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Sample records for endosomal cholesterol removal

  1. Late endosomal cholesterol accumulation leads to impaired intra-endosomal trafficking.

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    Komla Sobo

    Full Text Available BACKGROUND: Pathological accumulation of cholesterol in late endosomes is observed in lysosomal storage diseases such as Niemann-Pick type C. We here analyzed the effects of cholesterol accumulation in NPC cells, or as phenocopied by the drug U18666A, on late endosomes membrane organization and dynamics. METHODOLOGY/PRINCIPAL FINDINGS: Cholesterol accumulation did not lead to an increase in the raft to non-raft membrane ratio as anticipated. Strikingly, we observed a 2-3 fold increase in the size of the compartment. Most importantly, properties and dynamics of late endosomal intralumenal vesicles were altered as revealed by reduced late endosomal vacuolation induced by the mutant pore-forming toxin ASSP, reduced intoxication by the anthrax lethal toxin and inhibition of infection by the Vesicular Stomatitis Virus. CONCLUSIONS/SIGNIFICANCE: These results suggest that back fusion of intralumenal vesicles with the limiting membrane of late endosomes is dramatically perturbed upon cholesterol accumulation.

  2. Hepatitis C Virus Replication Depends on Endosomal Cholesterol Homeostasis.

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    Stoeck, Ina Karen; Lee, Ji-Young; Tabata, Keisuke; Romero-Brey, Inés; Paul, David; Schult, Philipp; Lohmann, Volker; Kaderali, Lars; Bartenschlager, Ralf

    2018-01-01

    Similar to other positive-strand RNA viruses, hepatitis C virus (HCV) causes massive rearrangements of intracellular membranes, resulting in a membranous web (MW) composed of predominantly double-membrane vesicles (DMVs), the presumed sites of RNA replication. DMVs are enriched for cholesterol, but mechanistic details on the source and recruitment of cholesterol to the viral replication organelle are only partially known. Here we focused on selected lipid transfer proteins implicated in direct lipid transfer at various endoplasmic reticulum (ER)-membrane contact sites. RNA interference (RNAi)-mediated knockdown identified several hitherto unknown HCV dependency factors, such as steroidogenic acute regulatory protein-related lipid transfer domain protein 3 (STARD3), oxysterol-binding protein-related protein 1A and -B (OSBPL1A and -B), and Niemann-Pick-type C1 (NPC1), all residing at late endosome and lysosome membranes and required for efficient HCV RNA replication but not for replication of the closely related dengue virus. Focusing on NPC1, we found that knockdown or pharmacological inhibition caused cholesterol entrapment in lysosomal vesicles concomitant with decreased cholesterol abundance at sites containing the viral replicase factor NS5A. In untreated HCV-infected cells, unesterified cholesterol accumulated at the perinuclear region, partially colocalizing with NS5A at DMVs, arguing for NPC1-mediated endosomal cholesterol transport to the viral replication organelle. Consistent with cholesterol being an important structural component of DMVs, reducing NPC1-dependent endosomal cholesterol transport impaired MW integrity. This suggests that HCV usurps lipid transfer proteins, such as NPC1, at ER-late endosome/lysosome membrane contact sites to recruit cholesterol to the viral replication organelle, where it contributes to MW functionality. IMPORTANCE A key feature of the replication of positive-strand RNA viruses is the rearrangement of the host cell

  3. An Essential Role of Hrs/Vps27 in Endosomal Cholesterol Trafficking

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    Ximing Du

    2012-01-01

    Full Text Available The endosomal sorting complex required for transport (ESCRT plays a crucial role in the degradation of ubiquitinated endosomal membrane proteins. Here, we report that Hrs, a key protein of the ESCRT-0 complex, is required for the transport of low-density lipoprotein-derived cholesterol from endosomes to the endoplasmic reticulum. This function of Hrs in cholesterol transport is distinct from its previously defined role in lysosomal sorting and downregulation of membrane receptors via the ESCRT pathway. In line with this, knocking down other ESCRT proteins does not cause prominent endosomal cholesterol accumulation. Importantly, the localization and biochemical properties of key cholesterol-sorting proteins, NPC1 and NPC2, appear to be unchanged upon Hrs knockdown. Our data identify Hrs as a regulator of endosomal cholesterol trafficking and provide additional insights into the budding of intralumenal vesicles.

  4. Cholesterol Regulates Syntaxin 6 Trafficking at trans-Golgi Network Endosomal Boundaries

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    Meritxell Reverter

    2014-05-01

    Full Text Available Inhibition of cholesterol export from late endosomes causes cellular cholesterol imbalance, including cholesterol depletion in the trans-Golgi network (TGN. Here, using Chinese hamster ovary (CHO Niemann-Pick type C1 (NPC1 mutant cell lines and human NPC1 mutant fibroblasts, we show that altered cholesterol levels at the TGN/endosome boundaries trigger Syntaxin 6 (Stx6 accumulation into VAMP3, transferrin, and Rab11-positive recycling endosomes (REs. This increases Stx6/VAMP3 interaction and interferes with the recycling of αVβ3 and α5β1 integrins and cell migration, possibly in a Stx6-dependent manner. In NPC1 mutant cells, restoration of cholesterol levels in the TGN, but not inhibition of VAMP3, restores the steady-state localization of Stx6 in the TGN. Furthermore, elevation of RE cholesterol is associated with increased amounts of Stx6 in RE. Hence, the fine-tuning of cholesterol levels at the TGN-RE boundaries together with a subset of cholesterol-sensitive SNARE proteins may play a regulatory role in cell migration and invasion.

  5. Routes and mechanisms of post-endosomal cholesterol trafficking: A story that never ends.

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    Luo, Jie; Jiang, Luyi; Yang, Hongyuan; Song, Bao-Liang

    2017-04-01

    Mammalian cells acquire most exogenous cholesterol through receptor-mediated endocytosis of low-density lipoproteins (LDLs). After internalization, LDL cholesteryl esters are hydrolyzed to release free cholesterol, which then translocates to late endosomes (LEs)/lysosomes (LYs) and incorporates into the membranes by co-ordinated actions of Niemann-Pick type C (NPC) 1 and NPC2 proteins. However, how cholesterol exits LEs/LYs and moves to other organelles remain largely unclear. Growing evidence has suggested that nonvesicular transport is critically involved in the post-endosomal cholesterol trafficking. Numerous sterol-transfer proteins (STPs) have been identified to mediate directional cholesterol transfer at membrane contact sites (MCSs) formed between 2 closely apposed organelles. In addition, a recent study reveals that lysosome-peroxisome membrane contact (LPMC) established by a non-STP synaptotagmin VII and a specific phospholipid phosphatidylinositol 4,5-bisphosphate also serves as a novel and important path for LDL-cholesterol trafficking. These findings highlight an essential role of MCSs in intracellular cholesterol transport, and further work is needed to unveil how various routes are regulated and integrated to maintain proper cholesterol distribution and homeostasis in eukaryotic cells. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  6. Cholesterol Removal from Whole Egg by Crosslinked β-Cyclodextrin

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    H. J. Jeong

    2014-04-01

    Full Text Available This study was carried out to optimize cholesterol removal in whole egg using crosslinked β-cyclodextrin (β-CD and to recycle the β-CD. Various factors for optimizing conditions were concentration of the β-CD, mixing temperature, mixing time, mixing speed and centrifugal speed. In the result of this study, the optimum conditions of cholesterol removal were 25% crosslinked β-CD, 40°C mixing temperature, 30 min mixing time, 1,200 rpm mixing speed and 2,810×g centrifugal speed. The recycling was repeated five times. The cholesterol removal was 92.76% when treated with the optimum conditions. After determining the optimum conditions, the recyclable yields of the crosslinked β-CD ranged from 86.66% to 87.60% in the recycling and the percentage of cholesterol removal was over 80% until third recycling. However, the cholesterol removal efficiency was decreased when the number of repeated recycling was increased. Based on the result of this study, it was concluded that the crosslinked β-CD was efficient for cholesterol removal in whole egg, and recycling is possible for only limited repeating times due to the interaction of the β-CD and egg protein.

  7. Apolipoprotein-mediated removal of cellular cholesterol and phospholipids.

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    Oram, J F; Yokoyama, S

    1996-12-01

    It is widely believed that high density lipoprotein (HDL) protects against cardiovascular disease by removing excess cholesterol from cells of the artery wall. Recent cell culture studies have provided evidence that a major pathway for removing cholesterol and phospholipids from cells is mediated by the direct interactions of HDL apolipoproteins (apo) with plasma membrane domains. These interactions efficiently clear cells of excess sterol by targeting for removal pools of cholesterol that feed into the cholesteryl ester cycle. The precursors for this pathway in vivo are likely to be lipid-free or lipid-poor apolipoproteins generated either by dissociation from the surface of HDL particles or by de novo synthesis. Fibroblasts from subjects with a severe HDL deficiency syndrome called Tangier disease have a cellular defect that prevents apolipoproteins from removing both cholesterol and phospholipids from cells. This defect is associated with a near absence of plasma HDL, markedly below normal low density lipoprotein (LDL) levels, and the appearance of macrophage foam cells in tissues. Thus, an inability of nascent apoA-I to acquire cellular lipids results in a rapid clearance of apoA-I from the plasma, decreased production and increased clearance of LDL, and sterol deposition in tissue macrophages. Although the molecular properties of this pathway are still poorly understood, these studies imply that the apolipoprotein-mediated pathway for removal of cellular lipids is a major source of plasma cholesterol and phospholipids and plays an important role in clearing excess cholesterol from macrophages in vivo.

  8. Removal of cholesterol from Cheddar cheese by beta-cyclodextrin.

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    Kwak, H S; Jung, C S; Shim, S Y; Ahn, J

    2002-12-04

    This study was carried out to determine the cholesterol removal rate and resulting changes in flavor, fatty acid and bitter amino acid production in reduced-cholesterol Cheddar cheese, made by cream separation followed by 10% beta-cyclodextrin (beta-CD) treatment. The cholesterol removal from the cheese was 92.1%. The production of short-chain free fatty acids (FFAs) increased the ripening time in control and cream-treated cheeses. The quantity of short-chain FFAs released between treatments during ripening was different, while not much difference was found in the production of neutral volatile compounds in the samples. Reduced-cholesterol cheese produced much higher levels of bitter amino acids than the control. In sensory analysis, the texture score of control Cheddar cheese increased significantly with ripening time; however, that of the cream treatment group decreased dramatically with ripening time. On the basis of our results, we conclude that the cheese made from beta-CD-treated cream had a higher rate of cholesterol removal and ripened rapidly.

  9. Ultrasound treatment enhances cholesterol removal ability of lactobacilli.

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    Lye, Huey-Shi; Alias, Karim Abdul; Rusul, Gulam; Liong, Min-Tze

    2012-05-01

    This study aimed to evaluate the effect of ultrasound treatment on the cholesterol removing ability of lactobacilli. Viability of lactobacilli cells was significantly increased (P ultrasound treatment at higher intensity and duration. Nevertheless, the effect of ultrasound on membrane properties was reversible, as the viability of ultrasound-treated lactobacilli was increased (P ultrasound-treated lactobacilli via assimilation and incorporation of cholesterol into the cellular membrane also increased significantly (P polar heads of the membrane bilayer. Copyright © 2011 Elsevier B.V. All rights reserved.

  10. Dietary cholesterol induces trafficking of intestinal Niemann-Pick Type C1 Like 1 from the brush border to endosomes

    DEFF Research Database (Denmark)

    Skov, Marianne; Tønnesen, Carina K; Hansen, Gert H

    2011-01-01

    The transmembrane protein Niemann-Pick C1 Like 1 (NPC1L1) belongs to the Niemann-Pick C1 (NPC1) family of cholesterol transporters and is mainly expressed in the liver and the small intestine. NPC1L1 is believed to be the main transporter responsible for the absorption of dietary cholesterol. Like...

  11. Recycling endosomes

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    Goldenring, James R

    2015-01-01

    The endosomal membrane recycling system represents a dynamic conduit for sorting and re-exporting internalized membrane constituents. The recycling system is composed of multiple tubulovesicular recycling pathways that likely confer distinct trafficking pathways for individual cargoes. In addition, elements of the recycling system are responsible for assembly and maintenance of apical membrane specializations including primary cilia and apical microvilli. The existence of multiple intersecting and diverging recycling tracks likely accounts for specificity in plasma membrane recycling trafficking. PMID:26022676

  12. Cholesterol

    Science.gov (United States)

    ... found in some meats, dairy products, chocolate, baked goods, and deep-fried and processed foods. Another type, trans fat, is in some fried and processed foods. Eating these fats can raise your LDL (bad) cholesterol. Lack of physical activity, with lots of ...

  13. Janus kinase 2 modulates the apolipoprotein interactions with ABCA1 required for removing cellular cholesterol.

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    Tang, Chongren; Vaughan, Ashley M; Oram, John F

    2004-02-27

    ATP-binding cassette transporter A1 (ABCA1) mediates transport of cellular cholesterol and phospholipids to high density lipoprotein (HDL) apolipoproteins, such as apoA-I. ABCA1 mutations can cause a severe HDL deficiency and atherosclerosis. Here we show that the protein-tyrosine kinase (TK) Janus kinase 2 (JAK2) modulates the apolipoprotein interactions with ABCA1 required for removing cellular lipids. The protein kinase A (PKA) inhibitor H89, the TK inhibitor genistein, and the JAK2 inhibitor AG490 suppressed apoA-I-mediated cholesterol and phospholipid efflux from ABCA1-expressing cells without altering the membrane ABCA1 content. Whereas PKA inhibition had no effect on apoA-I binding to cells or to ABCA1, TK and JAK2 inhibition greatly reduced these activities. Conversely, PKA but not JAK2 inhibition significantly reduced the intrinsic cholesterol translocase activity of ABCA1. Mutant cells lacking JAK2 had a severely impaired apoA-I-mediated cholesterol and phospholipid efflux and apoA-I binding despite normal ABCA1 protein levels and near normal cholesterol translocase activity. Thus, although PKA modulates ABCA1 lipid transport activity, JAK2 appears to selectively modulate apolipoprotein interactions with ABCA1. TK-mediated phosphorylation of ABCA1 was undetectable, implicating the involvement of another JAK2-targeted protein. Acute incubation of ABCA1-expressing cells with apoA-I had no effect on ABCA1 phosphorylation but stimulated JAK2 autophosphorylation. These results suggest that the interaction of apolipoproteins with ABCA1-expressing cells activates JAK2, which in turn activates a process that enhances apolipoprotein interactions with ABCA1 and lipid removal from cells.

  14. Lymphatic vessels are essential for the removal of cholesterol from peripheral tissues by SR-BI-mediated transport of HDL.

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    Lim, Hwee Ying; Thiam, Chung Hwee; Yeo, Kim Pin; Bisoendial, Radjesh; Hii, Chung Shii; McGrath, Kristine C Y; Tan, Kar Wai; Heather, Alison; Alexander, J Steven Jonathan; Angeli, Veronique

    2013-05-07

    Removal of cholesterol from peripheral tissues to the bloodstream via reverse cholesterol transport (RCT) is a process of major biological importance. Here we demonstrate that lymphatic drainage is required for RCT. We have previously shown that hypercholesterolemia in mice is associated with impaired lymphatic drainage and increased lipid accumulation in peripheral tissues. We now show that restoration of lymphatic drainage in these mice significantly improves cholesterol clearance. Conversely, obstruction of lymphatic vessels in wild-type mice significantly impairs RCT. Finally, we demonstrate using silencing RNA interference, neutralizing antibody, and transgenic mice that removal of cholesterol by lymphatic vessels is dependent on the uptake and transcytosis of HDL by scavenger receptor class B type I expressed on lymphatic endothelium. Collectively, this study challenges the current view that lymphatic endothelium is a passive exchange barrier for cholesterol transport and provides further evidence for its interplay with lipid biology in health and disease. Copyright © 2013 Elsevier Inc. All rights reserved.

  15. Comparison of Physicochemical and Sensory Properties between Cholesterol-removed Gouda Cheese and Gouda Cheese during Ripening

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    Jung, Ho-Jung; Ko, Eun-Jung; Kwak, Hae-Soo

    2013-01-01

    This study was performed to compare physicochemical and sensory properties of cholesterol-removed Gouda cheese (CRGC) and Gouda cheese made in the laboratory during ripening. Composition, short-chain free fatty acids (SCFFA), texture, color, and sensory properties were measured. In chemical composition analyses, moistures were significantly different between control cheeses (42.86%) and sample cheese (48.32%) (p0.05). The amount of cholesterol in control was 82.52 mg/100 g and the percentage ...

  16. Comparison of Physicochemical and Sensory Properties between Cholesterol-removed Gouda Cheese and Gouda Cheese during Ripening

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    Ho-Jung Jung

    2013-12-01

    Full Text Available This study was performed to compare physicochemical and sensory properties of cholesterol-removed Gouda cheese (CRGC and Gouda cheese made in the laboratory during ripening. Composition, short-chain free fatty acids (SCFFA, texture, color, and sensory properties were measured. In chemical composition analyses, moistures were significantly different between control cheeses (42.86% and sample cheese (48.32% (p0.05. The amount of cholesterol in control was 82.52 mg/100 g and the percentage of cholesterol removal was 90.7%. SCFFA increased gradually during ripening and its level of CRGC increased and significantly different from that of control (p0.05. In comparison of the control and sample cheeses, hardness, and springiness were not significantly different, but cohesiveness, gumminess, and chewiness were different (p0.05. However, L* value decreased, while a* and b* values tended to increase significantly (p0.05. Therefore, this study suggests that the quality of cholesterol-removed Gouda cheese is not different from the control cheese.

  17. Comparison of Physicochemical and Sensory Properties between Cholesterol-removed Gouda Cheese and Gouda Cheese during Ripening.

    Science.gov (United States)

    Jung, Ho-Jung; Ko, Eun-Jung; Kwak, Hae-Soo

    2013-12-01

    This study was performed to compare physicochemical and sensory properties of cholesterol-removed Gouda cheese (CRGC) and Gouda cheese made in the laboratory during ripening. Composition, short-chain free fatty acids (SCFFA), texture, color, and sensory properties were measured. In chemical composition analyses, moistures were significantly different between control cheeses (42.86%) and sample cheese (48.32%) (p0.05). The amount of cholesterol in control was 82.52 mg/100 g and the percentage of cholesterol removal was 90.7%. SCFFA increased gradually during ripening and its level of CRGC increased and significantly different from that of control (pcheeses during ripening periods (p>0.05). In comparison of the control and sample cheeses, hardness, and springiness were not significantly different, but cohesiveness, gumminess, and chewiness were different (p0.05). However, L* value decreased, while a* and b* values tended to increase significantly (pcheeses, and were not significantly different between the control and sample cheeses during ripening (p>0.05). Therefore, this study suggests that the quality of cholesterol-removed Gouda cheese is not different from the control cheese.

  18. Comparative study of flavor in cholesterol-removed Gouda cheese and Gouda cheese during ripening.

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    Jung, H J; Ganesan, P; Lee, S J; Kwak, H S

    2013-04-01

    This study was performed to compare the flavor compounds of cholesterol-removed Gouda cheese (CRGC) and those of Gouda cheese (control) during ripening. The CRGC was made using milk treated with cross-linked β-cyclodextrin (β-CD). The solid-phase microextraction (SPME) method was used to extract flavor compounds from Gouda cheese. In both CRGC and control cheese, 31 flavor compounds were identified, including 6 free fatty acids, 5 esters, 5 ketones, 1 aldehyde, 3 lactones, 5 alcohols, and 6 miscellaneous compounds. Free fatty acids were the most abundant flavor compounds quantified in CRGC and control cheese. In the early stage of ripening, concentrations of flavor compounds in CRGC and control cheese were 16.42 and 10.38 mg/kg, respectively. At 6 mo, they increased to 40.90 and 67.89 mg/kg, respectively. A group of esters was the second abundant flavor compound in CRGC and control cheese. At the initial stage of ripening, total concentrations of esters were 12.94 (CRGC) and 10.95 mg/kg (control) and they increased to 22.73 (CRGC) and 27.68 mg/kg (control). Total concentrations of ketones were 1.96 (CRGC) and 6.49 mg/kg (control) at the initial stage of ripening. After 6 mo of ripening, total concentrations reached 11.32 (CRGC) and 52.43 mg/kg (control). In the case of the lactones, at the early stage of ripening, total concentrations of CRGC and control cheese were 0.63 and 0.84mg/kg, respectively, and then increased to 1.73 (CRGC) and 3.25mg/kg (control) at the end of ripening. Based on the results of this study, the flavor compounds of CRGC and control showed slightly different profiles during ripening. Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  19. Ultrasound enhanced growth and cholesterol removal of Lactobacillus fermentum FTDC 1311 in the parent cells but not the subsequent passages.

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    Lye, H S; Khoo, B Y; Karim, A A; Rusul, G; Liong, M T

    2012-07-01

    The aim of this study was to evaluate the effect of ultrasound on the intestinal adherence ability, cell growth, and cholesterol removal ability of parent cells and subsequent passages of Lactobacillus fermentum FTDC 1311. Ultrasound significantly decreased the intestinal adherence ability of treated parent cells compared to that of the control by 11.32% (Pultrasound (0-4h) and showed an increase (P9.74%) was also observed for treated parent cells compared to that of the control, accompanied by increased (Ppolar regions of membrane phospholipids of parent cells compared to that of the control (Pultrasound treatment could be used to improve cholesterol removal ability of parent cells without inducing permanent damage/defects on treated cells of subsequent passages. Copyright © 2011 Elsevier B.V. All rights reserved.

  20. Effect of Cholesterol Removal Processing Using β-Cyclodextrin on Main Components of Milk

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    A. M. Maskooki

    2013-01-01

    Full Text Available Various concentrations (0%, 0.5%, 1% and 1.5% of β-CD were mixed with different fat contents (1%, 2.5% and 3% of raw (unhomogenized and homogenized milk at two mixing temperatures of 8 and 20°C. The cholesterol residue, fat, protein, lactose, solid nonfat (SNF, density, and ash content of milk were measured for each treatment. The results statistically analysed and showed that the cholesterol content of milk remarkably decreased as the β-CD was increased particularly in homogenized milk at 20°C. However, the reduction rate of cholesterol was decreased when extra β-CD was added due to its intermolecular reactions. The maximum cholesterol reduction was achieved at the level of 1% β-CD. The fat content, SNF, protein, lactose, and density content were decreased with increasing β-CD whereas it did not affect ash content.

  1. Positioning of AMPA Receptor-Containing Endosomes Regulates Synapse Architecture

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    Marta Esteves da Silva

    2015-11-01

    Full Text Available Lateral diffusion in the membrane and endosomal trafficking both contribute to the addition and removal of AMPA receptors (AMPARs at postsynaptic sites. However, the spatial coordination between these mechanisms has remained unclear, because little is known about the dynamics of AMPAR-containing endosomes. In addition, how the positioning of AMPAR-containing endosomes affects synapse organization and functioning has never been directly explored. Here, we used live-cell imaging in hippocampal neuron cultures to show that intracellular AMPARs are transported in Rab11-positive recycling endosomes, which frequently enter dendritic spines and depend on the microtubule and actin cytoskeleton. By using chemically induced dimerization systems to recruit kinesin (KIF1C or myosin (MyosinV/VI motors to Rab11-positive recycling endosomes, we controlled their trafficking and found that induced removal of recycling endosomes from spines decreases surface AMPAR expression and PSD-95 clusters at synapses. Our data suggest a mechanistic link between endosome positioning and postsynaptic structure and composition.

  2. Cholesterol Removal from Adult Skeletal Muscle impairs Excitation-Contraction Coupling and Aging reduces Caveolin-3 and alters the Expression of other Triadic Proteins

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    Genaro eBarrientos

    2015-04-01

    Full Text Available Cholesterol and caveolin are integral membrane components that modulate the function/location of many cellular proteins. Skeletal muscle fibers, which have unusually high cholesterol levels in transverse tubules, express the caveolin-3 isoform but its association with transverse tubules remains contentious. Cholesterol removal impairs excitation-contraction coupling in amphibian and mammalian fetal skeletal muscle fibers. Here, we show that treating single muscle fibers from adult mice with the cholesterol removing agent methyl-β-cyclodextrin decreased fiber cholesterol by 26%, altered the location pattern of caveolin-3 and of the voltage dependent calcium channel Cav1.1, and suppressed or reduced electrically evoked Ca2+ transients without affecting membrane integrity or causing sarcoplasmic reticulum calcium depletion. We found that transverse tubules from adult muscle and triad fractions that contain ~10% attached transverse tubules, but not sarcoplasmic reticulum membranes, contained caveolin-3 and Cav1.1; both proteins partitioned into detergent-resistant membrane fractions highly enriched in cholesterol. Aging entails significant deterioration of skeletal muscle function. We found that triad fractions from aged rats had similar cholesterol and RyR1 protein levels compared to triads from young rats, but had lower caveolin-3 and glyceraldehyde 3-phosphate dehydrogenase and increased Na+/K+-ATPase protein levels. Both triad fractions had comparable NADPH oxidase (NOX activity and protein content of NOX2 subunits (p47phox and gp91phox, implying that NOX activity does not increase during aging. These findings show that partial cholesterol removal impairs excitation-contraction coupling and alters caveolin-3 and Cav1.1 location pattern, and that aging reduces caveolin-3 protein content and modifies the expression of other triadic proteins. We discuss the possible implications of these findings for skeletal muscle function in young and aged

  3. Oriented immobilized anti-LDL antibody carrying poly(hydroxyethyl methacrylate) cryogel for cholesterol removal from human plasma

    Energy Technology Data Exchange (ETDEWEB)

    Bereli, Nilay [Department of Chemistry, Hacettepe University, Beytepe, Ankara (Turkey); Sener, Guelsu [Nanotechnology and Nanomedicine Division, Hacettepe University, Ankara (Turkey); Yavuz, Handan, E-mail: handany@hacettepe.edu.tr [Department of Chemistry, Hacettepe University, Beytepe, Ankara (Turkey); Denizli, Adil [Department of Chemistry, Hacettepe University, Beytepe, Ankara (Turkey)

    2011-07-20

    Low density lipoprotein (LDL) cholesterol is a major ingredient of the plaque that collects in the coronary arteries and causes coronary heart diseases. Among the methods used for the extracorporeal elimination of LDL from intravasal volume, immunoaffinity technique using anti-LDL antibody as a ligand offers superior selectivity and specificity. Proper orientation of the immobilized antibody is the main issue in immunoaffinity techniques. In this study, anti-human {beta}-lipoprotein antibody (anti-LDL antibody) molecules were immobilized and oriented through protein A onto poly(2-hydroxyethyl methacrylate) (PHEMA) cryogel in order to remove LDL from hypercholesterolemic human plasma. PHEMA cryogel was prepared by free radical polymerization initiated with N,N,N',N'-tetramethylene diamine (TEMED). PHEMA cryogel with a swelling degree of 8.89 g H{sub 2}O/g and 67% macro-porosity was characterized by swelling studies, scanning electron microscope (SEM) and blood compatibility tests. All the clotting times were increased when compared with control plasma. The maximum immobilized anti-LDL antibody amount was 63.2 mg/g in the case of random antibody immobilization and 19.6 mg/g in the case of oriented antibody immobilization (protein A loading was 57.0 mg/g). Random and oriented anti-LDL antibody immobilized PHEMA cryogels adsorbed 111 and 129 mg LDL/g cryogel from hypercholesterolemic human plasma, respectively. Up to 80% of the adsorbed LDL was desorbed. The adsorption-desorption cycle was repeated 6 times using the same cryogel. There was no significant loss of LDL adsorption capacity. - Research highlights: {yields} LDL cholesterol is a risk factor in the development of coronary heart diseases. {yields} Antibodies against LDL are used for the selective extracorporeal removal of LDL. {yields} Protein A is used for the oriented immobilization of anti LDL onto PHEMA cryogel. {yields} PHEMA cryogels are biocompatible, exhibit a low pressure drop, lack diffusion

  4. Positioning of AMPA Receptor-Containing Endosomes Regulates Synapse Architecture

    NARCIS (Netherlands)

    Esteves da Silva, Marta; Adrian, Max|info:eu-repo/dai/nl/369490959; Schätzle, Philipp; Lipka, Joanna|info:eu-repo/dai/nl/369403142; Watanabe, Takuya; Cho, Sukhee; Futai, Kensuke; Wierenga, Corette J|info:eu-repo/dai/nl/237383292; Kapitein, Lukas C|info:eu-repo/dai/nl/298806630; Hoogenraad, Casper C|info:eu-repo/dai/nl/227263502

    2015-01-01

    Lateral diffusion in the membrane and endosomal trafficking both contribute to the addition and removal of AMPA receptors (AMPARs) at postsynaptic sites. However, the spatial coordination between these mechanisms has remained unclear, because little is known about the dynamics of AMPAR-containing

  5. deep-orange and carnation define distinct stages in late endosomal biogenesis in Drosophila melanogaster.

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    Sriram, V; Krishnan, K S; Mayor, Satyajit

    2003-05-12

    Endosomal degradation is severely impaired in primary hemocytes from larvae of eye color mutants of Drosophila. Using high resolution imaging and immunofluorescence microscopy in these cells, products of eye color genes, deep-orange (dor) and carnation (car), are localized to large multivesicular Rab7-positive late endosomes containing Golgi-derived enzymes. These structures mature into small sized Dor-negative, Car-positive structures, which subsequently fuse to form tubular lysosomes. Defective endosomal degradation in mutant alleles of dor results from a failure of Golgi-derived vesicles to fuse with morphologically arrested Rab7-positive large sized endosomes, which are, however, normally acidified and mature with wild-type kinetics. This locates the site of Dor function to fusion of Golgi-derived vesicles with the large Rab7-positive endocytic compartments. In contrast, endosomal degradation is not considerably affected in car1 mutant; fusion of Golgi-derived vesicles and maturation of large sized endosomes is normal. However, removal of Dor from small sized Car-positive endosomes is slowed, and subsequent fusion with tubular lysosomes is abolished. Overexpression of Dor in car1 mutant aggravates this defect, implicating Car in the removal of Dor from endosomes. This suggests that, in addition to an independent role in fusion with tubular lysosomes, the Sec1p homologue, Car, regulates Dor function.

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

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

    2016-01-01

    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

  7. 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.

  8. About Cholesterol

    Science.gov (United States)

    ... Artery Disease Venous Thromboembolism Aortic Aneurysm More About Cholesterol Updated:Jul 5,2017 Whether you’ve just ... Quiz This content was last reviewed April 2017. Cholesterol • Home • About Cholesterol Introduction Atherosclerosis What Your Cholesterol ...

  9. GPCR sorting at multivesicular endosomes.

    Science.gov (United States)

    Dores, Michael Robert; Trejo, JoAnn

    2015-01-01

    The lysosomal degradation of G protein-coupled receptors (GPCRs) is essential for receptor signaling and down regulation. Once internalized, GPCRs are sorted within the endocytic pathway and packaged into intraluminal vesicles (ILVs) that bud inward to form the multivesicular endosome (MVE). The mechanisms that control GPCR sorting and ILV formation are poorly understood. Quantitative strategies are important for evaluating the function of adaptor and scaffold proteins that regulate sorting of GPCRs at MVEs. In this chapter, we outline two strategies for the quantification and visualization of GPCR sorting into the lumen of MVEs. The first protocol utilizes a biochemical approach to assay the sorting of GPCRs in a population of cells, whereas the second strategy examines GPCR sorting in individual cells using immunofluorescence confocal microscopy. Combined, these assays can be used to establish the kinetics of activated GPCR lysosomal trafficking in response to specific ligands, as well as evaluate the contribution of endosomal adaptors to GPCR sorting at MVEs. The protocols presented in this chapter can be adapted to analyze GPCR sorting in a myriad of cell types and tissues, and expanded to analyze the mechanisms that regulate MVE sorting of other cargoes. Copyright © 2015 Elsevier Inc. All rights reserved.

  10. The structure and function of presynaptic endosomes

    Energy Technology Data Exchange (ETDEWEB)

    Jähne, Sebastian, E-mail: sebastian.jaehne1@stud.uni-goettingen.de [Department of Neuro- and Sensory Physiology, University of Göttingen Medical Center, Cluster of Excellence Nanoscale Microscopy and Molecular Physiology of the Brain, Humboldtallee 23, 37073 Göttingen (Germany); International Max Planck Research School for Neurosciences, 37077 Göttingen (Germany); Rizzoli, Silvio O. [Department of Neuro- and Sensory Physiology, University of Göttingen Medical Center, Cluster of Excellence Nanoscale Microscopy and Molecular Physiology of the Brain, Humboldtallee 23, 37073 Göttingen (Germany); Helm, Martin S., E-mail: martin.helm@med.uni-goettingen.de [Department of Neuro- and Sensory Physiology, University of Göttingen Medical Center, Cluster of Excellence Nanoscale Microscopy and Molecular Physiology of the Brain, Humboldtallee 23, 37073 Göttingen (Germany); International Max Planck Research School for Molecular Biology, 37077 Göttingen (Germany)

    2015-07-15

    The function of endosomes and of endosome-like structures in the presynaptic compartment is still controversial. This is in part due to the absence of a consensus on definitions and markers for these compartments. Synaptic endosomes are sometimes seen as stable organelles, permanently present in the synapse. Alternatively, they are seen as short-lived intermediates in synaptic vesicle recycling, arising from the endocytosis of large vesicles from the plasma membrane, or from homotypic fusion of small vesicles. In addition, the potential function of the endosome is largely unknown in the synapse. Some groups have proposed that the endosome is involved in the sorting of synaptic vesicle proteins, albeit others have produced data that deny this possibility. In this review, we present the existing evidence for synaptic endosomes, we discuss their potential functions, and we highlight frequent technical pitfalls in the analysis of this elusive compartment. We also sketch a roadmap to definitely determine the role of synaptic endosomes for the synaptic vesicle cycle. Finally, we propose a common definition of synaptic endosome-like structures.

  11. 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.

  12. Cholesterol Levels

    Science.gov (United States)

    ... this page: https://medlineplus.gov/labtests/cholesterollevels.html Cholesterol Levels To use the sharing features on this page, please enable JavaScript. What is a Cholesterol Test? Cholesterol is a waxy, fat-like substance ...

  13. CCC- and WASH-mediated endosomal sorting of LDLR is required for normal clearance of circulating LDL

    NARCIS (Netherlands)

    Bartuzi, Paulina; Billadeau, Daniel D.; Favier, Robert; Rong, Shunxing; Dekker, Daphne; Fedoseienko, Alina; Fieten, Hille; Wijers, Melinde; Levels, Johannes H.; Huijkman, Nicolette; Kloosterhuis, Niels; van der Molen, Henk; Brufau, Gemma; Groen, Albert K.; Elliott, Alison M.; Kuivenhoven, Jan Albert; Plecko, Barbara; Grangl, Gernot; McGaughran, Julie; Horton, Jay D.; Burstein, Ezra; Hofker, Marten H.; van de Sluis, Bart

    The low-density lipoprotein receptor (LDLR) plays a pivotal role in clearing atherogenic circulating low-density lipoprotein (LDL) cholesterol. Here we show that the COMMD/CCDC22/CCDC93 (CCC) and the Wiskott-Aldrich syndrome protein and SCAR homologue (WASH) complexes are both crucial for endosomal

  14. PI(3,5)P2 controls endosomal branched actin dynamics by regulating cortactin–actin interactions

    Science.gov (United States)

    Hong, Nan Hyung; Qi, Aidong

    2015-01-01

    Branched actin critically contributes to membrane trafficking by regulating membrane curvature, dynamics, fission, and transport. However, how actin dynamics are controlled at membranes is poorly understood. Here, we identify the branched actin regulator cortactin as a direct binding partner of phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) and demonstrate that their interaction promotes turnover of late endosomal actin. In vitro biochemical studies indicated that cortactin binds PI(3,5)P2 via its actin filament-binding region. Furthermore, PI(3,5)P2 competed with actin filaments for binding to cortactin, thereby antagonizing cortactin activity. These findings suggest that PI(3,5)P2 formation on endosomes may remove cortactin from endosome-associated branched actin. Indeed, inhibition of PI(3,5)P2 production led to cortactin accumulation and actin stabilization on Rab7+ endosomes. Conversely, inhibition of Arp2/3 complex activity greatly reduced cortactin localization to late endosomes. Knockdown of cortactin reversed PI(3,5)P2-inhibitor–induced actin accumulation and stabilization on endosomes. These data suggest a model in which PI(3,5)P2 binding removes cortactin from late endosomal branched actin networks and thereby promotes net actin turnover. PMID:26323691

  15. Niacin to Boost Your HDL "Good" Cholesterol

    Science.gov (United States)

    Niacin can boost 'good' cholesterol Niacin is a B vitamin that may raise your HDL ("good") cholesterol. But side effects might outweigh benefits for most ... been used to increase high-density lipoprotein (HDL) cholesterol — the "good" cholesterol that helps remove low-density ...

  16. Cholesterol (image)

    Science.gov (United States)

    Cholesterol is a soft, waxy substance that is present in all parts of the body including the ... and obtained from animal products in the diet. Cholesterol is manufactured in the liver and is needed ...

  17. Cholesterol and lifestyle

    Science.gov (United States)

    Hyperlipidemia - cholesterol and lifestyle; CAD - cholesterol and lifestyle; Coronary artery disease - cholesterol and lifestyle; Heart disease - cholesterol and lifestyle; Prevention - cholesterol and lifestyle; ...

  18. What's Cholesterol?

    Science.gov (United States)

    ... the foods you eat. Meat, fish, eggs, butter, cheese, and whole or low-fat milk all have cholesterol in them. You Need a Little, Not a ... are some foods that have a lot of cholesterol? Meat, eggs, butter, cheese, and milk (and stuff that's made with some ...

  19. The novel endosomal membrane protein Ema interacts with the class C Vps-HOPS complex to promote endosomal maturation.

    Science.gov (United States)

    Kim, Sungsu; Wairkar, Yogesh P; Daniels, Richard W; DiAntonio, Aaron

    2010-03-08

    Endosomal maturation is critical for accurate and efficient cargo transport through endosomal compartments. Here we identify a mutation of the novel Drosophila gene, ema (endosomal maturation defective) in a screen for abnormal synaptic overgrowth and defective protein trafficking. Ema is an endosomal membrane protein required for trafficking of fluid-phase and receptor-mediated endocytic cargos. In the ema mutant, enlarged endosomal compartments accumulate as endosomal maturation fails, with early and late endosomes unable to progress into mature degradative late endosomes and lysosomes. Defective endosomal down-regulation of BMP signaling is responsible for the abnormal synaptic overgrowth. Ema binds to and genetically interacts with Vps16A, a component of the class C Vps-HOPS complex that promotes endosomal maturation. The human orthologue of ema, Clec16A, is a candidate susceptibility locus for autoimmune disorders, and its expression rescues the Drosophila mutant demonstrating conserved function. Characterizing this novel gene family identifies a new component of the endosomal pathway and provides insights into class C Vps-HOPS complex function.

  20. The novel endosomal membrane protein Ema interacts with the class C Vps–HOPS complex to promote endosomal maturation

    Science.gov (United States)

    Kim, Sungsu; Wairkar, Yogesh P.; Daniels, Richard W.

    2010-01-01

    Endosomal maturation is critical for accurate and efficient cargo transport through endosomal compartments. Here we identify a mutation of the novel Drosophila gene, ema (endosomal maturation defective) in a screen for abnormal synaptic overgrowth and defective protein trafficking. Ema is an endosomal membrane protein required for trafficking of fluid-phase and receptor-mediated endocytic cargos. In the ema mutant, enlarged endosomal compartments accumulate as endosomal maturation fails, with early and late endosomes unable to progress into mature degradative late endosomes and lysosomes. Defective endosomal down-regulation of BMP signaling is responsible for the abnormal synaptic overgrowth. Ema binds to and genetically interacts with Vps16A, a component of the class C Vps–HOPS complex that promotes endosomal maturation. The human orthologue of ema, Clec16A, is a candidate susceptibility locus for autoimmune disorders, and its expression rescues the Drosophila mutant demonstrating conserved function. Characterizing this novel gene family identifies a new component of the endosomal pathway and provides insights into class C Vps–HOPS complex function. PMID:20194640

  1. High blood cholesterol levels

    Science.gov (United States)

    Cholesterol - high; Lipid disorders; Hyperlipoproteinemia; Hyperlipidemia; Dyslipidemia; Hypercholesterolemia ... There are many types of cholesterol. The ones talked about most are: ... lipoprotein (HDL) cholesterol -- often called "good" cholesterol ...

  2. Intestinal cholesterol secretion : future clinical implications

    NARCIS (Netherlands)

    Jakulj, L.; Besseling, J.; Stroes, E. S. G.; Groen, A. K.

    2013-01-01

    Together with the liver, the intestine serves as a homeostatic organ in cholesterol metabolism. Recent evidence has substantiated the pivotal role of the intestine in reverse cholesterol transport (RCT). RCT is a fundamental antiatherogenic pathway, mediating the removal of cholesterol from tissues

  3. Cholesterol Depletion from a Ceramide/Cholesterol Mixed Monolayer: A Brewster Angle Microscope Study

    KAUST Repository

    Mandal, Pritam

    2016-06-01

    Cholesterol is crucial to the mechanical properties of cell membranes that are important to cells’ behavior. Its depletion from the cell membranes could be dramatic. Among cyclodextrins (CDs), methyl beta cyclodextrin (MβCD) is the most efficient to deplete cholesterol (Chol) from biomembranes. Here, we focus on the depletion of cholesterol from a C16 ceramide/cholesterol (C16-Cer/Chol) mixed monolayer using MβCD. While the removal of cholesterol by MβCD depends on the cholesterol concentration in most mixed lipid monolayers, it does not depend very much on the concentration of cholesterol in C16-Cer/Chol monolayers. The surface pressure decay during depletion were described by a stretched exponential that suggested that the cholesterol molecules are unable to diffuse laterally and behave like static traps for the MβCD molecules. Cholesterol depletion causes morphology changes of domains but these disrupted monolayers domains seem to reform even when cholesterol level was low.

  4. Dependence of the effects of dietary cholesterol and experimental conditions on serum lipids in man. III. The effect on serum cholesterol of removal of eggs from the diet of free-living habitually egg-eating people

    NARCIS (Netherlands)

    Bronsgeest-Schoute, D. C.; Hermus, R. J.; Dallinga-Thie, G. M.; Hautvast, J. G.

    1979-01-01

    Forty-four healthy free living volunteers were used to study the effect of the removal of eggs from a habitual egg-rich diet. The subjects, recruited by advertising, normally consumed at least 1 egg per day. During the 3-week experimental period they were not allowed to eat any eggs or products

  5. Cholesterol IQ Quiz

    Science.gov (United States)

    ... Peripheral Artery Disease Venous Thromboembolism Aortic Aneurysm More Cholesterol IQ Quiz Updated:Jul 5,2017 Begin the quiz Cholesterol • Home • About Cholesterol Introduction Atherosclerosis What Your Cholesterol ...

  6. High Blood Cholesterol

    Science.gov (United States)

    ... To Health Topics / High Blood Cholesterol High Blood Cholesterol Also known as Hypercholesterolemia High blood cholesterol is ... Lipid panel tests to check for healthy blood cholesterol levels Doctors use lipid panels to check whether ...

  7. Cholesterol and Your Child

    Science.gov (United States)

    ... Needs a Kidney Transplant Vision Facts and Myths Cholesterol KidsHealth > For Parents > Cholesterol Print A A A ... español El colesterol y su hijo What Is Cholesterol? Cholesterol is a waxy substance made by the ...

  8. Retromer guides STxB and CD8-M6PR from early to recycling endosomes, EHD1 guides STxB from recycling endosome to Golgi

    Science.gov (United States)

    McKenzie, Jenna E.; Raisley, Brent; Zhou, Xin; Naslavsky, Naava; Taguchi, Tomohiko; Caplan, Steve; Sheff, David

    2012-01-01

    Retrograde trafficking transports proteins, lipids and toxins from the plasma membrane to the Golgi and ER. To reach the Golgi, these cargos must transit the endosomal system, consisting of early endosomes, recycling endosomes, late endosomes and lysosomes. All cargos pass through early endosomes, but may take different routes to the Golgi. Retromer dependent cargos bypass the late endosomes to reach the Golgi. We compared how two very different retromer dependent cargos negotiate the endosomal sorting system. Shiga toxin B, bound to the external layer of the plasma membrane, and chimeric CD8-Mannose-6-Phosphate Receptor, which is anchored via a transmembrane domain. Both appear to pass through the recycling endosome. Ablation of the recycling endosome diverted both of these cargos to an aberrant compartment and prevented them from reaching the Golgi. Once in the recycling endosome, Shiga toxin required EHD1 to traffic to the TGN, while the CD8-Mannose-6-Phosphate Receptor was not significantly dependent on EHD1. Knockdown of retromer components left cargo in the early endosomes, suggesting that it is required for retrograde exit from this compartment. This work establishes the recycling endosome as a required step in retrograde traffic of at least these two retromer dependent cargos. Along this pathway, retromer is associated with EE to recycling endosome traffic, while EHD1 is associated with recycling endosome to TGN traffic of STxB. PMID:22540229

  9. Potential of BODIPY-cholesterol for analysis of cholesterol transport and diffusion in living cells

    DEFF Research Database (Denmark)

    Wüstner, Daniel; Lund, Frederik Wendelboe; Röhrl, Clemens

    2016-01-01

    Cholesterol is an abundant and important lipid component of cellular membranes. Analysis of cholesterol transport and diffusion in living cells is hampered by the technical challenge of designing suitable cholesterol probes which can be detected for example by optical microscopy. One strategy...... and collaborative efforts with Bob Bittman for studying diffusion in the plasma membrane (PM) and uptake of BChol in a quantitative manner. For that purpose, we used a variety of fluorescence approaches including fluorescence correlation spectroscopy and its imaging variants, fluorescence recovery after...... photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP). We also describe pulse-chase studies from the PM using BChol in direct comparison to DHE. Based on the gathered imaging data, we present a two-step kinetic model for sterol transport between PM and recycling endosomes. In addition, we...

  10. Measuring the role for Met endosomal signaling in tumorigenesis.

    Science.gov (United States)

    Barrow, Rachel; Joffre, Carine; Ménard, Ludovic; Kermorgant, Stéphanie

    2014-01-01

    Met is a receptor tyrosine kinase, often overexpressed or mutated in human cancer. Upon activation by its ligand, the hepatocyte growth factor, Met controls several cell functions such as proliferation, migration, and survival through the activation of multiple pathways. Upon ligand binding, Met rapidly internalizes and continues to signal from endosomal compartments prior to its degradation. Importantly, this "endosomal signaling" has recently been shown to be involved in tumorigenesis and experimental metastasis. Consequently, interfering with Met endosomal signaling may provide a novel therapeutic approach in cancer treatment. However, there is a need for additional studies in various experimental models to confirm this and find the most specific ways of achieving it. Thus, outlined in this review are the techniques and tools we have been using to study Met endocytosis and Met endosomal signaling. © 2014 Elsevier Inc. All rights reserved.

  11. Accumulation of rhodopsin in late endosomes triggers photoreceptor cell degeneration.

    Directory of Open Access Journals (Sweden)

    Yashodhan Chinchore

    2009-02-01

    Full Text Available Progressive retinal degeneration is the underlying feature of many human retinal dystrophies. Previous work using Drosophila as a model system and analysis of specific mutations in human rhodopsin have uncovered a connection between rhodopsin endocytosis and retinal degeneration. In these mutants, rhodopsin and its regulatory protein arrestin form stable complexes, and endocytosis of these complexes causes photoreceptor cell death. In this study we show that the internalized rhodopsin is not degraded in the lysosome but instead accumulates in the late endosomes. Using mutants that are defective in late endosome to lysosome trafficking, we were able to show that rhodopsin accumulates in endosomal compartments in these mutants and leads to light-dependent retinal degeneration. Moreover, we also show that in dying photoreceptors the internalized rhodopsin is not degraded but instead shows characteristics of insoluble proteins. Together these data implicate buildup of rhodopsin in the late endosomal system as a novel trigger of death of photoreceptor neurons.

  12. Endosomes: Emerging Platforms for Integrin-Mediated FAK Signalling.

    Science.gov (United States)

    Alanko, Jonna; Ivaska, Johanna

    2016-06-01

    Integrins are vital cell adhesion receptors with the ability to transmit extracellular matrix (ECM) cues to intracellular signalling pathways. ECM-integrin signalling regulates various cellular functions such as cell survival and movement. Integrin signalling has been considered to occur exclusively from adhesion sites at the plasma membrane (PM). However, recent data demonstrates integrin signalling also from endosomes. Integrin-mediated focal adhesion kinase (FAK) signalling is strongly dependent on integrin endocytosis, and endosomal FAK signalling facilitates cancer metastasis by supporting anchorage-independent growth and anoikis resistance. Here we discuss the possible mechanisms and functions of endosomal FAK signalling compared with its previously known roles in other cellular locations and discuss the potential of endosomal FAK as novel target for future cancer therapies. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. 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.

  14. Vps1 in the late endosome-to-vacuole traffic.

    Science.gov (United States)

    Hayden, Jacob; Williams, Michelle; Granich, Ann; Ahn, Hyoeun; Tenay, Brandon; Lukehart, Joshua; Highfill, Chad; Dobard, Sarah; Kim, Kyoungtae

    2013-03-01

    Vacuolar protein sorting 1 (Vps1), the yeast homolog to human dynamin, is a GTP hydrolyzing protein, which plays an important role in protein sorting and targeting between the Golgi and late endosomal compartments. In this study, we assessed the functional significance of Vps1 in the membrane traffic towards the vacuole. We show here that vps1 delta cells accumulated FM4-64 to a greater extent than wild-type (WT))cells, suggesting slower endocytic degradation traffic toward the vacuole. In addition, we observed that two endosome-to-vacuole traffic markers, DsRed-FYVE and Ste2-GFP, were highly accumulated in Vps1-deficient cells, further supporting Vps1's implication in efficient trafficking of endocytosed materials to the vacuole. Noteworthy, a simultaneous imaging analysis in conjunction with FM4-64 pulse-chase experiment further revealed that Vps1 plays a role in late endosome to the vacuole transport. Consistently, our subcellular localization analysis showed that Vps1 is present at the late endosome. The hyperaccumulation of endosomal intermediates in the vps1 mutant cells appears to be caused by the disruption of integrity of HOPS tethering complexes, manifested by mislocalization of Vps39 to the cytoplasm. Finally, we postulate that Vps1 functions together with the Endosomal Sorting Complex Required for Transport (ESCRT) complex at the late endosomal compartments, based on the observation that the double mutants, in which VPS1 along with singular ESCRT I, II and III genes have been disrupted, exhibited synthetic lethality. Together, we propose that Vps1 is required for correct and efficient trafficking from the late endosomal compartments to the vacuole.

  15. Inhibition of cholesterol recycling impairs cellular PrPSc propagation

    OpenAIRE

    Gilch, Sabine; Bach, Christian; Lutzny, Gloria; Vorberg, Ina; Sch?tzl, Hermann M.

    2009-01-01

    The infectious agent in prion diseases consists of an aberrantly folded isoform of the cellular prion protein (PrPc), termed PrPSc, which accumulates in brains of affected individuals. Studies on prion-infected cultured cells indicate that cellular cholesterol homeostasis influences PrPSc propagation. Here, we demonstrate that the cellular PrPSc content decreases upon accumulation of cholesterol in late endosomes, as induced by NPC-1 knock-down or treatment with U18666A. PrPc trafficking, lip...

  16. How much in vitro cholesterol reducing activity of lactobacilli predicts their in vivo cholesterol function?

    Directory of Open Access Journals (Sweden)

    Golnoush Madani

    2013-01-01

    Results: No cholesterol assimilation was detected by growth and incubation of the active culture in either of the medium. Thus, in vivo cholesterol function of LA7 was not caused by cholesterol consumption. A comprehensive review of literature on the related studies also showed that there are other documented studies which evidenced the uncertainty of the direct relation between in vitro and in vivo studies. Conclusion: Cholesterol removal from the cultured media may not be considered as an appropriate integral index for selection of Lactobacillus strains with cholesterol-lowering activity.

  17. What Is Cholesterol?

    Science.gov (United States)

    ... School Counselors Kidney Stones Brain and Nervous System Cholesterol KidsHealth > For Teens > Cholesterol Print A A A ... High Cholesterol? en español ¿Qué es el colesterol? Cholesterol Is a Fat in the Blood Cholesterol (kuh- ...

  18. Causes of High Cholesterol

    Science.gov (United States)

    ... Venous Thromboembolism Aortic Aneurysm More Causes of High Cholesterol Updated:Nov 16,2017 If you have high ... for a heart or stroke event? Find out . Cholesterol • Home • About Cholesterol • HDL, LDL, and Triglycerides • Causes ...

  19. Cholesterol Facts and Statistics

    Science.gov (United States)

    ... Blood Pressure Salt Million Hearts® WISEWOMAN Program High Cholesterol Facts Recommend on Facebook Tweet Share Compartir Find ... about high cholesterol in the United States. High Cholesterol in the United States In 2011–2012, 78 ...

  20. Redistribution of Endosomal Membranes to the African Swine Fever Virus Replication Site

    Directory of Open Access Journals (Sweden)

    Miguel Ángel Cuesta-Geijo

    2017-06-01

    Full Text Available African swine fever virus (ASFV infection causes endosomal reorganization. Here, we show that the virus causes endosomal congregation close to the nucleus as the infection progresses, which is necessary to build a compact viral replication organelle. ASFV enters the cell by the endosomal pathway and reaches multivesicular late endosomes. Upon uncoating and fusion, the virus should exit to the cytosol to start replication. ASFV remodels endosomal traffic and redistributes endosomal membranes to the viral replication site. Virus replication also depends on endosomal membrane phosphoinositides (PtdIns synthesized by PIKfyve. Endosomes could act as platforms providing membranes and PtdIns, necessary for ASFV replication. Our study has revealed that ASFV reorganizes endosome dynamics, in order to ensure a productive infection.

  1. Cholesterol Balance in Prion Diseases and Alzheimer’s Disease

    Directory of Open Access Journals (Sweden)

    Samia Hannaoui

    2014-11-01

    Full Text Available Prion diseases are transmissible and fatal neurodegenerative disorders of humans and animals. They are characterized by the accumulation of PrPSc, an aberrantly folded isoform of the cellular prion protein PrPC, in the brains of affected individuals. PrPC is a cell surface glycoprotein attached to the outer leaflet of the plasma membrane by a glycosyl-phosphatidyl-inositol (GPI anchor. Specifically, it is associated with lipid rafts, membrane microdomains enriched in cholesterol and sphinoglipids. It has been established that inhibition of endogenous cholesterol synthesis disturbs lipid raft association of PrPC and prevents PrPSc accumulation in neuronal cells. Additionally, prion conversion is reduced upon interference with cellular cholesterol uptake, endosomal export, or complexation at the plasma membrane. Altogether, these results demonstrate on the one hand the importance of cholesterol for prion propagation. On the other hand, growing evidence suggests that prion infection modulates neuronal cholesterol metabolism. Similar results were reported in Alzheimer’s disease (AD: whereas amyloid β peptide formation is influenced by cellular cholesterol, levels of cholesterol in the brains of affected individuals increase during the clinical course of the disease. In this review, we summarize commonalities of alterations in cholesterol homeostasis and discuss consequences for neuronal function and therapy of prion diseases and AD.

  2. Cholesterol Balance in Prion Diseases and Alzheimer’s Disease

    Science.gov (United States)

    Hannaoui, Samia; Shim, Su Yeon; Cheng, Yo Ching; Corda, Erica; Gilch, Sabine

    2014-01-01

    Prion diseases are transmissible and fatal neurodegenerative disorders of humans and animals. They are characterized by the accumulation of PrPSc, an aberrantly folded isoform of the cellular prion protein PrPC, in the brains of affected individuals. PrPC is a cell surface glycoprotein attached to the outer leaflet of the plasma membrane by a glycosyl-phosphatidyl-inositol (GPI) anchor. Specifically, it is associated with lipid rafts, membrane microdomains enriched in cholesterol and sphinoglipids. It has been established that inhibition of endogenous cholesterol synthesis disturbs lipid raft association of PrPC and prevents PrPSc accumulation in neuronal cells. Additionally, prion conversion is reduced upon interference with cellular cholesterol uptake, endosomal export, or complexation at the plasma membrane. Altogether, these results demonstrate on the one hand the importance of cholesterol for prion propagation. On the other hand, growing evidence suggests that prion infection modulates neuronal cholesterol metabolism. Similar results were reported in Alzheimer’s disease (AD): whereas amyloid β peptide formation is influenced by cellular cholesterol, levels of cholesterol in the brains of affected individuals increase during the clinical course of the disease. In this review, we summarize commonalities of alterations in cholesterol homeostasis and discuss consequences for neuronal function and therapy of prion diseases and AD. PMID:25419621

  3. Lipid peroxidation causes endosomal antigen release for cross-presentation

    Science.gov (United States)

    Dingjan, Ilse; Verboogen, Daniëlle RJ; Paardekooper, Laurent M; Revelo, Natalia H; Sittig, Simone P; Visser, Linda J; Mollard, Gabriele Fischer von; Henriet, Stefanie SV; Figdor, Carl G; ter Beest, Martin; van den Bogaart, Geert

    2016-01-01

    Dendritic cells (DCs) present foreign antigen in major histocompatibility complex (MHC) class I molecules to cytotoxic T cells in a process called cross-presentation. An important step in this process is the release of antigen from the lumen of endosomes into the cytosol, but the mechanism of this step is still unclear. In this study, we show that reactive oxygen species (ROS) produced by the NADPH-oxidase complex NOX2 cause lipid peroxidation, a membrane disrupting chain-reaction, which in turn results in antigen leakage from endosomes. Antigen leakage and cross-presentation were inhibited by blocking ROS production or scavenging radicals and induced when using a ROS-generating photosensitizer. Endosomal antigen release was impaired in DCs from chronic granulomatous disease (CGD) patients with dysfunctional NOX2. Thus, NOX2 induces antigen release from endosomes for cross-presentation by direct oxidation of endosomal lipids. This constitutes a new cellular function for ROS in regulating immune responses against pathogens and cancer. PMID:26907999

  4. Enhancing endosomal escape for nanoparticle mediated siRNA delivery

    Science.gov (United States)

    Ma, Da

    2014-05-01

    Gene therapy with siRNA is a promising biotechnology to treat cancer and other diseases. To realize siRNA-based gene therapy, a safe and efficient delivery method is essential. Nanoparticle mediated siRNA delivery is of great importance to overcome biological barriers for systemic delivery in vivo. Based on recent discoveries, endosomal escape is a critical biological barrier to be overcome for siRNA delivery. This feature article focuses on endosomal escape strategies used for nanoparticle mediated siRNA delivery, including cationic polymers, pH sensitive polymers, calcium phosphate, and cell penetrating peptides. Work has been done to develop different endosomal escape strategies based on nanoparticle types, administration routes, and target organ/cell types. Also, enhancement of endosomal escape has been considered along with other aspects of siRNA delivery to ensure target specific accumulation, high cell uptake, and low toxicity. By enhancing endosomal escape and overcoming other biological barriers, great progress has been achieved in nanoparticle mediated siRNA delivery.

  5. Endosomal sensing of fungi: current understanding and emerging concepts.

    Science.gov (United States)

    Bercusson, Amelia; de Boer, Leon; Armstrong-James, Darius

    2017-01-01

    Endosomal sensing represents a key strategy by which mammalian cells detect parasitization by invading pathogens. This is critical for the control of fungal pathogens, which are for the most part phagocytosed by effector cells of the innate immune system. Despite rapid overall progress in our understanding of endosomal responses in recent times, relatively little is known about how the endosomal sensing system detects fungi and the ensuing immunological consequences. Considering that many fungal pathogens must overcome and evade endosomal killing in order to survive in the host, understanding this key area of the early innate response is crucial for our understanding of fungal infection. In this review we present a summary of our current knowledge of endosomal sensing within the context of fungal pathogens, with a focus on the myeloid compartment. © The Author 2016. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  6. Cholesterol testing and results

    Science.gov (United States)

    Cholesterol test results; LDL test results; VLDL test results; HDL test results; Coronary risk profile results; Hyperlipidemia-results; Lipid disorder test results; Heart disease - cholesterol results

  7. Regulation of direct transintestinal cholesterol excretion in mice

    NARCIS (Netherlands)

    van der Velde, Astrid E.; Vrins, Carlos L. J.; van den Oever, Karin; Seemann, Ingar; Elferink, Ronald P. J. Oude; van Eck, Miranda; Kuipers, Folkert; Groen, Albert K.

    2008-01-01

    Biliary secretion is generally considered to be an obligate step in the pathway of excess cholesterol excretion from the body. We have recently shown that an alternative route exists. Direct transintestinal cholesterol efflux ( TICE) contributes significantly to cholesterol removal in mice. Our aim

  8. Free cholesterol and cholesterol esters in bovine oocytes: Implications in survival and membrane raft organization after cryopreservation.

    Science.gov (United States)

    Buschiazzo, Jorgelina; Ríos, Glenda L; Canizo, Jesica R; Antollini, Silvia S; Alberio, Ricardo H

    2017-01-01

    Part of the damage caused by cryopreservation of mammalian oocytes occurs at the plasma membrane. The addition of cholesterol to cell membranes as a strategy to make it more tolerant to cryopreservation has been little addressed in oocytes. In order to increase the survival of bovine oocytes after cryopreservation, we proposed not only to increase cholesterol level of oocyte membranes before vitrification but also to remove the added cholesterol after warming, thus recovering its original level. Results from our study showed that modulation of membrane cholesterol by methyl-β-cyclodextrin (MβCD) did not affect the apoptotic status of oocytes and improved viability after vitrification yielding levels of apoptosis closer to those of fresh oocytes. Fluorometric measurements based on an enzyme-coupled reaction that detects both free cholesterol (membrane) and cholesteryl esters (stored in lipid droplets), revealed that oocytes and cumulus cells present different levels of cholesterol depending on the seasonal period. Variations at membrane cholesterol level of oocytes were enough to account for the differences found in total cholesterol. Differences found in total cholesterol of cumulus cells were explained by the differences found in both the content of membrane cholesterol and of cholesterol esters. Cholesterol was incorporated into the oocyte plasma membrane as evidenced by comparative labeling of a fluorescent cholesterol. Oocytes and cumulus cells increased membrane cholesterol after incubation with MβCD/cholesterol and recovered their original level after cholesterol removal, regardless of the season. Finally, we evaluated the effect of vitrification on the putative raft molecule GM1. Cholesterol modulation also preserved membrane organization by maintaining ganglioside level at the plasma membrane. Results suggest a distinctive cholesterol metabolic status of cumulus-oocyte complexes (COCs) among seasons and a dynamic organizational structure of cholesterol

  9. Free cholesterol and cholesterol esters in bovine oocytes: Implications in survival and membrane raft organization after cryopreservation.

    Directory of Open Access Journals (Sweden)

    Jorgelina Buschiazzo

    Full Text Available Part of the damage caused by cryopreservation of mammalian oocytes occurs at the plasma membrane. The addition of cholesterol to cell membranes as a strategy to make it more tolerant to cryopreservation has been little addressed in oocytes. In order to increase the survival of bovine oocytes after cryopreservation, we proposed not only to increase cholesterol level of oocyte membranes before vitrification but also to remove the added cholesterol after warming, thus recovering its original level. Results from our study showed that modulation of membrane cholesterol by methyl-β-cyclodextrin (MβCD did not affect the apoptotic status of oocytes and improved viability after vitrification yielding levels of apoptosis closer to those of fresh oocytes. Fluorometric measurements based on an enzyme-coupled reaction that detects both free cholesterol (membrane and cholesteryl esters (stored in lipid droplets, revealed that oocytes and cumulus cells present different levels of cholesterol depending on the seasonal period. Variations at membrane cholesterol level of oocytes were enough to account for the differences found in total cholesterol. Differences found in total cholesterol of cumulus cells were explained by the differences found in both the content of membrane cholesterol and of cholesterol esters. Cholesterol was incorporated into the oocyte plasma membrane as evidenced by comparative labeling of a fluorescent cholesterol. Oocytes and cumulus cells increased membrane cholesterol after incubation with MβCD/cholesterol and recovered their original level after cholesterol removal, regardless of the season. Finally, we evaluated the effect of vitrification on the putative raft molecule GM1. Cholesterol modulation also preserved membrane organization by maintaining ganglioside level at the plasma membrane. Results suggest a distinctive cholesterol metabolic status of cumulus-oocyte complexes (COCs among seasons and a dynamic organizational structure

  10. Apolipoprotein E Regulates Amyloid Formation within Endosomes of Pigment Cells

    Directory of Open Access Journals (Sweden)

    Guillaume van Niel

    2015-10-01

    Full Text Available Accumulation of toxic amyloid oligomers is a key feature in the pathogenesis of amyloid-related diseases. Formation of mature amyloid fibrils is one defense mechanism to neutralize toxic prefibrillar oligomers. This mechanism is notably influenced by apolipoprotein E variants. Cells that produce mature amyloid fibrils to serve physiological functions must exploit specific mechanisms to avoid potential accumulation of toxic species. Pigment cells have tuned their endosomes to maximize the formation of functional amyloid from the protein PMEL. Here, we show that ApoE is associated with intraluminal vesicles (ILV within endosomes and remain associated with ILVs when they are secreted as exosomes. ApoE functions in the ESCRT-independent sorting mechanism of PMEL onto ILVs and regulates the endosomal formation of PMEL amyloid fibrils in vitro and in vivo. This process secures the physiological formation of amyloid fibrils by exploiting ILVs as amyloid nucleating platforms.

  11. Synthesis and characterization of a novel rhodamine labeled cholesterol reporter.

    Science.gov (United States)

    Maiwald, Alexander; Bauer, Olivia; Gimpl, Gerald

    2017-06-01

    We introduce the novel fluorescent cholesterol probe RChol in which a sulforhodamine group is linked to the sixth carbon atom of the steroid backbone of cholesterol. The same position has recently been selected to generate the fluorescent reporter 6-dansyl-cholestanol (DChol) and the photoreactive 6-azi-cholestanol. In comparison with DChol, RChol is brighter, much more photostable, and requires less energy for excitation, i.e. favorable conditions for microscopical imaging. RChol easily incorporates into methyl-β-cyclodextrin forming a water-soluble inclusion complex that acts as an efficient sterol donor for cells and membranes. Like cholesterol, RChol possesses a free 3'OH group, a prerequisite to undergo intracellular esterification. RChol was also able to support the growth of cholesterol auxotrophic cells and can therefore substitute for cholesterol as a major component of the plasma membrane. According to subcellular fractionation, slight amounts of RChol (~12%) were determined in low-density Triton-insoluble fractions whereas the majority of RChol was localized in non-rafts fractions. In phase-separated giant unilamellar vesicles, RChol preferentially partitions in liquid-disordered membrane domains. Intracellular RChol was transferred to extracellular sterol acceptors such as high density lipoproteins in a dose-dependent manner. Unlike DChol, RChol was not delivered to the cholesterol storage pathway. Instead, it translocated to endosomes/lysosomes with some transient contacts to peroxisomes. Thus, RChol is considered as a useful probe to study the endosomal/lysosomal pathway of cholesterol. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. MLN64 induces mitochondrial dysfunction associated with increased mitochondrial cholesterol content

    Directory of Open Access Journals (Sweden)

    Elisa Balboa

    2017-08-01

    Full Text Available MLN64 is a late endosomal cholesterol-binding membrane protein that has been implicated in cholesterol transport from endosomal membranes to the plasma membrane and/or mitochondria, in toxin-induced resistance, and in mitochondrial dysfunction. Down-regulation of MLN64 in Niemann-Pick C1 deficient cells decreased mitochondrial cholesterol content, suggesting that MLN64 functions independently of NPC1. However, the role of MLN64 in the maintenance of endosomal cholesterol flow and intracellular cholesterol homeostasis remains unclear. We have previously described that hepatic MLN64 overexpression increases liver cholesterol content and induces liver damage. Here, we studied the function of MLN64 in normal and NPC1-deficient cells and we evaluated whether MLN64 overexpressing cells exhibit alterations in mitochondrial function. We used recombinant-adenovirus-mediated MLN64 gene transfer to overexpress MLN64 in mouse liver and hepatic cells; and RNA interference to down-regulate MLN64 in NPC1-deficient cells. In MLN64-overexpressing cells, we found increased mitochondrial cholesterol content and decreased glutathione (GSH levels and ATPase activity. Furthermore, we found decreased mitochondrial membrane potential and mitochondrial fragmentation and increased mitochondrial superoxide levels in MLN64-overexpressing cells and in NPC1-deficient cells. Consequently, MLN64 expression was increased in NPC1-deficient cells and reduction of its expression restore mitochondrial membrane potential and mitochondrial superoxide levels. Our findings suggest that MLN64 overexpression induces an increase in mitochondrial cholesterol content and consequently a decrease in mitochondrial GSH content leading to mitochondrial dysfunction. In addition, we demonstrate that MLN64 expression is increased in NPC cells and plays a key role in cholesterol transport into the mitochondria.

