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Sample records for lysosomal thiol reductase

  1. Lysosome

    Directory of Open Access Journals (Sweden)

    Ursula Matte BSc, PhD

    2016-12-01

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

  2. Lysosome

    National Research Council Canada - National Science Library

    Ursula Matte BSc, PhD; Gabriela Pasqualim BSc, MSc

    2016-01-01

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

  3. Comparative study of the physiological roles of three peroxidases (NADH peroxidase, Alkyl hydroperoxide reductase and Thiol peroxidase) in oxidative stress response, survival inside macrophages and virulence of Enterococcus faecalis.

    Science.gov (United States)

    La Carbona, Stephanie; Sauvageot, Nicolas; Giard, Jean-Christophe; Benachour, Abdellah; Posteraro, Brunella; Auffray, Yanick; Sanguinetti, Maurizio; Hartke, Axel

    2007-12-01

    The opportunistic pathogen Enterococcus faecalis is well equipped with peroxidatic activities. It harbours three loci encoding a NADH peroxidase, an alkyl hydroperoxide reductase and a protein (EF2932) belonging to the AhpC/TSA family. We present results demonstrating that ef2932 does encode a thiol peroxidase (Tpx) and show that it is part of the regulon of the hydrogen peroxide regulator HypR. Characterization of unmarked deletion mutants showed that all three peroxidases are important for the defence against externally provided H(2)O(2). Exposure to internal generated H(2)O(2) by aerobic growth on glycerol, lactose, galactose or ribose showed that Npr was absolutely required for aerobic growth on glycerol and optimal growth on the other substrates. Growth on glycerol was also dependent on Ahp. Addition of catalase restored growth of the mutants, and therefore, extracellular H(2)O(2) concentrations have been determined. This showed that the time point of growth arrest of the Deltanpr mutant correlated with the highest H(2)O(2) concentration measured. Analysis of the survival of the different strains inside peritoneal macrophages revealed that Tpx was the most important antioxidant activity for protecting the cells against the hostile phagocyte environment. Finally, the Deltatpx and the triple mutant showed attenuated virulence in a mouse peritonitis model.

  4. Thiol-disulfide exchange between the PDI family of oxidoreductases negates the requirement for an oxidase or reductase for each enzyme.

    Science.gov (United States)

    Oka, Ojore B V; Yeoh, Hui Y; Bulleid, Neil J

    2015-07-15

    The formation of disulfides in proteins entering the secretory pathway is catalysed by the protein disulfide isomerase (PDI) family of enzymes. These enzymes catalyse the introduction, reduction and isomerization of disulfides. To function continuously they require an oxidase to reform the disulfide at their active site. To determine how each family member can be recycled to catalyse disulfide exchange, we have studied whether disulfides are transferred between individual PDI family members. We studied disulfide exchange either between purified proteins or by identifying mixed disulfide formation within cells grown in culture. We show that disulfide exchange occurs efficiently and reversibly between specific PDIs. These results have allowed us to define a hierarchy for members of the PDI family, in terms of ability to act as electron acceptors or donors during thiol-disulfide exchange reactions and indicate that there is no kinetic barrier to the exchange of disulfides between several PDI proteins. Such promiscuous disulfide exchange negates the necessity for each enzyme to be oxidized by Ero1 (ER oxidoreductin 1) or reduced by a reductive system. The lack of kinetic separation of the oxidative and reductive pathways in mammalian cells contrasts sharply with the equivalent systems for native disulfide formation within the bacterial periplasm.

  5. Arabidopsis CDS blastp result: AK071633 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK071633 J023102J23 At1g07080.1 gamma interferon responsive lysosomal thiol reducta...se family protein / GILT family protein similar to SP|P13284 Gamma-interferon inducible lysosomal thiol redu...ctase precursor {Homo sapiens}; contains Pfam profile PF03227: Gamma interferon inducible lysosomal thiol reductase (GILT) 6e-66 ...

  6. Arabidopsis CDS blastp result: AK106050 [KOME

    Lifescience Database Archive (English)

    Full Text Available AK106050 001-206-F01 At1g07080.1 gamma interferon responsive lysosomal thiol reduct...ase family protein / GILT family protein similar to SP|P13284 Gamma-interferon inducible lysosomal thiol red...uctase precursor {Homo sapiens}; contains Pfam profile PF03227: Gamma interferon inducible lysosomal thiol reductase (GILT) 6e-63 ...

  7. TRPML and lysosomal function.

    Science.gov (United States)

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

    2007-08-01

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

  8. The proteome of lysosomes.

    Science.gov (United States)

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

    2010-11-01

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

  9. Distribution and abundance of organic thiols

    Science.gov (United States)

    Fahey, R.

    1985-01-01

    The role of glutathione (GSH) in protecting against the toxicity of oxygen and oxygen by products is well established for all eukaryotes studied except Entamoeba histolytica which lacks mitochrondria, chloroplasts, and microtubules. The GSH is not universal among prokaryotes. Entamoeba histolytica does not produce GSH or key enzymes of GSH metabolism. A general method of thiol analysis based upon fluorescent labeling with monobromobimane and HPLC separation of the resulting thiol derivatives was developed to determine the occurrence of GSH and other low molecular weight thiols in bacteria. Glutathione is the major thiol in cyanobacteria and in most bacteria closely related to the purple photosynthetic bacteria, but GSH was not found in archaebacteria, green bacteria, or GRAM positive bacteria. It suggested that glutathione metabolism was incorporated into eukaryotes at the time that mitochondria and chloroplasts were acquired by endosymbiosis. In Gram positive aerobes, coenzyme A occurs at millimolar levels and CoA disulfide reductases are identified. The CoA, rather than glutathione, may function in the oxygen detoxification processes of these organisms.

  10. Lysosome Biogenesis and Autophagy

    NARCIS (Netherlands)

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

    2016-01-01

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

  11. Thiol biochemistry of prokaryotes

    Science.gov (United States)

    Fahey, Robert C.

    1986-01-01

    The present studies have shown that GSH metabolism arose in the purple bacteria and cyanobacteria where it functions to protect against oxygen toxicity. Evidence was obtained indicating that GSH metabolism was incorporated into eucaryotes via the endosymbiosis giving rise to mitochrondria and chloroplasts. Aerobic bacteria lacking GSH utilize other thiols for apparently similar functions, the thiol being coenzyme A in Gram positive bacteria and chi-glutamylcysteine in the halobacteria. The thiol biochemistry of prokaryotes is thus seen to be much more highly diversified than that of eucaryotes and much remains to be learned about this subject.

  12. The lysosome and neurodegenerative diseases

    Institute of Scientific and Technical Information of China (English)

    Lisha Zhang; Rui Sheng; Zhenghong Qin

    2009-01-01

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

  13. Electroanalysis of Plant Thiols

    Directory of Open Access Journals (Sweden)

    Rene Kizek

    2007-06-01

    Full Text Available Due to unique physico-chemical properties of –SH moiety thiols comprise widegroup of biologically important compounds. A review devoted to biological functions ofglutathione and phytochelatins with literature survey of methods used to analysis of thesecompounds and their interactions with cadmium(II ions and Murashige-Skoog medium ispresented. For these purposes electrochemical techniques are used. Moreover, we revealedthe effect of three different cadmium concentrations (0, 10 and 100 μM on cadmiumuptake and thiols content in maize plants during 192 hours long experiments usingdifferential pulse anodic stripping voltammetry to detect cadmium(II ions and highperformance liquid chromatography with electrochemical detection to determineglutathione. Cadmium concentration determined in tissues of the plants cultivated innutrient solution containing 10 μM Cd was very low up to 96 hours long exposition andthen the concentration of Cd markedly increased. On the contrary, the addition of 100 μMCd caused an immediate sharp increase in all maize plant parts to 96 hours Cd expositionbut subsequently the Cd concentration increased more slowly. A high performance liquidchromatography with electrochemical detection was used for glutathione determination intreated maize plants after 96 and 192 hours of treatment. The highest total content of glutathione per one plant was 6 μg (96 h, 10 μM Cd in comparison with non-treated plant (control where glutathione content was 1.5 μg. It can be concluded that electrochemical techniques have proved to be useful to analyse plant thiols.

  14. Cancer-associated lysosomal changes

    DEFF Research Database (Denmark)

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

    2013-01-01

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

  15. LYSOSOMAL DISRUPTION BY BACTERIAL TOXINS

    Science.gov (United States)

    Bernheimer, Alan W.; Schwartz, Lois L.

    1964-01-01

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

  16. Histochemical Localization of Glutathione Dependent NBT-Reductase in Mouse Skin

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Objective Localization of the glutathione dependent Nitroblue tetrazolium (NBT) reductase in fresh frozen sections of mouse skin and possible dependence of NBT reductase on tissue thiol levels has been investigated. Methods The fresh frozen tissue sections (8m thickness) were prepared and incubated in medium containing NBT, reduced glutathione (GSH) and phosphate buffer. The staining for GSH was performed with mercury orange. Results  The activity of the NBT-reductase in mouse skin has been found to be localized in the areas rich in glutathione and actively proliferating area of the skin. Conclusion The activity of the NBT-reductase seems to be dependent on the glutathione contents.

  17. Cancer-associated lysosomal changes

    DEFF Research Database (Denmark)

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

    2013-01-01

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

  18. Naegleria fowleri: a free-living highly pathogenic amoeba contains trypanothione/trypanothione reductase and glutathione/glutathione reductase systems.

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    Ondarza, Raúl N; Hurtado, Gerardo; Tamayo, Elsa; Iturbe, Angélica; Hernández, Eva

    2006-11-01

    This paper presents definitive data showing that the thiol-bimane compound isolated and purified by HPLC from Naegleria fowleri trophozoites unequivocally corresponds by matrix assisted laser-desorption ionization-time-of-flight MS, to the characteristic monoprotonated ion of trypanothione-(bimane)(2) [M(+)H(+)] of m/z 1104.57 and to the trypanothione-(bimane) of m/z 914.46. The trypanothione disulfide T(S)(2) was also found to have a molecular ion of m/z 723.37. Additionally HPLC demonstrated that thiol-bimane compounds corresponding to cysteine and glutathione were present in Naegleria. The ion patterns of the thiol-bimane compounds prepared from commercial trypanothione standard, Entamoeba histolytica and Crithidia luciliae are identical to the Naegleria thiol-bimane compound. Partially purified extracts from N. fowleri showed the coexistence of glutathione and trypanothione reductases activities. There is not doubt that the thiol compound trypanothione, which was previously thought to occur only in Kinetoplastida, is also present in the human pathogens E. histolytica and N. fowleri, as well as in the non-pathogenic euglenozoan E. gracilis. The presence of the trypanothione/trypanothione reductase system in N. fowleri creates the possibility of using this enzyme as a new "drug target" for rationally designed drugs to eliminate the parasite, without affecting the human host.

  19. Lysosomal cell death at a glance

    DEFF Research Database (Denmark)

    Aits, Sonja; Jaattela, Marja

    2013-01-01

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

  20. Low-molecular-mass thiol compounds from a free-living highly pathogenic amoeba, Naegleria fowleri.

    Science.gov (United States)

    Ondarza, Raúl N; Iturbe, Angélica; Hernández, Eva; Hurtado, Gerardo

    2003-04-01

    Acid extracts labelled with the fluorescent reagent monobromobimane and separated by HPLC have enabled the detection of low-molecular-mass thiol compounds in Naegleria fowleri for the first time. The amounts detected are expressed in nmol/1 x 10(6) trophozoites cultivated at various stages of growth in the appropriate culture medium. N. fowleri is a highly pathogenic free-living amoeba, in which we found important thiol compounds, some of them in their reduced and oxidized forms. Unlike cysteine and glutathione, a number of these are not represented in normal human lymphocytes. Some of these thiol compounds from Naegleria must have their respective disulphide reductases, although the presence of thiol-disulphide exchange reactions must be considered. Ovothiol A, with antioxidant properties, is an example of a compound that is kept reduced by trypanothione in trypanosomatids, although no disulphide reductase for ovothiol A has yet been discovered. In our case we were unable to detect this biothiol in Naegleria. The presence of thiol compounds that seem to be particular to this pathogen and which are not present in human lymphocytes opens the possibility of searching for disulphide-reducing enzymes that can serve as drug targets.

  1. The isolation of lysosomes from Ehrlich ascites tumor cells following pretreatment of mice with Triton WR-1339.

    Science.gov (United States)

    Horvat, A; Baxandall, J; Touster, O

    1969-08-01

    A method is described for obtaining highly purified lysosomes from Ehrlich ascites tumo cells grown in mice injected with Triton WR-1339. The isolated particles show a high specific activity for aryl sulfatase, representing an 80-90-fold purification over the homogenate, and a 15-18% yield of the total enzyme activity. Mitochondrial and microsomal marker enzymes are present in negligible amounts (0.2% of the activity of the homogenate). The biochemical evidence for a rather high degree of homogeneity of the fraction is supported by the electron microscopic examination of the purified lysosomes. The intracellular localizations of N-acetyl-beta-glucosaminidase, NADH-cytochrome c reductase and NADPH-cytochrome c reductase in Ehrlich ascites cells are also reported, the first two being present in highest concentration in the combined mitochondrial-lysosomal fraction and the third in the microsomal fraction.

  2. Lysosomal cell death mechanisms in aging.

    Science.gov (United States)

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

    2016-12-01

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

  3. Biomarkers in Lysosomal Storage Diseases

    Directory of Open Access Journals (Sweden)

    Joaquin Bobillo Lobato

    2016-12-01

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

  4. EXPRESSION AND DISTRIBUTION OF THIOL-REGULATING ENZYME GLUTAREDOXIN 2 (GRX2) IN PORCINE OCULAR TISSUES*

    OpenAIRE

    Upadhyaya, Bijaya; Tian, Xiaoli; Wu, Hongli; Lou, Marjorie F.

    2014-01-01

    Glutaredoxin2 (Grx2) is a mitochondrial isozyme of the cytosolic glutaredoxin1 (thioltransferase or TTase). Both belong to the large oxidoreductase family and play an important role in maintaining thiol/disulfide redox homeostasis in the cells. Grx2 is recently found in the lens where its activities of disulfide reductase and peroxidase, similar to TTase, can protect the lens against oxidative stress. Since other eye tissues are also highly sensitive to oxidative stress, and TTase’s distribut...

  5. Brief exposure to copper activates lysosomal exocytosis.

    Science.gov (United States)

    Peña, Karina; Coblenz, Jessica; Kiselyov, Kirill

    2015-04-01

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

  6. Inhibitors of lysosomal cysteine proteases

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    Lyanna O. L.

    2011-04-01

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

  7. Loss of Thiol Repair Systems in Human Cataractous Lenses

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    Wei, Min; Xing, Kui-Yi; Fan, Yin-Chuan; Libondi, Teodosio; Lou, Marjorie F.

    2015-01-01

    Purpose. The purpose of this study was to investigate the thiol repair systems of thioltransferase (TTase) and thioredoxin (Trx) and oxidation-damaged proteins in human cataractous lenses. Methods. Cataractous lenses in humans (57–85 years of age) were classified into cortical, nuclear, mixed, mature, and hypermature cataract types by using a lens opacity classification system, and were obtained by extracapsular cataract extraction (ECCE) procedure. Cortical and nuclear cataracts were grouped by decreasing order of visual acuity into optical chart reading (R), counting fingers (CF), hand motion (HM), and light perception (LP). ECCE lens homogenate was analyzed for glutathione (GSH) level and enzyme activities of TTase, glutathione reductase (GR), Trx, and thioredoxin reductase (TR). Cortical and nuclear cataractous lenses (8 of each) with visual acuity better than HM were each dissected into cortical and nuclear portions for measurement of glyceraldehyde 3-phosphate dehydrogenase (G3PD) activity. Clear lenses (in humans 49–71 years of age) were used as control. Results. Compared with control, all cataractous lenses lost more than 80% GSH and 70% GR; TR and Trx activity; and 40% to 70% TTase activity, corroborated with the loss in visual acuity. Among cataracts with R and CF visual acuity, cortical cataract lost more cortical G3PD activity (18% of control) than that of nuclear cataract (50% of control), whereas GSH depletion and TTase inactivation were similar in both cataracts. Conclusions. Thiol repair systems were damaged in all types of cataracts. Cortical and nuclear cataracts showed differential G3PD inactivation in the cortex, implying those 2 type of cataracts might be formed through different mechanisms. PMID:25537203

  8. GNeosomes: Highly Lysosomotropic Nanoassemblies for Lysosomal Delivery.

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

    2015-01-01

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

  9. Antioxidant Defense by Thioredoxin Can Occur Independently of Canonical Thiol-Disulfide Oxidoreductase Enzymatic Activity

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    Miryoung Song

    2016-03-01

    Full Text Available The thiol-disulfide oxidoreductase CXXC catalytic domain of thioredoxin contributes to antioxidant defense in phylogenetically diverse organisms. We find that although the oxidoreductase activity of thioredoxin-1 protects Salmonella enterica serovar Typhimurium from hydrogen peroxide in vitro, it does not appear to contribute to Salmonella’s antioxidant defenses in vivo. Nonetheless, thioredoxin-1 defends Salmonella from oxidative stress resulting from NADPH phagocyte oxidase macrophage expression during the innate immune response in mice. Thioredoxin-1 binds to the flexible linker, which connects the receiver and effector domains of SsrB, thereby keeping this response regulator in the soluble fraction. Thioredoxin-1, independently of thiol-disulfide exchange, activates intracellular SPI2 gene transcription required for Salmonella resistance to both reactive species generated by NADPH phagocyte oxidase and oxygen-independent lysosomal host defenses. These findings suggest that the horizontally acquired virulence determinant SsrB is regulated post-translationally by ancestrally present thioredoxin.

  10. Quinone Reductase 2 Is a Catechol Quinone Reductase

    Energy Technology Data Exchange (ETDEWEB)

    Fu, Yue; Buryanovskyy, Leonid; Zhang, Zhongtao (NYMEDCO)

    2008-09-05

    The functions of quinone reductase 2 have eluded researchers for decades even though a genetic polymorphism is associated with various neurological disorders. Employing enzymatic studies using adrenochrome as a substrate, we show that quinone reductase 2 is specific for the reduction of adrenochrome, whereas quinone reductase 1 shows no activity. We also solved the crystal structure of quinone reductase 2 in complexes with dopamine and adrenochrome, two compounds that are structurally related to catecholamine quinones. Detailed structural analyses delineate the mechanism of quinone reductase 2 specificity toward catechol quinones in comparison with quinone reductase 1; a side-chain rotational difference between quinone reductase 1 and quinone reductase 2 of a single residue, phenylalanine 106, determines the specificity of enzymatic activities. These results infer functional differences between two homologous enzymes and indicate that quinone reductase 2 could play important roles in the regulation of catecholamine oxidation processes that may be involved in the etiology of Parkinson disease.

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

    Science.gov (United States)

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

    2016-04-01

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

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

    Science.gov (United States)

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

    2015-03-17

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

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

    Science.gov (United States)

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

    2015-05-26

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

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

    Directory of Open Access Journals (Sweden)

    Hanna Appelqvist

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

  15. Histochemical Localization of Glutathione Dependent NBT—Reductase in Mouse Skin

    Institute of Scientific and Technical Information of China (English)

    YOESHWERSHUKLA

    2001-01-01

    Objective:Localization of the glutathione dependent Nitroblue tetrazolium(NBT) reductase in fresh frozen sections of mouse skin and possible dependence of NBT reductase on tissue thiol levels has been investigated.Methods:The fresh frozen tissue sections(8m thickness)were prepared and incuated in medium containing NBT,reduced glutathione(GSH) and Phosphate uffer,The staining for GSH was performed with mercury orange.Results:The activity of the NBT-reductase in mouse skin has een found to be localized in the areas rich in glutatione and actively proliferating area of the skin.Conclusion:The activity of the NBT-reductase seems to be dependent on the glutatione contents.

  16. 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.

  17. Thiol/disulfide homeostasis in asphalt workers.

    Science.gov (United States)

    Yilmaz, Ömer Hınç; Bal, Ceylan; Neşelioglu, Salim; Büyükşekerci, Murat; Gündüzöz, Meşide; Eren, Funda; Tutkun, Lutfiye; Yilmaz, Fatma Meric

    2016-09-02

    The aim of this study was to investigate thiol/disulfide homeostasis in asphalt workers who are exposed to polycyclic aromatic hydrocarbons occupationally. The study was carried out in 34 nonsmoker asphalt workers. Additionally, 35 healthy nonsmoker volunteers were recruited as control group. Thiol and disulfide concentrations were determined using the novel automated measurement method. Levels of urinary 1-OH-pyrene were analyzed by liquid chromatography. Disulfide/thiol ratio was significantly higher in exposed group (p = .034). Also, a positive correlation was detected between disulfide/thiol ratio and 1-OH-pyrene values (r = .249, p = .036). Thiol/disulfide homeostasis was found to be disturbed in asphalt workers. The novel test used in this study may be useful for evaluating the oxidative status in polycyclic aromatic hydrocarbon (PAH) exposure.

  18. UVA causes dual inactivation of cathepsin B and L underlying lysosomal dysfunction in human dermal fibroblasts.

    Science.gov (United States)

    Lamore, Sarah D; Wondrak, Georg T

    2013-06-05

    Cutaneous exposure to chronic solar UVA-radiation is a causative factor in photocarcinogenesis and photoaging. Recently, we have identified the thiol-dependent cysteine-protease cathepsin B as a novel UVA-target undergoing photo-oxidative inactivation upstream of autophagic-lysosomal dysfunction in fibroblasts. In this study, we examined UVA effects on a wider range of cathepsins and explored the occurrence of UVA-induced cathepsin inactivation in other cultured skin cell types. In dermal fibroblasts, chronic exposure to non-cytotoxic doses of UVA caused pronounced inactivation of the lysosomal cysteine-proteases cathepsin B and L, effects not observed in primary keratinocytes and occurring only to a minor extent in primary melanocytes. In order to determine if UVA-induced lysosomal impairment requires single or dual inactivation of cathepsin B and/or L, we used a genetic approach (siRNA) to selectively downregulate enzymatic activity of these target cathepsins. Monitoring an established set of protein markers (including LAMP1, LC3-II, and p62) and cell ultrastructural changes detected by electron microscopy, we observed that only dual genetic antagonism (targeting both CTSB and CTSL expression) could mimic UVA-induced autophagic-lysosomal alterations, whereas single knockdown (targeting CTSB or CTSL only) did not display 'UVA-mimetic' effects failing to reproduce the UVA-induced phenotype. Taken together, our data demonstrate that chronic UVA inhibits both cathepsin B and L enzymatic activity and that dual inactivation of both enzymes is a causative factor underlying UVA-induced impairment of lysosomal function in dermal fibroblasts.

  19. Mitochondrial Thioredoxin-Glutathione Reductase from Larval Taenia crassiceps (Cysticerci

    Directory of Open Access Journals (Sweden)

    Alberto Guevara-Flores

    2010-01-01

    Full Text Available Mitochondrial thioredoxin-glutathione reductase was purified from larval Taenia crassiceps (cysticerci. The preparation showed NADPH-dependent reductase activity with either thioredoxin or GSSG, and was able to perform thiol/disulfide exchange reactions. At 25∘C specific activities were 437  ±  27 mU mg-1 and 840  ±  49 mU mg-1 with thioredoxin and GSSG, respectively. Apparent Km values were 0.87  ±  0.04  μM, 41  ±  6  μM and 19  ±  10  μM for thioredoxin, GSSG and NADPH, respectively. Thioredoxin from eukaryotic sources was accepted as substrate. The enzyme reduced H2O2 in a NADPH-dependent manner, although with low catalytic efficiency. In the presence of thioredoxin, mitochondrial TGR showed a thioredoxin peroxidase-like activity. All disulfide reductase activities were inhibited by auranofin, suggesting mTGR is dependent on selenocysteine. The reductase activity with GSSG showed a higher dependence on temperature as compared with the DTNB reductase activity. The variation of the GSSG- and DTNB reductase activities on pH was dependent on the disulfide substrate. Like the cytosolic isoform, mTGR showed a hysteretic kinetic behavior at moderate or high GSSG concentrations, but it was less sensitive to calcium. The enzyme was able to protect glutamine synthetase from oxidative inactivation, suggesting that mTGR is competent to contend with oxidative stress.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2009-05-15

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

  1. Lysosomal exoglycosidases in nasal polyps.

    Science.gov (United States)

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

    2013-01-01

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

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Science.gov (United States)

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

    2012-03-13

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

  4. 100% thiol-functionalized ethylene PMOs prepared by "thiol acid-ene" chemistry.

    Science.gov (United States)

    Esquivel, Dolores; van den Berg, Otto; Romero-Salguero, Francisco J; Du Prez, Filip; Van der Voort, Pascal

    2013-03-21

    A novel thiol functionalized bis-silane PMO precursor was synthesized by highly efficient thiol acid-ene chemistry between the double bonds of 1,2-(E)-bis(triethoxysilyl)ethene and thioacetic acid. After aminolysis the self-assembly process of the formed SH-precursor with Pluronic P123 under acidic conditions yields the first 100% thiol-PMO material with good structural ordering.

  5. Hypochlorite-induced oxidation of thiols

    DEFF Research Database (Denmark)

    Davies, Michael Jonathan; Hawkins, C L

    2000-01-01

    -molecular-weight thiols such as reduced glutathione (GSH), and sulfur-containing amino acids in proteins, are major targets for HOCl. Radicals have not generally been implicated as intermediates in thiol oxidation by HOCl, though there is considerable literature evidence for the involvement of radicals in the metal ion...... to the thiol, thiyl radicals are the major species detected by EPR spin trapping. When the HOCl is present in excess over the thiol, additional radicals are detected with compounds which contain amine functions; these additional radicals are assigned to nitrogen-centered species. Evidence is presented...... for the involvement of sulfenyl chlorides (RSCl) in the formation of these radicals, and studies with an authentic sulfenyl chloride have demonstrated that this compound readily decomposes in thermal-, metal-ion- or light-catalysed reactions to give thiyl radicals. The formation of thiyl radicals on oxidation...

  6. Nucleophilic Addition of Thiols to Deoxynivalenol.

    Science.gov (United States)

    Stanic, Ana; Uhlig, Silvio; Solhaug, Anita; Rise, Frode; Wilkins, Alistair L; Miles, Christopher O

    2015-09-02

    Conjugation of deoxynivalenol (DON) with sulfur compounds is recognized as a significant reaction pathway, and putative DON-glutathione (DON-GSH) conjugates have been reported in planta. To understand and control the reaction of trichothecenes with biologically important thiols, we studied the reaction of DON, T-2 tetraol, and de-epoxy-DON with a range of model thiols. Reaction conditions were optimized for DON with 2-mercaptoethanol. Major reaction products were identified using HRMS and NMR spectroscopy. The results indicate that thiols react reversibly with the double bond (Michael addition) and irreversibly with the epoxide group in trichothecenes. These reactions occurred at different rates, and multiple isomers were produced including diconjugated forms. LC-MS analyses indicated that glutathione and cysteine reacted with DON in a similar manner to the model thiols. In contrast to DON, none of the tested mercaptoethanol adducts displayed toxicity in human monocytes or induced pro-inflammatory cytokines in human macrophages.

  7. Evolution of thiol protective systems in prokaryotes

    Science.gov (United States)

    Fahey, R. C.; Newton, G. L.

    1986-01-01

    Biological thiols are essential elements in most aspects of cell function but undergo rapid oxidation to disulfides in the presence of oxygen. The evolution of systems to protect against such oxygen toxicity was essential to the emergence of aerobic life. The protection system used by eukaryotes is based upon glutathione (GSH) and GSH-dependent enzymes but many bacteria lack GSH and apparently use other mechanisms. The objective of this research is to elaborate the thiol protective mechanisms employed by prokaryotes of widely divergent evolutionary origin and to understand why GSH became the central thiol employed in essentially all higher organisms. Thiol-selective fluorescent labeling and HPLC analysis has been used to determine key monothiol components.

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

    Science.gov (United States)

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

    2013-11-01

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

  9. Lysosomal Storage Disorders and Malignancy

    Directory of Open Access Journals (Sweden)

    Gregory M. Pastores

    2017-02-01

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

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

    DEFF Research Database (Denmark)

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

    2010-01-01

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

  11. ErbB2-associated changes in the lysosomal proteome

    DEFF Research Database (Denmark)

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

    2011-01-01

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

  12. Mitochondrial Dysfunction in Lysosomal Storage Disorders

    Directory of Open Access Journals (Sweden)

    Mario de la Mata

    2016-10-01

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

  13. Thiol-based redox switches in prokaryotes.

    Science.gov (United States)

    Hillion, Melanie; Antelmann, Haike

    2015-05-01

    Bacteria encounter reactive oxygen species (ROS) as a consequence of the aerobic life or as an oxidative burst of activated neutrophils during infections. In addition, bacteria are exposed to other redox-active compounds, including hypochloric acid (HOCl) and reactive electrophilic species (RES) such as quinones and aldehydes. These reactive species often target the thiol groups of cysteines in proteins and lead to thiol-disulfide switches in redox-sensing regulators to activate specific detoxification pathways and to restore the redox balance. Here, we review bacterial thiol-based redox sensors that specifically sense ROS, RES and HOCl via thiol-based mechanisms and regulate gene transcription in Gram-positive model bacteria and in human pathogens, such as Staphylococcus aureus and Mycobacterium tuberculosis. We also pay particular attention to emerging widely conserved HOCl-specific redox regulators that have been recently characterized in Escherichia coli. Different mechanisms are used to sense and respond to ROS, RES and HOCl by 1-Cys-type and 2-Cys-type thiol-based redox sensors that include versatile thiol-disulfide switches (OxyR, OhrR, HypR, YodB, NemR, RclR, Spx, RsrA/RshA) or alternative Cys phosphorylations (SarZ, MgrA, SarA), thiol-S-alkylation (QsrR), His-oxidation (PerR) and methionine oxidation (HypT). In pathogenic bacteria, these redox-sensing regulators are often important virulence regulators and required for adapation to the host immune defense.

  14. Protein-thiol substitution or protein dethiolation by thiol/disulfide exchange reactions: the albumin model.

    Science.gov (United States)

    Summa, Domenico; Spiga, Ottavia; Bernini, Andrea; Venditti, Vincenzo; Priora, Raffaella; Frosali, Simona; Margaritis, Antonios; Di Giuseppe, Danila; Niccolai, Neri; Di Simplicio, Paolo

    2007-11-01

    Dethiolation experiments of thiolated albumin with thionitrobenzoic acid and thiols (glutathione, cysteine, homocysteine) were carried out to understand the role of albumin in plasma distribution of thiols and disulfide species by thiol/disulfide (SH/SS) exchange reactions. During these experiments we observed that thiolated albumin underwent thiol substitution (Alb-SS-X+RSHAlb-SS-R+XSH) or dethiolation (Alb-SS-X+XSHAlb-SH+XSSX), depending on the different pK(a) values of thiols involved in protein-thiol mixed disulfides (Alb-SS-X). It appeared in these reactions that the compound with lower pK(a) in mixed disulfide was a good leaving group and that the pK(a) differences dictated the kind of reaction (substitution or dethiolation). Thionitrobenzoic acid, bound to albumin by mixed disulfide (Alb-TNB), underwent rapid substitution after thiol addition, forming the corresponding Alb-SS-X (peaks at 0.25-1 min). In turn, Alb-SS-X were dethiolated by the excess nonprotein SH groups because of the lower pK(a) value in mixed disulfide with respect to that of other thiols. Dethiolation of Alb-SS-X was accompanied by formation of XSSX and Alb-SH up to equilibrium levels at 35 min, which were different for each thiol. Structures by molecular simulation of thiolated albumin, carried out for understanding the role of sulfur exposure in mixed disulfides in dethiolation process, evidenced that the sulfur exposure is important for the rate but not for determining the kind of reaction (substitution or dethiolation). Our data underline the contribution of SH/SS exchanges to determine levels of various thiols as reduced and oxidized species in human plasma.

  15. Changes in plasma thiol levels induced by different phases of treatment in breast cancer; the role of commercial extract from black chokeberry.

    Science.gov (United States)

    Kędzierska, Magdalena; Głowacki, Rafał; Czernek, Urszula; Szydłowska-Pazera, Katarzyna; Potemski, Piotr; Piekarski, Janusz; Jeziorski, Arkadiusz; Olas, Beata

    2013-01-01

    Different low-molecular-weight thiols, including glutathione, cysteine, and cysteinylglycine are physiological free radical scavengers. On the other hand, homocysteine may play a role as an oxidant. The aim of our present study was to establish in vitro the effects of the commercial extract of Aronia melanocarpa (Aronox(®)) on the amount of selected low-molecular-weight thiols and the activity of antioxidative enzymes (superoxide dismutase, glutathione peroxidase, and glutathione reductase) in plasma obtained from patients with invasive breast cancer during different phases of treatment [before or after the surgery and patients after different phases of chemotherapy (doxorubicin and cyclophosphamide)] and from healthy subjects. Patients were hospitalized in Department of Oncological Surgery and Department of Chemotherapy, Medical University of Lodz, Poland. The level of low-molecular-weight thiols was determined by high-performance liquid chromatography. We observed that in the presence of the Aronia extract changes in amount of thiols in plasma from breast cancer patients (at all tested groups) were significantly reduced. Our results showed that tested commercial extract reduced modifications of antioxidative enzymes activity in plasma from patients during different phases of treatment, but this effect was not statistical significant. Our results suggest that the Aronia extract supplementation in breast cancer patients has a beneficial effect on thiols concentration in plasma. Plasma, as reported in this work, could be used as an experimental model to evaluate the beneficial action of plant supplements, including phenolic extracts on thiols or other molecules during different phases of treatment.

  16. Genomics and X-ray microanalysis indicate that Ca2+ and thiols mediate the aggregation and adhesion of Xylella fastidiosa

    Directory of Open Access Journals (Sweden)

    Leite B.

    2002-01-01

    Full Text Available The availability of the genome sequence of the bacterial plant pathogen Xylella fastidiosa, the causal agent of citrus variegated chlorosis, is accelerating important investigations concerning its pathogenicity. Plant vessel occlusion is critical for symptom development. The objective of the present study was to search for information that would help to explain the adhesion of X. fastidiosa cells to the xylem. Scanning electron microscopy revealed that adhesion may occur without the fastidium gum, an exopolysaccharide produced by X. fastidiosa, and X-ray microanalysis demonstrated the presence of elemental sulfur both in cells grown in vitro and in cells found inside plant vessels, indicating that the sulfur signal is generated by the pathogen surface. Calcium and magnesium peaks were detected in association with sulfur in occluded vessels. We propose an explanation for the adhesion and aggregation process. Thiol groups, maintained by the enzyme peptide methionine sulfoxide reductase, could be active on the surface of the bacteria and appear to promote cell-cell aggregation by forming disulfide bonds with thiol groups on the surface of adjacent cells. The enzyme methionine sulfoxide reductase has been shown to be an auxiliary component in the adhesiveness of some human pathogens. The negative charge conferred by the ionized thiol group could of itself constitute a mechanism of adhesion by allowing the formation of divalent cation bridges between the negatively charged bacteria and predominantly negatively charged xylem walls.

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

    Science.gov (United States)

    Lloyd-Evans, Emyr; Platt, Frances M

    2011-08-01

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

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

    Directory of Open Access Journals (Sweden)

    Ekaterina A Ivanova

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

  19. Imine reductases (IREDs).

    Science.gov (United States)

    Mangas-Sanchez, Juan; France, Scott P; Montgomery, Sarah L; Aleku, Godwin A; Man, Henry; Sharma, Mahima; Ramsden, Jeremy I; Grogan, Gideon; Turner, Nicholas J

    2017-04-01

    Imine reductases (IREDs) have emerged as a valuable new set of biocatalysts for the asymmetric synthesis of optically active amines. The development of bioinformatics tools and searchable databases has led to the identification of a diverse range of new IRED biocatalysts that have been characterised and employed in different synthetic processes. This review describes the latest developments in the structural and mechanistic aspects of IREDs, together with synthetic applications of these enzymes, and identifies ongoing and future challenges in the field. Copyright © 2016 Elsevier Ltd. All rights reserved.

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

    NARCIS (Netherlands)

    Gabriel, T.L.

    2017-01-01

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

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

    NARCIS (Netherlands)

    Gabriel, T.L.

    2017-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

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

    2013-01-01

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

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

    Science.gov (United States)

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

    2013-04-01

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

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

    Science.gov (United States)

    Miller, Austin; Schafer, Jessica; Upchurch, Cameron; Spooner, Ellen; Huynh, Julie; Hernandez, Sebastian; McLaughlin, Brooke; Oden, Liam; Fares, Hanna

    2015-03-01

    Lysosomes are dynamic organelles that undergo cycles of fusion and fission with themselves and with other organelles. Following fusion with late endosomes to form hybrid organelles, lysosomes are reformed as discrete organelles. This lysosome reformation or formation is a poorly understood process that has not been systematically analyzed and that lacks known regulators. In this study, we quantitatively define the multiple steps of lysosome formation and identify the first regulator of this process.

  5. Effects of ethanol, acetaldehyde and cholesteryl esters on pancreatic lysosomes.

    OpenAIRE

    Wilson, J S; Apte, M V; Thomas, M. C.; Haber, P S; Pirola, R C

    1992-01-01

    Recent studies indicate that altered lysosomal function may be involved in the early stages of pancreatic injury. Chronic consumption of ethanol increases rat pancreatic lysosomal fragility. The aim of this study is to determine whether the lysosomal fragility observed after chronic ethanol consumption is mediated by ethanol per se, its oxidative metabolite acetaldehyde or cholesteryl esters (substances which accumulate in the pancreas after ethanol consumption). Pancreatic lysosomes from cho...

  6. Lysosomal storage disease 2 - Pompe's disease

    NARCIS (Netherlands)

    van der Ploeg, Ans T.; Reuser, Arnold J. J.

    2008-01-01

    Pompe's disease, glycogen-storage disease type II, and acid maltase deficiency are alternative names for the same metabolic disorder. It is a pan-ethnic autosomal recessive trait characterised by acid alpha-glucosidase deficiency leading to lysosomal glycogen storage. Pompe's disease is also

  7. Transport of Lysosome-Related Organelles

    NARCIS (Netherlands)

    Jordens, Ingrid

    2005-01-01

    Many intracellular compartments, including (MHC class II-containing) lysosomes, melanosomes and phagosomes, move along microtubules in a bi-directional manner due to the alternating activities of the plus-end directed kinesin motor and the minus-end directed dynein-dynactin motor. However, it is lar

  8. Transport of Lysosome-Related Organelles

    NARCIS (Netherlands)

    Jordens, Ingrid

    2005-01-01

    Many intracellular compartments, including (MHC class II-containing) lysosomes, melanosomes and phagosomes, move along microtubules in a bi-directional manner due to the alternating activities of the plus-end directed kinesin motor and the minus-end directed dynein-dynactin motor. However, it is

  9. Lysosomal proteolysis: effects of aging and insulin.

    Science.gov (United States)

    Gromakova, I A; Konovalenko, O A

    2003-07-01

    Age-related characteristics of the effect of insulin on the activity of lysosomal proteolytic enzymes were studied. The relationship between the insulin effect on protein degradation and insulin degradation was analyzed. The effect of insulin on the activities of lysosomal enzymes was opposite in young and old rats (inhibitory in 3-month-old and stimulatory in 24-month-old animals). The activities of proteolytic enzymes were regulated by insulin in a glucose-independent manner: similar hypoglycemic effects of insulin in animals of different ages were accompanied by opposite changes in the activities of lysosomal enzymes. The inhibition of lysosomal enzymes by insulin in 3-month-old rats is consistent with a notion on the inhibitory effect of insulin on protein degradation. An opposite insulin effect in 24-month-old rats (i.e., stimulation of proteolytic activity by insulin) may be partly associated with attenuation of the degradation of insulin, resulting in disturbances in signaling that mediates the regulatory effects of insulin on protein degradation.

