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Sample records for lysosome membrane stability

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

    Science.gov (United States)

    Chen, Linhua; Lu, Xianting; Ma, Yun

    2012-12-01

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

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

    Science.gov (United States)

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

    2015-05-01

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

  3. Stabilization of lysosomal membrane and cell membrane glycoprotein profile by Semecarpus anacardium linn. nut milk extract in experimental hepatocellular carcinoma.

    Science.gov (United States)

    Premalatha, B; Sachdanandam, P

    2000-08-01

    Semecarpus anacardium Linn. nut milk extract administered orally at a dose of 200 mg/kg/day for 14 days exerted an in vivo stabilizing effect on lysosomal membrane and glycoprotein content in rat hepatocellular carcinoma. This was demonstrated in normal rats and in animals whose biomembranes were rendered fragile by induction of hepatocellular carcinoma with aflatoxin B(1) and subsequent treatment with Semecarpus anacardium nut extract. In this condition, the discharge of lysosomal enzymes increased significantly with a subsequent increase in glycoprotein components. The nut extract administration reversed these adverse changes to near normal in treated animals. The possible reason for this reversal is discussed. Such stabilization of biomembranes by Semecarpus anacardium nut extract may have a beneficial effect in the treatment of hepatocellular carcinoma and other cancers involving abnormal fragility of lysosomes and glycoprotein content providing the extract demonstrates safety in a full toxicity study. Copyright 2000 John Wiley & Sons, Ltd.

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

    Science.gov (United States)

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

    2006-08-01

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

  5. Lysosomal membrane stability and metallothioneins in digestive gland of mussels (Mytilus galloprovincialis Lam.) as biomarkers in a field study

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    Petrovic, S.; Ozretic, B.; Krajnovic-Ozretic, M. [Ruder Boskovic Inst., Center for Marine Research, Rovinj (Croatia); Bobinac, D. [Rijeka Univ., Dept. of Anatomy, Rijeka (Croatia)

    2001-07-01

    The lysosomal membrane destabilisation and the metallothionein content in the digestive gland cells of mussels (Mytilus galloprovincialis Lam.), collected along the east coast of the North Adriatic (Istrian and Kvarnerine coast, Croatia), were examined over a period of four years (1996-1999). The lysosomal membrane stability, as a biomarker of general stress, showed that the membrane labilisation period in mussels from polluted, urban- and industrial-related areas was significantly decreased (p<0.05) when compared to mussels from control, clean sea water sites. In the harbour of Rijeka, the most contaminated site, the lysosomal membrane stability was reduced by more than 70% compared to the control. This method also proved to be a useful biomarker for detection of additional stress caused by short-term hypoxia that occurred once during this study inside the polluted and periodically quite eutrophic Pula Harbour. The concentration of metallothioneins in the mussel digestive gland, as a specific biomarker of exposure to heavy metals, did not reveal significant differences (p<0.05) between sites covered by this study. (Author)

  6. Lysosomal membrane stability and metallothionein content in Mytilus galloprovincialis (L.), as biomarkers Combination with trace metal concentrations

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    Domouhtsidou, G.P.; Dailianis, S.; Kaloyianni, M.; Dimitriadis, V.K

    2004-03-01

    The simultaneous study of two biomarkers, the lysosomal membrane stability (LMS) of the digestive gland and the metallothionein (MT) content of the digestive gland, the gills and the mantle/gonad complex of the mussel Mytilus galloprovincialis was examined in an enclosed estuarine system in June and October 2001. Mussels were collected along the Gulf of Thermaikos (northern Greece) from stations displaying a pollution gradient, while Olympiada in the Gulf of Strymonikos was used as a reference station. In addition, the heavy metal (Cd, Pb, Cu and Zn) content, using atomic absorption spectrophotometry (AAS), were evaluated in the digestive gland, the gills and the mantle/gonad complex of mussels collected from the same sites and seasons. LMS values were significantly greater, and the MT content of the studied tissues were significantly less in mussels collected from the reference station compared to mussels from more polluted stations located in the Gulf of Thermaikos. Significant correlation was observed between the MT content of the gills and the mantle/gonad complex with the LMS values.

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

    Science.gov (United States)

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

    2016-07-01

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

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

    Science.gov (United States)

    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

  9. Lysosome

    National Research Council Canada - National Science Library

    Ursula Matte BSc, PhD; Gabriela Pasqualim BSc, MSc

    2016-01-01

    Since Christian de Duve first described the lysosome in the 1950s, it has been generally presented as a membrane-bound compartment containing acid hydrolases that enables the cell to degrade molecules...

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

    Science.gov (United States)

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

    2016-01-26

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

  11. Lysosome

    Directory of Open Access Journals (Sweden)

    Ursula Matte BSc, PhD

    2016-12-01

    Full Text Available Since Christian de Duve first described the lysosome in the 1950s, it has been generally presented as a membrane-bound compartment containing acid hydrolases that enables the cell to degrade molecules without being digested by autolysis. For those working on the field of lysosomal storage disorders, the lack of one such hydrolase would lead to undegraded or partially degraded substrate storage inside engorged organelles disturbing cellular function by yet poorly explored mechanisms. However, in recent years, a much more complex scenario of lysosomal function has emerged, beyond and above the cellular “digestive” system. Knowledge on how the impairment of this organelle affects cell functioning may shed light on signs and symptoms of lysosomal disorders and open new roads for therapy.

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

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

    2009-05-15

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

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

    Science.gov (United States)

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

    2013-12-01

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

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

    DEFF Research Database (Denmark)

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

    2010-01-01

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

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

    Science.gov (United States)

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

    1995-05-01

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

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

    Science.gov (United States)

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

    2012-01-01

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

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

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    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.

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

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    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.

  19. Septins as modulators of endo-lysosomal membrane traffic

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    Kyungyeun Song

    2016-11-01

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

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

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    Wilke Sonja

    2012-07-01

    Full Text Available Abstract Background The family of lysosome-associated membrane proteins (LAMP comprises the multifunctional, ubiquitous LAMP-1 and LAMP-2, and the cell type-specific proteins DC-LAMP (LAMP-3, BAD-LAMP (UNC-46, C20orf103 and macrosialin (CD68. LAMPs have been implicated in a multitude of cellular processes, including phagocytosis, autophagy, lipid transport and aging. LAMP-2 isoform A acts as a receptor in chaperone-mediated autophagy. LAMP-2 deficiency causes the fatal Danon disease. The abundant proteins LAMP-1 and LAMP-2 are major constituents of the glycoconjugate coat present on the inside of the lysosomal membrane, the 'lysosomal glycocalyx'. The LAMP family is characterized by a conserved domain of 150 to 200 amino acids with two disulfide bonds. Results The crystal structure of the conserved domain of human DC-LAMP was solved. It is the first high-resolution structure of a heavily glycosylated lysosomal membrane protein. The structure represents a novel β-prism fold formed by two β-sheets bent by β-bulges and connected by a disulfide bond. Flexible loops and a hydrophobic pocket represent possible sites of molecular interaction. Computational models of the glycosylated luminal regions of LAMP-1 and LAMP-2 indicate that the proteins adopt a compact conformation in close proximity to the lysosomal membrane. The models correspond to the thickness of the lysosomal glycoprotein coat of only 5 to 12 nm, according to electron microscopy. Conclusion The conserved luminal domain of lysosome-associated membrane proteins forms a previously unknown β-prism fold. Insights into the structure of the lysosomal glycoprotein coat were obtained by computational models of the LAMP-1 and LAMP-2 luminal regions.

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

    NARCIS (Netherlands)

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

    2008-01-01

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

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

    NARCIS (Netherlands)

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

    2008-01-01

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

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

    DEFF Research Database (Denmark)

    Groth-Pedersen, Line; Aits, Sonja; Corcelle-Termeau, Elisabeth

    2012-01-01

    Microtubule-disturbing drugs inhibit lysosomal trafficking and induce lysosomal membrane permeabilization followed by cathepsin-dependent cell death. To identify specific trafficking-related proteins that control cell survival and lysosomal stability, we screened a molecular motor siRNA library...... in human MCF7 breast cancer cells. SiRNAs targeting four kinesins (KIF11/Eg5, KIF20A, KIF21A, KIF25), myosin 1G (MYO1G), myosin heavy chain 1 (MYH1) and tropomyosin 2 (TPM2) were identified as effective inducers of non-apoptotic cell death. The cell death induced by KIF11, KIF21A, KIF25, MYH1 or TPM2 si......), increased dextran accumulation (KIF20A), or reduced autophagic flux (MYO1G, MYH1). Importantly, all seven siRNAs also killed human cervix cancer (HeLa) and osteosarcoma (U-2-OS) cells and sensitized cancer cells to other lysosome-destabilizing treatments, i.e. photo-oxidation, siramesine, etoposide...

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

    Directory of Open Access Journals (Sweden)

    Bárbara Hissa

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

  5. The lysosomal stability as a biomarker for the determination of pollution in aquatic environments

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    Maria Loreto Nazar

    2008-10-01

    Full Text Available This work studied the effects caused by five different formulae of gasoline on the stability of the lysosomes isolated from the liver of the tilapia fish (Oreochromis niloticus. The functional integrity of the lysosomal membranes was evaluated via the acid phosphatase activity. The results showed that there were significant changes in the stability of the lysosomes exposed to the presence of the hydrocarbons in the environment. Therefore, considering the method's simplicity, the sensitivity of the responses and its low cost the assessment of the lysosomal activity could be an important tool for the study of the effects of pollution in the aquatic environments.A procura de biomarcadores de agentes poluidores, mais simples e menos custosos, tem levado ao estudo dos lisossomos, isolados de animais componentes da biota nos ambientes contaminados, principalmente por poluentes com características lipofílicas, a exemplo dos hidrocarbonetos policíclicos e seus derivados. Este trabalho estudou os efeitos provocados por 05 diferentes formulações de gasolina sobre a estabilidade de lisossomos, isolados de fígado de tilápia (Oreochromis niloticus. A integridade funcional das membranas lisossômicas foi avaliada através da atividade da fosfatase ácida, expressa em mU/mg de proteínas totais. Os resultados obtidos mostraram que existem alterações significativas na estabilidade dos lisossomos isolados de fígado de tilápias submetidas aos efeitos de hidrocarbonetos presentes no meio ambiente. Portanto, levando em conta a simplicidade, a sensibilidade de resposta e o baixo custo, os autores recomendam a avaliação da atividade lisossômica, como uma importante ferramenta para o estudo dos efeitos da poluição dos meios aquáticos.

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

    Science.gov (United States)

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

    2016-04-25

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

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

    OpenAIRE

    Wilke Sonja; Krausze Joern; Büssow Konrad

    2012-01-01

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

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

    Science.gov (United States)

    Einck, K H; Norkin, L C

    1979-01-01

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

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

    DEFF Research Database (Denmark)

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

    Targeting lysosomes is a novel approach in cancer therapy providing a possible way of killing the otherwise apoptosis-resistant cancer cells. Recent research has thus shown that lysosome targeting compounds induce cell death in a cervix cancer cell line. Tumor stem cells in glioblastomas have...... recently been suggested to possess innate resistance mechanisms against radiation and chemotherapy possibly explaining the high level of therapeutic resistance of these tumors. Since the presence and distribution of lysosomes in tumor cells and especially in tumor stem cells in astrocytomas is unknown......, the aim of this study was to investigate the immunohistochemical expression of LAMP-1, a membrane bound protein in lysosomes, in formalin fixed paraffin embedded tumor tissue from 23 diffuse astrocytomas, 17 anaplastic astrocytomas and 72 glioblastomas. The LAMP-1 expression was scored and compared...

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2013-10-18

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

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

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

    2016-12-18

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

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

    Science.gov (United States)

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

    2013-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Hanna Appelqvist

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

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

    Science.gov (United States)

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

    2001-01-01

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

  16. Cytosolic peroxidases protect the lysosome of bloodstream African trypanosomes from iron-mediated membrane damage.

    Directory of Open Access Journals (Sweden)

    Corinna Hiller

    2014-04-01

    Full Text Available African trypanosomes express three virtually identical non-selenium glutathione peroxidase (Px-type enzymes which preferably detoxify lipid-derived hydroperoxides. As shown previously, bloodstream Trypanosoma brucei lacking the mitochondrial Px III display only a weak and transient proliferation defect whereas parasites that lack the cytosolic Px I and Px II undergo extremely fast lipid peroxidation and cell lysis. The phenotype can completely be rescued by supplementing the medium with the α-tocopherol derivative Trolox. The mechanism underlying the rapid cell death remained however elusive. Here we show that the lysosome is the origin of the cellular injury. Feeding the px I-II knockout parasites with Alexa Fluor-conjugated dextran or LysoTracker in the presence of Trolox yielded a discrete lysosomal staining. Yet upon withdrawal of the antioxidant, the signal became progressively spread over the whole cell body and was completely lost, respectively. T. brucei acquire iron by endocytosis of host transferrin. Supplementing the medium with iron or transferrin induced, whereas the iron chelator deferoxamine and apo-transferrin attenuated lysis of the px I-II knockout cells. Immunofluorescence microscopy with MitoTracker and antibodies against the lysosomal marker protein p67 revealed that disintegration of the lysosome precedes mitochondrial damage. In vivo experiments confirmed the negligible role of the mitochondrial peroxidase: Mice infected with px III knockout cells displayed only a slightly delayed disease development compared to wild-type parasites. Our data demonstrate that in bloodstream African trypanosomes, the lysosome, not the mitochondrion, is the primary site of oxidative damage and cytosolic trypanothione/tryparedoxin-dependent peroxidases protect the lysosome from iron-induced membrane peroxidation. This process appears to be closely linked to the high endocytic rate and distinct iron acquisition mechanisms of the infective

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Hornick John R

    2012-05-01

    Full Text Available Abstract 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

  19. Anti-ulcer and membrane stabilizing actions of zinc acexamate.

    Science.gov (United States)

    Pfeiffer, C J; Bulbena, O; Esplugues, J V; Escolar, G; Navarro, C; Esplugues, J

    1987-01-01

    The effects of zinc acexamate on stress and reserpine ulcers as well as on gastric mast cells degranulation and membrane stability were evaluated in the rat. Zinc acexamate (100 mg/kg) has demonstrated an inhibitory effect on cold-restraint stress and reserpine-induced ulcer in a dose-dependent manner. Pretreatment of rats, prior to cold restraint stress, reduced gastric mast cell degranulation. Zinc acexamate (10(-4) M) inhibits Triton X-100 release of beta-glucuronidase in isolated hepatic lysosomes. These observations suggest that ulcer protective actions of zinc acexamate may be exerted in part through enhancing gastric mucosal resistance by stabilizing biological membrane integrity.

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

    Science.gov (United States)

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

    2010-12-01

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

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

    Science.gov (United States)

    Grasso, E J; Calderón, R O

    2013-02-01

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

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

    Science.gov (United States)

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

    1990-07-15

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

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

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

    2016-07-01

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

  5. Lack of the Lysosomal Membrane Protein, GLMP, in Mice Results in Metabolic Dysregulation in Liver.

    Directory of Open Access Journals (Sweden)

    Xiang Yi Kong

    Full Text Available Ablation of glycosylated lysosomal membrane protein (GLMP, formerly known as NCU-G1 has been shown to cause chronic liver injury which progresses into liver fibrosis in mice. Both lysosomal dysfunction and chronic liver injury can cause metabolic dysregulation. Glmp gt/gt mice (formerly known as Ncu-g1gt/gt mice were studied between 3 weeks and 9 months of age. Body weight gain and feed efficiency of Glmp gt/gt mice were comparable to wild type siblings, only at the age of 9 months the Glmp gt/gt siblings had significantly reduced body weight. Reduced size of epididymal fat pads was accompanied by hepatosplenomegaly in Glmp gt/gt mice. Blood analysis revealed reduced levels of blood glucose, circulating triacylglycerol and non-esterified fatty acids in Glmp gt/gt mice. Increased flux of glucose, increased de novo lipogenesis and lipid accumulation were detected in Glmp gt/gt primary hepatocytes, as well as elevated triacylglycerol levels in Glmp gt/gt liver homogenates, compared to hepatocytes and liver from wild type mice. Gene expression analysis showed an increased expression of genes involved in fatty acid uptake and lipogenesis in Glmp gt/gt liver compared to wild type. Our findings are in agreement with the metabolic alterations observed in other mouse models lacking lysosomal proteins, and with alterations characteristic for advanced chronic liver injury.

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

    Energy Technology Data Exchange (ETDEWEB)

    Lieberman, Raquel L.; D’aquino, J. Alejandro; Ringe, Dagmar; Petsko, Gregory A.; (Harvard-Med); (Brandeis)

    2009-06-05

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

  7. Membrane cholesterol removal changes mechanical properties of cells and induces secretion of a specific pool of lysosomes.

    Directory of Open Access Journals (Sweden)

    Barbara Hissa

    Full Text Available 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.

  8. ErbB2-associated changes in the lysosomal proteome

    DEFF Research Database (Denmark)

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

    2011-01-01

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

  9. Effects of lysosomal membrane protein depletion on the Salmonella-containing vacuole.

    Directory of Open Access Journals (Sweden)

    Everett A Roark

    Full Text Available Salmonella is an intracellular bacterial pathogen that replicates within a membrane-bound vacuole in host cells. The major lysosomal membrane proteins 1 and 2 (LAMP-1 and LAMP-2 are recruited to the Salmonella-containing vacuole as well as Salmonella- associated filaments (Sifs that emerge from the vacuole. LAMP-1 is a dominant membrane marker for the vacuole and Sifs. Its colocalization with both is dependent on a major secreted bacterial virulence protein, SifA. Here, we show that SifA is required for the recruitment of LAMP-2 and can be used as a second independent marker for both the bacterial vacuolar membrane and Sifs. Further, RNAi studies revealed that in LAMP-1 depleted cells, the bacteria remain membrane bound as measured by their association with LAMP-2 protein. In contrast, LAMP-2 depletion increased the amount of LAMP-1 free bacteria. Together, the data suggests that despite its abundance, LAMP-1 is not essential, but LAMP-2 may be partially important for the Salmonella-containing vacuolar membrane.

  10. Characterization of lysosomal membrane proteins of Dictyostelium discoideum. A complex population of acidic integral membrane glycoproteins, Rab GTP-binding proteins and vacuolar ATPase subunits.

    Science.gov (United States)

    Temesvari, L; Rodriguez-Paris, J; Bush, J; Steck, T L; Cardelli, J

    1994-10-14

    Highly purified lysosomes, prepared by magnetic fractionation of homogenates from Dictyostelium discoideum cells fed colloidal iron, were lysed under hypoosmotic conditions, and the membrane-associated proteins were subjected to gel electrophoresis. Thirteen major membrane polypeptides, ranging in molecular weight from 25,000 to 100,000 were identified. The isoelectric points of these proteins ranged from below 3.8 to greater than 7.0. Most of these proteins were stripped from membranes exposed to a chaotropic agent, 3,5-diodo-2-hydroxybenzoic acid lithium salt, and were therefore classified as peripheral membrane proteins. Twenty five glycoprotein species were detected by lectin blot analysis; 19 were classified as integral membrane proteins, and were, in general, larger than 45 kDa and negatively charged due in part to the presence of mannose 6-sulfate. Western blot analysis also demonstrated that a Rab 4-like GTPase, a Rab 7-like GTPase, and at least three subunits of the vacuolar ATPase were associated with the lysosomal membrane; the ATPase subunits appeared to be major proteins in lysosomal membranes. Finally, based on N-terminal sequence analysis of a major 41-kDa lysosome-associated membrane protein, we cloned a cDNA that encodes a protein (DVA41) highly homologous to a yeast and a bovine vacuolar ATPase subunit of approximately 41 kDa. The D. discoideum DVA41 gene was apparently a single copy gene, expressed at constant levels during growth and development.

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

    Science.gov (United States)

    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. PMID:27057276

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

    Science.gov (United States)

    Wang, Zheng; Cheng, Xingan; Meng, Qianqian; Wang, Peidan; Shu, Benshui; Hu, Qiongbo; Hu, Meiying; Zhong, Guohua

    2015-07-01

    Azadirachtin as a kind of botanical insecticide has been widely used in pest control. We previously reported that azadirachtin could induce apoptosis of Spodoptera litura cultured cell line Sl-1, which involves in the up-regulation of P53 protein. However, the detailed mechanism of azadirachtin-induced apoptosis is not clearly understood in insect cultured cells. The aim of the present study was to address the involvement of lysosome and lysosomal protease in azadirachtin-induced apoptosis in Sf9 cells. The result confirmed that azadirachtin indeed inhibited proliferation and induced apoptosis. The lysosomes were divided into different types as time-dependent manner, which suggested that changes of lysosomes were necessarily physiological processes in azadirachtin-induced apoptosis in Sf9 cells. Interestingly, we noticed that azadirachtin could trigger lysosomal membrane permeabilization and cathepsin L releasing to cytosol. Z-FF-FMK (a cathepsin L inhibitor), but not CA-074me (a cathepsin B inhibitor), could effectively hinder the apoptosis induced by azadirachtin in Sf9 cells. Meanwhile, the activity of caspase-3 could also be inactivated by the inhibition of cathepsin L enzymatic activity induced by Z-FF-FMK. Taken together, our findings suggest that azadirachtin could induce apoptosis in Sf9 cells in a lysosomal pathway, and cathepsin L plays a pro-apoptosis role in this process through releasing to cytosol and activating caspase-3.

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Yanyan Li

    2016-01-01

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

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

    DEFF Research Database (Denmark)

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

    , the aim of this study was to investigate the immunohistochemical expression of LAMP-1, a membrane bound protein in lysosomes, in formalin fixed paraffin embedded tumor tissue from 23 diffuse astrocytomas, 17 anaplastic astrocytomas and 72 glioblastomas. The LAMP-1 expression was scored and compared...... with both tumor grade and patient survival. Moreover double immunofluorescence stainings with LAMP-1 and the stem cell marker CD133 as well as the macrophage marker CD68 were performed. The results showed that LAMP-1 was expressed in the vast majority of tumors being present in the cytoplasm of single tumor...... cells, cell clusters and in blood vessel endothelial cells. The LAMP-1 expression in glioblastomas was significantly higher than in diffuse and anaplastic astrocytomas (pLAMP-1 and patient overall survival was found. Double immunofluorescence staining...

  16. Stabilizing membrane domains antagonizes anesthesia

    CERN Document Server

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

    2016-01-01

    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 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 test the correlation between the anesthetic potency of n-alcohols and effects on $T_c$. First we show that hexadecanol acts oppositely to anesthetics on membrane mixing and antagonizes ethanol induced anesthesia in a tadpole behavioral assay. Second, we show that two previously described `intoxication reversers' raise $T_c$ in vesicles and counter ethanol's effects in vesicles, mimicking the findings of previous electrophysiological 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 an anesthetic at relevant concen...

  17. Enhanced thermal stability of lysosomal beta-D-galactosidase in parenchymal cells of tumour bearing mice.

    Science.gov (United States)

    Lenti, L; Lipari, M; Lombardi, D; Zicari, A; Dotta, A; Pontieri, G M

    1986-12-01

    The thermal stability of the enzyme beta-D-galactosidase varies among different organs in normal C57Bl/6 mice, and increases in the same organs in mice with Lewis Lung carcinoma. Thermal stability of this enzyme is also increased by treatment of the mice with cell-free extracts of tumour cells or with inflammatory compounds such as carrageenan or orosomucoid. After desialylation, orosomucoid more effectively increases the heat stability of the enzyme. By contrast talc, which has no galactosyl groups, is without effect on the stability of the enzyme in vivo. Macrophages of tumour bearing mice release into the culture medium a more heat resistant enzyme than macrophages from control mice. In both cases the heat resistance of the secreted enzyme is higher when fetal calf serum is present in the culture medium. Bovine serum does not modify the thermal stability of beta-D-galactosidase in this system. Incubation of lysosomal fractions of various organs with the synthetic beta-D-galactosidase substrate, p-nitrophenyl-galactopyranoside, also strongly increases the heat resistance of the enzyme. The results suggest that one factor influencing the heat resistance of this enzyme may be complex formation between the enzyme and its substrates, an example of substrate protection of the enzyme. This may not be the only factor involved in enzyme stabilization in vivo.

  18. 乌司他丁对双瓣置换患者围术期溶酶体膜稳定性及凝血功能的影响%Effects of ulinastatin on the stability of lysosomal membrane and blood coagulation functions in patients undergoing mitral and aortic valves replacement:a double blinded randomized controlled study

    Institute of Scientific and Technical Information of China (English)

    王臻; 程亮; 曹轶; 赵璧君; 张秀静; 金振晓

    2013-01-01

    目的观察体外循环中应用乌司他丁对双瓣置换患者围术期溶酶体膜稳定性及凝血功能的影响。方法在体外循环下行二尖瓣联合主动脉瓣置换患者20例,随机分为乌司他丁组及对照组,乌司他丁组患者按20000 U/kg总量给予乌司他丁,其中半量预充,半量于主动脉开放前加入到体外循环管路中,对照组患者给予等量生理盐水。分别于术前30 min,体外循环60 min、120 min及体外循环结束后30 min采集血样,ELISA方法检测血浆中组织蛋白酶D( CTSD)及N-乙酰-β-D-氨基葡萄糖苷酶( NAG)含量;采用血栓弹力图比较手术前后凝血功能。同时,记录术后ICU滞留时间、引流量、24 h尿量及呋塞米用量,以及术后呼吸机辅助时间、死亡等情况。结果两组患者均顺利出院,无死亡,无严重并发症;术后ICU时间、机械通气时间、术后第1个24小时尿量及呋塞米用量组间无显著差异( P>0.05);与对照组比较,凝血功能各项指标在手术结束后未见明显差异( P>0.05);体外循环过程中各时间点NAG及CTSD含量组间无显著差异( P>0.05)。结论体外循环中应用乌司他丁20000 U/kg对双瓣置换患者围术期溶酶体稳定性及凝血功能的保护作用与对照组没有明显差异。%Objective To investigate the effects of ulinastatin on the stability of lysosomal membrane and blood coagulation functions in patients undergoing mitral and aortic valves replacement .Methods Twenty consecutive patients scheduled for mitral and aortic valve replacement surgery under cardiopulmonary bypass (CPB) were randomized into two groups:ulinastatin group , who received 20 000 U/kg ulinastatin during CPB with half added into the prime solution and half added to the circuit upon aortic clamping removal;control group , who received same volume of saline .Blood sample were collected at 30 min before the operation ,60 min and 120

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

    NARCIS (Netherlands)

    Rood, I.M.; Merchant, M.L.; Wilkey, D.W.; Zhang, T.; Zabrouskov, V.; Vlag, J. van der; Dijkman, H.B.P.M.; Willemsen, B.K.; Wetzels, J.F.M.; Klein, J.B.; Deegens, J.K.J.

    2015-01-01

    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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2009-10-15

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

  1. Engineered nanomaterial-induced lysosomal membrane permeabilization and anti-cathepsin agents.

    Science.gov (United States)

    Bunderson-Schelvan, Melisa; Holian, Andrij; Hamilton, Raymond F

    2017-01-01

    Engineered nanomaterials (ENMs), or small anthropogenic particles approximately < 100 nm in size and of various shapes and compositions, are increasingly incorporated into commercial products and used for industrial and medical purposes. There is an exposure risk to both the population at large and individuals in the workplace with inhalation exposures to ENMs being a primary concern. Further, there is increasing evidence to suggest that certain ENMs may represent a significant health risk, and many of these ENMs exhibit distinct similarities with other particles and fibers that are known to induce adverse health effects, such as asbestos, silica, and particulate matter (PM). Evidence regarding the importance of lysosomal membrane permeabilization (LMP) and release of cathepsins in ENM toxicity has been accumulating. The aim of this review was to describe our current understanding of the mechanisms leading to ENM-associated pathologies, including LMP and the role of cathepsins with a focus on inflammation. In addition, anti-cathepsin agents, some of which have been tested in clinical trials and may prove useful for ameliorating the harmful effects of ENM exposure, are examined.

  2. Niemann-Pick C1 Functions Independently of Niemann-Pick C2 in the Initial Stage of Retrograde Transport of Membrane-impermeable Lysosomal Cargo*

    Science.gov (United States)

    Goldman, Stephen D. B.; Krise, Jeffrey P.

    2010-01-01

    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 [3H]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. PMID:20007703

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Chih-Peng Chang

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

  6. Lysosome associated membrane proteins maintain pancreatic acinar cell homeostasis : LAMP-2 deficient mice develop pancreatitis

    NARCIS (Netherlands)

    Mareninova, Olga A; Sendler, Matthias; Malla, Sudarshan Ravi; Yakubov, Iskandar; French, Samuel W; Tokhtaeva, Elmira; Vagin, Olga; Oorschot, Viola; Lüllmann-Rauch, Renate; Blanz, Judith; Dawson, David; Klumperman, Judith; Lerch, Markus M; Mayerle, Julia; Gukovsky, Ilya; Gukovskaya, Anna S

    2015-01-01

    BACKGROUND & AIMS: The pathogenic mechanism of pancreatitis is poorly understood. Recent evidence implicates defective autophagy in pancreatitis responses; however, the pathways mediating impaired autophagy in pancreas remain largely unknown. Here, we investigate the role of lysosome associated memb

  7. Lysosome associated membrane proteins maintain pancreatic acinar cell homeostasis : LAMP-2 deficient mice develop pancreatitis

    NARCIS (Netherlands)

    Mareninova, Olga A; Sendler, Matthias; Malla, Sudarshan Ravi; Yakubov, Iskandar; French, Samuel W; Tokhtaeva, Elmira; Vagin, Olga; Oorschot, Viola; Lüllmann-Rauch, Renate; Blanz, Judith; Dawson, David; Klumperman, Judith; Lerch, Markus M; Mayerle, Julia; Gukovsky, Ilya; Gukovskaya, Anna S

    2015-01-01

    BACKGROUND & AIMS: The pathogenic mechanism of pancreatitis is poorly understood. Recent evidence implicates defective autophagy in pancreatitis responses; however, the pathways mediating impaired autophagy in pancreas remain largely unknown. Here, we investigate the role of lysosome associated

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-10-01

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

  9. Nanoporous silica membranes with high hydrothermal stability

    DEFF Research Database (Denmark)

    Boffa, Vittorio; Magnacca, Giualiana; Yue, Yuanzheng

    Despite the use of sol-gel derived nanoporous silica membranes in substitution of traditional separation processes is expected leading to vast energy savings, their intrinsic poor steam-stability hampers their application at an industrial level. Transition metal ions can be used as dopant...... to improve the stability of nanoporous silica structure. This work is a quantitative study on the impact of type and concentration of transition metal ions on the microporous structure and stability of amorphous silica-based membranes, which provides information on how to design chemical compositions...... and synthetic paths for the fabrication of silica-based membranes with a well accessible and highly stabile nanoporous structure...

  10. Vertebrate scavenger receptor class B member 2 (SCARB2: comparative studies of a major lysosomal membrane glycoprotein

    Directory of Open Access Journals (Sweden)

    Roger Stephen Holmes

    2012-06-01

    Full Text Available Scavenger receptor class B member 2 (SCARB2 (also LIMP-2, CD36L2 or LGP85 is a major lysosomal membrane glycoprotein involved in endosomal and lysosomal biogenesis and maintenance. SCARB2 acts as a receptor for the lysosomal mannose-6-phosphate independent targeting of β-glucuronidase and enterovirus 71 and influences Parkinson’s disease and epilepsy. Genetic deficiency of this protein causes deafness and peripheral neuropathy in mice as well as myoclonic epilepsy and nephrotic syndrome in humans. Comparative SCARB2 amino acid sequences and structures and SCARB2 gene locations were examined using data from several vertebrate genome projects. Vertebrate SCARB2 sequences shared 43-100% identity as compared with 30-36% sequence identities with other CD36-like superfamily members, SCARB1 and CD36. At least 10 N-glycosylation sites were conserved among most vertebrate SCARB2 proteins examined. Sequence alignments, key amino acid residues and conserved predicted secondary structures were examined, including cytoplasmic, transmembrane and external lysosomal membrane sequences: cysteine disulfide residues, thrombospondin (THP1 binding sites and 16 proline and 20 glycine conserved residues, which may contribute to short loop formation within the exomembrane SCARB2 sequences. Vertebrate SCARB2 genes contained 12 coding exons. The human SCARB2 gene contained a CpG island (CpG100, ten microRNA-binding sites and several transcription factor binding sites (including PPARA which may contribute to a higher level (2.4 times average of gene expression. Phylogenetic analyses examined the relationships and potential evolutionary origins of the vertebrate SCARB2 gene with vertebrate SCARB1 and CD36 genes. These suggested that SCARB2 originated from duplications of the CD36 gene in an ancestral genome forming three vertebrate CD36 gene family members: SCARB1, SCARB2 and CD36.

  11. Erythrocyte stability, membrane protective and haematological ...

    African Journals Online (AJOL)

    Erythrocyte stability, membrane protective and haematological activities of Newbouldia ... The high prevalence rate of diabetes mellitus (DM) in the developing world and its ... It dose-dependently decreased the packed cell volume (PCV) from ...

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

    Science.gov (United States)

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

    2014-07-01

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

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

    Science.gov (United States)

    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.

  14. Tubular Steel Arch Stabilized by Textile Membranes

    Directory of Open Access Journals (Sweden)

    Ondrej Svoboda

    2016-10-01

    Full Text Available Tubular steel arch supporting textile membrane roofing is investigated experimentally and numerically. The stabilization effects of the textile membrane on in-plane and out-of-plane behavior of the arch is of primary interest. First a model of a large membrane structure tested in laboratory is described. Prestressed membranes of PVC coated polyester fabric Ferrari® Précontraint 702S were used as a currently standard and excellent material. The test arrangement, loading and resulting load/deflection values are presented. The supporting structure consisted of two steel arch tubes, outer at edge of the membrane and inner supporting interior of the membrane roofing. The stability and strength behavior of the inner tube under both symmetrical and asymmetrical loading was monitored and is shown in some details. Second the SOFiSTiK software was employed to analyze the structural behavior in 3D, using geometrically nonlinear analysis with imperfections (GNIA. The numerical analysis, FE mesh sensitivity, the membrane prestressing and common boundary conditions are validated by test results. Finally a parametrical study concerning stability of mid arch with various geometries in a membrane structure with several supporting arches is presented, with recommendations for a practical design.

  15. Isomeric Detergent Comparison for Membrane Protein Stability

    DEFF Research Database (Denmark)

    Cho, Kyung Ho; Hariharan, Parameswaran; Mortensen, Jonas S.;

    2016-01-01

    Membrane proteins encapsulated by detergent micelles are widely used for structural study. Because of their amphipathic property, detergents have the ability to maintain protein solubility and stability in an aqueous medium. However, conventional detergents have serious limitations in their scope...... and utility, particularly for eukaryotic membrane proteins and membrane protein complexes. Thus, a number of new agents have been devised; some have made significant contributions to membrane protein structural studies. However, few detergent design principles are available. In this study, we prepared meta...... and ortho isomers of the previously reported para-substituted xylene-linked maltoside amphiphiles (XMAs), along with alkyl chain-length variation. The isomeric XMAs were assessed with three membrane proteins, and the meta isomer with a C12 alkyl chain was most effective at maintaining solubility/stability...

  16. Changes in the morphology and lability of lysosomal subpopulations in caerulein-induced acute pancreatitis.

    Science.gov (United States)

    Sarmiento, Nancy; Sánchez-Yagüe, Jesús; Juanes, Pedro P; Pérez, Nieves; Ferreira, Laura; García-Hernández, Violeta; Mangas, Arturo; Calvo, José J; Sánchez-Bernal, Carmen

    2011-02-01

    Lysosomes play an important role in acute pancreatitis (AP). Here we developed a method for the isolation of lysosome subpopulations from rat pancreas and assessed the stability of lysosomal membranes. AP was induced by four subcutaneous injections of 20 μg caerulein/kg body weight at hourly intervals. The animals were killed 9h after the first injection. Marker enzymes [N-acetyl-β-D-glucosaminidase (NAG), cathepsin B and succinate dehydrogenase (SDH)] were assayed in subcellular fractions from control pancreas and in pancreatitis. Lysosomal subpopulations were separated by Percoll density gradient centrifugation and observed by electron microscopy. NAG molecular forms were determined by DEAE-cellulose chromatography. AP was associated with: (i) increases in the specific activity of lysosomal enzymes in the soluble fraction, (ii) changes in the size and alterations in the morphology of the organelles from the lysosomal subpopulations, (iii) the appearance of large vacuoles in the primary and secondary lysosome subpopulations, (iv) the increase in the amount of the NAG form associated with the pancreatic lysosomal membrane as well as its release towards the soluble fraction. Lysosome subpopulations are separated by a combination of differential and Percoll density gradient centrifugations. Primary lysosome membrane stability decreases in AP. Copyright © 2010 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.

