavirus (HPV) infection. Here, we show that bypassing the requirement for this cleavage failed to abrogate HD5 inhibition. Instead, HD5 altered HPV trafficking in the cell. In the presence of an inhibitory concentration of HD5, HPV was internalized and reached the early endosome. The internalized capsid became permeable to antibodies and proteases; however, HD5 prevented dissociation of the viral capsid from the genome, reduced viral trafficking to the trans-Golgi network, redirected the incoming viral particle to the lysosome, and accelerated the degradation of internalized capsid proteins. This mechanism is equivalent to the mechanism by which HD5 inhibits human adenovirus. Thus, our data support capsid stabilization and redirection to the lysosome during infection as a general antiviral mechanism of α-defensins against nonenveloped viruses. PMID:28119475

  15. Membrane-Associated RING-CH proteins associate with Bap31 and target CD81 and CD44 to lysosomes.

    PubMed

    Bartee, Eric; Eyster, Craig A; Viswanathan, Kasinath; Mansouri, Mandana; Donaldson, Julie G; Früh, Klaus

    2010-12-02

    Membrane-associated RING-CH (MARCH) proteins represent a family of transmembrane ubiquitin ligases modulating intracellular trafficking and turnover of transmembrane protein targets. While homologous proteins encoded by gamma-2 herpesviruses and leporipoxviruses have been studied extensively, limited information is available regarding the physiological targets of cellular MARCH proteins. To identify host cell proteins targeted by the human MARCH-VIII ubiquitin ligase we used stable isotope labeling of amino-acids in cell culture (SILAC) to monitor MARCH-dependent changes in the membrane proteomes of human fibroblasts. Unexpectedly, we observed that MARCH-VIII reduced the surface expression of Bap31, a chaperone that predominantly resides in the endoplasmic reticulum (ER). We demonstrate that Bap31 associates with the transmembrane domains of several MARCH proteins and controls intracellular transport of MARCH proteins. In addition, we observed that MARCH-VIII reduced the surface expression of the hyaluronic acid-receptor CD44 and both MARCH-VIII and MARCH-IV sequestered the tetraspanin CD81 in endo-lysosomal vesicles. Moreover, gene knockdown of MARCH-IV increased surface levels of endogenous CD81 suggesting a constitutive involvement of this family of ubiquitin ligases in the turnover of tetraspanins. Our data thus suggest a role of MARCH-VIII and MARCH-IV in the regulated turnover of CD81 and CD44, two ubiquitously expressed, multifunctional proteins.

  16. Thrombospondin expression in myofibers stabilizes muscle membranes

    PubMed Central

    Vanhoutte, Davy; Schips, Tobias G; Kwong, Jennifer Q; Davis, Jennifer; Tjondrokoesoemo, Andoria; Brody, Matthew J; Sargent, Michelle A; Kanisicak, Onur; Yi, Hong; Gao, Quan Q; Rabinowitz, Joseph E; Volk, Talila; McNally, Elizabeth M; Molkentin, Jeffery D

    2016-01-01

    Skeletal muscle is highly sensitive to mutations in genes that participate in membrane stability and cellular attachment, which often leads to muscular dystrophy. Here we show that Thrombospondin-4 (Thbs4) regulates skeletal muscle integrity and its susceptibility to muscular dystrophy through organization of membrane attachment complexes. Loss of the Thbs4 gene causes spontaneous dystrophic changes with aging and accelerates disease in 2 mouse models of muscular dystrophy, while overexpression of mouse Thbs4 is protective and mitigates dystrophic disease. In the myofiber, Thbs4 selectively enhances vesicular trafficking of dystrophin-glycoprotein and integrin attachment complexes to stabilize the sarcolemma. In agreement, muscle-specific overexpression of Drosophila Tsp or mouse Thbs4 rescues a Drosophila model of muscular dystrophy with augmented membrane residence of βPS integrin. This functional conservation emphasizes the fundamental importance of Thbs’ as regulators of cellular attachment and membrane stability and identifies Thbs4 as a potential therapeutic target for muscular dystrophy. DOI: http://dx.doi.org/10.7554/eLife.17589.001 PMID:27669143

  17. [Experimental models of lysosomal phase reactivity in blood leukocytes exposed to low doses of potassium cyanide].

    PubMed

    Dolgushin, M V; Khomuev, G D

    2013-01-01

    Cytochemical analysis of acid phosphatase was used to evaluate lysosomal membranes stability under oral intake of potassium cyanide by rats over one month in daily doses of 1.30 mg/kg (1/10 LD50) and 0.65 mg/kg (1/20 LD50). The authors demonstrated phase-related dose-dependent changes in the lysosomal state, and the main response feature was associated with functional activation that usually followed the membrane alteration.

  18. Polycyclic aromatic hydrocarbon body residues and lysosomal membrane destabilization in mussels exposed to the Dubai Star bunker fuel oil (intermediate fuel oil 380) spill in San Francisco Bay.

    PubMed

    Hwang, Hyun-Min; Stanton, Beckye; McBride, Toby; Anderson, Michael J

    2014-05-01

    Following the spill of bunker fuel oil (intermediate fuel oil 380, approximately 1500-3000 L) into San Francisco Bay in October 2009, polycyclic aromatic hydrocarbon (PAH) concentrations in mussels from moderately oiled areas increased up to 87 554 ng/g (dry wt) and, 3 mo later, decreased to concentrations found in mussels collected prior to oiling, with a biological half-life of approximately 16 d. Lysosomal membrane destabilization increased in mussels with higher PAH body burdens.

  19. Structure of transmembrane domain of lysosome-associated membrane protein type 2a (LAMP-2A) reveals key features for substrate specificity in chaperone-mediated autophagy.

    PubMed

    Rout, Ashok K; Strub, Marie-Paule; Piszczek, Grzegorz; Tjandra, Nico

    2014-12-19

    Chaperone-mediated autophagy (CMA) is a highly regulated cellular process that mediates the degradation of a selective subset of cytosolic proteins in lysosomes. Increasing CMA activity is one way for a cell to respond to stress, and it leads to enhanced turnover of non-critical cytosolic proteins into sources of energy or clearance of unwanted or damaged proteins from the cytosol. The lysosome-associated membrane protein type 2a (LAMP-2A) together with a complex of chaperones and co-chaperones are key regulators of CMA. LAMP-2A is a transmembrane protein component for protein translocation to the lysosome. Here we present a study of the structure and dynamics of the transmembrane domain of human LAMP-2A in n-dodecylphosphocholine micelles by nuclear magnetic resonance (NMR). We showed that LAMP-2A exists as a homotrimer in which the membrane-spanning helices wrap around each other to form a parallel coiled coil conformation, whereas its cytosolic tail is flexible and exposed to the cytosol. This cytosolic tail of LAMP-2A interacts with chaperone Hsc70 and a CMA substrate RNase A with comparable affinity but not with Hsp40 and RNase S peptide. Because the substrates and the chaperone complex can bind at the same time, thus creating a bimodal interaction, we propose that substrate recognition by chaperones and targeting to the lysosomal membrane by LAMP-2A are coupled. This can increase substrate affinity and specificity as well as prevent substrate aggregation, assist in the unfolding of the substrate, and promote the formation of the higher order complex of LAMP-2A required for translocation.

  20. The histone deacetylase inhibitor trichostatin A reduces lysosomal pH and enhances cisplatin-induced apoptosis.

    PubMed

    Eriksson, I; Joosten, M; Roberg, K; Ollinger, K

    2013-01-01

    High activity of histone deacetylases (HDACs) has been documented in several types of cancer and may be associated with survival advantage. In a head and neck squamous cell carcinoma cell line, cisplatin-induced apoptosis was augmented by pretreatment with the HDAC inhibitor trichostatin A. Apoptosis was accompanied by lysosomal membrane permeabilization (LMP), as shown by immunoblotting of the lysosomal marker protease cathepsin B in extracted cytosol and by immunofluorescence. Moreover, LAMP-2 (lysosomal associated membrane protein-2) was translocated from lysosomal membranes and found in a digitonin extractable fraction together with cytosolic proteins and pretreatment with trichostatin A potentiated the release. Overall, protein level of LAMP-2 was decreased during cell death and, interestingly, inhibition of cysteine cathepsins, by the pan-cysteine cathepsin inhibitor zFA-FMK, prevented loss of LAMP-2. The importance of LAMP-2 for lysosomal membrane stability, was confirmed by showing that LAMP-2 knockout MEFs (mouse embryonic fibroblasts) were more sensitive to cisplatin as compared to the corresponding wildtype cells. Trichostatin A reduced lysosomal pH from 4.46 to 4.25 and cell death was prevented when lysosomal pH was increased by NH(4)Cl, or when inhibiting the activity of lysosomal proteases. We conclude that trichostatin A enhances cisplatin induced cell death by decreasing lysosomal pH, which augments cathepsin activity resulting in reduced LAMP-2 level, and might promote LMP.

  1. The Antioxidant Profiles, Lysosomal and Membrane Enzymes Activity in Patients with Acute Pancreatitis

    PubMed Central

    Milnerowicz, Halina; Bukowski, Radosław; Jabłonowska, Monika; Ściskalska, Milena; Milnerowicz, Stanisław

    2014-01-01

    Oxidative stress and inflammatory mediators, such as IL-6, play an important role in the pathophysiology of acute pancreatitis. The study was aimed to assess the degree of the pro/antioxidative imbalance and estimate which antioxidant plays a role in the maintenance of pro/antioxidative balance during acute pancreatitis. The study was investigated in the blood of 32 patients with acute pancreatitis and 37 healthy subjects. IL-6 concentration as early marker of inflammation was determinated. The intensity of oxidative stress was assessed by TBARS concentration. To investigate antioxidative status, the GPx and Cu/Zn SOD activities and the levels of GSH, MT, SH groups, and TRAP were measured. The concentrations of Cu and Zn as ions participating in the maintenance of antioxidant enzymes stability and playing a role in the course of disease were determinated. The activities of GGT, AAP, NAG, and β-GD as markers of tissue damage were also measured. An increase in IL-6 concentration, which correlated with Ranson criteria, and an increase in GPx activity, levels of MT, TBARS, or GGT, and NAG activities in patients group compared to healthy subjects were demonstrated. A decrease in GSH level in patients group compared to control group was noted. The studies suggest that GPx/GSH and MT play the role of the first line of defence against oxidative stress and pro/antioxidant imbalance in the course of acute pancreatitis. PMID:25298618

  2. Synergistic salubrious effect of ferulic acid and ascorbic acid on membrane-bound phosphatases and lysosomal hydrolases during experimental myocardial infarction in rats.

    PubMed

    Yogeeta, Surinder Kumar; Gnanapragasam, Arunachalam; Senthilkumar, Subramanian; Subhashini, Rajakannu; Devaki, Thiruvengadam

    2006-12-23

    Altered membrane integrity has been suggested as a major factor in the development of cellular injury during myocardial necrosis. The present study was designed to investigate the effect of the combination of ferulic acid (FA) and ascorbic acid (AA) on lysosomal hydrolases and membrane-bound phosphatases during isoproterenol (ISO) induced myocardial necrosis in rats. Induction of rats with 1SO (150 mg/kg b.wt, i.p.) for 2 days resulted in a significant increase in the activities of lysosomal hydrolases (beta-D-glucuronidase, beta-D-galactosidase, beta-D-N-acetylglucosaminidase, acid phosphatase and cathepsin-D) in the heart and serum. A significant increase in plasma lactate level, cardiac levels of sodium, calcium and a decrease in cardiac level of potassium was also observed, which was paralleled by abnormal activities of membrane-bound phosphatases (Na(+)-K(+) ATPase, Ca(2+) ATPase and Mg(2+) ATPase) in the heart of ISO-administered rats. Pre-co-treatment with the combination of FA (20 mg/kg b.wt) and AA (80 mg/kg b.wt) orally for 6 days significantly attenuated these abnormalities and restored the levels to near normalcy when compared to individual drug treated groups. The combination of FA and AA preserved the membrane integrity by mitigating the oxidative stress and associated cellular damage more effectively when compared to individual treatment groups. In our study, the protection conferred by FA and AA might be through the nitric oxide pathway and by their ability of quenching free radicals. In conclusion, these findings indicate the synergistic modulation of lysosomal hydrolases and membrane phosphatases by the combination of FA and AA.

  3. Lysosomal cell death mechanisms in aging.

    PubMed

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

    2016-12-01

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

  4. Direct interaction with filamins modulates the stability and plasma membrane expression of CFTR

    PubMed Central

    Thelin, William R.; Chen, Yun; Gentzsch, Martina; Kreda, Silvia M.; Sallee, Jennifer L.; Scarlett, Cameron O.; Borchers, Christoph H.; Jacobson, Ken; Stutts, M. Jackson; Milgram, Sharon L.

    2007-01-01

    The role of the cystic fibrosis transmembrane conductance regulator (CFTR) as a cAMP-dependent chloride channel on the apical membrane of epithelia is well established. However, the processes by which CFTR is regulated on the cell surface are not clear. Here we report the identification of a protein-protein interaction between CFTR and the cytoskeletal filamin proteins. Using proteomic approaches, we identified filamins as proteins that associate with the extreme CFTR N terminus. Furthermore, we identified a disease-causing missense mutation in CFTR, serine 13 to phenylalanine (S13F), which disrupted this interaction. In cells, filamins tethered plasma membrane CFTR to the underlying actin network. This interaction stabilized CFTR at the cell surface and regulated the plasma membrane dynamics and confinement of the channel. In the absence of filamin binding, CFTR was internalized from the cell surface, where it prematurely accumulated in lysosomes and was ultimately degraded. Our data demonstrate what we believe to be a previously unrecognized role for the CFTR N terminus in the regulation of the plasma membrane stability and metabolic stability of CFTR. In addition, we elucidate the molecular defect associated with the S13F mutation. PMID:17235394

  5. Role of ubiquitylation and USP8-dependent deubiquitylation in the endocytosis and lysosomal targeting of plasma membrane KCa3.1.

    PubMed

    Balut, Corina M; Loch, Christian M; Devor, Daniel C

    2011-11-01

    We recently demonstrated that plasma membrane KCa3.1 is rapidly endocytosed and targeted for lysosomal degradation via a Rab7- and ESCRT-dependent pathway. Herein, we assess the role of ubiquitylation in this process. Using a biotin ligase acceptor peptide (BLAP)-tagged KCa3.1, in combination with tandem ubiquitin binding entities (TUBEs), we demonstrate that KCa3.1 is polyubiquitylated following endocytosis. Hypertonic sucrose inhibited KCa3.1 endocytosis and resulted in a significant decrease in channel ubiquitylation. Inhibition of the ubiquitin-activating enzyme (E1) with UBEI-41 resulted in reduced KCa3.1 ubiquitylation and internalization. The general deubiquitylase (DUB) inhibitor, PR-619 attenuated KCa3.1 degradation, indicative of deubiquitylation being required for lysosomal delivery. Using the DUB Chip, a protein microarray containing 35 DUBs, we demonstrate a time-dependent association between KCa3.1 and USP8 following endocytosis, which was confirmed by coimmunoprecipitation. Further, overexpression of wild-type USP8 accelerates channel deubiquitylation, while either a catalytically inactive mutant USP8 or siRNA-mediated knockdown of USP8 enhanced accumulation of ubiquitylated KCa3.1, thereby inhibiting channel degradation. In summary, by combining BLAP-tagged KCa3.1 with TUBEs and DUB Chip methodologies, we demonstrate that polyubiquitylation mediates the targeting of membrane KCa3.1 to the lysosomes and also that USP8 regulates the rate of KCa3.1 degradation by deubiquitylating KCa3.1 prior to lysosomal delivery.

  6. Loss of lysosomal membrane protein NCU-G1 in mice results in spontaneous liver fibrosis with accumulation of lipofuscin and iron in Kupffer cells

    PubMed Central

    Kong, Xiang Y.; Nesset, Cecilie Kasi; Damme, Markus; Løberg, Else-Marit; Lübke, Torben; Mæhlen, Jan; Andersson, Kristin B.; Lorenzo, Petra I.; Roos, Norbert; Thoresen, G. Hege; Rustan, Arild C.; Kase, Eili T.; Eskild, Winnie

    2014-01-01

    Human kidney predominant protein, NCU-G1, is a highly conserved protein with an unknown biological function. Initially described as a nuclear protein, it was later shown to be a bona fide lysosomal integral membrane protein. To gain insight into the physiological function of NCU-G1, mice with no detectable expression of this gene were created using a gene-trap strategy, and Ncu-g1gt/gt mice were successfully characterized. Lysosomal disorders are mainly caused by lack of or malfunctioning of proteins in the endosomal-lysosomal pathway. The clinical symptoms vary, but often include liver dysfunction. Persistent liver damage activates fibrogenesis and, if unremedied, eventually leads to liver fibrosis/cirrhosis and death. We demonstrate that the disruption of Ncu-g1 results in spontaneous liver fibrosis in mice as the predominant phenotype. Evidence for an increased rate of hepatic cell death, oxidative stress and active fibrogenesis were detected in Ncu-g1gt/gt liver. In addition to collagen deposition, microscopic examination of liver sections revealed accumulation of autofluorescent lipofuscin and iron in Ncu-g1gt/gt Kupffer cells. Because only a few transgenic mouse models have been identified with chronic liver injury and spontaneous liver fibrosis development, we propose that the Ncu-g1gt/gt mouse could be a valuable new tool in the development of novel treatments for the attenuation of fibrosis due to chronic liver damage. PMID:24487409

  7. Structure of LEP100, a glycoprotein that shuttles between lysosomes and the plasma membrane, deduced from the nucleotide sequence of the encoding cDNA

    PubMed Central

    1988-01-01

    LEP100, a membrane glycoprotein that has the unique property of shuttling from lysosomes to endosomes to plasma membrane and back, was purified from chicken brain. Its NH2-terminal amino acid sequence was determined, and an oligonucleotide encoding part of this sequence was used to clone the encoding cDNA. The deduced amino acid sequence consists of 414 residues of which the NH2-terminal 18 constitute a signal peptide. The sequence includes 17 sites for N-glycosylation in the NH2-terminal 75% of the polypeptide chain followed by a region lacking N-linked oligosaccharides, a single possible membrane-spanning segment, and a cytoplasmic domain of 11 residues, including three potential phosphorylation sites. Eight cysteine residues are spaced in a regular pattern through the lumenal (extracellular) domain, while a 32-residue sequence rich in proline, serine, and threonine occurs at its midpoint. Expression of the cDNA in mouse L cells resulted in targeting of LEP100 primarily to the mouse lysosomes. PMID:3339090

  8. Application of a battery of biomarkers in mussel digestive gland to assess long-term effects of the Prestige oil spill in Galicia and the Bay of Biscay: lysosomal responses.

    PubMed

    Garmendia, Larraitz; Izagirre, Urtzi; Cajaraville, Miren P; Marigómez, Ionan

    2011-04-01

    In order to assess the long-term lysosomal responses to the Prestige oil spill (POS), mussels, Mytilus galloprovincialis, were collected in 22 localities from Galicia and the Bay of Biscay (North Iberian peninsula) in July, and September 2003, April, July, and October 2004-2005 and April 2006. Lysosomal membrane stability (labilisation period, LP) and lysosomal structural changes (lysosomal volume density, Vv(L) and lysosomal surface-to-volume ratio, S/V(L)) were measured as general stress biomarkers. The most remarkable long-term effects after the POS were drastic changes in lysosomal size (lysosomal enlargement) and membrane stability (extremely low LP values) up to April-04. Later on, a recovery trend was envisaged all along the studied area after July-04, albeit membrane stability continued to be below 20 min throughout the studied period up to April-06, which indicates a "distress-to-moderate-stress" condition. Lysosomal Response Index (LRI) revealed that environmental stress was more marked in Galicia than in the Bay of Biscay, mainly in the first sampling year, although a "moderate-to-high-stress" condition persisted until July-05. Overall, although lysosomal size returned to reference values, membrane stability was not fully recovered indicating a stress situation throughout the studied period.

  9. Ceria nanoparticles stabilized by organic surface coatings activate the lysosome-autophagy system and enhance autophagic clearance.

    PubMed

    Song, Wensi; Soo Lee, Seung; Savini, Marzia; Popp, Lauren; Colvin, Vicki L; Segatori, Laura

    2014-10-28

    Cerium oxide nanoparticles (nanoceria) are widely used in a variety of industrial applications including UV filters and catalysts. The expanding commercial scale production and use of ceria nanoparticles have inevitably increased the risk of release of nanoceria into the environment as well as the risk of human exposure. The use of nanoceria in biomedical applications is also being currently investigated because of its recently characterized antioxidative properties. In this study, we investigated the impact of ceria nanoparticles on the lysosome-autophagy system, the main catabolic pathway that is activated in mammalian cells upon internalization of exogenous material. We tested a battery of ceria nanoparticles functionalized with different types of biocompatible coatings (N-acetylglucosamine, polyethylene glycol and polyvinylpyrrolidone) expected to have minimal effect on lysosomal integrity and function. We found that ceria nanoparticles promote activation of the transcription factor EB, a master regulator of lysosomal function and autophagy, and induce upregulation of genes of the lysosome-autophagy system. We further show that the array of differently functionalized ceria nanoparticles tested in this study enhance autophagic clearance of proteolipid aggregates that accumulate as a result of inefficient function of the lysosome-autophagy system. This study provides a mechanistic understanding of the interaction of ceria nanoparticles with the lysosome-autophagy system and demonstrates that ceria nanoparticles are activators of autophagy and promote clearance of autophagic cargo. These results provide insights for the use of nanoceria in biomedical applications, including drug delivery. These findings will also inform the design of engineered nanoparticles with safe and precisely controlled impact on the environment and the design of nanotherapeutics for the treatment of diseases with defective autophagic function and accumulation of lysosomal storage material.

  10. 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.

    PubMed

    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.

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

    PubMed

    Roy, Subhro Jyoti; Stanely Mainzen Prince, Ponnian

    2012-11-01

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

  12. Brief exposure to copper activates lysosomal exocytosis.

    PubMed

    Peña, Karina; Coblenz, Jessica; Kiselyov, Kirill

    2015-04-01

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

  13. The membrane-stabilizing action of zinc carnosine (Z-103) in stress-induced gastric ulceration in rats

    SciTech Connect

    Cho, C.H.; Luk, C.T.; Ogle, C.W. )

    1991-01-01

    Zinc compounds have been shown to antagonize various types of gastric ulceration in rats. Zinc carnosine (Z-103), a newly developed agent was, therefore, examined for its antiulcer effect in stress-induced ulceration and also its membrane stabilizing action in rat stomachs. Cold-restraint stress induced severe hemorrhagic lesions together with increased mast cell degranulation and {beta}-glucuronidase release in the gastric glandular mucosa. A-103 pretreatment with a single oral dose reversed these actions in a dose-dependent manner. When the compound was incubated in concentrations of 10{sup {minus}7}, 10{sup {minus}6}, 10{sup {minus}5} or 10{sup {minus}4} M, with isolated hepatic lysosomes, it significantly reduced the spontaneous release of {beta}-glucuronidase in the medium. The present study not only demonstrates the antiulcer effect of Z-103 but also indicates that the protective action is likely to be mediated by its membrane-stabilizing action on mast cells and lysosomes in the gastric glandular mucosa.

  14. Behaviour of Steel Arch Stabilized by a Textile Membrane

    NASA Astrophysics Data System (ADS)

    Svoboda, O.; Machacek, J.

    2015-11-01

    Behaviour of the slender steel arch supporting textile membranes in a membrane structure with respect to in-plane and out-of plane stability is investigated in the paper. In the last decades the textile membranes have been widely used to cover both common and exclusive structures due to progress in new membrane materials with eminent properties. Nevertheless, complex analysis of such membranes in interaction with steel structure (carbon/stainless steel perimeter or supporting elements) is rather demanding, even with specialized software. Laboratory model of a large membrane structure simulating a shelter roof of a concert stage was tested and the resulting stress/deflection values are presented. The model of a reasonable size was provided with prestressed membrane of PVC coated polyester fabric Ferrari® Précontraint 702S and tested under various loadings. The supporting steel structure consisted of two steel arch tubes from S355 grade steel and perimeter prestressed cables. The stability behaviour of the inner tube was the primary interest of the investigation. The SOFiSTiK software was used to analyse the structural behaviour in 3D. Numerical non-linear analysis of deflections and internal forces of the structure under symmetrical and asymmetrical loadings covers various membrane prestressing and specific boundary conditions. The numerical results are validated using test results. Finally, the preliminary recommendations for appropriate numerical modelling and stability design of the supporting structure are presented.

  15. A Molecular Mechanism to Regulate Lysosome Motility for Lysosome Positioning and Tubulation

    PubMed Central

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

    2016-01-01

    To mediate the degradation of bio-macromolecules, lysosomes must traffic towards cargo-carrying vesicles for subsequent membrane fusion or fission. Mutations of the lysosomal Ca2+ channel TRPML1 cause lysosome storage disease (LSD) characterized by disordered lysosomal membrane trafficking in cells. Here we show that TRPML1 activity is required to promote Ca2+-dependent centripetal movement of lysosomes towards the perinuclear region, where autophagosomes accumulate, upon autophagy induction. ALG-2, an EF-hand-containing protein, serves as a lysosomal Ca2+ sensor that associates physically with the minus-end directed dynactin-dynein motor, while PI(3,5)P2, a lysosome-localized phosphoinositide, acts upstream of TRPML1. Furthermore, the PI(3,5)P2-TRPML1-ALG-2-dynein signaling 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 caused by secondary cholesterol accumulation that constitutively activates Rab7-RILP-dependent retrograde transport. Collectively, Ca2+ release from lysosomes provides an on-demand mechanism regulating lysosome motility, positioning, and tubulation. PMID:26950892

  16. Stability and dynamics of membrane-spanning DNA nanopores

    PubMed Central

    Maingi, Vishal; Burns, Jonathan R.; Uusitalo, Jaakko J.; Howorka, Stefan; Marrink, Siewert J.; Sansom, Mark S. P.

    2017-01-01

    Recently developed DNA-based analogues of membrane proteins have advanced synthetic biology. A fundamental question is how hydrophilic nanostructures reside in the hydrophobic environment of the membrane. Here, we use multiscale molecular dynamics (MD) simulations to explore the structure, stability and dynamics of an archetypical DNA nanotube inserted via a ring of membrane anchors into a phospholipid bilayer. Coarse-grained MD reveals that the lipids reorganize locally to interact closely with the membrane-spanning section of the DNA tube. Steered simulations along the bilayer normal establish the metastable nature of the inserted pore, yielding a force profile with barriers for membrane exit due to the membrane anchors. Atomistic, equilibrium simulations at two salt concentrations confirm the close packing of lipid around of the stably inserted DNA pore and its cation selectivity, while revealing localized structural fluctuations. The wide-ranging and detailed insight informs the design of next-generation DNA pores for synthetic biology or biomedicine. PMID:28317903

  17. Stability and dynamics of membrane-spanning DNA nanopores

    NASA Astrophysics Data System (ADS)

    Maingi, Vishal; Burns, Jonathan R.; Uusitalo, Jaakko J.; Howorka, Stefan; Marrink, Siewert J.; Sansom, Mark S. P.

    2017-03-01

    Recently developed DNA-based analogues of membrane proteins have advanced synthetic biology. A fundamental question is how hydrophilic nanostructures reside in the hydrophobic environment of the membrane. Here, we use multiscale molecular dynamics (MD) simulations to explore the structure, stability and dynamics of an archetypical DNA nanotube inserted via a ring of membrane anchors into a phospholipid bilayer. Coarse-grained MD reveals that the lipids reorganize locally to interact closely with the membrane-spanning section of the DNA tube. Steered simulations along the bilayer normal establish the metastable nature of the inserted pore, yielding a force profile with barriers for membrane exit due to the membrane anchors. Atomistic, equilibrium simulations at two salt concentrations confirm the close packing of lipid around of the stably inserted DNA pore and its cation selectivity, while revealing localized structural fluctuations. The wide-ranging and detailed insight informs the design of next-generation DNA pores for synthetic biology or biomedicine.

  18. RADIATION STABILITY OF NAFION MEMBRANES USED FOR ISOTOPE SEPARATION BY PROTON EXCHANGE MEMBRANE ELECTROLYSIS

    SciTech Connect

    Fox, E

    2009-05-15

    Proton Exchange Membrane Electrolyzers have potential interest for use for hydrogen isotope separation from water. In order for PEME to be fully utilized, more information is needed on the stability of Nafion when exposed to radiation. This work examines Nafion 117 under varying exposure conditions, including dose rate, total dosage and atmospheric condition. Analytical tools, such as FT-IR, ion exchange capacity, DMA and TIC-TOC were used to characterize the exposed membranes. Analysis of the water from saturated membranes can provide important data on the stability of the membranes during radiation exposure. It was found that the dose rate of exposure plays an important role in membrane degradation. Potential mechanisms for membrane degradation include peroxide formation by free radicals.

  19. Regulated lysosomal exocytosis mediates cancer progression

    PubMed Central

    Machado, Eda; White-Gilbertson, Shai; van de Vlekkert, Diantha; Janke, Laura; Moshiach, Simon; Campos, Yvan; Finkelstein, David; Gomero, Elida; Mosca, Rosario; Qiu, Xiaohui; Morton, Christopher L.; Annunziata, Ida; d’Azzo, Alessandra

    2015-01-01

    Understanding how tumor cells transition to an invasive and drug-resistant phenotype is central to cancer biology, but the mechanisms underlying this transition remain unclear. We show that sarcomas gain these malignant traits by inducing lysosomal exocytosis, a ubiquitous physiological process. During lysosomal exocytosis, the movement of exocytic lysosomes along the cytoskeleton and their docking at the plasma membrane involve LAMP1, a sialylated membrane glycoprotein and target of the sialidase NEU1. Cleavage of LAMP1 sialic acids by NEU1 limits the extent of lysosomal exocytosis. We found that by down-regulation of NEU1 and accumulation of oversialylated LAMP1, tumor cells exacerbate lysosomal exocytosis of soluble hydrolases and exosomes. This facilitates matrix invasion and propagation of invasive signals, and purging of lysosomotropic chemotherapeutics. In Arf−⁄− mice, Neu1 haploinsufficiency fostered the development of invasive, pleomorphic sarcomas, expressing epithelial and mesenchymal markers, and lysosomal exocytosis effectors, LAMP1 and Myosin-11. These features are analogous to those of metastatic, pleomorphic human sarcomas, where low NEU1 levels correlate with high expression of lysosomal exocytosis markers. In a therapeutic proof of principle, we demonstrate that inhibiting lysosomal exocytosis reversed invasiveness and chemoresistance in aggressive sarcoma cells. Thus, we reveal that this unconventional, lysosome-regulated pathway plays a primary role in tumor progression and chemoresistance. PMID:26824057

  20. Liquid permeation and chemical stability of anodic alumina membranes

    PubMed Central

    Buldakov, Dmitrii A; Tishkin, Alexey A; Lukashin, Alexey V; Eliseev, Andrei A

    2017-01-01

    A study on the chemical stability of anodic alumina membranes and their performance in long-term water and organic solvent permeation experiments is reported. Anodic alumina possesses high stability for both protonic and aprotonic organic solvents. However, serious degradation of the membrane occurs in pure water, leading to a drastic decrease of permeance (over 20% of the initial value after the passing of 0.250 m3/m2 of pure water). The drying of the membrane induces further permeance drop-off. The rate of membrane degradation strongly depends on the pH of the penetrant solution and increases in basic media. According to 27Al NMR and thermogravimetry results, the degradation of the membranes is associated with the dissolution of water-soluble [Al13O4(OH)24(H2O)12]7+ polyhydroxocomplexes and their further redeposition in the form of [Al(OH)4]−, resulting in channels blocking. This process intensifies in basic pH due to the high positive charge of the anodic alumina surface. An approach for improving anodic aluminum oxide stability towards dissolution in water by carbon CVD coating of the membrane walls is suggested. PMID:28382245

  1. Stabilization of porous glass reverse-osmosis membranes

    NASA Technical Reports Server (NTRS)

    Ballou, E. V.; Leban, M. I.; Wydeven, T.

    1972-01-01

    Application of porous glass in form of capillary tubes for low capacity ion exchange in hyperfiltration experiments is discussed. Efficiency of desalination by process of reverse osmosis is described. Stabilization of porous glass membrane by presence of aluminum chloride is analyzed.

  2. A novel class of autoantigens of anti-neutrophil cytoplasmic antibodies in necrotizing and crescentic glomerulonephritis: the lysosomal membrane glycoprotein h-lamp-2 in neutrophil granulocytes and a related membrane protein in glomerular endothelial cells

    PubMed Central

    1995-01-01

    Necrotizing and crescentic glomerulonephritis (NCGN) is frequently associated with circulating antineutrophil cytoplasmic autoantibodies (ANCA). It is established that ANCA are specific for soluble enzymes of granules of polymorphonuclear neutrophil granulocytes (PMN), such as myeloperoxidase (MPO) or protease 3 (PR3). The purpose of this study was to identify membrane proteins of PMNs, and/or glomerular cells, as additional autoantigenic ANCA targets. When membrane protein fractions were prepared from PMNs and isolated human glomeruli, and immunoblotted with ANCA sera of NCGN patients, two bands with apparent molecular masses of 170 and 80-110 kD (gp170/80-110) were labeled in PMNs, and a 130-kD glycoprotein (gp130) in glomeruli. Gp130 was purified, and monoclonal and rabbit antibodies (Abs) were produced which showed the same double specificity as the patient's ANCA. Using these probes, evidence was provided that gp170/80-110 is identical with human lysosomal-associated membrane protein 2 (h-lamp-2), because both proteins were immunologically cross-reactive and screening of a cDNA expression library from human promyelocytic leukemia cells with anti- gp130 Ab yielded a clone derived from h-lamp-2. Gp170/80-110 was localized primarily in granule membranes of resting PMNs, and was translocated to the cell surfaces by activation with FMLP. By contrast, gp130 was localized in the surface membranes of endothelial cells of human glomerular and renal interstitial capillaries, rather than in lysosomes, as found for h-lamp-2. Potential clinical relevance of autoantibodies to gp170/80-110 and gp130 was assessed in a preliminary trial, in which ANCA sera of patients (n = 16) with NCGN were probed with purified or recombinant antigens. Specific reactivity was detected in approximately 90% of cases with active phases of NCGN, and frequently also in combination with autoantibodies specific for PR3 or MPO. Collectively, these data provide evidence that h-lamp-2 in PMNs and a

  3. Lysosomal responses to heat-shock of seasonal temperature extremes in Cd-exposed mussels.

    PubMed

    Múgica, M; Izagirre, U; Marigómez, I

    2015-07-01

    The present study was aimed at determining the effect of temperature extremes on lysosomal biomarkers in mussels exposed to a model toxic pollutant (Cd) at different seasons. For this purpose, temperature was elevated 10°C (from 12°C to 22°C in winter and from 18°C to 28°C in summer) for a period of 6h (heat-shock) in control and Cd-exposed mussels, and then returned back to initial one. Lysosomal membrane stability and lysosomal structural changes in digestive gland were investigated. In winter, heat-shock reduced the labilisation period (LP) of the lysosomal membrane, especially in Cd-exposed mussels, and provoked transient lysosomal enlargement. LP values recovered after the heat-shock cessation but lysosomal enlargement prevailed in both experimental groups. In summer, heat-shock induced remarkable reduction in LP and lysosomal enlargement (more markedly in Cd-exposed mussels), which recovered within 3 days. Besides, whilst heat-shock effects on LP were practically identical for Cd-exposed mussels in winter and summer, the effects were longer-lasting in summer than in winter for control mussels. Thus, lysosomal responsiveness after heat-shock was higher in summer than in winter but recovery was faster as well, and therefore the consequences of the heat shock seem to be more decisive in winter. In contrast, inter-season differences were attenuated in the presence of Cd. Consequently, mussels seem to be better prepared in summer than in winter to stand short periods of abrupt temperature change; this is, however, compromised when mussels are exposed to pollutants such as Cd.

  4. Stabilization of membranes upon interaction of amphipathic polymers with membrane proteins

    PubMed Central

    Picard, Martin; Duval-Terrié, Caroline; Dé, Emmanuelle; Champeil, Philippe

    2004-01-01

    Amphipathic polymers derived from polysaccharides, namely hydrophobically modified pullulans, were previously suggested to be useful as polymeric substitutes of ordinary surfactants for efficient and structure-conserving solubilization of membrane proteins, and one such polymer, 18C10, was optimized for solubilization of proteins derived from bacterial outer membranes (Duval-Terrié et al. 2003). We asked whether a similar ability to solubilize proteins could also be demonstrated in eukaryotic membranes, namely sarcoplasmic reticulum (SR) fragments, the major protein of which is SERCA1a, an integral membrane protein with Ca2+-dependent ATPase and Ca2+-pumping activity. We found that 18C10-mediated solubilization of these SR membranes did not occur. Simultaneously, however, we found that low amounts of this hydrophobically modified pullulan were very efficient at preventing long-term aggregation of these SR membranes. This presumably occurred because the negatively charged polymer coated the membranous vesicles with a hydrophilic corona (a property shared by many other amphipathic polymers), and thus minimized their flocculation. Reminiscent of the old Arabic gum, which stabilizes Indian ink by coating charcoal particles, the newly designed amphipathic polymers might therefore unintentionally prove useful also for stabilization of membrane suspensions. PMID:15459343

  5. Carotenoid incorporation into microsomes: yields, stability and membrane dynamics

    NASA Astrophysics Data System (ADS)

    Socaciu, Carmen; Jessel, Robert; Diehl, Horst A.

    2000-12-01

    The carotenoids β-carotene (BC), lycopene (LYC), lutein (LUT), zeaxanthin (ZEA), canthaxanthin (CTX) and astaxanthin (ASTA) have been incorporated into pig liver microsomes. Effective incorporation concentrations in the range of about 1-6 nmol/mg microsomal protein were obtained. A stability test at room temperature revealed that after 3 h BC and LYC had decayed totally whereas, gradually, CTX (46%), LUT (21%), ASTA (17%) and ZEA (5%) decayed. Biophysical parameters of the microsomal membrane were changed hardly by the incorporation of carotenoids. A small rigidification may occur. Membrane anisotropy seems to offer only a small tolerance for incorporation of carotenoids and seems to limit the achievable incorporation concentrations of the carotenoids into microsomes. Microsomes instead of liposomes should be preferred as a membrane model to study mutual effects of carotenoids and membrane dynamics.

  6. CDw78 defines MHC class II-peptide complexes that require Ii chain-dependent lysosomal trafficking, not localization to a specific tetraspanin membrane microdomain.

    PubMed

    Poloso, Neil J; Denzin, Lisa K; Roche, Paul A

    2006-10-15

    MHC class II molecules (MHC-II) associate with detergent-resistant membrane microdomains, termed lipid rafts, which affects the function of these molecules during Ag presentation to CD4+ T cells. Recently, it has been proposed that MHC-II also associates with another type of membrane microdomain, termed tetraspan microdomains. These microdomains are defined by association of molecules to a family of proteins that contain four-transmembrane regions, called tetraspanins. It has been suggested that MHC-II associated with tetraspanins are selectively identified by a mAb to a MHC-II determinant, CDw78. In this report, we have re-examined this issue of CDw78 expression and MHC-II-association with tetraspanins in human dendritic cells, a variety of human B cell lines, and MHC-II-expressing HeLa cells. We find no correlation between the expression of CDw78 and the expression of tetraspanins CD81, CD82, CD53, CD9, and CD37. Furthermore, we find that the relative amount of tetraspanins bound to CDw78-reactive MHC-II is indistinguishable from the amount bound to peptide-loaded MHC-II. We found that expression of CDw78 required coexpression of MHC-II together with its chaperone Ii chain. In addition, analysis of a panel of MHC-II-expressing B cell lines revealed that different alleles of HLA-DR express different amounts of CDw78 reactivity. We conclude that CDw78 defines a conformation of MHC-II bound to peptides that are acquired through trafficking to lysosomal Ag-processing compartments and not MHC-II-associated with tetraspanins.

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

    PubMed

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

    2014-04-01

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

  8. Capture-stabilize approach for membrane protein SPR assays.

    PubMed

    Chu, Ruiyin; Reczek, David; Brondyk, William

    2014-12-08

    Measuring the binding kinetics of antibodies to intact membrane proteins by surface plasmon resonance has been challenging largely because of the inherent difficulties in capturing membrane proteins on chip surfaces while retaining their native conformation. Here we describe a method in which His-tagged CXCR5, a GPCR, was purified and captured on a Biacore chip surface via the affinity tag. The captured receptor protein was then stabilized on the chip surface by limited cross-linking. The resulting chip surface retained ligand binding activity and was used for monoclonal antibody kinetics assays by a standard Biacore kinetics assay method with a simple low pH regeneration step. We demonstrate the advantages of this whole receptor assay when compared to available peptide-based binding assays. We further extended the application of the capture-stabilize approach to virus-like particles and demonstrated its utility analyzing antibodies against CD52, a GPI-anchored protein, in its native membrane environment. The results are the first demonstration of chemically stabilized chip surfaces for membrane protein SPR assays.

  9. Stability properties of elementary dynamic models of membrane transport.

    PubMed

    Hernández, Julio A

    2003-01-01

    Living cells are characterized by their capacity to maintain a stable steady state. For instance, cells are able to conserve their volume, internal ionic composition and electrical potential difference across the plasma membrane within values compatible with the overall cell functions. The dynamics of these cellular variables is described by complex integrated models of membrane transport. Some clues for the understanding of the processes involved in global cellular homeostasis may be obtained by the study of the local stability properties of some partial cellular processes. As an example of this approach, I perform, in this study, the neighborhood stability analysis of some elementary integrated models of membrane transport. In essence, the models describe the rate of change of the intracellular concentration of a ligand subject to active and passive transport across the plasma membrane of an ideal cell. The ligand can be ionic or nonionic, and it can affect the cell volume or the plasma membrane potential. The fundamental finding of this study is that, within the physiological range, the steady states are asymptotically stable. This basic property is a necessary consequence of the general forms of the expressions employed to describe the active and passive fluxes of the transported ligand.

  10. [Lysosomal system in hormonal mechanisms. Review].

    PubMed

    Duran Reyes, G; González Macías, G; Hicks, J J

    1995-02-01

    The role of lysosomes in the intracellular mechanism of action of several steroid an proteic hormones has been demonstrated. In presence of the specific hormone the target cell induce membranal changes and the lysosomes are moved toward the nucleus; after this the lysosomal enzymes are released in the perinuclear space. For the moment it is not possible to know the biochemical role of this enzymatic activities upon the nucleic acids function and des-repretion process of specific genes, but the inhibition of lysosomes movement utilizing hormone antagonist or dexamethasone inhibits some reproductive process like the implantation of the mammalian egg. We present herein a review related with the mode action of some hormones through the lysosomes in reproductive processes.

  11. Stabilization of concentration fluctuations in mixed membranes by hybrid lipids

    NASA Astrophysics Data System (ADS)

    Palmieri, Benoit; Safran, Samuel

    2012-02-01

    Finite-size domains have been observed at the surface of cells. These lipids ``rafts'' are stable nanodomains enriched in saturated lipids and cholesterol. While line tension favors macrodomains, one explanation for raft stabilization suggests that the membrane composition is tuned close to a spinodal temperature. From this point of view, rafts are long-lived concentration fluctuations in the mixed phase. We propose a ternary mixture model for the cell membrane that includes hybrid lipids which have one saturated and one unsaturated hydrocarbon chain. Finite amount of hybrid lipids reduces the packing incompatibility at the saturated/unsaturated lipid interface and stabilizes the concentration fluctuations. Hybrid-Hybrid interactions are included in the model and further increase the life-time of the rafts and decrease their length-scales. Moreover, the hybrid has extra orientational degrees of freedom that may lead to modulated phases.

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

    PubMed

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

    2014-08-01

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

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

    PubMed

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

    2014-05-01

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

  14. Stabilizing factors of phospholipid asymmetry in the erythrocyte membrane.

    PubMed

    Dressler, V; Haest, C W; Plasa, G; Deuticke, B; Erusalimsky, J D

    1984-08-22

    Transbilayer reorientation (flip) of exogenous lysophospholipids and changes of the transbilayer distribution of endogenous phospholipids were studied in human erythrocytes and membrane vesicles. (1) Exogenous lysophosphatidylserine irreversibly accumulates in the inner membrane layer of resealed ghosts of human erythrocytes. (2) This accumulation even occurs after complete loss of asymmetric distribution of endogenous phosphatidylethanolamine and partial loss of phosphatidylserine asymmetry in diamide-treated cells. (3) Formation of inside-out and right-side-out vesicles from erythrocyte membranes results in a loss of endogenous phospholipid asymmetry as well as of the ability to establish asymmetry of exogenous lysophosphatidylserine. Rates of transbilayer reorientation of lysophospholipids for the vesicles, however, are comparable to those for intact cells. (4) Loss of endogenous asymmetry of phosphatidylserine is also observed in vesicles isolated from erythrocytes after heat denaturation of spectrin. The asymmetry in the residual cells is maintained. (5) In contrast to the loss of asymmetry of phosphatidylethanolamine and of phosphatidylserine, the asymmetry of sphingomyelin is completely maintained in the vesicles. (6) The stability of phospholipid asymmetry in the native cell is discussed in terms of a limitation of access of phospholipids to hypothetical reorientation sites. Such a limitation may either be the result of interaction of phospholipids with the membrane skeleton as in case of phosphatidylserine and phosphatidylethanolamine, or the result of lipid-lipid interactions as in case of sphingomyelin.

  15. Emulsion liquid membrane for textile dye removal: Stability study

    NASA Astrophysics Data System (ADS)

    Kusumastuti, Adhi; Syamwil, Rodia; Anis, Samsudin

    2017-03-01

    Although textile dyes is basically available in very low concentration; it should be removed due to the toxicity to human body and environment. Among the existing methods, emulsion liquid membrane (ELM) is a promising method by providing high interfacial area and the ability to remove a very low concentration of the solute. The optimal emulsions were produced using commercially supplied homogeniser. The drop size was measured by the aid of microscope and image J software. Initially, methylene blue in simulated wastewater was extracted using a stirrer. Methylene blue concentration was determined using spectrophotometer. The research obtained optimal emulsion at surfactant concentration of 4 wt. %, kerosene as diluent, emulsification time of 30 min, emulsification speed of 2000 rpm. The lowest membrane breakage and the longest stability time were about 0.11% and 150 min, respectively.

  16. Sickled Erythrocytes Reversal and Membrane Stabilizing Compounds in Telfairia occidentalis

    PubMed Central

    Atabo, Samuel; Umar, Ismaila Alhaji; James, Dorcas Bolanle; Mamman, Aisha Indo

    2016-01-01

    Background and Purpose. Traditional management of sickle cell disease (SCD) is ubiquitous in Africa. In south-eastern Nigeria, Telfairia occidentalis (T. occidentalis) is strongly recommended for consumption by SCD patients, owing to its presumed therapeutic effect. This study investigates the antisickling and membrane regenerative potentials of T. occidentalis in sickled erythrocytes. Experimental Approach. Sickled erythrocytes obtained from SCD patients were treated with sodium metabisulphite (2%) to induce further sickling. Heat and hypotonic-induced lyses of red blood cells' membranes were also carried out. The RBCs were treated with varying concentration (10.0, 1.0, and 0.1 mg mL−1 and 0.5, 1.0, 1.5, 2.0, and 2.5 mg mL−1, resp.) of T. occidentalis extracts as treatment regimen for in vitro antisickling and membrane stabilizing assays. Extract with peak activity was purified and reused in antisickling assay. Key Results. The antisickling activity of aqueous and methanolic extracts of leaves, seeds, and stem of Telfairia occidentalis at 10.0, 1.0, and 0.1 mg mL−1 revealed that the aqueous leaves extract (10 mg mL−1) exhibited the highest antisickling activity (64.03%) which was significantly (p < 0.05) higher than that of the stem (47.30%) and seeds (37.50%). Partially purified fractions recorded improved antisickling effect (peak activity of 70%). Characterization (using GC-MS) of the most active fraction revealed some bioactive compounds. In the membrane stabilizing assay, methanolic and aqueous stem extracts of T. occidentalis showed the highest effect of 71.85% and 61.29%, respectively. Conclusions and Implications. The results provide scientific evidence for ethnopharmacological use of T. occidentalis in the management of SCD. PMID:27433373

  17. Stabilized phospholipid membranes in chromatography: toward membrane protein-functionalized stationary phases.

    PubMed

    Gallagher, Elyssia S; Mansfield, Elisabeth; Aspinwall, Craig A

    2014-04-01

    Transmembrane protein (TMP)-functionalized materials have resulted in powerful new methods in chemical analysis. Of particular interest is the development of high-throughput, TMP-functionalized stationary phases for affinity chromatography of complex mixtures of analytes. Several natural and synthetic phospholipids and lipid mimics have been used for TMP reconstitution, although the resulting membranes often lack the requisite chemical and temporal stability for long-term use, a problem that is exacerbated in flowing separation systems. Polymerizable lipids with markedly increased membrane stability and TMP functionality have been developed over the past two decades. More recently, these lipids have been incorporated into a range of analytical methods, including separation techniques, and are now poised to have a significant impact on TMP-based separations. Here, we describe current methods for preparing TMP-containing stationary phases and examine the potential utility of polymerizable lipids in TMP affinity chromatography.

  18. Tartrate-resistant acid phosphatase (TRAP) co-localizes with receptor activator of NF-KB ligand (RANKL) and osteoprotegerin (OPG) in lysosomal-associated membrane protein 1 (LAMP1)-positive vesicles in rat osteoblasts and osteocytes.

    PubMed

    Solberg, L B; Stang, E; Brorson, S-H; Andersson, G; Reinholt, F P

    2015-02-01

    Tartrate-resistant acid phosphatase (TRAP) is well known as an osteoclast marker; however, a recent study from our group demonstrated enhanced number of TRAP + osteocytes as well as enhanced levels of TRAP located to intracellular vesicles in osteoblasts and osteocytes in experimental osteoporosis in rats. Such vesicles were especially abundant in osteoblasts and osteocytes in cancellous bone as well as close to bone surface and intracortical remodeling sites. To further investigate TRAP in osteoblasts and osteocytes, long bones from young, growing rats were examined. Immunofluorescence confocal microscopy displayed co-localization of TRAP with receptor activator of NF-KB ligand (RANKL) and osteoprotegerin (OPG) in hypertrophic chondrocytes and diaphyseal osteocytes with Pearson's correlation coefficient ≥0.8. Transmission electron microscopy showed co-localization of TRAP and RANKL in lysosomal-associated membrane protein 1 (LAMP1) + vesicles in osteoblasts and osteocytes supporting the results obtained by confocal microscopy. Recent in vitro data have demonstrated OPG as a traffic regulator for RANKL to LAMP1 + secretory lysosomes in osteoblasts and osteocytes, which seem to serve as temporary storage compartments for RANKL. Our in situ observations indicate that TRAP is located to RANKL-/OPG-positive secretory lysosomes in osteoblasts and osteocytes, which may have implications for osteocyte regulation of osteoclastogenesis.