  10. Improvement of oxidized glutathione fermentation by thiol redox metabolism engineering in Saccharomyces cerevisiae.

    Science.gov (United States)

    Hara, Kiyotaka Y; Aoki, Naoko; Kobayashi, Jyumpei; Kiriyama, Kentaro; Nishida, Keiji; Araki, Michihiro; Kondo, Akihiko

    2015-11-01

    Glutathione is a valuable tripeptide widely used in the pharmaceutical, food, and cosmetic industries. In industrial fermentation, glutathione is currently produced primarily using the yeast Saccharomyces cerevisiae. Intracellular glutathione exists in two forms; the majority is present as reduced glutathione (GSH) and a small amount is present as oxidized glutathione (GSSG). However, GSSG is more stable than GSH and is a more attractive form for the storage of glutathione extracted from yeast cells after fermentation. In this study, intracellular GSSG content was improved by engineering thiol oxidization metabolism in yeast. An engineered strain producing high amounts of glutathione from over-expression of glutathione synthases and lacking glutathione reductase was used as a platform strain. Additional over-expression of thiol oxidase (1.8.3.2) genes ERV1 or ERO1 increased the GSSG content by 2.9-fold and 2.0-fold, respectively, compared with the platform strain, without decreasing cell growth. However, over-expression of thiol oxidase gene ERV2 showed almost no effect on the GSSG content. Interestingly, ERO1 over-expression did not decrease the GSH content, raising the total glutathione content of the cell, but ERV1 over-expression decreased the GSH content, balancing the increase in the GSSG content. Furthermore, the increase in the GSSG content due to ERO1 over-expression was enhanced by additional over-expression of the gene encoding Pdi1, whose reduced form activates Ero1 in the endoplasmic reticulum. These results indicate that engineering the thiol redox metabolism of S. cerevisiae improves GSSG and is critical to increasing the total productivity and stability of glutathione.

  11. Opposing regulation of histamine-induced calcium signaling by sodium selenite and ebselen via alterations of thiol redox status.

    Science.gov (United States)

    Zhang, Huihui; Zhong, Liangwei

    2010-01-25

    Elevated blood histamine plays a role in the pathogenesis of atherosclerosis. Calcium signaling mediates histamine action in endothelial cells. Selenium (Se) is a dietary essential trace element for humans. Se compounds in different oxidation states were found to exhibit an opposing effect on the histamine-induced calcium signaling in the ECV304 cell line. When Se in the form of sodium selenite was added in the cell culture, the reactivity of the histamine H(1)-receptor was increased as reported in our previous paper. We here show that as a culture supplement, sodium selenite enhanced the activity of selenoprotein thioredoxin reductase (TrxR) and the calcium response to histamine stimulation, which were reversed by treating the cells with gold thioglucose, a nucleophilic drug that selectively modifies thiolate/selenolate groups. Sodium selenite most likely caused a reductive shift in the thiol/disulfide redox balance through increasing TrxR activity. In contrast, when the cells were treated with Se in the form of ebselen, a thiol oxidant with peroxidase-like activity, histamine-induced calcium release and calcium entry were significantly suppressed. This effect appeared related to the thiol-directed modification rather than the peroxidase-like activity of ebselen, because this inhibitory effect was not replicated by increasing cellular peroxidase activity. Thus, the opposing effects of sodium selenite and ebselen on histamine-induced calcium signaling are achieved, at least in part, through their opposite actions in modulating the thiol/disulfide redox state.

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

    Science.gov (United States)

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

    2011-09-01

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

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

    DEFF Research Database (Denmark)

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

    2014-01-01

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

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

    Science.gov (United States)

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

    2015-04-29

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

  15. Neuroinflammatory paradigms in lysosomal storage diseases

    Directory of Open Access Journals (Sweden)

    Megan Elizabeth Bosch

    2015-10-01

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

  16. Mitigating role of baicalein on lysosomal enzymes and xenobiotic metabolizing enzyme status during lung carcinogenesis of Swiss albino mice induced by benzo(a)pyrene.

    Science.gov (United States)

    Naveenkumar, Chandrashekar; Raghunandakumar, Subramanian; Asokkumar, Selvamani; Binuclara, John; Rajan, Balan; Premkumar, Thandavamoorthy; Devaki, Thiruvengadam

    2014-06-01

    The lungs mainly serve as a primary site for xenobiotic metabolism and constitute an important defense mechanism against inhalation of carcinogens. Our current study aimed to evaluate the chemotherapeutic efficacy of baicalein (BE) in Swiss albino mice exposed to tobacco-specific carcinogen benzo(a)pyrene [B(a)P] for its ability to mitigate pulmonary carcinogenesis. Here, we report that altered activities/levels of lysosomal enzymes (cathepsin-D, cathepsin-B, acid phosphatase, β-D-galactosidase, β-D-glucuronidase, and β-D-N-acetyl glucosaminidase), phase I biotransformation enzymes (cytochrome P450, cytochrome b5, NADPH-cytochrome P450 reductase, and NADH-cytochrome b5 reductase), and phase II enzymes (glutathione S-transferase, UDP-glucuronyl transferase, and DT-diaphorase) were observed in the B(a)P-induced mice. Treatment with BE significantly restored back the activities/levels of lysosomal enzymes, phase I and phase II biotransformation enzymes. Moreover, assessment of lysosomal abnormalities by transmission electron microscopic examination revealed that BE treatment effectively counteract B(a)P-induced oxidative damages. Protein expression levels studied by immunohistochemistry, immunofluorescence, and immunoblot analysis of CYP1A1 revealed that BE treatment effectively negate B(a)P-induced upregulated expression of CYP1A1. Further analysis of scanning electron microscopic studies in lung was carried out to substantiate the anticarcinogenic effect of BE. The overall data suggest that BE treatment significantly inhibits lysosomal and microsomal dysfunction, thus revealing its potent anticarcinogenic effect.

  17. Total Thiols: Biomedical Importance And Their Alteration In Various Disorders

    Directory of Open Access Journals (Sweden)

    Mungli Prakash

    2009-09-01

    Full Text Available Thiols are the organic compounds that contain a sulphydryl group. Among all the antioxidants that are available in the body, thiols constitute the major portion of the total body antioxidants and they play a significant role in defense against reactive oxygen species. Total thiols composed of both intracellular and extracellular thiols either in the free form as oxidized or reduced glutathione, or thiols bound to proteins. Among the thiols that are bound to proteins, albumin makes the major portion of the protein bound thiols, which binds to sufhydryl group at its cysteine-34 portion. Apart from their role in defense against free radicals, thiols share significant role in detoxification, signal transduction, apoptosis and various other functions at molecular level. The thiol status in the body can be assessed easily by determining the serum levels of thiols. Decreased levels of thiols has been noted in various medical disorders including chronic renal failure and other disorders related to kidney, cardiovascular disorders, stroke and other neurological disorders, diabetes mellitus, alcoholic cirrhosis and various other disorders. Therapy using thiols has been under investigation for certain disorders.

  18. Fabrication and bonding of thiol-ene-based microfluidic devices

    DEFF Research Database (Denmark)

    Sikanen, Tiina M; Lafleur, Josiane P.; Moilanen, Maria-Elisa

    2013-01-01

    In this work, the bonding strength of microchips fabricated by thiol-ene free-radical polymerization was characterized in detail by varying the monomeric thiol/allyl composition from the stoichiometric ratio (1:1) up to 100% excess of thiol (2:1) or allyl (1:2) functional groups. Four different t...

  19. Chiral thiols. Synthesis and enantiomeric excess determination

    NARCIS (Netherlands)

    Strijtveen, Bernardus Wilhelmus Maria

    1987-01-01

    Chiral thiols are a class of chira compounds, which is gaining mone and nore attention the last decenniun. Especially α-mercaptocarboxylic acids are frequently found as a structunal unit in (biologically) important products. As a consequence, many synthetic analoga or derivatives of these natural pr

  20. A Search for Interstellar Monohydric Thiols

    Science.gov (United States)

    Gorai, Prasanta; Das, Ankan; Das, Amaresh; Sivaraman, Bhalamurugan; Etim, Emmanuel E.; Chakrabarti, Sandip K.

    2017-02-01

    It has been pointed out by various astronomers that a very interesting relationship exists between interstellar alcohols and the corresponding thiols (sulfur analog of alcohols) as far as the spectroscopic properties and chemical abundances are concerned. Monohydric alcohols such as methanol and ethanol are widely observed and 1-propanol was recently claimed to have been seen in Orion KL. Among the monohydric thiols, methanethiol (chemical analog of methanol) has been firmly detected in Orion KL and Sgr B2(N2) and ethanethiol (chemical analog of ethanol) has been observed in Sgr B2(N2), though the confirmation of this detection is yet to come. It is very likely that higher order thiols could be observed in these regions. In this paper, we study the formation of monohydric alcohols and their thiol analogs. Based on our quantum chemical calculation and chemical modeling, we find that the Tg conformer of 1-propanethiol is a good candidate of astronomical interest. We present various spectroscopically relevant parameters of this molecule to assist in its future detection in the interstellar medium.

  1. Evaluation of arene ruthenium(II) N-heterocyclic carbene complexes as organometallics interacting with thiol and selenol containing biomolecules.

    Science.gov (United States)

    Oehninger, Luciano; Stefanopoulou, Maria; Alborzinia, Hamed; Schur, Julia; Ludewig, Stephanie; Namikawa, Kazuhiko; Muñoz-Castro, Alvaro; Köster, Reinhard W; Baumann, Knut; Wölfl, Stefan; Sheldrick, William S; Ott, Ingo

    2013-02-07

    Metal complexes with N-heterocyclic carbene (NHC) ligands have been widely used in catalytic chemistry and are now increasingly considered for the development of new chemical tools and metal based drugs. Ruthenium complexes of the type (p-cymene)(NHC)RuCl(2) interacted with biologically relevant thiols and selenols, which resulted in the inhibition of enzymes such as thioredoxin reductase or cathepsin B. Pronounced antiproliferative effects could be obtained provided that an appropriate cellular uptake was achieved. Inhibition of tumor cell growth was accompanied by a perturbation of metabolic parameters such as cellular respiration.

  2. Activity of lysosomal exoglycosidases in human gliomas.

    Science.gov (United States)

    Wielgat, P; Walczuk, U; Szajda, S; Bień, M; Zimnoch, L; Mariak, Z; Zwierz, K

    2006-12-01

    There is a lot of data suggesting that modifications of cell glycoconjugates may be important in progression of cancer. In the present work we studied activities of lysosomal exoglycosidases: beta-hexosaminidase and its isoenzymes A and B, beta-galactosidase and alpha-mannosidase, in human gliomas. Enzyme activity was determined spectrophotometrically based on the release of p-nitrophenol from p-nitrophenyl-derivative of appropriate sugars. The activities of the exoglycosidases tested were significantly higher in malignant glial tumors than in control tissue (normal brain tissue) and non-glial tumors. The highest activities of exoglycosidases were observed in high-grade gliomas, and a positive correlation of enzyme activities and degree of malignancy was noted. Our results suggest that lysosomal exoglycosidases may participate in the progression and dynamical development of glial tumors.

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

    Science.gov (United States)

    Xiong, Jian; Zhu, Michael X

    2016-08-01

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

  4. Biphasic regulation of lysosomal exocytosis by oxidative stress.

    Science.gov (United States)

    Ravi, Sreeram; Peña, Karina A; Chu, Charleen T; Kiselyov, Kirill

    2016-11-01

    Oxidative stress drives cell death in a number of diseases including ischemic stroke and neurodegenerative diseases. A better understanding of how cells recover from oxidative stress is likely to lead to better treatments for stroke and other diseases. The recent evidence obtained in several models ties the process of lysosomal exocytosis to the clearance of protein aggregates and toxic metals. The mechanisms that regulate lysosomal exocytosis, under normal or pathological conditions, are only beginning to emerge. Here we provide evidence for the biphasic effect of oxidative stress on lysosomal exocytosis. Lysosomal exocytosis was measured using the extracellular levels of the lysosomal enzyme beta-hexosaminidase (ß-hex). Low levels or oxidative stress stimulated lysosomal exocytosis, but inhibited it at high levels. Deletion of the lysosomal ion channel TRPML1 eliminated the stimulatory effect of low levels of oxidative stress. The inhibitory effects of oxidative stress appear to target the component of lysosomal exocytosis that is driven by extracellular Ca(2+). We propose that while moderate oxidative stress promotes cellular repair by stimulating lysosomal exocytosis, at high levels oxidative stress has a dual pathological effect: it directly causes cell damage and impairs damage repair by inhibiting lysosomal exocytosis. Harnessing these adaptive mechanisms may point to pharmacological interventions for diseases involving oxidative proteotoxicity or metal toxicity.

  5. A potentially dynamic lysosomal role for the endogenous TRPML proteins.

    Science.gov (United States)

    Zeevi, David A; Frumkin, Ayala; Offen-Glasner, Vered; Kogot-Levin, Aviram; Bach, Gideon

    2009-10-01

    Lysosomal storage disorders (LSDs) constitute a diverse group of inherited diseases that result from lysosomal storage of compounds occurring in direct consequence to deficiencies of proteins implicated in proper lysosomal function. Pathology in the LSD mucolipidosis type IV (MLIV), is characterized by lysosomal storage of lipids together with water-soluble materials in cells from every tissue and organ of affected patients. Mutations in the mucolipin 1 (TRPML1) protein cause MLIV and TRPML1 has also been shown to interact with two of its paralogous proteins, mucolipin 2 (TRPML2) and mucolipin 3 (TRPML3), in heterologous expression systems. Heterogeneous lysosomal storage is readily identified in electron micrographs of MLIV patient cells, suggesting that proper TRPML1 function is essential for the maintenance of lysosomal integrity. In order to investigate whether TRPML2 and TRPML3 also play a role in the maintenance of lysosomal integrity, we conducted gene-specific knockdown assays against these protein targets. Ultrastructural analysis revealed lysosomal inclusions in both TRPML2 and TRPML3 knockdown cells, suggestive of a common mechanism for these proteins, in parallel with TRPML1, in the regulation of lysosomal integrity. However, co-immunoprecipitation assays revealed that physical interactions between each of the endogenous TRPML proteins are quite limited. In addition, we found that all three endogenous proteins only partially co-localize with each other in lysosomal as well as extra-lysosomal compartments. This suggests that native TRPML2 and TRPML3 might participate with native TRPML1 in a dynamic form of lysosomal regulation. Given that depletion of TRPML2/3 led to lysosomal storage typical to an LSD, we propose that depletion of these proteins might also underlie novel LSD pathologies not described hitherto.

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

    Science.gov (United States)

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

    2014-05-01

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

  7. Pathways of peroxynitrite oxidation of thiol groups.

    Science.gov (United States)

    Quijano, C; Alvarez, B; Gatti, R M; Augusto, O; Radi, R

    1997-02-15

    Peroxynitrite mediates the oxidation of the thiol group of both cysteine and glutathione. This process is associated with oxygen consumption. At acidic pH and a cysteine/peroxynitrite molar ratio of < or = 1.2, there was a single fast phase of oxygen consumption, which increased with increasing concentrations of both cysteine and oxygen. At higher molar ratios the profile of oxygen consumption became biphasic, with a fast phase (phase I) that decreased with increasing cysteine concentration, followed by a slow phase (phase II) whose rate of oxygen consumption increased with increasing cysteine concentration. Oxygen consumption in phase I was inhibited by desferrioxamine and 5,5-dimethyl-1-pyrroline N-oxide, but not by mannitol; superoxide dismutase also inhibited oxygen consumption in phase I, while catalase added during phase II decreased the rate of oxygen consumption. For both cysteine and glutathione, oxygen consumption in phase I was maximal at neutral to acidic pH: in contrast, total thiol oxidation was maximal at alkaline pH. EPR spin-trapping studies using N-tert-butyl-alpha-phenylnitrone indicated that the yield of thiyl radical adducts had a pH profile comparable with that found for oxygen consumption. The apparent second-order rate constants for the reactions of peroxynitrite with cysteine and glutathione were 1290 +/- 30 M-1.S-1 and 281 +/- 6 M-1.S-1 respectively at pH 5.75 and 37 degrees C. These results are consistent with two different pathways participating in the reaction of peroxynitrite with low-molecular-mass thiols: (a) the reaction of the peroxynitrite anion with the protonated thiol group, in a second-order process likely to involve a two-electron oxidation, and (b) the reaction of peroxynitrous acid, or a secondary species derived from it, with the thiolate in a one-electron transfer process that yields thiyl radicals capable of initiating an oxygen-dependent radical chain reaction.

  8. Detection of thiol modifications by hydrogen sulfide.

    Science.gov (United States)

    Williams, E; Pead, S; Whiteman, M; Wood, M E; Wilson, I D; Ladomery, M R; Teklic, T; Lisjak, M; Hancock, J T

    2015-01-01

    Hydrogen sulfide (H2S) is an important gasotransmitter in both animals and plants. Many physiological events, including responses to stress, have been suggested to involve H2S, at least in part. On the other hand, numerous responses have been reported following treatment with H2S, including changes in the levels of antioxidants and the activities of transcription factors. Therefore, it is important to understand and unravel the events that are taking place downstream of H2S in signaling pathways. H2S is known to interact with other reactive signaling molecules such as reactive oxygen species (ROS) and nitric oxide (NO). One of the mechanisms by which ROS and NO have effects in a cell is the modification of thiol groups on proteins, by oxidation or S-nitrosylation, respectively. Recently, it has been reported that H2S can also modify thiols. Here we report a method for the determination of thiol modifications on proteins following the treatment with biological samples with H2S donors. Here, the nematode Caenorhabditis elegans is used as a model system but this method can be used for samples from other animals or plants.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2012-01-27

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

  10. Quantitative modeling of selective lysosomal targeting for drug design

    DEFF Research Database (Denmark)

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

    2008-01-01

    Lysosomes are acidic organelles and are involved in various diseases, the most prominent is malaria. Accumulation of molecules in the cell by diffusion from the external solution into cytosol, lysosome and mitochondrium was calculated with the Fick–Nernst–Planck equation. The cell model considers....... This demonstrates that the cell model can be a useful tool for the design of effective lysosome-targeting drugs with minimal off-target interactions....

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

    OpenAIRE

    1980-01-01

    textabstractThe purpose of the experimental work described in this thesiswas to investigate some aspects of the release and uptake of lysosomal enzymes. The experiments involved the use of normal human and animal fibroblasts and some other cell types such as hepatocytes and hepatoma cells as sources of hydrolytic enzymes, and fibroblasts from patients with lysosomal storage diseases associated with a single lysosomal enzyme deficiency and with "1-cell" disease as recipient cells. In a number ...

  12. Factors and processes modulating phenotypes in neuronopathic lysosomal storage diseases

    OpenAIRE

    Jakóbkiewicz-Banecka, Joanna; Gabig-Cimińska, Magdalena; Banecka-Majkutewicz, Zyta; Banecki, Bogdan; Węgrzyn, Alicja; Węgrzyn, Grzegorz

    2013-01-01

    Lysosomal storage diseases are inherited metabolic disorders caused by genetic defects causing deficiency of various lysosomal proteins, and resultant accumulation of non-degraded compounds. They are multisystemic diseases, and in most of them (>70 %) severe brain dysfunctions are evident. However, expression of various phenotypes in particular diseases is extremely variable, from non-neuronopathic to severely neurodegenerative in the deficiency of the same enzyme. Although all lysosomal stor...

  13. Functional and Structural Characterization of a Thiol Peroxidase from Mycobacterium tuberculosis

    Energy Technology Data Exchange (ETDEWEB)

    Rho,B.; Hung, L.; Holton, J.; Vigil, D.; Kim, S.; Park, M.; Terwilliger, T.; Pedelacq, j.

    2006-01-01

    A thiol peroxidase (Tpx) from Mycobacterium tuberculosis was functionally analyzed. The enzyme shows NADPH-linked peroxidase activity using a thioredoxin-thioredoxin reductase system as electron donor, and anti-oxidant activity in a thiol-dependent metal-catalyzed oxidation system. It reduces H{sub 2}O{sub 2}, t-butyl hydroperoxide, and cumene hydroperoxide, and is inhibited by sulfhydryl reagents. Mutational studies revealed that the peroxidatic (Cys60) and resolving (Cys93) cysteine residues are critical amino acids for catalytic activity. The X-ray structure determined to a resolution of 1.75 Angstroms shows a thioredoxin fold similar to that of other peroxiredoxin family members. Superposition with structural homologues in oxidized and reduced forms indicates that the M. tuberculosis Tpx is a member of the atypical two-Cys peroxiredoxin family. In addition, the short distance that separates the Ca atoms of Cys60 and Cys93 and the location of these cysteine residues in unstructured regions may indicate that the M. tuberculosis enzyme is oxidized, though the side-chain of Cys60 is poorly visible. It is solely in the reduced Streptococcus pneumoniae Tpx structure that both residues are part of two distinct helical segments. The M. tuberculosis Tpx is dimeric both in solution and in the crystal structure. Amino acid residues from both monomers delineate the active site pocket.

  14. Differential response of oxidative stress and thiol metabolism in contrasting rice genotypes for arsenic tolerance.

    Science.gov (United States)

    Tripathi, Preeti; Mishra, Aradhana; Dwivedi, Sanjay; Chakrabarty, Debasis; Trivedi, Prabodh K; Singh, Rana Pratap; Tripathi, Rudra Deo

    2012-05-01

    The mechanism of arsenic (As) tolerance was investigated on two contrasting rice (Oryza sativa L.) genotypes, selected for As tolerance and accumulation. One tolerant (Triguna) and one sensitive (IET-4786) variety were exposed to various arsenate (0-50 μM) levels for 7 d for biochemical analyses. Arsenic induced oxidative stress was more pronounced in IET-4786 than Triguna especially in terms of reactive oxygen species, lipid peroxidation, EC and pro-oxidant enzymes (NADPH oxidase and ascorbate oxidase). However, Triguna tolerated As stress through the enhanced enzymes activities particularly pertaining to thiol metabolism such as serine acetyl transferase (SAT), cysteine synthase (CS), γ-glutamyl cysteine synthase (γ-ECS), γ-glutamyl transpeptidase (γ-GT), and glutathione-S-transferase (GST) as well as arsenate reductase (AR). Besides maintaining the ratio of redox couples GSH/GSSG and ASC/DHA, the level of phytochelatins (PCs) and phytochelatin synthase (PCS) activity were more pronounced in Triguna, in which harmonized responses of thiol metabolism was responsible for As tolerance in contrast to IET-4786 showing its susceptible nature towards As exposure.

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

    Science.gov (United States)

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

    2012-01-01

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

  16. A lysosome-centered view of nutrient homeostasis.

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

    Yang, Chonglin; Wang, Xiaochen

    2017-06-01

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

  18. Quantifying the global cellular thiol-disulfide status

    DEFF Research Database (Denmark)

    Hansen, Rosa E; Roth, Doris; Winther, Jakob R

    2009-01-01

    It is widely accepted that the redox status of protein thiols is of central importance to protein structure and folding and that glutathione is an important low-molecular-mass redox regulator. However, the total cellular pools of thiols and disulfides and their relative abundance have never been...... determined. In this study, we have assembled a global picture of the cellular thiol-disulfide status in cultured mammalian cells. We have quantified the absolute levels of protein thiols, protein disulfides, and glutathionylated protein (PSSG) in all cellular protein, including membrane proteins. These data...... cell types. However, when cells are exposed to a sublethal dose of the thiol-specific oxidant diamide, PSSG levels increase to >15% of all protein cysteine. Glutathione is typically characterized as the "cellular redox buffer"; nevertheless, our data show that protein thiols represent a larger active...

  19. Isolated menthone reductase and nucleic acid molecules encoding same

    Science.gov (United States)

    Croteau, Rodney B; Davis, Edward M; Ringer, Kerry L

    2013-04-23

    The present invention provides isolated menthone reductase proteins, isolated nucleic acid molecules encoding menthone reductase proteins, methods for expressing and isolating menthone reductase proteins, and transgenic plants expressing elevated levels of menthone reductase protein.

  20. Presenilin 1 Maintains Lysosomal Ca2+ Homeostasis via TRPML1 by Regulating vATPase-Mediated Lysosome Acidification

    Directory of Open Access Journals (Sweden)

    Ju-Hyun Lee

    2015-09-01

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

  1. Presenilin 1 Maintains Lysosomal Ca(2+) Homeostasis via TRPML1 by Regulating vATPase-Mediated Lysosome Acidification.

    Science.gov (United States)

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

    2015-09-01

    Presenilin 1 (PS1) deletion or Alzheimer's disease (AD)-linked mutations disrupt lysosomal acidification and proteolysis, which inhibits autophagy. Here, we establish that this phenotype stems from impaired glycosylation and instability of vATPase V0a1 subunit, causing deficient lysosomal vATPase assembly and function. We further demonstrate that elevated lysosomal pH in Presenilin 1 knockout (PS1KO) cells induces abnormal Ca(2+) efflux from lysosomes mediated by TRPML1 and elevates cytosolic Ca(2+). In WT cells, blocking vATPase activity or knockdown of either PS1 or the V0a1 subunit of vATPase reproduces all of these abnormalities. Normalizing lysosomal pH in PS1KO cells using acidic nanoparticles restores normal lysosomal proteolysis, autophagy, and Ca(2+) homeostasis, but correcting lysosomal Ca(2+) deficits alone neither re-acidifies lysosomes nor reverses proteolytic and autophagic deficits. Our results indicate that vATPase deficiency in PS1 loss-of-function states causes lysosomal/autophagy deficits and contributes to abnormal cellular Ca(2+) homeostasis, thus linking two AD-related pathogenic processes through a common molecular mechanism.

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

    Science.gov (United States)

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

    2010-10-01

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

  3. Gaucher disease: a lysosomal neurodegenerative disorder.

    Science.gov (United States)

    Huang, W J; Zhang, X; Chen, W W

    2015-04-01

    Gaucher disease is a multisystemic disorder that affects men and woman in equal numbers and occurs in all ethnic groups at any age with racial variations and an estimated worldwide incidence of 1/75,000. It is caused by a genetic deficient activity of the lysosomal enzyme glucocerebrosidase due to mutations in the β-glucocerebrosidase gene, and resulting in lack of glucocerebroside degradation. The subsequent accumulation of glucocerebroside in lysosomes of tissue macrophages primarily in the liver, bone marrow and spleen, causes damage in haematological, skeletal and nervous systems. The clinical manifestations show a high degree of variability with symptoms that varies according to organs involved. In many cases, these disorders do not correlate with mutations in the β-glucocerebrosidase gene. Although several mutations have been identified as responsible for the deficient activity of glucocerebrosidase, mechanisms by which this enzymatic defect leads to Gaucher disease remain poorly understood. Recent reports indicate the implication of complex mechanisms, including enzyme deficiency, substrate accumulation, unfolded protein response, and macrophage activation. Further elucidating these mechanisms will advance understanding of Gaucher disease and related disorders.

  4. Enhanced lysosomal activity by overexpressed aminopeptidase Y in Saccharomyces cerevisiae.

    Science.gov (United States)

    Yoon, Jihee; Sekhon, Simranjeet Singh; Kim, Yang-Hoon; Min, Jiho

    2016-06-01

    Saccharomyces cerevisiae contains vacuoles corresponding to lysosomes in higher eukaryotes. Lysosomes are dynamic (not silent) organelles in which enzymes can be easily integrated or released when exposed to stressful conditions. Changes in lysosomal enzymes have been observed due to oxidative stress, resulting in an increased function of lysosomes. The protein profiles from H2O2- and NH4Cl-treated lysosomes showed different expression patterns, observed with two-dimensional gel electrophoresis. The aminopeptidase Y protein (APE3) that conspicuously enhanced antimicrobial activity than other proteins was selected for further studies. The S. cerevisiae APE3 gene was isolated and inserted into pYES2.0 expression vector. The GFP gene was inserted downstream to the APE3 gene for confirmation of APE3 targeting to lysosomes, and S. cerevisiae was transformed to pYES2::APE3::GFP. The APE3 did not enter in lysosomes and formed an inclusion body at 30 °C, but it inserted to lysosomes as shown by the merger of GFP with lysosomes at 28 °C. Antimicrobial activity of the cloned S. cerevisiae increased about 5 to 10 % against eight strains, compared to normal cells, and galactose induction is increased more two folds than that of normal cells. Therefore, S. cerevisiae was transformed to pYES2::APE3::GFP, accumulating a large amount of APE3, resulting in increased lysosomal activity. Increase in endogenous levels of lysosomes and their activity following genetic modification can lead to its use in applications such as antimicrobial agents and apoptosis-inducing materials for cancer cells, and consequently, it may also be possible to use the organelles for improving in vitro functions.

  5. Lysosome/lipid droplet interplay in metabolic diseases.

    Science.gov (United States)

    Dugail, Isabelle

    2014-01-01

    Lysosomes and lipid droplets are generally considered as intracellular compartments with divergent roles in cell metabolism, lipid droplets serving as lipid reservoirs in anabolic pathways, whereas lysosomes are specialized in the catabolism of intracellular components. During the last few years, new insights in the biology of lysosomes has challenged this view by providing evidence for the importance of lysosome recycling as a sparing mechanism to maintain cellular fitness. On the other hand the understanding of lipid droplets has evolved from an inert intracellular deposit toward the status of an intracellular organelle with dynamic roles in cellular homeostasis beyond storage. These unrelated aspects have also recently converged in the finding of unexpected lipid droplet/lysosome communication through autophagy, and the discovery of lysosome-mediated lipid droplet degradation called lipopagy. Furthermore, adipocytes which are professional cells for lipid droplet formation were also shown to be active in peptide antigen presentation a pathway requiring lysosomal activity. The potential importance of lipid droplet/lysosome interplay is discussed in the context of metabolic diseases and the setting of chronic inflammation.

  6. [The blood-brain barrier and neurodegenerative lysosomal storage diseases].

    Science.gov (United States)

    Urayama, Akihiko

    2013-02-01

    Enzyme replacement therapy has been a very effective treatment for several lysosomal storage diseases. However, correcting central nervous system (CNS) storage has been challenging due to the presence of the blood-brain barrier (BBB), which hampers the entry of circulating lysosomal enzymes into the brain. In our previous studies, we discovered that luminally expressed cation-independent mannose 6-phosphate (M6P) receptor is a universal transporter for lysosomal enzymes that contain M6P moieties on the enzyme molecule. This receptor-mediated transport of lysosomal enzymes showed developmental down-regulation that resulted in a failure of delivery of lysosomal enzymes across the BBB in the adult brain. Conceptually, if one can re-induce M6P receptor-mediated transport of lysosomal enzymes in adult BBB, this could provide a novel brain targeting approach for treating abnormal storage in the CNS, regardless of the age of subjects. We found that systemic adrenergic stimuli restored functional transport of β-glucuronidase across the adult BBB. The concept of manipulating BBB transport activity by endogenous characteristics has also been demonstrated by another group who showed effective treatment in a Pompe disease model animal in vivo. It is intriguing that lysosomal enzymes utilize multiple mechanisms for their transport across the BBB. This review explores pharmacological manipulations for the delivery of lysosomal enzymes into the CNS, and the mechanisms of their transport across the BBB, based on existing evidence from studies of β-glucuronidase, sulfamidase, acid α-glucosidase, and arylsulfatase A.

  7. Photoaffinity labeling of the lysosomal neuraminidase from bovine testis

    NARCIS (Netherlands)

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

    1990-01-01

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

  8. Artesunate induces cell death in human cancer cells via enhancing lysosomal function and lysosomal degradation of ferritin.

    Science.gov (United States)

    Yang, Nai-Di; Tan, Shi-Hao; Ng, Shukie; Shi, Yin; Zhou, Jing; Tan, Kevin Shyong Wei; Wong, Wai-Shiu Fred; Shen, Han-Ming

    2014-11-28

    Artesunate (ART) is an anti-malaria drug that has been shown to exhibit anti-tumor activity, and functional lysosomes are reported to be required for ART-induced cancer cell death, whereas the underlying molecular mechanisms remain largely elusive. In this study, we aimed to elucidate the molecular mechanisms underlying ART-induced cell death. We first confirmed that ART induces apoptotic cell death in cancer cells. Interestingly, we found that ART preferably accumulates in the lysosomes and is able to activate lysosomal function via promotion of lysosomal V-ATPase assembly. Furthermore, we found that lysosomes function upstream of mitochondria in reactive oxygen species production. Importantly, we provided evidence showing that lysosomal iron is required for the lysosomal activation and mitochondrial reactive oxygen species production induced by ART. Finally, we showed that ART-induced cell death is mediated by the release of iron in the lysosomes, which results from the lysosomal degradation of ferritin, an iron storage protein. Meanwhile, overexpression of ferritin heavy chain significantly protected cells from ART-induced cell death. In addition, knockdown of nuclear receptor coactivator 4, the adaptor protein for ferritin degradation, was able to block ART-mediated ferritin degradation and rescue the ART-induced cell death. In summary, our study demonstrates that ART treatment activates lysosomal function and then promotes ferritin degradation, subsequently leading to the increase of lysosomal iron that is utilized by ART for its cytotoxic effect on cancer cells. Thus, our data reveal a new mechanistic action underlying ART-induced cell death in cancer cells.

  9. Simvastatin promotes NPC1-mediated free cholesterol efflux from lysosomes through CYP7A1/LXRα signalling pathway in oxLDL-loaded macrophages.

    Science.gov (United States)

    Xu, Xiaoyang; Zhang, Aolin; Halquist, Matthew S; Yuan, Xinxu; Henderson, Scott C; Dewey, William L; Li, Pin-Lan; Li, Ningjun; Zhang, Fan

    2017-02-01

    Statins, 3-hydroxyl-3-methylglutaryl coenzyme A reductase inhibitors, are the first-line medications prescribed for the prevention and treatment of coronary artery diseases. The efficacy of statins has been attributed not only to their systemic cholesterol-lowering actions but also to their pleiotropic effects that are unrelated to cholesterol reduction. These pleiotropic effects have been increasingly recognized as essential in statins therapy. This study was designed to investigate the pleiotropic actions of simvastatin, one of the most commonly prescribed statins, on macrophage cholesterol homeostasis with a focus on lysosomal free cholesterol egression. With simultaneous nile red and filipin staining, analysis of confocal/multi-photon imaging demonstrated that simvastatin markedly attenuated unesterified (free) cholesterol buildup in macrophages loaded with oxidized low-density lipoprotein but had little effect in reducing the sizes of cholesteryl ester-containing lipid droplets; the reduction in free cholesterol was mainly attributed to decreases in lysosome-compartmentalized cholesterol. Functionally, the egression of free cholesterol from lysosomes attenuated pro-inflammatory cytokine secretion. It was determined that the reduction of lysosomal free cholesterol buildup by simvastatin was due to the up-regulation of Niemann-Pick C1 (NPC1), a lysosomal residing cholesterol transporter. Moreover, the enhanced enzymatic production of 7-hydroxycholesterol by cytochrome P450 7A1 and the subsequent activation of liver X receptor α underscored the up-regulation of NPC1. These findings reveal a novel pleiotropic effect of simvastatin in affecting lysosomal cholesterol efflux in macrophages and the associated significance in the treatment of atherosclerosis. © 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

  10. Antibodies with thiol-S-transferase activity

    Energy Technology Data Exchange (ETDEWEB)

    Fan, E.; Oei, Yoko; Sweet, E.; Uno, Tetsuo; Schultz, P.G. [Univ. of California, Berkeley, CA (United States)

    1996-06-12

    A major detoxification pathway used by aerobic organisms involves the conjugation of the tripeptide glutathione (GSH) to the electrophilic center of toxic substances. This reaction is catalyzed by a class of enzymes referred to as the glutathione S-transferases (GST) (EC 2.5.1.18). These enzymes activate the cysteine thiol group of GSH for nucleophilic addition to a variety of substrates, including aryl halides, {alpha}{beta}-unsaturated aldehydes and ketones, and epoxides. Despite the availability of X-ray crystal structures, the mechanism whereby glutathione transferases catalyze these addition reactions remains unclear. In order to gain a greater understanding of this important biological transformation, as well as to generate new detoxification catalysts, we have asked whether antibodies can be generated that catalyze similar nucleophilic addition reactions. Our initial efforts focused on the addition reaction of thiol nucleophiles to the nitro-substituted styrene derivative 1. The ratio of k{sub cat}/K{sub m} reported for the reaction of the isozyme 4-4` of rat liver GST with the good substance, 1-chloro-2,4-dinitrobenzene, is approximately 10{sup 4} M{sup -1} s{sup -1} compared to a calculated pseudo-first-order rate constant for the uncatalyzed reaction of approximately 3 x 10{sup -2} s{sup -1} (60 mM GSH, pH = 80). These comparisons suggest that with further improvements in hapten design, catalytic antibodies may prove a good source of detoxification catalysts. 19 refs., 1 fig.

  11. Photoinduced formation of thiols in human hair.

    Science.gov (United States)

    Fedorkova, M V; Brandt, N N; Chikishev, A Yu; Smolina, N V; Balabushevich, N G; Gusev, S A; Lipatova, V A; Botchey, V M; Dobretsov, G E; Mikhalchik, E V

    2016-11-01

    Raman, scanning electron, and optical microscopy of hair and spectrophotometry of soluble hair proteins are used to study the effect of UV-vis radiation on white hair. The samples of a healthy subject are irradiated using a mercury lamp and compared with non-irradiated (control) hair. The cuticle damage with partial exfoliation is revealed with the aid of SEM and optical microscopy of semifine sections. Gel filtration chromatography shows that the molecular weight of soluble proteins ranges from 5 to 7kDa. Absorption spectroscopy proves an increase in amount of thiols in a heavier fraction of the soluble proteins of irradiated samples under study. Raman data indicate a decrease in the amount of SS and CS bonds in cystines and an increase in the amount of SH bonds due to irradiation. Such changes are more pronounced in peripheral regions of hair. Conformational changes of hair keratins presumably related to the cleavage of disulfide bonds, follow from variations in amide I and low-frequency Raman bands. An increase in the content of thiols in proteins revealed by both photometric data on soluble proteins and Raman microspectroscopy of hair cuts can be used to develop a protocol of the analysis of photoinduced hair modification. Copyright © 2016 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

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

    2016-01-01

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

  13. Trametes versicolor carboxylate reductase uncovered

    OpenAIRE

    Winkler, Margit; Winkler, Christoph K.