  17. Chromatographic finger print analysis and lysosomal membrane stabilisation activity of active fraction of Alstonia scholaris leaf extract in arthritic rats

    Directory of Open Access Journals (Sweden)

    Swapnil Goyal

    2014-01-01

    Full Text Available Object: The present study was aimed to assess the anti-arthritic activity of chloroform fraction of Alstonia scholaris leaf extract against Freund′s complete adjuvant (FCA-induced arthritis in rats. Materials and Methods: The anti-inflammatory activity of various fractions of ethanolic extract of Alstonia scholaris at concentration of 100 mg/kg was studied using the carrageenan-induced inflammatory models. The chloroform fraction shows significant anti-inflammatory activity. The chloroform fraction was further studied for anti-arthritic activity and HPTLC fingerprint analysis. For anti-arthritic activity, the active chloroform fraction was administered at the concentrations of 50 and 100 mg/kg body weight. The effect of chloroform fraction on liver ALP, ACP and LDH levels of lysosomal enzymes of FCA arthritic animals were studied. Indomethacin and prednisolone (10 mg/kg was used as standard. HPTLC studies were carried out using CAMAG HPTLC system equipped with linomat IV applicator, TLC scanner; Reprostar 3 and WIN CATS-4 software were used. Results: The chloroform fraction at 100 mg/kg, showed maximum inhibition (34.16% of inflammation induced by carrageenan. In FCA-induced arthritis, the chloroform fraction showed a highly significant reduction in paw volume (50 mg/kg-72.71%; 100 mg/kg-74.35%. The levels of lysosomal enzymes were significantly decreased in the chloroform fraction-treated groups. Conclusion: The possible mechanism of action of the chloroform fraction of Alstonia scholaris leaf extract may be through its stabilising action on lysosomal membranes. Future studies will provide new insights into the anti-arthritic activity of Alstonia scholaris and isolation of compound from it may eventually lead to development of a new class of anti-arthritic agent.

  18. The proteome of lysosomes.

    Science.gov (United States)

    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.

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

    OpenAIRE

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

    2015-01-01

    Para-toluenesulfonamide (PTS) has been implicated with anticancer effects against a variety of tumors. In the present study, we investigated the inhibitory effects of PTS on tongue squamous cell carcinoma (Tca-8113) and explored the lysosomal and mitochondrial changes after PTS treatment in vitro. High-performance liquid chromatography showed that PTS selectively accumulated in Tca-8113 cells with a relatively low concentration in normal fibroblasts. Next, the effects of PTS on cell viability...

  20. Lysosomal cell death at a glance

    DEFF Research Database (Denmark)

    Aits, Sonja; Jaattela, Marja

    2013-01-01

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

  1. Lysosomal membrane permeabilization is involved in oxidative stress-induced apoptotic cell death in LAMP2-deficient iPSCs-derived cerebral cortical neurons

    Directory of Open Access Journals (Sweden)

    Cheuk-Yiu Law

    2016-03-01

    Our results from cellular fractionation and inhibitor blockade experiments further revealed that oxidative stress-induced apoptosis in the LAMP2-deficient cortical neurons was caused by increased abundance of cytosolic cathepsin L. These results suggest the involvement of lysosomal membrane permeabilization in the LAMP2 deficiency associated neural injury.

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

    DEFF Research Database (Denmark)

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

    2013-01-01

    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......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...

  3. Polyunsaturated Lipids Regulate Membrane Domain Stability by Tuning Membrane Order.

    Science.gov (United States)

    Levental, Kandice R; Lorent, Joseph H; Lin, Xubo; Skinkle, Allison D; Surma, Michal A; Stockenbojer, Emily A; Gorfe, Alemayehu A; Levental, Ilya

    2016-04-26

    The plasma membrane (PM) serves as the functional interface between a cell and its environment, hosting extracellular signal transduction and nutrient transport among a variety of other processes. To support this extensive functionality, PMs are organized into lateral domains, including ordered, lipid-driven assemblies termed lipid rafts. Although the general requirements for ordered domain formation are well established, how these domains are regulated by cell-endogenous mechanisms or exogenous perturbations has not been widely addressed. In this context, an intriguing possibility is that dietary fats can incorporate into membrane lipids to regulate the properties and physiology of raft domains. Here, we investigate the effects of polyunsaturated fats on the organization of membrane domains across a spectrum of membrane models, including computer simulations, synthetic lipid membranes, and intact PMs isolated from mammalian cells. We observe that the ω-3 polyunsaturated fatty acid docosahexaenoic acid is robustly incorporated into membrane lipids, and this incorporation leads to significant remodeling of the PM lipidome. Across model systems, docosahexaenoic acid-containing lipids enhance the stability of ordered raft domains by increasing the order difference between them and coexisting nonraft domains. The relationship between interdomain order disparity and the stability of phase separation holds for a spectrum of different perturbations, including manipulation of cholesterol levels and high concentrations of exogenous amphiphiles, suggesting it as a general feature of the organization of biological membranes. These results demonstrate that polyunsaturated fats affect the composition and organization of biological membranes, suggesting a potential mechanism for the extensive effects of dietary fat on health and disease.

  4. Membrane chemical stability and seed longevity

    NARCIS (Netherlands)

    Golovina, E.A.; Hoekstra, F.A.; As, van H.

    2010-01-01

    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 chemi

  5. TRAIL death receptor 4 signaling via lysosome fusion and membrane raft clustering in coronary arterial endothelial cells: evidence from ASM knockout mice.

    Science.gov (United States)

    Li, Xiang; Han, Wei-Qing; Boini, Krishna M; Xia, Min; Zhang, Yang; Li, Pin-Lan

    2013-01-01

    Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptor, death receptor 4 (DR4), have been implicated in the development of endothelial dysfunction and atherosclerosis. However, the signaling mechanism mediating DR4 activation leading to endothelial injury remains unclear. We recently demonstrated that ceramide production via hydrolysis of membrane sphingomyelin by acid sphingomyelinase (ASM) results in membrane raft (MR) clustering and the formation of important redox signaling platforms, which play a crucial role in amplifying redox signaling in endothelial cells leading to endothelial dysfunction. The present study aims to investigate whether TRAIL triggers MR clustering via lysosome fusion and ASM activation, thereby conducting transmembrane redox signaling and changing endothelial function. Using confocal microscopy, we found that TRAIL induced MR clustering and co-localized with DR4 in coronary arterial endothelial cells (CAECs) isolated from wild-type (Smpd1 (+/+)) mice. Furthermore, TRAIL triggered ASM translocation, ceramide production, and NADPH oxidase aggregation in MR clusters in Smpd1 ( +/+ ) CAECs, whereas these observations were not found in Smpd1 (-/-) CAECs. Moreover, ASM deficiency reduced TRAIL-induced O(2) (-[Symbol: see text]) production in CAECs and abolished TRAIL-induced impairment on endothelium-dependent vasodilation in small resistance arteries. By measuring fluorescence resonance energy transfer, we found that Lamp-1 (lysosome membrane marker protein) and ganglioside G(M1) (MR marker) were trafficking together in Smpd1 (+/+) CAECs, which was absent in Smpd1 (-/-) CAECs. Consistently, fluorescence imaging of living cells with specific lysosome probes demonstrated that TRAIL-induced lysosome fusion with membrane was also absent in Smpd1 (-/-) CAECs. Taken together, these results suggest that ASM is essential for TRAIL-induced lysosomal trafficking, membrane fusion and formation of MR redox signaling platforms

  6. Membrane chemical stability and seed longevity.

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Mayim E. Wiens

    2017-01-01

    Full Text Available 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.

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

    Science.gov (United States)

    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

  9. Internalization, lysosomal degradation and new synthesis of surface membrane CD4 in phorbol ester-activated T-lymphocytes and U-937 cells

    DEFF Research Database (Denmark)

    Petersen, C M; Christensen, E I; Andresen, B S

    1992-01-01

    degradation was low in resting cells. Endocytosis and/or degradation of anti-CD4 mAb was suppressed by H7, and by inhibitors of membrane traffic (Monensin) and lysosome function (methylamine, chloroquine). Immunocytochemistry localized CD4 to the surface of unstimulated T-cells. Upon PMA stimulation...... occasional labeling was seen in endosomes but whole cell CD4 decreased dramatically. However, methylamine-treated PMA blasts showed accumulation of CD4 in lysosomes and accordingly, pulse-chase experiments in biolabeled cell cultures suggested a manifest reduction of CD4 half-life in response to PMA. Despite...... in activated cells was further evidenced by metabolic labeling and Northern blot analysis demonstrating unaltered or slightly increased CD4 protein and mRNA levels resulting from PMA. Our findings demonstrate that phorbol esters downregulate the cellular CD4 pool by endocytosis and subsequent lysosomal...

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

    Directory of Open Access Journals (Sweden)

    Eric Bartee

    Full Text Available 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.

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

    Science.gov (United States)

    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.

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

    DEFF Research Database (Denmark)

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

    2010-01-01

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

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

    OpenAIRE

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

    2016-01-01

    Prions are proteinaceous infectious particles which cause fatal neurodegenerative disorders in humans and animals. They consist of a mostly β-sheeted aggregated isoform (PrPSc) of the cellular prion protein (PrPc). Prions replicate autocatalytically in neurons and other cell types by inducing conformational conversion of PrPc into PrPSc. Within neurons, PrPSc accumulates at the plasma membrane and in vesicles of the endocytic pathway. To better understand the mechanisms underlying neuronal dy...

  14. Enhanced thermal stability of lysosomal beta-D-galactosidase in parenchymal cells of tumour bearing mice.

    OpenAIRE

    1986-01-01

    The thermal stability of the enzyme beta-D-galactosidase varies among different organs in normal C57Bl/6 mice, and increases in the same organs in mice with Lewis Lung carcinoma. Thermal stability of this enzyme is also increased by treatment of the mice with cell-free extracts of tumour cells or with inflammatory compounds such as carrageenan or orosomucoid. After desialylation, orosomucoid more effectively increases the heat stability of the enzyme. By contrast talc, which has no galactosyl...

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

    Directory of Open Access Journals (Sweden)

    Tatjana Mijatovic

    2006-05-01

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

  16. 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.

    Science.gov (United States)

    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.

  17. Molecular cloning of cDNAs encoding lamp A, a human lysosomal membrane glycoprotein with apparent M sub r approx 120,000

    Energy Technology Data Exchange (ETDEWEB)

    Viitala, J.; Carlsson, S.R.; Siebert, P.D.; Fukuda, M. (La Jolla Cancer Research Foundation, CA (USA))

    1988-06-01

    Although several lysosomal membrane glycoproteins have been characterized by using specific antibodies, none of the studies so far elucidated the amino acid sequence of a lysosomal membrane glycoprotein. Here we describe cDNA clones encoding for one of the lysosome-associated membrane proteins with apparent M{sub r} {approx} 120,000, lamp A. The amino acid sequence based on the fully coded cDNA shows that as many as 18 potential N-glycosylation sites can be found in the total of 385 amino acid residues. The results obtained by endoglycosidase F digestion support the conclusion that this glycoprotein contains 18 N-glycans. These N-glycosylation sites are clustered in two domains; one contains 10 and the other contains 8 N-glycosylation sites. These domains are separated by a (proline-serine)-rich region that has a distinct homology to the IgA hinge structure. The first N-glycosylated domain is elongated to a potential leader peptide toward the NH{sub 2}-terminal end. The second N-glycosylated domain, on the other hand, is connected to a putative transmembrane portion consisting of hydrophobic amino acids. This segment, in turn, is elongated to a short cytoplasmic segment composed of 11 amino acid residues at the COOH-terminal end.

  18. Stability of Model Membranes in Extreme Environments

    Science.gov (United States)

    Namani, Trishool; Deamer, David W.

    2008-08-01

    The first forms of cellular life required a source of amphiphilic compounds capable of assembling into stable boundary structures. Membranes composed of fatty acids have been proposed as model systems of primitive membranes, but their bilayer structure is stable only within a narrow pH range and low ionic strength. They are particularly sensitive to aggregating effects of divalent cations (Mg+2, Ca+2, Fe+2) that would be present in Archaean sea water. Here we report that mixtures of alkyl amines and fatty acids form vesicles at strongly basic and acidic pH ranges which are resistant to the effects of divalent cations up to 0.1 M. Vesicles formed by mixtures of decylamine and decanoic acid (1:1 mole ratio) are relatively permeable to pyranine, a fluorescent anionic dye, but permeability could be reduced by adding 2 mol% of a polycyclic aromatic hydrocarbon such as pyrene. Permeability to the dye was also reduced by increasing the chain length of the amphiphiles. For instance, 1:1 mole ratio mixtures of dodecylamine and dodecanoic acid were able to retain pyranine dye during and following gel filtration. We conclude that primitive cell membranes were likely to be composed of mixtures of amphiphilic and hydrophobic molecules that manifested increased stability over pure fatty acid membranes.

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

    Science.gov (United States)

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

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    Halina Milnerowicz

    2014-01-01

    Full Text Available 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.

  2. Anthrax lethal toxin induced lysosomal membrane permeabilization and cytosolic cathepsin release is Nlrp1b/Nalp1b-dependent.

    Directory of Open Access Journals (Sweden)

    Kathleen M Averette

    Full Text Available NOD-like receptors (NLRs are a group of cytoplasmic molecules that recognize microbial invasion or 'danger signals'. Activation of NLRs can induce rapid caspase-1 dependent cell death termed pyroptosis, or a caspase-1 independent cell death termed pyronecrosis. Bacillus anthracis lethal toxin (LT, is recognized by a subset of alleles of the NLR protein Nlrp1b, resulting in pyroptotic cell death of macrophages and dendritic cells. Here we show that LT induces lysosomal membrane permeabilization (LMP. The presentation of LMP requires expression of an LT-responsive allele of Nlrp1b, and is blocked by proteasome inhibitors and heat shock, both of which prevent LT-mediated pyroptosis. Further the lysosomal protease cathepsin B is released into the cell cytosol and cathepsin inhibitors block LT-mediated cell death. These data reveal a role for lysosomal membrane permeabilization in the cellular response to bacterial pathogens and demonstrate a shared requirement for cytosolic relocalization of cathepsins in pyroptosis and pyronecrosis.

  3. Lysosomal cell death mechanisms in aging.

    Science.gov (United States)

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

    2016-12-01

    Lysosomes are degradative organelles essential for cell homeostasis that regulate a variety of processes, from calcium signaling and nutrient responses to autophagic degradation of intracellular components. Lysosomal cell death is mediated by the lethal effects of cathepsins, which are released into the cytoplasm following lysosomal damage. This process of lysosomal membrane permeabilization and cathepsin release is observed in several physiopathological conditions and plays a role in tissue remodeling, the immune response to intracellular pathogens and neurodegenerative diseases. Many evidences indicate that aging strongly influences lysosomal activity by altering the physical and chemical properties of these organelles, rendering them more sensitive to stress. In this review we focus on how aging alters lysosomal function and increases cell sensitivity to lysosomal membrane permeabilization and lysosomal cell death, both in physiological conditions and age-related pathologies. Copyright © 2016 Elsevier B.V. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Xiang Y. Kong

    2014-03-01

    Full Text Available 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.

  5. Endocytosed 2-Microglobulin Amyloid Fibrils Induce Necrosis and Apoptosis of Rabbit Synovial Fibroblasts by Disrupting Endosomal/Lysosomal Membranes: A Novel Mechanism on the Cytotoxicity of Amyloid Fibrils.

    Directory of Open Access Journals (Sweden)

    Tadakazu Okoshi

    Full Text Available Dialysis-related amyloidosis is a major complication in long-term hemodialysis patients. In dialysis-related amyloidosis, β2-microglobulin (β2-m amyloid fibrils deposit in the osteoarticular tissue, leading to carpal tunnel syndrome and destructive arthropathy with cystic bone lesions, but the mechanism by which these amyloid fibrils destruct bone and joint tissue is not fully understood. In this study, we assessed the cytotoxic effect of β2-m amyloid fibrils on the cultured rabbit synovial fibroblasts. Under light microscopy, the cells treated with amyloid fibrils exhibited both necrotic and apoptotic changes, while the cells treated with β2-m monomers and vehicle buffer exhibited no morphological changes. As compared to β2-m monomers and vehicle buffer, β2-m amyloid fibrils significantly reduced cellular viability as measured by the lactate dehydrogenase release assay and the 3-(4,5-di-methylthiazol-2-yl-2,5-diphenyltetrazolium bromide reduction assay and significantly increased the percentage of apoptotic cells as measured by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling method. β2-m amyloid fibrils added to the medium adhered to cell surfaces, but did not disrupt artificial plasma membranes as measured by the liposome dye release assay. Interestingly, when the cells were incubated with amyloid fibrils for several hours, many endosomes/lysosomes filled with amyloid fibrils were observed under confocal laser microscopy and electron microscopy, Moreover, some endosomal/lysosomal membranes were disrupted by intravesicular fibrils, leading to the leakage of the fibrils into the cytosol and adjacent to mitochondria. Inhibition of actin-dependent endocytosis by cytochalasin D attenuated the toxicity of amyloid fibrils. These results suggest that endocytosed β2-m amyloid fibrils induce necrosis and apoptosis by disrupting endosomal/lysosomal membranes, and this novel mechanism on the cytotoxicity of amyloid

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

    Science.gov (United States)

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

    2014-10-28

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

  7. 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.

    Science.gov (United States)

    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.

  8. Hydrothermal stability of microporous silica and niobia-silica membranes

    NARCIS (Netherlands)

    Boffa, V.; Blank, David H.A.; ten Elshof, Johan E.

    2008-01-01

    The hydrothermal stability of microporous niobia–silica membranes was investigated and compared with silica membranes. The membranes were exposed to hydrothermal conditions at 150 and 200 °C for 70 h. The change of pore structure before and after exposure to steam was probed by single-gas permeation

  9. Evaluation of antioxidant capacity and membrane stabilizing ...

    African Journals Online (AJOL)

    Kemi Akinwunmi

    2015-05-27

    May 27, 2015 ... to protect red blood cell (RBC) membrane against hypotonic and heat induced lyses in a concentration dependent ... blood cells membrane by drugs against hypotonicity induced ..... Expert committee on diabetes mellitus.

  10. LYSOSOMAL DISRUPTION BY BACTERIAL TOXINS

    Science.gov (United States)

    Bernheimer, Alan W.; Schwartz, Lois L.

    1964-01-01

    Bernheimer, Alan W. (New York University School of Medicine, New York), and Lois L. Schwartz. Lysosomal disruption by bacterial toxins. J. Bacteriol. 87:1100–1104. 1964.—Seventeen bacterial toxins were examined for capacity (i) to disrupt rabbit leukocyte lysosomes as indicated by decrease in turbidity of lysosomal suspensions, and (ii) to alter rabbit liver lysosomes as measured by release of β-glucuronidase and acid phosphatase. Staphylococcal α-toxin, Clostridium perfringens α-toxin, and streptolysins O and S affected lysosomes in both systems. Staphylococcal β-toxin, leucocidin and enterotoxin, Shiga neurotoxin, Serratia endotoxin, diphtheria toxin, tetanus neurotoxin, C. botulinum type A toxin, and C. perfringens ε-toxin were not active in either system. Staphylococcal δ-toxin, C. histolyticum collagenase, crude C. perfringens β-toxin, and crude anthrax toxin caused lysosomal damage in only one of the test systems. There is a substantial correlation between the hemolytic property of a toxin and its capacity to disrupt lysosomes, lending support to the concept that erythrocytes and lysosomes are bounded by similar membranes. PMID:5874534

  11. Stability and rupture of archaebacterial cell membrane: a model study.

    Science.gov (United States)

    Li, Shuangyang; Zheng, Fengxian; Zhang, Xianren; Wang, Wenchuan

    2009-01-29

    It is known that the thermoacidophilic archaebacterium Sulfolobus acidocaldarius can grow in hot springs at 65-80 degrees C and live in acidic environments (pH 2-3); however, the origin of its unusual thermal stability remains unclear. In this work, using a vesicle as a model, we study the thermal stability and rupture of archaebacterial cell membrane. We perform a simulation investigation of the structure-property relationship of monolayer membrane formed by bolaform lipids and compare it with that of bilayer membrane formed by monopolar lipids. The origin of the unusually thermal stability of archaebacterial cell and the mechanism for its rupture are presented in molecular details.

  12. Impaired Lysosomal Integral Membrane Protein 2-dependent Peroxiredoxin 6 Delivery to Lamellar Bodies Accounts for Altered Alveolar Phospholipid Content in Adaptor Protein-3-deficient pearl Mice.

    Science.gov (United States)

    Kook, Seunghyi; Wang, Ping; Young, Lisa R; Schwake, Michael; Saftig, Paul; Weng, Xialian; Meng, Ying; Neculai, Dante; Marks, Michael S; Gonzales, Linda; Beers, Michael F; Guttentag, Susan

    2016-04-15

    The Hermansky Pudlak syndromes (HPS) constitute a family of disorders characterized by oculocutaneous albinism and bleeding diathesis, often associated with lethal lung fibrosis. HPS results from mutations in genes of membrane trafficking complexes that facilitate delivery of cargo to lysosome-related organelles. Among the affected lysosome-related organelles are lamellar bodies (LB) within alveolar type 2 cells (AT2) in which surfactant components are assembled, modified, and stored. AT2 from HPS patients and mouse models of HPS exhibit enlarged LB with increased phospholipid content, but the mechanism underlying these defects is unknown. We now show that AT2 in the pearl mouse model of HPS type 2 lacking the adaptor protein 3 complex (AP-3) fails to accumulate the soluble enzyme peroxiredoxin 6 (PRDX6) in LB. This defect reflects impaired AP-3-dependent trafficking of PRDX6 to LB, because pearl mouse AT2 cells harbor a normal total PRDX6 content. AP-3-dependent targeting of PRDX6 to LB requires the transmembrane protein LIMP-2/SCARB2, a known AP-3-dependent cargo protein that functions as a carrier for lysosomal proteins in other cell types. Depletion of LB PRDX6 in AP-3- or LIMP-2/SCARB2-deficient mice correlates with phospholipid accumulation in lamellar bodies and with defective intraluminal degradation of LB disaturated phosphatidylcholine. Furthermore, AP-3-dependent LB targeting is facilitated by protein/protein interaction between LIMP-2/SCARB2 and PRDX6 in vitro and in vivo Our data provide the first evidence for an AP-3-dependent cargo protein required for the maturation of LB in AT2 and suggest that the loss of PRDX6 activity contributes to the pathogenic changes in LB phospholipid homeostasis found HPS2 patients.

  13. Tandem Facial Amphiphiles for Membrane Protein Stabilization

    DEFF Research Database (Denmark)

    Chae, Pil Seok; Gotfryd, Kamil; Pacyna, Jennifer

    2010-01-01

    We describe a new type of synthetic amphiphile that is intended to support biochemical characterization of intrinsic membrane proteins. Members of this new family displayed favorable behavior with four of five membrane proteins tested, and these amphiphiles formed relatively small micelles....

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

    Science.gov (United States)

    Roy, Subhro Jyoti; Stanely Mainzen Prince, Ponnian

    2012-11-01

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

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

    OpenAIRE

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

    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 c...

  16. Copper Induced Lysosomal Membrane Destabilisation in Haemolymph Cells of Mediterranean Green Crab (Carcinus aestuarii, Nardo, 1847 from the Narta Lagoon (Albania

    Directory of Open Access Journals (Sweden)

    Valbona Aliko

    2015-10-01

    Full Text Available ABSTRACTDestabilisation of blood cell lysosomes in Mediterranean green crabCarcinus aestuarii was investigated using Neutral Red Retention Assay (NRRA. Crabs collected in Narta Lagoon, Vlora (Albania during May 2014 were exposed in the laboratory to sub-lethal, environmentally realistic concentrations of copper. Neutral Red Retention Time (NRRT and glucose concentration in haemolymph of animals were measured. The mean NRRT showed a significant reduction for the animals of the treatment group compared to the control one (from 118.6 ± 28.4 to 36.4 ± 10.48 min, p<0.05, indicating damage of lysosomal membrane. Haemolymph glucose concentration was significantly higher in the treatment group (from 37.8 ± 2.7 to 137.8.4 ± 16.2 mg/dL, p<0.05 than in control group, demonstrating the presence of stress on the animals. These results showed thatC. aestuarii could be used as a successful and reliable bioindicator for evaluating the exposure to contaminants in laboratory conditions. NRRA provides a successful tool for rapid assessment of heavy metal pollution effects on marine biota.

  17. Nanodomain stabilization dynamics in plasma membranes of biological cells

    Science.gov (United States)

    Das, Tamal; Maiti, Tapas K.; Chakraborty, Suman

    2011-02-01

    We discover that a synergistically amplifying role of stabilizing membrane proteins and continuous lipid recycling can explain the physics governing the stability, polydispersity, and dynamics of lipid raft domains in plasma membranes of biological cells. We establish the conjecture using a generalized order parameter based on theoretical formalism, endorsed by detailed scaling arguments and domain mapping. Quantitative agreements with morphological distributions of raft complexes, as obtained from Förster resonance energy transfer based visualization, support the present theoretical conjecture.

  18. Membrane and envelope virus proteins co-expressed as lysosome associated membrane protein (LAMP fused antigens: a potential tool to develop DNA vaccines against flaviviruses

    Directory of Open Access Journals (Sweden)

    Rafael Dhalia

    2009-12-01

    Full Text Available Vaccination is the most practical and cost-effective strategy to prevent the majority of the flavivirus infection to which there is an available vaccine. However, vaccines based on attenuated virus can potentially promote collateral side effects and even rare fatal reactions. Given this scenario, the developent of alternative vaccination strategies such as DNA-based vaccines encoding specific flavivirus sequences are being considered. Endogenous cytoplasmic antigens, characteristically plasmid DNA-vaccine encoded, are mainly presented to the immune system through Major Histocompatibility Complex class I - MHC I molecules. The MHC I presentation via is mostly associated with a cellular cytotoxic response and often do not elicit a satisfactory humoral response. One of the main strategies to target DNA-encoded antigens to the MHC II compartment is expressing the antigen within the Lysosome-Associated Membrane Protein (LAMP. The flavivirus envelope protein is recognized as the major virus surface protein and the main target for neutralizing antibodies. Different groups have demonstrated that co-expression of flavivirus membrane and envelope proteins in mammalian cells, fused with the carboxyl-terminal of LAMP, is able to induce satisfactory levels of neutralizing antibodies. Here we reviewed the use of the envelope flavivirus protein co-expression strategy as LAMP chimeras with the aim of developing DNA vaccines for dengue, West Nile and yellow fever viruses.A vacinação é a estratégia mais prática e o melhor custo-benefício para prevenir a maioria das infecções dos flavivirus, para os quais existe vacina disponível. Entretanto, as vacinas baseadas em vírus atenuados podem potencialmente promover efeitos colaterais e, mais raramente, reações fatais. Diante deste cenário, o desenvolvimento de estratégias alternativas de vacinação, como vacinas baseadas em DNA codificando seqüências específicas dos flavivirus, está sendo considerado

  19. Brief exposure to copper activates lysosomal exocytosis.

    Science.gov (United States)

    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.

  20. Behaviour of Steel Arch Stabilized by a Textile Membrane

    Science.gov (United States)

    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.

  1. The stability of supported liquid membranes

    NARCIS (Netherlands)

    Neplenbroek, A.M.; Bargeman, D.; Smolders, C.A.

    1990-01-01

    In this paper a new hypothesis about the instability mechanism of SLMs will be discussed: emulsion formation induced by lateral shear forces. Experimental results show that a water phase with a low salt concentration which flows along the membrane interface causes the removal of both solvent and car

  2. Stability and dynamics of membrane-spanning DNA nanopores

    Science.gov (United States)

    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.

  3. Role of cardiolipin in stability of integral membrane proteins.

    Science.gov (United States)

    Musatov, Andrej; Sedlák, Erik

    2017-08-23

    Cardiolipin (CL) is a unique phospholipid with a dimeric structure having four acyl chains and two phosphate groups found almost exclusively in certain membranes of bacteria and of mitochondria of eukaryotes. CL interacts with numerous proteins and has been implicated in function and stabilization of several integral membrane proteins (IMPs). While both functional and stabilization roles of CL in IMPs has been generally acknowledged, there are, in fact, only limited number of quantitative analysis that support this function of CL. This is likely caused by relatively complex determination of parameters characterizing stability of IMPs and particularly intricate assessment of role of specific PLs such as CL in IMPs stability. This review aims to summarize quantitative findings regarding stabilization role of CL in IMPs reported up to now. Copyright © 2017 Elsevier B.V. and Societe Francaise de Biochimie et Biologie Moleculaire (SFBBM). All rights reserved.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

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

    Science.gov (United States)

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

    2016-04-01

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

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

    Science.gov (United States)

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

    2015-05-26

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

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

    Science.gov (United States)

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

    2012-03-13

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

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

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

    2004-11-01

    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, 18C(10), was optimized for solubilization of proteins derived from bacterial outer membranes (Duval-Terrie 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 Ca(2+)-dependent ATPase and Ca(2+)-pumping activity. We found that 18C(10)-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.

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

    Science.gov (United States)

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

    2011-09-01

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

  11. Identification of differential anti-neoplastic activity of copper bis(thiosemicarbazones) that is mediated by intracellular reactive oxygen species generation and lysosomal membrane permeabilization.

    Science.gov (United States)

    Stefani, Christian; Al-Eisawi, Zaynab; Jansson, Patric J; Kalinowski, Danuta S; Richardson, Des R

    2015-11-01

    Bis(thiosemicarbazones) and their copper (Cu) complexes possess unique anti-neoplastic properties. However, their mechanism of action remains unclear. We examined the structure-activity relationships of twelve bis(thiosemicarbazones) to elucidate factors regarding their anti-cancer efficacy. Importantly, the alkyl substitutions at the diimine position of the ligand backbone resulted in two distinct groups, namely, unsubstituted/monosubstituted and disubstituted bis(thiosemicarbazones). This alkyl substitution pattern governed their: (1) Cu(II/I) redox potentials; (2) ability to induce cellular (64)Cu release; (3) lipophilicity; and (4) anti-proliferative activity. The potent anti-cancer Cu complex of the unsubstituted bis(thiosemicarbazone) analog, glyoxal bis(4-methyl-3-thiosemicarbazone) (GTSM), generated intracellular reactive oxygen species (ROS), which was attenuated by Cu sequestration by a non-toxic Cu chelator, tetrathiomolybdate, and the anti-oxidant, N-acetyl-l-cysteine. Fluorescence microscopy suggested that the anti-cancer activity of Cu(GTSM) was due, in part, to lysosomal membrane permeabilization (LMP). For the first time, this investigation highlights the role of ROS and LMP in the anti-cancer activity of bis(thiosemicarbazones).

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

    Science.gov (United States)

    Múgica, M; Izagirre, U; Marigómez, I

    2015-07-01

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

  13. The nucleotide sequence of a CpG island demonstrates the presence of the first exon of the gene encoding the human lysosomal membrane protein lamp2 and assigns the gene to Xq24.

    Science.gov (United States)

    Manoni, M; Tribioli, C; Lazzari, B; DeBellis, G; Patrosso, C; Pergolizzi, R; Pellegrini, M; Maestrini, E; Rivella, S; Vezzoni, P

    1991-03-01

    An EagI-EcoRI clone of human genomic DNA, p2-7, mapped to Xq24 has been sequenced. This analysis has confirmed the presence of a CpG island and has identified the first exon of the human LAMP2 gene, encoding a glycoprotein of the lysosomal membrane. Since the p2-7 clone corresponds to single-copy DNA, we can assign the human LAMP2 gene to Xq24.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  16. The role of interfacial lipids in stabilizing membrane protein oligomers.

    Science.gov (United States)

    Gupta, Kallol; Donlan, Joseph A C; Hopper, Jonathan T S; Uzdavinys, Povilas; Landreh, Michael; Struwe, Weston B; Drew, David; Baldwin, Andrew J; Stansfeld, Phillip J; Robinson, Carol V

    2017-01-19

    Oligomerization of membrane proteins in response to lipid binding has a critical role in many cell-signalling pathways but is often difficult to define or predict. Here we report the development of a mass spectrometry platform to determine simultaneously the presence of interfacial lipids and oligomeric stability and to uncover how lipids act as key regulators of membrane-protein association. Evaluation of oligomeric strength for a dataset of 125 α-helical oligomeric membrane proteins reveals an absence of interfacial lipids in the mass spectra of 12 membrane proteins with high oligomeric stability. For the bacterial homologue of the eukaryotic biogenic transporters (LeuT, one of the proteins with the lowest oligomeric stability), we found a precise cohort of lipids within the dimer interface. Delipidation, mutation of lipid-binding sites or expression in cardiolipin-deficient Escherichia coli abrogated dimer formation. Molecular dynamics simulation revealed that cardiolipin acts as a bidentate ligand, bridging across subunits. Subsequently, we show that for the Vibrio splendidus sugar transporter SemiSWEET, another protein with low oligomeric stability, cardiolipin shifts the equilibrium from monomer to functional dimer. We hypothesized that lipids are essential for dimerization of the Na(+)/H(+) antiporter NhaA from E. coli, which has the lowest oligomeric strength, but not for the substantially more stable homologous Thermus thermophilus protein NapA. We found that lipid binding is obligatory for dimerization of NhaA, whereas NapA has adapted to form an interface that is stable without lipids. Overall, by correlating interfacial strength with the presence of interfacial lipids, we provide a rationale for understanding the role of lipids in both transient and stable interactions within a range of α-helical membrane proteins, including G-protein-coupled receptors.

  17. Structuring detergents for extracting and stabilizing functional membrane proteins.

    Directory of Open Access Journals (Sweden)

    Rima Matar-Merheb

    Full Text Available BACKGROUND: Membrane proteins are privileged pharmaceutical targets for which the development of structure-based drug design is challenging. One underlying reason is the fact that detergents do not stabilize membrane domains as efficiently as natural lipids in membranes, often leading to a partial to complete loss of activity/stability during protein extraction and purification and preventing crystallization in an active conformation. METHODOLOGY/PRINCIPAL FINDINGS: Anionic calix[4]arene based detergents (C4Cn, n=1-12 were designed to structure the membrane domains through hydrophobic interactions and a network of salt bridges with the basic residues found at the cytosol-membrane interface of membrane proteins. These compounds behave as surfactants, forming micelles of 5-24 nm, with the critical micellar concentration (CMC being as expected sensitive to pH ranging from 0.05 to 1.5 mM. Both by 1H NMR titration and Surface Tension titration experiments, the interaction of these molecules with the basic amino acids was confirmed. They extract membrane proteins from different origins behaving as mild detergents, leading to partial extraction in some cases. They also retain protein functionality, as shown for BmrA (Bacillus multidrug resistance ATP protein, a membrane multidrug-transporting ATPase, which is particularly sensitive to detergent extraction. These new detergents allow BmrA to bind daunorubicin with a Kd of 12 µM, a value similar to that observed after purification using dodecyl maltoside (DDM. They preserve the ATPase activity of BmrA (which resets the protein to its initial state after drug efflux much more efficiently than SDS (sodium dodecyl sulphate, FC12 (Foscholine 12 or DDM. They also maintain in a functional state the C4Cn-extracted protein upon detergent exchange with FC12. Finally, they promote 3D-crystallization of the membrane protein. CONCLUSION/SIGNIFICANCE: These compounds seem promising to extract in a functional state

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

    Science.gov (United States)

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

    2014-04-01

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

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

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

    2014-08-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-04-01

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

  2. Thermal stability of nafion membranes under mechanical stress

    Energy Technology Data Exchange (ETDEWEB)

    Quintilii, M.; Struis, R. [Paul Scherrer Inst. (PSI), Villigen (Switzerland)

    1997-06-01

    The feasibility of adequately modified fluoro-ionomer membranes (NAFION{sup R}) is demonstrated for the selective separation of methanol synthesis products from the raw reactor gas at temperatures around 200{sup o}C. For an economically relevant application of this concept on a technical scale the Nafion membranes should be thin ({approx_equal}10 {mu}m) and thermally stable over a long period of time (1-2 years). In cooperation with industry (Methanol Casale SA, Lugano (CH)), we test the thermal stability of Nafion hollow fibers and supported Nafion thin sheet membranes at temperatures between 160 and 200{sup o}C under mechanical stress by applying a gas pressure difference over the membrane surface ({Delta}P{<=} 40 bar). Tests with the hollow fibers revealed that Nafion has visco-elastic properties. Tests with 50 {mu}m thin Nafion sheets supported by a porous metal carrier at 200{sup o}C and {Delta}P=39 bar showed no mechanical defects over a period of 92 days. (author) 5 figs., 4 refs.