  13. MLN64 induces mitochondrial dysfunction associated with increased mitochondrial cholesterol content.

    Science.gov (United States)

    Balboa, Elisa; Castro, Juan; Pinochet, María-José; Cancino, Gonzalo I; Matías, Nuria; Sáez, P J; Martínez, Alexis; Álvarez, Alejandra R; Garcia-Ruiz, Carmen; Fernandez-Checa, José C; Zanlungo, Silvana

    2017-08-01

    MLN64 is a late endosomal cholesterol-binding membrane protein that has been implicated in cholesterol transport from endosomal membranes to the plasma membrane and/or mitochondria, in toxin-induced resistance, and in mitochondrial dysfunction. Down-regulation of MLN64 in Niemann-Pick C1 deficient cells decreased mitochondrial cholesterol content, suggesting that MLN64 functions independently of NPC1. However, the role of MLN64 in the maintenance of endosomal cholesterol flow and intracellular cholesterol homeostasis remains unclear. We have previously described that hepatic MLN64 overexpression increases liver cholesterol content and induces liver damage. Here, we studied the function of MLN64 in normal and NPC1-deficient cells and we evaluated whether MLN64 overexpressing cells exhibit alterations in mitochondrial function. We used recombinant-adenovirus-mediated MLN64 gene transfer to overexpress MLN64 in mouse liver and hepatic cells; and RNA interference to down-regulate MLN64 in NPC1-deficient cells. In MLN64-overexpressing cells, we found increased mitochondrial cholesterol content and decreased glutathione (GSH) levels and ATPase activity. Furthermore, we found decreased mitochondrial membrane potential and mitochondrial fragmentation and increased mitochondrial superoxide levels in MLN64-overexpressing cells and in NPC1-deficient cells. Consequently, MLN64 expression was increased in NPC1-deficient cells and reduction of its expression restore mitochondrial membrane potential and mitochondrial superoxide levels. Our findings suggest that MLN64 overexpression induces an increase in mitochondrial cholesterol content and consequently a decrease in mitochondrial GSH content leading to mitochondrial dysfunction. In addition, we demonstrate that MLN64 expression is increased in NPC cells and plays a key role in cholesterol transport into the mitochondria. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  14. Biomechanics and Thermodynamics of Nanoparticle Interactions with Plasma and Endosomal Membrane Lipids in Cellular Uptake and Endosomal Escape

    Science.gov (United States)

    2015-01-01

    To be effective for cytoplasmic delivery of therapeutics, nanoparticles (NPs) taken up via endocytic pathways must efficiently transport across the cell membrane and subsequently escape from the secondary endosomes. We hypothesized that the biomechanical and thermodynamic interactions of NPs with plasma and endosomal membrane lipids are involved in these processes. Using model plasma and endosomal lipid membranes, we compared the interactions of cationic NPs composed of poly(d,l-lactide-co-glycolide) modified with the dichain surfactant didodecyldimethylammonium bromide (DMAB) or the single-chain surfactant cetyltrimethylammonium bromide (CTAB) vs anionic unmodified NPs of similar size. We validated our hypothesis in doxorubicin-sensitive (MCF-7, with relatively fluid membranes) and resistant breast cancer cells (MCF-7/ADR, with rigid membranes). Despite their cationic surface charges, DMAB- and CTAB-modified NPs showed different patterns of biophysical interaction: DMAB-modified NPs induced bending of the model plasma membrane, whereas CTAB-modified NPs condensed the membrane, thereby resisted bending. Unmodified NPs showed no effects on bending. DMAB-modified NPs also induced thermodynamic instability of the model endosomal membrane, whereas CTAB-modified and unmodified NPs had no effect. Since bending of the plasma membrane and destabilization of the endosomal membrane are critical biophysical processes in NP cellular uptake and endosomal escape, respectively, we tested these NPs for cellular uptake and drug efficacy. Confocal imaging showed that in both sensitive and resistant cells DMAB-modified NPs exhibited greater cellular uptake and escape from endosomes than CTAB-modified or unmodified NPs. Further, paclitaxel-loaded DMAB-modified NPs induced greater cytotoxicity even in resistant cells than CTAB-modified or unmodified NPs or drug in solution, demonstrating the potential of DMAB-modified NPs to overcome the transport barrier in resistant cells. In

  15. Cholesterol efflux is differentially regulated in neurons and astrocytes: implications for brain cholesterol homeostasis

    Science.gov (United States)

    Chen, Jing; Zhang, Xiaolu; Kusumo, Handojo; Costa, Lucio G.; Guizzetti, Marina

    2012-01-01

    Disruption of cholesterol homeostasis in the central nervous system (CNS) has been associated with neurological, neurodegenerative, and neurodevelopmental disorders. The CNS is a closed system with regard to cholesterol homeostasis, as cholesterol-delivering lipoproteins from the periphery cannot pass the blood-brain-barrier and enter the brain. Different cell types in the brain have different functions in the regulation of cholesterol homeostasis, with astrocytes producing and releasing apolipoprotein E and lipoproteins, and neurons metabolizing cholesterol to 24(S)-hydroxycholesterol. We present evidence that astrocytes and neurons adopt different mechanisms also in regulating cholesterol efflux. We found that in astrocytes cholesterol efflux is induced by both lipid-free apolipoproteins and lipoproteins, while cholesterol removal from neurons is triggered only by lipoproteins. The main pathway by which apolipoproteins induce cholesterol efflux is through ABCA1. By upregulating ABCA1 levels and by inhibiting its activity and silencing its expression, we show that ABCA1 is involved in cholesterol efflux from astrocytes but not from neurons. Furthermore, our results suggest that ABCG1 is involved in cholesterol efflux to apolipoproteins and lipoproteins from astrocytes but not from neurons, while ABCG4, whose expression is much higher in neurons than astrocytes, is involved in cholesterol efflux from neurons but not astrocytes. These results indicate that different mechanisms regulate cholesterol efflux from neurons and astrocytes, reflecting the different roles that these cell types play in brain cholesterol homeostasis. These results are important in understanding cellular targets of therapeutic drugs under development for the treatments of conditions associated with altered cholesterol homeostasis in the CNS. PMID:23010475

  16. A new Rab7 effector controls phosphoinositide conversion in endosome maturation.

    Science.gov (United States)

    Casanova, James E; Winckler, Bettina

    2017-10-02

    Endosome maturation requires a coordinated change in the Rab GTPase and phosphoinositide composition of the endosomal membrane. In this issue, Liu et al. (2017. J. Cell Biol. https://doi.org/10.1083/jcb.201705151) identify WDR91 as a ubiquitous Rab7 effector that inhibits phosphatidylinositol 3-kinase activity on endosomes and is critical for endosome maturation, viability, and dendrite growth of neurons in vivo. © 2017 Casanova and Winckler.

  17. Structural Basis for Endosomal Targeting by the Bro1 Domain

    Science.gov (United States)

    Kim, Jaewon; Sitaraman, Sujatha; Hierro, Aitor; Beach, Bridgette M.; Odorizzi, Greg; Hurley, James H.

    2010-01-01

    Summary Proteins delivered to the lysosome or the yeast vacuole via late endosomes are sorted by the ESCRT complexes and by associated proteins, including Alix and its yeast homolog Bro1. Alix, Bro1, and several other late endosomal proteins share a conserved 160 residue Bro1 domain whose boundaries, structure, and function have not been characterized. The crystal structure of the Bro1 domain of Bro1 reveals a folded core of 367 residues. The extended Bro1 domain is necessary and sufficient for binding to the ESCRT-III subunit Snf7 and for the recruitment of Bro1 to late endosomes. The structure resembles a boomerang with its concave face filled in and contains a triple tetratricopeptide repeat domain as a substructure. Snf7 binds to a conserved hydrophobic patch on Bro1 that is required for protein complex formation and for the protein-sorting function of Bro1. These results define a conserved mechanism whereby Bro1 domain-containing proteins are targeted to endosomes by Snf7 and its orthologs. PMID:15935782

  18. Endosomes: multipurpose designs for integrating housekeeping and specialized tasks

    NARCIS (Netherlands)

    Sachse, M.; Ramm, G.; Strous, G.J.; Klumperman, J.

    2002-01-01

    In most cells the endocytic system is organized following a common concept that allows for the integrative handling of a variety of housekeeping functions. In addition, variations on the general scheme provide for specialized endosome-based pathways that occur only in specific cell types. The

  19. Characterization of the Mammalian CORVET and HOPS Complexes and Their Modular Restructuring for Endosome Specificity

    NARCIS (Netherlands)

    van der Kant, Rik; Jonker, Caspar T. H.; Wijdeven, Ruud H.; Bakker, Jeroen; Janssen, Lennert; Klumperman, Judith; Neefjes, Jacques

    2015-01-01

    Trafficking of cargo through the endosomal system depends on endosomal fusion events mediated by SNARE proteins, Rab-GTPases, and multisubunit tethering complexes. The CORVET and HOPS tethering complexes, respectively, regulate early and late endosomal tethering and have been characterized in detail

  20. Glucose regulates clathrin adaptors at the trans-Golgi network and endosomes

    Science.gov (United States)

    Aoh, Quyen L.; Graves, Lee M.; Duncan, Mara C.

    2011-01-01

    Glucose is a rich source of energy and the raw material for biomass increase. Many eukaryotic cells remodel their physiology in the presence and absence of glucose. The yeast Saccharomyces cerevisiae undergoes changes in transcription, translation, metabolism, and cell polarity in response to glucose availability. Upon glucose starvation, translation initiation and cell polarity are immediately inhibited, and then gradually recover. In this paper, we provide evidence that, as in cell polarity and translation, traffic at the trans-Golgi network (TGN) and endosomes is regulated by glucose via an unknown mechanism that depends on protein kinase A (PKA). Upon glucose withdrawal, clathrin adaptors exhibit a biphasic change in localization: they initially delocalize from the membrane within minutes and later partially recover onto membranes. Additionally, the removal of glucose induces changes in posttranslational modifications of adaptors. Ras and Gpr1 signaling pathways, which converge on PKA, are required for changes in adaptor localization and changes in posttranslational modifications. Acute inhibition of PKA demonstrates that inhibition of PKA prior to glucose withdrawal prevents several adaptor responses to starvation. This study demonstrates that PKA activity prior to glucose starvation primes membrane traffic at the TGN and endosomes in response to glucose starvation. PMID:21832155

  1. Get Your Cholesterol Checked

    Science.gov (United States)

    ... cholesterol levels with a blood test called a lipid profile. For the test, a nurse will take a ... blood tests that can check cholesterol, but a lipid profile gives the most information. Find out more about ...

  2. Controlling Cholesterol with Statins

    Science.gov (United States)

    ... For Consumers Home For Consumers Consumer Updates Controlling Cholesterol with Statins Share Tweet Linkedin Pin it More ... not, the following tips can help keep your cholesterol in check: Talk with your healthcare provider about ...

  3. Low Cholesterol Diet

    Science.gov (United States)

    ... changes (TLC) and medicines. TLC includes a healthy diet, weight management, and regular physical activity. What is a low cholesterol diet? Therapeutic lifestyle changes (TLC) includes a low cholesterol ...

  4. LDL: The "Bad" Cholesterol

    Science.gov (United States)

    ... waxy, fat-like substance that's found in all the cells in your body. Your liver makes cholesterol, ... stands for low-density lipoproteins. It is called the "bad" cholesterol because a high LDL level leads ...

  5. Cholesterol oxides inhibit cholesterol esterification by lecithin: cholesterol acyl transferase

    Directory of Open Access Journals (Sweden)

    Eder de Carvalho Pincinato

    2009-09-01

    Full Text Available Cholesterol oxides are atherogenic and can affect the activity of diverse important enzymes for the lipidic metabolism. The effect of 7β-hydroxycholesterol, 7-ketocholesterol, 25-hydroxycholesterol, cholestan-3β,5α,6β-triol,5,6β-epoxycholesterol, 5,6α-epoxycholesterol and 7α-hydroxycholesterol on esterification of cholesterol by lecithin:cholesterol acyl transferase (LCAT, EC 2.3.1.43 and the transfer of esters of cholesterol oxides from high density lipoprotein (HDL to low density lipoproteins (LDL and very low density lipoproteins (VLDL by cholesteryl ester transfer protein (CETP was investigated. HDL enriched with increasing concentrations of cholesterol oxides was incubated with fresh plasma as source of LCAT. Cholesterol and cholesterol oxides esterification was followed by measuring the consumption of respective free sterol and oxysterols. Measurements of cholesterol and cholesterol oxides were done by gas-chromatography. 14C-cholesterol oxides were incorporated into HDL2 and HDL3 subfractions and then incubated with fresh plasma containing LCAT and CETP. The transfer of cholesterol oxide esters was followed by measuring the 14C-cholesterol oxide-derived esters transferred to LDL and VLDL. All the cholesterol oxides studied were esterified by LCAT after incorporation into HDL particles, competing with cholesterol by LCAT. Cholesterol esterification by LCAT was inversely related to the cholesterol oxide concentration. The esterification of 14C-cholesterol oxides was higher in HDL3 and the transfer of the derived esters was greater from HDL2 to LDL and VLDL. The results suggest that cholesterol esterification by LCAT is inhibited in cholesterol oxide-enriched HDL particles. Moreover, the cholesterol oxides-derived esters are efficiently transferred to LDL and VLDL. Therefore, we suggest that cholesterol oxides may exert part of their atherogenic effect by inhibiting cholesterol esterification on the HDL surface and thereby disturbing

  6. Endosomal generation of cAMP in GPCR signaling

    Science.gov (United States)

    Vilardaga, Jean-Pierre; Jean-Alphonse, Frederic G.; Gardella, Thomas J.

    2015-01-01

    It has been widely assumed that the production of the ubiquitous second messenger cyclic AMP, which is mediated by cell surface G protein–coupled receptors (GPCRs), and its termination take place exclusively at the plasma membrane. Recent studies reveal that diverse GPCRs do not always follow this conventional paradigm. In the new model, GPCRs mediate G-protein signaling not only from the plasma membrane but also from endosomal membranes. This model proposes that following ligand binding and activation, cell surface GPCRs internalize and redistribute into early endosomes, where trimeric G protein signaling can be maintained for an extended period of time. This Perspective discusses the molecular and cellular mechanistic subtleties as well as the physiological consequences of this unexpected process, which is considerably changing how we think about GPCR signaling and regulation and how we study drugs that target this receptor family. PMID:25271346

  7. Promyelocytic leukemia bodies tether to early endosomes during mitosis.

    Science.gov (United States)

    Palibrk, Vuk; Lång, Emma; Lång, Anna; Schink, Kay Oliver; Rowe, Alexander D; Bøe, Stig Ove

    2014-01-01

    During mitosis the nuclear envelope breaks down, leading to potential interactions between cytoplasmic and nuclear components. PML bodies are nuclear structures with tumor suppressor and antiviral functions. Early endosomes, on the other hand, are cytoplasmic vesicles involved in transport and growth factor signaling. Here we demonstrate that PML bodies form stable interactions with early endosomes immediately following entry into mitosis. The 2 compartments remain stably associated throughout mitosis and dissociate in the cytoplasm of newly divided daughter cells. We also show that a minor subset of PML bodies becomes anchored to the mitotic spindle poles during cell division. The study demonstrates a stable mitosis-specific interaction between a cytoplasmic and a nuclear compartment.

  8. ALIX and the multivesicular endosome: ALIX in Wonderland.

    Science.gov (United States)

    Bissig, Christin; Gruenberg, Jean

    2014-01-01

    In yeast and mammalian cells, endosomal sorting complexes required for transport (ESCRT) assist in sorting ubiquitinated proteins into intralumenal vesicles (ILVs) of multivesicular endosomes (MVEs) for degradation in the lysosome/vacuole. In mammalian cells, ESCRTs also drive other topologically identical membrane deformation processes, including cytokinesis, exosome release, and virus budding. Although the ESCRT-associated protein ALIX regulates these mammalian cell-specific functions, it was believed to be dispensable for receptor sorting into ILVs, unlike its yeast homolog Bro1. Despite these differences, recent evidence suggests ALIX and Bro1 share common properties in cargo sorting and ILV formation. We review these commonalities and discuss the role of ALIX in operating 'behind the mirror' during ILV back-fusion with the limiting membrane. We also propose models of how ALIX and some ESCRTs regulate the back-fusion process. Copyright © 2013 Elsevier Ltd. All rights reserved.

  9. Ebola virus entry requires the cholesterol transporter Niemann-Pick C1.

    Science.gov (United States)

    Carette, Jan E; Raaben, Matthijs; Wong, Anthony C; Herbert, Andrew S; Obernosterer, Gregor; Mulherkar, Nirupama; Kuehne, Ana I; Kranzusch, Philip J; Griffin, April M; Ruthel, Gordon; Dal Cin, Paola; Dye, John M; Whelan, Sean P; Chandran, Kartik; Brummelkamp, Thijn R

    2011-08-24

    Infections by the Ebola and Marburg filoviruses cause a rapidly fatal haemorrhagic fever in humans for which no approved antivirals are available. Filovirus entry is mediated by the viral spike glycoprotein (GP), which attaches viral particles to the cell surface, delivers them to endosomes and catalyses fusion between viral and endosomal membranes. Additional host factors in the endosomal compartment are probably required for viral membrane fusion; however, despite considerable efforts, these critical host factors have defied molecular identification. Here we describe a genome-wide haploid genetic screen in human cells to identify host factors required for Ebola virus entry. Our screen uncovered 67 mutations disrupting all six members of the homotypic fusion and vacuole protein-sorting (HOPS) multisubunit tethering complex, which is involved in the fusion of endosomes to lysosomes, and 39 independent mutations that disrupt the endo/lysosomal cholesterol transporter protein Niemann-Pick C1 (NPC1). Cells defective for the HOPS complex or NPC1 function, including primary fibroblasts derived from human Niemann-Pick type C1 disease patients, are resistant to infection by Ebola virus and Marburg virus, but remain fully susceptible to a suite of unrelated viruses. We show that membrane fusion mediated by filovirus glycoproteins and viral escape from the vesicular compartment require the NPC1 protein, independent of its known function in cholesterol transport. Our findings uncover unique features of the entry pathway used by filoviruses and indicate potential antiviral strategies to combat these deadly agents.

  10. Regulation of Liver Metabolism by the Endosomal GTPase Rab5

    Directory of Open Access Journals (Sweden)

    Anja Zeigerer

    2015-05-01

    Full Text Available The liver maintains glucose and lipid homeostasis by adapting its metabolic activity to the energy needs of the organism. Communication between hepatocytes and extracellular environment via endocytosis is key to such homeostasis. Here, we addressed the question of whether endosomes are required for gluconeogenic gene expression. We took advantage of the loss of endosomes in the mouse liver upon Rab5 silencing. Strikingly, we found hepatomegaly and severe metabolic defects such as hypoglycemia, hypercholesterolemia, hyperlipidemia, and glycogen accumulation that phenocopied those found in von Gierke’s disease, a glucose-6-phosphatase (G6Pase deficiency. G6Pase deficiency alone can account for the reduction in hepatic glucose output and glycogen accumulation as determined by mathematical modeling. Interestingly, we uncovered functional alterations in the transcription factors, which regulate G6Pase expression. Our data highlight a requirement of Rab5 and the endosomal system for the regulation of gluconeogenic gene expression that has important implications for metabolic diseases.

  11. What Your Cholesterol Levels Mean

    Science.gov (United States)

    ... Disease Venous Thromboembolism Aortic Aneurysm More What Your Cholesterol Levels Mean Updated:Nov 16,2017 Keeping your ... stroke. This content was last reviewed April 2017. Cholesterol • Home • About Cholesterol Introduction Atherosclerosis What Your Cholesterol ...

  12. Home-Use Tests - Cholesterol

    Science.gov (United States)

    ... Medical Procedures In Vitro Diagnostics Home Use Tests Cholesterol Share Tweet Linkedin Pin it More sharing options ... a home-use test kit to measure total cholesterol. What cholesterol is: Cholesterol is a fat (lipid) ...

  13. Endosome-mitochondria interactions are modulated by iron release from transferrin.

    Science.gov (United States)

    Das, Anupam; Nag, Sagarika; Mason, Anne B; Barroso, Margarida M

    2016-09-26

    Transient "kiss and run" interactions between endosomes containing iron-bound transferrin (Tf) and mitochondria have been shown to facilitate direct iron transfer in erythroid cells. In this study, we used superresolution three-dimensional (3D) direct stochastic optical reconstruction microscopy to show that Tf-containing endosomes directly interact with mitochondria in epithelial cells. We used live-cell time-lapse fluorescence microscopy, followed by 3D rendering, object tracking, and a distance transformation algorithm, to track Tf-endosomes and characterize the dynamics of their interactions with mitochondria. Quenching of iron sensor RDA-labeled mitochondria confirmed functional iron transfer by an interacting Tf-endosome. The motility of Tf-endosomes is significantly reduced upon interaction with mitochondria. To further assess the functional role of iron in the ability of Tf-endosomes to interact with mitochondria, we blocked endosomal iron release by using a Tf K206E/K534A mutant. Blocking intraendosomal iron release led to significantly increased motility of Tf-endosomes and increased duration of endosome-mitochondria interactions. Thus, intraendosomal iron regulates the kinetics of the interactions between Tf-containing endosomes and mitochondria in epithelial cells. © 2016 Das et al.

  14. Retromer revisited: Evolving roles for retromer in endosomal sorting.

    Science.gov (United States)

    Chamberland, John P; Ritter, Brigitte

    2017-11-06

    The highly conserved retromer complex has been linked to cargo retrieval from endosomes to the trans-Golgi network. In this issue, Kvainickas et al. (2017. J. Cell Biol. https://doi.org/10.1083/jcb.201702137) and Simonetti et al. (2017. J. Cell Biol. https://doi.org/10.1083/jcb.201703015) fundamentally question the current retromer model and demonstrate that in mammalian cells, the individual retromer subcomplexes have functionally diverged to organize multiple distinct sorting pathways. © 2017 Chamberland and Ritter.

  15. The ins and outs of reverse cholesterol transport

    NARCIS (Netherlands)

    Groen, AK; Elferink, RPJO; Verkade, HJ; Kuipers, F

    2004-01-01

    It is generally assumed that HDL is the obligate transport vehicle for 'reverse cholesterol transport'. the pathway for removal of excess cholesterol from peripheral tissues via the liver into bile and subsequent excretion via the feces. During the last few years, intensive research has generated

  16. Mitochondrial cholesterol import.

    Science.gov (United States)

    Elustondo, Pia; Martin, Laura A; Karten, Barbara

    2017-01-01

    All animal subcellular membranes require cholesterol, which influences membrane fluidity and permeability, fission and fusion processes, and membrane protein function. The distribution of cholesterol among subcellular membranes is highly heterogeneous and the cholesterol content of each membrane must be carefully regulated. Compared to other subcellular membranes, mitochondrial membranes are cholesterol-poor, particularly the inner mitochondrial membrane (IMM). As a result, steroidogenesis can be controlled through the delivery of cholesterol to the IMM, where it is converted to pregnenolone. The low basal levels of cholesterol also make mitochondria sensitive to changes in cholesterol content, which can have a relatively large impact on the biophysical and functional characteristics of mitochondrial membranes. Increased mitochondrial cholesterol levels have been observed in diverse pathological conditions including cancer, steatohepatitis, Alzheimer disease and Niemann-Pick Type C1-deficiency, and are associated with increased oxidative stress, impaired oxidative phosphorylation, and changes in the susceptibility to apoptosis, among other alterations in mitochondrial function. Mitochondria are not included in the vesicular trafficking network; therefore, cholesterol transport to mitochondria is mostly achieved through the activity of lipid transfer proteins at membrane contact sites or by cytosolic, diffusible lipid transfer proteins. Here we will give an overview of the main mechanisms involved in mitochondrial cholesterol import, focusing on the steroidogenic acute regulatory protein StAR/STARD1 and other members of the StAR-related lipid transfer (START) domain protein family, and we will discuss how changes in mitochondrial cholesterol levels can arise and affect mitochondrial function. This article is part of a Special Issue entitled: Lipids of Mitochondria edited by Guenther Daum. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. Transintestinal and Biliary Cholesterol Secretion Both Contribute to Macrophage Reverse Cholesterol Transport in RatsBrief Report

    NARCIS (Netherlands)

    Boer, de Jan Freark; Schonewille, Marleen; Dikkers, Arne; Koehorst, Martijn; Havinga, Rick; Kuipers, Folkert; Tietge, Uwe J F; Groen, Albert K

    Objective-Reverse cholesterol transport comprises efflux of cholesterol from macrophages and its subsequent removal from the body with the feces and thereby protects against formation of atherosclerotic plaques. Because of lack of suitable animal models that allow for evaluation of the respective

  18. Electrochemical oxidation of cholesterol

    Directory of Open Access Journals (Sweden)

    Jacek W. Morzycki

    2015-03-01

    Full Text Available Indirect cholesterol electrochemical oxidation in the presence of various mediators leads to electrophilic addition to the double bond, oxidation at the allylic position, oxidation of the hydroxy group, or functionalization of the side chain. Recent studies have proven that direct electrochemical oxidation of cholesterol is also possible and affords different products depending on the reaction conditions.

  19. National Cholesterol Education Month

    Centers for Disease Control (CDC) Podcasts

    2009-09-01

    Do you know your cholesterol numbers? Your doctor can do a simple test to check your cholesterol levels and help you make choices that lower your risk for heart disease and stroke.  Created: 9/1/2009 by National Center for Chronic Disease Prevention and Health Promotion (NCCDPHP).   Date Released: 9/9/2009.

  20. Cholesterol and Health

    Indian Academy of Sciences (India)

    cholesterol. In the gall bladder cholesterol crystalizes from the bile producing large crystalline aggregates. The causes of gall- stones are too complex for us to go into, but it can be stated that such factors as high levels of estrogens, multiple pregnancies, obesity, genetic factors and certain drugs influence the degree to.

  1. A role for endosomal proteins in alphavirus dissemination in mosquitoes

    Science.gov (United States)

    Campbell, Corey L.; Lehmann, Christopher J.; Gill, Sargeet S.; Dunn, W. A.; James, Anthony A.; Foy, Brian D.

    2011-01-01

    Little is known about endosomal pathway proteins involved in arthropod-borne virus (arbovirus) assembly and cell-to-cell spread in vector mosquitoes. UNC93A and Synaptic vesicle-2 (SV2) proteins are involved in intracellular transport in mammals. They show amino acid sequence conservation from mosquitoes to humans, and their transcripts are highly-enriched in Aedes aegypti during arbovirus infection. Transient gene silencing of SV2 or UNC93A in mosquitoes infected with the recombinant alphavirus Sindbis MRE16-eGFP (SINV; family Togaviridae) resulted in the accumulation of viral positive- and negative-strand RNA, congregation of virus envelope antigen in intracellular networks, and reduced virus dissemination outside of the midgut. Further, UNC93A silencing, but not SV2 silencing, resulted in a 10-fold reduction in viral titers at 4 days post-infection. Together, these data support a role for UNC93A and SV2 in virus assembly or budding. Cis-regulatory elements (CREs) were identified at the 5′-ends of genes from the original dataset in which SV2 and UNC93A were identified. Common CREs at the 5′-end genomic regions of a subset of enriched transcripts support the hypothesis that UNC93A transcription may be co-regulated with that of other ion transport and endosomal trafficking proteins. PMID:21496127

  2. [Involvement of the endosomal compartment in cellular insulin signaling].

    Science.gov (United States)

    Desbuquois, Bernard; Authier, François

    2014-01-01

    The insulin receptor and insulin signaling proteins downstream the receptor reside in different subcellular compartments and undergo redistribution within the cell upon insulin activation. Endocytosis of the insulin-receptor complex, by mediating ligand degradation and receptor dephosphorylation, is generally viewed as a mechanism which attenuates or arrests insulin signal transduction. However, several observations suggest that insulin receptor endocytosis and/or recruitement of insulin signaling proteins to endosomes are also involved in a positive regulation of insulin signaling: (1) upon internalization, the insulin receptor remains transiently phosphorylated and activated; (2) in insulin-stimulated cells or tissues, signaling proteins of the PI3K/Akt and Ras/Raf/Mek/Erk pathways are recruited to endosomes or other intracellular compartments, in which they undergo phosphorylation and/or activation; and (3) depletion or overexpression of proteins involved in the regulation of membrane trafficking and endocytosis interfere with insulin signaling. These observations support a spatial and temporal regulation of insulin signal transduction and reinforce the concept that, as for other membrane signaling receptors, endocytosis and signaling are functionally linked. © Société de Biologie, 2014.

  3. Evidence for condensed complexes of cholesterol in lipid membranes

    Science.gov (United States)

    Ratajczak, Maria K.

    Although cholesterol is a predominant lipid in the eukaryotic plasma membrane, its interactions with other lipids are still not well understood. Insights into the nature of lipid assembly can be gained from examining lipid-cholesterol interaction using model systems. A key observation was the discovery of liquid-liquid phase diagrams with two critical points in the binary mixtures of cholesterol and lipids. The shape of the phase diagrams can be explained by a thermodynamic model of "condensed complexes". In our quest to characterize cholesterol-lipid interactions, we determined phase diagrams of cholesterol and phospholipids that point to the existence of condensed complexes. This complex formation hypothesis was further supported by experiments involving cholesterol removal by cyclodextrin, grazing x-ray diffraction and x-ray reflectivity studies and isothermal calorimetry. Our study aimed at establishing a correlation (or the lack of) between domain formation and complex formation, as well as determining the mode of cholesterol association with different lipids based on their structural and physical properties. We established a displacement assay by which we were able to probe cholesterol-lipid interactions by perturbing them in the presence of an intercalator that competes with cholesterol for association with lipids. Our data support the condensed complex model between cholesterol and lipids, and cholesterol when complexed with lipids shows low activity whereas free, uncomplexed cholesterol exhibits high activity. We were successful in modulating cholesterol activity by varying the level of intercalator while keeping the cholesterol content fixed. In this thesis, not only have we shown that cholesterol can be displaced by intercalators in model systems, we have further established that such displacement can take place in membranes of live cell.

  4. Effect of diphtheria toxin T-domain on endosomal pH

    Directory of Open Access Journals (Sweden)

    A. J. Labyntsev

    2015-08-01

    Full Text Available A key step in the mode of cytotoxic action of diphtheria toxin (DT is the transfer of its catalytic domain (Cd from endosomes into the cytosol. The main activity in this process is performed by the transport domain (Td, but the molecular mechanism of its action remains unknown. We have previously shown that Td can have some influence on the endosomal transport of DT. The aim of this work was to study the effect of diphtheria toxin on the toxin compartmentalization in the intracellular transporting pathway and endosomal pH. We used recombinant fragments of DT, which differed only by the presence of Td in their structure, fused with fluorescent proteins. It was shown that the toxin fragment with Td moved slower by the pathway early-late endosomes-lysosomes, and had a slightly different pattern of colocalization with endosomal markers than DT fragment without Td. In addition, endosomes containing DT fragments with Td had a constant pH of about 6.5 from the 10th to 50th minute of observation, for the same time endosomes containing DT fragments without Td demons­trated a decrease in pH from 6.3 to 5.5. These results indicate that Td inhibits acidification of endosomal medium. One of possible explanations for this may be the effect of the ion channel formed by the T-domain on the process of the endosomal acidification. This property of Td may not only inhibit maturation of endosomes but also inhibit activation of endosomal pH-dependent proteases, and this promotes successful transport of Cd into the cell cytosol.

  5. Phosphatidylcholine: cholesterol phase diagrams.

    Science.gov (United States)

    Thewalt, J L; Bloom, M

    1992-10-01

    Two mono-cis-unsaturated phosphatidylcholine (PC) lipid molecules, having very different gel-liquid crystalline phase transition temperatures as a consequence of the relative positions of the double bond, exhibit PC:cholesterol phase diagrams that are very similar to each other and to that obtained previously for a fully saturated PC:cholesterol mixture (Vist, M. R., and J. H. Davis. 1990. Biochemistry 29:451-464). This leads to the conjecture that PC:cholesterol membrane phase diagrams have a universal form which is relatively independent of the precise chemical structure of the PC molecule. One feature of this phase diagram is the observation over a wide temperature range of a fluid but highly conformationally ordered phase at bilayer concentrations of more than approximately 25 mol% cholesterol. This ;liquid ordered' phase is postulated to be the relevant physical state for many biological membranes, such as the plasma membrane of eukaryotic cells, that contain substantial amounts of cholesterol or equivalent sterols.

  6. An Endosomal NAADP-Sensitive Two-Pore Ca2+ Channel Regulates ER-Endosome Membrane Contact Sites to Control Growth Factor Signaling

    Directory of Open Access Journals (Sweden)

    Bethan S. Kilpatrick

    2017-02-01

    Full Text Available Membrane contact sites are regions of close apposition between organelles that facilitate information transfer. Here, we reveal an essential role for Ca2+ derived from the endo-lysosomal system in maintaining contact between endosomes and the endoplasmic reticulum (ER. Antagonizing action of the Ca2+-mobilizing messenger NAADP, inhibiting its target endo-lysosomal ion channel, TPC1, and buffering local Ca2+ fluxes all clustered and enlarged late endosomes/lysosomes. We show that TPC1 localizes to ER-endosome contact sites and is required for their formation. Reducing NAADP-dependent contacts delayed EGF receptor de-phosphorylation consistent with close apposition of endocytosed receptors with the ER-localized phosphatase PTP1B. In accord, downstream MAP kinase activation and mobilization of ER Ca2+ stores by EGF were exaggerated upon NAADP blockade. Membrane contact sites between endosomes and the ER thus emerge as Ca2+-dependent hubs for signaling.

  7. Mouse polyomavirus enters early endosomes, requires their acidic pH for productive infection, and meets transferrin cargo in rab11-positive endosomes

    Czech Academy of Sciences Publication Activity Database

    Liebl, D.; Difato, F.; Horníková, L.; Mannová, P.; Štokrová, Jitka; Forstová, J.

    2006-01-01

    Roč. 80, č. 9 (2006), s. 4610-4622 ISSN 0022-538X R&D Projects: GA ČR(CZ) GA204/03/0593; GA MŠk(CZ) LC545 Institutional research plan: CEZ:AV0Z50520514 Keywords : Polyomavirus internalization and trafficking * Early endosomes * Dependence of infection on endosomal pH Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 5.341, year: 2006

  8. Bile acid sequestrants for cholesterol

    Science.gov (United States)

    ... ency/patientinstructions/000787.htm Bile acid sequestrants for cholesterol To use the sharing features on this page, ... are medicines that help lower your LDL (bad) cholesterol . Too much cholesterol in your blood can stick ...

  9. Endosomal Trafficking of HIV-1 Gag and Genomic RNAs Regulates Viral Egress

    DEFF Research Database (Denmark)

    Molle, Dorothée; Segura-Morales, Carollna; Camus, Gregory

    2009-01-01

    HIV-1 Gag can assemble and generate virions at the plasma membrane, but it is also present in endosomes where its role remains incompletely characterized. Here, we show that HIV-1 RNAs and Gag are transported on endosomal vesicles positive for TiVamp, a v-SNARE involved in fusion events with the ...

  10. Bilayered clathrin coats on endosomal vacuoles are involved in protein sorting toward lysosomes

    NARCIS (Netherlands)

    Sachse, M.; Urbé, S.; Oorschot, V.; Strous, G.J.; Klumperman, J.

    In many cells endosomal vacuoles show clathrin coats of which the function is unknown. Herein, we show that this coat is predominantly present on early endosomes and has a characteristic bilayered appearance in the electron microscope. By immunoelectron miscroscopy we show that the coat contains

  11. Membrane-associated cargo recycling by tubule-based endosomal sorting

    NARCIS (Netherlands)

    van Weering, J.R.T.; Cullen, P.J.

    2014-01-01

    The endosome system is a collection of organelles that sort membrane-associated proteins and lipids for lysosomal degradation or recycling back to their target organelle. Recycling cargo is captured in a network of membrane tubules emanating from endosomes where tubular carriers pinch off. These

  12. Evidence for a Golgi-to-endosome protein sorting pathway in Plasmodium falciparum.

    Directory of Open Access Journals (Sweden)

    Priscilla Krai

    Full Text Available During the asexual intraerythrocytic stage, the malaria parasite Plasmodium falciparum must traffic newly-synthesized proteins to a broad array of destinations within and beyond the parasite's plasma membrane. In this study, we have localized two well-conserved protein components of eukaryotic endosomes, the retromer complex and the small GTPase Rab7, to define a previously-undescribed endosomal compartment in P. falciparum. Retromer and Rab7 co-localized to a small number of punctate structures within parasites. These structures, which we refer to as endosomes, lie in close proximity to the Golgi apparatus and, like the Golgi apparatus, are inherited by daughter merozoites. However, the endosome is clearly distinct from the Golgi apparatus as neither retromer nor Rab7 redistributed to the endoplasmic reticulum upon brefeldin A treatment. Nascent rhoptries (specialized secretory organelles required for invasion developed adjacent to endosomes, an observation that suggests a role for the endosome in rhoptry biogenesis. A P. falciparum homolog of the sortilin family of protein sorting receptors (PfSortilin was localized to the Golgi apparatus. Together, these results elaborate a putative Golgi-to-endosome protein sorting pathway in asexual blood stage parasites and suggest that one role of retromer is to mediate the retrograde transport of PfSortilin from the endosome to the Golgi apparatus.

  13. Kinetic imaging of NPC1L1 and sterol trafficking between plasma membrane and recycling endosomes in hepatoma cells

    DEFF Research Database (Denmark)

    Hartwig Petersen, Nicole; Færgeman, Nils J; Yu, Liqing

    2008-01-01

    fluorescent protein (NPC1L1-EGFP) and cholesterol analogues in hepatoma cells. At steady state about 42% of NPC1L1 resided in the transferrin (Tf) positive, sterol enriched endocytic recycling compartment (ERC), while time-lapse microscopy demonstrated NPC1L1 traffic between plasma membrane and ERC....... Fluorescence recovery after photobleaching (FRAP) revealed rapid recovery (half-time t1/2 ~2.5 min) of about 35% of NPC1L1 in the ERC probably replenished from peripheral sorting endosomes. Acute cholesterol depletion blocked internalization of NPC1L1-EGFP and Tf and stimulated recycling of NPC1L1-EGFP from...... the ERC to the plasma membrane. NPC1L1-EGFP facilitated transport of fluorescent sterols from the plasma membrane to the ERC. Insulin induced translocation of vesicles containing NPC1L1 and fluorescent sterol from the ERC to the cell membrane. Upon polarization of hepatoma cells NPC1L1 resided almost...

  14. Enzymatic quantification of cholesterol and cholesterol esters from silicone hydrogel contact lenses.

    Science.gov (United States)

    Pucker, Andrew D; Thangavelu, Mirunalni; Nichols, Jason J

    2010-06-01

    The purpose of this work was to develop an enzymatic method of quantification of cholesterol and cholesterol esters derived from contact lenses, both in vitro and ex vivo. Lotrafilcon B (O2 Optix; CIBA Vision, Inc., Duluth, GA) and galyfilcon A (Acuvue Advance; Vistakon, Inc., Jacksonville, FL) silicone hydrogel contact lenses were independently incubated in cholesterol oleate solutions varying in concentrations. After incubation, the lenses were removed and underwent two separate 2:1 chloroform-methanol extractions. After in vitro studies, 10 human subjects wore both lotrafilcon B and galyfilcon A contact lenses for 7 days. The lenses also underwent two separate 2:1 chloroform-methanol extractions. All in vitro and ex vivo samples were quantified with a cholesterol esterase enzymatic reaction. Calibration curves from quantifications of in vitro contact lens samples soaked in successively decreasing concentrations of cholesterol oleate yielded coefficients of determination (R(2)) of 0.99 (lotrafilcon B) and 0.97 (galyfilcon A). For in vitro contact lens samples, galyfilcon A was associated with an average cholesterol oleate extraction of 39.85 +/- 48.65 microg/lens, whereas lotrafilcon B was associated with 5.86 +/- 3.36 microg/lens (P = 0.05) across both extractions and all incubation concentrations. For ex vivo contact lens samples, there was significantly more cholesterol and cholesterol esters deposited on galyfilcon A (5.77 +/- 1.87 microg/lens) than on lotrafilcon B (2.03 +/- 1.62 microg/lens; P = 0.0005). This is an efficient and simple method of quantifying total cholesterol extracted from silicone hydrogel contact lenses and, potentially, the meibum and/or tear film. Certain silicone hydrogel materials demonstrate more affinity for cholesterol and its esters than do others.

  15. Retrogradely Transported TrkA Endosomes Signal Locally within Dendrites to Maintain Sympathetic Neuron Synapses

    Directory of Open Access Journals (Sweden)

    Kathryn M. Lehigh

    2017-04-01

    Full Text Available Sympathetic neurons require NGF from their target fields for survival, axonal target innervation, dendritic growth and formation, and maintenance of synaptic inputs from preganglionic neurons. Target-derived NGF signals are propagated retrogradely, from distal axons to somata of sympathetic neurons via TrkA signaling endosomes. We report that a subset of TrkA endosomes that are transported from distal axons to cell bodies translocate into dendrites, where they are signaling competent and move bidirectionally, in close proximity to synaptic protein clusters. Using a strategy for spatially confined inhibition of TrkA kinase activity, we found that distal-axon-derived TrkA signaling endosomes are necessary within sympathetic neuron dendrites for maintenance of synapses. Thus, TrkA signaling endosomes have unique functions in different cellular compartments. Moreover, target-derived NGF mediates circuit formation and synapse maintenance through TrkA endosome signaling within dendrites to promote aggregation of postsynaptic protein complexes.

  16. Dengue virus ensures its fusion in late endosomes using compartment-specific lipids.

    Directory of Open Access Journals (Sweden)

    Elena Zaitseva

    2010-10-01

    Full Text Available Many enveloped viruses invade cells via endocytosis and use different environmental factors as triggers for virus-endosome fusion that delivers viral genome into cytosol. Intriguingly, dengue virus (DEN, the most prevalent mosquito-borne virus that infects up to 100 million people each year, fuses only in late endosomes, while activation of DEN protein fusogen glycoprotein E is triggered already at pH characteristic for early endosomes. Are there any cofactors that time DEN fusion to virion entry into late endosomes? Here we show that DEN utilizes bis(monoacylglycerophosphate, a lipid specific to late endosomes, as a co-factor for its endosomal acidification-dependent fusion machinery. Effective virus fusion to plasma- and intracellular- membranes, as well as to protein-free liposomes, requires the target membrane to contain anionic lipids such as bis(monoacylglycerophosphate and phosphatidylserine. Anionic lipids act downstream of low-pH-dependent fusion stages and promote the advance from the earliest hemifusion intermediates to the fusion pore opening. To reach anionic lipid-enriched late endosomes, DEN travels through acidified early endosomes, but we found that low pH-dependent loss of fusogenic properties of DEN is relatively slow in the presence of anionic lipid-free target membranes. We propose that anionic lipid-dependence of DEN fusion machinery protects it against premature irreversible restructuring and inactivation and ensures viral fusion in late endosomes, where the virus encounters anionic lipids for the first time during entry. Currently there are neither vaccines nor effective therapies for DEN, and the essential role of the newly identified DEN-bis(monoacylglycerophosphate interactions in viral genome escape from the endosome suggests a novel target for drug design.

  17. Unconventional secretion of FABP4 by endosomes and secretory lysosomes.

    Science.gov (United States)

    Villeneuve, Julien; Bassaganyas, Laia; Lepreux, Sebastien; Chiritoiu, Marioara; Costet, Pierre; Ripoche, Jean; Malhotra, Vivek; Schekman, Randy

    2017-12-06

    An appreciation of the functional properties of the cytoplasmic fatty acid binding protein 4 (FABP4) has advanced with the recent demonstration that an extracellular form secreted by adipocytes regulates a wide range of physiological functions. Little, however, is known about the mechanisms that mediate the unconventional secretion of FABP4. Here, we demonstrate that FABP4 secretion is mediated by a membrane-bounded compartment, independent of the conventional endoplasmic reticulum-Golgi secretory pathway. We show that FABP4 secretion is also independent of GRASP proteins, autophagy, and multivesicular bodies but involves enclosure within endosomes and secretory lysosomes. We highlight the physiological significance of this pathway with the demonstration that an increase in plasma levels of FABP4 is inhibited by chloroquine treatment of mice. These findings chart the pathway of FABP4 secretion and provide a potential therapeutic means to control metabolic disorders associated with its dysregulated secretion. © 2018 Villeneuve et al.

  18. Regulation of cholesterol homeostasis

    NARCIS (Netherlands)

    van der Wulp, Mariette Y. M.; Verkade, Henkjan J.; Groen, Albert K.

    2013-01-01

    Hypercholesterolemia is an important risk factor for cardiovascular disease. It is caused by a disturbed balance between cholesterol secretion into the blood versus uptake. The pathways involved are regulated via a complex interplay of enzymes, transport proteins, transcription factors and

  19. High Blood Cholesterol

    Science.gov (United States)

    ... cholesterol or other fats, such as triglycerides. Total testosterone and dehydroepiandrosterone sulphate tests can help rule out ... Intramural Research , which includes investigators in our Lipoprotein Metabolism Laboratory , is actively engaged in the study of ...

  20. Cholesterol - drug treatment

    Science.gov (United States)

    ... disease Heart attack Heart bypass surgery Heart bypass surgery - minimally invasive Heart disease and diet High blood cholesterol levels Peripheral artery bypass - leg Patient Instructions Abdominal aortic aneurysm repair - open - discharge Angina - discharge Angina - ...

  1. Cholesterol Domains Enhance Transfection

    Science.gov (United States)

    Betker, Jamie L.; Kullberg, Max; Gomez, Joe; Anchordoquy, Thomas J.

    2014-01-01

    The formation of cholesterol domains in lipoplexes has been associated with enhanced serum stability and transfection rates both in cell culture and in vivo. This study utilizes the ability of saturated phosphatidylcholines to promote the formation of cholesterol domains at much lower cholesterol contents than have been utilized in previous work. The results show that lipoplexes with identical cholesterol and cationic lipid contents exhibit significantly improved transfection efficiencies when a domain is present, consistent with previous work. In addition, studies assessing transfection rates in the absence of serum demonstrate that the ability of domains to enhance transfection is not dependent on interactions with serum proteins. Consistent with this hypothesis, characterization of the adsorbed proteins composing the corona of these lipoplex formulations did not reveal a correlation between transfection and the adsorption of a specific protein. Finally, we show that the interaction with serum proteins can promote domain formation in some formulations, and thereby result in enhanced transfection only after serum exposure. PMID:23557286

  2. High Blood Cholesterol Prevention

    Science.gov (United States)

    ... lots of foods high in saturated fat and trans fat may contribute to high cholesterol and related conditions, ... Choose foods that are low in saturated fat, trans fat, sodium (salt), and added sugars. These foods include ...

  3. Cytokinesis in plant and animal cells: endosomes 'shut the door'.

    Science.gov (United States)

    Baluska, Frantisek; Menzel, Diedrik; Barlow, Peter W

    2006-06-01

    For many years, cytokinesis in eukaryotic cells was considered to be a process that took a variety of forms. This is rather surprising in the face of an apparently conservative mitosis. Animal cytokinesis was described as a process based on an actomyosin-based contractile ring, assembling, and acting at the cell periphery. In contrast, cytokinesis of plant cells was viewed as the centrifugal generation of a new cell wall by fusion of Golgi apparatus-derived vesicles. However, recent advances in animal and plant cell biology have revealed that many features formerly considered as plant-specific are, in fact, valid also for cytokinetic animal cells. For example, vesicular trafficking has turned out to be important not only for plant but also for animal cytokinesis. Moreover, the terminal phase of animal cytokinesis based on midbody microtubule activity resembles plant cytokinesis in that interdigitating microtubules play a decisive role in the recruitment of cytokinetic vesicles and directing them towards the cytokinetic spaces which need to be plugged by fusing endosomes. Presently, we are approaching another turning point which brings cytokinesis in plant and animal cells even closer. As an unexpected twist, new studies reveal that both plant and animal cytokinesis is driven not so much by Golgi-derived vesicles but rather by homotypically and heterotypically fusing endosomes. These are generated from cytokinetic cortical sites defined by preprophase microtubules and contractile actomyosin ring, which induce local endocytosis of both the plasma membrane and cell wall material. Finally, plant and animal cytokinesis meet together at the physical separation of daughter cells despite obvious differences in their preparatory events.

  4. Rotational magnetic endosome microrheology: Viscoelastic architecture inside living cells

    Science.gov (United States)

    Wilhelm, C.; Gazeau, F.; Bacri, J.-C.

    2003-06-01

    The previously developed technique of magnetic rotational microrheology [Phys. Rev. E 67, 011504 (2003)] is proposed to investigate the rheological properties of the cell interior. An endogeneous magnetic probe is obtained inside living cells by labeling intracellular compartments with magnetic nanoparticles, following the endocytosis mechanism, the most general pathway used by eucaryotic cells to internalize substances from an extracellular medium. Primarily adsorbed on the plasma membrane, the magnetic nanoparticles are first internalized within submicronic membrane vesicles (100 nm diameter) to finally concentrate inside endocytotic intracellular compartments (0.6 μm diameter). These magnetic endosomes attract each other and form chains within the living cell when submitted to an external magnetic field. Here we demonstrate that these chains of magnetic endosomes are valuable tools to probe the intracellular dynamics at very local scales. The viscoelasticity of the chain microenvironment is quantified in terms of a viscosity η and a relaxation time τ by analyzing the rotational dynamics of each tested chain in response to a rotation of the external magnetic field. The viscosity η governs the long time flow of the medium surrounding the chains and the relaxation time τ reflects the proportion of solidlike versus liquidlike behavior (τ=η/G, where G is the high-frequency shear modulus). Measurements in HeLa cells show that the cell interior is a highly heterogeneous structure, with regions where chains are embedded inside a dense viscoelastic matrix and other domains where chains are surrounded by a less rigid viscoelastic material. When one compound of the cell cytoskeleton is disrupted (microfilaments or microtubules), the intracellular viscoelasticity becomes less heterogeneous and more fluidlike, in the sense of both a lower viscosity and a lower relaxation time.

  5. Up-regulation of cholesterol associated genes as novel resistance mechanism in glioblastoma cells in response to archazolid B.

    Science.gov (United States)

    Hamm, Rebecca; Zeino, Maen; Frewert, Simon; Efferth, Thomas

    2014-11-15

    Treatment of glioblastoma multiforme (GBM), the most common and aggressive lethal brain tumor, represents a great challenge. Despite decades of research, the survival prognosis of GBM patients is unfavorable and more effective therapeutics are sorely required. Archazolid B, a potent vacuolar H(+)-ATPase inhibitor influencing cellular pH values, is a promising new compound exerting cytotoxicity in the nanomolar range on wild-type U87MG glioblastoma cells and U87MG.∆EGFR cells transfected with a mutant epidermal growth factor receptor (EGFR) gene. Gene expression profiling using microarray technology showed that archazolid B caused drastic disturbances in cholesterol homeostasis. Cholesterol, a main component of cellular membranes, is known to be essential for GBM growth and cells bearing EGFRvIII mutation are highly dependent on exogenous cholesterol. Archazolid B caused excessive accumulation of free cholesterol within intracellular compartments thus depleting cellular cholesterol and leading to up-regulation of SREBP targeted genes, including LDLR and HMGCR, the key enzyme of cholesterol biosynthesis. This cholesterol response is considered to be a novel resistance mechanism induced by archazolid B. We surmise that re-elevation of cholesterol levels in archazolid B treated cells may be mediated by newly synthesized cholesterol, since the drug leads to endosomal/lysosomal malfunction and cholesterol accumulation. Copyright © 2014 Elsevier Inc. All rights reserved.

  6. Up-regulation of cholesterol associated genes as novel resistance mechanism in glioblastoma cells in response to archazolid B

    Energy Technology Data Exchange (ETDEWEB)

    Hamm, Rebecca; Zeino, Maen [Institute of Pharmacy and Biochemistry, Department of Pharmaceutical Biology, Johannes Gutenberg University, Staudinger Weg 5, 55128 Mainz (Germany); Frewert, Simon [Helmholtz Institute for Pharmaceutical Research Saarland, Helmholtz Centre for Infection Research and Department of Pharmaceutical Biotechnology, Saarland University, Saarbrücken (Germany); Efferth, Thomas, E-mail: efferth@uni-mainz.de [Institute of Pharmacy and Biochemistry, Department of Pharmaceutical Biology, Johannes Gutenberg University, Staudinger Weg 5, 55128 Mainz (Germany)

    2014-11-15

    Treatment of glioblastoma multiforme (GBM), the most common and aggressive lethal brain tumor, represents a great challenge. Despite decades of research, the survival prognosis of GBM patients is unfavorable and more effective therapeutics are sorely required. Archazolid B, a potent vacuolar H{sup +}-ATPase inhibitor influencing cellular pH values, is a promising new compound exerting cytotoxicity in the nanomolar range on wild-type U87MG glioblastoma cells and U87MG.∆EGFR cells transfected with a mutant epidermal growth factor receptor (EGFR) gene. Gene expression profiling using microarray technology showed that archazolid B caused drastic disturbances in cholesterol homeostasis. Cholesterol, a main component of cellular membranes, is known to be essential for GBM growth and cells bearing EGFRvIII mutation are highly dependent on exogenous cholesterol. Archazolid B caused excessive accumulation of free cholesterol within intracellular compartments thus depleting cellular cholesterol and leading to up-regulation of SREBP targeted genes, including LDLR and HMGCR, the key enzyme of cholesterol biosynthesis. This cholesterol response is considered to be a novel resistance mechanism induced by archazolid B. We surmise that re-elevation of cholesterol levels in archazolid B treated cells may be mediated by newly synthesized cholesterol, since the drug leads to endosomal/lysosomal malfunction and cholesterol accumulation.

  7. Reference intervals for serum total cholesterol, HDL cholesterol and ...

    African Journals Online (AJOL)

    Reference intervals of total cholesterol, HDL cholesterol and non-HDL cholesterol concentrations were determined on 309 blood donors from an urban and peri-urban population of Botswana. Using non-parametric methods to establish 2.5th and 97.5th percentiles of the distribution, the intervals were: total cholesterol 2.16 ...

  8. Cholesterol and ocular pathologies: focus on the role of cholesterol-24S-hydroxylase in cholesterol homeostasis

    Directory of Open Access Journals (Sweden)

    Fourgeux Cynthia

    2015-03-01

    Full Text Available The retina is responsible for coding the light stimulus into a nervous signal that is transferred to the brain via the optic nerve. The retina is formed by the association of the neurosensory retina and the retinal pigment epithelium that is supported by Bruch’s membrane. Both the physical and metabolic associations between these partners are crucial for the functioning of the retina, by means of nutrient intake and removal of the cell and metabolic debris from the retina. Dysequilibrium are involved in the aging processes and pathologies such as age-related macular degeneration, the leading cause of visual loss after the age of 50 years in Western countries. The retina is composed of several populations of cells including glia that is involved in cholesterol biosynthesis. Cholesterol is the main sterol in the retina. It is present as free form in cells and as esters in Bruch’s membrane. Accumulation of cholesteryl esters has been associated with aging of the retina and impairment of the retinal function. Under dietary influence and in situ synthesized, the metabolism of cholesterol is regulated by cell interactions, including neurons and glia via cholesterol-24S-hydroxylase. Several pathophysiological associations with cholesterol and its metabolism can be suggested, especially in relation to glaucoma and age-related macular degeneration.

  9. Cholesterol Assimilation with Isolated lactobacilli Strains of Fars’ Local Dairy Products

    Directory of Open Access Journals (Sweden)

    A Emami

    2008-12-01

    Full Text Available ABSTRACT: Introduction & Objective: Cholesterol is an important compound in most of the biological reactions which the excess of it can be seen as a harmful compound of causing heart diseases. The aim of the present study was to evaluate the cholesterol removal property and also its pathway by dairy lactobacillus in in vitro condition under different bile salts concentration. Materials & Methods: After isolation of lactobacillus strains from dairy products, they were identified with chemical tests and their growths were evaluated under presence of cholesterol and bile salts. The method of action of the bacillus in cholesterol removal was assayed by spectrophotometer method. Collected data was analyzed by SPSS software. Results: result of this study showed that any strains of the bacteria had the ability of cholesterol removal (7.82-34.69 µg/ml. L.casei had more competence for removal of cholesterol in compare to the rest of bacilli. The evaluation of cholesterol cell wall attachment revealed that most of removed cholesterols have been changed to the other products. Conclusion: Considering the result of this study, it can be concluded that cholesterol removal has a direct association with growth of bacteria where the L. casei with high growth rate had more capability of cholesterol removal. Whereas the Lactobacillus can remove the cholesterol with different methods, results of this study showed that dairy products, especially yogurt, can remove the harmful substances such as cholesterol using non chemical methods. The results of this study could be expanded on human use if more study and research could be carried out.

  10. Cholesterol through the Looking Glass

    Science.gov (United States)

    Kristiana, Ika; Luu, Winnie; Stevenson, Julian; Cartland, Sian; Jessup, Wendy; Belani, Jitendra D.; Rychnovsky, Scott D.; Brown, Andrew J.

    2012-01-01

    How cholesterol is sensed to maintain homeostasis has been explained by direct binding to a specific protein, Scap, or through altering the physical properties of the membrane. The enantiomer of cholesterol (ent-cholesterol) is a valuable tool in distinguishing between these two models because it shares nonspecific membrane effects with native cholesterol (nat-cholesterol), but not specific binding interactions. This is the first study to compare ent- and nat-cholesterol directly on major molecular parameters of cholesterol homeostasis. We found that ent-cholesterol suppressed activation of the master transcriptional regulator of cholesterol metabolism, SREBP-2, almost as effectively as nat-cholesterol. Importantly, ent-cholesterol induced a conformational change in the cholesterol-sensing protein Scap in isolated membranes in vitro, even when steps were taken to eliminate potential confounding effects from endogenous cholesterol. Ent-cholesterol also accelerated proteasomal degradation of the key cholesterol biosynthetic enzyme, squalene monooxygenase. Together, these findings provide compelling evidence that cholesterol maintains its own homeostasis not only via direct protein interactions, but also by altering membrane properties. PMID:22869373

  11. Defects in ER–endosome contacts impact lysosome function in hereditary spastic paraplegia

    Science.gov (United States)

    Newton, Timothy; Connell, James W.; Winner, Beate

    2017-01-01

    Contacts between endosomes and the endoplasmic reticulum (ER) promote endosomal tubule fission, but the mechanisms involved and consequences of tubule fission failure are incompletely understood. We found that interaction between the microtubule-severing enzyme spastin and the ESCRT protein IST1 at ER–endosome contacts drives endosomal tubule fission. Failure of fission caused defective sorting of mannose 6-phosphate receptor, with consequently disrupted lysosomal enzyme trafficking and abnormal lysosomal morphology, including in mouse primary neurons and human stem cell–derived neurons. Consistent with a role for ER-mediated endosomal tubule fission in lysosome function, similar lysosomal abnormalities were seen in cellular models lacking the WASH complex component strumpellin or the ER morphogen REEP1. Mutations in spastin, strumpellin, or REEP1 cause hereditary spastic paraplegia (HSP), a disease characterized by axonal degeneration. Our results implicate failure of the ER–endosome contact process in axonopathy and suggest that coupling of ER-mediated endosomal tubule fission to lysosome function links different classes of HSP proteins, previously considered functionally distinct, into a unifying pathway for axonal degeneration. PMID:28389476

  12. Defects in ER-endosome contacts impact lysosome function in hereditary spastic paraplegia.

    Science.gov (United States)

    Allison, Rachel; Edgar, James R; Pearson, Guy; Rizo, Tania; Newton, Timothy; Günther, Sven; Berner, Fiamma; Hague, Jennifer; Connell, James W; Winkler, Jürgen; Lippincott-Schwartz, Jennifer; Beetz, Christian; Winner, Beate; Reid, Evan

    2017-05-01

    Contacts between endosomes and the endoplasmic reticulum (ER) promote endosomal tubule fission, but the mechanisms involved and consequences of tubule fission failure are incompletely understood. We found that interaction between the microtubule-severing enzyme spastin and the ESCRT protein IST1 at ER-endosome contacts drives endosomal tubule fission. Failure of fission caused defective sorting of mannose 6-phosphate receptor, with consequently disrupted lysosomal enzyme trafficking and abnormal lysosomal morphology, including in mouse primary neurons and human stem cell-derived neurons. Consistent with a role for ER-mediated endosomal tubule fission in lysosome function, similar lysosomal abnormalities were seen in cellular models lacking the WASH complex component strumpellin or the ER morphogen REEP1. Mutations in spastin, strumpellin, or REEP1 cause hereditary spastic paraplegia (HSP), a disease characterized by axonal degeneration. Our results implicate failure of the ER-endosome contact process in axonopathy and suggest that coupling of ER-mediated endosomal tubule fission to lysosome function links different classes of HSP proteins, previously considered functionally distinct, into a unifying pathway for axonal degeneration. © 2017 Allison et al.

  13. The phosphoinositide-associated protein Rush hour regulates endosomal trafficking in Drosophila.

    Science.gov (United States)

    Gailite, Ieva; Egger-Adam, Diane; Wodarz, Andreas

    2012-02-01

    Endocytosis regulates multiple cellular processes, including the protein composition of the plasma membrane, intercellular signaling, and cell polarity. We have identified the highly conserved protein Rush hour (Rush) and show that it participates in the regulation of endocytosis. Rush localizes to endosomes via direct binding of its FYVE (Fab1p, YOTB, Vac1p, EEA1) domain to phosphatidylinositol 3-phosphate. Rush also directly binds to Rab GDP dissociation inhibitor (Gdi), which is involved in the activation of Rab proteins. Homozygous rush mutant flies are viable but show genetic interactions with mutations in Gdi, Rab5, hrs, and carnation, the fly homologue of Vps33. Overexpression of Rush disrupts progression of endocytosed cargo and increases late endosome size. Lysosomal marker staining is decreased in Rush-overexpressing cells, pointing to a defect in the transition between late endosomes and lysosomes. Rush also causes formation of endosome clusters, possibly by affecting fusion of endosomes via an interaction with the class C Vps/homotypic fusion and vacuole protein-sorting (HOPS) complex. These results indicate that Rush controls trafficking from early to late endosomes and from late endosomes to lysosomes by modulating the activity of Rab proteins.

  14. Stochastic acidification, activation of hemagglutinin and escape of influenza viruses from an endosome

    Science.gov (United States)

    Lagache, Thibault; Sieben, Christian; Meyer, Tim; Herrmann, Andreas; Holcman, David

    2017-06-01

    Influenza viruses enter the cell inside an endosome. During the endosomal journey, acidification triggers a conformational change of the virus spike protein hemagglutinin (HA) that results in escape of the viral genome from the endosome into the cytoplasm. It is still unclear how the interplay between acidification and HA conformation changes affects the kinetics of the viral endosomal escape. We develop here a stochastic model to estimate the change of conformation of HAs inside the endosome nanodomain. Using a Markov process, we model the arrival of protons to HA binding sites and compute the kinetics of their accumulation. We compute the Mean First Passage Time (MFPT) of the number of HA bound sites to a threshold, which is used to estimate the HA activation rate for a given pH concentration. The present analysis reveals that HA proton binding sites possess a high chemical barrier, ensuring a stability of the spike protein at sub-acidic pH. We predict that activating more than 3 adjacent HAs is necessary to trigger endosomal fusion and this configuration prevents premature release of viruses from early endosomes

  15. Roles of Dynein and Dynactin in Early Endosome Dynamics Revealed Using Automated Tracking and Global Analysis

    Science.gov (United States)

    Flores-Rodriguez, Neftali; Rogers, Salman S.; Kenwright, David A.; Waigh, Thomas A.

    2011-01-01

    Microtubule-dependent movement is crucial for the spatial organization of endosomes in most eukaryotes, but as yet there has been no systematic analysis of how a particular microtubule motor contributes to early endosome dynamics. Here we tracked early endosomes labeled with GFP-Rab5 on the nanometer scale, and combined this with global, first passage probability (FPP) analysis to provide an unbiased description of how the minus-end microtubule motor, cytoplasmic dynein, supports endosome motility. Dynein contributes to short-range endosome movement, but in particular drives 85–98% of long, inward translocations. For these, it requires an intact dynactin complex to allow membrane-bound p150Glued to activate dynein, since p50 over-expression, which disrupts the dynactin complex, inhibits inward movement even though dynein and p150Glued remain membrane-bound. Long dynein-dependent movements occur via bursts at up to ∼8 µms−1 that are linked by changes in rate or pauses. These peak speeds during rapid inward endosome movement are still seen when cellular dynein levels are 50-fold reduced by RNAi knock-down of dynein heavy chain, while the number of movements is reduced 5-fold. Altogether, these findings identify how dynein helps define the dynamics of early endosomes. PMID:21915335

  16. ER network homeostasis is critical for plant endosome streaming and endocytosis

    Science.gov (United States)

    Stefano, Giovanni; Renna, Luciana; Lai, YaShiuan; Slabaugh, Erin; Mannino, Nicole; Buono, Rafael A; Otegui, Marisa S; Brandizzi, Federica

    2015-01-01

    Eukaryotic cells internalize cargo at the plasma membrane via endocytosis, a vital process that is accomplished through a complex network of endosomal organelles. In mammalian cells, the ER is in close association with endosomes and regulates their fission. Nonetheless, the physiological role of such interaction on endocytosis is yet unexplored. Here, we probed the existence of ER–endosome association in plant cells and assayed its physiological role in endocytosis. Through live-cell imaging and electron microscopy studies, we established that endosomes are extensively associated with the plant ER, supporting conservation of interaction between heterotypic organelles in evolutionarily distant kingdoms. Furthermore, by analyzing ER–endosome dynamics in genetic backgrounds with defects in ER structure and movement, we also established that the ER network integrity is necessary for homeostasis of the distribution and streaming of various endosome populations as well as for efficient endocytosis. These results support a novel model that endocytosis homeostasis depends on a spatiotemporal control of the endosome dynamics dictated by the ER membrane network. PMID:27462431

  17. Herpes Simplex Virus 1 Envelope Cholesterol Facilitates Membrane Fusion

    Directory of Open Access Journals (Sweden)

    George A. Wudiri

    2017-12-01

    Full Text Available Methyl beta-cyclodextrin (MβCD treatment of herpes simplex virus 1 (HSV-1 reduced envelope cholesterol levels and inhibited viral entry and infectivity in several cell types, regardless of the dependence of entry on endocytosis or low pH. Viral protein composition was similar in MβCD-treated and untreated virions, and ultrastructural analysis by electron microscopy revealed that cholesterol removal did not grossly affect virion structure or integrity. Removal of envelope cholesterol greatly reduced virion fusion activity as measured by fusion-from-without, suggesting that virion cholesterol is critical for the step of membrane fusion. MβCD-treatment of HSV-1 did not reduce viral attachment to the cells nor endocytic uptake of HSV-1 from the cell surface. The pre-fusion form of gB present in the HSV-1 envelope undergoes conformational changes in response to mildly acidic pH. These gB changes occurred independently of envelope cholesterol. Removal of cholesterol compromised virion stability as measured by recovery of infectivity following cycles of freeze-thaw. Taken together, the data suggest that HSV-1 envelope cholesterol is important for viral entry and infectivity due to a critical role in membrane fusion.

  18. HDL cholesterol: atherosclerosis and beyond

    OpenAIRE

    Bochem, A.E.

    2013-01-01

    Cardiovascular disease (CVD) is the leading cause of death in the Western world. Myocardial infarction and stroke are the result of a compromised blood flow which may result from cholesterol accumulation in the vessel wall due to high plasma levels of LDL cholesterol. High plasma levels of HDL cholesterol, however, are inversely associated with CVD. This is commonly ascribed to a concept called "reverse cholesterol transport" a mechanism by which the HDL particle takes up cholesterol from the...

  19. Principles of membrane tethering and fusion in endosome and lysosome biogenesis.

    Science.gov (United States)

    Kümmel, Daniel; Ungermann, Christian

    2014-08-01

    Endosomes and lysosomes receive cargo via vesicular carriers that arrive along multiple trafficking routes. On both organelles, tethering proteins have been identified that interact specifically with Rab5 on endosomes and Rab7 on late endosomes/lysosomes and that facilitate the SNARE-driven membrane fusion. Even though the structure and stoichiometry of the involved proteins and protein complexes differ strongly, they may operate by similar principles. Within this review, we will provide insights into their common functions and discuss the open questions in the field. Copyright © 2014 Elsevier Ltd. All rights reserved.

  20. Transintestinal and Biliary Cholesterol Secretion Both Contribute to Macrophage Reverse Cholesterol Transport in Rats-Brief Report.

    Science.gov (United States)

    de Boer, Jan Freark; Schonewille, Marleen; Dikkers, Arne; Koehorst, Martijn; Havinga, Rick; Kuipers, Folkert; Tietge, Uwe J F; Groen, Albert K

    2017-04-01

    Reverse cholesterol transport comprises efflux of cholesterol from macrophages and its subsequent removal from the body with the feces and thereby protects against formation of atherosclerotic plaques. Because of lack of suitable animal models that allow for evaluation of the respective contributions of biliary cholesterol secretion and transintestinal cholesterol excretion (TICE) to macrophage reverse cholesterol transport under physiological conditions, the relative importance of both pathways in this process has remained controversial. To separate cholesterol traffic via the biliary route from TICE, bile flow was mutually diverted between rats, continuously, for 3 days. Groups of 2 weight-matched rats were designated as a pair, and both rats were equipped with cannulas in the bile duct and duodenum. Bile from rat 1 was diverted to the duodenum of rat 2, whereas bile from rat 2 was rerouted to the duodenum of rat 1. Next, rat 1 was injected with [ 3 H]cholesterol-loaded macrophages. [ 3 H]Cholesterol secreted via the biliary route was consequently diverted to rat 2 and could thus be quantified from the feces of that rat. On the other hand, [ 3 H]cholesterol tracer in the feces of rat 1 reflected macrophage-derived cholesterol excreted via TICE. Using this setup, we found that 63% of the label secreted with the fecal neutral sterols had travelled via the biliary route, whereas 37% was excreted via TICE. TICE and biliary cholesterol secretion contribute to macrophage reverse cholesterol transport in rats. The majority of macrophage-derived cholesterol is however excreted via the hepatobiliary route. © 2017 American Heart Association, Inc.

  1. Oxysterol-Binding Protein-Related Protein 1L Regulates Cholesterol Egress from the Endo-Lysosomal System

    Directory of Open Access Journals (Sweden)

    Kexin Zhao

    2017-05-01

    Full Text Available Lipoprotein cholesterol is delivered to the limiting membrane of late endosomes/lysosomes (LELs by Niemann-Pick C1 (NPC1. However, the mechanism of cholesterol transport from LELs to the endoplasmic reticulum (ER is poorly characterized. We report that oxysterol-binding protein-related protein 1L (ORP1L is necessary for this stage of cholesterol export. CRISPR-mediated knockout of ORP1L in HeLa and HEK293 cells reduced esterification of cholesterol to the level in NPC1 knockout cells, and it increased the expression of sterol-regulated genes and de novo cholesterol synthesis, indicative of a block in cholesterol transport to the ER. In the absence of this transport pathway, cholesterol-enriched LELs accumulated in the Golgi/perinuclear region. Cholesterol delivery to the ER required the sterol-, phosphatidylinositol 4-phosphate-, and vesicle-associated membrane protein-associated protein (VAP-binding activities of ORP1L, as well as NPC1 expression. These results suggest that ORP1L-dependent membrane contacts between LELs and the ER coordinate cholesterol transfer with the retrograde movement of endo-lysosomal vesicles.