  19. Lysosome-associated membrane glycoprotein 1 predicts fratricide amongst T cell receptor transgenic CD8+ T cells directed against tumor-associated antigens

    PubMed Central

    Kirschner, Andreas; Thiede, Melanie; Blaeschke, Franziska; Richter, Günther H.S.; Gerke, Julia S.; Baldauf, Michaela C.; Grünewald, Thomas G.P.; Busch, Dirk H.; Burdach, Stefan; Thiel, Uwe

    2016-01-01

    Aim Autologous as well as allogeneic CD8+ T cells transduced with tumor antigen specific T cell receptors (TCR) may cause significant tumor lysis upon adoptive transfer. Besides unpredictable life-threatening off-target effects, these TCRs may unexpectedly commit fratricide. We hypothesized lysosome-associated membrane glycoprotein 1 (LAMP1, CD107a) to be a marker for fratricide in TCR transgenic CD8+ T cells. Methods We identified HLA-A*02:01/peptide-restricted T cells directed against ADRB3295. After TCR identification, we generated HLA-A*02:01/peptide restricted TCR transgenic T cells by retroviral transduction and tested T cell expansion rates as well as A*02:01/peptide recognition and ES killing in ELISpot and xCELLigence assays. Expansion arrest was analyzed via Annexin and CD107a staining. Results were compared to CHM1319-TCR transgenic T cells. Results Beta-3-adrenergic receptor (ADRB3) as well as chondromodulin-1 (CHM1) are over-expressed in Ewing Sarcoma (ES) but not on T cells. TCR transgenic T cells demonstrated HLA-A*02:01/ADRB3295 mediated ES recognition and killing in ELISpot and xCELLigence assays. 24h after TCR transduction, CD107a expression correlated with low expansion rates due to apoptosis of ADRB3 specific T cells in contrast to CHM1 specific transgenic T cells. Amino-acid exchange scans clearly indicated the cross-reactive potential of HLA-A*02:01/ADRB3295- and HLA-A*02:01/CHM1319-TCR transgenic T cells. Comparison of peptide motive binding affinities revealed extended fratricide among ADRB3295 specific TCR transgenic T cells in contrast to CHM1319. Conclusion Amino-acid exchange scans alone predict TCR cross-reactivity with little specificity and thus require additional assessment of potentially cross-reactive HLA-A*02:01 binding candidates. CD107a positivity is a marker for fratricide of CD8+ TCR transgenic T cells. PMID:27447745

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

    PubMed

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

    2016-12-09

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

  1. Lysosomes, cholesterol and atherosclerosis

    PubMed Central

    Jerome, W Gray

    2011-01-01

    Cholesterol-engorged macrophage foam cells are a critical component of the atherosclerotic lesion. Reducing the sterol deposits in lesions reduces clinical events. Sterol accumulations within lysosomes have proven to be particularly hard to mobilize out of foam cells. Moreover, excess sterol accumulation in lysosomes has untoward effects, including a complete disruption of lysosome function. Recently, we demonstrated that treatment of sterol-engorged macrophages in culture with triglyceride-containing particles can reverse many of the effects of cholesterol on lysosomes and dramatically reduce the sterol burden in these cells. This article describes what is known about lysosomal sterol engorgement, discusses the possible mechanisms by which triglyceride could produce its effects, and evaluates the possible positive and negative effects of reducing the lysosomal cholesterol levels in foam cells. PMID:21643524

  2. Stability of membrane potential in heart mitochondria: Single mitochondrion imaging

    SciTech Connect

    Uechi, Yukiko; Yoshioka, Hisashi; Morikawa, Daisuke; Ohta, Yoshihiro . E-mail: ohta@cc.tuat.ac.jp

    2006-06-16

    Mitochondrial membrane potential ({delta}{psi} {sub m}) plays an important role in cellular activity. Although {delta}{psi} {sub m} of intracellular mitochondria are relatively stable, the recent experiments with isolated mitochondria demonstrate that individual mitochondria show frequent fluctuations of {delta}{psi} {sub m}. The current study is performed to investigate the factors that stabilize {delta}{psi} {sub m} in cells by observing {delta}{psi} {sub m} of individual isolated mitochondria with fluorescence microscopy. Here, we report that (1) the transient depolarizations are also induced for mitochondria in plasma membrane permeabilized cells, (2) almost all mitochondria isolated from porcine hearts show the transient depolarizations that is enhanced with the net efflux of protons from the matrix to the intermembrane space, and (3) ATP and ADP significantly inhibit the transient depolarizations by plural mechanisms. These results suggest that the suppression of acute alkalinization of the matrix together with the presence of ATP and ADP contributes to the stabilization of {delta}{psi} {sub m} in cells.

  3. Stabilization of composition fluctuations in mixed membranes by hybrid lipids

    NASA Astrophysics Data System (ADS)

    Safran, Samuel; Palmieri, Benoit

    2013-03-01

    A ternary mixture model is proposed to describe composition fluctuations in mixed membranes composed of saturated, unsaturated and hybrid lipids. The asymmetric hybrid lipid has one saturated and one unsaturated hydrocarbon chain and it can reduce the packing incompatibility between saturated and unsaturated lipids. A methodology to recast the free-energy of the lattice in terms of a continuous isotropic field theory is proposed and used to analyze composition fluctuations above the critical temperature. The effect of hybrid lipids on fluctuations domains rich in saturated/unsaturated lipids is predicted. The correlation length of such fluctuations decreases significantly with increasing amounts of hybrids even if the temperature is maintained close to the critical temperature. This provides an upper bound for the domain sizes expected in rafts stabilized by hybrids, above the critical temperature. When the hybrid composition of the membrane is increased further, a crossover value is found above which ``stripe-like'' fluctuations are observed. The wavelength of these fluctuations decreases with increasing hybrid fraction and tends toward a molecular size in a membrane that contains only hybrids.

  4. Injured astrocytes are repaired by Synaptotagmin XI-regulated lysosome exocytosis.

    PubMed

    Sreetama, S C; Takano, T; Nedergaard, M; Simon, S M; Jaiswal, J K

    2016-04-01

    Astrocytes are known to facilitate repair following brain injury; however, little is known about how injured astrocytes repair themselves. Repair of cell membrane injury requires Ca(2+)-triggered vesicle exocytosis. In astrocytes, lysosomes are the main Ca(2+)-regulated exocytic vesicles. Here we show that astrocyte cell membrane injury results in a large and rapid calcium increase. This triggers robust lysosome exocytosis where the fusing lysosomes release all luminal contents and merge fully with the plasma membrane. In contrast to this, receptor stimulation produces a small sustained calcium increase, which is associated with partial release of the lysosomal luminal content, and the lysosome membrane does not merge into the plasma membrane. In most cells, lysosomes express the synaptotagmin (Syt) isoform Syt VII; however, this isoform is not present on astrocyte lysosomes and exogenous expression of Syt VII on lysosome inhibits their exocytosis. Deletion of one of the most abundant Syt isoform in astrocyte--Syt XI--suppresses astrocyte lysosome exocytosis. This identifies lysosome as Syt XI-regulated exocytic vesicle in astrocytes. Further, inhibition of lysosome exocytosis (by Syt XI depletion or Syt VII expression) prevents repair of injured astrocytes. These results identify the lysosomes and Syt XI as the sub-cellular and molecular regulators, respectively of astrocyte cell membrane repair.

  5. Alpha-lipoic acid and alpha-lipoamide prevent oxidant-induced lysosomal rupture and apoptosis.

    PubMed

    Persson, H L; Svensson, A I; Brunk, U T

    2001-01-01

    Alpha-lipoic acid (LA) and its corresponding derivative, alpha-lipoamide (LM), have been described as antioxidants, but the mechanisms of their putative antioxidant effects remain largely uncharacterised. The vicinal thiols present in the reduced forms of these compounds suggest that they might possess metal chelating properties. We have shown previously that cell death caused by oxidants may be initiated by lysosomal rupture and that this latter event may involve intralysosomal iron which catalyzes Fenton-type chemistry and resultant peroxidative damage to lysosomal membranes. Here, using cultured J774 cells as a model, we show that both LA and LM stabilize lysosomes against oxidative stress, probably by chelating intralysosomal iron and, consequently, preventing intralysosomal Fenton reactions. In preventing oxidant-mediated apoptosis, LM is significantly more effective than LA, as would be expected from their differing capacities to enter cells and concentrate within the acidic lysosomal compartment. As previously reported, the powerful iron-chelator, desferrioxamine (Des) (which also locates within the lysosomal compartment), also provides protection against oxidant-mediated cell death. Interestingly, although Des enhances the partial protection afforded by LA, it confers no additional protection when added with LM. Therefore, the antioxidant actions of LA and LM may arise from intralysosomal iron chelation, with LM being more effective in this regard.

  6. Nanoporous membranes with electrochemically switchable, chemically stabilized ionic selectivity.

    PubMed

    Small, Leo J; Wheeler, David R; Spoerke, Erik D

    2015-10-28

    Nanopore size, shape, and surface charge all play important roles in regulating ionic transport through nanoporous membranes. The ability to control these parameters in situ provides a means to create ion transport systems tunable in real time. Here, we present a new strategy to address this challenge, utilizing three unique electrochemically switchable chemistries to manipulate the terminal functional group and control the resulting surface charge throughout ensembles of gold plated nanopores in ion-tracked polycarbonate membranes 3 cm(2) in area. We demonstrate the diazonium mediated surface functionalization with (1) nitrophenyl chemistry, (2) quinone chemistry, and (3) previously unreported trimethyl lock chemistry. Unlike other works, these chemistries are chemically stabilized, eliminating the need for a continuously applied gate voltage to maintain a given state and retain ionic selectivity. The effect of surface functionalization and nanopore geometry on selective ion transport through these functionalized membranes is characterized in aqueous solutions of sodium chloride at pH = 5.7. The nitrophenyl surface allows for ionic selectivity to be irreversibly switched in situ from cation-selective to anion-selective upon reduction to an aminophenyl surface. The quinone-terminated surface enables reversible changes between no ionic selectivity and a slight cationic selectivity. Alternatively, the trimethyl lock allows ionic selectivity to be reversibly switched by up to a factor of 8, approaching ideal selectivity, as a carboxylic acid group is electrochemically revealed or hidden. By varying the pore shape from cylindrical to conical, it is demonstrated that a controllable directionality can be imparted to the ionic selectivity. Combining control of nanopore geometry with stable, switchable chemistries facilitates superior control of molecular transport across the membrane, enabling tunable ion transport systems.

  7. Aspects of the digestive gland cells of the mussel Mytilus galloprovincialis, in relation to lysosomal enzymes, lipofuscin presence and shell size: contribution in the assessment of marine pollution biomarkers.

    PubMed

    Raftopoulou, E K; Dimitriadis, V K

    2012-02-01

    The present study investigates the histochemical localization of N-acetyl-β-hexozaminidase (Hex), acid phosphatase (AcP) and β-glucuronidase (β-Gus) in the digestive gland of mussels Mytilus galloprovincialis, as well as the clarification of suitable enzyme for biomarkers' application dealing with lysosomes. The results show more intense and homogenous localization of Hex, in relation to AcP and β-Gus and, thus, Hex histochemistry is supported as more suitable procedure for the evaluation of "lysosomal membrane stability" and "morphometrical alterations of lysosomes". The affection of lipofuscin granules on lysosomal enzymes' activity is also discussed. Additionally, the present study examines the response of small- and large-sized mussels M. galloprovincialis by assessing the "lysosomal membrane stability", "morphometrical alterations of lysosomes", "lysosomal response index (LRI)" and "structural epithelial changes in digestive tubules". The results indicate appreciable alterations of the above parameters in large-sized mussels, supporting their greater influence by the environmental factors, in relation to small-sized ones.

  8. Lysosomal and tissue-level biomarkers in mussels cross-transplanted among four estuaries with different pollution levels.

    PubMed

    Lekube, Xabier; Izagirre, Urtzi; Soto, Manu; Marigómez, Ionan

    2014-02-15

    A 3-4 wk cross-transplantation experiment was carried out in order to investigate the sensitivity, rapidity, durability and reversibility of lysosomal and tissue-level biomarkers in the digestive gland of mussels. Four localities in the Basque coast with different levels of chemical pollution and environmental stress were selected. Lysosomal membrane stability (LP) and lysosomal structural changes (VvL; S/VL; NvL) and changes in cell-type composition in digestive gland epithelium (VvBAS) were investigated to determine short (2d) and mid-term (3-4 wk) responses after cross-transplantation. Mussels from Txatxarramendi presented VvBAS<0.1 μm(3)/μm(3) (unstressed) whilst VvBAS>0.12 μm(3)/μm(3) was recorded in mussels from Plentzia (moderate stress) and VvBAS>0.2 μm(3)/μm(3) in Arriluze and Muskiz (high stress). Accordingly, LP<10 min (high stress) was recorded in mussels from Muskiz and Arriluze and LP~15 min (low-to-moderate stress) in those from Plentzia and Txatxarramendi. According to the VvL, S/VL and NvL data, a certain lysosomal enlargement was envisaged in mussels from Arriluze in comparison with those from Txatxarramendi and Plentzia. Mussels from Muskiz exhibited a peculiar endo-lysosomal system made of abundant tiny lysosomes (low VvL and high S/VL and NvL values). Lysosomal and tissue-level biomarkers were responsive after 2d cross-transplantation between the reference and the polluted localities, which indicated that these biomarkers were quickly induced and, to a large extent, reversible. Moreover, the tissue-level biomarker values were maintained during the entire period (3-4 wk) of cross-transplantation, which evidenced the durability of the responsiveness. In contrast, comparisons in the mid-term were unfeasible for lysosomal biomarkers as these exhibited a seasonal winter attenuation resulting from low food availability and low temperatures. In conclusion, lysosomal enlargement and membrane stability and changes in cell-type composition were

  9. Effect of antimalarials treatment on rat liver lysosomal function-Anin vivo study.

    PubMed

    Patel, Samir P; Katewa, Subhash D; Katyare, Surendra S

    2005-01-01

    Effects of treatmentin vivo with the antimalarials:chloroquine (CQ), primaquine (PQ) and quinine(Q) on lysosomal enzymes and lysosomal membrane integrity were examined. Treatment with the three antimalarials showed an apparent increase in the membrane stability. CQ treatment resulted in increase in both the 'free' and 'total' activities of all the enzymes i.e. acid phosphatase, RNase II, DNase II and cathepsin D. PQ treatment lowered the 'free' and 'total' activities of acid phosphatase and cathepsin D, but the DNase II activities increased. Treatment with Q resulted in increased 'free' and 'total' activities of RNase II and DNase II. While 'free' activities of acid phosphatase and cathepsin D were low; the 'total' activities increased significantly. Our results suggest that a generalized increase in free nucleases activities following prolonged treatment with antimalarials may lead to cell damage and/or necrosis.

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

    PubMed

    Xiong, Jian; Zhu, Michael X

    2016-08-01

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

  11. Involvement of lysosomes in the early stages of axon degeneration.

    PubMed

    Zheng, Jin; Yan, Tingting; Feng, Yan; Zhai, Qiwei

    2010-02-01

    Axon degeneration is a common hallmark of many neurodegenerative diseases, and the underlying mechanism remains largely unknown. Lysosomes are involved in some neurodegenerative diseases, including Alzheimer's disease and Parkinson's disease. Whether lysosomes are involved in axon degeneration is yet to be elucidated. In this study, we found only about 10% lysosomes remained in axons of cultured superior cervical ganglia (SCGs) after transection for 4h when stained with LysoTracker. Furthermore, we found that lysosomal disruption occurred earlier than morphological changes and loss of mitochondrial membrane potential. In addition, the well-known axon-protective protein Wld(S) delayed injury-induced axon degeneration from both morphological changes and lysosomal disruption. Lysosomal inhibitors including chloroquine and ammonium chloride induced axon degeneration in cultured SCGs, and Wld(S) also slowed down the axon degeneration induced by lysosomal inhibitors. All these data suggest that lysosomal disruption is an early marker of axon degeneration, and inhibition of lysosome induces axon degeneration in a Wld(S)-protectable way. Thus, maintenance of normal lysosomal function might be an important approach to delay axon degeneration in neurodegenerative diseases.

  12. Regulation of lysosomal ion homeostasis by channels and transporters

    PubMed Central

    Xiong, Jian; Zhu, Michael X.

    2016-01-01

    Lysosomes are the major organelles that carry out degradation functions. They integrate and digest materials compartmentalized by endocytosis, phagocytosis or autophagy. In addition to more than 60 hydrolases residing in the lysosomes, there are also ion channels and transporters that mediate the flux or transport of H+, Ca2+, Na+, K+, and Cl− across the lysosomal membranes. Defects in ionic exchange can lead to abnormal lysosome morphology, defective vesicle trafficking, impaired autophagy, and diseases such as neurodegeneration and lysosomal storage disorders. The latter are characterized by incomplete lysosomal digestion and accumulation of toxic materials inside enlarged intracellular vacuoles. In addition to degradation, recent studies have revealed the roles of lysosomes in metabolic pathways through kinases such as mechanistic target of rapamycin (mTOR) and transcriptional regulation through calcium signaling molecules such as transcription factor EB (TFEB) and calcineurin. Owing to the development of new approaches including genetically encoded fluorescence probes and whole endolysosomal patch clamp recording techniques, studies on lysosomal ion channels have made remarkable progress in recent years. In this review, we will focus on the current knowledge of lysosome-resident ion channels and transporters, discuss their roles in maintaining lysosomal function, and evaluate how their dysfunction can result in disease. PMID:27430889

  13. Preventive effects of p-coumaric acid on lysosomal dysfunction and myocardial infarct size in experimentally induced myocardial infarction.

    PubMed

    Jyoti Roy, Abhro; Stanely Mainzen Prince, P

    2013-01-15

    The present study was designed to evaluate the preventive effects of p-coumaric acid on lysosomal dysfunction and myocardial infarct size in isoproterenol induced myocardial infarcted rats. Male albino Wistar rats were pretreated with p-coumaric acid (8 mg/kg body weight) daily for a period of 7 days after which isoproterenol (100mg/kg body weight) was injected subcutaneously into rats twice at an interval of 24h (8th and 9th day).The activity/levels of serum cardiac diagnostic markers, heart lysosomal lipid peroxidation products and the activities of lysosomal enzymes (β-glucuronidase, β-galactosidase, cathepsin-B and cathepsin-D) were significantly (P<0.05) increased in the serum and heart of isoproterenol induced myocardial infarcted rats. Isoproterenol also lowered the activities of β-glucuronidase and cathepsin-D in the lysosomal fraction. The pretreatment with p-coumaric acid significantly (P<0.05) prevented the changes in the levels of lysosomal lipid peroxidation products and the activities of lysosomal enzymes. In addition, p-coumaric acid greatly reduced myocardial infarct size. p-Coumaric acid pretreatment (8 mg/kg body weight) to normal rats did not show any significant effect. Thus, this study showed that p-coumaric acid prevents lysosomal dysfunction against cardiac damage induced by isoproterenol and brings back the levels of lipid peroxidation products and activities of lysosomal enzymes to near normal levels. The in vitro study also revealed the free radical scavenging activity of p-coumaric acid. Thus, the observed effects are due to p-coumaric acid's free radical scavenging and membrane stabilizing properties.

  14. Endothelial Nlrp3 inflammasome activation associated with lysosomal destabilization during coronary arteritis.

    PubMed

    Chen, Yang; Li, Xiang; Boini, Krishna M; Pitzer, Ashley L; Gulbins, Erich; Zhang, Yang; Li, Pin-Lan

    2015-02-01

    Inflammasomes play a critical role in the development of vascular diseases. However, the molecular mechanisms activating the inflammasome in endothelial cells and the relevance of this inflammasome activation is far from clear. Here, we investigated the mechanisms by which an Nlrp3 inflammasome is activated to result in endothelial dysfunction during coronary arteritis by Lactobacillus casei (L. casei) cell wall fragments (LCWE) in a mouse model for Kawasaki disease. Endothelial dysfunction associated with increased vascular cell adhesion protein 1 (VCAM-1) expression and endothelial-leukocyte adhesion was observed during coronary arteritis in mice treated with LCWE. Accompanied with these changes, the inflammasome activation was also shown in coronary arterial endothelium, which was characterized by a marked increase in caspase-1 activity and IL-1β production. In cultured endothelial cells, LCWE induced Nlrp3 inflammasome formation, caspase-1 activation and IL-1β production, which were blocked by Nlrp3 gene silencing or lysosome membrane stabilizing agents such as colchicine, dexamethasone, and ceramide. However, a potassium channel blocker glibenclamide or an oxygen free radical scavenger N-acetyl-l-cysteine had no effects on LCWE-induced inflammasome activation. LCWE also increased endothelial cell lysosomal membrane permeability and triggered lysosomal cathepsin B release into cytosol. Silencing cathepsin B blocked LCWE-induced Nlrp3 inflammasome formation and activation in endothelial cells. In vivo, treatment of mice with cathepsin B inhibitor also abolished LCWE-induced inflammasome activation in coronary arterial endothelium. It is concluded that LCWE enhanced lysosomal membrane permeabilization and consequent release of lysosomal cathepsin B, resulting in activation of the endothelial Nlrp3 inflammasome, which may contribute to the development of coronary arteritis.

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

    PubMed

    Settembre, Carmine; Zoncu, Roberto; Medina, Diego L; Vetrini, Francesco; Erdin, Serkan; Erdin, SerpilUckac; Huynh, Tuong; Ferron, Mathieu; Karsenty, Gerard; Vellard, Michel C; Facchinetti, Valeria; Sabatini, David M; Ballabio, Andrea

    2012-03-07

    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 activity. Conversely, pharmacological inhibition of mTORC1, as well as starvation and lysosomal disruption, activates TFEB by promoting its nuclear translocation. In addition, the transcriptional response of lysosomal and autophagic genes to either lysosomal dysfunction or pharmacological inhibition of mTORC1 is suppressed in TFEB-/- cells. Interestingly, the Rag GTPase complex, which senses lysosomal amino acids and activates mTORC1, is both necessary and sufficient to regulate starvation- and stress-induced nuclear translocation of TFEB. These data indicate that the lysosome senses its content and regulates its own biogenesis by a lysosome-to-nucleus signalling mechanism that involves TFEB and mTOR.

  16. Endosome-lysosomes and neurodegeneration.

    PubMed

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

    1994-01-01

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

  17. P-selectin targeting to secretory lysosomes of Rbl-2H3 cells.

    PubMed

    Kaur, Jasber; Cutler, Daniel F

    2002-03-22

    The biogenesis of secretory lysosomes, which combine characteristics of both lysosomes and secretory granules, is currently of high interest. In particular, it is not clear whether delivery of membrane proteins to the secretory lysosome requires lysosomal, secretory granule, or some novel targeting determinants. Heterologous expression of P-selectin has established that this membrane protein contains targeting signals for both secretory granules and lysosomes. P-selectin is therefore an ideal probe with which to determine the signals required for targeting to secretory lysosomes. We have exploited subcellular fractionation and immunofluorescence microscopy to monitor targeting of transiently expressed wild-type and mutant horseradish peroxidase (HRP)-P-selectin chimeras to secretory lysosomes of Rbl-2H3 cells. The exposure of the HRP chimeras to intracellular proteolysis was also determined as a third monitor of secretory lysosome targeting. Our data show that HRP-P-selectin accumulates in secretory lysosomes of Rbl-2H3 cells using those cytoplasmic sequences previously found to be sufficient for targeting to conventional lysosomes. This work highlights the similar sorting signals used for targeting of membrane proteins to conventional lysosomes and secretory lysosomes.

  18. A lysosome-centered view of nutrient homeostasis.

    PubMed

    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.

  19. TFEB regulates lysosomal proteostasis.

    PubMed

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

    2013-05-15

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

  20. Misrouting of v-ATPase subunit V0a1 dysregulates lysosomal acidification in a neurodegenerative lysosomal storage disease model

    PubMed Central

    Bagh, Maria B.; Peng, Shiyong; Chandra, Goutam; Zhang, Zhongjian; Singh, Satya P.; Pattabiraman, Nagarajan; Liu, Aiyi; Mukherjee, Anil B.

    2017-01-01

    Defective lysosomal acidification contributes to virtually all lysosomal storage disorders (LSDs) and to common neurodegenerative diseases like Alzheimer's and Parkinson's. Despite its fundamental importance, the mechanism(s) underlying this defect remains unclear. The v-ATPase, a multisubunit protein complex composed of cytosolic V1-sector and lysosomal membrane-anchored V0-sector, regulates lysosomal acidification. Mutations in the CLN1 gene, encoding PPT1, cause a devastating neurodegenerative LSD, INCL. Here we report that in Cln1−/− mice, which mimic INCL, reduced v-ATPase activity correlates with elevated lysosomal pH. Moreover, v-ATPase subunit a1 of the V0 sector (V0a1) requires palmitoylation for interacting with adaptor protein-2 (AP-2) and AP-3, respectively, for trafficking to the lysosomal membrane. Notably, treatment of Cln1−/− mice with a thioesterase (Ppt1)-mimetic, NtBuHA, ameliorated this defect. Our findings reveal an unanticipated role of Cln1 in regulating lysosomal targeting of V0a1 and suggest that varying factors adversely affecting v-ATPase function dysregulate lysosomal acidification in other LSDs and common neurodegenerative diseases. PMID:28266544

  1. Lysosomal putative RNA transporter SIDT2 mediates direct uptake of RNA by lysosomes

    PubMed Central

    Aizawa, Shu; Fujiwara, Yuuki; Contu, Viorica Raluca; Hase, Katsunori; Takahashi, Masayuki; Kikuchi, Hisae; Kabuta, Chihana; Wada, Keiji; Kabuta, Tomohiro

    2016-01-01

    ABSTRACT Lysosomes are thought to be the major intracellular compartment for the degradation of macromolecules. We recently identified a novel type of autophagy, RNautophagy, where RNA is directly taken up by lysosomes in an ATP-dependent manner and degraded. However, the mechanism of RNA translocation across the lysosomal membrane and the physiological role of RNautophagy remain unclear. In the present study, we performed gain- and loss-of-function studies with isolated lysosomes, and found that SIDT2 (SID1 transmembrane family, member 2), an ortholog of the Caenorhabditis elegans putative RNA transporter SID-1 (systemic RNA interference deficient-1), mediates RNA translocation during RNautophagy. We also observed that SIDT2 is a transmembrane protein, which predominantly localizes to lysosomes. Strikingly, knockdown of Sidt2 inhibited up to ˜50% of total RNA degradation at the cellular level, independently of macroautophagy. Moreover, we showed that this impairment is mainly due to inhibition of lysosomal RNA degradation, strongly suggesting that RNautophagy plays a significant role in constitutive cellular RNA degradation. Our results provide a novel insight into the mechanisms of RNA metabolism, intracellular RNA transport, and atypical types of autophagy. PMID:27046251

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

    PubMed

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

    2015-04-29

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

  3. Control of lipid membrane stability by cholesterol content.

    PubMed Central

    Raffy, S; Teissié, J

    1999-01-01

    Cholesterol has a concentration-dependent effect on membrane organization. It is able to control the membrane permeability by inducing conformational ordering of the lipid chains. A systematic investigation of lipid bilayer permeability is described in the present work. It takes advantage of the transmembrane potential difference modulation induced in vesicles when an external electric field is applied. The magnitude of this modulation is under the control of the membrane electrical permeability. When brought to a critical value by the external field, the membrane potential difference induces a new membrane organization. The membrane is then permeable and prone to solubilized membrane protein back-insertion. This is obtained for an external field strength, which depends on membrane native permeability. This approach was used to study the cholesterol effect on phosphatidylcholine bilayers. Studies have been performed with lipids in gel and in fluid states. When cholesterol is present, it does not affect electropermeabilization and electroinsertion in lipids in the fluid state. When lipids are in the gel state, cholesterol has a dose-dependent effect. When present at 6% (mol/mol), cholesterol prevents electropermeabilization and electroinsertion. When cholesterol is present at more than 12%, electropermeabilization and electroinsertion are obtained under milder field conditions. This is tentatively explained by a cholesterol-induced alteration of the hydrophobic barrier of the bilayer core. Our results indicate that lipid membrane permeability is affected by the cholesterol content. PMID:10096902

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

    PubMed

    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.

  5. A Model for Prediction of Heat Stability of Photosynthetic Membranes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A previous study has revealed a positive correlation between heat-induced damage to photosynthetic membranes (thylakoid membranes) and chlorophyll loss. In this study, we exploited this correlation and developed a model for prediction of thermal damage to thylakoids. Prediction is based on estimat...

  6. Protein kinase C-δ isoform mediates lysosome labilization in DNA damage-induced apoptosis

    PubMed Central

    PARENT, NICOLAS; SCHERER, MAX; LIEBISCH, GERHARD; SCHMITZ, GERD; BERTRAND, RICHARD

    2013-01-01

    A lysosomal pathway, characterized by the partial rupture or labilization of lysosomal membranes (LLM) and cathepsin release into the cytosol, is evoked during the early events of 20-S-camptothecin lactone (CPT)-induced apoptosis in human cancer cells, including human histiocytic lymphoma U-937 cells. These lysosomal events begin rapidly and simultaneously with mitochondrial permeabilization and caspase activation within 3 h after drug treatment. Recently, in a comparative proteomics analysis performed on highly-enriched lysosomal extracts, we identified proteins whose translocation to lysosomes correlated with LLM induction after CPT treatment, including protein kinase C-δ (PKC-δ). In this study, we show that the PKC-δ translocation to lysosomes is required for LLM, as silencing its expression with RNA interference or suppressing its activity with the inhibitor, rottlerin, prevents CPT-induced LLM. PKC-δ translocation to lysosomes is associated with lysosomal acidic sphingomyelinase (ASM) phosphorylation and activation, which in turn leads to an increase in ceramide (CER) content in lysosomes. The accumulation of endogenous CER in lysosomes is a critical event for CPT-induced LLM as suppressing PKC-δ or ASM activity reduces both the CPT-mediated CER generation in lysosomes and CPT-induced LLM. These findings reveal a novel mechanism by which PKC-δ mediates ASM phosphorylation/activation and CER accumulation in lysosomes in CPT-induced LLM, rapidly activating the lysosomal pathway of apoptosis after CPT treatment. PMID:21174057

  7. Investigation of the anti-inflammatory and membrane-stabilizing potential of spiramycin in vitro.

    PubMed

    Theron, A J; Feldman, C; Anderson, R

    2000-08-01

    The effects of the 16-member macrolide spiramycin (2.5-80 mg/L) and the 14-member agent clarithromycin on the production of superoxide by activated human neutrophils were compared in vitro and related to membrane-stabilizing activity. Superoxide production was measured by lucigenin-enhanced chemiluminescence with N-formyl-L-methionyl-L-leucyl-L-phenylalanine (1 microM) as the stimulus, and membrane-stabilizing activity was measured by a haemolytic procedure. Clarithromycin, but not spiramycin, caused dose-related inhibition of superoxide production by activated neutrophils and also protected erythrocytes against haemolysis, while spiramycin possessed only weak membrane-stabilizing activity. These observations underscore the apparent association between the anti-inflammatory and membrane-stabilizing properties of macrolides.

  8. Hydrocarbon-Based Polymer Electrolyte Membranes: Importance of Morphology on Ion Transport and Membrane Stability.

    PubMed

    Shin, Dong Won; Guiver, Michael D; Lee, Young Moo

    2017-03-03

    A fundamental understanding of polymer microstructure is important in order to design novel polymer electrolyte membranes (PEMs) with excellent electrochemical performance and stabilities. Hydrocarbon-based polymers have distinct microstructure according to their chemical structure. The ionic clusters and/or channels play a critical role in PEMs, affecting ion conductivity and water transport, especially at medium temperature and low relative humidity (RH). In addition, physical properties such as water uptake and dimensional swelling behavior depend strongly on polymer morphology. Over the past few decades, much research has focused on the synthetic development and microstructural characterization of hydrocarbon-based PEM materials. Furthermore, blends, composites, pressing, shear field, electrical field, surface modification, and cross-linking have also been shown to be effective approaches to obtain/maintain well-defined PEM microstructure. This review summarizes recent work on developments in advanced PEMs with various chemical structures and architecture and the resulting polymer microstructures and morphologies that arise for potential application in fuel cell, lithium ion battery, redox flow battery, actuators, and electrodialysis.

  9. Regulation of HIV-Gag expression and targeting to the endolysosomal/secretory pathway by the luminal domain of lysosomal-associated membrane protein (LAMP-1) enhance Gag-specific immune response.

    PubMed

    Godinho, Rodrigo Maciel da Costa; Matassoli, Flavio Lemos; Lucas, Carolina Gonçalves de Oliveira; Rigato, Paula Ordonhez; Gonçalves, Jorge Luiz Santos; Sato, Maria Notomi; Maciel, Milton; Peçanha, Ligia Maria Torres; August, J Thomas; Marques, Ernesto Torres de Azevedo; de Arruda, Luciana Barros

    2014-01-01

    We have previously demonstrated that a DNA vaccine encoding HIV-p55gag in association with the lysosomal associated membrane protein-1 (LAMP-1) elicited a greater Gag-specific immune response, in comparison to a DNA encoding the native gag. In vitro studies have also demonstrated that LAMP/Gag was highly expressed and was present in MHCII containing compartments in transfected cells. In this study, the mechanisms involved in these processes and the relative contributions of the increased expression and altered traffic for the enhanced immune response were addressed. Cells transfected with plasmid DNA constructs containing p55gag attached to truncated sequences of LAMP-1 showed that the increased expression of gag mRNA required p55gag in frame with at least 741 bp of the LAMP-1 luminal domain. LAMP luminal domain also showed to be essential for Gag traffic through lysosomes and, in this case, the whole sequence was required. Further analysis of the trafficking pathway of the intact LAMP/Gag chimera demonstrated that it was secreted, at least in part, associated with exosome-like vesicles. Immunization of mice with LAMP/gag chimeric plasmids demonstrated that high expression level alone can induce a substantial transient antibody response, but targeting of the antigen to the endolysosomal/secretory pathways was required for establishment of cellular and memory response. The intact LAMP/gag construct induced polyfunctional CD4+ T cell response, which presence at the time of immunization was required for CD8+ T cell priming. LAMP-mediated targeting to endolysosomal/secretory pathway is an important new mechanistic element in LAMP-mediated enhanced immunity with applications to the development of novel anti-HIV vaccines and to general vaccinology field.

  10. A mechanism for overcoming P-glycoprotein-mediated drug resistance: novel combination therapy that releases stored doxorubicin from lysosomes via lysosomal permeabilization using Dp44mT or DpC

    PubMed Central

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

    2016-01-01

    The intracellular distribution of a drug can cause significant variability in both activity and selectivity. Herein, we investigate the mechanism by which the anti-cancer agents, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT) and the clinically trialed, di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC), re-instate the efficacy of doxorubicin (DOX), in drug-resistant P-glycoprotein (Pgp)-expressing cells. Both Dp44mT and DpC potently target and kill Pgp-expressing tumors, while DOX effectively kills non-Pgp-expressing cancers. Thus, the combination of these agents should be considered as an effective rationalized therapy for potently treating advanced and resistant tumors that are often heterogeneous in terms of Pgp-expression. These studies demonstrate that both Dp44mT and DpC are transported into lysosomes via Pgp transport activity, where they induce lysosomal-membrane permeabilization to release DOX trapped within lysosomes. This novel strategy of loading lysosomes with DOX, followed by permeabilization with Dp44mT or DpC, results in the relocalization of stored DOX from its lysosomal 'safe house' to its nuclear targets, markedly enhancing cellular toxicity against resistant tumor cells. Notably, the combination of Dp44mT or DpC with DOX showed a very high level of synergism in multiple Pgp-expressing cell types, for example, cervical, breast and colorectal cancer cells. These studies revealed that the level of drug synergy was proportional to Pgp activity. Interestingly, synergism was ablated by inhibiting Pgp using the pharmacological inhibitor, Elacridar, or by inhibiting Pgp-expression using Pgp-silencing, demonstrating the importance of Pgp in the synergistic interaction. Furthermore, lysosomal-membrane stabilization inhibited the relocalization of DOX from lysosomes to the nucleus upon combination with Dp44mT or DpC, preventing synergism. This latter observation demonstrated the importance of lysosomal-membrane

  11. The antimicrobial peptide microcin J25 stabilizes the gel phase of bacterial model membranes.

    PubMed

    Rintoul, M R; Morero, R D; Dupuy, F G

    2015-05-01

    The bacterial membrane interaction of the antimicrobial peptide microcin J25 was studied with the probe-free techniques Langmuir monolayers and infrared spectroscopy. Membrane model systems composed by phosphatidylethanolamine:phosphatidylglycerol 7:3, which mimic the cytoplasmic membrane of Gram negative bacteria, were used in both monolayer and bilayer approaches. The peptide reduced the transition surface pressure of the expanded-to-condensed lipid monolayer states, as well as increased the gel-to-liquid crystalline transition temperature in bilayers, indicating a stabilization of membrane ordered state. In addition, a reduction of the surface pressure at which condensed domains appeared was observed upon mixed monolayers compression after microcin J25 adsorption. The results indicate a favorable interaction of microcin J25 with bacterial membrane model systems. Also, the effects on the ordered phases stabilization are discussed in terms of the biological effects observed in membranes of sensitive cells.

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

    PubMed

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

    2016-10-01

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

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

    PubMed

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

    2015-04-17

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

  14. Proton-assisted amino acid transporter PAT1 complexes with Rag GTPases and activates TORC1 on late endosomal and lysosomal membranes.

    PubMed

    Ögmundsdóttir, Margrét H; Heublein, Sabine; Kazi, Shubana; Reynolds, Bruno; Visvalingam, Shivanthy M; Shaw, Michael K; Goberdhan, Deborah C I

    2012-01-01

    Mammalian Target of Rapamycin Complex 1 (mTORC1) is activated by growth factor-regulated phosphoinositide 3-kinase (PI3K)/Akt/Rheb signalling and extracellular amino acids (AAs) to promote growth and proliferation. These AAs induce translocation of mTOR to late endosomes and lysosomes (LELs), subsequent activation via mechanisms involving the presence of intralumenal AAs, and interaction between mTORC1 and a multiprotein assembly containing Rag GTPases and the heterotrimeric Ragulator complex. However, the mechanisms by which AAs control these different aspects of mTORC1 activation are not well understood. We have recently shown that intracellular Proton-assisted Amino acid Transporter 1 (PAT1)/SLC36A1 is an essential mediator of AA-dependent mTORC1 activation. Here we demonstrate in Human Embryonic Kidney (HEK-293) cells that PAT1 is primarily located on LELs, physically interacts with the Rag GTPases and is required for normal AA-dependent mTOR relocalisation. We also use the powerful in vivo genetic methodologies available in Drosophila to investigate the regulation of the PAT1/Rag/Ragulator complex. We show that GFP-tagged PATs reside at both the cell surface and LELs in vivo, mirroring PAT1 distribution in several normal mammalian cell types. Elevated PI3K/Akt/Rheb signalling increases intracellular levels of PATs and synergistically enhances PAT-induced growth via a mechanism requiring endocytosis. In light of the recent identification of the vacuolar H(+)-ATPase as another Rag-interacting component, we propose a model in which PATs function as part of an AA-sensing engine that drives mTORC1 activation from LEL compartments.

  15. The release of lysosomal arylsulfatase from liver lysosomes exposed to 2-chloroethylethyl sulfide.

    PubMed

    Shin, S; Choi, D S; Kim, Y B; Cha, S H; Sok, D E

    1995-08-18

    Treatment of a lysosome-rich fraction from liver with 2-chloroethylethyl sulfide resulted in a dose-dependent release of arylsulfatase. The inclusion of Ca2+ enhanced the enzyme release by approximately 2.3-fold. The enhancing effect of Ca2+, showing an EC50 value of 30 mM, was mimicked by neither Mg2+ nor Mn2+. Studies on a structural requirement and a time-dependent release suggest that the Ca(2+)-dependent release proceeds via a specific process involving the alkylation of lysosomal membranes by 2-chloroethylethyl sulfide. Furthermore, the Ca(2+)-dependent process was prevented partially by either leupeptin or gentamycin, but neither pepstatin nor PMSF, implying that the enzyme release may be partially mediated by lysosomal cysteine-protease or phospholipase. Meanwhile, the Ca(2+)-independent release seems to be expressed non-specifically by various compounds.

  16. Subcellular Trafficking of Mammalian Lysosomal Proteins: An Extended View

    PubMed Central

    Staudt, Catherine; Puissant, Emeline; Boonen, Marielle

    2016-01-01

    Lysosomes clear macromolecules, maintain nutrient and cholesterol homeostasis, participate in tissue repair, and in many other cellular functions. To assume these tasks, lysosomes rely on their large arsenal of acid hydrolases, transmembrane proteins and membrane-associated proteins. It is therefore imperative that, post-synthesis, these proteins are specifically recognized as lysosomal components and are correctly sorted to this organelle through the endosomes. Lysosomal transmembrane proteins contain consensus motifs in their cytosolic regions (tyrosine- or dileucine-based) that serve as sorting signals to the endosomes, whereas most lysosomal acid hydrolases acquire mannose 6-phosphate (Man-6-P) moieties that mediate binding to two membrane receptors with endosomal sorting motifs in their cytosolic tails. These tyrosine- and dileucine-based motifs are tickets for boarding in clathrin-coated carriers that transport their cargo from the trans-Golgi network and plasma membrane to the endosomes. However, increasing evidence points to additional mechanisms participating in the biogenesis of lysosomes. In some cell types, for example, there are alternatives to the Man-6-P receptors for the transport of some acid hydrolases. In addition, several “non-consensus” sorting motifs have been identified, and atypical transport routes to endolysosomes have been brought to light. These “unconventional” or “less known” transport mechanisms are the focus of this review. PMID:28036022

  17. Impact of Lysosome Status on Extracellular Vesicle Content and Release

    PubMed Central

    Eitan, Erez; Suire, Caitlin; Zhang, Shi; Mattson, Mark P.

    2016-01-01

    Extracellular vesicles (EVs) are nanoscale size bubble-like membranous structures released from cells. EVs contain RNA, lipids and proteins and are thought to serve various roles including intercellular communication and removal of misfolded proteins. The secretion of misfolded and aggregated proteins in EVs may be a cargo disposal alternative to the autophagy-lysosomal and ubiquitin-proteasome pathways. In this review we will discuss the importance of lysosome functionality for the regulation of EV secretion and content. Exosomes are a subtype of EVs that are released by the fusion of multivesicular bodies (MVB) with the plasma membrane. MVBs can also fuse with lysosomes, and the trafficking pathway of MVBs can therefore determine whether or not exosomes are released from cells. Here we summarize data from studies of the effects of lysosome inhibition on the secretion of EVs and on the possibility that cells compensate for lysosome malfunction by disposal of potentially toxic cargos in EVs. A better understanding of the molecular mechanisms that regulate trafficking of MVBs to lysosomes and the plasma membrane may advance an understanding of diseases in which pathogenic proteins, lipids or infectious agents accumulate within or outside of cells. PMID:27238186

  18. Progranulin regulates lysosomal function and biogenesis through acidification of lysosomes.

    PubMed

    Tanaka, Yoshinori; Suzuki, Genjiro; Matsuwaki, Takashi; Hosokawa, Masato; Serrano, Geidy; Beach, Thomas G; Yamanouchi, Keitaro; Hasegawa, Masato; Nishihara, Masugi

    2017-01-10

    Progranulin (PGRN) haploinsufficiency resulting from loss-of-function mutations in the PGRN gene causes frontotemporal lobar degeneration accompanied by TDP-43 accumulation, and patients with homozygous mutations in the PGRN gene present with neuronal ceroid lipofuscinosis. Although it remains unknown why PGRN deficiency causes neurodegenerative diseases, there is increasing evidence that PGRN is implicated in lysosomal functions. Here, we show PGRN is a secretory lysosomal protein that regulates lysosomal function and biogenesis by controlling the acidification of lysosomes. PGRN gene expression and protein levels increased concomitantly with the increase of lysosomal biogenesis induced by lysosome alkalizers or serum starvation. Down-regulation or insufficiency of PGRN led to the increased lysosomal gene expression and protein levels, while PGRN overexpression led to the decreased lysosomal gene expression and protein levels. In particular, the level of mature cathepsin D (CTSDmat) dramatically changed depending upon PGRN levels. The acidification of lysosomes was facilitated in cells transfected with PGRN. Then, this caused degradation of CTSDmat by cathepsin B. Secreted PGRN is incorporated into cells via sortilin or cation-independent mannose 6-phosphate receptor, and facilitated the acidification of lysosomes and degradation of CTSDmat Moreover, the change of PGRN levels led to a cell-type-specific increase of insoluble TDP-43. In the brain tissue of FTLD-TDP patients with PGRN deficiency, CTSD and phosphorylated TDP-43 accumulated in neurons. Our study provides new insights into the physiological function of PGRN and the role of PGRN insufficiency in the pathogenesis of neurodegenerative diseases.

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

    PubMed

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

    2015-11-01

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

  20. Mechanical and thermal stability of adhesive membranes with nonzero bending rigidity

    NASA Astrophysics Data System (ADS)

    Tallinen, Tuomas; Astrom, Jan; Kekalainen, Pekka; Timonen, Jussi

    2011-03-01

    Membranes at a microscopic scale are affected by thermal fluctuations and self-adhesion due to Van der Waals forces. Methods to prepare membranes of even molecular scale, e.g. graphene, have been recently developed, and the question of their mechanical and thermal stability is of crucial importance. To this end we modeled microscopic membranes with a short-range attractive interaction and applied Langevin dynamics. Their behavior was also analyzed under external loading. Even though these membranes folded during isotropic compression as a result of energy minimization, the process at high confinement did not differ much from crumpling of macroscopic thin sheets. The main difference appeared when the external load was released. In such cases, for membranes of sufficiently large size L , folded or scrolled conformations emerged. At high enough temperature T entropic effects made such conformations unfavorable, however. Possible conformations of free-standing membranes (``phase diagrams'') were determined in the TL -plane.

  1. Hydrophobic asymmetric ultrafiltration PVDF membranes: an alternative separator for VFB with excellent stability.

    PubMed

    Wei, Wenping; Zhang, Huamin; Li, Xianfeng; Zhang, Hongzhang; Li, Yun; Vankelecom, Ivo

    2013-02-14

    Polyvinylidene fluoride (PVDF) ultrafiltration membranes were investigated for the first time in vanadium redox flow battery (VFB) applications. Surprisingly, PVDF ultrafiltration membranes with hydrophobic pore walls and relatively large pore sizes of several tens of nanometers proved able to separate vanadium ions and protons efficiently, thus being suitable as a VFB separator. The ion selectivity of this new type of VFB membrane could be tuned readily by controlling the membrane morphology via changes in the composition of the membrane casting solution, and the casting thickness. The results showed that the PVDF membranes offered good performances and excellent stability in VFB applications, where it could, performance-wise, truly substitute Nafion in VFB applications, but at a much lower cost.

  2. Cholesterol expels ibuprofen from the hydrophobic membrane core and stabilizes lamellar phases in lipid membranes containing ibuprofen.

    PubMed

    Alsop, Richard J; Armstrong, Clare L; Maqbool, Amna; Toppozini, Laura; Dies, Hannah; Rheinstädter, Maikel C

    2015-06-28

    There is increasing evidence that common drugs, such as aspirin and ibuprofen, interact with lipid membranes. Ibuprofen is one of the most common over the counter drugs in the world, and is used for relief of pain and fever. It interacts with the cyclooxygenase pathway leading to inhibition of prostaglandin synthesis. From X-ray diffraction of highly oriented model membranes containing between 0 and 20 mol% ibuprofen, 20 mol% cholesterol, and dimyristoylphosphatidylcholine (DMPC), we present evidence for a non-specific interaction between ibuprofen and cholesterol in lipid bilayers. At a low ibuprofen concentrations of 2 mol%, three different populations of ibuprofen molecules were found: two in the lipid head group region and one in the hydrophobic membrane core. At higher ibuprofen concentrations of 10 and 20 mol%, the lamellar bilayer structure is disrupted and a lamellar to cubic phase transition was observed. In the presence of 20 mol% cholesterol, ibuprofen (at 5 mol%) was found to be expelled from the membrane core and reside solely in the head group region of the bilayers. 20 mol% cholesterol was found to stabilize lamellar membrane structure and the formation of a cubic phase at 10 and 20 mol% ibuprofen was suppressed. The results demonstrate that ibuprofen interacts with lipid membranes and that the interaction is strongly dependent on the presence of cholesterol.

  3. EMC1-dependent stabilization drives membrane penetration of a partially destabilized non-enveloped virus

    PubMed Central

    Bagchi, Parikshit; Inoue, Takamasa; Tsai, Billy

    2016-01-01

    Destabilization of a non-enveloped virus generates a membrane transport-competent viral particle. Here we probe polyomavirus SV40 endoplasmic reticulum (ER)-to-cytosol membrane transport, a decisive infection step where destabilization initiates this non-enveloped virus for membrane penetration. We find that a member of the ER membrane protein complex (EMC) called EMC1 promotes SV40 ER membrane transport and infection. Surprisingly, EMC1 does so by using its predicted transmembrane residue D961 to bind to and stabilize the membrane-embedded partially destabilized SV40, thereby preventing premature viral disassembly. EMC1-dependent stabilization enables SV40 to engage a cytosolic extraction complex that ejects the virus into the cytosol. Thus EMC1 acts as a molecular chaperone, bracing the destabilized SV40 in a transport-competent state. Our findings reveal the novel principle that coordinated destabilization-stabilization drives membrane transport of a non-enveloped virus. DOI: http://dx.doi.org/10.7554/eLife.21470.001 PMID:28012275

  4. Erythrocyte membrane stability to hydrogen peroxide is decreased in Alzheimer disease.

    PubMed

    Gilca, Marilena; Lixandru, Daniela; Gaman, Laura; Vîrgolici, Bogdana; Atanasiu, Valeriu; Stoian, Irina

    2014-01-01

    The brain and erythrocytes have similar susceptibility toward free radicals. Therefore, erythrocyte abnormalities might indicate the progression of the oxidative damage in Alzheimer disease (AD). The aim of this study was to investigate erythrocyte membrane stability and plasma antioxidant status in AD. Fasting blood samples (from 17 patients with AD and 14 healthy controls) were obtained and erythrocyte membrane stability against hydrogen peroxide and 2,2'-azobis-(2-amidinopropane) dihydrochloride (AAPH), serum Trolox equivalent antioxidant capacity (TEAC), residual antioxidant activity or gap (GAP), erythrocyte catalase activity (CAT), erythrocyte superoxide dismutase (SOD) activity, erythrocyte nonproteic thiols, and total plasma thiols were determined. A significant decrease in erythrocyte membrane stability to hydrogen peroxide was found in AD patients when compared with controls (P<0.05). On the contrary, CAT activity (P<0.0001) and total plasma thiols (P<0.05) were increased in patients with AD compared with controls. Our results indicate that the most satisfactory measurement of the oxidative stress level in the blood of patients with AD is the erythrocyte membrane stability to hydrogen peroxide. Reduced erythrocyte membrane stability may be further evaluated as a potential peripheral marker for oxidative damage in AD.