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

    Science.gov (United States)

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

    2014-05-01

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

  4. The role of cholesterol-sphingomyelin membrane nanodomains in the stability of intercellular membrane nanotubes

    Directory of Open Access Journals (Sweden)

    Veranič P

    2012-04-01

    Full Text Available Maruša Lokar1,*, Doron Kabaso1,2,*, Nataša Resnik3, Kristina Sepcic5, Veronika Kralj-Iglic4,6, Peter Veranic3, Robert Zorec2, Aleš Iglic1,6 1Laboratory of Biophysics, Faculty of Electrical Engineering, 2Laboratory of Neuroendocrinology-Molecular Cell Physiology, Faculty of Medicine, 3Institute of Cell Biology, Faculty of Medicine, 4Faculty of Health Sciences, 5Department of Biology, Biotechnical Faculty, 6Laboratory of Clinical Biophysics, Department of Orthopedic Surgery, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia*These authors equally share first authorshipAbstract: Intercellular membrane nanotubes (ICNs are highly curved tubular structures that connect neighboring cells. The stability of these structures depends on the inner cytoskeleton and the cell membrane composition. Yet, due to the difficulty in the extraction of ICNs, the cell membrane composition remains elusive. In the present study, a raft marker, ostreolysin, revealed the enrichment of cholesterol-sphingomyelin membrane nanodomains along ICNs in a T24 (malignant urothelial cancer cell line. Cholesterol depletion, due to the addition of methyl-β-cyclodextrin, caused the dispersion of cholesterol-sphingomyelin membrane nanodomains and the retraction of ICNs. The depletion of cholesterol also led to cytoskeleton reorganization and to formation of actin stress fibers. Live cell imaging data revealed the possible functional coupling between the change from polygonal to spherical shape, cell separation, and the disconnection of ICNs. The ICN was modeled as an axisymmetric tubular structure, enabling us to investigate the effects of cholesterol content on the ICN curvature. The removal of cholesterol was predicted to reduce the positive spontaneous curvature of the remaining membrane components, increasing their curvature mismatch with the tube curvature. The mechanisms by which the increased curvature mismatch could contribute to the disconnection of ICNs are

  5. Solid-Supported Lipid Membranes: Formation, Stability and Applications

    Science.gov (United States)

    Goh, Haw Zan

    This thesis presents a comprehensive investigation of the formation of supported lipid membranes with vesicle hemifusion, their stability under detergents and organic solvents and their applications in molecular biology. In Chapter 3, we describe how isolated patches of DOPC bilayers supported on glass surfaces are dissolved by various detergents (decyl maltoside, dodecyl maltoside, CHAPS, CTAB, SDS, TritonX-100 and Tween20) at their CMC, as investigated by fluorescence video microscopy. In general, detergents partition into distal leaflets of bilayers and lead to the expansion of the bilayers through a rolling motion of the distal over the proximal leaflets, in agreement with the first stage of the established 3-stage model of lipid vesicle solubilization by detergents. Subsequently, we study the partitioning of organic solvents (methanol, ethanol, isopropanol, propanol, acetone and chloroform) into isolated bilayer patches on glass in Chapter 4 with fluorescence microscopy. The area expansion of bilayers due to the partitioning of organic solvents is measured. From the titration of organic solvents, we measured the rate of area expansion as a function of the volume fraction of organic solvents, which is proposed to be a measure of strength of interactions between solvents and membranes. From the same experiments, we also measure the maximum expansion of bilayers (or the maximum binding stoichiometry between organic solvents and lipids) before structural breakdown, which depends on the depth of penetration of solvents to the membranes. In Chapter 5, we investigate the formation of sparsely-tethered bilayer lipid membranes (stBLMs) with vesicle hemifusion. In vesicle hemifusion, lipid vesicles in contact with a hydrophobic alkyl-terminated self-assembled monolayer (SAM) deposit a lipid monolayer to the SAM surface, thus completing the bilayer. Electrical Impedance Spectroscopy and Neutron Reflectivity are used to probe the integrity of stBLMs in terms of their

  6. Enzyme stabilization by linear chain polymers in ultrafiltration membrane reactors

    Energy Technology Data Exchange (ETDEWEB)

    Greco, G.; Gianfreda, L.

    1981-10-01

    The experimental results discussed in this article concern pi-nitrophenylphosphate hydrolysis by acid phosphatase in an ultrafiltration membrane reactor. The basic conclusions drawn are : 1) Linking the enzyme to a soluble support does not give rise to an increase in its stability while the chemical manipulations involved result in marked reductions in enzymic activity. 2) Enzyme entrapment within a proteic gel produces a considerable increase in its thermal stability as compared to the diluted native enzyme; this presumably stems from drastic reductions in enzyme mobility. 3) Correspondingly, considerable reductions occur in enzyme activity that depend on substrate mass transfer resistances within the gel layer. 4) Small amounts of linear chain water-soluble synthetic polymers (polyacrylamides) give rise to high macromolecular concentration levels in the reactor region where the enzyme is dynamically immobilized and produce the same enzyme stabilization as gel entrapment. 5) Only minor substrate mass transfer limitations take place in this region and hence enzyme activity is virtually unaffected. 6) Both effects (stabilization and slight activity reduction) seem not to depend strongly on the characteristics of the soluble polymer (molecular weight and ionic character). (Refs. 16).

  7. DNA encoding an HIV-1 Gag/human lysosome-associated membrane protein-1 chimera elicits a broad cellular and humoral immune response in Rhesus macaques.

    Directory of Open Access Journals (Sweden)

    Priya Chikhlikar

    Full Text Available Previous studies of HIV-1 p55Gag immunization of mice have demonstrated the usefulness of targeting antigens to the cellular compartment containing the major histocompatibility complex type II (MHC II complex molecules by use of a DNA antigen formulation encoding Gag as a chimera with the mouse lysosome-associated membrane protein (mLAMP/gag. In the present study, we have analyzed the magnitude and breadth of Gag-specific T-lymphocyte and antibody responses elicited in Rhesus macaques after immunization with DNA encoding a human LAMP/gag (hLAMP/gag chimera. ELISPOT analyses indicated that the average Gag-specific IFN-gamma response elicited by the hLAMP/gag chimera was detectable after only two or three naked DNA immunizations in all five immunized macaques and reached an average of 1000 spot-forming cells (SFC/10(6 PBMCs. High IFN-gamma ELISPOT responses were detected in CD8(+-depleted cells, indicating that CD4(+ T-cells play a major role in these responses. The T-cell responses of four of the macaques were also tested by use of ELISPOT to 12 overlapping 15-amino acids (aa peptide pools containing ten peptides each, encompassing the complete Gag protein sequence. The two Mamu 08 immunized macaques responded to eight and twelve of the pools, the Mamu B01 to six, and the other macaque to five pools indicating that the hLAMP/gag DNA antigen formulation elicits a broad T-cell response against Gag. Additionally, there was a strong HIV-1-specific IgG response. The IgG antibody titers increased after each DNA injection, indicating a strong amnestic B-cell response, and were highly elevated in all the macaques after three immunizations. Moreover, the serum of each macaque recognized 13 of the 49 peptides of a 20-aa peptide library covering the complete Gag amino acid sequence. In addition, HIV-1-specific IgA antibodies were present in the plasma and external secretions, including nasal washes. These data support the findings of increased

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

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

    2016-12-09

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

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

    Science.gov (United States)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-01-27

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

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

    Science.gov (United States)

    Jyoti Roy, Abhro; Stanely Mainzen Prince, P

    2013-01-15

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

  13. TRPML and lysosomal function.

    Science.gov (United States)

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

    2007-08-01

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

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

    DEFF Research Database (Denmark)

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

    2009-01-01

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

  15. Cell biology in China: Focusing on the lysosome.

    Science.gov (United States)

    Yang, Chonglin; Wang, Xiaochen

    2017-06-01

    The view that lysosomes are merely the recycling bins of the cell has changed greatly during recent years. Lysosomes are now known to play a central role in signal transduction, cellular adaptation, plasma membrane repair, immune responses and many other fundamental cellular processes. In conjunction with the seminal discoveries made by international colleagues, many important questions regarding lysosomes are being addressed by Chinese scientists. In this review, we briefly summarize recent exciting findings in China on lysosomal signaling, biogenesis, integrity and physiological functions. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

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

    Science.gov (United States)

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

    2012-01-01

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

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

    Science.gov (United States)

    Xiong, Jian; Zhu, Michael X

    2016-08-01

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

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

    Science.gov (United States)

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

    2015-02-01

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

  19. Supported liquid membrane stability in chiral resolution by chemically and physically modified membranes

    Energy Technology Data Exchange (ETDEWEB)

    Molinari, R.; Argurio, P. [Arcavata di Rende Univ. of Calabria, Arcavata di Rende, CS (Italy). Dept. of Chemical and Materials Engineering

    2001-04-01

    In the present work some stability studies on Supported Liquid Membranes (SLMs) to be used for chiral separations were realized. In particular, primary aim was to determine how a modification of the support surface influences the SLM stability. First, the procedure for support modification was optimised, making a screening of various compounds (sulphuric acid, nitric acid, chromic acid, sodium dodecyl sulphate (SDS), glycerol, oleic alcohol, propylene glycol (PPG), bovine serum albumin (BSA)) and testing their performance by means of contact angle measurements. Next, a second screening was realized by permeation tests in a stirred cell. Finally, to compare the stability of modified with unmodified support in a process of interest for chemical and/or biochemical industries, some permeation tests for resolution of DNB-DL-Leucine were realized in a re-circulation system. Results showed a better surface hydrophilization of chemically modified support and better stability of the sulphonated support. However, in operating conditions a little high stability of the unmodified support was obtained. [Italian] Nel presente lavoro sono stati realizzati degli studi di stabilita' di Membrane Liquide Supportate (SLMs) da impiegare in separazioni chirali. In particolare, obiettivo principale e' stato quello di determinare l'influenza che una modifica della superficie del supporto ha sulla stabilita' della SLM. Cosi', in un primo momento, e' stata ottimizzata le procedura di modifica del supporto, facendo una selezione tra vari composti (acido solforico, acido nitrico, acido cromico, sodio dodecil solfato (SDS), glicerolo, alcool oleico, glicole propilenico (PPG), siero di albumina bovina (BSA)) basata su misure dell'angolo di contatto. Successivamente, e' stata realizzata una seconda selezione mediante prove di permeazione in una cella agitata. Infine, con lo scopo di confrontare la stabilita' della SLM con supporto modificato rispetto

  20. Secondary Lysosomal Changes in Liver in Preclinical Drug Development

    Institute of Scientific and Technical Information of China (English)

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

    2005-01-01

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

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

    Science.gov (United States)

    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 (Perythrocyte membrane stability to hydrogen peroxide. Reduced erythrocyte membrane stability may be further evaluated as a potential peripheral marker for oxidative damage in AD.

  2. Lysosome Biogenesis and Autophagy

    NARCIS (Netherlands)

    Reggiori, Fulvio; Klumperman, Judith|info:eu-repo/dai/nl/075097273

    2016-01-01

    Lysosomes degrade biological components acquired by endocytosis, the major cellular pathway for internalization of extracellular material, and macroautophagy. This chapter presents an overview of these two major degradative intracellular pathways, and highlights the emerging cross talks between

  3. Endosome-lysosomes and neurodegeneration.

    Science.gov (United States)

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

    1994-01-01

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

  4. Insights into thermophilic archaebacterial membrane stability from simplified models of lipid membranes

    Science.gov (United States)

    Davis, Charles H.; Nie, Huifen; Dokholyan, Nikolay V.

    2007-05-01

    Lipid aggregation into fluid bilayers is an essential process for sustaining life. Simplified models of lipid structure, which allow for long time scales or large length scales not obtainable with all-atom simulations, have recently been developed and show promise for describing lipid dynamics in biological systems. Here, we describe two simplified models, a reduced-lipid model and a bola-lipid model for thermophilic bacterial membranes, developed for use with the rapid discrete molecular dynamics simulation method. In the reduced-lipid model, we represent the lipid chain by a series of three beads interacting through pairwise discrete potentials that model hydrophobic attractions between hydrocarbon tails in implicit solvent. Our phase diagram recapitulates those produced by continuous potential models with similar coarse-grained lipid representations. We also find that phase transition temperatures for our reduced-lipid model are dependent upon the flexibility of the lipid chain, giving an insight into archaebacterial membrane stability and prompting development of a bola-lipid model specific for archaebacteria lipids. With both the reduced-lipid and bola-lipid model, we find that the reduced flexibility inherent in archaebacteria lipids yields more stable bilayers as manifested by increased phase transition temperatures. The results of these studies provide a simulation methodology for lipid molecules in biological systems and show that discrete molecular dynamics is applicable to lipid aggregation and dynamics.

  5. Surface Modifications of Support Partitions for Stabilizing Biomimetic Membrane Arrays

    DEFF Research Database (Denmark)

    Perry, Mark; Hansen, Jesper Schmidt; Jensen, Karin Bagger Stibius;

    2011-01-01

    Black lipid membrane (BLM) formation across apertures in an ethylene tetra-fluoroethylene (ETFE) partition separating two aqueous compartments is an established technique for the creation of biomimetic membranes. Recently multi-aperture BLM arrays have attracted interest and in order to increase...... modified partitions were similar and significantly lower than for arrays formed using untreated ETFE partitions. For single side n-hexene modification average membrane array lifetimes were not significantly changed compared to untreated ETFE. Double-sided n-hexene modification greatly improved average...... membrane array lifetimes compared to membrane arrays formed across untreated ETFE partitions. n-hexene modifications resulted in BLM membrane arrays which over time developed significantly lower conductance (Gm) and higher capacitance (Cm) values compared to the other membranes with the strongest effect...

  6. A lysosome-centered view of nutrient homeostasis.

    Science.gov (United States)

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

    2016-01-01

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

  7. Membrane-stabilizing copolymers confer marked protection to dystrophic skeletal muscle in vivo

    Directory of Open Access Journals (Sweden)

    Evelyne M Houang

    2015-01-01

    Full Text Available Duchenne muscular dystrophy (DMD is a fatal disease of striated muscle deterioration. A unique therapeutic approach for DMD is the use of synthetic membrane stabilizers to protect the fragile dystrophic sarcolemma against contraction-induced mechanical stress. Block copolymer-based membrane stabilizer poloxamer 188 (P188 has been shown to protect the dystrophic myocardium. In comparison, the ability of synthetic membrane stabilizers to protect fragile DMD skeletal muscles has been less clear. Because cardiac and skeletal muscles have distinct structural and functional features, including differences in the mechanism of activation, variance in sarcolemma phospholipid composition, and differences in the magnitude and types of forces generated, we speculated that optimized membrane stabilization could be inherently different. Our objective here is to use principles of pharmacodynamics to evaluate membrane stabilization therapy for DMD skeletal muscles. Results show a dramatic differential effect of membrane stabilization by optimization of pharmacodynamic-guided route of poloxamer delivery. Data show that subcutaneous P188 delivery, but not intravascular or intraperitoneal routes, conferred significant protection to dystrophic limb skeletal muscles undergoing mechanical stress in vivo. In addition, structure-function examination of synthetic membrane stabilizers further underscores the importance of copolymer composition, molecular weight, and dosage in optimization of poloxamer pharmacodynamics in vivo.

  8. Mitochondrial Dysfunction in Lysosomal Storage Disorders

    Directory of Open Access Journals (Sweden)

    Mario de la Mata

    2016-10-01

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

  9. Documentation of an Imperative To Improve Methods for Predicting Membrane Protein Stability.

    Science.gov (United States)

    Kroncke, Brett M; Duran, Amanda M; Mendenhall, Jeffrey L; Meiler, Jens; Blume, Jeffrey D; Sanders, Charles R

    2016-09-13

    There is a compelling and growing need to accurately predict the impact of amino acid mutations on protein stability for problems in personalized medicine and other applications. Here the ability of 10 computational tools to accurately predict mutation-induced perturbation of folding stability (ΔΔG) for membrane proteins of known structure was assessed. All methods for predicting ΔΔG values performed significantly worse when applied to membrane proteins than when applied to soluble proteins, yielding estimated concordance, Pearson, and Spearman correlation coefficients of thermodynamic folding stability in membrane proteins.

  10. Stabilized composite membranes and membrane electrode assemblies for high temperature/low relative humidity polymer electrolyte fuel cell operation

    Science.gov (United States)

    Ramani, Vijay Krishna

    Polymer electrolyte membrane fuel cells (PEMFCs) have a variety of applications in the stationary power, mobile power and automotive power sectors. Existing membrane technology presently permits fuel cell operation at temperatures less than 100°C under fully saturated conditions. However, several advantages such as easier heat rejection rates and improved impurities tolerance by the anode electrocatalyst result by operating a PEMFC at elevated temperatures (above 100°C) and lower relative humidities. In an attempt to extend the operating range of the polymer electrolyte membrane, perfluorosulfonic acid (NafionRTM) based organic/inorganic (heteropolyacid) composite membranes were investigated in terms of thermal and electrochemical stability, additive stability and conductivity. Tungsten based heteropolyacids (HPAs) were found to be electrochemically stable as opposed to molybdenum based additives. The stability of the inorganic heteropolyacid additive in aqueous environments was enhanced by ion exchanging the protons of the HPAs with larger counter ions. An additional stabilization technique developed involved improving the interaction of HPA with NafionRTM by linking the particles to the sulfonic acid clusters via a sol-gel induced metal oxide linkage. The proton conductivity of the composite membranes was found to depend on the particle size of the HPA additive. A two order of magnitude change in additive particle size was attained by modification of the membrane preparation technique. This modification resulted in a nearly 50% increase in conductivity. The membranes prepared were characterized by thermal analysis, spectroscopy and microscopy. A technique was developed to incorporate existing MEA preparation and HPA stabilization techniques to the composite membranes with small HPA particles. All MEAs prepared were evaluated at high temperatures (120°C) and low relative humidities (35%) in an operating fuel cell, with membrane resistance and hence conductivity

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

    Science.gov (United States)

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

    2015-04-29

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

  12. Self-assembling peptides form nanodiscs that stabilize membrane proteins

    DEFF Research Database (Denmark)

    Midtgaard, Søren Roi; Pedersen, Martin Cramer; Kirkensgaard, Jacob Judas Kain;

    2014-01-01

    New methods to handle membrane bound proteins, e.g. G-protein coupled receptors (GPCRs), are highly desirable. Recently, apoliprotein A1 (ApoA1) based lipoprotein particles have emerged as a new platform for studying membrane proteins, and it has been shown that they can self-assemble in combinat...

  13. Textural stability of titania–alumina composite membranes

    NARCIS (Netherlands)

    Kumar, Krishnankutty-Nair P.; Keizer, Klaas; Burggraaf, Anthonie J.

    1993-01-01

    Textural evolution (porosity reduction, pore and crystallite growth) in titania–alumina composite membranes has been studied using thermal analysis, X-ray diffraction, field emission scanning electron microscopy and N2 physisorption techniques. The presence of alumina in the membranes improved the t

  14. Stabilization of membrane necks by adhesive particles, substrate surfaces, and constriction forces.

    Science.gov (United States)

    Agudo-Canalejo, Jaime; Lipowsky, Reinhard

    2016-10-21

    Membrane remodelling processes involving the formation and fission of small buds require the formation and closure of narrow membrane necks, both for biological membranes and for model membranes such as lipid bilayers. The conditions required for the stability of such necks are well understood in the context of budding of vesicles with bilayer asymmetry and/or intramembrane domains. In many cases, however, the necks form in the presence of an adhesive surface, such as a solid particle or substrate, or the cellular cortex itself. Examples of such processes in biological cells include endocytosis, exocytosis and phagocytosis of solid particles, the formation of extracellular and outer membrane vesicles by eukaryotic and prokaryotic cells, as well as the closure of the cleavage furrow in cytokinesis. Here, we study the interplay of curvature elasticity, membrane-substrate adhesion, and constriction forces to obtain generalized stability conditions for closed necks which we validate by numerical energy minimization. We then explore the consequences of these stability conditions in several experimentally accessible systems such as particle-filled membrane tubes, supported lipid bilayers, giant plasma membrane vesicles, bacterial outer membrane vesicles, and contractile rings around necks. At the end, we introduce an intrinsic engulfment force that directly describes the interplay between curvature elasticity and membrane-substrate adhesion.

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

    Science.gov (United States)

    Seggewiß, Nicole; Paulmann, Dajana; Dotzauer, Andreas

    2016-01-01

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

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

    Science.gov (United States)

    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.

  17. Improving stability and biocompatibility of alginate/chitosan microcapsule by fabricating bi-functional membrane.

    Science.gov (United States)

    Zheng, Guoshuang; Liu, Xiudong; Wang, Xiuli; Chen, Li; Xie, Hongguo; Wang, Feng; Zheng, Huizhen; Yu, Weiting; Ma, Xiaojun

    2014-05-01

    Cell encapsulation technology holds promise for the cell-based therapy. But poor mechanical strength and biocompatibility of microcapsule membrane are still obstacles for the clinical applications. A novel strategy is presented to prepare AC₁ C₂ A microcapsules with bi-functional membrane (that is, both desirable biocompatibility and membrane stability) by sequentially complexing chitosans with higher deacetylation degree (C₁) and lower deacetylation degree (C₂) on alginate (A) gel beads. Both in vitro and in vivo evaluation of AC₁C₂ A microcapsules demonstrate higher membrane stability and less cell adhesion, because the introduction of C₂ increases membrane strength and decreases surface roughness. Moreover, diffusion test of AC₁C₂ A microcapsules displays no inward permeation of IgG protein suggesting good immunoisolation function. The results demonstrate that AC₁C₂ A microcapsules with bi-functional membrane could be a promising candidate for microencapsulated cell implantation with cost effective usage of naturally biocompatible polysaccharides.

  18. 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.

    Directory of Open Access Journals (Sweden)

    Rodrigo Maciel da Costa Godinho

    Full Text Available 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.

  19. 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.

    Science.gov (United States)

    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.

  20. 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

    Science.gov (United States)

    Lucas, Carolina Gonçalves de Oliveira; Rigato, Paula Ordonhez; Gonçalves, Jorge Luiz Santos; Sato, Maria Notomi; Maciel, Milton; Peçanha, Ligia Maria Torres; August, J. Thomas; de Azevedo Marques, Ernesto Torres; 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. PMID:24932692

  1. Hollow-fiber-supported liquid membranes with improved stability for nitrate removal

    NARCIS (Netherlands)

    Kemperman, A.J.B.; Rolevink, H.H.M.; Boomgaard, van den Th.; Strathmann, H.

    1997-01-01

    This paper describes the development of a hollow-fiber-supported liquid membrane (HFSLM) for the removal of nitrate ions from water. Two different membrane modules were designed which differed in length of the fibers. In order to test the HFSLMs on nitrate flux and stability, two set-ups were used:

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

    Science.gov (United States)

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

    2016-10-01

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

  3. The effects of hydrocortisone and glycyrrhizine on the enzyme releases of arylsulfatase and hyaluronidase from lysosomes of liver.

    Science.gov (United States)

    Ozeki, T; Tokawa, Y; Ogasawara, T; Sato, K; Kan, M

    1978-03-15

    Hydrocortisone and glycyrrhizine act as both stabilizers and labilizers of the lysosomes of liver. The effect of both agents on the lysosomes is changeable according to the duration of their administration.

  4. On-line extractive separation in flow injection analysis based on polymer inclusion membranes: a study on membrane stability and approaches for improving membrane permeability.

    Science.gov (United States)

    Zhang, Lujia L; Cattrall, Robert W; Ashokkumar, Muthupandian; Kolev, Spas D

    2012-08-15

    The effect of temperature on the sensitivity and sampling rate is studied for a flow injection analysis (FIA) system that uses a membrane separation cell fitted with a polymer inclusion membrane (PIM) for the determination of Zn(II). A temperature of 50 °C for the flowing donor and acceptor solutions and the membrane separation cell improves the sensitivity and the sampling rate relative to 20 °C up to 10-fold and 2-fold, respectively. Studies on the stability of the PIM are reported that show a limited loss of the membrane liquid phase into the aqueous phases used in the FIA system but this has exhibited a negligible effect on the amount of Zn(II) transported across the membrane. Most importantly, the extent of leaching of the PIM components is shown to depend on the nature of the aqueous phase with the membrane eventually reaching a stable composition. It is also shown that the application of ultrasound to the membrane separation cell leads to a slight increase in sensitivity without affecting the long term membrane stability.

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

    Science.gov (United States)

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

    2015-04-17

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

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

    Directory of Open Access Journals (Sweden)

    Margrét H Ögmundsdóttir

    Full Text Available 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.

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

    Science.gov (United States)

    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.

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

    DEFF Research Database (Denmark)

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

    2013-01-01

    Lysosomal membrane permeabilization and subsequent cell death may prove useful in cancer treatment, provided that cancer cell lysosomes can be specifically targeted. Here, we identify acid sphingomyelinase (ASM) inhibition as a selective means to destabilize cancer cell lysosomes. Lysosome......-destabilizing experimental anticancer agent siramesine inhibits ASM by interfering with the binding of ASM to its essential lysosomal cofactor, bis(monoacylglycero)phosphate. Like siramesine, several clinically relevant ASM inhibitors trigger cancer-specific lysosomal cell death, reduce tumor growth in vivo, and revert...

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

    Directory of Open Access Journals (Sweden)

    Catherine Staudt

    2016-12-01

    Full Text Available 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.

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

    OpenAIRE

    2012-01-01

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

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

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

    2015-11-01

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

  13. Stability studies of immobilized lipase on rice husk and eggshell membrane

    Science.gov (United States)

    Abdulla, R.; Sanny, S. A.; Derman, E.

    2017-06-01

    Lipase immobilization for biodiesel production is gaining importance day by day. In this study, lipase from Burkholderia cepacia was immobilized on activated support materials namely rice husk and egg shell membrane. Both rice husk and eggshell membrane are natural wastes that holds a lot of potential as immobilization matrix. Rice husk and eggshell membrane were activated with glutaraldehyde. Lipase was immobilized on the glutaraldehyde-activated support material through adsorption. Immobilization efficiency together with enzyme activity was observed to choose the highest enzyme loading for further stability studies. Immobilization efficiency of lipase on rice husk was 81 as compared to an immobilization efficiency of 87 on eggshell membrane. Immobilized lipase on eggshell membrane exhibited higher enzyme activity as compared to immobilized lipase on rice husk. Eggshell membrane also reported higher stability than rice husk as immobilization matrix. Both types of immobilized lipase retatined its activity after ten cycles of reuse. In short, eggshell membrane showed to be a better immobilization platform for lipase as compared to rice husk. However, with further improvement in technique of immobilization, the stability of both types of immobilized lipase can be improved to a greater extent.

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

    Science.gov (United States)

    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

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Aljona Gaiko-Shcherbak

    Full Text Available 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.

  18. Anion exchange membranes for fuel cells and flow batteries : transport and stability of model systems

    OpenAIRE

    Marino, Michael G

    2015-01-01

    Polymeric anion exchange materials in membrane form can be key components in emerging energy storage and conversions systems such as the alkaline fuel cell and the RedOx flow battery. For these applications the membrane properties need to include good ionic conductivity and sufficient chemical stability, two aspects, that are not sufficiently understood in terms of materials science. Materials fulfilling both criteria are currently not available. The transport of ions and water in a model...

  19. Cross-linked comb-shaped anion exchange membranes with high base stability

    Energy Technology Data Exchange (ETDEWEB)

    Li, NW; Wang, LZ; Hickner, M

    2014-01-01

    A unique one-step cross-linking strategy that connects quaternary ammonium centers using Grubbs II-catalyzed olefin metathesis was developed. The cross-linked anion exchange membranes showed swelling ratios of less than 10% and hydroxide conductivities of 18 to 40 mS cm(- 1). Cross-linking improved the membranes' stability to hydroxide degradation compared to their non-cross-linked analogues.

  20. ELECTROCHEMICAL STABILITY OF STRONG BASIC ANION EXCHANGE MEMBRANES IN CONDITIONS OF HIGH INTENSIVE ELECTRODIALYSIS PROCESS

    OpenAIRE

    Zabolotskiy V. I.; Sharafan M. V.; Chermit R. H.; Vasilieva V. I.

    2014-01-01

    The stability of strongly basic anion-exchange membranes MA-41-2P (JSC "Schekino-Nitrogen", Russia) and AMX (Tokuyama Soda, Japan) under intensive current regimes was investigated in the current study. The process of water molecules dissociation at current densities above the limiting one in 0.01 M sodium chloride solution was studied in detail. The length of the electroconvective instability at the membrane / solution interface at currents exceeding the limiting current was measured by laser...

  1. Membrane protein thermodynamic stability may serve as the energy sink for sorting in the periplasm.

    Science.gov (United States)

    Moon, C Preston; Zaccai, Nathan R; Fleming, Patrick J; Gessmann, Dennis; Fleming, Karen G

    2013-03-12

    Thermodynamic stabilities are pivotal for understanding structure-function relationships of proteins, and yet such determinations are rare for membrane proteins. Moreover, the few measurements that are available have been conducted under very different experimental conditions, which compromises a straightforward extraction of physical principles underlying stability differences. Here, we have overcome this obstacle and provided structure-stability comparisons for multiple membrane proteins. This was enabled by measurements of the free energies of folding and the m values for the transmembrane proteins PhoP/PhoQ-activated gene product (PagP) and outer membrane protein W (OmpW) from Escherichia coli. Our data were collected in the same lipid bilayer and buffer system we previously used to determine those parameters for E. coli outer membrane phospholipase A (OmpLA). Biophysically, our results suggest that the stabilities of these proteins are strongly correlated to the water-to-bilayer transfer free energy of the lipid-facing residues in their transmembrane regions. We further discovered that the sensitivities of these membrane proteins to chemical denaturation, as judged by their m values, was consistent with that previously observed for water-soluble proteins having comparable differences in solvent exposure between their folded and unfolded states. From a biological perspective, our findings suggest that the folding free energies for these membrane proteins may be the thermodynamic sink that establishes an energy gradient across the periplasm, thus driving their sorting by chaperones to the outer membranes in living bacteria. Binding free energies of these outer membrane proteins with periplasmic chaperones support this energy sink hypothesis.

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

    Science.gov (United States)

    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

  3. The lysosome and neurodegenerative diseases

    Institute of Scientific and Technical Information of China (English)

    Lisha Zhang; Rui Sheng; Zhenghong Qin

    2009-01-01

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

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

    Science.gov (United States)

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

    2000-07-01

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

  5. Adhesion and receptor clustering stabilizes lateral heterogeneity in cell plasma membranes

    Science.gov (United States)

    Veatch, Sarah

    2013-03-01

    The thermodynamic properties of plasma membrane lipids play a vital role in many functions that initiate at the mammalian cell surface. Some functions are thought to occur, at least in part, because plasma membrane lipids have a tendency to separate into two distinct liquid phases, called liquid-ordered and liquid-disordered. We find that isolated cell plasma membranes are poised near a miscibility critical point separating these two liquid phases, and postulate that critical composition fluctuations provide the physical basis of functional membrane heterogeneity in intact cells. In this talk I will describe several possible mechanisms through which dynamic fluctuations can be stabilized in super-critical membranes, and will present some preliminary evidence suggesting that these structures can be visualized in intact cells using quantitative super-resolution fluorescence localization imaging.

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

    Science.gov (United States)

    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.

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

    OpenAIRE

    2013-01-01

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

  8. Benchmarking Membrane Protein Detergent Stability for Improving Throughput of High-Resolution X-ray Structures

    Science.gov (United States)

    Sonoda, Yo; Newstead, Simon; Hu, Nien-Jen; Alguel, Yilmaz; Nji, Emmanuel; Beis, Konstantinos; Yashiro, Shoko; Lee, Chiara; Leung, James; Cameron, Alexander D.; Byrne, Bernadette; Iwata, So; Drew, David

    2011-01-01

    Summary Obtaining well-ordered crystals is a major hurdle to X-ray structure determination of membrane proteins. To facilitate crystal optimization, we investigated the detergent stability of 24 eukaryotic and prokaryotic membrane proteins, predominantly transporters, using a fluorescent-based unfolding assay. We have benchmarked the stability required for crystallization in small micelle detergents, as they are statistically more likely to lead to high-resolution structures. Using this information, we have been able to obtain well-diffracting crystals for a number of sodium and proton-dependent transporters. By including in the analysis seven membrane proteins for which structures are already known, AmtB, GlpG, Mhp1, GlpT, EmrD, NhaA, and LacY, it was further possible to demonstrate an overall trend between protein stability and structural resolution. We suggest that by monitoring membrane protein stability with reference to the benchmarks described here, greater efforts can be placed on constructs and conditions more likely to yield high-resolution structures. PMID:21220112

  9. Digalactosyl-diacylglycerol-deficiency lowers the thermal stability of thylakoid membranes

    NARCIS (Netherlands)

    Krumova, S.K.B.; Laptenok, S.; Kovács, L.; Toth, T.; Hoek, van A.; Garab, G.; Amerongen, van H.

    2010-01-01

    We investigated the effects of digalactosyl-diacylglycerol (DGDG) on the organization and thermal stability of thylakoid membranes, using wild-type Arabidopsis thaliana and the DGDG-deficient mutant, dgd1. Circular-dichroism measurements reveal that DGDG-deficiency hampers the formation of the

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

    Science.gov (United States)

    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.

  11. Cancer-associated lysosomal changes

    DEFF Research Database (Denmark)

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

    2013-01-01

    Rapidly dividing and invasive cancer cells are strongly dependent on effective lysosomal function. Accordingly, transformation and cancer progression are characterized by dramatic changes in lysosomal volume, composition and cellular distribution. Depending on one's point of view, the cancer-asso......:10.1038/onc.2012.292....

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

    Science.gov (United States)

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

    2013-09-16

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

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

    NARCIS (Netherlands)

    R.J. Rottier (Robbert)

    1998-01-01

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

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

    Science.gov (United States)

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

    1999-01-01

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

  15. Chain ordering of hybrid lipids can stabilize domains in saturated/hybrid/cholesterol lipid membranes

    Science.gov (United States)

    Yamamoto, T.; Brewster, R.; Safran, S. A.

    2010-07-01

    We use a liquid-crystal model to predict that hybrid lipids (lipids that have one saturated and one unsaturated tail) can stabilize line interfaces between domains in mixed membranes of saturated lipids, hybrid lipids, and cholesterol (SHC membranes). The model predicts the phase separation of SHC membranes with both parabolic and loop binodals depending on the cholesterol concentration, modeled via an effective pressure. In some cases, the hybrid lipids can reduce the line tension to zero in SHC membranes at temperatures that approach the critical temperature as the pressure is increased. The differences in the hybrid saturated tail conformational order in bulk and at the interface are responsible for the reduction of the line tension.

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

    Directory of Open Access Journals (Sweden)

    Dang Hope

    2007-12-01

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

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

    Science.gov (United States)

    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

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

    Directory of Open Access Journals (Sweden)

    Janet To

    2015-08-01

    Full Text Available 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.

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

    Science.gov (United States)

    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.

  20. Presence of a lysosomal enzyme, arylsulfatase-A, in the prelysosome-endosome compartments of human cultured fibroblasts.

    Science.gov (United States)

    Kelly, B M; Yu, C Z; Chang, P L

    1989-02-01

    Although endosomes and lysosomes are associated with different subcellular functions, we present evidence that a lysosomal enzyme, arylsulfatase-A, is present in prelysosomal vesicles which constitute part of the endosomal compartment. When human cultured fibroblasts were subfractionated with Percoll gradients, arylsulfatase-A activity was enriched in three subcellular fractions: dense lysosomes, light lysosomes, and light membranous vesicles. Pulsing the cells for 1 to 10 min with the fluid-phase endocytic marker, horseradish peroxidase, showed that endosomes enriched with the marker were distributed partly in the light lysosome fraction but mainly in the light membranous fraction. By pulsing the fibroblasts for 10 min with horseradish peroxidase conjugated to colloidal gold and then staining the light membranous and light lysosomal fractions for arylsulfatase-A activity with a specific cytochemical technique, the endocytic marker was detected under the electron microscope in the same vesicles as the lysosomal enzyme. The origin of the lysosomal enzyme in this endosomal compartment was shown not to be acquired through mannose 6-phosphate receptor-mediated endocytosis of enzymes previously secreted from the cell. Together with our recent finding that the light membranous fraction contains prelysosomes distinct from bona fide lysosomes and was highly enriched with newly synthesized arylsulfatase-A molecules, these results demonstrate that prelysosomes also constitute part of the endosomal compartment to which intracellular lysosomal enzymes are targeted.