  2. Biogenesis of plasma membrane cholesterol

    Energy Technology Data Exchange (ETDEWEB)

    Lange, Y.

    1986-05-01

    A striking feature of the molecular organization of eukaryotic cells is the singular enrichment of their plasma membranes in sterols. The authors studies are directed at elucidating the mechanisms underlying this inhomogeneous disposition. Cholesterol oxidase catalyzes the oxidation of plasma membrane cholesterol in intact cells, leaving intracellular cholesterol pools untouched. With this technique, the plasma membrane was shown to contain 95% of the unesterified cholesterol of cultured human fibroblasts. Cholesterol synthesized from (/sup 3/H) acetate moved to the plasma membrane with a half-time of 1 h at 37/sup 0/C. They used equilibrium gradient centrifugation of homogenates of biosynthetically labeled, cholesterol oxidase treated cells to examine the distribution of newly synthesized sterols among intracellular pools. Surprisingly, lanosterol, a major precursor of cholesterol, and intracellular cholesterol both peaked at much lower buoyant density than did 3-hydroxy-3-methylglutaryl-CoA reductase. This suggests that cholesterol biosynthesis is not taken to completion in the endoplasmic reticulum. The cholesterol in the buoyant fraction eventually moved to the plasma membrane. Digitonin treatment increased the density of the newly synthesized cholesterol fractions, indicating that nascent cholesterol in transit is associated with cholesterol-rich membranes. The authors are testing the hypothesis that the pathway of cholesterol biosynthesis is spatially organized in various intracellular membranes such that the sequence of biosynthetic steps both concentrates the sterol and conveys it to the plasma membrane.

  3. Semiconductor quantum dot/albumin complex is a long-life and highly photostable endosome marker.

    Science.gov (United States)

    Hanaki, Ken-ichi; Momo, Asami; Oku, Taisuke; Komoto, Atsushi; Maenosono, Shinya; Yamaguchi, Yukio; Yamamoto, Kenji

    2003-03-14

    For the purpose of selecting the efficient dispersion condition of hydrophilic semiconductor quantum dots (QDs) in biological buffers, the dispersion of the QDs mixed with a serum albumin from 9 different species or an ovalbumin was compared by a fluorescence intensity analysis. The QDs mixed with sheep serum albumin (SSA) showed the highest fluorescence of all when the mixtures were dissolved in Dulbecco's MEM. QD/SSA complexes were accumulated in the endosome/lysosome of Vero cells and the fluorescence could be detected over a 5-day post-incubation period. The photostability of QD/SSA complexes associated with the endosomes was detectable, at least, 30 times as long as that of fluorescein-labeled dextran involved in endosomes. QD/SSA complex, therefore, can be used as a long-life and highly photostable endosome marker.

  4. Facilitation of Endosomal Recycling by an IRG Protein Homolog Maintains Apical Tubule Structure in Caenorhabditis elegans

    Science.gov (United States)

    Grussendorf, Kelly A.; Trezza, Christopher J.; Salem, Alexander T.; Al-Hashimi, Hikmat; Mattingly, Brendan C.; Kampmeyer, Drew E.; Khan, Liakot A.; Hall, David H.; Göbel, Verena; Ackley, Brian D.; Buechner, Matthew

    2016-01-01

    Determination of luminal diameter is critical to the function of small single-celled tubes. A series of EXC proteins, including EXC-1, prevent swelling of the tubular excretory canals in Caenorhabditis elegans. In this study, cloning of exc-1 reveals it to encode a homolog of mammalian IRG proteins, which play roles in immune response and autophagy and are associated with Crohn’s disease. Mutants in exc-1 accumulate early endosomes, lack recycling endosomes, and exhibit abnormal apical cytoskeletal structure in regions of enlarged tubules. EXC-1 interacts genetically with two other EXC proteins that also affect endosomal trafficking. In yeast two-hybrid assays, wild-type and putative constitutively active EXC-1 binds to the LIM-domain protein EXC-9, whose homolog, cysteine-rich intestinal protein, is enriched in mammalian intestine. These results suggest a model for IRG function in forming and maintaining apical tubule structure via regulation of endosomal recycling. PMID:27334269

  5. Mis-trafficking of endosomal urokinase proteins triggers drug-induced glioma nonapoptotic cell death.

    Science.gov (United States)

    Pasupuleti, Nagarekha; Grodzki, Ana Cristina; Gorin, Fredric

    2015-04-01

    5-Benzylglycinyl-amiloride (UCD38B) is the parent molecule of a class of anticancer small molecules that kill proliferative and nonproliferative high-grade glioma cells by programmed necrosis. UCD38B intracellularly triggers endocytosis, causing 40-50% of endosomes containing proteins of the urokinase plasminogen activator system (uPAS) to relocate to perinuclear mitochondrial regions. Endosomal "mis-trafficking" caused by UCD38B in human glioma cells corresponds to mitochondrial depolarization with the release and nuclear translocation of apoptotis-inducing factor (AIF) followed by irreversible caspase-independent cell demise. High-content quantification of immunocytochemical colocalization studies identified that UCD38B treatment increased endocytosis of the urokinase plasminogen activator (uPA), its receptor (uPAR), and plasminogen activator inhibitor-1 (PAI-1) into the early and late endosomes by 4- to 5-fold prior to AIF nuclear translocation and subsequent glioma demise. PAI-1 was found to comparably relocate with a subset of early and late endosomes in four different human glioma cell lines after UCD38B treatment, followed by caspase-independent, nonapoptotic cell death. Following UCD38B treatment, the receptor guidance protein LRP-1, which is required for endosomal recycling of the uPA receptor to the plasmalemma, remained abnormally associated with PAI-1 in early and late endosomes. The resultant aberrant endosomal recycling increased the total cellular content of the uPA-PAI-1 protein complex. Reversible inhibition of cellular endocytosis demonstrated that UCD38B bypasses the plasmalemmal uPAS complex and directly acts intracellularly to alter uPAS endocytotic trafficking. UCD38B represents a class of small molecules whose anticancer cytotoxicity is a consequence of causing the mis-trafficking of early and late endosomes containing uPAS cargo and leading to AIF-mediated necrotic cell death. Copyright © 2015 by The American Society for Pharmacology and

  6. deep-orange and carnation define distinct stages in late endosomal biogenesis in Drosophila melanogaster

    OpenAIRE

    Sriram, V.; K. S. Krishnan; Mayor, Satyajit

    2003-01-01

    Endosomal degradation is severely impaired in primary hemocytes from larvae of eye color mutants of Drosophila. Using high resolution imaging and immunofluorescence microscopy in these cells, products of eye color genes, deep-orange (dor) and carnation (car), are localized to large multivesicular Rab7-positive late endosomes containing Golgi-derived enzymes. These structures mature into small sized Dor-negative, Car-positive structures, which subsequently fuse to form tubular lysosomes. Defec...

  7. Erythroid cell mitochondria receive endosomal iron by a "kiss-and-run" mechanism.

    Science.gov (United States)

    Hamdi, Amel; Roshan, Tariq M; Kahawita, Tanya M; Mason, Anne B; Sheftel, Alex D; Ponka, Prem

    2016-12-01

    In erythroid cells, more than 90% of transferrin-derived iron enters mitochondria where ferrochelatase inserts Fe 2+ into protoporphyrin IX. However, the path of iron from endosomes to mitochondrial ferrochelatase remains elusive. The prevailing opinion is that, after its export from endosomes, the redox-active metal spreads into the cytosol and mysteriously finds its way into mitochondria through passive diffusion. In contrast, this study supports the hypothesis that the highly efficient transport of iron toward ferrochelatase in erythroid cells requires a direct interaction between transferrin-endosomes and mitochondria (the "kiss-and-run" hypothesis). Using a novel method (flow sub-cytometry), we analyze lysates of reticulocytes after labeling these organelles with different fluorophores. We have identified a double-labeled population definitively representing endosomes interacting with mitochondria, as demonstrated by confocal microscopy. Moreover, we conclude that this endosome-mitochondrion association is reversible, since a "chase" with unlabeled holotransferrin causes a time-dependent decrease in the size of the double-labeled population. Importantly, the dissociation of endosomes from mitochondria does not occur in the absence of holotransferrin. Additionally, mutated recombinant holotransferrin, that cannot release iron, significantly decreases the uptake of 59 Fe by reticulocytes and diminishes 59 Fe incorporation into heme. This suggests that endosomes, which are unable to provide iron to mitochondria, cause a "traffic jam" leading to decreased endocytosis of holotransferrin. Altogether, our results suggest that a molecular mechanism exists to coordinate the iron status of endosomal transferrin with its trafficking. Besides its contribution to the field of iron metabolism, this study provides evidence for a new intracellular trafficking pathway of organelles. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Cholesterol and Health

    Indian Academy of Sciences (India)

    Home; Journals; Resonance – Journal of Science Education; Volume 11; Issue 2. Cholesterol and Health. Pravina Piste Vidyadhar Patil. General Article Volume 11 Issue 2 February 2006 pp 74-77. Fulltext. Click here to view fulltext PDF. Permanent link: http://www.ias.ac.in/article/fulltext/reso/011/02/0074-0077. Keywords.

  9. γ-SNAP stimulates disassembly of endosomal SNARE complexes and regulates endocytic trafficking pathways.

    Science.gov (United States)

    Inoue, Hiroki; Matsuzaki, Yuka; Tanaka, Ayaka; Hosoi, Kaori; Ichimura, Kaoru; Arasaki, Kohei; Wakana, Yuichi; Asano, Kenichi; Tanaka, Masato; Okuzaki, Daisuke; Yamamoto, Akitsugu; Tani, Katsuko; Tagaya, Mitsuo

    2015-08-01

    Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) that reside in the target membranes and transport vesicles assemble into specific SNARE complexes to drive membrane fusion. N-ethylmaleimide-sensitive factor (NSF) and its attachment protein, α-SNAP (encoded by NAPA), catalyze disassembly of the SNARE complexes in the secretory and endocytic pathways to recycle them for the next round of fusion events. γ-SNAP (encoded by NAPG) is a SNAP isoform, but its function in SNARE-mediated membrane trafficking remains unknown. Here, we show that γ-SNAP regulates the endosomal trafficking of epidermal growth factor (EGF) receptor (EGFR) and transferrin. Immunoprecipitation and mass spectrometry analyses revealed that γ-SNAP interacts with a limited range of SNAREs, including endosomal ones. γ-SNAP, as well as α-SNAP, mediated the disassembly of endosomal syntaxin-7-containing SNARE complexes. Overexpression and small interfering (si)RNA-mediated depletion of γ-SNAP changed the morphologies and intracellular distributions of endosomes. Moreover, the depletion partially suppressed the exit of EGFR and transferrin from EEA1-positive early endosomes to delay their degradation and uptake. Taken together, our findings suggest that γ-SNAP is a unique SNAP that functions in a limited range of organelles - including endosomes - and their trafficking pathways. © 2015. Published by The Company of Biologists Ltd.

  10. Post-Golgi anterograde transport requires GARP-dependent endosome-to-TGN retrograde transport

    Science.gov (United States)

    Hirata, Tetsuya; Fujita, Morihisa; Nakamura, Shota; Gotoh, Kazuyoshi; Motooka, Daisuke; Murakami, Yoshiko; Maeda, Yusuke; Kinoshita, Taroh

    2015-01-01

    The importance of endosome-to–trans-Golgi network (TGN) retrograde transport in the anterograde transport of proteins is unclear. In this study, genome-wide screening of the factors necessary for efficient anterograde protein transport in human haploid cells identified subunits of the Golgi-associated retrograde protein (GARP) complex, a tethering factor involved in endosome-to-TGN transport. Knockout (KO) of each of the four GARP subunits, VPS51–VPS54, in HEK293 cells caused severely defective anterograde transport of both glycosylphosphatidylinositol (GPI)-anchored and transmembrane proteins from the TGN. Overexpression of VAMP4, v-SNARE, in VPS54-KO cells partially restored not only endosome-to-TGN retrograde transport, but also anterograde transport of both GPI-anchored and transmembrane proteins. Further screening for genes whose overexpression normalized the VPS54-KO phenotype identified TMEM87A, encoding an uncharacterized Golgi-resident membrane protein. Overexpression of TMEM87A or its close homologue TMEM87B in VPS54-KO cells partially restored endosome-to-TGN retrograde transport and anterograde transport. Therefore GARP- and VAMP4-dependent endosome-to-TGN retrograde transport is required for recycling of molecules critical for efficient post-Golgi anterograde transport of cell-surface integral membrane proteins. In addition, TMEM87A and TMEM87B are involved in endosome-to-TGN retrograde transport. PMID:26157166

  11. Enhanced Endosomal Escape by Light-Fueled Liquid-Metal Transformer.

    Science.gov (United States)

    Lu, Yue; Lin, Yiliang; Chen, Zhaowei; Hu, Quanyin; Liu, Yang; Yu, Shuangjiang; Gao, Wei; Dickey, Michael D; Gu, Zhen

    2017-04-12

    Effective endosomal escape remains as the "holy grail" for endocytosis-based intracellular drug delivery. To date, most of the endosomal escape strategies rely on small molecules, cationic polymers, or pore-forming proteins, which are often limited by the systemic toxicity and lack of specificity. We describe here a light-fueled liquid-metal transformer for effective endosomal escape-facilitated cargo delivery via a chemical-mechanical process. The nanoscale transformer can be prepared by a simple approach of sonicating a low-toxicity liquid-metal. When coated with graphene quantum dots (GQDs), the resulting nanospheres demonstrate the ability to absorb and convert photoenergy to drive the simultaneous phase separation and morphological transformation of the inner liquid-metal core. The morphological transformation from nanospheres to hollow nanorods with a remarkable change of aspect ratio can physically disrupt the endosomal membrane to promote endosomal escape of payloads. This metal-based nanotransformer equipped with GQDs provides a new strategy for facilitating effective endosomal escape to achieve spatiotemporally controlled drug delivery with enhanced efficacy.

  12. N-acyl phosphatidylethanolamines affect the lateral distribution of cholesterol in membranes

    DEFF Research Database (Denmark)

    Térová, B.; Slotte, J.P.; Petersen, G.

    2005-01-01

    -acyl-POPE) or N-acyl-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine (N-acyl-DPPE), and how the molecules interacted with cholesterol. The gel ¿ liquid crystalline transition temperature of sonicated N-acyl phosphatidylethanolamine vesicles in water correlated positively with the number of palmitic acyl chains...... in the molecules. Based on diphenylhexatriene steady state anisotropy measurements, the presence of 33 mol% cholesterol in the membranes removed the phase transition from N-oleoyl-POPE bilayers, but failed to completely remove it from N-palmitoyl-DPPE and N-palmitoyl-POPE bilayers, suggesting rather weak...... interaction of cholesterol with the N-saturated NAPEs. The rate of cholesterol desorption from mixed monolayers containing N-palmitoyl-DPPE and cholesterol (1:1 molar ratio) was much higher compared to cholesterol/DPPE binary monolayers, suggesting a weak cholesterol interaction with N-palmitoyl-DPPE also...

  13. Trust Your Gut: Galvanizing Nutritional Interest in Intestinal Cholesterol Metabolism for Protection Against Cardiovascular Diseases

    Directory of Open Access Journals (Sweden)

    Jiyoung Lee

    2013-01-01

    Full Text Available Recent studies have demonstrated that the intestine is a key target organ for overall health and longevity. Complementing these studies is the discovery of the trans-intestinal cholesterol efflux pathway and the emerging role of the intestine in reverse cholesterol transport. The surfacing dynamics of the regulation of cholesterol metabolism in the intestine provides an attractive platform for intestine-specific nutritional intervention strategies to lower blood cholesterol levels for protection against cardiovascular diseases. Notably, there is mounting evidence that stimulation of pathways associated with calorie restriction may have a large effect on the regulation of cholesterol removal by the intestine. However, intestinal energy metabolism, specifically the idiosyncrasies surrounding intestinal responses to energy deprivation, is poorly understood. The goal of this paper is to review recent insights into cholesterol regulation by the intestine and to discuss the potential for positive regulation of intestine-driven cholesterol removal through the nutritional induction of pathways associated with calorie restriction.

  14. Trust your gut: galvanizing nutritional interest in intestinal cholesterol metabolism for protection against cardiovascular diseases.

    Science.gov (United States)

    Wegner, Casey J; Kim, Bohkyung; Lee, Jiyoung

    2013-01-16

    Recent studies have demonstrated that the intestine is a key target organ for overall health and longevity. Complementing these studies is the discovery of the trans-intestinal cholesterol efflux pathway and the emerging role of the intestine in reverse cholesterol transport. The surfacing dynamics of the regulation of cholesterol metabolism in the intestine provides an attractive platform for intestine-specific nutritional intervention strategies to lower blood cholesterol levels for protection against cardiovascular diseases. Notably, there is mounting evidence that stimulation of pathways associated with calorie restriction may have a large effect on the regulation of cholesterol removal by the intestine. However, intestinal energy metabolism, specifically the idiosyncrasies surrounding intestinal responses to energy deprivation, is poorly understood. The goal of this paper is to review recent insights into cholesterol regulation by the intestine and to discuss the potential for positive regulation of intestine-driven cholesterol removal through the nutritional induction of pathways associated with calorie restriction.

  15. On the entry of an emerging arbovirus into host cells: Mayaro virus takes the highway to the cytoplasm through fusion with early endosomes and caveolae-derived vesicles

    Directory of Open Access Journals (Sweden)

    Carlos A.M. Carvalho

    2017-04-01

    Full Text Available Mayaro virus (MAYV is an emergent sylvatic alphavirus in South America, related to sporadic outbreaks of a chikungunya-like human febrile illness accompanied by severe arthralgia. Despite its high potential for urban emergence, MAYV is still an obscure virus with scarce information about its infection cycle, including the corresponding early events. Even for prototypical alphaviruses, the cell entry mechanism still has some rough edges to trim: although clathrin-mediated endocytosis is quoted as the putative route, alternative paths as distinct as direct virus genome injection through the cell plasma membrane seems to be possible. Our aim was to clarify crucial details on the entry route exploited by MAYV to gain access into the host cell. Tracking the virus since its first contact with the surface of Vero cells by fluorescence microscopy, we show that its entry occurs by a fast endocytic process and relies on fusion with acidic endosomal compartments. Moreover, blocking clathrin-mediated endocytosis or depleting cholesterol from the cell membrane leads to a strong inhibition of viral infection, as assessed by plaque assays. Following this clue, we found that early endosomes and caveolae-derived vesicles are both implicated as target membranes for MAYV fusion. Our findings unravel the very first events that culminate in a productive infection by MAYV and shed light on potential targets for a rational antiviral therapy, besides providing a better comprehension of the entry routes exploited by alphaviruses to get into the cell.

  16. The ESCRT regulator Did2 maintains the balance between long-distance endosomal transport and endocytic trafficking.

    Directory of Open Access Journals (Sweden)

    Carl Haag

    2017-04-01

    Full Text Available In highly polarised cells, like fungal hyphae, early endosomes function in both endocytosis as well as long-distance transport of various cargo including mRNA and protein complexes. However, knowledge on the crosstalk between these seemingly different trafficking processes is scarce. Here, we demonstrate that the ESCRT regulator Did2 coordinates endosomal transport in fungal hyphae of Ustilago maydis. Loss of Did2 results in defective vacuolar targeting, less processive long-distance transport and abnormal shuttling of early endosomes. Importantly, the late endosomal protein Rab7 and vacuolar protease Prc1 exhibit increased shuttling on these aberrant endosomes suggesting defects in endosomal maturation and identity. Consistently, molecular motors fail to attach efficiently explaining the disturbed processive movement. Furthermore, the endosomal mRNP linker protein Upa1 is hardly present on endosomes resulting in defects in long-distance mRNA transport. In conclusion, the ESCRT regulator Did2 coordinates precise maturation of endosomes and thus provides the correct membrane identity for efficient endosomal long-distance transport.

  17. Requirement of cholesterol in the viral envelope for dengue virus infection.

    Science.gov (United States)

    Carro, Ana C; Damonte, Elsa B

    2013-06-01

    The role of cholesterol in the virus envelope or in the cellular membranes for dengue virus (DENV) infection was examined by depletion with methyl-beta-cyclodextrin (MCD) or nystatin. Pretreatment of virions with MCD or nystatin significantly reduced virus infectivity in a dose-dependent manner. By contrast, pre-treatment of diverse human cell lines with MCD or nystatin did not affect DENV infection. The four DENV serotypes were similarly inactivated by cholesterol-extracting drugs and infectivity was partially rescued when virion suspensions were treated with MCD in the presence of bovine serum. The addition of serum or exogenous water-soluble cholesterol after MCD treatment did not produce a reversion of MCD inactivating effect. Furthermore, virion treatment with extra cholesterol exerted also a virucidal effect. Binding and uptake of cholesterol-deficient DENV into the host cell were not impaired, whereas the next step of fusion between virion envelope and endosome membrane leading to virion uncoating and release of nucleocapsids to the cytoplasm appeared to be prevented, as determined by the retention of capsid protein in cells infected with MCD inactivated-DENV virions. Thereafter, the infection was almost completely inhibited, given the failure of viral RNA synthesis and viral protein expression in cells infected with MCD-treated virions. These data suggest that envelope cholesterol is a critical factor in the fusion process for DENV entry. Copyright © 2013 Elsevier B.V. All rights reserved.

  18. Cholesterol binding to ion channels

    Directory of Open Access Journals (Sweden)

    Irena eLevitan

    2014-02-01

    Full Text Available Numerous studies demonstrated that membrane cholesterol is a major regulator of ion channel function. The goal of this review is to discuss significant advances that have been recently achieved in elucidating the mechanisms responsible for cholesterol regulation of ion channels. The first major insight that comes from growing number of studies that based on the sterol specificity of cholesterol effects, show that several types of ion channels (nAChR, Kir, BK, TRPV are regulated by specific sterol-protein interactions. This conclusion is supported by demonstrating direct saturable binding of cholesterol to a bacterial Kir channel. The second major advance in the field is the identification of putative cholesterol binding sites in several types of ion channels. These include sites at locations associated with the well-known cholesterol binding motif CRAC and its reversed form CARC in nAChR, BK, and TRPV, as well as novel cholesterol binding regions in Kir channels. Notably, in the majority of these channels, cholesterol is suggested to interact mainly with hydrophobic residues in non-annular regions of the channels being embedded in between transmembrane protein helices. We also discuss how identification of putative cholesterol binding sites is an essential step to understand the mechanistic basis of cholesterol-induced channel regulation. Clearly, however, these are only the first few steps in obtaining a general understanding of cholesterol-ion channels interactions and their roles in cellular and organ functions.

  19. Histone deacetylase inhibitors correct the cholesterol storage defect in most Niemann-Pick C1 mutant cells.

    Science.gov (United States)

    Pipalia, Nina H; Subramanian, Kanagaraj; Mao, Shu; Ralph, Harold; Hutt, Darren M; Scott, Samantha M; Balch, William E; Maxfield, Frederick R

    2017-04-01

    Niemann-Pick C (NPC) disease is an autosomal recessive disorder that leads to excessive storage of cholesterol and other lipids in late endosomes and lysosomes. The large majority of NPC disease is caused by mutations in NPC1, a large polytopic membrane protein that functions in late endosomes. There are many disease-associated mutations in NPC1, and most patients are compound heterozygotes. The most common mutation, NPC1I1061T, has been shown to cause endoplasmic reticulum-associated degradation of the NPC1 protein. Treatment of patient-derived NPC1I1061T fibroblasts with histone deacetylase inhibitors (HDACis) vorinostat or panobinostat increases expression of the mutant NPC1 protein and leads to correction of the cholesterol storage. Here, we show that several other human NPC1 mutant fibroblast cell lines can also be corrected by vorinostat or panobinostat and that treatment with vorinostat extends the lifetime of the NPC1I1061T protein. To test effects of HDACi on a large number of NPC1 mutants, we engineered a U2OS cell line to suppress NPC1 expression by shRNA and then transiently transfected these cells with 60 different NPC1 mutant constructs. The mutant NPC1 did not significantly reduce cholesterol accumulation, but approximately 85% of the mutants showed reduced cholesterol accumulation when treated with vorinostat or panobinostat. Copyright © 2017 by the American Society for Biochemistry and Molecular Biology, Inc.

  20. Endosomal WASH and exocyst complexes control exocytosis of MT1-MMP at invadopodia.

    Science.gov (United States)

    Monteiro, Pedro; Rossé, Carine; Castro-Castro, Antonio; Irondelle, Marie; Lagoutte, Emilie; Paul-Gilloteaux, Perrine; Desnos, Claire; Formstecher, Etienne; Darchen, François; Perrais, David; Gautreau, Alexis; Hertzog, Maud; Chavrier, Philippe

    2013-12-23

    Remodeling of the extracellular matrix by carcinoma cells during metastatic dissemination requires formation of actin-based protrusions of the plasma membrane called invadopodia, where the trans-membrane type 1 matrix metalloproteinase (MT1-MMP) accumulates. Here, we describe an interaction between the exocyst complex and the endosomal Arp2/3 activator Wiskott-Aldrich syndrome protein and Scar homolog (WASH) on MT1-MMP–containing late endosomes in invasive breast carcinoma cells. We found that WASH and exocyst are required for matrix degradation by an exocytic mechanism that involves tubular connections between MT1-MMP–positive late endosomes and the plasma membrane in contact with the matrix. This ensures focal delivery of MT1-MMP and supports pericellular matrix degradation and tumor cell invasion into different pathologically relevant matrix environments. Our data suggest a general mechanism used by tumor cells to breach the basement membrane and for invasive migration through fibrous collagen-enriched tissues surrounding the tumor.

  1. Cell polarity and patterning by PIN trafficking through early endosomal compartments in Arabidopsis thaliana.

    Directory of Open Access Journals (Sweden)

    Hirokazu Tanaka

    2013-05-01

    Full Text Available PIN-FORMED (PIN proteins localize asymmetrically at the plasma membrane and mediate intercellular polar transport of the plant hormone auxin that is crucial for a multitude of developmental processes in plants. PIN localization is under extensive control by environmental or developmental cues, but mechanisms regulating PIN localization are not fully understood. Here we show that early endosomal components ARF GEF BEN1 and newly identified Sec1/Munc18 family protein BEN2 are involved in distinct steps of early endosomal trafficking. BEN1 and BEN2 are collectively required for polar PIN localization, for their dynamic repolarization, and consequently for auxin activity gradient formation and auxin-related developmental processes including embryonic patterning, organogenesis, and vasculature venation patterning. These results show that early endosomal trafficking is crucial for cell polarity and auxin-dependent regulation of plant architecture.

  2. Thermoresponsive pegylated bubble liposome nanovectors for efficient siRNA delivery via endosomal escape

    KAUST Repository

    Alamoudi, Kholod

    2017-05-19

    Improving the delivery of siRNA into cancer cells via bubble liposomes. Designing a thermoresponsive pegylated liposome through the introduction of ammonium bicarbonate salt into liposomes so as to control their endosomal escape for gene therapy.A sub-200 nm nanovector was fully characterized and examined for cellular uptake, cytotoxicity, endosomal escape and gene silencing.The siRNA-liposomes were internalized into cancer cells within 5 min and then released siRNAs in the cytosol prior to lysosomal degradation upon external temperature elevation. This was confirmed by confocal bioimaging and gene silencing reaching up to 90% and further demonstrated by the protein inhibition of both target genes.The thermoresponsiveness of ammonium bicarbonate containing liposomes enabled the rapid endosomal escape of the particles and resulted in an efficient gene silencing.

  3. Neuronal Cholesterol Accumulation Induced by Cyp46a1 Down-Regulation in Mouse Hippocampus Disrupts Brain Lipid Homeostasis

    Directory of Open Access Journals (Sweden)

    Sophie Ayciriex

    2017-07-01

    Full Text Available Impairment in cholesterol metabolism is associated with many neurodegenerative disorders including Alzheimer's disease (AD. However, the lipid alterations underlying neurodegeneration and the connection between altered cholesterol levels and AD remains not fully understood. We recently showed that cholesterol accumulation in hippocampal neurons, induced by silencing Cyp46a1 gene expression, leads to neurodegeneration with a progressive neuronal loss associated with AD-like phenotype in wild-type mice. We used a targeted and non-targeted lipidomics approach by liquid chromatography coupled to high-resolution mass spectrometry to further characterize lipid modifications associated to neurodegeneration and cholesterol accumulation induced by CYP46A1 inhibition. Hippocampus lipidome of normal mice was profiled 4 weeks after cholesterol accumulation due to Cyp46a1 gene expression down-regulation at the onset of neurodegeneration. We showed that major membrane lipids, sphingolipids and specific enzymes involved in phosphatidylcholine and sphingolipid metabolism, were rapidly increased in the hippocampus of AAV-shCYP46A1 injected mice. This lipid accumulation was associated with alterations in the lysosomal cargoe, accumulation of phagolysosomes and impairment of endosome-lysosome trafficking. Altogether, we demonstrated that inhibition of cholesterol 24-hydroxylase, key enzyme of cholesterol metabolism leads to a complex dysregulation of lipid homeostasis. Our results contribute to dissect the potential role of lipids in severe neurodegenerative diseases like AD.

  4. Characterization of placental cholesterol transport

    DEFF Research Database (Denmark)

    Lindegaard, Marie L; Wassif, Christopher A; Vaisman, Boris

    2008-01-01

    Patients with Smith-Lemli-Opitz syndrome (SLOS) are born with multiple congenital abnormalities. Postnatal cholesterol supplementation is provided; however, it cannot correct developmental malformations due to in utero cholesterol deficit. Increased transport of cholesterol from maternal to fetal...... circulation might attenuate congenital malformations. The cholesterol transporters Abca1, Abcg1, and Sr-b1 are present in placenta; however, their potential role in placental transport remains undetermined. In mice, expression analyses showed that Abca1 and Abcg1 transcripts increased 2-3-fold between...... embryonic days 13.5 and 18.5 in placental tissue; whereas, Sr-b1 expression decreased. To examine the functional role of Abca1, Abcg1 and Sr-b1 we measured the maternal-fetal transfer of (14)C-cholesterol in corresponding mutant embryos. Disruption of either Abca1 or Sr-b1 decreased cholesterol transfer...

  5. NHX-5, an Endosomal Na+/H+ Exchanger, Is Associated with Metformin Action.

    Science.gov (United States)

    Kim, Jeongho; Lee, Hye-Yeon; Ahn, Jheesoo; Hyun, Moonjung; Lee, Inhwan; Min, Kyung-Jin; You, Young-Jai

    2016-08-26

    Diabetes is one of the most impactful diseases worldwide. The most commonly prescribed anti-diabetic drug is metformin. In this study, we identified an endosomal Na(+)/H(+) exchanger (NHE) as a new potential target of metformin from an unbiased screen in Caenorhabditis elegans The same NHE homolog also exists in flies, where it too mediates the effects of metformin. Our results suggest that endosomal NHEs could be a metformin target and provide an insight into a novel mechanism of action of metformin on regulating the endocytic cycle. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. NHX-5, an Endosomal Na+/H+ Exchanger, Is Associated with Metformin Action*

    OpenAIRE

    Kim, Jeongho; Lee,Hye-Yeon; Ahn, Jheesoo; Hyun, Moonjung; Lee, Inhwan; Min, Kyung-Jin; You, Young-Jai

    2016-01-01

    Diabetes is one of the most impactful diseases worldwide. The most commonly prescribed anti-diabetic drug is metformin. In this study, we identified an endosomal Na+/H+ exchanger (NHE) as a new potential target of metformin from an unbiased screen in Caenorhabditis elegans. The same NHE homolog also exists in flies, where it too mediates the effects of metformin. Our results suggest that endosomal NHEs could be a metformin target and provide an insight into a novel mechanism of action of metf...

  7. Structure of the GAT domain of the endosomal adapter protein Tom1

    Directory of Open Access Journals (Sweden)

    Shuyan Xiao

    2016-06-01

    Full Text Available Cellular homeostasis requires correct delivery of cell-surface receptor proteins (cargo to their target subcellular compartments. The adapter proteins Tom1 and Tollip are involved in sorting of ubiquitinated cargo in endosomal compartments. Recruitment of Tom1 to the endosomal compartments is mediated by its GAT domain’s association to Tollip’s Tom1-binding domain (TBD. In this data article, we report the solution NMR-derived structure of the Tom1 GAT domain. The estimated protein structure exhibits a bundle of three helical elements. We compare the Tom1 GAT structure with those structures corresponding to the Tollip TBD- and ubiquitin-bound states.

  8. Dendritic Spine Morphology: The Role of Micro tubules and Endosomes

    NARCIS (Netherlands)

    B.R. Dortland (Bjorn)

    2009-01-01

    markdownabstract__Abstract__ In Ancient Egypt, the function of the brain was regarded as not much more than cranial stuffing. The brain was regularly removed in preparation for mummification since the heart was assumed to be the organ of intelligence. Over the next five thousand years, this

  9. Recent advances in cholesterol chemistry.

    Science.gov (United States)

    Morzycki, Jacek W

    2014-05-01

    This review article presents advances in cholesterol chemistry since 2000. Various transformations (chemical, enzymatic, electrochemical, etc.) of cholesterol are presented. A special emphasis is given to cholesterol oxidation reactions, but also substitution of the 3β-hydroxyl group, addition to the C5-C6 double bond, C-H functionalization, and C-C bond forming reactions are discussed. Copyright © 2014 Elsevier Inc. All rights reserved.

  10. Laparoscopic sleeve gastrectomy modifies cholesterol synthesis but not cholesterol absorption.

    Science.gov (United States)

    De Vuono, S; Ricci, M A; Siepi, D; Boni, M; Gentili, A; Scavizzi, M; Daviddi, G; Labate, P; Roscini, A R; Lupattelli, G

    Each bariatric surgery procedure impacts differently on cholesterol synthesis and absorption. Although a restrictive procedure, sleeve gastrectomy resolves diabetes mellitus and, like mixed-type procedures, induces early changes in gastrointestinal hormones. To our knowledge the present study is the first to assess the effects of sleeve gastrectomy on cholesterol synthesis and absorption. 42 consecutive subjects with obesity and sleeve gastrectomy candidates were included in the study together with a control group of 20 subjects without obesity. Before sleeve gastrectomy and 10 months afterwards, all subjects underwent a clinical examination, blood tests, ultrasound visceral fat area estimation and determination of plasma lathosterol, campesterol and sitosterol concentrations. After sleeve gastrectomy, significant decreases were observed in BMI, waist circumference, visceral and subcutaneous fat, blood pressure, triglycerides, insulin and glucose levels, lathosterol and HOMA-IR. HDL-C and apolipoprotein AI levels increased significantly. No significant differences emerged in LDL-C, apolipoprotein B levels or cholesterol absorption markers. Lathosterol levels correlated significantly with BMI, visceral fat area and HOMA-IR. Differences in cholesterol intake after surgery were not significantly associated with differences in lathosterol, campesterol and sitosterol concentrations. Sleeve gastrectomy reduced the markers of cholesterol synthesis but did not modify cholesterol absorption. Changes in cholesterol synthesis and absorption were independent of variations in cholesterol intake, suggesting a specific sleeve gastrectomy-related effect. Copyright © 2016 Asia Oceania Association for the Study of Obesity. Published by Elsevier Ltd. All rights reserved.

  11. [The real measurement of non-HDL-cholesterol: Atherogenic cholesterol].

    Science.gov (United States)

    Millán, Jesús; Hernández-Mijares, Antonio; Ascaso, Juan F; Blasco, Mariano; Brea, Angel; Díaz, Ángel; González-Santos, Pedro; Mantilla, Teresa; Pedro-Botet, Juan; Pintó, Xavier

    Lowe density lipoproteins (LDL) are the causal agent of cardiovascular diseases. In practice, we identify LDL with cholesterol transported in LDL (cLDL). So, cLDL has become the major target for cardiovascular prevention. Howewer, we have progressive evidences about the role of triglycerides rich lipoproteins, particularly those very low density lipoprotein (VLDL) in promotion and progression of atherosclerosis, that leads cholesterol in VLDL and its remanents as a potential therapeutic target. This feature is particularly important and of a great magnitude, in patients with hypertiglyceridemia. We can to considere, that the non-HDL cholesterol -cLDL+cVLDL+c-remmants+Lp(a)- is the real measurement of atherogenic cholesterol. In addition, non-HDL-cholesterol do not show any variations between postprandial states. In fact, non-HDL-cholesterol should be an excellent marker of atherogenic cholesterol, and an major therapeutic target in patients with atherogenic dyslipidaemia. According with different clinical trials and with the epidemiological and mendelian studies, in patients with high cardiovascular risk, optimal level of cLDL will be under 70mg/dl, and under 100 ng/dl for non-HDL-cholesterol; and in high risk patients, 100mg/dl and 130mg/dl, respectively. Copyright © 2016. Publicado por Elsevier España, S.L.U.

  12. Cholesterol-Lowering Probiotics as Potential Biotherapeutics for Metabolic Diseases

    Science.gov (United States)

    Kumar, Manoj; Nagpal, Ravinder; Kumar, Rajesh; Hemalatha, R.; Verma, Vinod; Kumar, Ashok; Chakraborty, Chaitali; Singh, Birbal; Marotta, Francesco; Jain, Shalini; Yadav, Hariom

    2012-01-01

    Cardiovascular diseases are one of the major causes of deaths in adults in the western world. Elevated levels of certain blood lipids have been reported to be the principal cause of cardiovascular disease and other disabilities in developed countries. Several animal and clinical trials have shown a positive association between cholesterol levels and the risks of coronary heart disease. Current dietary strategies for the prevention of cardiovascular disease advocate adherence to low-fat/low-saturated-fat diets. Although there is no doubt that, in experimental conditions, low-fat diets offer an effective means of reducing blood cholesterol concentrations on a population basis, these appear to be less effective, largely due to poor compliance, attributed to low palatability and acceptability of these diets to the consumers. Due to the low consumer compliance, attempts have been made to identify other dietary components that can reduce blood cholesterol levels. Supplementation of diet with fermented dairy products or lactic acid bacteria containing dairy products has shown the potential to reduce serum cholesterol levels. Various approaches have been used to alleviate this issue, including the use of probiotics, especially Bifidobacterium spp. and Lactobacillus spp.. Probiotics, the living microorganisms that confer health benefits on the host when administered in adequate amounts, have received much attention on their proclaimed health benefits which include improvement in lactose intolerance, increase in natural resistance to infectious disease in gastrointestinal tract, suppression of cancer, antidiabetic, reduction in serum cholesterol level, and improved digestion. In addition, there are numerous reports on cholesterol removal ability of probiotics and their hypocholesterolemic effects. Several possible mechanisms for cholesterol removal by probiotics are assimilation of cholesterol by growing cells, binding of cholesterol to cellular surface, incorporation of

  13. Cholesterol-Lowering Probiotics as Potential Biotherapeutics for Metabolic Diseases

    Directory of Open Access Journals (Sweden)

    Manoj Kumar

    2012-01-01

    Full Text Available Cardiovascular diseases are one of the major causes of deaths in adults in the western world. Elevated levels of certain blood lipids have been reported to be the principal cause of cardiovascular disease and other disabilities in developed countries. Several animal and clinical trials have shown a positive association between cholesterol levels and the risks of coronary heart disease. Current dietary strategies for the prevention of cardiovascular disease advocate adherence to low-fat/low-saturated-fat diets. Although there is no doubt that, in experimental conditions, low-fat diets offer an effective means of reducing blood cholesterol concentrations on a population basis, these appear to be less effective, largely due to poor compliance, attributed to low palatability and acceptability of these diets to the consumers. Due to the low consumer compliance, attempts have been made to identify other dietary components that can reduce blood cholesterol levels. Supplementation of diet with fermented dairy products or lactic acid bacteria containing dairy products has shown the potential to reduce serum cholesterol levels. Various approaches have been used to alleviate this issue, including the use of probiotics, especially Bifidobacterium spp. and Lactobacillus spp.. Probiotics, the living microorganisms that confer health benefits on the host when administered in adequate amounts, have received much attention on their proclaimed health benefits which include improvement in lactose intolerance, increase in natural resistance to infectious disease in gastrointestinal tract, suppression of cancer, antidiabetic, reduction in serum cholesterol level, and improved digestion. In addition, there are numerous reports on cholesterol removal ability of probiotics and their hypocholesterolemic effects. Several possible mechanisms for cholesterol removal by probiotics are assimilation of cholesterol by growing cells, binding of cholesterol to cellular surface

  14. HDL cholesterol: atherosclerosis and beyond

    NARCIS (Netherlands)

    Bochem, A.E.

    2013-01-01

    Cardiovascular disease (CVD) is the leading cause of death in the Western world. Myocardial infarction and stroke are the result of a compromised blood flow which may result from cholesterol accumulation in the vessel wall due to high plasma levels of LDL cholesterol. High plasma levels of HDL

  15. Endosomal gene expression: a new indicator for prostate cancer patient prognosis?

    LENUS (Irish Health Repository)

    Johnson, Ian R D

    2015-11-10

    Prostate cancer continues to be a major cause of morbidity and mortality in men, but a method for accurate prognosis in these patients is yet to be developed. The recent discovery of altered endosomal biogenesis in prostate cancer has identified a fundamental change in the cell biology of this cancer, which holds great promise for the identification of novel biomarkers that can predict disease outcomes. Here we have identified significantly altered expression of endosomal genes in prostate cancer compared to non-malignant tissue in mRNA microarrays and confirmed these findings by qRT-PCR on fresh-frozen tissue. Importantly, we identified endosomal gene expression patterns that were predictive of patient outcomes. Two endosomal tri-gene signatures were identified from a previously published microarray cohort and had a significant capacity to stratify patient outcomes. The expression of APPL1, RAB5A, EEA1, PDCD6IP, NOX4 and SORT1 were altered in malignant patient tissue, when compared to indolent and normal prostate tissue. These findings support the initiation of a case-control study using larger cohorts of prostate tissue, with documented patient outcomes, to determine if different combinations of these new biomarkers can accurately predict disease status and clinical progression in prostate cancer patients.

  16. Inhibition of late endosomal maturation restores Wnt secretion in Caenorhabditis elegans vps-29 retromer mutants.

    Science.gov (United States)

    Lorenowicz, Magdalena J; Macurkova, Marie; Harterink, Martin; Middelkoop, Teije C; de Groot, Reinoud; Betist, Marco C; Korswagen, Hendrik C

    2014-01-01

    Secretion of Wnt proteins is mediated by the Wnt sorting receptor Wls, which transports Wnt from the Golgi to the cell surface for release. To maintain efficient Wnt secretion, Wls is recycled back to the trans-Golgi network (TGN) through a retromer dependent endosome to TGN retrieval pathway. It has recently been shown that this is mediated by an alternative retromer pathway in which the sorting nexin SNX3 interacts with the cargo-selective subcomplex of the retromer to sort Wls into a retrieval pathway that is morphologically distinct from the classical SNX-BAR dependent retromer pathway. Here, we investigated how sorting of Wls between the two different retromer pathways is specified. We found that when the function of the cargo-selective subcomplex of the retromer is partially disrupted, Wnt secretion can be restored by interfering with the maturation of late endosomes to lysosomes. This leads to an accumulation of Wls in late endosomes and facilitates the retrieval of Wls through a SNX-BAR dependent retromer pathway. Our results are consistent with a model in which spatial separation of the SNX3 and SNX-BAR retromer complexes along the endosomal maturation pathway as well as cargo-specific mechanisms contribute to the selective retrieval of Wls through the SNX3 retromer pathway. © 2013.

  17. Investigation of endosome and lysosome biology by ultra pH-sensitive nanoprobes.

    Science.gov (United States)

    Wang, Chensu; Zhao, Tian; Li, Yang; Huang, Gang; White, Michael A; Gao, Jinming

    2017-04-01

    Endosomes and lysosomes play a critical role in various aspects of cell physiology such as nutrient sensing, receptor recycling, protein/lipid catabolism, and cell death. In drug delivery, endosomal release of therapeutic payloads from nanocarriers is also important in achieving efficient delivery of drugs to reach their intracellular targets. Recently, we invented a library of ultra pH-sensitive (UPS) nanoprobes with exquisite fluorescence response to subtle pH changes. The UPS nanoprobes also displayed strong pH-specific buffer effect over small molecular bases with broad pH responses (e.g., chloroquine and NH 4 Cl). Tunable pH transitions from 7.4 to 4.0 of UPS nanoprobes cover the entire physiological pH of endocytic organelles (e.g., early and late endosomes) and lysosomes. These unique physico-chemical properties of UPS nanoprobes allowed a 'detection and perturbation' strategy for the investigation of luminal pH in cell signaling and metabolism, which introduces a nanotechnology-enabled paradigm for the biological studies of endosomes and lysosomes. Published by Elsevier B.V.

  18. Arabidopsis ALIX is required for the endosomal localization of the deubiquitinating enzyme AMSH3.

    Science.gov (United States)

    Kalinowska, Kamila; Nagel, Marie-Kristin; Goodman, Kaija; Cuyas, Laura; Anzenberger, Franziska; Alkofer, Angela; Paz-Ares, Javier; Braun, Pascal; Rubio, Vicente; Otegui, Marisa S; Isono, Erika

    2015-10-06

    Ubiquitination is a signal for various cellular processes, including for endocytic degradation of plasma membrane cargos. Ubiquitinating as well as deubiquitinating enzymes (DUBs) can regulate these processes by modifying the ubiquitination status of target protein. Although accumulating evidence points to the important regulatory role of DUBs, the molecular basis of their regulation is still not well understood. Associated molecule with the SH3 domain of signal transduction adaptor molecule (STAM) (AMSH) is a conserved metalloprotease DUB in eukaryotes. AMSH proteins interact with components of the endosomal sorting complex required for transport (ESCRT) and are implicated in intracellular trafficking. To investigate how the function of AMSH is regulated at the cellular level, we carried out an interaction screen for the Arabidopsis AMSH proteins and identified the Arabidopsis homolog of apoptosis-linked gene-2 interacting protein X (ALIX) as a protein interacting with AMSH3 in vitro and in vivo. Analysis of alix knockout mutants in Arabidopsis showed that ALIX is essential for plant growth and development and that ALIX is important for the biogenesis of the vacuole and multivesicular bodies (MVBs). Cell biological analysis revealed that ALIX and AMSH3 colocalize on late endosomes. Although ALIX did not stimulate AMSH3 activity in vitro, in the absence of ALIX, AMSH3 localization on endosomes was abolished. Taken together, our data indicate that ALIX could function as an important regulator for AMSH3 function at the late endosomes.

  19. Mutant Huntingtin Impairs Vesicle Formation from Recycling Endosomes by Interfering with Rab11 Activity▿ †

    Science.gov (United States)

    Li, Xueyi; Standley, Clive; Sapp, Ellen; Valencia, Antonio; Qin, Zheng-Hong; Kegel, Kimberly B.; Yoder, Jennifer; Comer-Tierney, Laryssa A.; Esteves, Miguel; Chase, Kathryn; Alexander, Jonathan; Masso, Nicholas; Sobin, Lindsay; Bellve, Karl; Tuft, Richard; Lifshitz, Lawrence; Fogarty, Kevin; Aronin, Neil; DiFiglia, Marian

    2009-01-01

    Huntingtin (Htt) localizes to endosomes, but its role in the endocytic pathway is not established. Recently, we found that Htt is important for the activation of Rab11, a GTPase involved in endosomal recycling. Here we studied fibroblasts of healthy individuals and patients with Huntington's disease (HD), which is a movement disorder caused by polyglutamine expansion in Htt. The formation of endocytic vesicles containing transferrin at plasma membranes was the same in control and HD patient fibroblasts. However, HD fibroblasts were delayed in recycling biotin-transferrin back to the plasma membrane. Membranes of HD fibroblasts supported less nucleotide exchange on Rab11 than did control membranes. Rab11-positive vesicular and tubular structures in HD fibroblasts were abnormally large, suggesting that they were impaired in forming vesicles. We used total internal reflection fluorescence imaging of living fibroblasts to monitor fluorescence-labeled transferrin-carrying transport intermediates that emerged from recycling endosomes. HD fibroblasts had fewer small vesicles and more large vesicles and long tubules than did control fibroblasts. Dominant active Rab11 expressed in HD fibroblasts normalized the recycling of biotin-transferrin. We propose a novel mechanism for cellular dysfunction by the HD mutation arising from the inhibition of Rab11 activity and a deficit in vesicle formation at recycling endosomes. PMID:19752198

  20. Natural Modulators of Endosomal Toll-Like Receptor-Mediated Psoriatic Skin Inflammation

    Directory of Open Access Journals (Sweden)

    Chao-Yang Lai

    2017-01-01

    Full Text Available Psoriasis is a chronic inflammatory autoimmune disease that can be initiated by excessive activation of endosomal toll-like receptors (TLRs, particularly TLR7, TLR8, and TLR9. Therefore, inhibitors of endosomal TLR activation are being investigated for their ability to treat this disease. The currently approved biological drugs adalimumab, etanercept, infliximab, ustekinumab, ixekizumab, and secukizumab are antibodies against effector cytokines that participate in the initiation and development of psoriasis. Several immune modulatory oligonucleotides and small molecular weight compounds, including IMO-3100, IMO-8400, and CPG-52364, that block the interaction between endosomal TLRs and their ligands are under clinical investigation for their effectiveness in the treatment of psoriasis. In addition, several chemical compounds, including AS-2444697, PF-05387252, PF-05388169, PF-06650833, ML120B, and PHA-408, can inhibit TLR signaling. Although these compounds have demonstrated anti-inflammatory activity in animal models, their therapeutic potential for the treatment of psoriasis has not yet been tested. Recent studies demonstrated that natural compounds derived from plants, fungi, and bacteria, including mustard seed, Antrodia cinnamomea extract, curcumin, resveratrol, thiostrepton, azithromycin, and andrographolide, inhibited psoriasis-like inflammation induced by the TLR7 agonist imiquimod in animal models. These natural modulators employ different mechanisms to inhibit endosomal TLR activation and are administered via different routes. Therefore, they represent candidate psoriasis drugs and might lead to the development of new treatment options.

  1. Crystal structure of subunit VPS25 of the endosomal trafficking complex ESCRT-II

    Directory of Open Access Journals (Sweden)

    Weissenhorn Winfried

    2004-12-01

    Full Text Available Abstract Background Down-regulation of plasma membrane receptors via the endocytic pathway involves their monoubiquitylation, transport to endosomal membranes and eventual sorting into multi vesicular bodies (MVB destined for lysosomal degradation. Successive assemblies of Endosomal Sorting Complexes Required for Transport (ESCRT-I, -II and III largely mediate sorting of plasma membrane receptors at endosomal membranes, the formation of multivesicular bodies and their release into the endosomal lumen. In addition, the human ESCRT-II has been shown to form a complex with RNA polymerase II elongation factor ELL in order to exert transcriptional control activity. Results Here we report the crystal structure of Vps25 at 3.1 Å resolution. Vps25 crystallizes in a dimeric form and each monomer is composed of two winged helix domains arranged in tandem. Structural comparisons detect no conformational changes between unliganded Vps25 and Vps25 within the ESCRT-II complex composed of two Vps25 copies and one copy each of Vps22 and Vps36 12. Conclusions Our structural analyses present a framework for studying Vps25 interactions with ESCRT-I and ESCRT-III partners. Winged helix domain containing proteins have been implicated in nucleic acid binding and it remains to be determined whether Vps25 has a similar activity which might play a role in the proposed transcriptional control exerted by Vps25 and/or the whole ESCRT-II complex.

  2. RECYCLING PATHWAYS OF GLUCOSYLCERAMIDE IN BHK CELLS - DISTINCT INVOLVEMENT OF EARLY AND LATE ENDOSOMES

    NARCIS (Netherlands)

    KOK, JW; HOEKSTRA, K; ESKELINEN, S; HOEKSTRA, D

    1992-01-01

    Recycling pathways of the sphingolipid glucosylceramide were studied by employing a fluorescent analog of glucosylceramide, 6-[N-(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]hexanoylglucosylsphingosine (C6-NBD-glucosylceramide). Direct recycling of the glycolipid from early endosomes to the plasma

  3. TLR2 ligands induce NF-κB activation from endosomal compartments of human monocytes.

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    Karim J Brandt

    Full Text Available Localization of Toll-like receptors (TLR in subcellular organelles is a major strategy to regulate innate immune responses. While TLR4, a cell-surface receptor, signals from both the plasma membrane and endosomal compartments, less is known about the functional role of endosomal trafficking upon TLR2 signaling. Here we show that the bacterial TLR2 ligands Pam3CSK4 and LTA activate NF-κB-dependent signaling from endosomal compartments in human monocytes and in a NF-κB sensitive reporter cell line, despite the expression of TLR2 at the cell surface. Further analyses indicate that TLR2-induced NF-κB activation is controlled by a clathrin/dynamin-dependent endocytosis mechanism, in which CD14 serves as an important upstream regulator. These findings establish that internalization of cell-surface TLR2 into endosomal compartments is required for NF-κB activation. These observations further demonstrate the need of endocytosis in the activation and regulation of TLR2-dependent signaling pathways.

  4. Epigenetic Regulation of Cholesterol Homeostasis

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    Steve eMeaney

    2014-09-01

    Full Text Available Although best known as a risk factor for cardiovascular disease, cholesterol is a vital component of all mammalian cells. In addition to key structural roles, cholesterol is a vital biochemical precursor for numerous biologically important compounds including oxysterols and bile acids, as well as acting as an activator of critical morphogenic systems (e.g. the Hedgehog system. A variety of sophisticated regulatory mechanisms interact to coordinate the overall level of cholesterol in cells, tissues and the entire organism. Accumulating evidence indicates that in additional to the more ‘traditional’ regulatory schemes, cholesterol homeostasis is also under the control of epigenetic mechanisms such as histone acetylation and DNA methylation. The available evidence supporting a role for these mechanisms in the control of cholesterol synthesis, elimination, transport and storage are the focus of this review.

  5. Quantitative comparison of the efficacy of various compounds in lowering intracellular cholesterol levels in Niemann-Pick type C fibroblasts.

    Directory of Open Access Journals (Sweden)

    Zachary T Wehrmann

    Full Text Available Niemann-Pick Type C disease (NPC is a lethal, autosomal recessive disorder caused by mutations in the NPC1 and NPC2 cholesterol transport proteins. NPC's hallmark symptoms include an accumulation of unesterified cholesterol and other lipids in the late endosomal and lysosomal cellular compartments, causing progressive neurodegeneration and death. Although the age of onset may vary in those affected, NPC most often manifests in juveniles, and is usually fatal before adolescence. In this study, we investigated the effects of various drugs, many of which modify the epigenetic control of NPC1/NPC2 gene expression, in lowering the otherwise harmful elevated intracellular cholesterol levels in NPC cells. Our studies utilized a previously described image analysis technique, which allowed us to make quantitative comparisons of the efficacy of these drugs in lowering cholesterol levels in a common NPC1 mutant model. Of the drugs analyzed, several that have been previously studied (vorinostat, panobinostat, and β-cyclodextrin significantly lowered the relative amount of unesterified cellular cholesterol, consistent with earlier observations. In addition, a novel potential treatment, rapamycin, likewise alleviated the NPC phenotype. We also studied combinations of effective compounds with β-cyclodextrin; the addition of β-cyclodextrin significantly enhanced the cholesterol-lowering activity of vorinostat and panobinostat, but had mixed effects with rapamycin. Collectively, these results may provide a basis for the eventual development of improved NPC therapies.

  6. How to Get Your Cholesterol Tested

    Science.gov (United States)

    ... Thromboembolism Aortic Aneurysm More How To Get Your Cholesterol Tested Updated:Nov 16,2017 High cholesterol usually ... diabetes and high blood pressure. How often should cholesterol be checked? The American Heart Association recommends that ...

  7. The cross-talk of LDL-cholesterol with cell motility: insights from the Niemann Pick Type C1 mutation and altered integrin trafficking.

    Science.gov (United States)

    Hoque, Monira; Rentero, Carles; Conway, James R; Murray, Rachael Z; Timpson, Paul; Enrich, Carlos; Grewal, Thomas

    2015-01-01

    Cholesterol is considered indispensible for the recruitment and functioning of integrins in focal adhesions for cell migration. However, the physiological cholesterol pools that control integrin trafficking and focal adhesion assembly remain unclear. Using Niemann Pick Type C1 (NPC) mutant cells, which accumulate Low Density lipoprotein (LDL)-derived cholesterol in late endosomes (LE), several recent studies indicate that LDL-cholesterol has multiple roles in regulating focal adhesion dynamics. Firstly, targeting of endocytosed LDL-cholesterol from LE to focal adhesions controls their formation at the leading edge of migrating cells. Other newly emerging literature suggests that this may be coupled to vesicular transport of integrins, Src kinase and metalloproteases from the LE compartment to focal adhesions. Secondly, our recent work identified LDL-cholesterol as a key factor that determines the distribution and ability of several Soluble NSF Attachment Protein (SNAP) Receptor (SNARE) proteins, key players in vesicle transport, to control integrin trafficking to the cell surface and extracellular matrix (ECM) secretion. Collectively, dietary, genetic and pathological changes in cholesterol metabolism may link with efficiency and speed of integrin and ECM cell surface delivery in metastatic cancer cells. This commentary will summarize how direct and indirect pathways enable LDL-cholesterol to modulate cell motility.

  8. Net cholesterol efflux capacity of HDL enriched serum and coronary atherosclerosis in rheumatoid arthritis.

    Science.gov (United States)

    Ormseth, Michelle J; Yancey, Patricia G; Yamamoto, Suguru; Oeser, Annette M; Gebretsadik, Tebeb; Shintani, Ayumi; Linton, MacRae F; Fazio, Sergio; Davies, Sean S; Roberts, L Jackson; Vickers, Kasey C; Raggi, Paolo; Kon, Valentina; Stein, C Michael

    2016-12-01

    Cardiovascular (CV) risk is increased in patients with rheumatoid arthritis (RA), but not fully explained by traditional risk factors such as LDL and HDL cholesterol concentrations. The cholesterol efflux capacity of HDL may be a better CV risk predictor than HDL concentrations. We hypothesized that HDL's cholesterol efflux capacity is impaired and inversely associated with coronary atherosclerosis in patients with RA. We measured the net cholesterol efflux capacity of apolipoprotein B depleted serum and coronary artery calcium score in 134 patients with RA and 76 control subjects, frequency-matched for age, race and sex. The relationship between net cholesterol efflux capacity and coronary artery calcium score and other clinical variables of interest was assessed in patients with RA. Net cholesterol efflux capacity was similar among RA (median [IQR]: 34% removal [28, 41%]) and control subjects (35% removal [27%, 39%]) (P=0.73). In RA, increasing net cholesterol efflux capacity was not significantly associated with decreased coronary calcium score (OR=0.78 (95% CI 0.51-1.19), P=0.24, adjusted for age, race and sex, Framingham risk score and presence of diabetes). Net cholesterol efflux capacity was not significantly associated with RA disease activity score, C-reactive protein, urinary F2-isoprostanes, or degree of insulin resistance in RA. Net cholesterol efflux capacity is not significantly altered in patients with relatively well-controlled RA nor is it significantly associated with coronary artery calcium score.

  9. Cholesterol depletion disorganizes oocyte membrane rafts altering mouse fertilization.

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    Jorgelina Buschiazzo

    Full Text Available Drastic membrane reorganization occurs when mammalian sperm binds to and fuses with the oocyte membrane. Two oocyte protein families are essential for fertilization, tetraspanins and glycosylphosphatidylinositol-anchored proteins. The firsts are associated to tetraspanin-enriched microdomains and the seconds to lipid rafts. Here we report membrane raft involvement in mouse fertilization assessed by cholesterol modulation using methyl-β-cyclodextrin. Cholesterol removal induced: (1 a decrease of the fertilization rate and index; and (2 a delay in the extrusion of the second polar body. Cholesterol repletion recovered the fertilization ability of cholesterol-depleted oocytes, indicating reversibility of these effects. In vivo time-lapse analyses using fluorescent cholesterol permitted to identify the time-point at which the probe is mainly located at the plasma membrane enabling the estimation of the extent of the cholesterol depletion. We confirmed that the mouse oocyte is rich in rafts according to the presence of the raft marker lipid, ganglioside GM1 on the membrane of living oocytes and we identified the coexistence of two types of microdomains, planar rafts and caveolae-like structures, by terms of two differential rafts markers, flotillin-2 and caveolin-1, respectively. Moreover, this is the first report that shows characteristic caveolae-like invaginations in the mouse oocyte identified by electron microscopy. Raft disruption by cholesterol depletion disturbed the subcellular localization of the signal molecule c-Src and the inhibition of Src kinase proteins prevented second polar body extrusion, consistent with a role of Src-related kinases in fertilization via signaling complexes. Our data highlight the functional importance of intact membrane rafts for mouse fertilization and its dependence on cholesterol.

  10. Human ClC-6 is a late endosomal glycoprotein that associates with detergent-resistant lipid domains.

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    Sofie Ignoul

    Full Text Available BACKGROUND: The mammalian CLC protein family comprises nine members (ClC-1 to -7 and ClC-Ka, -Kb that function either as plasma membrane chloride channels or as intracellular chloride/proton antiporters, and that sustain a broad spectrum of cellular processes, such as membrane excitability, transepithelial transport, endocytosis and lysosomal degradation. In this study we focus on human ClC-6, which is structurally most related to the late endosomal/lysomal ClC-7. PRINCIPAL FINDINGS: Using a polyclonal affinity-purified antibody directed against a unique epitope in the ClC-6 COOH-terminal tail, we show that human ClC-6, when transfected in COS-1 cells, is N-glycosylated in a region that is evolutionary poorly conserved between mammalian CLC proteins and that is located between the predicted helices K and M. Three asparagine residues (N410, N422 and N432 have been defined by mutagenesis as acceptor sites for N-glycosylation, but only two of the three sites seem to be simultaneously N-glycosylated. In a differentiated human neuroblastoma cell line (SH-SY5Y, endogenous ClC-6 colocalizes with LAMP-1, a late endosomal/lysosomal marker, but not with early/recycling endosomal markers such as EEA-1 and transferrin receptor. In contrast, when transiently expressed in COS-1 or HeLa cells, human ClC-6 mainly overlaps with markers for early/recycling endosomes (transferrin receptor, EEA-1, Rab5, Rab4 and not with late endosomal/lysosomal markers (LAMP-1, Rab7. Analogously, overexpression of human ClC-6 in SH-SY5Y cells also leads to an early/recycling endosomal localization of the exogenously expressed ClC-6 protein. Finally, in transiently transfected COS-1 cells, ClC-6 copurifies with detergent-resistant membrane fractions, suggesting its partitioning in lipid rafts. Mutating a juxtamembrane string of basic amino acids (amino acids 71-75: KKGRR disturbs the association with detergent-resistant membrane fractions and also affects the segregation of ClC-6

  11. Cholesterol worships a new idol.

    Science.gov (United States)

    Schulman, Ira G

    2009-12-01

    The growing worldwide epidemic of cardiovascular disease suggests that new therapeutic strategies are needed to complement statins in the lowering of cholesterol levels. In a recent paper in Science, Tontonoz and colleagues have identified Idol as a protein that can control cholesterol levels by regulating the stability of the low-density lipoprotein receptor; inhibiting the activity of Idol could provide novel approaches for the treatment of cardiovascular disease.

  12. Assimilation of cholesterol by yeast strains isolated from infant feces and Feta cheese.

    Science.gov (United States)

    Psomas, E I; Fletouris, D J; Litopoulou-Tzanetaki, E; Tzanetakis, N

    2003-11-01

    Eight yeast strains isolated from infant feces and the traditional Greek Feta cheese, selected for their probiotic properties, were tested along with a commercially available strain of Saccharomyces boulardii for their ability to remove cholesterol from a growth medium (yeast extract glucose peptone broth) supplemented with 0.3% Oxgall. The amount of cholesterol removed during 72 h of growth at 37 degrees C revealed significant variations among the yeast strains examined. Two isolates from infant feces, namely Saccharomyces cerevisiae KK1 and Isaatchenkia orientalis KK5.Y.1 and one isolate from Feta cheese, namely S. cerevisiae 832, along with the commercial strain S. boulardii, were able to remove cholesterol from the growth medium after 48 h of incubation at 37 degrees C. However, Saccharomyces strains proved to be able to remove cholesterol even after 24 h of growth at 37 degrees C. The cholesterol removed from the growth medium was not metabolically degraded but was rather assimilated into the yeast cells. The ability to assimilate cholesterol in vitro and to tolerate low pH levels, gastric juice, and bile indicate that S. cerevisiae 832, and especially S. cerevisiae KK1 and I. orientalis KK5.Y.1 (being more bile and gastric juice tolerant because of their human origin) may be promising candidate strains for use as probiotics.

  13. The Endosome-associated Deubiquitinating Enzyme USP8 Regulates BACE1 Enzyme Ubiquitination and Degradation.

    Science.gov (United States)

    Yeates, Eniola Funmilayo Aduke; Tesco, Giuseppina

    2016-07-22

    The β-site amyloid precursor protein-cleaving enzyme (BACE1) is the rate-limiting enzyme in the production of amyloid-β, the toxic peptide that accumulates in the brain of subjects affected by Alzheimer disease. Our previous studies have shown that BACE1 is degraded via the lysosomal pathway and that that depletion of the trafficking molecule Golgi-localized γ-ear-containing ARF-binding protein 3 (GGA3) results in increased BACE1 levels and activity because of impaired lysosomal degradation. We also determined that GGA3 regulation of BACE1 levels requires its ability to bind ubiquitin. Accordingly, we reported that BACE1 is ubiquitinated at lysine 501 and that lack of ubiquitination at lysine 501 produces BACE1 stabilization. Ubiquitin conjugation is a reversible process mediated by deubiquitinating enzymes. The ubiquitin-specific peptidase 8 (USP8), an endosome-associated deubiquitinating enzyme, regulates the ubiquitination, trafficking, and lysosomal degradation of several plasma membrane proteins. Here, we report that RNAi-mediated depletion of USP8 reduced levels of both ectopically expressed and endogenous BACE1 in H4 human neuroglioma cells. Moreover, USP8 depletion increased BACE1 ubiquitination, promoted BACE1 accumulation in the early endosomes and late endosomes/lysosomes, and decreased levels of BACE1 in the recycling endosomes. We also found that decreased BACE1 protein levels were accompanied by a decrease in BACE1-mediated amyloid precursor protein cleavage and amyloid-β levels. Our findings demonstrate that USP8 plays a key role in the trafficking and degradation of BACE1 by deubiquitinating lysine 501. These studies suggest that therapies able to accelerate BACE1 degradation (e.g. by increasing BACE1 ubiquitination) may represent a potential treatment for Alzheimer disease. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Imaging single retrovirus entry through alternative receptor isoforms and intermediates of virus-endosome fusion.

    Science.gov (United States)

    Jha, Naveen K; Latinovic, Olga; Martin, Erik; Novitskiy, Gennadiy; Marin, Mariana; Miyauchi, Kosuke; Naughton, John; Young, John A T; Melikyan, Gregory B

    2011-01-20

    A large group of viruses rely on low pH to activate their fusion proteins that merge the viral envelope with an endosomal membrane, releasing the viral nucleocapsid. A critical barrier to understanding these events has been the lack of approaches to study virus-cell membrane fusion within acidic endosomes, the natural sites of virus nucleocapsid capsid entry into the cytosol. Here we have investigated these events using the highly tractable subgroup A avian sarcoma and leukosis virus envelope glycoprotein (EnvA)-TVA receptor system. Through labeling EnvA pseudotyped viruses with a pH-sensitive fluorescent marker, we imaged their entry into mildly acidic compartments. We found that cells expressing the transmembrane receptor (TVA950) internalized the virus much faster than those expressing the GPI-anchored receptor isoform (TVA800). Surprisingly, TVA800 did not accelerate virus uptake compared to cells lacking the receptor. Subsequent steps of virus entry were visualized by incorporating a small viral content marker that was released into the cytosol as a result of fusion. EnvA-dependent fusion with TVA800-expressing cells occurred shortly after endocytosis and delivery into acidic endosomes, whereas fusion of viruses internalized through TVA950 was delayed. In the latter case, a relatively stable hemifusion-like intermediate preceded the fusion pore opening. The apparent size and stability of nascent fusion pores depended on the TVA isoforms and their expression levels, with TVA950 supporting more robust pores and a higher efficiency of infection compared to TVA800. These results demonstrate that surface receptor density and the intracellular trafficking pathway used are important determinants of efficient EnvA-mediated membrane fusion, and suggest that early fusion intermediates play a critical role in establishing low pH-dependent virus entry from within acidic endosomes.

  15. Imaging single retrovirus entry through alternative receptor isoforms and intermediates of virus-endosome fusion.

    Directory of Open Access Journals (Sweden)

    Naveen K Jha

    2011-01-01

    Full Text Available A large group of viruses rely on low pH to activate their fusion proteins that merge the viral envelope with an endosomal membrane, releasing the viral nucleocapsid. A critical barrier to understanding these events has been the lack of approaches to study virus-cell membrane fusion within acidic endosomes, the natural sites of virus nucleocapsid capsid entry into the cytosol. Here we have investigated these events using the highly tractable subgroup A avian sarcoma and leukosis virus envelope glycoprotein (EnvA-TVA receptor system. Through labeling EnvA pseudotyped viruses with a pH-sensitive fluorescent marker, we imaged their entry into mildly acidic compartments. We found that cells expressing the transmembrane receptor (TVA950 internalized the virus much faster than those expressing the GPI-anchored receptor isoform (TVA800. Surprisingly, TVA800 did not accelerate virus uptake compared to cells lacking the receptor. Subsequent steps of virus entry were visualized by incorporating a small viral content marker that was released into the cytosol as a result of fusion. EnvA-dependent fusion with TVA800-expressing cells occurred shortly after endocytosis and delivery into acidic endosomes, whereas fusion of viruses internalized through TVA950 was delayed. In the latter case, a relatively stable hemifusion-like intermediate preceded the fusion pore opening. The apparent size and stability of nascent fusion pores depended on the TVA isoforms and their expression levels, with TVA950 supporting more robust pores and a higher efficiency of infection compared to TVA800. These results demonstrate that surface receptor density and the intracellular trafficking pathway used are important determinants of efficient EnvA-mediated membrane fusion, and suggest that early fusion intermediates play a critical role in establishing low pH-dependent virus entry from within acidic endosomes.