  5. Poloxamer-188 and citicoline provide neuronal membrane integrity and protect membrane stability in cortical spreading depression.

    PubMed

    Yıldırım, Timur; Eylen, Alpaslan; Lule, Sevda; Erdener, Sefik Evren; Vural, Atay; Karatas, Hulya; Ozveren, Mehmet Faik; Dalkara, Turgay; Gursoy-Ozdemir, Yasemin

    2015-01-01

    Under pathological conditions such as brain trauma, subarachnoid hemorrhage and stroke, cortical spreading depression (CSD) or peri-infarct depolarizations contribute to brain damage in animal models of neurological disorders as well as in human neurological diseases. CSD causes transient megachannel opening on the neuronal membrane, which may compromise neuronal survival under pathological conditions. Poloxamer-188 (P-188) and citicoline are neuroprotectants with membrane sealing properties. The aim of this study is to investigate the effect of P-188 and citicoline on the neuronal megachannel opening induced by CSD in the mouse brain. We have monitored megachannel opening with propidium iodide, a membrane impermeable fluorescent dye and, demonstrate that P-188 and citicoline strikingly decreased CSD-induced neuronal PI influx in cortex and hippocampal dentate gyrus. Therefore, these agents may be providing neuroprotection by blocking megachannel opening, which may be related to their membrane sealing action and warrant further investigation for treatment of traumatic brain injury and ischemic stroke.

  6. Effect of L-carnitine and acetyl-L-carnitine on the human erythrocyte membrane stability and deformability.

    PubMed

    Arduini, A; Rossi, M; Mancinelli, G; Belfiglio, M; Scurti, R; Radatti, G; Shohet, S B

    1990-01-01

    In this study we examined the effect of carnitine and acetylcarnitine on the human erythrocyte membrane stability and membrane deformability. Since erythrocyte membranes are impermeable to these compounds, we resealed erythrocyte ghosts in the presence of different concentrations of carnitine or acetylcarnitine. Resealed ghosts can be adequately studied in their cellular deformability and membrane stability properties by means of ektacytometry. Both carnitine and acetylcarnitine alter the membrane stability but not membrane deformability of the red cell membrane. Resealed ghosts containing 20, 50, 150, and 300 microM carnitine had 1.1, 1.6, 0.9, and 0.7 times the normal stability. While resealed ghosts containing 20, 50, 150, and 300 microM acetylcarnitine had 1.1, 1.5, 1.3, and 1.2 times the normal stability. Such changes were found to be reversible. We also conducted SDS PAGE of cytoskeletal membrane proteins from membrane fragments and residual membranes produced during membrane stability analysis, and unsheared resealed membranes in those samples where we observed an increase or a decrease of membrane stability. No changes in the cytoskeletal membrane proteins were noticed, even when the samples, prior SDS PAGE analysis, were treated with or without dithiothreitol. In addition, fluorescence steady state anisotropy of DPH in the erythrocyte membrane treated with carnitine or acetylcarnitine shows no modification of the lipid order parameter. Our results would suggest that both carnitine and its acetyl-ester, at physiological concentrations, may increase membrane stability in mature erythrocytes, most likely via a specific interaction with one or more cytoskeletal proteins, and that this effect would manifest when the erythrocytes are subjected to high shear stress.

  7. The Acinar Cage: Basement Membranes Determine Molecule Exchange and Mechanical Stability of Human Breast Cell Acini

    PubMed Central

    Gaiko-Shcherbak, Aljona; Fabris, Gloria; Dreissen, Georg; Merkel, Rudolf; Hoffmann, Bernd; Noetzel, Erik

    2015-01-01

    The biophysical properties of the basement membrane that surrounds human breast glands are poorly understood, but are thought to be decisive for normal organ function and malignancy. Here, we characterize the breast gland basement membrane with a focus on molecule permeation and mechanical stability, both crucial for organ function. We used well-established and nature-mimicking MCF10A acini as 3D cell model for human breast glands, with ether low- or highly-developed basement membrane scaffolds. Semi-quantitative dextran tracer (3 to 40 kDa) experiments allowed us to investigate the basement membrane scaffold as a molecule diffusion barrier in human breast acini in vitro. We demonstrated that molecule permeation correlated positively with macromolecule size and intriguingly also with basement membrane development state, revealing a pore size of at least 9 nm. Notably, an intact collagen IV mesh proved to be essential for this permeation function. Furthermore, we performed ultra-sensitive atomic force microscopy to quantify the response of native breast acini and of decellularized basement membrane shells against mechanical indentation. We found a clear correlation between increasing acinar force resistance and basement membrane formation stage. Most important native acini with highly-developed basement membranes as well as cell-free basement membrane shells could both withstand physiologically relevant loads (≤ 20 nN) without loss of structural integrity. In contrast, low-developed basement membranes were significantly softer and more fragile. In conclusion, our study emphasizes the key role of the basement membrane as conductor of acinar molecule influx and mechanical stability of human breast glands, which are fundamental for normal organ function. PMID:26674091

  8. The Acinar Cage: Basement Membranes Determine Molecule Exchange and Mechanical Stability of Human Breast Cell Acini.

    PubMed

    Gaiko-Shcherbak, Aljona; Fabris, Gloria; Dreissen, Georg; Merkel, Rudolf; Hoffmann, Bernd; Noetzel, Erik

    2015-01-01

    The biophysical properties of the basement membrane that surrounds human breast glands are poorly understood, but are thought to be decisive for normal organ function and malignancy. Here, we characterize the breast gland basement membrane with a focus on molecule permeation and mechanical stability, both crucial for organ function. We used well-established and nature-mimicking MCF10A acini as 3D cell model for human breast glands, with ether low- or highly-developed basement membrane scaffolds. Semi-quantitative dextran tracer (3 to 40 kDa) experiments allowed us to investigate the basement membrane scaffold as a molecule diffusion barrier in human breast acini in vitro. We demonstrated that molecule permeation correlated positively with macromolecule size and intriguingly also with basement membrane development state, revealing a pore size of at least 9 nm. Notably, an intact collagen IV mesh proved to be essential for this permeation function. Furthermore, we performed ultra-sensitive atomic force microscopy to quantify the response of native breast acini and of decellularized basement membrane shells against mechanical indentation. We found a clear correlation between increasing acinar force resistance and basement membrane formation stage. Most important native acini with highly-developed basement membranes as well as cell-free basement membrane shells could both withstand physiologically relevant loads (≤ 20 nN) without loss of structural integrity. In contrast, low-developed basement membranes were significantly softer and more fragile. In conclusion, our study emphasizes the key role of the basement membrane as conductor of acinar molecule influx and mechanical stability of human breast glands, which are fundamental for normal organ function.

  9. What lysosomes actually tell us about Parkinson's disease?

    PubMed

    Bourdenx, Mathieu; Dehay, Benjamin

    2016-12-01

    Parkinson's disease is a common neurodegenerative disorder of unknown origin mainly characterized by the loss of neuromelanin-containing dopaminergic neurons in the substantia nigra pars compacta and the presence of intraneuronal proteinaceous inclusions called Lewy bodies. Lysosomes are dynamic organelles that degrade, in a controlled manner, cellular components delivered via the secretory, endocytic, autophagic and phagocytic membrane-trafficking pathways. Increasing amounts of evidence suggest a central role of lysosomal impairment in PD aetiology. This review provides an update on how genetic evidence support this connection and highlights how the neuropathologic and mechanistic evidence might relate to the disease process in sporadic forms of Parkinson's disease. Finally, we discuss the influence of ageing on lysosomal impairment and PD aetiology and therapeutic strategies targeting lysosomal function.

  10. Stability and Degradation Mechanisms of Radiation-Grafted Polymer Electrolyte Membranes for Water Electrolysis.

    PubMed

    Albert, Albert; Lochner, Tim; Schmidt, Thomas J; Gubler, L

    2016-06-22

    Radiation-grafted membranes are a promising alternative to commercial membranes for water electrolyzers, since they exhibit lower hydrogen crossover and area resistance, better mechanical properties, and are of potentially lower cost than perfluoroalkylsulfonic acid membranes, such as Nafion. Stability is an important factor in view of the expected lifetime of 40 000 h or more of an electrolyzer. In this study, combinations of styrene (St), α-methylstyrene (AMS), acrylonitrile (AN), and 1,3-diisopropenylbenzene (DiPB) are cografted into 50 μm preirradiated poly(ethylene-co-tetrafluoroethylene) (ETFE) base film, followed by sulfonation to produce radiation-grafted membranes. The stability of the membranes with different monomer combinations is compared under an accelerated stress test (AST), and the degradation mechanisms are investigated. To mimic the conditions in an electrolyzer, in which the membrane is always in contact with liquid water at elevated temperature, the membranes are immersed in water for 5 days at 90 °C, so-called thermal stress test (TST). In addition to testing in air atmosphere tests are also carried out under argon to investigate the effect of the absence of oxygen. The water is analyzed with UV-vis spectroscopy and ion chromatography. The ion exchange capacity (IEC), swelling degree, and Fourier transform infrared (FTIR) spectra of the membranes are compared before and after the test. Furthermore, energy-dispersive X-ray (EDX) spectroscopic analysis of the membrane cross-section is performed. Finally, the influence of the TST to the membrane area resistance and hydrogen crossover is measured. The stability increases along the sequence St/AN, St/AN/DiPB, AMS/AN, and AMS/AN/DiPB grafted membrane. The degradation at the weak-link, oxygen-induced degradation, and hydrothermal degradation are proposed in addition to the "swelling-induced detachment" reported in the literature. By mitigating the possible paths of degradation, the AMS

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

    SciTech Connect

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

    1984-02-01

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

  12. Preparation and characterization of titania-deposited silica composite hollow fiber membranes with high hydrothermal stability.

    PubMed

    Kwon, Young-Nam; Kim, In-Chul

    2013-11-01

    Hydrothermal stability of a porous nickel-supported silica membrane was successfully improved by deposition of titania multilayers on colloidal silica particles embedded in the porous nickel fiber support. Porous nickel-supported silica membranes were prepared by means of a dipping-freezing-fast drying (DFF) method. The titania layers were deposited on colloidal silica particles by repeating hydrolysis and condensation reactions of titanium isopropoxide on the silica particle surfaces. The deposition of thin titania layers on the nickel-supported silica membrane was verified by various analytical tools. The water flux and the solute rejection of the porous Ni fiber-supported silica membranes did not change after titania layer deposition, indicating that thickness of titania layers deposited on silica surface is enough thin not to affect the membrane performance. Moreover, improvement of the hydrothermal stability in the titania-deposited silica membranes was confirmed by stability tests, indicating that thin titania layers deposited on silica surface played an important role as a diffusion barrier against 90 degrees C water into silica particles.

  13. Kinetics of lysosomal storage of indigestible matter.

    PubMed Central

    Hurley, J; Alward, J

    1975-01-01

    In lysosomal storage diseases and in accumulation of lipofusion in the lysosomes there is a gradual eroding of the lysosomal system due to overloading the lysosomes by molecules which cannot be digested or expelled. The kinetics of this accumulation is examined for tissue cultures in terms of the cell growth rate, lysosomal production rate, and of generation of the indigestible element. PMID:1125388

  14. Structural and mechanical heterogeneity of the erythrocyte membrane reveals hallmarks of membrane stability.

    PubMed

    Picas, Laura; Rico, Félix; Deforet, Maxime; Scheuring, Simon

    2013-02-26

    The erythrocyte membrane, a metabolically regulated active structure that comprises lipid molecules, junctional complexes, and the spectrin network, enables the cell to undergo large passive deformations when passing through the microvascular system. Here we use atomic force microscopy (AFM) imaging and quantitative mechanical mapping at nanometer resolution to correlate structure and mechanics of key components of the erythrocyte membrane, crucial for cell integrity and function. Our data reveal structural and mechanical heterogeneity modulated by the metabolic state at unprecedented nanometer resolution. ATP-depletion, reducing skeletal junction phosphorylation in RBC cells, leads to membrane stiffening. Analysis of ghosts and shear-force opened erythrocytes show that, in the absence of cytosolic kinases, spectrin phosphorylation results in membrane stiffening at the extracellular face and a reduced junction remodeling in response to loading forces. Topography and mechanical mapping of single components at the cytoplasmic face reveal that, surprisingly, spectrin phosphorylation by ATP softens individual filaments. Our findings suggest that, besides the mechanical signature of each component, the RBC membrane mechanics is regulated by the metabolic state and the assembly of its structural elements.

  15. TFEB-mediated increase in peripheral lysosomes regulates store-operated calcium entry

    PubMed Central

    Sbano, Luigi; Bonora, Massimo; Marchi, Saverio; Baldassari, Federica; Medina, Diego L.; Ballabio, Andrea; Giorgi, Carlotta; Pinton, Paolo

    2017-01-01

    Lysosomes are membrane-bound organelles mainly involved in catabolic processes. In addition, lysosomes can expel their contents outside of the cell via lysosomal exocytosis. Some of the key steps involved in these important cellular processes, such as vesicular fusion and trafficking, require calcium (Ca2+) signaling. Recent data show that lysosomal functions are transcriptionally regulated by transcription factor EB (TFEB) through the induction of genes involved in lysosomal biogenesis and exocytosis. Given these observations, we investigated the roles of TFEB and lysosomes in intracellular Ca2+ homeostasis. We studied the effect of transient modulation of TFEB expression in HeLa cells by measuring the cytosolic Ca2+ response after capacitative Ca2+ entry activation and Ca2+ dynamics in the endoplasmic reticulum (ER) and directly in lysosomes. Our observations show that transient TFEB overexpression significantly reduces cytosolic Ca2+ levels under a capacitative influx model and ER re-uptake of calcium, increasing the lysosomal Ca2+ buffering capacity. Moreover, lysosomal destruction or damage abolishes these TFEB-dependent effects in both the cytosol and ER. These results suggest a possible Ca2+ buffering role for lysosomes and shed new light on lysosomal functions during intracellular Ca2+ homeostasis. PMID:28084445

  16. BK channel agonist represents a potential therapeutic approach for lysosomal storage diseases

    PubMed Central

    Zhong, Xi Zoë; Sun, Xue; Cao, Qi; Dong, Gaofeng; Schiffmann, Raphael; Dong, Xian-Ping

    2016-01-01

    Efficient lysosomal Ca2+ release plays an essential role in lysosomal trafficking. We have recently shown that lysosomal big conductance Ca2+-activated potassium (BK) channel forms a physical and functional coupling with the lysosomal Ca2+ release channel Transient Receptor Potential Mucolipin-1 (TRPML1). BK and TRPML1 forms a positive feedback loop to facilitate lysosomal Ca2+ release and subsequent lysosome membrane trafficking. However, it is unclear whether the positive feedback mechanism is common for other lysosomal storage diseases (LSDs) and whether BK channel agonists rescue abnormal lysosomal storage in LSDs. In this study, we assessed the effect of BK agonist, NS1619 and NS11021 in a number of LSDs including NPC1, mild cases of mucolipidosis type IV (ML4) (TRPML1-F408∆), Niemann-Pick type A (NPA) and Fabry disease. We found that TRPML1-mediated Ca2+ release was compromised in these LSDs. BK activation corrected the impaired Ca2+ release in these LSDs and successfully rescued the abnormal lysosomal storage of these diseases by promoting TRPML1-mediated lysosomal exocytosis. Our study suggests that BK channel activation stimulates the TRPML1-BK positive reinforcing loop to correct abnormal lysosomal storage in LSDs. Drugs targeting BK channel represent a potential therapeutic approach for LSDs. PMID:27670435

  17. Effect of membranes with various hydrophobic/hydrophilic properties on lipase immobilized activity and stability.

    PubMed

    Chen, Guan-Jie; Kuo, Chia-Hung; Chen, Chih-I; Yu, Chung-Cheng; Shieh, Chwen-Jen; Liu, Yung-Chuan

    2012-02-01

    In this study, three membranes: regenerated cellulose (RC), glass fiber (GF) and polyvinylidene fluoride (PVDF), were grafted with 1,4-diaminobutane (DA) and activated with glutaraldehyde (GA) for lipase covalent immobilization. The efficiencies of lipases immobilized on these membranes with different hydrophobic/hydrophilic properties were compared. The lipase immobilized on hydrophobic PVDF-DA-GA membrane exhibited more than an 11-fold increase in activity compared to its immobilization on a hydrophilic RC-DA-GA membrane. The relationship between surface hydrophobicity and immobilized efficiencies was investigated using hydrophobic/hydrophilic GF membranes which were prepared by grafting a different ratio of n-butylamine/1,4-diaminobutane (BA/DA). The immobilized lipase activity on the GF membrane increased with the increased BA/DA ratio. This means that lipase activity was exhibited more on the hydrophobic surface. Moreover, the modified PVDF-DA membrane was grafted with GA, epichlorohydrin (EPI) and cyanuric chloride (CC), respectively. The lipase immobilized on the PVDF-DA-EPI membrane displayed the highest specific activity compared to other membranes. This immobilized lipase exhibited more significant stability on pH, thermal, reuse, and storage than did the free enzyme. The results exhibited that the EPI modified PVDF is a promising support for lipase immobilization.

  18. Mechanical and Thermal Stability of Adhesive Membranes with Nonzero Bending Rigidity

    NASA Astrophysics Data System (ADS)

    Tallinen, T.; Åström, J. A.; Kekäläinen, P.; Timonen, J.

    2010-07-01

    Membranes at a microscopic scale are affected by thermal fluctuations and self-adhesion due to van der Waals forces. Methods to prepare membranes of even molecular scale, e.g., graphene, have recently been developed, and the question of their mechanical and thermal stability is of crucial importance. To this end we modeled microscopic membranes with an attractive interaction and applied Langevin dynamics. Their behavior was also analyzed under external loading. Even though these membranes folded during isotropic compression as a result of energy minimization, the process at high confinement was similar to crumpling of macroscopic nonadhesive sheets. The main difference appeared when the compression was released. In such cases, for membranes of sufficiently large size, folded or scrolled conformations emerged. At high temperature entropic effects made such conformations unfavorable, however.

  19. Genetics Home Reference: lysosomal acid lipase deficiency

    MedlinePlus

    ... Home Health Conditions lysosomal acid lipase deficiency lysosomal acid lipase deficiency Enable Javascript to view the expand/ ... Download PDF Open All Close All Description Lysosomal acid lipase deficiency is an inherited condition characterized by ...

  20. Early stabilization of traumatic aortic transection and mitral valve regurgitation with extracorporeal membrane oxygenation.

    PubMed

    Lambrechts, David L; Wellens, Francis; Vercoutere, Rik A; De Geest, Raf

    2003-01-01

    We report a case of life-threatening aortic transection with concomitant mitral papillary muscle rupture and severe lung contusion caused by a failed parachute jump. This blunt thoracic injury was treated by early stabilization with extracorporeal membrane oxygenation followed by successful delayed graft repair of the descending aorta and mitral valve replacement with a mechanical prosthesis.

  1. Protecting cells by protecting their vulnerable lysosomes: Identification of a new mechanism for preserving lysosomal functional integrity upon oxidative stress.

    PubMed

    Pascua-Maestro, Raquel; Diez-Hermano, Sergio; Lillo, Concepción; Ganfornina, Maria D; Sanchez, Diego

    2017-02-01

    Environmental insults such as oxidative stress can damage cell membranes. Lysosomes are particularly sensitive to membrane permeabilization since their function depends on intraluminal acidic pH and requires stable membrane-dependent proton gradients. Among the catalog of oxidative stress-responsive genes is the Lipocalin Apolipoprotein D (ApoD), an extracellular lipid binding protein endowed with antioxidant capacity. Within the nervous system, cell types in the defense frontline, such as astrocytes, secrete ApoD to help neurons cope with the challenge. The protecting role of ApoD is known from cellular to organism level, and many of its downstream effects, including optimization of autophagy upon neurodegeneration, have been described. However, we still cannot assign a cellular mechanism to ApoD gene that explains how this protection is accomplished. Here we perform a comprehensive analysis of ApoD intracellular traffic and demonstrate its role in lysosomal pH homeostasis upon paraquat-induced oxidative stress. By combining single-lysosome in vivo pH measurements with immunodetection, we demonstrate that ApoD is endocytosed and targeted to a subset of vulnerable lysosomes in a stress-dependent manner. ApoD is functionally stable in this acidic environment, and its presence is sufficient and necessary for lysosomes to recover from oxidation-induced alkalinization, both in astrocytes and neurons. This function is accomplished by preventing lysosomal membrane permeabilization. Two lysosomal-dependent biological processes, myelin phagocytosis by astrocytes and optimization of neurodegeneration-triggered autophagy in a Drosophila in vivo model, require ApoD-related Lipocalins. Our results uncover a previously unknown biological function of ApoD, member of the finely regulated and evolutionary conserved gene family of extracellular Lipocalins. They set a lipoprotein-mediated regulation of lysosomal membrane integrity as a new mechanism at the hub of many cellular

  2. Protecting cells by protecting their vulnerable lysosomes: Identification of a new mechanism for preserving lysosomal functional integrity upon oxidative stress

    PubMed Central

    Pascua-Maestro, Raquel

    2017-01-01

    Environmental insults such as oxidative stress can damage cell membranes. Lysosomes are particularly sensitive to membrane permeabilization since their function depends on intraluminal acidic pH and requires stable membrane-dependent proton gradients. Among the catalog of oxidative stress-responsive genes is the Lipocalin Apolipoprotein D (ApoD), an extracellular lipid binding protein endowed with antioxidant capacity. Within the nervous system, cell types in the defense frontline, such as astrocytes, secrete ApoD to help neurons cope with the challenge. The protecting role of ApoD is known from cellular to organism level, and many of its downstream effects, including optimization of autophagy upon neurodegeneration, have been described. However, we still cannot assign a cellular mechanism to ApoD gene that explains how this protection is accomplished. Here we perform a comprehensive analysis of ApoD intracellular traffic and demonstrate its role in lysosomal pH homeostasis upon paraquat-induced oxidative stress. By combining single-lysosome in vivo pH measurements with immunodetection, we demonstrate that ApoD is endocytosed and targeted to a subset of vulnerable lysosomes in a stress-dependent manner. ApoD is functionally stable in this acidic environment, and its presence is sufficient and necessary for lysosomes to recover from oxidation-induced alkalinization, both in astrocytes and neurons. This function is accomplished by preventing lysosomal membrane permeabilization. Two lysosomal-dependent biological processes, myelin phagocytosis by astrocytes and optimization of neurodegeneration-triggered autophagy in a Drosophila in vivo model, require ApoD-related Lipocalins. Our results uncover a previously unknown biological function of ApoD, member of the finely regulated and evolutionary conserved gene family of extracellular Lipocalins. They set a lipoprotein-mediated regulation of lysosomal membrane integrity as a new mechanism at the hub of many cellular

  3. Methane to syngas conversion. Part I. Equilibrium conditions and stability requirements of membrane materials

    NASA Astrophysics Data System (ADS)

    Frade, J. R.; Kharton, V. V.; Yaremchenko, A.; Naumovich, E.

    Thermodynamic data have been used to predict the dependence of methane conversion on temperature and oxygen partial pressure in mixed conducting membrane reactors, and the corresponding fractions of water vapor, H 2, CO and CO 2. The relations between methane conversion, gas composition and oxygen partial pressure were also used to formulate the oxygen balance in mixed conducting membrane reactors, with tubular reactor and continuous stirred tank reactor (CSTR) configurations. A single dimensionless parameter accounts for the combined effects of geometric parameters of the membrane reactor, the permeability of the membrane material, and flow rate at the entry of the reactor. Selected examples were calculated to illustrate the effects of steam to methane and inert to methane ratios in the gas entering the reactor. The values of oxygen partial pressure required to attain the highest yield of CO and H 2 were also used to estimate the stability requirements to be met by mixed conducting membrane materials. Suitable membrane designs might be needed to bridge the difference between the conditions inside the reactors and the stability limits of known mixed conductors.

  4. A Study of Lipid Bilayer Membrane Stability Using Precise Measurements of Specific Capacitance

    PubMed Central

    White, Stephen H.

    1970-01-01

    A method is described for measuring the specific capacitance (Cm) of lipid bilayer membranes with an estimated experimental error of only 1%. The gross capacitance was measured with an AC Wheatstone bridge and a photographic technique was used to determine the area of thin membrane. The results of measurements on oxidized cholesterol-decane membranes formed in 1 × 10-2 M KCl show that Cm depends upon temperature, voltage, time, and the age of the bulk membrane solutions. For a freshly thinned membrane (from 5 week old solution), Cm increases exponentially from an initial value of 0.432 ±0.021 (SD) μF/cm2 with a time constant of ∼15 min. A 100 mv potential applied across the membrane for 10-20 min prior to making measurements eliminated this time dependence and produced final-state membranes. Cm of final-state membranes depends upon applied voltage (Va) and obeys the equation Cm = C0 + βVa2 where Va ≃ VDC + VrmsAC. C0 and β depend upon temperature; C0 decreases linearly with temperature while β increases linearly. At 20°C, C0 = 0.559 ±0.01 (SD) μF/cm2 and β = 0.0123 ±0.0036 (SD) (μF/cm2)/(mv2) and at 34°C, C0 = 0.472 ±0.01 and β = 0.0382 ±0.0039. These variations in Cm are interpreted as resulting from thickness changes. The possibility that they result from diffuse layer and/or membrane dielectric phenomena is discussed and found to be unlikely. The results are discussed in terms of membrane stability by constructing hypothetical potential energy vs. thickness curves. ImagesFigure 2 PMID:5489777

  5. A time course of orchestrated endophilin action in sensing, bending, and stabilizing curved membranes

    PubMed Central

    Poudel, Kumud R.; Dong, Yongming; Yu, Hang; Su, Allen; Ho, Thuong; Liu, Yan; Schulten, Klaus; Bai, Jihong

    2016-01-01

    Numerous proteins act in concert to sculpt membrane compartments for cell signaling and metabolism. These proteins may act as curvature sensors, membrane benders, and scaffolding molecules. Here we show that endophilin, a critical protein for rapid endocytosis, quickly transforms from a curvature sensor into an active bender upon membrane association. We find that local membrane deformation does not occur until endophilin inserts its amphipathic helices into lipid bilayers, supporting an active bending mechanism through wedging. Our time-course studies show that endophilin continues to drive membrane changes on a seconds-to-minutes time scale, indicating that the duration of endocytosis events constrains the mode of endophilin action. Finally, we find a requirement of coordinated activities between wedging and scaffolding for endophilin to produce stable membrane tubules in vitro and to promote synaptic activity in vivo. Together these data demonstrate that endophilin is a multifaceted molecule that precisely integrates activities of sensing, bending, and stabilizing curvature to sculpt membranes with speed. PMID:27170174

  6. High-speed atomic force microscopy shows that annexin V stabilizes membranes on the second timescale

    NASA Astrophysics Data System (ADS)

    Miyagi, Atsushi; Chipot, Christophe; Rangl, Martina; Scheuring, Simon

    2016-09-01

    Annexins are abundant cytoplasmic proteins that can bind to negatively charged phospholipids in a Ca2+-dependent manner, and are known to play a role in the storage of Ca2+ and membrane healing. Little is known, however, about the dynamic processes of protein-Ca2+-membrane assembly and disassembly. Here we show that high-speed atomic force microscopy (HS-AFM) can be used to repeatedly induce and disrupt annexin assemblies and study their structure, dynamics and interactions. Our HS-AFM set-up is adapted for such biological applications through the integration of a pumping system for buffer exchange and a pulsed laser system for uncaging caged compounds. We find that biochemically identical annexins (annexin V) display different effective Ca2+ and membrane affinities depending on the assembly location, providing a wide Ca2+ buffering regime while maintaining membrane stabilization. We also show that annexin is membrane-recruited and forms stable supramolecular assemblies within ˜5 s in conditions that are comparable to a membrane lesion in a cell. Molecular dynamics simulations provide atomic detail of the role played by Ca2+ in the reversible binding of annexin to the membrane surface.

  7. Can Stabilization and Inhibition of Aquaporins Contribute to Future Development of Biomimetic Membranes?

    PubMed Central

    To, Janet; Torres, Jaume

    2015-01-01

    In recent years, the use of biomimetic membranes that incorporate membrane proteins, i.e., biomimetic-hybrid membranes, has increased almost exponentially. Key membrane proteins in these systems have been aquaporins, which selectively permeabilize cellular membranes to water. Aquaporins may be incorporated into synthetic lipid bilayers or to more stable structures made of block copolymers or solid-state nanopores. However, translocation of aquaporins to these alien environments has adverse consequences in terms of performance and stability. Aquaporins incorporated in biomimetic membranes for use in water purification and desalination should also withstand the harsh environment that may prevail in these conditions, such as high pressure, and presence of salt or other chemicals. In this respect, modified aquaporins that can be adapted to these new environments should be developed. Another challenge is that biomimetic membranes that incorporate high densities of aquaporin should be defect-free, and this can only be efficiently ascertained with the availability of completely inactive mutants that behave otherwise like the wild type aquaporin, or with effective non-toxic water channel inhibitors that are so far inexistent. In this review, we describe approaches that can potentially be used to overcome these challenges. PMID:26266425

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

    PubMed Central

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

    2015-01-01

    Preface 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 processes, with significant implications for health and disease. The identification of a master gene, transcription factor EB (TFEB), that regulates lysosomal biogenesis and autophagy, has revealed how the lysosome adapts to environmental cues, such as starvation, and suggests novel therapeutic strategies for modulating lysosomal function in human disease. PMID:23609508

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

    PubMed Central

    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 Ankrd13 Family of Ubiquitin-interacting Motif-bearing Proteins Regulates Valosin-containing Protein/p97 Protein-mediated Lysosomal Trafficking of Caveolin 1*

    PubMed Central

    Burana, Daocharad; Yoshihara, Hidehito; Tanno, Hidetaka; Yamamoto, Akitsugu; Saeki, Yasushi; Tanaka, Keiji; Komada, Masayuki

    2016-01-01

    Caveolin 1 (Cav-1) is an oligomeric protein that forms flask-shaped, lipid-rich pits, termed caveolae, on the plasma membrane. Cav-1 is targeted for lysosomal degradation in ubiquitination- and valosin-containing protein (VCP)-dependent manners. VCP, an ATPase associated with diverse cellular activities that remodels or segregates ubiquitinated protein complexes, has been proposed to disassemble Cav-1 oligomers on the endosomal membrane, facilitating the trafficking of Cav-1 to the lysosome. Genetic mutations in VCP compromise the lysosomal trafficking of Cav-1, leading to a disease called inclusion body myopathy with Paget disease of bone and/or frontotemporal dementia (IBMPFD). Here we identified the Ankrd13 family of ubiquitin-interacting motif (UIM)-containing proteins as novel VCP-interacting molecules on the endosome. Ankrd13 proteins formed a ternary complex with VCP and Cav-1 and exhibited high binding affinity for ubiquitinated Cav-1 oligomers in an UIM-dependent manner. Mass spectrometric analyses revealed that Cav-1 undergoes Lys-63-linked polyubiquitination, which serves as a lysosomal trafficking signal, and that the UIMs of Ankrd13 proteins bind preferentially to this ubiquitin chain type. The overexpression of Ankrd13 caused enlarged hollow late endosomes, which was reminiscent of the phenotype of the VCP mutations in IBMPFD. Overexpression of Ankrd13 proteins also stabilized ubiquitinated Cav-1 oligomers on the limiting membrane of enlarged endosomes. The interaction with Ankrd13 was abrogated in IMBPFD-associated VCP mutants. Collectively, our results suggest that Ankrd13 proteins cooperate with VCP to regulate the lysosomal trafficking of ubiquitinated Cav-1. PMID:26797118

  11. The Ankrd13 Family of Ubiquitin-interacting Motif-bearing Proteins Regulates Valosin-containing Protein/p97 Protein-mediated Lysosomal Trafficking of Caveolin 1.

    PubMed

    Burana, Daocharad; Yoshihara, Hidehito; Tanno, Hidetaka; Yamamoto, Akitsugu; Saeki, Yasushi; Tanaka, Keiji; Komada, Masayuki

    2016-03-18

    Caveolin 1 (Cav-1) is an oligomeric protein that forms flask-shaped, lipid-rich pits, termed caveolae, on the plasma membrane. Cav-1 is targeted for lysosomal degradation in ubiquitination- and valosin-containing protein (VCP)-dependent manners. VCP, an ATPase associated with diverse cellular activities that remodels or segregates ubiquitinated protein complexes, has been proposed to disassemble Cav-1 oligomers on the endosomal membrane, facilitating the trafficking of Cav-1 to the lysosome. Genetic mutations in VCP compromise the lysosomal trafficking of Cav-1, leading to a disease called inclusion body myopathy with Paget disease of bone and/or frontotemporal dementia (IBMPFD). Here we identified the Ankrd13 family of ubiquitin-interacting motif (UIM)-containing proteins as novel VCP-interacting molecules on the endosome. Ankrd13 proteins formed a ternary complex with VCP and Cav-1 and exhibited high binding affinity for ubiquitinated Cav-1 oligomers in an UIM-dependent manner. Mass spectrometric analyses revealed that Cav-1 undergoes Lys-63-linked polyubiquitination, which serves as a lysosomal trafficking signal, and that the UIMs of Ankrd13 proteins bind preferentially to this ubiquitin chain type. The overexpression of Ankrd13 caused enlarged hollow late endosomes, which was reminiscent of the phenotype of the VCP mutations in IBMPFD. Overexpression of Ankrd13 proteins also stabilized ubiquitinated Cav-1 oligomers on the limiting membrane of enlarged endosomes. The interaction with Ankrd13 was abrogated in IMBPFD-associated VCP mutants. Collectively, our results suggest that Ankrd13 proteins cooperate with VCP to regulate the lysosomal trafficking of ubiquitinated Cav-1.

  12. A mechanism for stabilization of membranes at low temperatures by an antifreeze protein.

    PubMed Central

    Tomczak, Melanie M; Hincha, Dirk K; Estrada, Sergio D; Wolkers, Willem F; Crowe, Lois M; Feeney, Robert E; Tablin, Fern; Crowe, John H

    2002-01-01

    Polar fish, cold hardy plants, and overwintering insects produce antifreeze proteins (AFPs), which lower the freezing point of solutions noncolligatively and inhibit ice crystal growth. Fish AFPs have been shown to stabilize membranes and cells in vitro during hypothermic storage, probably by interacting with the plasma membrane, but the mechanism of this stabilization has not been clear. We show here that during chilling to nonfreezing temperatures the alpha-helical AFP type I from polar fish inhibits leakage across model membranes containing an unsaturated chloroplast galactolipid. The mechanism involves binding of the AFP to the bilayer, which increases the phase transition temperature of the membranes and alters the molecular packing of the acyl chains. We suggest that this change in acyl chain packing results in the reduced membrane permeability. The data suggest a hydrophobic interaction between the peptide and the bilayer. Further, we suggest that the expression of AFP type I in transgenic plants may be significant for thermal adaptation of chilling-sensitive plants. PMID:11806929

  13. Ceramic membrane fouling during ultrafiltration of oil/water emulsions: roles played by stabilization surfactants of oil droplets.

    PubMed

    Lu, Dongwei; Zhang, Tao; Ma, Jun

    2015-04-07

    Oil/water (O/W) emulsion stabilized by surfactants is the part of oily wastewater that is most difficult to handle. Ceramic membrane ultrafiltration presently is an ideal process to treat O/W emulsions. However, little is known about the fouling mechanism of the ceramic membrane during O/W emulsion treatment. This paper investigated how stabilization surfactants of O/W emulsions influence the irreversible fouling of ceramic membranes during ultrafiltration. An unexpected phenomenon observed was that irreversible fouling was much less when the charge of the stabilization surfactant of O/W emulsions is opposite to the membrane. The less ceramic membrane fouling in this case was proposed to be due to a synergetic steric effect and demulsification effect which prevented the penetration of oil droplets into membrane pores and led to less pore blockage. This proposed mechanism was supported by cross section images of fouled and virgin ceramic membranes taken with scanning electron microscopy, regression results of classical fouling models, and analysis of organic components rejected by the membrane. Furthermore, this mechanism was also verified by the existence of a steric effect and demulsification effect. Our finding suggests that ceramic membrane oppositely charged to the stabilization surfactant should be applied in ultrafiltration of O/W emulsions to alleviate irreversible membrane fouling. It could be a useful rule for ceramic membrane ultrafiltration of oily wastewater.

  14. Cryoprotectin: a plant lipid-transfer protein homologue that stabilizes membranes during freezing.

    PubMed Central

    Hincha, Dirk K

    2002-01-01

    Plants from temperate and cold climates are able to increase their freezing tolerance during exposure to low non-freezing temperatures. It has been shown that several genes are induced in a coordinated manner during this process of cold acclimation. The functional role of most of the corresponding cold-regulated proteins is not yet known. We summarize our knowledge of those cold-regulated proteins that are able to stabilize membranes during a freeze-thaw cycle. Special emphasis is placed on cryoprotectin, a lipid-transfer protein homologue that was isolated from cold-acclimated cabbage leaves and that protects isolated chloroplast thylakoid membranes from freeze-thaw damage. PMID:12171654

  15. β-Glucuronidase, a Regulator of Lyme Arthritis Severity, Modulates Lysosomal Trafficking and MMP-9 Secretion in Response to Inflammatory Stimuli.

    PubMed

    Bramwell, Kenneth K C; Mock, Kelton; Ma, Ying; Weis, John H; Teuscher, Cory; Weis, Janis J

    2015-08-15

    The lysosomal enzyme β-glucuronidase (Gusb) is a key regulator of Lyme-associated and K/B×N-induced arthritis severity. The luminal enzymes present in lysosomes provide essential catabolic functions for the homeostatic degradation of a variety of macromolecules. In addition to this essential catabolic function, lysosomes play important roles in the inflammatory response following infection. Secretory lysosomes and related vesicles can participate in the inflammatory response through fusion with the plasma membrane and release of bioactive contents into the extracellular milieu. In this study, we show that GUSB hypomorphism potentiates lysosomal exocytosis following inflammatory stimulation. This leads to elevated secretion of lysosomal contents, including glycosaminoglycans, lysosomal hydrolases, and matrix metalloproteinase 9, a known modulator of Lyme arthritis severity. This mechanistic insight led us to test the efficacy of rapamycin, a drug known to suppress lysosomal exocytosis. Both Lyme and K/B×N-associated arthritis were suppressed by this treatment concurrent with reduced lysosomal release.

  16. A New Method for Measuring Edge Tensions and Stability of Lipid Bilayers: Effect of Membrane Composition

    PubMed Central

    Portet, Thomas; Dimova, Rumiana

    2010-01-01

    We report a novel and facile method for measuring edge tensions of lipid membranes. The approach is based on electroporation of giant unilamellar vesicles and analysis of the pore closure dynamics. We applied this method to evaluate the edge tension in membranes with four different compositions: egg phosphatidylcholine (eggPC), dioleoylphosphatidylcholine (DOPC), and mixtures of DOPC with cholesterol and dioleoylphosphatidylethanolamine. Our data confirm previous results for eggPC and DOPC. The addition of 17 mol % cholesterol to the DOPC membrane causes an increase in the membrane edge tension. On the contrary, when the same fraction of dioleoylphosphatidylethanolamine is added to the membrane, a decrease in the edge tension is observed, which is an unexpected result considering the inverted-cone shape geometry of the molecule. It is presumed that interlipid hydrogen bonding is the origin of this behavior. Furthermore, cholesterol was found to lower the lysis tension of DOPC bilayers. This behavior differs from that observed on bilayers made of stearoyloleoylphosphatidylcholine, suggesting that cholesterol influences the membrane mechanical stability in a lipid-specific manner. PMID:21081074

  17. Neuropathic Lysosomal Storage Disorders

    PubMed Central

    Pastores, Gregory M.; Maegawa, Gustavo H.B.

    2014-01-01

    The lysosomal storage disorders (LSDs) are a clinically heterogeneous group of inborn errors of metabolism, associated with the accumulation of incompletely degraded macromolecules within several cellular sites. Affected individuals present with a broad range of clinical problems, including hepatosplenomegaly and skeletal dysplasia. Onset of symptoms may range from birth to adulthood. The majority are associated with neurological features, including developmental delay, behavioral/psychiatric disturbances, seizures, acroparesthesia, motor weakness, cerebrovascular ischemic events and extra-pyramidal signs. It should be noted that later-onset forms are often misdiagnosed as symptoms, which might include psychiatric manifestations, are slowly progressive and may precede other neurologic or systemic features. Inheritance is primarily autosomal recessive. For all subtypes, diagnosis can be confirmed using a combination of biochemical and/or molecular assays. In a few LSDs, treatment with either hematopoietic stem cell transplantation, enzyme replacement or substrate reduction therapy is available. Genetic counseling is important, so patients and their families can be informed of reproductive risks, disease prognosis and therapeutic options. Investigations of disease mechanisms are providing insights into potential therapeutic approaches. Symptomatic care, which remains the mainstay for most subtypes, can lead to significant improvement in quality of life. PMID:24176423

  18. The maltose ABC transporter: action of membrane lipids on the transporter stability, coupling and ATPase activity.

    PubMed

    Bao, Huan; Dalal, Kush; Wang, Victor; Rouiller, Isabelle; Duong, Franck

    2013-08-01

    The coupling between ATP hydrolysis and substrate transport remains a key question in the understanding of ABC-mediated transport. We show using the MalFGK2 complex reconstituted into nanodiscs, that membrane lipids participate directly to the coupling reaction by stabilizing the transporter in a low energy conformation. When surrounded by short acyl chain phospholipids, the transporter is unstable and hydrolyzes large amounts of ATP without inducing maltose. The presence of long acyl chain phospholipids stabilizes the conformational dynamics of the transporter, reduces its ATPase activity and restores dependence on maltose. Membrane lipids therefore play an essential allosteric function, they restrict the transporter ATPase activity to increase coupling to the substrate. In support to the notion, we show that increasing the conformational dynamics of MalFGK2 with mutations in MalF increases the transporter ATPase activity but decreases the maltose transport efficiency.

  19. Highly stabilized, polymer-lipid membranes prepared on silica microparticles as stationary phases for capillary chromatography

    PubMed Central

    Gallagher, Elyssia S.; Adem, Seid M.; Baker, Christopher A.; Ratnayaka, Saliya N.; Jones, Ian W.; Hall, Henry K.; Saavedra, S. Scott; Aspinwall, Craig A.

    2015-01-01

    The ability to rapidly screen complex libraries of pharmacological modulators is paramount to modern drug discovery efforts. This task is particularly challenging for agents that interact with lipid bilayers or membrane proteins due to the limited chemical, physical, and temporal stability of conventional lipid-based chromatographic stationary phases. Here, we describe the preparation and characterization of a novel stationary phase material composed of highly stable, polymeric-phospholipid bilayers self-assembled onto silica microparticles. Polymer lipid membranes were prepared by photochemical or redox initiated polymerization of 1,2-bis[10-(2′,4′-hexadieoyloxy)decanoyl]-sn-glycero-2-phosphocholine (bis-SorbPC), a synthetic, polymerizable lipid. The resulting polymerized bis-SorbPC (poly(bis-SorbPC)) stationary phases exhibited enhanced stability compared to particles coated with 1,2-dioleoyl-sn-glycero-phosphocholine (unpolymerized) phospholipid bilayers when exposed to chemical (50mM triton X-100 or 50% acetonitrile) and physical (15 min sonication) insults after 30 days of storage. Further, poly(bis-SorbPC)-coated particles survived slurry packing into fused silica capillaries, compared to unpolymerized lipid membranes, where the lipid bilayer was destroyed during packing. Frontal chromatographic analyses of the lipophilic small molecules acetylsalicylic acid, benzoic acid, and salicylic acid showed > 44% increase in retention times (P < 0.0001) for all analytes on poly(bis-SorbPC)-functionalized stationary phase compared to bare silica microspheres, suggesting a lipophilic retention mechanism. Phospholipid membrane-functionalized stationary phases that withstand the chemical and physical rigors of capillary LC conditions can substantially increase the efficacy of lipid membrane affinity chromatography, and represents a key advance towards the development of robust membrane protein-functionalized chromatographic stationary phases. PMID:25670414

  20. Highly stabilized, polymer-lipid membranes prepared on silica microparticles as stationary phases for capillary chromatography.

    PubMed

    Gallagher, Elyssia S; Adem, Seid M; Baker, Christopher A; Ratnayaka, Saliya N; Jones, Ian W; Hall, Henry K; Saavedra, S Scott; Aspinwall, Craig A

    2015-03-13

    The ability to rapidly screen complex libraries of pharmacological modulators is paramount to modern drug discovery efforts. This task is particularly challenging for agents that interact with lipid bilayers or membrane proteins due to the limited chemical, physical, and temporal stability of conventional lipid-based chromatographic stationary phases. Here, we describe the preparation and characterization of a novel stationary phase material composed of highly stable, polymeric-phospholipid bilayers self-assembled onto silica microparticles. Polymer-lipid membranes were prepared by photochemical or redox initiated polymerization of 1,2-bis[10-(2',4'-hexadieoyloxy)decanoyl]-sn-glycero-2-phosphocholine (bis-SorbPC), a synthetic, polymerizable lipid. The resulting polymerized bis-SorbPC (poly(bis-SorbPC)) stationary phases exhibited enhanced stability compared to particles coated with 1,2-dioleoyl-sn-glycero-phosphocholine (unpolymerized) phospholipid bilayers when exposed to chemical (50 mM triton X-100 or 50% acetonitrile) and physical (15 min sonication) insults after 30 days of storage. Further, poly(bis-SorbPC)-coated particles survived slurry packing into fused silica capillaries, compared to unpolymerized lipid membranes, where the lipid bilayer was destroyed during packing. Frontal chromatographic analyses of the lipophilic small molecules acetylsalicylic acid, benzoic acid, and salicylic acid showed >44% increase in retention times (P<0.0001) for all analytes on poly(bis-SorbPC)-functionalized stationary phase compared to bare silica microspheres, suggesting a lipophilic retention mechanism. Phospholipid membrane-functionalized stationary phases that withstand the chemical and physical rigors of capillary LC conditions can substantially increase the efficacy of lipid membrane affinity chromatography, and represents a key advance toward the development of robust membrane protein-functionalized chromatographic stationary phases.

  1. Conformational Stability and Pathogenic Misfolding of the Integral Membrane Protein PMP22

    PubMed Central

    2016-01-01

    Despite broad biochemical relevance, our understanding of the physiochemical reactions that limit the assembly and cellular trafficking of integral membrane proteins remains superficial. In this work, we report the first experimental assessment of the relationship between the conformational stability of a eukaryotic membrane protein and the degree to which it is retained by cellular quality control in the secretory pathway. We quantitatively assessed both the conformational equilibrium and cellular trafficking of 12 variants of the α-helical membrane protein peripheral myelin protein 22 (PMP22), the intracellular misfolding of which is known to cause peripheral neuropathies associated with Charcot–Marie–Tooth disease (CMT). We show that the extent to which these mutations influence the energetics of Zn(II)-mediated PMP22 folding is proportional to the observed reduction in cellular trafficking efficiency. Strikingly, quantitative analyses also reveal that the reduction of motor nerve conduction velocities in affected patients is proportional to the extent of the mutagenic destabilization. This finding provides compelling evidence that the effects of these mutations on the energetics of PMP22 folding lie at the heart of the molecular basis of CMT. These findings highlight conformational stability as a key factor governing membrane protein biogenesis and suggest novel therapeutic strategies for CMT. PMID:26102530

  2. Receptor dimer stabilization by hierarchical plasma membrane microcompartments regulates cytokine signaling.

    PubMed

    You, Changjiang; Marquez-Lago, Tatiana T; Richter, Christian Paolo; Wilmes, Stephan; Moraga, Ignacio; Garcia, K Christopher; Leier, André; Piehler, Jacob

    2016-12-01

    The interaction dynamics of signaling complexes is emerging as a key determinant that regulates the specificity of cellular responses. We present a combined experimental and computational study that quantifies the consequences of plasma membrane microcompartmentalization for the dynamics of type I interferon receptor complexes. By using long-term dual-color quantum dot (QD) tracking, we found that the lifetime of individual ligand-induced receptor heterodimers depends on the integrity of the membrane skeleton (MSK), which also proved important for efficient downstream signaling. By pair correlation tracking and localization microscopy as well as by fast QD tracking, we identified a secondary confinement within ~300-nm-sized zones. A quantitative spatial stochastic diffusion-reaction model, entirely parameterized on the basis of experimental data, predicts that transient receptor confinement by the MSK meshwork allows for rapid reassociation of dissociated receptor dimers. Moreover, the experimentally observed apparent stabilization of receptor dimers in the plasma membrane was reproduced by simulations of a refined, hierarchical compartment model. Our simulations further revealed that the two-dimensional association rate constant is a key parameter for controlling the extent of MSK-mediated stabilization of protein complexes, thus ensuring the specificity of this effect. Together, experimental evidence and simulations support the hypothesis that passive receptor confinement by MSK-based microcompartmentalization promotes maintenance of signaling complexes in the plasma membrane.

  3. Receptor dimer stabilization by hierarchical plasma membrane microcompartments regulates cytokine signaling

    PubMed Central

    You, Changjiang; Marquez-Lago, Tatiana T.; Richter, Christian Paolo; Wilmes, Stephan; Moraga, Ignacio; Garcia, K. Christopher; Leier, André; Piehler, Jacob

    2016-01-01

    The interaction dynamics of signaling complexes is emerging as a key determinant that regulates the specificity of cellular responses. We present a combined experimental and computational study that quantifies the consequences of plasma membrane microcompartmentalization for the dynamics of type I interferon receptor complexes. By using long-term dual-color quantum dot (QD) tracking, we found that the lifetime of individual ligand-induced receptor heterodimers depends on the integrity of the membrane skeleton (MSK), which also proved important for efficient downstream signaling. By pair correlation tracking and localization microscopy as well as by fast QD tracking, we identified a secondary confinement within ~300-nm-sized zones. A quantitative spatial stochastic diffusion-reaction model, entirely parameterized on the basis of experimental data, predicts that transient receptor confinement by the MSK meshwork allows for rapid reassociation of dissociated receptor dimers. Moreover, the experimentally observed apparent stabilization of receptor dimers in the plasma membrane was reproduced by simulations of a refined, hierarchical compartment model. Our simulations further revealed that the two-dimensional association rate constant is a key parameter for controlling the extent of MSK-mediated stabilization of protein complexes, thus ensuring the specificity of this effect. Together, experimental evidence and simulations support the hypothesis that passive receptor confinement by MSK-based microcompartmentalization promotes maintenance of signaling complexes in the plasma membrane. PMID:27957535

  4. DNA as membrane-bound ligand-receptor pairs: duplex stability is tuned by intermembrane forces.

    PubMed

    Beales, Paul A; Vanderlick, T Kyle

    2009-02-18

    We use membrane-anchored DNA as model adhesion receptors between lipid vesicles. By studying the thermal stability of DNA duplex formation, which tethers the vesicles into superstructures, we show that the melting temperature of a 10-base DNA sequence is dependent on the lipid composition of the tethered vesicles. We propose a simple model that describes how the intermembrane interactions tilt the free energy landscape for DNA binding. From our model, we estimate the area per DNA in the binding sites between vesicles and also the total area of the adhesion plaques. We find that vesicles containing a small proportion of cationic lipid that are modified with membrane-anchored DNA can be reversibly tethered by specific DNA interactions and that the DNA also induces a small attraction between these membranes, which stabilizes the DNA duplex. By increasing the equilibrium intermembrane distance on binding, we show that intermembrane interactions become negligible for the binding thermodynamics of the DNA and hence the thermal stability of vesicle aggregates becomes independent of lipid composition at large enough intervesicle separations. We discuss the implications of our findings with regards to cell adhesion and fusion receptors, and the programmable self-assembly of nano-structured materials by DNA hybridization.