  1. On the Efficiency of NHS Ester Cross-Linkers for Stabilizing Integral Membrane Protein Complexes

    Science.gov (United States)

    Chen, Fan; Gerber, Sabina; Korkhov, Volodymyr M.; Mireku, Samantha; Bucher, Monika; Locher, Kaspar P.; Zenobi, Renato

    2015-03-01

    We have previously presented a straightforward approach based on high-mass matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) to study membrane proteins. In addition, the stoichiometry of integral membrane protein complexes could be determined by MALDI-MS, following chemical cross-linking via glutaraldehyde. However, glutaraldehyde polymerizes in solution and reacts nonspecifically with various functional groups of proteins, limiting its usefulness for structural studies of protein complexes. Here, we investigated the capability of N-hydroxysuccinimide (NHS) esters, which react much more specifically, to cross-link membrane protein complexes such as PglK and BtuC2D2. We present clear evidence that NHS esters are capable of stabilizing membrane protein complexes in situ, in the presence of detergents such as DDM, C12E8, and LDAO. The stabilization efficiency strongly depends on the membrane protein structure (i.e, the number of primary amine groups and the distances between primary amines). A minimum number of primary amine groups is required, and the distances between primary amines govern whether a cross-linker with a specific spacer arm length is able to bridge two amine groups.

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

    Science.gov (United States)

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

    2016-06-30

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

  3. Sedative, membrane stability, cytotoxic and antioxidant properties of methanol extract of leaves of Protium serratum Wall.

    Directory of Open Access Journals (Sweden)

    Md. Rafikul Islam

    2014-09-01

    Full Text Available Objective: To study the sedative, membrane stability, cytotoxic and antioxidant properties of the leaves of Protium serratum extracted using methanol. Methods: Sedative test was performed using hole cross and open field methods at 200 and 400 mg/kg. Membrane stability of red blood cell was used for anti-inflammatory test at different concentrations. Cytotoxic study was performed using brine shrimp lethality test. Total flavonoid contents, total phenol contents and reducing power were used to assess antioxidant properties of the extract. Results: Extract showed better sedative action at lower doses in both experiments. Maximum 73.33% locomotion reduction was found at 200 mg/kg at 1 20 min and that was 89.29% for diazepam in hole cross test. In membrane stability test, extract and standard drug diclofenac have 35.66% and 91.20% stability, respectively. LC50 value of the extract was 22.91 µg/mL. Total phenol and flavonoid contents were (55.53依14.63 mg gallic acid equivalent per gram of extract and (1 06.33依7.35 mg of quercetin equivalent per gram of extract, respectively per gram of extract. Significant reducing power was observed as compared to ascorbic acid. Conclusions: Extract possesses good pharmacological properties. Hence, further extensive study is essential to find out possible active constituents for the treatment of anxiety, inflammation or sickle cell disease, cancer and free radical mediated abnormalities.

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

    NARCIS (Netherlands)

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

    1992-01-01

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

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

    KAUST Repository

    Lu, Dongwei

    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. © 2015 American Chemical Society.

  6. TRPML1: an ion channel in the lysosome.

    Science.gov (United States)

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

    2014-01-01

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

  7. ELECTROCHEMICAL STABILITY OF STRONG BASIC ANION EXCHANGE MEMBRANES IN CONDITIONS OF HIGH INTENSIVE ELECTRODIALYSIS PROCESS

    Directory of Open Access Journals (Sweden)

    Zabolotskiy V. I.

    2014-12-01

    Full Text Available The stability of strongly basic anion-exchange membranes MA-41-2P (JSC "Schekino-Nitrogen", Russia and AMX (Tokuyama Soda, Japan under intensive current regimes was investigated in the current study. The process of water molecules dissociation at current densities above the limiting one in 0.01 M sodium chloride solution was studied in detail. The length of the electroconvective instability at the membrane / solution interface at currents exceeding the limiting current was measured by laser interferometry

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

    Science.gov (United States)

    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.

  9. Cancer-associated lysosomal changes

    DEFF Research Database (Denmark)

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

    2013-01-01

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

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

    Science.gov (United States)

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

    2012-09-01

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

  11. Influence of environmental conditions on the stability of oil in water emulsions containing droplets stabilized by lecithin-chitosan membranes.

    Science.gov (United States)

    Ogawa, Satoshi; Decker, Eric A; McClements, D Julian

    2003-08-27

    Oil-in-water emulsions containing cationic droplets stabilized by lecithin-chitosan membranes were produced using a two-stage process. A primary emulsion containing anionic lecithin-coated droplets was prepared by homogenizing oil and emulsifier solution using a high-pressure valve homogenizer (5 wt % corn oil, 1 wt % lecithin, 100 mM acetic acid, pH 3.0). A secondary emulsion containing cationic lecithin-chitosan-coated droplets was formed by diluting the primary emulsion with an aqueous chitosan solution (1 wt % corn oil, 0.2 wt % lecithin, 100 mM acetic acid, and 0.036 wt % chitosan). The stabilities of the primary and secondary emulsions with the same oil concentration to thermal processing, freeze-thaw cycling, high calcium chloride concentrations, and lipid oxidation were determined. The results showed that the secondary emulsions had better stability to droplet aggregation during thermal processing (30-90 degrees C for 30 min), freeze-thaw cycling (-10 degrees C for 22 h/30 degrees C for 2 h), and high calcium chloride contents (stability to environmental stresses.

  12. Thermal Stability of Silica-Zirconia Membranes%硅锆复合膜的热稳定性研究

    Institute of Scientific and Technical Information of China (English)

    刘伟; 张宝泉; 刘秀凤; 徐黎明

    2006-01-01

    The thermal stability, phase transformation, surface morphology, pore size distribution and permeation of the defect-free silica-zirconia membrane were investigated by using X-ray diffraction (XRD), atomic force microscopy (AFM), gas adsorption analyzer (BET), and gas permeation apparatus, respectively. Using silica as the stabilizing agent,the defect-free membrane was much more stable than pure zirconia. The crystal transformation of zirconia in the silica-stabilized membrane could be prohibited by the interaction between silica and zirconia. ZrO2 crystals were kept tetragonal below 900℃, the size of which did not change with temperature between 700℃ and 900℃. It was further verified by the AFM observation, pore size analysis and permeation study. This thermal stability makes the silica-zirconia membrane a good choice as the intermediate layer for zeolite and Pd-based membranes.

  13. Involvement of the Cdc42 pathway in CFTR post-translational turnover and in its plasma membrane stability in airway epithelial cells.

    Directory of Open Access Journals (Sweden)

    Romain Ferru-Clément

    Full Text Available Cystic fibrosis transmembrane conductance regulator (CFTR is a chloride channel that is expressed on the apical plasma membrane (PM of epithelial cells. The most common deleterious allele encodes a trafficking-defective mutant protein undergoing endoplasmic reticulum-associated degradation (ERAD and presenting lower PM stability. In this study, we investigated the involvement of the Cdc42 pathway in CFTR turnover and trafficking in a human bronchiolar epithelial cell line (CFBE41o- expressing wild-type CFTR. Cdc42 is a small GTPase of the Rho family that fulfils numerous cell functions, one of which is endocytosis and recycling process via actin cytoskeleton remodelling. When we treated cells with chemical inhibitors such as ML141 against Cdc42 and wiskostatin against the downstream effector N-WASP, we observed that CFTR channel activity was inhibited, in correlation with a decrease in CFTR amount at the cell surface and an increase in dynamin-dependent CFTR endocytosis. Anchoring of CFTR to the cortical cytoskeleton was then presumably impaired by actin disorganization. When we performed siRNA-mediated depletion of Cdc42, actin polymerization was not impacted, but we observed actin-independent consequences upon CFTR. Total and PM CFTR amounts were increased, resulting in greater activation of CFTR. Pulse-chase experiments showed that while CFTR degradation was slowed, CFTR maturation through the Golgi apparatus remained unaffected. In addition, we observed increased stability of CFTR in PM and reduction of its endocytosis. This study highlights the involvement of the Cdc42 pathway at several levels of CFTR biogenesis and trafficking: (i Cdc42 is implicated in the first steps of CFTR biosynthesis and processing; (ii it contributes to the stability of CFTR in PM via its anchoring to cortical actin; (iii it promotes CFTR endocytosis and presumably its sorting toward lysosomal degradation.

  14. Involvement of the Cdc42 pathway in CFTR post-translational turnover and in its plasma membrane stability in airway epithelial cells.

    Science.gov (United States)

    Ferru-Clément, Romain; Fresquet, Fleur; Norez, Caroline; Métayé, Thierry; Becq, Frédéric; Kitzis, Alain; Thoreau, Vincent

    2015-01-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that is expressed on the apical plasma membrane (PM) of epithelial cells. The most common deleterious allele encodes a trafficking-defective mutant protein undergoing endoplasmic reticulum-associated degradation (ERAD) and presenting lower PM stability. In this study, we investigated the involvement of the Cdc42 pathway in CFTR turnover and trafficking in a human bronchiolar epithelial cell line (CFBE41o-) expressing wild-type CFTR. Cdc42 is a small GTPase of the Rho family that fulfils numerous cell functions, one of which is endocytosis and recycling process via actin cytoskeleton remodelling. When we treated cells with chemical inhibitors such as ML141 against Cdc42 and wiskostatin against the downstream effector N-WASP, we observed that CFTR channel activity was inhibited, in correlation with a decrease in CFTR amount at the cell surface and an increase in dynamin-dependent CFTR endocytosis. Anchoring of CFTR to the cortical cytoskeleton was then presumably impaired by actin disorganization. When we performed siRNA-mediated depletion of Cdc42, actin polymerization was not impacted, but we observed actin-independent consequences upon CFTR. Total and PM CFTR amounts were increased, resulting in greater activation of CFTR. Pulse-chase experiments showed that while CFTR degradation was slowed, CFTR maturation through the Golgi apparatus remained unaffected. In addition, we observed increased stability of CFTR in PM and reduction of its endocytosis. This study highlights the involvement of the Cdc42 pathway at several levels of CFTR biogenesis and trafficking: (i) Cdc42 is implicated in the first steps of CFTR biosynthesis and processing; (ii) it contributes to the stability of CFTR in PM via its anchoring to cortical actin; (iii) it promotes CFTR endocytosis and presumably its sorting toward lysosomal degradation.

  15. Involvement of the Cdc42 Pathway in CFTR Post-Translational Turnover and in Its Plasma Membrane Stability in Airway Epithelial Cells

    Science.gov (United States)

    Ferru-Clément, Romain; Fresquet, Fleur; Norez, Caroline; Métayé, Thierry; Becq, Frédéric; Kitzis, Alain; Thoreau, Vincent

    2015-01-01

    Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that is expressed on the apical plasma membrane (PM) of epithelial cells. The most common deleterious allele encodes a trafficking-defective mutant protein undergoing endoplasmic reticulum-associated degradation (ERAD) and presenting lower PM stability. In this study, we investigated the involvement of the Cdc42 pathway in CFTR turnover and trafficking in a human bronchiolar epithelial cell line (CFBE41o-) expressing wild-type CFTR. Cdc42 is a small GTPase of the Rho family that fulfils numerous cell functions, one of which is endocytosis and recycling process via actin cytoskeleton remodelling. When we treated cells with chemical inhibitors such as ML141 against Cdc42 and wiskostatin against the downstream effector N-WASP, we observed that CFTR channel activity was inhibited, in correlation with a decrease in CFTR amount at the cell surface and an increase in dynamin-dependent CFTR endocytosis. Anchoring of CFTR to the cortical cytoskeleton was then presumably impaired by actin disorganization. When we performed siRNA-mediated depletion of Cdc42, actin polymerization was not impacted, but we observed actin-independent consequences upon CFTR. Total and PM CFTR amounts were increased, resulting in greater activation of CFTR. Pulse-chase experiments showed that while CFTR degradation was slowed, CFTR maturation through the Golgi apparatus remained unaffected. In addition, we observed increased stability of CFTR in PM and reduction of its endocytosis. This study highlights the involvement of the Cdc42 pathway at several levels of CFTR biogenesis and trafficking: (i) Cdc42 is implicated in the first steps of CFTR biosynthesis and processing; (ii) it contributes to the stability of CFTR in PM via its anchoring to cortical actin; (iii) it promotes CFTR endocytosis and presumably its sorting toward lysosomal degradation. PMID:25768293

  16. Antioxidant Enzymes, Membrane Stability and Essential Oil Percentage of Balm under Drought Stress

    Directory of Open Access Journals (Sweden)

    M Ardakani

    2011-01-01

    Full Text Available Abstract In this research, the effect of drought stress on antioxidant enzymes, membrane stability and essential oil percentage in Balm (Melissa officinalis L. has been evaluted under field conditions. Experimental design was complete randomized block design with 4 replications. The treatments included: T1 (control; non stress, T2 (80%FC, T3 (60%FC, T4 (40%FC and T5 (20%FC. Results showed that there was a significant differences (P

  17. Stability Limit of Water by Metastable Vapor-Liquid Equilibrium with Nanoporous Silicon Membranes.

    Science.gov (United States)

    Chen, I-Tzu; Sessoms, David A; Sherman, Zachary; Choi, Eugene; Vincent, Olivier; Stroock, Abraham D

    2016-06-16

    Liquid can sustain mechanical tension as its pressure drops below the vapor-liquid coexistence line and becomes less than zero, until it reaches the stability limit-the pressure at which cavitation inevitably occurs. For liquid water, its stability limit is still a subject of debate: the results obtained by researchers using a variety of techniques show discrepancies between the values of the stability limit and its temperature dependence as temperature approaches 0 °C. In this work, we present a study of the stability limit of water by the metastable vapor-liquid equilibrium (MVLE) method with nanoporous silicon membranes. We also report on an experimental system which enables tests of the temperature dependence of the stability limit with MVLE. The stability limit we found increases monotonically (larger tension) as temperature approaches 0 °C; this trend contradicts the centrifugal result of Briggs but agrees with the experiments by acoustic cavitation. This result confirms that a quasi-static method can reach stability values similar to that from the dynamic stretching technique, even close to 0 °C. Nevertheless, our results fall in the range of ∼ -20 to -30 MPa, a range that is consistent with the majority of experiments but is far less negative than the limit obtained in experiments involving quartz inclusions and that predicted for homogeneous nucleation.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  20. Salinomycin kills cancer stem cells by sequestering iron in lysosomes

    Science.gov (United States)

    Mai, Trang Thi; Hamaï, Ahmed; Hienzsch, Antje; Cañeque, Tatiana; Müller, Sebastian; Wicinski, Julien; Cabaud, Olivier; Leroy, Christine; David, Amandine; Acevedo, Verónica; Ryo, Akihide; Ginestier, Christophe; Birnbaum, Daniel; Charafe-Jauffret, Emmanuelle; Codogno, Patrice; Mehrpour, Maryam; Rodriguez, Raphaël

    2017-10-01

    Cancer stem cells (CSCs) represent a subset of cells within tumours that exhibit self-renewal properties and the capacity to seed tumours. CSCs are typically refractory to conventional treatments and have been associated to metastasis and relapse. Salinomycin operates as a selective agent against CSCs through mechanisms that remain elusive. Here, we provide evidence that a synthetic derivative of salinomycin, which we named ironomycin (AM5), exhibits a more potent and selective activity against breast CSCs in vitro and in vivo, by accumulating and sequestering iron in lysosomes. In response to the ensuing cytoplasmic depletion of iron, cells triggered the degradation of ferritin in lysosomes, leading to further iron loading in this organelle. Iron-mediated production of reactive oxygen species promoted lysosomal membrane permeabilization, activating a cell death pathway consistent with ferroptosis. These findings reveal the prevalence of iron homeostasis in breast CSCs, pointing towards iron and iron-mediated processes as potential targets against these cells.

  1. Mechanisms and stability of oxide-ion transport in homogenous and heterogeneous ceramic membranes

    Science.gov (United States)

    Tichy, Robin Sarah

    Solid oxide-ion conductors are basic components of several modern technologies. Oxide-ion electrolytes are oxide-ion conductors and electronic insulators; they are used in oxygen sensors and solid oxide fuel cells. The required oxide-ion conductivity is only achieved at higher temperatures. Commercialization of this technology demands the development of a better oxide-ion electrolyte and/or the ability to fabricate a large area ceramic membrane with a thickness of L membranes and methane conversion reactors that produce syn-gas. Structural and chemical stability of mixed conductors are a major problem for ceramic-membrane reactors because the material must exhibit good mixed conduction in both high and very low oxygen partial pressures and at operating temperatures, 600°C ≤ Top. ≤ 900°C. The material SrMnO3 is a high-temperature, oxygen-deficient, perovskite that may be preserved at room temperature. Although this material exhibits good mixed conduction, it reverts to its stable stoichiometric phase under oxidizing operating conditions. La2NiO4+delta has a tetragonal crystal structure that is closely related to the cubic perovskite structure. The ionic conduction occurs via the migration of interstitial oxygen, which is lost in reducing atmospheres. The stability of mixed conduction within one material proved difficult to achieve in both reducing and oxidizing conditions at high temperatures. Several oxides are known to exhibit stable ionic conduction in membrane operating conditions. A noble metal can provide a pathway for electronic conduction while the oxide phase conducts the oxygen ions. This heterogeneous composite configuration improves stability, but the exact nature of the conduction processes has not been determined. The performance of two composite materials, Ce 0.8Sm0.2O1.9/Pd and (Bi1.75Y0.25 O3)0.95(CeO2)0.05/Ag, was assessed through permeation studies.

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

    Science.gov (United States)

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

    2013-01-01

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

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

    Science.gov (United States)

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

    2009-06-01

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

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

    Science.gov (United States)

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

    2017-01-01

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

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

  8. Yttria-stabilized zirconia as membrane material for electrolytic deoxidation of CaO-CaCl{sub 2} melts

    Energy Technology Data Exchange (ETDEWEB)

    Martin, A.; Poignet, J. C.; Fouletier, J. [Univ Grenoble, LEPMI, CNRS, INPG, UJF, F-38402 St Martin Dheres (France); Allibert, M. [LPSC, F-38026 Grenoble 1 (France); Lambertin, D. [SPDE, CEA Marcoule, F-30207 Bagnols Sur Ceze (France); Bourges, G. [SRPU, CEA Valduc, F-21120 Is Sur Tille (France)

    2010-07-01

    This article is devoted to the study of the stability of an yttria-stabilized zirconia membrane used in the electrolysis of molten CaCl{sub 2}-CaO mixtures at 850 degrees C. Intentiostatic and potentiostatic electrolysis were carried for periods ranging from 10 to 20 h. Post-mortem composition profiles across the zirconia membrane were determined using Raman spectroscopy and microprobe analysis. The membrane degradation was analyzed in terms of synergetic parameters, i. e., chemical, electrochemical, and thermomechanical effects. (authors)

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

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

    2000-01-01

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

  11. High sphingomyelin levels induce lysosomal damage and autophagy dysfunction in Niemann Pick disease type A

    Science.gov (United States)

    Gabandé-Rodríguez, E; Boya, P; Labrador, V; Dotti, C G; Ledesma, M D

    2014-01-01

    Niemann Pick disease type A (NPA), which is caused by loss of function mutations in the acid sphingomyelinase (ASM) gene, is a lysosomal storage disorder leading to neurodegeneration. Yet, lysosomal dysfunction and its consequences in the disease are poorly characterized. Here we show that undegraded molecules build up in neurons of acid sphingomyelinase knockout mice and in fibroblasts from NPA patients in which autophagolysosomes accumulate. The latter is not due to alterations in autophagy initiation or autophagosome–lysosome fusion but because of inefficient autophago–lysosomal clearance. This, in turn, can be explained by lysosomal membrane permeabilization leading to cytosolic release of Cathepsin B. High sphingomyelin (SM) levels account for these effects as they can be induced in control cells on addition of the lipid and reverted on SM-lowering strategies in ASM-deficient cells. These results unveil a relevant role for SM in autophagy modulation and characterize autophagy anomalies in NPA, opening new perspectives for therapeutic interventions. PMID:24488099

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

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

    2010-08-01

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

  16. Cross-talk between TRPML1 channel, lipids and lysosomal storage diseases.

    Science.gov (United States)

    Weiss, Norbert

    2012-03-01

    Described by the Belgian cytologist Christian De Duve in 1949,(1) lysosomes (from the Greek "digestive bodies") are ubiquitous specialized intracellular organelles that ensure the degradation/recycling of macromolecules (proteins, lipids, membranes) through the activity of specific enzymes (i.e., acid hydrolases). They receive their substrates through different internalization pathways (i.e., endocytosis, phagocytosis and autophagy) and are involved in a wide range of physiological functions from cell death and signaling to cholesterol homeostasis and plasma membrane repair.(2) In Mammals, 50 soluble lysosomal hydrolases have been described, each targeting specific substrates. They are confined in the lumen of the lysosome and require an optimum pH (i.e., pH 4.5) to work. This acidic pH compared with the slightly alkaline pH of the cytosol (i.e., ~pH 7.2) is maintained by the activity of integral lysosomal membrane proteins (LMPs, that represent the second class of lysosomal proteins), including the V-type proton (H(+))-ATPase(3) and the chloride ion channel CLC7(4) that pumps protons from the cytosol across the lysosomal membrane.

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

    Science.gov (United States)

    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.

  18. Biomarkers in Lysosomal Storage Diseases

    Directory of Open Access Journals (Sweden)

    Joaquin Bobillo Lobato

    2016-12-01

    Full Text Available A biomarker is generally an analyte that indicates the presence and/or extent of a biological process, which is in itself usually directly linked to the clinical manifestations and outcome of a particular disease. The biomarkers in the field of lysosomal storage diseases (LSDs have particular relevance where spectacular therapeutic initiatives have been achieved, most notably with the introduction of enzyme replacement therapy (ERT. There are two main types of biomarkers. The first group is comprised of those molecules whose accumulation is directly enhanced as a result of defective lysosomal function. These molecules represent the storage of the principal macro-molecular substrate(s of a specific enzyme or protein, whose function is deficient in the given disease. In the second group of biomarkers, the relationship between the lysosomal defect and the biomarker is indirect. In this group, the biomarker reflects the effects of the primary lysosomal defect on cell, tissue, or organ functions. There is no “gold standard” among biomarkers used to diagnosis and/or monitor LSDs, but there are a number that exist that can be used to reasonably assess and monitor the state of certain organs or functions. A number of biomarkers have been proposed for the analysis of the most important LSDs. In this review, we will summarize the most promising biomarkers in major LSDs and discuss why these are the most promising candidates for screening systems.

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

    Directory of Open Access Journals (Sweden)

    Christine Burkard

    2014-11-01

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

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

    Science.gov (United States)

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

    2014-11-01

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

  1. Synthesis of mesh-shaped calcia partially stabilized zirconia using eggshell membrane template as filler composite

    Directory of Open Access Journals (Sweden)

    Gema Gempita

    2017-08-01

    Full Text Available This experiment was conducted experimentally to synthesize Calcia Partially Stabilized Zirconia (Ca-PSZ by sol-gel method using eggshell membrane template as a composite filler. The eggshell membrane was used to produce a mesh shaped structure, which hopefully can improve the mechanical properties of the composite. Ca-PSZ filler was synthesized from ZrOCl2 precursor and Ca(NO32 stabilizer with a 24 hours immersion time. Ca-PSZ of synthesis then mixed with the resin matrix to test its composite hardness. The EDS characterization results suggested that the sample contained elements of zirconia, calcium, and oxygen. Whereas, the XRD characterization identified that crystal structures that formed in the sample were nano scale tetragonal. Characterization of SEM showed Ca-PSZ with mesh structured. The average composite hardness value was 15.79 VHN. The composites with Ca-PSZ-synthesized filler could be prepared and its hardness value was higher than the composite with Ca-PSZ filler in spherical particles, but the hardness was still below the composite on the market.

  2. The lysosomal potassium channel TMEM175 adopts a novel tetrameric architecture

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Changkeun; Guo, Jiangtao; Zeng, Weizhong; Kim, Sunghoon; She, Ji; Cang, Chunlei; Ren, Dejian; Jiang, Youxing

    2017-07-19

    TMEM175 is a lysosomal K+ channel that is important for maintaining the membrane potential and pH stability in lysosomes1. It contains two homologous copies of a six-transmembrane-helix (6-TM) domain, which has no sequence homology to the canonical tetrameric K+ channels and lacks the TVGYG selectivity filter motif found in these channels2, 3, 4. The prokaryotic TMEM175 channel, which is present in a subset of bacteria and archaea, contains only a single 6-TM domain and functions as a tetramer. Here, we present the crystal structure of a prokaryotic TMEM175 channel from Chamaesiphon minutus, CmTMEM175, the architecture of which represents a completely different fold from that of canonical K+ channels. All six transmembrane helices of CmTMEM175 are tightly packed within each subunit without undergoing domain swapping. The highly conserved TM1 helix acts as the pore-lining inner helix, creating an hourglass-shaped ion permeation pathway in the channel tetramer. Three layers of hydrophobic residues on the carboxy-terminal half of the TM1 helices form a bottleneck along the ion conduction pathway and serve as the selectivity filter of the channel. Mutagenesis analysis suggests that the first layer of the highly conserved isoleucine residues in the filter is primarily responsible for channel selectivity. Thus, the structure of CmTMEM175 represents a novel architecture of a tetrameric cation channel whose ion selectivity mechanism appears to be distinct from that of the classical K+ channel family.

  3. The lysosomal potassium channel TMEM175 adopts a novel tetrameric architecture.

    Science.gov (United States)

    Lee, Changkeun; Guo, Jiangtao; Zeng, Weizhong; Kim, Sunghoon; She, Ji; Cang, Chunlei; Ren, Dejian; Jiang, Youxing

    2017-07-27

    TMEM175 is a lysosomal K(+) channel that is important for maintaining the membrane potential and pH stability in lysosomes. It contains two homologous copies of a six-transmembrane-helix (6-TM) domain, which has no sequence homology to the canonical tetrameric K(+) channels and lacks the TVGYG selectivity filter motif found in these channels. The prokaryotic TMEM175 channel, which is present in a subset of bacteria and archaea, contains only a single 6-TM domain and functions as a tetramer. Here, we present the crystal structure of a prokaryotic TMEM175 channel from Chamaesiphon minutus, CmTMEM175, the architecture of which represents a completely different fold from that of canonical K(+) channels. All six transmembrane helices of CmTMEM175 are tightly packed within each subunit without undergoing domain swapping. The highly conserved TM1 helix acts as the pore-lining inner helix, creating an hourglass-shaped ion permeation pathway in the channel tetramer. Three layers of hydrophobic residues on the carboxy-terminal half of the TM1 helices form a bottleneck along the ion conduction pathway and serve as the selectivity filter of the channel. Mutagenesis analysis suggests that the first layer of the highly conserved isoleucine residues in the filter is primarily responsible for channel selectivity. Thus, the structure of CmTMEM175 represents a novel architecture of a tetrameric cation channel whose ion selectivity mechanism appears to be distinct from that of the classical K(+) channel family.

  4. Membrane anchoring stabilizes and favors secretion of New Delhi metallo-β-lactamase.

    Science.gov (United States)

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

    2016-07-01

    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, because those enzymes are readily degraded in their nonmetallated form. However, the New Delhi metallo-β-lactamase (NDM-1) can persist under conditions of metal depletion. NDM-1 is a lipidated protein that anchors 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.

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

    Science.gov (United States)

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

    2016-10-01

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

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

    Directory of Open Access Journals (Sweden)

    Shinozawa,Shinya

    1980-09-01

    Full Text Available 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.

  7. Stability study and lyophilization of vitamin E-loaded nanocapsules prepared by membrane contactor.

    Science.gov (United States)

    Khayata, N; Abdelwahed, W; Chehna, M F; Charcosset, C; Fessi, H

    2012-12-15

    In this research, we studied the accelerated stability of vitamin E-loaded nanocapsules (NCs) prepared by the nanoprecipitation method. Vitamin E-loaded NCs were optimized firstly at the laboratory scale and then scaled up using the membrane contactor technique. The optimum conditions of the membrane contactor preparation (pilot scale) produced vitamin E-loaded NCs with an average size of 253 nm, polydispersity index 0.19 and a zeta potential -16 mV. The average size, polydispersity index and zeta potential values were 185 nm, 0.12 and -15 mV, respectively for the NCs prepared at laboratory scale. No significant changes were noticed in these values after 3 and 6 months of storage at high temperature (40±2 °C) and relative humidity (75±5%) in spite of vitamin E sensitivity to light, heat and oxygen. The entrapment efficiency of NCs prepared at pilot scale was 97% at the beginning of the stability study, and became (95%, 59%) after 3 and 6 months of storage, respectively. These values at lab-scale were (98%, 96%, and 89%) at time zero and after 3 and 6 months of storage, respectively. This confirms the ability of vitamin E encapsulation to preserve its stability, which is one major goal of our work. Lyophilization of the optimized formula at lab-scale was also performed. Four types of cryoprotectants were tested (poly(vinyl pyrrolidone), sucrose, mannitol, and glucose). Freeze-dried NCs prepared with sucrose were found acceptable. The other lyophilized NCs obtained at different conditions presented large aggregates.

  8. Research results on productivity stabilization by ultrasonic camera (plant with membrane ceramic elements during vine processing

    Directory of Open Access Journals (Sweden)

    V. T. Antufyev

    2016-01-01

    Full Text Available The article describes solutions to the problems of declining productivity of ceramic membrane elements for wine processing on the final manufacturing phase. A relative stabilization of filtration velocity, venting efficiency and wine lightening were experimentally confirmed during contacts with oscillation waves of ultrasonic transmitter on the ceramic filter. Which significantly reduced the cost of various preservatives to increase periods storage. To study the processes of wine processing by the proposed method it was made an experimental installation on the basis of pilot machine MRp-1/2 for bottling of quiet liquids and an ultrasonic device "Volna– M" UZTA-1/22-OM with a firmly, waveguide which transmits sound, fixed filter frame on the ultrasound emitter. To stabilize the performance of ultrasonic units with ceramic membrane elements without quality deterioration of wines it was empirically determined rational parameters of power of ultrasound input and pressure in the system. The given derived dependencies and graphs allow to define the time of relatively stable operating filter regime. It was revealed a significant cost reduction on filtration, as it allows escape from the contamination of the product by various preservatives, and increasing of storage duration in a sealed container during aseptic filling without a thermal sterilization. Ultrasonic emitter contact by superposition wave vibrations on the ceramic filter increases not only the efficiency of gas removal, but also improves the organoleptic characteristics, stabilizes the filters, improves their productivity. Gas removal creates unfavorable conditions for development of the yeast, which in turn increases the shelf life of semisweet wine.

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

    Directory of Open Access Journals (Sweden)

    William P Clafshenkel

    Full Text Available 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(sulfosuccinimidylsuberate (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.

  10. Stability of the micromachined membrane deformable mirror as a freeform optical element

    Science.gov (United States)

    Vdovin, Gleb; Soloviev, Oleg; Patlan, Seva

    2014-09-01

    Micromachined membrane deformable mirror (MMDM) can serve as an ad hoc" free-form optical element. To test the repeatability and stability of the standard MMDM, we have conducted the test of surface figure during multiple thermal cycling, test of figure drift at elevated temperatures, and a long-term 16-day stability test of actively formed mirror figure. The average rms error did not exceed λ =25 at λ = 633 nm, after repeated cycling from -14 to +70 C, with return to the room temperature. The existing design provides ~10° stability in the temperature range of ~10°. Optimization of the design, eliminating astigmatism, would allow one to extend the temperature range to about 30. The long-term mirror figure instability at a constant temperature reaches λ/20 rms in 16 days. The P-V error with respect to the nearest sphere changes from λ/20 in the first day, to about λ/10 in the 16-th day. The tests show that MMDM is stable enough to make a reasonable alternative to free-form optics in applications that require various optical shapes to be formed with a single element.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

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

    2011-01-01

    The antimalarial agent artesunate (ART) activates programmed cell death (PCD) in cancer cells in a manner dependent on the presence of iron and the generation of reactive oxygen species. In malaria parasites, ART cytotoxicity originates from interactions with heme-derived iron within the food vacuole. The analogous digestive compartment of mammalian cells, the lysosome, similarly contains high levels of redox-active iron and in response to specific stimuli can initiate mitochondrial apoptosis. We thus investigated the role of lysosomes in ART-induced PCD and determined that in MCF-7 breast cancer cells ART activates lysosome-dependent mitochondrial outer membrane permeabilization. ART impacted endolysosomal and autophagosomal compartments, inhibiting autophagosome turnover and causing perinuclear clustering of autophagosomes, early and late endosomes, and lysosomes. Lysosomal iron chelation blocked all measured parameters of ART-induced PCD, whereas lysosomal iron loading enhanced death, thus identifying lysosomal iron as the lethal source of reactive oxygen species upstream of mitochondrial outer membrane permeabilization. Moreover, lysosomal inhibitors chloroquine and bafilomycin A1 reduced ART-activated PCD, evidencing a requirement for lysosomal function during PCD signaling. ART killing did not involve activation of the BH3-only protein, Bid, yet ART enhanced TNF-mediated Bid cleavage. We additionally demonstrated the lysosomal PCD pathway in T47D and MDA-MB-231 breast cancer cells. Importantly, non-tumorigenic MCF-10A cells resisted ART-induced PCD. Together, our data suggest that ART triggers PCD via engagement of distinct, interconnected PCD pathways, with hierarchical signaling from lysosomes to mitochondria, suggesting a potential clinical use of ART for targeting lysosomes in cancer treatment. PMID:21149439

  13. Effect of Nd-doping on the Thermal Stability and Pore-structure of Al2O3 Membranes

    Institute of Scientific and Technical Information of China (English)

    YU Jian-Chang; XU Wei-Jun; HUANG Qing-Ming; HU Sheng-Wei

    2005-01-01

    Unsupported Nd-doped Al2O3 membranes have been prepared with a sol-gel treatnt by using aluminium isopropoxide and Nd(NO3)3 as the main raw materials. The properties of Nd-doped Al2O3 membranes were characterized by XRD, DTA-TG, IR and N2 adsorption. The effects of Nd-doping on the phase composition, thermal stability as well as applications of pore- structure of Nd-doped Al2O3 membranes at high temperature were discussed. The results show that Nd-doping can raise the transition temperature rom γ-Al2O3 to α-Al2O3, enhance the thermal stability of Al2O3 membranes, and evidently improve the pore-structural parameters of Al2O3 mem- branes applied at higher temperatures.

  14. Inhibitors of lysosomal cysteine proteases

    Directory of Open Access Journals (Sweden)

    Lyanna O. L.

    2011-04-01

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

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

    Science.gov (United States)

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

    2016-02-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

    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 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.

  18. The influence of a membrane environment on the structure and stability of a prokaryotic potassium channel, KcsA.

    Science.gov (United States)

    Encinar, J A; Molina, M L; Poveda, J A; Barrera, F N; Renart, M L; Fernández, A M; González-Ros, J M

    2005-09-26

    The lack of a membrane environment in membrane protein crystals is considered one of the major limiting factors to fully imply X-ray structural data to explain functional properties of ion channels [Gulbis, J.M. and Doyle, D. (2004) Curr. Opin. Struct. Biol. 14, 440-446]. Here, we provide infrared spectroscopic evidence that the structure and stability of the potassium channel KcsA and its chymotryptic derivative 1-125 KcsA reconstituted into native-like membranes differ from those exhibited by these proteins in detergent solution, the latter taken as an approximation of the mixed detergent-protein crystal conditions.

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

    Science.gov (United States)

    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.

  20. Structure and Stability of the Spinach Aquaporin SoPIP2;1 in Detergent Micelles and Lipid Membranes

    DEFF Research Database (Denmark)

    Plasencia, Ines; Survery, Sabeen; Ibragimova, Sania;

    2011-01-01

    PC), or reconstituted into lipid membranes formed from mixtures of 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPE), 1-palmitoyl-2-oleoyl-phosphatidylethanolamine (POPE), 1-palmitoyl-2-oleoyl-phosphatidylserine (POPS), and ergosterol. Generally, SoPIP2;1 secondary structure was found to be predominantly a...... by an increased melting temperature of up to 70 degrees C. Conclusion/Significance: The results of this study provide insights into SoPIP2;1 stability in various host membranes and suggest suitable choices of detergent and lipid composition for reconstitution of SoPIP2;1 into biomimetic membranes...

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Sung-Chih Hsieh

    2015-01-01

    Full Text Available 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.