  16. The Endosome-associated Deubiquitinating Enzyme USP8 Regulates BACE1 Enzyme Ubiquitination and Degradation*

    Science.gov (United States)

    Yeates, Eniola Funmilayo Aduke; Tesco, Giuseppina

    2016-01-01

    The β-site amyloid precursor protein-cleaving enzyme (BACE1) is the rate-limiting enzyme in the production of amyloid-β, the toxic peptide that accumulates in the brain of subjects affected by Alzheimer disease. Our previous studies have shown that BACE1 is degraded via the lysosomal pathway and that that depletion of the trafficking molecule Golgi-localized γ-ear-containing ARF-binding protein 3 (GGA3) results in increased BACE1 levels and activity because of impaired lysosomal degradation. We also determined that GGA3 regulation of BACE1 levels requires its ability to bind ubiquitin. Accordingly, we reported that BACE1 is ubiquitinated at lysine 501 and that lack of ubiquitination at lysine 501 produces BACE1 stabilization. Ubiquitin conjugation is a reversible process mediated by deubiquitinating enzymes. The ubiquitin-specific peptidase 8 (USP8), an endosome-associated deubiquitinating enzyme, regulates the ubiquitination, trafficking, and lysosomal degradation of several plasma membrane proteins. Here, we report that RNAi-mediated depletion of USP8 reduced levels of both ectopically expressed and endogenous BACE1 in H4 human neuroglioma cells. Moreover, USP8 depletion increased BACE1 ubiquitination, promoted BACE1 accumulation in the early endosomes and late endosomes/lysosomes, and decreased levels of BACE1 in the recycling endosomes. We also found that decreased BACE1 protein levels were accompanied by a decrease in BACE1-mediated amyloid precursor protein cleavage and amyloid-β levels. Our findings demonstrate that USP8 plays a key role in the trafficking and degradation of BACE1 by deubiquitinating lysine 501. These studies suggest that therapies able to accelerate BACE1 degradation (e.g. by increasing BACE1 ubiquitination) may represent a potential treatment for Alzheimer disease. PMID:27302062

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

    DEFF Research Database (Denmark)

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

    2007-01-01

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

  18. Rhinovirus stimulated IFN-? production: how important are plasmacytoid DCs, monocytes and endosomal pH?

    OpenAIRE

    Xi, Yang; Finlayson, Arvid; White, Oliva J; Carroll, Melanie L.; Upham, John W.

    2015-01-01

    Human rhinovirus (HRV) infection is a major cause of asthma exacerbations, which appears to be linked to a defective innate immune response to infection. Although the type I interferons (IFN-? and IFN-?) have a critical role in protecting against most viral infections, the cells responsible for IFN production in response to HRV and the relative importance of pattern recognition receptors located in endosomes has not been fully elucidated. In the current study we demonstrate that, using intrac...

  19. Endosome-based protein trafficking and Ca2+ homeostasis in the heart

    Directory of Open Access Journals (Sweden)

    Jerry eCurran

    2015-02-01

    Full Text Available The ability to dynamically regulate, traffic, retain, and recycle proteins within the cell membrane is fundamental to life and central to the normal function of the heart and cardiovascular system. In the heart, these systems are essential for the regulation of cardiac calcium, both at the level of the plasma membrane, but also at local domains of the endoplasmic reticulum, sarcoplasmic reticulum, mitochondria, nucleus, and nuclear envelope. One intracellular pathway often overlooked in relation to cardiovascular calcium regulation and signaling is the endosome-based trafficking pathway. Highlighting its importance, this system and its molecular components are evolutionarily conserved across all metazoans. However, remarkably little is known of how endosome-based protein trafficking and recycling functions within mammalian cells systems, especially in the heart. The vast majority of what is known has been derived from heterologous cell systems. However, recently, more appropriate cell and animal models been developed that have allowed researchers to begin to understand how this system functions within the intact physiological environment. All excitable cells, including cardiomyocytes, depend on the proper expression and organization of multiple ion channels, pumps, exchangers, and transporters within the plasma membrane. As the endosomal system acts to regulate the expression and localization of membrane proteins, understanding the in vivo function of this system in the heart is important. This review will focus on endosome-based protein trafficking in the heart in both health and disease. Special emphasis will be given to the role played by the family of endocytic regulatory proteins, C-terminal Eps15 homology domain -containing proteins (EHDs, as recent data demonstrates that this family of proteins is essential for the proper trafficking and localization and of key proteins involved in excitation-contraction coupling.

  20. Phosphatidylinositol 3,5-Bisphosphate-Rich Membrane Domains in Endosomes and Lysosomes.

    Science.gov (United States)

    Takatori, Sho; Tatematsu, Tsuyako; Cheng, Jinglei; Matsumoto, Jun; Akano, Takuya; Fujimoto, Toyoshi

    2016-02-01

    Phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2 ) has critical functions in endosomes and lysosomes. We developed a method to define nanoscale distribution of PtdIns(3,5)P2 using freeze-fracture electron microscopy. GST-ATG18-4×FLAG was used to label PtdIns(3,5)P2 and its binding to phosphatidylinositol 3-phosphate (PtdIns(3)P) was blocked by an excess of the p40(phox) PX domain. In yeast exposed to hyperosmotic stress, PtdIns(3,5)P2 was concentrated in intramembrane particle (IMP)-deficient domains in the vacuolar membrane, which made close contact with adjacent membranes. The IMP-deficient domain was also enriched with PtdIns(3)P, but was deficient in Vph1p, a liquid-disordered domain marker. In yeast lacking either PtdIns(3,5)P2 or its effector, Atg18p, the IMP-deficient, PtdIns(3)P-rich membranes were folded tightly to make abnormal tubular structures, thus showing where the vacuolar fragmentation process is arrested when PtdIns(3,5)P2 metabolism is defective. In HeLa cells, PtdIns(3,5)P2 was significantly enriched in the vesicular domain of RAB5- and RAB7-positive endosome/lysosomes of the tubulo-vesicular morphology. This biased distribution of PtdIns(3,5)P2 was also observed using fluorescence microscopy, which further showed enrichment of a retromer component, VPS35, in the tubular domain. This is the first report to show segregation of PtdIns(3,5)P2 -rich and -deficient domains in endosome/lysosomes, which should be important for endosome/lysosome functionality. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  1. Amperometric determination of serum total cholesterol with nanoparticles of cholesterol esterase and cholesterol oxidase.

    Science.gov (United States)

    Aggarwal, V; Malik, J; Prashant, A; Jaiwal, P K; Pundir, C S

    2016-05-01

    We describe the preparation of glutaraldehyde cross-linked and functionalized cholesterol esterase nanoparticles (ChENPs) and cholesterol oxidase nanoparticles (ChOxNPs) aggregates and their co-immobilization onto Au electrode for improved amperometric determination of serum total cholesterol. Transmission electron microscope (TEM) images of ChENPs and ChOxNPs showed their spherical shape and average size of 35.40 and 56.97 nm, respectively. Scanning electron microscope (SEM) studies of Au electrode confirmed the co-immobilization of enzyme nanoparticles (ENPs). The biosensor exhibited optimal response at pH 5.5 and 40°C within 5 s when polarized at +0.25 V versus Ag/AgCl. The working/linear range of the biosensor was 10-700 mg/dl for cholesterol. The sensor showed high sensitivity and measured total cholesterol as low as 0.1 mg/dl. The biosensor was evaluated and employed for total cholesterol determination in sera of apparently healthy and diseased persons. The analytical recovery of added cholesterol was 90%, whereas the within-batch and between-batch coefficients of variation (CVs) were less than 2% and less than 3%. There was a good correlation (r = 0.99) between serum cholesterol values as measured by the standard enzymic colorimetric method and the current method. The initial activity of ENPs/working electrode was reduced by 50% during its regular use (200 times) over a period of 60 days when stored dry at 4°C. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. Augmenting the Efficacy of Immunotoxins and Other Targeted Protein Toxins by Endosomal Escape Enhancers

    Directory of Open Access Journals (Sweden)

    Hendrik Fuchs

    2016-07-01

    Full Text Available The toxic moiety of almost all protein-based targeted toxins must enter the cytosol of the target cell to mediate its fatal effect. Although more than 500 targeted toxins have been investigated in the past decades, no antibody-targeted protein toxin has been approved for tumor therapeutic applications by the authorities to date. Missing efficacy can be attributed in many cases to insufficient endosomal escape and therefore subsequent lysosomal degradation of the endocytosed toxins. To overcome this drawback, many strategies have been described to weaken the membrane integrity of endosomes. This comprises the use of lysosomotropic amines, carboxylic ionophores, calcium channel antagonists, various cell-penetrating peptides of viral, bacterial, plant, animal, human and synthetic origin, other organic molecules and light-induced techniques. Although the efficacy of the targeted toxins was typically augmented in cell culture hundred or thousand fold, in exceptional cases more than million fold, the combination of several substances harbors new problems including additional side effects, loss of target specificity, difficulties to determine the therapeutic window and cell type-dependent variations. This review critically scrutinizes the chances and challenges of endosomal escape enhancers and their potential role in future developments.

  3. Role of the AP-5 adaptor protein complex in late endosome-to-Golgi retrieval.

    Directory of Open Access Journals (Sweden)

    Jennifer Hirst

    2018-01-01

    Full Text Available The AP-5 adaptor protein complex is presumed to function in membrane traffic, but so far nothing is known about its pathway or its cargo. We have used CRISPR-Cas9 to knock out the AP-5 ζ subunit gene, AP5Z1, in HeLa cells, and then analysed the phenotype by subcellular fractionation profiling and quantitative mass spectrometry. The retromer complex had an altered steady-state distribution in the knockout cells, and several Golgi proteins, including GOLIM4 and GOLM1, were depleted from vesicle-enriched fractions. Immunolocalisation showed that loss of AP-5 led to impaired retrieval of the cation-independent mannose 6-phosphate receptor (CIMPR, GOLIM4, and GOLM1 from endosomes back to the Golgi region. Knocking down the retromer complex exacerbated this phenotype. Both the CIMPR and sortilin interacted with the AP-5-associated protein SPG15 in pull-down assays, and we propose that sortilin may act as a link between Golgi proteins and the AP-5/SPG11/SPG15 complex. Together, our findings suggest that AP-5 functions in a novel sorting step out of late endosomes, acting as a backup pathway for retromer. This provides a mechanistic explanation for why mutations in AP-5/SPG11/SPG15 cause cells to accumulate aberrant endolysosomes, and highlights the role of endosome/lysosome dysfunction in the pathology of hereditary spastic paraplegia and other neurodegenerative disorders.

  4. Do Alix and ALG-2 really control endosomes for better or for worse?

    Science.gov (United States)

    Sadoul, Rémy

    2006-01-01

    Alix/AIP1 (ALG-2-interacting protein X/apoptosis-linked-gene-2-interacting protein 1) is an adaptor protein that was first described for its capacity to bind to the calcium-binding protein ALG-2 (apoptosis-linked gene 2), the expression of which seemed necessary for cell death. Over-expression of truncated forms of Alix blocks caspase-dependent and -independent mechanisms of cell death. Numerous observations in yeast and in mammalian cells suggest that Alix controls the making of and trafficking through endosomes called MVBs (multivesicular bodies), which are crucial intermediates within the endolysosomal system. In particular, deletion of Bro1, one of the yeast homologues of Alix, leads to an impairment in the function of MVBs, leading to mis-sorting of proteins normally destined to the vacuole. Mammalian Alix may have a similar function and has been shown to bind to lyso(bis)phosphatidic acid, ESCRT (endosomal sorting complex required for transport) proteins, endophilins and CIN85 (Cbl-interacting protein of 85 kDa), which are all main regulators of the endosomal system. EIAV (equine infectious anaemia virus) and HIV late domains use Alix to recruit the ESCRT machinery in order to bud from the cell surface, underscoring the crucial role of the protein in orchestrating membrane deformation. In this review I develop the hypothesis that the normal function of Alix in the endolysosomal system may be deviated by ALG-2 towards a destructive role during active cell death.

  5. ATG12-ATG3 interacts with Alix to promote basal autophagic flux and late endosome function.

    Science.gov (United States)

    Murrow, Lyndsay; Malhotra, Ritu; Debnath, Jayanta

    2015-03-01

    The ubiquitin-like molecule ATG12 is required for the early steps of autophagy. Recently, we identified ATG3, the E2-like enzyme required for LC3 lipidation during autophagy, as an ATG12 conjugation target. Here, we demonstrate that cells lacking ATG12-ATG3 have impaired basal autophagic flux, accumulation of perinuclear late endosomes, and impaired endolysosomal trafficking. Furthermore, we identify an interaction between ATG12-ATG3 and the ESCRT-associated protein Alix (also known as PDCD6IP) and demonstrate that ATG12-ATG3 controls multiple Alix-dependent processes including late endosome distribution, exosome biogenesis and viral budding. Similar to ATG12-ATG3, Alix is functionally required for efficient basal, but not starvation-induced, autophagy. Overall, these results identify a link between the core autophagy and ESCRT machineries and uncover a role for ATG12-ATG3 in late endosome function that is distinct from the canonical role of either ATG in autophagosome formation.

  6. Interferon-γ–inducible Rab20 regulates endosomal morphology and EGFR degradation in macrophages

    Science.gov (United States)

    Pei, Gang; Schnettger, Laura; Bronietzki, Marc; Repnik, Urska; Griffiths, Gareth; Gutierrez, Maximiliano Gabriel

    2015-01-01

    Little is known about the molecular players that regulate changes in the endocytic pathway during immune activation. Here we investigate the role of Rab20 in the endocytic pathway during activation of macrophages. Rab20 is associated with endocytic structures, but the function of this Rab GTPase in the endocytic pathway remains poorly characterized. We find that in macrophages, Rab20 expression and endosomal association significantly increase after interferon-γ (IFN-γ) treatment. Moreover, IFN-γ and Rab20 expression induce a dramatic enlargement of endosomes. These enlarged endosomes are the result of homotypic fusion promoted by Rab20 expression. The expression of Rab20 or the dominant-negative mutant Rab20T19N does not affect transferrin or dextran 70 kDa uptake. However, knockdown of Rab20 accelerates epidermal growth factor (EGF) trafficking to LAMP-2–positive compartments and EGF receptor degradation. Thus this work defines a function for Rab20 in the endocytic pathway during immune activation of macrophages. PMID:26157167

  7. Interferon-γ-inducible Rab20 regulates endosomal morphology and EGFR degradation in macrophages.

    Science.gov (United States)

    Pei, Gang; Schnettger, Laura; Bronietzki, Marc; Repnik, Urska; Griffiths, Gareth; Gutierrez, Maximiliano Gabriel

    2015-09-01

    Little is known about the molecular players that regulate changes in the endocytic pathway during immune activation. Here we investigate the role of Rab20 in the endocytic pathway during activation of macrophages. Rab20 is associated with endocytic structures, but the function of this Rab GTPase in the endocytic pathway remains poorly characterized. We find that in macrophages, Rab20 expression and endosomal association significantly increase after interferon-γ (IFN-γ) treatment. Moreover, IFN-γ and Rab20 expression induce a dramatic enlargement of endosomes. These enlarged endosomes are the result of homotypic fusion promoted by Rab20 expression. The expression of Rab20 or the dominant-negative mutant Rab20T19N does not affect transferrin or dextran 70 kDa uptake. However, knockdown of Rab20 accelerates epidermal growth factor (EGF) trafficking to LAMP-2-positive compartments and EGF receptor degradation. Thus this work defines a function for Rab20 in the endocytic pathway during immune activation of macrophages. © 2015 Pei, Schnettger, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  8. Neuronal retrograde transport of Borna disease virus occurs in signalling endosomes.

    Science.gov (United States)

    Charlier, Caroline M; Debaisieux, Solène; Foret, Charlotte; Thouard, Anne; Schiavo, Giampietro; Gonzalez-Dunia, Daniel; Malnou, Cécile E

    2016-12-01

    Long-range axonal retrograde transport is a key mechanism for the cellular dissemination of neuroinvasive viruses, such as Borna disease virus (BDV), for which entry and egress sites are usually distant from the nucleus, where viral replication takes place. Although BDV is known to disseminate very efficiently in neurons, both in vivo and in primary cultures, the modalities of its axonal transport are still poorly characterized. In this work, we combined different methodological approaches, such as confocal microscopy and biochemical purification of endosomes, to study BDV retrograde transport. We demonstrate that BDV ribonucleoparticles (composed of the viral genomic RNA, nucleoprotein and phosphoprotein), as well as the matrix protein, are transported towards the nucleus into endocytic carriers. These specialized organelles, called signalling endosomes, are notably used for the retrograde transport of neurotrophins and activated growth factor receptors. Signalling endosomes have a neutral luminal pH and thereby offer protection against degradation during long-range transport. This particularity could allow the viral particles to be delivered intact to the cell body of neurons, avoiding their premature release in the cytoplasm.

  9. CORVET and HOPS tethering complexes - coordinators of endosome and lysosome fusion.

    Science.gov (United States)

    Balderhaar, Henning J kleine; Ungermann, Christian

    2013-03-15

    Protein and lipid transport along the endolysosomal system of eukaryotic cells depends on multiple fusion and fission events. Over the past few years, the molecular constituents of both fission and fusion machineries have been identified. Here, we focus on the mechanism of membrane fusion at endosomes, vacuoles and lysosomes, and in particular on the role of the two homologous tethering complexes called CORVET and HOPS. Both complexes are heterohexamers; they share four subunits, interact with Rab GTPases and soluble NSF attachment protein receptors (SNAREs) and can tether membranes. Owing to the presence of specific subunits, CORVET is a Rab5 effector complex, whereas HOPS can bind efficiently to late endosomes and lysosomes through Rab7. Based on the recently described overall structure of the HOPS complex and a number of in vivo and in vitro analyses, important insights into their function have been obtained. Here, we discuss the general function of both complexes in yeast and in metazoan cells in the context of endosomal biogenesis and maturation.

  10. Endocytosis and Endosomal Trafficking of DNA After Gene Electrotransfer In Vitro

    Directory of Open Access Journals (Sweden)

    Christelle Rosazza

    2016-01-01

    Full Text Available DNA electrotransfer is a successful technique for gene delivery into cells and represents an attractive alternative to virus-based methods for clinical applications including gene therapy and DNA vaccination. However, little is currently known about the mechanisms governing DNA internalization and its fate inside cells. The objectives of this work were to investigate the role of endocytosis and to quantify the contribution of different routes of cellular trafficking during DNA electrotransfer. To pursue these objectives, we performed flow cytometry and single-particle fluorescence microscopy experiments using inhibitors of endocytosis and endosomal markers. Our results show that ≃50% of DNA is internalized by caveolin/raft-mediated endocytosis, 25% by clathrin-mediated endocytosis, and 25% by macropinocytosis. During active transport, DNA is routed through multiple endosomal compartments with, in the hour following electrotransfer, 70% found in Rab5 structures, 50% in Rab11-containing organelles and 30% in Rab9 compartments. Later, 60% of DNA colocalizes with Lamp1 vesicles. Because these molecular markers can overlap while following organelles through several steps of trafficking, the percentages do not sum up to 100%. We conclude that electrotransferred DNA uses the classical endosomal trafficking pathways. Our results are important for a generalized understanding of gene electrotransfer, which is crucial for its safe use in clinics.

  11. Nanoparticles Escaping RES and Endosome: Challenges for siRNA Delivery for Cancer Therapy

    Directory of Open Access Journals (Sweden)

    Shutao Guo

    2011-01-01

    Full Text Available Small interfering RNAs (siRNAs technology has emerged as a promising potential treatment for viral, genetic diseases and cancers. Despite the powerful therapeutic potential of siRNA, there are challenges for developing efficient and specific delivery systems for systemic administration. There are extracellular and intracellular barriers for nanoparticle-mediated delivery. First, nanoparticles are rapidly cleared from the circulation by the reticuloendothelial system (RES. Second, following their cellular uptake, nanoparticles are trapped in endosomes/lysosomes, where siRNA would be degraded by enzymes. In this review, we describe strategies for grafting a polyethylene glycol (PEG brush to the nanoparticles for evading RES, such that they may effectively accumulate in the tumor by the enhanced permeability and retention (EPR effect. PEG has to shed from the nanoparticles to allow close interaction with the tumor cells. Current strategies for facilitating endosome escape, such as ion pair formation, “proton sponge effect”, destabilizing endosome membrane, and hydrophobic modification of the vector, are discussed.

  12. Augmenting the Efficacy of Immunotoxins and Other Targeted Protein Toxins by Endosomal Escape Enhancers

    Science.gov (United States)

    Fuchs, Hendrik; Weng, Alexander; Gilabert-Oriol, Roger

    2016-01-01

    The toxic moiety of almost all protein-based targeted toxins must enter the cytosol of the target cell to mediate its fatal effect. Although more than 500 targeted toxins have been investigated in the past decades, no antibody-targeted protein toxin has been approved for tumor therapeutic applications by the authorities to date. Missing efficacy can be attributed in many cases to insufficient endosomal escape and therefore subsequent lysosomal degradation of the endocytosed toxins. To overcome this drawback, many strategies have been described to weaken the membrane integrity of endosomes. This comprises the use of lysosomotropic amines, carboxylic ionophores, calcium channel antagonists, various cell-penetrating peptides of viral, bacterial, plant, animal, human and synthetic origin, other organic molecules and light-induced techniques. Although the efficacy of the targeted toxins was typically augmented in cell culture hundred or thousand fold, in exceptional cases more than million fold, the combination of several substances harbors new problems including additional side effects, loss of target specificity, difficulties to determine the therapeutic window and cell type-dependent variations. This review critically scrutinizes the chances and challenges of endosomal escape enhancers and their potential role in future developments. PMID:27376327

  13. Atherogenic cholesterol-rich diet and periodontal disease.

    Science.gov (United States)

    Macri, Elisa; Lifshitz, Fima; Ramos, Cecilia; Orzuza, Ricardo; Costa, Osvaldo; Zago, Valeria; Boyer, Patricia; Friedman, Silvia

    2014-07-01

    This study investigated the effect of an atherogenic cholesterol-rich diet (AT) on the alveolar bone loss in rats with ligature-induced experimental periodontitis (EP). Female Wistar adult rats were assigned either a control (Co) or an AT diet fed for 9 weeks. The AT diet was high in saturated fat, cholesterol and energy. At week 2, animals were subjected to a unilateral ligature (L) around the left first molar (Co+L and AT+L). The contra lateral first right molar (not ligated) of both groups (Co and AT) were used as untreated controls. At week 9, blood was drawn, rats were euthanized, hemi-mandibles removed and stained digital photographs (buccal and lingual surfaces) and radiographs were obtained for quantification of alveolar bone loss (ABL). The ABL was determined by distance and area methods (mm(2)) and X-rays were used for periodontal bone support (PBS), (%). Rats in the AT group exhibited a 17% increase in energy intake, gained significant body weight and showed the highest serum total-cholesterol (T-C) and non-high density lipoprotein-cholesterol (HDL-C) levels (pperiodontal bone was the greatest in AT+L rats. AT feedings significantly increased the buccal area and distance of bone loss when compared with the unligated-teeth (pdiet high in saturated fatty acids and cholesterol. Copyright © 2014 Elsevier Ltd. All rights reserved.

  14. The Endosome Localized Arf-GAP AGAP1 Modulates Dendritic Spine Morphology Downstream of the Neurodevelopmental Disorder Factor Dysbindin

    Directory of Open Access Journals (Sweden)

    Miranda Arnold

    2016-09-01

    Full Text Available AGAP1 is an Arf1 GTPase activating protein that interacts with the vesicle-associated protein complexes adaptor protein 3 (AP-3 and Biogenesis of Lysosome Related Organelles Complex-1 (BLOC-1. Overexpression of AGAP1 in non-neuronal cells results in an accumulation of endosomal cargoes, which suggests a role in endosome-dependent traffic. In addition, AGAP1 is a candidate susceptibility gene for two neurodevelopmental disorders, autism spectrum disorder (ASD and schizophrenia (SZ; yet its localization and function in neurons have not been described. Here, we describe that AGAP1 localizes to axons, dendrites, dendritic spines, and synapses, colocalizing preferentially with markers of early and recycling endosomes. Functional studies reveal overexpression and down-regulation of AGAP1 affects both neuronal endosomal trafficking and dendritic spine morphology, supporting a role for AGAP1 in the recycling endosomal trafficking involved in their morphogenesis. Finally, we determined the sensitivity of AGAP1 expression to mutations in the DTNBP1 gene, which is associated with neurodevelopmental disorder, and found that AGAP1 mRNA and protein levels are selectively reduced in the null allele of the mouse orthologue of DTNBP1. We postulate that endosomal trafficking contributes to the pathogenesis of neurodevelopmental disorders affecting dendritic spine morphology, and thus excitatory synapse structure and function.

  15. NOK/STYK1 has a strong tendency towards forming aggregates and colocalises with epidermal growth factor receptor in endosomes.

    Science.gov (United States)

    Ding, Xue; Jiang, Qing-Bo; Li, Rui; Chen, Shaoyong; Zhang, Shuping

    2012-05-11

    Our previous studies showed that the overexpression of Novel Oncogene with Kinase-domain (NOK)/STYK1 led to cellular transformation, tumorigenesis and metastasis. This report characterises the subcellular distribution of NOK in HeLa cells and its localisation in early endosomes. Confocal immunolocalisation studies indicated that NOK had structural subtypes and was distributed into two distinct expression patterns: a dot pattern (DP) and an aggregation pattern (AP). The results of an immunohistochemistry (IHC) analysis of pathological tissues also showed that high expression level of endogenous NOK was expressed in an aggregate-like structure in vivo. Importantly, we found that NOK was localised in endosomes and colocalised with epidermal growth factor receptor (EGFR) in activated endosomal vesicles. However, as the stimulation time increased, NOK and EGFR began to progress through different pathways. EGFR was gradually degraded after treatment with EGF for approximately 20 min, whereas NOK levels were not reduced. This result suggests that NOK mainly plays a role in facilitating the trafficking of EGFR from early endosomes to later endosomes/lysosomes. Taken together, NOK has a strong tendency towards forming aggregates, which may have physiological implications and provide the first evidence that this novel receptor kinase is colocalised with EGFR in endosomes to participate in a post-internalisation step of EGFR. Copyright © 2012 Elsevier Inc. All rights reserved.

  16. Basolateral Endocytic Recycling Requires RAB-10 and AMPH-1 Mediated Recruitment of RAB-5 GAP TBC-2 to Endosomes

    Science.gov (United States)

    Liu, Ou; Grant, Barth D.

    2015-01-01

    The small GTPase RAB-5/Rab5 is a master regulator of the early endosome, required for a myriad of coordinated activities, including the degradation and recycling of internalized cargo. Here we focused on the recycling function of the early endosome and the regulation of RAB-5 by GAP protein TBC-2 in the basolateral C. elegans intestine. We demonstrate that downstream basolateral recycling regulators, GTPase RAB-10/Rab10 and BAR domain protein AMPH-1/Amphiphysin, bind to TBC-2 and help to recruit it to endosomes. In the absence of RAB-10 or AMPH-1 binding to TBC-2, RAB-5 membrane association is abnormally high and recycling cargo is trapped in early endosomes. Furthermore, the loss of TBC-2 or AMPH-1 leads to abnormally high spatial overlap of RAB-5 and RAB-10. Taken together our results indicate that RAB-10 and AMPH-1 mediated down-regulation of RAB-5 is an important step in recycling, required for cargo exit from early endosomes and regulation of early endosome–recycling endosome interactions. PMID:26393361

  17. Cholesterol-lowering effects of modified animal fats in postmenopausal women.

    Science.gov (United States)

    Labat, J B; Martini, M C; Carr, T P; Elhard, B M; Olson, B A; Bergmann, S D; Slavin, J L; Hayes, K C; Hassel, C A

    1997-12-01

    In an attempt to improve the nutritional value of animal fats (including milkfat and lard), two technological approaches (i.e., cholesterol removal by steam distillation and linoleic acid enrichment by addition of safflower oil) were tested for cholesterolemic effects in a cohort of 29 older women (age 68 +/- 7 years). Test fat sources were incorporated into crackers, cookies, cheese, ice cream, whipped topping, sour cream, baking shortening, and table spreads. Subjects were permanent residents of a convent where meals were prepared in a centralized kitchen, allowing test fats to be provided in daily food menu items. The foods containing test fats were introduced into three sequential dietary treatment periods, each lasting 4 weeks, in the following order: cholesterol-reduced animal fat (CRAF): fatty-acid modified, cholesterol-reduced animal fat (FAMCRAF); and-unaltered animal fat (AF). Subjects were offered menu items cafeteria style and encouraged to make food selections consistent with their habitual diets, which were recorded daily. Fasted blood lipid profiles determined at the end of each treatment period showed that FAMCRAF reduced mean plasma total cholesterol, LDL cholesterol, and apolipoprotein B concentrations relative to AF (p < 0.05). Mean HDL cholesterol concentrations were not influenced by diet. Relative to native products, animal fats modified by cholesterol removal and linoleic acid enrichment reduced plasma total and LDL cholesterol concentrations in a predictable manner similar to that based on studies of men.

  18. Short-term Acipimox decreases the ability of plasma from Type 2 diabetic patients and healthy subjects to stimulate cellular cholesterol efflux : a potentially adverse effect on reverse cholesterol transport

    NARCIS (Netherlands)

    Dullaart, RPF; van Tol, A

    Aims To evaluate the effect of short-term administration of the anti-lipolytic agent, Acipimox, on the ability of plasma to stimulate cellular cholesterol removal, which represents one of the first steps in the anti-atherogenic process of reverse cholesterol transport. Methods Eight male Type 2

  19. Short-term Acipimox decreases the ability of plasma from type 2 diabetic patients and healthy subjects to stimulate cellular cholesterol efflux: A potentially adverse effect on reverse cholesterol transport

    NARCIS (Netherlands)

    R.P.F. Dullaart (Robin); A. van Tol (Arie)

    2001-01-01

    textabstractAims: To evaluate the effect of short-term administration of the anti-lipolytic agent, Acipimox, on the ability of plasma to stimulate cellular cholesterol removal, which represents one of the first steps in the anti-atherogenic process of reverse cholesterol transport. Methods: Eight

  20. Enzymatic oxidation of cholesterol: properties and functional effects of cholestenone in cell membranes.

    Directory of Open Access Journals (Sweden)

    Maarit Neuvonen

    Full Text Available Bacterial cholesterol oxidase is commonly used as an experimental tool to reduce cellular cholesterol content. That the treatment also generates the poorly degradable metabolite 4-cholesten-3-one (cholestenone has received less attention. Here, we investigated the membrane partitioning of cholestenone using simulations and cell biological experiments and assessed the functional effects of cholestenone in human cells. Atomistic simulations predicted that cholestenone reduces membrane order, undergoes faster flip-flop and desorbs more readily from membranes than cholesterol. In primary human fibroblasts, cholestenone was released from membranes to physiological extracellular acceptors more avidly than cholesterol, but without acceptors it remained in cells over a day. To address the functional effects of cholestenone, we studied fibroblast migration during wound healing. When cells were either cholesterol oxidase treated or part of cellular cholesterol was exchanged for cholestenone with cyclodextrin, cell migration during 22 h was markedly inhibited. Instead, when a similar fraction of cholesterol was removed using cyclodextrin, cells replenished their cholesterol content in 3 h and migrated similarly to control cells. Thus, cholesterol oxidation produces long-term functional effects in cells and these are in part due to the generated membrane active cholestenone.

  1. Regulation of biliary cholesterol secretion and reverse cholesterol transport

    NARCIS (Netherlands)

    Dikkers, Arne

    2016-01-01

    According to the World Health Organization the number one cause of death throughout the world is cardiovascular disease. Therefore, there is an urgent need for new therapeutic strategies to prevent and treat cardiovascular disease. One possible way is to target the HDL-driven reverse cholesterol

  2. The effect of partial replacements of membrane cholesterol by other steroids on the osmotic fragility and glycerol permeability of erythrocytes

    NARCIS (Netherlands)

    Bruckdorfer, K.R.; Demel, R.A.; Gier, J. de; Deenen, L.L.M. van

    1969-01-01

    1. 1. (A) Sonicated dispersions of lecithins were incubated with aliquots of washed human erythrocytes, leading to the removal of part of the cholesterol complement, eventually followed by lysis. (B) Sonicated dispersions of lecithin and cholesterol with one of a range of other steroids were

  3. Remnant cholesterol and ischemic heart disease

    DEFF Research Database (Denmark)

    Varbo, Anette; Nordestgaard, Børge G

    2014-01-01

    PURPOSE OF REVIEW: To review recent advances in the field of remnant cholesterol as a contributor to the development of ischemic heart disease (IHD). RECENT FINDINGS: Epidemiologic, mechanistic, and genetic studies all support a role for elevated remnant cholesterol (=cholesterol in triglyceride......-rich lipoproteins) as a contributor to the development of atherosclerosis and IHD. Observational studies show association between elevated remnant cholesterol and IHD, and mechanistic studies show remnant cholesterol accumulation in the arterial wall like LDL-cholesterol (LDL-C) accumulation. Furthermore, large...... genetic studies show evidence of remnant cholesterol as a causal risk factor for IHD independent of HDL-cholesterol levels. Genetic studies also show that elevated remnant cholesterol is associated with low-grade inflammation, whereas elevated LDL-C is not. There are several pharmacologic ways of lowering...

  4. Intracellular mediators of transforming growth factor β superfamily signaling localize to endosomes in chicken embryo and mouse lenses in vivo

    Directory of Open Access Journals (Sweden)

    Ishii Shunsuke

    2007-06-01

    Full Text Available Abstract Background Endocytosis is a key regulator of growth factor signaling pathways. Recent studies showed that the localization to endosomes of intracellular mediators of growth factor signaling may be required for their function. Although there is substantial evidence linking endocytosis and growth factor signaling in cultured cells, there has been little study of the endosomal localization of signaling components in intact tissues or organs. Results Proteins that are downstream of the transforming growth factor-β superfamily signaling pathway were found on endosomes in chicken embryo and postnatal mouse lenses, which depend on signaling by members of the TGFβ superfamily for their normal development. Phosphorylated Smad1 (pSmad1, pSmad2, Smad4, Smad7, the transcriptional repressors c-Ski and TGIF and the adapter molecules Smad anchor for receptor activation (SARA and C184M, localized to EEA-1- and Rab5-positive vesicles in chicken embryo and/or postnatal mouse lenses. pSmad1 and pSmad2 also localized to Rab7-positive late endosomes. Smad7 was found associated with endosomes, but not caveolae. Bmpr1a conditional knock-out lenses showed decreased nuclear and endosomal localization of pSmad1. Many of the effectors in this pathway were distributed differently in vivo from their reported distribution in cultured cells. Conclusion Based on the findings reported here and data from other signaling systems, we suggest that the localization of activated intracellular mediators of the transforming growth factor-β superfamily to endosomes is important for the regulation of growth factor signaling.

  5. Serum lipid concentrations in patients with cholesterol and pigment gallstones.

    Science.gov (United States)

    Weerakoon, Harshi Thilanka Welegedara; Ranasinghe, Shirani; Navaratne, Ayanthi; Sivakanesan, Ramaiah; Galketiya, Kuda Banda; Rosairo, Shanthini

    2014-08-19

    Gallstones (GS) are formed as a result of impaired metabolic regulation of the human body. Abnormal lipid metabolism is partly responsible for the pathogenesis of GS mainly rich in cholesterol. Thus abnormalities of serum lipids would reflect the possibilities of the formation of cholesterol GS. This study aims to identify the significance of serum lipids on the development of GS disease. Serum lipid profiles were estimated in 73 patients with symptomatic GS, admitted to the Teaching Hospital, Peradeniya, Sri Lanka for GS removal surgeries from May 2011 to December 2012. Patients with normal serum bilirubin level and not being on lipid lowering drugs were recruited for the study. Serum lipid profile of each patient was analyzed by enzymatic kit assays and the chemical composition of GS was analyzed by Fourier Transform Infrared spectroscopy. Of the 73 patients, 37 (51%) had cholesterol GS while 36 (49%) had pigment GS. Serum lipid parameters of a majority of patients were within the normal range. Body mass index values of the patients with two types of GS were not significantly different (Two sample t test, p = 0.335). Out of the lipid parameters tested, only serum triglyceride concentration was significantly high in patients with cholesterol GS than that of pigment GS (Two sample t test, p = 0.038). None of the lipid parameters were significantly different between males and females (Two sample t test, p > 0.05). Compared to the patients with pigment GS who were aged below 50 years, mean total cholesterol and triglyceride concentrations were higher in the same age category patients with cholesterol GS. Abnormal serum lipid profiles doesn't seem to be an essential feature in patients with cholesterol GS. However when the two groups of patients with cholesterol and pigment GS with no significant difference of body mass indexes were compared, patients with cholesterol GS are more likely to have serum lipid parameters towards the undesirable cutoff levels

  6. THE INTERACTION BETWEEN SOME VEGETAL POLYPHENOLIC EXTRACTS AND CHOLESTEROL AT THE LEVEL OF THE CELLULAR MEMBRANE

    Directory of Open Access Journals (Sweden)

    Pincu Rotinberg

    2007-08-01

    Full Text Available “In vitro” treatment of the fibers of sartoruis muscle of frog (Rana ridibunda, Pall with 0,1 mM cholesterol causes on increase of the membranary resting potential (hyperpolarization, seen as irreversible on washing of the preparations with Ringer physiological solution without cholesterol. The polyphenolic extracts from bilberry (Vaccinum sp. fruits, red peony (Poeonia sp. flowers, black grapes and pink flowers of Hibiscus sp. (2 mg % active substance remove the cholesterol deposited in the structure of the cellular membranes, thus re-establishing the membranary properties and the redox potential (rH affected by cholesterol. Such effects are similar to those induced by similarine – a polyphenolic product, and by chlophenate – a drug recommended in therapeutics. The results obtained evidence the properties of the pholyphenolic extracts, as both normalizing agents of the cholesterol proportion from biomembranes and redox modulators, which recommends them for possible pharmacological utilization.

  7. Membrane Cholesterol Modulates Superwarfarin Toxicity

    Energy Technology Data Exchange (ETDEWEB)

    Marangoni, M. Natalia; Martynowycz, Michael W.; Kuzmenko, Ivan; Braun, David; Polak, Paul E.; Weinberg, Guy; Rubinstein, Israel; Gidalevitz, David; Feinstein, Douglas L.

    2016-04-26

    Superwarfarins are modified analogs of warfarin with additional lipophilic aromatic rings, up to 100-fold greater potency, and longer biological half-lives. We hypothesized that increased hydrophobicity allowed interactions with amphiphilic membranes and modulation of biological responses. We find that superwarfarins brodifacoum and difenacoum increase lactate production and cell death in neuroblastoma cells. In contrast, neither causes changes in glioma cells that have higher cholesterol content. After choleterol depletion, lactate production was increased and cell viability was reduced. Drug-membrane interactions were examined by surface X-ray scattering using Langmuir monolayers of dipalmitoylphosphatidylcholine and/or cholesterol. Specular X-ray reflectivity data revealed that superwarfarins, but not warfarin, intercalate between dipalmitoylphosphatidylcholine molecules, whereas grazing incidence X-ray diffraction demonstrated changes in lateral crystalline order of the film. Neither agent showed significant interactions with monolayers containing >20% cholesterol. These findings demonstrate an affinity of superwarfarins to biomembranes and suggest that cellular responses to these agents are regulated by cholesterol content.

  8. Parkinson Disease-linked Vps35 R524W Mutation Impairs the Endosomal Association of Retromer and Induces α-Synuclein Aggregation*

    OpenAIRE

    Follett, Jordan; Bugarcic, Andrea; Yang, Zhe; Ariotti, Nicholas; Norwood, Suzanne J.; Collins, Brett M.; Parton, Robert G.; Teasdale, Rohan D.

    2016-01-01

    Endosomal sorting is a highly orchestrated cellular process. Retromer is a heterotrimeric complex that associates with endosomal membranes and facilitates the retrograde sorting of multiple receptors, including the cation-independent mannose 6-phosphate receptor for lysosomal enzymes. The cycling of retromer on and off the endosomal membrane is regulated by a network of retromer-interacting proteins. Here, we find that Parkinson disease-associated Vps35 variant, R524W, but not P316S, is a los...

  9. ESCRT (Endosomal Sorting Complex Required for Transport) Machinery Is Essential for Acrosomal Exocytosis in Human Sperm.

    Science.gov (United States)

    Pocognoni, Cristian A; Berberián, María Victoria; Mayorga, Luis S

    2015-11-01

    The sperm acrosome reaction is a unique, regulated exocytosis characterized by the secretion of the acrosomal content and the release of hybrid vesicles formed by patches of the outer acrosomal and plasma membranes. In previous reports, we have shown that inward invaginations of the acrosomal membrane delineate ring-shaped membrane microdomains that contact the plasma membrane. We have postulated that the opening and expansion of fusion pores along these rings trigger acrosomal exocytosis. The invaginations of the acrosomal membrane topologically resemble the deformations of the endosomal membrane leading to the assembly of luminal vesicles in multivesicular bodies. In fact, intra-acrosomal vesicles are also formed during acrosomal exocytosis. Endosomal sorting complex required for transport (ESCRT) participates in the organization of membrane microdomains that are invaginated and released as intraluminal vesicles in endosomes. We report here that members of ESCRT I (TSG101), ESCRT III (CHMP4), and the AAA ATPase VPS4 are present in the acrosomal region of the human sperm. Perturbing the function of these factors with antibodies or recombinant proteins inhibited acrosomal exocytosis in permeabilized cells. A similar effect was observed with a dominant-negative mutant of VPS4A cross-linked to a cell-penetrating peptide in nonpermeabilized sperm stimulated with a calcium ionophore. When the function of ESCRTs was inhibited, acrosomes showed abnormal deformation of the acrosomal membrane, and SNARE proteins that participate in acrosomal exocytosis failed to be stabilized in neurotoxin-resistant complexes. However, the growing of membrane invaginations was not blocked, and numerous intra-acrosomal vesicles were observed. These observations indicate that ESCRT-mediated processes are essential for acrosomal secretion, implicating these multifunctional complexes in an exocytic event crucial for sperm-egg fusion. © 2015 by the Society for the Study of Reproduction, Inc.

  10. LRRK2 delays degradative receptor trafficking by impeding late endosomal budding through decreasing Rab7 activity.

    Science.gov (United States)

    Gómez-Suaga, Patricia; Rivero-Ríos, Pilar; Fdez, Elena; Blanca Ramírez, Marian; Ferrer, Isidro; Aiastui, Ana; López De Munain, Adolfo; Hilfiker, Sabine

    2014-12-20

    Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene cause late-onset autosomal dominant Parkinson's disease (PD), and sequence variations at the LRRK2 locus are associated with increased risk for sporadic PD. LRRK2 contains both GTPase and kinase domains flanked by protein interaction motifs, and mutations associated with familial PD have been described for both catalytic domains. LRRK2 has been implicated in diverse cellular processes, and recent evidence pinpoints to an important role for LRRK2 in modulating a variety of intracellular membrane trafficking pathways. However, the underlying mechanisms are poorly understood. Here, by studying the classical, well-understood, degradative trafficking pathway of the epidermal growth factor receptor (EGFR), we show that LRRK2 regulates endocytic membrane trafficking in an Rab7-dependent manner. Mutant LRRK2 expression causes a slight delay in early-to-late endosomal trafficking, and a pronounced delay in trafficking out of late endosomes, which become aberrantly elongated into tubules. This is accompanied by a delay in EGFR degradation. The LRRK2-mediated deficits in EGFR trafficking and degradation can be reverted upon coexpression of active Rab7 and of a series of proteins involved in bridging the EGFR to Rab7 on late endosomes. Effector pulldown assays indicate that pathogenic LRRK2 decreases Rab7 activity both in cells overexpressing LRRK2, as well as in fibroblasts from pathogenic mutant LRRK2 PD patients when compared with healthy controls. Together, these findings provide novel insights into a previously unknown regulation of Rab7 activity by mutant LRRK2 which impairs membrane trafficking at very late stages of the endocytic pathway. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  11. Structure and Function of Vps15 in the Endosomal G Protein Signaling Pathway

    Energy Technology Data Exchange (ETDEWEB)

    Heenan, Erin J.; Vanhooke, Janeen L.; Temple, Brenda R.; Betts, Laurie; Sondek, John E.; Dohlman, Henrik G.; (UNC)

    2009-09-11

    G protein-coupled receptors mediate cellular responses to a wide variety of stimuli, including taste, light, and neurotransmitters. In the yeast Saccharomyces cerevisiae, activation of the pheromone pathway triggers events leading to mating. The view had long been held that the G protein-mediated signal occurs principally at the plasma membrane. Recently, it has been shown that the G protein {alpha} subunit Gpa1 can promote signaling at endosomes and requires two components of the sole phosphatidylinositol-3-kinase in yeast, Vps15 and Vps34. Vps15 contains multiple WD repeats and also binds to Gpa1 preferentially in the GDP-bound state; these observations led us to hypothesize that Vps15 may function as a G protein {beta} subunit at the endosome. Here we show an X-ray crystal structure of the Vps15 WD domain that reveals a seven-bladed propeller resembling that of typical G{beta} subunits. We show further that the WD domain is sufficient to bind Gpa1 as well as to Atg14, a potential G{gamma} protein that exists in a complex with Vps15. The Vps15 kinase domain together with the intermediate domain (linking the kinase and WD domains) also contributes to Gpa1 binding and is necessary for Vps15 to sustain G protein signaling. These findings reveal that the Vps15 G{beta}-like domain serves as a scaffold to assemble Gpa1 and Atg14, whereas the kinase and intermediate domains are required for proper signaling at the endosome.

  12. Augmented cellular trafficking and endosomal escape of porous silicon nanoparticles via zwitterionic bilayer polymer surface engineering.

    Science.gov (United States)

    Shahbazi, Mohammad-Ali; Almeida, Patrick V; Mäkilä, Ermei M; Kaasalainen, Martti H; Salonen, Jarno J; Hirvonen, Jouni T; Santos, Hélder A

    2014-08-01

    The development of a stable vehicle with low toxicity, high cellular internalization, efficient endosomal escape, and optimal drug release profile is a key bottleneck in nanomedicine. To overcome all these problems, we have developed a successful layer-by-layer method to covalently conjugate polyethyleneimine (PEI) and poly(methyl vinyl ether-co-maleic acid) (PMVE-MA) copolymer on the surface of undecylenic acid functionalized thermally hydrocarbonized porous silicon nanoparticles (UnTHCPSi NPs), forming a bilayer zwitterionic nanocomposite containing free positive charge groups of hyper-branched PEI disguised by the PMVE-MA polymer. The surface smoothness, charge and hydrophilicity of the developed NPs considerably improved the colloidal and plasma stabilities via enhanced suspensibility and charge repulsion. Furthermore, despite the surface negative charge of the bilayer polymer-conjugated NPs, the cellular trafficking and endosomal escape were significantly increased in both MDA-MB-231 and MCF-7 breast cancer cells. Remarkably, we also showed that the conjugation of surface free amine groups of the highly toxic UnTHCPSi-PEI (Un-P) NPs to the carboxylic groups of PMVE-MA renders acceptable safety features to the system and preserves the endosomal escape properties via proton sponge mechanism of the free available amine groups located inside the hyper-branched PEI layer. Moreover, the double layer protection not only controlled the aggregation of the NPs and reduced the toxicity, but also sustained the drug release of an anticancer drug, methotrexate, with further improved cytotoxicity profile of the drug-loaded particles. These results provide a proof-of-concept evidence that such zwitterionic polymer-based PSi nanocomposites can be extensively used as a promising candidate for cytosolic drug delivery. Copyright © 2014 Elsevier Ltd. All rights reserved.

  13. Structural Studies of Adeno-Associated Virus Serotype 8 Capsid Transitions Associated with Endosomal Trafficking

    Energy Technology Data Exchange (ETDEWEB)

    Nam, Hyun-Joo; Gurda, Brittney L.; McKenna, Robert; Potter, Mark; Byrne, Barry; Salganik, Maxim; Muzyczka, Nicholas; Agbandje-McKenna, Mavis (Florida)

    2012-09-17

    The single-stranded DNA (ssDNA) parvoviruses enter host cells through receptor-mediated endocytosis, and infection depends on processing in the early to late endosome as well as in the lysosome prior to nuclear entry for replication. However, the mechanisms of capsid endosomal processing, including the effects of low pH, are poorly understood. To gain insight into the structural transitions required for this essential step in infection, the crystal structures of empty and green fluorescent protein (GFP) gene-packaged adeno-associated virus serotype 8 (AAV8) have been determined at pH values of 6.0, 5.5, and 4.0 and then at pH 7.5 after incubation at pH 4.0, mimicking the conditions encountered during endocytic trafficking. While the capsid viral protein (VP) topologies of all the structures were similar, significant amino acid side chain conformational rearrangements were observed on (i) the interior surface of the capsid under the icosahedral 3-fold axis near ordered nucleic acid density that was lost concomitant with the conformational change as pH was reduced and (ii) the exterior capsid surface close to the icosahedral 2-fold depression. The 3-fold change is consistent with DNA release from an ordering interaction on the inside surface of the capsid at low pH values and suggests transitions that likely trigger the capsid for genome uncoating. The surface change results in disruption of VP-VP interface interactions and a decrease in buried surface area between VP monomers. This disruption points to capsid destabilization which may (i) release VP1 amino acids for its phospholipase A2 function for endosomal escape and nuclear localization signals for nuclear targeting and (ii) trigger genome uncoating.

  14. MHC class I endosomal and lysosomal trafficking coincides with exogenous antigen loading in dendritic cells.

    Directory of Open Access Journals (Sweden)

    Genc Basha

    Full Text Available BACKGROUND: Cross-presentation by dendritic cells (DCs is a crucial prerequisite for effective priming of cytotoxic T-cell responses against bacterial, viral and tumor antigens; however, this antigen presentation pathway remains poorly defined. METHODOLOGY/PRINCIPAL FINDINGS: In order to develop a comprehensive understanding of this process, we tested the hypothesis that the internalization of MHC class I molecules (MHC-I from the cell surface is directly involved in cross-presentation pathway and the loading of antigenic peptides. Here we provide the first examination of the internalization of MHC-I in DCs and we demonstrate that the cytoplasmic domain of MHC-I appears to act as an addressin domain to route MHC-I to both endosomal and lysosomal compartments of DCs, where it is demonstrated that loading of peptides derived from exogenously-derived proteins occurs. Furthermore, by chasing MHC-I from the cell surface of normal and transgenic DCs expressing mutant forms of MHC-I, we observe that a tyrosine-based endocytic trafficking motif is required for the constitutive internalization of MHC-I molecules from the cell surface into early endosomes and subsequently deep into lysosomal peptide-loading compartments. Finally, our data support the concept that multiple pathways of peptide loading of cross-presented antigens may exist depending on the chemical nature and size of the antigen requiring processing. CONCLUSIONS/SIGNIFICANCE: We conclude that DCs have 'hijacked' and adapted a common vacuolar/endocytic intracellular trafficking pathway to facilitate MHC I access to the endosomal and lysosomal compartments where antigen processing and loading and antigen cross-presentation takes place.

  15. Hepatic ZIP14-mediated Zinc Transport Contributes to Endosomal Insulin Receptor Trafficking and Glucose Metabolism.

    Science.gov (United States)

    Aydemir, Tolunay Beker; Troche, Catalina; Kim, Min-Hyun; Cousins, Robert J

    2016-11-11

    Zinc influences signaling pathways through controlled targeted zinc transport. Zinc transporter Zip14 KO mice display a phenotype that includes impaired intestinal barrier function with low grade chronic inflammation, hyperinsulinemia, and increased body fat, which are signatures of diet-induced diabetes (type 2 diabetes) and obesity in humans. Hyperglycemia in type 2 diabetes and obesity is caused by insulin resistance. Insulin resistance results in inhibition of glucose uptake by liver and other peripheral tissues, principally adipose and muscle and with concurrently higher hepatic glucose production. Therefore, modulation of hepatic glucose metabolism is an important target for antidiabetic treatment approaches. We demonstrate that during glucose uptake, cell surface abundance of zinc transporter ZIP14 and mediated zinc transport increases. Zinc is distributed to multiple sites in hepatocytes through sequential translocation of ZIP14 from plasma membrane to early and late endosomes. Endosomes from Zip14 KO mice were zinc-deficient because activities of the zinc-dependent insulin-degrading proteases insulin-degrading enzyme and cathepsin D were impaired; hence insulin receptor activity increased. Transient increases in cytosolic zinc levels are concurrent with glucose uptake and suppression of glycogen synthesis. In contrast, Zip14 KO mice exhibited greater hepatic glycogen synthesis and impaired gluconeogenesis and glycolysis related to low cytosolic zinc levels. We can conclude that ZIP14-mediated zinc transport contributes to regulation of endosomal insulin receptor activity and glucose homeostasis in hepatocytes. Therefore, modulation of ZIP14 transport activity presents a new target for management of diabetes and other glucose-related disorders. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. Cytoplasmic Trafficking of Minute Virus of Mice: Low-pH Requirement, Routing to Late Endosomes, and Proteasome Interaction

    Science.gov (United States)

    Ros, Carlos; Burckhardt, Christoph J.; Kempf, Christoph

    2002-01-01

    The cytoplasmic trafficking of the prototype strain of minute virus of mice (MVMp) was investigated by analyzing and quantifying the effect of drugs that reduce or abolish specific cellular functions on the accumulation of viral macromolecules. With this strategy, it was found that a low endosomal pH is required for the infection, since bafilomycin A1 and chloroquine, two pH-interfering drugs, were similarly active against MVMp. Disruption of the endosomal network by brefeldin A interfered with MVMp infection, indicating that viral particles are routed farther than the early endocytic compartment. Pulse experiments with endosome-interfering drugs showed that the bulk of MVMp particles remained in the endosomal compartment for several hours before its release to the cytosol. Drugs that block the activity of the proteasome by different mechanisms, such as MG132, lactacystin, and epoxomicin, all strongly blocked MVMp infection. Pulse experiments with the proteasome inhibitor MG132 indicated that MVMp interacts with cellular proteasomes after endosomal escape. The chymotrypsin-like but not the trypsin-like activity of the proteasome is required for the infection, since the chymotrypsin inhibitors N-tosyl-l-phenylalanine chloromethyl ketone and aclarubicin were both effective in blocking MVMp infection. However, the trypsin inhibitor Nα-p-tosyl-l-lysine chloromethyl ketone had no effect. These results suggest that the ubiquitin-proteasome pathway plays an essential role in the MVMp life cycle, probably assisting at the stages of capsid disassembly and/or nuclear translocation. PMID:12438589

  17. Direct binding of retromer to human papillomavirus type 16 minor capsid protein L2 mediates endosome exit during viral infection.

    Directory of Open Access Journals (Sweden)

    Andreea Popa

    2015-02-01

    Full Text Available Trafficking of human papillomaviruses to the Golgi apparatus during virus entry requires retromer, an endosomal coat protein complex that mediates the vesicular transport of cellular transmembrane proteins from the endosome to the Golgi apparatus or the plasma membrane. Here we show that the HPV16 L2 minor capsid protein is a retromer cargo, even though L2 is not a transmembrane protein. We show that direct binding of retromer to a conserved sequence in the carboxy-terminus of L2 is required for exit of L2 from the early endosome and delivery to the trans-Golgi network during virus entry. This binding site is different from known retromer binding motifs and can be replaced by a sorting signal from a cellular retromer cargo. Thus, HPV16 is an unconventional particulate retromer cargo, and retromer binding initiates retrograde transport of viral components from the endosome to the trans-Golgi network during virus entry. We propose that the carboxy-terminal segment of L2 protein protrudes through the endosomal membrane and is accessed by retromer in the cytoplasm.

  18. Healthy Dietary Fats Help Beat High Cholesterol

    Science.gov (United States)

    ... 166625.html Healthy Dietary Fats Help Beat High Cholesterol Eating them can reduce your risk of heart ... ones found in some vegetable oils can reduce cholesterol levels and heart disease risk as much as ...

  19. High Cholesterol: Medicines to Help You

    Science.gov (United States)

    ... Consumers Consumer Information by Audience For Women High Cholesterol--Medicines To Help You Share Tweet Linkedin Pin ... side effects for each drug, check Drugs@FDA . Cholesterol Absorption Inhibitors Brand Name Generic Name Zetia Ezetimibe ...

  20. New Cholesterol Fighting Meds Target Key Gene

    Science.gov (United States)

    ... https://medlineplus.gov/news/fullstory_165942.html New Cholesterol Fighting Meds Target Key Gene Two trials show ... New gene-based therapies appear to significantly decrease cholesterol levels in people, and could even cut down ...

  1. Do You Know Your Cholesterol Levels?

    Science.gov (United States)

    ... The Health Information Center Do You Know Your Cholesterol Levels? Print-friendly Version (PDF, 6.1 MB) ... Eat Smart Did you know that high blood cholesterol is a serious problem among Latinos? About one ...

  2. Cholesterol: the debate should be terminated.

    Science.gov (United States)

    Nathan, David G

    2017-07-01

    Here, I offer personal perspectives on cholesterol homeostasis that reflect my belief that certain aspects of the debate have been overstated.-Nathan, D. G. Cholesterol: the debate should be terminated. © FASEB.

  3. Cholesterol 24-hydroxylase: Brain cholesterol metabolism and beyond.

    Science.gov (United States)

    Moutinho, Miguel; Nunes, Maria João; Rodrigues, Elsa

    2016-12-01

    Dysfunctions in brain cholesterol homeostasis have been extensively related to brain disorders. The major elimination pathway of brain cholesterol is its hydroxylation into 24 (S)-hydroxycholesterol by the cholesterol 24-hydroxylase (CYP46A1). Interestingly, there seems to be an association between CYP46A1 and high-order brain functions, in a sense that increased expression of this hydroxylase improves cognition, while a reduction leads to a poor cognitive performance. Moreover, increasing amount of epidemiological, biochemical and molecular evidence, suggests that CYP46A1 has a role in the pathogenesis or progression of neurodegenerative disorders, in which up-regulation of this enzyme is clearly beneficial. However, the mechanisms underlying these effects are poorly understood, which highlights the importance of studies that further explore the role of CYP46A1 in the central nervous system. In this review we summarize the major findings regarding CYP46A1, and highlight the several recently described pathways modulated by this enzyme from a physiological and pathological perspective, which might account for novel therapeutic strategies for neurodegenerative disorders. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. Cholesterol depletion induces large scale domain segregation in living cell membranes

    Science.gov (United States)

    Hao, Mingming; Mukherjee, Sushmita; Maxfield, Frederick R.

    2001-11-01

    Local inhomogeneities in lipid composition play a crucial role in regulation of signal transduction and membrane traffic. Nevertheless, most evidence for microdomains in cells remains indirect, and the nature of membrane inhomogeneities has been difficult to characterize. We used lipid analogs and lipid-anchored proteins with varying fluidity preferences to examine the effect of modulating cellular cholesterol on domain formation. We show that lowering cholesterol levels induces formation of visible micrometer-scale domains in the plasma membrane of several mammalian cell types with complementary distributions of fluorescent lipid analogs with preferences for fluid or ordered domains. A uniform distribution is restored by cholesterol repletion. Unexpectedly, cholesterol depletion does not visibly alter the distribution of a crosslinked or uncrosslinked glycosylphosphatidylinositol-anchored protein (the folate receptor). We also examined the effect of varying cholesterol content on the cold Triton X-100 solubility of several membrane constituents. Although a cholesterol analog, dehydroergosterol, and a glycosylphosphatidylinositol-anchored protein are largely retained after extraction, a lipid analog with saturated 16-carbon acyl chains is largely removed when the cellular cholesterol level is lowered. This result indicates that after cholesterol depletion molecules in the more ordered domains can be extracted differentially by cold nonionic detergents.

  5. Nanoscale Membrane Domain Formation Driven by Cholesterol

    DEFF Research Database (Denmark)

    Javanainen, Matti; Martinez-Seara, Hector; Vattulainen, Ilpo

    2017-01-01

    . The complex nanodomain substructure forms when cholesterol positions itself in the domain boundary region. Here cholesterol can also readily flip-flop across the membrane. Most importantly, replacing cholesterol with a sterol characterized by a less asymmetric ring region impairs the emergence of nanodomains...

  6. Isolation of Cholesterol from an Egg Yolk

    Science.gov (United States)

    Taber, Douglass F.; Li, Rui; Anson, Cory M.

    2011-01-01

    A simple procedure for the isolation of the cholesterol, by hydrolysis and extraction followed by column chromatography, is described. The cholesterol can be further purified by complexation with oxalic acid. It can also be oxidized and conjugated to cholestenone. The source of the cholesterol is one egg yolk, which contains about 200 mg of…

  7. Public health aspects of serum cholesterol

    NARCIS (Netherlands)

    S. Houterman (Saskia)

    2001-01-01

    textabstractIn the beginning of this century Anitschkow and De Langen started pioneering work concerning the relation between cholesterol and coronary heart disease. Both showed that there was a possible relation between cholesterol in the diet, blood cholesterol levels and atherosclerosis. It took

  8. Cholesterol Level: Can It Be Too Low?

    Science.gov (United States)

    ... D. A high blood cholesterol level increases your risk of coronary artery disease. Lower cholesterol is usually better, but in rare cases having ... in individuals with heart disease or at high risk of heart disease or stroke. If ... about your cholesterol level, consult your doctor. If you're taking ...

  9. Prevention and Treatment of High Cholesterol (Hyperlipidemia)

    Science.gov (United States)

    ... t is the first step in lowering your risk of heart disease. Cooking for Lower Cholesterol A heart-healthy eating plan can help you ... lowers HDL cholesterol. When a person with high cholesterol also smokes, their risk of coronary heart disease increases more than it otherwise would. ...

  10. Quantitation of the rates of hepatic and intestinal cholesterol synthesis in lysosomal acid lipase-deficient mice before and during treatment with ezetimibe.

    Science.gov (United States)

    Chuang, Jen-Chieh; Lopez, Adam M; Turley, Stephen D

    2017-07-01

    Esterified cholesterol (EC) and triglycerides, contained within lipoproteins taken up by cells, are hydrolysed by lysosomal acid lipase (LAL) in the late endosomal/lysosomal (E/L) compartment. The resulting unesterified cholesterol (UC) is transported via Niemann-Pick type C2 and C1 into the cytosolic compartment where it enters a putative pool of metabolically active cholesterol that is utilized in accordance with cellular needs. Loss-of-function mutations in LIPA, the gene encoding LAL, result in dramatic increases in tissue concentrations of EC, a hallmark feature of Wolman disease and cholesteryl ester storage disease (CESD). The lysosomal sequestration of EC causes cells to respond to a perceived deficit of sterol by increasing their rate of cholesterol synthesis, particularly in the liver. A similar compensatory response occurs with treatments that disrupt the enterohepatic movement of cholesterol or bile acids. Here we measured rates of cholesterol synthesis in vivo in the liver and small intestine of a mouse model for CESD given the cholesterol absorption inhibitor ezetimibe from weaning until early adulthood. Consistent with previous findings, this treatment significantly reduced the amount of EC sequestered in the liver (from 132.43±7.35 to 70.07±6.04mg/organ) and small intestine (from 2.78±0.21 to 1.34±0.09mg/organ) in the LAL-deficient mice even though their rates of hepatic and intestinal cholesterol synthesis were either comparable to, or exceeded those in matching untreated Lal -/- mice. These data reveal the role of intestinal cholesterol absorption in driving the expansion of tissue EC content and disease progression in LAL deficiency. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Desipramine induces disorder in cholesterol-rich membranes

    DEFF Research Database (Denmark)

    Pakkanen, Kirsi; Salonen, Emppu; Mäkelä, Anna R

    2009-01-01

    canine parvovirus (CPV), a virus known to interact with endosomal membranes and sphingomyelin, as an intracellular probe. DMI was found to cause retention of the virus in intracellular vesicular structures leading to the inhibition of viral proliferation. This implies that DMI has a deleterious effect...... on the penetration of the virus through an endosomal membrane....

  12. Endosomal pH in Neuronal Signaling and Synaptic Transmission: Role of Na+/H+ Exchangers

    Directory of Open Access Journals (Sweden)

    Graham H Diering

    2014-01-01

    Full Text Available Neuronal precursor cells extend multiple neurites during development, one of which extends to form an axon whereas others develop into dendrites. Chemical stimulation of N-methyl D-aspartate (NMDA receptor in fully-differentiated neurons induces projection of dendritic spines, small spikes protruding from dendrites, thereby establishing another layer of polarity within the dendrite. Neuron-enriched Na+/H+ exchanger NHE5 contributes to both neurite growth and dendritic spine formation. In resting neurons and neuro-endocrine cells, neuron-enriched NHE5 is predominantly associated with recycling endosomes where it colocalizes with nerve growth factor (NGF receptor TrkA. NHE5 potently acidifies the lumen of TrkA-positive recycling endosomes and regulates cell-surface targeting of TrkA, whereas chemical stimulation of NMDA receptor rapidly recruits NHE5 to dendritic spines, alkalinizes dendrites and down-regulates the dendritic spine formation. Possible roles of NHE5 in neuronal signaling via proton movement in subcellular compartments are discussed.

  13. Structural and functional analysis of FIP2 binding to the endosome-localised Rab25 GTPase.

    Science.gov (United States)

    Lall, Patrick; Horgan, Conor P; Oda, Shunichiro; Franklin, Edward; Sultana, Azmiri; Hanscom, Sara R; McCaffrey, Mary W; Khan, Amir R

    2013-12-01

    Rab small GTPases are the master regulators of intracellular trafficking in eukaryotes. They mediate spatial and temporal recruitment of effector proteins to distinct cellular compartments through GTP-induced changes in their conformation. Despite numerous structural studies, the molecular basis for Rab/effector specificity and subsequent biological activity remains poorly understood. Rab25, also known as Rab11c, which is epithelial-specific, has been heavily implicated in ovarian cancer development and independently appears to act as a tumour suppressor in the context of a distinct subset of carcinomas. Here, we show that Rab25 associates with FIP2 and can recruit this effector protein to endosomal membranes. We report the crystal structure of Rab25 in complex with the C-terminal region of FIP2, which consists of a central dimeric FIP2 coiled-coil that mediates a heterotetrameric Rab25-(FIP2)2-Rab25 complex. Thermodynamic analyses show that, despite a relatively conserved interface, FIP2 binds to Rab25 with an approximate 3-fold weaker affinity than to Rab11a. Reduced affinity is mainly associated with lower enthalpic gains for Rab25:FIP2 complex formation, and can be attributed to subtle differences in the conformations of switch 1 and switch 2. These cellular, structural and thermodynamic studies provide insight into the Rab11/Rab25 subfamily of small GTPases that regulate endosomal trafficking pathways in eukaryotes. © 2013.

  14. GRASP1 Regulates Synaptic Plasticity and Learning through Endosomal Recycling of AMPA Receptors.

    Science.gov (United States)

    Chiu, Shu-Ling; Diering, Graham Hugh; Ye, Bing; Takamiya, Kogo; Chen, Chih-Ming; Jiang, Yuwu; Niranjan, Tejasvi; Schwartz, Charles E; Wang, Tao; Huganir, Richard L

    2017-03-22

    Learning depends on experience-dependent modification of synaptic efficacy and neuronal connectivity in the brain. We provide direct evidence for physiological roles of the recycling endosome protein GRASP1 in glutamatergic synapse function and animal behavior. Mice lacking GRASP1 showed abnormal excitatory synapse number, synaptic plasticity, and hippocampal-dependent learning and memory due to a failure in learning-induced synaptic AMPAR incorporation. We identified two GRASP1 point mutations from intellectual disability (ID) patients that showed convergent disruptive effects on AMPAR recycling and glutamate uncaging-induced structural and functional plasticity. Wild-type GRASP1, but not ID mutants, rescued spine loss in hippocampal CA1 neurons in Grasp1 knockout mice. Together, these results demonstrate a requirement for normal recycling endosome function in AMPAR-dependent synaptic function and neuronal connectivity in vivo, and suggest a potential role for GRASP1 in the pathophysiology of human cognitive disorders. Copyright © 2017 Elsevier Inc. All rights reserved.

  15. Altered neurological function in mice immunized with early endosome antigen 1

    Directory of Open Access Journals (Sweden)

    Fritzler Marvin J

    2004-01-01

    Full Text Available Abstract Background Autoantibodies directed against the 160 kDa endosome protein early endosome antigen 1 (EEA1 are seen in patients with neurological diseases. To determine if antibodies to EEA1 have a neuropathological effect, mice from three major histocompatability haplotype backgrounds (H2q, H2b and H2d were immunized with EEA1 (amino acids 82–1411 that was previously shown to contain the target EEA1 epitopes. The mice were then subjected to five neuro-behavioural tests: grid walking, forelimb strength, open field, reaching and rotarod. Results The immunized SWR/J mice with sustained anti-EEA1 antibodies had significantly reduced forelimb strength than the control non-immune mice of the same strain, and BALB/CJ immune mice demonstrated significantly more forelimb errors on the grid walk test than the control group. Conclusions Antibodies to recombinant EEA1 in mice may mediate neurological deficits that are consistent with clinical features of some humans that spontaneously develop anti-EEA1 autoantibodies.