    2016-01-01

    Abstract The first carboxylate reductase from Trametes versicolor was identified, cloned, and expressed in Escherichia coli. The enzyme reduces aromatic acids such as benzoic acid and derivatives, cinnamic acid, and 3-phenylpropanoic acid, but also aliphatic acids such as octanoic acid are reduced. Graphical abstract

  14. Colorimetric and fluorescent detection of biological thiols in aqueous solution

    Institute of Scientific and Technical Information of China (English)

    Yin-Hui Li; Jin-Feng Yang; Chang-Hui Liu; Ji-Shan Li; Rong-Hua Yang

    2013-01-01

    A new colorimetric and fluorescent probe,2-(2,4-dinitrostyryl)-1,3,3-trimethyl-3H-indolium iodide (DTI),for selective and sensitive detection of biological thiols is reported.In aqueous solution at physiological pH 7.4,biological thiols react with DTI via Michael addition to give the brownish red adduct concomitant with fluorescence emission decrease.

  15. Biologically important thiols in various vegetables and fruits.

    Science.gov (United States)

    Demirkol, Omca; Adams, Craig; Ercal, Nuran

    2004-12-29

    Biological thiols are important antioxidants, and recent studies showed that their contents vary depending on the groups of foodstuffs. Therefore, we investigated the levels of some biological thiols in various vegetables and fruits by using a sensitive high-performance liquid chromatography (HPLC) technique. Biological thiols measured in some vegetables and fruits include glutathione (L-glutamyl-L-cysteinly glycine, GSH), N-acetylcysteine (NAC), captopril [CAP (C9H15NO3S)], homocysteine (HCYS), cysteine (CYS), and gamma-glutamyl cysteine (GGC). Our results show that biological thiol contents are between 3-349 nM/g wet weight in vegetables and 4-136 nM/g wet weight in fruits. CAP is only found in asparagus (28 nM/g wet weight). Furthermore, none of the biological thiols analyzed were found in cabbages, red grapes, blackberries, apples, and peaches. Therefore, various vegetables and fruits differ significantly in their thiol contents. Oxidation of these important thiols may occur and result in the production of toxic byproducts, if they are exposed to radiation and ozone treatment for sterilization purposes. Further studies should be performed to monitor the levels of these biological thiols.

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

    Science.gov (United States)

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

    2015-04-17

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

  17. Effects of copper on induction of thiol-compounds and antioxidant enzymes by the fruiting body of Oudemansiella radicata.

    Science.gov (United States)

    Jiang, Juan; Qin, Chuixin; Shu, Xueqin; Chen, Rong; Song, Haihai; Li, Qiao; Xu, Heng

    2015-01-01

    Oudemansiella radicata has been found to have ability to tolerate and accumulate heavy metals. In this study, to know about the metal tolerance and detoxification strategy of O. radicata, the tolerance responses in both cap and stipe of the fruiting body, including the copper content, the changes of thiol compounds production and antioxidant enzymes activities, caused by various copper stress (150-600 mg kg(-1)) during 2-6 days were investigated. Results showed that Cu content in the fruiting bodies increased with the increasing Cu concentrations and growing time, which was higher in cap than that in stipe. For thiols contents, the maximum level was in the sample at 300 mg kg(-1) Cu after 2 d both in cap and stipe, in accordance with superoxide dismutase (SOD) and glutathione reductase (GR) activities. Guaicol peroxidase (POD) activities reached maximum at 150 mg kg(-1) Cu after 4 d and 6 d, respectively in cap and stipe, while the maximum of catalase (CAT) activities was recorded at 300 and 600 mg kg(-1) Cu after 4 d in the cap and stipe, respectively. As a whole, low concentration of Cu stimulated the production of thiols and activated the antioxidant enzymes activities in the fruiting body of O. radicata after 2/4 d, while high-level Cu decreased the thiols production and enzymes activities after 4/6 d. Furthermore, the cap was more sensitive than the stipe to Cu exposure. Different indicators showed different responses to copper accumulation and the different fruiting part (cap and stipe) of O. radicata had ability to response the oxidative stress caused by Cu. Considering the metal accumulation and its own detoxification with short growing time, mushroom might have the potential to be used as bio-accumulator to deal with Cu exposure in the Cu-contaminated farmland soil.

  18. Serum paraoxonase activity and protein thiols in patients with hyperlipidemia

    Institute of Scientific and Technical Information of China (English)

    Mungli Prakash; Jeevan K Shetty; Sudeshna Tripathy; Pannuri Vikram; Manish Verma

    2009-01-01

    Objective: In the present study we evaluated the paraoxonase activity and protein thiols level in south Indian population with newly diagnosed hyperlipidemia. Methods: The study was conducted on 55 newly diagnosed hyperlipidemic pa-tients and 57 healthy controls. Serum paraoxonase activity and protein thiols were estimated by spectrophotometeric method and lipid profile by enzymatic kinetic assay method. Results: Serum paraoxonase activity, protein thiols and high density lipoprotein levels were low and total cholesterol, triglycerides and low density lipoprutein levels were high in patients with hyperlipidemia compared to healthy controls ( P < 0.01 ). Serum paranxonase activity correlated positively with protein thiols and high density lipoprotein (P<0.01). Conclusion: Decreased paraoxonase activity and protein thiols were found in patients with hyperlipi-demia. This may indicate the susceptibility of this population to accelerated atherogenesis and protein oxidation.

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

    Science.gov (United States)

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

    2009-06-01

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

  20. Efficient Functionalization of Oxide-Free Silicon(111) Surfaces: Thiol-yne versus Thiol-ene Click Chemistry

    NARCIS (Netherlands)

    Bhairamadgi, N.S.; Gangarapu, S.; Caipa Campos, M.A.; Paulusse, J.M.J.; Rijn, van C.J.M.; Zuilhof, H.

    2013-01-01

    Thiol-yne click (TYC) chemistry was utilized as a copper-free click reaction for the modification of alkyne-terminated monolayers on oxide-free Si(111) surfaces, and the results were compared with the analogous thiol–ene click (TEC) chemistry. A wide range of thiols such as 9-fluorenylmethoxy-carbon

  1. Selenium in Thioredoxin Reductase: A Mechanistic Perspective†

    Science.gov (United States)

    Lacey, Brian M.; Eckenroth, Brian E.; Flemer, Stevenson; Hondal, Robert J.

    2013-01-01

    Most high Mr thioredoxin reductases (TRs) have the unusual feature of utilizing a vicinal disulfide bond (Cys1-Cys2) which form an eight-membered ring during the catalytic cycle. Many eukaryotic TRs have replaced the Cys2 position of the dyad with the rare amino acid selenocysteine (Sec). Here we demonstrate that Cys- and Sec-containing TRs are distinguished by the importance each class of enzymes places on the 8-membered ring structure in the catalytic cycle. This hypothesis was explored by studying the truncated enzyme missing the C-terminal ring structure in conjunction with oxidized peptide substrates to investigate the reduction and opening of this dyad. The peptide substrates were identical in sequence to the missing part of the enzyme, containing either a disulfide or selenylsulfide linkage, but were differentiated by the presence (cyclic) and absence (acyclic) of the ring structure. The ratio of these turnover rates informs that the ring is only of modest importance for the truncated mouse mitochondrial Sec-TR (ring/no ring = 32), while the ring structure is highly important for the truncated Cys-TRs from D. melanogaster and C. elegans (ring/no ring > 1000). All three enzymes exhibit a similar dependence upon leaving group pKa as shown by the use of the acyclic peptides as substrates. These two factors can be reconciled for Cys-TRs if the ring functions to simultaneously allow for attack by a nearby thiolate while correctly positioning the leaving group sulfur atom to accept a proton from the enzymic general acid. For Sec-TRs the ring is unimportant because the lower pKa of the selenol relative to a thiol obviates its need to be protonated upon S-Se bond scission and permits physical separation of the selenol and the general acid. Further study of the biochemical properties of the truncated Cys and Sec TR enzymes demonstrates that the chemical advantage conferred on the eukaryotic enzyme by a selenol is the ability to function at acidic pH. PMID:18986163

  2. The binding sites on human heme oxygenase-1 for cytochrome p450 reductase and biliverdin reductase.

    Science.gov (United States)

    Wang, Jinling; de Montellano, Paul R Ortiz

    2003-05-30

    Human heme oxygenase-1 (hHO-1) catalyzes the NADPH-cytochrome P450 reductase-dependent oxidation of heme to biliverdin, CO, and free iron. The biliverdin is subsequently reduced to bilirubin by biliverdin reductase. Earlier kinetic studies suggested that biliverdin reductase facilitates the release of biliverdin from hHO-1 (Liu, Y., and Ortiz de Montellano, P. R. (2000) J. Biol. Chem. 275, 5297-5307). We have investigated the binding of P450 reductase and biliverdin reductase to truncated, soluble hHO-1 by fluorescence resonance energy transfer and site-specific mutagenesis. P450 reductase and biliverdin reductase bind to truncated hHO-1 with Kd = 0.4 +/- 0.1 and 0.2 +/- 0.1 microm, respectively. FRET experiments indicate that biliverdin reductase and P450 reductase compete for binding to truncated hHO-1. Mutation of surface ionic residues shows that hHO-1 residues Lys18, Lys22, Lys179, Arg183, Arg198, Glu19, Glu127, and Glu190 contribute to the binding of cytochrome P450 reductase. The mutagenesis results and a computational analysis of the protein surfaces partially define the binding site for P450 reductase. An overlapping binding site including Lys18, Lys22, Lys179, Arg183, and Arg185 is similarly defined for biliverdin reductase. These results confirm the binding of biliverdin reductase to hHO-1 and define binding sites of the two reductases.

  3. Autophagy and lysosomal dysfunction as emerging mechanisms of nanomaterial toxicity

    Directory of Open Access Journals (Sweden)

    Stern Stephan T

    2012-06-01

    Full Text Available Abstract The study of the potential risks associated with the manufacture, use, and disposal of nanoscale materials, and their mechanisms of toxicity, is important for the continued advancement of nanotechnology. Currently, the most widely accepted paradigms of nanomaterial toxicity are oxidative stress and inflammation, but the underlying mechanisms are poorly defined. This review will highlight the significance of autophagy and lysosomal dysfunction as emerging mechanisms of nanomaterial toxicity. Most endocytic routes of nanomaterial cell uptake converge upon the lysosome, making the lysosomal compartment the most common intracellular site of nanoparticle sequestration and degradation. In addition to the endo-lysosomal pathway, recent evidence suggests that some nanomaterials can also induce autophagy. Among the many physiological functions, the lysosome, by way of the autophagy (macroautophagy pathway, degrades intracellular pathogens, and damaged organelles and proteins. Thus, autophagy induction by nanoparticles may be an attempt to degrade what is perceived by the cell as foreign or aberrant. While the autophagy and endo-lysosomal pathways have the potential to influence the disposition of nanomaterials, there is also a growing body of literature suggesting that biopersistent nanomaterials can, in turn, negatively impact these pathways. Indeed, there is ample evidence that biopersistent nanomaterials can cause autophagy and lysosomal dysfunctions resulting in toxicological consequences.

  4. Autophagy and lysosomal dysfunction as emerging mechanisms of nanomaterial toxicity.

    Science.gov (United States)

    Stern, Stephan T; Adiseshaiah, Pavan P; Crist, Rachael M

    2012-06-14

    The study of the potential risks associated with the manufacture, use, and disposal of nanoscale materials, and their mechanisms of toxicity, is important for the continued advancement of nanotechnology. Currently, the most widely accepted paradigms of nanomaterial toxicity are oxidative stress and inflammation, but the underlying mechanisms are poorly defined. This review will highlight the significance of autophagy and lysosomal dysfunction as emerging mechanisms of nanomaterial toxicity. Most endocytic routes of nanomaterial cell uptake converge upon the lysosome, making the lysosomal compartment the most common intracellular site of nanoparticle sequestration and degradation. In addition to the endo-lysosomal pathway, recent evidence suggests that some nanomaterials can also induce autophagy. Among the many physiological functions, the lysosome, by way of the autophagy (macroautophagy) pathway, degrades intracellular pathogens, and damaged organelles and proteins. Thus, autophagy induction by nanoparticles may be an attempt to degrade what is perceived by the cell as foreign or aberrant. While the autophagy and endo-lysosomal pathways have the potential to influence the disposition of nanomaterials, there is also a growing body of literature suggesting that biopersistent nanomaterials can, in turn, negatively impact these pathways. Indeed, there is ample evidence that biopersistent nanomaterials can cause autophagy and lysosomal dysfunctions resulting in toxicological consequences.

  5. Purification and kinetic analysis of cytosolic and mitochondrial thioredoxin glutathione reductase extracted from Taenia solium cysticerci.

    Science.gov (United States)

    Plancarte, Agustin; Nava, Gabriela

    2015-02-01

    Thioredoxin glutathione reductases (TGRs) (EC 1.8.1.9) were purified to homogeneity from the cytosolic (cTsTGR) and mitochondrial (mTsTGR) fractions of Taenia solium, the agent responsible for neurocysticercosis, one of the major central nervous system parasitic diseases in humans. TsTGRs had a relative molecular weight of 132,000, while the corresponding value per subunit obtained under denaturing conditions, was of 62,000. Specific activities for thioredoxin reductase and glutathione reductase substrates for both TGRs explored were in the range or lower than values obtained for other platyhelminths and mammalian TGRs. cTsTGR and mTsTGR also showed hydroperoxide reductase activity using hydroperoxide as substrate. Km(DTNB) and Kcat(DTNB) values for cTsTGR and mTsTGR (88 µM and 1.9 s(-1); 45 µM and 12.6 s(-1), respectively) and Km(GSSG) and Kcat(GSSG) values for cTsTGR and mTsTGR (6.3 µM and 0.96 s(-1); 4 µM and 1.62 s(-1), respectively) were similar to or lower than those reported for mammalian TGRs. Mass spectrometry analysis showed that 12 peptides from cTsTGR and seven from mTsTGR were a match for gi|29825896 thioredoxin glutathione reductase [Echinococcus granulosus], confirming that both enzymes are TGRs. Both T. solium TGRs were inhibited by the gold compound auranofin, a selective inhibitor of thiol-dependent flavoreductases (I₅₀ = 3.25, 2.29 nM for DTNB and GSSG substrates, respectively for cTsTGR; I₅₀ = 5.6, 25.4 nM for mTsTGR toward the same substrates in the described order). Glutathione reductase activity of cTsTGR and mTsTGR exhibited hysteretic behavior with moderate to high concentrations of GSSG; this result was not observed either with thioredoxin, DTNB or NADPH. However, the observed hysteretic kinetics was suppressed with increasing amounts of both parasitic TGRs. These data suggest the existence of an effective substitute which may account for the lack of the detoxification enzymes glutathione reductase

  6. Nitrate Reductase: Properties and Regulation

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Nitrate Reductase (NR) is a rating-limit and key enzyme of nitrate assimilation in plants ,so ,NR activity is important for growth,development and the dry matter accumulation of plants. The regulation of NR activity appears to be rather complex and many studies have been devoted to the description of regulation and properties,but in this paper we focus on the properties and regulation of NR in higher plants.

  7. Two pore channel 2 (TPC2) inhibits autophagosomal-lysosomal fusion by alkalinizing lysosomal pH.

    Science.gov (United States)

    Lu, Yingying; Hao, Bai-Xia; Graeff, Richard; Wong, Connie W M; Wu, Wu-Tian; Yue, Jianbo

    2013-08-16

    Autophagy is an evolutionarily conserved lysosomal degradation pathway, yet the underlying mechanisms remain poorly understood. Nicotinic acid adenine dinucleotide phosphate (NAADP), one of the most potent Ca(2+) mobilizing messengers, elicits Ca(2+) release from lysosomes via the two pore channel 2 (TPC2) in many cell types. Here we found that overexpression of TPC2 in HeLa or mouse embryonic stem cells inhibited autophagosomal-lysosomal fusion, thereby resulting in the accumulation of autophagosomes. Treatment of TPC2 expressing cells with a cell permeant-NAADP agonist, NAADP-AM, further induced autophagosome accumulation. On the other hand, TPC2 knockdown or treatment of cells with Ned-19, a NAADP antagonist, markedly decreased the accumulation of autophagosomes. TPC2-induced accumulation of autophagosomes was also markedly blocked by ATG5 knockdown. Interestingly, inhibiting mTOR activity failed to increase TPC2-induced autophagosome accumulation. Instead, we found that overexpression of TPC2 alkalinized lysosomal pH, and lysosomal re-acidification abolished TPC2-induced autophagosome accumulation. In addition, TPC2 overexpression had no effect on general endosomal-lysosomal degradation but prevented the recruitment of Rab-7 to autophagosomes. Taken together, our data demonstrate that TPC2/NAADP/Ca(2+) signaling alkalinizes lysosomal pH to specifically inhibit the later stage of basal autophagy progression.

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

    Science.gov (United States)

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

    1996-10-14

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

  9. Secondary Lysosomal Changes in Liver in Preclinical Drug Development

    Institute of Scientific and Technical Information of China (English)

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

    2005-01-01

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

  10. 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.

  11. Fatty acyl-CoA reductase

    Energy Technology Data Exchange (ETDEWEB)

    Reiser, Steven E.; Somerville, Chris R.

    1998-12-01

    The present invention relates to bacterial enzymes, in particular to an acyl-CoA reductase and a gene encoding an acyl-CoA reductase, the amino acid and nucleic acid sequences corresponding to the reductase polypeptide and gene, respectively, and to methods of obtaining such enzymes, amino acid sequences and nucleic acid sequences. The invention also relates to the use of such sequences to provide transgenic host cells capable of producing fatty alcohols and fatty aldehydes.

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

    Science.gov (United States)

    Kumar, Nadimpalli Siva; Bhamidimarri, Poorna M

    2015-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Catherine Staudt

    2016-12-01

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

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

    Directory of Open Access Journals (Sweden)

    Dang Hope

    2007-12-01

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

  15. Thiol/disulphide homeostasis as a novel indicator of oxidative stress in sudden sensorineural hearing loss.

    Science.gov (United States)

    Dinc, M E; Ulusoy, S; Is, A; Ayan, N N; Avincsal, M O; Bicer, C; Erel, O

    2016-05-01

    To investigate a novel oxidative stress marker, thiol/disulphide literature homeostasis, in patients with idiopathic sudden sensorineural hearing loss, and to compare the results with healthy controls for the first time. Thirty-two patients with idiopathic sudden sensorineural hearing loss and 30 healthy individuals were included in the study. Serum native thiol, total thiol and disulphide levels were measured, and disulphide/native thiol and disulphide/total thiol ratios were determined in all subjects. Serum native thiol and total thiol levels were significantly lower in patients with sudden sensorineural hearing loss compared with controls (p sudden sensorineural hearing loss in those patients.

  16. Thiol-based redox signaling in the nitrogen-fixing symbiosis

    Directory of Open Access Journals (Sweden)

    Pierre eFrendo

    2013-09-01

    Full Text Available In nitrogen poor soils legumes establish a symbiotic interaction with rhizobia that results in the formation of root nodules. These are unique plant organs where bacteria differentiate into bacteroids, which express the nitrogenase enzyme complex that reduces atmospheric N2 to ammonia. Nodule metabolism requires a tight control of the concentrations of reactive oxygen and nitrogen species (RONS so that they can perform useful signaling roles while avoiding nitro-oxidative damage. In nodules a thiol-dependent regulatory network that senses, transmits and responds to redox changes is starting to be elucidated. A combination of enzymatic, immunological, pharmacological and molecular analyses has allowed to conclude that glutathione and its legume-specific homolog, homoglutathione, are abundant in meristematic and infected cells, their spatio-temporally distribution is correlated with the corresponding (homoglutathione synthetase activities, and are crucial for nodule development and function. Glutathione is at high concentrations in the bacteroids and at moderate amounts in the mitochondria, cytosol and nuclei. Less information is available on other components of the network. The expression of multiple isoforms of glutathione peroxidases, peroxiredoxins, thioredoxins, glutaredoxins and NADPH-thioredoxin reductases has been detected in nodule cells using antibodies and proteomics. Peroxiredoxins and thioredoxins are essential to regulate and in some cases to detoxify RONS in nodules. Further research is necessary to clarify the regulation of the expression and activity of thiol redox-active proteins in response to abiotic, biotic and developmental cues, their interactions with downstream targets by disulfide-exchange reactions, and their participation in signaling cascades. The availability of mutants and transgenic lines will be crucial to facilitate systematic investigations into the function of the various proteins in the legume

  17. Facially amphiphilic thiol capped gold and silver nanoparticles

    Indian Academy of Sciences (India)

    Shreedhar Bhata; Uday Maitra

    2008-11-01

    A series of bile acid-derived facially amphiphilic thiols have been used to cap sliver and gold nanoparticles. The self-assembling properties of these steroid-capped nanoparticles have been investigated and reported in this article.

  18. Data on the catalytic mechanism of thiol peroxidase mimics

    Directory of Open Access Journals (Sweden)

    B. Zadehvakili

    2016-09-01

    Full Text Available We have recently reported SAR data describing the pharmacological activity of a series of phenyl alkyl selenides and tellurides which catalyse the oxidation of thiols by hydrogen peroxide (H2O2, “The design of redox active thiol peroxidase mimics: dihydrolipoic acid recognition correlates with cytotoxicity and prooxidant action” B. Zadehvakili, S.M. McNeill, J.P. Fawcett, G.I. Giles (2016 [1]. This thiol peroxidase (TPx activity is potentially useful for a number of therapeutic applications, as it can alter the outcome of oxidative stress related pathologies and modify redox signalling. This article presents data describing the molecular changes that occur to a TPx mimic upon exposure to H2O2, and then the thiol mercaptoethanol, as characterised by UV–vis spectroscopy and HPLC retention time.

  19. Fast and Highly Efficient Solid State Oxidation of Thiols

    Directory of Open Access Journals (Sweden)

    Nasrin Haghighat

    2007-03-01

    Full Text Available A fast and efficient solid state method for the chemoselective room temperature oxidative coupling of thiols to afford their corresponding disulfides using inexpensive and readily available moist sodiumperiodate as the reagent is described. The reaction was applicable to a variety of thiols giving high yields after short reaction times. Comparison of yield/time ratios of this method with some of those reported in the literature shows the superiority of this reagent over others under these conditions.

  20. Thiol oxidation by nitrosative stress: Cellular localization in human spermatozoa.

    Science.gov (United States)

    Cabrillana, María E; Uribe, Pamela; Villegas, Juana V; Álvarez, Juan; Sánchez, Raúl; Fornés, Miguel W

    2016-10-01

    Peroxynitrite is a highly reactive nitrogen species and when it is generated at high levels it causes nitrosative stress, an important cause of impaired sperm function. High levels of peroxynitrite have been shown to correlate with decreased semen quality in infertile men. Thiol groups in sperm are mainly found in enzymes, antioxidant molecules, and structural proteins in the axoneme. Peroxynitrite primarily reacts with thiol groups of cysteine-containing proteins. Although it is well known that peroxynitrite oxidizes sulfhydryl groups in sperm, the subcellular localization of this oxidation remains unknown. The main objective of this study was to establish the subcellular localization of peroxynitrite-induced nitrosative stress in thiol groups and its relation to sperm motility in human spermatozoa. For this purpose, spermatozoa from healthy donors were exposed in vitro to 3-morpholinosydnonimine (SIN-1), a compound which generates peroxynitrite. In order to detect peroxynitrite and reduced thiol groups, the fluorescent probes, dihydrorhodamine 123 and monobromobimane (mBBr), were used respectively. Sperm viability was analyzed by propidium iodide staining. Peroxynitrite generation and thiol redox state were monitored by confocal microscopy whereas sperm viability was evaluated by flow cytometry. Sperm motility was analyzed by CASA using the ISAS(®) system. The results showed that exposure of human spermatozoa to peroxynitrite results in increased thiol oxidation which is mainly localized in the sperm head and principal piece regions. Thiol oxidation was associated with motility loss. The high susceptibility of thiol groups to peroxynitrite-induced oxidation could explain, at least in part, the negative effect of reactive nitrogen species on sperm motility. DHR: dihydrorhodamine 123; mBBr: monobromobimane ONOO(-): peroxynitrite RNS: reactive nitrogen species RFI: relative fluorescence intensity SIN-1: 3-morpholinosydnonimine CASA: Computer-Aided Sperm Analysis

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

    Directory of Open Access Journals (Sweden)

    Wilke Sonja

    2012-07-01

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

  2. Cell-type specific requirements for thiol/disulfide exchange during HIV-1 entry and infection.

    Science.gov (United States)

    Stantchev, Tzanko S; Paciga, Mark; Lankford, Carla R; Schwartzkopff, Franziska; Broder, Christopher C; Clouse, Kathleen A

    2012-12-03

    The role of disulfide bond remodeling in HIV-1 infection is well described, but the process still remains incompletely characterized. At present, the data have been predominantly obtained using established cell lines and/or CXCR4-tropic laboratory-adapted virus strains. There is also ambiguity about which disulfide isomerases/reductases play a major role in HIV-1 entry, as protein disulfide isomerase (PDI) and/or thioredoxin (Trx) have emerged as the two enzymes most often implicated in this process. We have extended our previous findings and those of others by focusing on CCR5-using HIV-1 strains and their natural targets--primary human macrophages and CD4+ T lymphocytes. We found that the nonspecific thiol/disulfide exchange inhibitor, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), significantly reduced HIV-1 entry and infection in cell lines, human monocyte-derived macrophages (MDM), and also phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMC). Subsequent studies were performed using specific anti-PDI or Trx monoclonal antibodies (mAb) in HIV-1 envelope pseudotyped and wild type (wt) virus infection systems. Although human donor-to-donor variability was observed as expected, Trx appeared to play a greater role than PDI in HIV-1 infection of MDM. In contrast, PDI, but not Trx, was predominantly involved in HIV-1 entry and infection of the CD4+/CCR5+ T cell line, PM-1, and PHA-stimulated primary human T lymphocytes. Intriguingly, both PDI and Trx were present on the surface of MDM, PM-1 and PHA-stimulated CD4+ T cells. However, considerably lower levels of Trx were detected on freshly isolated CD4+ lymphocytes, compared to PHA-stimulated cells. Our findings clearly demonstrate the role of thiol/disulfide exchange in HIV-1 entry in primary T lymphocytes and MDM. They also establish a cell-type specificity regarding the involvement of particular disulfide isomerases/reductases in this process and may provide an explanation for differences

  3. Cell-type specific requirements for thiol/disulfide exchange during HIV-1 entry and infection

    Directory of Open Access Journals (Sweden)

    Stantchev Tzanko S

    2012-12-01

    Full Text Available Abstract Background The role of disulfide bond remodeling in HIV-1 infection is well described, but the process still remains incompletely characterized. At present, the data have been predominantly obtained using established cell lines and/or CXCR4-tropic laboratory-adapted virus strains. There is also ambiguity about which disulfide isomerases/ reductases play a major role in HIV-1 entry, as protein disulfide isomerase (PDI and/or thioredoxin (Trx have emerged as the two enzymes most often implicated in this process. Results We have extended our previous findings and those of others by focusing on CCR5-using HIV-1 strains and their natural targets - primary human macrophages and CD4+ T lymphocytes. We found that the nonspecific thiol/disulfide exchange inhibitor, 5,5'-dithiobis(2-nitrobenzoic acid (DTNB, significantly reduced HIV-1 entry and infection in cell lines, human monocyte-derived macrophages (MDM, and also phytohemagglutinin (PHA-stimulated peripheral blood mononuclear cells (PBMC. Subsequent studies were performed using specific anti-PDI or Trx monoclonal antibodies (mAb in HIV-1 envelope pseudotyped and wild type (wt virus infection systems. Although human donor-to-donor variability was observed as expected, Trx appeared to play a greater role than PDI in HIV-1 infection of MDM. In contrast, PDI, but not Trx, was predominantly involved in HIV-1 entry and infection of the CD4+/CCR5+ T cell line, PM-1, and PHA-stimulated primary human T lymphocytes. Intriguingly, both PDI and Trx were present on the surface of MDM, PM-1 and PHA-stimulated CD4+ T cells. However, considerably lower levels of Trx were detected on freshly isolated CD4+ lymphocytes, compared to PHA-stimulated cells. Conclusions Our findings clearly demonstrate the role of thiol/disulfide exchange in HIV-1 entry in primary T lymphocytes and MDM. They also establish a cell-type specificity regarding the involvement of particular disulfide isomerases/reductases in this

  4. A maize gene encoding an NADPH binding enzyme highly homologous to isoflavone reductases is activated in response to sulfur starvation.

    Science.gov (United States)

    Petrucco, S; Bolchi, A; Foroni, C; Percudani, R; Rossi, G L; Ottonello, S

    1996-01-01

    we isolated a novel gene that is selectively induced both in roots and shoots in response to sulfur starvation. This gene encodes a cytosolic, monomeric protein of 33 kD that selectively binds NADPH. The predicted polypeptide is highly homologous ( > 70%) to leguminous isoflavone reductases (IFRs), but the maize protein (IRL for isoflavone reductase-like) belongs to a novel family of proteins present in a variety of plants. Anti-IRL antibodies specifically recognize IFR polypeptides, yet the maize protein is unable to use various isoflavonoids as substrates. IRL expression is correlated closely to glutathione availability: it is persistently induced in seedlings whose glutathione content is about fourfold lower than controls, and it is down-regulated rapidly when control levels of glutathione are restored. This glutathione-dependent regulation indicates that maize IRL may play a crucial role in the establishment of a thiol-independent response to oxidative stress under glutathione shortage conditions.

  5. Evaluation of thiol-disulphide homeostasis in radiation workers.

    Science.gov (United States)

    Koc, Ural; Tan, Sinan; Ertem, Ahmet Goktug; Gumus, Mehmet; Ozbek, Betul; Erel, Ozcan

    2017-07-01

    To evaluate thiol-disulphide homeostasis - a novel, easily calculated, readily available, and relatively cheap oxidative stress marker - in radiation workers and compare the results with healthy controls. A total of 108 participants were enrolled in the study including 63 hospital workers occupationally exposed to ionizing radiation in the units of interventional radiology, interventional cardiology and nuclear medicine. A control group consisted of 45 individuals staff in the same hospital. Serum thiol-disulphide homeostasis measurement was investigated via the spectrophotometric method newly described by Erel and Neşelioğlu. The mean serum native thiol levels of radiation workers (528.96 ± 86.42 μmol/l) was significantly lower than control subjects (561.05 ± 104.83 μmol/l) (p = .045). The mean serum total thiol levels of radiation workers (547.70 ± 91.50 μmol/l) was lower than control subjects (580.36 ± 112.24 μmol/l). Nevertheless, there was no significant difference between total thiol of exposed workers and controls. The results show that long-term low dose ionizing radiation may lead to oxidative stress and have side-effects in antioxidant thiol groups. We may suggest supporting radiation workers by safe antioxidant nutritional formulations and following up via both physical dosimetry and biodosimetric methods.

  6. Protein Thiols as an Indication of Oxidative Stress

    Directory of Open Access Journals (Sweden)

    Yousef Rezaei Chianeh

    2014-06-01

    Full Text Available Thiol is an organic compound that contain sulphhydryl group that have a critical role in preventing any involvement of oxidative stress in the cell. These defensive functions are generally considered to be carried out by the low molecular weight thiol glutathione and by cysteine residues in the active sites of proteins such as thioredoxin and peroxiredoxin. In addition, there are thiols exposed on protein surfaces that are not directly involved with protein function, although they can interact with the intracellular environment.The process of protection of the cell against an oxidative damage occur by thiol and cystein residue that has a low molecular weight. These residue are present in the active sites of a protein like, peroxiredoxin and thioredoxin. Apart from intracellular antioxidant defense mechanism by protein thiol, there are presence of thiol in outer surface of protein that are not involved with the function of protein, even though they can interact with intracellular part of the cell. [Archives Medical Review Journal 2014; 23(3.000: 443-456

  7. Investigation of thiol-disulphide balance in patients with acute urticaria and chronic spontaneous urticaria.

    Science.gov (United States)

    Akbas, Ayse; Kilinc, Fadime; Sener, Sertac; Aktaş, Akın; Baran, Pervin; Ergin, Merve

    2017-09-01

    Thiol-disulphide balance plays a major role in health and diseases. This balance may be disrupted by various diseases. We aimed to determine status of the effect of thiol-disulphide balance in urticaria. We aimed to investigate the thiol-disulphide balance in patients with acute urticaria (AUP) and chronic spontaneous urticaria (CSU). Study included 53 AUP and 47 healthy controls plus 57 patients with chronic spontaneous urticaria (CSUP) and 57 healthy controls. Levels of native thiols, disulphides and total thiols were evaluated in plasma using a new and automated spectrophotometric method. Ratios of disulphides/total thiols, disulphides/native thiols and native thiols/total thiols were calculated. For AU, there was no statistical difference compared to control group in levels of native thiols, disulphides and total thiols. For CSU, however, there was an increase in levels of native thiols, disulphides and total thiols and the ratio of thiol/disulphide in favour of disulphide. Thiol-disulphide balance was not affected by AU but shifted towards to disulphide in CSU indicating the presence of oxidative stress (OS).

  8. Lysosomal storage disorders: Molecular basis and laboratory testing

    Directory of Open Access Journals (Sweden)

    Filocamo Mirella

    2011-03-01

    Full Text Available Abstract Lysosomal storage disorders (LSDs are a large group of more than 50 different inherited metabolic diseases which, in the great majority of cases, result from the defective function of specific lysosomal enzymes and, in cases, of non-enzymatic lysosomal proteins or non-lysosomal proteins involved in lysosomal biogenesis. The progressive lysosomal accumulation of undegraded metabolites results in generalised cell and tissue dysfunction, and, therefore, multi-systemic pathology. Storage may begin during early embryonic development, and the clinical presentation for LSDs can vary from an early and severe phenotype to late-onset mild disease. The diagnosis of most LSDs--after accurate clinical/paraclinical evaluation, including the analysis of some urinary metabolites--is based mainly on the detection of a specific enzymatic deficiency. In these cases, molecular genetic testing (MGT can refine the enzymatic diagnosis. Once the genotype of an individual LSD patient has been ascertained, genetic counselling should include prediction of the possible phenotype and the identification of carriers in the family at risk. MGT is essential for the identification of genetic disorders resulting from non-enzymatic lysosomal protein defects and is complementary to biochemical genetic testing (BGT in complex situations, such as in cases of enzymatic pseudodeficiencies. Prenatal diagnosis is performed on the most appropriate samples, which include fresh or cultured chorionic villus sampling or cultured amniotic fluid. The choice of the test--enzymatic and/or molecular--is based on the characteristics of the defect to be investigated. For prenatal MGT, the genotype of the family index case must be known. The availability of both tests, enzymatic and molecular, enormously increases the reliability of the entire prenatal diagnostic procedure. To conclude, BGT and MGT are mostly complementary for post- and prenatal diagnosis of LSDs. Whenever genotype

  9. Efficient functionalization of oxide-free silicon(111) surfaces: thiol-yne versus thiol-ene click chemistry.

    Science.gov (United States)

    Bhairamadgi, Nagendra S; Gangarapu, Satesh; Caipa Campos, Mabel A; Paulusse, Jos M J; van Rijn, Cees J M; Zuilhof, Han

    2013-04-09

    Thiol-yne click (TYC) chemistry was utilized as a copper-free click reaction for the modification of alkyne-terminated monolayers on oxide-free Si(111) surfaces, and the results were compared with the analogous thiol-ene click (TEC) chemistry. A wide range of thiols such as 9-fluorenylmethoxy-carbonyl cysteine, thio-β-d-glucose tetraacetate, thioacetic acid, thioglycerol, thioglycolic acid, and 1H,1H,2H,2H-perfluorodecanethiol was immobilized using TYC under photochemical conditions, and all modified surfaces were characterized by static water contact angle measurements, X-ray photoelectron spectroscopy (including a simulation thereof by density functional calculations), and infrared absorption reflection spectroscopy. Surface-bound TYC proceeds with an efficiency of up to 1.5 thiols per alkyne group. This high surface coverage proceeds without oxidizing the Si surface. TYC yielded consistently higher surface coverages than TEC, due to double addition of thiols to alkyne-terminated monolayers. This also allows for the sequential and highly efficient attachment of two different thiols onto an alkyne-terminated monolayer.

  10. The Link Between Lysosomal Storage Disorders and More Common Diseases

    Directory of Open Access Journals (Sweden)

    Michael Beck MD

    2016-12-01

    Full Text Available In the last decades, it has become more and more evident that lysosomal storage disorders and common neurodegenerative diseases such as Alzheimer and Parkinson diseases have clinical, neuropathological, and genetic features in common, including lysosomal dysfunction and impaired autophagy. Patients with Gaucher and even carriers of Gaucher disease have an increased risk to develop Parkinson disease. Likewise, individuals who are heterozygous for a mutation of a gene that causes an adult form of neuronal ceroid lipofuscinosis are more likely to be affected by a form of frontotemporal dementia in their later life. A further example is the gene NAGLU encoding the enzyme α- N -acetylglucosaminidase, which is deficient in patients with mucopolysaccharidosis type IIIB. Mutations of the NAGLU gene have been observed in patients affected by an axonal neuropathy. An interesting unexpected finding was the link between stuttering and genes that are essential for the function of all lysosomal enzymes. This review will present some example of the association of lysosomal storage disorders and neurodegenerative disease and discuss possible pathogenic mechanisms that are common to both conditions. The understanding of the pathophysiology of the endosomal–lysosomal–autophagic system may help to develop drugs, which might provide benefit not only for patients with rare lysosomal storage disorders but also for individuals affected by more common diseases.

  11. TRPML1: an ion channel in the lysosome.

    Science.gov (United States)

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

    2014-01-01

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

  12. The Link Between Lysosomal Storage Disorders and More Common Diseases

    Directory of Open Access Journals (Sweden)

    Michael Beck MD

    2016-12-01

    Full Text Available In the last decades, it has become more and more evident that lysosomal storage disorders and common neurodegenerative diseases such as Alzheimer and Parkinson diseases have clinical, neuropathological, and genetic features in common, including lysosomal dysfunction and impaired autophagy. Patients with Gaucher and even carriers of Gaucher disease have an increased risk to develop Parkinson disease. Likewise, individuals who are heterozygous for a mutation of a gene that causes an adult form of neuronal ceroid lipofuscinosis are more likely to be affected by a form of frontotemporal dementia in their later life. A further example is the gene NAGLU encoding the enzyme α-N-acetylglucosaminidase, which is deficient in patients with mucopolysaccharidosis type IIIB. Mutations of the NAGLU gene have been observed in patients affected by an axonal neuropathy. An interesting unexpected finding was the link between stuttering and genes that are essential for the function of all lysosomal enzymes. This review will present some example of the association of lysosomal storage disorders and neurodegenerative disease and discuss possible pathogenic mechanisms that are common to both conditions. The understanding of the pathophysiology of the endosomal–lysosomal–autophagic system may help to develop drugs, which might provide benefit not only for patients with rare lysosomal storage disorders but also for individuals affected by more common diseases.