  3. Enhancing Structural Stability and Pervaporation Performance of Composite Membranes by Coating Gelatin onto Hydrophilically Modified Support Layer

    Institute of Scientific and Technical Information of China (English)

    吴洪; 芦霞; 李宪实; 李奕帆; 赵翠红; 姜忠义

    2014-01-01

    The interfacial compatibility of composite membrane is an important factor to its structural stability and separation performance. In this study, poly (ether sulfone) (PES) support layer was first hydrophilically modified with poly(vinyl alcohol) (PVA) via surface segregation during the phase inversion process. Gelatin (GE) was then cast on the PVA-modified PES support layer as the active layer followed by crosslinking to fabricate composite membranes for ethanol dehydration. The enrichment of PVA on the surface of support layer improved interfacial compatibility of the as-prepared GE/PVA-PES composite membrane. The water contact angle measurement and X-ray photoelectron spectroscopy (XPS) data confirmed the surface segregation of PVA with a surface coverage density of~80%. T-peel test showed that the maximal force to separate the support layer and the active layer was enhanced by 3 times compared with the GE/PES membrane. The effects of PVA content in the support layer, crosslinking of GE active layer and operating parameters on the pervaporative dehydration performance were in-vestigated. The operational stability of the composite membrane was tested by immersing the membrane in ethanol aqueous solution for a period of time. Stable pervaporation performance for dehydration of 90%ethanol solution was obtained for GE/PVA-PES membrane with a separation factor of~60 and a permeation flux of~1910 g·m-2·h-1 without peeling over 28 days immersion.

  4. Endosome-lysosomes, ubiquitin and neurodegeneration.

    Science.gov (United States)

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

    1996-01-01

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

  5. Membraner

    DEFF Research Database (Denmark)

    Bach, Finn

    2009-01-01

    Notatet giver en kort introduktion til den statiske virkemåde af membraner og membrankonstruktioner......Notatet giver en kort introduktion til den statiske virkemåde af membraner og membrankonstruktioner...

  6. Evidence for lysosomal exocytosis and release of aggrecan-degrading hydrolases from hypertrophic chondrocytes, in vitro and in vivo

    Directory of Open Access Journals (Sweden)

    Edward R. Bastow

    2012-02-01

    The abundant proteoglycan, aggrecan, is resorbed from growth plate cartilage during endochondral bone ossification, yet mice with genetically-ablated aggrecan-degrading activity have no defects in bone formation. To account for this apparent anomaly, we propose that lysosomal hydrolases degrade extracellular, hyaluronan-bound aggrecan aggregates in growth plate cartilage, and that lysosomal hydrolases are released from hypertrophic chondrocytes into growth plate cartilage via Ca2+-dependent lysosomal exocytosis. In this study we confirm that hypertrophic chondrocytes release hydrolases via lysosomal exocytosis in vitro and we show in vivo evidence for lysosomal exocytosis in hypertrophic chondrocytes during skeletal development. We show that lysosome-associated membrane protein 1 (LAMP1 is detected at the cell surface following in vitro treatment of epiphyseal chondrocytes with the calcium ionophore, ionomycin. Furthermore, we show that in addition to the lysosomal exocytosis markers, cathepsin D and β-hexosaminidase, ionomycin induces release of aggrecan- and hyaluronan-degrading activity from cultured epiphyseal chondrocytes. We identify VAMP-8 and VAMP7 as v-SNARE proteins with potential roles in lysosomal exocytosis in hypertrophic chondrocytes, based on their colocalisation with LAMP1 at the cell surface in secondary ossification centers in mouse tibiae. We propose that resorbing growth plate cartilage involves release of destructive hydrolases from hypertrophic chondrocytes, via lysosomal exocytosis.

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

    Science.gov (United States)

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

    2017-02-23

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

  8. Preparation of Cu{sub 2}O nanowire-blended polysulfone ultrafiltration membrane with improved stability and antimicrobial activity

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Zehai; Ye, Shuaiju; Fan, Zheng; Ren, Fanghua; Gao, Congjie [Zhejiang University of Technology, Institute of Oceanic and Environmental Chemical Engineering, College of Chemical Engineering and Material Science and College of Ocean, and State Key Lab Breeding Base of Green Chemical Synthesis Technology and Zhejiang Collaborative Innovation Center of Membrane Separation and Water Treatment (China); Li, Qingbiao; Li, Guoqing [Quanzhou Normal University, College of Chemistry and Life Science (China); Zhang, Guoliang, E-mail: membrane86571@163.com, E-mail: guoliangz@zjut.edu.cn [Zhejiang University of Technology, Institute of Oceanic and Environmental Chemical Engineering, College of Chemical Engineering and Material Science and College of Ocean, and State Key Lab Breeding Base of Green Chemical Synthesis Technology and Zhejiang Collaborative Innovation Center of Membrane Separation and Water Treatment (China)

    2015-10-15

    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 Cu{sub 2}O nanowires (NWs) to improve their stability and antifouling activity. Synthesis of novel Cu{sub 2}O NWs/PSF-blended ultrafiltration membrane was achieved via phase inversion method by dispersing one-dimensional Cu{sub 2}O 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 Cu{sub 2}O 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.

  9. On Stabilization of PVPA/PVA Electrospun Nanofiber Membrane and Its Effect on Material Properties and Biocompatibility

    Directory of Open Access Journals (Sweden)

    Rose Ann Franco

    2012-01-01

    Full Text Available A novel nanofiber membrane was fabricated by electrospinning composed of polyvinyl phosphonic acid (PVPA and polyvinyl alcohol (PVA. Stabilization was done due to the high dissolvability of the membrane when in contact with water. Physical treatment was done by exposure to heat at 150°C in a vacuum environment at different periods of time. Chemical crosslinking was done by immersion in methanol and methanol/ glutaraldehyde. A heat-exposed membrane was also further crosslinked chemically. All conditions were compared with regards to its effect on the material properties of the membranes and its biological response in vitro with MG-63 osteoblast-like cell line. Visual examination and dimensional analyses showed that heat treatment produced discoloration on the membrane surface and chemical crosslinking reduced membrane dimensions. Tensile strength and strain improved in crosslinked membranes compared to noncrosslinked counterpart. Swelling and degradation was also investigated and was seen to vary depending on the crosslinking condition. Biocompatibility was observed to be more favorable in heat-treated membranes.

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

    Science.gov (United States)

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

    2017-03-01

    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. Copyright © 2017 Elsevier GmbH. All rights reserved.

  11. Microvillar membrane microdomains exist at physiological temperature. Role of galectin-4 as lipid raft stabilizer revealed by "superrafts"

    DEFF Research Database (Denmark)

    Braccia, Anita; Villani, Maristella; Immerdal, Lissi

    2003-01-01

    and the transmembrane aminopeptidase N, whereas the peripheral lipid raft protein annexin 2 was essentially absent. In conclusion, in the microvillar membrane, galectin-4, functions as a core raft stabilizer/organizer for other, more loosely raft-associated proteins. The superraft analysis might be applicable to other...

  12. Oxygen permeation and thermo-chemical stability of oxygen separation membrane materials for the oxyfuel process

    Energy Technology Data Exchange (ETDEWEB)

    Ellett, Anna Judith

    2009-07-01

    analysis (TGA) and thermo mechanical analysis (TMA). An increase in thermal expansion and oxygen permeation associated with an increase in oxygen vacancy concentration, observed also in the TGA curves, occurs during heating. BSCF50 exhibits permeation fluxes well above those of LSCF58, PSCF58 and La{sub 2}NiO{sub 4+{delta}}, which are quite similar to each other. After exposure, no degradation of LSCF58, La{sub 2}NiO{sub 4+{delta}} and PSCF58 occurs. On the other hand BSCF50 is found to be unstable in CO{sub 2}- and/or H{sub 2}O-containing atmospheres and also to exhibit a chemical demixing. The thermo-chemical stability and the oxygen permeation performances are both crucial factors in the selection of high purity oxygen separation membranes for the oxyfuel process, thus making LSCF58, PSCF58 and La{sub 2}NiO{sub 4+{delta}} in this study the most suitable materials for this application. Serious issues arise, however, from the fact that secondary non-ion conducting oxide phases are formed in the bulk of every material, forming obstacles for oxygen ion migration, and also that a reaction with chromia occurs, preventing their use without protection. (orig.)

  13. GNeosomes: Highly Lysosomotropic Nanoassemblies for Lysosomal Delivery.

    Science.gov (United States)

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

    2015-01-01

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

  14. Distinct Structural Elements Govern the Folding, Stability, and Catalysis in the Outer Membrane Enzyme PagP.

    Science.gov (United States)

    Iyer, Bharat Ramasubramanian; Mahalakshmi, Radhakrishnan

    2016-09-06

    The outer membrane enzyme PagP is indispensable for lipid A palmitoylation in Gram-negative bacteria and has been implicated in resistance to host immune defenses. PagP possesses an unusual structure for an integral membrane protein, with a highly dynamic barrel domain that is tilted with respect to the membrane normal. In addition, it contains an N-terminal amphipathic helix. Recent functional and structural studies have shown that these molecular factors are critical for PagP to carry out its function in the challenging environment of the bacterial outer membrane. However, the precise contributions of the N-helix to folding and stability and residues that can influence catalytic rates remain to be addressed. Here, we identify a sequence-dependent stabilizing role for the N-terminal helix of PagP in the measured thermodynamic stability of the barrel. Using chimeric barrel sequences, we show that the Escherichia coli PagP N-terminal helix confers 2-fold greater stability to the Salmonella typhimurium barrel. Further, we find that the W78F substitution in S. typhimurium causes a nearly 20-fold increase in the specific activity in vitro for the phospholipase reaction, compared to that of E. coli PagP. Here, phenylalanine serves as a key regulator of catalysis, possibly by increasing the reaction rate. Through coevolution analysis, we detect an interaction network between seemingly unrelated segments of this membrane protein. Exchanging the structural and functional features between homologous PagP enzymes from E. coli and S. typhimurium has provided us with an understanding of the molecular factors governing PagP stability and function.

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

    Science.gov (United States)

    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. Crown Copyright © 2016. Published by Elsevier Ltd. All rights reserved.

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

    Science.gov (United States)

    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.

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

    Science.gov (United States)

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

    2015-05-01

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

  18. Membrane Protein Stability Analyses by Means of Protein Energy Profiles in Case of Nephrogenic Diabetes Insipidus

    Directory of Open Access Journals (Sweden)

    Florian Heinke

    2012-01-01

    Full Text Available 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.

  19. Membrane protein stability analyses by means of protein energy profiles in case of nephrogenic diabetes insipidus.

    Science.gov (United States)

    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.

  20. Electrochemical stability of carbon nanofibers in proton exchange membrane fuel cells

    Energy Technology Data Exchange (ETDEWEB)

    Alvarez, Garbine [Energy Department, CIDETEC-IK4, Po Miramon, 196, 20009 San Sebastian (Spain); Alcaide, Francisco, E-mail: falcaide@cidetec.es [Energy Department, CIDETEC-IK4, Po Miramon, 196, 20009 San Sebastian (Spain); Miguel, Oscar [Energy Department, CIDETEC-IK4, Po Miramon, 196, 20009 San Sebastian (Spain); Cabot, Pere L. [Laboratori d' Electroquimica de Materials i del Medi Ambient, Dept. Quimica Fisica, Universitat de Barcelona, Marti i Franques, 1-11, 08028 Barcelona (Spain); Martinez-Huerta, M.V.; Fierro, J.L.G. [Instituto de Catalisis y Petroleoquimica (CSIC), Marie Curie 2, Cantoblanco, 28049 Madrid (Spain)

    2011-10-30

    This fundamental study deals with the electrochemical stability of several non-conventional carbon based catalyst supports, intended for low temperature proton exchange membrane fuel cell (PEMFC) cathodes. Electrochemical surface oxidation of raw and functionalized carbon nanofibers, and carbon black for comparison, was studied following a potential step treatment at 25.0 deg. C in acid electrolyte, which mimics the operating conditions of low temperature PEMFCs. Surface oxidation was characterized using cyclic voltammetry, X-ray photoelectron spectroscopy (XPS), and contact angle measurements. Cyclic voltammograms clearly showed the presence of the hydroquinone/quinone couple. Furthermore, identification of carbonyl, ether, hydroxyl and carboxyl surface functional groups were made by deconvolution of the XPS spectra. The relative increase in surface oxides on carbon nanofibers during the electrochemical oxidation treatment is significantly smaller than that on carbon black. This suggests that carbon nanofibers are more resistant to the electrochemical corrosion than carbon black under the experimental conditions used in this work. This behaviour could be attributed to the differences found in the microstructure of both kinds of carbons. According to these results, carbon nanofibers possess a high potential as catalyst support to increase the durability of catalysts used in low temperature PEMFC applications.

  1. Anti-inflammatory activity of leaves ofJatropha gossypifolia L. by hrbc membrane stabilization method

    Institute of Scientific and Technical Information of China (English)

    Yerramsetty Nagaharika; Valluri kalyani; Shaik Rasheed; Ramadosskarthikeyan

    2013-01-01

    Object:To evaluate the anti inflammatory activity of leaves extracts ofJatropha gossypifolia(J. gossypifolia)L.Methods:The plantJ. gossypifoliaL.(Eurphorbiaceae) is known as belly ache bush.The plant originated fromBrazil and it is now cultivated in tropical countries throughout the world.The roots, stems, leaves, seeds and fruits of the plant have been widely used in traditional folk medicine in many parts ofWestAfrica.The young stem of the plant is used as tooth brush as well as to clean tongue in the treatment thrush.The tuber of the plant grinded into a paste is also locally used in the treatment of hemorrhoids.The present study aimed to evaluate the anti inflammatory activity of aqueous and alcoholic extract ofJ. gossypifolia leaves byin vitroHRBC membrane stabilization method.Results:Thein vitro method showed significant anti inflammatory property of different extracts tested.Conclusion:The aqueous extract at a concentration of200 μg/mL showed significant activity when compared with the standard drug Diclofenac sodium.

  2. Cytotoxic, thrombolytic, membrane stabilizing and anti-oxidant activities of Hygrophila schulli

    Directory of Open Access Journals (Sweden)

    Md. Abu Sufian

    2015-08-01

    Full Text Available The crude methanol extract of Hygrophila schulli and its petroleum ether, chloroform, ethyl acetate and aqueous soluble Kupchan partitionates were investigated for in vitro cytotoxic, thrombolytic, membrane stabilizing and anti-oxidant activities. In the brine shrimp lethality bioassay, the petroleum ether soluble extract of H. schulli showed significant cytotoxicity (LC50 = 0.1 µg/mL and LC90 = 15 µg/mL as compared to vincristine sulfate (LC50 = 0.4 µg/mL and LC90 = 9 µg/mL. Among all the partitionates, chloroform soluble fraction demonstrated the highest thrombolytic activity with 10.5% clot lyses. Moreover, in hypotonic- and heat-induced conditions, the chloroform soluble extractive inhibited hemolysis of human erythrocyte by 113.7% and 14.3%, respectively as compared to 71.9% and 42.2% demonstrated by standard acetylsalicylic acid. On the other hand, in anti-oxidant activity test, chloroform soluble fraction revealed mild antioxidant activity (IC50 = 195.1 µg/mL as compared to standard tert-butyl-1-hydroxytoluene (IC50 = 27.5 µg/mL.

  3. Reliable characteristics and stabilization of on-membrane SOI MOSFET-based components heated up to 335 °C

    Science.gov (United States)

    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.

  4. 溶酶体膜蛋白LAMP3对肝癌转移影响的研究进展%Progress of metastasis in hepatic cell carcinoma effected by lysosome-associated mem-brane proteins(LAMP3)

    Institute of Scientific and Technical Information of China (English)

    任庆; 谭宁

    2015-01-01

    原发性肝细胞肝癌( hepatic cell carcinoma,HCC)是广西区域性高发恶性肿瘤,其预后与肝癌是否转移密切相关。目前有研究发现溶酶体膜相关蛋白LAMP3在肝癌细胞稳定过表达后,可促进肝癌细胞的转移,同时诱导细胞骨架F-actin重排并上调Cdc42活性,提示LAMP3可能是肝癌转移的促进因子。本文就溶酶体膜蛋白LAMP3对肝癌转移的意义作一综述。%Hepatic cell carcinoma( HCC)was regional and high incidence of malignant tumor in Guangxi Province, the prognosis was closely related with metastasis whether or not. At present,some studies have discovered that lyso-some-associated membrane proteins( LAMP3 )stable over expression in hepatocellular carcinoma cells,which could promote the metastasis,at the same time induced cytoskeletal F-actin rearrangement and upregulation of Cdc42 ac-tivity. Results suggest that LAMP3 is a metastasis promoting factor. The lysosomal membrane protein LAMP3 on liver metastasis were reviewed in this article.

  5. Cancer cell injury by cytotoxins from cobra venom is mediated through lysosomal damage.

    Science.gov (United States)

    Feofanov, Alexei V; Sharonov, George V; Astapova, Maria V; Rodionov, Dmitriy I; Utkin, Yuriy N; Arseniev, Alexander S

    2005-08-15

    Cytotoxins from cobra venom are known to manifest cytotoxicity in various cell types. It is widely accepted that the plasma membrane is a target of cytotoxins, but the mechanism of their action remains obscure. Using the confocal spectral imaging technique, we show for the first time that cytotoxins from cobra venom penetrate readily into living cancer cells and accumulate markedly in lysosomes. Cytotoxins CT1 and CT2 from Naja oxiana, CT3 from Naja kaouthia and CT1 from Naja haje are demonstrated to possess this property with respect to human lung adenocarcinoma A549 and promyelocytic leukaemia HL60 cells. Immobilized plasma membrane binding accompanies the internalization of CT3 from Naja kaouthia in the HL60 cells, but it is very weak for other cytotoxins. Detectable membrane binding is not a property of any of the cytotoxins tested in A549 cells. The kinetics and concentration-dependence of cytotoxin accumulation in lysosomes correlate well with their cytotoxic effects. On the basis of the results obtained, we propose that lysosomes are a primary target of the lytic action of cytotoxins. Plasma membrane permeabilization seems to be a downstream event relative to lysosome rupture. Direct damage to the plasma membrane may be a complementary mechanism, but its relative contribution to the cytotoxic action depends on the cytotoxin structure and cell type.

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

    Science.gov (United States)

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

    2015-03-17

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

  7. Innovative methods to stabilize liquid membranes for removal of radionuclides from groundwater

    Energy Technology Data Exchange (ETDEWEB)

    Lokhandwala, K. [Membrane Technology and Research, Inc., Menlo Park, CA (United States)

    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.

  8. Hollow fibre microporous silica membranes for gas separation and pervaporation: synthesis, performance and stability

    NARCIS (Netherlands)

    Soest-Vercammen, E.L.J. van; Peters, T.A.; Fontalvo, J.; Vorstman, M.A.G.; Benes, N.E.; Dam, R.A. van; Vroon, Z.A.E.P.; Keurentjes, J.T.F.

    2005-01-01

    Thin microporous silica membranes were prepared on the outer surface of hollow fibre ceramic substrates. In principle this enables relatively fast and inexpensive production of large membrane surface area, combined with a low support resistance and a high membrane surface area/module volume ratio (>

  9. Stability of membranous nanostructures: a possible key mechanism in cancer progression

    Directory of Open Access Journals (Sweden)

    Kralj-Iglic V

    2012-07-01

    Full Text Available Veronika Kralj-IglicBiomedical Research Group, Faculty of Health Sciences, University of Ljubljana, Zdravstvena 5, Ljubljana, SloveniaAbstract: Membranous nanostructures, such as nanovesicles and nanotubules, are an important pool of biological membranes. Recent results indicate that they constitute cell-cell communication systems and that cancer development is influenced by these systems. Nanovesicles that are pinched off from cancer cells can move within the circulation and interact with distant cells. It has been suggested and indicated by experimental evidence that nanovesicles can induce metastases from the primary tumor in this way. Therefore, it is of importance to understand better the mechanisms of membrane budding and vesiculation. Here, a theoretical description is presented concerning consistently related lateral membrane composition, orientational ordering of membrane constituents, and a stable shape of nanovesicles and nanotubules. It is shown that the character of stable nanostructures reflects the composition of the membrane and the intrinsic shape of its constituents. An extension of the fluid mosaic model of biological membranes is suggested by taking into account curvature-mediated orientational ordering of the membrane constituents on strongly anisotropically curved regions. Based on experimental data for artificial membranes, a possible antimetastatic effect of plasma constituents via mediation of attractive interaction between membranous structures is suggested. This mediated attractive interaction hypothetically suppresses nanovesiculation by causing adhesion of buds to the mother membrane and preventing them from being pinched off from the membrane.Keywords: nanovesicles, nanotubules, nanotubes, microvesicles, exosomes, metastasis

  10. Residual stress and buckling patterns of free-standing yttria-stabilized-zirconia membranes fabricated by pulsed laser deposition

    Energy Technology Data Exchange (ETDEWEB)

    Evans, A.; Prestat, M.; Toelke, R.; Schlupp, M.V.F.; Gauckler, L.J. [ETH Zurich, Nonmetallic Inorganic Materials, Zurich (Switzerland); Safa, Y.; Hocker, T. [ZHAW Winterthur, Institute of Computational Physics, Winterthur (Switzerland); Courbat, J.; Briand, D.; Rooij, N.F. de [Ecole Polytechnique Federale de Lausanne (EPFL), Institute of Microengineering, Neuchatel (Switzerland); Courty, D. [ETH Zurich, Laboratory for Nanometallurgy, Zurich (Switzerland)

    2012-08-15

    The residual stress and buckling patterns of free-standing 8 mol.% yttria-stabilized-zirconia (8YSZ) membranes prepared by pulsed laser deposition and microfabrication techniques on silicon substrates are investigated by wafer curvature, light microscopy, white light interferometry, and nanoindentation. The 300 nm thin 8YSZ membranes (390 {mu}m x 390 {mu}m) deposited at 25 C are almost flat after free-etching, whereas deposition at 700 C yields strongly buckled membranes with a compressive stress of -1,100 {+-} 150 MPa and an out-of-plane-displacement of 6.5 {mu}m. These latter membranes are mechanically stable during thermal cycling up to 500 C. Numerical simulations of the buckling shape using the Rayleigh-Ritz-method and a Young's modulus of 200 GPa are in good agreement with the experimental data. The simulated buckling patterns are used to extract the local stress distribution within the free-standing membrane which consists of tensile and compressive stress regions that are below the failure stresses. This is important regarding the application in, e.g., microsolid oxide fuel cell membranes which must be thermomechanically stable during microfabrication and device operation. (Copyright copyright 2012 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim)

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

    Science.gov (United States)

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

    2010-10-01

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

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

    Science.gov (United States)

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

    2016-03-18

    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.

  13. Biochemical and lysosomal biomarkers in the mussel Mytilus galloprovincialis from the Mar Piccolo of Taranto (Ionian Sea, Southern Italy).

    Science.gov (United States)

    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.

  14. Effect of integral proteins in the phase stability of a lipid bilayer: Application to raft formation in cell membranes

    Science.gov (United States)

    Gómez, Jordi; Sagués, Francesc; Reigada, Ramon

    2010-04-01

    The existence of lipid rafts is a controversial issue. The affinity of cholesterol for saturated lipids is manifested in macroscopic phase separation in model membranes, and is believed to be the thermodynamic driving force for raft formation. However, there is no clear reason to explain the small (nanometric) size of raft domains in cell membranes. In a recent paper Yethiraj and Weisshaar [Biophys. J. 93, 3113 (2007)] proposed that the effect of neutral integral membrane proteins may prevent from the formation of large lipid domains. In this paper we extend this approach by studying the effect of the protein size, as well as the lipid-protein interaction. Depending on these factors, two different mechanisms for nanodomain stabilization are shown to be possible for static proteins. The application of these results to a biological context is discussed.

  15. Streptococcus oralis Induces Lysosomal Impairment of Macrophages via Bacterial Hydrogen Peroxide.

    Science.gov (United States)

    Okahashi, Nobuo; Nakata, Masanobu; Kuwata, Hirotaka; Kawabata, Shigetada

    2016-07-01

    Streptococcus oralis, an oral commensal, belongs to the mitis group of streptococci and occasionally causes opportunistic infections, such as bacterial endocarditis and bacteremia. Recently, we found that the hydrogen peroxide (H2O2) produced by S. oralis is sufficient to kill human monocytes and epithelial cells, implying that streptococcal H2O2 is a cytotoxin. In the present study, we investigated whether streptococcal H2O2 impacts lysosomes, organelles of the intracellular digestive system, in relation to cell death. S. oralis infection induced the death of RAW 264 macrophages in an H2O2-dependent manner, which was exemplified by the fact that exogenous H2O2 also induced cell death. Infection with either a mutant lacking spxB, which encodes pyruvate oxidase responsible for H2O2 production, or Streptococcus mutans, which does not produce H2O2, showed less cytotoxicity. Visualization of lysosomes with LysoTracker revealed lysosome deacidification after infection with S. oralis or exposure to H2O2, which was corroborated by acridine orange staining. Similarly, fluorescent labeling of lysosome-associated membrane protein-1 gradually disappeared during infection with S. oralis or exposure to H2O2 The deacidification and the following induction of cell death were inhibited by chelating iron in lysosomes. Moreover, fluorescent staining of cathepsin B indicated lysosomal destruction. However, treatment of infected cells with a specific inhibitor of cathepsin B had negligible effects on cell death; instead, it suppressed the detachment of dead cells from the culture plates. These results suggest that streptococcal H2O2 induces cell death with lysosomal destruction and then the released lysosomal cathepsins contribute to the detachment of the dead cells.

  16. Eps8 is recruited to lysosomes and subjected to chaperone-mediated autophagy in cancer cells.

    Science.gov (United States)

    Welsch, Thilo; Younsi, Alexander; Disanza, Andrea; Rodriguez, Jose Antonio; Cuervo, Ana Maria; Scita, Giorgio; Schmidt, Jan

    2010-07-15

    Eps8 controls actin dynamics directly through its barbed end capping and actin-bundling activity, and indirectly by regulating Rac-activation when engaged into a trimeric complex with Eps8-Abi1-Sos1. Recently, Eps8 has been associated with promotion of various solid malignancies, but neither its mechanisms of action nor its regulation in cancer cells have been elucidated. Here, we report a novel association of Eps8 with the late endosomal/lysosomal compartment, which is independent from actin polymerization and specifically occurs in cancer cells. Endogenous Eps8 localized to large vesicular lysosomal structures in metastatic pancreatic cancer cell lines, such as AsPC-1 and Capan-1 that display high Eps8 levels. Additionally, ectopic expression of Eps8 increased the size of lysosomes. Structure-function analysis revealed that the region encompassing the amino acids 184-535 of Eps8 was sufficient to mediate lysosomal recruitment. Notably, this fragment harbors two KFERQ-like motifs required for chaperone-mediated autophagy (CMA). Furthermore, Eps8 co-immunoprecipitated with Hsc70 and LAMP-2, which are key elements for the CMA degradative pathway. Consistently, in vitro, a significant fraction of Eps8 bound to (11.9+/-5.1%) and was incorporated into (5.3+/-6.5%) lysosomes. Additionally, Eps8 binding to lysosomes was competed by other known CMA-substrates. Fluorescence recovery after photobleaching revealed that Eps8 recruitment to the lysosomal membrane was highly dynamic. Collectively, these results indicate that Eps8 in certain human cancer cells specifically localizes to lysosomes, and is directed to CMA. These results open a new field for the investigation of how Eps8 is regulated and contributes to tumor promotion in human cancers.

  17. Effect of intrinsic curvature and edge tension on the stability of binary mixed-membrane three-junctions

    Science.gov (United States)

    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.

  18. Evaluation of Membrane Stabilizing Activity, Total Phenolic Content, Brine Shrimp Lethality Bioassay, Thrombolytic and Antimicrobial Activities of Tagetes patula L.

    Directory of Open Access Journals (Sweden)

    Md. Ruhul Kuddus

    2012-11-01

    Full Text Available The methanol extract of leaf of Tagetes patula L. as well as its n-hexane, carbon tetrachloride, chloroform and aqueous soluble partitionates were subjected to screening for total phenolic content, brine shrimp lethality, membrane stabilizing, thrombolytic and antimicrobial activity. The membrane stabilizing activity was assessed by hypotonic solution-and heat-induced methods and was compared with acetyl salicylic acid. In the present studies, the n-hexane soluble fraction demonstrated strong membrane stabilizing activity in both hypotonic solution-and heat-induced methods with 44.48% and 42.68% inhibition of haemolysis, respectively. The total phenolic content was also determined and expressed in gallic acid equivalent. In brine shrimp bioassay, the crude methanol extract of leaf showed strong cytotoxic activity with LC50 value of 8.58 μg/ml compared to that of 0.451 μg/ml exhibited by standard vincristine sulphate. During assay for thrombolytic activity, the n-hexane soluble fraction revealed 43.7% lysis of clot while standard streptokinase and water, used as positive and negative controls, demonstrated 65.8% and 3.62% lysis of clot, respectively. In antimicrobial assay by disc diffusion method, all the samples exhibited moderate to significant antimicrobial activity (zone of inhibition = 9.0-22.0 mm against all the test organisms. Among all the samples, the carbon tetrachloride soluble fraction displayed strong antimicrobial activity against Escherichia coli (22.0 mm.

  19. Effect of operation parameters on the flux stabilization of gravity-driven membrane (GDM) filtration system for decentralized water supply.

    Science.gov (United States)

    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.

  20. Effect of intrinsic curvature and edge tension on the stability of binary mixed-membrane three-junctions.

    Science.gov (United States)

    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.

  1. Membrane stability of sickle erythrocytes incubated in extracts of three medicinal plants: Anacardium occidentale, Psidium guajava, and Terminalia catappa

    Directory of Open Access Journals (Sweden)

    Paul Chidoka Chikezie

    2011-01-01

    Full Text Available Background: Many reports showed that medicinal plant extracts cause alterations on the shape and physiology of erythrocytes. Objective: The present study seeks to ascertain the osmotic stability of sickle erythrocytes incubated in aqueous extracts of Anacardium occidentale, Psidium guajava, and Terminalia catappa. Materials and Methods: The fraction of erythrocytes lysed when suspended in saline solution of varying concentrations was investigated by spectrophotometric method. The percentage hemolysis of erythrocytes in the control and test samples showed a sigmoidal relationship with increasing concentrations of saline solution. Membrane stability was ascertained as mean corpuscular fragility (MCF index of erythrocytes incubated in 400 and 800 mg/dL aqueous concentrations of the three plant extracts. Results: The two experimental concentrations of P. guajava and T. catappa protected the erythrocytes against osmotic stress, as evidenced by decreases in the values of MCF compared with the control sample (P < 0.05. However, 800 mg/dL of A. occidentale promoted significant (P < 0.05 distabilization of sickle erythrocytes. Conclusion: Whereas the two experimental concentrations of aqueous extracts of P. guajava and T. catappa stabilized erythrocyte membrane, higher concentration (800 mg/dL of A. occidentale exhibited no membrane protective effect.

  2. Stability of a nanofiltration membrane after contact with a low-level liquid radioactive waste

    Directory of Open Access Journals (Sweden)

    Elizabeth Eugenio de Mello Oliveira

    2013-01-01

    Full Text Available This study investigated the treatment of a liquid radioactive waste containing uranium (235U + 238U using nanofiltration membranes. The membranes were immersed in the waste for 24-5000 h, and their transport properties were evaluated before and after the immersion. Surface of the membranes changed after immersion in the waste. The SW5000 h specimen lost its coating layer of polyvinyl alcohol, and its rejection of sulfate ions and uranium decreased by about 35% and 30%, respectively. After immersion in the waste, the polyamide selective layer of the membranes became less thermally stable than that before immersion.

  3. Dicarboxylic acids with limited numbers of hydrocarbons stabilize cell membrane and increase osmotic resistance in rat erythrocytes.

    Science.gov (United States)

    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.

  4. Sulphonated imidized graphene oxide (SIGO) based polymer electrolyte membrane for improved water retention, stability and proton conductivity

    Science.gov (United States)

    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. Low concentration of DMSO stabilizes the bilayer gel phase rather than the interdigitated gel phase in dihexadecylphosphatidylcholine membrane.

    Science.gov (United States)

    Yamashita, Y; Kinoshita, K; Yamazaki, M

    2000-08-25

    We have investigated effects of dimethylsulfoxide (DMSO) on the phase stability of multilamellar vesicles of the ether-linked 1,2-dihexadecyl-sn-glycero-3-phosphatidylcholine (DHPC-MLV), which is known to be in the interdigitated gel (LbetaI) phase in excess water at 20 degrees C. The results of X-ray diffraction experiments indicate that the DHPC membrane was in the Lbeta, phase at X> or =0.12 (X=mole fraction of DMSO in DMSO/water mixture). The result of differential scanning calorimetry indicate that the gel to liquid-crystalline phase transition temperature increased, but the LbetaI to Pbeta, phase transition temperature decreased with an increase in DMSO concentration. These results show that DMSO stabilizes the bilayer gel phase rather than the LbetaI phase at its low concentration. The solubility of phosphorylcholine, which is the same structure as the headgroup of DHPC, decreased with an increase in DMSO concentration, indicating that the interaction free energy of the hydrophilic segments of the membrane with solvents increases with an increase in DMSO concentration. On the basis of the thermodynamic analysis, the mechanism of the stabilization of the bilayer gel phase of DHPC-MLV by DMSO is discussed. The decrease in the repulsive interaction between the headgroups of the phospholipid induced by the low concentrations of DMSO in water plays an important role in this stabilization.

  6. Aquifex aeolicus membrane hydrogenase for hydrogen biooxidation: Role of lipids and physiological partners in enzyme stability and activity

    Energy Technology Data Exchange (ETDEWEB)

    Infossi, Pascale; Lojou, Elisabeth; Giudici-Orticoni, Marie-Therese [Unite de Bioenergetique et Ingenierie des Proteines, UPR 9036, Institut de Microbiologie de la Mediterranee - CNRS, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20 (France); Chauvin, Jean-Paul [Institut de Biologie du developpement de Marseille Luminy, UMR 6216, Parc Scientifique de Luminy, 163 Avenue de Luminy, BP 907, 13009 Marseille (France); Herbette, Gaetan [Spectropole FI 1739, Aix-Marseille Universite case 511, Faculte de St Jerome Avenue Escadrille Normandie Niemen, 13397 Marseille Cedex 20 (France); Brugna, Myriam [Unite de Bioenergetique et Ingenierie des Proteines, UPR 9036, Institut de Microbiologie de la Mediterranee - CNRS, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20 (France); Universite de Provence, 3 Place Victor Hugo, 13331 Marseille Cedex 03 (France)

    2010-10-15

    Hydrogenase I from the hyperthermophilic bacterium Aquifex aeolicus is a good candidate for biotechnological devices thanks to its ability to oxidize hydrogen at high temperature, even in the presence of oxygen and CO. In order to enhance the enzyme stability and the catalytic efficiency, we investigated the hydrogen oxidation process with hydrogenase I embedded in a physiological-like environment. Hydrogenase I partners in the metabolic chain, namely membrane quinone and cytochrome b, were purified and fully characterized. The complex hydrogenase I-cytochrome b was inserted into liposomes. Surface Plasmon Resonance revealed that quinone took part in the stabilization of the complex. By use of molecular modelization and electrochemistry analysis, enzyme stability has been demonstrated to be stronger and enzymatic efficiency to be five times higher when hydrogenase is embedded into the liposomes. This result raises the possibility of using hydrogenases as biocatalysts in fuel cells. (author)

  7. A New Class of Amphiphiles Bearing Rigid Hydrophobic Groups for Solubilization and Stabilization of Membrane Proteins

    DEFF Research Database (Denmark)

    Chae, Pil Seok; Rasmussen, Søren G F; Rana, Rohini R;

    2012-01-01

    Non-traditional amphiphiles: Conferring aqueous solubility on membrane proteins generally requires the use of a detergent or other amphiphilic agent. A new class of amphiphiles was synthesized, based on steroidal lipophilic groups, and evaluated with several membrane proteins. The results show th...

  8. Structure of anti-FLAG M2 Fab domain and its use in the stabilization of engineered membrane proteins

    Energy Technology Data Exchange (ETDEWEB)

    Roosild, Tarmo P.; Castronovo, Samantha; Choe, Senyon, E-mail: choe@salk.edu [Structural Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, California 92037 (United States)

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

    CERN Document Server

    Portet, Thomas; 10.1016/j.bpj.2010.09.032

    2011-01-01

    We report a new 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 the latter with cholesterol and dioleoylphosphatidylethanolamine (DOPE). 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 DOPE 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. Presumably, interlipid hydrogen bonding lies in the origin of this behavior. Furthermore, cholesterol was found to lower the lysis tension of DOPC bilayers. This behavior differs from that observed on...