  16. An Inside Job: How Endosomal Na+/H+ Exchangers Link to Autism and Neurological Disease

    Directory of Open Access Journals (Sweden)

    Kalyan C. Kondapalli

    2014-06-01

    Full Text Available Autism imposes a major impediment to childhood development and a huge emotional and financial burden on society. In recent years, there has been rapidly accumulating genetic evidence that links the eNHE, a subset of Na+/H+ exchangers that localize to intracellular vesicles, to a variety of neurological conditions including autism, attention deficit hyperactivity disorder, intellectual disability and epilepsy. By providing a leak pathway for protons pumped by the V-ATPase, eNHE determine luminal pH and regulate cation (Na+, K+ content in early and recycling endosomal compartments. Loss-of-function mutations in eNHE cause hyperacidification of endosomal lumen, as a result of imbalance in pump and leak pathways. Two isoforms, NHE6 and NHE9 are highly expressed in brain, including hippocampus and cortex. Here, we summarize evidence for the importance of luminal cation content and pH on processing, delivery and fate of cargo and on the surface expression and function of membrane receptors and neurotransmitter transporters, drawing upon insights from model organisms and mammalian cells. These studies lead to cellular models of eNHE activity in pre- and post-synaptic neurons and astrocytes, where they could impact synapse development and plasticity. The study of eNHE has provided new insight on the mechanism of autism and other debilitating neurological disorders and opened up new possibilities for therapeutic intervention.

  17. Apolipoprotein E4 Impairs Neuronal Insulin Signaling by Trapping Insulin Receptor in the Endosomes.

    Science.gov (United States)

    Zhao, Na; Liu, Chia-Chen; Van Ingelgom, Alexandra J; Martens, Yuka A; Linares, Cynthia; Knight, Joshua A; Painter, Meghan M; Sullivan, Patrick M; Bu, Guojun

    2017-09-27

    Diabetes and impaired brain insulin signaling are linked to the pathogenesis of Alzheimer's disease (AD). The association between diabetes and AD-associated amyloid pathology is stronger among carriers of the apolipoprotein E (APOE) ε4 gene allele, the strongest genetic risk factor for late-onset AD. Here we report that apoE4 impairs neuronal insulin signaling in human apoE-targeted replacement (TR) mice in an age-dependent manner. High-fat diet (HFD) accelerates these effects in apoE4-TR mice at middle age. In primary neurons, apoE4 interacts with insulin receptor and impairs its trafficking by trapping it in the endosomes, leading to impaired insulin signaling and insulin-stimulated mitochondrial respiration and glycolysis. In aging brains, the increased apoE4 aggregation and compromised endosomal function further exacerbate the inhibitory effects of apoE4 on insulin signaling and related functions. Together, our study provides novel mechanistic insights into the pathogenic mechanisms of apoE4 and insulin resistance in AD. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. High-Content Imaging Reveals Expansion of the Endosomal Compartment during Coxiella burnetii Parasitophorous Vacuole Maturation.

    Science.gov (United States)

    Larson, Charles L; Heinzen, Robert A

    2017-01-01

    Coxiella burnetii is an obligate intracellular pathogen and the causative agent of human Q fever. Replication of the bacterium within a large parasitophorous vacuole (PV) resembling a host phagolysosome is required for pathogenesis. PV biogenesis is a pathogen driven process that requires engagement of several host cell vesicular trafficking pathways to acquire vacuole components. The goal of this study was to determine if infection by C. burnetii modulates endolysosomal flux to potentially benefit PV formation. HeLa cells, infected with C. burnetii or left uninfected, were incubated with fluorescent transferrin (Tf) for 0-30 min, and the amount of Tf internalized by cells quantitated by high-content imaging. At 3 and 5 days, but not 1 day post-infection, the maximal amounts of fluorescent Tf internalized by infected cells were significantly greater than uninfected cells. The rates of Tf uptake and recycling were the same for infected and uninfected cells; however, residual Tf persisted in EEA.1 positive compartments adjacent to large PV after 30 min of recycling in the absence of labeled Tf. On average, C. burnetii-infected cells contained significantly more CD63-positive endosomes than uninfected cells. In contrast, cells containing large vacuoles generated by Chlamydia trachomatis exhibited increased rates of Tf internalization without increased CD63 expression. Our results suggest that C. burnetii infection expands the endosomal system to increase capacity for endocytic material. Furthermore, this study demonstrates the power of high-content imaging for measurement of cellular responses to infection by intracellular pathogens.

  19. Dual Effects of Lactobacilli as a Cholesterol Assimilator and an Inhibitor ofGastrointestinal Pathogenic Bacteria

    Directory of Open Access Journals (Sweden)

    Amir Emami

    2014-02-01

    Full Text Available Background: Probiotics are live microbial supplements which can improve the healthy intestinal microbial balance. Lactobacilli are a group of lactic acid producing bacteria (LAB that are known as natural probiotics found in the dairy products. Objectives: In this study, we aimed to detect the most potent Lactobacillus isolates of the Fars province local dairy products in cholesterol removal and investigate their antibacterial properties against some gastrointestinal pathogens. Materials and Methods: Fifteen locally produced yogurt samples of the Fars province were collected and characterized with routine microbiology methods. Cholesterol removal ability of the Lactobacilli isolates were determined, and their growth inhibitory effect on some standard pathogenic strains pathogen was evaluated using the well-diffusion method. Results: In this study, five common strains of Lactobacilli including L. acidophilus, L. casei, L. fermentum, L. lactis, and L. bulgaricus were identified in the samples obtained from the locally produced yogurt in the Fars province. L. lactis and L. acidophilus were determined as the two most active strains with the maximum rate of cholesterol assimilation (5.6 and 4.5 mg/mL, respectively in the process of cholesterol removal. In the antibacterial activity assay, the two mentioned strains had significant inhibitory effect on all of the tested bacteria except for B. subtilis. Conclusions: Cholesterol removal ability had a direct relation with bacterial growth, so it is suggested to use the probiotic bacteria in the growth phase to achieve better results.

  20. Peptide mediators of cholesterol efflux

    Energy Technology Data Exchange (ETDEWEB)

    Bielicki, John K.; Johansson, Jan

    2013-04-09

    The present invention provides a family of non-naturally occurring polypeptides having cholesterol efflux activity that parallels that of full-length apolipoproteins (e.g., Apo AI and Apo E), and having high selectivity for ABAC1 that parallels that of full-length apolipoproteins. The invention also provides compositions comprising such polypeptides, methods of identifying, screening and synthesizing such polypeptides, and methods of treating, preventing or diagnosing diseases and disorders associated with dyslipidemia, hypercholesterolemia and inflammation.

  1. Beclin 1 is required for neuron viability and regulates endosome pathways via the UVRAG-VPS34 complex.

    Directory of Open Access Journals (Sweden)

    Nicole C McKnight

    2014-10-01

    Full Text Available Deficiency of autophagy protein beclin 1 is implicated in tumorigenesis and neurodegenerative diseases, but the molecular mechanism remains elusive. Previous studies showed that Beclin 1 coordinates the assembly of multiple VPS34 complexes whose distinct phosphatidylinositol 3-kinase III (PI3K-III lipid kinase activities regulate autophagy at different steps. Recent evidence suggests a function of beclin 1 in regulating multiple VPS34-mediated trafficking pathways beyond autophagy; however, the precise role of beclin 1 in autophagy-independent cellular functions remains poorly understood. Herein we report that beclin 1 regulates endocytosis, in addition to autophagy, and is required for neuron viability in vivo. We find that neuronal beclin 1 associates with endosomes and regulates EEA1/early endosome localization and late endosome formation. Beclin 1 maintains proper cellular phosphatidylinositol 3-phosphate (PI(3P distribution and total levels, and loss of beclin 1 causes a disruption of active Rab5 GTPase-associated endosome formation and impairment of endosome maturation, likely due to a failure of Rab5 to recruit VPS34. Furthermore, we find that Beclin 1 deficiency causes complete loss of the UVRAG-VPS34 complex and associated lipid kinase activity. Interestingly, beclin 1 deficiency impairs p40phox-linked endosome formation, which is rescued by overexpressed UVRAG or beclin 1, but not by a coiled-coil domain-truncated beclin 1 (a UVRAG-binding mutant, Atg14L or RUBICON. Thus, our study reveals the essential role for beclin 1 in neuron survival involving multiple membrane trafficking pathways including endocytosis and autophagy, and suggests that the UVRAG-beclin 1 interaction underlies beclin 1's function in endocytosis.

  2. Early to Late Endosome Trafficking Controls Secretion and Zymogen Activation in Rodent and Human Pancreatic Acinar Cells.

    Science.gov (United States)

    Messenger, Scott W; Thomas, Diana Dh; Cooley, Michelle M; Jones, Elaina K; Falkowski, Michelle A; August, Benjamin K; Fernandez, Luis A; Gorelick, Fred S; Groblewski, Guy E

    2015-11-01

    Pancreatic acinar cells have an expanded apical endosomal system, the physiological and pathophysiological significance of which is still emerging. Phosphatidylinositol-3,5-bisphosphate (PI(3,5)P 2 ) is an essential phospholipid generated by PIKfyve, which phosphorylates phosphatidylinositol-3-phosphate (PI(3)P). PI(3,5)P 2 is necessary for maturation of early endosomes (EE) to late endosomes (LE). Inhibition of EE to LE trafficking enhances anterograde endosomal trafficking and secretion at the plasma membrane by default through a recycling endosome (RE) intermediate. We assessed the effects of modulating PIKfyve activity on apical trafficking and pancreatitis responses in pancreatic acinar cells. Inhibition of EE to LE trafficking was achieved using pharmacological inhibitors of PIKfyve, expression of dominant negative PIKfyve K1877E, or constitutively active Rab5-GTP Q79L. Anterograde endosomal trafficking was manipulated by expression of constitutively active and dominant negative Rab11a mutants. The effects of these agents on secretion, endolysosomal exocytosis of lysosome associated membrane protein (LAMP1), and trypsinogen activation in response to high-dose CCK-8, bile acids and cigarette toxin was determined. PIKfyve inhibition increased basal and stimulated secretion. Adenoviral overexpression of PIKfyve decreased secretion leading to cellular death. Expression of Rab5-GTP Q79L or Rab11a-GTP Q70L enhanced secretion. Conversely, dominant-negative Rab11a-GDP S25N reduced secretion. High-dose CCK inhibited endolysosomal exocytosis that was reversed by PIKfyve inhibition. PIKfyve inhibition blocked intracellular trypsin accumulation and cellular damage responses to high CCK-8, tobacco toxin, and bile salts in both rodent and human acini. These data demonstrate that EE-LE trafficking acutely controls acinar secretion and the intracellular activation of zymogens leading to the pathogenicity of acute pancreatitis.

  3. Cataract removal

    Science.gov (United States)

    ... eye diseases and eye surgery. Adults are usually awake for the procedure. Numbing medicine (local anesthesia) is ... removed. Tips for recovering after cataract surgery: Wear dark sunglasses outside after you remove the patch. Wash ...

  4. Nevus Removal

    Science.gov (United States)

    ... find the answers you seek. What are the Negative Effects of Nevus Removal? Removal procedures are major ... Reunited Donor Challenge Met! Find Nevus Outreach on Facebook To New Parents of a Child With a ...

  5. A fluorescent glycolipid-binding peptide probe traces cholesterol dependent microdomain-derived trafficking pathways.

    Directory of Open Access Journals (Sweden)

    Steffen Steinert

    Full Text Available BACKGROUND: The uptake and intracellular trafficking of sphingolipids, which self-associate into plasma membrane microdomains, is associated with many pathological conditions, including viral and toxin infection, lipid storage disease, and neurodegenerative disease. However, the means available to label the trafficking pathways of sphingolipids in live cells are extremely limited. In order to address this problem, we have developed an exogenous, non-toxic probe consisting of a 25-amino acid sphingolipid binding domain, the SBD, derived from the amyloid peptide Abeta, and conjugated by a neutral linker with an organic fluorophore. The current work presents the characterization of the sphingolipid binding and live cell trafficking of this novel probe, the SBD peptide. SBD was the name given to a motif originally recognized by Fantini et al in a number of glycolipid-associated proteins, and was proposed to interact with sphingolipids in membrane microdomains. METHODOLOGY/PRINCIPAL FINDINGS: In accordance with Fantini's model, optimal SBD binding to membranes depends on the presence of sphingolipids and cholesterol. In synthetic membrane binding assays, SBD interacts preferentially with raft-like lipid mixtures containing sphingomyelin, cholesterol, and complex gangliosides in a pH-dependent manner, but is less glycolipid-specific than Cholera toxin B (CtxB. Using quantitative time-course colocalization in live cells, we show that the uptake and intracellular trafficking route of SBD is unlike that of either the non-raft marker Transferrin or the raft markers CtxB and Flotillin2-GFP. However, SBD traverses an endolysosomal route that partially intersects with raft-associated pathways, with a major portion being diverted at a late time point to rab11-positive recycling endosomes. Trafficking of SBD to acidified compartments is strongly disrupted by cholesterol perturbations, consistent with the regulation of sphingolipid trafficking by cholesterol

  6. Analysis of Cholesterol Trafficking with Fluorescent Probes

    DEFF Research Database (Denmark)

    Maxfield, Frederick R.; Wustner, Daniel

    2012-01-01

    Cholesterol plays an important role in determining the biophysical properties of biological membranes, and its concentration is tightly controlled by homeostatic processes. The intracellular transport of cholesterol among organelles is a key part of the homeostatic mechanism, but sterol transport...... that can bind to cholesterol to reveal its distribution in cells. We also discuss the use of intrinsically fluorescent sterols that closely mimic cholesterol, as well as some minimally modified fluorophore-labeled sterols. Methods for imaging these sterols by conventional fluorescence microscopy...... and by multiphoton microscopy are described. Some label-free methods for imaging cholesterol itself are also discussed briefly....

  7. Biliary cholesterol secretion : More than a simple ABC

    NARCIS (Netherlands)

    Dikkers, Arne; Tietge, Uwe J. F.

    2010-01-01

    Biliary cholesterol secretion is a process important for 2 major disease complexes, atherosclerotic cardiovascular disease and cholesterol gallstone disease With respect to cardiovascular disease, biliary cholesterol secretion is regarded as the final step for the elimination of cholesterol

  8. The NKG2D ligand ULBP2 is specifically regulated through an invariant chain-dependent endosomal pathway

    DEFF Research Database (Denmark)

    Uhlenbrock, Franziska Katharina; Hagemann-Jensen, Michael Henrik; Kehlet, Stephanie

    2014-01-01

    by affecting endosomal/lysosomal integrity and protein kinase C activity. The invariant chain was further essential for endosomal transport of ULBP2. This novel pathway was identified through screening experiments by which methylselenic acid was found to possess notable NKG2D ligand regulatory properties....... The protein kinase C inhibitor methylselenic acid induced MICA/B surface expression but dominantly blocked ULBP2 surface transport. Remarkably, by targeting this novel pathway we could specifically block the production of soluble ULBP2 from different, primary melanomas. Our findings strongly suggest...

  9. Intracellular transport of cholesterol in mammalian cells

    Energy Technology Data Exchange (ETDEWEB)

    Brasaemle, D.L.

    1989-01-01

    The erythrocyte was selected as a simple cell for the study of transbilayer movement of cholesterol. Cholesterol oxidase was used to measure the distribution of ({sup 3}H)cholesterol across the erythrocyte membrane. Cholesterol oxidase was also used to estimate the rate of transport of low density lipoprotein (LDL) cholesterol to the plasma membrane of cultured Chinese hamster ovary (CHO) fibroblasts; the half-time of this process was 42 minutes. The rate of transport of LDL cholesterol to the plasma membrane was confirmed by a second procedure using amphotericin B. Amphotericin B was also used to estimate the rate of transport of endogenously synthesized cholesterol to the plasma membrane of CHO cells. New methodology was developed including improvements of the previously published cholesterol oxidase assay for plasma membrane cholesterol. A new method for detecting transport of cholesterol to the plasma membrane in cultured cells was developed using amphotericin B. Preliminary studies investigated the use of fluorescent polyenes, pimaricin and etruscomycin, as probes for plasma membrane cholesterol in transport studies. Finally, a modification of a previously published cell staining protocol yielded a simple, quantitative assay for cell growth.

  10. Cholesterol

    Science.gov (United States)

    ... Mental Health Sex and Birth Control Sex and Sexuality Birth Control Family HealthInfants and Toddlers Kids and Teens Pregnancy and ... Mental Health Sex and Birth Control Sex and Sexuality Birth Control Family HealthInfants and Toddlers Kids and Teens Pregnancy and ...

  11. Class II MHC molecules are spontaneously internalized in acidic endosomes by activated B cells.

    Science.gov (United States)

    Weber, D A; Buck, L B; Delohery, T M; Agostino, N; Pernis, B

    1990-01-01

    The antibody response to protein antigens requires specific cooperation between B and T cells. In order to deliver the helper signal, T cells must recognize, in the context of Class II MHC, processed antigen on the membrane of B cells. Processed antigen is in the form of peptides bound in a given site of the Class II MHC molecule; in order to address the question of where, in the B cell, the complex of Class II MHC and processed antigen is formed, we studied the subcellular localization of these two molecules. Since the formation of this complex is the crucial step in antigen processing and presentation, the answer to this question is central to the whole problem of the physiology of antigen handling by B cells. To collect information pertinent to the question, we have compared, in B cells, the intracellular traffic of Class II MHC and of monovalent and divalent anti-immunoglobulin antibodies used as protein ligands of the membrane immunoglobulins. We have done so by two-color immunofluorescence microscopy, and we have detected extensive confluence of Class II MHC molecules with the immunoglobulin ligand, both mono- and bi-valent, in the endosomes of LPS-activated murine B cells. Whereas the ligand clearly reaches the endosomes by internalization from the cell membrane, the Class II MHC molecules could reach the same location either by endocytosis from the membrane or through targeting to the endosomes of newly synthesized Class II MHC molecules. We have collected quantitative evidence for endocytosis of Class II MHC by following, with the fluorescence activated cell sorter, the quenching of the fluorescence of fluoresceinated Fab' anti Class II MHC in LPS-activated murine B cells; this quenching indicates the entry of the label into an acidic intracellular compartment. Together with the results of others, obtained with different methods, our observations support the concept that, at least in mature activated B cells, Class II MHC molecules reach the organelles

  12. Cholesterol-enriched diet causes age-related macular degeneration-like pathology in rabbit retina

    Directory of Open Access Journals (Sweden)

    Singh Brij B

    2011-08-01

    Full Text Available Abstract Background Alzheimer's disease (AD and age-related macular degeneration (AMD share several pathological hallmarks including β-amyloid (Aβ accumulation, oxidative stress, and apoptotic cell death. The causes of AD and AMD are likely multi-factorial with several factors such as diet, environment, and genetic susceptibility participating in the pathogenesis of these diseases. Epidemiological studies correlated high plasma cholesterol levels with high incidence of AD, and feeding rabbits with a diet rich in cholesterol has been shown to induce AD-like pathology in rabbit brain. High intake of cholesterol and saturated fat were also long been suspected to increase the risk for AMD. However, the extent to which cholesterol-enriched diet may also cause AMD-like features in rabbit retinas is not well known. Methods Male New Zealand white rabbits were fed normal chow or a 2% cholesterol-enriched diet for 12 weeks. At necropsy, animals were perfused with Dulbecco's phosphate-buffered saline and the eyes were promptly removed. One eye of each animal was used for immunohistochemistry and retina dissected from the other eye was used for Western blot, ELISA assays, spectrophotometry and mass spectrometry analyses. Results Increased levels of Aβ, decreased levels of the anti-apoptotic protein Bcl-2, increased levels of the pro-apoptotic Bax and gadd153 proteins, emergence of TUNEL-positive cells, and increased generation of reactive oxygen species were found in retinas from cholesterol-fed compared to normal chow-fed rabbits. Additionally, astrogliosis, drusen-like debris and cholesterol accumulations in retinas from cholesterol-fed rabbits were observed. As several lines of evidence suggest that oxidized cholesterol metabolites (oxysterols may be the link by which cholesterol contributes to the pathogenesis of AMD, we determined levels of oxysterols and found a dramatic increase in levels of oxysterols in retinas from cholesterol-fed rabbits

  13. Cholesterol selectively regulates IL-5 induced mitogen activated protein kinase signaling in human eosinophils.

    Directory of Open Access Journals (Sweden)

    Mandy E Burnham

    Full Text Available Eosinophils function contributes to human allergic and autoimmune diseases, many of which currently lack curative treatment. Development of more effective treatments for eosinophil-related diseases requires expanded understanding of eosinophil signaling and biology. Cell signaling requires integration of extracellular signals with intracellular responses, and is organized in part by cholesterol rich membrane microdomains (CRMMs, commonly referred to as lipid rafts. Formation of these organizational membrane domains is in turn dependent upon the amount of available cholesterol, which can fluctuate widely with a variety of disease states. We tested the hypothesis that manipulating membrane cholesterol content in primary human peripheral blood eosinophils (PBEos would selectively alter signaling pathways that depend upon membrane-anchored signaling proteins localized within CRMMs (e.g., mitogen activated protein kinase [MAPK] pathway, while not affecting pathways that signal through soluble proteins, like the Janus Kinase/Signal Transducer and Activator of Transcription [JAK/STAT] pathway. Cholesterol levels were increased or decreased utilizing cholesterol-chelating methyl-β-cyclodextrin (MβCD, which can either extract membrane cholesterol or add exogenous membrane cholesterol depending on whether MβCD is preloaded with cholesterol. Human PBEos were pretreated with MβCD (cholesterol removal or MβCD+Cholesterol (MβCD+Chol; cholesterol delivery; subsequent IL-5-stimulated signaling and physiological endpoints were assessed. MβCD reduced membrane cholesterol in PBEos, and attenuated an IL-5-stimulated p38 and extracellular-regulated kinase 1/2 phosphorylation (p-p38, p-ERK1/2, and an IL-5-dependent increase in interleukin-1β (IL-1β mRNA levels. In contrast, MβCD+Chol treatment elevated PBEos membrane cholesterol levels and basal p-p38, but did not alter IL-5-stimulated phosphorylation of ERK1/2, STAT5, or STAT3. Furthermore, M

  14. Serum glycomarkers of endoplasmic reticulum and lysosomal-endosomal system stress in human healthy aging and diseases

    Directory of Open Access Journals (Sweden)

    I. U. Pismenetskaya

    2017-02-01

    Full Text Available To verify the idea that extracellular free oligosaccharides might be able to reflect the functional status of the endoplasmic reticulum (ER and lysosomal-endosomal system, HPLC-profiles of serum-derived free oligosaccharides (FOS in human healthy aging, acute myeloproliferative neoplasms, and cardiovascular pathologies were compared with intracellular glycans. After plasma deproteinization and FOS purification the oligosaccharides were labelled with anthranilic acid, separated into the neutral and charged with QAE Sephadex (Q25-120 chromatography and analysed using high-performance liquid chromatography (HPLC. The charged FOS were digested with a sialidase and compared with free oligosaccharides from transferrin for structural decoding. HPLC-profiles of serum-derived FOS revealed mild delay of the dolichol phosphate cycle in ER, moderate intensification of ER-associated degradation (ERAD and degradation in endosomal-lysosomal system with aging; an inhibition of the dolichol phosphate cycle, intensification of ERAD and increasing of lysosomal exocytosis in acute myeloproliferative neoplasms; intensification of ERAD and glycocojugate degradation with endosomal-lysosomal system in cardiovascular diseases. As serum free oligosaccharides are able to reflect specifically perturbations in ER and endosomal-lysosomal system under wide range of stressors they can serve as extracellular markers of functionality of these organelles.

  15. Munc13-4 functions as a Ca2+ sensor for homotypic secretory granule fusion to generate endosomal exocytic vacuoles

    Science.gov (United States)

    Woo, Sang Su; James, Declan J.; Martin, Thomas F. J.

    2017-01-01

    Munc13-4 is a Ca2+-dependent SNARE (soluble N-ethylmaleimide–sensitive factor attachment protein receptor)- and phospholipid-binding protein that localizes to and primes secretory granules (SGs) for Ca2+-evoked secretion in various secretory cells. Studies in mast cell–like RBL-2H3 cells provide direct evidence that Munc13–4 with its two Ca2+-binding C2 domains functions as a Ca2+ sensor for SG exocytosis. Unexpectedly, Ca2+ stimulation also generated large (>2.4 μm in diameter) Munc13-4+/Rab7+/Rab11+ endosomal vacuoles. Vacuole generation involved the homotypic fusion of Munc13-4+/Rab7+ SGs, followed by a merge with Rab11+ endosomes, and depended on Ca2+ binding to Munc13-4. Munc13-4 promoted the Ca2+-stimulated fusion of VAMP8-containing liposomes with liposomes containing exocytic or endosomal Q-SNAREs and directly interacted with late endosomal SNARE complexes. Thus Munc13-4 is a tethering/priming factor and Ca2+ sensor for both heterotypic SG-plasma membrane and homotypic SG-SG fusion. Total internal reflection fluorescence microscopy imaging revealed that vacuoles were exocytic and mediated secretion of β-hexosaminidase and cytokines accompanied by Munc13-4 diffusion onto the plasma membrane. The results provide new molecular insights into the mechanism of multigranular compound exocytosis commonly observed in various secretory cells. PMID:28100639

  16. The protein transportation pathway from Golgi to vacuoles via endosomes plays a role in enhancement of methylmercury toxicity

    Science.gov (United States)

    Hwang, Gi-Wook; Murai, Yasutaka; Takahashi, Tsutomu; Naganuma, Akira

    2014-07-01

    Methylmercury causes serious damage to the central nervous system, but the molecular mechanisms of methylmercury toxicity are only marginally understood. In this study, we used a gene-deletion mutant library of budding yeast to conduct genome-wide screening for gene knockouts affecting the sensitivity of methylmercury toxicity. We successfully identified 31 genes whose deletions confer resistance to methylmercury in yeast, and 18 genes whose deletions confer hypersensitivity to methylmercury. Yeast genes whose deletions conferred resistance to methylmercury included many gene encoding factors involved in protein transport to vacuoles. Detailed examination of the relationship between the factors involved in this transport system and methylmercury toxicity revealed that mutants with loss of the factors involved in the transportation pathway from the trans-Golgi network (TGN) to the endosome, protein uptake into the endosome, and endosome-vacuole fusion showed higher methylmercury resistance than did wild-type yeast. The results of our genetic engineering study suggest that this vesicle transport system (proteins moving from the TGN to vacuole via endosome) is responsible for enhancing methylmercury toxicity due to the interrelationship between the pathways. There is a possibility that there may be proteins in the cell that enhance methylmercury toxicity through the protein transport system.

  17. ANTIGEN PROCESSING BY ENDOSOMAL PROTEASES DETERMINES WHICH SITES OF SPERM-WHALE MYOGLOBIN ARE EVENTUALLY RECOGNIZED BY T-CELLS

    NARCIS (Netherlands)

    VANNOORT, JM; BOON, J; VANDERDRIFT, ACM; WAGENAAR, JPA; BOOTS, AMH; BOOG, CJP; Boots, Annemieke

    This study reports an identification of the major processing products of an exogenous protein antigen, viz. sperm-whale myoglobin, as obtained after cell-free processing with partially purified macrophage endosomes. It is demonstrated that such a system yields fragments that are indistinguishable by

  18. Kinesin-3 and dynein cooperate in long-range retrograde endosome motility along a nonuniform microtubule array

    NARCIS (Netherlands)

    Schuster, M.; Kilaru, S.; Fink, G.; Collemare, J.A.R.; Roger, Y.; Steinberg, G.

    2011-01-01

    The polarity of microtubules (MTs) determines the motors for intracellular motility, with kinesins moving to plus ends and dynein to minus ends. In elongated cells of Ustilago maydis, dynein is thought to move early endosomes (EEs) toward the septum (retrograde), whereas kinesin-3 transports them to

  19. COMMD1 is linked to the WASH complex and regulates endosomal trafficking of the copper transporter ATP7A

    NARCIS (Netherlands)

    Phillips-Krawczak, Christine A.; Singla, Amika; Starokadomskyy, Petro; Deng, Zhihui; Osborne, Douglas G.; Li, Haiying; Dick, Christopher J.; Gomez, Timothy S.; Koenecke, Megan; Zhang, Jin-San; Dai, Haiming; Sifuentes-Dominguez, Luis F.; Geng, Linda N.; Kaufmann, Scott H.; Hein, Marco Y.; Wallis, Mathew; McGaughran, Julie; Gecz, Jozef; De Sluis, Bart van; Billadeau, Daniel D.; Burstein, Ezra

    2015-01-01

    COMMD1 deficiency results in defective copper homeostasis, but the mechanism for this has remained elusive. Here we report that COMMD1 is directly linked to early endosomes through its interaction with a protein complex containing CCDC22, CCDC93, and C16orf62. This COMMD/CCDC22/CCDC93 (CCC) complex

  20. Association with AflR in Endosomes Reveals New Functions for AflJ in Aflatoxin Biosynthesis

    Directory of Open Access Journals (Sweden)

    John E. Linz

    2012-12-01

    Full Text Available Aflatoxins are the most potent naturally occurring carcinogens of fungal origin. Biosynthesis of aflatoxin involves the coordinated expression of more than 25 genes. The function of one gene in the aflatoxin gene cluster, aflJ, is not entirely understood but, because previous studies demonstrated a physical interaction between the Zn2Cys6 transcription factor AflR and AflJ, AflJ was proposed to act as a transcriptional co-activator. Image analysis revealed that, in the absence of aflJ in A. parasiticus, endosomes cluster within cells and near septa. AflJ fused to yellow fluorescent protein complemented the mutation in A. parasiticus ΔaflJ and localized mainly in endosomes. We found that AflJ co-localizes with AflR both in endosomes and in nuclei. Chromatin immunoprecipitation did not detect AflJ binding at known AflR DNA recognition sites suggesting that AflJ either does not bind to these sites or binds to them transiently. Based on these data, we hypothesize that AflJ assists in AflR transport to or from the nucleus, thus controlling the availability of AflR for transcriptional activation of aflatoxin biosynthesis cluster genes. AflJ may also assist in directing endosomes to the cytoplasmic membrane for aflatoxin export.

  1. The CORVET subunit Vps8 cooperates with the Rab5 homolog Vps21 to induce clustering of late endosomal compartments.

    Science.gov (United States)

    Markgraf, Daniel F; Ahnert, Franziska; Arlt, Henning; Mari, Muriel; Peplowska, Karolina; Epp, Nadine; Griffith, Janice; Reggiori, Fulvio; Ungermann, Christian

    2009-12-01

    Membrane tethering, the process of mediating the first contact between membranes destined for fusion, requires specialized multisubunit protein complexes and Rab GTPases. In the yeast endolysosomal system, the hexameric HOPS tethering complex cooperates with the Rab7 homolog Ypt7 to promote homotypic fusion at the vacuole, whereas the recently identified homologous CORVET complex acts at the level of late endosomes. Here, we have further functionally characterized the CORVET-specific subunit Vps8 and its relationship to the remaining subunits using an in vivo approach that allows the monitoring of late endosome biogenesis. In particular, our results indicate that Vps8 interacts and cooperates with the activated Rab5 homolog Vps21 to induce the clustering of late endosomal membranes, indicating that Vps8 is the effector subunit of the CORVET complex. This clustering, however, requires Vps3, Vps16, and Vps33 but not the remaining CORVET subunits. These data thus suggest that the CORVET complex is built of subunits with distinct activities and potentially, their sequential assembly could regulate tethering and successive fusion at the late endosomes.

  2. Characterization of PEBBLEs as a Tool for Real-Time Measurement of Dictyostelium discoideum Endosomal pH

    Directory of Open Access Journals (Sweden)

    Everett Moding

    2009-01-01

    Full Text Available The measurement of intracellular ion concentration change is important for understanding the cellular mechanisms for communication. Recently developed nanosensors, (Photonic Explorers for Biomedical use with Biologically Localized Embedding PEBBLEs, have a number of advantages for measuring ions in cells over established methods using microelectrodes, unbound fluorescent dyes, or NMR. PEBBLE sensors have been shown to work in principle for measuring dynamic ion changes, but few in vivo applications have been demonstrated. We modified the protocol for the fabrication of pH sensing PEBBLEs and developed a protocol for the utilization of these sensors for the monitoring of dynamic pH changes in the endosomes of slime mold Dictyostelium discoideum (D. discoideum. Oregon Green 514-CdSe Quantum Dot PEBBLEs were used to measure real-time pH inside D. discoideum endosomes during cAMP stimulation. Endosomal pH was shown to decrease during cAMP signaling, demonstrating a movement of protons into the endosomes of D. discoideum amoebae.

  3. Dendronized Mesoporous Silica Nanoparticles Provide an Internal Endosomal Escape Mechanism for Successful Cytosolic Drug Release

    CERN Document Server

    Weiss, Veronika; Torrano, Adriano A; Strobel, Claudia; Mackowiak, Stephan A; Gatzenmeier, Tim; Hilger, Ingrid; Braeuchle, Christoph; Bein, Thomas

    2015-01-01

    Mesoporous silica nanoparticles (MSNs) attract increasing interest in the field of gene and drug delivery due to their versatile features as a multifunctional drug delivery platform. Here, we describe poly(amidoamine) (PAMAM) dendron-functionalized MSNs that fulfill key prerequisites for a controllable intracellular drug release. In addition to high loading capacity, they offer 1) low cytotoxicity, showing no impact on the metabolism of endothelial cells, 2) specific cancer cell targeting due to receptor-mediated cell uptake, 3) a redox-driven cleavage of disulfide bridges allowing for stimuli-responsive cargo release, and most importantly, 4) a specific internal trigger based on the high buffering capacity of PAMAM dendrons to provide endosomal escape.

  4. Retromer's Role in Endosomal Trafficking and Impaired Function in Neurodegenerative Diseases.

    Science.gov (United States)

    Follett, Jordan; Bugarcic, Andrea; Collins, Brett M; Teasdale, Rohan D

    2017-01-01

    The retromer complex is a highly conserved membrane trafficking assembly composed of three proteins - Vps26, Vps29 and Vps35 - that were identified over a decade ago in Saccharomyces cerevisiae (S. cerevisiae). Initially, mammalian retromer was shown to sort transmembrane proteins from the endosome to the trans-Golgi network (TGN), though recent work has identified a critical role for retromer in multiple trafficking pathways, including recycling to the plasma membrane and regulation of cell polarity. In recent years, genetic, cellular, pharmacological and animal model studies have identified retromer and its interacting proteins as being linked to familial forms of neurodegenerative diseases such as Alzheimer's (AD) and Parkinson's (PD). Here, this commentary will summarize recently identified point mutations in retromer linked to PD, and explore the molecular functions of retromer that may be relevant to disease progression. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  5. Parkin Modulates Endosomal Organization and Function of the Endo-Lysosomal Pathway.

    Science.gov (United States)

    Song, Pingping; Trajkovic, Katarina; Tsunemi, Taiji; Krainc, Dimitri

    2016-02-24

    Mutations in PARK2 (parkin), which encodes Parkin protein, an E3 ubiquitin ligase, are associated with autosomal recessive early-onset Parkinson's disease (PD). While several studies implicated Parkin in the regulation of mitophagy and proteasomal degradation, the precise mechanism leading to neurodegeneration upon Parkin loss of function remains incompletely understood. In this study, we found that Parkin modulates the endocytic pathway through the regulation of endosomal structure and function. We showed that loss of Parkin function led to decreased endosomal tubulation and membrane association of vesicle protein sorting 35 (VPS35) and sorting nexin 1 (SNX1), as well as decreased mannose 6 phosphate receptor (M6PR), suggesting the impairment of retromer pathway in Parkin-deficient cells. We also found increased formation of intraluminal vesicles coupled with enhanced release of exosomes in the presence of mutant Parkin. To elucidate the molecular mechanism of these alterations in the endocytic pathway in Parkin-deficient cells, we found that Parkin regulates the levels and activity of Rab7 by promoting its ubiquitination on lysine 38 residue. Both endogenous Rab7 in Parkin-deficient cells and overexpressed K38 R-Rab7 mutant displayed decreased effector binding and membrane association. Furthermore, overexpression of K38R-Rab7 in HEK293 cells phenocopied the increased secretion of exosomes observed in Parkin-deficient cells, suggesting that Rab7 deregulation may be at least partially responsible for the endocytic phenotype observed in Parkin-deficient cells. These findings establish a role for Parkin in regulating the endo-lysosomal pathway and retromer function and raise the possibility that alterations in these pathways contribute to the development of pathology in Parkin-linked Parkinson's disease. Copyright © 2016 the authors 0270-6474/16/362425-13$15.00/0.

  6. Atg8 is involved in endosomal and phagosomal acidification in the parasitic protist Entamoeba histolytica.

    Science.gov (United States)

    Picazarri, Karina; Nakada-Tsukui, Kumiko; Tsuboi, Kumiko; Miyamoto, Eri; Watanabe, Naoko; Kawakami, Eiryo; Nozaki, Tomoyoshi

    2015-10-01

    Autophagy is one of two major bulk protein degradation systems and is conserved throughout eukaryotes. The protozoan Entamoeba histolytica, which is a human intestinal parasite, possesses a restricted set of autophagy-related (Atg) proteins compared with other eukaryotes and thus represents a suitable model organism for studying the minimal essential components and ancestral functions of autophagy. E. histolytica possesses two conjugation systems: Atg8 and Atg5/12, although a gene encoding Atg12 is missing in the genome. Atg8 is considered to be the central and authentic marker of autophagosomes, but recent studies have demonstrated that Atg8 is not exclusively involved in autophagy per se, but other fundamental mechanisms of vesicular traffic. To investigate this question in E. histolytica, we studied on Atg8 during the proliferative stage. Atg8 was constitutively expressed in both laboratory-maintained and recently established clinical isolates and appeared to be lipid-modified in logarithmic growth phase, suggesting a role of Atg8 in non-stress and proliferative conditions. These findings are in contrast to those for Entamoeba invadens, in which autophagy is markedly induced during an early phase of differentiation from the trophozoite into the cyst. The repression of Atg8 gene expression in En. histolytica by antisense small RNA-mediated transcriptional gene silencing resulted in growth retardation, delayed endocytosis and reduced acidification of endosomes and phagosomes. Taken together, these results suggest that Atg8 and the Atg8 conjugation pathway have some roles in the biogenesis of endosomes and phagosomes in this primitive eukaryote. © 2015 The Authors. Cellular Microbiology published by John Wiley & Sons Ltd.

  7. Raising HDL cholesterol in women

    Directory of Open Access Journals (Sweden)

    Danny J Eapen

    2009-11-01

    Full Text Available Danny J Eapen1, Girish L Kalra1, Luay Rifai1, Christina A Eapen2, Nadya Merchant1, Bobby V Khan11Emory University School of Medicine, Atlanta, GA, USA; 2University of South Florida School of Medicine, Tampa, FL, USAAbstract: High-density lipoprotein cholesterol (HDL-C concentration is essential in the determination of coronary heart disease (CHD risk in women. This is especially true in the postmenopausal state, where lipid profiles and CHD risk mimic that of age-matched men. Thus, interventions designed to reduce CHD risk by raising HDL-C levels may have particular significance during the transition to menopause. This review discusses HDL-C-raising therapies and the role of HDL in the primary prevention of CHD in women. Lifestyle-based interventions such as dietary change, aerobic exercise regimens, and smoking cessation are initial steps that are effective in raising HDL-C, and available data suggest women respond similarly to men with these interventions. When combined with pharmacotherapy, the effects of these lifestyle alterations are further amplified. Though studies demonstrating gender-specific differences in therapy are limited, niacin continues to be the most effective agent in raising HDL-C levels, especially when used in combination with fibrate or statin therapy. Emerging treatments such as HDL mimetic therapy show much promise in further raising HDL-C levels and improving cardiovascular outcomes.Keywords: high-density lipoprotein, HDL, women, cholesterol, heart disease

  8. The conserved SNARE SEC-22 localizes to late endosomes and negatively regulates RNA interference in Caenorhabditis elegans.

    Science.gov (United States)

    Zhao, Yani; Holmgren, Benjamin T; Hinas, Andrea

    2017-03-01

    Small RNA pathways, including RNA interference (RNAi), play crucial roles in regulation of gene expression. Initially considered to be cytoplasmic, these processes have later been demonstrated to associate with membranes. For example, maturation of late endosomes/multivesicular bodies (MVBs) is required for efficient RNAi, whereas fusion of MVBs to lysosomes appears to reduce silencing efficiency. SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) mediate membrane fusion and are thus at the core of membrane trafficking. In spite of this, no SNARE has previously been reported to affect RNAi. Here, we demonstrate that in Caenorhabditis elegans, loss of the conserved SNARE SEC-22 results in enhanced RNAi upon ingestion of double-stranded RNA. Furthermore, SEC-22 overexpression inhibits RNAi in wild-type animals. We find that overexpression of SEC-22 in the target tissue (body wall muscle) strongly suppresses the sec-22(-) enhanced RNAi phenotype, supporting a primary role for SEC-22 in import of RNAi silencing signals or cell autonomous RNAi. A functional mCherry::SEC-22 protein localizes primarily to late endosomes/MVBs and these compartments are enlarged in animals lacking sec-22 SEC-22 interacts with late endosome-associated RNA transport protein SID-5 in a yeast two-hybrid assay and functions in a sid-5-dependent manner. Taken together, our data indicate that SEC-22 reduces RNAi efficiency by affecting late endosome/MVB function, for example, by promoting fusion between late endosomes/MVBs and lysosomes. To our knowledge, this is the first report of a SNARE with a function in small RNA-mediated gene silencing. © 2017 Zhao et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  9. A sensitive assay for ABCA1-mediated cholesterol efflux using BODIPY-cholesterol

    Science.gov (United States)

    Sankaranarayanan, Sandhya; Kellner-Weibel, Ginny; de la Llera-Moya, Margarita; Phillips, Michael C.; Asztalos, Bela F.; Bittman, Robert; Rothblat, George H.

    2011-01-01

    Studies have shown a negative association between cellular cholesterol efflux and coronary artery disease (CAD). Standard protocol for quantitating cholesterol efflux involves labeling cells with [3H]cholesterol and measuring release of the labeled sterol. Using [3H]cholesterol is not ideal for the development of a high-throughput assay to screen large numbers of serum as would be required in studying the link between efflux and CAD. We compared efflux using a fluorescent sterol (boron dipyrromethene difluoride linked to sterol carbon-24, BODIPY-cholesterol) with that of [3H]cholesterol in J774 macrophages. Fractional efflux of BODIPY-cholesterol was significantly higher than that of [3H]cholesterol when apo A-I, HDL3, or 2% apoB-depleted human serum were used as acceptors. BODIPY-cholesterol efflux correlated significantly with [3H]cholesterol efflux (p cholesterol efflux correlated significantly with preβ-1 (r2 = 0.6) but not with total HDL-cholesterol. Reproducibility of the BODIPY-cholesterol efflux assay was excellent between weeks (r2 = 0.98, inter-assay CV = 3.31%). These studies demonstrate that BODIPY-cholesterol provides an efficient measurement of efflux compared with [3H]cholesterol and is a sensitive probe for ABCA1-mediated efflux. The increased sensitivity of BODIPY-cholesterol assay coupled with the simplicity of measuring fluorescence results in a sensitive, high-throughput assay that can screen large numbers of sera, and thus establish the relationship between cholesterol efflux and atherosclerosis. PMID:21957199

  10. NPC2 regulates biliary cholesterol secretion via stimulation of ABCG5/G8-mediated cholesterol transport.

    Science.gov (United States)

    Yamanashi, Yoshihide; Takada, Tappei; Yoshikado, Takashi; Shoda, Jun-Ichi; Suzuki, Hiroshi

    2011-05-01

    Biliary cholesterol secretion helps maintain cholesterol homeostasis; it is regulated by the cholesterol exporter adenosine triphosphate-binding cassettes G5 and G8 (ABCG5/G8) and the cholesterol importer Niemann-Pick C1-like 1 (NPC1L1). We studied another putative regulator of cholesterol secretion into bile, Niemann-Pick C2 (NPC2)--a cholesterol-binding protein secreted by the biliary system--and determined its effects on transporter-mediated biliary secretion of cholesterol. Mice with hepatic knockdown of Npc2 or that overexpressed NPC2 were created using adenovirus-mediated gene transfer; biliary lipids were characterized. The effects of secreted NPC2 on cholesterol transporter activity were examined in vitro using cells that overexpressed ABCG5/G8 or NPC1L1. Studies of mice with altered hepatic expression of NPC2 revealed that this expression positively regulates the biliary secretion of cholesterol, supported by the correlation between levels of NPC2 protein and cholesterol in human bile. In vitro analysis showed that secreted NPC2 stimulated ABCG5/G8-mediated cholesterol efflux but not NPC1L1-mediated cholesterol uptake. Consistent with these observations, no significant changes in biliary cholesterol secretion were observed on hepatic overexpression of NPC2 in ABCG5/G8-null mice, indicating that NPC2 requires ABCG5/G8 to stimulate cholesterol secretion. Analyses of NPC2 mutants showed that the stimulatory effect of biliary NPC2 was independent of the function of lysosomal NPC2 as a regulator of intracellular cholesterol trafficking. NPC2 is a positive regulator of biliary cholesterol secretion via stimulation of ABCG5/G8-mediated cholesterol transport. Copyright © 2011 AGA Institute. Published by Elsevier Inc. All rights reserved.

  11. Cholesterol and late-life cognitive decline.

    Science.gov (United States)

    van Vliet, Peter

    2012-01-01

    High cholesterol levels are a major risk factor for cardiovascular disease, but their role in dementia and cognitive decline is less clear. This review highlights current knowledge on the role of cholesterol in late-life cognitive function, cognitive decline, and dementia. When measured in midlife, high cholesterol levels associate with an increased risk of late-life dementia and cognitive decline. However, when measured in late-life, high cholesterol levels show no association with cognitive function, or even show an inverse relation. Although statin treatment has been shown to associate with a lower risk of dementia and cognitive decline in observational studies, randomized controlled trials show no beneficial effect of statin treatment on late-life cognitive function. Lowering cholesterol levels may impair brain function, since cholesterol is essential for synapse formation and maturation and plays an important role in the regulation of signal transduction through its function as a component of the cell membrane. However, membrane cholesterol also plays a role in the formation and aggregation of amyloid-β. Factors that influence cholesterol metabolism, such as dietary intake, are shown to play a role in late-life cognitive function and the risk of dementia. In conclusion, cholesterol associates with late-life cognitive function, but the association is strongly age-dependent. There is no evidence that treatment with statins in late-life has a beneficial effect on cognitive function.

  12. TEMPORARY REMOVAL

    DEFF Research Database (Denmark)

    Calkins, Hugh; Hindricks, Gerhard; Cappato, Riccardo

    2017-01-01

    The publisher regrets that this article has been temporarily removed. A replacement will appear as soon as possible in which the reason for the removal of the article will be specified, or the article will be reinstated. The full Elsevier Policy on Article Withdrawal can be found at https://www.e...

  13. High Density Lipoproteins and Arteriosclerosis: Role of Cholesterol Efflux and Reverse Cholesterol Transport

    National Research Council Canada - National Science Library

    von Eckardstein, Arnold; Nofer, Jerzy Roch; Assmann, Gerd

    2001-01-01

    Abstract—High density lipoprotein (HDL) cholesterol is an important risk factor for coronary heart disease, and HDL exerts various potentially antiatherogenic properties, including the mediation of reverse transport of cholesterol...

  14. Factors associated with serum cholesterol level in a pediatric practice. Cholesterol screening in a pediatric practice.

    Science.gov (United States)

    Donker, G A; Goff, D C; Ragan, J D; Killinger, R P; Harrist, R B; Labarthe, D R

    1993-01-01

    The associations between age, sex, height, Quetelet index, blood pressure, and serum cholesterol level were examined among 1406 routinely screened children, aged 4 to 19 years, in a pediatric practice. After adjustment for sex and age, height and Quetelet index were associated with serum cholesterol levels. Quetelet index was shown by multiple linear regression to be positively related to cholesterol levels (b = 0.780, P Quetelet index was marginal. Clustering of elevated serum cholesterol level, Quetelet index, and systolic blood pressure was observed. Familial aggregation of cholesterol levels was demonstrated using analysis of variance for 742 children from 342 families included in the regression analysis (F341,400 = 1.56, P Quetelet index, and familial aggregation accounted for 10.6% of the variance in serum cholesterol levels. Siblings of children with high cholesterol levels are a high-yield group in cholesterol screening.

  15. Black pepper and piperine reduce cholesterol uptake and enhance translocation of cholesterol transporter proteins.

    Science.gov (United States)

    Duangjai, Acharaporn; Ingkaninan, Kornkanok; Praputbut, Sakonwun; Limpeanchob, Nanteetip

    2013-04-01

    Black pepper (Piper nigrum L.) lowers blood lipids in vivo and inhibits cholesterol uptake in vitro, and piperine may mediate these effects. To test this, the present study aimed to compare actions of black pepper extract and piperine on (1) cholesterol uptake and efflux in Caco-2 cells, (2) the membrane/cytosol distribution of cholesterol transport proteins in these cells, and (3) the physicochemical properties of cholesterol micelles. Piperine or black pepper extract (containing the same amount of piperine) dose-dependently reduced cholesterol uptake into Caco-2 cells in a similar manner. Both preparations reduced the membrane levels of NPC1L1 and SR-BI proteins but not their overall cellular expression. Micellar cholesterol solubility of lipid micelles was unaffected except by 1 mg/mL concentration of black pepper extract. These data suggest that piperine is the active compound in black pepper and reduces cholesterol uptake by internalizing the cholesterol transporter proteins.

  16. Oxidised LDL, HDL cholesterol, LDL cholesterol levels in patients of coronary artery disease

    OpenAIRE

    Ghosh, Joya; Mishra, T. K.; Rao, Y. N.; Aggarwal, S K

    2006-01-01

    Coronary artery disease is a major cause of morbidity and has various risk factors. Lipid profile i.e. low HDL-cholesterol, high LDL cholesterol, high total cholesterol, high triglycerides playing important role in its causation. Recently interest has been shown in the oxidized fraction of LDL as one of the risk factors. In the present study 60 age and sex matched normal healthy individuals were taken as controls and 60 patients of CAD were taken. Cholesterol was measured by enzymatic method,...

  17. Cholesterol Transport Revisited: A New Turbo Mechanism to Drive Cholesterol Excretion

    NARCIS (Netherlands)

    de Boer, Jan Freark; Kuipers, Folkert; Groen, Albert K.

    2017-01-01

    A fine-tuned balance between cholesterol uptake and excretion by the body is pivotal to maintain health and to remain free from the deleterious consequences of cholesterol accumulation such as cardiovascular disease. The pathways involved in intracellular and extracellular cholesterol transport are

  18. Cholesterol Transport Revisited: A New Turbo Mechanism to Drive Cholesterol Excretion

    NARCIS (Netherlands)

    de Boer, Jan Freark; Kuipers, Folkert; Groen, Albert K.

    2018-01-01

    A fine-tuned balance between cholesterol uptake and excretion by the body is pivotal to maintain health and to remain free from the deleterious consequences of cholesterol accumulation such as cardiovascular disease. The pathways involved in intracellular and extracellular cholesterol transport are

  19. Statins increase hepatic cholesterol synthesis and stimulate fecal cholesterol elimination in mice

    NARCIS (Netherlands)

    Schonewille, Marleen; de Boer, Jan Freark; Mele, Laura; Wolters, Henk; Bloks, Vincent W.; Wolters, Justina C.; Kuivenhoven, Jan A.; Tietge, Uwe J. F.; Brufau, Gemma; Groen, Albert K.

    Statins are competitive inhibitors of HMG-CoA reductase, the rate-limiting enzyme of cholesterol synthesis. Statins reduce plasma cholesterol levels, but whether this is actually caused by inhibition of de novo cholesterol synthesis has not been clearly established. Using three different statins, we

  20. Dietary cholesterol and fats at a young age : do they influence cholesterol metabolism in adult life?

    NARCIS (Netherlands)

    Temmerman, A.M.; Vonk, R.J.; Niezen-Koning, K.; Berger, R.; Fernandes, J.

    1989-01-01

    The effects of dietary cholesterol and fats on cholesterol metabolism later in life were studied in Mongolian gerbils. Three groups were given a basic diet with soybean oil, palm kernel oil amounting to 8.75% (w/w), or the basic diet only. In three other groups, cholesterol (0.05%) was added to the

  1. Statins increase hepatic cholesterol synthesis and stimulate fecal cholesterol elimination in mice

    NARCIS (Netherlands)

    Schonewille, Marleen; de Boer, Jan Freark; Mele, Laura; Wolters, Henk; Bloks, Vincent W.; Wolters, Justina C.; Kuivenhoven, Jan A.; Tietge, Uwe J. F.; Brufau, Gemma; Groen, Albert K.

    2016-01-01

    Statins are competitive inhibitors of HMG-CoA reductase, the rate-limiting enzyme of cholesterol synthesis. Statins reduce plasma cholesterol levels, but whether this is actually caused by inhibition of de novo cholesterol synthesis has not been clearly established. Using three different statins, we

  2. ACAT inhibitors: the search for novel cholesterol lowering agents.

    Science.gov (United States)

    Pal, Palash; Gandhi, Hardik; Giridhar, Rajani; Yadav, Mange Ram

    2013-06-01

    Increased level of serum cholesterol (hyperlipidemia) is the most significant risk factor for the development of atherosclerosis. Cholesterol levels are affected by factors such as rate of endogenous cholesterol synthesis, biliary cholesterol excretion and dietary cholesterol absorption. Acyl CoA: Cholesterol O-acyl transferases (ACAT) are a small family of enzymes that catalyze cholesterol esterification and cholesterol absorption in intestinal mucosal cells and maintain the cholesterol homeostasis in the blood. Inhibition of the ACAT enzymes is one of the attractive targets to treat hyperlipidemia. Literature survey shows that structurally diverse compounds possess ACAT inhibitory properties. In this review, a comprehensive presentation of the literature on diverse ACAT inhibitors has been given.

  3. Rab35 GTPase Triggers Switch-like Recruitment of the Lowe Syndrome Lipid Phosphatase OCRL on Newborn Endosomes.

    Science.gov (United States)

    Cauvin, Clothilde; Rosendale, Morgane; Gupta-Rossi, Neetu; Rocancourt, Murielle; Larraufie, Pierre; Salomon, Rémi; Perrais, David; Echard, Arnaud

    2016-01-11

    Phosphoinositide (PtdIns) homeostasis requires a tight spatial and temporal regulation during the endocytic process [1]. Indeed, PtdIns(4,5)P2 plays a crucial role in endocytosis by controlling clathrin-coated pit formation, whereas its conversion into PtdIns4P right after scission of clathrin-coated vesicles (CCVs) is essential for successful uncoating and cargo sorting [1-6]. In non-neuronal cells, endosomal PtdIns(4,5)P2 hydrolysis critically relies on the lipid phosphatase OCRL [7-9], the inactivation of which causes the Oculo-Cerebro-Renal syndrome of Lowe [10, 11]. To understand the coupling between PtdIns(4,5)P2 hydrolysis and endosome formation, a key issue is thus to unravel the mechanism by which OCRL is recruited on CCVs precisely after their scission from the plasma membrane. Here we found that the Rab35 GTPase, which plays a fundamental but poorly understood role in endosomal trafficking after cargo internalization [12-21], directly recruits the OCRL phosphatase immediately after scission of the CCVs. Consistent with Rab35 and OCRL acting together, depletion of either Rab35 or OCRL leads to retention of internalized receptors such as the endogenous cation-independent mannose-6-phosphate receptor (CI-MPR) in peripheral clathrin-positive endosomes that display abnormal association with PtdIns(4,5)P2- and actin-binding proteins. Remarkably, Rab35 loading on CCVs rapidly follows the recruitment of the AP2-binding Rab35 GEF/activator DENND1A (connecdenn 1) and the disappearance of the Rab35 GAP/inhibitor EPI64B. We propose that the precise spatial and temporal activation of Rab35 acts as a major switch for OCRL recruitment on newborn endosomes, post-scission PtdIns(4,5)P2 hydrolysis, and subsequent endosomal trafficking. Copyright © 2016 Elsevier Ltd. All rights reserved.

  4. Nuclear receptors in control of cholesterol transport

    NARCIS (Netherlands)

    van der Veen, Jelske Nynke

    2007-01-01

    Cholesterol is een structurele component van celmembranen en een grondstof voor de aanmaak van steroïde hormonen en galzouten en vervult dus een aantal essentiële fysiologische functies. Een goede balans van cholesterol opname, synthese, afbraak en uitscheiding is noodzakelijk, omdat verhoogde

  5. Chemical activity of cholesterol in membranes.

    Science.gov (United States)

    Radhakrishnan, A; McConnell, H M

    2000-07-18

    Measurements are reported for the rate constants for the release of cholesterol (and dihydrocholesterol) to beta-cyclodextrin from mixtures with phospholipids in homogeneous monolayers at constant pressure at the air-water interface. In each mixture, it is found that the release rate shows a sharp decrease as the cholesterol concentration in the monolayer decreases through a composition corresponding to the stoichiometry of a cholesterol-phospholipid complex. The stoichiometry of the complex was established previously by the position of a sharp cusp in the thermodynamic phase diagram of each mixture and also by a minimum in average molecular area versus composition measurements. A theoretical model used earlier to account for the phase diagrams predicts the chemical potential and chemical activity of cholesterol in these mixtures. The calculated chemical activity also shows a sharp change at the complex stoichiometry in homogeneous monolayers. The similarities in change of observed release rate and calculated chemical activity are expected from reaction rate theory where the release rate is proportional to the cholesterol chemical activity. The chemical activity of cholesterol as determined by complex formation between some phospholipids and cholesterol in the plasma membrane of cells may serve a regulatory function with respect to intracellular cholesterol transport and biosynthesis.

  6. Evaluating computational models of cholesterol metabolism

    NARCIS (Netherlands)

    Paalvast, Yared; Kuivenhoven, Jan Albert; Groen, Albert K.

    2015-01-01

    Regulation of cholesterol homeostasis has been studied extensively during the last decades. Many of the metabolic pathways involved have been discovered. Yet important gaps in our knowledge remain. For example, knowledge on intracellular cholesterol traffic and its relation to the regulation of

  7. HDL (Good), LDL (Bad) Cholesterol and Triglycerides

    Science.gov (United States)

    ... Artery Disease Venous Thromboembolism Aortic Aneurysm More HDL (Good), LDL (Bad) Cholesterol and Triglycerides Updated:Feb 19,2018 Cholesterol isn’t just something that sits in your body like fat around your waist. It’s carried through your bloodstream ...

  8. Role of cholesterol in Mycobacterium tuberculosis infection.

    Science.gov (United States)

    Miner, Maurine D; Chang, Jennifer C; Pandey, Amit K; Sassetti, Christopher M; Sherman, David R

    2009-06-01

    Mycobacterium tuberculosis (MTB) acquisition and utilization of nutrients within the host cell is poorly understood, although it has been hypothesized that host lipids probably play an important role in MTB survival. Cholesterol has recently been identified as an important lipid for mycobacterial infection. The mce4 transport system is required for cholesterol import into bacterial cells, and deletion of mce4 locus resulted in severe attenuation in a chronic mouse model of infection. However, it has remained unclear what additional bacterial functions were required for utilization of this sterol. We have found that the igr locus, which was previously found essential for intracellular growth and virulence of MTB, is required for cholesterol metabolism: igr-deficient bacteria cannot grow using cholesterol as a primary carbon source. The growth-inhibitory effect of cholesterol in vitro depends on cholesterol import, as the delta igr mutant growth defect during the early phase of disease is completely suppressed by mutating mce4, implicating cholesterol intoxication as the primary mechanism of attenuation. We conclude that M. tuberculosis metabolizes cholesterol throughout the course of infection, and that degradation of this sterol is crucial for bacterial persistence.

  9. Cholesterol: a Century of Research and Debate

    Directory of Open Access Journals (Sweden)

    Mathew B C

    2008-01-01

    Full Text Available To The Editor: The excellent article by Dr Elmehdawi RR entitled “Hypolipidemia: A word of caution” has once again shown the multifaceted properties of cholesterol which is the most highly decorated molecule in biology [1]. Thirteen nobel prizes have been awarded to scientists who devoted major parts of their careers to cholesterol [2].

  10. Cholesterol Absorption and Synthesis in Vegetarians and Omnivores.

    Science.gov (United States)

    Lütjohann, Dieter; Meyer, Sven; von Bergmann, Klaus; Stellaard, Frans

    2018-02-10

    Vegetarian diets are considered health-promoting; however, a plasma cholesterol lowering effect is not always observed. We investigate the link between vegetarian-diet-induced alterations in cholesterol metabolism. We study male and female omnivores, lacto-ovo vegetarians, lacto vegetarians, and vegans. Cholesterol intake, absorption, and fecal sterol excretion are measured as well as plasma concentrations of cholesterol and noncholesterol sterols. These serve as markers for cholesterol absorption, synthesis, and catabolism. The biliary cholesterol secretion rate is estimated. Flux data are related to body weight. Individual vegetarian diet groups are statistically compared to the omnivore group. Lacto vegetarians absorb 44% less dietary cholesterol, synthesized 22% more cholesterol, and show no differences in plasma total and LDL cholesterol. Vegan subjects absorb 90% less dietary cholesterol, synthesized 35% more cholesterol, and have a similar plasma total cholesterol, but a 13% lower plasma LDL cholesterol. No diet-related differences in biliary cholesterol secretion and absorption are observed. Total cholesterol absorption is lower only in vegans. Total cholesterol input is similar under all vegetarian diets. Unaltered biliary cholesterol secretion and higher cholesterol synthesis blunt the lowered dietary cholesterol intake in vegetarians. LDL cholesterol is significantly lower only in vegans. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Cholesterol-lowering effect of plant sterols.

    Science.gov (United States)

    AbuMweis, Suhad S; Jones, Peter J H

    2008-12-01

    Plant sterols are plant components that have a chemical structure similar to cholesterol except for the addition of an extra methyl or ethyl group; however, plant sterol absorption in humans is considerably less than that of cholesterol. In fact, plant sterols reduce cholesterol absorption and thus reduce circulating levels of cholesterol. Earlier studies that have tested the efficacy of plant sterols as cholesterol-lowering agents incorporated plant sterols into fat spreads. Later on, plant sterols were added to other food matrices, including juices, nonfat beverages, milk and yogurt, cheese, meat, croissants and muffins, and cereal and chocolate bars. The beneficial physiologic effects of plant sterols could be further enhanced by combining them with other beneficial substances, such as olive and fish oils, fibers, and soy proteins, or with exercise. The addition of plant sterols to the diet is suggested by health experts as a safe and effective way to reduce the risk of coronary heart disease.

  12. Trapping crystal nucleation of cholesterol monohydrate

    DEFF Research Database (Denmark)

    Solomonov, I.; Weygand, M.J.; Kjær, K.

    2005-01-01

    Crystalline nucleation of cholesterol at the air-water interface has been studied via grazing incidence x-ray diffraction using synchrotron radiation. The various stages of cholesterol molecular assembly from monolayer to three bilayers incorporating interleaving hydrogen-bonded water layers...... in a monoclinic cholesterol . H2O phase, has been monitored and their structures characterized to near atomic resolution. Crystallographic evidence is presented that this multilayer phase is similar to that of a reported metastable cholesterol phase of undetermined structure obtained from bile before...... transformation to the triclinic phase of cholesterol . H2O, the thermodynamically stable macroscopic form. According to grazing incidence x-ray diffraction measurements and crystallographic data, a transformation from the monoclinic film structure to a multilayer of the stable monohydrate phase involves...

  13. Exocytosis of Varicella-Zoster Virus Virions Involves a Convergence of Endosomal and Autophagy Pathways.

    Science.gov (United States)

    Buckingham, Erin M; Jarosinski, Keith W; Jackson, Wallen; Carpenter, John E; Grose, Charles

    2016-10-01

    Varicella-zoster virus (VZV) is an extremely cell-associated herpesvirus with limited egress of viral particles. The induction of autophagy in VZV-infected monolayers is easily detectable; inhibition of autophagy leads to decreased VZV glycoprotein biosynthesis and diminished viral titers. To explain how autophagic flux could exert a proviral effect on the VZV infectious cycle, we postulated that the VZV exocytosis pathway following secondary envelopment may converge with the autophagy pathway. This hypothesis depended on known similarities between VZV gE and autophagy-related (Atg) Atg9/Atg16L1 trafficking pathways. Investigations were carried out with highly purified fractions of VZV virions. When the virion fraction was tested for the presence of autophagy and endosomal proteins, microtubule-associated protein 1 light chain (MAP1LC3B) and Ras-like GTPase 11 (Rab11) were detected. By two-dimensional (2D) and 3D imaging after immunolabeling, both proteins also colocalized with VZV gE in a proportion of cytoplasmic vesicles. When purified VZV virions were enumerated after immunoelectron microscopy, gold beads were detected on viruses following incubation with antibodies to VZV gE (∼100%), Rab11 (50%), and LC3B (30%). Examination of numerous electron micrographs demonstrated that enveloped virions were housed in single-membraned vesicles; viral particles were not observed in autophagosomes. Taken together, our data suggested that some viral particles after secondary envelopment accumulated in a heterogeneous population of single-membraned vesicular compartments, which were decorated with components from both the endocytic pathway (Rab11) and the autophagy pathway (LC3B). The latter cytoplasmic viral vesicles resembled an amphisome. VZV infection leads to increased autophagic flux, while inhibition of autophagy leads to a marked reduction in virus spread. In this investigation of the proviral role of autophagy, we found evidence for an intersection of viral

  14. Effects of psyllium on plasma total and lipoprotein cholesterol and hepatic cholesterol in hamsters fed n-3 PUFA or n-6 PUFA with high cholesterol levels.

    Science.gov (United States)

    Liu, Young-Chau; Liu, Shyun-Yeu; Lin, Mei-Huei

    2004-01-01

    This study was conducted to determine whether psyllium is known to alter cholesterol metabolism modulate the hypercholesterolemic effect of a high cholesterol, n-3 polyunsaturated fatty acids (PUFA) diet in hamsters. Concentrations of plasma, hepatic total cholesterol and lipoprotein cholesterol were measured in male hamsters fed an n-3 PUFA plus psyllium (8%, wt/wt) diet combined with variable levels of cholesterol (0, 0.05, 0.1%, wt/wt) or a cholesterol-enriched (0.2%, wt/wt) n-3 PUFA or n-6 PUFA diet that contained either 8% methyl cellulose or psyllium for 4 weeks. In the n-3 PUFA-fed hamsters, we have found that psyllium was able to reduce plasma total cholesterol and low density lipoprotein (LDL)-cholesterol significantly when 0.1% cholesterol was added to the diet. In contrast, the effects of psyllium were not seen in the n-3 PUFA-fed hamsters without dietary cholesterol or with 0.05% dietary cholesterol. However, no matter in the presence of psyllium or not, the increase of plasma total cholesterol, very-low-density lipoprotein (VLDL)-cholesterol, LDL-cholesterol and high-density lipoprotein (HDL)-cholesterol levels was depend on the content of dietary cholesterol. Although the cholesterol diet increased the liver total cholesterol level, 80 g psyllium/kg diet resulted in a significantly lower concentration of liver total cholesterol in the cholesterol-fed hamsters. In the second experiment, we have also found that psyllium feeding lowered significantly plasma total cholesterol and VLDL-cholesterol concentrations in hamsters fed n-3 PUFA but not in those fed n-6 PUFA. However, the levels of plasma total cholesterol, VLDL-cholesterol and LDL-cholesterol levels of the (n-6) PUFA-fed hamsters were significantly lower than those in the (n-3) PUFA-fed hamsters in the absence or presence of dietary psyllium. Our data also showed that hamsters fed both high-cholesterol n-3 PUFA and n-6 PUFA diets had a significant decrease in hepatic cholesterol with intake of

  15. Tattoo removal.

    Science.gov (United States)

    Adatto, Maurice A; Halachmi, Shlomit; Lapidoth, Moshe

    2011-01-01

    Over 50,000 new tattoos are placed each year in the United States. Studies estimate that 24% of American college students have tattoos and 10% of male American adults have a tattoo. The rising popularity of tattoos has spurred a corresponding increase in tattoo removal. Not all tattoos are placed intentionally or for aesthetic reasons though. Traumatic tattoos due to unintentional penetration of exogenous pigments can also occur, as well as the placement of medical tattoos to mark treatment boundaries, for example in radiation therapy. Protocols for tattoo removal have evolved over history. The first evidence of tattoo removal attempts was found in Egyptian mummies, dated to have lived 4,000 years BC. Ancient Greek writings describe tattoo removal with salt abrasion or with a paste containing cloves of white garlic mixed with Alexandrian cantharidin. With the advent of Q-switched lasers in the late 1960s, the outcomes of tattoo removal changed radically. In addition to their selective absorption by the pigment, the extremely short pulse duration of Q-switched lasers has made them the gold standard for tattoo removal. Copyright © 2011 S. Karger AG, Basel.

  16. Continuous transport of a small fraction of plasma membrane cholesterol to endoplasmic reticulum regulates total cellular cholesterol.

    Science.gov (United States)

    Infante, Rodney Elwood; Radhakrishnan, Arun

    2017-04-17

    Cells employ regulated transport mechanisms to ensure that their plasma membranes (PMs) are optimally supplied with cholesterol derived from uptake of low-density lipoproteins (LDL) and synthesis. To date, all inhibitors of cholesterol transport block steps in lysosomes, limiting our understanding of post-lysosomal transport steps. Here, we establish the cholesterol-binding domain 4 of anthrolysin O (ALOD4) as a reversible inhibitor of cholesterol transport from PM to endoplasmic reticulum (ER). Using ALOD4, we: (1) deplete ER cholesterol without altering PM or overall cellular cholesterol levels; (2) demonstrate that LDL-derived cholesterol travels from lysosomes first to PM to meet cholesterol needs, and subsequently from PM to regulatory domains of ER to suppress activation of SREBPs, halting cholesterol uptake and synthesis; and (3) determine that continuous PM-to-ER cholesterol transport allows ER to constantly monitor PM cholesterol levels, and respond rapidly to small declines in cellular cholesterol by activating SREBPs, increasing cholesterol uptake and synthesis.