  13. Designed Chemical Intervention with Thiols for Prophylactic Contraception.

    Directory of Open Access Journals (Sweden)

    Monika Sharma

    Full Text Available Unlike somatic cells, sperm have several-fold more available-thiols that are susceptible to redox-active agents. The present study explains the mechanism behind the instant sperm-immobilizing and trichomonacidal activities of pyrrolidinium pyrrolidine-1-carbodithioate (PPC, a novel thiol agent rationally created for prophylactic contraception by minor chemical modifications of some known thiol drugs. PPC, and its three derivatives (with potential active-site blocked by alkylation, were synthesized and evaluated against live human sperm and metronidazole-susceptible and resistant Trichomonas vaginalis, in vitro. Sperm hexokinase activity was evaluated by coupled enzyme assay. PPC irreversibly immobilized 100% human sperm in ∼30 seconds and totally eliminated Trichomonas vaginalis more efficiently than nonoxynol-9 and metronidazole. It significantly inhibited (P<0.001 thiol-sensitive sperm hexokinase. However, the molecule completely lost all its biological activities once its thiol group was blocked by alkylation. PPC was subsequently formulated into a mucoadhesive vaginal film using GRaS excipients and evaluated for spermicidal and microbicidal activities (in vitro, and contraceptive efficacy in rabbits. PPC remained fully active in quick-dissolving, mucoadhesive vaginal-film formulation, and these PPC-films significantly reduced pregnancy and fertility rates in rabbits. The films released ∼90% of PPC in simulated vaginal fluid (pH 4.2 at 37°C in 5 minutes, in vitro. We have thus discovered a common target (reactive thiols on chiefly-anaerobic, redox-sensitive cells like sperm and Trichomonas, which is susceptible to designed chemical interference for prophylactic contraception. The active thiol in PPC inactivates sperm and Trichomonas via interference with crucial sulfhydryl-disulfide based reactions, e.g. hexokinase activation in human sperm. In comparison to non-specific surfactant action of OTC spermicide nonoxynol-9, the action of

  14. Copper-induced changes in intracellular thiols in two marine diatoms: Phaeodactylum tricornutum and Ceratoneis closterium.

    Science.gov (United States)

    Smith, Cassandra L; Steele, Jessica E; Stauber, Jennifer L; Jolley, Dianne F

    2014-11-01

    Phytochelatins and glutathione (reduced (GSH) and oxidised (GSSG)) are important intracellular ligands involved in metal sequestration and detoxification in algae. Intracellular ratios of GSH:GSSG are sensitive indicators of metal stress in algae, and like phytochelatin production are influenced by metal speciation, concentration, exposure time and the biological species. This study investigated the effect of copper exposure on phytochelatin and glutathione content in two marine diatoms Phaeodactylum tricornutum and Ceratoneis closterium at various time intervals between 0.5 and 72h. Liberation of cellular glutathione and phytochelatins was optimised using freeze/thaw cycles and chemical extraction, respectively. Extracted phytochelatins were derivatised (by fluorescent tagging of thiol compounds), separated and quantified using HPLC with fluorescence detection. Glutathione ratios were determined using a commercially available kit, which uses the enzyme glutathione reductase to measure total and oxidised glutathione. Despite similarities in size and shape between the two diatoms, differences in internalised copper, phytochelatin production (both chain length and quantity) and reduced glutathione concentrations were observed. P. tricornutum maintained reduced glutathione at between 58 and 80% of total glutathione levels at all time points, which would indicate low cellular stress. In C. closterium reduced glutathione constituted <10% of total glutathione after 48h. P. tricornutum also produced more phytochelatins and phytochelatins of longer chain length than C. closterium despite the latter species internalising significantly more copper. Copyright © 2014 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

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

    2017-10-01

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

  16. Rab2 promotes autophagic and endocytic lysosomal degradation.

    Science.gov (United States)

    Lőrincz, Péter; Tóth, Sarolta; Benkő, Péter; Lakatos, Zsolt; Boda, Attila; Glatz, Gábor; Zobel, Martina; Bisi, Sara; Hegedűs, Krisztina; Takáts, Szabolcs; Scita, Giorgio; Juhász, Gábor

    2017-07-03

    Rab7 promotes fusion of autophagosomes and late endosomes with lysosomes in yeast and metazoan cells, acting together with its effector, the tethering complex HOPS. Here we show that another small GTPase, Rab2, is also required for autophagosome and endosome maturation and proper lysosome function in Drosophila melanogaster We demonstrate that Rab2 binds to HOPS, and that its active, GTP-locked form associates with autolysosomes. Importantly, expression of active Rab2 promotes autolysosomal fusions unlike that of GTP-locked Rab7, suggesting that its amount is normally rate limiting. We also demonstrate that RAB2A is required for autophagosome clearance in human breast cancer cells. In conclusion, we identify Rab2 as a key factor for autophagic and endocytic cargo delivery to and degradation in lysosomes. © 2017 Lőrincz et al.

  17. Parkinson's Disease Shares the Lysosome with Gaucher's Disease

    Science.gov (United States)

    Dawson, Ted M.; Dawson, Valina L.

    2015-01-01

    Summary The second most common neurodegenerative disorder, Parkinson's disease (PD) is an age dependent progressive neurodegenerative disorder that affects movement. While many of the causes of PD remain unclear, a consistent finding in PD is the abnormal accumulation of α-synuclein that has lead to the widely held notion that PD is a synucleinopathy. In a recent Cell manuscript Mazzuli et al., provide a potential mechanistic link between Gaucher's disease, a glycolipid lysosomal storage disorder due to Glucocerebrocidase (GBA) deficiency and PD. The authors reveal a reciprocal connection between the loss of GBA activity and accumulation of α-synuclein in the lysosome establishing a bidirectional positive feed back loop with pathologic consequences. These findings should stimulate further work on role of the lysosome in PD pathogenesis and the identification of new treatment strategies for PD. PMID:21753118

  18. The impact of thiol peroxidases on redox regulation.

    Science.gov (United States)

    Flohé, Leopold

    2016-01-01

    The biology of glutathione peroxidases and peroxiredoxins is reviewed with emphasis on their role in metabolic regulation. Apart from their obvious function in balancing oxidative challenge, these thiol peroxidases are not only implicated in orchestrating the adaptive response to oxidative stress, but also in regulating signaling triggered by hormones, growth factors and cytokines. The mechanisms presently discussed comprise dampening of redox-sensitive regulatory processes by elimination of hydroperoxides, suppression of lipoxygenase activity, committing suicide to save H2O2 for signaling, direct binding to receptors or regulatory proteins in a peroxidase activity-independent manner, or acting as sensors for hydroperoxides and as transducers of oxidant signals. The various mechanistic proposals are discussed in the light of kinetic data, which unfortunately are scarce. Taking into account pivotal criteria of a meaningful regulatory circuit, kinetic plausibility and specificity, the mechanistic concepts implying a direct sensor/transducer function of the thiol peroxidases appear most appealing. With rate constants for the reaction with hydroperoxide of 10(5)-10(8) M(-1) s(-1), thiol peroxidases are qualified as kinetically preferred hydroperoxide sensors, and the ability of the oxidized enzymes to react with defined protein thiols lends specificity to the transduction process. The versatility of thiol peroxidases, however, allows multiple ways of interaction with regulatory pathways.

  19. Surface functionalized thiol-ene waveguides for fluorescence biosensing in microfluidic devices

    DEFF Research Database (Denmark)

    Feidenhans'l, Nikolaj Agentoft; Lafleur, Josiane P.; Jensen, Thomas Glasdam;

    2013-01-01

    . The reactive functional groups present at the surface of the thiol-ene polymer are subsequently used for the rapid, one step, site-specific functionalization of the waveguide with biological recognition molecules. It was found that while the bulk properties and chemical surface properties of thiol......-ene waveguides were fabricated from 40% excess thiol thiol-ene to ensure the presence of thiol functional groups at the surface of the waveguide. Biotin alkyne was photografted at specific locations using a photomask, directly at the interface between the microfluidic channel and the thiol-ene waveguide prior...

  20. Importance of lysosomal cysteine proteases in lung disease

    Directory of Open Access Journals (Sweden)

    Chapman Harold A

    2000-11-01

    Full Text Available Abstract The human lysosomal cysteine proteases are a family of 11 proteases whose members include cathepsins B, C, H, L, and S. The biology of these proteases was largely ignored for decades because of their lysosomal location and the belief that their function was limited to the terminal degradation of proteins. In the past 10 years, this view has changed as these proteases have been found to have specific functions within cells. This review highlights some of these functions, specifically their roles in matrix remodeling and in regulating the immune response, and their relationship to lung diseases.

  1. PDT: loss of autophagic cytoprotection after lysosomal photodamage

    Science.gov (United States)

    Kessel, David; Price, Michael

    2012-02-01

    Photodynamic therapy is known to evoke both autophagy and apoptosis. Apoptosis is an irreversible death pathway while autophagy can serve a cytoprotective function. In this study, we examined two photosensitizing agents that target lysosomes, although they differ in the reactive oxygen species (ROS) formed during irradiation. With both agents, the 'shoulder' on the PDT dose-response curve was substantially attenuated, consistent with loss of a cytoprotective pathway. In contrast, this 'shoulder' is commonly observed when PDT targets mitochondria or the ER. We propose that lysosomal targets may offer the possibility of promoting PDT efficacy by eliminating a potentially protective pathway.

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

    Science.gov (United States)

    Einck, K H; Norkin, L C

    1979-01-01

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

  3. Analyzing Lysosome-Related Organelles by Electron Microscopy

    KAUST Repository

    Hurbain, Ilse

    2017-04-29

    Intracellular organelles have a particular morphological signature that can only be appreciated by ultrastructural analysis at the electron microscopy level. Optical imaging and associated methodologies allow to explore organelle localization and their dynamics at the cellular level. Deciphering the biogenesis and functions of lysosomes and lysosome-related organelles (LROs) and their dysfunctions requires their visualization and detailed characterization at high resolution by electron microscopy. Here, we provide detailed protocols for studying LROs by transmission electron microscopy. While conventional electron microscopy and its recent improvements is the method of choice to investigate organelle morphology, immunoelectron microscopy allows to localize organelle components and description of their molecular make up qualitatively and quantitatively.

  4. The endoplasmic reticulum, not the pH gradient, drives calcium refilling of lysosomes.

    Science.gov (United States)

    Garrity, Abigail G; Wang, Wuyang; Collier, Crystal Md; Levey, Sara A; Gao, Qiong; Xu, Haoxing

    2016-05-23

    Impaired homeostasis of lysosomal Ca(2+) causes lysosome dysfunction and lysosomal storage diseases (LSDs), but the mechanisms by which lysosomes acquire and refill Ca(2+) are not known. We developed a physiological assay to monitor lysosomal Ca(2+) store refilling using specific activators of lysosomal Ca(2+) channels to repeatedly induce lysosomal Ca(2+) release. In contrast to the prevailing view that lysosomal acidification drives Ca(2+) into the lysosome, inhibiting the V-ATPase H(+) pump did not prevent Ca(2+) refilling. Instead, pharmacological depletion or chelation of Endoplasmic Reticulum (ER) Ca(2+) prevented lysosomal Ca(2+) stores from refilling. More specifically, antagonists of ER IP3 receptors (IP3Rs) rapidly and completely blocked Ca(2+) refilling of lysosomes, but not in cells lacking IP3Rs. Furthermore, reducing ER Ca(2+) or blocking IP3Rs caused a dramatic LSD-like lysosome storage phenotype. By closely apposing each other, the ER may serve as a direct and primary source of Ca(2+)for the lysosome.

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

    Science.gov (United States)

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

    2016-12-09

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

  6. Highly tailorable thiol-ene based emulsion-templated monoliths

    DEFF Research Database (Denmark)

    Lafleur, J. P.; Kutter, J. P.

    2014-01-01

    The attractive surface properties of thiol-ene polymers combined with their ease of processing make them ideal substrates in many bioanalytical applications. We report the synthesis of highly tailorable emulsion-templated porous polymers and beads in microfluidic devices based on off-stoichiometr......The attractive surface properties of thiol-ene polymers combined with their ease of processing make them ideal substrates in many bioanalytical applications. We report the synthesis of highly tailorable emulsion-templated porous polymers and beads in microfluidic devices based on off......-stoichiometry thiolene chemistry. The method allows monolith synthesis and anchoring inside thiol-ene microchannels in a single step. Variations in the monomer stoichiometric ratios and/or amount of porogen used allow for the creation of extremely varied polymer morphologies, from foam-like materials to dense networks...

  7. Suspended hybrid films assembled from thiol-capped gold nanoparticles.

    Science.gov (United States)

    Zhang, Yu Xin; Huang, Ming; Hao, Xiao Dong; Dong, Meng; Li, Xin Lu; Huang, Jia Mu

    2012-01-01

    In this work, we explored the formation processes of suspended hybrid thin films of thiol-capped Au nanoparticles (AuNPs) inside metal oxide tubular structures. We found that a balance between in-film interactions of the AuNPs and boundary interactions with metal oxides is a key in making these special organic-inorganic thin films. The hybrid films process many processing advantages and flexibilities, such as controllable film thickness, interfacial shape and inter-AuNPs distance, tuning of particle sizes, thiol population, chain lengths, and other new properties by introducing functional groups to thiol chains. Among their many unique features, the assembly-disassembly property may be useful for future on-off or store-release applications.

  8. Cysteine-functional polymers via thiol-ene conjugation.

    Science.gov (United States)

    Kuhlmann, Matthias; Reimann, Oliver; Hackenberger, Christian P R; Groll, Jürgen

    2015-03-01

    A thiofunctional thiazolidine is introduced as a new low-molar-mass building block for the introduction of cysteine residues via a thiol-ene reaction. Allyl-functional polyglycidol (PG) is used as a model polymer to demonstrate polymer-analogue functionalization through reaction with the unsaturated side-chains. A modified trinitrobenzenesulfonic acid (TNBSA) assay is used for the redox-insensitive quantification and a precise final cysteine content can be predetermined at the polymerization stage. Native chemical ligation at cysteine-functional PG is performed as a model reaction for a chemoselective peptide modification of this polymer. The three-step synthesis of the thiofunctional thiazolidine reactant, together with the standard thiol-ene coupling and the robust quantification assay, broadens the toolbox for thiol-ene chemistry and offers a generic and straightforward approach to cysteine-functional materials. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Genomic expression analyses reveal lysosomal, innate immunity proteins, as disease correlates in murine models of a lysosomal storage disorder.

    Directory of Open Access Journals (Sweden)

    Md Suhail Alam

    Full Text Available Niemann-Pick Type C (NPC disease is a rare, genetic, lysosomal disorder with progressive neurodegeneration. Poor understanding of the pathophysiology and a lack of blood-based diagnostic markers are major hurdles in the treatment and management of NPC and several additional, neurological lysosomal disorders. To identify disease severity correlates, we undertook whole genome expression profiling of sentinel organs, brain, liver, and spleen of Balb/c Npc1(-/- mice relative to Npc1(+/- at an asymptomatic stage, as well as early- and late-symptomatic stages. Unexpectedly, we found prominent up regulation of innate immunity genes with age-dependent change in their expression, in all three organs. We shortlisted a set of 12 secretory genes whose expression steadily increased with age in both brain and liver, as potential plasma correlates of neurological and/or liver disease. Ten were innate immune genes with eight ascribed to lysosomes. Several are known to be elevated in diseased organs of murine models of other lysosomal diseases including Gaucher's disease, Sandhoff disease and MPSIIIB. We validated the top candidate lysozyme, in the plasma of Npc1(-/- as well as Balb/c Npc1(nmf164 mice (bearing a point mutation closer to human disease mutants and show its reduction in response to an emerging therapeutic. We further established elevation of innate immunity in Npc1(-/- mice through multiple functional assays including inhibition of bacterial infection as well as cellular analysis and immunohistochemistry. These data revealed neutrophil elevation in the Npc1(-/- spleen and liver (where large foci were detected proximal to damaged tissue. Together our results yield a set of lysosomal, secretory innate immunity genes that have potential to be developed as pan or specific plasma markers for neurological diseases associated with lysosomal storage and where diagnosis is a major problem. Further, the accumulation of neutrophils in diseased organs

  10. General and practical formation of thiocyanates from thiols.

    Science.gov (United States)

    Frei, Reto; Courant, Thibaut; Wodrich, Matthew D; Waser, Jerome

    2015-02-02

    A new method for the cyanation of thiols and disulfides using cyanobenziodoxol(on)e hypervalent iodine reagents is described. Both aliphatic and aromatic thiocyanates can be accessed in good yields in a few minutes at room temperature starting from a broad range of thiols with high chemioselectivity. The complete conversion of disulfides to thiocyanates was also possible. Preliminary computational studies indicated a low energy concerted transition state for the cyanation of the thiolate anion or radical. The developed thiocyanate synthesis has broad potential for various applications in synthetic chemistry, chemical biology and materials science.

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

    Science.gov (United States)

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

    2000-07-01

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

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

    Science.gov (United States)

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

    2015-11-01

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

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

    NARCIS (Netherlands)

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

    1992-01-01

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

  14. Recent advances in gene therapy for lysosomal storage disorders

    Directory of Open Access Journals (Sweden)

    Rastall DP

    2015-06-01

    Full Text Available David PW Rastall,1 Andrea Amalfitano1,2 1Department of Microbiology and Molecular Genetics, 2Department of Pediatrics, College of Osteopathic Medicine, Michigan State University, East Lansing, MI, USA Abstract: Lysosomal storage disorders (LSDs are a group of genetic diseases that result in metabolic derangements of the lysosome. Most LSDs are due to the genetic absence of a single catabolic enzyme, causing accumulation of the enzyme's substrate within the lysosome. Over time, tissue-specific substrate accumulations result in a spectrum of symptoms and disabilities that vary by LSD. LSDs are promising targets for gene therapy because delivery of a single gene into a small percentage of the appropriate target cells may be sufficient to impact the clinical course of the disease. Recently, there have been several significant advancements in the potential for gene therapy of these disorders, including the first human trials. Future clinical trials will build upon these initial attempts, with an improved understanding of immune system responses to gene therapy, the obstacle that the blood–brain barrier poses for neuropathic LSDs, as well other biological barriers that, when overcome, may facilitate gene therapy for LSDs. In this manuscript, we will highlight the recent innovations in gene therapy for LSDs and discuss the clinical limitations that remain to be overcome, with the goal of fostering an understanding and further development of this important field. Keywords: human trials, clinical trials, gene therapy, lysosomal storage disease, blood-brain barrier, adeno-associated virus, lentivirus, adenovirus 

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

    Science.gov (United States)

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

    2016-01-26

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

  16. The frequency of lysosomal storage diseases in The Netherlands

    NARCIS (Netherlands)

    Poorthuis, BJHM; Wevers, RA; Kleijer, WJ; Groener, JEM; de Jong, JGN; van Weely, S; Niezen-Koning, KE; van Diggelen, OP

    1999-01-01

    We have calculated the relative frequency and the birth prevalence of lysosomal storage diseases (LSDs) in The Netherlands based on all 963 enzymatically confirmed cases diagnosed during the period 1970-1996. The combined birth prevalence for all LSDs is 14 per 100,000 live births. Glycogenosis type

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

    NARCIS (Netherlands)

    A.J.J. Reuser (Arnold)

    1977-01-01

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

  18. The frequency of lysosomal storage diseases in The Netherlands

    NARCIS (Netherlands)

    Poorthuis, BJHM; Wevers, RA; Kleijer, WJ; Groener, JEM; de Jong, JGN; van Weely, S; Niezen-Koning, KE; van Diggelen, OP

    1999-01-01

    We have calculated the relative frequency and the birth prevalence of lysosomal storage diseases (LSDs) in The Netherlands based on all 963 enzymatically confirmed cases diagnosed during the period 1970-1996. The combined birth prevalence for all LSDs is 14 per 100,000 live births. Glycogenosis type

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

    NARCIS (Netherlands)

    D.J.J. Halley (Dicky)

    1980-01-01

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

  20. Neuronopathic Lysosomal Storage Diseases: Clinical and Pathologic Findings

    Science.gov (United States)

    Prada, Carlos E.; Grabowski, Gregory A.

    2013-01-01

    Background: The lysosomal--autophagocytic system diseases (LASDs) affect multiple body systems including the central nervous system (CNS). The progressive CNS pathology has its onset at different ages, leading to neurodegeneration and early death. Methods: Literature review provided insight into the current clinical neurological findings,…

  1. 5-Furan-2yl[1,3,4]oxadiazole-2-thiol, 5-Furan-2yl-4H [1,2,4] triazole-3-thiol and Their Thiol-Thione Tautomerism

    Directory of Open Access Journals (Sweden)

    A. Cansız

    2005-02-01

    Full Text Available 5-Furan-2-yl[1,3,4]oxadiazole-2-thiol (Ia and 5-furan-2-yl-4H-[1,2,4]-triazole-3-thiol (Ib were synthesized from furan-2-carboxylic acid hydrazide. Mannich basesand methyl derivatives were then prepared. The structures of the synthesized compoundswere confirmed by elemental analyses, IR and 1H-NMR spectra. Their thiol-thione tautomericequilibrium is described.

  2. A fluorescent probe which allows highly specific thiol labeling at low pH

    DEFF Research Database (Denmark)

    Nielsen, Jonas W.; Jensen, Kristine Steen; Hansen, Rosa E.

    2012-01-01

    and properties of a thiol-specific reagent, fluorescent cyclic activated disulfide (FCAD), which includes the fluorescein moiety as fluorophore and utilizes a variation of thiol-disulfide exchange chemistry. The leaving-group character of FCAD makes it reactive at pH 3, allowing modification at low pH, limiting...... thiol-disulfide exchange. Different applications are demonstrated including picomolar thiol detection, determination of redox potentials, and in-gel detection of labeled proteins....

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

    NARCIS (Netherlands)

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

    2008-01-01

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

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

    NARCIS (Netherlands)

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

    2008-01-01

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

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

    Science.gov (United States)

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

    2016-02-01

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

  6. Thiol-Disulfide Exchange between Glutaredoxin and Glutathione

    DEFF Research Database (Denmark)

    Iversen, Rasmus; Andersen, Peter Anders; Jensen, Kristine Steen

    2010-01-01

    Glutaredoxins are ubiquitous thiol-disulfide oxidoreductases which catalyze the reduction of glutathione-protein mixed disulfides. Belonging to the thioredoxin family, they contain a conserved active site CXXC motif. The N-proximal active site cysteine can form a mixed disulfide with glutathione ...

  7. Preparation of Novel Hydrolyzing Urethane Modified Thiol-Ene Networks

    Directory of Open Access Journals (Sweden)

    Bridget S. Confait

    2011-10-01

    Full Text Available Novel tetra-functional hydrolyzing monomers were prepared from the reaction of TEOS and select alkene-containing alcohols, ethylene glycol vinyl ether or 2-allyloxy ethanol, and combined with trimethylolpropane tris(3-mercaptopropionate (tri-thiol in a thiol-ene “click” polymerization reaction to produce clear, colorless thiol-ene networks using both radiation and thermal-cure techniques. These networks were characterized for various mechanical characteristics, and found to posses Tg’s (DSC, hardness, tack, and thermal stability (TGA consistent with their molecular structures. A new ene-modified urethane oligomer was prepared based on the aliphatic polyisocyanate Desmodur® N 3600 and added to the thiol-ene hydrolyzable network series in increasing amounts, creating a phase-segregated material having two Tg’s. An increase in water absorption in the ene-modified urethane formulations leading to a simultaneous increase in the rate of hydrolysis was supported by TGA data, film hardness measurements, and an NMR study of closely related networks. This phenomenon was attributed to the additional hydrogen bonding elements and polar functionality brought to the film with the addition of the urethane segment. SEM was utilized for visual analysis of topographical changes in the film’s surface upon hydrolysis and provides support for surface-driven erosion. Coatings prepared in this study are intended for use as hydrolyzing networks for marine coatings to protect against ship fouling.

  8. Thiol reactive nitroimidazoles: radiosensitization studies in vitro and in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Stratford, I.J.; Adams, G.E.; Hardy, C.; Hoe, S.; O' Neill, P.; Sheldon, P.W. (Medical Research Council, Harwell (UK). Radiobiological Research Unit)

    1984-12-01

    Using Chinese hamster V79 cells in vitro a study was made of the radiosensitizing properties of 4- or 5-nitroimidazoles substituted in the 2,5 or 4 position with various halo, sulphur ether, sulphonamide, sulphonate, ether or nitro groups. Values of E/sub 7//sup 1/ (the one-electron reduction potential measured versus the normal hydrogen electrode at pH7) vary in the range -178 to -565 mV. All the compounds, with one exception, are more efficient radiosensitizers than would be predicted from redox potentials, and the factor Csub(1.6)/Csub(1.6), by which a compound is more efficient has been calculated. The second-order rate constants, k/sub 2/, for reaction of these nitroimidazoles with glutathione and/or dithiothreitol tended to increase with increasing redox potential. However, there is no clear trend between k/sub 2/ and Csub(1.6)/Csub(1.6). The concentration required to cause a 50% depletion of intracellular glutathione was determined for selected compounds, as was the ability of glutathione-S-transferase to catalyse reaction with thiols. These observations suggested the relative thiol reactivity measured under chemically controlled conditions does not necessarily indicate thiol reactivity intracellularly. Studies using MT tumour in mice showed that high levels of radiosensitization seen in vitro could not be duplicated in vivo (attributed to thiol reactivity).

  9. Are free radicals involved in thiol-based redox signaling?

    Science.gov (United States)

    Winterbourn, Christine C

    2015-03-01

    Cells respond to many stimuli by transmitting signals through redox-regulated pathways. It is generally accepted that in many instances signal transduction is via reversible oxidation of thiol proteins, although there is uncertainty about the specific redox transformations involved. The prevailing view is that thiol oxidation occurs by a two electron mechanism, most commonly involving hydrogen peroxide. Free radicals, on the other hand, are considered as damaging species and not generally regarded as important in cell signaling. This paper examines whether it is justified to dismiss radicals or whether they could have a signaling role. Although there is no direct evidence that radicals are involved in transmitting thiol-based redox signals, evidence is presented that they are generated in cells when these signaling pathways are activated. Radicals produce the same thiol oxidation products as two electron oxidants, although by a different mechanism, and at this point radical-mediated pathways should not be dismissed. There are unresolved issues about how radical mechanisms could achieve sufficient selectivity, but this could be possible through colocalization of radical-generating and signal-transducing proteins. Colocalization is also likely to be important for nonradical signaling mechanisms and identification of such associations should be a priority for advancing the field.

  10. Differing views of the role of selenium in thioredoxin reductase

    Science.gov (United States)

    Ruggles, Erik L.

    2010-01-01

    This review covers three different chemical explanations that could account for the requirement of selenium in the form of selenocysteine in the active site of mammalian thioredoxin reductase. These views are the following: (1) the traditional view of selenocysteine as a superior nucleophile relative to cysteine, (2) the superior leaving group ability of a selenol relative to a thiol due to its significantly lower pKa and, (3) the superior ability of selenium to accept electrons (electrophilicity) relative to sulfur. We term these chemical explanations as the “chemico-enzymatic” function of selenium in an enzyme. We formally define the chemico-enzymatic function of selenium as its specific chemical property that allows a selenoenzyme to catalyze its individual reaction. However we, and others, question whether selenocysteine is chemically necessary to catalyze an enzymatic reaction since cysteine-homologs of selenocysteine-containing enzymes catalyze their specific enzymatic reactions with high catalytic efficiency. There must be a unique chemical reason for the presence of selenocysteine in enzymes that explains the biological pressure on the genome to maintain the complex selenocysteine-insertion machinery. We term this biological pressure the “chemico-biological” function of selenocysteine. We discuss evidence that this chemico-biological function is the ability of selenoenzymes to resist inactivation by irreversible oxidation. The way in which selenocysteine confers resistance to oxidation could be due to the superior ability of the oxidized form of selenocysteine (Sec-SeO2−, seleninic acid) to be recycled back to its parent form (Sec-SeH, selenocysteine) in comparison to the same cycling of cysteine-sulfinic acid to cysteine (Cys-SO2− to Cys-SH). PMID:20397034

  11. Acute effects of the sigma-2 receptor agonist siramesine on lysosomal and extra-lysosomal proteolytic systems in lens epithelial cells

    OpenAIRE

    Jonhede, S.; Petersen, A; Zetterberg, M.; Karlsson, J-O

    2010-01-01

    Purpose The aim of the present study was to examine the effects of the sigma-2 receptor agonist, siramesine, on morphology, growth, cell death, lysosomal function, and effects on extra-lysosomal proteolytic systems in human lens epithelial cells. Methods Human lens epithelial cells in culture were exposed to siramesine and examined for morphological changes using Nomarski optics or calcein. Lysosomes were evaluated using acridine orange and Magic Red (RR-cresyl violet). Nuclear morphology was...

  12. New drug target in protozoan parasites: the role of thioredoxin reductase.

    Science.gov (United States)

    Andrade, Rosa M; Reed, Sharon L

    2015-01-01

    Amebiasis causes approximately 70,000 deaths annually and is the third cause of death due to parasites worldwide. It is treated primarily with metronidazole, which has adverse side effects, is mutagenic and carcinogenic, and emergence of resistance is an increasing concern. Unfortunately, better therapeutic alternatives are lacking. Re-purposing of older FDA approved drugs is advantageous to drug discovery since safety and pharmacokinetic effects in humans are already known. In high throughput screening studies, we recently demonstrated that auranofin, a gold containing compound originally approved to treat rheumatoid arthritis, has activity against trophozoites of E. histolytica, the causative agent of amebiasis. Auranofin's anti-parasitic activity is attributed to its monovalent gold molecule that readily inhibits E. histolytica thioredoxin reductase. This anti-oxidant enzyme is the only thiol-dependent flavo-reductase present in E. histolytica. Auranofin has also shown promising activity against other protozoans of significant public health importance. Altogether, this evidence suggests that auranofin has the potential to become a broad spectrum alternative therapeutic agent for diseases with a large global burden.

  13. Corynebacterium diphtheriae methionine sulfoxide reductase a exploits a unique mycothiol redox relay mechanism.

    Science.gov (United States)

    Tossounian, Maria-Armineh; Pedre, Brandán; Wahni, Khadija; Erdogan, Huriye; Vertommen, Didier; Van Molle, Inge; Messens, Joris

    2015-05-01

    Methionine sulfoxide reductases are conserved enzymes that reduce oxidized methionines in proteins and play a pivotal role in cellular redox signaling. We have unraveled the redox relay mechanisms of methionine sulfoxide reductase A of the pathogen Corynebacterium diphtheriae (Cd-MsrA) and shown that this enzyme is coupled to two independent redox relay pathways. Steady-state kinetics combined with mass spectrometry of Cd-MsrA mutants give a view of the essential cysteine residues for catalysis. Cd-MsrA combines a nucleophilic cysteine sulfenylation reaction with an intramolecular disulfide bond cascade linked to the thioredoxin pathway. Within this cascade, the oxidative equivalents are transferred to the surface of the protein while releasing the reduced substrate. Alternatively, MsrA catalyzes methionine sulfoxide reduction linked to the mycothiol/mycoredoxin-1 pathway. After the nucleophilic cysteine sulfenylation reaction, MsrA forms a mixed disulfide with mycothiol, which is transferred via a thiol disulfide relay mechanism to a second cysteine for reduction by mycoredoxin-1. With x-ray crystallography, we visualize two essential intermediates of the thioredoxin relay mechanism and a cacodylate molecule mimicking the substrate interactions in the active site. The interplay of both redox pathways in redox signaling regulation forms the basis for further research into the oxidative stress response of this pathogen.

  14. New drug target in protozoan parasites: the role of thioredoxin reductase

    Directory of Open Access Journals (Sweden)

    Rosa M. Andrade

    2015-09-01

    Full Text Available Amebiasis causes approximately 70,000 deaths annually and is the third cause of death due to parasites worldwide. It is treated primarily with metronidazole, which has adverse side effects, is mutagenic and carcinogenic, and emergence of resistance is an increasing concern. Unfortunately, better therapeutic alternatives are lacking. Re-purposing of older FDA approved drugs is advantageous to drug discovery since safety and pharmacokinetic effects in humans are already known. In high throughput screening studies, we recently demonstrated that auranofin, a gold containing compound originally approved to treat rheumatoid arthritis, has activity against trophozoites of E. histolytica, the causative agent of amebiasis. Auranofin’s anti-parasitic activity is attributed to its monovalent gold molecule that readily inhibits E.histolytica thioredoxin reductase. This anti-oxidant enzyme is the only thiol-dependent flavo-reductase present in E.histolytica. Auranofin has also shown promising activity against other protozoans of significant public health importance. Altogether, this evidence suggests that auranofin has the potential to become a broad spectrum alternative therapeutic agent for diseases with a large global burden.

  15. The aldo-keto reductase superfamily homepage.

    Science.gov (United States)

    Hyndman, David; Bauman, David R; Heredia, Vladi V; Penning, Trevor M

    2003-02-01

    The aldo-keto reductases (AKRs) are one of the three enzyme superfamilies that perform oxidoreduction on a wide variety of natural and foreign substrates. A systematic nomenclature for the AKR superfamily was adopted in 1996 and was updated in September 2000 (visit www.med.upenn.edu/akr). Investigators have been diligent in submitting sequences of functional proteins to the Web site. With the new additions, the superfamily contains 114 proteins expressed in prokaryotes and eukaryotes that are distributed over 14 families (AKR1-AKR14). The AKR1 family contains the aldose reductases, the aldehyde reductases, the hydroxysteroid dehydrogenases and steroid 5beta-reductases, and is the largest. Other families of interest include AKR6, which includes potassium channel beta-subunits, and AKR7 the aflatoxin aldehyde reductases. Two new families include AKR13 (yeast aldose reductase) and AKR14 (Escherichia coli aldehyde reductase). Crystal structures of many AKRs and their complexes with ligands are available in the PDB and accessible through the Web site. Each structure has the characteristic (alpha/beta)(8)-barrel motif of the superfamily, a conserved cofactor binding site and a catalytic tetrad, and variable loop structures that define substrate specificity. Although the majority of AKRs are monomeric proteins of about 320 amino acids in length, the AKR2, AKR6 and AKR7 family may form multimers. To expand the nomenclature to accommodate multimers, we recommend that the composition and stoichiometry be listed. For example, AKR7A1:AKR7A4 (1:3) would designate a tetramer of the composition indicated. The current nomenclature is recognized by the Human Genome Project (HUGO) and the Web site provides a link to genomic information including chromosomal localization, gene boundaries, human ESTs and SNPs and much more.

  16. Influence of the microenvironment of thiol groups in low molecular mass thiols and serum albumin on the reaction with methylglyoxal.

    Science.gov (United States)

    Aćimović, Jelena M; Stanimirović, Bojana D; Todorović, Nina; Jovanović, Vesna B; Mandić, Ljuba M

    2010-10-06

    Methylglyoxal (MG), a reactive alpha-oxoaldehyde that is produced in higher quantities in diabetes, uremia, oxidative stress, aging and inflammation, reacts with the thiol groups (in addition to the amino and guanidino groups) of proteins. This causes protein modification, formation of advanced glycated end products (AGEs) and cross-linking. Low molecular mass thiols can be used as competitive targets for MG, preventing the reactions mentioned above. Therefore, this paper investigated how the microenvironment of the thiol group in low molecular mass thiols (cysteine, N-acetylcysteine (NAcCys), carboxymethylcysteine (CMC) and glutathione (GSH)) and human serum albumin (HSA) affected the thiol reaction with MG. The SH group reaction course was monitored by (1)H-NMR spectroscopy and spectrophotometric quantification. Changes in the HSA molecules were monitored by SDS-PAGE. The microenvironment of the SH group had a major effect on its reactivity and on the product yield. The reactivity of SH groups decreased in the order Cys>GSH>NAcCys. CMC did not react. The percentages of the reacted SH groups in the equilibrium state were almost equal, regardless of the ratio of thiol compound/MG (1:1, 1:2, 1:5): 38.1 + or - 0.9%; 38.2 + or - 0.7% and 39.0 + or - 0.8% for Cys; 26.5 + or - 0.6%; 26.6 + or - 2.6% and 27.4 + or - 2.5% for GSH; 10.8 + or - 0.9%; and 11.2 + or - 0.7% and 12.2 + or - 0.9% for NAcCys, respectively. Our results explain why substances containing alpha-amino-beta-mercapto-ethane as a pharmacophore are successful scavengers of MG. In equilibrium, HSA SH reacted in high percentages both with an insufficient amount and with an excess of MG (55% and 65%, respectively). An analysis of the hydrophobicity of the microenvironment of the SH group on the HSA surface showed that it could contribute to high levels of SH modification, leading to an increase in the scavenging activity of the albumin thiol.

  17. Autophagic flux promotes cisplatin resistance in human ovarian carcinoma cells through ATP-mediated lysosomal function.

    Science.gov (United States)

    Ma, Liwei; Xu, Ye; Su, Jing; Yu, Huimei; Kang, Jinsong; Li, Hongyan; Li, Xiaoning; Xie, Qi; Yu, Chunyan; Sun, Liankun; Li, Yang

    2015-11-01

    Lysosomes are involved in promoting resistance of cancer cells to chemotherapeutic agents. However, the mechanisms underlying lysosomal influence of cisplatin resistance in ovarian cancer remain incompletely understood. We report that, compared with cisplatin-sensitive SKOV3 cells, autophagy increases in cisplatin-resistant SKOV3/DDP cells treated with cisplatin. Inhibition of early-stage autophagy enhanced cisplatin-mediated cytotoxicity in SKOV3/DDP cells, but autophagy inhibition at a later stage by disturbing autophagosome-lysosome fusion is more effective. Notably, SKOV3/DDP cells contained more lysosomes than cisplatin-sensitive SKOV3 cells. Abundant lysosomes and lysosomal cathepsin D activity were required for continued autolysosomal degradation and maintenance of autophagic flux in SKOV3/DDP cells. Furthermore, SKOV3/DDP cells contain abundant lysosomal ATP required for lysosomal function, and inhibition of lysosomal ATP accumulation impaired lysosomal function and blocked autophagic flux. Therefore, our findings suggest that lysosomes at least partially contribute to cisplatin resistance in ovarian cancer cells through their role in cisplatin-induced autophagic processes, and provide insight into the mechanism of cisplatin resistance in tumors.