  10. Lysosomal exoglycosidases in nasal polyps.

    Science.gov (United States)

    Chojnowska, Sylwia; Minarowska, Alina; Knaś, Małgorzata; Niemcunowicz-Janica, Anna; Kołodziejczyk, Paweł; Zalewska-Szajda, Beata; Kępka, Alina; Minarowski, Łukasz; Waszkiewicz, Napoleon; Zwierz, Krzysztof; Szajda, Sławomir Dariusz

    2013-01-01

    Nasal polyps are smooth outgrowths assuming a shape of grapes, formed from the nasal mucosa, limiting air flow by projecting into a lumen of a nasal cavity. Up to now the surgical resection is the best method of their treatment, but etiology and pathogenesis of the nasal polyps is not yet fully established. The aim of the study was the assessment of the selected lysosomal exoglycosidases activity in the nasal polyps. In this study the activity of β-galactosidase, α-mannosidase and α-fucosidase was determined in the tissue of the nasal polyps obtained from 40 patients (10F, 30M) and control tissues derived from mucosa of lower nasal conchas obtained during mucotomy from 20 patients (10F, 10M). We observed significant lower values of GAL, FUC and tendency to decrease of MAN and GLU concentration in nasal polyps (P) in comparison to control healthy nasal mucosa (C). In nasal polyp tissue (P) no differences of GAL, MAN and FUC specific activity in comparison to control mucosa (C) were found. Our research supports bioelectrical theory of the nasal polyps pathogenesis and directs attention at research on glycoconjugates and glycosidases of the nasal mucosa extracellular matrix. Copyright © 2013 Polish Otorhinolaryngology - Head and Neck Surgery Society. Published by Elsevier Urban & Partner Sp. z.o.o. All rights reserved.

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

    Science.gov (United States)

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

    2015-01-01

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

  12. Comparing the short and long term stability of biodegradable, ceramic and cation exchange membranes in microbial fuel cells.

    Science.gov (United States)

    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. FBAR syndapin 1 recognizes and stabilizes highly curved tubular membranes in a concentration dependent manner

    DEFF Research Database (Denmark)

    Ramesh, Pradeep; Baroji, Younes F.; Seyyed Reihani, Seyyed Nader;

    2013-01-01

    Syndapin 1 FBAR, a member of the Bin-amphiphysin-Rvs (BAR) domain protein family, is known to induce membrane curvature and is an essential component in biological processes like endocytosis and formation and growth of neurites. We quantify the curvature sensing of FBAR on reconstituted porcine...... at low protein densities. Interestingly, FBAR from syndapin 1 has a large affinity for tubular membranes with curvatures larger than its own intrinsic concave curvature. Finally, we studied the effect of FBAR on membrane relaxation kinetics with high temporal resolution and found that the protein...

  14. Polymer-stabilized palladium nanoparticles for catalytic membranes: ad hoc polymer fabrication

    Directory of Open Access Journals (Sweden)

    Macanás Jorge

    2011-01-01

    Full Text Available Abstract Metal nanoparticles are known as highly effective catalysts although their immobilization on solid supports is frequently required to prevent aggregation and to facilitate the catalyst application, recovery, and reuse. This paper reports the intermatrix synthesis of Pd0 nanoparticles in sulfonated polyethersulfone with Cardo group membranes and their use as nanocomposite catalytic membrane reactors. The synthesized polymer and the corresponding nanocomposite were characterized by spectroscopic and microscopic techniques. The catalytic efficiency of catalytic membranes was evaluated by following the reduction of p-nitrophenol in the presence of NaBH4.

  15. VOLTAMMETRY AND ELECTROCHEMICAL IMPEDANCE SPECTROSCOPY OF MEMBRANE SYSTEMS UNDER STABILIZED DIFFUSION LAYER THICKNESS

    Directory of Open Access Journals (Sweden)

    Sharafan M. V.

    2015-11-01

    Full Text Available The current-voltage characteristics and the number of effective ion transfer, as well as the frequency spectrum of the electrochemical impedance of multilayer ion-exchange membranes in a stable and controllable thickness of the diffusion layer were measured, using the of rotating membrane disk complex. The article presents a comparative analysis of the frequency spectra of the electrochemical impedance of the source and a surface-modified monopolar anion exchange membranes in 0.01 M sodium chloride was made. The process of water molecules dissociation at current densities above the limiting one in 0.01 M sodium chloride solution was studied in detail

  16. Phototoxic effects of lysosome-associated genetically encoded photosensitizer KillerRed

    Science.gov (United States)

    Serebrovskaya, Ekaterina O.; Ryumina, Alina P.; Boulina, Maria E.; Shirmanova, Marina V.; Zagaynova, Elena V.; Bogdanova, Ekaterina A.; Lukyanov, Sergey A.; Lukyanov, Konstantin A.

    2014-07-01

    KillerRed is a unique phototoxic red fluorescent protein that can be used to induce local oxidative stress by green-orange light illumination. Here we studied phototoxicity of KillerRed targeted to cytoplasmic surface of lysosomes via fusion with Rab7, a small GTPase that is known to be attached to membranes of late endosomes and lysosomes. It was found that lysosome-associated KillerRed ensures efficient light-induced cell death similar to previously reported mitochondria- and plasma membrane-localized KillerRed. Inhibitory analysis demonstrated that lysosomal cathepsins play an important role in the manifestation of KillerRed-Rab7 phototoxicity. Time-lapse monitoring of cell morphology, membrane integrity, and nuclei shape allowed us to conclude that KillerRed-Rab7-mediated cell death occurs via necrosis at high light intensity or via apoptosis at lower light intensity. Potentially, KillerRed-Rab7 can be used as an optogenetic tool to direct target cell populations to either apoptosis or necrosis.

  17. Nanoporous membrane robustness / stability in small form factor microfluidic filtration system.

    Science.gov (United States)

    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.

  18. Polymer-stabilized palladium nanoparticles for catalytic membranes: ad hoc polymer fabrication

    OpenAIRE

    2011-01-01

    Metal nanoparticles are known as highly effective catalysts although their immobilization on solid supports is frequently required to prevent aggregation and to facilitate the catalyst application, recovery, and reuse. This paper reports the intermatrix synthesis of Pd0 nanoparticles in sulfonated polyethersulfone with Cardo group membranes and their use as nanocomposite catalytic membrane reactors. The synthesized polymer and the corresponding nanocomposite were characterized by spectr...

  19. RNS60, a charge-stabilized nanostructure saline alters Xenopus Laevis oocyte biophysical membrane properties by enhancing mitochondrial ATP production.

    Science.gov (United States)

    Choi, Soonwook; Yu, Eunah; Kim, Duk-Soo; Sugimori, Mutsuyuki; Llinás, Rodolfo R

    2015-03-01

    We have examined the effects of RNS60, a 0.9% saline containing charge-stabilized oxygen nanobubble-based structures. RNS60 is generated by subjecting normal saline to Taylor-Couette-Poiseuille (TCP) flow under elevated oxygen pressure. This study, implemented in Xenopus laevis oocytes, addresses both the electrophysiological membrane properties and parallel biological processes in the cytoplasm. Intracellular recordings from defolliculated X. laevis oocytes were implemented in: (1) air oxygenated standard Ringer's solution, (2) RNS60-based Ringer's solution, (3) RNS10.3 (TCP-modified saline without excess oxygen)-based Ringer's, and (4) ONS60 (saline containing high pressure oxygen without TCP modification)-based Ringer's. RNS60-based Ringer's solution induced membrane hyperpolarization from the resting membrane potential. This effect was prevented by: (1) ouabain (a blocker of the sodium/potassium ATPase), (2) rotenone (a mitochondrial electron transfer chain inhibitor preventing usable ATP synthesis), and (3) oligomycin A (an inhibitor of ATP synthase) indicating that RNS60 effects intracellular ATP levels. Increased intracellular ATP levels following RNS60 treatment were directly demonstrated using luciferin/luciferase photon emission. These results indicate that RNS60 alters intrinsic the electrophysiological properties of the X. laevis oocyte membrane by increasing mitochondrial-based ATP synthesis. Ultrastructural analysis of the oocyte cytoplasm demonstrated increased mitochondrial length in the presence of RNS60-based Ringer's solution. It is concluded that the biological properties of RNS60 relate to its ability to optimize ATP synthesis.

  20. Innexin7a forms junctions that stabilize the basal membrane during cellularization of the blastoderm in Tribolium castaneum.

    Science.gov (United States)

    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.

  1. Lysosomal Storage Disorders and Malignancy

    Directory of Open Access Journals (Sweden)

    Gregory M. Pastores

    2017-02-01

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

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

    Science.gov (United States)

    Oh, Doo-Byoung

    2015-08-01

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

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

    Directory of Open Access Journals (Sweden)

    Alberto Canfrán-Duque

    2016-03-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

  5. A novel approach to analyze lysosomal dysfunctions through subcellular proteomics and lipidomics: the case of NPC1 deficiency

    Science.gov (United States)

    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.

  6. A novel approach to analyze lysosomal dysfunctions through subcellular proteomics and lipidomics: the case of NPC1 deficiency

    Science.gov (United States)

    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

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

    Science.gov (United States)

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

    2011-10-01

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

  8. The Phosphoinositide-Gated Lysosomal Ca(2+) Channel, TRPML1, Is Required for Phagosome Maturation.

    Science.gov (United States)

    Dayam, Roya M; Saric, Amra; Shilliday, Ryan E; Botelho, Roberto J

    2015-09-01

    Macrophages internalize and sequester pathogens into a phagosome. Phagosomes then sequentially fuse with endosomes and lysosomes, converting into degradative phagolysosomes. Phagosome maturation is a complex process that requires regulators of the endosomal pathway including the phosphoinositide lipids. Phosphatidylinositol-3-phosphate and phosphatidylinositol-3,5-bisphosphate (PtdIns(3,5)P2 ), which respectively control early endosomes and late endolysosomes, are both required for phagosome maturation. Inhibition of PIKfyve, which synthesizes PtdIns(3,5)P2 , blocked phagosome-lysosome fusion and abated the degradative capacity of phagosomes. However, it is not known how PIKfyve and PtdIns(3,5)P2 participate in phagosome maturation. TRPML1 is a PtdIns(3,5)P2 -gated lysosomal Ca(2+) channel. Because Ca(2+) triggers membrane fusion, we postulated that TRPML1 helps mediate phagosome-lysosome fusion. Using Fcγ receptor-mediated phagocytosis as a model, we describe our research showing that silencing of TRPML1 hindered phagosome acquisition of lysosomal markers and reduced the bactericidal properties of phagosomes. Specifically, phagosomes isolated from TRPML1-silenced cells were decorated with lysosomes that docked but did not fuse. We could rescue phagosome maturation in TRPML1-silenced and PIKfyve-inhibited cells by forcible Ca(2+) release with ionomycin. We also provide evidence that cytosolic Ca(2+) concentration increases upon phagocytosis in a manner dependent on TRPML1 and PIKfyve. Overall, we propose a model where PIKfyve and PtdIns(3,5)P2 activate TRPML1 to induce phagosome-lysosome fusion.

  9. Adaptor Protein Complexes AP-1 and AP-3 Are Required by the HHV-7 Immunoevasin U21 for Rerouting of Class I MHC Molecules to the Lysosomal Compartment

    Science.gov (United States)

    Kimpler, Lisa A.; Glosson, Nicole L.; Downs, Deanna; Gonyo, Patrick; May, Nathan A.; Hudson, Amy W.

    2014-01-01

    The human herpesvirus-7 (HHV-7) U21 gene product binds to class I major histocompatibility complex (MHC) molecules and reroutes them to a lysosomal compartment. Trafficking of integral membrane proteins to lysosomes is mediated through cytoplasmic sorting signals that recruit heterotetrameric clathrin adaptor protein (AP) complexes, which in turn mediate protein sorting in post-Golgi vesicular transport. Since U21 can mediate rerouting of class I molecules to lysosomes even when lacking its cytoplasmic tail, we hypothesize the existence of a cellular protein that contains the lysosomal sorting information required to escort class I molecules to the lysosomal compartment. If such a protein exists, we expect that it might recruit clathrin adaptor protein complexes as a means of lysosomal sorting. Here we describe experiments demonstrating that the μ adaptins from AP-1 and AP-3 are involved in U21-mediated trafficking of class I molecules to lysosomes. These experiments support the idea that a cellular protein(s) is necessary for U21-mediated lysosomal sorting of class I molecules. We also examine the impact of transient versus chronic knockdown of these adaptor protein complexes, and show that the few remaining μ subunits in the cells are eventually able to reroute class I molecules to lysosomes. PMID:24901711

  10. Thrombin-induced lysosomal exocytosis in human platelets is dependent on secondary activation by ADP and regulated by endothelial-derived substances.

    Science.gov (United States)

    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.

  11. On the edge energy of lipid membranes and the thermodynamic stability of pores

    Energy Technology Data Exchange (ETDEWEB)

    Pera, H.; Kleijn, J. M.; Leermakers, F. A. M., E-mail: Frans.leermakers@wur.nl [Laboratory of Physical Chemistry and Colloid Science, W ageningen University, Dreijenplein 6, 6307 HB Wageningen (Netherlands)

    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.

  12. Lysosomal and mitochondrial permeabilization mediates zinc(II) cationic phthalocyanine phototoxicity.

    Science.gov (United States)

    Marino, Julieta; García Vior, María C; Furmento, Verónica A; Blank, Viviana C; Awruch, Josefina; Roguin, Leonor P

    2013-11-01

    In order to find a novel photosensitizer to be used in photodynamic therapy for cancer treatment, we have previously showed that the cationic zinc(II) phthalocyanine named Pc13, the sulfur-linked dye 2,9(10),16(17),23(24)-tetrakis[(2-trimethylammonium) ethylsulfanyl]phthalocyaninatozinc(II) tetraiodide, exerts a selective phototoxic effect on human nasopharynx KB carcinoma cells and induces an apoptotic response characterized by an increase in the activity of caspase-3. Since the activation of an apoptotic pathway by chemotherapeutic agents contributes to the elimination of malignant cells, in this study we investigated the molecular mechanisms underlying the antitumor action of Pc13. We found that after light exposure, Pc13 induced the production of reactive oxygen species (ROS), which are mediating the resultant cytotoxic action on KB cells. ROS led to an early permeabilization of lysosomal membranes as demonstrated by the reduction of lysosome fluorescence with acridine orange and the release of lysosomal proteases to cytosol. Treatment with antioxidants inhibited ROS generation, preserved the integrity of lysosomal membrane and increased cell proliferation in a concentration-dependent manner. Lysosome disruption was followed by mitochondrial depolarization, cytosolic release of cytochrome C and caspases activation. Although no change in the total amount of Bax was observed, the translocation of Bax from cytosol to mitochondria, the cleavage of the pro-apoptotic protein Bid, together with the decrease of the anti-apoptotic proteins Bcl-XL and Bcl-2 indicated the involvement of Bcl-2 family proteins in the induction of the mitochondrial pathway. It was also demonstrated that cathepsin D, but not caspase-8, contributed to Bid cleavage. In conclusion, Pc13-induced cell photodamage is triggered by ROS generation and activation of the mitochondrial apoptotic pathway through the release of lysosomal proteases. In addition, our results also indicated that Pc13 induced

  13. Role of interfacial protein membrane in oxidative stability of vegetable oil substitution emulsions applicable to nutritionally modified sausage.

    Science.gov (United States)

    Jiang, Jiang; Xiong, Youling L

    2015-11-01

    The potential health risk associated with excessive dietary intake of fat and cholesterol has led to a renewed interest in replacing animal fat with nutritionally-balanced unsaturated oil in processed meats. However, as oils are more fluid and unsaturated than fats, one must overcome the challenge of maintaining both physical and chemical (oxidative) stabilities of prepared emulsions. Apart from physical entrapments, an emulsion droplet to be incorporated into a meat protein gel matrix (batter) should be equipped with an interactive protein membrane rather than a small surfactant, and the classical DLVO stabilization theory becomes less applicable. This review paper describes the steric effects along with chemical roles (radical scavenging and metal ion chelation) of proteins and their structurally modified derivatives as potential interface-building materials for oxidatively stable meat emulsions.

  14. High stability of the hinge region in the membrane-active peptide helix of zervamicin: paramagnetic relaxation enhancement studies.

    Science.gov (United States)

    Shenkarev, Zakhar O; Paramonov, Alexander S; Balashova, Tamara A; Yakimenko, Zoya A; Baru, Michael B; Mustaeva, Leila G; Raap, Jan; Ovchinnikova, Tatyana V; Arseniev, Alexander S

    2004-12-17

    Zervamicin IIB is a 16 amino acid peptaibol that forms voltage dependent ion channels with multilevel conductance states in planar lipid bilayers and vesicular systems. Stability of the hinge region and intermolecular interactions were investigated in the N- and C-terminally spin-labelled peptide analogues. Intermolecular and intramolecular paramagnetic enhancement indicates that zervamicin behaves as a rigid helical rod in methanol solution. There are no high amplitude hinge-bending motions, and the peptaibol is monomeric up to concentration 1.5 mM. Stability of the hinge region illustrates the helix stabilising propensity of the Pro residue in membrane mimic environments and implies absence of significant conformational rearrangement due to voltage peptaibol activation.

  15. LAMP-2 is required for incorporating syntaxin-17 into autophagosomes and for their fusion with lysosomes.

    Science.gov (United States)

    Hubert, Virginie; Peschel, Andrea; Langer, Brigitte; Gröger, Marion; Rees, Andrew; Kain, Renate

    2016-10-15

    Autophagy is an evolutionarily conserved process used for removing surplus and damaged proteins and organelles from the cytoplasm. The unwanted material is incorporated into autophagosomes that eventually fuse with lysosomes, leading to the degradation of their cargo. The fusion event is mediated by the interaction between the Qa-SNARE syntaxin-17 (STX17) on autophagosomes and the R-SNARE VAMP8 on lysosomes. Cells deficient in lysosome membrane-associated protein-2 (LAMP-2) have increased numbers of autophagosomes but the underlying mechanism is poorly understood. By transfecting LAMP-2-deficient and LAMP-1/2--double-deficient mouse embryonic fibroblasts (MEFs) with a tandem fluorescent-tagged LC3 we observed a failure of fusion between the autophagosomes and the lysosomes that could be rescued by complementation with LAMP-2A. Although we observed no change in expression and localization of VAMP8, its interacting partner STX17 was absent from autophagosomes of LAMP-2-deficient cells. Thus, LAMP-2 is essential for STX17 expression by the autophagosomes and this absence is sufficient to explain their failure to fuse with lysosomes. The results have clear implications for situations associated with a reduction of LAMP-2 expression.

  16. Dynamics of membrane protein/amphipol association studied by Förster resonance energy transfer: implications for in vitro studies of amphipol-stabilized membrane proteins.

    Science.gov (United States)

    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.

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

    Science.gov (United States)

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

    2016-01-01

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

  18. Regulation of high-voltage-activated Ca(2+) channel function, trafficking, and membrane stability by auxiliary subunits.

    Science.gov (United States)

    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. Tailoring of porosity of yttria-stabilized zirconia tubes as supports for oxygen separation membranes

    DEFF Research Database (Denmark)

    Bjørnetun Haugen, Astri; Kothanda Ramachandran, Dhavanesan; Gurauskis, Jonas

    deformation during debinding. The influence of the amount of pore formers (relative to the amount of ceramic and thermoplastics) on the microstructure of sintered samples, as well as the extrudability and ease of debinding of the feedstock, has been studied. Ceramics with 1-20 μm pores, open porosities......Pure oxygen gas supplied by ceramic oxygen transport membranes can facilitate reduced CO2 emissions through more efficient gasification processes and CO2 capture and storage. Tubular membranes have some advantages compared to planar membranes, such as better resistance to thermal gradients and more...... exceeding 55 % and gas permeabilities close to 10-14 m2 could be produced, demonstrating that thermoplastic extrusion is suitable for fabrication of porous and permeable tubes....

  20. Visualization of ceramide channels in lysosomes following endogenous palmitoyl-ceramide accumulation as an initial step in the induction of necrosis

    Directory of Open Access Journals (Sweden)

    Mototeru Yamane

    2017-09-01

    Full Text Available In this study, we showed that the dual addition of glucosyl ceramide synthase and ceramidase inhibitors to A549 cell culture led to the possibility of ceramide channel formation via endogenous palmitoyl-ceramide accumulation with an increase in cholesterol contents in the lysosome membrane as an initial step prior to initiation of necrotic cell death. In addition, the dual addition led to black circular structures of 10–20 nm, interpreted as stain-filled cylindrical channels on transmission electron microscopy. The formation of palmitoyl-ceramide channels in the lysosome membrane causes the liberation of cathepsin B from lysosomes for necrotic cell death. On the other hand, necrotic cell death in the dual addition was not caused by oxidative stress or cathepsin B activity, and the cell death was free from the contribution of the translation of Bax protein to the lysosome membrane.

  1. Stability and structure of the membrane protein transporter Ffh is modulated by substrates and lipids

    DEFF Research Database (Denmark)

    Reinau, Marika Ejby; Otzen, Daniel

    2009-01-01

    The cytosolic protein Ffh transports membrane proteins from the ribosome to the inner membrane in complex with 4.5S RNA. Here we show that native Ffh binds to the hydrophobic probe ANS in a 1 Ffh:3 ANS stoichiometry, revealing a hydrophobic binding site. Thermal precipitation of Ffh is shifted...... upwards by ∼10 °C by ANS or substrate protein, suggesting that the hydrophobic binding site makes the protein aggregation prone. Chemical denaturation confirm that Ffh is a rather unstable protein. 4.5S RNA destabilizes Ffh further, suggesting it keeps the protein in a more open conformation than...

  2. Fast determination of operational stability of the soluble acetylacetone-cleaving enzyme Dke1 in an enzyme membrane reactor.

    Science.gov (United States)

    Hofer, Hannes; Steiner, Walter

    2005-11-01

    The main aim of this study was the determination of the operational stability of soluble Dke1 (EC 1.13.11.50) in an enzyme membrane reactor. In order to calculate the half-life of soluble Dke1, the K (M) of oxygen must be known. The determination of this constant was done using progress curve analysis (K (M) = 260 micromol l(-1)). In a next step, the reactor system was studied by building a mathematical model for calculation of the reactor system, using Berkeley Madonna ver. 8.0.1 software. After that, the determination of the half-life of Dke1 under operational conditions at different temperatures (5, 10, 15, 25, 30, 35 degrees C) was performed. The quantitative criterion for stability was the value of the first-order rate constant of monomolecular inactivation. The experiments showed that soluble Dke1 is poorly stable. The half-life ranged from 308 min at 5 degrees C to 9 min at 35 degrees C. This method for determining the half-life is quite applicable for enzymes which are poorly stable. In addition, both the storage stability and the operational stability can be determined.

  3. Maltose-neopentyl glycol (MNG) amphiphiles for solubilization, stabilization and crystallization of membrane proteins

    DEFF Research Database (Denmark)

    Chae, Pil Seok; Rasmussen, Søren G F; Rana, Rohini R;

    2010-01-01

    The understanding of integral membrane protein (IMP) structure and function is hampered by the difficulty of handling these proteins. Aqueous solubilization, necessary for many types of biophysical analysis, generally requires a detergent to shield the large lipophilic surfaces of native IMPs. Ma...

  4. High-performance hybrid pervaporation membranes with superior hydrothermal and acid stability

    NARCIS (Netherlands)

    H.L. Castricum; R. Kreiter; H.M. van Veen; D.H.A. Blank; J.F. Vente; J.E. ten Elshof

    2008-01-01

    A new organic-inorganic hybrid membrane has been prepared with exceptional performance in dewatering applications. The only precursor used in the sol-gel synthesis of the selective layer was organically linked 1,2-bis(triethoxysilyl)ethane (BTESE). The microporous structure of this layer enables sel

  5. A Stability Study of Alkali Doped PBI Membranes for Alkaline Electrolyzer Cells

    DEFF Research Database (Denmark)

    Jensen, Jens Oluf; Aili, David; Hansen, Martin Kalmar

    2014-01-01

    conductivity was similar to a commercial Zirfon membrane and suitable for a water electrolyzer. Some chemical degradation was seen during the aging period, but the crosslinked and the cured materials were both integral after 176 days of aging. A simplified electrolyzer test cell was operated successfully....

  6. Antibacterial Activities and In Vitro Anti-Inflammatory (Membrane Stability Properties of Methanolic Extracts of Gardenia coronaria Leaves

    Directory of Open Access Journals (Sweden)

    Amin Chowdhury

    2014-01-01

    Full Text Available This work is carried out with Gardenia coronaria leaves that belong to the family Rubiaceae, which is a small-to-medium-sized but tall, deciduous tree, 7.6–9 m high on an average. Leaves are used for the treatment of rheumatic pain and bronchitis. The leaf of the plant consists of coronalolide, coronalolic acid, coronalolide methyl ester, ethyl coronalolate acetate triterpenes (secocycloartanes, and so forth. Methanol extract from the leaves of Gardenia coronaria was completely screened for membrane stability and antibacterial activity. The lower concentrations of Methanolic leaf extract of Gardenia coronaria gave good antimicrobial and anti-inflammatory activity, but higher concentrations gave relatively more projecting antibacterial activity in vitro as compared with Kanamycin. The crude drug’s anti-inflammatory effects were compared with those of Aspirin as positive control. The Methanolic extracts of Gardenia coronaria leaves possessed a broad spectrum antibacterial activity against a variety of both Gram-negative and Gram-positive organisms like Streptococcus agalactiae, Escherichia coli, Pseudomonas aeruginosa, Bacillus cereus, Shigella sonnei, Shigella boydii, and Proteus mirabilis, with a zone of inhibition from 10 to 16 mm. The extract also showed good membrane stability to be considered as having significant anti-inflammatory action.

  7. Fibrous Support Stabilizes Nitrification Performance of a Membrane-Aerated Biofilm: The Effect of Liquid Flow Perturbation

    DEFF Research Database (Denmark)

    Terada, Akihiko; Ito, J; Matsumoto, S

    2009-01-01

    Nitrification stability and biofilm robustness were examined by comparing a fibrous support membrane-aerated biofilm reactor (FS-MABR), where a woven fibrous support was surrounded on a silicone tube, with an MABR. The overall mass transfer coefficient of oxygen for the FS-MABR, assuming no bound......Nitrification stability and biofilm robustness were examined by comparing a fibrous support membrane-aerated biofilm reactor (FS-MABR), where a woven fibrous support was surrounded on a silicone tube, with an MABR. The overall mass transfer coefficient of oxygen for the FS-MABR, assuming...... liquid flow rate condition was 49% and 75% in the FS-MABR and MABR, exhibiting robust biofilms grown on the fibrous support. The FS-MABR provided more stable nitrification performance than the MABR irrespective of a high liquid flow rate. Both reactors have deteriorated ammonium (NH4+-N) removal without...... a high liquid flow rate condition to eliminate excessive biomass, indicating that regular maintenance is essential to eliminate excessive biofilm from a MABR for nitrification, which potentially acts as a NH4+ diffusion barrier....

  8. Stability and Membrane Orientation of the Fukutin Transmembrane Domain: A Combined Multiscale Molecular Dynamics and Circular Dichroism Study

    Science.gov (United States)

    2010-01-01

    The N-terminal domain of fukutin-I has been implicated in the localization of the protein in the endoplasmic reticulum and Golgi Apparatus. It has been proposed to mediate this through its interaction with the thinner lipid bilayers found in these compartments. Here we have employed multiscale molecular dynamics simulations and circular dichroism spectroscopy to explore the structure, stability, and orientation of the short 36-residue N-terminus of fukutin-I (FK1TMD) in lipids with differing tail lengths. Our results show that FK1TMD adopts a stable helical conformation in phosphatidylcholine lipids when oriented with its principal axis perpendicular to the bilayer plane. The stability of the helix is largely insensitive to the lipid tail length, preventing hydrophobic mismatch by virtue of its mobility and ability to tilt within the lipid bilayers. This suggests that changes in FK1TMD tilt in response to bilayer properties may be implicated in the regulation of its trafficking. Coarse-grained simulations of the complex Golgi membrane suggest the N-terminal domain may induce the formation of microdomains in the surrounding membrane through its preferential interaction with 1,2-dipalmitoyl-sn-glycero-3-phosphatidylinositol 4,5-bisphosphate lipids. PMID:21105749

  9. Crucial roles of membrane stability and its related proteins in the tolerance of peach fruit to chilling injury.

    Science.gov (United States)

    Zhang, Changfeng; Ding, Zhansheng; Xu, Xiangbing; Wang, Qing; Qin, Guozheng; Tian, Shiping

    2010-06-01

    Proteome patterns in peach fruit (Prunus persica L.) stored at different low temperatures were examined in order to gain a better understanding why peach fruit is less prone to chilling injury when stored at 0 degrees C than at 5 degrees C. Some differently expressed proteins in peach fruit stored at 0 and 5 degrees C were identified using electrospray ionization quadrupole time-of-flight tandem mass spectrometry. Among these proteins, four membrane stability related proteins, i.e., enolase, temperature-induced lipocalin, major allergen Pru p 1, and type II SK2 dehydrin were enhanced, but three proteins related to phenolic compounds metabolization, cinnamyl-alcohol dehydrogenase 5, cinnamyl-alcohol dehydrogenase 1, and chorismate mutase, were repressed in peach fruit at 0 degrees C as compared to that at 5 degrees C. The abundance of glucose-6-phosphate dehydrogenase, NADP-dependent isocitrate dehydrogenase, and NADP-dependent malic enzyme, which catalyze the reactions during sugar metabolism and energy pathways, was found to decrease in peach fruit stored at 0 degrees C. In addition, our data revealed that low temperature of 0 degrees C might regulate the endogenous H(2)O(2) level, resulting in activating the transcriptional level of genes encoding the proteins related to membrane stability. These results provide a comprehensive knowledge to understand the mechanisms by which peach fruit stored at 0 degrees C showed a higher chilling tolerance than that at 5 degrees C.

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

    Directory of Open Access Journals (Sweden)

    Viorica Ivan

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

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

    Science.gov (United States)

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

    2015-11-01

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

  12. Citreoviridin Induces Autophagy-Dependent Apoptosis through Lysosomal-Mitochondrial Axis in Human Liver HepG2 Cells.

    Science.gov (United States)

    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.

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

    Science.gov (United States)

    Lloyd-Evans, Emyr; Platt, Frances M

    2011-08-01

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

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

    Directory of Open Access Journals (Sweden)

    Ekaterina A Ivanova

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

  15. New generation of membrane efficient water-based drilling fluids: pragmatic and cost-effective solutions to borehole stability problems

    Energy Technology Data Exchange (ETDEWEB)

    Tare, U.A. [Haliburton, Calgary, AB (Canada); Mody, F.K. [Shell International E and P Inc., Calgary, AB (Canada); Tan, C.P. [CSIRO Petroleum, Kensington, WA (Australia)

    2002-06-01

    Drilling and completion operations in shales often suffer as a result of wellbore instability. Mechanical failure of the rock around a wellbore is the primary cause of shale instability. This process can be exacerbated by physico-chemical interactions between drilling fluids and shales. Water-based drilling fluids are used more and more due to environmental awareness that becomes more prevalent. Wellbore instability problems can however result from an improper application of water-based drilling fluids in those cases where drilling occurs in sensitive clay-rich formations. To meet the requirements of the petroleum industry, considerable collaborative efforts were expanded in the development of innovative environmentally acceptable water-based drilling fluids. In this paper, the authors describe the process that leads to the development of these drilling fluids. It is possible to achieve shale stability through an osmotic outflow of pore fluid and prevention/minimization of mud pressure penetration, as laboratory experiments on shale samples under realistic downhole conditions exposed to these drilling fluids prove. High membrane efficiencies, in excess of 80 per cent, were generated by this new generation of membrane efficient water-based drilling fluids. Drilling objectives resulting from an improved application of water-based drilling fluids are made possible by a fundamental understanding of the main drilling fluid-shale interaction mechanisms for shale stability and the application of experimental data to field conditions. The authors indicate that the achievement of trouble-free drilling of shales and notable reductions in non-productive time is accomplished by following the practical guidelines included in this paper for maintaining shale stability with the new generation of water-based drilling fluids. 8 refs., 2 tabs., 4 figs.

  16. Crystallization around solid-like nanosized docks can explain the specificity, diversity, and stability of membrane microdomains

    Science.gov (United States)

    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

  17. Molecularly Designed Stabilized Asymmetric Hollow Fiber Membranes for Aggressive Natural Gas Separation.

    Science.gov (United States)

    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. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Lipid composition determines the effects of arbutin on the stability of membranes.

    OpenAIRE

    Hincha, D K; Oliver, A E; Crowe, J H

    1999-01-01

    Arbutin (hydroquinone-beta-D-glucopyranoside) is an abundant solute in the leaves of many freezing- or desiccation-tolerant plants. Its physiological role in plants, however, is not known. Here we show that arbutin protects isolated spinach (Spinacia oleracea L.) thylakoid membranes from freeze-thaw damage. During freezing of liposomes, the presence of only 20 mM arbutin led to complete leakage of a soluble marker from egg PC (EPC) liposomes. When the nonbilayer-forming chloroplast lipid mono...

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

    NARCIS (Netherlands)

    Gabriel, T.L.

    2017-01-01

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

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

    NARCIS (Netherlands)

    Gabriel, T.L.

    2017-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2013-01-01

    Lysosome is a key subcellular organelle in the execution of the autophagic process and at present little is known whether lysosomal function is controlled in the process of autophagy.In this study,we first found that suppression of mammalian target of rapamycin (mTOR) activity by starvation or two mTOR catalytic inhibitors (PP242 and Torinl),but not by an allosteric inhibitor (rapamycin),leads to activation of lysosomal function.Second,we provided evidence that activation of lysosomal function is associated with the suppression of mTOR complex 1 (mTORC1),but not mTORC2,and the mTORC1 localization to lysosomes is not directly correlated to its regulatory role in lysosomal function.Third,we examined the involvement of transcription factor EB (TFEB) and demonstrated that TFEB activation following mTORC1 suppression is necessary but not sufficient for lysosomal activation.Finally,Atg5 or Atg7deletion or blockage of the autophagosome-lysosome fusion process effectively diminished lysosomal activation,suggesting that lysosomal activation occurring in the course of autophagy is dependent on antophagosome-lysosome fusion.Taken together,this study demonstrates that in the course of autophagy,lysosomal function is upregulated via a dual mechanism involving mTORC1 suppression and autophagosome-lysosome fusion.

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

    Science.gov (United States)

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

    2013-04-01

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

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

    Science.gov (United States)

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

    2015-03-01

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

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

    OpenAIRE

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

    1992-01-01

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

  5. Lysosomal storage disease 2 - Pompe's disease

    NARCIS (Netherlands)

    van der Ploeg, Ans T.; Reuser, Arnold J. J.

    2008-01-01

    Pompe's disease, glycogen-storage disease type II, and acid maltase deficiency are alternative names for the same metabolic disorder. It is a pan-ethnic autosomal recessive trait characterised by acid alpha-glucosidase deficiency leading to lysosomal glycogen storage. Pompe's disease is also

  6. Transport of Lysosome-Related Organelles

    NARCIS (Netherlands)

    Jordens, Ingrid

    2005-01-01

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

  7. Transport of Lysosome-Related Organelles

    NARCIS (Netherlands)

    Jordens, Ingrid

    2005-01-01

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

  8. Lysosomal proteolysis: effects of aging and insulin.

    Science.gov (United States)

    Gromakova, I A; Konovalenko, O A

    2003-07-01

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

  9. Invited: A Stability Study of Alkali Doped PBI Membranes for Alkaline Electrolyzer Cells

    DEFF Research Database (Denmark)

    Jensen, Jens Oluf; Aili, David; Hansen, Martin Kalmar

    2014-01-01

    lost less that 10 mass %. Viscosity measurement on the linear PBI (the only one soluble) showed a decreasing molecular weight over time of storage. The materials were also examined by IR, NMR and TGA. Finally, electrolysis tests were made including comparison with a commercial diaphragm material...... polarization characteristics. [1] G. Merle, M. Wessling and K. Nijmeijer. J. Membrane Sci. 377 (2011) 1– 35 [2] Q. F. Li, J. O. Jensen, R. F. Savinell and N. J. Bjerrum. Prog. Polym. Sci. 34(2009) 449 [3] B. Xing, O. Savadogo, Electrochemistry Communications 2(2000) 697-702 [4] D. Aili, M. K. Hansen, R...

  10. TPC1 has two variant isoforms, and their removal has different effects on endo-lysosomal functions compared to loss of TPC2.

    Science.gov (United States)

    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.

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

    Science.gov (United States)

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

    2016-04-01

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

  12. ESCRT-Dependent Cell Death in a Caenorhabditis elegans Model of the Lysosomal Storage Disorder Mucolipidosis Type IV.