  5. Influence of acute exercise on the osmotic stability of the human erythrocyte membrane.

    PubMed

    Paraiso, L F; de Freitas, M V; Gonçalves-E-Oliveira, A F M; de Almeida Neto, O P; Pereira, E A; Mascarenhas Netto, R C; Cunha, L M; Bernardino Neto, M; de Agostini, G G; Resende, E S; Penha-Silva, N

    2014-12-01

    This study evaluated the effects of 2 different types of acute aerobic exercise on the osmotic stability of human erythrocyte membrane and on different hematological and biochemical variables that are associated with this membrane property. The study population consisted of 20 healthy and active men. Participants performed single sessions of 2 types of exercise. The first session consisted of 60 min of moderate-intensity continuous exercise (MICE). The second session, executed a week later, consisted of high-intensity interval exercise (HIIE) until exhaustion. The osmotic stability of the erythrocyte membrane was represented by the inverse of the salt concentration (1/H50) at the midpoint of the sigmoidal curve of dependence between the absorbance of hemoglobin and the NaCl concentration. The values of 1/H50 changed from 2.29±0.1 to 2.33±0.09 after MICE and from 2.30±0.08 to 2.23±0.12 after HIIE. During MICE mean corpuscular volume increased, probably due to in vivo lysis of older erythrocytes, with preservation of cells that were larger and more resistant to in vitro lysis. The study showed that a single bout of acute exercise affected erythrocyte stability, which increased after MICE and decreased after HIIE.

  6. Enhanced Stability of Laminated Graphene Oxide Membranes for Nanofiltration via Interstitial Amide Bonding.

    PubMed

    Nam, Yoon Tae; Choi, Junghoon; Kang, Kyoung Min; Kim, Dae Woo; Jung, Hee-Tae

    2016-10-03

    Laminated graphene oxide (GO) has promising use as a membrane because of its high permeance, chemical and mechanical stability, as well as the molecular sieving effect of its interlayers. However, the hydrophilic surface of GO, which is highly decorated with oxygen groups, easily induces delamination of stacked GO films in aqueous media, thereby limiting the practical application. To stabilize GO films in aqueous media, we functionalized a polymer support with branched polyethylene-imine (BPEI). BPEI adsorbed intercalated into the stacked GO sheets via diffusion during filtration. The GO/BPEI membrane obtained exhibits high stability during sonication (>1 h duration, 40 kHz frequency) in water within a broad pH range (2-12). In contrast, the GO film spontaneously delaminated upon sonication. Furthermore, BPEI treatment did not affect the filtration performance of the GO film, as evidenced by the high rejection rates (>90%) for the dye molecules methylene blue, rose bengal, and brilliant blue and by their permeation rates of ca. 124, 34.8, 12.2, and 5.1%, respectively, relative to those of a typical GO membrane.

  7. The influence of plasma membrane electrostatic properties on the stability of cell ionic composition.

    PubMed Central

    Genet, S; Costalat, R; Burger, J

    2001-01-01

    An electro-osmotic model is developed to examine the influence of plasma membrane superficial charges on the regulation of cell ionic composition. Assuming membrane osmotic equilibrium, the ion distribution predicted by Gouy-Chapman-Grahame (GCG) theory is introduced into ion transport equations, which include a kinetic model of the Na/K-ATPase based on the stimulation of this ion pump by internal Na(+) ions. The algebro-differential equation system describing dynamics of the cell model has a unique resting state, stable with respect to finite-sized perturbations of various types. Negative charges on the membrane are found to greatly enhance relaxation toward steady state following these perturbations. We show that this heightened stability stems from electrostatic interactions at the inner membrane side that shift resting state coordinates along the sigmoidal activation curve of the sodium pump, thereby increasing the pump sensitivity to internal Na(+) fluctuations. The accuracy of electrostatic potential description with GCG theory is proved using an alternate formalism, based on irreversible thermodynamics, which shows that pressure contribution to ion potential energy is negligible in electrostatic double layers formed at the surfaces of biological membranes. We discuss implications of the results regarding a reliable operation of ionic process coupled to the transmembrane electrochemical gradient of Na(+) ions. PMID:11606261

  8. Template-particle stabilized bicontinuous emulsion yielding controlled assembly of hierarchical high-flux filtration membranes.

    PubMed

    Hess, Samuel C; Kohll, A Xavier; Raso, Renzo A; Schumacher, Christoph M; Grass, Robert N; Stark, Wendelin J

    2015-01-14

    A novel solvent-evaporation-based process that exploits template-particle stabilized bicontinuous emulsions for the formation of previously unreached membrane morphologies is reported in this article. Porous membranes have a wide range of applications spanning from water filtration, pharmaceutical purification, and battery separators to scaffolds for tissue engineering. Different situations require different membrane morphologies including various pore sizes and pore gradients. However, most of the previously reported membrane preparation procedures are restricted to specific morphologies and morphology alterations require an extensive optimization process. The tertiary system presented in this article, which consists of a poly(ether sulfone)/dimethylacetamide (PES/DMAc) solution, glycerol, and ZnO-nanoparticles, allows simple and exact tuning of pore diameters ranging from sub-20 nm, up to 100 nm. At the same time, the pore size gradient is controlled from 0 up to 840%/μm yielding extreme asymmetry. In addition to structural analysis, water flux rates of over 5600 L m(-2) h(-1) are measured for membranes retaining 45 nm silica beads.

  9. Deficiency of ATP13A2 leads to lysosomal dysfunction, α-synuclein accumulation, and neurotoxicity.

    PubMed

    Usenovic, Marija; Tresse, Emilie; Mazzulli, Joseph R; Taylor, J Paul; Krainc, Dimitri

    2012-03-21

    The autophagy-lysosomal pathway plays an important role in the clearance of long-lived proteins and dysfunctional organelles. Lysosomal dysfunction has been implicated in several neurodegenerative disorders including Parkinson's disease and related synucleinopathies that are characterized by accumulations of α-synuclein in Lewy bodies. Recent identification of mutations in genes linked to lysosomal function and neurodegeneration has offered a unique opportunity to directly examine the role of lysosomes in disease pathogenesis. Mutations in lysosomal membrane protein ATP13A2 (PARK9) cause familial Kufor-Rakeb syndrome characterized by early-onset parkinsonism, pyramidal degeneration and dementia. While previous data suggested a role of ATP13A2 in α-synuclein misfolding and toxicity, the mechanistic link has not been established. Here we report that loss of ATP13A2 in human fibroblasts from patients with Kufor-Rakeb syndrome or in mouse primary neurons leads to impaired lysosomal degradation capacity. This lysosomal dysfunction results in accumulation of α-synuclein and toxicity in primary cortical neurons. Importantly, silencing of endogenous α-synuclein attenuated the toxicity in ATP13A2-depleted neurons, suggesting that loss of ATP13A2 mediates neurotoxicity at least in part via the accumulation of α-synuclein. Our findings implicate lysosomal dysfunction in the pathogenesis of Kufor-Rakeb syndrome and suggest that upregulation of lysosomal function and downregulation of α-synuclein represent important therapeutic strategies for this disorder.

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

    PubMed

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

    2013-01-01

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

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

    PubMed Central

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

    2013-01-01

    Lysosomes are cytoplasmic organelles present in almost all eukaryotic cells, which play a fundamental role in key aspects of cellular homeostasis such as membrane repair, autophagy, endocitosis and protein metabolism. The characterization of the genes and enzymes constituting the lysosome represents a central issue to be addressed toward a better understanding of the biology of this organelle. In humans, mutations that cause lysosomal enzyme deficiencies result in >50 different disorders and severe pathologies. So far, many experimental efforts using different methodologies have been carried out to identity lysosomal genes. The Human Lysosome Gene Database (hLGDB) is the first resource that provides a comprehensive and accessible census of the human genes belonging to the lysosomal system. This database was developed by collecting and annotating gene lists from many different sources. References to the studies that have identified each gene are provided together with cross databases gene related information. Special attention has been given to the regulation of the genes through microRNAs and the transcription factor EB. The hLGDB can be easily queried to retrieve, combine and analyze information on different lists of lysosomal genes and their regulation by microRNA (binding sites predicted by five different algorithms). The hLGDB is an open access dynamic project that will permit in the future to collapse in a unique publicly accessible resource all the available biological information about lysosome genes and their regulation. Database URL: http://lysosome.unipg.it/ PMID:23584836

  12. Failure of lysosome clustering and positioning in the juxtanuclear region in cells deficient in rapsyn

    PubMed Central

    Aittaleb, Mohamed; Chen, Po-Ju; Akaaboune, Mohammed

    2015-01-01

    ABSTRACT Rapsyn, a scaffold protein, is required for the clustering of acetylcholine receptors (AChRs) at contacts between motor neurons and differentiating muscle cells. Rapsyn is also expressed in cells that do not express AChRs. However, its function in these cells remains unknown. Here, we show that rapsyn plays an AChR-independent role in organizing the distribution and mobility of lysosomes. In cells devoid of AChRs, rapsyn selectively induces the clustering of lysosomes at high density in the juxtanuclear region without affecting the distribution of other intracellular organelles. However, when the same cells overexpress AChRs, rapsyn is recruited away from lysosomes to colocalize with AChR clusters on the cell surface. In rapsyn-deficient (Rapsn−/−) myoblasts or cells overexpressing rapsyn mutants, lysosomes are scattered within the cell and highly dynamic. The increased mobility of lysosomes in Rapsn−/− cells is associated with a significant increase in lysosomal exocytosis, as evidenced by increased release of lysosomal enzymes and plasma membrane damage when cells were challenged with the bacterial pore-forming toxin streptolysin-O. These findings uncover a new link between rapsyn, lysosome positioning, exocytosis and plasma membrane integrity. PMID:26330529

  13. Plant plasma membrane aquaporins in natural vesicles as potential stabilizers and carriers of glucosinolates.

    PubMed

    Martínez-Ballesta, Maria Del Carmen; Pérez-Sánchez, Horacio; Moreno, Diego A; Carvajal, Micaela

    2016-07-01

    Their biodegradable nature and ability to target cells make biological vesicles potential nanocarriers for bioactives delivery. In this work, the interaction between proteoliposomes enriched in aquaporins derived from broccoli plants and the glucosinolates was evaluated. The vesicles were stored at different temperatures and their integrity was studied. Determination of glucosinolates, showed that indolic glucosinolates were more sensitive to degradation in aqueous solution than aliphatic glucosinolates. Glucoraphanin was stabilized by leaf and root proteoliposomes at 25°C through their interaction with aquaporins. An extensive hydrogen bond network, including different aquaporin residues, and hydrophobic interactions, as a consequence of the interaction between the linear alkane chain of glucoraphanin and Glu31 and Leu34 protein residues, were established as the main stabilizing elements. Combined our results showed that plasma membrane vesicles from leaf and root tissues of broccoli plants may be considered as suitable carriers for glucosinolate which stabilization can be potentially attributed to aquaporins.

  14. Stability of aneurysm solutions in a fluid-filled elastic membrane tube

    NASA Astrophysics Data System (ADS)

    Il'ichev, A. T.; Fu, Y.-B.

    2012-08-01

    When a hyperelastic membrane tube is inflated by an internal pressure, a localized bulge will form when the pressure reaches a critical value. As inflation continues the bulge will grow until it reaches a maximum size after which it will then propagate in both directions to form a hat-like profile. The stability of such bulging solutions has recently been studied by neglecting the inertia of the inflating fluid and it was shown that such bulging solutions are unstable under pressure control. In this paper we extend this recent study by assuming that the inflation is by an inviscid fluid whose inertia we take into account in the stability analysis. This reflects more closely the situation of aneurysm formation in human arteries which motivates the current series of studies. It is shown that fluid inertia would significantly reduce the growth rate of the unstable mode and thus it has a strong stabilizing effect.

  15. Removal of seminal plasma enhances membrane stability on fresh and cooled stallion spermatozoa.

    PubMed

    Barrier-Battut, I; Bonnet, C; Giraudo, A; Dubois, C; Caillaud, M; Vidament, M

    2013-02-01

    Fertility is reduced after semen cooling for a considerable number of stallions. The main hypotheses include alterations in plasma membrane following cooling and deleterious influence of seminal plasma. However, interindividual variability is controversial. We hypothesized that the removal of seminal plasma could enhance motility in some 'poor cooler' stallions, but could also affect, negatively or positively, membrane quality in some stallions. This study examined the effect of centrifugation, followed or not by removal of seminal plasma, on parameters indicating semen quality after 48 h at 4 °C: motility, plasma membrane integrity as evaluated by hypo-osmotic swelling test, acrosome integrity and response to a pharmacological induction of acrosome reaction using ionophore A23187. Sixty-six ejaculates from 14 stallions were used, including stallions showing high or low sperm motility after cooled storage. Centrifugation without removal of seminal plasma did not affect sperm parameters. Removal of seminal plasma did not affect motility, but significantly stabilized sperm membranes, as demonstrated by a higher response to the osmotic challenge, and a reduced reactivity of the acrosome. Moreover, for the same semen sample, the response to an induction of acrosome reaction was significantly higher when the induction was performed in the presence of seminal plasma, compared with the induction in the absence of seminal plasma. This was observed both for fresh and cooled semen. When the induction of acrosome reaction with ionophore A23187 is used to evaluate sperm quality, care must therefore be taken to standardize the proportion of seminal plasma between samples. For the 10 stallions serving at least 25 mares, the only variable significantly correlated with fertility was motility. The influence of membrane stabilization regarding fertility requires further investigations.

  16. Enhancing lysosomal biogenesis and autophagic flux by activating the transcription factor EB protects against cadmium-induced neurotoxicity

    PubMed Central

    Pi, Huifeng; Li, Min; Tian, Li; Yang, Zhiqi; Yu, Zhengping; Zhou, Zhou

    2017-01-01

    Cadmium (Cd), a highly ubiquitous heavy metal, is a well-known inducer of neurotoxicity. However, the mechanism underlying cadmium-induced neurotoxicity remains unclear. In this study, we found that Cd inhibits autophagosome-lysosome fusion and impairs lysosomal function by reducing the levels of lysosomal-associated membrane proteins, inhibiting lysosomal proteolysis and altering lysosomal pH, contributing to defects in autophagic clearance and subsequently leading to nerve cell death. In addition, Cd decreases transcription factor EB (TFEB) expression at both the mRNA and protein levels. Furthermore, Cd induces the nuclear translocation of TFEB and TFEB target-gene expression, associated with compromised lysosomal function or a compensatory effect after the impairment of the autophagic flux. Notably, restoration of the levels of lysosomal-associated membrane protein, lysosomal proteolysis, lysosomal pH and autophagic flux through Tfeb overexpression protects against Cd-induced neurotoxicity, and this protective effect is incompletely dependent on TFEB nuclear translocation. Moreover, gene transfer of the master autophagy regulator TFEB results in the clearance of toxic proteins and the correction of Cd-induced neurotoxicity in vivo. Our study is the first to demonstrate that Cd disrupts lysosomal function and autophagic flux and manipulation of TFEB signalling may be a therapeutic approach for antagonizing Cd-induced neurotoxicity. PMID:28240313

  17. Fructans from oat and rye: composition and effects on membrane stability during drying.

    PubMed

    Hincha, Dirk K; Livingston, David P; Premakumar, Ramaswamy; Zuther, Ellen; Obel, Nicolai; Cacela, Constança; Heyer, Arnd G

    2007-06-01

    Fructans have been implicated in the abiotic stress tolerance of many plant species, including grasses and cereals. To elucidate the possibility that cereal fructans may stabilize cellular membranes during dehydration, we used liposomes as a model system and isolated fructans from oat (Avena sativa) and rye (Secale cereale). Fructans were fractionated by preparative size exclusion chromatography into five defined size classes (degree of polymerization (DP) 3 to 7) and two size classes containing high DP fructans (DP>7 short and long). They were characterized by high performance liquid chromatography (HPLC) and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The effects of the fructans on liposome stability during drying and rehydration were assessed as the ability of the sugars to prevent leakage of a soluble marker from liposomes and liposome fusion. Both species contain highly complex mixtures of fructans, with a DP up to 17. The two DP>7 fractions from both species were unable to protect liposomes, while the fractions containing smaller fructans were protective to different degrees. Protection showed an optimum at DP 4 and the DP 3, 4, and 5 fractions from oat were more protective than all other fractions from both species. In addition, we found evidence for synergistic effects in membrane stabilization in mixtures of low DP with DP>7 fructans. The data indicate that cereal fructans have the ability to stabilize membranes under stress conditions and that there are size and species dependent differences between the fructans. In addition, mixtures of fructans, as they occur in living cells may have protective properties that differ significantly from those of the purified fractions.

  18. Size-controlled nanopores in lipid membranes with stabilizing electric fields.

    PubMed

    Fernández, M Laura; Risk, Marcelo; Reigada, Ramon; Vernier, P Thomas

    2012-06-29

    Molecular dynamics (MD) has been shown to be a useful tool for unveiling many aspects of pore formation in lipid membranes under the influence of an applied electric field. However, the study of the structure and transport properties of electropores by means of MD has been hampered by difficulties in the maintenance of a stable electropore in the typically small simulated membrane patches. We describe a new simulation scheme in which an initially larger porating field is systematically reduced after pore formation to lower stabilizing values to produce stable, size-controlled electropores, which can then be characterized at the molecular level. A new method allows the three-dimensional modeling of the irregular shape of the pores obtained as well as the quantification of its volume. The size of the pore is a function of the value of the stabilizing field. At lower fields the pore disappears and the membrane recovers its normal shape, although in some cases long-lived, fragmented pores containing unusual lipid orientations in the bilayer are observed.

  19. Sphingosine-induced apoptosis is dependent on lysosomal proteases.

    PubMed Central

    Kågedal, K; Zhao, M; Svensson, I; Brunk, U T

    2001-01-01

    We propose a new mechanism for sphingosine-induced apoptosis, involving relocation of lysosomal hydrolases to the cytosol. Owing to its lysosomotropic properties, sphingosine, which is also a detergent, especially when protonated, accumulates by proton trapping within the acidic vacuolar apparatus, where most of its action as a detergent would be exerted. When sphingosine was added in low-to-moderate concentrations to Jurkat and J774 cells, partial lysosomal rupture occurred dose-dependently, starting within a few minutes. This phenomenon preceded caspase activation, as well as changes of mitochondrial membrane potential. High sphingosine doses rapidly caused extensive lysosomal rupture and ensuing necrosis, without antecedent apoptosis or caspase activation. The sphingosine effect was prevented by pre-treatment with another, non-toxic, lysosomotropic base, ammonium chloride, at 10 mM. The lysosomal protease inhibitors, pepstatin A and epoxysuccinyl-L-leucylamido-3-methyl-butane ethyl ester ('E-64d'), inhibited markedly sphingosine-induced caspase activity to almost the same degree as the general caspase inhibitor benzyloxycarbonyl-Val-Ala-DL-Asp-fluoromethylketone ('Z-VAD-FMK'), although they did not by themselves inhibit caspases. We conclude that cathepsin D and one or more cysteine proteases, such as cathepsins B or L, are important mediators of sphingosine-induced apoptosis, working upstream of the caspase cascade and mitochondrial membrane-potential changes. PMID:11583579

  20. Effect of the compatible solute ectoine on the stability of the membrane proteins.

    PubMed

    Roychoudhury, Arpita; Haussinger, Dieter; Oesterhelt, Filipp

    2012-08-01

    Mechanical single molecule techniques offer exciting possibilities for investigating protein folding and stability in native environments at sub-nanometer resolutions. Compatible solutes show osmotic activity which even at molar concentrations do not interfere with cell metabolism. They are known to protect proteins against external stress like temperature, high salt concentrations and dehydrating conditions. We studied the impact of the compatible solute ectoine (1M) on membrane proteins by analyzing the mechanical properties of Bacteriorhodopsin (BR) in its presence and absence by single molecule force spectroscopy. The unfolding experiments on BR revealed that ectoine decreases the persistence length of its polypeptide chain thereby increasing its tendency to coil up. In addition, we found higher unfolding forces indicating strengthening of those intra molecular interactions which are crucial for stability. This shows that force spectroscopy is well suited to study the effect of compatible solutes to stabilize membrane proteins against unfolding. In addition, it may lead to a better understanding of their detailed mechanism of action.

  1. Polyacrylamide-Polydivinylbenzene Decorated Membrane for Sundry Ionic Stabilized Emulsions Separation via a Facile Solvothermal Method.

    PubMed

    Zhang, Weifeng; Liu, Na; Cao, Yingze; Chen, Yuning; Zhang, Qingdong; Lin, Xin; Qu, Ruixiang; Li, Haifang; Feng, Lin

    2016-08-24

    Aiming to solve the worldwide challenge of stabilized oil-in-water emulsion separation, a PAM-PDVB decorated nylon membrane is fabricated via a facile solvothermal route in our group. The main composition is PAM, while the PDVB plays a role as cross-linker in order to improve the interaction between the polymer and the substrate. By the combination of the superhydrophilic and underwater superoleophobic wettability of the PAM polymer with the micropore size of the substrate, the as-prepared material is able to achieve the separation of various stabilized oil-in-water emulsions including cationic type, nonionic type, and anionic type. Compared with previous works, the emulsions used in this case are more stable and can stay for several days. Besides, the solvothermal method is facile, cost saving, and relatively environmentally friendly in this experiment. Moreover, the PAM-PDVB modified membrane exhibits excellent pH stability, recyclability, and high separation efficiency (above 99%), which can be scaled up and used in the practical industrial field.

  2. Stability in alkaline aqueous electrolyte of air electrode protected with fluorinated interpenetrating polymer network membrane

    NASA Astrophysics Data System (ADS)

    Bertolotti, Bruno; Messaoudi, Houssam; Chikh, Linda; Vancaeyzeele, Cédric; Alfonsi, Séverine; Fichet, Odile

    2015-01-01

    We developed original anion exchange membranes to protect air electrodes operating in aqueous lithium-air battery configuration, i.e. supplied with atmospheric air and in concentrated aqueous lithium hydroxide. These protective membranes have an interpenetrating polymer network (IPN) architecture combining a hydrogenated cationic polyelectrolyte network based on poly(epichlorohydrin) (PECH) and a fluorinated neutral network based on perfluoropolyether (Fluorolink® MD700). Two phases, each one rich in one of the polymer, are co-continuous in the materials. This morphology allows combining their properties according to the weight proportions of each polymer. Thus, PECH/Fluorolink IPNs show ionic conductivity varying from 1 to 2 mS cm-1, water uptake from 30 to 90 wt.% and anionic transport number from 0.65 to 0.80 when the PECH proportion varies from 40 to 90 wt.%. These membranes have been systematically assembled on air electrodes. Air electrode protected with PECH/Fluorolink 70/30 IPN shows outstanding stability higher than 1000 h, i.e. a 20-fold increase in the lifetime of the non-modified electrode. This efficient membrane/air electrode assembly is promising for development of alkaline electrolyte based storage or production energy systems, such as metal air batteries or alkaline fuel cells.

  3. Conditions that Stabilize Membrane Domains Also Antagonize n-Alcohol Anesthesia

    NASA Astrophysics Data System (ADS)

    Machta, Benjamin B.; Gray, Ellyn; Nouri, Mariam; McCarthy, Nicola L. C.; Gray, Erin M.; Miller, Ann L.; Brooks, Nicholas J.; Veatch, Sarah L.

    2016-08-01

    Diverse molecules induce general anesthesia with potency strongly correlated both with their hydrophobicity and their effects on certain ion channels. We recently observed that several n-alcohol anesthetics inhibit heterogeneity in plasma membrane derived vesicles by lowering the critical temperature ($T_c$) for phase separation. Here we exploit conditions that stabilize membrane heterogeneity to further test the correlation between the anesthetic potency of n-alcohols and effects on $T_c$. First we show that hexadecanol acts oppositely to n-alcohol anesthetics on membrane mixing and antagonizes ethanol induced anesthesia in a tadpole behavioral assay. Second, we show that two previously described `intoxication reversers' raise $T_c$ and counter ethanol's effects in vesicles, mimicking the findings of previous electrophysiological and behavioral measurements. Third, we find that hydrostatic pressure, long known to reverse anesthesia, also raises $T_c$ in vesicles with a magnitude that counters the effect of butanol at relevant concentrations and pressures. Taken together, these results demonstrate that $\\Delta T_c$ predicts anesthetic potency for n-alcohols better than hydrophobicity in a range of contexts, supporting a mechanistic role for membrane heterogeneity in general anesthesia.

  4. Lysosome-mediated processing of chromatin in senescence.

    PubMed

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

    2013-07-08

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

  5. Molecular basis of multiple sulfatase deficiency, mucolipidosis II/III and Niemann-Pick C1 disease - Lysosomal storage disorders caused by defects of non-lysosomal proteins.

    PubMed

    Dierks, Thomas; Schlotawa, Lars; Frese, Marc-André; Radhakrishnan, Karthikeyan; von Figura, Kurt; Schmidt, Bernhard

    2009-04-01

    Multiple sulfatase deficiency (MSD), mucolipidosis (ML) II/III and Niemann-Pick type C1 (NPC1) disease are rare but fatal lysosomal storage disorders caused by the genetic defect of non-lysosomal proteins. The NPC1 protein mainly localizes to late endosomes and is essential for cholesterol redistribution from endocytosed LDL to cellular membranes. NPC1 deficiency leads to lysosomal accumulation of a broad range of lipids. The precise functional mechanism of this membrane protein, however, remains puzzling. ML II, also termed I cell disease, and the less severe ML III result from deficiencies of the Golgi enzyme N-acetylglucosamine 1-phosphotransferase leading to a global defect of lysosome biogenesis. In patient cells, newly synthesized lysosomal proteins are not equipped with the critical lysosomal trafficking marker mannose 6-phosphate, thus escaping from lysosomal sorting at the trans Golgi network. MSD affects the entire sulfatase family, at least seven members of which are lysosomal enzymes that are specifically involved in the degradation of sulfated glycosaminoglycans, sulfolipids or other sulfated molecules. The combined deficiencies of all sulfatases result from a defective post-translational modification by the ER-localized formylglycine-generating enzyme (FGE), which oxidizes a specific cysteine residue to formylglycine, the catalytic residue enabling a unique mechanism of sulfate ester hydrolysis. This review gives an update on the molecular bases of these enigmatic diseases, which have been challenging researchers since many decades and so far led to a number of surprising findings that give deeper insight into both the cell biology and the pathobiochemistry underlying these complex disorders. In case of MSD, considerable progress has been made in recent years towards an understanding of disease-causing FGE mutations. First approaches to link molecular parameters with clinical manifestation have been described and even therapeutical options have been

  6. Alteration of epithelial cell lysosomal integrity induced by bacterial cholesterol‐dependent cytolysins

    PubMed Central

    Malet, Julien Karim

    2016-01-01

    Abstract Bacterial pathogens can interfere during infection with host cell organelles, such as mitochondria, the endoplasmic reticulum‐Golgi system or nuclei. As important cellular functions are often compartmentalized in these organelles, their targeting allows pathogens to manipulate key host functions during infection. Here, we identify lysosomes as a new class of organelles targeted by the pathogenic bacterium Listeria monocytogenes. We demonstrate that extracellular Listeria, via secretion of the pore‐forming toxin listeriolysin O, alters lysosomal integrity in epithelial cells but not in macrophages. Listeriolysin O induces lysosomal membrane permeabilization and release of lysosomal content, such as cathepsins proteases, which remain transiently active in the host cytosol. We furthermore show that other bacterial pore‐forming toxins, such as perfringolysin O and pneumolysin, also induce lysosomes alteration. Together, our data unveil a novel activity of bacterial cholesterol‐dependent cytolysins. PMID:27739224

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

    PubMed Central

    2016-01-01

    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

  8. Preparation of uniform particle-stabilized emulsions using SPG membrane emulsification.

    PubMed

    Sun, Guanqing; Qi, Feng; Wu, Jie; Ma, Guanghui; Ngai, To

    2014-06-24

    Various aspects of particle-stabilized emulsions (or so-called Pickering emulsions) have been extensively investigated during the last two decades, but the preparation of uniform Pickering emulsion droplets via a simple and scalable method has been sparingly realized. We report the preparation of uniform Pickering emulsions by Shirasu porous glass (SPG) membrane emulsification. The size of the emulsion droplets ranging from 10-50 μm can be precisely controlled by the size of the membrane pore. The emulsion droplets have a high monodispersity with coefficients of variation (CV) lower than 15% in all of the investigated systems. We further demonstrate the feasibility of locking the assembled particles at the interface, and emulsion droplets have been shown to be excellent templates for the preparation of monodisperse colloidosomes that are necessary in drug-delivery systems.

  9. The SM protein Car/Vps33A regulates SNARE-mediated trafficking to lysosomes and lysosome-related organelles.

    PubMed

    Akbar, Mohammed A; Ray, Sanchali; Krämer, Helmut

    2009-03-01

    The SM proteins Vps33A and Vps33B are believed to act in membrane fusions in endosomal pathways, but their specific roles are controversial. In Drosophila, Vps33A is the product of the carnation (car) gene. We generated a null allele of car to test its requirement for trafficking to different organelles. Complete loss of car function is lethal during larval development. Eye-specific loss of Car causes late, light-independent degeneration of photoreceptor cells. Earlier in these cells, two distinct phenotypes were detected. In young adults, autophagosomes amassed indicating that their fusion with lysosomes requires Car. In eye discs, endocytosed receptors and ligands accumulate in Rab7-positive prelysosomal compartments. The requirement of Car for late endosome-to-lysosome fusion in imaginal discs is specific as early endosomes are unaffected. Furthermore, lysosomal delivery is not restored by expression of dVps33B. This specificity reflects the distinct pattern of binding to different Syntaxins in vitro: dVps33B predominantly binds the early endosomal Avl and Car to dSyntaxin16. Consistent with a role in Car-mediated fusion, dSyntaxin16 is not restricted to Golgi membranes but also present on lysosomes.

  10. The SM Protein Car/Vps33A Regulates SNARE-mediated Trafficking to Lysosomes and Lysosome-related Organelles

    PubMed Central

    Akbar, Mohammed A.; Ray, Sanchali

    2009-01-01

    The SM proteins Vps33A and Vps33B are believed to act in membrane fusions in endosomal pathways, but their specific roles are controversial. In Drosophila, Vps33A is the product of the carnation (car) gene. We generated a null allele of car to test its requirement for trafficking to different organelles. Complete loss of car function is lethal during larval development. Eye-specific loss of Car causes late, light-independent degeneration of photoreceptor cells. Earlier in these cells, two distinct phenotypes were detected. In young adults, autophagosomes amassed indicating that their fusion with lysosomes requires Car. In eye discs, endocytosed receptors and ligands accumulate in Rab7-positive prelysosomal compartments. The requirement of Car for late endosome-to-lysosome fusion in imaginal discs is specific as early endosomes are unaffected. Furthermore, lysosomal delivery is not restored by expression of dVps33B. This specificity reflects the distinct pattern of binding to different Syntaxins in vitro: dVps33B predominantly binds the early endosomal Avl and Car to dSyntaxin16. Consistent with a role in Car-mediated fusion, dSyntaxin16 is not restricted to Golgi membranes but also present on lysosomes. PMID:19158398

  11. Haematopoietic, Antioxidant and Membrane Stabilizing Property of Diallyl Disulphide in Irradiated Mice

    PubMed Central

    Tenkanidiyoor, Yogish Somayaji; Vasudeva, Vidya; Rao, Shama; Gowda, Damodara; Rao, Chandrika; Sanjeev, Ganesh

    2016-01-01

    Introduction Diallyl disulphide is an organo-sulphur compound which is present in garlic and responsible for the characteristic odor of garlic. It is known for its anticancer and invitro membrane stabilizing properties. Aim The main aim was to evaluate the haematopoietic, antioxidant and membrane stabilizing property of diallyl disulfide in irradiated mice. Materials and Methods Mice were grouped into 6 groups as control, drug control, radiation control and drug pre-treatment groups (i.e. drug administration + radiation group) The mice were fed orally for 15 consecutive days and on the 15th day, one hour after drug administration, the mice were irradiated with 6Gy electron beam radiation. The changes in blood cell count, total antioxidant levels, malondialdehyde and reduced glutathione levels were determined. The immunomodulatory response of DADS to the radiological effects was determined by the estimation of IL-6 levels. Results A significant improvement in pre-drug treatment group when compared to control groups in the haemoglobin, red blood cell count, white blood cell count, haematocrit and platelet counts was observed. There is an increased level of interleukin-6 in the drug treated groups compared to the radiation control. An increase in the malondialdehyde levels and decrease in the glutathione levels in the irradiated group indicate increased lipid peroxidation and oxidative stress, whereas, there is a significant reduction in the malondialdehyde levels and increased glutathione levels in the drug pre-treatment groups showing membrane stabilization. Conclusion Thus DADS proves to be an effective haematopoietic and antioxidative agent to counter radiation induced haematopoietic suppression and oxidative stress. PMID:27042448

  12. Insertion stability of poly(ethylene glycol)-cholesteryl-based lipid anchors in liposome membranes.

    PubMed

    Molnar, Daniel; Linders, Jürgen; Mayer, Christian; Schubert, Rolf

    2016-06-01

    Liposomes consist of a hydrophilic core surrounded by a phospholipid (PL) bilayer. In human blood, the half-life of such artificial vesicles is limited. To prolong their stability in the circulation, liposomal bilayers can be modified by inserting poly(ethylene glycol) (PEG) molecules using either PL or sterols as membrane anchors. This establishes a hydrophilic steric barrier, reducing the adsorption of serum proteins, recognition and elimination by cells of the immune system. In addition, targeting ligands (such as antibodies) are frequently coupled to the distal end of the PEG chains to direct the vesicles (then called 'immuno-liposomes') to specific cell types, such as tumor cells. To our knowledge, experiments on the stability of ligand anchoring have so far only been conducted with PL-based PEGs and not with sterol-based PEGs after insertion via the sterol-based post-insertion technique (SPIT). Therefore, our study examines the insertion stability of PEG-cholesteryl ester (Chol-PEG) molecules with PEG chains of 1000, 1500 and 2000Da molecular mass which have been inserted into the membranes of liposomes using SPIT. For this study we used different acceptor media and multiple analytical techniques, including pulsed-field-gradient nuclear magnetic resonance (PFG-NMR), free-flow electrophoresis, size exclusion chromatography and ultracentrifugation. The obtained data consistently showed that a higher molar mass of PEG chains positively correlates with higher release from the liposome membranes. Furthermore, we could detect and quantify the migration of Chol-PEG molecules from radioactively double-labeled surface-modified liposomes to negatively charged acceptor liposomes via free-flow electrophoresis. Insertion of Chol-PEG molecules into the membrane of preformed liposomes using SPIT is an essential step for the functionalization of liposomes with the aim of specific targeting. For the first time, we present a kinetic analysis of this insertion process using PFG

  13. Graphene Oxide Nanofiltration Membranes Stabilized by Cationic Porphyrin for High Salt Rejection.

    PubMed

    Xu, Xiao-Ling; Lin, Fu-Wen; Du, Yong; Zhang, Xi; Wu, Jian; Xu, Zhi-Kang

    2016-05-25

    Swelling has great influences on the structure stability and separation performance of graphene oxide laminate membranes (GOLMs) for water desalination and purification. Herein, we report cross-linked GOLMs from GO assembled with cationic tetrakis(1-methyl-pyridinium-4-yl)porphyrin (TMPyP) by a vacuum-assisted strategy. The concave nonoxide regions (G regions) of GO are used as cross-linking sites for the first time to precisely control the channel size for water permeation and salt ion retention. Channels around 1 nm are constructed by modulating the assembly ratio of TMPyP/GO, and these cross-linked GOLMs show high salt rejection.

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

    PubMed

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

    2010-08-01

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

  15. Muscle intermediate filaments and their links to membranes and membranous organelles.

    PubMed

    Capetanaki, Yassemi; Bloch, Robert J; Kouloumenta, Asimina; Mavroidis, Manolis; Psarras, Stelios

    2007-06-10

    Intermediate filaments (IFs) play a key role in the integration of structure and function of striated muscle, primarily by mediating mechanochemical links between the contractile apparatus and mitochondria, myonuclei, the sarcolemma and potentially the vesicle trafficking apparatus. Linkage of all these membranous structures to the contractile apparatus, mainly through the Z-disks, supports the integration and coordination of growth and energy demands of the working myocyte, not only with force transmission, but also with de novo gene expression, energy production and efficient protein and lipid trafficking and targeting. Desmin, the most abundant and intensively studied muscle intermediate filament protein, is linked to proper costamere organization, myoblast and stem cell fusion and differentiation, nuclear shape and positioning, as well as mitochondrial shape, structure, positioning and function. Similar links have been established for lysosomes and lysosome-related organelles, consistent with the presence of widespread links between IFs and membranous structures and the regulation of their fusion, morphology and stabilization necessary for cell survival.

  16. Muscle intermediate filaments and their links to membranes and membranous organelles

    SciTech Connect

    Capetanaki, Yassemi . E-mail: ycapetanaki@bioacademy.gr; Bloch, Robert J.; Kouloumenta, Asimina; Mavroidis, Manolis; Psarras, Stelios

    2007-06-10

    Intermediate filaments (IFs) play a key role in the integration of structure and function of striated muscle, primarily by mediating mechanochemical links between the contractile apparatus and mitochondria, myonuclei, the sarcolemma and potentially the vesicle trafficking apparatus. Linkage of all these membranous structures to the contractile apparatus, mainly through the Z-disks, supports the integration and coordination of growth and energy demands of the working myocyte, not only with force transmission, but also with de novo gene expression, energy production and efficient protein and lipid trafficking and targeting. Desmin, the most abundant and intensively studied muscle intermediate filament protein, is linked to proper costamere organization, myoblast and stem cell fusion and differentiation, nuclear shape and positioning, as well as mitochondrial shape, structure, positioning and function. Similar links have been established for lysosomes and lysosome-related organelles, consistent with the presence of widespread links between IFs and membranous structures and the regulation of their fusion, morphology and stabilization necessary for cell survival.

  17. Membrane potential stabilization in amphibian skeletal muscle fibres in hypertonic solutions

    PubMed Central

    Ferenczi, Emily A; Fraser, James A; Chawla, Sangeeta; Skepper, Jeremy N; Schwiening, Christof J; Huang, Christopher L-H

    2004-01-01

    This study investigated membrane transport mechanisms influencing relative changes in cell volume (V) and resting membrane potential (Em) following osmotic challenge in amphibian skeletal muscle fibres. It demonstrated a stabilization of Em despite cell shrinkage, which was attributable to elevation of intracellular [Cl−] above electrochemical equilibrium through Na+–Cl− and Na+−K+−2Cl− cotransporter action following exposures to extracellular hypertonicity. Fibre volumes (V) determined by confocal microscope xz-scanning of cutaneous pectoris muscle fibres varied linearly with [1/extracellular osmolarity], showing insignificant volume corrections, in fibres studied in Cl−-free, normal and Na+-free Ringer solutions and in the presence of bumetanide, chlorothiazide and ouabain. The observed volume changes following increases in extracellular tonicity were compared with microelectrode measurements of steady-state resting potentials (Em). Fibres in isotonic Cl−-free, normal and Na+-free Ringer solutions showed similar Em values consistent with previously reported permeability ratios PNa/PK(0.03–0.05) and PCl/PK (∼2.0) and intracellular [Na+], [K+] and [Cl−]. Increased extracellular osmolarities produced hyperpolarizing shifts in Em in fibres studied in Cl−-free Ringer solution consistent with the Goldman-Hodgkin-Katz (GHK) equation. In contrast, fibres exposed to hypertonic Ringer solutions of normal ionic composition showed no such Em shifts, suggesting a Cl−-dependent stabilization of membrane potential. This stabilization of Em was abolished by withdrawing extracellular Na+ or by the combined presence of the Na+–Cl− cotransporter (NCC) inhibitor chlorothiazide (10 μm) and the Na+−K+−2Cl− cotransporter (NKCC) inhibitor bumetanide (10 μm), or the Na+−K+-ATPase inhibitor ouabain (1 or 10 μm) during alterations in extracellular osmolarity. Application of such agents after such increases in tonicity only produced a

  18. Effects of acute and chronic exercise on the osmotic stability of erythrocyte membrane of competitive swimmers

    PubMed Central

    2017-01-01

    This study aimed to evaluate the influence of acute and chronic exercise on erythrocyte membrane stability and various blood indices in a population consisting of five national-level male swimmers, over 18 weeks of training. The evaluations were made at the beginning and end of the 1st, 7th, 13th and 18th weeks, when volume and training intensity have changed. The effects manifested at the beginning of those weeks were considered due to chronic adaptations, while the effects observed at the end of the weeks were considered due to acute manifestations of the exercise load of that week. Acute changes resulting from the exercise comprised increases in creatine kinase activity (CK) and leukocyte count (Leu), and decrease in hematocrit (Ht) and mean corpuscular volume (MCV), at the end of the first week; increase in the activities of CK and lactate dehydrogenase (LDH), in the uric acid (UA) concentration and Leu count, at the end of the seventh week; increases in CK and LDH activities and in the mean corpuscular hemoglobin concentration (MCHC), at the end of the 13th week; and decrease in the value of the osmotic stability index 1/H50 and increases in the CK activity and platelets (Plt) count, at the end of the 18th week. Chronic changes due to training comprised increase in the values of 1/H50, CK, LDH, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), serum iron (Fe), MCV and Plt. Although acute training has resulted in decrease in the osmotic stability of erythrocytes, possibly associated with exacerbation of the oxidative processes during intense exercise, chronic training over 18 weeks resulted in increased osmotic stability of erythrocytes, possibly by modulation in the membrane cholesterol content by low and high density lipoproteins. PMID:28151958

  19. Effects of acute and chronic exercise on the osmotic stability of erythrocyte membrane of competitive swimmers.

    PubMed

    Paraiso, Lara Ferreira; Gonçalves-E-Oliveira, Ana Flávia Mayrink; Cunha, Lucas Moreira; de Almeida Neto, Omar Pereira; Pacheco, Adriana Garcia; Araújo, Karinne Beatriz Gonçalves; Garrote-Filho, Mário da Silva; Bernardino Neto, Morun; Penha-Silva, Nilson

    2017-01-01

    This study aimed to evaluate the influence of acute and chronic exercise on erythrocyte membrane stability and various blood indices in a population consisting of five national-level male swimmers, over 18 weeks of training. The evaluations were made at the beginning and end of the 1st, 7th, 13th and 18th weeks, when volume and training intensity have changed. The effects manifested at the beginning of those weeks were considered due to chronic adaptations, while the effects observed at the end of the weeks were considered due to acute manifestations of the exercise load of that week. Acute changes resulting from the exercise comprised increases in creatine kinase activity (CK) and leukocyte count (Leu), and decrease in hematocrit (Ht) and mean corpuscular volume (MCV), at the end of the first week; increase in the activities of CK and lactate dehydrogenase (LDH), in the uric acid (UA) concentration and Leu count, at the end of the seventh week; increases in CK and LDH activities and in the mean corpuscular hemoglobin concentration (MCHC), at the end of the 13th week; and decrease in the value of the osmotic stability index 1/H50 and increases in the CK activity and platelets (Plt) count, at the end of the 18th week. Chronic changes due to training comprised increase in the values of 1/H50, CK, LDH, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), serum iron (Fe), MCV and Plt. Although acute training has resulted in decrease in the osmotic stability of erythrocytes, possibly associated with exacerbation of the oxidative processes during intense exercise, chronic training over 18 weeks resulted in increased osmotic stability of erythrocytes, possibly by modulation in the membrane cholesterol content by low and high density lipoproteins.

  20. Intrinsic stability of Brassicaceae plasma membrane in relation to changes in proteins and lipids as a response to salinity.

    PubMed

    Chalbi, Najla; Martínez-Ballesta, Ma Carmen; Youssef, Nabil Ben; Carvajal, Micaela

    2015-03-01

    Changes in plasma membrane lipids, such as sterols and fatty acids, have been observed as a result of salt stress. These alterations, together with modification of the plasma membrane protein profile, confer changes in the physical properties of the membrane to be taken into account for biotechnological uses. In our experiments, the relationship between lipids and proteins in three different Brassicaceae species differing in salinity tolerance (Brassica oleracea, B. napus and Cakile maritima) and the final plasma membrane stability were studied. The observed changes in the sterol (mainly an increase in sitosterol) and fatty acid composition (increase in RUFA) in each species led to physical adaptation of the plasma membrane to salt stress. The in vitro vesicles stability was higher in the less tolerant (B. oleracea) plants together with low lipoxygenase activity. These results indicate that the proteins/lipids ratio and lipid composition is an important aspect to take into account for the use of natural vesicles in plant biotechnology.

  1. Specific lysosomal transport of small neutral amino acids

    SciTech Connect

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

    1986-05-01

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

  2. UV-visible spectroscopy method for screening the chemical stability of potential antioxidants for proton exchange membrane fuel cells

    NASA Astrophysics Data System (ADS)

    Banham, Dustin; Ye, Siyu; Knights, Shanna; Stewart, S. Michael; Wilson, Mahlon; Garzon, Fernando

    2015-05-01

    A novel method based on UV-visible spectroscopy is reported for screening the chemical stability of potential antioxidant additives for proton exchange membrane fuel cells, and the chemical stabilities of three CeOx samples of varying crystallite sizes (6, 13, or 25 nm) are examined. The chemical stabilities predicted by this new screening method are compared to in-situ membrane electrode assembly (MEA) accelerated stress testing, with the results confirming that this rapid and inexpensive method can be used to accurately predict performance impacts of antioxidants.

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

    PubMed

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

    2014-11-01

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

  4. Coronavirus Cell Entry Occurs through the Endo-/Lysosomal Pathway in a Proteolysis-Dependent Manner

    PubMed Central

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

    2014-01-01

    Enveloped viruses need to fuse with a host cell membrane in order to deliver their genome into the host cell. While some viruses fuse with the plasma membrane, many viruses are endocytosed prior to fusion. Specific cues in the endosomal microenvironment induce conformational changes in the viral fusion proteins leading to viral and host membrane fusion. In the present study we investigated the entry of coronaviruses (CoVs). Using siRNA gene silencing, we found that proteins known to be important for late endosomal maturation and endosome-lysosome fusion profoundly promote infection of cells with mouse hepatitis coronavirus (MHV). Using recombinant MHVs expressing reporter genes as well as a novel, replication-independent fusion assay we confirmed the importance of clathrin-mediated endocytosis and demonstrated that trafficking of MHV to lysosomes is required for fusion and productive entry to occur. Nevertheless, MHV was shown to be less sensitive to perturbation of endosomal pH than vesicular stomatitis virus and influenza A virus, which fuse in early and late endosomes, respectively. Our results indicate that entry of MHV depends on proteolytic processing of its fusion protein S by lysosomal proteases. Fusion of MHV was severely inhibited by a pan-lysosomal protease inhibitor, while trafficking of MHV to lysosomes and processing by lysosomal proteases was no longer required when a furin cleavage site was introduced in the S protein immediately upstream of the fusion peptide. Also entry of feline CoV was shown to depend on trafficking to lysosomes and processing by lysosomal proteases. In contrast, MERS-CoV, which contains a minimal furin cleavage site just upstream of the fusion peptide, was negatively affected by inhibition of furin, but not of lysosomal proteases. We conclude that a proteolytic cleavage site in the CoV S protein directly upstream of the fusion peptide is an essential determinant of the intracellular site of fusion. PMID:25375324

  5. Reconstruction of the Distal Oblique Bundle of the Interosseous Membrane: A Technique to Restore Distal Radioulnar Joint Stability.

    PubMed

    Riggenbach, Michael D; Wright, Thomas W; Dell, Paul C

    2015-11-01

    The distal radioulnar ligament reconstruction is a technique that may be used for distal radioulnar joint instability without arthritis and failed nonsurgical management; clinical results demonstrate resolved or improved stability. Recent literature has focused on the distal oblique bundle of the interosseous membrane and its contributions to stability. This article describes a technically simple surgical technique to reconstruct the distal oblique bundle and restore distal radioulnar joint stability.