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

    Science.gov (United States)

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

    2016-05-01

    Abnormalities in lysosomal function have been reported in diabetes, aging, and age-related degenerative diseases. These lysosomal abnormalities are an early manifestation of neurodegenerative diseases and often precede the onset of clinical symptoms such as learning and memory deficits; however, the mechanism underlying lysosomal dysfunction is not known. In the current study, we investigated the mechanism underlying lysosomal dysfunction in the cortex and hippocampi, key structures involved in learning and memory, of a type 2 diabetes (T2D) mouse model, the leptin receptor deficient db/db mouse. We demonstrate for the first time that diabetes leads to destabilization of lysosomes as well as alterations in the protein expression, activity, and/or trafficking of two lysosomal enzymes, hexosaminidase A and cathepsin D, in the hippocampus of db/db mice. Pioglitazone, a thiazolidinedione (TZD) commonly used in the treatment of diabetes due to its ability to improve insulin sensitivity and reverse hyperglycemia, was ineffective in reversing the diabetes-induced changes on lysosomal enzymes. Our previous work revealed that pioglitazone does not reverse hypercholesterolemia; thus, we investigated whether cholesterol plays a role in diabetes-induced lysosomal changes. In vitro, cholesterol promoted the destabilization of lysosomes, suggesting that lysosomal-related changes associated with diabetes are due to elevated levels of cholesterol. Since lysosome dysfunction precedes neurodegeneration, cognitive deficits, and Alzheimer's disease neuropathology, our results may provide a potential mechanism that links diabetes with complications of the central nervous system.

  19. Myelin lesions associated with lysosomal and peroxisomal disorders.

    Science.gov (United States)

    Faust, Phyllis L; Kaye, Edward M; Powers, James M

    2010-09-01

    Abnormalities of myelin are common in lysosomal and peroxisomal disorders. Most display a primary loss of myelin in which the myelin sheath and/or oligodendrocytes are selectively targeted by diverse pathogenetic processes. The most severe and, hence, clinically relevant are heritable diseases predominantly of infants and children, the leukodystrophies: metachromatic, globoid cell (Krabbe disease) and adreno-leukodystrophy. Our still limited understanding of these diseases has derived from multiple sources: originally, neurological-neuropathologic-neurochemical correlative studies of the natural disease in humans or other mammals, which has been enhanced by more sophisticated and contemporary techniques of cell and molecular biology. Transgenic mouse models seem to be the most promising methodology, allowing the examination of the cellular role of lysosomes and peroxisomes for formation and maintenance of both myelin and axons, and providing initial platforms to evaluate therapies. Treatment options are woefully inadequate and in their nascent stages, but still inspire some hope for the future.

  20. Induced pluripotent stem cell models of lysosomal storage disorders

    Directory of Open Access Journals (Sweden)

    Daniel K. Borger

    2017-06-01

    Full Text Available Induced pluripotent stem cells (iPSCs have provided new opportunities to explore the cell biology and pathophysiology of human diseases, and the lysosomal storage disorder research community has been quick to adopt this technology. Patient-derived iPSC models have been generated for a number of lysosomal storage disorders, including Gaucher disease, Pompe disease, Fabry disease, metachromatic leukodystrophy, the neuronal ceroid lipofuscinoses, Niemann-Pick types A and C1, and several of the mucopolysaccharidoses. Here, we review the strategies employed for reprogramming and differentiation, as well as insights into disease etiology gleaned from the currently available models. Examples are provided to illustrate how iPSC-derived models can be employed to develop new therapeutic strategies for these disorders. We also discuss how models of these rare diseases could contribute to an enhanced understanding of more common neurodegenerative disorders such as Parkinson’s disease, and discuss key challenges and opportunities in this area of research.

  1. Immune response hinders therapy for lysosomal storage diseases.

    Science.gov (United States)

    Ponder, Katherine P

    2008-08-01

    Enzyme replacement therapy (ERT) for the lysosomal storage disease mucopolysaccharidosis I (MPS I) involves i.v. injection of alpha-l-iduronidase, which can be taken up by cells throughout the body. While a significant immune response to ERT has been shown in patients with MPS I, little is known about what effect anti-enzyme antibodies have on treatment efficacy. In this issue of the JCI, Dickson et al. demonstrate that anti-enzyme antibodies inhibit enzyme uptake and substantially limit the therapeutic efficacy of ERT in canines with MPS I (see the related article beginning on page 2868). Furthermore, the induction of immune tolerance--via oral delivery of cyclosporine A and azathioprine for two months at the time of initiation of ERT with recombinant human alpha-L-iduronidase--improved enzyme uptake in organs. Therefore, transient immunosuppression may enhance ERT for lysosomal storage diseases.

  2. Thiol/disulfide homeostasis as a novel indicator of oxidative stress in children with simple febrile seizures.

    Science.gov (United States)

    Elmas, Bahri; Erel, Özcan; Ersavaş, Dilek; Yürümez, Yusuf

    2017-08-14

    Simple febrile seizures are generally benign, but during the seizure, elevated levels of glutamate and high levels of oxygen use due to the high metabolic brain activity result in oxidative stress. However, the relationship between febrile seizures and oxidative stress remains unclear. In this study, we investigated thiol/disulfide homeostasis as a new oxidative stress parameter in patients with simple febrile seizures. This study was performed between February 2016 and May 2016 at the Pediatric Emergency Unit. The study population consisted of 40 patients with a diagnosis of simple febrile seizure and 30 control participants aged 8-59 months. Total thiol, native thiol and disulfide levels, disulfide/native thiol, disulfide/total thiol, and native thiol/total thiol ratios were used as thiol/disulfide homeostasis parameters and were quantified in patient and control groups. Furthermore, correlations with seizure duration were investigated. In the patient group, native and total thiol levels and native thiol/total thiol ratios were low, and disulfide levels, disulfide/native thiol, and disulfide/total thiol ratios were significantly higher than in the control group. Negative correlations were observed between seizure duration, total and native thiol levels, and native thiol/total thiol ratio, whereas positive correlations were observed between seizure duration and disulfide/native thiol and disulfide/total thiol ratio. The sensitivities of both disulfide/native thiol and disulfide/total thiol ratios were high for simple febrile seizures. Simple febrile seizures may cause impairment in favor of disulfide bonds in thiol/disulfide homeostasis. Overall, these changes may contribute to neuronal cell damage after simple febrile seizures.

  3. Targeting Androgen Receptor by Lysosomal Degradation in Prostate Cancer

    Science.gov (United States)

    2014-09-01

    were done as described.13 Protein Sample Preparation and Mass Spectrometry Tandem Affinity Purification of FLAG-His-EWS-Fli-1- Interacting Proteins . Forty...incubated with Ni-NTA agarose (Qiagen), FLAG-His-EWS-Fli-1 and its interacting proteins were collected by centrifugation, washed three times with TN buffer...the lysosome fraction was loaded at 100x compared to the input. ■ RESULTS AND DISCUSSION Proteomic Analysis of the EWS-Fli-1- Interacting Proteins To

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

    Science.gov (United States)

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

    1999-01-01

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

  5. Doxorubicin Blocks Cardiomyocyte Autophagic Flux by Inhibiting Lysosome Acidification.

    Science.gov (United States)

    Li, Dan L; Wang, Zhao V; Ding, Guanqiao; Tan, Wei; Luo, Xiang; Criollo, Alfredo; Xie, Min; Jiang, Nan; May, Herman; Kyrychenko, Viktoriia; Schneider, Jay W; Gillette, Thomas G; Hill, Joseph A

    2016-04-26

    The clinical use of doxorubicin is limited by cardiotoxicity. Histopathological changes include interstitial myocardial fibrosis and the appearance of vacuolated cardiomyocytes. Whereas dysregulation of autophagy in the myocardium has been implicated in a variety of cardiovascular diseases, the role of autophagy in doxorubicin cardiomyopathy remains poorly defined. Most models of doxorubicin cardiotoxicity involve intraperitoneal injection of high-dose drug, which elicits lethargy, anorexia, weight loss, and peritoneal fibrosis, all of which confound the interpretation of autophagy. Given this, we first established a model that provokes modest and progressive cardiotoxicity without constitutional symptoms, reminiscent of the effects seen in patients. We report that doxorubicin blocks cardiomyocyte autophagic flux in vivo and in cardiomyocytes in culture. This block was accompanied by robust accumulation of undegraded autolysosomes. We go on to localize the site of block as a defect in lysosome acidification. To test the functional relevance of doxorubicin-triggered autolysosome accumulation, we studied animals with diminished autophagic activity resulting from haploinsufficiency for Beclin 1. Beclin 1(+/-) mice exposed to doxorubicin were protected in terms of structural and functional changes within the myocardium. Conversely, animals overexpressing Beclin 1 manifested an amplified cardiotoxic response. Doxorubicin blocks autophagic flux in cardiomyocytes by impairing lysosome acidification and lysosomal function. Reducing autophagy initiation protects against doxorubicin cardiotoxicity. © 2016 American Heart Association, Inc.

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

    Science.gov (United States)

    Tripathi, Vijay; Gupta, Dwijendra Kumar

    2014-01-01

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

  7. Recent advances in gene therapy for lysosomal storage disorders.

    Science.gov (United States)

    Rastall, David Pw; Amalfitano, Andrea

    2015-01-01

    Lysosomal storage disorders (LSDs) are a group of genetic diseases that result in metabolic derangements of the lysosome. Most LSDs are due to the genetic absence of a single catabolic enzyme, causing accumulation of the enzyme's substrate within the lysosome. Over time, tissue-specific substrate accumulations result in a spectrum of symptoms and disabilities that vary by LSD. LSDs are promising targets for gene therapy because delivery of a single gene into a small percentage of the appropriate target cells may be sufficient to impact the clinical course of the disease. Recently, there have been several significant advancements in the potential for gene therapy of these disorders, including the first human trials. Future clinical trials will build upon these initial attempts, with an improved understanding of immune system responses to gene therapy, the obstacle that the blood-brain barrier poses for neuropathic LSDs, as well other biological barriers that, when overcome, may facilitate gene therapy for LSDs. In this manuscript, we will highlight the recent innovations in gene therapy for LSDs and discuss the clinical limitations that remain to be overcome, with the goal of fostering an understanding and further development of this important field.

  8. Respiratory arsenate reductase as a bidirectional enzyme

    Science.gov (United States)

    Richey, C.; Chovanec, P.; Hoeft, S.E.; Oremland, R.S.; Basu, P.; Stolz, J.F.

    2009-01-01

    The haloalkaliphilic bacterium Alkalilimnicola ehrlichii is capable of anaerobic chemolithoautotrophic growth by coupling the oxidation of arsenite (As(III)) to the reduction of nitrate and carbon dioxide. Analysis of its complete genome indicates that it lacks a conventional arsenite oxidase (Aox), but instead possesses two operons that each encode a putative respiratory arsenate reductase (Arr). Here we show that one homolog is expressed under chemolithoautotrophic conditions and exhibits both arsenite oxidase and arsenate reductase activity. We also demonstrate that Arr from two arsenate respiring bacteria, Alkaliphilus oremlandii and Shewanella sp. strain ANA-3, is also biochemically reversible. Thus Arr can function as a reductase or oxidase. Its physiological role in a specific organism, however, may depend on the electron potentials of the molybdenum center and [Fe–S] clusters, additional subunits, or constitution of the electron transfer chain. This versatility further underscores the ubiquity and antiquity of microbial arsenic metabolism.

  9. Adenanthin, a new inhibitor of thiol-dependent antioxidant enzymes, impairs the effector functions of human natural killer cells.

    Science.gov (United States)

    Siernicka, Marta; Winiarska, Magdalena; Bajor, Malgorzata; Firczuk, Malgorzata; Muchowicz, Angelika; Bobrowicz, Malgorzata; Fauriat, Cyril; Golab, Jakub; Olive, Daniel; Zagozdzon, Radoslaw

    2015-09-01

    Natural killer (NK) cells are considered critical components of the innate and adaptive immune responses. Deficiencies in NK cell activity are common, such as those that occur in cancer patients, and they can be responsible for dysfunctional immune surveillance. Persistent oxidative stress is intrinsic to many malignant tumours, and numerous studies have focused on the effects of reactive oxygen species on the anti-tumour activity of NK cells. Indeed, investigations in animal models have suggested that one of the most important thiol-dependent antioxidant enzymes, peroxiredoxin 1 (PRDX1), is essential for NK cell function. In this work, our analysis of the transcriptomic expression pattern of antioxidant enzymes in human NK cells has identified PRDX1 as the most prominently induced transcript out of the 18 transcripts evaluated in activated NK cells. The change in PRDX1 expression was followed by increased expression of two other enzymes from the PRDX-related antioxidant chain: thioredoxin and thioredoxin reductase. To study the role of thiol-dependent antioxidants in more detail, we applied a novel compound, adenanthin, to induce an abrupt dysfunction of the PRDX-related antioxidant chain in NK cells. In human primary NK cells, we observed profound alterations in spontaneous and antibody-dependent NK cell cytotoxicity against cancer cells, impaired degranulation, and a decreased expression of activation markers under these conditions. Collectively, our study pinpoints the unique role for the antioxidant activity of the PRDX-related enzymatic chain in human NK cell functions. Further understanding this phenomenon will prospectively lead to fine-tuning of the novel NK-targeted therapeutic approaches to human disease. © 2015 John Wiley & Sons Ltd.

  10. Thiol metabolism and antioxidant systems complement each other during arsenate detoxification in Ceratophyllum demersum L.

    Science.gov (United States)

    Mishra, Seema; Srivastava, Sudhakar; Tripathi, Rudra D; Trivedi, Prabodh K

    2008-01-31

    Ceratophyllum demersum L. is known to be a potential accumulator of arsenic (As), but mechanisms of As detoxification have not been investigated so far. In the present study, we analyzed the biochemical responses of Ceratophyllum plants to arsenate (As(V); 0-250 microM) exposure to explore the underlying mechanisms of As detoxification. Plants efficiently tolerated As toxicity up to concentrations of 50 microM As(V) and durations of 4 d with no significant effect on growth by modulating various pathways in a coordinated and complementary manner and accumulated about 76 microg As g(-1)dw. Significant increases were observed in the levels of various thiols including phytochelatins (PCs), the activities of enzymes of thiolic metabolism as well as arsenate reductase (AR). These primary responses probably enabled plants to detoxify at least some part of As(V) through its reduction and subsequent complexation. The maximum proportion of As chelated by PCs was found to be about 30% (at 50 microM As(V) after 2 d). Simultaneously, a significant increase in the activities of antioxidant enzymes was observed and hence plants did not experience oxidative stress when exposed to 50 microM As(V) for 4 d. Exposure of plants to higher concentrations (250 microM As(V)) and/or for longer durations (7 d) resulted in a significant increase in the level of As (maximum 525 microgg(-1)dw at 250 microM after 7 d) and an inverse relationship between As accumulation and various detoxification strategies was observed that lead to enhanced oxidative stress and hampered growth.

  11. Radical Scavenging Efficacy of Thiol Capped Silver Nanoparticles

    Indian Academy of Sciences (India)

    Kumudini Chandraker; Sandeep Kumar Vaishanav; Rekha Nagwanshi; Manmohan L Satnami

    2015-12-01

    Radical scavenging efficacy of L-cysteine (L-Cys), glutathione (GSH) and thioctic acid (TA) in the presence of silver nanoparticles (AgNPs) were determined by 1,1-diphenyl 2-picryl hydrazil (DPPH), nitric oxide (NO) and hydroxyl (OH) radicals as spectrophotometric assay. The hydrogen peroxide (H2O2) scavenging efficacy has been determined by titration method. Ascorbic acid has been used as standard for all radical scavenging efficacies. In general, antioxidant activity decreases in the presence of AgNPs. The covalent interactions of thiols (-SH) were found to be a key factor for the decreases in scavenging activity. The effect of thiol concentrations has been discussed. The size and shape of the nanoparticles and AgNP-SR interactions have been characterized through Transmission Electron Microscopy (TEM) and Fourier Transform Infrared (FTIR) spectroscopy, respectively.

  12. Asymmetric Ketone Reduction by Imine Reductases.

    Science.gov (United States)

    Lenz, Maike; Meisner, Jan; Quertinmont, Leann; Lutz, Stefan; Kästner, Johannes; Nestl, Bettina M

    2017-02-01

    The rapidly growing area of asymmetric imine reduction by imine reductases (IREDs) has provided alternative routes to chiral amines. Here we report the expansion of the reaction scope of IREDs by showing the stereoselective reduction of 2,2,2-trifluoroacetophenone. Assisted by an in silico analysis of energy barriers, we evaluated asymmetric hydrogenations of carbonyls and imines while considering the influence of substrate reactivity on the chemoselectivity of this novel class of reductases. We report the asymmetric reduction of C=N as well as C=O bonds catalysed by members of the IRED enzyme family. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Evolution of plant defense mechanisms. Relationships of phenylcoumaran benzylic ether reductases to pinoresinol-lariciresinol and isoflavone reductases.

    Science.gov (United States)

    Gang, D R; Kasahara, H; Xia, Z Q; Vander Mijnsbrugge, K; Bauw, G; Boerjan, W; Van Montagu, M; Davin, L B; Lewis, N G

    1999-03-12

    Pinoresinol-lariciresinol and isoflavone reductase classes are phylogenetically related, as is a third, the so-called "isoflavone reductase homologs." This study establishes the first known catalytic function for the latter, as being able to engender the NADPH-dependent reduction of phenylcoumaran benzylic ethers. Accordingly, all three reductase classes are involved in the biosynthesis of important and related phenylpropanoid-derived plant defense compounds. In this investigation, the phenylcoumaran benzylic ether reductase from the gymnosperm, Pinus taeda, was cloned, with the recombinant protein heterologously expressed in Escherichia coli. The purified enzyme reduces the benzylic ether functionalities of both dehydrodiconiferyl alcohol and dihydrodehydrodiconiferyl alcohol, with a higher affinity for the former, as measured by apparent Km and Vmax values and observed kinetic 3H-isotope effects. It abstracts the 4R-hydride of the required NADPH cofactor in a manner analogous to that of the pinoresinol-lariciresinol reductases and isoflavone reductases. A similar catalytic function was observed for the corresponding recombinant reductase whose gene was cloned from the angiosperm, Populus trichocarpa. Interestingly, both pinoresinol-lariciresinol reductases and isoflavone reductases catalyze enantiospecific conversions, whereas the phenylcoumaran benzylic ether reductase only shows regiospecific discrimination. A possible evolutionary relationship among the three reductase classes is proposed, based on the supposition that phenylcoumaran benzylic ether reductases represent the progenitors of pinoresinol-lariciresinol and isoflavone reductases.

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

    Science.gov (United States)

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

    2011-02-01

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

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

    Science.gov (United States)

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

    2016-06-30

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

  16. Dynamic Cyclic Thiodepsipeptide Libraries from Thiol-Thioester Exchange

    Science.gov (United States)

    2010-04-01

    the reaction dynamics are discussed. Cyclic peptides have been described as “privileged structures” for drug design because so many natural and...inhibitors, which hold promise for treatment of cancer.4 As drug scaffolds, cyclic peptides are advantageous because they mimic native protein structure...but instead an influence of chirality on the accessibility of the thioester or thiol. We also investigated the effect of positively charged amino

  17. Investigation of thiol derivatized gold nanoparticle sensors for gas analysis

    Science.gov (United States)

    Stephens, Jared S.

    Analysis of volatile organic compounds (VOCs) in air and exhaled breath by sensor array is a very useful testing technique. It can provide non-invasive, fast, inexpensive testing for many diseases. Breath analysis has been very successful in identifying cancer and other diseases by using a chemiresistor sensor or array with gold nanoparticles to detect biomarkers. Acetone is a biomarker for diabetes and having a portable testing device could help to monitor diabetic and therapeutic progress. An advantage to this testing method is it is conducted at room temperature instead of 200 degrees Celsius. 3. The objective of this research is to determine the effect of thiol derivatized gold nanoparticles based on sensor(s) detection of VOCs. The VOCs to be tested are acetone, ethanol, and a mixture of acetone and ethanol. Each chip is tested under all three VOCs and three concentration levels (0.1, 1, and 5.0 ppm). VOC samples are used to test the sensors' ability to detect and differentiate VOCs. Sensors (also referred to as a chip) are prepared using several types of thiol derivatized gold nanoparticles. The factors are: thiol compound and molar volume loading of the thiol in synthesis. The average resistance results are used to determine the VOC selectivity of the sensors tested. The results show a trend of increasing resistance as VOC concentration is increased relative to dry air; which is used as baseline for VOCs. Several sensors show a high selectivity to one or more VOCs. Overall the 57 micromoles of 4-methoxy-toluenethiol sensor shows the strongest selectivity for VOCs tested. 3. Gerfen, Kurt. 2012. Detection of Acetone in Air Using Silver Ion Exchanged ZSM-5 and Zinc Oxide Sensing Films. Master of Science thesis, University of Louisville.

  18. Selenocysteine in thiol/disulfide-like exchange reactions.

    Science.gov (United States)

    Hondal, Robert J; Marino, Stefano M; Gladyshev, Vadim N

    2013-05-01

    Among trace elements used as cofactors in enzymes, selenium is unique in that it is incorporated into proteins co-translationally in the form of an amino acid, selenocysteine (Sec). Sec differs from cysteine (Cys) by only one atom (selenium versus sulfur), yet this switch dramatically influences important aspects of enzyme reactivity. The main focus of this review is an updated and critical discussion on how Sec might be used to accelerate thiol/disulfide-like exchange reactions in natural selenoenzymes, compared with their Cys-containing homologs. We discuss in detail three major aspects associated with thiol/disulfide exchange reactions: (i) nucleophilicity of the attacking thiolate (or selenolate); (ii) electrophilicity of the center sulfur (or selenium) atom; and (iii) stability of the leaving group (sulfur or selenium). In all these cases, we analyze the benefits that selenium might provide in these types of reactions. It is the biological thiol oxidoreductase-like function that benefits from the use of Sec, since Sec functions to chemically accelerate the rate of these reactions. We review various hypotheses that could help explain why Sec is used in enzymes, particularly with regard to competitive chemical advantages provided by the presence of the selenium atom in enzymes. Ultimately, these chemical advantages must be connected to biological functions of Sec.

  19. Differential stress induced by thiol adsorption on facetted nanocrystals.

    Science.gov (United States)

    Watari, Moyu; McKendry, Rachel A; Vögtli, Manuel; Aeppli, Gabriel; Soh, Yeong-Ah; Shi, Xiaowen; Xiong, Gang; Huang, Xiaojing; Harder, Ross; Robinson, Ian K

    2011-09-25

    Polycrystalline gold films coated with thiol-based self-assembled monolayers (SAM) form the basis of a wide range of nanomechanical sensor platforms. The detection of adsorbates with such devices relies on the transmission of mechanical forces, which is mediated by chemically derived stress at the organic-inorganic interface. Here, we show that the structure of a single 300-nm-diameter facetted gold nanocrystal, measured with coherent X-ray diffraction, changes profoundly after the adsorption of one of the simplest SAM-forming organic molecules. On self-assembly of propane thiol, the crystal's flat facets contract radially inwards relative to its spherical regions. Finite-element modelling indicates that this geometry change requires large stresses that are comparable to those observed in cantilever measurements. The large magnitude and slow kinetics of the contraction can be explained by an intermixed gold-sulphur layer that has recently been identified crystallographically. Our results illustrate the importance of crystal edges and grain boundaries in interface chemistry and have broad implications for the application of thiol-based SAMs, ranging from nanomechanical sensors to coating technologies.

  20. Organized thiol functional groups in mesoporous core shell colloids

    Energy Technology Data Exchange (ETDEWEB)

    Marchena, Martin H. [Gerencia Quimica, Centro Atomico Constituyentes, Comision Nacional de Energia Atomica (CNEA), Avda. Gral. Paz 1499, B1650KNA Buenos Aires (Argentina); Granada, Mara [Centro Atomico Bariloche-CNEA, 8400 San Carlos de Bariloche (Argentina); Instituto Balseiro-Centro Atomico Bariloche-CNEA, San Carlos de Bariloche 8400 (Argentina); Bordoni, Andrea V. [Gerencia Quimica, Centro Atomico Constituyentes, Comision Nacional de Energia Atomica (CNEA), Avda. Gral. Paz 1499, B1650KNA Buenos Aires (Argentina); Joselevich, Maria [Asociacion Civil Expedicion Ciencia, Cabrera 4948, C1414BGP Buenos Aires (Argentina); Troiani, Horacio [Centro Atomico Bariloche-CNEA, 8400 San Carlos de Bariloche (Argentina); Instituto Balseiro-Centro Atomico Bariloche-CNEA, San Carlos de Bariloche 8400 (Argentina); Williams, Federico J. [DQIAQyF-INQUIMAE FCEN, Universidad de Buenos Aires, Ciudad Universitaria, Pabellon II, C1428EHA Buenos Aires (Argentina); Wolosiuk, Alejandro, E-mail: wolosiuk@cnea.gov.ar [Gerencia Quimica, Centro Atomico Constituyentes, Comision Nacional de Energia Atomica (CNEA), Avda. Gral. Paz 1499, B1650KNA Buenos Aires (Argentina)

    2012-03-15

    The co-condensation in situ of tetraethoxysilane (TEOS) and mercaptopropyltrimethoxysilane (MPTMS) using cetyltrimethylammonium bromide (CTAB) as a template results in the synthesis of multilayered mesoporous structured SiO{sub 2} colloids with 'onion-like' chemical environments. Thiol groups were anchored to an inner selected SiO{sub 2} porous layer in a bilayered core shell particle producing different chemical regions inside the colloidal layered structure. X-Ray Photoelectron Spectroscopy (XPS) shows a preferential anchoring of the -SH groups in the double layer shell system, while porosimetry and simple chemical modifications confirm that pores are accessible. We can envision the synthesis of interesting colloidal objects with defined chemical environments with highly controlled properties. - Graphical abstract: Mesoporous core shell SiO{sub 2} colloids with organized thiol groups. Highlights: Black-Right-Pointing-Pointer Double shell mesoporous silica colloids templated with CTAB. Black-Right-Pointing-Pointer Sequential deposition of mesoporous SiO{sub 2} layers with different chemistries. Black-Right-Pointing-Pointer XPS shows the selective functionalization of mesoporous layers with thiol groups.

  1. Occurrence of polyfunctional thiols in fresh lager beers.

    Science.gov (United States)

    Vermeulen, C; Lejeune, I; Tran, T T H; Collin, S

    2006-07-12

    Polyfunctional thiols are known to have a strong impact on the overall aroma of many fermented foods. Surprisingly, very little data is available on their occurrence in beer. A specific extraction with p-hydroxymercuribenzoic acid was performed on four different fresh light-protected lager beers. gas chromatography-olfactometry, gas chromatography-mass spectrometry, and gas chromatography-pulsed-flame photometer detector analyses of the extracts revealed the presence of more than 10 polyfunctional thiols. All of them were absent from wort, suggesting a key role of the H(2)S excreted by yeasts. 3-Methyl-2-buten-1-thiol, 2-mercapto-3-methylbutanol, 3-mercapto-3-methylbutanol seem to be created from hop allylic alcohols via four different mechanisms: nucleophilic substitution, addition-elimination, and radical anti-Markovnikov or electrophilic Markovnikov additions. 1,4 Addition of hydrogen sulfide to wort alpha,beta-unsaturated aldehydes or ketones may explain the synthesis of 1-mercapto-3-pentanol, 3-mercaptohexanol, and 4-mercapto-4-methyl-2-pentanone through fermentation. Finally, 2-mercaptoethanol, 3-mercaptopropanol, and their corresponding acetates may derive from Ehrlich degradation of sulfur amino acids, while 2-methyl-3-furanthiol should be logically issued from Maillard reactions.

  2. Isoquinoline-mediated S-vinylation and N-vinylation of benzo[d]oxazole-2-thiol and benzo[d]thiazole-2-thiol

    Institute of Scientific and Technical Information of China (English)

    Issa Yavari; Samira Nasiri-Gheidari; Anvar Mirzaei

    2012-01-01

    An effective route to S-vinylated andN-vinylated benzo[d]oxazole-2(3H)-thiones and benzo[d]thiazole-2(3H)-thionesis described via reaction ofacetylenic esters and benzo[d] oxazole-2-thiol and benzo [d]thiazole-2-thiol in the presence of 15 mol% of isoquinoline.

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

    Science.gov (United States)

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

    2000-01-01

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

  4. Thermal and Mechanical Properties of Sequential and Simultaneous Thiol-Ene-Isocyanate Networks

    Science.gov (United States)

    McNair, Olivia; Brent, Davis; Savin, Daniel

    2011-03-01

    Ternary networks containing having stoichiometrically balanced thiol /(ene+isocyanate) ranging from 0 to 20 mol% isocyanate were synthesized via sequential or simultaneous thiol/ene and thiol/isocyanate click reactions. The effects of cross-link density were studied using three thiols, GDMP (difunctional), 3T (trifunctional) and 4T (tetrafunctional) respectively. TEA catalyzes the isocyanate-thiol coupling and chain extension, while the photoinitiator DMPA initiates a radical thiol-ene crosslinking process. Real-time FTIR was used to study kinetics of both light and dark reactions utilizing thiol, ene and isocyanate peaks which appear independently. It was found that difunctional thiols and isocyanates reacted initially, forming chain extended prepolymers end-capped with thiol functionalities. Upon UV irradiation, thiol functionalized prepolymers reacted with TTT, a trifunctional ene, forming networks containing incorporated thiourethane linkages. Initial DSC results indicated higher Tgs for higher cross-linked networks; however, isocyanate content has significant effects on each system. Films were also be thermally characterized via DMA and mechanical properties measured using MTS.

  5. Cathepsin B modulates lysosomal biogenesis and host defense against Francisella novicida infection.

    Science.gov (United States)

    Qi, Xiaopeng; Man, Si Ming; Malireddi, R K Subbarao; Karki, Rajendra; Lupfer, Christopher; Gurung, Prajwal; Neale, Geoffrey; Guy, Clifford S; Lamkanfi, Mohamed; Kanneganti, Thirumala-Devi

    2016-09-19

    Lysosomal cathepsins regulate an exquisite range of biological functions, and their deregulation is associated with inflammatory, metabolic, and degenerative diseases in humans. In this study, we identified a key cell-intrinsic role for cathepsin B as a negative feedback regulator of lysosomal biogenesis and autophagy. Mice and macrophages lacking cathepsin B activity had increased resistance to the cytosolic bacterial pathogen Francisella novicida Genetic deletion or pharmacological inhibition of cathepsin B down-regulated mechanistic target of rapamycin activity and prevented cleavage of the lysosomal calcium channel TRPML1. These events drove transcription of lysosomal and autophagy genes via transcription factor EB, which increased lysosomal biogenesis and activation of autophagy initiation kinase ULK1 for clearance of the bacteria. Our results identified a fundamental biological function of cathepsin B in providing a checkpoint for homeostatic maintenance of lysosome populations and basic recycling functions in the cell.

  6. TFEB and TFE3: Linking Lysosomes to Cellular Adaptation to Stress.

    Science.gov (United States)

    Raben, Nina; Puertollano, Rosa

    2016-10-06

    In recent years, our vision of lysosomes has drastically changed. Formerly considered to be mere degradative compartments, they are now recognized as key players in many cellular processes. The ability of lysosomes to respond to different stimuli revealed a complex and coordinated regulation of lysosomal gene expression. This review discusses the participation of the transcription factors TFEB and TFE3 in the regulation of lysosomal function and biogenesis, as well as the role of the lysosomal pathway in cellular adaptation to a variety of stress conditions, including nutrient deprivation, mitochondrial dysfunction, protein misfolding, and pathogen infection. We also describe how cancer cells make use of TFEB and TFE3 to promote their own survival and highlight the potential of these transcription factors as therapeutic targets for the treatment of neurological and lysosomal diseases.

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

    OpenAIRE

    2013-01-01

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

  8. Disruption of Lysosome Function Promotes Tumor Growth and Metastasis in Drosophila *

    OpenAIRE

    Chi, Congwu; Zhu, Huanhu; Han,Min; Zhuang, Yuan; Wu, Xiaohui; Xu, Tian

    2010-01-01

    Lysosome function is essential to many physiological processes. It has been suggested that deregulation of lysosome function could contribute to cancer. Through a genetic screen in Drosophila, we have discovered that mutations disrupting lysosomal degradation pathway components contribute to tumor development and progression. Loss-of-function mutations in the Class C vacuolar protein sorting (VPS) gene, deep orange (dor), dramatically promote tumor overgrowth and invasion of the RasV12 cells....

  9. Thiol switches in redox regulation of chloroplasts: balancing redox state, metabolism and oxidative stress.

    Science.gov (United States)

    Dietz, Karl-Josef; Hell, Rüdiger

    2015-05-01

    In photosynthesizing chloroplasts, rapidly changing energy input, intermediate generation of strong reductants as well as oxidants and multiple participating physicochemical processes and pathways, call for efficient regulation. Coupling redox information to protein function via thiol modifications offers a powerful mechanism to activate, down-regulate and coordinate interdependent processes. Efficient thiol switching of target proteins involves the thiol-disulfide redox regulatory network, which is highly elaborated in chloroplasts. This review addresses the features of this network. Its conditional function depends on specificity of reduction and oxidation reactions and pathways, thiol redox buffering, but also formation of heterogeneous milieus by microdomains, metabolite gradients and macromolecular assemblies. One major player is glutathione. Its synthesis and function is under feedback redox control. The number of thiol-controlled processes and involved thiol switched proteins is steadily increasing, e.g., in tetrapyrrole biosynthesis, plastid transcription and plastid translation. Thus chloroplasts utilize an intricate and versatile redox regulatory network for intraorganellar and retrograde communication.

  10. A new lysosomal storage disorder resembling Morquio syndrome in sibs.

    Science.gov (United States)

    Perrin, Laurence; Fenneteau, Odile; Ilharreborde, Brice; Capri, Yline; Gérard, Marion; Quoc, Emmanuel Bui; Passemard, Sandrine; Ghoumid, Jamal; Caillaud, Catherine; Froissart, Roseline; Tabet, Anne-Claude; Lebon, Sophie; El Ghouzzi, Vincent; Mazda, Keyvan; Verloes, Alain

    2012-03-01

    We report two male sibs, born from unrelated French Caribbean parents, presenting with an unclassifiable storage disorder. Pregnancy and delivery were uneventful. Stunted growth was noted during the first year of life. Both children have short stature (below - 4SD) with short trunk, barrel chest, micromelia with rhizomelic shortening, severe kyphoscoliosis, pectus carinatum, short hands and feet with metatarsus adductus, and excessive joint laxity of the small joints. Learning difficulties with borderline intelligence quotient (IQ) were noted in one of them. They had no hepatomegaly, no splenomegaly, and no dysmorphism. Skeletal X-rays survey demonstrated generalized platyspondyly with tongue-like deformity of the anterior part of the vertebral bodies, hypoplasia of the odontoid process, generalized epiphyseal dysplasia and abnormally shaped metaphyses. The acetabular roofs had a trident aspect. Ophthalmologic and cardiac examinations were normal. Spine deformity required surgical correction in one of the patient at age 4 years. Lysosomal enzymes assays including N-acetylgalactosamine-6-sulfate sulfatase and β-galactosidase were normal, excluding mucopolysaccharidoses type IV A and IV B (Morquio syndrome), respectively. Qualitative analysis found traces of dermatan and chondroitin-sulfates in urine, but quantitative glycosaminoglycan excretion fell within normal limits. They were no vacuolated lymphocytes. Abnormal coarse inclusions were present in eosinophils. Mild Alder anomaly was observed in polymorphonuclears. Granulations were discretely metachromatic with toluidine blue. Those morphological anomalies are in favor of a lysosomal storage disease. No inclusions were found in skin fibroblasts. We hypothesize that these two boys have a distinct autosomal recessive or X-linked lysosomal storage disorder of unknown origin that shares clinical and radiological features with Morquio disease.

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

    Science.gov (United States)

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

    2016-12-18

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

  12. Reporter Assay for Endo/Lysosomal Escape of Toxin-Based Therapeutics

    Directory of Open Access Journals (Sweden)

    Roger Gilabert-Oriol

    2014-05-01

    Full Text Available Protein-based therapeutics with cytosolic targets are capable of exhibiting their therapeutic effect once they have escaped from the endosomes or lysosomes. In this study, the reporters—horseradish peroxidase (HRP, Alexa Fluor 488 (Alexa and ricin A-chain (RTA—were investigated for their capacity to monitor the endo/lysosomal escape of the ribosome-inactivating protein, saporin. The conjugates—saporin-HRP, Alexasaporin and saporin-KQ-RTA—were constructed, and the endo/lysosomal escape of these conjugates alone (lack of endo/lysosomal release or in combination with certain structurally-specific triterpenoidal saponins (efficient endo/lysosomal escape was characterized. HRP failed in reporting the endo/lysosomal escape of saporin. Contrastingly, Alexa Fluor 488 successfully allowed the report of the process at a toxin concentration of 1000 nM. In addition, single endo/lysosome analysis facilitated the determination of the amount of Alexasaporin released from each vesicle. RTA was also successful in reporting the endo/lysosomal escape of the enzymatically inactive mutant, saporin-KQ, but in this case, the sensitivity of the method reached a toxin concentration of 10 nM. In conclusion, the simultaneous usage of Alexa Fluor 488 and RTA as reporters may provide the possibility of monitoring the endo/lysosomal escape of protein-based therapeutics in the concentration range of 10–1000 nM.

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

    Science.gov (United States)

    Seggewiß, Nicole; Paulmann, Dajana; Dotzauer, Andreas

    2016-01-01

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

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

    OpenAIRE

    2012-01-01

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

  15. Reporter assay for endo/lysosomal escape of toxin-based therapeutics.

    Science.gov (United States)

    Gilabert-Oriol, Roger; Thakur, Mayank; von Mallinckrodt, Benedicta; Bhargava, Cheenu; Wiesner, Burkhard; Eichhorst, Jenny; Melzig, Matthias F; Fuchs, Hendrik; Weng, Alexander

    2014-05-22

    Protein-based therapeutics with cytosolic targets are capable of exhibiting their therapeutic effect once they have escaped from the endosomes or lysosomes. In this study, the reporters-horseradish peroxidase (HRP), Alexa Fluor 488 (Alexa) and ricin A-chain (RTA)-were investigated for their capacity to monitor the endo/lysosomal escape of the ribosome-inactivating protein, saporin. The conjugates-saporin-HRP, (Alexa)saporin and saporin-KQ-RTA-were constructed, and the endo/lysosomal escape of these conjugates alone (lack of endo/lysosomal release) or in combination with certain structurally-specific triterpenoidal saponins (efficient endo/lysosomal escape) was characterized. HRP failed in reporting the endo/lysosomal escape of saporin. Contrastingly, Alexa Fluor 488 successfully allowed the report of the process at a toxin concentration of 1000 nM. In addition, single endo/lysosome analysis facilitated the determination of the amount of (Alexa)saporin released from each vesicle. RTA was also successful in reporting the endo/lysosomal escape of the enzymatically inactive mutant, saporin-KQ, but in this case, the sensitivity of the method reached a toxin concentration of 10 nM. In conclusion, the simultaneous usage of Alexa Fluor 488 and RTA as reporters may provide the possibility of monitoring the endo/lysosomal escape of protein-based therapeutics in the concentration range of 10-1000 nM.