  17. Role of cholesterol in the biophysical dysfunction of surfactant in ventilator-induced lung injury.

    Science.gov (United States)

    Vockeroth, Dan; Gunasekara, Lasantha; Amrein, Matthias; Possmayer, Fred; Lewis, James F; Veldhuizen, Ruud A W

    2010-01-01

    Mechanical ventilation may lead to an impairment of the endogenous surfactant system, which is one of the mechanisms by which this intervention contributes to the progression of acute lung injury. The most extensively studied mechanism of surfactant dysfunction is serum protein inhibition. However, recent studies indicate that hydrophobic components of surfactant may also contribute. It was hypothesized that elevated levels of cholesterol significantly contribute to surfactant dysfunction in ventilation-induced lung injury. Sprague-Dawley rats (n = 30) were randomized to either high-tidal volume or low-tidal volume ventilation and monitored for 2 h. Subsequently, the lungs were lavaged, surfactant was isolated, and the biophysical properties of this isolated surfactant were analyzed on a captive bubble surfactometer with and without the removal of cholesterol using methyl-beta-cyclodextrin. The results showed lower oxygenation values in the high-tidal volume group during the last 30 min of ventilation compared with the low-tidal volume group. Surfactant obtained from the high-tidal volume animals had a significant impairment in function compared with material from the low-tidal volume group. Removal of cholesterol from the high-tidal volume group improved the ability of the surfactant to reduce the surface tension to low values. Subsequent reconstitution of high-cholesterol values led to an impairment in surface activity. It is concluded that increased levels of cholesterol associated with endogenous surfactant represent a major contributor to the inhibition of surfactant function in ventilation-induced lung injury.

  18. Membrane fluidity changes in goat sperm induced by cholesterol depletion using beta-cyclodextrin.

    Science.gov (United States)

    Companyó, Mònica; Iborra, Antoni; Villaverde, Joaquim; Martínez, Paz; Morros, Antoni

    2007-09-01

    Cholesterol efflux from membranes promotes acrosome reaction in goat spermatozoa. In 1 h of incubation of sperm in the presence of beta-cyclodextrin (beta CD), all the interchangeable cholesterol is desorbed from sperm membranes, although acrosome reaction is fully accomplished only after 3-4 h of incubation, as previously published. In the present paper we investigate the effect of cholesterol removal from mature goat spermatozoa on the overall membrane "fluidity" of live cell membranes and of liposomes from sperm lipid extracts. Using steady state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH), we studied the average thermotropic behaviour of membrane lipids, after incubation of live sperm for 1 h in BSA-free medium with the presence/absence of 8 mM beta-cyclodextrin, as a cholesterol acceptor. Unimodal and bimodal theoretical sigmoids fitted best to the experimental thermotropic profiles of liposomes and whole cells, respectively. In the case of whole sperm, two phase transitions, attributable to different lipid domains, were clearly separated by using the fitting parameters. After cholesterol removal, important changes in the relative anisotropy range of the two transitions were found, indicating an increase in the "fluidity" of some of the lipid microdomains of sperm membranes. These changes in sperm lipid dynamics are produced before the onset of sperm acrosome reaction.

  19. Selective cholesterol adsorption by molecular imprinted polymeric nanospheres and application to GIMS.

    Science.gov (United States)

    Inanan, Tülden; Tüzmen, Nalan; Akgöl, Sinan; Denizli, Adil

    2016-11-01

    Molecular imprinted polymers (MIPs) are tailor-made materials with selective recognition to the target. The goals of this study were to prepare cholesterol imprinted polymeric nanospheres (CIPNs) and optimize their adsorption parameters and also to use CIPNs for adsorption of cholesterol (CHO), which is an important physiological biomacromolecule, from gastrointestinal mimicking solution (GIMS). Pre-polymerization complex was prepared using CHO as template and N-methacryloylamido-(l)-phenylalanine methyl ester (MAPA). This complex was polymerized with 2-hydroxyethyl methacrylate (HEMA). CHO was removed by MeOH and tetrahydrofuran (THF). Adsorption studies were performed after chacterization studies to interrogate the effects of time, initial concentration, temperature, and ionic strength on CHO adsorption onto CIPNs. Maximum adsorption capacity (714.17mg/g) was higher than that of cholesterol imprinted polymers in literature. Pseudo-second-order kinetics and Langmuir isotherm fitted best with the adsorption onto CIPNs. 86% of adsorbed cholesterol was desorbed with MeOH:HAc (80:20, v/v) and CIPNs were used in adsorption-desorption cycle for 5-times with a decrease as 12.28%. CHO analogues; estron, estradiol, testosterone, and progesterone were used for competitive adsorption. The relative selectivity coefficients of CINPs for cholesterol/estron and cholesterol/testosterone were 3.84 and 10.47 times greater than the one of non-imprinted polymeric nanospheres (NIPNs) in methanol, respectively. Copyright © 2016 Elsevier B.V. All rights reserved.

  20. High Sensitivity Electrochemical Cholesterol Sensor Utilizing a Vertically Aligned Carbon Nanotube Electrode with Electropolymerized Enzyme Immobilization

    Directory of Open Access Journals (Sweden)

    Ditsayut Phokharatkul

    2009-10-01

    Full Text Available In this report, a new cholesterol sensor is developed based on a vertically aligned CNT electrode with two-step electrochemical polymerized enzyme immobilization. Vertically aligned CNTs are selectively grown on a 1 mm2 window of gold coated SiO2/Si substrate by thermal chemical vapor deposition (CVD with gravity effect and water-assisted etching. CNTs are then simultaneously functionalized and enzyme immobilized by electrochemical polymerization of polyaniline and cholesterol enzymes. Subsequently, ineffective enzymes are removed and new enzymes are electrochemically recharged. Scanning electron microscopic characterization indicates polymer-enzyme nanoparticle coating on CNT surface. Cyclic voltammogram (CV measurements in cholesterol solution show the oxidation and reduction peaks centered around 450 and −220 mV, respectively. An approximately linear relationship between the cholesterol concentration and the response current could be observed in the concentration range of 50–300 mg/dl with a sensitivity of approximately 0.22 μA/mg·dl−1, which is considerably higher compared to previously reported CNT bioprobe. In addition, good specificity toward glucose, uric acid acetaminophen and ascorbic acid have been obtained. Moreover, sensors have satisfactory stability, repeatability and life time. Therefore, the electropolymerized CNT bioprobe is promising for cholesterol detection in normal cholesterol concentration in human blood.

  1. Cholesterol biosynthesis by the cornea. Comparison of rates of sterol synthesis with accumulation during early development

    Energy Technology Data Exchange (ETDEWEB)

    Cenedella, R.J.; Fleschner, C.R. (Kirksville College of Osteopathic Medicine, MO (USA))

    1989-07-01

    The origin of the cholesterol needed by the cornea for growth and cell turnover was addressed by comparing absolute rates of sterol synthesis with rates of sterol accumulation during early development of the rabbit. Linearity of incorporation of {sup 3}H{sub 2}O and ({sup 14}C)mevalonate into digitonin-precipitable sterols with time of incubation in vitro and a lack of accumulation of {sup 14}C in intermediates of sterol biosynthesis indicated that tritiated water can validly be used to measure rates of sterol synthesis by the cornea. The rate of sterol synthesis per unit weight of rabbit cornea was constant between 14 and 60 days of age at an average 1.03 nmol of {sup 3}H of {sup 3}H{sub 2}O incorporated/mg dry cornea per 8 h. Essentially all of the synthesized cholesterol and most of the cholesterol mass was present in corneal epithelium. The cumulative sterol synthesized over the 46-day period studied exceeded the observed rate of cholesterol accumulation by sixfold. Cholesterol synthesized in excess of the growth requirement was likely used to support turnover of the epithelium which was estimated at 9 days. Removal of cholesterol from the cornea by excretion into tear fluid and clearance by high density lipoproteins are also considered.

  2. The accelerated ripening of cholesterol-reduced Cheddar cheese by crosslinked beta-cyclodextrin.

    Science.gov (United States)

    Seon, K H; Ahn, J; Kwak, H S

    2009-01-01

    This study was carried out to investigate the influence of salt content on cholesterol-reduced Cheddar cheese obtained by a treatment with crosslinked beta-cyclodextrin (beta-CD) and to find if the ripening process was accelerated in cholesterol-reduced cheese. The crosslinked beta-CD used was made by adipic acid. A primary study indicated that the chemical and rheological properties were not changed by the salt addition and the composition of Cheddar cheese treated with crosslinked beta-CD was similar to untreated Cheddar cheese. Approximately 91 to 92% cholesterol reduction was observed in the cheeses that were treated using beta-CD. In a subsequent study, we found accelerated ripening by the crosslinked beta-CD based on the productions of short-chain free fatty acids and free amino acids. In rheological properties, elasticity, cohesiveness, and gumminess scores in the cholesterol-reduced Cheddar cheese were significantly greater at 5 wk ripening than those in the control at 4 mo ripening. At the early stage of ripening, most flavor properties such as rancidity, bitterness, and off-flavor in the cholesterol-reduced cheese were greater. With ripening, however, those scores changed to similar or lower scores than those in the control. The present study indicated that the crosslinked beta-CD treatment for cholesterol removal showed accelerated ripening effect on the properties of Cheddar cheese.

  3. Brain Control of Plasma Cholesterol Involves Polysialic Acid Molecules in the Hypothalamus

    Directory of Open Access Journals (Sweden)

    Xavier Brenachot

    2017-05-01

    Full Text Available The polysialic acid (PSA is a large glycan that is added to cell-surface proteins during their post-translational maturation. In the brain, PSA modulates distances between cells and controls the plasticity of the nervous system. In the hypothalamus, PSA is involved in many aspects of energy balance including food intake, osmoregulation, circadian rhythm, and sleep. In this work, we investigated the role of hypothalamic PSA in the regulation of plasma cholesterol levels and distribution. We report that HFD consumption in mice rapidly increased plasma cholesterol, including VLDL, LDL, and HDL-cholesterol. Although plasma VLDL-cholesterol was normalized within the first week, LDL and HDL were still elevated after 2 weeks upon HFD. Importantly, we found that hypothalamic PSA removal aggravated LDL elevation and reduced HDL levels upon HFD. These results indicate that hypothalamic PSA controls plasma lipoprotein profile by circumventing the rise of LDL-to-HDL cholesterol ratio in plasma during overfeeding. Although mechanisms by which hypothalamic PSA controls plasma cholesterol homeostasis remains to be elucidated, these findings also suggest that low level of hypothalamic PSA might be a risk factor for dyslipidemia and cardiovascular diseases.

  4. Ligand Conformation Dictates Membrane and Endosomal Trafficking of Arginine-Glycine-Aspartate (RGD)-Functionalized Mesoporous Silica Nanoparticles

    Energy Technology Data Exchange (ETDEWEB)

    Fang, I-Ju; Slowing, Igor I; Wu, Kevin C.W.; Lin, Victor S.Y.; Trewyn, Brian

    2012-05-15

    Recent breakthrough research on mesoporous silica nanoparticle (MSN) materials has illustrated their significant potential in biological applications due to their excellent drug delivery and endocytotic behavior. We set out to determine if MSN, covalently functionalized with conformation specific bioactive molecules (either linear or cyclic RGD ligands), behave towards mammalian cells in a similar manner as the free ligands. We discovered that RGD immobilized on the MSN surface did not influence the integrity of the porous matrix and improved the endocytosis efficiency of the MSN materials. Through competition experiments with free RGD ligands, we also discovered a conformation specific receptor–integrin association. The interaction between RGD immobilized on the MSN surface and integrins plays an important role in endosome trafficking, specifically dictating the kinetics of endosomal escape. Thus, covalent functionalization of biomolecules on MSN assists in the design of a system for controlling the interface with cancer cells.

  5. Unrestricted synaptic growth in spinster-a late endosomal protein implicated in TGF-beta-mediated synaptic growth regulation.

    Science.gov (United States)

    Sweeney, Sean T; Davis, Graeme W

    2002-10-24

    In a genetic screen for genes that control synapse development, we have identified spinster (spin), which encodes a multipass transmembrane protein. spin mutant synapses reveal a 200% increase in bouton number and a deficit in presynaptic release. We demonstrate that spin is expressed in both nerve and muscle and is required both pre- and postsynaptically for normal synaptic growth. We have localized Spin to a late endosomal compartment and present evidence for altered endosomal/lysosomal function in spin. We also present evidence that synaptic overgrowth in spin is caused by enhanced/misregulated TGF-beta signaling. TGF-beta receptor mutants show dose-dependent suppression of synaptic overgrowth in spin. Furthermore, mutations in Dad, an inhibitory Smad, cause synapse overgrowth. We present a model for synaptic growth control with implications for the etiology of lysosomal storage and neurodegenerative disease.

  6. Cholesterol: Top Five Foods to Lower Your Numbers

    Science.gov (United States)

    ... store-bought cookies, crackers and cakes, are particularly bad for your cholesterol levels. Trans fats raise LDL cholesterol, and lower high-density lipoprotein (HDL), the "good" cholesterol. Food labels report the content of trans ...

  7. Cholesterol Test: MedlinePlus Lab Test Information

    Science.gov (United States)

    ... this page: https://medlineplus.gov/labtests/cholesteroltest.html Cholesterol Test To use the sharing features on this page, please enable JavaScript. What is a Cholesterol Test? Cholesterol is a waxy, fat-like substance ...

  8. High Cholesterol and Complementary Health Practices: What the Science Says

    Science.gov (United States)

    ... Health NCCIH Clinical Digest for health professionals High Cholesterol and Complementary Health Practices: What the Science Says ... chemically identical to the active ingredient in the cholesterol-lowering drug lovastatin. Available evidence on the cholesterol- ...

  9. Major Risk Factors for Heart Disease: High Blood Cholesterol

    Science.gov (United States)

    ... Major Risk Factors for Heart Disease High Blood Cholesterol High blood cholesterol is another major risk factor for heart disease ... can do something about. The higher your blood cholesterol level, the greater your risk for developing heart ...

  10. CD44 Receptor Targeting and Endosomal pH-Sensitive Dual Functional Hyaluronic Acid Micelles for Intracellular Paclitaxel Delivery.

    Science.gov (United States)

    Liu, Yanhua; Zhou, Chengming; Wang, Wenping; Yang, Jianhong; Wang, Hao; Hong, Wei; Huang, Yu

    2016-12-05

    A novel CD44 receptor targeting and endosome pH-sensitive dual functional hyaluronic acid-deoxycholic acid-histidine (HA-DOCA-His) micellar system was designed for intracellular paclitaxel (PTX) delivery. The HA-DOCA-His micelles exhibited desirable endosome pH (5.0-6.0)-induced aggregation and deformation behavior verified by size distribution, critical micellar concentration, and zeta potential changes. The HA-DOCA-His micelles presented excellent encapsulation efficiency and loading capacity of 90.0% and 18.9% for PTX, respectively. The PTX release from HA-DOCA-His micelles was pH-dependent, with more rapid PTX release at pH 6.0 and 5.0 than those at pH 7.4 and 6.5. The cellular uptake performance of HA-DOCA-His micelles was enhanced comparing with pH-insensitive HA-DOCA micelles by qualitative and quantitative measurements. HA-DOCA-His micelles could be taken up via CD44-receptor mediated endocytosis, transported into endosomes, and triggered drug release to cytoplasm. In vitro cytotoxicity study exhibited PTX-loaded HA-DOCA-His micelles were more active in tumor cell growth inhibition in MCF-7 cells at pH 5.8 than those at pH 6.8 and pH 7.4. A superior antitumor efficacy was demonstrated with HA-DOCA-His micelles in a MCF-7 breast tumor model. These indicated that the dual functional HA-DOCA-His micelles combined targeted intracellular delivery and endosomal release strategies could be developed as a promising nanocarrier for anticancer efficacy improvement of PTX.

  11. Self-Assembly of Porphyrin-Paclitaxel Conjugates Into Nanomedicines: Enhanced Cytotoxicity due to Endosomal Escape.

    Science.gov (United States)

    Zheng, Xiaohua; Li, Zhensheng; Chen, Li; Xie, Zhigang; Jing, Xiabin

    2016-06-21

    Nanomedicines assembled directly from drug molecules possess several advantages, including precise molecular structure and high content of drugs. Herein, porphyrin-paclitaxel conjugates (Py-s-s-PTX) were synthesized by using a disulfide bond as a linker. The Py-s-s-PTX could self-assemble into nanoparticles (Py-s-s-PTX NPs) with a size of about 100 nm via disulfide-induced assembly. Py-s-s-PTX NPs are highly stable under biological conditions and could be destroyed in the presence of reducing agents as revealed by dynamic light scattering. The obtained Py-s-s-PTX NPs could be internalized by cancer cells via endocytosis and disassociated in the reducing cytoplasm, thus releasing PTX in cancer cells. Endosomal escape triggered upon irradiation could enhance the cytotoxicity of paclitaxel, and Py-s-s-PTX NPs possess cytotoxicity comparable to that of free PTX. We believe that this disulfide-assembled nanomedicine represents a new and important development for chemotherapy in cancer therapy. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Peroxisomes, lipid droplets, and endoplasmic reticulum “hitchhike” on motile early endosomes

    Science.gov (United States)

    Guimaraes, Sofia C.; Schuster, Martin; Bielska, Ewa; Dagdas, Gulay; Kilaru, Sreedhar; Meadows, Ben R.A.; Schrader, Michael

    2015-01-01

    Intracellular transport is mediated by molecular motors that bind cargo to be transported along the cytoskeleton. Here, we report, for the first time, that peroxisomes (POs), lipid droplets (LDs), and the endoplasmic reticulum (ER) rely on early endosomes (EEs) for intracellular movement in a fungal model system. We show that POs undergo kinesin-3– and dynein-dependent transport along microtubules. Surprisingly, kinesin-3 does not colocalize with POs. Instead, the motor moves EEs that drag the POs through the cell. PO motility is abolished when EE motility is blocked in various mutants. Most LD and ER motility also depends on EE motility, whereas mitochondria move independently of EEs. Covisualization studies show that EE-mediated ER motility is not required for PO or LD movement, suggesting that the organelles interact with EEs independently. In the absence of EE motility, POs and LDs cluster at the growing tip, whereas ER is partially retracted to subapical regions. Collectively, our results show that moving EEs interact transiently with other organelles, thereby mediating their directed transport and distribution in the cell. PMID:26620910

  13. Niclosamide is a proton carrier and targets acidic endosomes with broad antiviral effects.

    Directory of Open Access Journals (Sweden)

    Andreas Jurgeit

    Full Text Available Viruses use a limited set of host pathways for infection. These pathways represent bona fide antiviral targets with low likelihood of viral resistance. We identified the salicylanilide niclosamide as a broad range antiviral agent targeting acidified endosomes. Niclosamide is approved for human use against helminthic infections, and has anti-neoplastic and antiviral effects. Its mode of action is unknown. Here, we show that niclosamide, which is a weak lipophilic acid inhibited infection with pH-dependent human rhinoviruses (HRV and influenza virus. Structure-activity studies showed that antiviral efficacy and endolysosomal pH neutralization co-tracked, and acidification of the extracellular medium bypassed the virus entry block. Niclosamide did not affect the vacuolar H(+-ATPase, but neutralized coated vesicles or synthetic liposomes, indicating a proton carrier mode-of-action independent of any protein target. This report demonstrates that physico-chemical interference with host pathways has broad range antiviral effects, and provides a proof of concept for the development of host-directed antivirals.

  14. The coordinating role of IQGAP1 in the regulation of local, endosome-specific actin networks

    Directory of Open Access Journals (Sweden)

    Edward B. Samson

    2017-06-01

    Full Text Available IQGAP1 is a large, multi-domain scaffold that helps orchestrate cell signaling and cytoskeletal mechanics by controlling interactions among a spectrum of receptors, signaling intermediates, and cytoskeletal proteins. While this coordination is known to impact cell morphology, motility, cell adhesion, and vesicular traffic, among other functions, the spatiotemporal properties and regulatory mechanisms of IQGAP1 have not been fully resolved. Herein, we describe a series of super-resolution and live-cell imaging analyses that identified a role for IQGAP1 in the regulation of an actin cytoskeletal shell surrounding a novel membranous compartment that localizes selectively to the basal cortex of polarized epithelial cells (MCF-10A. We also show that IQGAP1 appears to both stabilize the actin coating and constrain its growth. Loss of compartmental IQGAP1 initiates a disassembly mechanism involving rapid and unconstrained actin polymerization around the compartment and dispersal of its vesicle contents. Together, these findings suggest IQGAP1 achieves this control by harnessing both stabilizing and antagonistic interactions with actin. They also demonstrate the utility of these compartments for image-based investigations of the spatial and temporal dynamics of IQGAP1 within endosome-specific actin networks.

  15. The coordinating role of IQGAP1 in the regulation of local, endosome-specific actin networks.

    Science.gov (United States)

    Samson, Edward B; Tsao, David S; Zimak, Jan; McLaughlin, R Tyler; Trenton, Nicholaus J; Mace, Emily M; Orange, Jordan S; Schweikhard, Volker; Diehl, Michael R

    2017-06-15

    IQGAP1 is a large, multi-domain scaffold that helps orchestrate cell signaling and cytoskeletal mechanics by controlling interactions among a spectrum of receptors, signaling intermediates, and cytoskeletal proteins. While this coordination is known to impact cell morphology, motility, cell adhesion, and vesicular traffic, among other functions, the spatiotemporal properties and regulatory mechanisms of IQGAP1 have not been fully resolved. Herein, we describe a series of super-resolution and live-cell imaging analyses that identified a role for IQGAP1 in the regulation of an actin cytoskeletal shell surrounding a novel membranous compartment that localizes selectively to the basal cortex of polarized epithelial cells (MCF-10A). We also show that IQGAP1 appears to both stabilize the actin coating and constrain its growth. Loss of compartmental IQGAP1 initiates a disassembly mechanism involving rapid and unconstrained actin polymerization around the compartment and dispersal of its vesicle contents. Together, these findings suggest IQGAP1 achieves this control by harnessing both stabilizing and antagonistic interactions with actin. They also demonstrate the utility of these compartments for image-based investigations of the spatial and temporal dynamics of IQGAP1 within endosome-specific actin networks. © 2017. Published by The Company of Biologists Ltd.

  16. Endogenous cholesterol synthesis, fecal steroid excretion and serum lanosterol in subjects with high or low response of serum cholesterol to dietary cholesterol

    NARCIS (Netherlands)

    Beynen, A.C.; Katan, M.B.; Gent, van C.M.

    1986-01-01

    In this study we addressed the question whether hypo- and hyper-responders to dietary cholesterol differ with regard to the flexibility of endogenous cholesterol synthesis after changes in cholesterol intake. Whole-body cholesterol synthesis was measured as faecal excretion of neutral steroids and

  17. Genetic therapies to lower cholesterol.

    Science.gov (United States)

    Khoo, Bernard

    2015-01-01

    This review surveys the state-of-the-art in genetic therapies for familial hypercholesterolaemia (FH), caused most commonly by mutations in the LDL receptor (LDLR) gene. FH manifests as highly elevated low density lipoprotein (LDL) cholesterol levels and consequently accelerated atherosclerosis. Modern pharmacological therapies for FH are insufficiently efficacious to prevent premature cardiovascular disease, can cause significant adverse effects and can be expensive. Genetic therapies for FH have been mooted since the mid 1990s but gene replacement strategies using viral vectors have so far been unsuccessful. Other strategies involve knocking down the expression of Apolipoprotein B100 (APOB100) and the protease PCSK9 which designates LDLR for degradation. The antisense oligonucleotide mipomersen, which knocks down APOB100, is currently marketed (with restrictions) in the USA, but is not approved in Europe due to its adverse effects. To address this problem, we have devised a novel therapeutic concept, APO-skip, which is based on modulation of APOB splicing, and which has the potential to deliver a cost-effective, efficacious and safe therapy for FH. Copyright © 2014 Elsevier Inc. All rights reserved.

  18. Physiological and pathological implications of cholesterol.

    Science.gov (United States)

    Cortes, Victor A; Busso, Dolores; Maiz, Alberto; Arteaga, Antonio; Nervi, Flavio; Rigotti, Attilio

    2014-01-01

    Cholesterol has evolved to fulfill sophisticated biophysical, cell signaling and endocrine requirements of animal systems. At a cellular level, cholesterol is found in membranes, where it increases both bilayer stiffness and impermeability to water and ions. Furthermore, cholesterol is integrated into specialized lipid-protein membrane microdomains with critical topographical and signaling functions. At an organismal level, cholesterol is the precursor for all steroid hormones, including gluco- and mineralo-corticoids, sex hormones and vitamin D, all of which regulate carbohydrate, sodium, reproductive and bone homeostasis, respectively. This sterol is also the precursor for bile acids, which are important for intestinal absorption of dietary lipids as well as energy and glucose metabolic regulation. Importantly, complex mechanisms maintain cholesterol within physiological ranges and the disregulation of these mechanisms results in embryonic or adult diseases, caused by either excessive or reduced tissue cholesterol levels. The causative role of cholesterol in these diseases has been demonstrated by diverse genetic and pharmacologic animal models that are commented in this review.

  19. African Swine Fever Virus Undergoes Outer Envelope Disruption, Capsid Disassembly and Inner Envelope Fusion before Core Release from Multivesicular Endosomes.

    Directory of Open Access Journals (Sweden)

    Bruno Hernáez

    2016-04-01

    Full Text Available African swine fever virus (ASFV is a nucleocytoplasmic large DNA virus (NCLDV that causes a highly lethal disease in domestic pigs. As other NCLDVs, the extracellular form of ASFV possesses a multilayered structure consisting of a genome-containing nucleoid successively wrapped by a thick protein core shell, an inner lipid membrane, an icosahedral protein capsid and an outer lipid envelope. This structural complexity suggests an intricate mechanism of internalization in order to deliver the virus genome into the cytoplasm. By using flow cytometry in combination with pharmacological entry inhibitors, as well as fluorescence and electron microscopy approaches, we have dissected the entry and uncoating pathway used by ASFV to infect the macrophage, its natural host cell. We found that purified extracellular ASFV is internalized by both constitutive macropinocytosis and clathrin-mediated endocytosis. Once inside the cell, ASFV particles move from early endosomes or macropinosomes to late, multivesicular endosomes where they become uncoated. Virus uncoating requires acidic pH and involves the disruption of the outer membrane as well as of the protein capsid. As a consequence, the inner viral membrane becomes exposed and fuses with the limiting endosomal membrane to release the viral core into the cytosol. Interestingly, virus fusion is dependent on virus protein pE248R, a transmembrane polypeptide of the inner envelope that shares sequence similarity with some members of the poxviral entry/fusion complex. Collective evidence supports an entry model for ASFV that might also explain the uncoating of other multienveloped icosahedral NCLDVs.

  20. ABCA1-mediated transport of cellular cholesterol and phospholipids to HDL apolipoproteins.

    Science.gov (United States)

    Oram, J F; Vaughan, A M

    2000-06-01

    Lipid-poor apolipoproteins remove cellular cholesterol and phospholipids by an active transport pathway controlled by an ATP binding cassette transporter called ABCA1 (formerly ABC1). Mutations in ABCA1 cause Tangier disease, a severe HDL deficiency syndrome characterized by a rapid turnover of plasma apolipoprotein A-I, accumulation of sterol in tissue macrophages, and prevalent atherosclerosis. This implies that lipidation of apolipoprotein A-I by the ABCA1 pathway is required for generating HDL particles and clearing sterol from macrophages. Thus, the ABCA1 pathway has become an important therapeutic target for mobilizing excess cholesterol from tissue macrophages and protecting against atherosclerosis.

  1. Acyl-coenzyme A:cholesterol acyltransferases

    OpenAIRE

    Chang, Ta-Yuan; Li, Bo-Liang; Chang, Catherine C. Y.; Urano, Yasuomi

    2009-01-01

    The enzymes acyl-coenzyme A (CoA):cholesterol acyltransferases (ACATs) are membrane-bound proteins that utilize long-chain fatty acyl-CoA and cholesterol as substrates to form cholesteryl esters. In mammals, two isoenzymes, ACAT1 and ACAT2, encoded by two different genes, exist. ACATs play important roles in cellular cholesterol homeostasis in various tissues. This chapter summarizes the current knowledge on ACAT-related research in two areas: 1) ACAT genes and proteins and 2) ACAT enzymes as...

  2. Biotechniques in Electrochemical Determination of Cholesterol: Review

    Directory of Open Access Journals (Sweden)

    VIKAS

    2007-09-01

    Full Text Available With rising healthcare costs and to improve patient care, diagnostic laboratories have been challenged to develop new tests that are reliable, cost–effective and accurate and to optimize existing protocols by making them faster and more economical. Determination of serum total cholesterol is one of the most vital biochemical parameters in healthcare. With the availability of new materials associated with new sensing techniques has led to remarkable innovations in the design and construction of cholesterol biosensors. The present review describes the specifications of most of the electrochemical cholesterol biosensors reported till date.

  3. Cholesterol homeostasis in cardiovascular disease and recent advances in measuring cholesterol signatures.

    Science.gov (United States)

    Seo, Hong Seog; Choi, Man Ho

    2015-09-01

    Despite the biochemical importance of cholesterol, its abnormal metabolism has serious cellular consequences that lead to endocrine disorders such as cardiovascular disease (CVD). Nevertheless, the impact of blood cholesterol as a CVD risk factor is still debated, and treatment with cholesterol-lowering drugs remains controversial, particularly in older patients. Although, the prevalence of CVD increases with age, the underlying mechanisms for this phenomenon are not well understood, and metabolic changes have not been confirmed as predisposing factors of atherogenesis. The quantification of circulating biomarkers for cholesterol homeostasis is therefore warranted, and reference values for cholesterol absorption and synthesis should be determined in order to establish CVD risk factors. The traditional lipid profile is often derived rather than directly measured and lacks a universal standard to interpret the results. In contrast, mass spectrometry-based cholesterol profiling can accurately measure free cholesterol as a biologically active component. This approach allows to detect alterations in various metabolic pathways that control cholesterol homeostasis, by quantitative analysis of cholesterol and its precursors/metabolites as well as dietary sterols. An overview of the mechanism of cholesterol homeostasis under different physiological conditions may help to identify predictive biomarkers of concomitant atherosclerosis and conventional CVD risk factors. Copyright © 2015 Elsevier Ltd. All rights reserved.

  4. Cellular Cholesterol Regulates Ubiquitination and Degradation of the Cholesterol Export Proteins ABCA1 and ABCG1*

    Science.gov (United States)

    Hsieh, Victar; Kim, Mi-Jurng; Gelissen, Ingrid C.; Brown, Andrew J.; Sandoval, Cecilia; Hallab, Jeannette C.; Kockx, Maaike; Traini, Mathew; Jessup, Wendy; Kritharides, Leonard

    2014-01-01

    The objective of this study was to examine the influence of cholesterol in post-translational control of ABCA1 and ABCG1 protein expression. Using CHO cell lines stably expressing human ABCA1 or ABCG1, we observed that the abundance of these proteins is increased by cell cholesterol loading. The response to increased cholesterol is rapid, is independent of transcription, and appears to be specific for these membrane proteins. The effect is mediated through cholesterol-dependent inhibition of transporter protein degradation. Cell cholesterol loading similarly regulates degradation of endogenously expressed ABCA1 and ABCG1 in human THP-1 macrophages. Turnover of ABCA1 and ABCG1 is strongly inhibited by proteasomal inhibitors and is unresponsive to inhibitors of lysosomal proteolysis. Furthermore, cell cholesterol loading inhibits ubiquitination of ABCA1 and ABCG1. Our findings provide evidence for a rapid, cholesterol-dependent, post-translational control of ABCA1 and ABCG1 protein levels, mediated through a specific and sterol-sensitive mechanism for suppression of transporter protein ubiquitination, which in turn decreases proteasomal degradation. This provides a mechanism for acute fine-tuning of cholesterol transporter activity in response to fluctuations in cell cholesterol levels, in addition to the longer term cholesterol-dependent transcriptional regulation of these genes. PMID:24500716

  5. Tissue storage and control of cholesterol metabolism in man on high cholesterol diets.

    Science.gov (United States)

    Quintão, E C; Brumer, S; Stechhahn, K

    1977-03-01

    The possibility of accumulation of tissue cholesterol in human beings submitted to high cholesterol feeding was investigated in liver biopsies and through fecal sterol balance studies. Feeding to 10 individuals 3.1 to 3.4 g/day of cholesterol for 3 weeks raised the mean serum level from 293 to 349 mg/100 ml, namely 19%, whereas the liver cholesterol content was 417 mg/100 g of wet weight. In 10 control cases eating 0.1--0.4 g/day of cholesterol serum cholesterol remained stable throughout the experimental period and the liver cholesterol content was 256 mg/100 g. Difference of liver colesterol level between the two groups was 62%. In 7 patients submitted to two periods of balance investigation on a cholesterol-free synthetic formula diet respectively prior to (PI) and after (PIII) eating the high cholesterol solid food from 4 to 15 weeks (PII), fecal steroid excretion in PIII exceeded PI in 3 patients. Such data are a direct evidence for the existence of an efficient system to release acutely stored cholesterol. In one patient bile acid excretion accounted for the difference between PIII and PI.

  6. Triglycerides, total cholesterol, high density lipoprotein cholesterol and low density lipoprotein cholesterol in rats exposed to premium motor spirit fumes.

    Science.gov (United States)

    Aberare, Ogbevire L; Okuonghae, Patrick; Mukoro, Nathaniel; Dirisu, John O; Osazuwa, Favour; Odigie, Elvis; Omoregie, Richard

    2011-06-01

    Deliberate and regular exposure to premium motor spirit fumes is common and could be a risk factor for liver disease in those who are occupationally exposed. A possible association between premium motor spirit fumes and plasma levels of triglyceride, total cholesterol, high density lipoprotein cholesterol and low density lipoprotein cholesterol using a rodent model could provide new insights in the pathology of diseases where cellular dysfunction is an established risk factor. The aim of this study was to evaluate the possible effect of premium motor spirit fumes on lipids and lipoproteins in workers occupationally exposed to premium motor spirit fumes using rodent model. Twenty-five Wister albino rats (of both sexes) were used for this study between the 4(th) of August and 7(th) of September, 2010. The rats were divided into five groups of five rats each. Group 1 rats were not exposed to premium motor spirit fumes (control group), group 2 rats were exposed for 1 hour daily, group 3 for 3 hours daily, group 4 for 5 hours daily and group 5 for 7 hours daily. The experiment lasted for a period of 4 weeks. Blood samples obtained from all the groups after 4 weeks of exposure were used for the estimation of plasma levels of triglyceride, total cholesterol, high density lipoprotein- cholesterol and low density lipoprotein- cholesterol. Results showed significant increase in means of plasma total cholesterol and low density lipoprotein levels (Plevel of high density lipoprotein, the ratio of low density lipoprotein to high density lipoprotein and the ratio of total cholesterol to high density lipoprotein did not differ significantly in exposed subjects when compared with the control group. These results showed that frequent exposure to petrol fumes may be highly deleterious to the liver cells.

  7. Skin lesion removal

    Science.gov (United States)

    Shave excision - skin; Excision of skin lesions - benign; Skin lesion removal - benign; Cryosurgery - skin, benign; BCC - removal; Basal cell cancer - removal; Actinic keratosis - removal; Wart - removal; Squamous cell - removal; ...

  8. Decoupling internalization, acidification and phagosomal-endosomal/lysosomal fusion during phagocytosis of InlA coated beads in epithelial cells.

    Directory of Open Access Journals (Sweden)

    Craig D Blanchette

    Full Text Available BACKGROUND: Phagocytosis has been extensively examined in 'professional' phagocytic cells using pH sensitive dyes. However, in many of the previous studies, a separation between the end of internalization, beginning of acidification and completion of phagosomal-endosomal/lysosomal fusion was not clearly established. In addition, very little work has been done to systematically examine phagosomal maturation in 'non-professional' phagocytic cells. Therefore, in this study, we developed a simple method to measure and decouple particle internalization, phagosomal acidification and phagosomal-endosomal/lysosomal fusion in Madin-Darby Canine Kidney (MDCK and Caco-2 epithelial cells. METHODOLOGY/PRINCIPAL FINDINGS: Our method was developed using a pathogen mimetic system consisting of polystyrene beads coated with Internalin A (InlA, a membrane surface protein from Listeria monocytogenes known to trigger receptor-mediated phagocytosis. We were able to independently measure the rates of internalization, phagosomal acidification and phagosomal-endosomal/lysosomal fusion in epithelial cells by combining the InlA-coated beads (InlA-beads with antibody quenching, a pH sensitive dye and an endosomal/lysosomal dye. By performing these independent measurements under identical experimental conditions, we were able to decouple the three processes and establish time scales for each. In a separate set of experiments, we exploited the phagosomal acidification process to demonstrate an additional, real-time method for tracking bead binding, internalization and phagosomal acidification. CONCLUSIONS/SIGNIFICANCE: Using this method, we found that the time scales for internalization, phagosomal acidification and phagosomal-endosomal/lysosomal fusion ranged from 23-32 min, 3-4 min and 74-120 min, respectively, for MDCK and Caco-2 epithelial cells. Both the static and real-time methods developed here are expected to be readily and broadly applicable, as they simply

  9. Hair Removal

    Science.gov (United States)

    ... in girls who need it. Deciding to remove body hair is a personal choice. Getting rid of body hair doesn't make a person healthier, and you ... you don't want to. Some cultures view body hair as beautiful and natural, so do what feels ...

  10. Decoupling Internalization, Acidification and Phagosmal-Endosomal/Iysosomal Phagocytosis of Internalin A coated Beads in epithelial cells

    Energy Technology Data Exchange (ETDEWEB)

    Blanchette, C D; Woo, Y; Thomas, C; Shen, N; Sulchek, T A; Hiddessen, A L

    2008-12-22

    Phagocytosis has been extensively examined in 'professional' phagocytic cells using pH sensitive dyes. However, in many of the previous studies, a separation between the end of internalization, beginning of acidification and completion of phagosomal-endosomal/lysosomal fusion was not clearly established, and in several cases, it was treated as a one-step process. In addition, very little work has been done to systematically examine phagosomal maturation in 'non-professional' phagocytic cells, such as epithelial cells. Therefore, in this study, we developed a simple and novel method to decouple and accurately measure particle internalization, phagosomal acidification and phagosomal-endosomal/lysosomal fusion in Madin-Darby Canine Kidney (MDCK) and Caco-2 epithelial cells. Our method was developed using a pathogen mimetic system consisting of polystyrene beads coated with Internalin A (InlA), a membrane surface protein from Listeria monocytogenes known to trigger receptor-mediated internalization. We achieved independent measurements of the rates of internalization, phagosomal acidification and phagosomal-endosomal/lysosomal fusion in epithelial cells by combining the InlA-coated beads (InlA-beads) with antibody quenching, pH sensitive dyes and endosomal/lysosomal dyes, as follows: the rate of InlA bead internalization was measured via antibody quenching of a pH independent dye (Alexa488) conjugated to InlA-beads, the rate at which phagosomes containing internalized InlA beads became acidified was measured using a pH dependent dye (FITC) conjugated to the beads and the rate of phagosomal-endosomal/lysosomal fusion was measured using a combination of unlabeled InlA-beads and an endosomal/lysosomal dye. By performing these independent measurements under identical experimental conditions, we were able to decouple the three processes and establish time scales for each. In a separate set of experiments, we also exploited the phagosomal acidification

  11. estimations of cholesterol, triglycerides and fractionation

    African Journals Online (AJOL)

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    . 2Department of Science Laboratory .... clinical-chemistry parameters directly from whole blood, plasma or serum. The direct use of ..... Research suggests that men with high cholesterol levels when they are young. (mean age of 22 years) ...

  12. [Cholesterol and atherosclerosis. Historical considerations and treatment].

    Science.gov (United States)

    Zárate, Arturo; Manuel-Apolinar, Leticia; Basurto, Lourdes; De la Chesnaye, Elsa; Saldívar, Iván

    2016-01-01

    Cholesterol is a precursor of steroid hormones and an essential component of the cell membrane, however, altered regulation of the synthesis, absorption and excretion of cholesterol predispose to cardiovascular diseases of atherosclerotic origin. Despite, the recognition of historical events for 200 years, starting with Michel Chevreul naming «cholesterol»; later on, Lobstein coining the term atherosclerosis and Marchand introducing it, Anichkov identifying cholesterol in atheromatous plaque, and Brown and Goldstein discovering LDL receptor; as well as the emerging of different drugs, such as fibrates, statins and cetrapibs this decade, promising to increase HDL and the most recent ezetimibe and anti-PCSK9 to inhibit the degradation of LDL receptor, however morbidity has not been reduced in cardiovascular disease. Copyright © 2016. Published by Masson Doyma México S.A.

  13. What Are High Blood Cholesterol and Triglycerides?

    Science.gov (United States)

    ... in your blood. How can I control my cholesterol? • Cut down on foods high in saturated and trans fats. These include fatty meats, organ meats such as liver, shellfish, cheese, whole-milk dairy products, and solid fats such ...

  14. Proximate composition and cholesterol concentrations of ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-05-18

    DWB) for raw and fried samples, respectively, but decreased to 295.20 ... Key words: Rhynchophorus phoenicis, Oryctes monoceros, proximate composition, cholesterol, heat treatment. INTRODUCTION. Insects have played ...

  15. Hair removal.

    Science.gov (United States)

    Haedersdal, Merete; Haak, Christina S

    2011-01-01

    Hair removal with optical devices has become a popular mainstream treatment that today is considered the most efficient method for the reduction of unwanted hair. Photothermal destruction of hair follicles constitutes the fundamental concept of hair removal with red and near-infrared wavelengths suitable for targeting follicular and hair shaft melanin: normal mode ruby laser (694 nm), normal mode alexandrite laser (755 nm), pulsed diode lasers (800, 810 nm), long-pulse Nd:YAG laser (1,064 nm), and intense pulsed light (IPL) sources (590-1,200 nm). The ideal patient has thick dark terminal hair, white skin, and a normal hormonal status. Currently, no method of lifelong permanent hair eradication is available, and it is important that patients have realistic expectations. Substantial evidence has been found for short-term hair removal efficacy of up to 6 months after treatment with the available systems. Evidence has been found for long-term hair removal efficacy beyond 6 months after repetitive treatments with alexandrite, diode, and long-pulse Nd:YAG lasers, whereas the current long-term evidence is sparse for IPL devices. Treatment parameters must be adjusted to patient skin type and chromophore. Longer wavelengths and cooling are safer for patients with darker skin types. Hair removal with lasers and IPL sources are generally safe treatment procedures when performed by properly educated operators. However, safety issues must be addressed since burns and adverse events do occur. New treatment procedures are evolving. Consumer-based treatments with portable home devices are rapidly evolving, and presently include low-level diode lasers and IPL devices. Copyright © 2011 S. Karger AG, Basel.

  16. Wheat alkylresorcinols reduce micellar solubility of cholesterol in vitro and increase cholesterol excretion in mice.

    Science.gov (United States)

    Horikawa, Kazumasa; Hashimoto, Chiaki; Kikuchi, Yosuke; Makita, Miki; Fukudome, Shin-Ichi; Okita, Kimiko; Wada, Naoyuki; Oishi, Katsutaka

    2017-03-01

    Epidemiological studies have shown that the consumption of whole grains can reduce risk for metabolic disorders. We recently showed that chronic supplementation with wheat alkylresorcinols (ARs) prevents glucose intolerance and insulin resistance with hepatic lipid accumulation induced in mice by a high-fat high-sucrose diet (HFHSD). This study examines the effects of ARs on the micellar solubility of cholesterol in vitro, as well as the effects of transient AR supplementation on faecal lipid excretion and plasma lipid levels in mice. We found that ARs formed bile micelles with taurocholate independently of phospholipids, and dose-dependently decreased the micellar solubility of cholesterol in a biliary micelle model. Transient AR supplementation with HFHSD increased faecal cholesterol and triglyceride contents and decreased plasma cholesterol concentrations. These suggest that one underlying mechanism through which ARs suppress diet-induced obesity is by interfering with the micellar cholesterol solubilisation in the digestive tract, which subsequently decreases cholesterol absorption.

  17. SNAREs and cholesterol movement for steroidogenesis.

    Science.gov (United States)

    Kraemer, Fredric B; Shen, Wen-Jun; Azhar, Salman

    2017-02-05

    Steroidogenesis is a complex process through which cholesterol traffics to mitochondria and is converted via a series of enzymatic steps to steroid hormones. Although the rate-limiting step in this process is the movement of cholesterol from the outer to the inner mitochondrial membrane via the actions of StAR, a continuous supply of cholesterol must be delivered to the outer mitochondrial membrane during active steroidogenesis and this is derived from multiple sources, including lipoprotein uptake, endogenous cholesterol synthesis and release from stores within cytoplasmic lipid droplets. A number of mechanisms have been suggested to contribute to cholesterol trafficking to mitochondria; however, there is no definitive consensus and this is particularly so in regards to trafficking from cytoplasmic lipid droplets. In this paper we review experiments in which we have surveyed the expression of SNARE proteins in steroidogenic tissue and cells and examined the role of SNAREs in mediating cholesterol movement from lipid droplets to the mitochondria based on multiple studies that identified SNAREs as components of cytoplasmic lipid droplets. We established and characterized an in vitro mitochondria reconstitution assay system that enabled us to examine the impact of adding recombinant SNARE proteins specifically on the movement of cholesterol from model lipid droplets to the outer mitochondrial membrane. Using this reconstitution assay system in combination with siRNA knockdown experiments in rat primary granulosa cells or in steroidogenic cell lines, we showed that several SNARE proteins are important components in the trafficking of cholesterol from lipid droplets to the mitochondria for steroidogenesis. Published by Elsevier B.V.

  18. Assessing possible hazards of reducing serum cholesterol.

    Science.gov (United States)

    Law, M R; Thompson, S G; Wald, N J

    1994-02-05

    To assess whether low serum cholesterol concentration increases mortality from any cause. Systematic review of published data on mortality from causes other than ischaemic heart disease derived from the 10 largest cohort studies, two international studies, and 28 randomised trials, supplemented by unpublished data on causes of death obtained when necessary. Excess cause specific mortality associated with low or lowered serum cholesterol concentration. The only cause of death attributable to low serum cholesterol concentration was haemorrhagic stroke. The excess risk was associated only with concentrations below about 5 mmol/l (relative risk 1.9, 95% confidence interval 1.4 to 2.5), affecting about 6% of people in Western populations. For noncirculatory causes of death there was a pronounced difference between cohort studies of employed men, likely to be healthy at recruitment, and cohort studies of subjects in community settings, necessarily including some with existing disease. The employed cohorts showed no excess mortality. The community cohorts showed associations between low cholesterol concentration and lung cancer, haemopoietic cancers, suicide, chronic bronchitis, and chronic liver and bowel disease; these were most satisfactorily explained by early disease or by factors that cause the disease lowering serum cholesterol concentration (depression causes suicide and lowers cholesterol concentration, for example). In the randomised trials nine deaths (from a total of 687 deaths not due to ischaemic heart disease in treated subjects) were attributed to known adverse effects of the specific treatments, but otherwise there was no evidence of an increased mortality from any cause arising from reduction in cholesterol concentration. There is no evidence that low or reduced serum cholesterol concentration increases mortality from any cause other than haemorrhagic stroke. This risk affects only those people with a very low concentration and even in these will be

  19. Biodynamics of cholesterol and bile acids in the lithiasic hamster.

    Science.gov (United States)

    Khallou, J; Riottot, M; Parquet, M; Verneau, C; Lutton, C

    1991-11-01

    By using the isotopic equilibrium method in the young male Syrian hamster, the rates of cholesterol turnover processes, i.e. dietary cholesterol absorption, cholesterol synthesis, cholesterol excretion in the faeces and urine and cholesterol transformation into bile acids, were determined in the hamster receiving a control (C) or a lithogenic diet (L) for 7 weeks. At the end of this period the gall bladder of all animals in group L contained cholesterol gallstones. The coefficient of dietary cholesterol absorption was reduced by 26%, cholesterol synthesis and cholesterol faecal excretion were twofold higher in group L than in group C. Bile acid content in the small intestine was diminished in group L, but bile acid composition was similar in the two groups. The increase in cholesterogenesis in lithiasic animals essentially took place in the liver. Bile acid biosynthesis did not significantly differ in the two groups, but represented only 35% of total cholesterol input (dietary absorption + internal secretion) in group L v. 52% in group C. Thus, in the lithiasic hamster, hepatic synthesis of cholesterol and bile acids are not coupled. The molar percentage of cholesterol in bile was twofold higher in group L than in group C but those of bile acids and of phospholipids were not modified. In the lithiasic hamster the specific activity of biliary cholesterol was similar to that in plasma and liver. Consequently, biliary cholesterol does not derive directly from cholesterol newly synthesized in the liver but from hepatic cholesterol rapidly exchangeable with plasma cholesterol.

  20. Evaluation of LDL-Cholesterol / HDL-Cholesterol Ratio as Predictor of Dyslipidemia in Subclinical Hypothyroidism

    Directory of Open Access Journals (Sweden)

    Smita S. Kottagi

    2014-01-01

    Full Text Available Background: Subclinical hypothyroidism is defined as a serum TSH concentration above the upper limit of the reference range when serum T3 and T4 concentrations are within reference ranges. Subclinical thyroid disease is a laboratory diagnosis. Patients with subclinical disease have few or no definitive clinical signs or symptoms of thyroid dysfunction. It has been associated with higher levels of some cardiovascular risk factors. Despite some conflicting results, many studies have found that subjects with subclinical hypothyroidism have total cholesterol and low density lipoprotein cholesterol levels higher than euthyroid subjects. The association between subclinical hypothyroidism and dyslipidemia is well known. Aims and Objectives: This study is an attempt to find the importance of Low Density Lipoprotein – Cholesterol / Higher Density Lipoprotein - Cholesterol (LDL-C/HDL-C ratio rather than measurement of individual lipid profile parameters in bringing to light the dyslipidemic state associated with subclinical hypothyroidism. Materials and Methods: We studied 30 subclinical hypothyroid cases with age above 35 yrs and 30 age matched euthyroid controls. Serum T3, T4, TSH were estimated by ELISA method, serum total cholesterol, HDL Cholesterol by enzymatic CHOD-PAP method, and LDL cholesterol using Friedewald formula. Results: We found the significant increase in the serum levels of TSH (p < 0.001, Total cholesterol (p<0.001, LDL cholesterol (p<0.001, and LDL-C/HDL-C (p<0.001, Systolic blood pressure and diastolic blood pressure (p<0.001. There was no significant change in the levels of serum T3, T4, HDL- cholesterol. Conclusion: Increased levels of total cholesterol, LDL cholesterol and increased LDL-C/HDL-C ratio are seen in patients with subclinical hypothyroidism. LDL-C/HDLC ratio is a better indicator for dyslipidemia in subclinical hypothyroid cases.

  1. Recycling endosomes undergo rapid closure of a fusion pore on exocytosis in neuronal dendrites.

    Science.gov (United States)

    Jullié, Damien; Choquet, Daniel; Perrais, David

    2014-08-13

    Exocytosis of recycling endosomes (REs) represents the last step of receptor and membrane recycling, a fundamental process involved in many aspects of cell physiology. In neurons, it is involved in the control of cell polarity and synaptic plasticity and is locally and tightly regulated. However, its molecular mechanisms are still poorly understood. We have imaged single exocytosis events of REs in rat hippocampal neurons in culture transfected with three types of receptors tagged with the pH-sensitive GFP mutant superecliptic phluorin. We found that exocytosis events are grouped into two categories: (1) short burst events in which receptors diffuse into the plasma membrane in a few seconds; and (2) long display events in which receptors remain visible and clustered after exocytosis for many seconds. Display events are much rarer in non-neuronal cells, such as fibroblasts and astrocytes. Using two-color imaging and fast extracellular solution changes, we show that display events correspond to the rapid opening and closing of a fusion pore (or "kiss-and-run") with a median opening time of 2.6 s, which restricts the diffusion of multiple receptor types and bound cargo. Moreover, the RE marker Rab11 remains enriched after display exocytosis events and controls the mode of RE exocytosis. Finally, a given RE can undergo multiple rounds of display exocytosis. The last step of recycling can thus be controlled in neurons for the selective delivery of receptors at the cell surface. Copyright © 2014 the authors 0270-6474/14/3411106-13$15.00/0.

  2. Eps homology domain endosomal transport proteins differentially localize to the neuromuscular junction

    Directory of Open Access Journals (Sweden)

    Mate Suzanne E

    2012-09-01

    Full Text Available Abstract Background Recycling of endosomes is important for trafficking and maintenance of proteins at the neuromuscular junction (NMJ. We have previously shown high expression of the endocytic recycling regulator Eps15 homology domain-containing (EHD1 proteinin the Torpedo californica electric organ, a model tissue for investigating a cholinergic synapse. In this study, we investigated the localization of EHD1 and its paralogs EHD2, EHD3, and EHD4 in mouse skeletal muscle, and assessed the morphological changes in EHD1−/− NMJs. Methods Localization of the candidate NMJ protein EHD1 was assessed by confocal microscopy analysis of whole-mount mouse skeletal muscle fibers after direct gene transfer and immunolabeling. The potential function of EHD1 was assessed by specific force measurement and α-bungarotoxin-based endplate morphology mapping in EHD1−/− mouse skeletal muscle. Results Endogenous EHD1 localized to primary synaptic clefts of murine NMJ, and this localization was confirmed by expression of recombinant green fluorescent protein labeled-EHD1 in murine skeletal muscle in vivo. EHD1−/− mouse skeletal muscle had normal histology and NMJ morphology, and normal specific force generation during muscle contraction. The EHD 1–4 proteins showed differential localization in skeletal muscle: EHD2 to muscle vasculature, EHD3 to perisynaptic regions, and EHD4 to perinuclear regions and to primary synaptic clefts, but at lower levels than EHD1. Additionally, specific antibodies raised against mammalian EHD1-4 recognized proteins of the expected mass in the T. californica electric organ. Finally, we found that EHD4 expression was more abundant in EHD1−/− mouse skeletal muscle than in wild-type skeletal muscle. Conclusion EHD1 and EHD4 localize to the primary synaptic clefts of the NMJ. Lack of obvious defects in NMJ structure and muscle function in EHD1−/− muscle may be due to functional compensation by other EHD paralogs.

  3. Dietary cholesterol modulates pathogen blocking by Wolbachia.

    Directory of Open Access Journals (Sweden)

    Eric P Caragata

    Full Text Available The bacterial endosymbiont Wolbachia pipientis protects its hosts from a range of pathogens by limiting their ability to form infections inside the insect. This "pathogen blocking" could be explained by innate immune priming by the symbiont, competition for host-derived resources between pathogens and Wolbachia, or the direct modification of the cell or cellular environment by Wolbachia. Recent comparative work in Drosophila and the mosquito Aedes aegypti has shown that an immune response is not required for pathogen blocking, implying that there must be an additional component to the mechanism. Here we have examined the involvement of cholesterol in pathogen blocking using a system of dietary manipulation in Drosophila melanogaster in combination with challenge by Drosophila C virus (DCV, a common fly pathogen. We observed that flies reared on cholesterol-enriched diets infected with the Wolbachia strains wMelPop and wMelCS exhibited reduced pathogen blocking, with viral-induced mortality occurring 2-5 days earlier than flies reared on Standard diet. This shift toward greater virulence in the presence of cholesterol also corresponded to higher viral copy numbers in the host. Interestingly, an increase in dietary cholesterol did not have an effect on Wolbachia density except in one case, but this did not directly affect the strength of pathogen blocking. Our results indicate that host cholesterol levels are involved with the ability of Wolbachia-infected flies to resist DCV infections, suggesting that cholesterol contributes to the underlying mechanism of pathogen blocking.

  4. Cholesterol suppresses antimicrobial effect of statins

    Directory of Open Access Journals (Sweden)

    Mohammad Reza Haeri

    2015-12-01

    Full Text Available Objective(s:Isoprenoid biosynthesis is a key metabolic pathway to produce a wide variety of biomolecules such as cholesterol and carotenoids, which target cell membranes. On the other hand, it has been reported that statins known as inhibitors of isoprenoid biosynthesis and cholesterol lowering agents, may have a direct antimicrobial effect on the some bacteria. The exact action of statins in microbial metabolism is not clearly understood. It is possible that statins inhibit synthesis or utilization of some sterol precursor necessary for bacterial membrane integrity. Accordingly, this study was designed in order to examine if statins inhibit the production of a compound, which can be used in the membrane, and whether cholesterol would replace it and rescue bacteria from toxic effects of statins. Materials and Methods: To examine the possibility we assessed antibacterial effect of statins with different classes; lovastatin, simvastatin, and atorvastatin, alone and in combination with cholesterol on two Gram-positive (Staphylococcus aureus and Enterococcus faecalis and two Gram-negative (Pseudomonas aeruginosa and Escherichia coli bacteria using gel diffusion assay. Results: Our results showed that all of the statins except for lovastatin had significant antibacterial property in S. aureus, E. coli, and Enter. faecalis. Surprisingly, cholesterol nullified the antimicrobial action of effective statins in statin-sensitive bacteria. Conclusion: It is concluded that statins may deprive bacteria from a metabolite responsible for membrane stability, which is effectively substituted by cholesterol.

  5. Methotrexate in Atherogenesis and Cholesterol Metabolism

    Directory of Open Access Journals (Sweden)

    Eric Coomes

    2011-01-01

    Full Text Available Methotrexate is a disease-modifying antirheumatic drug commonly used to treat inflammatory conditions such as rheumatoid arthritis which itself is linked to increased cardiovascular risk. Treatments that target inflammation may also impact the cardiovascular system. While methotrexate improves cardiovascular risk, inhibition of the cyclooxygenase (COX-2 enzyme promotes atherosclerosis. These opposing cardiovascular influences may arise from differing effects on the expression of proteins involved in cholesterol homeostasis. These proteins, ATP-binding cassette transporter (ABC A1 and cholesterol 27-hydroxylase, facilitate cellular cholesterol efflux and defend against cholesterol overload. Methotrexate upregulates expression of cholesterol 27-hydroxylase and ABCA1 via adenosine release, while COX-2 inhibition downregulates these proteins. Adenosine, acting through the A2A and A3 receptors, may upregulate proteins involved in reverse cholesterol transport by cAMP-PKA-CREB activation and STAT inhibition, respectively. Elucidating underlying cardiovascular mechanisms of these drugs provides a framework for developing novel cardioprotective anti-inflammatory medications, such as selective A2A receptor agonists.

  6. Dietary cholesterol impairs memory and memory increases brain cholesterol and sulfatide levels.

    Science.gov (United States)

    Darwish, Deya S; Wang, Desheng; Konat, Gregory W; Schreurs, Bernard G

    2010-02-01

    Cholesterol and sulfatides play many important roles in learning and memory. To date, our observations about the effects of cholesterol on learning have been assessed during response acquisition; that is, the learning of a new memory. Here, we report for the first time to our knowledge, on the effect of a cholesterol diet on a previously formed memory. Rabbits were given trace conditioning of the nictitating membrane response for 10 days, then fed a 2% cholesterol diet for 8 weeks, and then assessed for memory recall of the initially learned task. We show that dietary cholesterol had an adverse effect on memory recall. Second, we investigated whether dietary cholesterol caused an increase in brain cholesterol and sulfatide levels in four major brain structures (hippocampus, frontal lobe, brainstem, and cerebellum) using a technique for analyzing myelin and myelin-free fractions separately. Although our data confirm previous findings that dietary cholesterol does not directly affect cholesterol and establish that it does not affect sulfatide levels in the brain, these levels did increase rather significantly in the hippocampus and frontal lobe as a function of learning and memory. (c) 2009 APA, all rights reserved.

  7. Cholesterol aided etching of tomatine gold nanoparticles: a non-enzymatic blood cholesterol monitor.

    Science.gov (United States)

    Raj, Vidya; Johnson, Teslin; Joseph, Kuruvilla

    2014-10-15

    Colloidal gold is extensively used for molecular sensing because of the wide flexibilities it offers in terms of modifications of the gold nanoparticles (GNPs) surface with a variety of functional groups. We describe a simple, enzyme free assay for the detection of cholesterol, and demonstrate its applicability by estimating cholesterol in human serum samples. To enable cholesterol detection, we functionalized GNPs with tomatine, a glycoalkaloid found in the leaves and stem of tomato plants. The binding of cholesterol onto tomatine functionalized gold nanoparticles (TGNPs) was characterized by a blue shift in the plasmon absorption spectra (SPR) followed by reduction in the particle size. The TGNPs have been core etched with increasing concentration of cholesterol and with 800 ng/mL of cholesterol particles in the size range of 10-12 nm have been obtained. This behavior was attributed to the enhanced hydrophobicity of the surface acquired by cholesterol binding resulting in the folding or shrinkage of molecule in turn leading to core etching. The method was successfully applied for the detection of cholesterol in real samples and agrees well with values obtained from the conventional method. Because of its significant plasmonic shift and simplicity, this biosensor could be used for cholesterol detection as it does not demand either any hazardous and costly chemicals or any complex synthetic routes. Copyright © 2014. Published by Elsevier B.V.

  8. Plasma membrane cholesterol is a key molecule in shear stress-dependent activation of extracellular signal-regulated kinase.

    Science.gov (United States)

    Park, H; Go, Y M; St John, P L; Maland, M C; Lisanti, M P; Abrahamson, D R; Jo, H

    1998-11-27

    Shear stress, the dragging force generated by fluid flow, differentially activates extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) in bovine aortic endothelial cells (BAEC) (Jo, H., Sipos, K., Go, Y. M., Law, R., Rong, J., and McDonald, J. M. (1997) J. Biol. Chem. 272, 1395-1401). Here, we examine whether cholesterol-enriched compartments in the plasma membrane are responsible for such differential regulation. Pretreatment of BAEC with a cholesterol-binding antibiotic, filipin, did not inhibit shear-dependent activation of JNK. In contrast, filipin and other membrane-permeable cholesterol-binding agents (digitonin and nystatin), but not the lipid-binding agent xylazine, inhibited shear-dependent activation of ERK. The effect of cholesterol-binding drugs did not appear to be due to membrane permeabilization, since treatment of BAEC with a detergent, Triton X-100 which also permeabilizes membranes, did not inhibit shear-dependent activation of ERK. Furthermore, shear-dependent activation of ERK, but not JNK, was inhibited by cyclodextrin, a membrane-impermeable cholesterol-binding agent, which removes cell-surface cholesterol. Moreover, the effects of cyclodextrin were prevented by adding cholesterol during the incubation. These results indicate that cholesterol or cholesterol-sensitive compartments in the plasma membrane play a selective and essential role in activation of ERK, but not JNK, by shear stress. Although exposure to shear stress (1 h) increased the number of caveolae by 3-fold, treatment with filipin had no effect in either control or shear-exposed cells suggesting that caveolae density per se is not a crucial determinant in shear-dependent ERK activation. In summary, the current study suggests that cholesterol-sensitive microdomains in the plasma membrane, such as caveolae-like domains, play a critical role in differential activation of ERK and JNK by shear stress.

  9. Blackcurrant anthocyanins stimulated cholesterol transport via post-transcriptional induction of LDL receptor in Caco-2 cells.

    Science.gov (United States)

    Kim, Bohkyung; Bae, Minkyung; Park, Young-Ki; Ma, Hang; Yuan, Tao; Seeram, Navindra P; Lee, Ji-Young

    2017-07-17

    We previously showed that polyphenol-rich blackcurrant extract (BCE) showed a hypocholesterolemic effect in mice fed a high fat diet. As direct cholesterol removal from the body via the intestine has been recently appreciated, we investigated the effect of BCE on the modulation of genes involved in intestinal cholesterol transport using Caco-2 cells as an in vitro model. Caco-2 cells were treated with BCE to determine its effects on mRNA and protein expression of genes important for intestinal cholesterol transport, low-density lipoprotein (LDL) uptake, cellular cholesterol content, and cholesterol transport from basolateral to apical membrane of Caco-2 cell monolayers. Cells were also treated with anthocyanin-rich or -poor fraction of BCE to determine the role of anthocyanin on BCE effects. BCE significantly increased protein levels of LDL receptor (LDLR) without altering its mRNA, which consequently increased LDL uptake into Caco-2 cells. This post-transcriptional induction of LDLR by BCE was markedly attenuated in the presence of rapamycin, an inhibitor of mechanistic target of rapamycin complex 1 (mTORC1). In addition, BCE altered genes involved in cholesterol transport in the enterocytes, including apical and basolateral cholesterol transporters, in such a way that could enhance cholesterol flux from the basolateral to apical side of the enterocytes. Indeed, BCE significantly increased the flux of LDL-derived cholesterol from the basolateral to the apical chamber of Caco-2 monolayer. LDLR protein levels were markedly increased by anthocyanin-rich fraction, but not by anthocyanin-free fraction. mTORC1-dependent post-transcriptional induction of LDLR by BCE anthocyanins drove the transport of LDL-derived cholesterol to the apical side of the enterocytes. This may represent a potential mechanism for the hypocholesterolemic effect of BCE.

  10. Dairy products and plasma cholesterol levels

    Directory of Open Access Journals (Sweden)

    Lena Ohlsson

    2010-08-01

    Full Text Available Cholesterol synthesized in the body or ingested is an essential lipid component for human survival from our earliest life. Newborns ingest about 3–4 times the amount per body weight through mother's milk compared to the dietary intake of adults. A birth level of 1.7 mmol/L plasma total cholesterol will increase to 4–4.5 mmol/L during the nursing period and continue to increase from adulthood around 40% throughout life. Coronary artery disease and other metabolic disorders are strongly associated with low-density lipoprotein (LDL and high-density lipoprotein (HDL cholesterol as well as triacylglycerol concentration. Milk fat contains a broad range of fatty acids and some have a negative impact on the cholesterol rich lipoproteins. The saturated fatty acids (SFAs, such as palmitic acid (C16:0, myristic acid (C14:0, and lauric acid (C12:0, increase total plasma cholesterol, especially LDL, and constitute 11.3 g/L of bovine milk, which is 44.8% of total fatty acid in milk fat. Replacement of dairy SFA and trans-fatty acids with polyunsaturated fatty acids decreases plasma cholesterol, especially LDL cholesterol, and is associated with a reduced risk of cardiovascular disease. Available data shows different effects on lipoproteins for different dairy products and there is uncertainty as to the impact a reasonable intake amount of dairy items has on cardiovascular risk. The aim of this review is to elucidate the effect of milk components and dairy products on total cholesterol, LDL, HDL, and the LDL/HDL quotients. Based on eight recent randomized controlled trials of parallel or cross-over design and recent reviews it can be concluded that replacement of saturated fat mainly (but not exclusively derived from high-fat dairy products with low-fat dairy products lowers LDL/HDL cholesterol and total/HDL cholesterol ratios. Whey, dairy fractions enriched in polar lipids, and techniques such as fermentation, or fortification of cows feeding can be used

  11. Inhibition of rat mammary tumorigenesis by dietary cholesterol.

    Science.gov (United States)

    el-Sohemy, A; Bruce, W R; Archer, M C

    1996-01-01

    The effects of dietary cholesterol and oxidized cholesterol on mammary tumor development were examined in female Sprague-Dawley rats exposed to the carcinogen N-methyl-N-nitrosourea (MNU). Animals were administered 50 mg/kg MNU at 50 days of age and fed either a control (AIN-76) diet or the control diet supplemented with 0.3% cholesterol or 0.3% oxidized cholesterol for up to 26 weeks. The oxidized cholesterol was prepared by heating cholesterol at 110 degrees C for 48 h. Gas chromatographic analysis of the oxidized cholesterol revealed a 2% yield of oxidation products in addition to a large amount of unchanged cholesterol (> 96%). Tumor incidence in the cholesterol group (67%) was significantly lower than in the control group (96%, P < 0.05), but the oxidized cholesterol group (79%) was not significantly different from the control or cholesterol groups. Average number of tumors per animal was lower in the cholesterol group (1.5) than in the control (2.8) or oxidized cholesterol groups (2.3, P < 0.005). Serum low density lipoprotein (LDL) cholesterol was greater in the cholesterol (185 +/- 38 mg/dl) and the oxidized cholesterol groups (160 +/- 34 mg/dl) than in the controls (55 +/- 4 mg/dl, P < 0.05), although there was no difference between the cholesterol and the oxidized cholesterol groups. These results show that dietary cholesterol inhibits mammary tumor development in this model. Elevated serum LDL cholesterol may inhibit de novo cholesterol synthesis in preneoplastic and/or tumor cells, thereby inhibiting their proliferation.

  12. The usefulness of total cholesterol and high density lipoprotein ...

    African Journals Online (AJOL)

    Objective: To determine the usefulness of total cholesterol/high-density lipoprotein cholesterol and/or highdensity lipoprotein cholesterol/total cholesterol ratios in the interpretation of lipid profile result in clinical practice. Methods: This is a prospective case-control study involving 109 diabetics, 98 diabetic hypertensives, 102 ...

  13. Cholesterol granuloma of the breast: a case report

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Jung Eun; Park, In Seo; Lee, Kyung Hee; Kim, Mi Young; Kim, Youn Jeong [College of Medicine, Inha University, Incheon (Korea, Republic of)

    2008-06-15

    Cholesterol granuloma is histologically characterized as fibrous granulation tissue containing cholesterol crystals within surrounding giant cells. Cases of cholesterol granuloma of the breast are rare. In fact, only eight cases have been previously described, and of these, an ultrasonography was performed in only. Here, we report the ultrasonographic findings of a breast cholesterol granuloma accompanied with a literature review.

  14. Dietary cholesterol - the role of eggs in the prudent diet

    African Journals Online (AJOL)

    eggs from the diet should be weighed against the effect of deprivation of these nutrients. Dietary cholesterol, serum cholesterol and CHD. Metabolic ward and controlled studies have demonstrated that an increase in dietary cholesterol as the only dietary variable, taken either as crystalline cholesterol, egg yolk or whole egg ...

  15. Cholesterol-Lowering Therapy Interventions: A Pharmacoeconomic Assessment

    OpenAIRE

    Michael A. Kortt; Armstrong, Edward P.