  6. Activation of Peroxisome Proliferator-activated Receptor α Induces Lysosomal Biogenesis in Brain Cells

    PubMed Central

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

    2015-01-01

    Lysosomes are ubiquitous membrane-enclosed organelles filled with an acidic interior and are central to the autophagic, endocytic, or phagocytic pathway. In contrast to its classical function as the waste management machinery, lysosomes are now considered to be an integral part of various cellular signaling processes. The diverse functionality of this single organelle requires a very complex and coordinated regulation of its activity with transcription factor EB (TFEB), a master regulator of lysosomal biogenesis, at its core. However, mechanisms by which TFEB is regulated are poorly understood. This study demonstrates that gemfibrozil, an agonist of peroxisome proliferator-activated receptor (PPAR) α, alone and in conjunction with all-trans-retinoic acid is capable of enhancing TFEB in brain cells. We also observed that PPARα, but not PPARβ and PPARγ, is involved in gemfibrozil-mediated up-regulation of TFEB. Reporter assay and chromatin immunoprecipitation studies confirmed the recruitment of retinoid X receptor α, PPARα, and PGC1α on the PPAR-binding site on the Tfeb promoter as well. Subsequently, the drug-mediated induction of TFEB caused an increase in lysosomal protein and the lysosomal abundance in cell. Collectively, this study reinforces the link between lysosomal biogenesis and lipid metabolism with TFEB at the crossroads. Furthermore, gemfibrozil may be of therapeutic value in the treatment of lysosomal storage disorders in which autophagy-lysosome pathway plays an important role. PMID:25750174

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

    PubMed Central

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

    2012-01-01

    Multidrug resistance (MDR) remains a primary hindrance to curative cancer therapy. Thus, introduction of novel strategies to overcome MDR is of paramount therapeutic significance. Sequestration of chemotherapeutics in lysosomes is an established mechanism of drug resistance. Here, we show that MDR cells display a marked increase in lysosome number. We further demonstrate that imidazoacridinones (IAs), which are cytotoxic fluorochromes, undergo a dramatic compartmentalization in lysosomes because of their hydrophobic weak base nature. We hence developed a novel photoactivation-based pharmacological Trojan horse approach to target and eradicate MDR cancer cells based on photo-rupture of IA-loaded lysosomes and tumor cell lysis via formation of reactive oxygen species. Illumination of IA-loaded cells resulted in lysosomal photodestruction and restoration of parental cell drug sensitivity. Lysosomal photodestruction of MDR cells overexpressing the key MDR efflux transporters ABCG2, ABCB1 or ABCC1 resulted in 10- to 52-fold lower IC50 values of various IAs, thereby restoring parental cell sensitivity. Finally, in vivo application of this photodynamic therapy strategy after i.v. injection of IAs in human ovarian tumor xenografts in the chorioallantoic membrane model revealed selective destruction of tumors and their associated vasculature. These findings identify lysosomal sequestration of IAs as an Achilles heel of MDR cells that can be harnessed to eradicate MDR tumor cells via lysosomal photodestruction. PMID:22476101

  8. Multivesicular endosomes containing internalized EGF-EGF receptor complexes mature and then fuse directly with lysosomes

    PubMed Central

    1996-01-01

    We have followed the transfer of EGF-EGF receptor (EGFR) complexes from endosomal vacuoles that contain transferrin receptors (TfR) to lysosome vacuoles identified by their content of HRP loaded as a 15-min pulse 4 h previously. We show that the HRP-loaded lysosomes are lysosomal- associated membrane protein-1 (LAMP-1) positive, mannose-6-phosphate receptor (M6PR) negative. and contain active acid hydrolase. EGF-EGFR complexes are delivered to these lysosomes intact and are then rapidly degraded. Preactivating the HRP contained within the preloaded lysosomes inhibits the delivery of EGFR and degradation of EGF, and results in the accumulation of EGFR-containing multivesicular bodies (MVB). With time these accumulating MVB undergo a series of maturation changes that include the loss of TfR, the continued recruitment of EGFR, and the accumulation of internal vesicles, but they remain LAMP-1 and M6PR negative. The mature MVB are often seen to make direct contact with lysosomes containing preactivated HRP, but their perimeter membranes remain intact. Together our observations suggest that the transfer of EGF-EGFR complexes from the TfR-containing endosome compartment to the lysosomes that degrade them employs a single vacuolar intermediate, the maturing MVB, and can be achieved by a single heterotypic fusion step. PMID:8601581

  9. The small GTPase Arl8b regulates assembly of the mammalian HOPS complex on lysosomes

    PubMed Central

    Khatter, Divya; Raina, Vivek B.; Dwivedi, Devashish; Sindhwani, Aastha; Bahl, Surbhi; Sharma, Mahak

    2015-01-01

    The homotypic fusion and protein sorting (HOPS) complex is a multi-subunit complex conserved from yeast to mammals that regulates late endosome and lysosome fusion. However, little is known about how the HOPS complex is recruited to lysosomes in mammalian cells. Here, we report that the small GTPase Arl8b, but not Rab7 (also known as RAB7A), is essential for membrane localization of the human (h)Vps41 subunit of the HOPS complex. Assembly of the core HOPS subunits to Arl8b- and hVps41-positive lysosomes is guided by their subunit–subunit interactions. RNA interference (RNAi)-mediated depletion of hVps41 resulted in the impaired degradation of EGFR that was rescued upon expression of wild-type but not an Arl8b-binding-defective mutant of hVps41, suggesting that Arl8b-dependent lysosomal localization of hVps41 is required for its endocytic function. Furthermore, we have also identified that the Arl8b effector SKIP (also known as PLEKHM2) interacts with and recruits HOPS subunits to Arl8b and kinesin-positive peripheral lysosomes. Accordingly, RNAi-mediated depletion of SKIP impaired lysosomal trafficking and degradation of EGFR. These findings reveal that Arl8b regulates the association of the human HOPS complex with lysosomal membranes, which is crucial for the function of this tethering complex in endocytic degradation. PMID:25908847

  10. Isolation of Spheroplast Membranes and Stability of Spheroplasts of Bacillus stearothermophilus

    PubMed Central

    Bodman, Hollis; Welker, N. E.

    1969-01-01

    Spheroplasts were prepared by lysozyme digestion of the cell wall and ruptured by suspension in 0.15 m NaCl, followed by centrifugation at 30,900 × g for 35 min, and by a final suspension in 0.05 m NaCl for 12 to 16 hr at 5 C. The membrane ghosts were washed four times in tris(hydroxylmethyl)aminomethane (Tris) magnesium buffer and once in distilled water. The intact membranes resembled empty sacs with narrow slits in which the cytoplasm was extruded. A 92% recovery of cell membrane was obtained with all membrane preparations. The spheroplasts do not require a stabilizing medium to keep them from rupturing, and they are stable for 2 to 3 hr when exposed to a temperature of 65 C. The membrane content of the cell increases with age of culture (mid-log, 16.5%; late-log, 17.0%; and stationary, 17.6%) and temperature of growth (55 C, 16.5%; and 65 C, 17.8%), and it is unaffected by composition of the growth medium. The ratio of the protein to lipid content of the membrane increases with the complexity of the medium, age of culture (mid-log, 3.65; late-log, 3.91; and stationary, 4.15), and temperature of growth (55 C, 3.65; and 65 C, 5.22). The ribonucleic acid (RNA) and deoxyribonucleic acid (DNA) content of the membranes was 9.0 to 13.7% and 0.3 to 0.8%, respectively. Reducing sugar (determined as glucose) amounts to 0.9 to 1.0% of the membrane weight and did not significantly vary for the different membrane preparations. Medium composition, age of culture, and temperature of growth have no significant effect on the amount of each amino acid in the membrane. Aspartic acid, glutamic acid, alanine, leucine, and lysine are present in the greatest amount and represent 12.9 to 14.1%, 10.4 to 11.3%, 9.6 to 10.3%, 7.7 to 8.8%, and 7.6 to 8.5% of the membrane peptide, respectively. Prior to the rupture of the spheroplasts, 25.0, 15.7, and 50.0% of the protein, RNA, and DNA, respectively, is lost. In potassium phosphate-magnesium buffer without sucrose, 90% of the protein and

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

    PubMed

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

    2016-10-01

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

  12. Ferritin-stimulated lipid peroxidation, lysosomal leak, and macroautophagy promote lysosomal "metastability" in primary hepatocytes determining in vitro cell survival.

    PubMed

    Krenn, Margit A; Schürz, Melanie; Teufl, Bernhard; Uchida, Koji; Eckl, Peter M; Bresgen, Nikolaus

    2015-03-01

    Several pathologies are associated with elevated levels of serum ferritin, for which growth inhibitory properties have been reported; however, the underlying mechanisms are still poorly defined. Previously we have described cytotoxic properties of isoferritins released from primary hepatocytes in vitro, which induce apoptosis in an iron and oxidative stress-dependent mode. Here we show that this ferritin species stimulates endosome clustering and giant endosome formation in primary hepatocytes accompanied by enhanced lysosomal membrane permeability (LMP). In parallel, protein modification by lipid peroxidation-derived 4-hydroxynonenal (HNE) is strongly promoted by ferritin, the HNE-modified proteins (HNE-P) showing remarkable aggregation. Emphasizing the prooxidant context, GSH is rapidly depleted and the GSH/GSSG ratio is substantially declining in ferritin-treated cells. Furthermore, ferritin triggers a transient upregulation of macroautophagy which is abolished by iron chelation and apparently supports HNE-P clearance. Macroautophagy inhibition by 3-methyladenine strongly amplifies ferritin cytotoxicity in a time- and concentration-dependent mode, suggesting an important role of macroautophagy on cellular responses to ferritin endocytosis. Moreover, pointing at an involvement of lysosomal proteolysis, ferritin cytotoxicity and lysosome fragility are aggravated by the protease inhibitor leupeptin. In contrast, EGF which suppresses ferritin-induced cell death attenuates ferritin-mediated LMP. In conclusion, we propose that HNE-P accumulation, lysosome dysfunction, and macroautophagy stimulated by ferritin endocytosis provoke lysosomal "metastability" in primary hepatocytes which permits cell survival as long as in- and extrinsic determinants (e.g., antioxidant availability, damage repair, EGF signaling) keep the degree of lysosomal destabilization below cell death-inducing thresholds.

  13. Differential stability of photosynthetic membranes and fatty acid composition at elevated temperature in Symbiodinium

    NASA Astrophysics Data System (ADS)

    Díaz-Almeyda, E.; Thomé, P. E.; El Hafidi, M.; Iglesias-Prieto, R.

    2011-03-01

    Coral reefs are threatened by increasing surface seawater temperatures resulting from climate change. Reef-building corals symbiotic with dinoflagellates in the genus Symbiodinium experience dramatic reductions in algal densities when exposed to temperatures above the long-term local summer average, leading to a phenomenon called coral bleaching. Although the temperature-dependent loss in photosynthetic function of the algal symbionts has been widely recognized as one of the early events leading to coral bleaching, there is considerable debate regarding the actual damage site. We have tested the relative thermal stability and composition of membranes in Symbiodinium exposed to high temperature. Our results show that melting curves of photosynthetic membranes from different symbiotic dinoflagellates substantiate a species-specific sensitivity to high temperature, while variations in fatty acid composition under high temperature rather suggest a complex process in which various modifications in lipid composition may be involved. Our results do not support the role of unsaturation of fatty acids of the thylakoid membrane as being mechanistically involved in bleaching nor as being a dependable tool for the diagnosis of thermal susceptibility of symbiotic reef corals.

  14. Membrane-anchoring stabilizes and favors secretion of New Delhi Metallo-β-lactamase

    PubMed Central

    González, Lisandro J.; Bahr, Guillermo; Nakashige, Toshiki G.; Nolan, Elizabeth M.; Bonomo, Robert A.; Vila, Alejandro J.

    2016-01-01

    Carbapenems, “last resort” β-lactam antibiotics, are inactivated by zinc-dependent metallo-β-lactamases (MBLs). The host innate immune response withholds nutrient metal ions from microbial pathogens by releasing metal-chelating proteins such as calprotectin. We show that metal sequestration is detrimental for the accumulation of MBLs in the bacterial periplasm, since these enzymes are readily degraded in their non-metallated form. However, the New Delhi Metallo-β-lactamase (NDM-1) is able to persist under conditions of metal depletion. NDM-1 is a lipidated protein anchored to the outer membrane of Gram-negative bacteria. Membrane-anchoring contributes to the unusual stability of NDM-1 and favors secretion of this enzyme in outer membrane vesicles (OMVs). OMVs containing NDM-1 can protect nearby populations of bacteria from otherwise lethal antibiotic levels, and OMVs from clinical pathogens expressing NDM-1 can carry this MBL and the blaNDM gene. We show that protein export into OMVs can be targeted, providing possibilities of new antibacterial therapeutic strategies. PMID:27182662

  15. A fluorescence resonance energy transfer-based approach for investigating late endosome-lysosome retrograde fusion events.

    PubMed

    Kaufmann, A M; Goldman, S D B; Krise, J P

    2009-03-01

    Traditionally, lysosomes have been considered to be a terminal endocytic compartment. Recent studies suggest that lysosomes are quite dynamic, being able to fuse with other late endocytic compartments as well as with the plasma membrane. Here we describe a quantitative fluorescence energy transfer (FRET)-based method for assessing rates of retrograde fusion between terminal lysosomes and late endosomes in living cells. Late endosomes were specifically labeled with 800-nm latex beads that were conjugated with streptavidin and Alexa Fluor 555 (FRET donor). Terminal lysosomes were specifically labeled with 10,000-MW dextran polymers conjugated with biotin and Alexa Fluor 647 (FRET acceptor). Following late endosome-lysosome fusion, the strong binding affinity between streptavidin and biotin brought the donor and acceptor fluorophore molecules into close proximity, thereby facilitating the appearance of a FRET emission signal. Because apparent size restrictions in the endocytic pathway do not permit endocytosed latex beads from reaching terminal lysosomes in an anterograde fashion, the appearance of the FRET signal is consistent with retrograde transport of lysosomal cargo back to late endosomes. We assessed the efficiency of this transport step in fibroblasts affected by different lysosome storage disorders-Niemann-Pick type C, mucolipidosis type IV, and Sandhoff's disease, all of which have a similar lysosomal lipid accumulation phenotype. We report here, for the first time, that these disorders can be distinguished by their rate of transfer of lysosome cargos to late endosomes, and we discuss the implications of these findings for developing new therapeutic strategies.

  16. Association of myosin I alpha with endosomes and lysosomes in mammalian cells.

    PubMed

    Raposo, G; Cordonnier, M N; Tenza, D; Menichi, B; Dürrbach, A; Louvard, D; Coudrier, E

    1999-05-01

    Myosin Is, which constitute a ubiquitous monomeric subclass of myosins with actin-based motor properties, are associated with plasma membrane and intracellular vesicles. Myosin Is have been proposed as key players for membrane trafficking in endocytosis or exocytosis. In the present paper we provide biochemical and immunoelectron microscopic evidence indicating that a pool of myosin I alpha (MMIalpha) is associated with endosomes and lysosomes. We show that the overproduction of MMIalpha or the production of nonfunctional truncated MMIalpha affects the distribution of the endocytic compartments. We also show that truncated brush border myosin I proteins, myosin Is that share 78% homology with MMIalpha, promote the dissociation of MMIalpha from vesicular membranes derived from endocytic compartments. The analysis at the ultrastructural level of cells producing these brush border myosin I truncated proteins shows that the delivery of the fluid phase markers from endosomes to lysosomes is impaired. MMIalpha might therefore be involved in membrane trafficking occurring between endosomes and lysosomes.

  17. Association of Myosin I Alpha with Endosomes and Lysosomes in Mammalian Cells

    PubMed Central

    Raposo, Graça; Cordonnier, Marie-Neige; Tenza, Danièle; Menichi, Bernadette; Dürrbach, Antoine; Louvard, Daniel; Coudrier, Evelyne

    1999-01-01

    Myosin Is, which constitute a ubiquitous monomeric subclass of myosins with actin-based motor properties, are associated with plasma membrane and intracellular vesicles. Myosin Is have been proposed as key players for membrane trafficking in endocytosis or exocytosis. In the present paper we provide biochemical and immunoelectron microscopic evidence indicating that a pool of myosin I alpha (MMIα) is associated with endosomes and lysosomes. We show that the overproduction of MMIα or the production of nonfunctional truncated MMIα affects the distribution of the endocytic compartments. We also show that truncated brush border myosin I proteins, myosin Is that share 78% homology with MMIα, promote the dissociation of MMIα from vesicular membranes derived from endocytic compartments. The analysis at the ultrastructural level of cells producing these brush border myosin I truncated proteins shows that the delivery of the fluid phase markers from endosomes to lysosomes is impaired. MMIα might therefore be involved in membrane trafficking occurring between endosomes and lysosomes. PMID:10233157

  18. Stabilizing effects of coenzyme Q10 on potassium ion release, membrane potential and fluidity of rabbit red blood cells.

    PubMed

    Shinozawa, S; Araki, Y; Oda, T

    1980-09-01

    The effects of coenzyme Q10 (Co Q10) on potassium ion release, membrane potential and fluidity of rabbit red blood cells were studied. Co Q10 inhibited the increased potassium ion release induced by cetylamine or lysolecithin from the cells. Co Q10 slightly decreased the membrane potential monitored by changes in fluorescence intensity of cyanine dye, 3,3'-dipropyl-2,2'-thiodicarbocyanine iodide [diS-C3-(5)], and also slightly decreased the membrane fluidity measured by using 1,6-diphenyl-1,3,5-hexatriene (DPH). These effects of Co Q10 on the membrane are considered to be due to its membrane stabilizing activity by interaction with lipid bilayers of the membrane.

  19. The Effect of Covalently-Attached ATRP-Synthesized Polymers on Membrane Stability and Cytoprotection in Human Erythrocytes

    PubMed Central

    Clafshenkel, William P.; Murata, Hironobu; Andersen, Jill; Creeger, Yehuda; Russell, Alan J.

    2016-01-01

    Erythrocytes have been described as advantageous drug delivery vehicles. In order to ensure an adequate circulation half-life, erythrocytes may benefit from protective enhancements that maintain membrane integrity and neutralize oxidative damage of membrane proteins that otherwise facilitate their premature clearance from circulation. Surface modification of erythrocytes using rationally designed polymers, synthesized via atom-transfer radical polymerization (ATRP), may further expand the field of membrane-engineered red blood cells. This study describes the fate of ATRP-synthesized polymers that were covalently attached to human erythrocytes as well as the effect of membrane engineering on cell stability under physiological and oxidative conditions in vitro. The biocompatible, membrane-reactive polymers were homogenously retained on the periphery of modified erythrocytes for at least 24 hours. Membrane engineering stabilized the erythrocyte membrane and effectively neutralized oxidative species, even in the absence of free-radical scavenger-containing polymers. The targeted functionalization of Band 3 protein by NHS-pDMAA-Cy3 polymers stabilized its monomeric form preventing aggregation in the presence of the crosslinking reagent, bis(sulfosuccinimidyl)suberate (BS3). A free radical scavenging polymer, NHS-pDMAA-TEMPO˙, provided additional protection of surface modified erythrocytes in an in vitro model of oxidative stress. Preserving or augmenting cytoprotective mechanisms that extend circulation half-life is an important consideration for the use of red blood cells for drug delivery in various pathologies, as they are likely to encounter areas of imbalanced oxidative stress as they circuit the vascular system. PMID:27331401

  20. Proteomic analysis of enriched lysosomes at early phase of camptothecin-induced apoptosis in human U-937 cells

    PubMed Central

    Parent, Nicolas; Winstall, Eric; Beauchemin, Myriam; Paquet, Claudie; Poirier, Guy G.; Bertrand, Richard

    2013-01-01

    A lysosomal pathway, characterized by partial rupture or labilization of lysosomal membranes and cathepsin activation, is evoked during camptothecin-induced apoptosis in human cancer cells, including human histiocytic lymphoma U-937 cells. These lysosomal events begin rapidly and simultaneously with mitochondrial permeabilization and caspase activation within 3 h after drug treatment. In this study, comparative and quantitative proteome analyses were performed to identify early changes in lysosomal protein expression/localization from U-937 cells undergoing apoptosis. In 2 independent experiments, among a total of more than 538 proteins putatively identified and quantitated by iTRAQ isobaric labeling and LC-ESI-MS/MS, 18 proteins were found to be upregulated and 9 downregulated in lysosomes purified from early apoptotic compared to control cells. Protein expression was validated by Western blotting on enriched lysosome fractions, and protein localization confirmed by fluorescence confocal microscopy of representative protein candidates, whose functions are associated with lysosomal membrane fluidity and dynamics. These include sterol-4-alpha-carboxylate 3-dehydrogenase (NSDHL), prosaposin (PSAP) and protein kinase C delta (PKC-δ). This comparative proteome analysis provides the basis for novel hypothesis and rationale functional experimentation, where the 3 validated candidate proteins are associated with lysosomal membrane fluidity and dynamics, particularly cholesterol, sphingolipid and glycosphingolipid metabolism. PMID:19393779

  1. Model Systems of Precursor Cellular Membranes: Long-Chain Alcohols Stabilize Spontaneously Formed Oleic Acid Vesicles

    PubMed Central

    Rendón, Adela; Carton, David Gil; Sot, Jesús; García-Pacios, Marcos; Montes, Ruth; Valle, Mikel; Arrondo, José-Luis R.; Goñi, Felix M.; Ruiz-Mirazo, Kepa

    2012-01-01

    Oleic acid vesicles have been used as model systems to study the properties of membranes that could be the evolutionary precursors of more complex, stable, and impermeable phospholipid biomembranes. Pure fatty acid vesicles in general show high sensitivity to ionic strength and pH variation, but there is growing evidence that this lack of stability can be counterbalanced through mixtures with other amphiphilic or surfactant compounds. Here, we present a systematic experimental analysis of the oleic acid system and explore the spontaneous formation of vesicles under different conditions, as well as the effects that alcohols and alkanes may have in the process. Our results support the hypothesis that alcohols (in particular 10- to 14-C-atom alcohols) contribute to the stability of oleic acid vesicles under a wider range of experimental conditions. Moreover, studies of mixed oleic-acid-alkane and oleic-acid-alcohol systems using infrared spectroscopy and Langmuir trough measurements indicate that precisely those alcohols that increased vesicle stability also decreased the mobility of oleic acid polar headgroups, as well as the area/molecule of lipid. PMID:22339864

  2. Alkaline stability of quaternary ammonium cations for alkaline fuel cell membranes and ionic liquids.

    PubMed

    Marino, M G; Kreuer, K D

    2015-02-01

    The alkaline stability of 26 different quaternary ammonium groups (QA) is investigated for temperatures up to 160 °C and NaOH concentrations up to 10 mol L(-1) with the aim to provide a basis for the selection of functional groups for hydroxide exchange membranes in alkaline fuel cells and of ionic-liquid cations stable in basic conditions. Most QAs exhibit unexpectedly high alkaline stability with the exception of aromatic cations. β-Protons are found to be far less susceptible to nucleophilic attack than previously suggested, whereas the presence of benzyl groups, nearby hetero-atoms, or other electron-withdrawing species promote degradation reactions significantly. Cyclic QAs proved to be exceptionally stable, with the piperidine-based 6-azonia-spiro[5.5]undecane featuring the highest half-life at the chosen conditions. Absolute and relative stabilities presented herein stand in contrast to literature data, the differences being ascribed to solvent effects on degradation.

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

    PubMed Central

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

    1998-01-01

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

  4. Lysosomal Membrane Glycoproteins: Properties of LAMP-1 (Lysosome Associated Membrane Protein) and LAMP-2

    DTIC Science & Technology

    1985-01-01

    Hank’s Buffered Salt Solution " NP- 10, Nonidet - P40 - .A, Bovine Serum Albumin, FBS, Fetal Bovine Serum. *Present address: University of California... Nonidet -P-"- ( ’P- ,9,Particle Dta Inc., Elmhurst, IL), 5 mM EDTA, I mM phenyl7.eth/ls,,!f, nv fluori , . .nd 0.15 711 ,NaCl (Hughes and August, 1982...for 3 -ii n the cells were centrifuged at 100.000 x n for . e supernatants were dialyzed exhaustively auainst IK’is putter i. , 5 percent Nonidet P-40

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

    PubMed

    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.

  6. [Clinico-functional efficacy of medicinal and photon stabilization of cell membrane in patients with angina pectoris].

    PubMed

    Vasil'ev, A P; Senatorov, Iu N; Strel'tsova, N N; Gorbunova, T Iu

    2003-01-01

    Modification of erythrocytic membrane and the trend in clinicofunctional indices were studied in 90 patients with angina of effort (FC I-IV) in the course of treatment with a combination of membranoprotective drugs (group 1), magneto-laser radiation (group 2) and imitation of laser radiation (group 3). In patients of groups 1 and 2 the treatment resulted in stabilization of cell membrane accompanied with a hypotensive effect and increased exercise tolerance due to more effective cardiac performance.

  7. Membrane protein stability can be compromised by detergent interactions with the extramembranous soluble domains

    PubMed Central

    Yang, Zhengrong; Wang, Chi; Zhou, Qingxian; An, Jianli; Hildebrandt, Ellen; Aleksandrov, Luba A; Kappes, John C; DeLucas, Lawrence J; Riordan, John R; Urbatsch, Ina L; Hunt, John F; Brouillette, Christie G

    2014-01-01

    Detergent interaction with extramembranous soluble domains (ESDs) is not commonly considered an important determinant of integral membrane protein (IMP) behavior during purification and crystallization, even though ESDs contribute to the stability of many IMPs. Here we demonstrate that some generally nondenaturing detergents critically destabilize a model ESD, the first nucleotide-binding domain (NBD1) from the human cystic fibrosis transmembrane conductance regulator (CFTR), a model IMP. Notably, the detergents show equivalent trends in their influence on the stability of isolated NBD1 and full-length CFTR. We used differential scanning calorimetry (DSC) and circular dichroism (CD) spectroscopy to monitor changes in NBD1 stability and secondary structure, respectively, during titration with a series of detergents. Their effective harshness in these assays mirrors that widely accepted for their interaction with IMPs, i.e., anionic > zwitterionic > nonionic. It is noteworthy that including lipids or nonionic detergents is shown to mitigate detergent harshness, as will limiting contact time. We infer three thermodynamic mechanisms from the observed thermal destabilization by monomer or micelle: (i) binding to the unfolded state with no change in the native structure (all detergent classes); (ii) native state binding that alters thermodynamic properties and perhaps conformation (nonionic detergents); and (iii) detergent binding that directly leads to denaturation of the native state (anionic and zwitterionic). These results demonstrate that the accepted model for the harshness of detergents applies to their interaction with an ESD. It is concluded that destabilization of extramembranous soluble domains by specific detergents will influence the stability of some IMPs during purification. PMID:24652590

  8. Stability of Mitochondrial Membrane Proteins in Terrestrial Vertebrates Predicts Aerobic Capacity and Longevity

    PubMed Central

    Kitazoe, Yasuhiro; Kishino, Hirohisa; Hasegawa, Masami; Matsui, Atsushi; Lane, Nick; Tanaka, Masashi

    2011-01-01

    The cellular energy produced by mitochondria is a fundamental currency of life. However, the extent to which mitochondrial (mt) performance (power and endurance) is adapted to habitats and life strategies of vertebrates is not well understood. A global analysis of mt genomes revealed that hydrophobicity (HYD) of mt membrane proteins (MMPs) is much lower in terrestrial vertebrates than in fishes and shows a strong negative correlation with serine/threonine composition (STC). Here, we present evidence that this systematic feature of MMPs was crucial for the evolution of large terrestrial vertebrates with high aerobic capacity. An Arrhenius-type equation gave positive correlations between STC and maximum life span (MLS) in terrestrial vertebrates (with a few exceptions relating to the lifestyle of small animals with a high resting metabolic rate [RMR]) and negative correlations in secondary marine vertebrates, such as cetaceans and alligators (which returned from land to water, utilizing buoyancy with increased body size). In particular, marked STC increases in primates (especially hominoids) among placentals were associated with very high MLS values. We connected these STC increases in MMPs with greater stability of respiratory complexes by estimating the degradation of the Arrhenius plot given by accelerating mtRMR up to mt maximum metabolic rate. Both mtRMR and HYD in terrestrial vertebrates decreased with increasing body mass. Decreases in mtRMR raise MMP stability when high mobility is not required, whereas decreased HYD may weaken this stability under the hydrophobic environment of lipid bilayer. High maximal metabolic rates (5–10 RMR), which we postulate require high MMP mobility, presumably render MMPs more unstable. A marked rise in STC may therefore be essential to stabilize MMPs, perhaps as dynamic supercomplexes, via hydrogen bonds associated with serine/threonine motifs. PMID:21824868

  9. Membrane protein stability can be compromised by detergent interactions with the extramembranous soluble domains.

    PubMed

    Yang, Zhengrong; Wang, Chi; Zhou, Qingxian; An, Jianli; Hildebrandt, Ellen; Aleksandrov, Luba A; Kappes, John C; DeLucas, Lawrence J; Riordan, John R; Urbatsch, Ina L; Hunt, John F; Brouillette, Christie G

    2014-06-01

    Detergent interaction with extramembranous soluble domains (ESDs) is not commonly considered an important determinant of integral membrane protein (IMP) behavior during purification and crystallization, even though ESDs contribute to the stability of many IMPs. Here we demonstrate that some generally nondenaturing detergents critically destabilize a model ESD, the first nucleotide-binding domain (NBD1) from the human cystic fibrosis transmembrane conductance regulator (CFTR), a model IMP. Notably, the detergents show equivalent trends in their influence on the stability of isolated NBD1 and full-length CFTR. We used differential scanning calorimetry (DSC) and circular dichroism (CD) spectroscopy to monitor changes in NBD1 stability and secondary structure, respectively, during titration with a series of detergents. Their effective harshness in these assays mirrors that widely accepted for their interaction with IMPs, i.e., anionic > zwitterionic > nonionic. It is noteworthy that including lipids or nonionic detergents is shown to mitigate detergent harshness, as will limiting contact time. We infer three thermodynamic mechanisms from the observed thermal destabilization by monomer or micelle: (i) binding to the unfolded state with no change in the native structure (all detergent classes); (ii) native state binding that alters thermodynamic properties and perhaps conformation (nonionic detergents); and (iii) detergent binding that directly leads to denaturation of the native state (anionic and zwitterionic). These results demonstrate that the accepted model for the harshness of detergents applies to their interaction with an ESD. It is concluded that destabilization of extramembranous soluble domains by specific detergents will influence the stability of some IMPs during purification.

  10. Pluronic F127 as a cell encapsulation material: utilization of membrane-stabilizing agents.

    PubMed

    Khattak, Sarwat F; Bhatia, Surita R; Roberts, Susan C

    2005-01-01

    Thermoreversible gelation of the copolymer Pluronic F127 (generic name, poloxamer 407) in water makes it a unique candidate for cell encapsulation applications, either alone or to promote cell seeding and attachment in tissue scaffolds. At concentrations of 15-20% (w/w), aqueous Pluronic F127 (F127) solutions gel at physiological temperatures. The effect of F127 on viability and proliferation of human liver carcinoma cells (HepG2) was determined for both liquid and gel formulations. Cell concentration and viability over a 5-day period were measured by the trypan blue assay via hemocytometry and results were confirmed in both the MTT and LDH assays. With 0.1-5% (w/w) F127 (liquid), cells proliferated and maintained high viability over 5 days. However, at 10% (w/w) F127 (liquid), there was a significant decrease in cell viability and no cell proliferation was evident. HepG2 cell encapsulation in F127 concentrations ranging from 15 to 20% (w/w) (gel) resulted in complete cell death by 5 days. This was also true for the HMEC-1 (endothelial) and L6 (muscle) cell lines evaluated. Cell-seeding density did not affect cell survival or proliferation. Membrane-stabilizing agents (hydrocortisone, glucose, and glycerol) were added to the F127 gel formulations to improve cell viability. The steroid hydrocortisone demonstrated the most significant improvement in viability, from <2% (in F127 alone) to >70% (with 60 nM hydrocortisone added). These results suggest that F127 formulations supplemented with membrane-stabilizing agents can serve as viable cell encapsulation materials. In addition, hydrocortisone may be generally useful in the promotion of cell viability for a wide range of encapsulation materials.

  11. Enhanced stability of Zr-doped Ba(CeTb)O(3-δ)-Ni cermet membrane for hydrogen separation.

    PubMed

    Wei, Yanying; Xue, Jian; Fang, Wei; Chen, Yan; Wang, Haihui; Caro, Jürgen

    2015-07-25

    A mixed protonic and electronic conductor material BaCe(0.85)Tb(0.05)Zr(0.1)O(3-δ) (BCTZ) is prepared and a Ni-BCTZ cermet membrane is synthesized for hydrogen separation. Stable hydrogen permeation fluxes can be obtained for over 100 h through the Ni-BCTZ membrane in both dry and humid conditions, which exhibits an excellent stability compared with Ni-BaCe(0.95)Tb(0.05)O(3-δ) membrane due to the Zr doping.

  12. Stabilized liquid membrane device (SLMD) for the passive, integrative sampling of labile metals in water

    USGS Publications Warehouse

    Brumbaugh, W.G.; Petty, J.D.; Huckins, J.N.; Manahan, S.E.

    2002-01-01

    A stabilized liquid membrane device (SLMD) is described for potential use as an in situ, passive, integrative sampler for cadmium (Cd), cobalt (Co), copper (Cu), nickel (Ni), lead (Pb), and zinc (Zn) in natural waters. The SLMD (patent pending) consists of a 2.5-cm-wide by 15-cm-long strip of low-density polyethylene (LDPE) layflat tubing containing 1 mL of an equal mixture (v/v) of oleic acid (cis-9-octadecenoic acid) and EMO-8Q (7-[4-ethyl-1-methyloctyl]-8-quinolinol). The reagent mixture continuously diffuses to the exterior surface of the LDPE membrane, and provides for sequestration of several divalent metals for up to several weeks. Depending on sampler configuration, concentration factors of several thousand can be realized for these metal ions after just a few days. In addition to in situ deployment, the SLMD may be useful for laboratory determination of labile metal species in grab samples. Methods for minimizing the effects of water flow on the sampling rate are currently under investigation.

  13. Enhanced stability of multilayer graphene-supported catalysts for polymer electrolyte membrane fuel cell cathodes

    NASA Astrophysics Data System (ADS)

    Marinkas, A.; Hempelmann, R.; Heinzel, A.; Peinecke, V.; Radev, I.; Natter, H.

    2015-11-01

    One of the biggest challenges in the field of polymer electrolyte membrane fuel cells (PEMFC) is to enhance the lifetime and the long-term stability of PEMFC electrodes, especially of cathodes, furthermore, to reduce their platinum loading, which could lead to a cost reduction for efficient PEMFCs. These demands could be achieved with a new catalyst support architecture consisting of a composite of carbon structures with significant different morphologies. A highly porous cathode catalyst support layer is prepared by addition of various carbon types (carbon black particles, multi-walled carbon nanotubes (MWCNT)) to multilayer graphene (MLG). The reported optimized cathodes shows extremely high durability and similar performance to commercial standard cathodes but with 89% lower Pt loading. The accelerated aging protocol (AAP) on the membrane electrode assemblies (MEA) shows that the presence of MLG increases drastically the durability and the Pt-extended electrochemical surface area (ECSA). In fact, after the AAP slightly enhanced performance can be observed for the MLG-containing cathodes instead of a performance loss, which is typical for the commercial carbon-based cathodes. Furthermore, the presence of MLG drastically decreases the ECSA loss rate. The MLG-containing cathodes show up to 6.8 times higher mass-normalized Pt-extended ECSA compared to the commercial standard systems.

  14. Static Magnetic Field Attenuates Lipopolysaccharide-Induced Inflammation in Pulp Cells by Affecting Cell Membrane Stability

    PubMed Central

    Tsao, Jeng-Ting; Lee, Lin-Wen; Lin, Che-Tong

    2015-01-01

    One of the causes of dental pulpitis is lipopolysaccharide- (LPS-) induced inflammatory response. Following pulp tissue inflammation, odontoblasts, dental pulp cells (DPCs), and dental pulp stem cells (DPSCs) will activate and repair damaged tissue to maintain homeostasis. However, when LPS infection is too serious, dental repair is impossible and disease may progress to irreversible pulpitis. Therefore, the aim of this study was to examine whether static magnetic field (SMF) can attenuate inflammatory response of dental pulp cells challenged with LPS. In methodology, dental pulp cells were isolated from extracted teeth. The population of DPSCs in the cultured DPCs was identified by phenotypes and multilineage differentiation. The effects of 0.4 T SMF on DPCs were observed through MTT assay and fluorescent anisotropy assay. Our results showed that the SMF exposure had no effect on surface markers or multilineage differentiation capability. However, SMF exposure increases cell viability by 15%. In addition, SMF increased cell membrane rigidity which is directly related to higher fluorescent anisotropy. In the LPS-challenged condition, DPCs treated with SMF demonstrated a higher tolerance to LPS-induced inflammatory response when compared to untreated controls. According to these results, we suggest that 0.4 T SMF attenuates LPS-induced inflammatory response to DPCs by changing cell membrane stability. PMID:25884030

  15. Endosome-lysosomes, ubiquitin and neurodegeneration.

    PubMed

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

    1996-01-01

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

  16. The role of intraorganellar Ca(2+) in late endosome-lysosome heterotypic fusion and in the reformation of lysosomes from hybrid organelles.

    PubMed

    Pryor, P R; Mullock, B M; Bright, N A; Gray, S R; Luzio, J P

    2000-05-29

    We have investigated the requirement for Ca(2+) in the fusion and content mixing of rat hepatocyte late endosomes and lysosomes in a cell-free system. Fusion to form hybrid organelles was inhibited by 1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA), but not by EGTA, and this inhibition was reversed by adding additional Ca(2+). Fusion was also inhibited by methyl ester of EGTA (EGTA-AM), a membrane permeable, hydrolyzable ester of EGTA, and pretreatment of organelles with EGTA-AM showed that the chelation of lumenal Ca(2+) reduced the amount of fusion. The requirement for Ca(2+) for fusion was a later event than the requirement for a rab protein since the system became resistant to inhibition by GDP dissociation inhibitor at earlier times than it became resistant to BAPTA. We have developed a cell-free assay to study the reformation of lysosomes from late endosome-lysosome hybrid organelles that were isolated from the rat liver. The recovery of electron dense lysosomes was shown to require ATP and was inhibited by bafilomycin and EGTA-AM. The data support a model in which endocytosed Ca(2+) plays a role in the fusion of late endosomes and lysosomes, the reformation of lysosomes, and the dynamic equilibrium of organelles in the late endocytic pathway.

  17. Glucagon-like Peptide-1 Protects Pancreatic Beta-cells from Death by Increasing Autophagic Flux and Restoring Lysosomal Function.

    PubMed

    Zummo, Francesco P; Cullen, Kirsty S; Honkanen-Scott, Minna; Shaw, James Am; Lovat, Penny E; Arden, Catherine

    2017-02-23

    Studies in animal models of type 2 diabetes have shown that glucagon-like peptide-1 (GLP-1) receptor agonists prevent β-cell loss. Whether GLP-1 mediates β-cell survival via the key lysosomal-mediated process of autophagy is unknown.Here we report that treatment of INS-1E β-cells and primary islets with glucolipotoxicity (0.5mmol/l palmitate, 25mmol/l glucose) increases LC3 II, a marker of autophagy. Further analysis indicates a blockage in autophagic flux associated with lysosomal dysfunction. Accumulation of defective lysosomes leads to lysosomal membrane permeabilisation (LMP) and release of Cathepsin D, which contributes to cell death. Our data further demonstrated defects in autophagic flux and lysosomal staining in human samples of type 2 diabetes. Co-treatment with the GLP-1 receptor agonist exendin-4 reversed the lysosomal dysfunction, relieving the impairment in autophagic flux and further stimulated autophagy. siRNA knockdown showed the restoration of autophagic flux is also essential for the protective effects of exendin-4.Collectively, our data highlights lysosomal dysfunction as a critical mediator of β-cell loss and shows that exendin-4 improves cell survival via restoration of lysosomal function and autophagic flux. Modulation of autophagy / lysosomal homeostasis may thus define a novel therapeutic strategy for type 2 diabetes, with the GLP-1 signalling pathway as a potential focus.

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

    PubMed

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

    2017-01-16

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

  19. Toxoplasma gondii sequesters lysosomes from mammalian hosts in the vacuolar space.

    PubMed

    Coppens, Isabelle; Dunn, Joe Dan; Romano, Julia D; Pypaert, Marc; Zhang, Hui; Boothroyd, John C; Joiner, Keith A

    2006-04-21

    The intracellular compartment harboring Toxoplasma gondii satisfies the parasite's nutritional needs for rapid growth in mammalian cells. We demonstrate that the parasitophorous vacuole (PV) of T. gondii accumulates material coming from the host mammalian cell via the exploitation of the host endo-lysosomal system. The parasite actively recruits host microtubules, resulting in selective attraction of endo-lysosomes to the PV. Microtubule-based invaginations of the PV membrane serve as conduits for the delivery of host endo-lysosomes within the PV. These tubular conduits are decorated by a parasite coat, including the tubulogenic protein GRA7, which acts like a garrote that sequesters host endocytic organelles in the vacuolar space. These data define an unanticipated process allowing the parasite intimate and concentrated access to a diverse range of low molecular weight components produced by the endo-lysosomal system. More generally, they identify a unique mechanism for unidirectional transport and sequestration of host organelles.

  20. GNeosomes: Highly Lysosomotropic Nanoassemblies for Lysosomal Delivery.

    PubMed

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

    2015-01-01

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

  1. Preparation of Cu2O nanowire-blended polysulfone ultrafiltration membrane with improved stability and antimicrobial activity

    NASA Astrophysics Data System (ADS)

    Xu, Zehai; Ye, Shuaiju; Fan, Zheng; Ren, Fanghua; Gao, Congjie; Li, Qingbiao; Li, Guoqing; Zhang, Guoliang

    2015-10-01

    Polysulfone (PSF) membranes have been widely applied in water and wastewater treatment, food-processing and biomedical fields. In this study, we report the preparation of modified PSF membranes by blending PSF with Cu2O nanowires (NWs) to improve their stability and antifouling activity. Synthesis of novel Cu2O NWs/PSF-blended ultrafiltration membrane was achieved via phase inversion method by dispersing one-dimensional Cu2O nanowires in PSF casting solutions. Various techniques such as XRD, SEM, TEM, and EDS were applied to characterize and investigate the properties of nanowires and membranes. The introduced Cu2O nanowires can firmly be restricted into micropores of PSF membranes, and therefore, they can effectively prevent the serious leaking problem of inorganic substances in separation process. The blended PSF membranes also provided enhanced antimicrobial activity and superior permeation property compared to pure PSF membrane. The overall work can not only provide a new way for preparation of novel blended membranes with multidimensional nanomaterials, but can also be beneficial to solve the annoying problem of biofouling.

  2. Expression and purification of the recombinant membrane protein YidC: a case study for increased stability and solubility.

    PubMed

    Martinez Molina, Daniel; Lundbäck, Anna-Karin; Niegowski, Damian; Eshaghi, Said

    2008-11-01

    YidC is an inner membrane protein from Escherichia coli and is an essential component in insertion, translocation and assembly of membrane proteins in the membranes. Previous purification attempts resulted in heavy aggregates and precipitated protein at later stages of purification. Here we present a rapid and straightforward stability screening strategy based on gel filtration chromatography, which requires as little as 10 microg of protein and takes less than 15 min to perform. With this technique, we could rapidly screen several buffers in order to identify an optimum condition that stabilizes purified YidC. After optimization we could obtain several milligrams of purified YidC that could be easily prepared at high concentrations and that was stable for weeks at +4 degrees C. The isolated protein is thus well suited for structural studies.

  3. Positive Lysosomal Modulation As a Unique Strategy to Treat Age-Related Protein Accumulation Diseases

    PubMed Central

    Wisniewski, Meagan L.; Butler, David

    2012-01-01

    Abstract Lysosomes are involved in degrading and recycling cellular ingredients, and their disruption with age may contribute to amyloidogenesis, paired helical filaments (PHFs), and α-synuclein and mutant huntingtin aggregation. Lysosomal cathepsins are upregulated by accumulating proteins and more so by the modulator Z-Phe-Ala-diazomethylketone (PADK). Such positive modulators of the lysosomal system have been studied in the well-characterized hippocampal slice model of protein accumulation that exhibits the pathogenic cascade of tau aggregation, tubulin breakdown, microtubule destabilization, transport failure, and synaptic decline. Active cathepsins were upregulated by PADK; Rab proteins were modified as well, indicating enhanced trafficking, whereas lysosome-associated membrane protein and proteasome markers were unchanged. Lysosomal modulation reduced the pre-existing PHF deposits, restored tubulin structure and transport, and recovered synaptic components. Further proof-of-principle studies used Alzheimer disease mouse models. It was recently reported that systemic PADK administration caused dramatic increases in cathepsin B protein and activity levels, whereas neprilysin, insulin-degrading enzyme, α-secretase, and β-secretase were unaffected by PADK. In the transgenic models, PADK treatment resulted in clearance of intracellular amyloid beta (Aβ) peptide and concomitant reduction of extracellular deposits. Production of the less pathogenic Aβ1–38 peptide corresponded with decreased levels of Aβ1–42, supporting the lysosome's antiamyloidogenic role through intracellular truncation. Amelioration of synaptic and behavioral deficits also indicates a neuroprotective function of the lysosomal system, identifying lysosomal modulation as an avenue for disease-modifying therapies. From the in vitro and in vivo findings, unique lysosomal modulators represent a minimally invasive, pharmacologically controlled strategy against protein accumulation disorders

  4. Bivariate and multivariate analyses of the correlations between stability of the erythrocyte membrane, serum lipids and hematological variables.

    PubMed

    Bernardino Neto, M; de Avelar, E B; Arantes, T S; Jordão, I A; da Costa Huss, J C; de Souza, T M T; de Souza Penha, V A; da Silva, S C; de Souza, P C A; Tavares, M; Penha-Silva, N

    2013-01-01

    The observation that the fluidity must remain within a critical interval, outside which the stability and functionality of the cell tends to decrease, shows that stability, fluidity and function are related and that the measure of erythrocyte stability allows inferences about the fluidity or functionality of these cells. This study determined the biochemical and hematological variables that are directly or indirectly related to erythrocyte stability in a population of 71 volunteers. Data were evaluated by bivariate and multivariate analysis. The erythrocyte stability showed a greater association with hematological variables than the biochemical variables. The RDW stands out for its strong correlation with the stability of erythrocyte membrane, without being heavily influenced by other factors. Regarding the biochemical variables, the erythrocyte stability was more sensitive to LDL-C. Erythrocyte stability was significantly associated with RDW and LDL-C. Thus, the level of LDL-C is a consistent link between stability and functionality, suggesting that a measure of stability could be more one indirect parameter for assessing the risk of degenerative processes associated with high levels of LDL-C.

  5. Study of catalysts with high stability for proton exchange membrane fuel cells

    NASA Astrophysics Data System (ADS)

    Yang, Fan

    The innovation and investigation of catalysts in proton exchange membrane fuel cells are included in this thesis. In the first part of this work, stability of the catalyst support of PEMFC catalyst is investigated. Nanoscale platinum particles were loaded on two different kinds of carbon supports, nano graphene sheets and functionalized carbon black/graphene hybrid were developed by the liquid phase reaction. The crystal structure of two kinds of catalysts was characterized by X-ray diffractometer (XRD). The morphology and particle size were characterized by scanning electron microscope (SEM) and transmission electron microscope (TEM). Pt loading was measured by thermal gravimetric analysis (TGA). The Brunauer, Emmett and Teller (BET) method was applied to test the surface area of the catalysts. The electrochemical surface area (ECSA) and mass activity during oxygen reduction reaction (ORR) process for two kinds of catalyst were tested by cyclic voltammetry method under different conditions. The stability of the catalysts were tested by accelerated durability test (ADT). The results show that although the mass activity of Pt/graphene is much lower, the stability of it is much better than that of the commercial catalyst. After adding functionalized carbon black (FCB) as spacer, the stability of the catalyst is preserved and at the meantime, the mass activity becomes higher than 20% Pt/XC72 catalyst. The lower mass activity of both catalysts are due to the limitation of the electrolyte diffusion into the carbon support because of the aggregation nature of graphene nano-sheets. After introducing functional carbon black as spacer, the mass activity and ECSA increased dramatically which proved that FCB can be applied to prevent the restacking of graphene and hence solved the diffusion problem. In the meantime, the durability was still keeping the same as Pt/graphene catalyst. In the second part of the work, the restacking problem was solved by introducing FCB as spacers

  6. Osmotic versus conventional membrane bioreactors integrated with reverse osmosis for water reuse: Biological stability, membrane fouling, and contaminant removal.

    PubMed

    Luo, Wenhai; Phan, Hop V; Xie, Ming; Hai, Faisal I; Price, William E; Elimelech, Menachem; Nghiem, Long D

    2017-02-01

    This study systematically compares the performance of osmotic membrane bioreactor - reverse osmosis (OMBR-RO) and conventional membrane bioreactor - reverse osmosis (MBR-RO) for advanced wastewater treatment and water reuse. Both systems achieved effective removal of bulk organic matter and nutrients, and almost complete removal of all 31 trace organic contaminants investigated. They both could produce high quality water suitable for recycling applications. During OMBR-RO operation, salinity build-up in the bioreactor reduced the water flux and negatively impacted the system biological treatment by altering biomass characteristics and microbial community structure. In addition, the elevated salinity also increased soluble microbial products and extracellular polymeric substances in the mixed liquor, which induced fouling of the forward osmosis (FO) membrane. Nevertheless, microbial analysis indicated that salinity stress resulted in the development of halotolerant bacteria, consequently sustaining biodegradation in the OMBR system. By contrast, biological performance was relatively stable throughout conventional MBR-RO operation. Compared to conventional MBR-RO, the FO process effectively prevented foulants from permeating into the draw solution, thereby significantly reducing fouling of the downstream RO membrane in OMBR-RO operation. Accumulation of organic matter, including humic- and protein-like substances, as well as inorganic salts in the MBR effluent resulted in severe RO membrane fouling in conventional MBR-RO operation.

  7. Not nanocarbon but dispersant induced abnormality in lysosome in macrophages in vivo

    NASA Astrophysics Data System (ADS)

    Yudasaka, Masako; Zhang, Minfang; Matsumura, Sachiko; Yuge, Ryota; Ichihashi, Toshinari; Irie, Hiroshi; Shiba, Kiyotaka; Iijima, Sumio

    2015-05-01

    The properties of nanocarbons change from hydrophobic to hydrophilic as a result of coating them with dispersants, typically phospholipid polyethylene glycols, for biological studies. It has been shown that the dispersants remain attached to the nanocarbons when they are injected in mice and influence the nanocarbons’ biodistribution in vivo. We show in this report that the effects of dispersants also appear at the subcellular level in vivo. Carbon nanohorns (CNHs), a type of nanocarbon, were dispersed with ceramide polyethylene glycol (CPEG) and intravenously injected in mice. Histological observations and electron microscopy with energy dispersive x-ray analysis revealed that, in liver and spleen, the lysosome membranes were damaged, and the nanohorns formed a complex with hemosiderin in the lysosomes of the macrophages. It is inferred that the lysosomal membrane was damaged by sphigosine generated as a result of CPEG decomposition, which changed the intra lysosomal conditions, inducing the formation of the CPEG-CNH and hemosiderin complex. For comparison, when glucose was used instead of CPEG, neither the nanohorn-hemosiderin complex nor lysosomal membrane damage was found. Our results suggest that surface functionalization can control the behavior of nancarbons in cells in vivo and thereby improve their suitability for medical applications.

  8. Cellulose nanocrystal-based composite electrolyte with superior dimensional stability for alkaline fuel cell membranes

    DOE PAGES

    Lu, Yuan; Artmentrout, Aaron A.; Li, Juchuan; ...