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

    Science.gov (United States)

    Jung, Minjeong; Lee, Jaemeun; Seo, Hye-Young; Lim, Ji Sun; Kim, Eun-Kyoung

    2015-01-01

    Autophagy is a lysosomal degradative pathway that plays an important role in maintaining cellular homeostasis. We previously showed that the inhibition of autophagy causes pancreatic β-cell apoptosis, suggesting that autophagy is a protective mechanism for the survival of pancreatic β-cells. The current study demonstrates that treatment with inhibitors and knockdown of the lysosomal cysteine proteases such as cathepsins B and L impair autophagy, enhancing the caspase-dependent apoptosis of INS-1 cells and islets upon exposure to high concentration of glucose. Interestingly, treatment with cathepsin B and L inhibitors prevented the proteolytic processing of cathepsins B, D and L, as evidenced by gradual accumulation of the respective pro-forms. Of note, inhibition of aspartic cathepsins had no effect on autophagy and cell viability, suggesting the selective role of cathepsins B and L in the regulation of β-cell autophagy and apoptosis. Lysosomal localization of accumulated pro-cathepsins in the presence of cathepsin B and L inhibitors was verified via immunocytochemistry and lysosomal fractionation. Lysotracker staining indicated that cathepsin B and L inhibitors led to the formation of severely enlarged lysosomes in a time-dependent manner. The abnormal accumulation of pro-cathepsins following treatment with inhibitors of cathepsins B and L suppressed normal lysosomal degradation and the processing of lysosomal enzymes, leading to lysosomal dysfunction. Collectively, our findings suggest that cathepsin defects following the inhibition of cathepsin B and L result in lysosomal dysfunction and consequent cell death in pancreatic β-cells.

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

    Science.gov (United States)

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

    2013-01-01

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

  18. Appel-reagent-mediated transformation of glycosyl hemiacetal derivatives into thioglycosides and glycosyl thiols

    Directory of Open Access Journals (Sweden)

    Tamashree Ghosh

    2013-05-01

    Full Text Available A series of glycosyl hemiacetal derivatives have been transformed into thioglycosides and glycosyl thiols in a one-pot two-step reaction sequence mediated by Appel reagent (carbon tetrabromide and triphenylphosphine. 1,2-trans-Thioglycosides and β-glycosyl thiol derivatives were stereoselectively formed by the reaction of the in situ generated glycosyl bromides with thiols and sodium carbonotrithioate. The reaction conditions are reasonably simple and yields were very good.

  19. Methods for the determination and quantification of the reactive thiol proteome

    OpenAIRE

    Hill, Bradford G.; Reily, Colin; Oh, Joo-Yeun; Johnson, Michelle S.; Landar, Aimee

    2009-01-01

    Protein thiol modifications occur under both physiological and pathological conditions and have been shown to contribute to changes in protein structure, function, and redox signaling. The majority of protein thiol modifications occur on cysteine residues that have a low pKa; these nucleophilic proteins comprise the “reactive thiol proteome.” The most reactive members of this proteome are typically low abundance proteins. Therefore, sensitive and quantitative methods are needed to detect and ...

  20. Neutron-gamma irradiation and protein thiols: development of a protein thiol evaluation micro-method and application to irradiated baboons; Irradiation neutron-gamma et groupements thiols proteiques: developpement d`une micromethode d`evaluation des thiols proteiques et application au babouin irradie

    Energy Technology Data Exchange (ETDEWEB)

    Chancerelle, Y.; Lafond, J.L.; Della-Maura, L.; Faure, P.; Mathieu, J.; Costa, P.; Mestries, J.C.; Kergonou, J.F.

    1994-12-31

    The essential non-protein sulfhydryl compound implicated in cellular radioprotection is glutathione. Protein thiols seem to be also involved in this protection and might be scavengers for free radical injury. We developed an analytical procedure for protein thiols measurement and we applied this method in neutron-gamma irradiated baboons. Our results demonstrated the reliability and sensitivity of the procedure. They also a drastic decrease of in vivo protein thiols after irradiation. (author). 5 refs.

  1. Multi-chamber and multi-layer thiol-ene microchip for cell culture

    DEFF Research Database (Denmark)

    Tan, H. Y.; Hemmingsen, Mette; Lafleur, Josiane P.

    2014-01-01

    We present a multi-layer and multi-chamber microfluidic chip fabricated using two different thiol-ene mixtures. Sandwiched between the thiol-ene chip layers is a commercially available membrane whose morphology has been altered with coatings of thiol-ene mixtures. Experiments have been conducted ...... with the microchip and shown that the fabricated microchip is suitable for long term cell culture....

  2. Quantification of protein-derived thiols during atmosphere-controlled brewing in laboratory scale

    DEFF Research Database (Denmark)

    Murmann, Anne Nordmark; Andersen, Preben; Mauch, Alexander

    2016-01-01

    An atmosphere-controlled brewing system was built to study thiol oxidation during brewing in laboratory scale under conditions with limited oxygen exposure. Quantification of free and total thiols and protein showed that thiols were lost during wort boiling possibly owing to protein precipitation...... was more pronounced at longer incubation times. However, the reduction of the pool of oxidized thiols by sulfite was inefficient for sulfite concentrations typically found in beer, and the reaction was found to be relatively slow compared with reduction by tris(carboxyethyl)phosphine....

  3. Distributions of dissolved and particulate biogenic thiols in the subartic Pacific Ocean

    Science.gov (United States)

    Dupont, Christopher L.; Moffett, James. W.; Bidigare, Robert R.; Ahner, Beth A.

    2006-12-01

    Dissolved and particulate concentrations of the biogenic thiols cysteine (Cys), arginine-cysteine (Arg-Cys), glutamine-cysteine (Gln-Cys), γ-glutamate-cysteine ( γ-Glu-Cys) and glutathione (GSH) were measured in the subartic Pacific Ocean in the summer of 2003 using high performance liquid chromatography (HPLC) with precolumn derivatization as reported in previous work. In this study, a preconcentration protocol for the derivatized thiols was utilized to extend detection limits of dissolved thiols to picomolar levels. The measured concentrations of particulate and dissolved thiols were uncoupled, with distinctive depth profiles and large differences in the particulate to dissolved ratios between individual compounds. Glutathione was the most abundant particulate thiol whereas the most abundant dissolved thiol was γ-Glu-Cys, with concentrations as high as 15 nM. Given the relatively small pool of intracellular γ-Glu-Cys and the very low dissolved concentrations of GSH, we hypothesize that glutathione released from cells is rapidly converted to the potentially degradation resistant γ-Glu-Cys outside the cell. The relatively high concentrations of other dissolved thiols compared to particulate concentrations implies both biological exudation and slow degradation rates. Some thiols appear to vary with changes in nutrient availability but this effect is difficult to decouple from changes in community structure inferred from pigment analyses. Dissolved thiol concentrations also exceed typical metal concentrations in the subartic Pacific, supporting previous arguments that they may be important in metal speciation.

  4. Chemical interaction between polyphenols and a cysteinyl thiol under radical oxidation conditions.

    Science.gov (United States)

    Fujimoto, Aya; Masuda, Toshiya

    2012-05-23

    Chemical interaction between polyphenols and thiols was investigated under radical oxidation conditions using a model cysteinyl thiol derivative, N-benzoylcysteine methyl ester. The radical oxidation was carried out with a stoichiometric amount of 2,2-diphenyl-1-picrylhydrazyl (DPPH), and the decreases in the amounts of polyphenols and the thiol were measured by HPLC analysis. Cross-coupling products between various polyphenols and the thiol were examined by LC-MS in reactions that showed decreases in both the polyphenols and the thiol. The LC-MS results indicated that three phenolic acid esters (methyl caffeate, methyl dihydrocaffeate, and methyl protocatechuate) and six flavonoids (kaempferol, myricetin, luteolin, morin, taxifolin, and catechin) gave corresponding thiol adducts, whereas three polyphenols (methyl ferulate, methyl sinapate, and quercetin) gave only dimers or simple oxidation products without thiol substituents. Thiol adducts of the structurally related compounds methyl caffeate and methyl dihydrocaffeate were isolated, and their chemical structures were determined by NMR analysis. The mechanism for the thiol addition was discussed on the basis of the structures of the products.

  5. Thiol-chromene click chemistry: a coumarin-based derivative and its use as regenerable thiol probe and in bioimaging applications.

    Science.gov (United States)

    Yang, Yutao; Huo, Fangjun; Yin, Caixia; Zheng, Anmin; Chao, Jianbin; Li, Yingqi; Nie, Zongxiu; Martínez-Máñez, Ramón; Liu, Diansheng

    2013-09-15

    The synthesis and characterization of a coumarin-chromene (8, 9-dihydro-2H-cyclopenta[b]pyrano[2,3-f]chromene-2,10(7aH)-dione) (1) derivative and its use for thiol chemosensing in water was reported. Experimental details showed 1 acts as a probe for the detection of thiols including cysteine (Cys), homocysteine (Hcy) and glutathione (GSH), whereas amino acids which do not contain thiols induced no changes in UV-vis spectra and fluorescence emission properties of 1. A possible detection mechanism is a nucleophilic attack of thiols to the α,β-unsaturated ketone in 1 that resulted in a fluorescent coumarin derivative. Further studies showed that 1-thiol derivatives can be applied to the design of regenerative chemodosimeters for Cu(2+), Hg(2+) and Cd(2+) in water based on M(n+)-promoted desulfurization and recovery of 1. Furthermore, the optical properties of the probe and its Cys-addition product were theoretically studied. The ability of probe 1 to detect thiols in living cells (HepG2 cells) via an enhancement of the fluorescence was proved. Moreover, the applicability of 1 for the direct determination of biorelevant thiols in a complex matrix such as human plasma was also demonstrated. Copyright © 2013 Elsevier B.V. All rights reserved.

  6. Integration between anticipatory blocking and redox signaling by the peroxiredoxin/thioredoxin/thioredoxin-reductase system.

    Science.gov (United States)

    Selvaggio, Gianluca; Coelho, Pedro M B M; Salvador, Armindo

    2014-10-01

    Cells are occasionally exposed to high H2O2 concentrations, often preceding exposure to other electrophylic compounds. Both H2O2 and these compounds can irreversibly modify protein thiols, with deleterious consequences. Induction of enzymatic defenses against those agents is too slow to avoid significant damage. Cells may solve this conundrum by reversibly "blocking" the thiols once H2O2 concentrations begin to increase. We term this mechanism "anticipatory blocking" because it acts in anticipation of irreversible damage upon detection of early signs of stress. Here we examine the design requirements for the Peroxiredoxin/Thioredoxin/Thioredoxin-Reductase/Protein-Dithiol System (PTTRDS) to effectively integrate H2O2 signaling and anticipatory blocking of protein dithiols as disulfides, and we compared them to the designs found in cells. To that effect, we developed a minimal model of the PTTRDS, and we defined a set of quantitative performance criteria that embody the requirements for (a) efficient scavenging capacity, (b) low NADPH consumption, (c) effective signal propagation, and (d) effective anticipatory blocking. We then sought the design principles (relationships among rate constants and species concentrations) that warrant fulfillment of all these criteria. Experimental data indicates that the design of the PTTRDS in human erythrocytes fulfills these principles and thus accomplishes effective integration between anticipatory blocking, antioxidant protection and redox signaling. A more general analysis suggests that the same principles hold in a wide variety of cell types and organisms. We acknowledge grants PEst-C/SAU/LA0001/2013-2014, PEst-OE/QUI/UI0612/2013, FCOMP-01-0124-FEDER-020978 (PTDC/QUI-BIQ/119657/2010) financed by FEDER through the "Programa Operacional Factores de Competitividade, COMPETE" and by national funds through "FCT, Fundação para a Ciência e a Tecnologia".

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

    DEFF Research Database (Denmark)

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

    2013-01-01

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

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

    OpenAIRE

    Wilke Sonja; Krausze Joern; Büssow Konrad

    2012-01-01

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

  9. Transsulfuration pathway thiols and methylated arginines: the Hunter Community Study.

    Directory of Open Access Journals (Sweden)

    Arduino A Mangoni

    Full Text Available BACKGROUND: Serum homocysteine, when studied singly, has been reported to be positively associated both with the endogenous nitric oxide synthase inhibitor asymmetric dimethylarginine [ADMA, via inhibition of dimethylarginine dimethylaminohydrolase (DDAH activity] and with symmetric dimethylarginine (SDMA. We investigated combined associations between transsulfuration pathway thiols, including homocysteine, and serum ADMA and SDMA concentrations at population level. METHODS: Data on clinical and demographic characteristics, medication exposure, C-reactive protein, serum ADMA and SDMA (LC-MS/MS, and thiols (homocysteine, cysteine, taurine, glutamylcysteine, total glutathione, and cysteinylglycine; capillary electrophoresis were collected from a sample of the Hunter Community Study on human ageing [n = 498, median age (IQR = 64 (60-70 years]. RESULTS: REGRESSION ANALYSIS SHOWED THAT: a age (P = 0.001, gender (P = 0.03, lower estimated glomerular filtration rate (eGFR, P = 0.08, body mass index (P = 0.008, treatment with beta-blockers (P = 0.03, homocysteine (P = 0.02, and glutamylcysteine (P = 0.003 were independently associated with higher ADMA concentrations; and b age (P = 0.001, absence of diabetes (P = 0.001, lower body mass index (P = 0.01, lower eGFR (P<0.001, cysteine (P = 0.007, and glutamylcysteine (P < 0.001 were independently associated with higher SDMA concentrations. No significant associations were observed between methylated arginines and either glutathione or taurine concentrations. CONCLUSIONS: After adjusting for clinical, demographic, biochemical, and pharmacological confounders the combined assessment of transsulfuration pathway thiols shows that glutamylcysteine has the strongest and positive independent associations with ADMA and SDMA. Whether this reflects a direct effect of glutamylcysteine on DDAH activity (for ADMA and/or cationic amino acid transport requires further investigations.

  10. Electrodeposition of gold templated by patterned thiol monolayers

    Energy Technology Data Exchange (ETDEWEB)

    She, Zhe [EaStCHEM School of Chemistry, University of St. Andrews, KY16 9ST (United Kingdom); Di Falco, Andrea [SUPA, School of Physics and Astronomy, University of St. Andrews, KY16 9SS (United Kingdom); Hähner, Georg [EaStCHEM School of Chemistry, University of St. Andrews, KY16 9ST (United Kingdom); Buck, Manfred, E-mail: mb45@st-andrews.ac.uk [EaStCHEM School of Chemistry, University of St. Andrews, KY16 9ST (United Kingdom)

    2016-06-15

    Graphical abstract: - Highlights: • First demonstration of electrodeposition/lift-off of gold using thiol monolayers. • Microelectrode structures with large length to width ratio were generated. • Performance of two different patterning techniques was investigated. • Conditions for achieving good contrast in the electrodeposition were established. - Abstract: The electrochemical deposition of Au onto Au substrates modified by self-assembled monolayers (SAMs) was studied by linear sweep voltammetry (LSV), atomic force microscopy (AFM) and scanning electron microscopy (SEM). Patterned SAMs exhibiting electrochemical contrast were prepared by two different methods. One used microcontact printing (μCP) to generate a binary SAM of ω-(4′-methyl-biphenyl-4-yl)-propane thiol (CH{sub 3}-C{sub 6}H{sub 4}-C{sub 6}H{sub 4}-(CH{sub 2}){sub 3}-SH, MBP3) and octadecane thiol (CH{sub 3}(CH{sub 2}){sub 17}SH, ODT). Templated by the SAM, a gold microelectrode structure was electrodeposited featuring a line 15 μm wide and 3 mm long. After transfer to an epoxy substrate the structure proved to be electrically conductive across the full length. The other patterning method applied electron beam lithography (EBL) where electrochemical contrast was achieved by crosslinking molecules in a single component SAM of MBP3. An electron dose above 250 mC/cm{sup 2} results in a high deposition contrast. The choice of parameters for the deposition/lift-off process is found to be more critical for Au compared to Cu studied previously. The origin of the differences and implications for nanoscale patterning are discussed.

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

    Science.gov (United States)

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

    2015-11-01

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

  12. From Lysosomal Storage Diseases to NKT Cell Activation and Back

    Science.gov (United States)

    Pereira, Cátia S.; Ribeiro, Helena; Macedo, M. Fatima

    2017-01-01

    Lysosomal storage diseases (LSDs) are inherited metabolic disorders characterized by the accumulation of different types of substrates in the lysosome. With a multisystemic involvement, LSDs often present a very broad clinical spectrum. In many LSDs, alterations of the immune system were described. Special emphasis was given to Natural Killer T (NKT) cells, a population of lipid-specific T cells that is activated by lipid antigens bound to CD1d (cluster of differentiation 1 d) molecules at the surface of antigen-presenting cells. These cells have important functions in cancer, infection, and autoimmunity and were altered in a variety of LSDs’ mouse models. In some cases, the observed decrease was attributed to defects in either lipid antigen availability, trafficking, processing, or loading in CD1d. Here, we review the current knowledge about NKT cells in the context of LSDs, including the alterations detected, the proposed mechanisms to explain these defects, and the relevance of these findings for disease pathology. Furthermore, the effect of enzyme replacement therapy on NKT cells is also discussed. PMID:28245613

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

    Science.gov (United States)

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

    2013-12-01

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

  14. From Lysosomal Storage Diseases to NKT Cell Activation and Back

    Directory of Open Access Journals (Sweden)

    Cátia S. Pereira

    2017-02-01

    Full Text Available Lysosomal storage diseases (LSDs are inherited metabolic disorders characterized by the accumulation of different types of substrates in the lysosome. With a multisystemic involvement, LSDs often present a very broad clinical spectrum. In many LSDs, alterations of the immune system were described. Special emphasis was given to Natural Killer T (NKT cells, a population of lipid-specific T cells that is activated by lipid antigens bound to CD1d (cluster of differentiation 1 d molecules at the surface of antigen-presenting cells. These cells have important functions in cancer, infection, and autoimmunity and were altered in a variety of LSDs’ mouse models. In some cases, the observed decrease was attributed to defects in either lipid antigen availability, trafficking, processing, or loading in CD1d. Here, we review the current knowledge about NKT cells in the context of LSDs, including the alterations detected, the proposed mechanisms to explain these defects, and the relevance of these findings for disease pathology. Furthermore, the effect of enzyme replacement therapy on NKT cells is also discussed.

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

    Directory of Open Access Journals (Sweden)

    Arnaud M. Labrousse

    2011-10-01

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

  16. Regulative roles of glutathione reductase and four glutaredoxins in glutathione redox, antioxidant activity, and iron homeostasis of Beauveria bassiana.

    Science.gov (United States)

    Zhang, Long-Bin; Tang, Li; Ying, Sheng-Hua; Feng, Ming-Guang

    2016-07-01

    Multiple glutaredoxins (Grx) and glutathione reductase (Glr) are vital for the thiol-disulfide redox system in budding yeast but generally unexplored in filamentous fungi. Here we characterized the Beauveria bassiana redox system comprising dithiol Grx1, monothiol Grx2-4, Grx-like Grx5, and Glr orthologue. Each grx or glr deletion was compensated by increased transcripts of some other grx genes in normal cultures. Particularly, grx3 compensated the absence of grx1, grx2, grx5, or glr under oxidative stress while its absence was compensated only by undeletable grx4 under normal conditions but by most of other undeleted grx and glr genes in response to menadione. Consequently, the redox state was disturbed in Δglr more than in Δgrx3 but not in Δgrx1/2/5. Superoxide dismutases were more active in normal Δgrx1-3 cultures but less in Δgrx5 or Δglr response to menadione. Total catalase activity increased differentially in all the mutant cultures stressed with or without H2O2 while total peroxidase activity decreased more in the normal or H2O2-stressed culture of Δglr than of Δgrx3. Among the mutants, Δgrx3 showed slightly increased sensitivity to menadione or H2O2; Δglr exhibited greater sensitivity to thiol-oxidizing diamide than thiol-reducing 1-chloro-2,4-dinitrobenzene as well as increased sensitivity to the two oxidants. Intriguingly, all the mutants grew slower in a Fe(3+)-inclusive medium perhaps due to elevated transcripts of two Fe(3+) transporter genes. More or fewer phenotypes linked with biocontrol potential were altered in four deletion mutants excluding Δgrx5. All the changes were restored by targeted gene complementation. Overall, Grx3 played more critical role than other Grx homologues in the Glr-dependent redox system of the fungal entomopathogen.

  17. Characterization of human platelet glutathione reductase.

    Science.gov (United States)

    Moroff, G; Kosow, D P

    1978-12-08

    Glutathione reductase (NAD(P)h:oxidized glutathione oxidoreductase, EC 1.6.4.2) has been purified 1000-fold from the cytoplasmic fraction of human platelets. Salts, including the heretofore unreported effect of sodium citrate, activate the NADPH-dependent reduction of oxidized glutathione. Sodium citrate and monovalent salt activation appears to involve multiple sites having different binding affinities. At sub-saturating sodium phosphate, non-linear double reciprocal plots indicative of substrate activation by oxidized glutathione were observed. Initial velocity double reciprocal plots at sub-saturating and saturating concentrations of phosphate generate a family of converging lines. NADP+ is a partial inhibitor, indicating that the reduction of oxidized glutathione can proceed by more than one pathway. FMN, FAD, and riboflavin inhibit platelet glutathione reductase by influencing only the V while nitrofurantoin inhibition is associated with an increase Koxidized glutathione and a decreased V.

  18. Selenium as an electron acceptor during the catalytic mechanism of thioredoxin reductase.

    Science.gov (United States)

    Lothrop, Adam P; Snider, Gregg W; Ruggles, Erik L; Patel, Amar S; Lees, Watson J; Hondal, Robert J

    2014-02-04

    Mammalian thioredoxin reductase (TR) is a pyridine nucleotide disulfide oxidoreductase that uses the rare amino acid selenocysteine (Sec) in place of the more commonly used amino acid cysteine (Cys) in the redox-active tetrapeptide Gly-Cys-Sec-Gly motif to catalyze thiol/disulfide exchange reactions. Sec can accelerate the rate of these exchange reactions (i) by being a better nucleophile than Cys, (ii) by being a better electrophile than Cys, (iii) by being a better leaving group than Cys, or (iv) by using a combination of all three of these factors, being more chemically reactive than Cys. The role of the selenolate as a nucleophile in the reaction mechanism was recently demonstrated by creating a mutant of human thioredoxin reductase-1 in which the Cys497-Sec498 dyad of the C-terminal redox center was mutated to either a Ser497-Cys498 dyad or a Cys497-Ser498 dyad. Both mutant enzymes were incubated with human thioredoxin (Trx) to determine which mutant formed a mixed disulfide bond complex. Only the mutant containing the Ser497-Cys498 dyad formed a complex, and this structure has been determined by X-ray crystallography [Fritz-Wolf, K., Kehr, S., Stumpf, M., Rahlfs, S., and Becker, K. (2011) Crystal structure of the human thioredoxin reductase-thioredoxin complex. Nat. Commun. 2, 383]. This experimental observation most likely means that the selenolate is the nucleophile initially attacking the disulfide bond of Trx because a complex resulted only when Cys was present in the second position of the dyad. As a nucleophile, the selenolate of Sec helps to accelerate the rate of this exchange reaction relative to Cys in the Sec → Cys mutant enzyme. Another thiol/disulfide exchange reaction that occurs in the enzymatic cycle of the enzyme is the transfer of electrons from the thiolate of the interchange Cys residue of the N-terminal redox center to the eight-membered selenosulfide ring of the C-terminal redox center. The selenium atom of the selenosulfide could

  19. A mycothiol synthase mutant of Mycobacterium smegmatis produces novel thiols and has an altered thiol redox status.

    Science.gov (United States)

    Newton, Gerald L; Ta, Philong; Fahey, Robert C

    2005-11-01

    Mycobacteria and other actinomycetes do not produce glutathione but make mycothiol (MSH; AcCys-GlcN-Ins) that has functions similar to those of glutathione and is essential for growth of Mycobacterium tuberculosis. Mycothiol synthase (MshD) catalyzes N acetylation of Cys-GlcN-Ins to produce MSH in Mycobacterium smegmatis mc2155, and Cys-GlcN-Ins is maintained at a low level. The mycothiol synthase mutant, the mshD::Tn5 mutant, produces high levels of Cys-GlcN-Ins along with two novel thiols, N-formyl-Cys-GlcN-Ins and N-succinyl-Cys-GlcN-Ins, and a small amount of MSH. The nonenzymatic reaction of acyl-coenzyme A (CoA) with Cys-GlcN-Ins to produce acyl-Cys-GlcN-Ins is a facile reaction under physiologic conditions, with succinyl-CoA being an order of magnitude more reactive than acetyl-CoA. The uncatalyzed reaction rates are adequate to account for the observed production of N-succinyl-Cys-GlcN-Ins and MSH under physiologic conditions. It was shown that the N-acyl-Cys-GlcN-Ins compounds are maintained in a substantially reduced state in the mutant but that Cys-GlcN-Ins exists in disulfide forms at 5 to 40% at different stages of growth. MSH was able to facilitate reduction of N-succinyl-Cys-GlcN-Ins disulfide through thiol-disulfide exchange, but N-formyl-Cys-GlcN-Ins was ineffective. The oxidized state of Cys-GlcN-Ins in cells appears to result from a high susceptibility to autoxidation and a low capacity of the cell to reduce its disulfide forms. The mutant exhibited no enhanced sensitivity to hydrogen peroxide, tert-butyl hydroperoxide, or cumene hydroperoxide relative to the parent strain, suggesting that the most abundant thiol, N-formyl-Cys-GlcN-Ins, functions as a substitute for MSH.

  20. The protonation state of thiols in self-assembled monolayers on roughened Ag/Au surfaces and nanoparticles.

    Science.gov (United States)

    Bandyopadhyay, Sabyasachi; Chattopadhyay, Samir; Dey, Abhishek

    2015-10-14

    The protonation state of thiols in self-assembled monolayers (SAMs) on Ag and Au surfaces and nanoparticles (NPs) has been an issue of contestation. It has been recently demonstrated that deuterating the thiol proton produces ostentatious changes in the Raman spectra of thiols and can be used to detect the presence of the thiol functional group. Surface enhanced Raman spectroscopy (SERS) of H/D substituted aliphatic thiols on Ag surfaces clearly shows the presence of S-H vibration between 2150-2200 cm(-1) which shifts by 400 cm(-1) upon deuteration and a simultaneous >20 cm(-1) shift in the C-S vibration of thiol deuteration. Large shifts (>15 cm(-1)) in the C-S vibration are also observed for alkyl thiol SAMs on Au surfaces. Alternatively, neither the S-H vibration nor the H/D isotope effect on the C-S vibration is observed for alkyl thiol SAMs on Ag/Au NPs. XPS data on Ag/Au surfaces bearing aliphatic thiol SAMs show the presence of both protonated and deprotonated thiols while on Ag/Au NPs only deprotonated thiols are detected. These data suggest that aliphatic thiol SAMs on Au/Ag surfaces are partially protonated whereas they are totally deprotonated on Au/Ag NPs. Aromatic PhSH SAMs on Ag/Au surfaces and Ag/Au NPs do not show these vibrations or H/D shifts as well indicating that the thiols are deprotonated at these interfaces.

  1. Discovery of pinoresinol reductase genes in sphingomonads.

    Science.gov (United States)

    Fukuhara, Y; Kamimura, N; Nakajima, M; Hishiyama, S; Hara, H; Kasai, D; Tsuji, Y; Narita-Yamada, S; Nakamura, S; Katano, Y; Fujita, N; Katayama, Y; Fukuda, M; Kajita, S; Masai, E

    2013-01-10

    Bacterial genes for the degradation of major dilignols produced in lignifying xylem are expected to be useful tools for the structural modification of lignin in plants. For this purpose, we isolated pinZ involved in the conversion of pinoresinol from Sphingobium sp. strain SYK-6. pinZ showed 43-77% identity at amino acid level with bacterial NmrA-like proteins of unknown function, a subgroup of atypical short chain dehydrogenases/reductases, but revealed only 15-21% identity with plant pinoresinol/lariciresinol reductases. PinZ completely converted racemic pinoresinol to lariciresinol, showing a specific activity of 46±3 U/mg in the presence of NADPH at 30°C. In contrast, the activity for lariciresinol was negligible. This substrate preference is similar to a pinoresinol reductase, AtPrR1, of Arabidopsis thaliana; however, the specific activity of PinZ toward (±)-pinoresinol was significantly higher than that of AtPrR1. The role of pinZ and a pinZ ortholog of Novosphingobium aromaticivorans DSM 12444 were also characterized.

  2. Characterization of storage material in cultured fibroblasts by specific lectin binding in lysosomal storage diseases.

    Science.gov (United States)

    Virtanen, I; Ekblom, P; Laurila, P; Nordling, S; Raivio, K O; Aula, P

    1980-11-01

    The lysosomal storage material in cultured fibroblasts from patients with various lysosomal storage diseases was characterized by fluorescence microscopy using lectins specific for different saccharide moieties. In normal fibroblasts and cultured amniotic fluid cells lectins specific for mannosyl and glucosyl moieties, Con A and LcA gave a bright perinuclear cytoplasmic staining corresponding to the localization of endoplasmic reticulum in the cells. All other lectins stained the Golgi apparatus as a juxtanuclear reticular structure. In fucosidosis fibroblasts, only lectins specific for fucosyl groups LTA and UEA, distinctly stained the lysosomal inclusions. The lysosomes in mannosidosis fibroblasts did not react with Con A and LcA, both specific for mannosyl moieties of glycoconjugates, but were brightly labeled with WGA, a lectin specific for N-acetyl glucosaminyl moieties. In I-cell fibroblasts, the numerous perinuclear phase-dense granules, representing abnormal lysosomes, were labeled with every lectin used. In fibroblasts from patients with Salla disease, a newly discovered lysosomal storage disorder, the lysosomes were brightly stained only with LPA, indicating the presence of increased amounts of sialic acid residues in the lysosomal inclusions.

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

    Science.gov (United States)

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

    2013-09-16

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

  4. Autophagy-lysosomal pathway is involved in lipid degradation in rat liver.

    Science.gov (United States)

    Skop, V; Cahová, M; Papáčková, Z; Páleníčková, E; Daňková, H; Baranowski, M; Zabielski, P; Zdychová, J; Zídková, J; Kazdová, L

    2012-01-01

    We present data supporting the hypothesis that the lysosomal-autophagy pathway is involved in the degradation of intracellular triacylglycerols in the liver. In primary hepatocytes cultivated in the absence of exogenous fatty acids (FFA), both inhibition of autophagy flux (asparagine) or lysosomal activity (chloroquine) decreased secretion of VLDL (very low density lipoproteins) and formation of FFA oxidative products while the stimulation of autophagy by rapamycine increased some of these parameters. Effect of rapamycine was completely abolished by inactivation of lysosomes. Similarly, when autophagic activity was influenced by cultivating the hepatocytes in "starving" (amino-acid poor medium) or "fed" (serum-supplemented medium) conditions, VLDL secretion and FFA oxidation mirrored the changes in autophagy being higher in starvation and lower in fed state. Autophagy inhibition as well as lysosomal inactivation depressed FFA and DAG (diacylglycerol) formation in liver slices in vitro. In vivo, intensity of lysosomal lipid degradation depends on the formation of autophagolysosomes, i.e. structures bringing the substrate for degradation and lysosomal enzymes into contact. We demonstrated that lysosomal lipase (LAL) activity in liver autophagolysosomal fraction was up-regulated in fasting and down-regulated in fed state together with the increased translocation of LAL and LAMP2 proteins from lysosomal pool to this fraction. Changes in autophagy intensity (LC3-II/LC3-I ratio) followed a similar pattern.

  5. The phytoestrogen genistein modulates lysosomal metabolism and transcription factor EB (TFEB) activation.

    Science.gov (United States)

    Moskot, Marta; Montefusco, Sandro; Jakóbkiewicz-Banecka, Joanna; Mozolewski, Paweł; Węgrzyn, Alicja; Di Bernardo, Diego; Węgrzyn, Grzegorz; Medina, Diego L; Ballabio, Andrea; Gabig-Cimińska, Magdalena

    2014-06-13

    Genistein (5,7-dihydroxy-3-(4-hydroxyphenyl)-4H-1-benzopyran-4-one) has been previously proposed as a potential drug for use in substrate reduction therapy for mucopolysaccharidoses, a group of inherited metabolic diseases caused by mutations leading to inefficient degradation of glycosaminoglycans (GAGs) in lysosomes. It was demonstrated that this isoflavone can cross the blood-brain barrier, making it an especially desirable potential drug for the treatment of neurological symptoms present in most lysosomal storage diseases. So far, no comprehensive genomic analyses have been performed to elucidate the molecular mechanisms underlying the effect elicited by genistein. Therefore, the aim of this work was to identify the genistein-modulated gene network regulating GAG biosynthesis and degradation, taking into consideration the entire lysosomal metabolism. Our analyses identified over 60 genes with known roles in lysosomal biogenesis and/or function whose expression was enhanced by genistein. Moreover, 19 genes whose products are involved in both GAG synthesis and degradation pathways were found to be remarkably differentially regulated by genistein treatment. We found a regulatory network linking genistein-mediated control of transcription factor EB (TFEB) gene expression, TFEB nuclear translocation, and activation of TFEB-dependent lysosome biogenesis to lysosomal metabolism. Our data indicate that the molecular mechanism of genistein action involves not only impairment of GAG synthesis but more importantly lysosomal enhancement via TFEB. These findings contribute to explaining the beneficial effects of genistein in lysosomal storage diseases as well as envisage new therapeutic approaches to treat these devastating diseases.

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

    Science.gov (United States)

    Roy, Subhro Jyoti; Stanely Mainzen Prince, Ponnian

    2012-11-01

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

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

    Science.gov (United States)

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

    2016-04-25

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

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

    NARCIS (Netherlands)

    R.J. Rottier (Robbert)

    1998-01-01

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

  9. Vps33B is required for delivery of endocytosed cargo to lysosomes

    NARCIS (Netherlands)

    Galmes, Romain; ten Brink, Corlinda; Oorschot, Viola; Veenendaal, Tineke; Jonker, Caspar; van der Sluijs, Peter; Klumperman, Judith

    2015-01-01

    In mammalian cells Vps33B forms a complex with VIPAS-39 that is recruited to recycling endosomes. Here we show that when Vps33B is expressed together with Rab7-interacting lysosomal protein (RILP) it is recruited to late endosomes-lysosomes and that depletion of Vps33B impairs late

  10. Glycogenosis type II : cloning and characterization of the human lysosomal α-glucosidase gene

    NARCIS (Netherlands)

    E.H. Hoefsloot (Lies)

    1991-01-01

    textabstractGlycogenosis type II is a lysosomal storage disorder. Characteristic features are heart failure and generalized muscle weakness. The disease is caused by the inherited deficiency of acid α-glucosidase, the enzyme responsible for the degradation of lysosomal glycogen. The aim of the work

  11. Lysosomal cholesterol accumulation : driver on the road to inflammation during atherosclerosis and non-alcoholic steatohepatitis

    NARCIS (Netherlands)

    Hendrikx, T.; Walenbergh, S. M. A.; Hofker, M. H.; Shiri-Sverdlov, R.

    2014-01-01

    Many studies show an association between the accumulation of cholesterol inside lysosomes and the progression towards inflammatory disease states that are closely related to obesity. While in the past, the knowledge regarding lysosomal cholesterol accumulation was limited to its association with pla

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

    Science.gov (United States)

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

    2012-09-01

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

  13. Impairment of thiol-disulfide homeostasis in preeclampsia.

    Science.gov (United States)

    Korkmaz, Vakkas; Kurdoglu, Zehra; Alisik, Murat; Cetin, Orkun; Korkmaz, Hilal; Surer, Hatice; Erel, Ozcan

    2016-12-01

    To investigate the effects of severity of preeclampsia on thiol-disulfide homeostasis (TDH). A total of 108 participants were divided into three groups: Group 1 was composed of pregnant women with no obstetric complications, Group 2 included pregnant women with mild preeclampsia, and Group 3 consisted of pregnant women with severe preeclampsia. TDH parameters were determined, and comparisons of clinical and routine laboratory test findings were made in all groups. The serum native thiol level was 347.9 ± 27.4 in the control group, 237.2 ± 44.2 in the mild preeclampsia group, and 227.9 ± 53.1 in the severe preeclampsia group (p preeclampsia group, and 248.3 ± 57.4 in the severe preeclampsia group (p preeclampsia group, and 10.2 ± 4.8 in the severe preeclampsia group (p = 0.001). A significant correlation between impairment in degree of TDH and severity of preeclampsia was observed. TDH was impaired in women with preeclampsia, and this impairment increased with disease severity. Therefore, impaired TDH may have a role in the etiopathogenesis of the disease.

  14. Electrodeposition of gold templated by patterned thiol monolayers

    Science.gov (United States)

    She, Zhe; Di Falco, Andrea; Hähner, Georg; Buck, Manfred

    2016-06-01

    The electrochemical deposition of Au onto Au substrates modified by self-assembled monolayers (SAMs) was studied by linear sweep voltammetry (LSV), atomic force microscopy (AFM) and scanning electron microscopy (SEM). Patterned SAMs exhibiting electrochemical contrast were prepared by two different methods. One used microcontact printing (μCP) to generate a binary SAM of ω-(4‧-methyl-biphenyl-4-yl)-propane thiol (CH3-C6H4-C6H4-(CH2)3-SH, MBP3) and octadecane thiol (CH3(CH2)17SH, ODT). Templated by the SAM, a gold microelectrode structure was electrodeposited featuring a line 15 μm wide and 3 mm long. After transfer to an epoxy substrate the structure proved to be electrically conductive across the full length. The other patterning method applied electron beam lithography (EBL) where electrochemical contrast was achieved by crosslinking molecules in a single component SAM of MBP3. An electron dose above 250 mC/cm2 results in a high deposition contrast. The choice of parameters for the deposition/lift-off process is found to be more critical for Au compared to Cu studied previously. The origin of the differences and implications for nanoscale patterning are discussed.

  15. Purification and characterization of a pineapple crown leaf thiol protease.

    Science.gov (United States)

    Singh, L Rupachandra; Devi, Th Premila; Devi, S Kunjeshwori

    2004-02-01

    A thiol protease was isolated and purified from the crown leaf of pineapple, Ananas comosus (L.) Merr. cv. Queen, by an immunoaffinity procedure. After the purification to electrophoretic homogeneity, the enzyme was characterized with respect to some of its physico-chemical and kinetic properties. The molecular weight of the protease (22.4-22.9 kDa), Km (97 microM) and kcat (8.8 s(-1)) for its esterolytic cleavage of the synthetic protease substrate N(alpha)-CBZ-L-lysine p-nitrophenyl ester, the concentration of its thiol activator L-cysteine required for half maximal activation A0.5 (9.9 microM), optimum pH (6.5) for its proteolytic action on azocasein, T(1/2) (60 degrees C) for inactivation by heating the enzyme (35.5 microg protein/mL) in citrate buffer pH 6.0 for 15 min, and SH-group content (0.98 mol/mol enzyme) were determined. Most of these physicochemical and kinetic properties were found to be similar to those of the already well-characterized stem bromelain (EC 3.4.22.32). Thus, the immunoaffinity purified crown leaf protease appeared to be closely related to stem bromelain.