    1998-01-01

    Elevated cholesterol levels are associated with an increased risk of cardiovascular diseases. Treatment strategies promoting the associated health benefits from a reduction in elevated cholesterol levels have been outlined in guidelines published by the National Cholesterol Education Program. Clinicians and researchers have also examined the economic benefits associated with reducing elevated cholesterol levels. Most of these studies have employed traditional pharmacoeconomic techniques like ...

  16. The PDZ-interaction of the intestinal anion exchanger downregulated in adenoma (DRA; SLC26A3) facilitates its movement into Rab11a-positive recycling endosomes.

    Science.gov (United States)

    Lissner, S; Hsieh, C-J; Nold, L; Bannert, K; Bodammer, P; Sultan, A; Seidler, U; Graeve, L; Lamprecht, G

    2013-06-01

    Electroneutral NaCl absorption in the ileum and colon is mediated by downregulated in adenoma (DRA) (Cl⁻/HCO₃⁻ exchanger; SLC26A3) and Na⁺/H⁺ exchanger 3 (NHE3, SLC9A3). Surface expression of transport proteins undergoes basal and regulated recycling by endo- and exocytosis. Expression and activity of DRA in the plasma membrane depend on intact lipid rafts, phosphatidylinositol 3-kinase (PI3-kinase), and the PDZ interaction of DRA. However, it is unknown how the PDZ interaction of DRA affects its trafficking to the cell surface. Therefore, the (re)cycling pathway of DRA was investigated in HEK cells stably expressing enhanced green fluorescent protein (EGFP)-DRA or EGFP-DRA-ETKFminus (a mutant lacking the PDZ interaction motif). Early, late, and recycling endosomes were immunoisolated by precipitating stably transfected mCherry-hemagglutinin (HA)-Rab5a, -7a, or -11a. EGFP-DRA and EGFP-DRA-ETKFminus were equally present in early endosomes. In recycling endosomes, wild-type DRA was preferentially present, whereas, in late endosomes, DRA-ETKF-minus dominated. Correspondingly, EGFP-DRA colocalized with mCherry-HA-Rab11a in recycling endosomes, whereas EGFP-DRA-ETKFminus colocalized with mCherry-HA-Rab7a in late endosomes. Functionally, this different distribution was reflected by a shorter half-life of the mutant DRA. Transient expression of dominant-negative Rab11a(S25N) inhibited the activity (-17%, P recycling pathway. Taken together, the PDZ interaction of DRA facilitates its movement into Rab11a-positive recycling endosomes, from where it is inserted in the plasma membrane. A scenario emerges where specific PDZ adaptor proteins are present along several compartments of the endocytosis-recycling pathway.

  17. Intracellular cholesterol-binding proteins enhance HDL-mediated cholesterol uptake in cultured primary mouse hepatocytes

    Science.gov (United States)

    Storey, Stephen M.; McIntosh, Avery L.; Huang, Huan; Landrock, Kerstin K.; Martin, Gregory G.; Landrock, Danilo; Payne, H. Ross; Atshaves, Barbara P.; Kier, Ann B.

    2012-01-01

    A major gap in our knowledge of rapid hepatic HDL cholesterol clearance is the role of key intracellular factors that influence this process. Although the reverse cholesterol transport pathway targets HDL to the liver for net elimination of free cholesterol from the body, molecular details governing cholesterol uptake into hepatocytes are not completely understood. Therefore, the effects of sterol carrier protein (SCP)-2 and liver fatty acid-binding protein (L-FABP), high-affinity cholesterol-binding proteins present in hepatocyte cytosol, on HDL-mediated free cholesterol uptake were examined using gene-targeted mouse models, cultured primary hepatocytes, and 22-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-amino]-23,24-bisnor-5-cholen-3β-ol (NBD-cholesterol). While SCP-2 overexpression enhanced NBD-cholesterol uptake, counterintuitively, SCP-2/SCP-x gene ablation also 1) enhanced the rapid molecular phase of free sterol uptake detectable in cholesterol and 2) differentially enhanced free cholesterol uptake mediated by the HDL3, rather than the HDL2, subfraction. The increased HDL free cholesterol uptake was not due to increased expression or distribution of the HDL receptor [scavenger receptor B1 (SRB1)], proteins regulating SRB1 [postsynaptic density protein (PSD-95)/Drosophila disk large tumor suppressor (dlg)/tight junction protein (ZO1) and 17-kDa membrane-associated protein], or other intracellular cholesterol trafficking proteins (steroidogenic acute response protein D, Niemann Pick C, and oxysterol-binding protein-related proteins). However, expression of L-FABP, the single most prevalent hepatic cytosolic protein that binds cholesterol, was upregulated twofold in SCP-2/SCP-x null hepatocytes. Double-immunogold electron microscopy detected L-FABP sufficiently close to SRB1 for direct interaction, similar to SCP-2. These data suggest a role for L-FABP in HDL cholesterol uptake, a finding confirmed with SCP-2/SCP-x/L-FABP null mice and hepatocytes. Taken together

  18. The Role of Macrophage Lipophagy in Reverse Cholesterol Transport

    Directory of Open Access Journals (Sweden)

    Se-Jin Jeong

    2017-03-01

    Full Text Available Macrophage cholesterol efflux is a central step in reverse cholesterol transport, which helps to maintain cholesterol homeostasis and to reduce atherosclerosis. Lipophagy has recently been identified as a new step in cholesterol ester hydrolysis that regulates cholesterol efflux, since it mobilizes cholesterol from lipid droplets of macrophages via autophagy and lysosomes. In this review, we briefly discuss recent advances regarding the mechanisms of the cholesterol efflux pathway in macrophage foam cells, and present lipophagy as a therapeutic target in the treatment of atherosclerosis.

  19. Statins, PCSK9 inhibitors and cholesterol homeostasis: a view from within the hepatocyte.

    Science.gov (United States)

    Sniderman, Allan D; Kiss, Robert Scott; Reid, Thomas; Thanassoulis, George; Watts, Gerald F

    2017-05-01

    Statins and PCSK9 inhibitors dramatically lower plasma LDL levels and dramatically increase LDL receptor number within hepatocyte cell membranes. It seems self-evident that total clearance of LDL particles from plasma and total delivery of cholesterol to the liver must increase in consequence. However, based on the results of stable isotope tracer studies, this analysis demonstrates the contrary to be the case. Statins do not change the production rate of LDL particles. Accordingly, at steady state, the clearance rate cannot change. Because LDL particles contain less cholesterol on statin therapy, the delivery of cholesterol to the liver must, therefore, be reduced. PCSK9 inhibitors reduce the production of LDL particles and this further reduces cholesterol delivery to the liver. With both agents, a larger fraction of a smaller pool is removed per unit time. These findings are inconsistent with the conventional model of cholesterol homeostasis within the liver, but are consistent with a new model of regulation, the multi-channel model, which postulates that different lipoprotein particles enter the hepatocyte by different routes and have different metabolic fates within the hepatocyte. The multi-channel model, but not the conventional model, may explain how statins and PCSK9 inhibitors can produce sustained increases in LDL receptor number. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

  20. Increased maternal and fetal cholesterol efflux capacity and placental CYP27A1 expression in preeclampsia.

    Science.gov (United States)

    Mistry, Hiten D; Kurlak, Lesia O; Mansour, Yosef T; Zurkinden, Line; Mohaupt, Markus G; Escher, Geneviève

    2017-06-01

    Preeclampsia is a pregnancy-specific condition that leads to increased cardiovascular risk in later life. A decrease in cholesterol efflux capacity is linked to CVD. We hypothesized that in preeclampsia there would be a disruption of maternal/fetal plasma to efflux cholesterol, as well as differences in the concentrations of both placental sterol 27-hydroxylase (CYP27A1) and apoA1 binding protein (AIBP). Total, HDL-, and ABCA1-mediated cholesterol effluxes were performed with maternal and fetal plasma from women with preeclampsia and normotensive controls (both n = 17). apoA1 and apoE were quantified by chemiluminescence, and 27-hydroxycholesterol (27-OHC) by GC-MS. Immunohistochemistry was used to determine placental expression/localization of CYP27A1, AIBP, apoA1, apoE, and SRB1. Maternal and fetal total and HDL-mediated cholesterol efflux capacities were increased in preeclampsia (by 10-20%), but ABCA1-mediated efflux was decreased (by 20-35%; P preeclampsia. Fetal plasma 27-OHC levels were decreased in preeclamptic samples ( P preeclampsia ( P = 0.04). Placental 27-OHC concentrations were also raised in preeclampsia ( P preeclampsia, to remove cholesterol from cells to limit lipid peroxidation and increase placental angiogenesis. Copyright © 2017 by the American Society for Biochemistry and Molecular Biology, Inc.

  1. Isolation and characterization of Chinese hamster ovary (CHO) cells deficient in acyl coenzyme A: cholesterol acyltransferase (ACAT) activity

    Energy Technology Data Exchange (ETDEWEB)

    Cadigan, K.M.; Heider, J.G.; Chang, T.Y.

    1986-05-01

    The specific ACAT inhibitor compound 58-035 has been used to mimic the phenotype of an ACAT deficient mutant in 25-RA cells. 25-RA is a CHO cell line resistant to 25-hydroxycholesterol and contains five times more cholesterol ester than wild-type (WT) cells. 25-RA cells preincubated with 58-035 are 100 to 500 times more resistant to amphotericin B killing than untreated 25-RA. 100 x 10/sup 6/ mutagenized 25-RA cells underwent three rounds of amphotericin B killing and two rounds of 25-hydroxycholesterol killing (to remove WT revertants which are amphotericin B resistant). Thus far, three biochemically distinct mutants have been isolated containing 33% (AC27), 25% (AC90), and 10% (AC232) of the parental ACAT activity as measured by an /sup 3/H-oleate pulse in intact cells. When parental and mutant cell extracts are reconstituted into cholesterol containing liposomes the differences in ACAT activity remain. They have also found that 25-RA cells can survive in cholesterol free medium containing TMD, an inhibitor of cholesterol biosynthesis, presumably because of adequate supply of endogenous cholesterol from hydrolysis of its stored cholesterol ester. In contrast, under the same conditions, mutant AC232 is effectively killed ( greater than or equal to 99%) by cholesterol starvation, thus providing a potential selection procedure for isolating revertants of ACAT mutants.

  2. Enhancement of liposome mediated gene transfer by adding cholesterol and cholesterol modulating drugs.

    Science.gov (United States)

    Bae, Yun-Ui; Huh, Jae-Wan; Kim, Bieong-Kil; Park, Hyeon Young; Seu, Young-Bae; Doh, Kyung-Oh

    2016-12-01

    Cholesterol is an important cell membrane component and has been used as co-lipid for cationic liposome to enhance gene delivery. However, the role of cholesterol in transfection efficiency has not been fully understood. In this study, transfection efficiency of liposome was measured after cholesterol was added to the cell culture medium. As a result, addition of cholesterol increased transfection efficiency of several liposomes consisting of different lipid components in various cells (AGS, CHO, COS7 and, MCF7). Furthermore, treatment of cells with cholesterol modulating drugs, imipramine and U18666A, also increased transfection efficiency of liposomes. To elucidate the role of added cholesterol in gene transfer, endocytotic mechanism was studied and also revealed that adding cholesterol in culture media induced participation of caveolae-mediated endocytosis and micropinocytosis in CHO cell. Therefore, the results of this work suggest that modulation of intracellular cholesterol can be an important method to enhance gene delivery. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. Prevention of cholesterol gallstones by inhibiting hepatic biosynthesis and intestinal absorption of cholesterol

    Science.gov (United States)

    Wang, Helen H; Portincasa, Piero; de Bari, Ornella; Liu, Kristina J; Garruti, Gabriella; Neuschwander-Tetri, Brent A; Wang, David Q.-H

    2013-01-01

    Cholesterol cholelithiasis is a multifactorial disease influenced by a complex interaction of genetic and environmental factors, and represents a failure of biliary cholesterol homeostasis in which the physical-chemical balance of cholesterol solubility in bile is disturbed. The primary pathophysiologic event is persistent hepatic hypersecretion of biliary cholesterol, which has both hepatic and small intestinal components. The majority of the environmental factors are probably related to Western-type dietary habits, including excess cholesterol consumption. Laparoscopic cholecystectomy, one of the most commonly performed surgical procedures in the US, is nowadays a major treatment for gallstones. However, it is invasive and can cause surgical complications, and not all patients with symptomatic gallstones are candidates for surgery. The hydrophilic bile acid, ursodeoxycholic acid (UDCA) has been employed as first-line pharmacological therapy in a subgroup of symptomatic patients with small, radiolucent cholesterol gallstones. Long-term administration of UDCA can promote the dissolution of cholesterol gallstones. However, the optimal use of UDCA is not always achieved in clinical practice because of failure to titrate the dose adequately. Therefore, the development of novel, effective, and noninvasive therapies is crucial for reducing the costs of health care associated with gallstones. In this review, we summarize recent progress in investigating the inhibitory effects of ezetimibe and statins on intestinal absorption and hepatic biosynthesis of cholesterol, respectively, for the treatment of gallstones, as well as in elucidating their molecular mechanisms by which combination therapy could prevent this very common liver disease worldwide. PMID:23419155

  4. Alcohol consumption stimulates early steps in reverse cholesterol transport

    OpenAIRE

    Gaag, M.S.; Tol, A; Vermunt, S.H.F.; Scheek, L.M.; Schaafsma, G.; Hendriks, H.F.J.

    2001-01-01

    Alcohol consumption is associated with increased HDL cholesterol levels, which may indicate stimulated reverse cholesterol transport. The mechanism is, however, not known. The aim of this study was to evaluate the effects of alcohol consumption on the first two steps of the reverse cholesterol pathway: cellular cholesterol efflux and plasma cholesterol esterification. Eleven healthy middle-aged men consumed four glasses (40 g of alcohol) of red wine, beer, spirits (Dutch gin), or carbonated m...

  5. Arf6-Dependent Intracellular Trafficking of Pasteurella multocida Toxin and pH-Dependent Translocation from Late Endosomes

    Directory of Open Access Journals (Sweden)

    Tracy P. M. Chong

    2011-03-01

    Full Text Available The potent mitogenic toxin from Pasteurella multocida (PMT is the major virulence factor associated with a number of epizootic and zoonotic diseases caused by infection with this respiratory pathogen. PMT is a glutamine-specific protein deamidase that acts on its intracellular G-protein targets to increase intracellular calcium, cytoskeletal, and mitogenic signaling. PMT enters cells through receptor-mediated endocytosis and then translocates into the cytosol through a pH-dependent process that is inhibited by NH4Cl or bafilomycin A1. However, the detailed mechanisms that govern cellular entry, trafficking, and translocation of PMT remain unclear. Co-localization studies described herein revealed that while PMT shares an initial entry pathway with transferrin (Tfn and cholera toxin (CT, the trafficking pathways of Tfn, CT, and PMT subsequently diverge, as Tfn is trafficked to recycling endosomes, CT is trafficked retrograde to the ER, and PMT is trafficked to late endosomes. Our studies implicate the small regulatory GTPase Arf6 in the endocytic trafficking of PMT. Translocation of PMT from the endocytic vesicle occurs through a pH-dependent process that is also dependent on both microtubule and actin dynamics, as evidenced by inhibition of PMT activity in our SRE-based reporter assay, with nocodazole and cytochalasin D, respectively, suggesting that membrane translocation and cytotoxicity of PMT is dependent on its transfer to late endosomal compartments. In contrast, disruption of Golgi-ER trafficking with brefeldin A increased PMT activity, suggesting that inhibiting PMT trafficking to non-productive compartments that do not lead to translocation, while promoting formation of an acidic tubulovesicle system more conducive to translocation, enhances PMT translocation and activity.

  6. Accumulation of dsRNA in endosomes contributes to inefficient RNA interference in the fall armyworm, Spodoptera frugiperda.

    Science.gov (United States)

    Yoon, June-Sun; Gurusamy, Dhandapani; Palli, Subba Reddy

    2017-11-01

    RNA interference (RNAi) efficiency varies among insects studied. The barriers for successful RNAi include the presence of double-stranded ribonucleases (dsRNase) in the lumen and hemolymph that could potentially digest double-stranded RNA (dsRNA) and the variability in the transport of dsRNA into and within the cells. We recently showed that the dsRNAs are transported into lepidopteran cells, but they are not processed into small interference RNAs (siRNAs) because they are trapped in acidic bodies. In the current study, we focused on the identification of acidic bodies in which dsRNAs accumulate in Sf9 cells. Time-lapse imaging studies showed that dsRNAs enter Sf9 cells and accumulate in acidic bodies within 20 min after their addition to the medium. CypHer-5E-labeled dsRNA also accumulated in the midgut and fat body dissected from Spodoptera frugiperda larvae with similar patterns observed in Sf9 cells. Pharmacological inhibitor assays showed that the dsRNAs use clathrin mediated endocytosis pathway for transport into the cells. We investigated the potential dsRNA accumulation sites employing LysoTracker and double labeling experiments using the constructs to express a fusion of green fluorescence protein with early or late endosomal marker proteins and CypHer-5E-labeled dsRNA. Interestingly, CypHer-5E-labeled dsRNA accumulated predominantly in early and late endosomes. These data suggest that entrapment of internalized dsRNA in endosomes is one of the major factors contributing to inefficient RNAi response in lepidopteran insects. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. The late endosomal HOPS complex anchors active G-protein signaling essential for pathogenesis in magnaporthe oryzae.

    Directory of Open Access Journals (Sweden)

    Ravikrishna Ramanujam

    Full Text Available In Magnaporthe oryzae, the causal ascomycete of the devastating rice blast disease, the conidial germ tube tip must sense and respond to a wide array of requisite cues from the host in order to switch from polarized to isotropic growth, ultimately forming the dome-shaped infection cell known as the appressorium. Although the role for G-protein mediated Cyclic AMP signaling in appressorium formation was first identified almost two decades ago, little is known about the spatio-temporal dynamics of the cascade and how the signal is transmitted through the intracellular network during cell growth and morphogenesis. In this study, we demonstrate that the late endosomal compartments, comprising of a PI3P-rich (Phosphatidylinositol 3-phosphate highly dynamic tubulo-vesicular network, scaffold active MagA/GαS, Rgs1 (a GAP for MagA, Adenylate cyclase and Pth11 (a non-canonical GPCR in the likely absence of AKAP-like anchors during early pathogenic development in M. oryzae. Loss of HOPS component Vps39 and consequently the late endosomal function caused a disruption of adenylate cyclase localization, cAMP signaling and appressorium formation. Remarkably, exogenous cAMP rescued the appressorium formation defects associated with VPS39 deletion in M. oryzae. We propose that sequestration of key G-protein signaling components on dynamic late endosomes and/or endolysosomes, provides an effective molecular means to compartmentalize and control the spatio-temporal activation and rapid downregulation (likely via vacuolar degradation of cAMP signaling amidst changing cellular geometry during pathogenic development in M. oryzae.

  8. Arf6-dependent intracellular trafficking of Pasteurella multocida toxin and pH-dependent translocation from late endosomes.

    Science.gov (United States)

    Repella, Tana L; Ho, Mengfei; Chong, Tracy P M; Bannai, Yuka; Wilson, Brenda A

    2011-03-01

    The potent mitogenic toxin from Pasteurella multocida (PMT) is the major virulence factor associated with a number of epizootic and zoonotic diseases caused by infection with this respiratory pathogen. PMT is a glutamine-specific protein deamidase that acts on its intracellular G-protein targets to increase intracellular calcium, cytoskeletal, and mitogenic signaling. PMT enters cells through receptor-mediated endocytosis and then translocates into the cytosol through a pH-dependent process that is inhibited by NH(4)Cl or bafilomycin A1. However, the detailed mechanisms that govern cellular entry, trafficking, and translocation of PMT remain unclear. Co-localization studies described herein revealed that while PMT shares an initial entry pathway with transferrin (Tfn) and cholera toxin (CT), the trafficking pathways of Tfn, CT, and PMT subsequently diverge, as Tfn is trafficked to recycling endosomes, CT is trafficked retrograde to the ER, and PMT is trafficked to late endosomes. Our studies implicate the small regulatory GTPase Arf6 in the endocytic trafficking of PMT. Translocation of PMT from the endocytic vesicle occurs through a pH-dependent process that is also dependent on both microtubule and actin dynamics, as evidenced by inhibition of PMT activity in our SRE-based reporter assay, with nocodazole and cytochalasin D, respectively, suggesting that membrane translocation and cytotoxicity of PMT is dependent on its transfer to late endosomal compartments. In contrast, disruption of Golgi-ER trafficking with brefeldin A increased PMT activity, suggesting that inhibiting PMT trafficking to non-productive compartments that do not lead to translocation, while promoting formation of an acidic tubulovesicle system more conducive to translocation, enhances PMT translocation and activity.

  9. Genotypic distribution of common variants of endosomal toll like receptors in healthy Spanish women. A comparative study with other populations.

    Science.gov (United States)

    Martínez-Robles, Elena; Yebra-Bango, Miguel; Mellor-Pita, Susana; Tutor-Ureta, Pablo; Vargas, Juan A; Citores, Maria J

    2016-03-01

    Genetic variants of endosomal toll like receptors (TLR) have been associated with many infectious, autoimmune and inflammatory diseases, but few studies have been reported in the Spanish population. The aim of this study was to describe the allelic and genotypic distributions of some common nucleotide substitutions of endosomal TLRs in healthy Spanish women and to compare them with those already published in other population groups. Nine substitutions were analysed in 150 DNA samples from 150 Spanish, non-related healthy females: TLR3 rs3775291 and rs5743305; TLR7 rs179008 and rs5743781; TLR8 rs3764880 and TLR9 rs187084, rs5743836, rs352139 and rs352140. Genotyping was carried out by real time PCR and melting curve analysis in a LightCycler 480. A systematic review was performed in order to compare the genotypic distributions in our cohort with those previously published in other population groups. The comparative study was performed with the two tailed Fisher's test or the Yates continuity correction for the Chi-square test when appropriate. No homozygotes for rs5743781 in TLR7 were found, and rs352139 and rs352140 of TLR9 were in strong linkage disequilibrium. Genotype distributions in endosomal TLR are similar to other Spanish series previously reported. As expected, most differences were found when comparing our distributions with Asiatics, but differences were also found with other Caucasian populations. Since there are significant variations in genotypic distributions of TLRs in both interracial groups and within the same ethnic group, to carry out studies of disease susceptibility in more restricted groups is mandatory. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. Endosomal accumulation of APP in wobbler motor neurons reflects impaired vesicle trafficking: Implications for human motor neuron disease

    Directory of Open Access Journals (Sweden)

    Troakes Claire

    2011-03-01

    Full Text Available Abstract Background The cause of sporadic amyotrophic lateral sclerosis (ALS is largely unknown but hypotheses about disease mechanisms include oxidative stress, defective axonal transport, mitochondrial dysfunction and disrupted RNA processing. Whereas familial ALS is well represented by transgenic mutant SOD1 mouse models, the mouse mutant wobbler (WR develops progressive motor neuron degeneration due to a point mutation in the Vps54 gene, and provides an animal model for sporadic ALS. VPS54 protein as a component of a protein complex is involved in vesicular Golgi trafficking; impaired vesicle trafficking might also be mechanistic in the pathogenesis of human ALS. Results In motor neurons of homozygous symptomatic WR mice, a massive number of endosomal vesicles significantly enlarged (up to 3 μm in diameter were subjected to ultrastructural analysis and immunohistochemistry for the endosome-specific small GTPase protein Rab7 and for amyloid precursor protein (APP. Enlarged vesicles were neither detected in heterozygous WR nor in transgenic SOD1(G93A mice; in WR motor neurons, numerous APP/Rab7-positive vesicles were observed which were mostly LC3-negative, suggesting they are not autophagosomes. Conclusions We conclude that endosomal APP/Rab7 staining reflects impaired vesicle trafficking in WR mouse motor neurons. Based on these findings human ALS tissues were analysed for APP in enlarged vesicles and were detected in spinal cord motor neurons in six out of fourteen sporadic ALS cases. These enlarged vesicles were not detected in any of the familial ALS cases. Thus our study provides the first evidence for wobbler-like aetiologies in human ALS and suggests that the genes encoding proteins involved in vesicle trafficking should be screened for pathogenic mutations.

  11. The effect of lowering LDL cholesterol on vascular access patency

    DEFF Research Database (Denmark)

    Herrington, William; Emberson, Jonathan; Staplin, Natalie

    2014-01-01

    BACKGROUND AND OBJECTIVES: Reducing LDL cholesterol (LDL-C) with statin-based therapy reduces the risk of major atherosclerotic events among patients with CKD, including dialysis patients, but the effect of lowering LDL-C on vascular access patency is unclear. DESIGN, SETTING, PARTICIPANTS......, & MEASUREMENTS: The Study of Heart and Renal Protection (SHARP) randomized patients with CKD to 20 mg simvastatin plus 10 mg ezetimibe daily versus matching placebo. This study aimed to explore the effects of treatment on vascular access occlusive events, defined as any access revision procedure, access...... thrombosis, removal of an old dialysis access, or formation of new permanent dialysis access. RESULTS: Among 2353 SHARP participants who had functioning vascular access at randomization, allocation to simvastatin plus ezetimibe resulted in a 13% proportional reduction in vascular access occlusive events (355...

  12. Milk removal

    OpenAIRE

    Ferneborg, Sabine

    2016-01-01

    Milk from dairy cows is a staple dietary component for humans all over the world. Regardless of whether milk is consumed in its purest, unaltered form or as high-end products such as fine cheese or ice cream, it needs to be of high quality when taken from the cow, produced at a low price and produced in a system that consider aspects such as animal health, animal welfare and sustainability. This thesis investigated the role of milk removal and the importance of residual milk on milk yield...

  13. Inhibition of intestinal cholesterol absorption with ezetimibe increases components of reverse cholesterol transport in humans.

    Science.gov (United States)

    Davidson, Michael H; Voogt, Jason; Luchoomun, Jayraz; Decaris, Julie; Killion, Salena; Boban, Drina; Glass, Alexander; Mohammad, Hussein; Lu, Yun; Villegas, Deona; Neese, Richard; Hellerstein, Marc; Neff, David; Musliner, Thomas; Tomassini, Joanne E; Turner, Scott

    2013-10-01

    Reverse cholesterol transport (RCT) can be defined as a pathway of flux of cholesterol from peripheral tissues to the liver for potential excretion into feces. This prospective, placebo-controlled, double-blind crossover study assessed the effect of ezetimibe on several RCT parameters in hyperlipidemic patients. Following 7 weeks of treatment (ezetimibe 10 mg/day or placebo), 26 patients received 24-h continuous IV infusions of [3,4-(13)C2]-cholesterol, then took heavy water ((2)H2O) by mouth. Cholesterol excretion was measured by quantification of neutral/acid sterols in stool and blood samples during 7 days post-infusion with continued treatment. Plasma de novo cholesterol synthesis was assessed by (2)H-labeling from (2)H2O. Ezetimibe significantly reduced levels of low-density lipoprotein cholesterol (22%, P < 0.001) without significant changes in triglycerides and high-density lipoprotein cholesterol and significantly increased the flux of plasma-derived cholesterol into fecal neutral sterols by 52% (P = 0.04) without change in flux into fecal bile acids. Total fecal neutral sterol output increased by 23% (P = 0.02). Plasma de novo cholesterol synthesis increased by 57% (P < 0.001). The fractional clearance rate (FCR) of plasma cholesteryl-ester trended higher (7%; P = 0.055) with a reduction in absolute cholesteryl-ester production rate (9%, P < 0.01). Whole-body free cholesterol efflux rate from extra-hepatic tissues into plasma was not measurably changed by ezetimibe. Ezetimibe treatment approximately doubled the flux of plasma-derived cholesterol into fecal neutral sterols, in association with increases in total fecal neutral sterol excretion, FCR of plasma cholesterol ester, and plasma de novo cholesterol synthesis. These effects are consistent with increased cholesterol transport through the plasma compartment and excretion from the body, in response to ezetimibe treatment in hyperlipidemic humans. Clintrials.gov: NCT00701727. Copyright

  14. A whole-body mathematical model of cholesterol metabolism and its age-associated dysregulation.

    Science.gov (United States)

    Mc Auley, Mark T; Wilkinson, Darren J; Jones, Janette J L; Kirkwood, Thomas B L

    2012-10-10

    Global demographic changes have stimulated marked interest in the process of aging. There has been, and will continue to be, an unrelenting rise in the number of the oldest old ( >85 years of age). Together with an ageing population there comes an increase in the prevalence of age related disease. Of the diseases of ageing, cardiovascular disease (CVD) has by far the highest prevalence. It is regarded that a finely tuned lipid profile may help to prevent CVD as there is a long established relationship between alterations to lipid metabolism and CVD risk. In fact elevated plasma cholesterol, particularly Low Density Lipoprotein Cholesterol (LDL-C) has consistently stood out as a risk factor for having a cardiovascular event. Moreover it is widely acknowledged that LDL-C may rise with age in both sexes in a wide variety of groups. The aim of this work was to use a whole-body mathematical model to investigate why LDL-C rises with age, and to test the hypothesis that mechanistic changes to cholesterol absorption and LDL-C removal from the plasma are responsible for the rise. The whole-body mechanistic nature of the model differs from previous models of cholesterol metabolism which have either focused on intracellular cholesterol homeostasis or have concentrated on an isolated area of lipoprotein dynamics. The model integrates both current and previously published data relating to molecular biology, physiology, ageing and nutrition in an integrated fashion. The model was used to test the hypothesis that alterations to the rate of cholesterol absorption and changes to the rate of removal of LDL-C from the plasma are integral to understanding why LDL-C rises with age. The model demonstrates that increasing the rate of intestinal cholesterol absorption from 50% to 80% by age 65 years can result in an increase of LDL-C by as much as 34 mg/dL in a hypothetical male subject. The model also shows that decreasing the rate of hepatic clearance of LDL-C gradually to 50% by age 65

  15. A whole-body mathematical model of cholesterol metabolism and its age-associated dysregulation

    Directory of Open Access Journals (Sweden)

    Mc Auley Mark T

    2012-10-01

    Full Text Available Abstract Background Global demographic changes have stimulated marked interest in the process of aging. There has been, and will continue to be, an unrelenting rise in the number of the oldest old ( >85 years of age. Together with an ageing population there comes an increase in the prevalence of age related disease. Of the diseases of ageing, cardiovascular disease (CVD has by far the highest prevalence. It is regarded that a finely tuned lipid profile may help to prevent CVD as there is a long established relationship between alterations to lipid metabolism and CVD risk. In fact elevated plasma cholesterol, particularly Low Density Lipoprotein Cholesterol (LDL-C has consistently stood out as a risk factor for having a cardiovascular event. Moreover it is widely acknowledged that LDL-C may rise with age in both sexes in a wide variety of groups. The aim of this work was to use a whole-body mathematical model to investigate why LDL-C rises with age, and to test the hypothesis that mechanistic changes to cholesterol absorption and LDL-C removal from the plasma are responsible for the rise. The whole-body mechanistic nature of the model differs from previous models of cholesterol metabolism which have either focused on intracellular cholesterol homeostasis or have concentrated on an isolated area of lipoprotein dynamics. The model integrates both current and previously published data relating to molecular biology, physiology, ageing and nutrition in an integrated fashion. Results The model was used to test the hypothesis that alterations to the rate of cholesterol absorption and changes to the rate of removal of LDL-C from the plasma are integral to understanding why LDL-C rises with age. The model demonstrates that increasing the rate of intestinal cholesterol absorption from 50% to 80% by age 65 years can result in an increase of LDL-C by as much as 34 mg/dL in a hypothetical male subject. The model also shows that decreasing the rate of hepatic

  16. Effect of Inhibition of Intestinal Cholesterol Absorption on the Prevention of Cholesterol Gallstone Formation.

    Science.gov (United States)

    Portincasa, Piero; Wang, David Q-H

    2017-01-01

    Cholesterol cholelithiasis is a multifactorial hepatobiliary disease. Interactions between genetic and environmental factors play a critical role in biliary cholesterol homeostasis and its imbalance enhances cholelithogenesis. In patients developing symptoms or complications of gallstone disease, laparoscopic cholecystectomy is recommended for treatment of gallstones. In a subgroup of patients with small, radiolucent pure cholesterol gallstones, the hydrophilic bile acid, ursodeoxycholic acid (UDCA) is still considered the only pharmacological therapy able to induce oral litholysis. Identifying novel and effective pharmacological therapies is being investigated. We propose that the specific intestinal Niemann-Pick C1-like 1 protein inhibitor ezetimibe is a potential agent for preventing gallstone formation by reducing bioavailability of intestine- derived cholesterol to the liver for biliary secretion and desaturating bile through the inhibition of intestinal absorption of cholesterol. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  17. Sphingomyelin synthase overexpression increases cholesterol accumulation and decreases cholesterol secretion in liver cells

    Directory of Open Access Journals (Sweden)

    Li Yue

    2011-03-01

    Full Text Available Abstract Background Studies have shown that plasma high density lipoprotein cholesterol levels are negatively correlated with the development of atherosclerosis, whereas epidemiological studies have also shown that plasma sphingomyelin level is an independent risk factor for atherosclerosis. Methods To evaluate the relationship between cellular sphingomyelin level and cholesterol metabolism, we created two cell lines that overexpressed sphingomyelin synthase 1 or 2 (SMS1 or SMS2, using the Tet-off expression system. Results We found that SMS1 or SMS2 overexpression in Huh7 cells, a human hepatoma cell line, significantly increased the levels of intracellular sphingomyelin, cholesterol, and apolipoprotein A-I and decreased levels of apolipoprotein A-I and cholesterol in the cell culture medium, implying a defect in both processes. Conclusions Our findings indicate that the manipulation of sphingomyelin synthase activity could influence the metabolism of sphingomyelin, cholesterol and apolipoprotein A-I.

  18. Ca2+ homeostasis in microvascular endothelial cells from an insulin-dependent diabetic model: role of endosomes/lysosomes

    Science.gov (United States)

    Sanka, Shankar C.; Bennett, David C.; Rojas, Jose D.; Tasby, Geraldine B.; Meininger, Cynthia J.; Wu, Guoyao; Wesson, Donald E.; Pfarr, Curtis M.; Martinez-Zaguilan, Raul

    2000-04-01

    Cytosolic Ca2+ ([Ca2+]cyt) regulates several cellular functions, e.g. cell growth, contraction, secretion, etc. In many cell types, ion homeostasis appears to be coupled with glucose metabolism. In certain cell types, a strict coupling between glycolysis and the activity of Sarcoplasmic/Endoplasmic Reticulum Ca2+-ATPases (SERCA) has been suggested. Glucose metabolism is altered in diabetes. We hypothesize that: (1) Ca2+ homeostasis is altered in microvascular endothelial cells from diabetic animals due to the dysfunction of glycolysis coupling the activity of SERCA; (2) endosomal/lysosomal compartments expressing SERCA are involved in the dysfunction associated with diabetes.

  19. Structure and flexibility of the endosomal Vps34 complex reveals the basis of its function on membranes

    OpenAIRE

    Rostislavleva, Ksenia; Soler, Nicolas; Ohashi, Yohei; Zhang, Lufei; Pardon, Els; Burke, John E.; Masson, Glenn?R.; Johnson, Chris; Steyaert, Jan; Ktistakis, Nicholas T.; Williams, Roger L.

    2015-01-01

    Phosphatidylinositol 3-kinase Vps34 complexes regulate intracellular membrane trafficking in endocytic sorting, cytokinesis and autophagy. We present the 4.4 Å crystal structure of the 385 kDa endosomal complex II (PIK3C3-CII), consisting of Vps34, Vps15 (p150), Vps30/Atg6 (Beclin 1) and Vps38 (UVRAG). The subunits form a Y-shaped complex, centered on the Vps34 C2 domain. Vps34 and Vps15 intertwine in one arm where the Vps15 kinase domain engages the Vps34 activation loop to regulate its acti...

  20. Vps26p, a Component of Retromer, Directs the Interactions of Vps35p in Endosome-to-Golgi Retrieval

    OpenAIRE

    Reddy, Jonathan V.; Seaman, Matthew N.J.

    2001-01-01

    Endosome-to-Golgi retrieval of the carboxypeptidase Y receptor Vps10p is mediated by a recently discovered membrane coat complex termed retromer. Retromer comprises five conserved proteins: Vps35p, Vps29p, Vps5p, Vps17p, and Vps26p. Vps35p recognizes cargo molecules such as Vps10p and interacts strongly with Vps29p. Vps5p forms a subcomplex with Vps17p and has been proposed to play a structural role by self-assembling into large multimeric structures. The function of Vps26p is currently unkno...

  1. Genetic reconstitution of the human Adenovirus type 2 temperature-sensitive 1 mutant defective in endosomal escape

    Directory of Open Access Journals (Sweden)

    Gastaldelli Michele

    2009-10-01

    Full Text Available Abstract Human Adenoviruses infect the upper and lower respiratory tracts, the urinary and digestive tracts, lymphoid systems and heart, and give rise to epidemic conjunctivitis. More than 51 human serotypes have been identified to-date, and classified into 6 species A-F. The species C Adenoviruses Ad2 and Ad5 (Ad2/5 cause upper and lower respiratory disease, but how viral structure relates to the selection of particular infectious uptake pathways is not known. An adenovirus mutant, Ad2-ts1 had been isolated upon chemical mutagenesis in the past, and shown to have unprocessed capsid proteins. Ad2-ts1 fails to package the viral protease L3/p23, and Ad2-ts1 virions do not efficiently escape from endosomes. It had been suggested that the C22187T point mutation leading to the substitution of the conserved proline 137 to leucine (P137L in the L3/p23 protease was at least in part responsible for this phenotype. To clarify if the C22187T mutation is necessary and sufficient for the Ad2-ts1 phenotype, we sequenced the genes encoding the structural proteins of Ad2-ts1, and confirmed that the Ad2-ts1 DNA carries the point mutation C22187T. Introduction of C22187T to the wild-type Ad2 genome in a bacterial artificial chromosome (Ad2-BAC gave Ad2-BAC46 virions with the full Ad2-ts1 phenotype. Reversion of Ad2-BAC46 gave wild-type Ad2 particles indicating that P137L is necessary and sufficient for the Ad2-ts1 phenotype. The kinetics of Ad2-ts1 uptake into cells were comparable to Ad2 suggesting similar endocytic uptake mechanisms. Surprisingly, infectious Ad2 or Ad5 but not Ad2-ts1 uptake required CALM (clathrin assembly lymphoid myeloid protein, which controls clathrin-mediated endocytosis and membrane transport between endosomes and the trans-Golgi-network. The data show that no other mutations than P137L in the viral protease are necessary to give rise to particles that are defective in capsid processing and endosomal escape. This provides a basis for

  2. Lamp1 Increases the Efficiency of Lassa Virus Infection by Promoting Fusion in Less Acidic Endosomal Compartments.

    Science.gov (United States)

    Hulseberg, Christine E; Fénéant, Lucie; Szymańska, Katarzyna M; White, Judith M

    2018-01-02

    Lassa virus (LASV) is an arenavirus whose entry into host cells is mediated by a glycoprotein complex (GPC) comprised of a receptor binding subunit, GP1, a fusogenic transmembrane subunit, GP2, and a stable signal peptide. After receptor-mediated internalization, arenaviruses converge in the endocytic pathway, where they are thought to undergo low-pH-triggered, GPC-mediated fusion with a late endosome membrane. A unique feature of LASV entry is a pH-dependent switch from a primary cell surface receptor (α-dystroglycan) to an endosomal receptor, lysosomal-associated membrane protein (Lamp1). Despite evidence that the interaction between LASV GP1 and Lamp1 is critical, the function of Lamp1 in promoting LASV infection remains poorly characterized. Here we used wild-type (WT) and Lamp1 knockout (KO) cells to show that Lamp1 increases the efficiency of, but is not absolutely required for, LASV entry and infection. We then used cell-cell and pseudovirus-cell surface fusion assays to demonstrate that LASV GPC-mediated fusion occurs at a significantly higher pH when Lamp1 is present compared to when Lamp1 is missing. Correspondingly, we found that LASV entry occurs through less acidic endosomes in WT (Lamp1-positive) versus Lamp1 KO cells. We propose that, by elevating the pH threshold for fusion, Lamp1 allows LASV particles to exit the endocytic pathway before they encounter an increasingly acidic and harsh proteolytic environment, which could inactivate a significant percentage of incoming viruses. In this manner Lamp1 increases the overall efficiency of LASV entry and infection. IMPORTANCE Lassa virus is the most clinically important member of the Arenaviridae , a family that includes six additional biosafety level 4 (BSL4) hemorrhagic fever viruses. The lack of specific antiviral therapies for Lassa fever drives an urgent need to identify druggable targets, and interventions that block infection at the entry stage are particularly attractive. Lassa virus is only the

  3. The Processed Amino-Terminal Fragment of Human TLR7 Acts as a Chaperone To Direct Human TLR7 into Endosomes

    Science.gov (United States)

    Shepherd, Dawn; Booth, Sarah; Waithe, Dominic; Reis e Sousa, Caetano

    2015-01-01

    TLR7 mediates innate immune responses to viral RNA in endocytic compartments. Mouse and human (h)TLR7 undergo proteolytic cleavage, resulting in the generation of a C-terminal fragment that accumulates in endosomes and associates with the signaling adaptor MyD88 upon receptor triggering by TLR7 agonists. Although mouse TLR7 is cleaved in endosomes by acidic proteases, hTLR7 processing can occur at neutral pH throughout the secretory pathway through the activity of furin-like proprotein convertases. However, the mechanisms by which cleaved hTLR7 reaches the endosomal compartment remain unclear. In this study, we demonstrate that, after hTLR7 proteolytic processing, the liberated amino (N)-terminal fragment remains bound to the C terminus through disulfide bonds and provides key trafficking information that ensures correct delivery of the complex to endosomal compartments. In the absence of the N-terminal fragment, the C-terminal fragment is redirected to the cell surface, where it is functionally inactive. Our data reveal a novel role for the N terminus of hTLR7 as a molecular chaperone that provides processed hTLR7 with the correct targeting instructions to reach the endosomal compartment, hence ensuring its biological activity and preventing inadvertent cell surface responses to self-RNA. PMID:25917086

  4. Structure of cholesterol/ceramide monolayer mixtures

    DEFF Research Database (Denmark)

    Scheffer, L.; Solomonov, I.; Weygand, M.J.

    2005-01-01

    The structure of monolayers of cholesterol/ ceramide mixtures was investigated using grazing incidence x-ray diffraction, immunofluorescence, and atomic force microscopy techniques. Grazing incidence x-ray diffraction measurements showed the existence of a crystalline mixed phase of the two...... that in uncompressed ceramide monolayers, the highly crystalline phase coexists with a disordered loosely packed phase. In contrast, no disordered phase coexists with the new crystalline mixed phase. We conclude that the new mixed phase represents a stable homogeneous arrangement of cholesterol with ceramide...... components within a range of compositions of cholesterol/ ceramide between 100: 0 and 67: 33. The mixed phase coexists with the ceramide crystalline phase in the range of compositions between 50: 50 and 30: 70; between 30: 70 and 0: 100 only the highly crystalline phase of ceramide was detected. The latter...

  5. Ordering effects of cholesterol and its analogues

    DEFF Research Database (Denmark)

    Róg, Tomasz; Pasenkiewicz-Gierula, Marta; Vattulainen, Ilpo

    2009-01-01

    Without any exaggeration, cholesterol is one of the most important lipid species in eukaryotic cells. Its effects on cellular membranes and functions range from purely mechanistic to complex metabolic ones, besides which it is also a precursor of the sex hormones (steroids) and several vitamins....... In this review, we discuss the biophysical effects of cholesterol on the lipid bilayer, in particular the ordering and condensing effects, concentrating on the molecular level or inter-atomic interactions perspective, starting from two-component systems and proceeding to many-component ones e.g., modeling lipid...... rafts. Particular attention is paid to the roles of the methyl groups in the cholesterol ring system, and their possible biological function. Although our main research methodology is computer modeling, in this review we make extensive comparisons between experiments and different modeling approaches....

  6. CHOBIMALT: A Cholesterol-Based Detergent†

    Science.gov (United States)

    Howell, Stanley C.; Mittal, Ritesh; Huang, Lijun; Travis, Benjamin; Breyer, Richard M.; Sanders, Charles R.

    2010-01-01

    Cholesterol and its hemisuccinate and sulfate derivatives are widely used in studies of purified membrane proteins, but are difficult to solubilize in aqueous solution, even in the presence of detergent micelles. Other cholesterol derivatives do not form conventional micelles and lead to viscous solutions. To address these problems a cholesterol-based detergent, CHOBIMALT, has been synthesized and characterized. At concentrations above 3–4μM, CHOBIMALT forms micelles without the need for elevated temperatures or sonic disruption. Diffusion and fluorescence measurements indicated that CHOBIMALT micelles are large (210 ± 30 kDa). The ability to solubilize a functional membrane protein was explored using a G-protein coupled receptor, the human kappa opioid receptor type 1 (hKOR1). While CHOBIMALT alone was not found to be effective as a surfactant for membrane extraction, when added to classical detergent micelles CHOBIMALT was observed to dramatically enhance the thermal stability of solubilized hKOR1. PMID:20919740

  7. ATP-binding cassette transporters A1 and G1, HDL metabolism, cholesterol efflux, and inflammation: important targets for the treatment of atherosclerosis.

    Science.gov (United States)

    Ye, Dan; Lammers, Bart; Zhao, Ying; Meurs, Illiana; Van Berkel, T J; Van Eck, Miranda

    2011-05-01

    Atherosclerosis has been characterized as a chronic inflammatory response to cholesterol deposition in arteries. Plasma high density lipoprotein (HDL) levels bear a strong independent inverse relationship with atherosclerotic cardiovascular disease. One central antiatherogenic role of HDL is believed to be its ability to remove excessive peripheral cholesterol back to the liver for subsequent catabolism and excretion, a physiologic process termed reverse cholesterol transport (RCT). Cholesterol efflux from macrophage foam cells, the initial step of RCT is the most relevant step with respect to atherosclerosis. The ATP-binding cassette (ABC) transporters ABCA1 and ABCG1 play crucial roles in the efflux of cellular cholesterol to HDL and its apolipoproteins. Moreover, ABCA1 and ABCG1 affect cellular inflammatory cytokine secretion by modulating cholesterol content in the plasma membrane and within intracellular compartments. In humans, ABCA1 mutations can cause a severe HDL-deficiency syndrome characterized by cholesterol deposition in tissue macrophages and prevalent atherosclerosis. Disrupting Abca1 or Abcg1 in mice promotes accumulation of excessive cholesterol in macrophages, and physiological manipulation of ABCA1 expression affects atherogenesis. Here we review recent advances in the role of ABCA1 and ABCG1 in HDL metabolism, macrophage cholesterol efflux, inflammation, and atherogenesis. Next, we summarize the structure, expression, and regulation of ABCA1 and ABCG1. Finally, we give an update on the progress and pitfalls of therapeutic approaches that target ABCA1 and ABCG1 to stimulate the flux of lipids through the RCT pathway.

  8. [Screening and optimization of cholesterol conversion strain].

    Science.gov (United States)

    Fan, Dan; Xiong, Bingjian; Pang, Cuiping; Zhu, Xiangdong

    2014-10-04

    Bacterial strain SE-1 capable of transforming cholesterol was isolated from soil and characterized. The transformation products were identified. Fermentation conditions were optimized for conversion. Cholesterol was used as sole carbon source to isolate strain SE-1. Morphology, physiological and biochemical characteristics of strain SE-1 were studied. 16S rRNA gene was sequenced and subjected to phylogenetic analysis. Fermentation supernatants were extracted with chloroform, the transformation products were analyzed by silica gel thin layer chromatography and Sephadex LH20. Their structures were identified by 1H-NMR and 13C-NMR. Fermentation medium including carbon and nitrogen, methods of adding substrates and fermentation conditions for Strain SE-1 were optimized. Strain SE-1 was a Gram-negative bacterium, exhibiting the highest homologs to Burkholderia cepacia based on the physiological analysis. The sequence analysis of 16S rRNA gene of SE-1 strain and comparison with related Burkholderia show that SE-1 strain was very close to B. cepacia (Genbank No. U96927). The similarity was 99%. The result of silica gel thin layer chromatography shows that strain SE-1 transformed cholesterol to two products, 7beta-hydroxycholesterol and the minor product was 7-oxocholesterol. The optimum culture conditions were: molasses 5%, (NH4 )2SO4 0.3%, 4% of inoculation, pH 7.5 and 36 degrees C. Under the optimum culture condition, the conversion rate reached 34.4% when concentration of cholesterol-Tween 80 was 1 g/L. Cholesterol 7beta-hydroxylation conversion rate under optimal conditions was improved by 20.8%. Strain SE-1 isolated from soil is capable of converting cholesterol at lab-scale.

  9. Tympanomastoid cholesterol granulomas: Immunohistochemical evaluation of angiogenesis.

    Science.gov (United States)

    Iannella, Giannicola; Di Gioia, Cira; Carletti, Raffaella; Magliulo, Giuseppe

    2017-08-01

    This study investigates the immunohistochemical expression of vascular endothelial growth factor (VEGF) and CD34 in patients treated for middle ear and mastoid cholesterol granulomas to evaluate the angiogenesis and vascularization of this type of lesion. A correlation between the immunohistochemical data and the radiological and intraoperative evidence of temporal bone marrow invasion and blood source connection was performed to validate this hypothesis. Retrospective study. Immunohistochemical expression of VEGF and CD34 in a group of 16 patients surgically treated for cholesterol granuloma was examined. Middle ear cholesteatomas with normal middle ear mucosa and external auditory canal skin were used as the control groups. The radiological and intraoperative features of cholesterol granulomas were also examined. In endothelial cells, there was an increased expression of angiogenetic growth factor receptors in all the cholesterol granulomas in this study. The quantitative analysis of VEGF showed a mean value of 37.5, whereas the CD34 quantitative analysis gave a mean value of 6.8. Seven patients presented radiological or intraoperative evidence of bone marrow invasion, hematopoietic potentialities, or blood source connections that might support the bleeding theory. In all of these cases there was computed tomography or intraoperative evidence of bone erosion of the middle ear and/or temporal bone structures. The mean values of VEGF and CD34 were 41.1 and 7.7, respectively. High values of VEGF and CD34 are present in patients with cholesterol granulomas. Upregulation of VEGF and CD34 is indicative of a remarkable angiogenesis and a widespread vascular concentration in cholesterol granulomas. 3b. Laryngoscope, 127:E283-E290, 2017. © 2017 The American Laryngological, Rhinological and Otological Society, Inc.

  10. Dietary cholesterol from eggs increases the ratio of total cholesterol to high-density lipoprotein cholesterol in humans : a meta-analysis

    NARCIS (Netherlands)

    Weggemans, R.M.; Zock, P.L.; Katan, M.B.

    2001-01-01

    Several epidemiologic studies found no effect of egg consumption on the risk of coronary heart disease. It is possible that the adverse effect of eggs on LDL-cholesterol is offset by their favorable effect on HDL cholesterol. Objective: The objective was to review the effect of dietary cholesterol

  11. Cholesterol mediates membrane curvature during fusion events.

    Science.gov (United States)

    Ivankin, Andrey; Kuzmenko, Ivan; Gidalevitz, David

    2012-06-08

    Biomembranes undergo extensive shape changes as they perform vital cellular functions. The mechanisms by which lipids and proteins control membrane curvature remain unclear. We use x-ray reflectivity, grazing incidence x-ray diffraction, and epifluorescence microscopy to study binding of HIV-1 glycoprotein gp41's membrane-bending domain to DPPC/cholesterol monolayers of various compositions at the air-liquid interface. The results offer a new insight into how membrane curvature could be regulated by cholesterol during fusion of the viral lipid envelope and the host cell membranes.

  12. Cholesterol oxidation products and their biological importance

    DEFF Research Database (Denmark)

    Kulig, Waldemar; Cwiklik, Lukasz; Jurkiewicz, Piotr

    2016-01-01

    and precisely regulated level, with an excess of cholesterol. Like cholesterol, many oxysterols are hydrophobic and hence confined to cell membranes. However, small chemical differences between the sterols can significantly affect how they interact with other membrane components, and this in turn can have...... a substantial effect on membrane properties. In this spirit, this review describes the biological importance and the roles of oxysterols in the human body. We focus primarily on the effect of oxysterols on lipid membranes, but we also consider other issues such as enzymatic and nonenzymatic synthesis processes...

  13. 2013 Cholesterol Guidelines Revisited: Percent LDL Cholesterol Reduction or Attained LDL Cholesterol Level or Both for Prognosis?

    NARCIS (Netherlands)

    Bangalore, Sripal; Fayyad, Rana; Kastelein, John J.; Laskey, Rachel; Amarenco, Pierre; Demicco, David A.; Waters, David D.

    2016-01-01

    The 2013 American College of Cardiology (ACC)/American Heart Association (AHA) guideline on the treatment of blood cholesterol recommends moderate- to high-intensity statins for patients with atherosclerotic cardiovascular disease but departs from the traditional treat-to-target approach. Whether

  14. Aspirin Increases the Solubility of Cholesterol in Lipid Membranes

    Science.gov (United States)

    Alsop, Richard; Barrett, Matthew; Zheng, Sonbo; Dies, Hannah; Rheinstadter, Maikel

    2014-03-01

    Aspirin (ASA) is often prescribed for patients with high levels of cholesterol for the secondary prevention of myocardial events, a regimen known as the Low-Dose Aspirin Therapy. We have recently shown that Aspirin partitions in lipid bilayers. However, a direct interplay between ASA and cholesterol has not been investigated. Cholesterol is known to insert itself into the membrane in a dispersed state at moderate concentrations (under ~37.5%) and decrease fluidity of membranes. We prepared model lipid membranes containing varying amounts of both ASA and cholesterol molecules. The structure of the bilayers as a function of ASA and cholesterol concentration was determined using high-resolution X-ray diffraction. At cholesterol levels of more than 40mol%, immiscible cholesterol plaques formed. Adding ASA to the membranes was found to dissolve the cholesterol plaques, leading to a fluid lipid bilayer structure. We present first direct evidence for an interaction between ASA and cholesterol on the level of the cell membrane.

  15. [Basic mechanisms: absorption and excretion of cholesterol and other sterols].

    Science.gov (United States)

    Cofan Pujol, Montserrat

    2014-01-01

    Cholesterol is of vital importance for vertebrate cell membrane structure and function. It is obvious that adequate regulation of cholesterol homeostasis is essential. Hypercholesterolemia promotes atherosclerosis and thereby represents a major risk factor for cardiovascular disease. The liver has been considered the major site of control in maintenance of cholesterol homeostasis. The liver facilitates clearance of (very) low density lipoprotein particles and cholesterol-containing chylomicron remnants, synthesizes cholesterol, synthesizes and secretes (nascent) high density lipoprotein particles, secretes cholesterol and bile salts to bile, and is involved in reverse cholesterol transport. In recent years, however, the importance of the intestine in many aspects of cholesterol physiology is increasingly recognized. It has become apparent that direct secretion of cholesterol from the blood compartment into the intestine, or transintestinal cholesterol excretion, plays a major role in disposal of cholesterol via the feces. This review will discuss current knowledge on the physiology of cholesterol homeostasis, with emphasis on cholesterol absorption, cholesterol synthesis and fecal excretion, and therapeutic options for hypercholesterolemia. Copyright © 2013 Elsevier España, S.L. y SEA. All rights reserved.

  16. Toxoplasma gondii Syntaxin 6 Is Required for Vesicular Transport Between Endosomal-Like Compartments and the Golgi Complex

    Science.gov (United States)

    Jackson, Allison J; Clucas, Caroline; Mamczur, Nicola J; Ferguson, David J; Meissner, Markus

    2013-01-01

    Apicomplexans are obligate intracellular parasites that invade the host cell in an active process that relies on unique secretory organelles (micronemes, rhoptries and dense granules) localized at the apical tip of these highly polarized eukaryotes. In order for the contents of these specialized organelles to reach their final destination, these proteins are sorted post-Golgi and it has been speculated that they pass through endosomal-like compartments (ELCs), where they undergo maturation. Here, we characterize a Toxoplasma gondii homologue of Syntaxin 6 (TgStx6), a well-established marker for the early endosomes and trans Golgi network (TGN) in diverse eukaryotes. Indeed, TgStx6 appears to have a role in the retrograde transport between ELCs, the TGN and the Golgi, because overexpression of TgStx6 results in the development of abnormally shaped parasites with expanded ELCs, a fragmented Golgi and a defect in inner membrane complex maturation. Interestingly, other organelles such as the micronemes, rhoptries and the apicoplast are not affected, establishing the TGN as a major sorting compartment where several transport pathways intersect. It therefore appears that Toxoplasma has retained a plant-like secretory pathway. PMID:23962112

  17. Reorganization of the Endosomal System in Salmonella-Infected Cells: The Ultrastructure of Salmonella-Induced Tubular Compartments

    Science.gov (United States)

    Krieger, Viktoria; Liebl, David; Zhang, Yuying; Rajashekar, Roopa; Chlanda, Petr; Giesker, Katrin; Chikkaballi, Deepak; Hensel, Michael

    2014-01-01

    During the intracellular life of Salmonella enterica, a unique membrane-bound compartment termed Salmonella-containing vacuole, or SCV, is formed. By means of translocated effector proteins, intracellular Salmonella also induce the formation of extensive, highly dynamic membrane tubules termed Salmonella-induced filaments or SIF. Here we report the first detailed ultrastructural analyses of the SCV and SIF by electron microscopy (EM), EM tomography and live cell correlative light and electron microscopy (CLEM). We found that a subset of SIF is composed of double membranes that enclose portions of host cell cytosol and cytoskeletal filaments within its inner lumen. Despite some morphological similarities, we found that the formation of SIF double membranes is independent from autophagy and requires the function of the effector proteins SseF and SseG. The lumen of SIF network is accessible to various types of endocytosed material and our CLEM analysis of double membrane SIF demonstrated that fluid phase markers accumulate only between the inner and outer membrane of these structures, a space continual with endosomal lumen. Our work reveals how manipulation of the endosomal membrane system by an intracellular pathogen results in a unique tubular membrane compartmentalization of the host cell, generating a shielded niche permissive for intracellular proliferation of Salmonella. PMID:25254663

  18. Nuclear transport of cancer extracellular vesicle-derived biomaterials through nuclear envelope invagination-associated late endosomes

    Science.gov (United States)

    Rappa, Germana; Santos, Mark F.; Green, Toni M.; Karbanová, Jana; Hassler, Justin; Bai, Yongsheng; Barsky, Sanford H.; Corbeil, Denis; Lorico, Aurelio

    2017-01-01

    Extracellular membrane vesicles (EVs) function as vehicles of intercellular communication, but how the biomaterials they carry reach the target site in recipient cells is an open question. We report that subdomains of Rab7+ late endosomes and nuclear envelope invaginations come together to create a sub-nuclear compartment, where biomaterials associated with CD9+ EVs are delivered. EV-derived biomaterials were also found in the nuclei of host cells. The inhibition of nuclear import and export pathways abrogated the nuclear localization of EV-derived biomaterials or led to their accumulation therein, respectively, suggesting that their translocation is dependent on nuclear pores. Nuclear envelope invagination-associated late endosomes were observed in ex vivo biopsies in both breast carcinoma and associated stromal cells. The transcriptome of stromal cells exposed to cancer cell-derived CD9+ EVs revealed that the regulation of eleven genes, notably those involved in inflammation, relies on the nuclear translocation of EV-derived biomaterials. Our findings uncover a new cellular pathway used by EVs to reach nuclear compartment. PMID:28129640

  19. Multi-layered nanoparticles for penetrating the endosome and nuclear membrane via a step-wise membrane fusion process.

    Science.gov (United States)

    Akita, Hidetaka; Kudo, Asako; Minoura, Arisa; Yamaguti, Masaya; Khalil, Ikramy A; Moriguchi, Rumiko; Masuda, Tomoya; Danev, Radostin; Nagayama, Kuniaki; Kogure, Kentaro; Harashima, Hideyoshi

    2009-05-01

    Efficient targeting of DNA to the nucleus is a prerequisite for effective gene therapy. The gene-delivery vehicle must penetrate through the plasma membrane, and the DNA-impermeable double-membraned nuclear envelope, and deposit its DNA cargo in a form ready for transcription. Here we introduce a concept for overcoming intracellular membrane barriers that involves step-wise membrane fusion. To achieve this, a nanotechnology was developed that creates a multi-layered nanoparticle, which we refer to as a Tetra-lamellar Multi-functional Envelope-type Nano Device (T-MEND). The critical structural elements of the T-MEND are a DNA-polycation condensed core coated with two nuclear membrane-fusogenic inner envelopes and two endosome-fusogenic outer envelopes, which are shed in stepwise fashion. A double-lamellar membrane structure is required for nuclear delivery via the stepwise fusion of double layered nuclear membrane structure. Intracellular membrane fusions to endosomes and nuclear membranes were verified by spectral imaging of fluorescence resonance energy transfer (FRET) between donor and acceptor fluorophores that had been dually labeled on the liposome surface. Coating the core with the minimum number of nucleus-fusogenic lipid envelopes (i.e., 2) is essential to facilitate transcription. As a result, the T-MEND achieves dramatic levels of transgene expression in non-dividing cells.

  20. Intra-endosomal trafficking mediated by lysobisphosphatidic acid contributes to intracellular release of phosphorothioate-modified antisense oligonucleotides

    Science.gov (United States)

    Sun, Hong; Tanowitz, Michael; Liang, Xue-hai; Crooke, Stanley T.

    2017-01-01

    Abstract Antisense oligonucleotides (ASOs) with phosphorothioate (PS) linkages are broadly used as research tools and therapeutic agents. Chemically modified PS-ASOs can mediate efficient target reduction by site-specific cleavage of RNA through RNase H1. PS-ASOs are known to be internalized via a number of endocytotic pathways and are released from membrane-enclosed endocytotic organelles, mainly late endosomes (LEs). This study was focused on the details of PS-ASO trafficking through endocytic pathways. It was found that lysobisphosphatidic acid (LBPA) is required for release of PS-ASOs from LEs. PS-ASOs exited early endosomes (EEs) rapidly after internalization and became co-localized with LBPA by 2 hours in LEs. Inside LEs, PS-ASOs and LBPA were co-localized in punctate, dot-like structures, likely intraluminal vesicles (ILVs). Deactivation of LBPA using anti-LBPA antibody significantly decreased PS-ASO activities without affecting total PS-ASO uptake. Reduction of Alix also substantially decreased PS-ASO activities without affecting total PS-ASO uptake. Furthermore, Alix reduction decreased LBPA levels and limited co-localization of LBPA with PS-ASOs at ILVs inside LEs. Thus, the fusion properties of ILVs, which are supported by LBPA, contribute to PS-ASO intracellular release from LEs. PMID:28379543

  1. Abnormal Localization of Leucine-Rich Repeat Kinase 2 to the Endosomal-Lysosomal Compartment in Lewy Body Disease

    Science.gov (United States)

    Higashi, Shinji; Moore, Darren J; Yamamoto, Ryoko; Minegishi, Michiko; Sato, Kiyoshi; Togo, Takashi; Katsuse, Omi; Uchikado, Hirotake; Furukawa, Yoshiko; Hino, Hiroaki; Kosaka, Kenji; Emson, Piers C.; Wada, Keiji; Dawson, Valina L; Dawson, Ted M.; Arai, Heii; Iseki, Eizo

    2009-01-01

    Missense mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common causes of both familial and sporadic forms of Parkinson disease (PD) and are also associated with a diverse pathological alterations. The mechanisms whereby LRRK2 mutations cause these pathological phenotypes are unknown. We employed immunohistochemistry with 3 distinct anti-LRRK2 antibodies to characterize the expression of LRRK2 in the brains of 21 subjects with various neurodegenerative disorders and 7 controls. LRRK2 immunoreactivity was localized in a subset of brainstem-type Lewy bodies (LBs) but not in cortical-type LBs, tau-positive inclusions or TAR-DNA binding protein-43-positive inclusions. LRRK2 immunoreactivity frequently appeared as enlarged granules or vacuoles within neurons of affected brain regions, including the substantia nigra, amygdala and entorhinal cortex in patients with PD or dementia with Lewy bodies (DLB). The volumes of LRRK2-positive granular structures in neurons of the entorhinal cortex were significantly increased in DLB brains compared to aged-matched control brains (p<0.05). Double immunolabeling demonstrated that these LRRK2-positive granular structures frequently colocalized with the late-endosomal marker Rab7B and occasionally with the lysosomal marker, LAMP2. These results suggest that LRRK2 normally localizes to the endosomal-lysosomal compartment within morphologically altered neurons in neurodegenerative diseases, particularly in the brains of patients with LB diseases. PMID:19680143

  2. Rhinovirus stimulated IFN-α production: how important are plasmacytoid DCs, monocytes and endosomal pH?

    Science.gov (United States)

    Xi, Yang; Finlayson, Arvid; White, Oliva J; Carroll, Melanie L; Upham, John W

    2015-10-01

    Human rhinovirus (HRV) infection is a major cause of asthma exacerbations, which appears to be linked to a defective innate immune response to infection. Although the type I interferons (IFN-α and IFN-β) have a critical role in protecting against most viral infections, the cells responsible for IFN production in response to HRV and the relative importance of pattern recognition receptors located in endosomes has not been fully elucidated. In the current study we demonstrate that, using intracellular flow cytometry, >90% of the IFN-α-producing cells in human blood mononuclear cells following HRV16 exposure are plasmacytoid dendritic cells, whereas monocytes and myeloid dendritic cells contribute only 10% and effectively suppressed HRV16-stimulated IFN-α and IP-10 production, whereas neither bafilomycin or chloroquine inhibited HRV16-stimulated interleukin-6 release. Attempts to block IFN-α production with commercially available TLR-specific oligonucleotides were unsuccessful due to major 'off-target' effects. These findings suggest that among circulating haemopoietic cells, plasmacytoid dendritic cells and TLRs located within endosomes are critical for inducing efficient IFN-I production in response to HRVs.

  3. ESCRT-II/Vps25 constrains digit number by endosome-mediated selective modulation of FGF-SHH signaling

    Science.gov (United States)

    Handschuh, Karen; Feenstra, Jennifer; Koss, Matthew; Ferretti, Elisabetta; Risolino, Maurizio; Zewdu, Rediet; Sahai, Michelle A.; Bénazet, Jean-Denis; Peng, Xiao P.; Depew, Michael J.; Quintana, Laura; Sharpe, James; Wang, Baolin; Alcorn, Heather; Rivi, Roberta; Butcher, Stephen; Manak, J Robert; Vaccari, Thomas; Weinstein, Harel; Anderson, Kathryn V.; Lacy, Elizabeth; Selleri, Licia

    2014-01-01

    Summary Sorting and degradation of receptors and associated signaling molecules maintain homeostasis of conserved signaling pathways during cell specification and tissue development. Yet, whether machineries that sort signaling proteins act preferentially on different receptors and ligands in different contexts remains mysterious. Here we show that Vacuolar protein sorting 25, Vps25, a component of ESCRT-II (Endosomal Sorting Complex Required for Transport II), directs preferential endosome-mediated modulation of FGF signaling in limbs. By ENU-induced mutagenesis we isolated a polydactylous mouse line carrying a hypomorphic mutation of Vps25 (Vps25ENU). Unlike Vps25-null embryos we generated, Vps25ENU/ENU mutants survive until late gestation. Their limbs display FGF signaling enhancement and consequent hyper-activation of the FGF-SHH feedback loop causing polydactyly, whereas WNT and BMP signaling remain unperturbed. Notably, Vps25ENU/ENU Mouse Embryonic Fibroblasts exhibit aberrant FGFR trafficking and degradation; however SHH signaling is unperturbed. These studies establish that the ESCRT-II machinery selectively limits FGF signaling in vertebrate skeletal patterning. PMID:25373905

  4. The role of endosomal escape and mitogen-activated protein kinases in adenoviral activation of the innate immune response.

    Directory of Open Access Journals (Sweden)

    Jeffrey S Smith

    Full Text Available Adenoviral vectors (AdV activate multiple signaling pathways associated with innate immune responses, including mitogen-activated protein kinases (MAPKs. In this study, we investigated how systemically-injected AdVs activate two MAPK pathways (p38 and ERK and the contribution of these kinases to AdV-induced cytokine and chemokine responses in mice. Mice were injected intravenously either with a helper-dependent Ad2 vector that does not express viral genes or transgenes, or with the Ad2 mutant ts1, which is defective in endosomal escape. We found that AdV induced rapid phosphorylation of p38 and ERK as well as a significant cytokine response, but ts1 failed to activate p38 or ERK and induced only a limited cytokine response. These results demonstrate that endosomal escape of virions is a critical step in the induction of these innate pathways and responses. We then examined the roles of p38 and ERK pathways in the innate cytokine response by administering specific kinase inhibitors to mice prior to AdV. The cytokine and chemokine response to AdV was only modestly suppressed by a p38 inhibitor, while an ERK inhibitor has mixed effects, lowering some cytokines and elevating others. Thus, even though p38 and ERK are rapidly activated after i.v. injection of AdV, cytokine and chemokine responses are mostly independent of these kinases.

  5. An amperometric cholesterol biosensor based on epoxy resin membrane bound cholesterol oxidase.

    Science.gov (United States)

    Pundir, C S; Narang, Jagriti; Chauhan, Nidhi; Sharma, Preety; Sharma, Renu

    2012-10-01

    The use of epoxy resin membrane as a support for immobilization of enzyme has resulted into improved sensitivity and stability of biosensors for uric acid, ascorbic acid and polyphenols. The present work was aimed to prepare an improved amperometric biosensor for determination of serum cholesterol required in the diagnostics and management of certain pathological conditions. Epoxy resin membrane with immobilized cholesterol oxidase was mounted on the cleaned platinum (Pt) electrode with a parafilm to construct a working electrode. This working electrode along with Ag/AgCl as reference and Ag wire as an auxiliary electrode were connected through a three terminal electrometer to construct a cholesterol biosensor. The sensor showed optimum response within 25 sec at pH 7.0 and 45°C. The linear working range of biosensor was 1.0 to 8.0 mM cholesterol. K m and I max for cholesterol were 5.0 mM and 9.09 μA, respectively. The biosensor measured serum cholesterol. The minimum detection limit of the sensor was 1.0 mM. The mean analytical recoveries of added cholesterol in serum (2.84 and 4.13 mM) were 91.4 ± 2.8 and 92.3 ± 3.1 per cent (n=6), respectively. Within and between assay coefficient of variation (CV) were epoxy resin membrane as a support for immobilization of cholesterol oxidase has resulted into an improved amperometric cholesterol biosensor. The present biosensor had an advantage over the existing biosensors as it worked at comparatively lower potential.