    2015-01-01

    Cellulose nanocrystal (CNC)-based composite films were prepared as a solid electrolyte for alkaline fuel cells. Poly (vinyl alcohol) (PVA) and silica gel hybrid was used to bind the CNCs to form a robust composite film. The mass ratio (i.e., 1 : 1, 1 : 2) of PVA and silica gel was tuned to control the hydrophobicity of the resulting films. Composite films with a range of CNC content (i.e., 20 to 60%) were prepared to demonstrate the impact of CNC on the performance of these materials as a solid electrolyte for alkaline fuel cells. Different from previously reported cross-linked polymermore » films, CNC-based composite films with 40% hydrophobic binder (i.e., PVA : silica gel=1 : 2) exhibited simultaneous low water swelling (e.g., ~5%) and high water uptake (e.g., ~80%) due to the hydrophilicity and extraordinary dimensional stability of CNC. It also showed a conductivity of 0.044 and 0.065 S/cm at 20 and 60 oC, respectively. To the best of our knowledge, the film with 60% CNC and 40% binder is characterized by the lowest hydroxide conductivity-normalized swelling ratio. Decreased CNC content (i.e., 40 and 20%) resulted in comparable hydroxide conductivity but a greater swelling ratio. These results demonstrate the advantage of CNC as a key component for a solid electrolyte for alkaline fuel cells over conventional polymers, suggesting the great potential of CNCs in improving the dimensional stability while maintaining the conductivity of existing anion exchange membranes.« less

  9. All-atom and coarse-grained molecular dynamics simulations of a membrane protein stabilizing polymer.

    PubMed

    Perlmutter, Jason D; Drasler, William J; Xie, Wangshen; Gao, Jiali; Popot, Jean-Luc; Sachs, Jonathan N

    2011-09-06

    Amphipathic polymers called amphipols (APols) have been developed as an alternative to detergents for stabilizing membrane proteins (MPs) in aqueous solutions. APols provide MPs with a particularly mild environment and, as a rule, keep them in a native functional state for longer periods than do detergents. Amphipol A8-35, a derivative of polyacrylate, is widely used and has been particularly well studied experimentally. In aqueous solutions, A8-35 molecules self-assemble into well-defined globular particles with a mass of ∼40 kDa and a R(g) of ∼2.4 nm. As a first step towards describing MP/A8-35 complexes by molecular dynamics (MD), we present three sets of simulations of the pure APol particle. First, we performed a series of all-atom MD (AAMD) simulations of the particle in solution, starting from an arbitrary initial configuration. Although AAMD simulations result in stable cohesive particles over a 45 ns simulation, the equilibration of the particle organization is limited. This motivated the use of coarse-grained MD (CGMD), allowing us to investigate processes on the microsecond time scale, including de novo particle assembly. We present a detailed description of the parametrization of the CGMD model from the AAMD simulations and a characterization of the resulting CGMD particles. Our third set of simulations utilizes reverse coarse-graining (rCG), through which we obtain all-atom coordinates from a CGMD simulation. This allows a higher-resolution characterization of a configuration determined by a long-timescale simulation. Excellent agreement is observed between MD models and experimental, small-angle neutron scattering data. The MD data provides new insight into the structure and dynamics of A8-35 particles, which is possibly relevant to the stabilizing effects of APols on MPs, as well as a starting point for modeling MP/A8-35 complexes.

  10. Cellulose nanocrystal-based composite electrolyte with superior dimensional stability for alkaline fuel cell membranes

    SciTech Connect

    Lu, Yuan; Artmentrout, Aaron A.; Li, Juchuan; Tekinalp, Halil L.; Nanda, Jagjit; Ozcan, Soydan

    2015-01-01

    Cellulose nanocrystal (CNC)-based composite films were prepared as a solid electrolyte for alkaline fuel cells. Poly (vinyl alcohol) (PVA) and silica gel hybrid was used to bind the CNCs to form a robust composite film. The mass ratio (i.e., 1 : 1, 1 : 2) of PVA and silica gel was tuned to control the hydrophobicity of the resulting films. Composite films with a range of CNC content (i.e., 20 to 60%) were prepared to demonstrate the impact of CNC on the performance of these materials as a solid electrolyte for alkaline fuel cells. Different from previously reported cross-linked polymer films, CNC-based composite films with 40% hydrophobic binder (i.e., PVA : silica gel=1 : 2) exhibited simultaneous low water swelling (e.g., ~5%) and high water uptake (e.g., ~80%) due to the hydrophilicity and extraordinary dimensional stability of CNC. It also showed a conductivity of 0.044 and 0.065 S/cm at 20 and 60 oC, respectively. To the best of our knowledge, the film with 60% CNC and 40% binder is characterized by the lowest hydroxide conductivity-normalized swelling ratio. Decreased CNC content (i.e., 40 and 20%) resulted in comparable hydroxide conductivity but a greater swelling ratio. These results demonstrate the advantage of CNC as a key component for a solid electrolyte for alkaline fuel cells over conventional polymers, suggesting the great potential of CNCs in improving the dimensional stability while maintaining the conductivity of existing anion exchange membranes.

  11. All-Atom and Coarse-Grained Molecular Dynamics Simulations of a Membrane Protein Stabilizing Polymer

    PubMed Central

    Perlmutter, Jason D.; Drasler, William J.; Xie, Wangshen; Gao, Jiali; Popot, Jean-Luc; Sachs, Jonathan N.

    2011-01-01

    Amphipathic polymers called amphipols (APols) have been developed as an alternative to detergents for stabilizing membrane proteins (MPs) in aqueous solutions. APols provide MPs with a particularly mild environment and, as a rule, keep them in a native and functional state for longer periods than detergents do. Amphipol A8-35, a derivative of polyacrylate, is widely used and has been particularly well studied experimentally. In aqueous solutions, A8-35 molecules self-assemble into well-defined globular particles, with a mass of ~40 kDa and a Rg of ~2.4 nm. As a first step towards describing MP/A8-35 complexes by molecular dynamics (MD), we present three sets of simulations of the pure APol particle. First, we performed a series of all-atom MD (AAMD) simulations of the particle in solution, starting from an arbitrary initial configuration. While AAMD simulations result in cohesive and stable particles over a 45-ns simulation, the equilibration of the particle organization is limited. This motivated the use of coarse-grained MD (CGMD), allowing us to investigate processes on the microsecond timescale, including de novo particle assembly. We present a detailed description of the parametrization of the CGMD model from the AAMD simulations, and a characterization of the resulting CGMD particles. Our third set of simulations utilizes reverse coarse-graining (rCG), through which we obtain all-atom coordinates from a CGMD simulation. This allows higher-resolution characterization of a configuration determined by a long-timescale simulation. An excellent agreement is observed between MD models and experimental, small angle neutron scattering data. The MD data provides new insights into the structure and dynamics of A8-35 particles, possibly relevant to the stabilizing effects of APols on MPs, as well as a starting point for modeling MP/A8-35 complexes. PMID:21806035

  12. LYSOSOMAL ACTIVITY ASSOCIATED WITH DEVELOPMENTAL AXON PRUNING

    PubMed Central

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

    2009-01-01

    Clearance of cellular debris is a critical feature of the developing nervous system, as evidenced by the severe neurological consequences of lysosomal storage diseases in children. An important developmental process, that generates considerable cellular debris, is synapse elimination in which many axonal branches are pruned. The fate of these pruned branches is not known. Here, we investigate the role of lysosomal activity in neurons and glia in the removal of axon branches during early postnatal life. Using a probe for lysosomal activity, we observed robust staining associated with retreating motor axons. Lysosomal function was involved in axon removal because retreating axons were cleared more slowly in a mouse model of a lysosomal storage disease. In addition, we found lysosomal activity in the cerebellum at the time of, and at sites where, climbing fibers are eliminated. We propose that lysosomal activity is a central feature of synapse elimination. Moreover, staining for lysosomal activity may serve as a marker for regions of the developing nervous system undergoing axon pruning. PMID:18768693

  13. Membrane Protein Stability Analyses by Means of Protein Energy Profiles in Case of Nephrogenic Diabetes Insipidus

    PubMed Central

    Heinke, Florian; Labudde, Dirk

    2012-01-01

    Diabetes insipidus (DI) is a rare endocrine, inheritable disorder with low incidences in an estimated one per 25,000–30,000 live births. This disease is characterized by polyuria and compensatory polydypsia. The diverse underlying causes of DI can be central defects, in which no functional arginine vasopressin (AVP) is released from the pituitary or can be a result of defects in the kidney (nephrogenic DI, NDI). NDI is a disorder in which patients are unable to concentrate their urine despite the presence of AVP. This antidiuretic hormone regulates the process of water reabsorption from the prourine that is formed in the kidney. It binds to its type-2 receptor (V2R) in the kidney induces a cAMP-driven cascade, which leads to the insertion of aquaporin-2 water channels into the apical membrane. Mutations in the genes of V2R and aquaporin-2 often lead to NDI. We investigated a structure model of V2R in its bound and unbound state regarding protein stability using a novel protein energy profile approach. Furthermore, these techniques were applied to the wild-type and selected mutations of aquaporin-2. We show that our results correspond well to experimental water ux analysis, which confirms the applicability of our theoretical approach to equivalent problems. PMID:22474537

  14. A theoretical formalism for aggregation of peroxidized lipids and plasma membrane stability during photolysis.

    PubMed Central

    Busch, N A; Yarmush, M L; Toner, M

    1998-01-01

    The objective of this investigation was to examine, from a theoretical perspective, the mechanism underlying the lysis of plasma membranes by photoinduced, chemically mediated damage such as is found in photolysis. Toward this end, a model is presented which relates the membrane lifetime to the thermodynamic parameters of the membrane components based upon the kinetic theory of aggregate formation. The formalism includes a standard birth/death process for the formation of damaged membrane components (i.e., peroxidized lipids) as well as a terminating condensation process for the formation of aggregates of peroxidized plasma membrane lipids. Our theory predicts that 1) the membrane lifetime is inversely correlated with predicted rate of membrane damage; 2) an upper limit on the duration of membrane damage exists, above which the mean and variance of the membrane lifetime is independent of further membrane damage; and 3) both the mean and variance of the time of membrane lifetime distribution are correlated with the number of sites that may be damaged to form a single membrane defect. The model provides a framework to optimize the lysis of cell membranes by photodynamic therapy. PMID:9826616

  15. Reliable characteristics and stabilization of on-membrane SOI MOSFET-based components heated up to 335 °C

    NASA Astrophysics Data System (ADS)

    Amor, S.; André, N.; Gérard, P.; Ali, S. Z.; Udrea, F.; Tounsi, F.; Mezghani, B.; Francis, L. A.; Flandre, D.

    2017-01-01

    In this work we investigate the characteristics and critical operating temperatures of on-membrane embedded MOSFETs from an experimental and analytical point of view. This study permits us to conclude the possibility of integrating electronic circuitry in the close vicinity of micro-heaters and hot operation transducers. A series of calibrations and measurements has been performed to examine the behaviors of transistors, inverters and diodes, actuated at high temperature, on a membrane equipped with an on-chip integrated micro-heater. The studied n- and p-channel body-tied partially-depleted MOSFETs and CMOS inverter are embedded in a 5 μm-thick membrane fabricated by back-side MEMS micromachining using SOI technology. It has been noted that a pre-stabilization step after the harsh post-CMOS processing, through an in situ high-temperature annealing using the micro-heater, is mandatory in order to stabilize the MOSFETs characteristics. The electrical characteristics and performance of the on-membrane MOS components are discussed when heated up to 335 °C. This study supports the possibility of extending the potential of the micro-hotplate concept, under certain conditions, by embedding more electronic functionalities on the interface of on-membrane-based sensors leading to better sensing and actuation performances and a total area reduction, particularly for environmental or industrial applications.

  16. Pregnancy-Induced Amelioration of Muscular Dystrophy Phenotype in mdx Mice via Muscle Membrane Stabilization Effect of Glucocorticoid

    PubMed Central

    Shimizu-Motohashi, Yuko; Asakura, Yoko; Motohashi, Norio; Belur, Nandkishore R.; Baumrucker, Michael G.; Asakura, Atsushi

    2015-01-01

    Duchenne muscular dystrophy (DMD), the most common and severe type of dystrophinopathy, is an X-linked recessive genetic disease caused by the absence of dystrophin, which leads to fragility and vulnerability of the sarcolemma to mechanical stretching with increased membrane permeability. Currently, glucocorticoids such as prednisolone are the only medication available for DMD. However, molecular pathways responsible for this effect are still unclear. In addition, it remains unclear whether sex-related factors, including pregnancy and the postpartum period, affect the phenotype of dystrophinopathy. Here, we report the amelioration of muscle membrane permeability in the diaphragm muscle of pregnant and postpartum, but not in nulliparous, mdx mice, an animal model for DMD, during the physiological surge of corticosterone, the most abundant glucocorticoid in rodents. Cultures of single muscle fibers and myotubes isolated from mdx mouse diaphragm demonstrate resistance to hypo-osmotic shock when treated with corticosterone but not with estradiol or progesterone. This corticosterone-mediated resistance was diminished by an antagonist of corticosterone, indicating that the glucocorticoid-glucocorticoid receptor axis plays a role in this membrane stabilization effect on muscle. Moreover, subcutaneous injection of corticosterone into mdx mice showed decreased membrane permeability. This is the first report to demonstrate that pregnancy-related resistance to muscle fiber damage in mdx mice due to the membrane stabilization effect of corticosterone. We also propose that this membrane stabilization effect is exerted through annexin A1 up-regulation as the molecular mechanisms of glucocorticoid effects on DMD muscle. Furthermore, single muscle fiber culture studies provide a sensitive chemical screening platform for muscular dystrophies. PMID:25775477

  17. Monitoring Autophagy in Lysosomal Storage Disorders

    PubMed Central

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

    2009-01-01

    Lysosomes are the final destination of the autophagic pathway. It is in the acidic milieu of the lysosomes that autophagic cargo is metabolized and recycled. One would expect that diseases with primary lysosomal defects would be among the first systems in which autophagy would be studied. In reality, this is not the case. Lysosomal storage diseases, a group of more than 60 diverse inherited disorders, have only recently become a focus of autophagic research. Studies of these clinically severe conditions promise not only to clarify pathogenic mechanisms, but also to expand our knowledge of autophagy itself. In this chapter, we will describe the lysosomal storage diseases in which autophagy has been explored, and present the approaches used to evaluate this essential cellular pathway. PMID:19216919

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

    PubMed Central

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

    2013-01-01

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

  19. Residual stress of free-standing membranes of yttria-stabilized zirconia for micro solid oxide fuel cell applications.

    PubMed

    Tarancón, Albert; Sabaté, Neus; Cavallaro, Andrea; Gràcia, Isabel; Roqueta, Jaume; Garbayo, Iñigo; Esquivel, Juan P; Garcia, Gemma; Cané, Carles; Santiso, José

    2010-02-01

    The present study is devoted to analyze the compatibility of yttria-stabilized zirconia thin films prepared by pulsed laser deposition and metalorganic chemical vapor deposition techniques, with microfabrication processes based on silicon technologies for micro solid oxide fuel cells applications. Deposition of yttria-stabilized zirconia on Si/SiO2/Si3N4 substrates was optimized for both techniques in order to obtain high density and homogeneity, as well as a good crystallinity for film thicknesses ranging from 60 to 240 nm. In addition, stabilized zirconia free-standing membranes were fabricated from the deposited films with surface areas between 50 x 50 microm2 and 820 x 820 microm2. Particular emphasis was made on the analysis of the effect of the nature of the deposition technique and the different design and fabrication parameters (membrane area, thickness and substrate deposition temperature) on the residual stress of the membranes in order to control their thermomechanical stability for application as electrolyte in micro solid oxide fuel cells.

  20. Innovative methods to stabilize liquid membranes for removal of radionuclides from groundwater

    SciTech Connect

    Lokhandwala, K.

    1997-10-01

    In this Phase I Small Business Innovation Research program, Membrane Technology Research, Inc., is developing a stable liquid membrane for extracting uranium and other radionuclides from groundwater. The improved membrane can also be applied to separation of other metal ions from aqueous streams in industrial operations.

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

    PubMed

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

    2010-10-01

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

  2. Physicochemical stability, microrheological properties and microstructure of lutein emulsions stabilized by multilayer membranes consisting of whey protein isolate, flaxseed gum and chitosan.

    PubMed

    Xu, Duoxia; Aihemaiti, Zulipiya; Cao, Yanping; Teng, Chao; Li, Xiuting

    2016-07-01

    The impact of chitosan (CTS) on the physicochemical stability, microrheological property and microstructure of whey protein isolate (WPI)-flaxseed gum (FG) stabilized lutein emulsions at pH 3.0 was studied. A layer-by-layer electrostatic deposition method was used to prepare multilayered lutein emulsions. Droplet size, zeta-potential, instability index, microstructure and microrheological behavior of lutein emulsions were measured. The influences of interfacial layer, metal chelator and free radical scavenger on the chemical stability of lutein emulsions were also investigated. It was found that multilayer emulsions had better physical stability showing the pronounced effect of 1wt% CTS. The mean square displacement analysis demonstrated that CTS led to increases of macroscopic viscosity and elasticity index for WPI-FG stabilized lutein emulsions due to CTS embedding in the network. CTS also helped to chemically stabilize the lutein emulsions against degradation. The combination of interfacial membrane and prooxidative metal chelator or free radical scavenger was an effective method to control lutein degradation.

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

    PubMed

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

    2016-03-18

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

  4. ATP-containing vesicles in stria vascular marginal cell cytoplasms in neonatal rat cochlea are lysosomes

    PubMed Central

    Liu, Jun; Liu, Wenjing; Yang, Jun

    2016-01-01

    We confirmed that ATP is released from cochlear marginal cells in the stria vascular but the cell organelle in which ATP stores was not identified until now. Thus, we studied the ATP-containing cell organelles and suggest that these are lysosomes. Primary cultures of marginal cells of Sprague-Dawley rats aged 1–3 days was established. Vesicles within marginal cells stained with markers were identified under confocal laser scanning microscope and transmission electron microscope (TEM). Then ATP release from marginal cells was measured after glycyl-L-phenylalanine-ß- naphthylamide (GPN) treatment using a bioluminescent assay. Quinacrine-stained granules within marginal cells were labeled with LysoTracker, a lysosome tracer, and lysosomal-associated membrane protein 1(LAMP1), but not labeled with the mitochondrial tracer MitoTracker. Furthermore, LysoTracker-labelled puncta showed accumulation of Mant-ATP, an ATP analog. Treatment with 200 μM GPN quenched fluorescently labeled puncta after incubation with LysoTracker or quinacrine, but not MitoTracker. Quinacrine-labeled organelles observed by TEM were lysosomes, and an average 27.7 percent increase in ATP luminescence was observed in marginal cells extracellular fluid after GPN treatment. ATP-containing vesicles in cochlear marginal cells of the stria vascular from neonatal rats are likely lysosomes. ATP release from marginal cells may be via Ca2+-dependent lysosomal exocytosis. PMID:26864824

  5. Distinct Protein Sorting and Localization to Premelanosomes, Melanosomes, and Lysosomes in Pigmented Melanocytic Cells✪

    PubMed Central

    Raposo, Graça; Tenza, Danielle; Murphy, Diane M.; Berson, Joanne F.; Marks, Michael S.

    2001-01-01

    Melanosomes and premelanosomes are lysosome-related organelles with a unique structure and cohort of resident proteins. We have positioned these organelles relative to endosomes and lysosomes in pigmented melanoma cells and melanocytes. Melanosome resident proteins Pmel17 and TRP1 localized to separate vesicular structures that were distinct from those enriched in lysosomal proteins. In immunogold-labeled ultrathin cryosections, Pmel17 was most enriched along the intralumenal striations of premelanosomes. Increased pigmentation was accompanied by a decrease in Pmel17 and by an increase in TRP1 in the limiting membrane. Both proteins were largely excluded from lysosomal compartments enriched in LAMP1 and cathepsin D. By kinetic analysis of fluid phase uptake and immunogold labeling, premelanosomal proteins segregated from endocytic markers within an unusual endosomal compartment. This compartment contained Pmel17, was accessed by BSA–gold after 15 min, was acidic, and displayed a cytoplasmic planar coat that contained clathrin. Our results indicate that premelanosomes and melanosomes represent a distinct lineage of organelles, separable from conventional endosomes and lysosomes within pigmented cells. Furthermore, they implicate an unusual clathrin-coated endosomal compartment as a site from which proteins destined for premelanosomes and lysosomes are sorted. PMID:11266471

  6. Distinct protein sorting and localization to premelanosomes, melanosomes, and lysosomes in pigmented melanocytic cells.

    PubMed

    Raposo, G; Tenza, D; Murphy, D M; Berson, J F; Marks, M S

    2001-02-19

    Melanosomes and premelanosomes are lysosome-related organelles with a unique structure and cohort of resident proteins. We have positioned these organelles relative to endosomes and lysosomes in pigmented melanoma cells and melanocytes. Melanosome resident proteins Pmel17 and TRP1 localized to separate vesicular structures that were distinct from those enriched in lysosomal proteins. In immunogold-labeled ultrathin cryosections, Pmel17 was most enriched along the intralumenal striations of premelanosomes. Increased pigmentation was accompanied by a decrease in Pmel17 and by an increase in TRP1 in the limiting membrane. Both proteins were largely excluded from lysosomal compartments enriched in LAMP1 and cathepsin D. By kinetic analysis of fluid phase uptake and immunogold labeling, premelanosomal proteins segregated from endocytic markers within an unusual endosomal compartment. This compartment contained Pmel17, was accessed by BSA-gold after 15 min, was acidic, and displayed a cytoplasmic planar coat that contained clathrin. Our results indicate that premelanosomes and melanosomes represent a distinct lineage of organelles, separable from conventional endosomes and lysosomes within pigmented cells. Furthermore, they implicate an unusual clathrin-coated endosomal compartment as a site from which proteins destined for premelanosomes and lysosomes are sorted.

  7. Lysosome abnormalities and lipofucsin content of nerve cells of oedematous human cerebral cortex.

    PubMed

    Castejón, O J

    2004-01-01

    Lysosome alterations and lipofucsin content of nerve cells, capillary endothelial cells and pericytes were examined in the anoxic-ischaemic brain parenchyma of thirty two patients with congenital hydrocephalus, complicated brain traumatic injuries, brain tumours and vascular anomalies. Cortical biopsies of frontal, parietal and temporal cortex were processed for transmission electron microscopy. In oedematous non pyramidal and pyramidal nerve cells, lysosomes showed fragmentation of their limiting membranes and an associated dense granulation. Areas of cytoplasmic focal necrosis were observed surrounding the lysosomes. Lipofucsin granules were also observed in neonate and infant patients with congenital hydrocephalus, suggesting that lipofucsin formation is a life span process. Lysosomes coexisting with an increased amount of lipofucsin granules were observed in young and adult patients with brain trauma, tumours and vascular anomalies. Phagocytic astrocytes and activated oligodendroglial cells showed the overall spectrum of an altered endosomal/lysosomal system. Lipofucsin granules and multivesicular bodies also were distinguished in endothelial and pericyte cells. The role of released and activated lysosomal enzymes is discussed in relation with the cytoplasmatic focal necrosis of nerve cells and the genesis of moderate and severe oedema.

  8. The protonophore CCCP interferes with lysosomal degradation of autophagic cargo in yeast and mammalian cells.

    PubMed

    Padman, Benjamin S; Bach, Markus; Lucarelli, Giuseppe; Prescott, Mark; Ramm, Georg

    2013-11-01

    Mitophagy is a selective pathway, which targets and delivers mitochondria to the lysosomes for degradation. Depolarization of mitochondria by the protonophore CCCP is a strategy increasingly used to experimentally trigger not only mitophagy, but also bulk autophagy. Using live-cell fluorescence microscopy we found that treatment of HeLa cells with CCCP caused redistribution of mitochondrially targeted dyes, including DiOC6, TMRM, MTR, and MTG, from mitochondria to the cytosol, and subsequently to lysosomal compartments. Localization of mitochondrial dyes to lysosomal compartments was caused by retargeting of the dye, rather than delivery of mitochondrial components to the lysosome. We showed that CCCP interfered with lysosomal function and autophagosomal degradation in both yeast and mammalian cells, inhibited starvation-induced mitophagy in mammalian cells, and blocked the induction of mitophagy in yeast cells. PARK2/Parkin-expressing mammalian cells treated with CCCP have been reported to undergo high levels of mitophagy and clearance of all mitochondria during extensive treatment with CCCP. Using correlative light and electron microscopy in PARK2-expressing HeLa cells, we showed that mitochondrial remnants remained present in the cell after 24 h of CCCP treatment, although they were no longer easily identifiable as such due to morphological alterations. Our results showed that CCCP inhibits autophagy at both the initiation and lysosomal degradation stages. In addition, our data demonstrated that caution should be taken when using organelle-specific dyes in conjunction with strategies affecting membrane potential.

  9. Biochemical and lysosomal biomarkers in the mussel Mytilus galloprovincialis from the Mar Piccolo of Taranto (Ionian Sea, Southern Italy).

    PubMed

    Moschino, Vanessa; Da Ros, Luisa

    2016-07-01

    Biomarkers are internationally recognized as useful tools in marine coastal biomonitoring, in particular, as early-warning signals at the level of individual organisms to assess biological effects of pollutants and other stressors. In the present study, Mytilus galloprovincialis has been employed as a sentinel organism to assess biological pollution effects in the Mar Piccolo of Taranto (Southern Italy), a coastal lagoon divided into two small inlets, connected to the open sea through one natural and one artificial narrow openings. Mussels were collected in June 2013 at three sites located within each of the two inlets of the Mar Piccolo. Biological effects were investigated through a suite of biomarkers suitable to reflect effects and/or exposure to contaminants at biochemical and cellular levels. Biochemical biomarkers included glutathione-S-transferase (GST) and acetylcholinesterase (AChE) enzyme activities; as histochemical biomarkers, lysosomal membrane stability, lipofuscin and neutral lipid accumulation, and lysosomal structural changes were considered. As a whole, results highlighted differences among the three study sites, particularly for GST, AChE, and lipofuscins, which are consistent with the variations of the chemical pollutants in sediments. The applied biomarkers showed that a stress syndrome likely to be ascribed to environmental pollutants is occurring in mussels living in the Mar Piccolo of Taranto, in particular, the ones inhabiting the first inlet.

  10. Effects of substituents and substitution positions on alkaline stability of imidazolium cations and their corresponding anion-exchange membranes.

    PubMed

    Si, Zhihong; Qiu, Lihua; Dong, Huilong; Gu, Fenglou; Li, Youyong; Yan, Feng

    2014-03-26

    Imidazolium cations with butyl groups at various substitution positions (N1-, C2-, and N3-), 1-butyl-2,3-dimethylimidazolium ([N1-BDMIm](+)), 2-butyl-1,3-dimethylimidazolium ([C2-BDMIm](+)), and 3-butyl-1,2-dimethylimidazolium ([N3-BDMIm](+)), were synthesized. Quantitative (1)H NMR spectra and density functional theory calculation were applied to investigate the chemical stability of the imidazolium cations in alkaline solutions. The results suggested that the alkaline stability of the imidazolium cations was drastically affected by the C2-substitution groups. The alkaline stability of imidazolium cations with various substitution groups at the C2-position, including 2-ethyl-1-butyl-3-methylimidazolium ([C2-EBMIm](+)), 1,2-dibutyl-3-methylimidazolium ([C2-BBMIm](+)), and 2-hydroxymethyl-1-butyl-3-methylimidazolium ([C2-HMBMIm](+)), was further studied. The butyl group substituted imidazolium cation ([C2-BBMIm](+)) exhibited the highest alkaline stability at the elevated temperatures. The synthesized anion-exchange membranes based on the [C2-BBMIm](+) cation showed promising alkaline stability. These observations should pave the way to the practical application of imidazolium-based anion exchange membrane fuel cells.

  11. Streptococcus oralis Induces Lysosomal Impairment of Macrophages via Bacterial Hydrogen Peroxide

    PubMed Central

    Okahashi, Nobuo; Kuwata, Hirotaka; Kawabata, Shigetada

    2016-01-01

    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

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

    PubMed Central

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

    2016-01-01

    ABSTRACT 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

  13. Covalent conjugation of tetrameric bovine liver catalase to liposome membranes for stabilization of the enzyme tertiary and quaternary structures.

    PubMed

    Yoshimoto, Makoto; Sakamoto, Hideyuki; Shirakami, Hiroshi

    2009-03-01

    Tetrameric bovine liver catalase (BLC) is unstable because of its dissociation into subunits at low enzyme concentrations and the conformational change of the subunits at high temperatures. In this work, for stabilization of BLC, the enzyme was covalently conjugated with liposome membranes composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), cholesterol and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-glutaryl (NGPE). The NGPE, which was responsible for the BLC/membrane coupling, was altered from 0.05 to 0.2 in its liposomal mole fraction f(G). The catalase-conjugated liposome (CCL) with f(G) of 0.15 showed the maximum number of the conjugated BLC molecules of 28 per liposome. The reactivity of CCLs to H(2)O(2) was as high as that of free BLC at 25 degrees C in Tris-HCl buffer of pH 7.4. Among the CCLs, the catalyst with f(G) of 0.15 was the most stable at 55 degrees C in its enzyme activity in the buffer because the appropriate number of BLC/liposome covalent bonding prevented the dissociation-induced enzyme deactivation. Furthermore, the CCL showed much higher stability at 55 degrees C than the free BLC/enzyme-free liposome mixture and free BLC at the low BLC concentration of 340ng/mL. This was because BLC in the CCL was located in the vicinity of the host membrane regardless of the catalyst concentration, which could induce the effective stabilization effect of the membrane on the enzyme tertiary structure as indicated by the intrinsic tryptophan fluorescence analysis. The results obtained demonstrate the high structural stability of BLC in the CCL system, which was derived from the covalent bonding and interaction between BLC and liposomes.

  14. The self-crosslinked ufasome of conjugated linoleic acid: investigation of morphology, bilayer membrane and stability.

    PubMed

    Fan, Ye; Fang, Yun; Ma, Lin

    2014-11-01

    Unsaturated fatty acid liposomes (Ufasomes) have attracted interests because of the ready availability of unsaturated fatty acids and the simple assembly strategy. However, the colloidal instability of the ufasomes hinders them from applying in the fields of drug delivery and food additives. In the present work, conjugated linoleic acid (CLA) with triple activities of bioactive, assembling and crosslinking was employed as a new molecular building block to construct ufasome and afterwards crosslinked ufasome. First, CLA ufasome was self-assembled from CLA molecules in response to pH variation, and the suitable CLA concentrations and pH ranges were determined by surface tension measurement and acid-base titration. Subsequently, the self-crosslinked CLA ufasome was prepared by intra-ufasomal crosslinking of conjugated double bonds in the CLA molecules. The morphologies of the self-crosslinked CLA ufasomes were imaged using transmission electron microscopy (TEM), from which the size of 20-50 nm and the bilayer thickness of 2.7±0.5 nm were detected. Most importantly, based on the comparison of the bilayer thicknesses of the different fatty acids, the molecular arrangement in the bilayer membrane of the self-crosslinked CLA ufasome is named "side-by-side" model contrary to the ordinary "tail-to-tail" model. The pH stability of the self-crosslinked CLA ufasome was examined in virtue of dynamic light scattering tests. Finally, in vitro release results of 5-fluorouracil from the self-crosslinked CLA ufasome showed that the process was slow and sustainable.

  15. Lysosomal cross-correction by hematopoietic stem cell-derived macrophages via tunneling nanotubes

    PubMed Central

    Naphade, Swati; Sharma, Jay; Chevronnay, Héloïse P. Gaide; Shook, Michael A.; Yeagy, Brian A.; Rocca, Celine J.; Ur, Sarah N.; Lau, Athena J.; Courtoy, Pierre J.; Cherqui, Stephanie

    2014-01-01

    Despite controversies on the potential of hematopoietic stem cells (HSCs) to promote tissue repair, we previously showed that HSC transplantation could correct cystinosis, a multi-systemic lysosomal storage disease, caused by a defective lysosomal membrane cystine transporter, cystinosin (CTNS). Addressing the cellular mechanisms, we here report vesicular cross-correction after HSC differentiation into macrophages. Upon co-culture with cystinotic fibroblasts, macrophages produced tunneling nanotubes (TNTs) allowing transfer of cystinosin-bearing lysosomes into Ctns-deficient cells, which exploited the same route to retrogradely transfer cystine-loaded lysosomes to macrophages, providing a bidirectional correction mechanism. TNT formation was enhanced by contact with diseased cells. In vivo, HSCs grafted to cystinotic kidneys also generated nanotubular extensions resembling invadopodia that crossed the dense basement membranes and delivered cystinosin into diseased proximal tubular cells. This is the first report of correction of a genetic lysosomal defect by bidirectional vesicular exchange via TNTs and suggests broader potential for HSC transplantation for other disorders due to defective vesicular proteins. PMID:25186209

  16. Brief reports: Lysosomal cross-correction by hematopoietic stem cell-derived macrophages via tunneling nanotubes.

    PubMed

    Naphade, Swati; Sharma, Jay; Gaide Chevronnay, Héloïse P; Shook, Michael A; Yeagy, Brian A; Rocca, Celine J; Ur, Sarah N; Lau, Athena J; Courtoy, Pierre J; Cherqui, Stephanie

    2015-01-01

    Despite controversies on the potential of hematopoietic stem cells (HSCs) to promote tissue repair, we previously showed that HSC transplantation could correct cystinosis, a multisystemic lysosomal storage disease, caused by a defective lysosomal membrane cystine transporter, cystinosin (CTNS gene). Addressing the cellular mechanisms, we here report vesicular cross-correction after HSC differentiation into macrophages. Upon coculture with cystinotic fibroblasts, macrophages produced tunneling nanotubes (TNTs) allowing transfer of cystinosin-bearing lysosomes into Ctns-deficient cells, which exploited the same route to retrogradely transfer cystine-loaded lysosomes to macrophages, providing a bidirectional correction mechanism. TNT formation was enhanced by contact with diseased cells. In vivo, HSCs grafted to cystinotic kidneys also generated nanotubular extensions resembling invadopodia that crossed the dense basement membranes and delivered cystinosin into diseased proximal tubular cells. This is the first report of correction of a genetic lysosomal defect by bidirectional vesicular exchange via TNTs and suggests broader potential for HSC transplantation for other disorders due to defective vesicular proteins.

  17. Defects in lysosomal maturation facilitate the activation of innate sensors in systemic lupus erythematosus

    PubMed Central

    Monteith, Andrew J.; Kang, SunAh; Scott, Eric; Hillman, Kai; Rajfur, Zenon; Jacobson, Ken; Costello, M. Joseph; Vilen, Barbara J.

    2016-01-01

    Defects in clearing apoptotic debris disrupt tissue and immunological homeostasis, leading to autoimmune and inflammatory diseases. Herein, we report that macrophages from lupus-prone MRL/lpr mice have impaired lysosomal maturation, resulting in heightened ROS production and attenuated lysosomal acidification. Impaired lysosomal maturation diminishes the ability of lysosomes to degrade apoptotic debris contained within IgG–immune complexes (IgG-ICs) and promotes recycling and the accumulation of nuclear self-antigens at the membrane 72 h after internalization. Diminished degradation of IgG-ICs prolongs the intracellular residency of nucleic acids, leading to the activation of Toll-like receptors. It also promotes phagosomal membrane permeabilization, allowing dsDNA and IgG to leak into the cytosol and activate AIM2 and TRIM21. Collectively, these events promote the accumulation of nuclear antigens and activate innate sensors that drive IFNα production and heightened cell death. These data identify a previously unidentified defect in lysosomal maturation that provides a mechanism for the chronic activation of intracellular innate sensors in systemic lupus erythematosus. PMID:27035940

  18. Effect of operation parameters on the flux stabilization of gravity-driven membrane (GDM) filtration system for decentralized water supply.

    PubMed

    Tang, Xiaobin; Ding, An; Qu, Fangshu; Jia, Ruibao; Chang, Haiqing; Cheng, Xiaoxiang; Liu, Bin; Li, Guibai; Liang, Heng

    2016-08-01

    A pilot-scale gravity-driven membrane (GDM) filtration system under low gravitational pressure without any pre-treatment, backwash, flushing, or chemical cleaning was carried out to investigate the effect of operation parameters (including operation pressure, aeration mode, and intermittent filtration) on the effluent quality and permeability development. The results revealed that GDM system exhibited an efficient performance for the removal of suspended substances and organic compounds. The stabilization of flux occurred and the average values of stable flux were 6.6, 8.1, and 8.6 Lm(-2) h(-1) for pressures of 65, 120, and 200 mbar, respectively. In contrast, flux stabilization was not observed under continuous and intermittent aeration conditions. However, aeration (especially continuous aeration) was effective to improve flux and alleviate membrane fouling during 1-month operation. Moreover, intermittent filtration would influence the stabilization of permeate flux, resulting in a higher stable flux (ranging from 6 to 13 Lm(-2) h(-1)). The stable flux significantly improved with the increase of intermittent period. Additionally, GDM systems exhibited an efficient recovery of flux after simple physical cleaning and the analyses of resistance reversibility demonstrated that most of the total resistance was hydraulic reversible resistance (50-75 %). Therefore, it is expected that the results of this study can develop strategies to increase membrane permeability and reduce energy consumption in GDM systems for decentralized water supply.

  19. Effect of intrinsic curvature and edge tension on the stability of binary mixed-membrane three-junctions.

    PubMed

    Gardner, Jasmine M; Deserno, Markus; Abrams, Cameron F

    2016-08-21

    We use a combination of coarse-grained molecular dynamics simulations and theoretical modeling to examine three-junctions in mixed lipid bilayer membranes. These junctions are localized defect lines in which three bilayers merge in such a way that each bilayer shares one monolayer with one of the other two bilayers. The resulting local morphology is non-lamellar, resembling the threefold symmetric defect lines in inverse hexagonal phases, but it regularly occurs during membrane fission and fusion events. We realize a system of junctions by setting up a honeycomb lattice, which in its primitive cell contains two hexagons and four three-line junctions, permitting us to study their stability as well as their line tension. We specifically consider the effects of lipid composition and intrinsic curvature in binary mixtures, which contain a fraction of negatively curved lipids in a curvature-neutral background phase. Three-junction stability results from a competition between the junction and an open edge, which arises if one of the three bilayers detaches from the other two. We show that the stable phase is the one with the lower defect line tension. The strong and opposite monolayer curvatures present in junctions and edges enhance the mole fraction of negatively curved lipids in junctions and deplete it in edges. This lipid sorting affects the two line tensions and in turn the relative stability of the two phases. It also leads to a subtle entropic barrier for the transition between junction and edge that is absent in uniform membranes.

  20. Effect of intrinsic curvature and edge tension on the stability of binary mixed-membrane three-junctions

    NASA Astrophysics Data System (ADS)

    Gardner, Jasmine M.; Deserno, Markus; Abrams, Cameron F.

    2016-08-01

    We use a combination of coarse-grained molecular dynamics simulations and theoretical modeling to examine three-junctions in mixed lipid bilayer membranes. These junctions are localized defect lines in which three bilayers merge in such a way that each bilayer shares one monolayer with one of the other two bilayers. The resulting local morphology is non-lamellar, resembling the threefold symmetric defect lines in inverse hexagonal phases, but it regularly occurs during membrane fission and fusion events. We realize a system of junctions by setting up a honeycomb lattice, which in its primitive cell contains two hexagons and four three-line junctions, permitting us to study their stability as well as their line tension. We specifically consider the effects of lipid composition and intrinsic curvature in binary mixtures, which contain a fraction of negatively curved lipids in a curvature-neutral background phase. Three-junction stability results from a competition between the junction and an open edge, which arises if one of the three bilayers detaches from the other two. We show that the stable phase is the one with the lower defect line tension. The strong and opposite monolayer curvatures present in junctions and edges enhance the mole fraction of negatively curved lipids in junctions and deplete it in edges. This lipid sorting affects the two line tensions and in turn the relative stability of the two phases. It also leads to a subtle entropic barrier for the transition between junction and edge that is absent in uniform membranes.

  1. mTOR and lysosome regulation

    PubMed Central

    2014-01-01

    Lysosomes are key cellular organelles that play a crucial role in catabolism by degrading extracellular and intracellular material. It is, therefore, very intriguing that mTORC1 (mechanistic target of rapamycin complex 1), a major promoter of anabolic processes, localizes in its active form to the surface of lysosomes. In recent years, many exciting observations have revealed a tightly regulated crosstalk between mTORC1 activity and lysosomal function. These findings highlight the complex regulatory network that modulates energy metabolism in cells. PMID:25184042

  2. Dicarboxylic acids with limited numbers of hydrocarbons stabilize cell membrane and increase osmotic resistance in rat erythrocytes.

    PubMed

    Mineo, Hitoshi; Amita, Nozomi; Kawawake, Megumi; Higuchi, Ayaka

    2013-11-01

    We examined the effect of dicarboxylic acids having 0 to 6 hydrocarbons and their corresponding monocarboxylic or tricarboxylic acids in changing the osmotic fragility (OF) in rat red blood cells (RBCs). Malonic, succinic, glutaric and adipic acids, which are dicarboxylic acids with 1, 2, 3 and 4 straight hydrocarbons located between two carboxylic groups, decreased the OF in a concentration-dependent manner. Other long-chain dicarboxylic acids did not change the OF in rat RBCs. The benzoic acid derivatives, isophthalic and terephthalic acids, but not phthalic acid, decreased the OF in a concentration-dependent manner. Benzene-1,2,3-tricarboxylic acid, but not benzene-1,3,5-tricarboxylic acid, also decreased the OF in rat RBCs. On the other hand, monocarboxylic acids possessing 2 to 7 straight hydrocarbons and benzoic acid increased the OF in rat RBCs. In short-chain dicarboxylic acids, a limited number of hydrocarbons between the two carboxylic groups are thought to form a V- or U-shaped structure and interact with phospholipids in the RBC membrane. In benzene dicarboxylic and tricarboxylic acids, a part of benzene nucleus between the two carboxylic groups is thought to enter the plasma membrane and act on acyl-chain in phospholipids in the RBC membrane. For dicarboxylic and tricarboxylic acids, limited numbers of hydrocarbons in molecules are speculated to enter the RBC membrane with the hydrophilic carboxylic groups remaining outside, stabilizing the structure of the cell membrane and resulting in an increase in osmotic resistance in rat RBCs.

  3. Allometric dependence of the life span of mammal erythrocytes on thermal stability and sphingomyelin content of plasma membranes.

    PubMed

    Ivanov, Ivan Tanev

    2007-08-01

    Thermal stability of erythrocyte membrane is a measure for its ability to maintain permeability barrier at deleterious conditions. Hence, it could impact the resistance of erythrocytes against detrimental factors in circulation. In this study the thermostability of erythrocyte membranes was expressed by the temperature, T(go), at which the transmembrane gradient of ion concentration rapidly dissipated during transient heating. T(go) is the inducing temperature of the membrane transition that activated passive ion permeability at hyperthermia causing thermal hemolysis. A good allometric correlation of T(go) to the resistance against thermal hemolysis and the life span of erythrocytes were found for 13 mammals; sheep, cow, goat, dog, horse, man, rabbit, pig, cat, hamster, guinea pig, rat, and mouse. For the same group, the values of T(go) were strictly related to the sphingomyelin content of erythrocyte membranes. The residual ion permeability, P, was temperature activated from 38 to 57 degrees C with activation energy of 250+/-15 kJ/mol that strongly differed from that below 37 degrees C. The projected value of P at 37 degrees C was about half that of residual physiological permeability for Na+ and K+ that build ground for possible explanation of the life span vs membrane thermostability allometric correlation.

  4. Sulphonated imidized graphene oxide (SIGO) based polymer electrolyte membrane for improved water retention, stability and proton conductivity

    NASA Astrophysics Data System (ADS)

    Pandey, Ravi P.; Shahi, Vinod K.

    2015-12-01

    Sulphonated imidized graphene oxide (SIGO) (graphene oxide (GO) tethered sulphonated polyimide) has been successfully synthesized by polycondensation reaction using dianhydride and sulphonated diamine. Polymer electrolyte membranes (PEMs) are prepared by using SIGO (different wt%) and sulphonated poly(imide) (SPI). Resultant SPI/SIGO composite PEMs exhibit improved stabilities (thermal, mechanical and oxidative) and good water-retention properties (high bound water content responsible for proton conduction at high temperature by internal self-humidification). Incorporation of covalent bonded SIGO into SPI matrix results hydrophobic-hydrophilic phase separation and facile architecture of proton conducting path. Well optimized sulphonated poly(imide)/sulphonated imidized graphene oxide (15 wt%) (SPI/SIGO-15) composite membrane shows 2.24 meq g-1 ion-exchange capacity (IEC); 11.38 × 10-2 S cm-1 proton conductivity; 5.12% bound water content; and 10.52 × 10-7 cm2 s-1 methanol permeability. Maximum power density for pristine SPI membrane (57.12 mW cm-2) improves to 78.53 mW cm-2 for SPI/SIGO-15 membrane, in single-cell direct methanol fuel cell (DMFC) test at 70 °C using 2 M methanol fuel. Under similar experimental conditions, Nafion 117 membrane exhibits 62.40 mW cm-2 maximum power density. Reported strategy for the preparation of PEMs, offers a useful protocol for grafting of functionalized inorganic materials with in organic polymer chain by imidization.

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

    PubMed Central

    Hafner Česen, M; Repnik, U; Turk, V; Turk, B

    2013-01-01

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

  6. Mutagenesis of Paramyxovirus Hemagglutinin-Neuraminidase Membrane-Proximal Stalk Region Influences Stability, Receptor Binding, and Neuraminidase Activity

    PubMed Central

    Adu-Gyamfi, Emmanuel; Kim, Lori S.; Jardetzky, Theodore S.

    2016-01-01

    ABSTRACT Paramyxoviridae consist of a large family of enveloped, negative-sense, nonsegmented single-stranded RNA viruses that account for a significant number of human and animal diseases. The fusion process for nearly all paramyxoviruses involves the mixing of the host cell plasma membrane and the virus envelope in a pH-independent fashion. Fusion is orchestrated via the concerted action of two surface glycoproteins: an attachment protein called hemagglutinin-neuraminidase (HN [also called H or G depending on virus type and substrate]), which acts as a receptor binding protein, and a fusion (F) protein, which undergoes a major irreversible refolding process to merge the two membranes. Recent biochemical evidence suggests that receptor binding by HN is dispensable for cell-cell fusion. However, factors that influence the stability and/or conformation of the HN 4-helix bundle (4HB) stalk have not been studied. Here, we used oxidative cross-linking as well as functional assays to investigate the role of the structurally unresolved membrane-proximal stalk region (MPSR) (residues 37 to 58) of HN in the context of headless and full-length HN membrane fusion promotion. Our data suggest that the receptor binding head serves to stabilize the stalk to regulate fusion. Moreover, we found that the MPSR of HN modulates receptor binding and neuraminidase activity without a corresponding regulation of F triggering. IMPORTANCE Paramyxoviruses require two viral membrane glycoproteins, the attachment protein variously called HN, H, or G and the fusion protein (F), to couple host receptor recognition to virus-cell fusion. The HN protein has a globular head that is attached to a membrane-anchored flexible stalk of ∼80 residues and has three activities: receptor binding, neuraminidase, and fusion activation. In this report, we have identified the functional significance of the membrane-proximal stalk region (MPSR) (HN, residues 37 to 56) of the paramyxovirus parainfluenza virus

  7. Improvement in the long-term stability of an amperometric glucose sensor system by introducing a cellulose membrane of bacterial origin.

    PubMed

    Ammon, H P; Ege, W; Oppermann, M; Gŏpel, W; Eisele, S

    1995-01-15

    Classical amperometric glucose sensors that use cellulose membranes of wood origin (Cuprophan) suffer from the fact that their long-term stability in blood is short; therefore, their clinical use is limited. In the present study, a classical amperometric glucose sensor was covered with a bacterial cellulose (BC) membrane. Its surface in comparison to that of the classical glucose sensor (Cuprophan) and its long-term stability were tested in vitro and in vivo. The surface element composition was approximately 44% oxygen and approximately 56% carbon in both membranes and thus typical for cellulose. BC membranes exhibited fiber structure, whereas cup membranes did not. There was also a qualitative difference in protein adsorption between both membranes on exposure to bovine serum albumin. Treatment with Trogamid of one site of the BC membranes allowed linear glucose detection between 0 and 40 mM. Hemocompatibility of BC membranes was improved in comparison to cup membranes on the basis of complement activation (C3a and C5a). In diluted blood (1:10), the BC-covered sensor exhibited a long-term stability of more than 200 h; in undiluted blood it was stable for about 24 h, which is about 6-7 times longer than the stability of the classical Cup membrane-covered sensor. In in vivo studies, where the BC membrane-covered sensors were connected to the jugular vein of rats, blood glucose levels could be monitored for at least 24 h. In summary, the use of a modified bacterial cellulose membrane to cover the classical amperometric glucose sensor significantly improves the sensor's long-term stability both in vitro and in vivo.

  8. Structure of anti-FLAG M2 Fab domain and its use in the stabilization of engineered membrane proteins

    SciTech Connect

    Roosild, Tarmo P.; Castronovo, Samantha; Choe, Senyon

    2006-09-01

    The X-ray crystallographic analysis of anti-FLAG M2 Fab is reported and the implications of the structure on FLAG epitope binding are described as a first step in the development of a tool for the structural and biophysical study of membrane proteins. The inherent difficulties of stabilizing detergent-solubilized integral membrane proteins for biophysical or structural analysis demand the development of new methodologies to improve success rates. One proven strategy is the use of antibody fragments to increase the ‘soluble’ portion of any membrane protein, but this approach is limited by the difficulties and expense associated with producing monoclonal antibodies to an appropriate exposed epitope on the target protein. Here, the stabilization of a detergent-solubilized K{sup +} channel protein, KvPae, by engineering a FLAG-binding epitope into a known loop region of the protein and creating a complex with Fab fragments from commercially available anti-FLAG M2 monoclonal antibodies is reported. Although well diffracting crystals of the complex have not yet been obtained, during the course of crystallization trials the structure of the anti-FLAG M2 Fab domain was solved to 1.86 Å resolution. This structure, which should aid future structure-determination efforts using this approach by facilitating molecular-replacement phasing, reveals that the binding pocket appears to be specific only for the first four amino acids of the traditional FLAG epitope, namely DYKD. Thus, the use of antibody fragments for improving the stability of target proteins can be rapidly applied to the study of membrane-protein structure by placing the short DKYD motif within a predicted peripheral loop of that protein and utilizing commercially available anti-FLAG M2 antibody fragments.

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

    PubMed Central

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

    2015-01-01

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

  10. Lysosomal Storage Disorders in the Newborn

    PubMed Central

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

    2009-01-01

    Lysosomal storage disorders are rare inborn errors of metabolism, with a combined incidence of 1 in 1500 to 7000 live births. These relatively rare disorders are seldom considered when evaluating a sick newborn. A significant number of the >50 different lysosomal storage disorders, however, do manifest in the neonatal period and should be part of the differential diagnosis of several perinatal phenotypes. We review the earliest clinical features, diagnostic tests, and treatment options for lysosomal storage disorders that can present in the newborn. Although many of the lysosomal storage disorders are characterized by a range in phenotypes, the focus of this review is on the specific symptoms and clinical findings that present in the perinatal period, including neurologic, respiratory, endocrine, and cardiovascular manifestations, dysmorphic features, hepatosplenomegaly, skin or ocular involvement, and hydrops fetalis/congenital ascites. A greater awareness of these features may help to reduce misdiagnosis and promote the early detection of lysosomal storage disorders. Implementing therapy at the earliest stage possible is crucial for several of the lysosomal storage disorders; hence, an early appreciation of these disorders by physicians who treat newborns is essential. PMID:19336380

  11. Phototoxic effects of lysosome-associated genetically encoded photosensitizer KillerRed

    NASA Astrophysics Data System (ADS)

    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.

  12. Comparing the short and long term stability of biodegradable, ceramic and cation exchange membranes in microbial fuel cells.