  16. Novel Thiol-Ene Hybrid Coating for Metal Protection

    Directory of Open Access Journals (Sweden)

    Mona Taghavikish

    2016-04-01

    Full Text Available A novel hybrid anticorrosion coating with dual network of inorganic (Si–O–Si and organic bonds (C–S–C was prepared on metal through an in situ sol-gel and thiol-ene click reaction. This novel interfacial thin film coating incorporates (3-mercaptopropyl trimethoxysilane (MPTS and 1,4-di(vinylimidazolium butane bisbromide based polymerizable ionic liquid (PIL to form a thiol-ene based photo-polymerized film, which on subsequent sol-gel reaction forms a thin hybrid interfacial layer on metal surface. On top of this PIL hybrid film, a self-assembled nanophase particle (SNAP coating was employed to prepare a multilayer thin film coating for better corrosion protection and barrier performance. The novel PIL hybrid film was characterised for structure and properties using Fourier transform infrared spectroscopy (FTIR, differential scanning calorimetry (DSC, and thermogravimetric analysis (TGA. The corrosion protection performance of the multilayer coating was examined using potentiodynamic polarization and electrochemical impedance spectroscopy (EIS. The results reveal that this novel double layer coating on metal offers excellent protection against corrosion and has remarkably improved the barrier effect of the coating.

  17. Molecular modeling, structural analysis and identification of ligand binding sites of trypanothione reductase from Leishmania mexicana

    Directory of Open Access Journals (Sweden)

    Ozal Mutlu

    2013-01-01

    Full Text Available Background & objectives: Trypanothione reductase (TR is a member of FAD-dependent NADPH oxidoreductase protein family and it is a key enzyme which connects the NADPH and the thiol-based redox system. Inhibition studies indicate that TR is an essential enzyme for parasite survival. Therefore, it is an attractive target enzyme for novel drug candidates. There is no structural model for TR of Leishmania mexicana (LmTR in the protein databases. In this work, 3D structure of TR from L. mexicana was identified by template-based in silico homology modeling method, resultant model was validated, structurally analyzed and possible ligand binding pockets were identified. Methods: For computational molecular modeling study, firstly, template was identified by BLAST search against PDB database. Multiple alignments were achieved by ClustalW2. Molecular modeling of LmTR was done and possible drug targeting sites were identified. Refinement of the model was done by performing local energy minimization for backbone, hydrogen and side chains. Model was validated by web-based servers. Results: A reliable 3D model for TR from L. mexicana was modeled by using L. infantum trypanothione reductase (LiTR as a template. RMSD results according to C-alpha, visible atoms and backbone were 0.809 Å, 0.732 Å and 0.728 Å respectively. Ramachandran plot indicates that model shows an acceptable stereochemistry. Conclusion: Modeled structure of LmTR shows high similarity with LiTR based on overall structural features like domains and folding patterns. Predicted structure will provide a source for the further docking studies of various peptide-based inhibitors.

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

    Science.gov (United States)

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

    2014-01-01

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

  19. Ubiquitin trafficking to the lysosome: keeping the house tidy and getting rid of unwanted guests.

    Science.gov (United States)

    Purdy, Georgiana E; Russell, David G

    2007-01-01

    Bacterial killing by autophagic delivery to the lysosomal compartment has been shown for Mycobacteria, Streptococcus, Shigella, Legionella and Salmonella, indicating an important role for this conserved trafficking pathway for the control of intracellular bacterial pathogens.(1-5) In a recent study we found that solubilized lysosomes isolated from bone marrow-derived macrophages had potent antibacterial properties against M. tuberculosis and M. smegmatis that were associated with ubiquitin and ubiquitin-derived peptides. We propose that ubiquitinated proteins are delivered to the lysosomal compartment, where degradation by lysosomal proteinases generates ubiquitin-derived peptides with antimycobacterial properties. This surprising finding provokes a number of questions regarding the nature and trafficking of ubiquitin and ubiquitin-modified proteins in mammalian cells. We discuss the possible role(s) that the multivesicular body (MVB), the late endosome and the autophagosome may play in trafficking of ubiquitinated proteins to the lysosome.

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

    Science.gov (United States)

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

    1978-03-15

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

  1. Conservation of thiol-oxidative stress responses regulated by SigR orthologues in actinomycetes

    Science.gov (United States)

    Kim, Min-Sik; Dufour, Yann S.; Yoo, Ji Sun; Cho, Yoo-Bok; Park, Joo-Hong; Nam, Gi-Baeg; Kim, Hae Min; Lee, Kang-Lok; Donohue, Timothy J.; Roe, Jung-Hye

    2015-01-01

    Summary Numerous thiol-reactive compounds cause oxidative stress where cells counteract by activation of survival strategies regulated by thiol-based sensors. In Streptomyces coelicolor, a model actinomycete, a sigma/antisigma pair SigR/RsrA controls the response to thiol-oxidative stress. To unravel its full physiological functions, chromatin immuno-precipitation combined with sequence and transcript analyses were employed to identify 108 SigR target genes in S. coelicolor and to predict orthologous regulons across actinomycetes. In addition to reported genes for thiol homeostasis, protein degradation and ribosome modulation, 64 additional operons were identified suggesting new functions of this global regulator. We demonstrate that SigR maintains the level and activity of the housekeeping sigma factor HrdB during thiol-oxidative stress, a novel strategy for stress responses. We also found that SigR defends cells against UV and thiol-reactive damages, in which repair UvrA takes a part. Using a refined SigR-binding sequence model, SigR orthologues and their targets were predicted in 42 actinomycetes. This revealed a conserved core set of SigR targets to function for thiol homeostasis, protein quality control, possible modulation of transcription and translation, flavin-mediated redox reactions, and Fe-S delivery. The composition of the SigR regulon reveals a robust conserved physiological mechanism to deal with thiol-oxidative stress from bacteria to human. PMID:22651816

  2. Spectrophotometric Determination of Phenolic Antioxidants in the Presence of Thiols and Proteins

    Directory of Open Access Journals (Sweden)

    Aslı Neslihan Avan

    2016-08-01

    Full Text Available Development of easy, practical, and low-cost spectrophotometric methods is required for the selective determination of phenolic antioxidants in the presence of other similar substances. As electron transfer (ET-based total antioxidant capacity (TAC assays generally measure the reducing ability of antioxidant compounds, thiols and phenols cannot be differentiated since they are both responsive to the probe reagent. In this study, three of the most common TAC determination methods, namely cupric ion reducing antioxidant capacity (CUPRAC, 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid diammonium salt/trolox equivalent antioxidant capacity (ABTS/TEAC, and ferric reducing antioxidant power (FRAP, were tested for the assay of phenolics in the presence of selected thiol and protein compounds. Although the FRAP method is almost non-responsive to thiol compounds individually, surprising overoxidations with large positive deviations from additivity were observed when using this method for (phenols + thiols mixtures. Among the tested TAC methods, CUPRAC gave the most additive results for all studied (phenol + thiol and (phenol + protein mixtures with minimal relative error. As ABTS/TEAC and FRAP methods gave small and large deviations, respectively, from additivity of absorbances arising from these components in mixtures, mercury(II compounds were added to stabilize the thiol components in the form of Hg(II-thiol complexes so as to enable selective spectrophotometric determination of phenolic components. This error compensation was most efficient for the FRAP method in testing (thiols + phenols mixtures.

  3. Isothiazolones; thiol-reactive inhibitors of cysteine protease cathepsin B and histone acetyltransferase PCAF

    NARCIS (Netherlands)

    Wisastra, Rosalina; Ghizzoni, Massimo; Maarsingh, Harm; Minnaard, Adriaan J.; Haisma, Hidde J.; Dekker, Frank J.

    2011-01-01

    Isothiazolones and 5-chloroisothiazolones react chemoselectively with thiols by cleavage of the weak nitrogen-sulfur bond to form disulfides. They show selectivity for inhibition of the thiol-dependent cysteine protease cathepsin B and the histone acetyltransferase p300/CBP associated factor (PCAF)

  4. A method for site-specific labeling of multiple protein thiols

    NARCIS (Netherlands)

    Kuiper, Johanna M.; Pluta, Radek; Huibers, Wim H. C.; Fusetti, Fabrizia; Geertsma, Eric R.; Poolman, Bert

    We present a generic method for the site-specific and differential labeling of multiple cysteine residues in one protein. Phenyl arsenic oxide has been employed as a protecting group of two closely spaced thiols, allowing first labeling of a single thiol. Subsequently, the protecting group is

  5. Rapid photochemical surface patterning of proteins in thiol-ene based microfluidic devices

    DEFF Research Database (Denmark)

    Lafleur, Josiane P.; Kwapiszewski, Radoslaw; Jensen, Thomas Glasdam;

    2012-01-01

    ” and “ene” monomers present in the microfluidic chip bulk material provides a simple and efficient way of tuning the chip’s surface chemistry. Here, thiol-ene chips displaying an excess of functional thiol groups at their surfaces are functionalized with biotin and streptavidin in a controlled fashion using...

  6. EFFECTS OF ATMOSPHERIC H2S ON THIOL COMPOSITION OF CROP PLANTS

    NARCIS (Netherlands)

    BUWALDA, F; DE KOK, LJ; Stulen, I.

    1993-01-01

    Exposure of crop plants to H2S resulted in an increase in thiol level and a change in the composition of the thiol pool. Non-leguminous species accumulated cysteine and glutathione in the light, whereas in the dark, substantial amounts of gamma-glutamyl-cysteine were also detected. In leguminous spe

  7. Surfactant-free coating of thiols on gold nanoparticles using sonochemistry: a study of competing processes.

    Science.gov (United States)

    Pallipurath, Anuradha; Nicoletti, Olivia; Skelton, Jonathan M; Mahajan, Sumeet; Midgley, Paul A; Elliott, Stephen R

    2014-09-01

    A method for the surfactant-free coating of gold nanoparticles with thiols using sonochemistry is presented. The gold nanoparticles were prepared by a modified Zsigmondy method, affording good control over the particle-size distribution, and the thiol coating was performed by the sonication of a biphasic system consisting of a nanoparticle suspension in water and thiols in toluene. The effects of two important reaction parameters on the particle morphology, viz. sonication time and thiol concentration, were investigated in detail using transmission electron microscopy. The effect of the thiol chain length was also studied. We show that the morphology of the coated particles is determined through a competition between two opposing effects: particle fusion, due to the sonication conditions, and digestive ripening, due to the action of the thiols. Additionally, we illustrate the utility of our technique for various applications, including surface-enhanced Raman scattering from bound molecules, and further functionalization using a thiol-exchange reaction. Our technique paves the way for an efficient synthesis of thiol-coated AuNPs of different shapes and sizes, suitable for a range of diverse applications. Copyright © 2014 Elsevier B.V. All rights reserved.

  8. ENANTIOMERICALLY PURE BETA-AMINO SULFIDES AND BETA-AMINO THIOLS FROM EPHEDRINE

    NARCIS (Netherlands)

    POELERT, MA; HOF, RP; PEPER, NCMW; KELLOGG, RM

    1994-01-01

    Ephedrine and pseudoephedrine are converted by means of a Mitsunobu reaction to respectively trans- and cis-aziridines, which can be ring-opened at the benzylic center with inversion of configuration by thiols and thiol acids. The trans-aziridine from ephedrine reacts also with H2S in acetone under

  9. Serum Protein Thiol Levels in Patients with Hospital-Acquired Acute Kidney Injury

    Directory of Open Access Journals (Sweden)

    Jing Qian

    2015-12-01

    Full Text Available Background/Aims: This study aimed to examine antioxidants in patients with acute kidney injury (AKI and determine whether serum protein thiol levels are associated with all-cause 90-day mortality in patients with hospital-acquired AKI. Methods: According to the RIFLE criteria, 160 patients with hospital-acquired AKI were enrolled in our prospective cohort study. As controls, 72 critically ill patients without AKI and 72 age and sex-matched healthy subjects were also recruited. Serum protein thiol levels were analyzed in relation to all-cause mortality of patients with AKI. Results: Serum protein thiol levels in AKI patients were lower than those in healthy people (p=0.010. Protein thiol levels showed a weak but significant positive correlation with serum albumin levels. The 90-day overall mortality rate was higher in AKI patients with high serum protein thiol levels than in those with low levels (p=0.032 by log rank test. In multivariate analysis (Cox regression, serum protein thiol levels (p=0.031 were independently associated with 90-day overall mortality after adjustment for age, sex, sepsis, and the Acute Physiology and Chronic Health Evaluation II score. Conclusions: Patients with hospital-acquired AKI have remarkably low serum protein thiol levels. Elevated protein thiol levels are associated with 90-day overall mortality in hospital-acquired AKI.

  10. Resonance Light-Scattering Spectroscopy Study on Interaction between Gold Colloid and Thiol Containing Pharmaceutical

    Institute of Scientific and Technical Information of China (English)

    Liu Xiao-ling; Cai Ru-xiu; Yuan Hong

    2003-01-01

    In this paper, we used resonance light-scattering (RLS) spectroscopy to study the interaction between thiol-containing pharmaceutical and gold colloid. And for the first time, we proposed that this highly sensitive, gold colloid-based assay using RLS technique may have potential application in detecting thiol-containing substances.

  11. Spectrophotometric Determination of Phenolic Antioxidants in the Presence of Thiols and Proteins

    Science.gov (United States)

    Avan, Aslı Neslihan; Demirci Çekiç, Sema; Uzunboy, Seda; Apak, Reşat

    2016-01-01

    Development of easy, practical, and low-cost spectrophotometric methods is required for the selective determination of phenolic antioxidants in the presence of other similar substances. As electron transfer (ET)-based total antioxidant capacity (TAC) assays generally measure the reducing ability of antioxidant compounds, thiols and phenols cannot be differentiated since they are both responsive to the probe reagent. In this study, three of the most common TAC determination methods, namely cupric ion reducing antioxidant capacity (CUPRAC), 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt/trolox equivalent antioxidant capacity (ABTS/TEAC), and ferric reducing antioxidant power (FRAP), were tested for the assay of phenolics in the presence of selected thiol and protein compounds. Although the FRAP method is almost non-responsive to thiol compounds individually, surprising overoxidations with large positive deviations from additivity were observed when using this method for (phenols + thiols) mixtures. Among the tested TAC methods, CUPRAC gave the most additive results for all studied (phenol + thiol) and (phenol + protein) mixtures with minimal relative error. As ABTS/TEAC and FRAP methods gave small and large deviations, respectively, from additivity of absorbances arising from these components in mixtures, mercury(II) compounds were added to stabilize the thiol components in the form of Hg(II)-thiol complexes so as to enable selective spectrophotometric determination of phenolic components. This error compensation was most efficient for the FRAP method in testing (thiols + phenols) mixtures. PMID:27529232

  12. The first peripherally masked thiol dendrimers: a facile and highly efficient functionalization strategy of polyester dendrimers via one-pot xanthate deprotection/thiol-acrylate Michael addition reactions.

    Science.gov (United States)

    Auty, Sam E R; Andrén, Oliver; Malkoch, Michael; Rannard, Steven P

    2014-06-25

    Introducing multiple reactive functional groups at the periphery of dendrimer materials presents considerable challenges if the functionality is able to self-react. An efficient and facile approach to introducing masked thiols at the surface of polyester dendrimers is presented. One-pot, deprotection/thiol-acrylate Michael addition from the xanthate-functional dendritic substrates (generation zero to two) has been achieved for the first time, with high efficiency demonstrated using three acrylates of varying chemistry and avoiding disulfide formation.

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

    Science.gov (United States)

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

    2010-08-01

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

  14. Mild MPP(+) exposure impairs autophagic degradation through a novel lysosomal acidity-independent mechanism.

    Science.gov (United States)

    Miyara, Masatsugu; Kotake, Yaichiro; Tokunaga, Wataru; Sanoh, Seigo; Ohta, Shigeru

    2016-10-01

    Parkinson's disease (PD) is the second most common neurodegenerative disorder, but its underlying cause remains unknown. Although recent studies using PD-related neurotoxin MPP(+) suggest autophagy involvement in the pathogenesis of PD, the effect of MPP(+) on autophagic processes under mild exposure, which mimics the slow progressive nature of PD, remains largely unclear. We examined the effect of mild MPP(+) exposure (10 and 200 μM for 48 h), which induces a more slowly developing cell death, on autophagic processes and the mechanistic differences with acute MPP(+) toxicity (2.5 and 5 mM for 24 h). In SH-SY5Y cells, mild MPP(+) exposure predominantly inhibited autophagosome degradation, whereas acute MPP(+) exposure inhibited both autophagosome degradation and basal autophagy. Mild MPP(+) exposure reduced lysosomal hydrolase cathepsin D activity without changing lysosomal acidity, whereas acute exposure decreased lysosomal density. Lysosome biogenesis enhancers trehalose and rapamycin partially alleviated mild MPP(+) exposure induced impaired autophagosome degradation and cell death, but did not prevent the pathogenic response to acute MPP(+) exposure, suggesting irreversible lysosomal damage. We demonstrated impaired autophagic degradation by MPP(+) exposure and mechanistic differences between mild and acute MPP(+) toxicities. Mild MPP(+) toxicity impaired autophagosome degradation through novel lysosomal acidity-independent mechanisms. Sustained mild lysosomal damage may contribute to PD. We examined the effects of MPP(+) on autophagic processes under mild exposure, which mimics the slow progressive nature of Parkinson's disease, in SH-SY5Y cells. This study demonstrated impaired autophagic degradation through a reduction in lysosomal cathepsin D activity without altering lysosomal acidity by mild MPP(+) exposure. Mechanistic differences between acute and mild MPP(+) toxicity were also observed. Sustained mild damage of lysosome may be an underlying cause

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

    Science.gov (United States)

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

    2012-01-01

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

  16. Oxidant-induced autophagy and ferritin degradation contribute to epithelial–mesenchymal transition through lysosomal iron

    Science.gov (United States)

    Sioutas, Apostolos; Vainikka, Linda K; Kentson, Magnus; Dam-Larsen, Sören; Wennerström, Urban; Jacobson, Petra; Persson, Hans Lennart

    2017-01-01

    Purpose Transforming growth factor (TGF)-β1 triggers epithelial–mesenchymal transition (EMT) through autophagy, which is partly driven by reactive oxygen species (ROS). The aim of this study was to determine whether leaking lysosomes and enhanced degradation of H-ferritin could be involved in EMT and whether it could be possible to prevent EMT by iron chelation targeting of the lysosome. Materials and methods EMT, H-ferritin, and autophagy were evaluated in TGF-β1-stimulated A549 human lung epithelial cells cultured in vitro using Western blotting, with the additional morphological assessment of EMT. By using immunofluorescence and flow cytometry, lysosomes and ROS were assessed by acridine orange and 6-carboxy-2′,7′-dichlorodihydrofluorescein acetate assays, respectively. Results TGF-β1-stimulated cells demonstrated a loss of H-ferritin, which was prevented by the antioxidant N-acetyl-L-cysteine (NAC) and inhibitors of lysosomal degradation. TGF-β1 stimulation generated ROS and autophagosome formation and led to EMT, which was further promoted by the additional ROS-generating cytokine, tumor necrosis factor-α. Lysosomes of TGF-β1-stimulated cells were sensitized to oxidants but also completely protected by lysosomal loading with dextran-bound deferoxamine (DFO). Autophagy and EMT were prevented by NAC, DFO, and inhibitors of autophagy and lysosomal degradation. Conclusion The findings of this study support the role of enhanced autophagic degradation of H-ferritin as a mechanism for increasing the vulnerability of lysosomes to iron-driven oxidant injury that triggers further autophagy during EMT. This study proposes that lysosomal leakage is a novel pathway of TGF-β1-induced EMT that may be prevented by iron-chelating drugs that target the lysosome.

  17. Purification and characterization of Taenia crassiceps cysticerci thioredoxin: insight into thioredoxin-glutathione-reductase (TGR) substrate recognition.

    Science.gov (United States)

    Martínez-González, J J; Guevara-Flores, A; Rendón, J L; Sosa-Peinado, A; Del Arenal Mena, I P

    2015-04-01

    Thioredoxin (Trx) is an oxidoreductase central to redox homeostasis in cells and is involved in the regulation of protein activity through thiol/disulfide exchanges. Based on these facts, our goal was to purify and characterize cytosolic thioredoxin from Taenia crassiceps cysticerci, as well as to study its behavior as a substrate of thioredoxin-glutathione reductase (TGR). The enzyme was purified >133-fold with a total yield of 9.7%. A molecular mass of 11.7kDa and a pI of 4.84 were measured. Native electrophoresis was used to identify the oxidized and reduced forms of the monomer as well as the presence of a homodimer. In addition to the catalytic site cysteines, cysticerci thioredoxin contains Cys28 and Cys65 residues conserved in previously sequenced cestode thioredoxins. The following kinetic parameters were obtained for the substrate of TGR: a Km of 3.1μM, a kcat of 10s(-1) and a catalytic efficiency of 3.2×10(6)M(-1)s(-1). The negative patch around the α3-helix of Trx is involved in the interaction with TGR and suggests variable specificity and catalytic efficiency of the reductase toward thioredoxins of different origins.

  18. Activity assays of mammalian thioredoxin and thioredoxin reductase: fluorescent disulfide substrates, mechanisms, and use with tissue samples.

    Science.gov (United States)

    Montano, Sergio J; Lu, Jun; Gustafsson, Tomas N; Holmgren, Arne

    2014-03-15

    Thioredoxin (Trx) is a protein disulfide reductase that, together with nicotinamide adenine dinucleotide phosphate (NADPH) and thioredoxin reductase (TrxR), controls oxidative stress or redox signaling via thiol redox control. Human cytosolic Trx1 has Cys32 and Cys35 as the active site and three additional cysteine residues (Cys62, Cys69, and Cys73), which by oxidation generates inactive Cys62 to Cys69 two-disulfide Trx. This, combined with TrxR with a broad substrate specificity, complicates assays of mammalian Trx and TrxR. We sought to understand the autoregulation of Trx and TrxR and to generate new methods for quantification of Trx and TrxR. We optimized the synthesis of two fluorescent substrates, di-eosin-glutathione disulfide (Di-E-GSSG) and fluorescein isothiocyanate-labeled insulin (FiTC-insulin), which displayed higher fluorescence on disulfide reduction. Di-E-GSSG showed a very large increase in fluorescence quantum yield but had a relatively low affinity for Trx and was also a weak direct substrate for TrxR, in contrast to GSSG. FiTC-insulin was used to develop highly sensitive assays for TrxR and Trx. Reproducible conditions were developed for reactivation of modified Trx, commonly present in frozen or oxidized samples. Trx in cell extracts and tissue samples, including plasma and serum, were subsequently analyzed, showing highly reproducible results and allowing measurement of trace amounts of Trx.

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

    Science.gov (United States)

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

    1998-01-01

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

  20. Septins as modulators of endo-lysosomal membrane traffic

    Directory of Open Access Journals (Sweden)

    Kyungyeun Song

    2016-11-01

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

  1. Lysosomal exoglycosidases and cathepsin D in colon adenocarcinoma.

    Science.gov (United States)

    Waszkiewicz, Napoleon; Zalewska-Szajda, Beata; Szajda, Sławomir D; Kępka, Alina; Waszkiewicz, Magdalena; Roszkowska-Jakimiec, Wiesława; Wojewódzka-Żeleźniakowicz, Marzena; Milewska, Anna J; Dadan, Jacek; Szulc, Agata; Zwierz, Krzysztof; Ladny, Jerzy R

    2012-01-01

    Changes in the structure of membrane glycoconjugates and activity of glycosidases and proteases are important in tumor formation. The aim of the study was to compare the specific activity of lysosomal exoglycosidases: N-acetyl-β-D-hexosaminidase (HEX), its isoenzymes A (HEX A) and B (HEX B), β-D-galactosidase (GAL), α-fucosidase (FUC), and α-mannosidase (MAN) with the activity of cathepsin D (CD) in serum, urine, and carcinoma tissue of patients with colon adenocarcinoma. The specific activity of HEX, HEX A, HEX B, GAL, FUC, MAN, and CD was assayed in serum, urine, and carcinoma tissue of 12 patients with colon adenocarcinoma. Lysosomal exoglycosidases and CD have similar specific activity in colon adenocarcinoma tissue and urine, which is higher than their activity in serum (with the exception of the highest specific activity of CD in urine). A positive correlation was observed between the specific activity of CD and that of HEX, HEX A, FUC, and MAN in the carcinoma tissue and urine as well as between CD and GAL in the urine of patients with colon adenocarcinoma. Negative correlations were observed between protein levels and the specific activity of HEX, HEX A, FUC, MAN, and CD in the carcinoma tissue and urine, and between protein levels and GAL in urine. Increased degradation and remodeling of glycoconjugates in the colon adenocarcinoma tissue is reflected by increased specific activity of exoglycosidases and CD. The results suggest a strong effect of exoglycosidase action on tissue degradation and a potential role of exoglycosidases in the initiation of proteolysis.

  2. EXPRESSION AND DISTRIBUTION OF THIOL-REGULATING ENZYME GLUTAREDOXIN 2 (GRX2) IN PORCINE OCULAR TISSUES*

    Science.gov (United States)

    Upadhyaya, Bijaya; Tian, Xiaoli; Wu, Hongli; Lou, Marjorie F.

    2014-01-01

    Glutaredoxin2 (Grx2) is a mitochondrial isozyme of the cytosolic glutaredoxin1 (thioltransferase or TTase). Both belong to the large oxidoreductase family and play an important role in maintaining thiol/disulfide redox homeostasis in the cells. Grx2 is recently found in the lens where its activities of disulfide reductase and peroxidase, similar to TTase, can protect the lens against oxidative stress. Since other eye tissues are also highly sensitive to oxidative stress, and TTase’s distribution in the eye is known, we focused on this study by investigating the Grx2 distribution in the ocular tissues in comparison to the lens. Fresh porcine eyes were dissected into cornea, iris, ciliary body, the lens, vitreous humor, retina, and optic nerve. Each tissue (pooled from three eyes) was homogenized and processed for mitochondrial isolation. The mitochondrial fraction was analyzed for Grx2 protein using Western blotting with anti-Grx2 antibody, and Grx2 activity using the published procedure. The eye tissues were also measured for Grx2 mRNA expression by RT-PCR with GAPDH as the control. Grx2-rich mouse liver and purified recombinant mouse Grx2 were used as positive controls for the above analyses. It was found that Grx2 was present in all the tested ocular tissues, except vitreous humor. In comparison with the mouse liver, the protein levels of Grx2 in porcine ciliary body and the lens were 27-fold and 0.75-fold, respectively. Comparing to the lens, Grx2 protein was highest in the ciliary body (13.5-fold), followed by retina (9.2-fold), iris and optic nerve (2-fold), and cornea (1.2-fold). Enzyme activity assays showed that the retina had the highest Grx2 specific activity (3.9 mU/mg protein), followed by ciliary body (3.1 mU/mg), the lens (0.58 mU/mg), and optic nerve (0.32 mU/mg). Grx2 gene expression in these ocular tissues was further confirmed by RT-PCR analysis. Grx2 mRNA expression showed the highest in ciliary body, followed by retina, optic nerve, cornea

  3. Ester-free Thiol-ene Dental Restoratives – Part A: Resin Development

    Science.gov (United States)

    Podgórski, Maciej; Becka, Eftalda; Claudino, Mauro; Flores, Alexander; Shah, Parag K.; Stansbury, Jeffrey W.; Bowman, Christopher N.

    2015-01-01

    Objectives To detail the development of ester-free thiol-ene dental resins with enhanced mechanical performance, limited potential for water uptake/leachables/degradation and low polymerization shrinkage stress. Methods Thiol-terminated oligomers were prepared via a thiol-Michael reaction and a bulky tetra-allyl monomer containing urethane linkages was synthesized. The experimental oligomers and/or monomers were photopolymerized using visible light activation. Several thiol-ene formulations were investigated and their performance ranked by comparisons of the thermo-mechanical properties, polymerization shrinkage stress, water sorption/solubility, and reactivity with respect to a control comprising a conventional BisGMA/TEGDMA dental resin. Results The ester-free thiol-ene formulations had significantly lower viscosities, water sorption and solubility than the BisGMA/TEGDMA control. Depending on the resin, the limiting functional conversions were equivalent to or greater than that of BisGMA/TEGDMA. At comparable conversions, lower shrinkage stress values were achieved by the thiol-ene systems. The polymerization shrinkage stress was dramatically reduced when the tetra-allyl monomer was used as the ene in ester-free thiol-ene mixtures. Although exhibiting lower Young’s modulus, flexural strength, and glass transition temperatures, the toughness values associated with thiol-ene resins were greater than that of the BisGMA/TEGDMA control. In addition, the thiol-ene polymerization resulted in highly uniform polymer networks as indicated by the narrow tan delta peak widths. Significance Employing the developed thiol-ene resins in dental composites will reduce shrinkage stress and moisture absorption and form tougher materials. Furthermore, their low viscosities are expected to enable higher loadings of functionalized micro/nano-scale filler particles relevant for practical dental systems. PMID:26360013

  4. HPLC analysis of nonprotein thiols in planktonic diatoms: Pool size, redox state and response to copper and cadmium exposure

    NARCIS (Netherlands)

    Rijstenbil, J.W.; Wijnholds, J.A.

    1996-01-01

    A sensitive method was developed to analyze low molecular weight thiols involved in metal homeostasis and detoxification in phytoplankton. The aims of this study were to (1) separate and measure all relevant thiols in a single HPLC run; (2) measure redox states of the thiols and (3) identify specifi

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

    Science.gov (United States)

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

    2010-12-01

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

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

    Science.gov (United States)

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

    2014-04-01

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

  7. Investigations of the catalytic mechanism of thioredoxin glutathione reductase from Schistosoma mansoni.

    Science.gov (United States)

    Huang, Hsin-Hung; Day, Latasha; Cass, Cynthia L; Ballou, David P; Williams, Charles H; Williams, David L

    2011-07-05

    Thioredoxin glutathione reductase from Schistosoma mansoni (SmTGR) catalyzes the reduction of both thioredoxin and glutathione disulfides (GSSG), thus playing a crucial role in maintaining redox homeostasis in the parasite. In line with this role, previous studies have demonstrated that SmTGR is a promising drug target for schistosomiasis. To aid in the development of efficacious drugs that target SmTGR, it is essential to understand the catalytic mechanism of SmTGR. SmTGR is a dimeric flavoprotein in the glutathione reductase family and has a head-to-tail arrangement of its monomers; each subunit has the components of both a thioredoxin reductase (TrxR) domain and a glutaredoxin (Grx) domain. However, the active site of the TrxR domain is composed of residues from both subunits: FAD and a redox-active Cys-154/Cys-159 pair from one subunit and a redox-active Cys-596'/Sec-597' pair from the other; the active site of the Grx domain contains a redox-active Cys-28/Cys-31 pair. Via its Cys-28/Cys-31 dithiol and/or its Cys-596'/Sec-597' thiol-selenolate, SmTGR can catalyze the reduction of a variety of substrates by NADPH. It is presumed that SmTGR catalyzes deglutathionylation reactions via the Cys-28/Cys-31 dithiol. Our anaerobic titration data suggest that reducing equivalents from NADPH can indeed reach the Cys-28/Cys-31 disulfide in the Grx domain to facilitate reductions effected by this cysteine pair. To clarify the specific chemical roles of each redox-active residue with respect to its various reactivities, we generated variants of SmTGR. Cys-28 variants had no Grx deglutathionylation activity, whereas Cys-31 variants retained partial Grx deglutathionylation activity, indicating that the Cys-28 thiolate is the nucleophile initiating deglutathionylation. Lags in the steady-state kinetics, found when wild-type SmTGR was incubated at high concentrations of GSSG, were not present in Grx variants, indicating that this cysteine pair is in some way responsible for the

  8. Investigations on the Catalytic Mechanism of Thioredoxin Glutathione Reductase (TGR) from Schistosoma mansoni

    Science.gov (United States)

    Huang, Hsin-Hung; Day, Latasha; Cass, Cynthia L.; Ballou, David P.; Williams, Charles H.; Williams, David L.

    2013-01-01

    Thioredoxin glutathione reductase from Schistosoma mansoni (SmTGR) catalyzes the reduction of both thioredoxin and glutathione disulfides (GSSG), thus playing a crucial role in maintaining redox homeostasis in the parasite. In line with this role, previous studies have demonstrated that SmTGR is a promising drug target for schistosomiasis. To aid in the development of efficacious drugs that target SmTGR, it is essential to understand the catalytic mechanism of SmTGR. SmTGR is a dimeric flavoprotein in the glutathione reductase family and it has a head-to-tail arrangement of its monomers; each subunit has the components of both a thioredoxin reductase (TrxR) domain and a glutaredoxin (Grx) domain. However, the active site of the TrxR domain is composed of residues from both subunits: FAD and a redox-active Cys-154/Cys-159 pair from one subunit and a redox-active Cys-596′/Sec-597′ pair from the other; the active site of the Grx domain contains a redox-active Cys-28/Cys-31 pair. Via its Cys-28/Cys-31 dithiol and/or its Cys-596′/Sec-597′ thiol-selenolate, SmTGR can catalyze the reduction of a variety of substrates by NADPH. It is presumed that SmTGR catalyzes deglutathionylation reactions via the Cys-28/Cys-31 dithiol. Our anaerobic titration data suggest that reducing equivalents from NADPH can indeed reach the Cys-28/Cys-31 disulfide in the Grx domain to facilitate reductions effected by this cysteine pair. To clarify the specific chemical roles of each redox-active residue with respect to its various reactivities, we generated variants of SmTGR. Cys-28 variants had no Grx glutathionylation activity whereas Cys-31 variants retained partial Grx glutathionylation activity, indicating that the Cys-28 thiolate is the nucleophile initiating deglutathionylation. Lags in the steady-state kinetics, found when wild-type (WT) SmTGR was incubated at high concentrations of GSSG, were not present in Grx variants, indicating that this cysteine pair is in some way

  9. Functional thioredoxin reductase from pathogenic and free-living Leptospira spp.

    Science.gov (United States)

    Sasoni, Natalia; Iglesias, Alberto A; Guerrero, Sergio A; Arias, Diego G

    2016-08-01

    Low molecular mass thiols and antioxidant enzymes have essential functions to detoxify reactive oxygen and nitrogen species maintaining cellular redox balance. The metabolic pathways for redox homeostasis in pathogenic (Leptospira interrogans) and free-living (Leptospira biflexa) leptospires species were not functionally characterized. We performed biochemical studies on recombinantly produced proteins to in depth analyze kinetic and structural properties of thioredoxin reductase (LinTrxR) and thioredoxin (LinTrx) from L. interrogans, and two TrxRs (LbiTrxR1 and LbiTrxR2) from L. biflexa. All the TrxRs were characterized as homodimeric flavoproteins, with LinTrxR and LbiTrxR1 catalyzing the NADPH dependent reduction of LinTrx and DTNB. The thioredoxin system from L. interrogans was able to use glutathione disulfide, lipoamide disulfide, cystine and bis-γ-glutamyl cysteine and homologous peroxiredoxin as substrates. Classic TrxR activity of LinTrxR2 had not been evidenced in vitro, but recombinant Escherichia coli cells overexpressing LbiTrxR2 showed high tolerance to oxidative stress. The enzymatic systems herein characterized could play a key role for the maintenance of redox homeostasis and the function of defense mechanisms against reactive oxidant species in Leptospira spp. Our results contribute to the general knowledge about redox biochemistry in these bacteria, positioning TrxR as a critical molecular target for the development of new anti-leptospiral drugs.

  10. Binding to large enzyme pockets: small-molecule inhibitors of trypanothione reductase.

    Science.gov (United States)

    Persch, Elke; Bryson, Steve; Todoroff, Nickolay K; Eberle, Christian; Thelemann, Jonas; Dirdjaja, Natalie; Kaiser, Marcel; Weber, Maria; Derbani, Hassan; Brun, Reto; Schneider, Gisbert; Pai, Emil F; Krauth-Siegel, R Luise; Diederich, François

    2014-08-01

    The causative agents of the parasitic disease human African trypanosomiasis belong to the family of trypanosomatids. These parasitic protozoa exhibit a unique thiol redox metabolism that is based on the flavoenzyme trypanothione reductase (TR). TR was identified as a potential drug target and features a large active site that allows a multitude of possible ligand orientations, which renders rational structure-based inhibitor design highly challenging. Herein we describe the synthesis, binding properties, and kinetic analysis of a new series of small-molecule inhibitors of TR. The conjunction of biological activities, mutation studies, and virtual ligand docking simulations led to the prediction of a binding mode that was confirmed by crystal structure analysis. The crystal structures revealed that the ligands bind to the hydrophobic wall of the so-called "mepacrine binding site". The binding conformation and potency of the inhibitors varied for TR from Trypanosoma brucei and T. cruzi. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Bacillithiol: a key protective thiol in Staphylococcus aureus.

    Science.gov (United States)

    Perera, Varahenage R; Newton, Gerald L; Pogliano, Kit

    2015-01-01

    Bacillithiol is a low-molecular-weight thiol analogous to glutathione and is found in several Firmicutes, including Staphylococcus aureus. Since its discovery in 2009, bacillithiol has been a topic of interest because it has been found to contribute to resistance during oxidative stress and detoxification of electrophiles, such as the antibiotic fosfomycin, in S. aureus. The rapid increase in resistance of methicillin-resistant Staphylococcus aureus (MRSA) to available therapeutic agents is a great health concern, and many research efforts are focused on identifying new drugs and targets to combat this organism. This review describes the discovery of bacillithiol, studies that have elucidated the physiological roles of this molecule in S. aureus and other Bacilli, and the contribution of bacillithiol to S. aureus fitness during pathogenesis. Additionally, the bacillithiol biosynthesis pathway is evaluated as a novel drug target that can be utilized in combination with existing therapies to treat S. aureus infections.

  12. GILT expression in B cells diminishes cathepsin S steady-state protein expression and activity

    OpenAIRE

    Phipps-Yonas, Hannah; Semik, Vikki; Hastings, Karen Taraszka

    2012-01-01

    MHC class II-restricted Ag processing requires protein degradation in the endocytic pathway for the activation of CD4+ T cells. Gamma-interferon-inducible lysosomal thiol reductase (GILT) facilitates Ag processing by reducing protein disulfide bonds in this compartment. Lysosomal cysteine protease cathepsin S (CatS) contains disulfide bonds and mediates essential steps in MHC class II-restricted processing, including proteolysis of large polypeptides and cleavage of the invariant chain. We so...