    Science.gov (United States)

    Huynh, Julie M; Dang, Hope; Munoz-Tucker, Isabel A; O'Ketch, Marvin; Liu, Ian T; Perno, Savannah; Bhuyan, Natasha; Crain, Allison; Borbon, Ivan; Fares, Hanna

    2016-02-01

    Mutations in MCOLN1, which encodes the cation channel protein TRPML1, result in the neurodegenerative lysosomal storage disorder Mucolipidosis type IV. Mucolipidosis type IV patients show lysosomal dysfunction in many tissues and neuronal cell death. The ortholog of TRPML1 in Caenorhabditis elegans is CUP-5; loss of CUP-5 results in lysosomal dysfunction in many tissues and death of developing intestinal cells that results in embryonic lethality. We previously showed that a null mutation in the ATP-Binding Cassette transporter MRP-4 rescues the lysosomal defect and embryonic lethality of cup-5(null) worms. Here we show that reducing levels of the Endosomal Sorting Complex Required for Transport (ESCRT)-associated proteins DID-2, USP-50, and ALX-1/EGO-2, which mediate the final de-ubiquitination step of integral membrane proteins being sequestered into late endosomes, also almost fully suppresses cup-5(null) mutant lysosomal defects and embryonic lethality. Indeed, we show that MRP-4 protein is hypo-ubiquitinated in the absence of CUP-5 and that reducing levels of ESCRT-associated proteins suppresses this hypo-ubiquitination. Thus, increased ESCRT-associated de-ubiquitinating activity mediates the lysosomal defects and corresponding cell death phenotypes in the absence of CUP-5.

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

    DEFF Research Database (Denmark)

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

    2014-01-01

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

  14. Neuroinflammatory paradigms in lysosomal storage diseases

    Directory of Open Access Journals (Sweden)

    Megan Elizabeth Bosch

    2015-10-01

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

  15. 溶酶体相关膜蛋白2基因新突变导致Danon病的临床病理特点%Clinical and pathological features of Danon disease associated with a novel lysosome-associated membrane protein-2B mutation

    Institute of Scientific and Technical Information of China (English)

    洪道俊; 石志鸿; 张巍; 王朝霞; 袁云

    2010-01-01

    目的 报道1例Danon病患者的临床表现、病理改变特点和基因突变位点.方法 16岁男性患者表现为缓慢进展的骨骼肌无力和萎缩,以近端为主,伴随轻度脊柱强直.心电图示Ⅰ度房室传导阻滞,心脏超声示二尖瓣局限性增厚伴舒张功能损害,肌电图提示神经源性合并肌源性损害.患者左腓肠肌活体组织检查后,进行组织学、酶组织化学、电镜观察及抗肌营养不良素蛋白、层黏连蛋白α2、C5b9等免疫组织化学染色.患者及其父母进行溶酶体相关膜蛋白2(LAMP2)B基因的直接测序.结果 骨骼肌纤维内出现大小不一的自嗜空泡和镶边空泡,空泡内缺乏糖原,免疫组织化学显示多数肌纤维内的空泡边缘出现肌营养不良素蛋白、层黏连蛋白α2和C5b9的表达.电镜显示肌纤维内大量膜性空泡和溶酶体聚集.患者的LAMP2B基因9号外显子存在K402X突变,患者母亲无此突变,50名健康对照无此突变.结论 LAMP2B的9号外显子羧基末端的截断突变可以导致相对良性的Danon病,其心脏损害相对轻微.%Objectives To report the clinical and myopathological features in a case with Danon disease caused by a novel mutation in the lysosome-associated membrane protein-2 ( LAMP2 ) B gene.Methods A 16-year-old boy presenting progressive muscle weakness and atrophy, accompanied with spinal ankylosis was clinically evaluated including electrocardiogram, echocardiogram and electromyogram.Muscle biopsy was carried out in the patient.The histological staining, ultrastructural examination, and immunohistochemical staining with antibodies against dystrophin, merosin and C5b9 were performed in frozen sections.LAMP2B sequence was analyzed in the patient and his parents.Results Electrocardiogram in the patient showed Ⅰ atrioventricular block; echocardiogram revealed focal hypertrophy in mitral valve with mild cardiac diastolic dysfunction; electromyogram indicated myogenic and neurogenic

  16. Administration of flutamide alters sperm ultrastructure, sperm plasma membrane integrity and its stability, and sperm mitochondrial oxidative capability in the boar: in vivo and in vitro approach.

    Science.gov (United States)

    Lydka, M; Piasecka, M; Gaczarzewicz, D; Koziorowski, M; Bilinska, B

    2012-08-01

    Our previous work has shown that an anti-androgen flutamide administered pre- and post-natally induced adverse effects on the epididymal morphology and function of adult boars. The present investigation is aimed to understand the effect of flutamide and its metabolite on changes in sperm plasma membrane integrity and its stability, changes in mitochondrial oxidative capability and frequency of abnormal sperm. In vivo effects of flutamide (50 mg/kg b.w.) on sperm ultrastructure were examined by electron microscopic observations. In vitro effects of 5, 50 and 100 μg/ml hydroxyflutamide, administered for 2 and 24 h, on sperm plasma membrane integrity were measured by LIVE/DEAD Sperm Vitality kit, while those on sperm membrane stability and mitochondrial oxidoreductive activity were investigated using Merocyanine 540 and NADH tests, respectively. The incidence of abnormal spermatozoa increased significantly (p boars compared with controls. In an in vitro approach, low dose of hydroxyflutamide in 2-h incubations appeared less effective in altering the sperm plasma membrane integrity and its stability than two higher doses used (p sperm membrane destabilization and mitochondrial oxidoreductive activity was strengthened after 24 h of hydroxyflutamide administration (p sperm parameters with regard to oxidative capability of mitochondria, plasma membrane changes and sperm ultrastructure provides novel data on the boar sperm sensitivity to anti-androgen action. Results indicate high sensitivity of boar spermatozoa to androgen withdrawal.

  17. Enhanced stability of Zr-doped Ba(CeTb)O3−δ-Ni cermet membrane for hydrogen separation

    OpenAIRE

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

    2015-01-01

    A mixed protonic and electronic conductor material BaCe0.85Tb0.05Zr0.1O3−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-BaCe0.95Tb0.05O3−δ membrane due to the Zr doping.

  18. Artificial-enzyme gel membrane-based biosurveillance sensor with high reproducibility and long-term storage stability.

    Science.gov (United States)

    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.

  19. EPAC1 activation by cAMP stabilizes CFTR at the membrane by promoting its interaction with NHERF1.

    Science.gov (United States)

    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.

  20. Turning the gun on cancer: Utilizing lysosomal P-glycoprotein as a new strategy to overcome multi-drug resistance.

    Science.gov (United States)

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

    2016-07-01

    Oxidative stress plays a role in the development of drug resistance in cancer cells. Cancer cells must constantly and rapidly adapt to changes in the tumor microenvironment, due to alterations in the availability of nutrients, such as glucose, oxygen and key transition metals (e.g., iron and copper). This nutrient flux is typically a consequence of rapid growth, poor vascularization and necrosis. It has been demonstrated that stress factors, such as hypoxia and glucose deprivation up-regulate master transcription factors, namely hypoxia inducible factor-1α (HIF-1α), which transcriptionally regulate the multi-drug resistance (MDR), transmembrane drug efflux transporter, P-glycoprotein (Pgp). Interestingly, in addition to the established role of plasma membrane Pgp in MDR, a new paradigm of intracellular resistance has emerged that is premised on the ability of lysosomal Pgp to transport cytotoxic agents into this organelle. This mechanism is enabled by the topological inversion of Pgp via endocytosis resulting in the transporter actively pumping agents into the lysosome. In this way, classical Pgp substrates, such as doxorubicin (DOX), can be actively transported into this organelle. Within the lysosome, DOX becomes protonated upon acidification of the lysosomal lumen, causing its accumulation. This mechanism efficiently traps DOX, preventing its cytotoxic interaction with nuclear DNA. This review discusses these effects and highlights a novel mechanism by which redox-active and protonatable Pgp substrates can utilize lysosomal Pgp to gain access to this compartment, resulting in catastrophic lysosomal membrane permeabilization and cell death. Hence, a key MDR mechanism that utilizes Pgp (the "gun") to sequester protonatable drug substrates safely within lysosomes can be "turned on" MDR cancer cells to destroy them from within.

  1. PBI-based polymer electrolyte membranes fuel cells. Temperature effects on cell performance and catalyst stability

    Energy Technology Data Exchange (ETDEWEB)

    Lobato, Justo; Canizares, Pablo; Rodrigo, Manuel A.; Linares, Jose J. [Chemical Engineering Department, University of Castilla-La Mancha, Campus Universitario s/n, 13004 Ciudad Real (Spain)

    2007-03-10

    In this work, it has been shown that the temperature (ranging from 100 to 175 C) greatly influences the performance of H{sub 3}PO{sub 4}-doped polybenzimidazole-based high-temperature polymer electrolyte membrane fuel cells by several and complex processes. The temperature, by itself, increases H{sub 3}PO{sub 4}-doped PBI conductivity and enhances the electrodic reactions as it rises. Nevertheless, high temperatures reduce the level of hydration of the membrane, above 130-140 C accelerate the self-dehydration of H{sub 3}PO{sub 4}, and they may boost the process of catalyst particle agglomeration that takes place in strongly acidic H{sub 3}PO{sub 4} medium (as checked by multi-cycling sweep voltammetry), reducing the overall electrochemical active surface. The first process seems to have a rapid response to changes in the temperature and controls the cell performance immediately after them. The second process seems to develop slower, and influences the cell performance in the 'long-term'. The predominant processes, at each moment and temperature, determine the effect of the temperature on the cell performance, as potentiostatic curves display. 'Long-term' polarization curves grow up to 150 C and decrease at 175 C. 'Short-term' ones continuously increase as the temperature does after 'conditioning' the cell at 125 C. On the contrary, when compared the polarization curves at 175 C a continuous decrease is observed with the 'conditioning' temperature. A discussion of the observed trends is proposed in this work. (author)

  2. Assembly of BODIPY-carbazole dyes with liposomes to fabricate fluorescent nanoparticles for lysosomal bioimaging in living cells.

    Science.gov (United States)

    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.

  3. Id1 interacts and stabilizes the Epstein-Barr virus latent membrane protein 1 (LMP1 in nasopharyngeal epithelial cells.

    Directory of Open Access Journals (Sweden)

    Pok Man Hau

    Full Text Available The EBV-encoded latent membrane protein 1 (LMP1 functions as a constitutive active form of tumor necrosis factor receptor (TNFR and activates multiple downstream signaling pathways similar to CD40 signaling in a ligand-independent manner. LMP1 expression in EBV-infected cells has been postulated to play an important role in pathogenesis of nasopharyngeal carcinoma. However, variable levels of LMP1 expression were detected in nasopharyngeal carcinoma. At present, the regulation of LMP1 levels in nasopharyngeal carcinoma is poorly understood. Here we show that LMP1 mRNAs are transcribed in an EBV-positive nasopharyngeal carcinoma (NPC cell line (C666-1 and other EBV-negative nasopharyngeal carcinoma cells stably re-infected with EBV. The protein levels of LMP1 could readily be detected after incubation with proteasome inhibitor, MG132 suggesting that LMP1 protein is rapidly degraded via proteasome-mediated proteolysis. Interestingly, we observed that Id1 overexpression could stabilize LMP1 protein in EBV-infected cells. In contrary, Id1 knockdown significantly reduced LMP1 levels in cells. Co-immunoprecipitation studies revealed that Id1 interacts with LMP1 by binding to the CTAR1 domain of LMP1. N-terminal region of Id1 is required for the interaction with LMP1. Furthermore, binding of Id1 to LMP1 suppressed polyubiquitination of LMP1 and may be involved in stabilization of LMP1 in EBV-infected nasopharyngeal epithelial cells.

  4. New insights into non-precious metal catalyst layer designs for proton exchange membrane fuel cells: Improving performance and stability

    Science.gov (United States)

    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.

  5. Impact of Solvent pH on Direct Immobilization of Lysosome-Related Cell Organelle Extracts on TiO₂ for Melanin Treatment.

    Science.gov (United States)

    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.

  6. Stability of transmembrane amyloid β-peptide and membrane integrity tested by molecular modeling of site-specific Aβ42 mutations.

    Directory of Open Access Journals (Sweden)

    Chetan Poojari

    Full Text Available Interactions of the amyloid β-protein (Aβ with neuronal cell membranes, leading to the disruption of membrane integrity, are considered to play a key role in the development of Alzheimer's disease. Natural mutations in Aβ42, such as the Arctic mutation (E22G have been shown to increase Aβ42 aggregation and neurotoxicity, leading to the early-onset of Alzheimer's disease. A correlation between the propensity of Aβ42 to form protofibrils and its effect on neuronal dysfunction and degeneration has been established. Using rational mutagenesis of the Aβ42 peptide it was further revealed that the aggregation of different Aβ42 mutants in lipid membranes results in a variety of polymorphic aggregates in a mutation dependent manner. The mutant peptides also have a variable ability to disrupt bilayer integrity. To further test the connection between Aβ42 mutation and peptide-membrane interactions, we perform molecular dynamics simulations of membrane-inserted Aβ42 variants (wild-type and E22G, D23G, E22G/D23G, K16M/K28M and K16M/E22G/D23G/K28M mutants as β-sheet monomers and tetramers. The effects of charged residues on transmembrane Aβ42 stability and membrane integrity are analyzed at atomistic level. We observe an increased stability for the E22G Aβ42 peptide and a decreased stability for D23G compared to wild-type Aβ42, while D23G has the largest membrane-disruptive effect. These results support the experimental observation that the altered toxicity arising from mutations in Aβ is not only a result of the altered aggregation propensity, but also originates from modified Aβ interactions with neuronal membranes.

  7. Effects of induced breeding with various ion concentrations of cadmium on the lysosomal membrane of coelomocytes in Pheretima aspergillum%不同镉离子浓度诱导饲养对参环毛蚓体腔细胞溶酶体膜的影响

    Institute of Scientific and Technical Information of China (English)

    吴波; 李薇; 喻良文; 付玉梅

    2010-01-01

    目的 观察不同浓度镉离子诱导饲养对参环毛蚓体腔细胞溶酶体膜的毒性效应.方法 选取健康参环毛蚓70只,按照饲养土壤镉离子浓度的不同随机分为7组,每组10只.各组土壤镉离子浓度分别为0.28(对照组)、3.28、6.28、12.28、18.28、24.28和30.28 mg/kg,采用不同浓度镉离子诱导参环毛蚓染毒并饲养14 d.诱导饲养7 d和14 d时观察参环毛蚓体腔细胞的形态变化,测定其体腔细胞溶酶体中性红保留时间.结果 高浓度镉离子诱导参环毛蚓体腔细胞溶酶体的损害明显.诱导饲养7 d,对照组、3.28、6.28、12.28、18.28、24.28和30.28 mg/kg镉离子浓度组参环毛蚓体腔细胞溶酶体中性红保留时间随着镉离子浓度的增加而逐渐缩短,分别为(71.3±2.4)、(60.5±1.6)、(55.1±2.9)、(51.9±3.6)、(46.0±2.5)、(38.8±1.8)、(34.2±2.0)min.与对照组比较,诱导饲养7 d、14d时各实验组参环毛蚓溶酶体中性红保留时间明显缩短(均P<0.05).结论 体腔细胞溶酶体中性红保留时间可用于评价土壤中重金属镉对参环毛蚓的毒性效应.%Objective To investigate the toxicity effects of induced breeding with various ion concentrations of cadmium on the lysosomal membrane of coelomocytes in Pheretima aspergillum. Methods Seventy healthy Pheretima aspergilla were selected and randomized into 7 groups (n=10 each) according to different levels of cadmium ion in the soils [0.28 mg/kg (control group) , 3.28 mg/kg, 6.28 mg/kg, 12.28 mg/kg, 18.28 mg/kg, 24.28 mg/kg and 30.28 mg/kg, respectively] and were bred with the contaminating cadmium ions for 14 days. On day 7 and day 14, the morphological changes in coelomocytes were studied and neutral red retention time (NRRT) of coelomocyte lysosome was determined in Pheretima aspergilla.Results High levels of cadmium ion were associated with apparent damages to the coelomocyte lysosome in Pheretima asperillum. NRRT decreased along with increasing levels of

  8. Modeling error and stability of endothelial cytoskeletal membrane parameters based on modeling transendothelial impedance as resistor and capacitor in series.

    Science.gov (United States)

    Bodmer, James E; English, Anthony; Brady, Megan; Blackwell, Ken; Haxhinasto, Kari; Fotedar, Sunaina; Borgman, Kurt; Bai, Er-Wei; Moy, Alan B

    2005-09-01

    Transendothelial impedance across an endothelial monolayer grown on a microelectrode has previously been modeled as a repeating pattern of disks in which the electrical circuit consists of a resistor and capacitor in series. Although this numerical model breaks down barrier function into measurements of cell-cell adhesion, cell-matrix adhesion, and membrane capacitance, such solution parameters can be inaccurate without understanding model stability and error. In this study, we have evaluated modeling stability and error by using a chi(2) evaluation and Levenberg-Marquardt nonlinear least-squares (LM-NLS) method of the real and/or imaginary data in which the experimental measurement is compared with the calculated measurement derived by the model. Modeling stability and error were dependent on current frequency and the type of experimental data modeled. Solution parameters of cell-matrix adhesion were most susceptible to modeling instability. Furthermore, the LM-NLS method displayed frequency-dependent instability of the solution parameters, regardless of whether the real or imaginary data were analyzed. However, the LM-NLS method identified stable and reproducible solution parameters between all types of experimental data when a defined frequency spectrum of the entire data set was selected on the basis of a criterion of minimizing error. The frequency bandwidth that produced stable solution parameters varied greatly among different data types. Thus a numerical model based on characterizing transendothelial impedance as a resistor and capacitor in series and as a repeating pattern of disks is not sufficient to characterize the entire frequency spectrum of experimental transendothelial impedance.

  9. Preparation and thermal stability of porous alumina membranes with nano-pore arrays

    Science.gov (United States)

    Wang, Xue Hua; Li, Cheng Yong; Chen, Gui; He, Lei; Cao, Hong

    2010-03-01

    Porous alumina membranes (PAMs) were fabricated using a two-step oxidization method in oxalic acid. Polycrystalline PAMs have been prepared after annealing at different temperatures. The high-temperature properties of the PAMs were investigated using scanning electron microscopy, thermal analysis, X-ray diffraction and infrared spectrometer. At 870°C the amorphous alumina crystallizes to γ-Al2O3. Finally at 1228°C the alumina converts into the thermodynamically preferred phase, α-Al2O3. Differential thermal analysis showed that there was a gradual weight loss at about 900°C due to decomposed of oxalate. A mass of carboxylate ions and carboxylic groups were found in the infrared spectrum of PAMs, and reduced with the increasing of annealing temperature. Characteristic analysis with scanning electron microscopy shows that the pore structure of the PAMs was very stable and there was no detectable structural modification below 900°C, and microstructures of the pores changed slightly during the transformation from α-Al2O3 to γ-Al2O3 while increasing the annealing temperature.

  10. A novel membrane distillation-thermophilic bioreactor system: biological stability and trace organic compound removal.

    Science.gov (United States)

    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. Crown Copyright © 2014. Published by Elsevier Ltd. All rights reserved.

  11. The role of the interosseous membrane and triangular fibrocartilage complex in forearm stability.

    Science.gov (United States)

    Rabinowitz, R S; Light, T R; Havey, R M; Gourineni, P; Patwardhan, A G; Sartori, M J; Vrbos, L

    1994-05-01

    This study investigated the relative roles of the interosseous membrane (IOM) and triangular fibrocartilage complex (TFCC) in the transmission of force from the hand to the humerus. Our findings suggest a spectrum of forearm destabilizing injuries. The intact radius abutting the capitellum provides the primary restraint to proximal migration of the radius. After radial head excision, up to 7 mm of proximal radial migration can occur under axial compression. If the TFCC or the IOM alone is disrupted, little alteration in load or displacement is evident. When both the midportion of the IOM and TFCC are incompetent, however, further proximal radial migration occurs, the radial stump abuts the humerus, and load is shifted back to the radial column. These data suggest that the central portion of the IOM is the crucial structural subdivision within the IOM acting as a restraint to proximal radial migration. The TFCC also resists proximal radial migration and participates in load transfer. We propose that clinical migration of the radius under an axial load greater than 7 mm implies disruption of both the midportion of the IOM and TFCC.

  12. The research about microscopic structure of emulsion membrane in O/W emulsion by NMR and its influence to emulsion stability.

    Science.gov (United States)

    Xie, Yiqiao; Chen, Jisheng; Zhang, Shu; Fan, Kaiyan; Chen, Gang; Zhuang, Zerong; Zeng, Mingying; Chen, De; Lu, Longgui; Yang, Linlin; Yang, Fan

    2016-03-16

    This paper discussed the influence of microstructure of emulsion membrane on O/W emulsion stability. O/W emulsions were emulsified with equal dosage of egg yolk lecithin and increasing dosage of co-emulsifier (oleic acid or HS15). The average particle size and centrifugal stability constant of emulsion, as well as interfacial tension between oil and water phase were determined. The microstructure of emulsion membrane had been studied by (1)H/(13)C NMR, meanwhile the emulsion droplets were visually presented with TEM and IFM. With increasing dosage of co-emulsifier, emulsions showed two stable states, under which the signal intensity of characteristic group (orient to lipophilic core) of egg yolk lecithin disappeared in NMR of emulsions, but that (orient to aqueous phase) of co-emulsifiers only had some reduction at the second stable state. At the two stable states, the emulsion membranes were neater in TEM and emulsion droplets were rounder in IFM. Furthermore, the average particle size of emulsions at the second stable state was bigger than that at the first stable state. Egg yolk lecithin and co-emulsifier respectively arranged into monolayer and bilayer emulsion membrane at the two stable states. The microstructure of emulsion membrane was related to the stability of emulsion. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. Activity of lysosomal exoglycosidases in human gliomas.

    Science.gov (United States)

    Wielgat, P; Walczuk, U; Szajda, S; Bień, M; Zimnoch, L; Mariak, Z; Zwierz, K

    2006-12-01

    There is a lot of data suggesting that modifications of cell glycoconjugates may be important in progression of cancer. In the present work we studied activities of lysosomal exoglycosidases: beta-hexosaminidase and its isoenzymes A and B, beta-galactosidase and alpha-mannosidase, in human gliomas. Enzyme activity was determined spectrophotometrically based on the release of p-nitrophenol from p-nitrophenyl-derivative of appropriate sugars. The activities of the exoglycosidases tested were significantly higher in malignant glial tumors than in control tissue (normal brain tissue) and non-glial tumors. The highest activities of exoglycosidases were observed in high-grade gliomas, and a positive correlation of enzyme activities and degree of malignancy was noted. Our results suggest that lysosomal exoglycosidases may participate in the progression and dynamical development of glial tumors.

  14. Effects of three stabilizing agents--proline, betaine, and trehalose--on membrane phospholipids.

    Science.gov (United States)

    Rudolph, A S; Crowe, J H; Crowe, L M

    1986-02-15

    We have studied the interaction between three compounds which accumulate in organisms under hydration stress--proline, betaine, and trehalose--and the membrane phospholipids dimyristoylphosphatidylcholine (DMPC), palmitoyloleoylphosphatidylcholine (POPC), and dimyristoylphosphatidylethanolamine in bulk solution. Film balance studies reveal that these compounds increase the area/molecule of these lipids. Differential scanning calorimetry has been employed to investigate the effect these agents have on the gel-to-liquid crystalline phase transition of multilamellar and small unilamellar vesicles of DMPC, dipalmitoylphosphatidylcholine, and POPC:phosphatidylserine (90:10 mole ratio) in bulk solution. In the presence of 1 M proline, trehalose, or betaine, the midtransition temperature in small unilamellar vesicles is reduced (up to 7 degrees C in 1 M trehalose), and the transition broadened. In contrast, multilamellar vesicles of similar lipid composition show an increased transition temperature in the presence of the same concentration of these compounds. This result suggests that the inner lamellae in multilamellar vesicles may be dehydrated with only a few outer lamellae exposed to the protective compound. Finally, we have used stereomodels of phosphatidylcholine to investigate the mechanism of action of these agents. Hydrogen bonding of trehalose to the head group region results in an increase in the distance between head groups of 6.9 A. This amount of spreading compares well with data from the monolayer experiments which indicate that maximal spreading of DMPC monolayers by trehalose is 6.5 A. Molecular models of proline and betaine have also been constructed, and these models suggest potential interactions between these compounds and phosphatidylcholines. For the amphipath proline, this interaction may involve intercalation between phospholipid head groups.

  15. Self-Assembling Peptide Surfactants A6K and A6D Adopt a-Helical Structures Useful for Membrane Protein Stabilization

    Directory of Open Access Journals (Sweden)

    Furen Zhuang

    2011-10-01

    Full Text Available Elucidation of membrane protein structures have been greatly hampered by difficulties in producing adequately large quantities of the functional protein and stabilizing them. A6D and A6K are promising solutions to the problem and have recently been used for the rapid production of membrane-bound G protein-coupled receptors (GPCRs. We propose that despite their short lengths, these peptides can adopt α-helical structures through interactions with micelles formed by the peptides themselves. These α-helices are then able to stabilize α-helical motifs which many membrane proteins contain. We also show that A6D and A6K can form β-sheets and appear as weak hydrogels at sufficiently high concentrations. Furthermore, A6D and A6K together in sodium dodecyl sulfate (SDS can form expected β-sheet structures via a surprising α-helical intermediate.

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

    Science.gov (United States)

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

    2016-02-01

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

  17. Modulation of LAT1 (SLC7A5) transporter activity and stability by membrane cholesterol

    Science.gov (United States)

    Dickens, David; Chiduza, George N.; Wright, Gareth S. A.; Pirmohamed, Munir; Antonyuk, Svetlana V.; Hasnain, S. Samar

    2017-01-01

    LAT1 (SLC7A5) is a transporter for both the uptake of large neutral amino acids and a number of pharmaceutical drugs. It is expressed in numerous cell types including T-cells, cancer cells and brain endothelial cells. However, mechanistic knowledge of how it functions and its interactions with lipids are unknown or limited due to inability of obtaining stable purified protein in sufficient quantities. Our data show that depleting cellular cholesterol reduced the Vmax but not the Km of the LAT1 mediated uptake of a model substrate into cells (L-DOPA). A soluble cholesterol analogue was required for the stable purification of the LAT1 with its chaperon CD98 (4F2hc,SLC3A2) and that this stabilised complex retained the ability to interact with a substrate. We propose cholesterol interacts with the conserved regions in the LAT1 transporter that have been shown to bind to cholesterol/CHS in Drosophila melanogaster dopamine transporter. In conclusion, LAT1 is modulated by cholesterol impacting on its stability and transporter activity. This novel finding has implications for other SLC7 family members and additional eukaryotic transporters that contain the LeuT fold. PMID:28272458

  18. Insight into the Mechanism of Human Herpesvirus 7 U21-mediated Diversion of Class I MHC Molecules to Lysosomes*

    Science.gov (United States)

    Glosson, Nicole L.; Gonyo, Patrick; May, Nathan A.; Schneider, Christine L.; Ristow, Laura C.; Wang, Qiuhong; Hudson, Amy W.

    2010-01-01

    The U21 open reading frame from human herpesvirus-7 encodes a membrane protein that associates with and redirects class I MHC molecules to the lysosomal compartment. The mechanism by which U21 accomplishes this trafficking excursion is unknown. Here we have examined the contribution of localization, glycosylation, domain structure, and the absence of substrate class I MHC molecules on the ability of U21 to traffic to lysosomes. Our results suggest the existence of a cellular protein necessary for U21-mediated rerouting of class I MHC molecules. PMID:20833720

  19. 前列腺素E1对移植肝的保护作用%Experiment study on the relationship between the protective effect of prostaglandin E1 and the lysosome in orthotopic liver transplantation in rats

    Institute of Scientific and Technical Information of China (English)

    阚彤; 杨甲梅; 严以群; 陈汉; 吴孟超

    2001-01-01

    Objective To study the protective effect of prostaglandin E1 (PGE1) on the liver in orthotopic liver transplantation rats. Methods PGE1 was infused intravenously during orthotopic liver transplantation in recipient rats. Sham-operated and normal saline groups served as control groups. One-week survival rate and ultrastructure pathological changes in liver were observed. Results The survival rate in the PGE1-treated group was obviously increased as compared with that in the control group. Electron microscopic examination revealed a dramatic elevation of hepatocellular lysosome number in the control groups, and bleb and rupture of lysosome membrane were also observed in the PGE1-trated group. Conclusions PGE1 treatment during recipient operation could increase one-week survival rate, which might be related with its stabilizing effect on lysosome membrane.%目的探讨术中应用前列腺素E1(PGE1)对移植肝脏的保护作用。方法大鼠原位肝移植术中经颈内静脉灌注PGE1,设盐水和空白对照,观察术后1周存活率和肝脏超微结构变化。结果 PGE1治疗组术后1周存活率较对照组明显提高,电镜证实PGE1治疗组肝细胞内溶酶体膜无鼓泡和破溃现象。结论术中应用PGE1能显著提高大鼠肝移植后的1周存活率,其机制可能与PGE1稳定溶酶体膜的作用有关。

  20. Lysosomal exoglycosidases and cathepsin D in colon adenocarcinoma.

    Science.gov (United States)

    Waszkiewicz, Napoleon; Zalewska-Szajda, Beata; Szajda, Sławomir D; Kępka, Alina; Waszkiewicz, Magdalena; Roszkowska-Jakimiec, Wiesława; Wojewódzka-Żeleźniakowicz, Marzena; Milewska, Anna J; Dadan, Jacek; Szulc, Agata; Zwierz, Krzysztof; Ladny, Jerzy R

    2012-01-01

    Changes in the structure of membrane glycoconjugates and activity of glycosidases and proteases are important in tumor formation. The aim of the study was to compare the specific activity of lysosomal exoglycosidases: N-acetyl-β-D-hexosaminidase (HEX), its isoenzymes A (HEX A) and B (HEX B), β-D-galactosidase (GAL), α-fucosidase (FUC), and α-mannosidase (MAN) with the activity of cathepsin D (CD) in serum, urine, and carcinoma tissue of patients with colon adenocarcinoma. The specific activity of HEX, HEX A, HEX B, GAL, FUC, MAN, and CD was assayed in serum, urine, and carcinoma tissue of 12 patients with colon adenocarcinoma. Lysosomal exoglycosidases and CD have similar specific activity in colon adenocarcinoma tissue and urine, which is higher than their activity in serum (with the exception of the highest specific activity of CD in urine). A positive correlation was observed between the specific activity of CD and that of HEX, HEX A, FUC, and MAN in the carcinoma tissue and urine as well as between CD and GAL in the urine of patients with colon adenocarcinoma. Negative correlations were observed between protein levels and the specific activity of HEX, HEX A, FUC, MAN, and CD in the carcinoma tissue and urine, and between protein levels and GAL in urine. Increased degradation and remodeling of glycoconjugates in the colon adenocarcinoma tissue is reflected by increased specific activity of exoglycosidases and CD. The results suggest a strong effect of exoglycosidase action on tissue degradation and a potential role of exoglycosidases in the initiation of proteolysis.

  1. A low pressure gravity-driven membrane filtration (GDM) system for rainwater recycling: Flux stabilization and removal performance.

    Science.gov (United States)

    Ding, An; Wang, Jinlong; Lin, Dachao; Tang, Xiaobin; Cheng, Xiaoxiang; Wang, Hui; Bai, Langming; Li, Guibai; Liang, Heng

    2017-04-01

    Rainwater is a nature resource, which can be widely used for non-potable and potable applications in water scared countries after appropriate treatment. Gravity-driven membrane filtration (GDM) process is a promising technology for decentralized rainwater treatment due to no backwashing, flushing and chemical cleaning. In this study, we established a single lab-scale GDM system for the stored rainwater (simulative cellar rainwater) treatment with two months operation, and a stored tap water was used as a compared system to evaluate the permeability and organics removal performance. Results showed that GDM exhibited a good performance for bacteria and turbidity removals, but the removal performance of DOC was undesirable due to the low rejection of low molecular-weight fulvic. Additionally, the permeate flux reached stable with the value of 6-6.5 L/m(2)h during 60 days operation in the rainwater system, however, the tap water system stabilized only at 4 L/m(2)h. Hydraulically reversible resistance accounted for large proportions (90%) of the total resistance, which indicated that the flux could be recovered by simple physical flushing. The bio-fouling layer adhered on the membrane surface was characterized at the end of the filtration experiment. Higher bio-activity with lower EPS (polysaccharides and proteins) contents of the fouling layer were found in the rainwater system compared with the control system, which was the main reason for the higher flux. These results show that rainwater can be treated in a single GDM process with low maintenance, which makes the process suitable for decentralized water supply.

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

    Science.gov (United States)

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

    2017-01-01

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

  3. Biphasic regulation of lysosomal exocytosis by oxidative stress.

    Science.gov (United States)

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

    2016-11-01

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

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

    Science.gov (United States)

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

    2009-10-01

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

  5. Stabilization

    Directory of Open Access Journals (Sweden)

    Muhammad H. Al-Malack

    2016-07-01

    Full Text Available Fuel oil flyash (FFA produced in power and water desalination plants firing crude oils in the Kingdom of Saudi Arabia is being disposed in landfills, which increases the burden on the environment, therefore, FFA utilization must be encouraged. In the current research, the effect of adding FFA on the engineering properties of two indigenous soils, namely sand and marl, was investigated. FFA was added at concentrations of 5%, 10% and 15% to both soils with and without the addition of Portland cement. Mixtures of the stabilized soils were thoroughly evaluated using compaction, California Bearing Ratio (CBR, unconfined compressive strength (USC and durability tests. Results of these tests indicated that stabilized sand mixtures could not attain the ACI strength requirements. However, marl was found to satisfy the ACI strength requirement when only 5% of FFA was added together with 5% of cement. When the FFA was increased to 10% and 15%, the mixture’s strength was found to decrease to values below the ACI requirements. Results of the Toxicity Characteristics Leaching Procedure (TCLP, which was performed on samples that passed the ACI requirements, indicated that FFA must be cautiously used in soil stabilization.

  6. The Effect of Membrane Environment on Surfactant Protein C Stability Studied by Constant-pH Molecular Dynamics.

    Science.gov (United States)

    Carvalheda, Catarina A; Campos, Sara R R; Baptista, António M

    2015-10-26

    Pulmonary surfactant protein C (SP-C) is a small peptide with two covalently linked fatty acyl chains that plays a crucial role in the formation and stabilization of the pulmonary surfactant reservoirs during the compression and expansion steps of the respiratory cycle. Although its function is known to be tightly related to its highly hydrophobic character and key interactions maintained with specific lipid components, much is left to understand about its molecular mechanism of action. Also, although it adopts a mainly helical structure while associated with the membrane, factors as pH variation and deacylation have been shown to affect its stability and function. In this work, the conformational behavior of both the acylated and deacylated SP-C isoforms was studied in a DPPC bilayer under different pH conditions using constant-pH molecular dynamics simulations. Our findings show that both protein isoforms are remarkably stable over the studied pH range, even though the acylated isoform exhibits a labile helix-turn-helix motif rarely observed in the other isoform. We estimate similar tilt angles for the two isoforms over the studied pH range, with a generally higher degree of internalization of the basic N-terminal residues in the deacylated case, and observe and discuss some protonation-conformation coupling effects. Both isoforms establish contacts with the surrounding lipid molecules (preferentially with the sn-2 ester bonds) and have a local effect on the conformational behavior of the surrounding lipid molecules, the latter being more pronounced for acylated SP-C.

  7. N370S-GBA1 mutation causes lysosomal cholesterol accumulation in Parkinson's disease.

    Science.gov (United States)

    García-Sanz, Patricia; Orgaz, Lorena; Bueno-Gil, Guillermo; Espadas, Isabel; Rodríguez-Traver, Eva; Kulisevsky, Jaime; Gutierrez, Antonia; Dávila, José C; González-Polo, Rosa A; Fuentes, José M; Mir, Pablo; Vicario, Carlos; Moratalla, Rosario

    2017-08-05

    Heterozygous mutations in the GBA1 gene, which encodes the lysosomal enzyme β-glucocerebrosidase-1, increase the risk of developing Parkinson's disease, although the underlying mechanisms remain unclear. The aim of this study was to explore the impact of the N370S-GBA1 mutation on cellular homeostasis and vulnerability in a patient-specific cellular model of PD. We isolated fibroblasts from 4 PD patients carrying the N370S/wild type GBA1 mutation and 6 controls to study the autophagy-lysosome pathway, endoplasmic reticulum stress, and Golgi apparatus structure by Western blot, immunofluorescence, LysoTracker and Filipin stainings, mRNA analysis, and electron microscopy. We evaluated cell vulnerability by apoptosis, reactive oxygen species and mitochondrial membrane potential with flow cytometry. The N370S mutation produced a significant reduction in β-glucocerebrosidase-1 protein and enzyme activity and β-glucocerebrosidase-1 retention within the endoplasmic reticulum, which interrupted its traffic to the lysosome. This led to endoplasmic reticulum stress activation and triggered unfolded protein response and Golgi apparatus fragmentation. Furthermore, these alterations resulted in autophagosome and p62/SQSTM1 accumulation. This impaired autophagy was a result of dysfunctional lysosomes, indicated by multilamellar body accumulation probably caused by increased cholesterol, enlarged lysosomal mass, and reduced enzyme activity. This phenotype impaired the removal of damaged mitochondria and reactive oxygen species production and enhanced cell death. Our results support a connection between the loss of β-glucocerebrosidase-1 function, cholesterol accumulation, and the disruption of cellular homeostasis in GBA1-PD. Our work reveals new insights into the cellular pathways underlying PD pathogenesis, providing evidence that GBA1-PD shares common features with lipid-storage diseases. © 2017 International Parkinson and Movement Disorder Society. © 2017 International

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

    Science.gov (United States)

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

    2015-01-01

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

  9. Involvement of the heterodimeric interface region of the nucleotide binding domain-2 (NBD2) in the CFTR quaternary structure and membrane stability.