    PubMed

    Winfield, Jonathan; Chambers, Lily D; Rossiter, Jonathan; Ieropoulos, Ioannis

    2013-11-01

    The long and short-term stability of two porous dependent ion exchange materials; starch-based compostable bags (BioBag) and ceramic, were compared to commercially available cation exchange membrane (CEM) in microbial fuel cells. Using bi-directional polarisation methods, CEM exhibited power overshoot during the forward sweep followed by significant power decline over the reverse sweep (38%). The porous membranes displayed no power overshoot with comparably smaller drops in power during the reverse sweep (ceramic 8%, BioBag 5.5%). The total internal resistance at maximum power increased by 64% for CEM compared to 4% (ceramic) and 6% (BioBag). Under fixed external resistive loads, CEM exhibited steeper pH reductions than the porous membranes. Despite its limited lifetime, the BioBag proved an efficient material for a stable microbial environment until failing after 8 months, due to natural degradation. These findings highlight porous separators as ideal candidates for advancing MFC technology in terms of cost and operation stability.

  13. De novo design of transmembrane helix-helix interactions and measurement of stability in a biological membrane.

    PubMed

    Nash, Anthony; Notman, Rebecca; Dixon, Ann M

    2015-05-01

    Membrane proteins regulate a large number of cellular functions, and have great potential as tools for manipulation of biological systems. Developing these tools requires a robust and quantitative understanding of membrane protein folding and interactions within the bilayer. With this in mind, we have designed a series of proteins to probe the net thermodynamic contribution of well-known sequence motifs to transmembrane helix-helix association in a biological membrane. The proteins were designed from first principles (de novo) using current knowledge about membrane insertion and stabilizing interaction motifs. A simple poly-Leu "scaffold" was decorated with individual helix interaction motifs (G-XXX-G, polar residues, heptad repeat) to create transmembrane helix-helix interactions of increasing strength. The GALLEX assay, an in vivo assay for measurement of transmembrane helix self-association, was combined with computational methods to characterize the relative strength and mode of interaction for each sequence. In addition, the apparent free energy contribution (ΔΔGapp) of each motif to transmembrane helix self-association was measured in a biological membrane, results that are the first of their kind for these de novo designed sequences, and suggest that the free energy barrier to overcoming weak association is quite small (<1.4 kcal mol(-1)) in a natural membrane. By quantifying and rationalizing the contribution of key motifs to transmembrane helix association, our work offers a route to direct the design of novel sequences for use in biotechnology or synthetic biology (e.g. molecular switches) and to predict the effects of sequence modification in known transmembrane domains (for control of cellular processes).

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

    PubMed Central

    Oh, Doo-Byoung

    2015-01-01

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

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

    PubMed Central

    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

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

    PubMed

    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.

  17. A novel approach to analyze lysosomal dysfunctions through subcellular proteomics and lipidomics: the case of NPC1 deficiency

    NASA Astrophysics Data System (ADS)

    Tharkeshwar, Arun Kumar; Trekker, Jesse; Vermeire, Wendy; Pauwels, Jarne; Sannerud, Ragna; Priestman, David A.; Te Vruchte, Danielle; Vints, Katlijn; Baatsen, Pieter; Decuypere, Jean-Paul; Lu, Huiqi; Martin, Shaun; Vangheluwe, Peter; Swinnen, Johannes V.; Lagae, Liesbet; Impens, Francis; Platt, Frances M.; Gevaert, Kris; Annaert, Wim

    2017-01-01

    Superparamagnetic iron oxide nanoparticles (SPIONs) have mainly been used as cellular carriers for genes and therapeutic products, while their use in subcellular organelle isolation remains underexploited. We engineered SPIONs targeting distinct subcellular compartments. Dimercaptosuccinic acid-coated SPIONs are internalized and accumulate in late endosomes/lysosomes, while aminolipid-SPIONs reside at the plasma membrane. These features allowed us to establish standardized magnetic isolation procedures for these membrane compartments with a yield and purity permitting proteomic and lipidomic profiling. We validated our approach by comparing the biomolecular compositions of lysosomes and plasma membranes isolated from wild-type and Niemann-Pick disease type C1 (NPC1) deficient cells. While the accumulation of cholesterol and glycosphingolipids is seen as a primary hallmark of NPC1 deficiency, our lipidomics analysis revealed the buildup of several species of glycerophospholipids and other storage lipids in selectively late endosomes/lysosomes of NPC1-KO cells. While the plasma membrane proteome remained largely invariable, we observed pronounced alterations in several proteins linked to autophagy and lysosomal catabolism reflecting vesicular transport obstruction and defective lysosomal turnover resulting from NPC1 deficiency. Thus the use of SPIONs provides a major advancement in fingerprinting subcellular compartments, with an increased potential to identify disease-related alterations in their biomolecular compositions.

  18. A novel approach to analyze lysosomal dysfunctions through subcellular proteomics and lipidomics: the case of NPC1 deficiency

    PubMed Central

    Tharkeshwar, Arun Kumar; Trekker, Jesse; Vermeire, Wendy; Pauwels, Jarne; Sannerud, Ragna; Priestman, David A.; te Vruchte, Danielle; Vints, Katlijn; Baatsen, Pieter; Decuypere, Jean-Paul; Lu, Huiqi; Martin, Shaun; Vangheluwe, Peter; Swinnen, Johannes V.; Lagae, Liesbet; Impens, Francis; Platt, Frances M.; Gevaert, Kris; Annaert, Wim

    2017-01-01

    Superparamagnetic iron oxide nanoparticles (SPIONs) have mainly been used as cellular carriers for genes and therapeutic products, while their use in subcellular organelle isolation remains underexploited. We engineered SPIONs targeting distinct subcellular compartments. Dimercaptosuccinic acid-coated SPIONs are internalized and accumulate in late endosomes/lysosomes, while aminolipid-SPIONs reside at the plasma membrane. These features allowed us to establish standardized magnetic isolation procedures for these membrane compartments with a yield and purity permitting proteomic and lipidomic profiling. We validated our approach by comparing the biomolecular compositions of lysosomes and plasma membranes isolated from wild-type and Niemann-Pick disease type C1 (NPC1) deficient cells. While the accumulation of cholesterol and glycosphingolipids is seen as a primary hallmark of NPC1 deficiency, our lipidomics analysis revealed the buildup of several species of glycerophospholipids and other storage lipids in selectively late endosomes/lysosomes of NPC1-KO cells. While the plasma membrane proteome remained largely invariable, we observed pronounced alterations in several proteins linked to autophagy and lysosomal catabolism reflecting vesicular transport obstruction and defective lysosomal turnover resulting from NPC1 deficiency. Thus the use of SPIONs provides a major advancement in fingerprinting subcellular compartments, with an increased potential to identify disease-related alterations in their biomolecular compositions. PMID:28134274

  19. A novel approach to analyze lysosomal dysfunctions through subcellular proteomics and lipidomics: the case of NPC1 deficiency.

    PubMed

    Tharkeshwar, Arun Kumar; Trekker, Jesse; Vermeire, Wendy; Pauwels, Jarne; Sannerud, Ragna; Priestman, David A; Te Vruchte, Danielle; Vints, Katlijn; Baatsen, Pieter; Decuypere, Jean-Paul; Lu, Huiqi; Martin, Shaun; Vangheluwe, Peter; Swinnen, Johannes V; Lagae, Liesbet; Impens, Francis; Platt, Frances M; Gevaert, Kris; Annaert, Wim

    2017-01-30

    Superparamagnetic iron oxide nanoparticles (SPIONs) have mainly been used as cellular carriers for genes and therapeutic products, while their use in subcellular organelle isolation remains underexploited. We engineered SPIONs targeting distinct subcellular compartments. Dimercaptosuccinic acid-coated SPIONs are internalized and accumulate in late endosomes/lysosomes, while aminolipid-SPIONs reside at the plasma membrane. These features allowed us to establish standardized magnetic isolation procedures for these membrane compartments with a yield and purity permitting proteomic and lipidomic profiling. We validated our approach by comparing the biomolecular compositions of lysosomes and plasma membranes isolated from wild-type and Niemann-Pick disease type C1 (NPC1) deficient cells. While the accumulation of cholesterol and glycosphingolipids is seen as a primary hallmark of NPC1 deficiency, our lipidomics analysis revealed the buildup of several species of glycerophospholipids and other storage lipids in selectively late endosomes/lysosomes of NPC1-KO cells. While the plasma membrane proteome remained largely invariable, we observed pronounced alterations in several proteins linked to autophagy and lysosomal catabolism reflecting vesicular transport obstruction and defective lysosomal turnover resulting from NPC1 deficiency. Thus the use of SPIONs provides a major advancement in fingerprinting subcellular compartments, with an increased potential to identify disease-related alterations in their biomolecular compositions.

  20. Nanoporous membrane robustness / stability in small form factor microfluidic filtration system.

    PubMed

    Johnson, Dean G; Pan, Sabrina; Hayden, Andrew; McGrath, James L

    2016-08-01

    The development of wearable hemodialysis (HD) devices that replace center-based HD holds the promise to improve both outcomes and quality-of-life for patients with end-stage-renal disease (ERD). A prerequisite for these devices is the development of highly efficient membranes that can achieve high toxin clearance in small footprints. The ultrathin nanoporous membrane material developed by our group is orders of magnitude more permeable than conventional HD membranes. We report on our progress making a prototype wearable dialysis unit. First, we present data from benchtop studies confirming that clinical levels of urea clearance can be obtained in a small animal model with low blood flow rates. Second, we report on efforts to improve the mechanical robustness of high membrane area dialysis devices.

  1. A semi-interpenetrating network approach for dimensionally stabilizing highly-charged anion exchange membranes for alkaline fuel cells.

    PubMed

    He, Steve S; Strickler, Alaina L; Frank, Curtis W

    2015-04-24

    There is a delicate balance between ion exchange capacity (IEC), conductivity, and dimensional stability in anion exchange membranes as higher charge content can lead to increased water uptake, causing excessive swelling and charge dilution. Using highly-charged benzyltrimethylammonium polysulfone (IEC=2.99 mEq g(-1) ) as a benchmark (which ruptured in water even at room temperature), we report the ability to dramatically decrease water uptake using a semi-interpenetrating network wherein we reinforced the linear polyelectrolyte with a crosslinked poly(styrene-co-divinylbenzene) network. These membranes show enhanced dimensional stability as a result of lower water uptake (75 % vs. 301 % at 25 °C) while maintaining excellent hydroxide conductivity (up to 50 mS cm(-1) at 25 °C). These improvements produced an enhanced alkaline fuel cell capable of generating 236 mW cm(-2) peak power density at 80 °C. This method is easily adaptable and can be a viable strategy for stabilizing existing systems.

  2. Mycosins Are Required for the Stabilization of the ESX-1 and ESX-5 Type VII Secretion Membrane Complexes

    PubMed Central

    van Winden, Vincent J. C.; Ummels, Roy; Piersma, Sander R.; Jiménez, Connie R.; Korotkov, Konstantin V.; Bitter, Wilbert

    2016-01-01

    ABSTRACT Pathogenic mycobacteria contain up to five type VII secretion (T7S) systems, ESX-1 to ESX-5. One of the conserved T7S components is the serine protease mycosin (MycP). Strikingly, whereas MycP is essential for secretion, the protease activity of MycP1 in Mycobacterium tuberculosis has been shown to be dispensable for secretion. The essential role of MycP therefore remains unclear. Here we show that MycP1 and MycP5 of M. marinum have similar phenotypes, confirming that MycP has a second unknown function that is essential for its T7S system. To investigate whether this role is related to proper functioning of the T7S membrane complex, we first analyzed the composition of the ESX-1 membrane complex and showed that this complex consists of EccBCDE1, similarly to what was previously shown for ESX-5. Surprisingly, while mycosins are not an integral part of these purified core complexes, we noticed that the stability of both the ESX-1 complex and the ESX-5 complex is compromised in the absence of their MycP subunit. Additional interaction studies showed that, although mycosins are not part of the central ESX membrane complex, they loosely associate with this complex. We hypothesize that this MycP association with the core membrane complex is crucial for the integrity and functioning of the T7S machinery. PMID:27795391

  3. Innexin7a forms junctions that stabilize the basal membrane during cellularization of the blastoderm in Tribolium castaneum.

    PubMed

    van der Zee, Maurijn; Benton, Matthew A; Vazquez-Faci, Tania; Lamers, Gerda E M; Jacobs, Chris G C; Rabouille, Catherine

    2015-06-15

    In insects, the fertilized egg undergoes a series of rapid nuclear divisions before the syncytial blastoderm starts to cellularize. Cellularization has been extensively studied in Drosophila melanogaster, but its thick columnar blastoderm is unusual among insects. We therefore set out to describe cellularization in the beetle Tribolium castaneum, the embryos of which exhibit a thin blastoderm of cuboidal cells, like most insects. Using immunohistochemistry, live imaging and transmission electron microscopy, we describe several striking differences to cellularization in Drosophila, including the formation of junctions between the forming basal membrane and the yolk plasmalemma. To identify the nature of this novel junction, we used the parental RNAi technique for a small-scale screen of junction proteins. We find that maternal knockdown of Tribolium innexin7a (Tc-inx7a), an ortholog of the Drosophila gap junction gene Innexin 7, leads to failure of cellularization. In Inx7a-depleted eggs, the invaginated plasma membrane retracts when basal cell closure normally begins. Furthermore, transiently expressed tagged Inx7a localizes to the nascent basal membrane of the forming cells in wild-type eggs. We propose that Inx7a forms the newly identified junctions that stabilize the forming basal membrane and enable basal cell closure. We put forward Tribolium as a model for studying a more ancestral mode of cellularization in insects.

  4. Thrombin-induced lysosomal exocytosis in human platelets is dependent on secondary activation by ADP and regulated by endothelial-derived substances.

    PubMed

    Södergren, Anna L; Svensson Holm, Ann-Charlotte B; Ramström, Sofia; Lindström, Eva G; Grenegård, Magnus; Öllinger, Karin

    2016-01-01

    Exocytosis of lysosomal contents from platelets has been speculated to participate in clearance of thrombi and vessel wall remodelling. The mechanisms that regulate lysosomal exocytosis in platelets are, however, still unclear. The aim of this study was to identify the pathways underlying platelet lysosomal secretion and elucidate how this process is controlled by platelet inhibitors. We found that high concentrations of thrombin induced partial lysosomal exocytosis as assessed by analysis of the activity of released N-acetyl-β-glucosaminidase (NAG) and by identifying the fraction of platelets exposing the lysosomal-associated membrane protein (LAMP)-1 on the cell surface by flow cytometry. Stimulation of thrombin receptors PAR1 or PAR4 with specific peptides was equally effective in inducing LAMP-1 surface expression. Notably, lysosomal exocytosis in response to thrombin was significantly reduced if the secondary activation by ADP was inhibited by the P2Y12 antagonist cangrelor, while inhibition of thromboxane A2 formation by treatment with acetylsalicylic acid was of minor importance in this regard. Moreover, the NO-releasing drug S-nitroso-N-acetyl penicillamine (SNAP) or the cyclic AMP-elevating eicosanoid prostaglandin I2 (PGI2) significantly suppressed lysosomal exocytosis. We conclude that platelet inhibitors that mimic functional endothelium such as PGI2 or NO efficiently counteract lysosomal exocytosis. Furthermore, we suggest that secondary release of ADP and concomitant signaling via PAR1/4- and P2Y12 receptors is important for efficient platelet lysosomal exocytosis by thrombin.

  5. Mutant Huntingtin Impairs Post-Golgi Trafficking to Lysosomes by Delocalizing Optineurin/Rab8 Complex from the Golgi Apparatus

    PubMed Central

    del Toro, Daniel; Alberch, Jordi; Lázaro-Diéguez, Francisco; Martín-Ibáñez, Raquel; Xifró, Xavier; Egea, Gustavo

    2009-01-01

    Huntingtin regulates post-Golgi trafficking of secreted proteins. Here, we studied the mechanism by which mutant huntingtin impairs this process. Colocalization studies and Western blot analysis of isolated Golgi membranes showed a reduction of huntingtin in the Golgi apparatus of cells expressing mutant huntingtin. These findings correlated with a decrease in the levels of optineurin and Rab8 in the Golgi apparatus that can be reverted by overexpression of full-length wild-type huntingtin. In addition, immunoprecipitation studies showed reduced interaction between mutant huntingtin and optineurin/Rab8. Cells expressing mutant huntingtin produced both an accumulation of clathrin adaptor complex 1 at the Golgi and an increase of clathrin-coated vesicles in the vicinity of Golgi cisternae as revealed by electron microscopy. Furthermore, inverse fluorescence recovery after photobleaching analysis for lysosomal-associated membrane protein-1 and mannose-6-phosphate receptor showed that the optineurin/Rab8-dependent post-Golgi trafficking to lysosomes was impaired in cells expressing mutant huntingtin or reducing huntingtin levels by small interfering RNA. Accordingly, these cells showed a lower content of cathepsin D in lysosomes, which led to an overall reduction of lysosomal activity. Together, our results indicate that mutant huntingtin perturbs post-Golgi trafficking to lysosomal compartments by delocalizing the optineurin/Rab8 complex, which, in turn, affects the lysosomal function. PMID:19144827

  6. Ii Chain Controls the Transport of Major Histocompatibility Complex Class II Molecules to and from Lysosomes

    PubMed Central

    Brachet, Valérie; Raposo, Graça; Amigorena, Sebastian; Mellman, Ira

    1997-01-01

    Major histocompatibility complex class II molecules are synthesized as a nonameric complex consisting of three αβ dimers associated with a trimer of invariant (Ii) chains. After exiting the TGN, a targeting signal in the Ii chain cytoplasmic domain directs the complex to endosomes where Ii chain is proteolytically processed and removed, allowing class II molecules to bind antigenic peptides before reaching the cell surface. Ii chain dissociation and peptide binding are thought to occur in one or more postendosomal sites related either to endosomes (designated CIIV) or to lysosomes (designated MIIC). We now find that in addition to initially targeting αβ dimers to endosomes, Ii chain regulates the subsequent transport of class II molecules. Under normal conditions, murine A20 B cells transport all of their newly synthesized class II I-Ab αβ dimers to the plasma membrane with little if any reaching lysosomal compartments. Inhibition of Ii processing by the cysteine/serine protease inhibitor leupeptin, however, blocked transport to the cell surface and caused a dramatic but selective accumulation of I-Ab class II molecules in lysosomes. In leupeptin, I-Ab dimers formed stable complexes with a 10-kD NH2-terminal Ii chain fragment (Ii-p10), normally a transient intermediate in Ii chain processing. Upon removal of leupeptin, Ii-p10 was degraded and released, I-Ab dimers bound antigenic peptides, and the peptide-loaded dimers were transported slowly from lysosomes to the plasma membrane. Our results suggest that alterations in the rate or efficiency of Ii chain processing can alter the postendosomal sorting of class II molecules, resulting in the increased accumulation of αβ dimers in lysosome-like MIIC. Thus, simple differences in Ii chain processing may account for the highly variable amounts of class II found in lysosomal compartments of different cell types or at different developmental stages. PMID:9105036

  7. Clathrin heavy chain functions in sorting and secretion of lysosomal enzymes in Dictyostelium discoideum

    PubMed Central

    1994-01-01

    The clathrin heavy chain is a major component of clathrin-coated vesicles that function in selective membrane traffic in eukaryotic cells. We disrupted the clathrin heavy chain gene (chcA) in Dictyostelium discoideum to generate a stable clathrin heavy chain- deficient cell line. Measurement of pinocytosis in the clathrin-minus mutant revealed a four-to five-fold deficiency in the internalization of fluid-phase markers. Once internalized, these markers recycled to the cell surface of mutant cells at wild-type rates. We also explored the involvement of clathrin heavy chain in the trafficking of lysosomal enzymes. Pulse chase analysis revealed that clathrin-minus cells processed most alpha-mannosidase to mature forms, however, approximately 20-25% of the precursor molecules remained uncleaved, were missorted, and were rapidly secreted by the constitutive secretory pathway. The remaining intracellular alpha-mannosidase was successfully targeted to mature lysosomes. Standard secretion assays showed that the rate of secretion of alpha-mannosidase was significantly less in clathrin-minus cells compared to control cells in growth medium. Interestingly, the secretion rates of another lysosomal enzyme, acid phosphatase, were similar in clathrin-minus and wild-type cells. Like wild-type cells, clathrin-minus mutants responded to starvation conditions with increased lysosomal enzyme secretion. Our study of the mutant cells provide in vivo evidence for roles for the clathrin heavy chain in (a) the internalization of fluid from the plasma membrane; (b) sorting of hydrolase precursors from the constitutive secretory pathway to the lysosomal pathway; and (c) secretion of mature hydrolases from lysosomes to the extracellular space. PMID:8034739

  8. Lysosomal and mitochondrial permeabilization mediates zinc(II) cationic phthalocyanine phototoxicity.

    PubMed

    Marino, Julieta; García Vior, María C; Furmento, Verónica A; Blank, Viviana C; Awruch, Josefina; Roguin, Leonor P

    2013-11-01

    In order to find a novel photosensitizer to be used in photodynamic therapy for cancer treatment, we have previously showed that the cationic zinc(II) phthalocyanine named Pc13, the sulfur-linked dye 2,9(10),16(17),23(24)-tetrakis[(2-trimethylammonium) ethylsulfanyl]phthalocyaninatozinc(II) tetraiodide, exerts a selective phototoxic effect on human nasopharynx KB carcinoma cells and induces an apoptotic response characterized by an increase in the activity of caspase-3. Since the activation of an apoptotic pathway by chemotherapeutic agents contributes to the elimination of malignant cells, in this study we investigated the molecular mechanisms underlying the antitumor action of Pc13. We found that after light exposure, Pc13 induced the production of reactive oxygen species (ROS), which are mediating the resultant cytotoxic action on KB cells. ROS led to an early permeabilization of lysosomal membranes as demonstrated by the reduction of lysosome fluorescence with acridine orange and the release of lysosomal proteases to cytosol. Treatment with antioxidants inhibited ROS generation, preserved the integrity of lysosomal membrane and increased cell proliferation in a concentration-dependent manner. Lysosome disruption was followed by mitochondrial depolarization, cytosolic release of cytochrome C and caspases activation. Although no change in the total amount of Bax was observed, the translocation of Bax from cytosol to mitochondria, the cleavage of the pro-apoptotic protein Bid, together with the decrease of the anti-apoptotic proteins Bcl-XL and Bcl-2 indicated the involvement of Bcl-2 family proteins in the induction of the mitochondrial pathway. It was also demonstrated that cathepsin D, but not caspase-8, contributed to Bid cleavage. In conclusion, Pc13-induced cell photodamage is triggered by ROS generation and activation of the mitochondrial apoptotic pathway through the release of lysosomal proteases. In addition, our results also indicated that Pc13 induced

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

    PubMed Central

    Colombo, Federico; Trombetta, Elena; Cetrangolo, Paola; Maggioni, Marco; Razini, Paola; De Santis, Francesca; Torrente, Yvan; Prati, Daniele; Torresani, Erminio; Porretti, Laura

    2014-01-01

    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. PMID:25493932

  10. HIV-1 Tat Promotes Lysosomal Exocytosis in Astrocytes and Contributes to Astrocyte-mediated Tat Neurotoxicity.

    PubMed

    Fan, Yan; He, Johnny J

    2016-10-21

    Tat interaction with astrocytes has been shown to be important for Tat neurotoxicity and HIV/neuroAIDS. We have recently shown that Tat expression leads to increased glial fibrillary acidic protein (GFAP) expression and aggregation and activation of unfolded protein response/endoplasmic reticulum (ER) stress in astrocytes and causes neurotoxicity. However, the exact molecular mechanism of astrocyte-mediated Tat neurotoxicity is not defined. In this study, we showed that neurotoxic factors other than Tat protein itself were present in the supernatant of Tat-expressing astrocytes. Two-dimensional gel electrophoresis and mass spectrometry revealed significantly elevated lysosomal hydrolytic enzymes and plasma membrane-associated proteins in the supernatant of Tat-expressing astrocytes. We confirmed that Tat expression and infection of pseudotyped HIV.GFP led to increased lysosomal exocytosis from mouse astrocytes and human astrocytes. We found that Tat-induced lysosomal exocytosis was tightly coupled to astrocyte-mediated Tat neurotoxicity. In addition, we demonstrated that Tat-induced lysosomal exocytosis was astrocyte-specific and required GFAP expression and was mediated by ER stress. Taken together, these results show for the first time that Tat promotes lysosomal exocytosis in astrocytes and causes neurotoxicity through GFAP activation and ER stress induction in astrocytes and suggest a common cascade through which aberrant astrocytosis/GFAP up-regulation potentiates neurotoxicity and contributes to neurodegenerative diseases.

  11. LAMP-2 is required for incorporating syntaxin-17 into autophagosomes and for their fusion with lysosomes

    PubMed Central

    Peschel, Andrea; Langer, Brigitte; Gröger, Marion; Rees, Andrew; Kain, Renate

    2016-01-01

    ABSTRACT Autophagy is an evolutionarily conserved process used for removing surplus and damaged proteins and organelles from the cytoplasm. The unwanted material is incorporated into autophagosomes that eventually fuse with lysosomes, leading to the degradation of their cargo. The fusion event is mediated by the interaction between the Qa-SNARE syntaxin-17 (STX17) on autophagosomes and the R-SNARE VAMP8 on lysosomes. Cells deficient in lysosome membrane-associated protein-2 (LAMP-2) have increased numbers of autophagosomes but the underlying mechanism is poorly understood. By transfecting LAMP-2-deficient and LAMP-1/2­-double-deficient mouse embryonic fibroblasts (MEFs) with a tandem fluorescent-tagged LC3 we observed a failure of fusion between the autophagosomes and the lysosomes that could be rescued by complementation with LAMP-2A. Although we observed no change in expression and localization of VAMP8, its interacting partner STX17 was absent from autophagosomes of LAMP-2-deficient cells. Thus, LAMP-2 is essential for STX17 expression by the autophagosomes and this absence is sufficient to explain their failure to fuse with lysosomes. The results have clear implications for situations associated with a reduction of LAMP-2 expression. PMID:27628032

  12. An essential role for functional lysosomes in ferroptosis of cancer cells.

    PubMed

    Torii, Seiji; Shintoku, Ryosuke; Kubota, Chisato; Yaegashi, Makoto; Torii, Ryoko; Sasaki, Masaya; Suzuki, Toshinobu; Mori, Masanobu; Yoshimoto, Yuhei; Takeuchi, Toshiyuki; Yamada, Keiichi

    2016-03-15

    Pharmacological challenges to oncogenic Ras-expressing cancer cells have shown a novel type of cell death, ferroptosis, which requires intracellular iron. In the present study, we assessed ferroptosis following treatment of human fibrosarcoma HT1080 cells with several inhibitors of lysosomal activity and found that they prevented cell death induced by the ferroptosis-inducing compounds erastin and RSL3. Fluorescent analyses with a reactive oxygen species (ROS) sensor revealed constitutive generation of ROS in lysosomes, and treatment with lysosome inhibitors decreased both lysosomal ROS and a ferroptotic cell-death-associated ROS burst. These inhibitors partially prevented intracellular iron provision by attenuating intracellular transport of transferrin or autophagic degradation of ferritin. Furthermore, analyses with a fluorescent sensor that detects oxidative changes in cell membranes revealed that formation of lipid ROS in perinuclear compartments probably represented an early event in ferroptosis. These results suggest that lysosomal activity is involved in lipid ROS-mediated ferroptotic cell death through regulation of cellular iron equilibria and ROS generation.

  13. LAMP-2 is required for incorporating syntaxin-17 into autophagosomes and for their fusion with lysosomes.

    PubMed

    Hubert, Virginie; Peschel, Andrea; Langer, Brigitte; Gröger, Marion; Rees, Andrew; Kain, Renate

    2016-10-15

    Autophagy is an evolutionarily conserved process used for removing surplus and damaged proteins and organelles from the cytoplasm. The unwanted material is incorporated into autophagosomes that eventually fuse with lysosomes, leading to the degradation of their cargo. The fusion event is mediated by the interaction between the Qa-SNARE syntaxin-17 (STX17) on autophagosomes and the R-SNARE VAMP8 on lysosomes. Cells deficient in lysosome membrane-associated protein-2 (LAMP-2) have increased numbers of autophagosomes but the underlying mechanism is poorly understood. By transfecting LAMP-2-deficient and LAMP-1/2--double-deficient mouse embryonic fibroblasts (MEFs) with a tandem fluorescent-tagged LC3 we observed a failure of fusion between the autophagosomes and the lysosomes that could be rescued by complementation with LAMP-2A. Although we observed no change in expression and localization of VAMP8, its interacting partner STX17 was absent from autophagosomes of LAMP-2-deficient cells. Thus, LAMP-2 is essential for STX17 expression by the autophagosomes and this absence is sufficient to explain their failure to fuse with lysosomes. The results have clear implications for situations associated with a reduction of LAMP-2 expression.

  14. Nitric Oxide Deficiency Accelerates Chlorophyll Breakdown and Stability Loss of Thylakoid Membranes during Dark-Induced Leaf Senescence in Arabidopsis

    PubMed Central

    Liu, Fang; Guo, Fang-Qing

    2013-01-01

    Nitric oxide (NO) has been known to preserve the level of chlorophyll (Chl) during leaf senescence. However, the mechanism by which NO regulates Chl breakdown remains unknown. Here we report that NO negatively regulates the activities of Chl catabolic enzymes during dark-induced leaf senescence. The transcriptional levels of the major enzyme genes involving Chl breakdown pathway except for RED CHL CATABOLITE REDUCTASE (RCCR) were dramatically up-regulated during dark-induced Chl degradation in the leaves of Arabidopsis NO-deficient mutant nos1/noa1 that exhibited an early-senescence phenotype. The activity of pheide a oxygenase (PAO) was higher in the dark-induced senescent leaves of nos1/noa1 compared with wild type. Furthermore, the knockout of PAO in nos1/noa1 background led to pheide a accumulation in the double mutant pao1 nos1/noa1, which retained the level of Chl during dark-induced leaf senescence. The accumulated pheide a in darkened leaves of pao1 nos1/noa1 was likely to inhibit the senescence-activated transcriptional levels of Chl catabolic genes as a feed-back inhibitory effect. We also found that NO deficiency led to decrease in the stability of photosynthetic complexes in thylakoid membranes. Importantly, the accumulation of pheide a caused by PAO mutations in combination with NO deficiency had a synergistic effect on the stability loss of thylakoid membrane complexes in the double mutant pao1 nos1/noa1 during dark-induced leaf senescence. Taken together, our findings have demonstrated that NO is a novel negative regulator of Chl catabolic pathway and positively functions in maintaining the stability of thylakoid membranes during leaf senescence. PMID:23418559

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

    PubMed Central

    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

  16. On the edge energy of lipid membranes and the thermodynamic stability of pores

    SciTech Connect

    Pera, H.; Kleijn, J. M.; Leermakers, F. A. M.

    2015-01-21

    To perform its barrier function, the lipid bilayer membrane requires a robust resistance against pore formation. Using a self-consistent field (SCF) theory and a molecularly detailed model for membranes composed of charged or zwitterionic lipids, it is possible to predict structural, mechanical, and thermodynamical parameters for relevant lipid bilayer membranes. We argue that the edge energy in membranes is a function of the spontaneous lipid monolayer curvature, the mean bending modulus, and the membrane thickness. An analytical Helfrich-like model suggests that most bilayers should have a positive edge energy. This means that there is a natural resistance against pore formation. Edge energies evaluated explicitly in a two-gradient SCF model are consistent with this. Remarkably, the edge energy can become negative for phosphatidylglycerol (e.g., dioleoylphosphoglycerol) bilayers at a sufficiently low ionic strength. Such bilayers become unstable against the formation of pores or the formation of lipid disks. In the weakly curved limit, we study the curvature dependence of the edge energy and evaluate the preferred edge curvature and the edge bending modulus. The latter is always positive, and the former increases with increasing ionic strength. These results point to a small window of ionic strengths for which stable pores can form as too low ionic strengths give rise to lipid disks. Higher order curvature terms are necessary to accurately predict relevant pore sizes in bilayers. The electric double layer overlap across a small pore widens the window of ionic strengths for which pores are stable.

  17. Dynamics of membrane protein/amphipol association studied by Förster resonance energy transfer: implications for in vitro studies of amphipol-stabilized membrane proteins.

    PubMed

    Zoonens, Manuela; Giusti, Fabrice; Zito, Francesca; Popot, Jean-Luc

    2007-09-11

    Amphipols (APols) are short amphipathic polymers that can substitute for detergents to keep membrane proteins (MPs) water-soluble while stabilizing them biochemically. We have examined the factors that determine the size and dispersity of MP/APol complexes and studied the dynamics of the association, taking as a model system the transmembrane domain of Escherichia coli outer membrane protein A (tOmpA) trapped by A8-35, a polyacrylate-based APol. Molecular sieving indicates that the solution properties of the APol largely determine those of tOmpA/APol complexes. Achieving monodispersity depends on using amphipols that themselves form monodisperse particles, on working in neutral or basic solutions, and on the presence of free APols. In order to investigate the role of the latter, a fluorescently labeled version of A8-35 has been synthesized. Förster resonance energy transfer measurements show that extensive dilution of tOmpA/A8-35 particles into an APol-free medium does not entail any detectable desorption of A8-35, even after extended periods of time (hours-days). The fluorescent APol, on the other hand, readily exchanges for other surfactants, be they detergent or unlabeled APol. These findings are discussed in the contexts of sample optimization for MP solution studies and of APol-mediated MP functionalization.

  18. Regulation of high-voltage-activated Ca(2+) channel function, trafficking, and membrane stability by auxiliary subunits.

    PubMed

    Felix, Ricardo; Calderón-Rivera, Aida; Andrade, Arturo

    2013-09-01

    Voltage-gated Ca(2+) (CaV) channels mediate Ca(2+) ions influx into cells in response to depolarization of the plasma membrane. They are responsible for initiation of excitation-contraction and excitation-secretion coupling, and the Ca(2+) that enters cells through this pathway is also important in the regulation of protein phosphorylation, gene transcription, and many other intracellular events. Initial electrophysiological studies divided CaV channels into low-voltage-activated (LVA) and high-voltage-activated (HVA) channels. The HVA CaV channels were further subdivided into L, N, P/Q, and R-types which are oligomeric protein complexes composed of an ion-conducting CaVα1 subunit and auxiliary CaVα2δ, CaVβ, and CaVγ subunits. The functional consequences of the auxiliary subunits include altered functional and pharmacological properties of the channels as well as increased current densities. The latter observation suggests an important role of the auxiliary subunits in membrane trafficking of the CaVα1 subunit. This includes the mechanisms by which CaV channels are targeted to the plasma membrane and to appropriate regions within a given cell. Likewise, the auxiliary subunits seem to participate in the mechanisms that remove CaV channels from the plasma membrane for recycling and/or degradation. Diverse studies have provided important clues to the molecular mechanisms involved in the regulation of CaV channels by the auxiliary subunits, and the roles that these proteins could possibly play in channel targeting and membrane Stabilization.

  19. Lysosomal Function Is Involved in 17β-Estradiol-Induced Estrogen Receptor α Degradation and Cell Proliferation

    PubMed Central

    Marino, Maria; Acconcia, Filippo

    2014-01-01

    The homeostatic control of the cellular proteome steady-state is dependent either on the 26S proteasome activity or on the lysosome function. The sex hormone 17β-estradiol (E2) controls a plethora of biological functions by binding to the estrogen receptor α (ERα), which is both a nuclear ligand-activated transcription factor and also an extrinsic plasma membrane receptor. Regulation of E2-induced physiological functions (e.g., cell proliferation) requires the synergistic activation of both transcription of estrogen responsive element (ERE)-containing genes and rapid extra-nuclear phosphorylation of many different signalling kinases (e.g., ERK/MAPK; PI3K/AKT). Although E2 controls ERα intracellular content and activity via the 26S proteasome-mediated degradation, biochemical and microscopy-based evidence suggests a possible cross-talk among lysosomes and ERα activities. Here, we studied the putative localization of endogenous ERα to lysosomes and the role played by lysosomal function in ERα signalling. By using confocal microscopy and biochemical assays, we report that ERα localizes to lysosomes and to endosomes in an E2-dependent manner. Moreover, the inhibition of lysosomal function obtained by chloroquine demonstrates that, in addition to 26S proteasome-mediated receptor elimination, lysosome-based degradation also contributes to the E2-dependent ERα breakdown. Remarkably, the lysosome function is further involved in those ERα activities required for E2-dependent cell proliferation while it is dispensable for ERα-mediated ERE-containing gene transcription. Our discoveries reveal a novel lysosome-dependent degradation pathway for ERα and show a novel biological mechanism by which E2 regulates ERα cellular content and, as a consequence, cellular functions. PMID:24736371

  20. The role of lysosomes in BDE 47-mediated activation of mitochondrial apoptotic pathway in HepG2 cells.

    PubMed

    Liu, Xiaohui; Wang, Jian; Lu, Chengquan; Zhu, Chunyan; Qian, Bo; Li, Zhenwei; Liu, Chang; Shao, Jing; Yan, Jinsong

    2015-04-01

    Polybrominated diphenyl ethers (PBDEs) are a group of widely used flame retardants. The rising presence of PBDEs in human tissues has received considerable concerns with regard to potential health risks. While the mitochondrial-apoptotic pathway has been suggested in PBDEs-induced apoptosis, the role of lysosomes is yet to be understood. In the present study, HepG2 cells were exposed to BDE 47 at various concentrations and durations to establish the causal and temporal relationships among various cellular events, such as cell viability, reactive oxygen species (ROS), mitochondrial membrane potential (MMP), apoptosis, and expression of cytochrome C and caspase 3. The involvement of lysosomes was simultaneously studied by evaluating lysosomal membrane permeability (LMP) and changes in the expression of cathepsin B, a lysosome hydrolase. In addition, a cathepsin B inhibitor (10 μM CA-074) was used to determine the involvement of lysosomes and potential interactions between lysosomes and mitochondria. Our results showed that ROS production was an initial response of HepG2 to BDE 47 exposure, followed by a decreased MMP; a loss of MMP caused additional ROS generation which acted to induce LMP; an increased LMP resulted in a release of cathepsin B which aggravated the loss of MMP leading to release of cytochrome C and caspase 3 and subsequent apoptosis. Pretreatment with CA-074 did not abolish the initial ROS generation, however, all downstream events were dramatically alleviated. Taken together, our data indicate that lysosomes might be involved in BDE 47-mediated mitochondrial-apoptotic pathway in HepG2 cells, possibly through feedback interactions between mitochondria and lysosomes.

  1. Lysosomes can fuse with a late endosomal compartment in a cell-free system from rat liver

    PubMed Central

    1994-01-01

    The passage of pulse doses of asialoglycoproteins through the endosomal compartments of rat liver hepatocytes was studied by subcellular fractionation and EM. The kinetics of disappearance of radiolabeled asialofetuin from light endosomes prepared on Ficoll gradients were the same as the kinetics of disappearance of asialoorosomucoid-horse radish peroxidase reaction products from intracellular membrane-bound structures in the blood sinusoidal regions of hepatocytes. The light endosomes were therefore identifiable as being derived from the peripheral early endosome compartment. In contrast, the labeling of dense endosomes from the middle of the Ficoll gradient correlated with EM showing large numbers of reaction product-containing structures in the nonsinusoidal parts of the hepatocyte. In cell-free, postmitochondrial supernatants, we have previously observed that dense endosomes, but not light endosomes, interact with lysosomes. Cell-free interaction between isolated dense endosomes and lysosomes has now been reconstituted and analyzed in three ways: by transfer of radiolabeled ligand from endosomal to lysosomal densities, by a fluorescence dequenching assay which can indicate membrane fusion, and by measurement of content mixing. Maximum transfer of radiolabel to lysosomal densities required ATP and GTP plus cytosolic components, including N-ethylmaleimide-sensitive factor(s). Dense endosomes incubated in the absence of added lysosomes did not mature into vesicles of lysosomal density. Content mixing, and hence fusion, between endosomes and lysosomes was maximal in the presence of cytosol and ATP and also showed inhibition by N-ethyl-maleimide. Thus, we have demonstrated that a fusion step is involved in the transfer of radiolabeled ligand from an isolated endosome fraction derived from the nonsinusoidal regions of the hepatocyte to preexisting lysosomes in a cell-free system. PMID:7520447

  2. Defective macroautophagic turnover of brain lipids in the TgCRND8 Alzheimer mouse model: prevention by correcting lysosomal proteolytic deficits.

    PubMed

    Yang, Dun-Sheng; Stavrides, Philip; Saito, Mitsuo; Kumar, Asok; Rodriguez-Navarro, Jose A; Pawlik, Monika; Huo, Chunfeng; Walkley, Steven U; Saito, Mariko; Cuervo, Ana M; Nixon, Ralph A

    2014-12-01

    Autophagy, the major lysosomal pathway for the turnover of intracellular organelles is markedly impaired in neurons in Alzheimer's disease and Alzheimer mouse models. We have previously reported that severe lysosomal and amyloid neuropathology and associated cognitive deficits in the TgCRND8 Alzheimer mouse model can be ameliorated by restoring lysosomal proteolytic capacity and autophagy flux via genetic deletion of the lysosomal protease inhibitor, cystatin B. Here we present evidence that macroautophagy is a significant pathway for lipid turnover, which is defective in TgCRND8 brain where lipids accumulate as membranous structures and lipid droplets within giant neuronal autolysosomes. Levels of multiple lipid species including several sphingolipids (ceramide, ganglioside GM3, GM2, GM1, GD3 and GD1a), cardiolipin, cholesterol and cholesteryl esters are elevated in autophagic vacuole fractions and lysosomes isolated from TgCRND8 brain. Lipids are localized in autophagosomes and autolysosomes by double immunofluorescence analyses in wild-type mice and colocalization is increased in TgCRND8 mice where abnormally abundant GM2 ganglioside-positive granules are detected in neuronal lysosomes. Cystatin B deletion in TgCRND8 significantly reduces the number of GM2-positive granules and lowers the levels of GM2 and GM3 in lysosomes, decreases lipofuscin-related autofluorescence, and eliminates giant lipid-containing autolysosomes while increasing numbers of normal-sized autolysosomes/lysosomes with reduced content of undigested components. These findings have identified macroautophagy as a previously unappreciated route for delivering membrane lipids to lysosomes for turnover, a function that has so far been considered to be mediated exclusively through the endocytic pathway, and revealed that autophagic-lysosomal dysfunction in TgCRND8 brain impedes lysosomal turnover of lipids as well as proteins. The amelioration of lipid accumulation in TgCRND8 by removing cystatin B

  3. Lysosomal solute carrier transporters gain momentum in research.

    PubMed

    Bissa, B; Beedle, A M; Govindarajan, R

    2016-11-01

    Emerging evidence indicates that lysosome function extends beyond macromolecular degradation. Genetic and functional defects in components of the lysosomal transport machinery cause lysosomal storage disorders implicating the lysosomal solute carrier (SLC) transporters as essential to vital cell processes. The pathophysiology and therapeutic potential of lysosomal SLC transporters are highlighted here, focusing on recent discoveries in autophagic amino acid sensing (SLC38A9), phagocytic regulation in macrophages (SLC29A3, SLC15A3/A4), adenosine triphosphate (ATP) exocytosis in neurotransmission (SLC17A9), and lysosomal transport of maytansine catabolites into the cytoplasm (SLC46A3).

  4. Lysosomal solute carrier transporters gain momentum in research

    PubMed Central

    Beedle, AM; Govindarajan, R

    2016-01-01

    Emerging evidence indicates that lysosome function extends beyond macromolecular degradation. Genetic and functional defects in components of the lysosomal transport machinery cause lysosomal storage disorders implicating the lysosomal solute carrier (SLC) transporters as essential to vital cell processes. The pathophysiology and therapeutic potential of lysosomal SLC transporters are highlighted here, focusing on recent discoveries in autophagic amino acid sensing (SLC38A9), phagocytic regulation in macrophages (SLC29A3, SLC15A3/A4), adenosine triphosphate (ATP) exocytosis in neurotransmission (SLC17A9), and lysosomal transport of maytansine catabolites into the cytoplasm (SLC46A3). PMID:27530302

  5. Biogenesis of the avian erythroid membrane skeleton: receptor-mediated assembly and stabilization of ankyrin (goblin) and spectrin.

    PubMed

    Moon, R T; Lazarides, E

    1984-05-01

    association with their respective membrane receptor(s). The existence in the detergent-soluble compartment of newly synthesized ankyrin and alpha- and beta-spectrin that are catabolized, rather than assembled, suggests that ankyrin and spectrin are synthesized in excess of available respective membrane binding sites, and that the assembly of these polypeptides, while rapid, is not tightly coupled to their synthesis. We hypothesize that the availability of the high affinity receptor(s) localized on the membrane mediates posttranslationally the extent of assembly of the three cytoskeletal proteins in the correct stoichiometry, their stability, and their spatial localization.

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

    NASA Astrophysics Data System (ADS)

    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.

  7. AP-3 and Rabip4’ Coordinately Regulate Spatial Distribution of Lysosomes

    PubMed Central

    Ivan, Viorica; Martinez-Sanchez, Emma; Sima, Livia E.; Oorschot, Viola; Klumperman, Judith; Petrescu, Stefana M.; van der Sluijs, Peter

    2012-01-01

    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. PMID:23144738

  8. Molecularly Designed Stabilized Asymmetric Hollow Fiber Membranes for Aggressive Natural Gas Separation.

    PubMed

    Liu, Gongping; Li, Nanwen; Miller, Stephen J; Kim, Danny; Yi, Shouliang; Labreche, Ying; Koros, William J

    2016-10-24

    New rigid polyimides with bulky CF3 groups were synthesized and engineered into high-performance hollow fiber membranes. The enhanced rotational barrier provided by properly positioned CF3 side groups prohibited fiber transition layer collapse during cross-linking, thereby greatly improving CO2 /CH4 separation performance compared to conventional materials for aggressive natural gas feeds.

  9. Lysosomal Storage Diseases—Regulating Neurodegeneration

    PubMed Central

    Onyenwoke, Rob U.; Brenman, Jay E.

    2015-01-01

    Autophagy is a complex pathway regulated by numerous signaling events that recycles macromolecules and can be perturbed in lysosomal storage diseases (LSDs). The concept of LSDs, which are characterized by aberrant, excessive storage of cellular material in lysosomes, developed following the discovery of an enzyme deficiency as the cause of Pompe disease in 1963. Great strides have since been made in better understanding the biology of LSDs. Defective lysosomal storage typically occurs in many cell types, but the nervous system, including the central nervous system and peripheral nervous system, is particularly vulnerable to LSDs, being affected in two-thirds of LSDs. This review provides a summary of some of the better characterized LSDs and the pathways affected in these disorders. PMID:27081317

  10. Immunomodulatory gene therapy in lysosomal storage disorders

    PubMed Central

    Koeberl, D.D.; Kishnani, P.S.

    2010-01-01

    Significant advances in therapy for lysosomal storage disorders have occurred with an accelerating pace over the past decade. Although enzyme replacement therapy has improved the outcome of lysosomal storage disorders, antibody responses have occurred and sometimes prevented efficacy, especially in cross-reacting immune material negative patients with Pompe disease. Preclinical gene therapy experiments have revealed the relevance of immune responses to long-term efficacy. The choice of regulatory cassette played a critical role in evading humoral and cellular immune responses to gene therapy in knockout mouse models, at least in adult animals. Liver-specific regulatory cassettes prevented antibody formation and enhanced the efficacy of gene therapy. Regulatory T cells prevented transgene directed immune responses, as shown by adoptive transfer of antigen-specific immune tolerance to enzyme therapy. Immunomodulatory gene therapy with a very low vector dose could enhance the efficacy of enzyme therapy in Pompe disease and other lysosomal storage disorders. PMID:19807648

  11. Immunomodulatory gene therapy in lysosomal storage disorders.

    PubMed

    Koeberl, Dwight D; Kishnani, Priya S

    2009-12-01

    Significant advances in therapy for lysosomal storage disorders have occurred with an accelerating pace over the past decade. Although enzyme replacement therapy has improved the outcome of lysosomal storage disorders, antibody responses have occurred and sometimes prevented efficacy, especially in cross-reacting immune material negative patients with Pompe disease. Preclinical gene therapy experiments have revealed the relevance of immune responses to long-term efficacy. The choice of regulatory cassette played a critical role in evading humoral and cellular immune responses to gene therapy in knockout mouse models, at least in adult animals. Liver-specific regulatory cassettes prevented antibody formation and enhanced the efficacy of gene therapy. Regulatory T cells prevented transgene directed immune responses, as shown by adoptive transfer of antigen-specific immune tolerance to enzyme therapy. Immunomodulatory gene therapy with a very low vector dose could enhance the efficacy of enzyme therapy in Pompe disease and other lysosomal storage disorders.

  12. Secretion from Myeloid Cells: Secretory Lysosomes.

    PubMed

    Griffiths, Gillian M

    2016-08-01

    Many cells of the myeloid lineage use an unusual secretory organelle to deliver their effector mechanisms. In these cells, the lysosomal compartment is often modified not only to fulfill the degradative functions of a lysosome but also as a mechanism for secreting additional proteins that are found in the lysosomes of each specialized cell type. These extra proteins vary from one cell type to another according to the specialized function of the cell. For example, mast cells package histamine; cytotoxic T cells express perforin; azurophilic granules in neutrophils express antimicrobial peptides, and platelets von Willebrand factor. Upon release, these very different proteins can trigger inflammation, cell lysis, microbial death, and clotting, respectively, and hence deliver the very different effector mechanisms of these different myeloid cells.

  13. Performance and Long-Term Stability of Pd/PSS and Pd/Al2O3 Membranes for Hydrogen Separation

    PubMed Central

    Liguori, Simona; Iulianelli, Adolfo; Dalena, Francesco; Pinacci, Pietro; Drago, Francesca; Broglia, Maria; Huang, Yan; Basile, Angelo

    2014-01-01

    The present work is focused on the investigation of the performance and long-term stability of two composite palladium membranes under different operating conditions. One membrane (Pd/porous stainless steel (PSS)) is characterized by a ~10 µm-thick palladium layer on a porous stainless steel substrate, which is pretreated by means of surface modification and oxidation; the other membrane (Pd/Al2O3) is constituted by a ~7 µm-thick palladium layer on an asymmetric microporous Al2O3 substrate. The operating temperature and pressure ranges, used for studying the performance of these two kinds of membranes, are 350–450 °C and 200–800 kPa, respectively. The H2 permeances and the H2/N2 selectivities of both membranes were investigated and compared with literature data. At 400 °C and 200 kPa as pressure difference, Pd/PSS and Pd/Al2O3 membranes exhibited an H2/N2 ideal selectivity equal to 11700 and 6200, respectively, showing stability for 600 h. Thereafter, H2/N2 selectivity of both membranes progressively decreased and after around 2000 h, dropped dramatically to 55 and 310 for the Pd/PSS and Pd/Al2O3 membranes, respectively. As evidenced by Scanning Electron Microscope (SEM) analyses, the pinholes appear on the whole surface of the Pd/PSS membrane and this is probably due to release of sulphur from the graphite seal rings. PMID:24957126

  14. Parallel damage in mitochondrial and lysosomal compartments promotes efficient cell death with autophagy: The case of the pentacyclic triterpenoids

    PubMed Central

    Martins, Waleska K.; Costa, Érico T.; Cruz, Mário C.; Stolf, Beatriz S.; Miotto, Ronei; Cordeiro, Rodrigo M.; Baptista, Maurício S.