  6. Inhibiting Cholesterol Absorption During Lactation Programs Future Intestinal Absorption of Cholesterol in Adult Mice.

    Science.gov (United States)

    Dimova, Lidiya G; de Boer, Jan Freark; Plantinga, Josee; Plösch, Torsten; Hoekstra, Menno; Verkade, Henkjan J; Tietge, Uwe J F

    2017-08-01

    In nematodes, the intestine senses and integrates early life dietary cues that lead to lifelong epigenetic adaptations to a perceived nutritional environment-it is not clear whether this process occurs in mammals. We aimed to establish a mouse model of reduced dietary cholesterol availability from maternal milk and investigate the consequences of decreased milk cholesterol availability, early in life, on the metabolism of cholesterol in adult mice. We blocked intestinal absorption of cholesterol in milk fed to newborn mice by supplementing the food of dams (for 3 weeks between birth and weaning) with ezetimibe, which is secreted into milk. Ezetimibe interacts with the intestinal cholesterol absorption transporter NPC1l1 to block cholesterol uptake into enterocytes. Characterization of these offspring at 24 weeks of age showed a 27% decrease in cholesterol absorption (P < .001) and reduced levels of Npc1l1 messenger RNA and protein, but not other cholesterol transporters, in the proximal small intestine. We observed increased histone H3K9me3 methylation at positions -423 to -607 of the proximal Npc1l1 promoter in small intestine tissues from 24-week-old offspring fed ezetimibe during lactation, compared with controls. These findings show that the early postnatal mammalian intestine functions as an environmental sensor of nutritional conditions, responding to conditions such as low cholesterol levels by epigenetic modifications of genes. Further studies are needed to determine how decreased sterol absorption for a defined period might activate epigenetic regulators; the findings of our study might have implications for human infant nutrition and understanding and preventing cardiometabolic disease. Copyright © 2017 AGA Institute. Published by Elsevier Inc. All rights reserved.

  7. Elevated Remnant Cholesterol Causes Both Low-Grade Inflammation and Ischemic Heart Disease, Whereas Elevated Low-Density Lipoprotein Cholesterol Causes Ischemic Heart Disease Without Inflammation

    DEFF Research Database (Denmark)

    Varbo, Anette; Benn, Marianne; Tybjærg-Hansen, Anne

    2013-01-01

    Elevated nonfasting remnant cholesterol and low-density lipoprotein (LDL) cholesterol are causally associated with ischemic heart disease (IHD), but whether elevated nonfasting remnant cholesterol and LDL cholesterol both cause low-grade inflammation is currently unknown.......Elevated nonfasting remnant cholesterol and low-density lipoprotein (LDL) cholesterol are causally associated with ischemic heart disease (IHD), but whether elevated nonfasting remnant cholesterol and LDL cholesterol both cause low-grade inflammation is currently unknown....

  8. The Na+/H+ exchanger NHE6 modulates endosomal pH to control processing of amyloid precursor protein in a cell culture model of Alzheimer disease.

    Science.gov (United States)

    Prasad, Hari; Rao, Rajini

    2015-02-27

    Early intervention may be key to safe and effective therapies in patients with Alzheimer disease. Endosomal dysfunction is an early step in neurodegeneration. Endosomes are a major site of production of Aβ peptide from the processing of amyloid precursor protein (APP) by clipping enzymes (β- and γ-secretases). The β-secretase enzyme BACE1 requires acidic lumen pH for optimum function, and acid pH promotes Aβ aggregation. The Na(+)/H(+) exchanger NHE6 provides a leak pathway for protons, limiting luminal acidification by proton pumps. Like APP, NHE6 expression was induced upon differentiation of SH-SY5Y neuroblastoma cells and localized to an endosomal compartment. Therefore, we investigated whether NHE6 expression altered APP localization and processing in a stably transfected cell culture model of human APP expression. We show that co-expression with NHE6 or treatment with the Na(+)/H(+) ionophore monensin shifted APP away from the trans-Golgi network into early and recycling endosomes in HEK293 cells. NHE6 alkalinized the endosomal lumen, similar to monensin, and significantly attenuated APP processing and Aβ secretion. In contrast, Aβ production was elevated upon NHE6 knockdown. We show that NHE6 transcript and protein levels are lowered in Alzheimer brains relative to control. These findings, taken together with emerging genetic evidence linking endosomal Na(+)/H(+) exchangers with Alzheimer disease, suggest that proton leak pathways may regulate Aβ generation and contribute to disease etiology. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Old world arenaviruses enter the host cell via the multivesicular body and depend on the endosomal sorting complex required for transport.

    Directory of Open Access Journals (Sweden)

    Giulia Pasqual

    2011-09-01

    Full Text Available The highly pathogenic Old World arenavirus Lassa virus (LASV and the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV use α-dystroglycan as a cellular receptor and enter the host cell by an unusual endocytotic pathway independent of clathrin, caveolin, dynamin, and actin. Upon internalization, the viruses are delivered to acidified endosomes in a Rab5-independent manner bypassing classical routes of incoming vesicular trafficking. Here we sought to identify cellular factors involved in the unusual and largely unknown entry pathway of LASV and LCMV. Cell entry of LASV and LCMV required microtubular transport to late endosomes, consistent with the low fusion pH of the viral envelope glycoproteins. Productive infection with recombinant LCMV expressing LASV envelope glycoprotein (rLCMV-LASVGP and LCMV depended on phosphatidyl inositol 3-kinase (PI3K as well as lysobisphosphatidic acid (LBPA, an unusual phospholipid that is involved in the formation of intraluminal vesicles (ILV of the multivesicular body (MVB of the late endosome. We provide evidence for a role of the endosomal sorting complex required for transport (ESCRT in LASV and LCMV cell entry, in particular the ESCRT components Hrs, Tsg101, Vps22, and Vps24, as well as the ESCRT-associated ATPase Vps4 involved in fission of ILV. Productive infection with rLCMV-LASVGP and LCMV also critically depended on the ESCRT-associated protein Alix, which is implicated in membrane dynamics of the MVB/late endosomes. Our study identifies crucial cellular factors implicated in Old World arenavirus cell entry and indicates that LASV and LCMV invade the host cell passing via the MVB/late endosome. Our data further suggest that the virus-receptor complexes undergo sorting into ILV of the MVB mediated by the ESCRT, possibly using a pathway that may be linked to the cellular trafficking and degradation of the cellular receptor.

  10. High Cholesterol/Low Cholesterol: Effects in Biological Membranes: A Review.

    Science.gov (United States)

    Subczynski, Witold K; Pasenkiewicz-Gierula, Marta; Widomska, Justyna; Mainali, Laxman; Raguz, Marija

    2017-12-01

    Lipid composition determines membrane properties, and cholesterol plays a major role in this determination as it regulates membrane fluidity and permeability, as well as induces the formation of coexisting phases and domains in the membrane. Biological membranes display a very diverse lipid composition, the lateral organization of which plays a crucial role in regulating a variety of membrane functions. We hypothesize that, during biological evolution, membranes with a particular cholesterol content were selected to perform certain functions in the cells of eukaryotic organisms. In this review, we discuss the major membrane properties induced by cholesterol, and their relationship to certain membrane functions.

  11. Histone deacetylase inhibition decreases cholesterol levels in neuronal cells by modulating key genes in cholesterol synthesis, uptake and efflux.

    Directory of Open Access Journals (Sweden)

    Maria João Nunes

    Full Text Available Cholesterol is an essential component of the central nervous system and increasing evidence suggests an association between brain cholesterol metabolism dysfunction and the onset of neurodegenerative disorders. Interestingly, histone deacetylase inhibitors (HDACi such as trichostatin A (TSA are emerging as promising therapeutic approaches in neurodegenerative diseases, but their effect on brain cholesterol metabolism is poorly understood. We have previously demonstrated that HDACi up-regulate CYP46A1 gene transcription, a key enzyme in neuronal cholesterol homeostasis. In this study, TSA was shown to modulate the transcription of other genes involved in cholesterol metabolism in human neuroblastoma cells, namely by up-regulating genes that control cholesterol efflux and down-regulating genes involved in cholesterol synthesis and uptake, thus leading to an overall decrease in total cholesterol content. Furthermore, co-treatment with the amphipathic drug U18666A that can mimic the intracellular cholesterol accumulation observed in cells of Niemman-Pick type C patients, revealed that TSA can ameliorate the phenotype induced by pathological cholesterol accumulation, by restoring the expression of key genes involved in cholesterol synthesis, uptake and efflux and promoting lysosomal cholesterol redistribution. These results clarify the role of TSA in the modulation of neuronal cholesterol metabolism at the transcriptional level, and emphasize the idea of HDAC inhibition as a promising therapeutic tool in neurodegenerative disorders with impaired cholesterol metabolism.

  12. Garbanzo diet lowers cholesterol in hamsters

    Science.gov (United States)

    Cholesterol-lowering potential of diets with 22% protein from Chickpea (Cicer arietinum, European variety of Garbanzo, Kabuli Chana), Bengal gram (Cicer arietinum, Asian variety of Garbanzo, Desi Chana, smaller in size, yellow to black color), lentils, soy protein isolate, hydrolyzed salmon protein...

  13. Cholesterol oxidation products and their biological importance

    Czech Academy of Sciences Publication Activity Database

    Kulig, W.; Cwiklik, Lukasz; Jurkiewicz, Piotr; Rog, T.; Vattulainen, I.

    2016-01-01

    Roč. 199, SI (2016), s. 144-160 ISSN 0009-3084 R&D Projects: GA ČR(CZ) GBP208/12/G016; GA ČR GA15-14292S Institutional support: RVO:61388955 Keywords : cholesterol * oxidation * oxysterols Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 3.361, year: 2016

  14. Cholesterol levels in fragile X syndrome.

    Science.gov (United States)

    Berry-Kravis, Elizabeth; Levin, Rebecca; Shah, Haroon; Mathur, Shaguna; Darnell, Jennifer C; Ouyang, Bichun

    2015-02-01

    Fragile X syndrome (FXS) is associated with intellectual disability and behavioral dysfunction, including anxiety, ADHD symptoms, and autistic features. Although individuals with FXS are largely considered healthy and lifespan is not thought to be reduced, very little is known about the long-term medical health of adults with FXS and no systematically collected information is available on standard laboratory measures from metabolic screens. During the course of follow up of a large cohort of patients with FXS we noted that many patients had low cholesterol and high density lipoprotein (HDL) values and thus initiated a systematic chart review of all cholesterol values present in charts from a clinic cohort of over 500 patients with FXS. Total cholesterol (TC), low density lipoprotein (LDL) and HDL were all significantly reduced in males from the FXS cohort relative to age-adjusted population normative data. This finding has relevance for health monitoring in individuals with FXS, for treatments with cholesterol-lowering agents that have been proposed to target the underlying CNS disorder in FXS based on work in animal models, and for potential biomarker development in FXS. © 2014 Wiley Periodicals, Inc.

  15. Estimations of cholesterol, triglycerides and fractionation of ...

    African Journals Online (AJOL)

    cholesterol (VLDL-C) in 53 female subjects in Warri, Delta State, Nigeria using the Reflotron® (an auto analyser), supported with the use of questionnaire to get information on age and sex. Age range of the subjects was 18–80 years. The TG levels in ...

  16. globulins and cholesterol levels in Ibadan, Nigeria.

    African Journals Online (AJOL)

    rum total protein, globulin and cholesterol levels were sig- nificantly increased in oral contraceptive and their control counterparts. The albumin/globulin ratio in subjects on oral contraceptives users is significantly decreased compared with controls. In view of the findings of this study, it is suggested that the biochemical ...

  17. Resveratrol protects rabbits against cholesterol diet- induced ...

    African Journals Online (AJOL)

    The excessive consumption of high cholesterol diet has been associated with an increased incidence of lipidaemia. Lipidaemia is enhanced by formation of oxidative stress, lipid peroxidation and hyperglycaemia. The aim of these experiments was to investigate the protective effect of resveratrol co-administered with ...

  18. Expression and comparison of recombinant cholesterol oxidases ...

    African Journals Online (AJOL)

    The structure and bio-activity of an endogenous cholesterol oxidase from Brevibacterium sp. was compared to the same enzyme exogenously expressed in Escherichia coli BL21 (DE3) with and without N- or C-terminal his-tags. The different proteins were purified with affinity and subtractive protocols. The specific activity of ...

  19. Blood cholesterol : a public health perspective

    NARCIS (Netherlands)

    Verschuren, W.M.M.

    1995-01-01

    Changes in total cholesterol levels (TC) were studied using data from three epidemiological studies: about 30,000 men and women aged 37-43 were examined between 1974 and 1980 (CB Project), about 80,000 men aged 33-37 between 1981 and 1986 (RIFOH Project) and 42,000 men and women aged 20-59 from 1987

  20. Top Five Lifestyle Changes to Reduce Cholesterol

    Science.gov (United States)

    ... protein is one of two proteins in dairy products — the other is casein. Whey protein may account for many of the health benefits attributed to dairy. Studies have shown that whey protein given as a supplement lowers both LDL and total cholesterol. You can ...

  1. Proximate composition and cholesterol concentrations of ...

    African Journals Online (AJOL)

    Proximate composition and cholesterol concentrations of Rhynchophorus phoenicis and Oryctes monoceros larvae subjected to different heat treatments. ... 514.63 mg/100g dry weight basis (DWB) for raw and fried samples, respectively, but decreased to 295.20 mg/100 g DWB in the smoke-dried samples. Similarly, the ...

  2. Dietary cholesterol increases paraoxonase 1 enzyme activity

    National Research Council Canada - National Science Library

    Kim, Daniel S; Burt, Amber A; Ranchalis, Jane E; Richter, Rebecca J; Marshall, Julieann K; Nakayama, Karen S; Jarvik, Ella R; Eintracht, Jason F; Rosenthal, Elisabeth A; Furlong, Clement E; Jarvik, Gail P

    2012-01-01

    .... Five dietary components, cholesterol (P < 2.0 × 10(-16)), alcohol (P = 8.51 × 10(-8)), vitamin C (P = 7.97 × 10(-5)), iron (P = 0.0026), and folic acid (0.037) were independently predictive of PON1 activity...

  3. Compared with Acyl-CoA:cholesterol O-acyltransferase (ACAT) 1 and lecithin:cholesterol acyltransferase, ACAT2 displays the greatest capacity to differentiate cholesterol from sitosterol.

    Science.gov (United States)

    Temel, Ryan E; Gebre, Abraham K; Parks, John S; Rudel, Lawrence L

    2003-11-28

    The capacity of acyl-CoA:cholesterol O-acyltransferase (ACAT) 2 to differentiate cholesterol from the plant sterol, sitosterol, was compared with that of the sterol esterifying enzymes, ACAT1 and lecithin:cholesterol acyltransferase (LCAT). Cholesterol-loaded microsomes from transfected cells containing either ACAT1 or ACAT2 exhibited significantly more ACAT activity than their sitosterol-loaded counterparts. In sitosterol-loaded microsomes, both ACAT1 and ACAT2 were able to esterify sitosterol albeit with lower efficiencies than cholesterol. The mass ratios of cholesterol ester to sitosterol ester formed by ACAT1 and ACAT2 were 1.6 and 7.2, respectively. Compared with ACAT1, ACAT2 selectively esterified cholesterol even when sitosterol was loaded into the microsomes. To further characterize the difference in sterol specificity, ACAT1 and ACAT2 were compared in intact cells loaded with either cholesterol or sitosterol. Despite a lower level of ACAT activity, the ACAT1-expressing cells esterified 4-fold more sitosterol than the ACAT2 cells. The data showed that compared with ACAT1, ACAT2 displayed significantly greater selectively for cholesterol compared with sitosterol. The plasma cholesterol esterification enzyme lecithin:cholesterol acyltransferase was also compared. With recombinant high density lipoprotein particles, the esterification rate of cholesterol by LCAT was only 15% greater than for sitosterol. Thus, LCAT was able to efficiently esterify both cholesterol and sitosterol. In contrast, ACAT2 demonstrated a strong preference for cholesterol rather than sitosterol. This sterol selectivity by ACAT2 may reflect a role in the sorting of dietary sterols during their absorption by the intestine in vivo.

  4. Transbilayer movement and net flux of cholesterol and cholesterol sulfate between liposomal membranes.

    Science.gov (United States)

    Rodrigueza, W V; Wheeler, J J; Klimuk, S K; Kitson, C N; Hope, M J

    1995-05-09

    The kinetics of cholesterol and cholesterol sulfate (CS) movement between vesicles have been investigated. CS is widely distributed in cell membranes, plasma and skin and is similar in structure to cholesterol, but possesses an ionizable sulfate moiety at the 3 beta-position which imparts a negative charge at physiological pHs. Donor vesicles of various sizes ranging from 40 to 250 nm, composed of egg phosphatidylcholine (EPC)/sterol/N-palmitoyldihydrolactosylcerebroside (75:10:15 mole ratio) containing trace amounts of [3H]sterol, were used to monitor sterol transfer into a 10-fold excess of large unilamellar vesicles (LUV) composed of EPC with a diameter of 100 nm. The two populations of vesicles were separated by centrifugation following the addition of a lectin which caused the aggregation of donor vesicles. Both cholesterol and CS exhibited biphasic kinetics of exchange. The rate constants for efflux and transbilayer diffusion for both sterol molecules were determined after fitting kinetic data, using numerical integration, to a three-compartment model, which includes the inner and outer monolayers of donor vesicles and the acceptor bilayer. The rate of intermembrane exchange for CS was approximately 10-fold faster than for cholesterol in all liposomes tested. Using the kinetic model, a rate of transbilayer movement for cholesterol and CS was estimated. In both cases, it was found to be slower than the rate of efflux from the surface of vesicles. For vesicles containing CS, the surface charge was monitored to demonstrate that the slowly exchanging pool was located in the inner monolayer, and the rapidly exchanging pool in the outer half of the bilayer. For cholesterol, it was not possible to distinguish between this model and one where lateral domains of cholesterol within the plane of the bilayer may influence the kinetics of exchange.

  5. Lactobacillus gasseri [corrected] CHO-220 and inulin reduced plasma total cholesterol and low-density lipoprotein cholesterol via alteration of lipid transporters.

    Science.gov (United States)

    Ooi, L-G; Ahmad, R; Yuen, K-H; Liong, M-T

    2010-11-01

    This randomized, double-blind, placebo-controlled, and parallel-designed study was conducted to investigate the effect of a synbiotic product containing Lactobacillus gasseri [corrected] CHO-220 and inulin on lipid profiles of hypercholesterolemic men and women. Thirty-two hypercholesterolemic men and women with initial mean plasma cholesterol levels of 5.7±0.32 mmol/L were recruited for the 12-wk study. The subjects were randomly allocated to 2 groups; namely the treatment group (synbiotic product) and the control group (placebo), and each received 4 capsules of synbiotic or placebo daily. Our results showed that the mean body weight, energy, and nutrient intake of the subjects did not differ between the 2 groups over the study period. The supplementation of synbiotic reduced plasma total cholesterol and low-density lipoprotein (LDL)-cholesterol by 7.84 and 9.27%, respectively, compared with the control over 12 wk. Lipoproteins were subsequently subfractionated and characterized. The synbiotic supplementation resulted in a lower concentration of triglycerides in the very low, intermediate, low, and high-density lipoprotein particles compared with the control over 12 wk. The concentration of triglycerides in lipoproteins is positively correlated with an increased risk of atherosclerosis. Our results showed that the synbiotic might exhibit an atheropreventive characteristic. Cholesteryl ester (CE) in the high-density lipoprotein particles of the synbiotic group was also higher compared with the control, indicating greater transport of cholesterol in the form of CE to the liver for hydrolysis. This may have led to the reduced plasma total cholesterol level of the synbiotic group. The supplementation of synbiotic also reduced the concentration of CE in the LDL particles compared with the control, leading to the formation of smaller and denser particles that are more easily removed from blood. This supported the reduced LDL-cholesterol level of the synbiotic group

  6. Trans Fat Now Listed With Saturated Fat and Cholesterol

    Science.gov (United States)

    ... Trans Fat Now Listed With Saturated Fat and Cholesterol Share Tweet Linkedin Pin it More sharing options ... I Do About Saturated Fat, Trans Fat, and Cholesterol? When comparing foods, look at the Nutrition Facts ...

  7. Short communication Fatty acid and cholesterol content, chemical ...

    African Journals Online (AJOL)

    user

    This study aimed to determine the fatty acid and chemical composition and cholesterol ... It is an essential component of cell membranes and lipoproteins, and a ... present in large quantities, cholesterol may cause cardiovascular diseases, ...

  8. Plasma Ubiquinone, Alpha-Tocopherol and Cholesterol in Man

    DEFF Research Database (Denmark)

    Karlsson, Jan; Diamant, Bertil; Edlund, Per Olof

    1992-01-01

    Farmakologi, Coenzyme Q10, free cholesterol, vitamin E, antioxidants, Alpha-Tocopherol, vitamin Q, plasma, LDL-particle......Farmakologi, Coenzyme Q10, free cholesterol, vitamin E, antioxidants, Alpha-Tocopherol, vitamin Q, plasma, LDL-particle...

  9. Are You Taking the Right Treatment for Your High Cholesterol?

    Science.gov (United States)

    ... You Taking the Right Treatment For Your High Cholesterol? Our analysis and new guidelines could change your ... and 75 years old and have diabetes, high cholesterol, high blood pressure, or if you smoke: • If ...

  10. Remnant cholesterol as a cause of ischemic heart disease

    DEFF Research Database (Denmark)

    Varbo, Anette; Benn, Marianne; Nordestgaard, Børge G

    2014-01-01

    This review focuses on remnant cholesterol as a causal risk factor for ischemic heart disease (IHD), on its definition, measurement, atherogenicity, and levels in high risk patient groups; in addition, present and future pharmacological approaches to lowering remnant cholesterol levels...... are considered. Observational studies show association between elevated levels of remnant cholesterol and increased risk of cardiovascular disease, even when remnant cholesterol levels are defined, measured, or calculated in different ways. In-vitro and animal studies also support the contention that elevated...... levels of remnant cholesterol may cause atherosclerosis same way as elevated levels of low-density lipoprotein (LDL) cholesterol, by cholesterol accumulation in the arterial wall. Genetic studies of variants associated with elevated remnant cholesterol levels show that an increment of 1mmol/L (39mg...

  11. Nonfasting triglycerides, cholesterol, and ischemic stroke in the general population

    DEFF Research Database (Denmark)

    Varbo, Anette; Nordestgaard, Børge G; Tybjaerg-Hansen, Anne

    2011-01-01

    Current guidelines on stroke prevention have recommendations on desirable cholesterol levels, but not on nonfasting triglycerides. We compared stepwise increasing levels of nonfasting triglycerides and cholesterol for their association with risk of ischemic stroke in the general population....

  12. Implementation of cellulomonas cholesterol oxidase for total serum cholesterol determination by the endpoint method.

    Science.gov (United States)

    Srisawasdi, Pornpen; Chaichanajarernkul, Upsorn; Teerakranjana, Narumon; Kroll, Martin H

    2008-01-01

    Cellulomonas has been shown to be a good source of cholesterol oxidase in addition to Streptomyces for serum cholesterol determination by the endpoint method, inexpensive in cost, and showing excellent performance. For clinical use, we have assessed the reliability of Cellulomonas reagent for cholesterol determination. We constructed the user-defined endpoint methods on three automated analyzers. The analytical performances (linearity, precision, recovery, interference, stability, and comparison with the standardized method) of Cellulomonas cholesterol reagents were evaluated and compared to those of Streptomyces reagents. Linearity (18.1-23.3 mmol/L) and stability of reagents (6-11 weeks) depended on the analyzers being used. The average within-run and between-day % coefficients of variation (CVs) ranged from 1.44 to 2.45 and 1.98 to 2.99, respectively, and were within National Cholesterol Education Program analytical criteria (Cellulomonas enzyme is analytically reliable when used for serum cholesterol determination by the endpoint method. Its analytical performance is equivalent to Streptomyces enzymes and meets the analytical goals. It has an advantage over the other enzymes in that it does not ship in the frozen state. (c) 2008 Wiley-Liss, Inc.

  13. Novel Regulation of Ski Protein Stability and Endosomal Sorting by Actin Cytoskeleton Dynamics in Hepatocytes*

    Science.gov (United States)

    Vázquez-Victorio, Genaro; Caligaris, Cassandre; Del Valle-Espinosa, Eugenio; Sosa-Garrocho, Marcela; González-Arenas, Nelly R.; Reyes-Cruz, Guadalupe; Briones-Orta, Marco A.; Macías-Silva, Marina

    2015-01-01

    TGF-β-induced antimitotic signals are highly regulated during cell proliferation under normal and pathological conditions, such as liver regeneration and cancer. Up-regulation of the transcriptional cofactors Ski and SnoN during liver regeneration may favor hepatocyte proliferation by inhibiting TGF-β signals. In this study, we found a novel mechanism that regulates Ski protein stability through TGF-β and G protein-coupled receptor (GPCR) signaling. Ski protein is distributed between the nucleus and cytoplasm of normal hepatocytes, and the molecular mechanisms controlling Ski protein stability involve the participation of actin cytoskeleton dynamics. Cytoplasmic Ski is partially associated with actin and localized in cholesterol-rich vesicles. Ski protein stability is decreased by TGF-β/Smads, GPCR/Rho signals, and actin polymerization, whereas GPCR/cAMP signals and actin depolymerization promote Ski protein stability. In conclusion, TGF-β and GPCR signals differentially regulate Ski protein stability and sorting in hepatocytes, and this cross-talk may occur during liver regeneration. PMID:25561741

  14. Novel regulation of Ski protein stability and endosomal sorting by actin cytoskeleton dynamics in hepatocytes.

    Science.gov (United States)

    Vázquez-Victorio, Genaro; Caligaris, Cassandre; Del Valle-Espinosa, Eugenio; Sosa-Garrocho, Marcela; González-Arenas, Nelly R; Reyes-Cruz, Guadalupe; Briones-Orta, Marco A; Macías-Silva, Marina

    2015-02-13

    TGF-β-induced antimitotic signals are highly regulated during cell proliferation under normal and pathological conditions, such as liver regeneration and cancer. Up-regulation of the transcriptional cofactors Ski and SnoN during liver regeneration may favor hepatocyte proliferation by inhibiting TGF-β signals. In this study, we found a novel mechanism that regulates Ski protein stability through TGF-β and G protein-coupled receptor (GPCR) signaling. Ski protein is distributed between the nucleus and cytoplasm of normal hepatocytes, and the molecular mechanisms controlling Ski protein stability involve the participation of actin cytoskeleton dynamics. Cytoplasmic Ski is partially associated with actin and localized in cholesterol-rich vesicles. Ski protein stability is decreased by TGF-β/Smads, GPCR/Rho signals, and actin polymerization, whereas GPCR/cAMP signals and actin depolymerization promote Ski protein stability. In conclusion, TGF-β and GPCR signals differentially regulate Ski protein stability and sorting in hepatocytes, and this cross-talk may occur during liver regeneration. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. Retrograde Transport from Early Endosomes to the trans-Golgi Network Enables Membrane Wrapping and Egress of Vaccinia Virus Virions.

    Science.gov (United States)

    Sivan, Gilad; Weisberg, Andrea S; Americo, Jeffrey L; Moss, Bernard

    2016-10-01

    The anterograde pathway, from the endoplasmic reticulum through the trans-Golgi network to the cell surface, is utilized by trans-membrane and secretory proteins. The retrograde pathway, which directs traffic in the opposite direction, is used following endocytosis of exogenous molecules and recycling of membrane proteins. Microbes exploit both routes: viruses typically use the anterograde pathway for envelope formation prior to exiting the cell, whereas ricin and Shiga-like toxins and some nonenveloped viruses use the retrograde pathway for cell entry. Mining a human genome-wide RNA interference (RNAi) screen revealed a need for multiple retrograde pathway components for cell-to-cell spread of vaccinia virus. We confirmed and extended these results while discovering that retrograde trafficking was required for virus egress rather than entry. Retro-2, a specific retrograde trafficking inhibitor of protein toxins, potently prevented spread of vaccinia virus as well as monkeypox virus, a human pathogen. Electron and confocal microscopy studies revealed that Retro-2 prevented wrapping of virions with an additional double-membrane envelope that enables microtubular transport, exocytosis, and actin polymerization. The viral B5 and F13 protein components of this membrane, which are required for wrapping, normally colocalize in the trans-Golgi network. However, only B5 traffics through the secretory pathway, suggesting that F13 uses another route to the trans-Golgi network. The retrograde route was demonstrated by finding that F13 was largely confined to early endosomes and failed to colocalize with B5 in the presence of Retro-2. Thus, vaccinia virus makes novel use of the retrograde transport system for formation of the viral wrapping membrane. Efficient cell-to-cell spread of vaccinia virus and other orthopoxviruses depends on the wrapping of infectious particles with a double membrane that enables microtubular transport, exocytosis, and actin polymerization

  16. Diet-induced dyslipidemia impairs reverse cholesterol transport in hamsters.

    Science.gov (United States)

    Tréguier, Morgan; Briand, François; Boubacar, Adamou; André, Agnès; Magot, Thierry; Nguyen, Patrick; Krempf, Michel; Sulpice, Thierry; Ouguerram, Khadija

    2011-09-01

    Reverse cholesterol transport (RCT) is an anti-atherogenic process by which cholesterol is effluxed from peripheral tissues by high-density lipoprotein (HDL) and returned to the liver for excretion into the bile and faeces. Dyslipidemia is thought to impair RCT through higher triglyceride-rich lipoprotein (TRL), low HDL-cholesterol and higher activity of cholesteryl ester transfer protein (CETP), which transfers cholesteryl esters from HDL to TRL for further hepatic uptake. As CETP pathway would represent a major route in human RCT, we therefore investigated whether diet-induced dyslipidemia impairs RCT in hamster, a CETP-expressing species. Golden Syrian hamsters were fed a chow or chow+0·3% cholesterol diet over 4 weeks. Biochemical parameters and in vivo VLDL-triglycerides secretion (Triton WR-1339 injection) were then measured. In vitro macrophage cholesterol efflux was measured, and in vivo macrophage-to-faeces RCT was also assessed after an intraperitoneal injection of (3) H-cholesterol-labelled hamster primary macrophages. Cholesterol-enriched diet increased plasma total cholesterol (144%), triglycerides (101%), VLDL-triglycerides secretion (175%), CETP activity (44%) and reduced HDL-cholesterol/total cholesterol ratio by 20% (P diet significantly increased hepatic total cholesterol and triglycerides by 459 and 118% and increased aortic total cholesterol content by 304%. In vitro cholesterol efflux from macrophages to plasma was significantly reduced by 25% with plasma from cholesterol-fed hamsters. In vivo RCT experiments showed a significant 75% reduction of macrophage-derived cholesterol faecal excretion in cholesterol-fed hamsters. Overall, these data demonstrate that diet-induced dyslipidemia severely impairs in vivo RCT in hamsters. © 2011 The Authors. European Journal of Clinical Investigation © 2011 Stichting European Society for Clinical Investigation Journal Foundation.

  17. Cholesterol and Copper Affect Learning and Memory in the Rabbit

    OpenAIRE

    Schreurs, Bernard G.

    2013-01-01

    A rabbit model of Alzheimer’s disease based on feeding a cholesterol diet for eight weeks shows sixteen hallmarks of the disease including beta amyloid accumulation and learning and memory changes. Although we have shown that feeding 2% cholesterol and adding copper to the drinking water can retard learning, other studies have shown that feeding dietary cholesterol before learning can improve acquisition and feeding cholesterol after learning can degrade long-term memory. We explore the devel...

  18. Cholesterol in the retina: the best is yet to come

    OpenAIRE

    Pikuleva, Irina A.; Curcio, Christine A.

    2014-01-01

    Historically understudied, cholesterol in the retina is receiving more attention now because of genetic studies showing that several cholesterol-related genes are risk factors for age-related macular degeneration (AMD) and because eye pathology studies showing high cholesterol content of drusen, aging Bruch's membrane, and newly found subretinal lesions. The challenge before us is determining how the cholesterol-AMD link is realized. Meeting this challenge will require an excellent understand...

  19. Planar Optical Nanoantennas Resolve Cholesterol-Dependent Nanoscale Heterogeneities in the Plasma Membrane of Living Cells

    Science.gov (United States)

    Regmi, Raju; Winkler, Pamina M.; Flauraud, Valentin; Borgman, Kyra J. E.; Manzo, Carlo; Brugger, Jürgen; Rigneault, Hervé; Wenger, Jérôme; García-Parajo, María F.

    2017-10-01

    Optical nanoantennas can efficiently confine light into nanoscopic hotspots, enabling single-molecule detection sensitivity at biological relevant conditions. This innovative approach to breach the diffraction limit offers a versatile platform to investigate the dynamics of individual biomolecules in living cell membranes and their partitioning into cholesterol-dependent lipid nanodomains. Here, we present optical nanoantenna arrays with accessible surface hotspots to study the characteristic diffusion dynamics of phosphoethanolamine (PE) and sphingomyelin (SM) in the plasma membrane of living cells at the nanoscale. Fluorescence burst analysis and fluorescence correlation spectroscopy performed on nanoantennas of different gap sizes show that, unlike PE, SM is transiently trapped in cholesterol-enriched nanodomains of 10 nm diameter with short characteristic times around 100 {\\mu}s. The removal of cholesterol led to the free diffusion of SM, consistent with the dispersion of nanodomains. Our results are consistent with the existence of highly transient and fluctuating nanoscale assemblies enriched by cholesterol and sphingolipids in living cell membranes, also known as lipid rafts. Quantitative data on sphingolipids partitioning into lipid rafts is crucial to understand the spatiotemporal heterogeneous organization of transient molecular complexes on the membrane of living cells at the nanoscale. The proposed technique is fully biocompatible and thus provides various opportunities for biophysics and live cell research to reveal details that remain hidden in confocal diffraction-limited measurements.

  20. Antagonism of miR-33 in mice promotes reverse cholesterol transport and regression of atherosclerosis.

    Science.gov (United States)

    Rayner, Katey J; Sheedy, Frederick J; Esau, Christine C; Hussain, Farah N; Temel, Ryan E; Parathath, Saj; van Gils, Janine M; Rayner, Alistair J; Chang, Aaron N; Suarez, Yajaira; Fernandez-Hernando, Carlos; Fisher, Edward A; Moore, Kathryn J

    2011-07-01

    Plasma HDL levels have a protective role in atherosclerosis, yet clinical therapies to raise HDL levels have remained elusive. Recent advances in the understanding of lipid metabolism have revealed that miR-33, an intronic microRNA located within the SREBF2 gene, suppresses expression of the cholesterol transporter ABC transporter A1 (ABCA1) and lowers HDL levels. Conversely, mechanisms that inhibit miR-33 increase ABCA1 and circulating HDL levels, suggesting that antagonism of miR-33 may be atheroprotective. As the regression of atherosclerosis is clinically desirable, we assessed the impact of miR-33 inhibition in mice deficient for the LDL receptor (Ldlr-/- mice), with established atherosclerotic plaques. Mice treated with anti-miR33 for 4 weeks showed an increase in circulating HDL levels and enhanced reverse cholesterol transport to the plasma, liver, and feces. Consistent with this, anti-miR33-treated mice showed reductions in plaque size and lipid content, increased markers of plaque stability, and decreased inflammatory gene expression. Notably, in addition to raising ABCA1 levels in the liver, anti-miR33 oligonucleotides directly targeted the plaque macrophages, in which they enhanced ABCA1 expression and cholesterol removal. These studies establish that raising HDL levels by anti-miR33 oligonucleotide treatment promotes reverse cholesterol transport and atherosclerosis regression and suggest that it may be a promising strategy to treat atherosclerotic vascular disease.

  1. 25-Hydroxycholesterol Increases the Availability of Cholesterol in Phospholipid Membranes

    Energy Technology Data Exchange (ETDEWEB)

    Olsen, Brett N.; Schlesinger, Paul H.; Ory, Daniel S.; Baker, Nathan A.

    2011-02-01

    Side-chain oxysterols are enzymatically generated oxidation products of cholesterol that serve a central role in mediating cholesterol homeostasis. Recent work has shown that side-chain oxysterols, such as 25-hydroxycholesterol (25-HC), alter membrane structure in very different ways from cholesterol, suggesting a possible mechanism for how these oxysterols regulate cholesterol homeostasis. Here we extend our previous work, using molecular dynamics simulations of 25-HC and cholesterol mixtures in 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) bilayers to examine interactions between 25-HC and cholesterol in the same bilayer. When added to cholesterol-containing membranes, 25-HC causes larger changes in membrane structure than when added to cholesterol-free membranes, demonstrating interactions between the two sterols. We also find that the presence of 25-HC changes the position, orientation, and solvent accessibility of cholesterol, shifting it into the water interface and therefore its availability to external acceptors. This is consistent with experimental results showing that oxysterols can trigger cholesterol trafficking from the plasma membrane to the endoplasmic reticulum. These interactions provide a potential mechanism for 25-HC-mediated regulation of cholesterol trafficking and homeostasis through direct modulation of cholesterol availability.

  2. Sex Differences in the Hepatic Cholesterol Sensing Mechanisms in Mice

    Directory of Open Access Journals (Sweden)

    Ingemar Björkhem

    2013-09-01

    Full Text Available Cholesterol is linked to many multifactorial disorders, including different forms of liver disease where development and severity depend on the sex. We performed a detailed analysis of cholesterol and bile acid synthesis pathways at the level of genes and metabolites combined with the expression studies of hepatic cholesterol uptake and transport in female and male mice fed with a high-fat diet with or without cholesterol. Lack of dietary cholesterol led to a stronger response of the sterol sensing mechanism in females, resulting in higher expression of cholesterogenic genes compared to males. With cholesterol in the diet, the genes were down-regulated in both sexes; however, males maintained a more efficient hepatic metabolic flux through the pathway. Females had higher content of hepatic cholesterol but this was likely not due to diminished excretion but rather due to increased synthesis and absorption. Dietary cholesterol and sex were not important for gallbladder bile acids composition. Neither sex up-regulated Cyp7a1 upon cholesterol loading and there was no compensatory up-regulation of Abcg5 or Abcg8 transporters. On the other hand, females had higher expression of the Ldlr and Cd36 genes. These findings explain sexual dimorphism of cholesterol metabolism in response to dietary cholesterol in a high-fat diet in mice, which contributes to understanding the sex-basis of cholesterol-associated liver diseases.

  3. diagnostic potential of serum vitamin e tocopherol and cholesterol ...

    African Journals Online (AJOL)

    hi-tech

    2003-08-01

    Aug 1, 2003 ... 8 August 2003. DIAGNOSTIC POTENTIAL OF SERUM VITAMIN E TOCOPHEROL AND CHOLESTEROL LEVELS IN CHILDREN WITH PROTEIN ENERGY .... taken as significant. Cholesterol assay of serum: Cholesterol was determined after enzymatic hydrolysis and oxidation of the EDTA-serum samples.

  4. Understanding Lipoproteins as Transporters of Cholesterol and Other Lipids

    Science.gov (United States)

    Biggerstaff, Kyle D.; Wooten, Joshua S.

    2004-01-01

    A clear picture of lipoprotein metabolism is essential for understanding the pathophysiology of atherosclerosis. Many students are taught that low-density lipoprotein-cholesterol is "bad" and high-density lipoprotein-cholesterol is "good." This misconception leads to students thinking that lipoproteins are types of cholesterol rather than…

  5. Immuno-histochemical localization of cholesterol binding proteins in ...

    African Journals Online (AJOL)

    SAM

    2014-07-09

    Jul 9, 2014 ... Positive control tissue sections were stained with Sudan Black-B for microscopic visualization of cholesterol .... 6 cm) by holes made. .... Sudan black-B specially stained the cholesterol droplets in form of black granules, which gave positive binding of cholesterol at appropriate binding sites (Figure 8).

  6. Tuberculosis treatment raises total cholesterol level and restores ...

    African Journals Online (AJOL)

    The aim of this study was to determine whether tuberculosis (TB) treatment normalizes the lipid profile strongly affected by pulmonary TB. Serum levels of total cholesterol (TC), high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C) and triglycerides (TG) were determined in 83 patients with ...

  7. Beyond Low-Density Lipoprotein Cholesterol Respective Contributions of Non-High-Density Lipoprotein Cholesterol Levels, Triglycerides, and the Total Cholesterol/High-Density Lipoprotein Cholesterol Ratio to Coronary Heart Disease Risk in Apparently Healthy Men and Women

    NARCIS (Netherlands)

    Arsenault, Benoit J.; Rana, Jamal S.; Stroes, Erik S. G.; Després, Jean-Pierre; Shah, Prediman K.; Kastelein, John J. P.; Wareham, Nicholas J.; Boekholdt, S. Matthijs; Khaw, Kay-Tee

    2009-01-01

    OBJECTIVES: This study was designed to test the hypothesis that at any low-density lipoprotein cholesterol (LDL-C) level, other lipid parameters such as non-high-density lipoprotein cholesterol (HDL-C) levels, triglyceride (TG) levels, and the total cholesterol (TC)/HDL-C are still associated with

  8. Alcohol consumption stimulates early stemps in reverse cholesterol transport

    NARCIS (Netherlands)

    Gaag, van der M.S.; Tol, van A.; Vermunt, S.H.F.; Scheek, L.M.; Schaafsma, G.; Hendriks, H.F.J.

    2001-01-01

    Alcohol consumption is associated with increased HDL cholesterol levels, which may indicate stimulated reverse cholesterol transport. The mechanism is, however, not known. The aim of this study was to evaluate the effects of alcohol consumption on the first two steps of the reverse cholesterol

  9. CHROMATOGRAPHIC METHODS IN THE ANALYSIS OF CHOLESTEROL AND RELATED LIPIDS

    NARCIS (Netherlands)

    HOVING, EB

    1995-01-01

    Methods using thin-layer chromatography, solid-phase extraction, gas chromatography, high-performance liquid chromatography and supercritical fluid chromatography are described for the analysis of single cholesterol, esterified and sulfated cholesterol, and for cholesterol in the context of other

  10. HDL Cholesterol and Risk of Type 2 Diabetes

    DEFF Research Database (Denmark)

    Haase, Christiane L; Tybjærg-Hansen, Anne; Nordestgaard, Børge G

    2015-01-01

    associated with type 2 diabetes is unknown. In a prospective study of the general population (n = 47,627), we tested whether HDL cholesterol-related genetic variants were associated with low HDL cholesterol levels and, in turn, with an increased risk of type 2 diabetes. HDL cholesterol-decreasing gene scores...

  11. Emerging roles of the intestine in control of cholesterol metabolism

    NARCIS (Netherlands)

    Kruit, Janine K.; Groen, Albert K.; van Berkel, Theo J.; Kuipers, Folkert

    2006-01-01

    The liver is considered the major "control center" for maintenance of whole body cholesterol homeostasis. This organ is the main site for de novo cholesterol synthesis, clears cholesterol-containing chylomicron remnants and low density lipoprotein particles from plasma and is the major contributor

  12. Cholesterol Profile of Adults Resident in Eastern Nigeria | Igweh ...

    African Journals Online (AJOL)

    Objective: The present study aims to determine a cholesterol profile for people living in this part of Eastern Nigeria. This will enable recommendation of a range of normal Cholesterol levels for the people living in this part of the world. Method: Total serum cholesterol, HDL, LDL, VLDL and triglycerides levels were determined ...

  13. The treatment of cholesterol: issues, effects and targets

    African Journals Online (AJOL)

    Review: The treatment of cholesterol: issues, effects and targets. 523. Vol 52 No 6. SA Fam Pract 2010. Statins: what are they? Statins are the most powerful cholesterol lowering drugs currently available. Statins inhibit 3-hydroxy-3-methyl- glutaryl-coenzyme A (HMG CoA) reductase, which leads to reduced cholesterol ...

  14. Alcohol consumption stimulates early steps in reverse cholesterol transport

    NARCIS (Netherlands)

    Gaag, M.S. van der; Tol, A. van; Vermunt, S.H.F.; Scheek, L.M.; Schaafsma, G.; Hendriks, H.F.J.

    2001-01-01

    Alcohol consumption is associated with increased HDL cholesterol levels, which may indicate stimulated reverse cholesterol transport. The mechanism is, however, not known. The aim of this study was to evaluate the effects of alcohol consumption on the first two steps of the reverse cholesterol

  15. Diet and lifestyle: its association with cholesterol levels among ...

    African Journals Online (AJOL)

    Dietary patterns, physical activities related with work, habit of consuming liquor were noted while cholesterol level, blood glucose levels, blood pressure, height, weight, waist girth, and hip circumference were measured. Results: The overall prevalence rate of borderline high cholesterol level (200-239) and high cholesterol ...

  16. Moderate alcohol consumption increases cholesterol efflux mediated by ABCA1

    NARCIS (Netherlands)

    Beulens, J.W.J.; Sierksma, A.; Tol, van A.; Fournier, C.

    2004-01-01

    Moderate alcohol consumption increases HDL cholesterol, which is involved in reverse cholesterol transport (RCT). The aim of this study was to investigate the effect of moderate alcohol consumption on cholesterol efflux, using J774 mouse macrophages and Fu5AH cells, and on other parameters in the

  17. Cholesterol-Lowering Supplements: Lower Your Numbers without Prescription Medication

    Science.gov (United States)

    ... fat protein sources May reduce LDL None Whey protein May reduce total cholesterol, LDL cholesterol and triglycerides May cause nausea, constipation, diarrhea or gas Another popular cholesterol-lowering supplement is red yeast rice. There is some evidence that red yeast rice ...

  18. Strategies for the Activation and Release of the Membranolytic Peptide Melittin from Liposomes Using Endosomal pH as a Trigger

    NARCIS (Netherlands)

    Oude Blenke, E.; Sleszynska, M.; Evers, M.J.W.; Storm, Gerrit; Martin, N.I.; Mastrobattista, E.

    2017-01-01

    Endosomolytic peptides are often coupled to drug delivery systems to enhance endosomal escape, which is crucial for the delivery of macromolecular drugs that are vulnerable to degradation in the endolysosomal pathway. Melittin is a 26 amino acid peptide derived from bee venom that has a very high

  19. Rabaptin-5 alpha/rabaptin-4 serves as a linker between rab4 and gamma(1)-adaptin in membrane recycling from endosomes

    NARCIS (Netherlands)

    Deneka, M; Neeft, M; Popa, [No Value; van Oort, M; Sprong, H; Oorschot, [No Value; Klumperman, J; Schu, P; van der Sluijs, P

    2003-01-01

    Rab4 regulates recycling from early endosomes. We investigated the role of the rab4 effector rabaptin-5alpha and its putative partner gamma(1)-adaptin in membrane recycling. We found that rabaptin-5alpha forms a ternary complex with the gamma(1)-sigma(1) subcomplex of AP-1, via a direct interaction

  20. Endosomal Escape and Delivery of CRISPR/Cas9 Genome Editing Machinery Enabled by Nanoscale Zeolitic Imidazolate Framework

    KAUST Repository

    Alsaiari, Shahad K.

    2017-12-22

    CRISPR/Cas9 is a combined protein (Cas9) and an engineered single guide RNA (sgRNA) genome editing platform that offers revolutionary solutions to genetic diseases. It has, however, a double delivery problem owning to the large protein size and the highly charged RNA component. In this work, we report the first example of CRISPR/Cas9 encapsulated by nanoscale zeolitic imidazole frameworks (ZIFs) with a loading efficiency of 17% and enhanced endosomal escape promoted by the protonated imidazole moieties. The gene editing potential of CRISPR/Cas9 encapsulated by ZIF-8 (CC-ZIFs) is further verified by knocking down the gene expression of green fluorescent protein by 37% over 4 days employing CRISPR/Cas9 machinery. The nanoscale CC-ZIFs are biocompatible and easily scaled-up offering excellent loading capacity and controlled co-delivery of intact Cas9 protein and sgRNA.

  1. The abnormal isoform of the prion protein accumulates in late-endosome-like organelles in scrapie-infected mouse brain.

    Science.gov (United States)

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

    1995-08-01

    The prion encephalopathies are characterized by accumulation in the brain of the abnormal form PrPsc of a normal host gene product PrPc. The mechanism and site of formation of PrPsc from PrPc are currently unknown. In this study, ME7 scrapie-infected mouse brain was used to show, both biochemically and by double-labelled immunogold electron microscopy, that proteinase K-resistant PrPsc is enriched in subcellular structures which contain the cation-independent mannose 6-phosphate receptor, ubiquitin-protein conjugates, beta-glucuronidase, and cathepsin B, termed late endosome-like organelles. The glycosylinositol phospholipid membrane-anchored PrPc will enter such compartment for normal degradation and the organelles may therefore act as chambers for the conversion of PrPc into infectious PrPsc in this murine model of scrapie.

  2. Plasma membrane Toll-like receptor activation increases bacterial uptake but abrogates endosomal Lactobacillus acidophilus induction of interferon-β

    DEFF Research Database (Denmark)

    Boye, Louise; Welsby, Iain; Lund, Lisbeth Drozd

    2016-01-01

    Lactobacillus acidophilus induces a potent interferon-β (IFN-β) response in dendritic cells (DCs) by a Toll-like receptor 2 (TLR2) -dependent mechanism, in turn leading to strong interleukin-12 (IL-12) production. In the present study, we investigated the involvement of different types...... of endocytosis in the L. acidophilus-induced IFN-β and IL-12 responses and how TLR2 or TLR4 ligation by lipopolysaccharide and Pam3/4CSK4 influenced endocytosis of L. acidophilus and the induced IFN-β and IL-12 production. Lactobacillus acidophilus was endocytosed by constitutive macropinocytosis taking place....... acidophilus enhanced the uptake of the bacteria. However, in these experimental conditions, induction of IFN-β and IL-12 was strongly inhibited. As L. acidophilus-induced IFN-β depends on endocytosis and endosomal degradation before signalling and as TLR stimulation from the plasma membrane leading...

  3. [Cholesterol bound to high density lipoproteins: critical review of the methods of analysis and personal data].

    Science.gov (United States)

    Orso Giacone, G

    1982-01-01

    It is widely known that atherosclerosis through its complication, i.e. heart and brain infarction, is at the present the main cause of death. The atherosclerotic process has been shown in correlation with hyperlipemia especially as far as the plasma lipoprotein cholesterol level is concerned. A preminent role in removing cholesterol from tissues and arterial walls then in preventing atherosclerosis is played by a specific class of plasma lipoproteins, the high density lipoproteins (HDL). Since the HDL-colesterol level seems to have an inverse correlation with the atherosclerotic disease it is of primary importance to define a reliable and reproducible technique to measure it. One of the aims of this paper was to examine the different methods now available for such a determination. This analysis has underlined the discrepancy among the reference values reported in the literature. However, all the authors agree that only the simultaneous measurement of total and HDL-colesterol levels is of prognostic value. Personal studies are here reported on the relationship between total and HDL-colesterol levels and risk factor of cardiovascular diseases. The two mentioned laboratory analyses have been performed on blood samples from 250 between male and female human subjects of different age. The obtained results show that the highest HDL-colesterol concentrations determined by a lipoprotein precipitation procedure with dextran sulphate, are typical in the first ten years of life both in male and in female, while the lowest levels of plasma HDL-cholesterol have been evintiated during the fifth decade of life, when the total cholesterol and the risk of cardiovascular complications rich the highest values. In a following set of investigations, the already examined blood parameters together with the risk factor values have been examined in two groups of subjects, the first one represented by adult healthy persons the second one by patients of similar age from a cardiovascular

  4. Effect of testosterone deficiency on cholesterol metabolism in pigs fed a high-fat and high-cholesterol diet

    OpenAIRE

    Cai, Zhaowei; Xi, Haitao; Pan, Yongming; Jiang, Xiaoling; Chen, Liang; Cai, Yueqin; Zhu, Keyan; Chen, Cheng; Xu, Xiaoping; Chen, Minli

    2015-01-01

    Background Testosterone deficiency is associated with increased serum cholesterol levels. However, how testosterone deficiency precisely affects cholesterol metabolism remains unclear. Therefore, in the current study, we examined the effect of testosterone deficiency on cholesterol metabolism and liver gene expression in pigs fed a high-fat and high-cholesterol (HFC) diet. Methods Sexually mature male miniature pigs (6?7 months old) were randomly divided into 3 groups as follows: intact male ...

  5. Potent and selective mediators of cholesterol efflux

    Energy Technology Data Exchange (ETDEWEB)

    Bielicki, John K; Johansson, Jan

    2015-03-24

    The present invention provides a family of non-naturally occurring polypeptides having cholesterol efflux activity that parallels that of full-length apolipoproteins (e.g., Apo AI and Apo E), and having high selectivity for ABAC1 that parallels that of full-length apolipoproteins. The invention also provides compositions comprising such polypeptides, methods of identifying, screening and synthesizing such polypeptides, and methods of treating, preventing or diagnosing diseases and disorders associated with dyslipidemia, hypercholesterolemia and inflammation.

  6. EVALUATION OF SERUM CHOLESTEROL, AMINO TRANSFERASES

    Directory of Open Access Journals (Sweden)

    Anantha Babu

    2016-01-01

    Full Text Available BACKGROUND AND AIMS The purpose of this study was to determine the efficacy of red yeast rice (Monascus purpureus-fermented rice in lowering cholesterol in the blood. At the same time, alanine aminotranferase (ALT, aspartate aminotransferase (AST and gamma-glutamyl transferase (γ-GT were measured for notable side effects in the liver. Possible muscle damage was determined by measuring creatine kinase (CK. METHODS The cholesterol lowering effect in serum of red yeast rice-fed rats were studied over a 42-day feeding period. A total of 16 male Sprague-Dawley rats were randomised into 8 per group: control and treated. Treated rats were administered 1.35g/kg/day. Control rats were maintained on ordinary rat chow. RESULTS Serum cholesterol levels were significantly decreased by 19.13% in treated group compared to controls. This treatment also showed increase in serum ALT and AST activities by 41.90% and 21.53%, respectively. Mean CK activity in treated rats showed an increase by 32.32% when compared with control rats. γ-GT is the only enzyme that showed a decrease of 15.16% in sera of treated rats. Body weights of control and treated rats increased significantly by 10% end of feeding period but were not due to treatment. CONCLUSION Red yeast rice significantly decreased serum cholesterol level at a dosage of 1.35g/kg/day. However, the differences in serum enzyme activities between control and treated rats were not significant.

  7. Cholesterol impairment contributes to neuroserpin aggregation

    Science.gov (United States)

    Giampietro, Costanza; Lionetti, Maria Chiara; Costantini, Giulio; Mutti, Federico; Zapperi, Stefano; La Porta, Caterina A. M.

    2017-03-01

    Intraneural accumulation of misfolded proteins is a common feature of several neurodegenerative pathologies including Alzheimer’s and Parkinson’s diseases, and Familial Encephalopathy with Neuroserpin Inclusion Bodies (FENIB). FENIB is a rare disease due to a point mutation in neuroserpin which accelerates protein aggregation in the endoplasmic reticulum (ER). Here we show that cholesterol depletion induced either by prolonged exposure to statins or by inhibiting the sterol reg-ulatory binding-element protein (SREBP) pathway also enhances aggregation of neuroserpin proteins. These findings can be explained considering a computational model of protein aggregation under non-equilibrium conditions, where a decrease in the rate of protein clearance improves aggregation. Decreasing cholesterol in cell membranes affects their biophysical properties, including their ability to form the vesicles needed for protein clearance, as we illustrate by a simple mathematical model. Taken together, these results suggest that cholesterol reduction induces neuroserpin aggregation, even in absence of specific neuroserpin mutations. The new mechanism we uncover could be relevant also for other neurodegenerative diseases associated with protein aggregation.

  8. Fibroblast cholesterol efflux to plasma from metabolic syndrome subjects is not defective despite low high-density lipoprotein cholesterol

    NARCIS (Netherlands)

    Dullaart, Robin P. F.; Groen, Albert K.; Dallinga-Thie, Geesje M.; de Vries, Rindert; Sluiter, Wim J.; van Tol, Arie

    Objective: We tested whether in metabolic syndrome (MetS) subjects the ability of plasma to stimulate cellular cholesterol efflux. an early step in the anti-atherogenic reverse cholesterol transport pathway. is maintained despite low high-density lipoprotein (HDL) cholesterol. Design: In 76 subjects

  9. Fibroblast cholesterol efflux to plasma from metabolic syndrome subjects is not defective despite low high-density lipoprotein cholesterol

    NARCIS (Netherlands)

    Dullaart, Robin P. F.; Groen, Albert K.; Dallinga-Thie, Geesje M.; de Vries, Rindert; Sluiter, Wim J.; van Tol, Arie

    2008-01-01

    OBJECTIVE: We tested whether in metabolic syndrome (MetS) subjects the ability of plasma to stimulate cellular cholesterol efflux, an early step in the anti-atherogenic reverse cholesterol transport pathway, is maintained despite low high-density lipoprotein (HDL) cholesterol. DESIGN: In 76 subjects

  10. Fibroblast cholesterol efflux to plasma from metabolic syndrome subjects is not defective despite low high-density lipoprotein cholesterol

    NARCIS (Netherlands)

    R.P.F. Dullaart (Robin); A. Groen (Albert); G.M. Dallinga-Thie (Geesje); R. de Vries (Rindert); W. Sluiter (Wim); A. van Tol (Arie)

    2008-01-01

    textabstractObjective: We tested whether in metabolic syndrome (MetS) subjects the ability of plasma to stimulate cellular cholesterol efflux, an early step in the anti-atherogenic reverse cholesterol transport pathway, is maintained despite low high-density lipoprotein (HDL) cholesterol. Design: In

  11. Protein replacement therapy partially corrects the cholesterol-storage phenotype in a mouse model of Niemann-Pick type C2 disease.

    Directory of Open Access Journals (Sweden)

    Gitte Krogh Nielsen

    Full Text Available Niemann-Pick type C2 (NPC2 disease is a fatal autosomal recessive neurovisceral degenerative disorder characterized by late endosomal-lysosomal sequestration of low-density lipoprotein derived cholesterol. The breach in intracellular cholesterol homeostasis is caused by deficiency of functional NPC2, a soluble sterol binding protein targeted to the lysosomes by binding the mannose-6-phosphate receptor. As currently there is no effective treatment for the disorder, we have investigated the efficacy of NPC2 replacement therapy in a murine gene-trap model of NPC2-disease generated on the 129P2/OlaHsd genetic background. NPC2 was purified from bovine milk and its functional competence assured in NPC2-deficient fibroblasts using the specific cholesterol fluorescent probe filipin. For evaluation of phenotype correction in vivo, three-week-old NPC2(-/- mice received two weekly intravenous injections of 5 mg/kg NPC2 until trial termination 66 days later. Whereas the saline treated NPC2(-/- mice exhibited massive visceral cholesterol storage as compared to their wild-type littermates, administration of NPC2 caused a marked reduction in cholesterol build up. The histological findings, indicating an amelioration of the disease pathology in liver, spleen, and lungs, corroborated the biochemical results. Little or no difference in the overall cholesterol levels was observed in the kidneys, blood, cerebral cortex and hippocampus when comparing NPC2(-/- and wild type mice. However, cerebellum cholesterol was increased about two fold in NPC2(-/- mice compared with wild-type littermates. Weight gain performance was slightly improved as a result of the NPC2 treatment but significant motor coordination deficits were still observed. Accordingly, ultrastructural cerebellar abnormalities were detected in both saline treated and NPC2 treated NPC2(-/- animals 87 days post partum. Our data indicate that protein replacement may be a beneficial therapeutic approach in the

  12. Endoscopic Transnasal Approach for Cholesterol Granuloma of the Petrous Apex

    Directory of Open Access Journals (Sweden)

    Mohammad Samadian

    2015-01-01

    Full Text Available Cholesterol granulomas are rare round or ovoid cysts. They contain cholesterol crystals surrounded by foreign bodies of giant cells and are characterized by chronic inflammation. Large cholesterol granuloma can compress surrounding tissue especially cranial nerves. There are several types of surgery for the resection of cholesterol granuloma. We describe 4 cases of cholesterol granuloma operated on via transnasal endoscopic approach. In this report, we describe radiologic and pathologic features of this lesion and explain the advantages and disadvantages of transsphenoidal endoscopic approach for these rare lesions.

  13. Cholesterol monohydrate nucleation in ultrathin films on water

    DEFF Research Database (Denmark)

    Rapaport, H.; Kuzmenko, I.; Lafont, S.

    2001-01-01

    The growth of a cholesterol crystalline phase, three molecular layers thick at the air-water interface, was monitored by grazing incidence x-ray diffraction and x-ray reflectivity. Upon compression, a cholesterol film transforms from a monolayer of trigonal symmetry and low crystallinity...... to the triclinic 3-D crystal structure of cholesterol . H(2)O. By comparison, the cholesterol derivative stigmasterol transforms, upon compression, directly into a crystalline trilayer in the rectangular lattice. These results may contribute to an understanding of the onset of cholesterol crystallization...

  14. Biochemical and Bioimaging Evidence of Cholesterol in Acquired Cholesteatoma

    DEFF Research Database (Denmark)

    Thorsted, Bjarne; Bloksgaard, Maria; Groza, Alexandra

    2016-01-01

    : The results show that the total lipid content of the cholesteatoma matrix is similar to that of stratum corneum from skin and that the cholesteatoma matrix unquestionably contains cholesterol. The cholesterol content in the cholesteatoma matrix is increased by over 30% (w/w dry weight) compared to the control....... The cholesterol sulfate content is below 1% of the total lipids in both the cholesteatoma and the control. Cholesterol ester was reduced by over 30% when compared to the control. CONCLUSIONS: The content of cholesterol in the cholesteatoma matrix is significantly different from that in stratum corneum from skin...

  15. LITAF mutations associated with Charcot-Marie-Tooth disease 1C show mislocalization from the late endosome/lysosome to the mitochondria.

    Directory of Open Access Journals (Sweden)

    Andressa Ferreira Lacerda

    Full Text Available Charcot-Marie-Tooth (CMT disease is one of the most common heritable neuromuscular disorders, affecting 1 in every 2500 people. Mutations in LITAF have been shown to be causative for CMT type 1C disease. In this paper we explore the subcellular localization of wild type LITAF and mutant forms of LITAF known to cause CMT1C (T49M, A111G, G112S, T115N, W116G, L122V and P135T. The results show that LITAF mutants A111G, G112S, W116G, and T115N mislocalize from the late endosome/lysosome to the mitochondria while the mutants T49M, L122V, and P135T show partial mislocalization with a portion of the total protein present in the late endosome/lysosome and the remainder of the protein localized to the mitochondria. This suggests that different mutants of LITAF will produce differing severity of disease. We also explored the effect of the presence of mutant LITAF on wild-type LITAF localization. We showed that in cells heterozygous for LITAF, CMT1C mutants T49M and G112S are dominant since wild-type LITAF localized to the mitochondria when co-transfected with a LITAF mutant. Finally, we demonstrated how LITAF transits to the endosome and mitochondria compartments of the cell. Using Brefeldin A to block ER to Golgi transport we demonstrated that wild type LITAF traffics through the secretory pathway to the late endosome/lysosome while the LITAF mutants transit to the mitochondria independent of the secretory pathway. In addition, we demonstrated that the C-terminus of LITAF is necessary and sufficient for targeting of wild-type LITAF to the late endosome/lysosome and the mutants to the mitochondria. Together these data provide insight into how mutations in LITAF cause CMT1C disease.

  16. Effect of Moderate Alcohol Consumption on Parameters of Reverse Cholesterol Transport in Postmenopausal Women

    NARCIS (Netherlands)

    Sierksma, A.; Vermunt, S.H.F.; Lankhuizen, I.M.; Gaag, M.S. van der; Scheek, L.M.; Grobbee, D.E.; Tol, A. van; Hendriks, H.F.J.

    2004-01-01

    Background: Alcohol consumption is associated with increased high-density lipoprotein (HDL) cholesterol levels. One of the main antiatherogenic functions of HDL is reverse cholesterol transport. Three early steps of reverse cholesterol transport are (1) cellular cholesterol efflux, (2) plasma

  17. HDL Cholesterol, LDL Cholesterol, and Triglycerides as Risk Factors for CKD: A Mendelian Randomization Study.

    Science.gov (United States)

    Lanktree, Matthew B; Thériault, Sébastien; Walsh, Michael; Paré, Guillaume

    2017-07-26

    High-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, and triglyceride concentrations are heritable risk factors for vascular disease, but their role in the progression of chronic kidney disease (CKD) is unclear. 2-sample Mendelian randomization analysis of data derived from the largest published lipid and CKD studies. Effect of independent genetic variants significantly associated with lipid concentrations was obtained from the Global Lipids Genetics Consortium (n=188,577), and the effect of these same variants on estimated glomerular filtration rate (eGFR), CKD (defined as eGFRGenetics Consortium (n=133,814). Using conventional, multivariable, and Egger Mendelian randomization approaches, we assessed the causal association between genetically determined lipid concentrations and kidney traits. eGFR, dichotomous eGFRGenetically higher triglyceride concentrations appeared associated with higher eGFRs, but this finding was driven by a single pleiotropic variant in the glucokinase regulator gene (GCKR). After exclusion, genetically higher triglyceride concentration was not associated with any kidney trait. Individual patient-level phenotype and genotype information were unavailable. 2-sample Mendelian randomization analysis of data from the largest lipid and CKD cohorts supports genetically higher HDL cholesterol concentration as causally associated with better kidney function. There was no association between genetically altered LDL cholesterol or triglyceride concentration and kidney function. Further analysis of CKD outcomes in HDL cholesterol intervention trials is warranted. Copyright © 2017 National Kidney Foundation, Inc. Published by Elsevier Inc. All rights reserved.

  18. Preparation of cholesterol oxidase nanoparticles and their application in amperometric determination of cholesterol

    Science.gov (United States)

    Chawla, Sheetal; Rawal, Rachna; Sonia; Ramrati; Pundir, C. S.

    2013-09-01

    The nanoparticle (NP) aggregates of commercial cholesterol oxidase (ChOx) were prepared by desolvation method. The formation and characterization of ChOxNP aggregates were studied by transmission electron microscopy and scanning electron microscopy. NP aggregates were more stable, active and had a higher shelf life than that of free enzyme. An amperometric cholesterol biosensor was constructed by immobilizing ChOxNPs onto Au electrode. The biosensor showed optimum response within 8 s at pH 6.0 and 35 °C, when polarized at +0.27 V versus Ag/AgCl. The biosensor possesses high sensitivity and measures cholesterol concentrations as low as 1.56 mg/dl. The working linear range was 12.5-700 mg/dl for cholesterol. The biosensor was evaluated and employed for measurement of total cholesterol in human serum. The enzyme electrode lost 50 % of its initial activity during its regular use for 180 times over a period of 90 days when stored in 0.1 M sodium phosphate buffer, pH 7.0 at 4 °C.

  19. Preparation of cholesterol oxidase nanoparticles and their application in amperometric determination of cholesterol

    Energy Technology Data Exchange (ETDEWEB)

    Chawla, Sheetal; Rawal, Rachna; Sonia; Ramrati; Pundir, C. S., E-mail: pundircs@rediffmail.com [M. D. University, Department of Biochemistry (India)

    2013-09-15

    The nanoparticle (NP) aggregates of commercial cholesterol oxidase (ChOx) were prepared by desolvation method. The formation and characterization of ChOxNP aggregates were studied by transmission electron microscopy and scanning electron microscopy. NP aggregates were more stable, active and had a higher shelf life than that of free enzyme. An amperometric cholesterol biosensor was constructed by immobilizing ChOxNPs onto Au electrode. The biosensor showed optimum response within 8 s at pH 6.0 and 35 Degree-Sign C, when polarized at +0.27 V versus Ag/AgCl. The biosensor possesses high sensitivity and measures cholesterol concentrations as low as 1.56 mg/dl. The working linear range was 12.5-700 mg/dl for cholesterol. The biosensor was evaluated and employed for measurement of total cholesterol in human serum. The enzyme electrode lost 50 % of its initial activity during its regular use for 180 times over a period of 90 days when stored in 0.1 M sodium phosphate buffer, pH 7.0 at 4 Degree-Sign C.

  20. THE REDUCTION OF CHOLESTEROL WITH CUPPING THERAPY ON CHOLESTEROL REDUCTION IN PATIENTS WITH HYPERCHOLESTEROLEMIA

    Directory of Open Access Journals (Sweden)

    Zahid Fikri

    2017-04-01

    Full Text Available Introduction: Hypercholesterolemia is a risk factor causes of death at younger ages. Hypercholesterolemia may increase the risk of atherosclerosis, coronary heart disease, pancreatitis (pancreas inflammation in organs, diabetes mellitus, thyroid disorders, liver disease and kidney disease. Many patients with hypercholesterolemia using cupping therapy. Cupping therapy is alternative treatment process of throwing dirty blood from the body through the skin surface. The objective of this study was to determine the effect of cupping therapy to decrease cholesterol levels in patients with hypercholesterolemia. Method: Design used in this study was quasy experimental design. The population is all patients with hypercholesterolemia in the health center plaza Gresik. The total sample is 18 respondents, taken according to inclusion criteria. Independent variable is the cupping therapy. The dependent variable was the decrease in cholesterol levels. Data were collected using a questionnaire and observation of cholesterol. Data were analyzed using independent t-test and paired t tests with signi fi cance level α < 0.05. Result: The results show that cholesterol levels in patients with hypercholesterolemia treated groups decreased majority. Independent statistical analysis using t-test showed p = 0.001 and with the Paired t-test p value = 0.003. Discussion: This result means that there are significant effects of cupping therapy on cholesterol reduction in patients with hypercholesterolemia aged 45 years and over. Further research needs to be done in control diet, lifestyle and daily activities for the success of cupping therapy.