    Science.gov (United States)

    Micoud, Julien; Chauvet, Sylvain; Scheckenbach, Klaus Ernst Ludwig; Alfaidy, Nadia; Chanson, Marc; Benharouga, Mohamed

    2015-10-01

    The cystic fibrosis transmembrane conductance regulator (CFTR) is the only member of the ATP-binding cassette (ABC) superfamily that functions as a chloride channel. The predicted structure of CFTR protein contains two membrane-spanning domains (MSDs), each followed by a nucleotide binding domain (NBD1 and NBD2). The opening of the Cl- channel is directly linked to ATP-driven tight dimerization of CFTR's NBD1 and NBD2 domains. The presence of a heterodimeric interfaces (HI) region in NBD1 and NBD2 generated a head to tail orientation necessary for channel activity. This process was also suggested to promote important conformational changes in the associated transmembrane domains of CFTR, which may impact the CFTR plasma membrane stability. To better understand the role of the individual HI region in this process, we generated recombinant CFTR protein with suppressed HI-NBD1 and HI-NBD2. Our results indicate that HI-NBD2 deletion leads to the loss of the dimerization profile of CFTR that affect its plasma membrane stability. We conclude that, in addition to its role in Cl- transport, HI-NBD2 domain confers membrane stability of CFTR by consolidating its quaternary structure through interactions with HI-NBD1 region.

  10. Fast Pyrolysis Oil Stabilization: An Integrated Catalytic and Membrane Approach for Improved Bio-oils. Final Report

    Energy Technology Data Exchange (ETDEWEB)

    Huber, George W.; Upadhye, Aniruddha A.; Ford, David M.; Bhatia, Surita R.; Badger, Phillip C.

    2012-10-19

    This University of Massachusetts, Amherst project, "Fast Pyrolysis Oil Stabilization: An Integrated Catalytic and Membrane Approach for Improved Bio-oils" started on 1st February 2009 and finished on August 31st 2011. The project consisted following tasks: Task 1.0: Char Removal by Membrane Separation Technology The presence of char particles in the bio-oil causes problems in storage and end-use. Currently there is no well-established technology to remove char particles less than 10 micron in size. This study focused on the application of a liquid-phase microfiltration process to remove char particles from bio-oil down to slightly sub-micron levels. Tubular ceramic membranes of nominal pore sizes 0.5 and 0.8m were employed to carry out the microfiltration, which was conducted in the cross-flow mode at temperatures ranging from 38 to 45 C and at three different trans-membrane pressures varying from 1 to 3 bars. The results demonstrated the removal of the major quantity of char particles with a significant reduction in overall ash content of the bio-oil. The results clearly showed that the cake formation mechanism of fouling is predominant in this process. Task 2.0 Acid Removal by Membrane Separation Technology The feasibility of removing small organic acids from the aqueous fraction of fast pyrolysis bio-oils using nanofiltration (NF) and reverse osmosis (RO) membranes was studied. Experiments were carried out with a single solute solutions of acetic acid and glucose, binary solute solutions containing both acetic acid and glucose, and a model aqueous fraction of bio-oil (AFBO). Retention factors above 90% for glucose and below 0% for acetic acid were observed at feed pressures near 40 bar for single and binary solutions, so that their separation in the model AFBO was expected to be feasible. However, all of the membranes were irreversibly damaged when experiments were conducted with the model AFBO due to the presence of guaiacol in the feed solution. Experiments

  11. Fast Pyrolysis Oil Stabilization: An Integrated Catalytic and Membrane Approach for Improved Bio-oils. Final Report

    Energy Technology Data Exchange (ETDEWEB)

    Huber, George W.; Upadhye, Aniruddha A.; Ford, David M.; Bhatia, Surita R.; Badger, Phillip C.

    2012-10-19

    This University of Massachusetts, Amherst project, "Fast Pyrolysis Oil Stabilization: An Integrated Catalytic and Membrane Approach for Improved Bio-oils" started on 1st February 2009 and finished on August 31st 2011. The project consisted following tasks: Task 1.0: Char Removal by Membrane Separation Technology The presence of char particles in the bio-oil causes problems in storage and end-use. Currently there is no well-established technology to remove char particles less than 10 micron in size. This study focused on the application of a liquid-phase microfiltration process to remove char particles from bio-oil down to slightly sub-micron levels. Tubular ceramic membranes of nominal pore sizes 0.5 and 0.8m were employed to carry out the microfiltration, which was conducted in the cross-flow mode at temperatures ranging from 38 to 45 C and at three different trans-membrane pressures varying from 1 to 3 bars. The results demonstrated the removal of the major quantity of char particles with a significant reduction in overall ash content of the bio-oil. The results clearly showed that the cake formation mechanism of fouling is predominant in this process. Task 2.0 Acid Removal by Membrane Separation Technology The feasibility of removing small organic acids from the aqueous fraction of fast pyrolysis bio-oils using nanofiltration (NF) and reverse osmosis (RO) membranes was studied. Experiments were carried out with a single solute solutions of acetic acid and glucose, binary solute solutions containing both acetic acid and glucose, and a model aqueous fraction of bio-oil (AFBO). Retention factors above 90% for glucose and below 0% for acetic acid were observed at feed pressures near 40 bar for single and binary solutions, so that their separation in the model AFBO was expected to be feasible. However, all of the membranes were irreversibly damaged when experiments were conducted with the model AFBO due to the presence of guaiacol in the feed solution. Experiments

  12. MitoNEET Is a Uniquely Folded 2Fe-2S Outer Mitochondrial Membrane Protein Stabilized By Pioglitazone

    Energy Technology Data Exchange (ETDEWEB)

    Paddock, M.L.; Wiley, S.E.; Axelrod, H.L.; Cohen, A.E.; Roy, M.; Abresch, E.C.; Capraro, D.; Murphy, A.N.; Nechushtai, R.; Dixon, J.E.; Jennings, P.A.; /UC, San Diego /SLAC, SSRL /Hebrew U.

    2007-10-19

    Iron-sulfur (Fe-S) proteins are key players in vital processes involving energy homeostasis and metabolism from the simplest to most complex organisms. We report a 1.5 Angstrom x-ray crystal structure of the first identified outer mitochondrial membrane Fe-S protein, mitoNEET. Two protomers intertwine to form a unique dimeric structure that constitutes a new fold to not only the {approx}650 reported Fe-S protein structures but also to all known proteins. We name this motif the NEET fold. The protomers form a two-domain structure: a {beta}-cap domain and a cluster-binding domain that coordinates two acid-labile 2Fe-2S clusters. Binding of pioglitazone, an insulin-sensitizing thiazolidinedione used in the treatment of type 2 diabetes, stabilizes the protein against 2Fe-2S cluster release. The biophysical properties of mitoNEET suggest that it may participate in a redox-sensitive signaling and/or in Fe-S cluster transfer.

  13. Key Role for Intracellular K+ and Protein Kinases Sat4/Hal4 and Hal5 in the Plasma Membrane Stabilization of Yeast Nutrient Transporters▿

    Science.gov (United States)

    Pérez-Valle, Jorge; Jenkins, Huw; Merchan, Stephanie; Montiel, Vera; Ramos, José; Sharma, Sukesh; Serrano, Ramón; Yenush, Lynne

    2007-01-01

    K+ transport in living cells must be tightly controlled because it affects basic physiological parameters such as turgor, membrane potential, ionic strength, and pH. In yeast, the major high-affinity K+ transporter, Trk1, is inhibited by high intracellular K+ levels and positively regulated by two redundant “halotolerance” protein kinases, Sat4/Hal4 and Hal5. Here we show that these kinases are not required for Trk1 activity; rather, they stabilize the transporter at the plasma membrane under low K+ conditions, preventing its endocytosis and vacuolar degradation. High concentrations (0.2 M) of K+, but not Na+ or sorbitol, transported by undefined low-affinity systems, maintain Trk1 at the plasma membrane in the hal4 hal5 mutant. Other nutrient transporters, such as Can1 (arginine permease), Fur4 (uracil permease), and Hxt1 (low-affinity glucose permease), are also destabilized in the hal4 hal5 mutant under low K+ conditions and, in the case of Can1, are stabilized by high K+ concentrations. Other plasma membrane proteins such as Pma1 (H+-pumping ATPase) and Sur7 (an eisosomal protein) are not regulated by halotolerance kinases or by high K+ levels. This novel regulatory mechanism of nutrient transporters may participate in the quiescence/growth transition and could result from effects of intracellular K+ and halotolerance kinases on membrane trafficking and/or on the transporters themselves. PMID:17548466

  14. Quantitative modeling of selective lysosomal targeting for drug design

    DEFF Research Database (Denmark)

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

    2008-01-01

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

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

    OpenAIRE

    1980-01-01

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

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

    OpenAIRE

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

    2013-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Magdalena L Circu

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

  18. Lysosomal degradation of receptor-bound urokinase-type plasminogen activator is enhanced by its inhibitors in human trophoblastic choriocarcinoma cells

    DEFF Research Database (Denmark)

    Jensen, Poul Henning; Christensen, Erik Ilsø; Ebbesen, P.

    1990-01-01

    in an apparently intact form in the medium or was still cell associated. The degradation could be inhibited by inhibitors of vesicle transport and lysosomal hydrolases. By electron microscopic autoradiography, both 125I-u-PA and 125I-u-PA-inhibitor complexes were located over the cell membrane at 4 degrees C......, with the highest density of grains over the membrane at cell-cell interphases, but, after incubation at 37 degrees C, 17 and 27% of the grains for u-PA and u-PA-PAI-1 complexes, respectively, appeared over lysosomal-like bodies. These findings suggest that the u-PA receptor possesses a clearance function...

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

    Directory of Open Access Journals (Sweden)

    Ju-Hyun Lee

    2015-09-01

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

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

    Science.gov (United States)

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

    2015-09-01

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

  1. Gaucher disease: a lysosomal neurodegenerative disorder.

    Science.gov (United States)

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

    2015-04-01

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

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

    Science.gov (United States)

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

    2016-06-01

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

  3. Stability

    Directory of Open Access Journals (Sweden)

    Nada S. Abdelwahab

    2017-05-01

    Full Text Available The present work concerns with the development of stability indicating the RP-HPLC method for simultaneous determination of guaifenesin (GUF and pseudoephedrine hydrochloride (PSH in the presence of guaifenesin related substance (Guaiacol. GUC, and in the presence of syrup excepients with minimum sample pre-treatment. In the developed RP-HPLC method efficient chromatographic separation was achieved for GUF, PSH, GUC and syrup excepients using ODS column as a stationary phase and methanol: water (50:50, v/v, pH = 4 with orthophosphoric acid as a mobile phase with a flow rate of 1 mL min−1 and UV detection at 210 nm. The chromatographic run time was approximately 10 min. Calibration curves were drawn relating the integrated area under peak to the corresponding concentrations of PSH, GUF and GUC in the range of 1–8, 1–20, 0.4–8 μg mL−1, respectively. The developed method has been validated and met the requirements delineated by ICH guidelines with respect to linearity, accuracy, precision, specificity and robustness. The validated method was successfully applied for determination of the studied drugs in triaminic chest congestion® syrup; moreover its results were statistically compared with those obtained by the official method and no significant difference was found between them.

  4. A Polybasic Plasma Membrane Binding Motif in the I-II Linker Stabilizes Voltage-gated CaV1.2 Calcium Channel Function.

    Science.gov (United States)

    Kaur, Gurjot; Pinggera, Alexandra; Ortner, Nadine J; Lieb, Andreas; Sinnegger-Brauns, Martina J; Yarov-Yarovoy, Vladimir; Obermair, Gerald J; Flucher, Bernhard E; Striessnig, Jörg

    2015-08-21

    L-type voltage-gated Ca(2+) channels (LTCCs) regulate many physiological functions like muscle contraction, hormone secretion, gene expression, and neuronal excitability. Their activity is strictly controlled by various molecular mechanisms. The pore-forming α1-subunit comprises four repeated domains (I-IV), each connected via an intracellular linker. Here we identified a polybasic plasma membrane binding motif, consisting of four arginines, within the I-II linker of all LTCCs. The primary structure of this motif is similar to polybasic clusters known to interact with polyphosphoinositides identified in other ion channels. We used de novo molecular modeling to predict the conformation of this polybasic motif, immunofluorescence microscopy and live cell imaging to investigate the interaction with the plasma membrane, and electrophysiology to study its role for Cav1.2 channel function. According to our models, this polybasic motif of the I-II linker forms a straight α-helix, with the positive charges facing the lipid phosphates of the inner leaflet of the plasma membrane. Membrane binding of the I-II linker could be reversed after phospholipase C activation, causing polyphosphoinositide breakdown, and was accelerated by elevated intracellular Ca(2+) levels. This indicates the involvement of negatively charged phospholipids in the plasma membrane targeting of the linker. Neutralization of four arginine residues eliminated plasma membrane binding. Patch clamp recordings revealed facilitated opening of Cav1.2 channels containing these mutations, weaker inhibition by phospholipase C activation, and reduced expression of channels (as quantified by ON-gating charge) at the plasma membrane. Our data provide new evidence for a membrane binding motif within the I-II linker of LTCC α1-subunits essential for stabilizing normal Ca(2+) channel function. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  5. Stability of polydopamine and poly(DOPA) melanin-like films on the surface of polymer membranes under strongly acidic and alkaline conditions.

    Science.gov (United States)

    Wei, Houliang; Ren, Jun; Han, Bo; Xu, Li; Han, Lulu; Jia, Lingyun

    2013-10-01

    This study investigated the stability of polydopamine and poly(3,4-dihydroxyphenylalanine) (poly(DOPA)) melanin-like films on the surface of polymer substrates. Three polymer membranes, polypropylene (PP), poly(vinylidenefluoride) (PVDF) and nylon, were modified with polydopamine or poly(DOPA), and then immersed in 0.1M HCl or NaOH, followed by UV-vis spectrometry analysis to detect the presence of film detachment. The results showed that the outer parts of both polydopamine and poly(DOPA) films were detached, probably due to electrostatic repulsion between the polymers within the film, when the modified membranes were washed in HCl or NaOH solution. These two films were more stable in strongly acidic solution, but the stability of poly(DOPA) film was better than that of polydopamine film. Compared to the films on the surface of PVDF or nylon membrane, films on PP surface showed the lowest stability, possibly because of the hydrophobic property of PP. The process of film detachment was analyzed by GPC, which showed that unreacted dopamine or DOPA monomers were still present in the freshly formed films. The unreacted monomers, as well as polydopamine or poly(DOPA) that were incorporated in the film via noncovalent interactions, became detached when the film was exposed to strongly acidic or alkaline solution. Oxidation of freshly formed films could significantly enhance their stability. The results therefore provide us with a better understanding of the stability of melanin-like films, and allow us to develop an effective strategy for constructing stable films.

  6. Lysosome/lipid droplet interplay in metabolic diseases.

    Science.gov (United States)

    Dugail, Isabelle

    2014-01-01

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

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

    Science.gov (United States)

    Urayama, Akihiko

    2013-02-01

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

  8. Photoaffinity labeling of the lysosomal neuraminidase from bovine testis

    NARCIS (Netherlands)

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

    1990-01-01

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

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

    Science.gov (United States)

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

    2014-11-28

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

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

    Science.gov (United States)

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

    2016-01-01

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

  11. Thermal stability, complexing behavior, and ionic transport of polymeric gel membranes based on polymer PVdF-HFP and ionic liquid, [BMIM][BF4].

    Science.gov (United States)

    Shalu; Chaurasia, S K; Singh, R K; Chandra, S

    2013-01-24

    PVdF-HFP + IL(1-butyl-3-methylimidazolium tetrafluoroborate; [BMIM][BF(4)]) polymeric gel membranes containing different amounts of ionic liquid have been synthesized and characterized by X-ray diffraction, scanning electron microscopy, Fourier transform infrared (FTIR), differential scanning calorimetry, thermogravimetric analysis (TGA), and complex impedance spectroscopic techniques. Incorporation of IL in PVdF-HFP polymer changes different physicochemical properties such as melting temperature (T(m)), thermal stability, structural morphology, amorphicity, and ionic transport. It is shown by FTIR, TGA (also first derivative of TGA, "DTGA") that IL partly complexes with the polymer PVdF-HFP and partly remains dispersed in the matrix. The ionic conductivity of polymeric gel membranes has been found to increase with increasing concentration of IL and attains a maximum value of 1.6 × 10(-2) S·cm(-1) for polymer gel membrane containing 90 wt % IL at room temperature. Interestingly, the values of conductivity of membranes with 80 and 90 wt % of IL were higher than that of pure IL (100 wt %). The polymer chain breathing model has been suggested to explain it. The variation of ionic conductivity with temperature of these gel polymeric membranes follows Arrhenius type thermally activated behavior.

  12. Lysosomal storage disease upon disruption of the neuronal chloride transport protein ClC-6.

    Science.gov (United States)

    Poët, Mallorie; Kornak, Uwe; Schweizer, Michaela; Zdebik, Anselm A; Scheel, Olaf; Hoelter, Sabine; Wurst, Wolfgang; Schmitt, Anja; Fuhrmann, Jens C; Planells-Cases, Rosa; Mole, Sara E; Hübner, Christian A; Jentsch, Thomas J

    2006-09-12

    Mammalian CLC proteins function as Cl(-) channels or as electrogenic Cl(-)/H(+) exchangers and are present in the plasma membrane and intracellular vesicles. We now show that the ClC-6 protein is almost exclusively expressed in neurons of the central and peripheral nervous systems, with a particularly high expression in dorsal root ganglia. ClC-6 colocalized with markers for late endosomes in neuronal cell bodies. The disruption of ClC-6 in mice reduced their pain sensitivity and caused moderate behavioral abnormalities. Neuronal tissues showed autofluorescence at initial axon segments. At these sites, electron microscopy revealed electron-dense storage material that caused a pathological enlargement of proximal axons. These deposits were positive for several lysosomal proteins and other marker proteins typical for neuronal ceroid lipofuscinosis (NCL), a lysosomal storage disease. However, the lysosomal pH of Clcn6(-/-) neurons appeared normal. CLCN6 is a candidate gene for mild forms of human NCL. Analysis of 75 NCL patients identified ClC-6 amino acid exchanges in two patients but failed to prove a causative role of CLCN6 in that disease.

  13. Abeta42-induced neurodegeneration via an age-dependent autophagic-lysosomal injury in Drosophila.

    Directory of Open Access Journals (Sweden)

    Daijun Ling

    Full Text Available The mechanism of widespread neuronal death occurring in Alzheimer's disease (AD remains enigmatic even after extensive investigation during the last two decades. Amyloid beta 42 peptide (Abeta(1-42 is believed to play a causative role in the development of AD. Here we expressed human Abeta(1-42 and amyloid beta 40 (Abeta(1-40 in Drosophila neurons. Abeta(1-42 but not Abeta(1-40 causes an extensive accumulation of autophagic vesicles that become increasingly dysfunctional with age. Abeta(1-42-induced impairment of the degradative function, as well as the structural integrity, of post-lysosomal autophagic vesicles triggers a neurodegenerative cascade that can be enhanced by autophagy activation or partially rescued by autophagy inhibition. Compromise and leakage from post-lysosomal vesicles result in cytosolic acidification, additional damage to membranes and organelles, and erosive destruction of cytoplasm leading to eventual neuron death. Neuronal autophagy initially appears to play a pro-survival role that changes in an age-dependent way to a pro-death role in the context of Abeta(1-42 expression. Our in vivo observations provide a mechanistic understanding for the differential neurotoxicity of Abeta(1-42 and Abeta(1-40, and reveal an Abeta(1-42-induced death execution pathway mediated by an age-dependent autophagic-lysosomal injury.

  14. Towards stabilization of supported liquid membranes: preparation and characterization of polysulfone support and sulfonated poly (ether ether ketone) coated composite hollow fiber membranes

    NARCIS (Netherlands)

    He, T.

    2008-01-01

    A supported liquid membrane (SLM) contains organic solvent and organic extractant as one organic phase, and a porous support structure. It has been widely investigated for separation and purification of various chemical compounds. SLM has high permeability and selectivity, being regarded as one of t

  15. Dynein Clusters into Lipid Microdomains on Phagosomes to Drive Rapid Transport toward Lysosomes

    Science.gov (United States)

    Rai, Ashim; Pathak, Divya; Thakur, Shreyasi; Singh, Shampa; Dubey, Alok Kumar; Mallik, Roop

    2016-01-01

    Summary Diverse cellular processes are driven by motor proteins that are recruited to and generate force on lipid membranes. Surprisingly little is known about how membranes control the force from motors and how this may impact specific cellular functions. Here, we show that dynein motors physically cluster into microdomains on the membrane of a phagosome as it matures inside cells. Such geometrical reorganization allows many dyneins within a cluster to generate cooperative force on a single microtubule. This results in rapid directed transport of the phagosome toward microtubule minus ends, likely promoting phagolysosome fusion and pathogen degradation. We show that lipophosphoglycan, the major molecule implicated in immune evasion of Leishmania donovani, inhibits phagosome motion by disrupting the clustering and therefore the cooperative force generation of dynein. These findings appear relevant to several pathogens that prevent phagosome-lysosome fusion by targeting lipid microdomains on phagosomes. PMID:26853472

  16. Fluence- and time-dependant lysosomal and mitochondrial damage induced by LS11 PDT characterized with light scattering

    Science.gov (United States)

    Wilson, Jeremy D.; Foster, Thomas H.

    2007-02-01

    Light scattering from cells originates from sub-cellular organelles. Our measurements of angularly resolved light scattering have demonstrated that at 633 nm, the dominant scattering centers within EMT6 cells are mitochondria and lysosomes. To assess their specific contributions, we have used photodynamic therapy (PDT) to induce organelle-specific perturbations within intact cells. We have developed a coated sphere scattering model for mitochondrial swelling in response to ALA- and Pc 4-PDT, and in the case of Pc 4-PDT we have used this model to map the scattering responses into clonogenic cell survival. More recently, we demonstrated the ability to measure the size, scattering contribution, and refractive index of lysosomes within cells by exploiting the localization and high extinction of the photosensitizer LS11 and an absorbing sphere scattering model. Here we report on time- and fluence-dependant scattering measurements from cells treated with LS11-PDT. LS11-PDT causes rapid lysosomal disruption, as quantified by uptake of acridine orange, and can induce downstream effects including release of mitochondrial cytochrome c preceding the loss of mitochondrial membrane potential (Reiners et al., Cell Death Differ. 9:934, 2002). Using scattering and these various methods of analysis, we observed that the induction of lysosomal morphology changes requires a fluence significantly higher than that reported for cell killing. At lower fluences, we observe that at 1 h after irradiation there is significant mitochondrial swelling, consistent with the onset of cytochrome c-induced cell death, while the morphology of lysosomes remains unchanged. We also expand on the ideas of lysosomal staining to demonstrate the sensitivity of scattering measurements at different wavelengths to different organelle populations.

  17. Abnormal lysosomal trafficking and enhanced exosomal export of cisplatin in drug-resistant human ovarian carcinoma cells.

    Science.gov (United States)

    Safaei, Roohangiz; Larson, Barrett J; Cheng, Timothy C; Gibson, Michael A; Otani, Shinji; Naerdemann, Wiltrud; Howell, Stephen B

    2005-10-01

    Previous work has shown that cisplatin (CDDP) becomes concentrated in lysosomes, and that acquired resistance to CDDP is associated with abnormalities of protein trafficking and secretion. The lysosomal compartment in CDDP-sensitive 2008 human ovarian carcinoma cells was compared with that in CDDP-resistant 2008/C13*5.25 subline using deconvoluting imaging and specific dyes and antibodies. The lysosomal compartment in CDDP-resistant cells was reduced to just 40% of that in the parental CDDP-sensitive cells (P<0.002). This was accompanied by a reduced expression of the lysosome-associated proteins 1 and 2 (LAMP1 and LAMP2) as determined by both microscopy and Western blot analysis. The CDDP-resistant cells released more protein as exosomes and Western blot analysis revealed that these exosomes contained substantially more LAMP1 than those released by the CDDP-sensitive cells. Following loading of the whole cell with CDDP, the exosomes released from 2008/C13*5.25 cells contained 2.6-fold more platinum than those released from sensitive cells. Enhanced exosomal export was accompanied by higher exosomal levels of the putative CDDP export transporters MRP2, ATP7A, and ATP7B. Expression profiling identified significant increases in the expression of several genes whose products function in membrane fusion and vesicle trafficking. This study shows that the lysosomal compartment of human ovarian carcinoma cells selected for stable resistance to CDDP is markedly reduced in size, and that these cells abnormally sort some lysosomal proteins and the putative CDDP transporters into an exosomal pathway that also exports CDDP.

  18. Bacterial reaction centers purified with styrene maleic acid copolymer retain native membrane functional properties and display enhanced stability

    NARCIS (Netherlands)

    Swainsbury, David J K; Scheidelaar, Stefan; Van Grondelle, Rienk; Killian, J. Antoinette; Jones, Michael R.

    2014-01-01

    Integral membrane proteins often present daunting challenges for biophysical characterization, a fundamental issue being how to select a surfactant that will optimally preserve the individual structure and functional properties of a given membrane protein. Bacterial reaction centers offer a rare opp

  19. Hydration, Ionic Valence and Cross-Linking Propensities of Cations Determine the Stability of Lipopolysaccharide (LPS) Membranes

    Energy Technology Data Exchange (ETDEWEB)

    Nascimento, Agrinaldo; Pontes, Frederico J.; Lins, Roberto D.; Soares, Thereza A.

    2013-10-29

    The supra-molecular structure of LPS aggregates governs outer membrane permeability and activation of the host immune response during Gram-negative bacterial infections. Molecular dynamics simulations unveil at atomic resolution 10 the subtle balance between cation hydration and cross-link ability in modulating phase transitions of LPS membranes.

  20. Stability of radicals in electron-irradiated fluoropolymer film for the preparation ofgraft copolymer fuel cell electrolyte membranes

    DEFF Research Database (Denmark)

    Larsen, Mikkel Juul; Ma, Yue; Qian, Huan;

    2010-01-01

    The content of radicals in the base polymer film before and after grafting is an important issue in theproduction and use of membranes for electrochemical devices by radiation grafting. In this work a methodhas been developed for determination of relative radical content in fluoropolymer films...... in the grafting process, and no significant amount of potentiallyharmful radicals is thus left in the final membrane....

  1. The serotonin transporter undergoes constitutive internalization and is primarily sorted to late endosomes and lysosomal degradation

    DEFF Research Database (Denmark)

    Rahbek-Clemmensen, Troels; Bay, Tina; Eriksen, Jacob

    2014-01-01

    The serotonin transporter (SERT) plays a critical role in regulating serotonin signaling by mediating reuptake of serotonin from the extracellular space. The molecular and cellular mechanisms controlling SERT levels in the membrane remain poorly understood. To study trafficking of surface resident....... Furthermore, internalized SERT co-localized with the lysosomal marker LysoTracker and not with transferrin. The sorting pattern was further confirmed by visualizing internalization of SERT using the fluorescent cocaine analogue JHC1-64, and by reversible and pulse chase biotinylation assays showing evidence...

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

    Science.gov (United States)

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

    2008-02-01

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

  3. Syntaxin 7 is localized to late endosome compartments, associates with Vamp 8, and Is required for late endosome-lysosome fusion.

    Science.gov (United States)

    Mullock, B M; Smith, C W; Ihrke, G; Bright, N A; Lindsay, M; Parkinson, E J; Brooks, D A; Parton, R G; James, D E; Luzio, J P; Piper, R C

    2000-09-01

    Protein traffic from the cell surface or the trans-Golgi network reaches the lysosome via a series of endosomal compartments. One of the last steps in the endocytic pathway is the fusion of late endosomes with lysosomes. This process has been reconstituted in vitro and has been shown to require NSF, alpha and gamma SNAP, and a Rab GTPase based on inhibition by Rab GDI. In Saccharomyces cerevisiae, fusion events to the lysosome-like vacuole are mediated by the syntaxin protein Vam3p, which is localized to the vacuolar membrane. In an effort to identify the molecular machinery that controls fusion events to the lysosome, we searched for mammalian homologues of Vam3p. One such candidate is syntaxin 7. Here we show that syntaxin 7 is concentrated in late endosomes and lysosomes. Coimmunoprecipitation experiments show that syntaxin 7 is associated with the endosomal v-SNARE Vamp 8, which partially colocalizes with syntaxin 7. Importantly, we show that syntaxin 7 is specifically required for the fusion of late endosomes with lysosomes in vitro, resulting in a hybrid organelle. Together, these data identify a SNARE complex that functions in the late endocytic system of animal cells.

  4. Syntaxin 7 Is Localized to Late Endosome Compartments, Associates with Vamp 8, and Is Required for Late Endosome–Lysosome Fusion

    Science.gov (United States)

    Mullock, Barbara M.; Smith, Chez W.; Ihrke, Gudrun; Bright, Nicholas A.; Lindsay, Margaret; Parkinson, Emma J.; Brooks, Doug A.; Parton, Robert G.; James, David E.; Luzio, J. Paul; Piper, Robert C.

    2000-01-01

    Protein traffic from the cell surface or the trans-Golgi network reaches the lysosome via a series of endosomal compartments. One of the last steps in the endocytic pathway is the fusion of late endosomes with lysosomes. This process has been reconstituted in vitro and has been shown to require NSF, α and γ SNAP, and a Rab GTPase based on inhibition by Rab GDI. In Saccharomyces cerevisiae, fusion events to the lysosome-like vacuole are mediated by the syntaxin protein Vam3p, which is localized to the vacuolar membrane. In an effort to identify the molecular machinery that controls fusion events to the lysosome, we searched for mammalian homologues of Vam3p. One such candidate is syntaxin 7. Here we show that syntaxin 7 is concentrated in late endosomes and lysosomes. Coimmunoprecipitation experiments show that syntaxin 7 is associated with the endosomal v-SNARE Vamp 8, which partially colocalizes with syntaxin 7. Importantly, we show that syntaxin 7 is specifically required for the fusion of late endosomes with lysosomes in vitro, resulting in a hybrid organelle. Together, these data identify a SNARE complex that functions in the late endocytic system of animal cells. PMID:10982406

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2005-12-11

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

  6. Gravity-driven membrane filtration as pretreatment for seawater reverse osmosis: linking biofouling layer morphology with flux stabilization.

    Science.gov (United States)

    Akhondi, Ebrahim; Wu, Bing; Sun, Shuyang; Marxer, Brigit; Lim, Weikang; Gu, Jun; Liu, Linbo; Burkhardt, Michael; McDougald, Diane; Pronk, Wouter; Fane, Anthony G

    2015-03-01

    In this study gravity-driven membrane (GDM) ultrafiltration is investigated for the pretreatment of seawater before reverse osmosis (RO). The impacts of temperature (21 ± 1 and 29 ± 1 °C) and hydrostatic pressure (40 and 100 mbar) on dynamic flux development and biofouling layer structure were studied. The data suggested pore constriction fouling was predominant at the early stage of filtration, during which the hydrostatic pressure and temperature had negligible effects on permeate flux. With extended filtration time, cake layer fouling played a major role, during which higher hydrostatic pressure and temperature improved permeate flux. The permeate flux stabilized in a range of 3.6 L/m(2) h (21 ± 1 °C, 40 mbar) to 7.3 L/m(2) h (29 ± 1 °C, 100 mbar) after slight fluctuations and remained constant for the duration of the experiments (almost 3 months). An increase in biofouling layer thickness and a variable biofouling layer structure were observed over time by optical coherence tomography and confocal laser scanning microscopy. The presence of eukaryotic organisms in the biofouling layer was observed by light microscopy and the microbial community structure of the biofouling layer was analyzed by sequences of 16S rRNA genes. The magnitude of permeate flux was associated with the combined effect of the biofouling layer thickness and structure. Changes in the biofouling layer structure were attributed to (1) the movement and predation behaviour of the eukaryotic organisms which increased the heterogeneous nature of the biofouling layer; (2) the bacterial debris generated by eukaryotic predation activity which reduced porosity; (3) significant shifts of the dominant bacterial species over time that may have influenced the biofouling layer structure. As expected, most of the particles and colloids in the feed seawater were removed by the GDM process, which led to a lower RO fouling potential. However, the dissolved organic carbon in the

  7. Stabilization of membrane bound ATPases and lipid peroxidation by carotenoids from Chlorococcum humicola in Benzo(a)pyrene induced toxicity

    Institute of Scientific and Technical Information of China (English)

    Bhagavathy S; Sumathi P

    2012-01-01

    Objective: To identify the alteration of the membrane potential and the effect of carotenoid extracts from Chlorococcum humicola (C. humicola) on membrane bound ATPases and lipid peroxidation. Methods: The total carotenoids were extracted from C. humicola. Four groups of Swiss albino mice were treated as control, Benzo(a)pyrene [B(a)P], total carotenoids, B(a)P +total carotenoids respectively for a period of 60 days. Membrane lipid peroxidation and ATPases (Total ATPases, Ca2+- ATPases, Mg2+ - ATPases, Na+K+ - ATPase) were determined in lung, liver and erythrocyte samples. Results: The activity of total ATPase was found to be significantly increased in the B(a)P treated liver and lung tissue. Erythrocyte membrane also showed higher ATPase activity which was significantly reverted on total carotenoid treatment. Conclusions:It can be concluded that the changes in membrane potential favour the functional deterioration of physiological system. The overall findings demonstrates that the animals post treated with carotenoid extract from C. humicola may maintains the alterations in membrane bound ATPase and lipid peroxidation in tissues against the carcinogenic chemical and hence aid in establishing the membrane potential action. Therefore C. humicola can be further extended to exploits its possible application for various health benefits as neutraceuticals and food additives.

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

    Directory of Open Access Journals (Sweden)

    Stern Stephan T

    2012-06-01

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

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

    Science.gov (United States)

    Stern, Stephan T; Adiseshaiah, Pavan P; Crist, Rachael M

    2012-06-14

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

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

    Science.gov (United States)

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

    2014-08-25

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

  11. Cyclosporin a, but not FK506, induces osmotic lysis of pancreas zymogen granules, intra-acinar enzyme release, and lysosome instability by activating K+ channel.

    Science.gov (United States)

    Lee, Wing-Kee; Braun, Matthias; Langelüddecke, Christian; Thévenod, Frank

    2012-05-01

    The immunosuppressant tacrolimus (FK506) has improved pancreas allograft survival compared with cyclosporin A (CsA), possibly because of reduced acute pancreatitis following ischemia-reperfusion injury. Ion permeabilities in zymogen granule (ZG) membranes, including a KCNQ1 K channel, promote hormone-stimulated enzyme secretion. We investigated whether a differential modulation of ZG and lysosomal ion permeabilities and enzyme secretion by CsA/FK506 contributes to pancreatitis. Rat ZGs and lysosomes were isolated by gradient centrifugation, ion permeabilities assayed by osmotic lysis, and single-channel currents recorded in a planar lipid bilayer. Amylase release was measured in permeabilized acini and lysosomal cathepsin B release detected by immunoblotting. CsA (1-10 μM), but not FK506, enhanced ZGs osmotic lysis by selectively increasing K permeability up to 5-fold. Zymogen granule membrane K channels showed ∼2-fold increased single-channel open probability with CsA only. Cyclosporin A selectively increased basal (∼2-fold), but not cholecystokinin-octapeptide (1 nM)-induced amylase secretion in K medium only. Cyclosporin A (5 μM), but not FK506, increased cathepsin B release from lysosomes. Cyclosporin A selectively opens the ZG K channel and induces cathepsin B release from lysosomes, which cause increased in situ lysis of ZGs and may aggravate or fuel acute allograft pancreatitis following hypoxia-reperfusion injury.

  12. Critical role of CAV1/caveolin-1 in cell stress responses in human breast cancer cells via modulation of lysosomal function and autophagy.

    Science.gov (United States)

    Shi, Yin; Tan, Shi-Hao; Ng, Shukie; Zhou, Jing; Yang, Na-Di; Koo, Gi-Bang; McMahon, Kerrie-Ann; Parton, Robert G; Hill, Michelle M; Del Pozo, M