    2015-01-01

    The role of autophagy in cell death is still controversial and a lot of debate has concerned the transition from its pro-survival to its pro-death roles. The similar structure of the triterpenoids Betulinic (BA) and Oleanolic (OA) acids allowed us to prove that this transition involves parallel damage in mitochondria and lysosome. After treating immortalized human skin keratinocytes (HaCaT) with either BA or OA, we evaluated cell viability, proliferation and mechanism of cell death, function and morphology of mitochondria and lysosomes, and the status of the autophagy flux. We also quantified the interactions of BA and OA with membrane mimics, both in-vitro and in-silico. Essentially, OA caused mitochondrial damage that relied on autophagy to rescue cellular homeostasis, which failed upon lysosomal inhibition by Chloroquine or Bafilomycin-A1. BA caused parallel damage on mitochondria and lysosome, turning autophagy into a destructive process. The higher cytotoxicity of BA correlated with its stronger efficiency in damaging membrane mimics. Based on these findings, we underlined the concept that autophagy will turn into a destructive outcome when there is parallel damage in mitochondrial and lysosomal membranes. We trust that this concept will help the development of new drugs against aggressive cancers. PMID:26213355

  15. Citreoviridin Induces Autophagy-Dependent Apoptosis through Lysosomal-Mitochondrial Axis in Human Liver HepG2 Cells

    PubMed Central

    Wang, Yuexia; Liu, Yanan; Liu, Xiaofang; Jiang, Liping; Yang, Guang; Sun, Xiance; Geng, Chengyan; Li, Qiujuan; Yao, Xiaofeng; Chen, Min

    2015-01-01

    Citreoviridin (CIT) is a mycotoxin derived from fungal species in moldy cereals. In our previous study, we reported that CIT stimulated autophagosome formation in human liver HepG2 cells. Here, we aimed to explore the relationship of autophagy with lysosomal membrane permeabilization and apoptosis in CIT-treated cells. Our data showed that CIT increased the expression of LC3-II, an autophagosome biomarker, from the early stage of treatment (6 h). After treatment with CIT for 12 h, lysosomal membrane permeabilization occurred, followed by the release of cathepsin D in HepG2 cells. Inhibition of autophagosome formation with siRNA against Atg5 attenuated CIT-induced lysosomal membrane permeabilization. In addition, CIT induced collapse of mitochondrial transmembrane potential as assessed by JC-1 staining. Furthermore, caspase-3 activity assay showed that CIT induced apoptosis in HepG2 cells. Inhibition of autophagosome formation attenuated CIT-induced apoptosis, indicating that CIT-induced apoptosis was autophagy-dependent. Cathepsin D inhibitor, pepstatin A, relieved CIT-induced apoptosis as well, suggesting the involvement of the lysosomal-mitochondrial axis in CIT-induced apoptosis. Taken together, our data demonstrated that CIT induced autophagy-dependent apoptosis through the lysosomal-mitochondrial axis in HepG2 cells. The study thus provides essential mechanistic insight, and suggests clues for the effective management and treatment of CIT-related diseases. PMID:26258792

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

    PubMed

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

    2017-03-16

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

  17. Citreoviridin Induces Autophagy-Dependent Apoptosis through Lysosomal-Mitochondrial Axis in Human Liver HepG2 Cells.

    PubMed

    Wang, Yuexia; Liu, Yanan; Liu, Xiaofang; Jiang, Liping; Yang, Guang; Sun, Xiance; Geng, Chengyan; Li, Qiujuan; Yao, Xiaofeng; Chen, Min

    2015-08-06

    Citreoviridin (CIT) is a mycotoxin derived from fungal species in moldy cereals. In our previous study, we reported that CIT stimulated autophagosome formation in human liver HepG2 cells. Here, we aimed to explore the relationship of autophagy with lysosomal membrane permeabilization and apoptosis in CIT-treated cells. Our data showed that CIT increased the expression of LC3-II, an autophagosome biomarker, from the early stage of treatment (6 h). After treatment with CIT for 12 h, lysosomal membrane permeabilization occurred, followed by the release of cathepsin D in HepG2 cells. Inhibition of autophagosome formation with siRNA against Atg5 attenuated CIT-induced lysosomal membrane permeabilization. In addition, CIT induced collapse of mitochondrial transmembrane potential as assessed by JC-1 staining. Furthermore, caspase-3 activity assay showed that CIT induced apoptosis in HepG2 cells. Inhibition of autophagosome formation attenuated CIT-induced apoptosis, indicating that CIT-induced apoptosis was autophagy-dependent. Cathepsin D inhibitor, pepstatin A, relieved CIT-induced apoptosis as well, suggesting the involvement of the lysosomal-mitochondrial axis in CIT-induced apoptosis. Taken together, our data demonstrated that CIT induced autophagy-dependent apoptosis through the lysosomal-mitochondrial axis in HepG2 cells. The study thus provides essential mechanistic insight, and suggests clues for the effective management and treatment of CIT-related diseases.

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

    PubMed

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

    2015-11-01

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

  19. Stability of the Shab K+ channel conductance in 0 K+ solutions: the role of the membrane potential.

    PubMed

    Gómez-Lagunas, Froylán

    2007-12-15

    Shab channels are fairly stable with K(+) present on only one side of the membrane. However, on exposure to 0 K(+) solutions on both sides of the membrane, the Shab K(+) conductance (G(K)) irreversibly drops while the channels are maintained undisturbed at the holding potential. Herein it is reported that the drop of G(K) follows first-order kinetics, with a voltage-dependent decay rate r. Hyperpolarized potentials drastically inhibit the drop of G(K). The G(K) drop at negative potentials cannot be explained by a shift in the voltage dependence of activation. At depolarized potentials, where the channels undergo a slow inactivation process, G(K) drops in 0 K(+) with rates slower than those predicted based on the behavior of r at negative potentials, endowing the r-V(m) relationship with a maximum. Regardless of voltage, r is very small compared with the rate of ion permeation. Observations support the hypothesized presence of a stabilizing K(+) site (or sites) located either within the pore itself or in its external vestibule, at an inactivation-sensitive location. It is argued that part of the G(K) stabilization achieved at hyperpolarized potentials could be the result of a conformational change in the pore itself.

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

    PubMed

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

    2016-01-15

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

  1. Endo-Lysosomal Dysfunction in Human Proximal Tubular Epithelial Cells Deficient for Lysosomal Cystine Transporter Cystinosin

    PubMed Central

    Van Den Heuvel, Lambertus; Pastore, Anna; Dijkman, Henry; De Matteis, Maria Antonietta; Levtchenko, Elena N.

    2015-01-01

    Nephropathic cystinosis is a lysosomal storage disorder caused by mutations in the CTNS gene encoding cystine transporter cystinosin that results in accumulation of amino acid cystine in the lysosomes throughout the body and especially affects kidneys. Early manifestations of the disease include renal Fanconi syndrome, a generalized proximal tubular dysfunction. Current therapy of cystinosis is based on cystine-lowering drug cysteamine that postpones the disease progression but offers no cure for the Fanconi syndrome. We studied the mechanisms of impaired reabsorption in human proximal tubular epithelial cells (PTEC) deficient for cystinosin and investigated the endo-lysosomal compartments of cystinosin-deficient PTEC by means of light and electron microscopy. We demonstrate that cystinosin-deficient cells had abnormal shape and distribution of the endo-lysosomal compartments and impaired endocytosis, with decreased surface expression of multiligand receptors and delayed lysosomal cargo processing. Treatment with cysteamine improved surface expression and lysosomal cargo processing but did not lead to a complete restoration and had no effect on the abnormal morphology of endo-lysosomal compartments. The obtained results improve our understanding of the mechanism of proximal tubular dysfunction in cystinosis and indicate that impaired protein reabsorption can, at least partially, be explained by abnormal trafficking of endosomal vesicles. PMID:25811383

  2. Uncovering the Stabilization Mechanism in Bimetallic Ruthenium-Iridium Anodes for Proton Exchange Membrane Electrolyzers.

    PubMed

    Saveleva, Viktoriia A; Wang, Li; Luo, Wen; Zafeiratos, Spyridon; Ulhaq-Bouillet, Corinne; Gago, Aldo S; Friedrich, K Andreas; Savinova, Elena R

    2016-08-18

    Proton exchange membrane (PEM) electrolyzers are attracting an increasing attention as a promising technology for the renewable electricity storage. In this work, near ambient pressure X-ray photoelectron spectroscopy (NAP-XPS) is applied for in situ monitoring of the surface state of membrane electrode assemblies with RuO2 and bimetallic Ir0.7Ru0.3O2 anodes during water splitting. We demonstrate that Ir protects Ru from the formation of an unstable hydrous Ru(IV) oxide thereby rendering bimetallic Ru-Ir oxide electrodes with higher corrosion resistance. We further show that the water splitting occurs through a surface Ru(VIII) intermediate, and, contrary to common opinion, the presence of Ir does not hinder its formation.

  3. Long-Term Stability of a Vaccine Formulated with the Amphipol-Trapped Major Outer Membrane Protein from Chlamydia trachomatis

    PubMed Central

    Feinstein, H. Eric; Tifrea, Delia; Sun, Guifeng; Popot, Jean-Luc; de la Maza, Luis M.

    2014-01-01

    Chlamydia trachomatis is a major bacterial pathogen throughout the world. Although antibiotic therapy can be implemented in the case of early detection, a majority of the infections are asymptomatic, requiring the development of preventive measures. Efforts have focused on the production of a vaccine using the C. trachomatis major outer membrane protein (MOMP). MOMP is purified in its native (n) trimeric form using the zwitterionic detergent Z3–14, but its stability in detergent solutions is limited. Amphipols (APols) are synthetic polymers that can stabilize membrane proteins (MPs) in detergent-free aqueous solutions. Preservation of protein structure and optimization of exposure of the most effective antigenic regions can avoid vaccination with misfolded, poorly protective protein. Previously, we showed that APols maintain nMOMP secondary structure and that nMOMP/APol vaccine formulations elicit better protection than formulations using either recombinant or nMOMP solubilized in Z3–14. To achieve a greater understanding of the structural behavior and stability of nMOMP in APols, we have used several spectroscopic techniques to characterize its secondary structure (circular dichroism), tertiary and quaternary structures (immunochemistry and gel electrophoresis) and aggregation state (light scattering) as a function of temperature and time. We have also recorded NMR spectra of 15N-labeled nMOMP and find that the exposed loops are detectable in APols but not in detergent. Our analyses show that APols protect nMOMP much better than Z3–14 against denaturation due to continuous heating, repeated freeze/thaw cycles, or extended storage at room temperature. These results indicate that APols can help improve MP-based vaccine formulations. PMID:24942817

  4. Crystallization around solid-like nanosized docks can explain the specificity, diversity, and stability of membrane microdomains

    PubMed Central

    de Almeida, Rodrigo F. M.; Joly, Etienne

    2014-01-01

    To date, it is widely accepted that microdomains do form in the biological membranes of all eukaryotic cells, and quite possibly also in prokaryotes. Those sub-micrometric domains play crucial roles in signaling, in intracellular transport, and even in inter-cellular communications. Despite their ubiquitous distribution, and the broad and lasting interest invested in those microdomains, their actual nature and composition, and even the physical rules that regiment their assembly still remain elusive and hotly debated. One of the most often considered models is the raft hypothesis, i.e., the partition of lipids between liquid disordered and ordered phases (Ld and Lo, respectively), the latter being enriched in sphingolipids and cholesterol. Although it is experimentally possible to obtain the formation of microdomains in synthetic membranes through Ld/Lo phase separation, there is an ever increasing amount of evidence, obtained with a wide array of experimental approaches, that a partition between domains in Ld and Lo phases cannot account for many of the observations collected in real cells. In particular, it is now commonly perceived that the plasma membrane of cells is mostly in Lo phase and recent data support the existence of gel or solid ordered domains in a whole variety of live cells under physiological conditions. Here, we present a model whereby seeds comprised of oligomerised proteins and/or lipids would serve as crystal nucleation centers for the formation of diverse gel/crystalline nanodomains. This could confer the selectivity necessary for the formation of multiple types of membrane domains, as well as the stability required to match the time frames of cellular events, such as intra- or inter-cellular transport or assembly of signaling platforms. Testing of this model will, however, require the development of new methods allowing the clear-cut discrimination between Lo and solid nanoscopic phases in live cells. PMID:24634670

  5. Ankyrin Repeat-rich Membrane Spanning/Kidins220 protein regulates dendritic branching and spine stability in vivo.

    PubMed

    Wu, Synphen H; Arévalo, Juan Carlos; Sarti, Federica; Tessarollo, Lino; Gan, Wen-Biao; Chao, Moses V

    2009-08-01

    The development of nervous system connectivity depends upon the arborization of dendritic fields and the stabilization of dendritic spine synapses. It is well established that neuronal activity and the neurotrophin BDNF modulate these correlated processes. However, the downstream mechanisms by which these extrinsic signals regulate dendritic development and spine stabilization are less well known. Here we report that a substrate of BDNF signaling, the Ankyrin Repeat-rich Membrane Spanning (ARMS) protein or Kidins220, plays a critical role in the branching of cortical and hippocampal dendrites and in the turnover of cortical spines. In the barrel somatosensory cortex and the dentate gyrus, regions where ARMS/Kidins220 is highly expressed, no difference in the complexity of dendritic arbors was observed in 1-month-old adolescent ARMS/Kidins220(+/-) mice compared to wild-type littermates. However, at 3 months of age, young adult ARMS/Kidins220(+/-) mice exhibited decreased dendritic complexity. This suggests that ARMS/Kidins220 does not play a significant role in the initial formation of dendrites but, rather, is involved in the refinement or stabilization of the arbors later in development. In addition, at 1 month of age, the rate of spine elimination was higher in ARMS/Kidins220(+/-) mice than in wild-type mice, suggesting that ARMS/Kidins220(+/-) levels regulate spine stability. Taken together, these data suggest that ARMS/Kidins220 is important for the growth of dendritic arbors and spine stability during an activity- and BDNF-dependent period of development.

  6. Stabilization of mitochondrial membrane potential prevents doxorubicin-induced cardiotoxicity in isolated rat heart

    SciTech Connect

    Montaigne, David; Marechal, Xavier; Baccouch, Riadh; Modine, Thomas; Preau, Sebastien; Zannis, Konstantinos; Marchetti, Philippe; Lancel, Steve; Neviere, Remi

    2010-05-01

    The present study was undertaken to examine the effects of doxorubicin on left ventricular function and cellular energy state in intact isolated hearts, and, to test whether inhibition of mitochondrial membrane potential dissipation would prevent doxorubicin-induced mitochondrial and myocardial dysfunction. Myocardial contractile performance and mitochondrial respiration were evaluated by left ventricular tension and its first derivatives and cardiac fiber respirometry, respectively. NADH levels, mitochondrial membrane potential and glucose uptake were monitored non-invasively via epicardial imaging of the left ventricular wall of Langendorff-perfused rat hearts. Heart performance was reduced in a time-dependent manner in isolated rat hearts perfused with Krebs-Henseleit solution containing 1 muM doxorubicin. Compared with controls, doxorubicin induced acute myocardial dysfunction (dF/dt{sub max} of 105 +- 8 mN/s in control hearts vs. 49 +- 7 mN/s in doxorubicin-treated hearts; *p < 0.05). In cardiac fibers prepared from perfused hearts, doxorubicin induced depression of mitochondrial respiration (respiratory control ratio of 4.0 +- 0.2 in control hearts vs. 2.2 +- 0.2 in doxorubicin-treated hearts; *p < 0.05) and cytochrome c oxidase kinetic activity (24 +- 1 muM cytochrome c/min/mg in control hearts vs. 14 +- 3 muM cytochrome c/min/mg in doxorubicin-treated hearts; *p < 0.05). Acute cardiotoxicity induced by doxorubicin was accompanied by NADH redox state, mitochondrial membrane potential, and glucose uptake reduction. Inhibition of mitochondrial permeability transition pore opening by cyclosporine A largely prevented mitochondrial membrane potential dissipation, cardiac energy state and dysfunction. These results suggest that in intact hearts an impairment of mitochondrial metabolism is involved in the development of doxorubicin cardiotoxicity.

  7. Stability of purple membranes from Halobacterium salinarum toward surfactants: inkjet printing of a retinal protein.

    PubMed

    Imhof, Martin; Pudewills, Jens; Rhinow, Daniel; Chizhik, Ivan; Hampp, Norbert

    2012-08-16

    Inkjet printing is a versatile technique widely applied in biological microarray technology. Because of its photochemical and photophysical properties, bacteriorhodopsin (BR) from Halobacterium salinarum holds promise for applications in nanotechnology, and inkjet printing would simplify the transfer of BR to suitable substrates. Surfactants are essential parts of inkjet formulations tuning viscosity, rheology, and spreading behavior of the solution. However, many surfactants destabilize the structure of proteins and often cause denaturation accompanied by a complete loss of function. Inkjet printing of membrane proteins is particularly challenging and special care must be taken in the choice of the surfactant. For BR, the situation is complicated by the fact that the structural integrity of BR depends on its native membrane environment, the so-called purple membrane (PM). PM contains 10 lipid molecules per BR monomer and is very sensitive toward surfactants. In this work, we identified surfactants suitable for inkjet formulations containing PM. Initially, we screened a variety of technically relevant surfactants for compatibility with PM using the UV-vis absorption of the retinal chromophore as a natural probe. Promising candidates were selected, and their impact on the structure of PM and BR was analyzed using UV-vis spectroscopy, CD spectroscopy, and small-angle X-ray scattering (SAXS). We identified two surfactants compatible with PM and suitable for inkjet formulations. An inkjet formulation containing PM as dye component was developed. We demonstrate that the photochromic properties of BR are maintained upon inkjet printing.

  8. Drug induced phospholipidosis: an acquired lysosomal storage disorder.

    PubMed

    Shayman, James A; Abe, Akira

    2013-03-01

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

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

    PubMed

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

    2013-04-01

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

  10. Neutrophil lysosomal dysfunctions in mutant C57 Bl/6J mice: interstrain variations in content of lysosomal elastase, cathepsin G and their inhibitors.

    PubMed Central

    Gardi, C; Cavarra, E; Calzoni, P; Marcolongo, P; de Santi, M; Martorana, P A; Lungarella, G

    1994-01-01

    In this paper we report the serum antiprotease screening and the biochemical and functional characteristics of neutrophils in a variety of mouse strains with different susceptibilities for developing a protease-mediated injury. C57Bl/6J mice and their mutants tight-skin and pallid have a lower serum elastase inhibitory capacity (-30, -65 and -70% respectively) than other inbred strains (i.e. NMRI and Balb/c, which both have similar values). We demonstrate that these values are a consequence of a decreased concentration of the alpha 1-protease inhibitor for elastase [PI(E)], which is the major serum inhibitor of elastase and cathepsin G. In addition, neutrophil lysosomal dysfunctions characterized by abnormally high contents of elastase and cathepsin G, or defective lysosomal secretion are observed in tight-skin and pallid mice respectively. Another C57Bl/6J mutant with lysosomal abnormalities is the beige mouse. Negligible amounts of elastase and cathepsin G, as well as defective neutrophil degranulation, have been described previously in this strain. We found, however, discrete amounts of a latent form of neutrophil elastase that undergoes a spontaneous activation by a protease-dependent mechanism. We also report that neutrophil cathepsin G in this mouse is tightly bound to lysosomal membranes, but is released in near normal quantities during exocytosis. Cytosolic elastase and cathepsin G inhibitors, which were previously reported as being specific for the beige neutrophils, have also been detected in all the examined strains. Neutrophil functions, lysosomal enzyme content and serum antiprotease screening may represent key elements in the protease-antiprotease balance and may explain the different interstrain susceptibility to developing lesions in which an elastolytic activity has been implicated. Images Figure 1 Figure 2 Figure 3 Figure 4 PMID:8166647

  11. Lysosomal protease expression in mature enamel.

    PubMed

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

    2009-01-01

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

  12. Lysosomal proteolysis: effects of aging and insulin.

    PubMed

    Gromakova, I A; Konovalenko, O A

    2003-07-01

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

  13. TPC1 has two variant isoforms, and their removal has different effects on endo-lysosomal functions compared to loss of TPC2.

    PubMed

    Ruas, Margarida; Chuang, Kai-Ting; Davis, Lianne C; Al-Douri, Areej; Tynan, Patricia W; Tunn, Ruth; Teboul, Lydia; Galione, Antony; Parrington, John

    2014-11-01

    Organelle ion homeostasis within the endo-lysosomal system is critical for physiological functions. Two-pore channels (TPCs) are cation channels that reside in endo-lysosomal organelles, and overexpression results in endo-lysosomal trafficking defects. However, the impact of a lack of TPC expression on endo-lysosomal trafficking is unknown. Here, we characterize Tpcn1 expression in two transgenic mouse lines (Tpcn1(XG716) and Tpcn1(T159)) and show expression of a novel evolutionarily conserved Tpcn1B transcript from an alternative promoter, raising important questions regarding the status of Tpcn1 expression in mice recently described to be Tpcn1 knockouts. We show that the transgenic Tpcn1(T159) line lacks expression of both Tpcn1 isoforms in all tissues analyzed. Using mouse embryonic fibroblasts (MEFs) from Tpcn1(-/-) and Tpcn2(-/-) animals, we show that a lack of Tpcn1 or Tpcn2 expression has no significant impact on resting endo-lysosomal pH or morphology. However, differential effects in endo-lysosomal function were observed upon the loss of Tpcn1 or Tpcn2 expression; thus, while Tpcn1(-/-) MEFs have impaired trafficking of cholera toxin from the plasma membrane to the Golgi apparatus, Tpcn2(-/-) MEFs show slower kinetics of ligand-induced platelet-derived growth factor receptor β (PDGFRβ) degradation, which is dependent on trafficking to lysosomes. Our findings indicate that TPC1 and TPC2 have important but distinct roles in the endo-lysosomal pathway.

  14. ESCRT-Dependent Cell Death in a Caenorhabditis elegans Model of the Lysosomal Storage Disorder Mucolipidosis Type IV.

    PubMed

    Huynh, Julie M; Dang, Hope; Munoz-Tucker, Isabel A; O'Ketch, Marvin; Liu, Ian T; Perno, Savannah; Bhuyan, Natasha; Crain, Allison; Borbon, Ivan; Fares, Hanna

    2016-02-01

    Mutations in MCOLN1, which encodes the cation channel protein TRPML1, result in the neurodegenerative lysosomal storage disorder Mucolipidosis type IV. Mucolipidosis type IV patients show lysosomal dysfunction in many tissues and neuronal cell death. The ortholog of TRPML1 in Caenorhabditis elegans is CUP-5; loss of CUP-5 results in lysosomal dysfunction in many tissues and death of developing intestinal cells that results in embryonic lethality. We previously showed that a null mutation in the ATP-Binding Cassette transporter MRP-4 rescues the lysosomal defect and embryonic lethality of cup-5(null) worms. Here we show that reducing levels of the Endosomal Sorting Complex Required for Transport (ESCRT)-associated proteins DID-2, USP-50, and ALX-1/EGO-2, which mediate the final de-ubiquitination step of integral membrane proteins being sequestered into late endosomes, also almost fully suppresses cup-5(null) mutant lysosomal defects and embryonic lethality. Indeed, we show that MRP-4 protein is hypo-ubiquitinated in the absence of CUP-5 and that reducing levels of ESCRT-associated proteins suppresses this hypo-ubiquitination. Thus, increased ESCRT-associated de-ubiquitinating activity mediates the lysosomal defects and corresponding cell death phenotypes in the absence of CUP-5.

  15. Site-1 protease-activated formation of lysosomal targeting motifs is independent of the lipogenic transcription control[S

    PubMed Central

    Klünder, Sarah; Heeren, Jörg; Markmann, Sandra; Santer, René; Braulke, Thomas; Pohl, Sandra

    2015-01-01

    Site-1 protease (S1P) cleaves membrane-bound lipogenic sterol regulatory element-binding proteins (SREBPs) and the α/β-subunit precursor protein of the N-acetylglucosamine-1-phosphotransferase forming mannose 6-phosphate (M6P) targeting markers on lysosomal enzymes. The translocation of SREBPs from the endoplasmic reticulum (ER) to the Golgi-resident S1P depends on the intracellular sterol content, but it is unknown whether the ER exit of the α/β-subunit precursor is regulated. Here, we investigated the effect of cholesterol depletion (atorvastatin treatment) and elevation (LDL overload) on ER-Golgi transport, S1P-mediated cleavage of the α/β-subunit precursor, and the subsequent targeting of lysosomal enzymes along the biosynthetic and endocytic pathway to lysosomes. The data showed that the proteolytic cleavage of the α/β-subunit precursor into mature and enzymatically active subunits does not depend on the cholesterol content. In either treatment, lysosomal enzymes are normally decorated with M6P residues, allowing the proper sorting to lysosomes. In addition, we found that, in fibroblasts of mucolipidosis type II mice and Niemann-Pick type C patients characterized by aberrant cholesterol accumulation, the proteolytic cleavage of the α/β-subunit precursor was not impaired. We conclude that S1P substrate-dependent regulatory mechanisms for lipid synthesis and biogenesis of lysosomes are different. PMID:26108224

  16. ESCRT-Dependent Cell Death in a Caenorhabditis elegans Model of the Lysosomal Storage Disorder Mucolipidosis Type IV

    PubMed Central

    Huynh, Julie M.; Dang, Hope; Munoz-Tucker, Isabel A.; O’Ketch, Marvin; Liu, Ian T.; Perno, Savannah; Bhuyan, Natasha; Crain, Allison; Borbon, Ivan; Fares, Hanna

    2016-01-01

    Mutations in MCOLN1, which encodes the cation channel protein TRPML1, result in the neurodegenerative lysosomal storage disorder Mucolipidosis type IV. Mucolipidosis type IV patients show lysosomal dysfunction in many tissues and neuronal cell death. The ortholog of TRPML1 in Caenorhabditis elegans is CUP-5; loss of CUP-5 results in lysosomal dysfunction in many tissues and death of developing intestinal cells that results in embryonic lethality. We previously showed that a null mutation in the ATP-Binding Cassette transporter MRP-4 rescues the lysosomal defect and embryonic lethality of cup-5(null) worms. Here we show that reducing levels of the Endosomal Sorting Complex Required for Transport (ESCRT)-associated proteins DID-2, USP-50, and ALX-1/EGO-2, which mediate the final de-ubiquitination step of integral membrane proteins being sequestered into late endosomes, also almost fully suppresses cup-5(null) mutant lysosomal defects and embryonic lethality. Indeed, we show that MRP-4 protein is hypo-ubiquitinated in the absence of CUP-5 and that reducing levels of ESCRT-associated proteins suppresses this hypo-ubiquitination. Thus, increased ESCRT-associated de-ubiquitinating activity mediates the lysosomal defects and corresponding cell death phenotypes in the absence of CUP-5. PMID:26596346

  17. [Fabry disease and cystinosis, two lysosomal diseases: similarities and differences].

    PubMed

    Grünfeld, J-P; Servais, A

    2010-12-01

    Fabry disease and cystinosis are both lysosomal diseases. Some clinical features (such as renal and corneal involvement) are shared by both diseases whereas many other features are different (mode of inheritance, rate of progression, mechanism of lysosomal storage, therapeutic modalities etc.). Intermediary mechanisms that lead from lysosomal overload to lesions and disease are still incompletely understood.

  18. Inositol phosphosphingolipid phospholipase C1 regulates plasma membrane ATPase (Pma1) stability in Cryptococcus neoformans.

    PubMed

    Farnoud, Amir M; Mor, Visesato; Singh, Ashutosh; Del Poeta, Maurizio

    2014-11-03

    Cryptococcus neoformans is a facultative intracellular pathogen, which can replicate in the acidic environment inside phagolysosomes. Deletion of the enzyme inositol-phosphosphingolipid-phospholipase-C (Isc1) makes C. neoformans hypersensitive to acidic pH likely by inhibiting the function of the proton pump, plasma membrane ATPase (Pma1). In this work, we examined the role of Isc1 on Pma1 transport and oligomerization. Our studies showed that Isc1 deletion did not affect Pma1 synthesis or transport, but significantly inhibited Pma1 oligomerization. Interestingly, Pma1 oligomerization could be restored by supplementing the medium with phytoceramide. These results offer insight into the mechanism of intracellular survival of C. neoformans.

  19. Neuroinflammatory paradigms in lysosomal storage diseases.

    PubMed

    Bosch, Megan E; Kielian, Tammy

    2015-01-01

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

  20. Neuroinflammatory paradigms in lysosomal storage diseases

    PubMed Central

    Bosch, Megan E.; Kielian, Tammy

    2015-01-01

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

  1. Influences of the Structure of Lipids on Thermal Stability of Lipid Membranes

    NASA Astrophysics Data System (ADS)

    Hai, Nan-Nan; Zhou, Xin; Li, Ming

    2015-08-01

    The binding free energy (BFE) of lipid to lipid bilayer is a critical factor to determine the thermal or mechanical stability of the bilayer. Although the molecular structure of lipids has significant impacts on BFE of the lipid, there lacks a systematic study on this issue. In this paper we use coarse-grained molecular dynamics simulation to investigate this problem for several typical phospholipids. We find that both the tail length and tail unsaturation can significantly affect the BFE of lipids but in opposite way, namely, BFE decreases linearly with increasing length, but increases linearly with addition of unsaturated bonds. Inspired by the specific structure of cholesterol which is a crucial component of biomembrane, we also find that introduction of carbo-ring-like structures to the lipid tail or to the bilayer may greatly enhance the stability of the bilayer. Our simulation also shows that temperature can influence the bilayer stability and this effect can be significant when the bilayer undergoes phase transition. These results may be helpful to the design of liposome or other self-assembled lipid systems. Support by the National Natural Science Foundation of China under Grant Nos. 91027046 and 11105218.

  2. Oxidized phagosomal NOX2 is replenished from lysosomes.

    PubMed

    Dingjan, Ilse; Linders, Peter T A; van den Bekerom, Luuk; Baranov, Maksim V; Halder, Partho; Ter Beest, Martin; van den Bogaart, Geert

    2017-02-15

    In dendritic cells, the NADPH oxidase 2 (NOX2) is recruited to the phagosomal membrane during antigen uptake. NOX2 produces reactive oxygen species (ROS) in the lumen of the phagosome which kill ingested pathogens, delay antigen breakdown and alter the peptide repertoire for presentation to T cells. How the integral membrane component of NOX2, cytochrome b558, traffics to phagosomes is incompletely understood. In this study, we show in dendritic cells derived from human blood-isolated monocytes that cytochrome b558 is initially recruited to the phagosome from the plasma membrane during phagosome formation. Cytochrome b558 also traffics from a lysosomal pool to phagosomes and this is required to replenish oxidatively damaged NOX2. We identified syntaxin-7, SNAP23 and VAMP8 as the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins mediating this process. Our data describe a key mechanism of how dendritic cells sustain ROS production after antigen uptake required to initiate T cell responses.

  3. Turning the gun on cancer: Utilizing lysosomal P-glycoprotein as a new strategy to overcome multi-drug resistance.

    PubMed

    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.

  4. Assembly of BODIPY-carbazole dyes with liposomes to fabricate fluorescent nanoparticles for lysosomal bioimaging in living cells.

    PubMed

    Lv, Hai-Juan; Zhang, Xiao-Tai; Wang, Shu; Xing, Guo-Wen

    2017-01-31

    Two BODIPY-carbazole dye based fluorescent probes BCA and BCAS were designed, synthesized and encapsulated by liposomes to obtain fluorescent nanoparticles BCA-FNP and BCAS-FNP. The fluorescence imaging showed that BCA-FNP was membrane-permeable and capable of localizing lysosomes in living cells.

  5. Disordered Cold Regulated15 Proteins Protect Chloroplast Membranes during Freezing through Binding and Folding, But Do Not Stabilize Chloroplast Enzymes in Vivo1[W][OPEN

    PubMed Central

    Thalhammer, Anja; Bryant, Gary; Sulpice, Ronan; Hincha, Dirk K.

    2014-01-01

    Freezing can severely damage plants, limiting geographical distribution of natural populations and leading to major agronomical losses. Plants native to cold climates acquire increased freezing tolerance during exposure to low nonfreezing temperatures in a process termed cold acclimation. This involves many adaptative responses, including global changes in metabolite content and gene expression, and the accumulation of cold-regulated (COR) proteins, whose functions are largely unknown. Here we report that the chloroplast proteins COR15A and COR15B are necessary for full cold acclimation in Arabidopsis (Arabidopsis thaliana). They protect cell membranes, as indicated by electrolyte leakage and chlorophyll fluorescence measurements. Recombinant COR15 proteins stabilize lactate dehydrogenase during freezing in vitro. However, a transgenic approach shows that they have no influence on the stability of selected plastidic enzymes in vivo, although cold acclimation results in increased enzyme stability. This indicates that enzymes are stabilized by other mechanisms. Recombinant COR15 proteins are disordered in water, but fold into amphipathic α-helices at high osmolyte concentrations in the presence of membranes, a condition mimicking molecular crowding induced by dehydration during freezing. X-ray scattering experiments indicate protein-membrane interactions specifically under such crowding conditions. The COR15-membrane interactions lead to liposome stabilization during freezing. Collectively, our data demonstrate the requirement for COR15 accumulation for full cold acclimation of Arabidopsis. The function of these intrinsically disordered proteins is the stabilization of chloroplast membranes during freezing through a folding and binding mechanism, but not the stabilization of chloroplastic enzymes. This indicates a high functional specificity of these disordered plant proteins. PMID:25096979

  6. Enhanced performance and stability of high temperature proton exchange membrane fuel cell by incorporating zirconium hydrogen phosphate in catalyst layer

    NASA Astrophysics Data System (ADS)

    Barron, Olivia; Su, Huaneng; Linkov, Vladimir; Pollet, Bruno G.; Pasupathi, Sivakumar

    2015-03-01

    Zirconium hydrogen phosphate (ZHP) together with polytetrafluoroethylene (PTFE) polymer binder is incorporated into the catalyst layers (CLs) of ABPBI (poly(2,5-benzimidazole))-based high temperature polymer electrolyte membrane fuel cell (HT-PEMFCs) to improve its performance and durability. The influence of ZHP content (normalised with respect to dry PTFE) on the CL properties are structurally characterised by scanning electron microscopy (SEM) and mercury intrusion porosimetry. Electrochemical analyses of the resultant membrane electrode assemblies (MEAs) are performed by recording polarisation curves and impedance spectra at 160 °C, ambient pressure and humidity. The result show that a 30 wt.% ZHP/PTFE content in the CL is optimum for improving fuel cell performance, the resultant MEA delivers a peak power of 592 mW cm-2 at a cell voltage of 380 mV. Electrochemical impedance spectra (EIS) indicate that 30% ZHP in the CL can increase the proton conductivity compared to the pristine PTFE-gas diffusion electrode (GDE). A short term stability test (∼500 h) on the 30 wt.% ZHP/PTFE-GDE shows a remarkable high durability with a degradation rate as low as ∼19 μV h-1 at 0.2 A cm-2, while 195 μV h-1 was obtained for the pristine GDE.

  7. Artificial-enzyme gel membrane-based biosurveillance sensor with high reproducibility and long-term storage stability.

    PubMed

    Ikeno, Shinya; Yoshida, Tetsuya; Haruyama, Tetsuya

    2009-02-01

    We propose that the most sophisticated strategy for primary biosurveillance is to exploit structural commonality through the detection of biologically relevant phosphoric substances. A novel assay, an artificial-enzyme membrane was designed and synthesized for sensor fabrication. This artificial-enzyme catalyzes the hydrolysis of the diphosphoric acid anhydride structure. This structure-selective, albeit not molecule-selective, catalytic hydrolysis was successfully coupled with amperometric detection. Since the catalytic reaction produces a dephosphorylation product (PO(4)(3-)), it can be reduced by an electrode potential of -250 mV vs. Ag/AgCl. Owing to the structural selectivity of the artificial-enzyme membrane, the sensor can detect biological phosphoric substances comprehensively that have the diphosphoric acid anhydride structure. The sensor successfully determined various biological phosphoric substances at concentrations in the micromolar (microM) to millimolar (mM) range, and it showed good functional stability and reproducibility in terms of sensor responses. This sensor was used to detect Escherichia coli lysed by heat treatment, and the response increased with increasing bacterial numbers. This unique technique for analyzing molecular commonality can be applied to the surveillance of biocontaminants, e.g. microorganisms, spores and viruses. Artificial-enzyme-based detection is a novel strategy for practical biosurveillance in the front line.

  8. EPAC1 activation by cAMP stabilizes CFTR at the membrane by promoting its interaction with NHERF1.

    PubMed

    Lobo, Miguel J; Amaral, Margarida D; Zaccolo, Manuela; Farinha, Carlos M

    2016-07-01

    Cyclic AMP (cAMP) activates protein kinase A (PKA) but also the guanine nucleotide exchange factor 'exchange protein directly activated by cAMP' (EPAC1; also known as RAPGEF3). Although phosphorylation by PKA is known to regulate CFTR channel gating - the protein defective in cystic fibrosis - the contribution of EPAC1 to CFTR regulation remains largely undefined. Here, we demonstrate that in human airway epithelial cells, cAMP signaling through EPAC1 promotes CFTR stabilization at the plasma membrane by attenuating its endocytosis, independently of PKA activation. EPAC1 and CFTR colocalize and interact through protein adaptor NHERF1 (also known as SLC9A3R1). This interaction is promoted by EPAC1 activation, triggering its translocation to the plasma membrane and binding to NHERF1. Our findings identify a new CFTR-interacting protein and demonstrate that cAMP activates CFTR through two different but complementary pathways - the well-known PKA-dependent channel gating pathway and a new mechanism regulating endocytosis that involves EPAC1. The latter might constitute a novel therapeutic target for treatment of cystic fibrosis.

  9. Breakdown of the reciprocal stabilization of QBRICK/Frem1, Fras1, and Frem2 at the basement membrane provokes Fraser syndrome-like defects.

    PubMed

    Kiyozumi, Daiji; Sugimoto, Nagisa; Sekiguchi, Kiyotoshi

    2006-08-08

    An emerging family of extracellular matrix proteins characterized by 12 consecutive CSPG repeats and the presence of Calx-beta motif(s) includes Fras1, QBRICK/Frem1, and Frem2. Mutations in the genes encoding these proteins have been associated with mouse models of Fraser syndrome, which is characterized by subepidermal blistering, cryptophthalmos, syndactyly, and renal dysmorphogenesis. Here, we report that all of these proteins are localized to the basement membrane, and that their basement membrane localization is simultaneously impaired in Fraser syndrome model mice. In Frem2 mutant mice, not only Frem2 but Fras1 and QBRICK/Frem1 were depleted from the basement membrane zone. This coordinated reduction in basement membrane deposition was also observed in another Fraser syndrome model mouse, in which GRIP1, a Fras1- and Frem2-interacting adaptor protein, is primarily affected. Targeted disruption of Qbrick/Frem1 also resulted in diminished expression of Fras1 and Frem2 at the epidermal basement membrane, confirming the reciprocal stabilization of QBRICK/Frem1, Fras1, and Frem2 in this location. When expressed and secreted by transfected cells, these proteins formed a ternary complex, raising the possibility that their reciprocal stabilization at the basement membrane is due to complex formation. Given the close association of Fraser syndrome phenotypes with defective epidermal-dermal interactions, the coordinated assembly of three Fraser syndrome-associated proteins at the basement membrane appears to be instrumental in epidermal-dermal interactions during morphogenetic processes.

  10. A novel membrane distillation-thermophilic bioreactor system: biological stability and trace organic compound removal.

    PubMed

    Wijekoon, Kaushalya C; Hai, Faisal I; Kang, Jinguo; Price, William E; Guo, Wenshan; Ngo, Hao H; Cath, Tzahi Y; Nghiem, Long D

    2014-05-01

    The removal of trace organic compounds (TrOCs) by a novel membrane distillation-thermophilic bioreactor (MDBR) system was examined. Salinity build-up and the thermophilic conditions to some extent adversely impacted the performance of the bioreactor, particularly the removal of total nitrogen and recalcitrant TrOCs. While most TrOCs were well removed by the thermophilic bioreactor, compounds containing electron withdrawing functional groups in their molecular structure were recalcitrant to biological treatment and their removal efficiency by the thermophilic bioreactor was low (0-53%). However, the overall performance of the novel MDBR system with respect to the removal of total organic carbon, total nitrogen, and TrOCs was high and was not significantly affected by the conditions of the bioreactor. All TrOCs investigated here were highly removed (>95%) by the MDBR system. Biodegradation, sludge adsorption, and rejection by MD contribute to the removal of TrOCs by MDBR treatment.

  11. Impact of Solvent pH on Direct Immobilization of Lysosome-Related Cell Organelle Extracts on TiO₂ for Melanin Treatment.

    PubMed

    Bang, Seung Hyuck; Kim, Pil; Oh, Suk-Jung; Kim, Yang-Hoon; Min, Jiho

    2015-05-01

    Techniques for immobilizing effective enzymes on nanoparticles for stabilization of the activity of free enzymes have been developing as a pharmaceutical field. In this study, we examined the effect of three different pH conditions of phosphate buffer, as a dissolving solvent for lysosomal enzymes, on the direct immobilization of lysosomal enzymes extracted from Hen's egg white and Saccharomyces cerevisiae. Titanium(IV) oxide (TiO2) nanoparticles, which are extensively used in many research fields, were used in this study. The lysosomal enzymes immobilized on TiO2 under each pH condition were evaluated to maintain the specific activity of lysosomal enzymes, so that we can determine the degree of melanin treatment in lysosomal enzymes immobilized on TiO2. We found that the immobilization efficiency and melanin treatment activity in both lysosomal enzymes extracted from Hen's egg white and S. cerevisiae were the highest in an acidic condition of phosphate buffer (pH 4). However, the immobilization efficiency and melanin treatment activity were inversely proportional to the increase in pH under alkaline conditions. In addition, enhanced immobilization efficiency was shown in TiO2 pretreated with a divalent, positively charged ion, Ca(2+), and the melanin treatment activity of immobilized lysosomal enzymes on TiO2 pretreated with Ca(2+) was also increased. Therefore, this result suggests that the immobilization efficiency and melanin treatment activity of lysosomal enzymes can be enhanced according to the pH conditions of the dissolving solvent.

  12. Silica nanoparticles induce autophagy dysfunction via lysosomal impairment and inhibition of autophagosome degradation in hepatocytes

    PubMed Central

    Wang, Ji; Yu, Yongbo; Lu, Ke; Yang, Man; Li, Yang; Zhou, Xianqing; Sun, Zhiwei

    2017-01-01

    Autophagy dysfunction is considered as a potential toxic mechanism of nanomaterials. Silica nanoparticles (SiNPs) can induce autophagy, but the specific mechanism involved remains unclear. Therefore, the aim of this study was to confirm the effects of SiNPs on autophagy dysfunction and explore the possible underlying mechanism. In this article, we reported that cell-internalized SiNPs exhibited dose- and time-dependent cytotoxicity in both L-02 and HepG2 cells. Multiple methods verified that SiNPs induced autophagy even at the noncytotoxic level and blocked the autophagic flux at the high-dose level. Notably, SiNPs impaired the lysosomal function through damaging lysosomal ultrastructures, increasing membrane permeability, and downregulating the expression of lysosomal proteases, cathepsin B, as evidenced by transmission electron microscopy, acridine orange staining, quantitative reverse transcription-polymerase chain reaction, and Western blot assays. Collectively, these data concluded that SiNPs inhibited autophagosome degradation via lysosomal impairment in hepatocytes, resulting in autophagy dysfunction. The current study not only discloses a potential mechanism of autophagy dysfunction induced by SiNPs but also provides novel evidence for the study of toxic effect and safety evaluation of SiNPs. PMID:28182147

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

    PubMed Central

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

    2016-01-01

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

  14. α-Synuclein Oligomers Stabilize Pre-Existing Defects in Supported Bilayers and Propagate Membrane Damage in a Fractal-Like Pattern.

    PubMed

    Chaudhary, Himanshu; Iyer, Aditya; Subramaniam, Vinod; Claessens, Mireille M A E

    2016-11-15

    Phospholipid vesicles are commonly used to get insights into the mechanism by which oligomers of amyloidogenic proteins damage membranes. Oligomers of the protein α-synuclein (αS) are thought to create pores in phospholipid vesicles containing a high amount of anionic phospholipids but fail to damage vesicle membranes at low surface charge densities. The current understanding of how αS oligomers damage the membranes is thus incomplete. This incomplete understanding may, in part, result from the choice of model membrane systems. The use of free-standing membranes such as vesicles may interfere with the unraveling of some damage mechanisms because the line tension at the edge of a membrane defect or pore ensures defect closure. Here, we have used supported lipid bilayers (SLBs) of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine (POPC/POPS) to study the membrane damage caused by αS oligomers. Although αS oligomers were not able to initiate the disruption of POPC/POPS vesicles or intact SLBs, oligomers did stabilize and enlarge pre-existing SLB defects. The increased exposure of lipid acyl chains at the edges of defects very likely facilitates membrane-oligomer interactions, resulting in the growth of fractal domains devoid of lipids. Concomitant with the appearance of the fractal membrane damage patterns, lipids appear in solution, directly implicating αS oligomers in the observed lipid extraction. The growth of the membrane damage patterns is not limited by the binding of lipids to the oligomer. The analysis of the shape and growth of the lipid-free domains suggests the involvement of an oligomer-dependent diffusion-limited extraction mechanism. The observed αS oligomer-induced propagation of membrane defects offers new insights into the mechanisms by which αS oligomers can contribute to the loss in membrane integrity.

  15. Role of the N-terminal transmembrane domain in the endo-lysosomal targeting and function of the human ABCB6 protein

    PubMed Central

    Kiss, Katalin; Kucsma, Nora; Brozik, Anna; Tusnady, Gabor E.; Bergam, Ptissam; vanNiel, Guillaume; Szakacs, Gergely

    2015-01-01

    ATP-binding cassette, subfamily B (ABCB) 6 is a homodimeric ATP-binding cassette (ABC) transporter present in the plasma membrane and in the intracellular organelles. The intracellular localization of ABCB6 has been a matter of debate, as it has been suggested to reside in the mitochondria and the endo-lysosomal system. Using a variety of imaging modalities, including confocal microscopy and EM, we confirm the endo-lysosomal localization of ABCB6 and show that the protein is internalized from the plasma membrane through endocytosis, to be distributed to multivesicular bodies and lysosomes. In addition to the canonical nucleotide-binding domain (NBD) and transmembrane domain (TMD), ABCB6 contains a unique N-terminal TMD (TMD0), which does not show sequence homology to known proteins. We investigated the functional role of these domains through the molecular dissection of ABCB6. We find that the folding, dimerization, membrane insertion and ATP binding/hydrolysis of the core–ABCB6 complex devoid of TMD0 are preserved. However, in contrast with the full-length transporter, the core–ABCB6 construct is retained at the plasma membrane and does not appear in Rab5-positive endosomes. TMD0 is directly targeted to the lysosomes, without passage to the plasma membrane. Collectively, our results reveal that TMD0 represents an independently folding unit, which is dispensable for catalysis, but has a crucial role in the lysosomal targeting of ABCB6. PMID:25627919

  16. Mechanisms of Dendritic Cell Lysosomal Killing of Cryptococcus

    NASA Astrophysics Data System (ADS)

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

    2012-10-01

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

  17. Size-Controlled AgI/Ag Heteronanowires in Highly Ordered Alumina Membranes: Superionic Phase Stabilization and Conductivity.

    PubMed

    Zhang, Hemin; Tsuchiya, Takashi; Liang, Changhao; Terabe, Kazuya

    2015-08-12

    Nanoscaled ionic conductors are crucial for future nanodevices. A well-known ionic conductor, AgI, exhibited conductivity greater than 1 Ω(-1) cm(-1) in α-phase and transformed into poorly conducting β-/γ-phase below 147 °C, thereby limiting applications. Here, we report that transition temperatures both from the β-/γ- to α-phase (Tc↑) and the α- to β-/γ-phase (Tc↓) are tuned by AgI/Ag heteronanowires embedded in anodic aluminum oxide (AAO) membranes with 10-30 nm pores. Tc↑ and Tc↓ shift to correspondingly higher and lower temperature as pore size decreases, generating a progressively enlarged thermal hysteresis. Tc↑ and Tc↓ specifically achieve 185 and 52 °C in 10 nm pores, and the final survived conductivity reaches ∼8.3 × 10(-3) Ω(-1) cm(-1) at room temperature. Moreover, the low-temperature stabilizing α-phase (down to 21 °C, the lowest in state of the art temperatures) is reproducible and survives further thermal cycling. The low-temperature phase stabilization and enhancement conductivity reported here suggest promising applications in silver-ion-based future nanodevices.

  18. New insights into non-precious metal catalyst layer designs for proton exchange membrane fuel cells: Improving performance and stability

    NASA Astrophysics Data System (ADS)

    Banham, Dustin; Kishimoto, Takeaki; Sato, Tetsutaro; Kobayashi, Yoshikazu; Narizuka, Kumi; Ozaki, Jun-ichi; Zhou, Yingjie; Marquez, Emil; Bai, Kyoung; Ye, Siyu

    2017-03-01

    The activity of non-precious metal catalysts (NPMCs) has now reached a stage at which they can be considered as possible alternatives to Pt for some proton exchange membrane fuel cell (PEMFC) applications. However, despite significant efforts over the past 50 years on catalyst development, only limited studies have been performed on NPMC-based cathode catalyst layer (CCL) designs. In this work, an extensive ionomer study is performed to investigate the impact of ionomer equivalent weight on performance, which has uncovered two crucial findings. Firstly, it is demonstrated that beyond a critical CCL conductance, no further improvement in performance is observed. The procedure used to determine this critical conductance can be used by other researchers in this field to aid in their design of high performing NPMC-based CCLs. Secondly, it is shown that the stability of NPMC-based CCLs can be improved through the use of low equivalent weight ionomers. This represents a completely unexplored pathway for further stability improvements, and also provides new insights into the possible degradation mechanisms occurring in NPMC-based CCLs. These findings have broad implications on all future NPMC-based CCL designs.

  19. Lysosomal adaptation: How cells respond to lysosomotropic compounds

    PubMed Central

    Lu, Shuyan; Sung, Tae; Lin, Nianwei; Abraham, Robert T.; Jessen, Bart A.

    2017-01-01

    Lysosomes are acidic organelles essential for degradation and cellular homoeostasis and recently lysosomes have been shown as signaling hub to respond to the intra and extracellular changes (e.g. amino acid availability). Compounds including pharmaceutical drugs that are basic and lipophilic will become sequestered inside