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

    Science.gov (United States)

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

    2016-01-01

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

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

    Science.gov (United States)

    Opperdoes, F R; Van Roy, J

    1982-09-01

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

  15. TRPML cation channels regulate the specialized lysosomal compartment of vertebrate B-lymphocytes.

    Science.gov (United States)

    Song, Yumei; Dayalu, Rashmi; Matthews, Sharon A; Scharenberg, Andrew M

    2006-12-01

    B-lymphocytes possess a specialized lysosomal compartment, the regulated transformation of which has been implicated in B-cell antigen presentation. Members of the mucolipin (TRPML) family of cation channels have been implicated in regulated vesicular transport in several tissues, but a role for TRPML function in lymphocyte vesicular transport physiology has not been previously described. To address the role of TRPML proteins in lymphocyte vesicular transport, we analyzed the lysosomal compartment in cultured B-lymphocytes engineered to lack TRPML1 or after expression of N- or C-terminal GFP fusion proteins of TRPML1 or TRPML2. Consistent with previous analyses of lymphocytes derived from human patients with mutations in TRPML1, we were not able to detect abnormalities in the lysosomes of TRPML1-deficient DT40 B-lymphocytes. However, while N-terminal GFP fusions of TRPML2 localized to normal appearing lysosomes, C-terminal GFP fusions of either TRPML1 or TRPML2 acted to antagonize endogenous TRPML function, localizing to large vesicular structures, the histological properties of which were indistinguishable from the enlarged lysosomes observed in affected tissues of TRPML1-deficient humans. Endocytosed B-cell receptors were delivered to these enlarged lysosomes, demonstrating that a TRPML-dependent process is required for normal regulation of the specialized lysosome compartment of vertebrate B-lymphocytes.

  16. Chlamydia species-dependent differences in the growth requirement for lysosomes.

    Directory of Open Access Journals (Sweden)

    Scot P Ouellette

    Full Text Available Genome reduction is a hallmark of obligate intracellular pathogens such as Chlamydia, where adaptation to intracellular growth has resulted in the elimination of genes encoding biosynthetic enzymes. Accordingly, chlamydiae rely heavily on the host cell for nutrients yet their specific source is unclear. Interestingly, chlamydiae grow within a pathogen-defined vacuole that is in close apposition to lysosomes. Metabolically-labeled uninfected host cell proteins were provided as an exogenous nutrient source to chlamydiae-infected cells, and uptake and subsequent labeling of chlamydiae suggested lysosomal degradation as a source of amino acids for the pathogen. Indeed, Bafilomycin A1 (BafA1, an inhibitor of the vacuolar H(+/ATPase that blocks lysosomal acidification and functions, impairs the growth of C. trachomatis and C. pneumoniae, and these effects are especially profound in C. pneumoniae. BafA1 induced the marked accumulation of material within the lysosomal lumen, which was due to the inhibition of proteolytic activities, and this response inhibits chlamydiae rather than changes in lysosomal acidification per se, as cathepsin inhibitors also inhibit the growth of chlamydiae. Finally, the addition of cycloheximide, an inhibitor of eukaryotic protein synthesis, compromises the ability of lysosomal inhibitors to block chlamydial growth, suggesting chlamydiae directly access free amino acids in the host cytosol as a preferred source of these nutrients. Thus, chlamydiae co-opt the functions of lysosomes to acquire essential amino acids.

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

    Science.gov (United States)

    Lee, Geum-Hwa; Lee, Mi-Rin; Lee, Hwa-Young; Kim, Seung Hyun; Kim, Hye-Kyung; Kim, Hyung-Ryong; Chae, Han-Jung

    2014-01-01

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

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

    Science.gov (United States)

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

    2016-10-01

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

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

    Science.gov (United States)

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

    1995-05-01

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

  20. Lysosomal cholesterol accumulation: driver on the road to inflammation during atherosclerosis and non-alcoholic steatohepatitis.

    Science.gov (United States)

    Hendrikx, T; Walenbergh, S M A; Hofker, M H; Shiri-Sverdlov, R

    2014-05-01

    Many studies show an association between the accumulation of cholesterol inside lysosomes and the progression towards inflammatory disease states that are closely related to obesity. While in the past, the knowledge regarding lysosomal cholesterol accumulation was limited to its association with plaque severity during atherosclerosis, recently, a growing body of evidence indicates a causal link between lysosomal cholesterol accumulation and inflammation. These findings make lysosomal cholesterol accumulation an important target for intervention in metabolic diseases that are characterized by the presence of an inflammatory response. In this review, we aim to show the importance of cholesterol trapping inside lysosomes to the development of inflammation by focusing upon cardiovascular disease and non-alcoholic steatohepatitis (NASH) in particular. We summarize current data supporting the hypothesis that lysosomal cholesterol accumulation plays a key role in the development of inflammation during atherosclerosis and NASH. In addition, potential mechanisms by which disturbed lysosomal function can trigger the inflammatory response, the challenges in improving cholesterol trafficking in macrophages and recent successful research directions will be discussed.

  1. Two mutations of dihydropteridine reductase deficiency.

    Science.gov (United States)

    Ponzone, A; Guardamagna, O; Ferraris, S; Bracco, G; Niederwieser, A; Cotton, R G

    1988-02-01

    Two patients with dihydropteridine reductase (DHPR) deficiency, in one case due to the absence of any enzyme protein (DHPR- cross reactive material (CRM)-) and in the other case due to the production of a mutant type devoid of catalytic activity (DHPR- CRM+) were examined. This latter form of malignant phenylketonuria, whose relative frequency seems to be higher in the Italian population, possibly has a worse prognosis. The earlier onset and the greater severity of clinical symptoms are associated with a more pronounced hydroxylation defect, as shown by higher degree of neonatal hyperphenylalaninaemia, unresponsiveness to an oral tetrahydrobiopterin load, lower concentrations of neurotransmitter metabolites, and reduced tyrosine production after an oral phenylalanine load.

  2. Autophagic lysosome reformation dysfunction in glucocerebrosidase deficient cells: relevance to Parkinson disease.

    Science.gov (United States)

    Magalhaes, Joana; Gegg, Matthew E; Migdalska-Richards, Anna; Doherty, Mary K; Whitfield, Phillip D; Schapira, Anthony H V

    2016-08-15

    Glucocerebrosidase (GBA1) gene mutations increase the risk of Parkinson disease (PD). While the cellular mechanisms associating GBA1 mutations and PD are unknown, loss of the glucocerebrosidase enzyme (GCase) activity, inhibition of autophagy and increased α-synuclein levels have been implicated. Here we show that autophagy lysosomal reformation (ALR) is compromised in cells lacking functional GCase. ALR is a cellular process controlled by mTOR which regenerates functional lysosomes from autolysosomes formed during macroautophagy. A decrease in phopho-S6K levels, a marker of mTOR activity, was observed in models of GCase deficiency, including primary mouse neurons and the PD patient derived fibroblasts with GBA1 mutations, suggesting that ALR is compromised. Importantly Rab7, a GTPase crucial for endosome-lysosome trafficking and ALR, accumulated in GCase deficient cells, supporting the notion that lysosomal recycling is impaired. Recombinant GCase treatment reversed ALR inhibition and lysosomal dysfunction. Moreover, ALR dysfunction was accompanied by impairment of macroautophagy and chaperone-mediated autophagy, increased levels of total and phosphorylated (S129) monomeric α-synuclein, evidence of amyloid oligomers and increased α-synuclein release. Concurrently, we found increased cholesterol and altered glucosylceramide homeostasis which could compromise ALR. We propose that GCase deficiency in PD inhibits lysosomal recycling. Consequently neurons are unable to maintain the pool of mature and functional lysosomes required for the autophagic clearance of α-synuclein, leading to the accumulation and spread of pathogenic α-synuclein species in the brain. Since GCase deficiency and lysosomal dysfunction occur with ageing and sporadic PD pathology, the decrease in lysosomal reformation may be a common feature in PD. © The Author 2016. Published by Oxford University Press.

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

    Directory of Open Access Journals (Sweden)

    Christine Burkard

    2014-11-01

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

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

    Science.gov (United States)

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

    2014-11-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-04-01

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

  6. Disulfide-Linked Dinitroxides for Monitoring Cellular Thiol Redox Status through Electron Paramagnetic Resonance Spectroscopy.

    Science.gov (United States)

    Legenzov, Eric A; Sims, Stephen J; Dirda, Nathaniel D A; Rosen, Gerald M; Kao, Joseph P Y

    2015-12-01

    Intracellular thiol-disulfide redox balance is crucial to cell health, and may be a key determinant of a cancer's response to chemotherapy and radiation therapy. The ability to assess intracellular thiol-disulfide balance may thus be useful not only in predicting responsiveness of cancers to therapy, but in assessing predisposition to disease. Assays of thiols in biology have relied on colorimetry or fluorimetry, both of which require UV-visible photons, which do not penetrate the body. Low-frequency electron paramagnetic resonance imaging (EPRI) is an emerging magnetic imaging technique that uses radio waves, which penetrate the body well. Therefore, in combination with tailored imaging agents, EPRI affords the opportunity to image physiology within the body. In this study, we have prepared water-soluble and membrane-permeant disulfide-linked dinitroxides, at natural isotopic abundance, and with D,(15)N-substitution. Thiols such as glutathione cleave the disulfides, with simple bimolecular kinetics, to yield the monomeric nitroxide species, with distinctive changes in the EPR spectrum. Using the D,(15)N-substituted disulfide-dinitroxide and EPR spectroscopy, we have obtained quantitative estimates of accessible intracellular thiol in cultured human lymphocytes. Our estimates are in good agreement with published measurements. This suggests that in vivo EPRI of thiol-disulfide balance is feasible. Finally, we discuss the constraints on the design of probe molecules that would be useful for in vivo EPRI of thiol redox status.

  7. Studies of Aqueous U(IV) Complexation under Thiol-rich Conditions

    Energy Technology Data Exchange (ETDEWEB)

    Cha, Wansik; Cho, Hyeryun; Jung, Euo Chang [Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of)

    2013-05-15

    Organic thiol compounds and hydrogen sulfide (H{sub 2}S) are electron donors and metabolic products of sulfate reducing bacteria. In addition, they are among redox potential (Eh) determinants of groundwater systems due to their redox characteristics. The low values of acid dissociation constants for .SH (pK{sub a}, 7-9) compared to those of aliphatic or phenolic .OH, impart greater anionic and metal-binding properties to the molecules. Recently, we demonstrated that a thiol compound (i. e., thiosalicylate) enhances the solubility of U(VI) at higher pH levels (< ∼9). In this study, to have a better knowledge of the behaviors of U(IV) species under anaerobic conditions, the U(IV)-OH complex formation in the presence of thiol was examined using UV-Vis spectrophotometry and TRLFS (time-resolved laser-induced fluorescence spectroscopy). A TRLFS-based U(IV) quantification methodology developed earlier was applied to examine the effects of thiol species on the dissolution behaviors. Based on UV-Vis absorption monitoring, the presence of thiol does not result in a significant changes in the low-pH hydrolysis behaviors of U(IV). However, the concentration of U(IV) dissolved in bulk phase of aqueous solutions increased with the increase of thiol concentration. The formation of soluble thiol complexes or the stabilization of UO{sub 2} nanoparticles may explain the observed solubility increase.

  8. Thiol compounds from a free-living pathogenic opportunistic amoeba, Acanthamoeba polyphaga.

    Science.gov (United States)

    Ondarza, Raúl N; Iturbe, Angélica; Hernández, Eva; Hurtado, Gerardo

    2002-12-01

    New bimane-reacting compounds from perchloric acid extracts have been detected by HPLC from Acanthamoeba polyphaga. The main compounds detected are cysteine, glutathione and other novel thiol compounds. All of these compounds must be thiols, since they disappear or decrease substantially when treated by N -ethylmaleimide prior to acetonitrile/bimane derivatization. Cysteine and glutathione increase in quantity when dithiothreitol reduction is applied to the fresh extract. This means that they are likely to be present in their oxidized and reduced form and indicates the possible presence of a corresponding thiol/disulphide enzymic system. There are other compounds that have a different behaviour, since although they can react with bimane, they do not disappear if treated previously by N -ethylmaleimide. This shows that they are not thiols but can react with bimane. The main thiol compounds found to be present, in both the parasite and the host lymphocyte cells, were cysteine and glutathione. We were unable to detect ovothiol A in Acanthamoeba but instead we found another thiol compound that could be structurally related to trypanothione. The new thiol compounds unique to this parasite and not present in lymphocytes will permit the study of disulphide-reducing enzymes as potential drug targets.

  9. Redox regulation of sperm surface thiols modulates adhesion to the fallopian tube epithelium.

    Science.gov (United States)

    Talevi, Riccardo; Zagami, Maria; Castaldo, Marianna; Gualtieri, Roberto

    2007-04-01

    Sperm that adhere to the fallopian tube epithelium are of superior quality and adhesion extends their fertile life. It has been postulated that periovulatory signals, as yet undefined, promote sperm release. In the in vitro studies described here, we examined the effects of several antioxidants, reportedly present within oviductal fluid, on the modulation of sperm-oviduct adhesion in bovine species. Results showed that 1) the cell-permeant thiols (penicillamine, beta mercaptoethanol, cysteine, and dithiotreitol), as well as the nonpermeant thiol, reduced glutathione, cause adhering spermatozoa to release from the epithelium; 2) thiol action is exerted on spermatozoa; and 3) oxidized glutathione, as well as the non-thiol antioxidants (dimethylthiourea, trolox, superoxide dismutase, and catalase) have no effect. Sperm surface sulfhydryls labeled with iodoacetamide fluorescein showed that spermatozoa devoid of sulfhydryls on the head surface adhered to the fallopian epithelium in vitro, whereas thiol-induced release increased the exposure of sulfhydryls on the sperm head surface. Finally, analysis of capacitation status demonstrated that uncapacitated spermatozoa adhered to the oviduct, and that thiol-induced release of spermatozoa was accompanied by capacitation. In conclusion, thiol-reducing agents in the oviductal fluid may modulate the redox status of sperm surface proteins, leading to the release of spermatozoa selected and stored through adhesion to the fallopian tube epithelium in the bovine species.

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

    Science.gov (United States)

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

    2016-03-01

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

  11. Beer thiol-containing compounds and redox stability: kinetic study of 1-hydroxyethyl radical scavenging ability.

    Science.gov (United States)

    de Almeida, Natália E C; Lund, Marianne N; Andersen, Mogens L; Cardoso, Daniel R

    2013-10-02

    The 1-hydroxyethyl radical is a central intermediate in oxidative reactions occurring in beer. The reactivity of thiol-containing compounds toward 1-hydroxyethyl radical was evaluated in beer model solutions using a competitive kinetic approach, employing the spin-trap 4-POBN as a probe and by using electron paramagnetic resonance to detect the generated 1-hydroxyethyl/4-POBN spin adduct. Thiol-containing compounds were highly reactive toward the 1-hydroxyethyl radical with apparent second-order rate constants close to the diffusion limit in water and ranging from 0.5 × 10⁹ L mol⁻¹ s⁻¹ for the His-Cys-Lys-Phe-Trp-Trp peptide to 6.1 × 10⁹ L mol⁻¹ s⁻¹ for the reduced lipid transfer protein 1 (LTP1) isolated from beer. The reactions gave rise to a moderate kinetic isotope effect (k(H)/k(D) = 2.3) suggesting that reduction of the 1-hydroxyethyl radical by thiol-containing compounds takes place by hydrogen atom abstraction from the RSH group rather than electron transfer. The content of reduced thiols in different beers was determined using a previously established method based on ThioGlo-1 as the thiol derivatization reagent and detection of the derivatized thiols by reverse-phase liquid chromatography coupled to a fluorescence detector. The total level of thiol in beer (oxidized and reduced) was determined after a reduction step employing 3,3',3″-phosphanetriyltripropanoic acid (TCEP) as the disulfide reductant. A good correlation among total protein and total thiol content in different beers was observed. The results suggest a similar ratio between reduced thiols and disulfides in all of the tested beers, which indicates a similar redox state.

  12. Enzyme toolbox: novel enantiocomplementary imine reductases.

    Science.gov (United States)

    Scheller, Philipp N; Fademrecht, Silvia; Hofelzer, Sebastian; Pleiss, Jürgen; Leipold, Friedemann; Turner, Nicholas J; Nestl, Bettina M; Hauer, Bernhard

    2014-10-13

    Reducing reactions are among the most useful transformations for the generation of chiral compounds in the fine-chemical industry. Because of their exquisite selectivities, enzymatic approaches have emerged as the method of choice for the reduction of C=O and activated C=C bonds. However, stereoselective enzymatic reduction of C=N bonds is still in its infancy-it was only recently described after the discovery of enzymes capable of imine reduction. In our work, we increased the spectrum of imine-reducing enzymes by database analysis. By combining the currently available knowledge about the function of imine reductases with the experimentally uncharacterized diversity stored in protein sequence databases, three novel imine reductases with complementary enantiopreference were identified along with amino acids important for catalysis. Furthermore, their reducing capability was demonstrated by the reduction of the pharmaceutically relevant prochiral imine 2-methylpyrroline. These novel enzymes exhibited comparable to higher catalytic efficiencies than previously described enzymes, and their biosynthetic potential is highlighted by the full conversion of 2-methylpyrroline in whole cells with excellent selectivities. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Iron-mediated effects on nitrate reductase in marine phytoplankton

    NARCIS (Netherlands)

    Timmermans, K.R.; Stolte, W.; Baar, H.J.W. de

    1994-01-01

    The potential activity of nitrate reductase was determined in uni-algal cultures in the laboratory and in natural marine phytoplankton assemblages. In the laboratory bioassays, distinct differences in nitrate reductase activity were observed in iron replete versus depleted cultures for Emiliania hux

  14. Visible-Light-Mediated Thiol-Ene Reactions through Organic Photoredox Catalysis.

    Science.gov (United States)

    Zhao, Gaoyuan; Kaur, Sarbjeet; Wang, Ting

    2017-06-16

    Synthetically useful radical thiol-ene reactions can be initiated by visible-light irradiation in the presence of an organic photocatalyst, 9-mesityl-10-methylacridinum tetrafluoroborate. The key thiyl radical intermediates are generated upon quenching of the photoexcited catalyst with a variety of thiols. The success of this method requires only the use of near-stoichiometric levels of alkene coupling partners. Using these highly efficient metal-free conditions, thiol-ene reactions between carbohydrates and peptides can be accomplished in excellent yields.

  15. Strategies for creating antifouling surfaces using selfassembled poly(ethylene glycol) thiol molecules

    DEFF Research Database (Denmark)

    Lokanathan, Arcot R.

    2011-01-01

    conditions for the reversible, initial attachment of microbial cells. This effect can be obtained by grafting hydrophilic polymeric chains onto surfaces and thereby provide a steric barrier between the substrate surface and the microbial cell. Poly (ethylene glycol) (PEG) is one of the most widely used....... The work focuses on novel strategies to self assemble PEG thiol monolayers with high graft density. One of the strategies investigated involved backfilling a self assembled layer of 2000 Da PEG thiol with shorter oligo (ethylene glycol) (OEG) thiol molecules to form a mixed monolayer. Detailed quantitative...

  16. Quinoline-2-thiol Derivatives as Fluorescent Sensors for Metals, pH and HNO

    Directory of Open Access Journals (Sweden)

    Naphtali A. O’Connor

    2014-06-01

    Full Text Available A tautomeric equilibrium exists for quinoline-2-thiol and quinoline-2(1H-thione. Quantum mechanical calculations predict the thione is the major tautomer and this is confirmed by the absorption spectra. The utility of quinolone-2-thiol/quinoline-2(1H-thione as a chromophore for developing fluorescent sensors is explored. No fluorescence is observed when excited at absorption maxima, however a fluorescence increase is observed when exposed to HNO, a molecule of import as a cardiovascular therapeutic. Alkylated quinoline-2-thiol derivatives are found to be fluorescent and show a reduction in fluorescence when exposed to metals and changes in pH.

  17. Readout fidelity of coaxial holographic digital data page recording in nanoparticle-(thiol-ene) polymer composites

    Science.gov (United States)

    Nagaya, Kohta; Hata, Eiji; Tomita, Yasuo

    2016-09-01

    We report on an experimental investigation of nanoparticle-concentration and thiol-to-ene stoichiometric ratio dependences of symbol error rates (SERs) and signal-to-noise ratios (SNRs) of digital data pages recorded at a wavelength of 532 nm in thiol-ene based nanoparticle-polymer composite (NPC) films by using a coaxial holographic digital data storage method. We show that SERs and SNRs at the optimized material condition can be lower than 1 × 10-4 and higher than 10, respectively, without error correction coding. These results show the usefulness of thiol-ene based NPCs as coaxial holographic data storage media.

  18. The effect of a cross-bridging thiol reagent on the catecholamine fluxes of adrenal medulla vesicles

    Science.gov (United States)

    Hasselbach, W.; Taugner, G.

    1970-01-01

    The thiol groups of the vesicular protein of bovine adrenal medulla were allowed to react with the bifunctional thiol reagent bis-(N-maleimidomethyl) ether and with the monofunctional thiol reagent N-ethylmaleimide, and the ATP-dependent and -independent catecholamine fluxes of the modified preparations were studied. 1. During the initial phase of the reaction bis-(N-maleimidomethyl) ether blocks twice as many thiol groups as does N-ethylmaleimide at equimolar concentrations. 2. Labelling of the bis-(N-maleimidomethyl) ether–protein compound with [14C]-cysteine shows that 70–80% of the blocked thiol groups are interconnected by the bifunctional thiol reagent. 3. At a low extent of reaction (1.5mol of thiol groups/106g of protein) the catecholamine efflux is diminished. If more than 2mol of thiol groups/106g of protein are blocked, the efflux is enhanced whichever thiol reagent is applied. 4. If 2–4mol of thiol groups/106g of protein are blocked the inhibition of the catecholamine influx increases linearly with the proportion of the thiol groups blocked. 5. ATP protects the catecholamine influx and the adenosine triphosphatase activity against bis-(N-maleimidomethyl) ether poisoning somewhat less effectively than against N-ethylmaleimide poisoning. PMID:4249860

  19. Preparation and Preliminary Dielectric Characterization of Structured C60-Thiol-Ene Polymer Nanocomposites Assembled Using the Thiol-Ene Click Reaction

    Directory of Open Access Journals (Sweden)

    Hanaa M. Ahmed

    2015-11-01

    Full Text Available Fullerene-containing materials have the ability to store and release electrical energy. Therefore, fullerenes may ultimately find use in high-voltage equipment devices or as super capacitors for high electric energy storage due to this ease of manipulating their excellent dielectric properties and their high volume resistivity. A series of structured fullerene (C60 polymer nanocomposites were assembled using the thiol-ene click reaction, between alkyl thiols and allyl functionalized C60 derivatives. The resulting high-density C60-urethane-thiol-ene (C60-Thiol-Ene networks possessed excellent mechanical properties. These novel networks were characterized using standard techniques, including infrared spectroscopy (FTIR, differential scanning calorimetry (DSC, dynamic mechanical analysis (DMA, and thermal gravimetric analysis (TGA. The dielectric spectra for the prepared samples were determined over a broad frequency range at room temperature using a broadband dielectric spectrometer and a semiconductor characterization system. The changes in thermo-mechanical and electrical properties of these novel fullerene-thiol-ene composite films were measured as a function of the C60 content, and samples characterized by high dielectric permittivity and low dielectric loss were produced. In this process, variations in chemical composition of the networks were correlated to performance characteristics.

  20. Preparation and Preliminary Dielectric Characterization of Structured C60-Thiol-Ene Polymer Nanocomposites Assembled Using the Thiol-Ene Click Reaction.

    Science.gov (United States)

    Ahmed, Hanaa M; Windham, Amber D; Al-Ejji, Maryam M; Al-Qahtani, Noora H; Hassan, Mohammad K; Mauritz, Kenneth A; Buchanan, Randy K; Buchanan, J Paige

    2015-11-18

    Fullerene-containing materials have the ability to store and release electrical energy. Therefore, fullerenes may ultimately find use in high-voltage equipment devices or as super capacitors for high electric energy storage due to this ease of manipulating their excellent dielectric properties and their high volume resistivity. A series of structured fullerene (C60) polymer nanocomposites were assembled using the thiol-ene click reaction, between alkyl thiols and allyl functionalized C60 derivatives. The resulting high-density C60-urethane-thiol-ene (C60-Thiol-Ene) networks possessed excellent mechanical properties. These novel networks were characterized using standard techniques, including infrared spectroscopy (FTIR), differential scanning calorimetry (DSC), dynamic mechanical analysis (DMA), and thermal gravimetric analysis (TGA). The dielectric spectra for the prepared samples were determined over a broad frequency range at room temperature using a broadband dielectric spectrometer and a semiconductor characterization system. The changes in thermo-mechanical and electrical properties of these novel fullerene-thiol-ene composite films were measured as a function of the C60 content, and samples characterized by high dielectric permittivity and low dielectric loss were produced. In this process, variations in chemical composition of the networks were correlated to performance characteristics.

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

    Directory of Open Access Journals (Sweden)

    Fares Hanna

    2010-06-01

    Full Text Available Abstract Background CUP-5 is a Transient Receptor Potential protein in C. elegans that is the orthologue of mammalian TRPML1. Loss of TRPML1 results in the lysosomal storage disorder Mucolipidosis type IV. Loss of CUP-5 results in embryonic lethality and the accumulation of enlarged yolk granules in developing intestinal cells. The embryonic lethality of cup-5 mutants is rescued by mutations in mrp-4, which is required for gut granule differentiation. Gut granules are intestine-specific lysosome-related organelles that accumulate birefringent material. This link between CUP-5 and gut granules led us to determine the roles of CUP-5 in lysosome and gut granule biogenesis in developing intestinal cells. Results We show that CUP-5 protein localizes to lysosomes, but not to gut granules, in developing intestinal cells. Loss of CUP-5 results in defects in endo-lysosomal transport in developing intestinal cells of C. elegans embryos. This ultimately leads to the appearance of enlarged terminal vacuoles that show defective lysosomal degradation and that have lysosomal and endosomal markers. In contrast, gut granule biogenesis is normal in the absence of CUP-5. Furthermore, loss of CUP-5 does not result in inappropriate fusion or mixing of content between lysosomes and gut granules. Conclusions Using an in vivo model of MLIV, we show that there is a defect in lysosomal transport/biogenesis that is earlier than the presumed function of TRPML1 in terminal lysosomes. Our results indicate that CUP-5 is required for the biogenesis of lysosomes but not of gut granules. Thus, cellular phenotypes in Mucolipidosis type IV are likely not due to defects in lysosome-related organelle biogenesis, but due to progressive defects in lysosomal transport that lead to severe lysosomal dysfunction.

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

    NARCIS (Netherlands)

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

    2015-01-01

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

  3. Lysosome dysfunction enhances oxidative stress-induced apoptosis through ubiquitinated protein accumulation in Hela cells.

    Science.gov (United States)

    Yu, Chunyan; Huang, Xiaowei; Xu, Ye; Li, Hongyan; Su, Jing; Zhong, Jiateng; Kang, Jinsong; Liu, Yuhe; Sun, Liankun

    2013-01-01

    The role of lysosomal system in oxidative stress-induced apoptosis in cancer cells is not fully understood. Menadione is frequently used as oxidative stress model. It is indicated that menadione could induce autophagy in Hela cells. In the present study, we examined whether the lysosomal inhibitor, ammonium chloride (NH(4)Cl) could prevent the autophagy flux by inhibiting the fusion of autophagosomes with lysosomes and enhance apoptosis induced by menadione via mitochondrial pathway. The results demonstrated generation and accumulation of reactive oxygen species and increased levels of ubiquitinated proteins and GRP78 in cells treated with both menadione and NH(4)Cl. Our data indicates that lysosomal system through autophagy plays an important role in preventing menadione-induced apoptosis in Hela cells by clearing misfolded proteins, which alleviates endoplasmic reticulum stress.

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

    Directory of Open Access Journals (Sweden)

    Joshua A Dubland

    2015-02-01

    Full Text Available Unregulated cellular uptake of apolipoprotein B-containing lipoproteins in the arterial intima leads to the formation of foam cells in atherosclerosis. Lysosomal acid lipase (LAL plays a crucial role in both lipoprotein lipid catabolism and excess lipid accumulation as it is the primary enzyme that hydrolyzes cholesteryl esters derived from both low density lipoprotein (LDL and modified forms of LDL. Evidence suggests that as atherosclerosis progresses, accumulation of excess free cholesterol in lysosomes leads to impairment of LAL activity, resulting in accumulation of cholesteryl esters in the lysosome as well as the cytosol in foam cells. Impaired metabolism and release of cholesterol from lysosomes can lead to downstream defects in ATP-binding cassette transporter A1 regulation, needed to offload excess cholesterol from plaque foam cells. This review focuses on the role LAL plays in normal cholesterol metabolism and how the associated changes in its enzymatic activity may ultimately contribute to atherosclerosis progression.

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

    Science.gov (United States)

    Chen, Linhua; Lu, Xianting; Ma, Yun

    2012-12-01

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

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

    DEFF Research Database (Denmark)

    Kjøller, Lars; Engelholm, Lars H; Høyer-Hansen, Maria

    2004-01-01

    transmembrane glycoprotein urokinase plasminogen activator receptor-associated protein (uPARAP/endo180) directs collagen IV for lysosomal delivery and degradation. In wild-type fibroblasts, fluorescently labeled collagen IV was first internalized into vesicular structures with diffuse fluorescence eventually...... appearing uniformly within the wild-type cells after longer incubation times. In these cells, some collagen-containing vesicles were identified as lysosomes by staining for LAMP-1. In contrast, collagen IV remained extracellular and associated with fiber-like structures on uPARAP/endo180-deficient...... fibroblasts. Blocking lysosomal cysteine proteases with the inhibitor E64d resulted in strong accumulation of collagen IV in lysosomes in wild-type cells, but only very weak intracellular fluorescence accumulation in uPARAP/endo180-deficient fibroblasts. We conclude that uPARAP/endo180 is critical...

  7. High resolution crystal structure of human β-glucuronidase reveals structural basis of lysosome targeting

    National Research Council Canada - National Science Library

    Hassan, Md Imtaiyaz; Waheed, Abdul; Grubb, Jeffery H; Klei, Herbert E; Korolev, Sergey; Sly, William S

    2013-01-01

    ...). Here we report a high resolution crystal structure of human GUS at 1.7 Å resolution and present an extensive analysis of the structural features, unifying recent findings in the field of lysosome targeting and glycosyl hydrolases...

  8. High Resolution Crystal Structure of Human [beta]-Glucuronidase Reveals Structural Basis of Lysosome Targeting

    National Research Council Canada - National Science Library

    Hassan, Md; Waheed, Abdul; Grubb, Jeffery; Klei, Herbert; Korolev, Sergey; Sly, William

    2013-01-01

    ...). Here we report a high resolution crystal structure of human GUS at 1.7 Å resolution and present an extensive analysis of the structural features, unifying recent findings in the field of lysosome targeting and glycosyl hydrolases...

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

    DEFF Research Database (Denmark)

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

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

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

    NARCIS (Netherlands)

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

    2015-01-01

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

  11. Emerging therapies for neurodegenerative lysosomal storage disorders - from concept to reality.

    Science.gov (United States)

    Hemsley, Kim M; Hopwood, John J

    2011-10-01

    Lysosomal storage disorders are inherited metabolic diseases in which a mutation in a gene encoding a lysosomal enzyme or lysosome-related protein results in the intra-cellular accumulation of substrate and reduced cell/tissue function. Few patients with neurodegenerative lysosomal storage disorders have access to safe and effective treatments although many therapeutic strategies have been or are presently being studied in vivo thanks to the availability of a large number of animal models. This review will describe the comparative advancement of a variety of therapeutic strategies through the 'research pipeline'. Our goal is to provide information for clinicians, researchers and patients/families alike on the leading therapeutic candidates at this point in time, and also to provide information on emerging approaches that may provide a safe and effective treatment in the future. The length of the pipeline represents the significant and sustained effort required to move a novel concept from the laboratory into the clinic.

  12. Magnesium Modulates Doxorubicin Activity through Drug Lysosomal Sequestration and Trafficking.

    Science.gov (United States)

    Trapani, Valentina; Luongo, Francesca; Arduini, Daniela; Wolf, Federica I

    2016-03-21

    Magnesium is directly involved in the control of cell growth and survival, but its role in cancer biology and therapy is multifaceted; in particular, it is highly controversial whether magnesium levels can affect therapy outcomes. Here we investigated whether magnesium availability can modulate cellular responses to the widely used chemotherapeutic doxorubicin. We used an in vitro model consisting of mammary epithelial HC11 cells and found that high magnesium availability was correlated with diminished sensitivity both in cells chronically adapted to high magnesium concentrations and in acutely magnesium-supplemented cells. This decrease in sensitivity resulted from reduced intracellular doxorubicin accumulation in the face of a similar drug uptake rate. We observed that high-magnesium conditions caused a decrease in intracellular drug retention by altering drug lysosomal sequestration and trafficking. In our model, magnesium supplementation correspondingly modulated expression of the TRPM7 channel, which is known to control cytoskeletal organization and dynamics and may be involved in the proposed mechanism. Our findings suggest that magnesium supplementation in hypomagnesemic cancer patients may hinder response to therapy.

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

    Directory of Open Access Journals (Sweden)

    Maria Francisca Coutinho

    2016-07-01

    Full Text Available Lysosomal storage diseases (LSDs are a group of rare, life-threatening genetic disorders, usually caused by a dysfunction in one of the many enzymes responsible for intralysosomal digestion. Even though no cure is available for any LSD, a few treatment strategies do exist. Traditionally, efforts have been mainly targeting the functional loss of the enzyme, by injection of a recombinant formulation, in a process called enzyme replacement therapy (ERT, with no impact on neuropathology. This ineffectiveness, together with its high cost and lifelong dependence is amongst the main reasons why additional therapeutic approaches are being (and have to be investigated: chaperone therapy; gene enhancement; gene therapy; and, alternatively, substrate reduction therapy (SRT, whose aim is to prevent storage not by correcting the original enzymatic defect but, instead, by decreasing the levels of biosynthesis of the accumulating substrate(s. Here we review the concept of substrate reduction, highlighting the major breakthroughs in the field and discussing the future of SRT, not only as a monotherapy but also, especially, as complementary approach for LSDs.

  14. Lysosomal Changes in Renal Proximal Tubular Epithelial Cells of Male Sprague Dawley Rats Following Decalin Exposure

    Science.gov (United States)

    1990-01-01

    decalin-treated animal. Note large, pale, rcd-staining lysosome (-). An exfoliated epithelial cell can iu- seen in the tubular lumen containing large...photomicrograph contains an exfoliated epithelial cell (-) with enlarged, intact lysosomes. The tubule on the left half of the photomicrograph contains an...metabolism of proteins. In: Cytology , GH Bourne and JF Danielli (eds). Academ- The Kidney: Physiology and Pathophysiology, DW ic Press, NY, pp. 251-300. - ~- i :- d .L n .- 2

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

    Directory of Open Access Journals (Sweden)

    Vasileios A Stamelos

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

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

    Science.gov (United States)

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

    2016-07-01

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

  17. Reduction of mutant huntingtin accumulation and toxicity by lysosomal cathepsins D and B in neurons

    OpenAIRE

    Ouyang Xiaosen; Liang Qiuli; Schneider Lonnie; Zhang Jianhua

    2011-01-01

    Abstract Background Huntington's disease is caused by aggregation of mutant huntingtin (mHtt) protein containing more than a 36 polyQ repeat. Upregulation of macroautophagy was suggested as a neuroprotective strategy to degrade mutant huntingtin. However, macroautophagy initiation has been shown to be highly efficient in neurons whereas lysosomal activities are rate limiting. The role of the lysosomal and other proteases in Huntington is not clear. Some studies suggest that certain protease a...

  18. Action of polystyrene nanoparticles of different sizes on lysosomal function and integrity

    OpenAIRE

    Fröhlich Eleonore; Meindl Claudia; Roblegg Eva; Ebner Birgit; Absenger Markus; Pieber Thomas R

    2012-01-01

    Abstract Background Data from environmental exposure to nanoparticles (NPs) suggest that chronic exposure may increase the incidence of lung, cardiovascular and neurodegenerative diseases. Impairment of cell function by intracellular accumulation of NPs is also suspected. Many types of NPs have been detected in the endosomal-lysosomal system and, upon repeated exposure, alterations of the endosomal-lysosomal system may occur. To identify such effects we compared the effect of carboxyl polysty...

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

    Science.gov (United States)

    Múgica, M; Izagirre, U; Marigómez, I

    2015-07-01

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

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

    DEFF Research Database (Denmark)

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

    2007-01-01

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

  1. Observation of intracellular interactions between DNA origami and lysosomes by the fluorescence localization method.

    Science.gov (United States)

    Fu, Meifang; Dai, Luru; Jiang, Qiao; Tang, Yunqing; Zhang, Xiaoming; Ding, Baoquan; Li, Junbai

    2016-07-28

    We obtained the fluorescence localization images of tube DNA origami nanostructures in NIH 3T3 cells for the first time. The fluorescence localization images of tube DNA origami nanostructures and TIRF images of lysosomes were combined and they revealed the detailed interactions between the two structures. Quantitative analysis illustrated that the tube origami can be captured as well as degraded by lysosomes with time.

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

    Science.gov (United States)

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

    2010-07-15

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

  3. Atmospheric reactivity of alcohols, thiols and fluoroalcohols with chlorine atoms

    Science.gov (United States)

    Garzon Ruiz, Andres

    Alcohols, thiols and fluoroalcohols are volatile organic compounds (VOCs) which are emitted to the atmosphere from both natural (vegetation, oceans, volcanoes, etc.) and anthropogenic sources (fuels, solvents, wastewater, incinerators, refrigerants, etc.). These pollutants can be eliminated from the troposphere by deposition on the terrestrial surface, direct photolysis or reaction with different tropospheric oxidants. Reactions of VOCs with tropospheric oxidants are involved in the well-known atmospheric phenomenon of photochemical smog or the production of tropospheric ozone. The oxidation of these VOCs in the troposphere is mainly initiated by reaction with OH radicals during the daytime and with NO radicals at night. However, in recent years, the oxidation by chlorine atoms (Cl) has gained great importance in the study of atmospheric reactions because they may exert some influence in the boundary layer, particularly in marine and coastal environments. In general, Cl atoms are much more reactive species than OH and NO; radicals and therefore low concentrations of Cl may compete with OH and NO3 in hydrocarbon oxidation processes. The main source of tropospheric Cl atoms is believed to be the photolysis of chlorine-containing molecules generated by heterogeneous reactions of sea salt aerosols. It has also been proposed that Cl atoms, produced in the photolysis of Cl2 emitted from industrial processes, may enhance hydrocarbon oxidation rates and ozone production in urban environments. In this work, a kinetic, theoretical and mechanistic study of the reaction of several alcohols, thiols, and fluoroalcohols with Cl atoms has been carried out. Pulsed laser photolysis-fluorescence resonance (PLP-RF) technique was used for the kinetic study as a function of temperature and pressure. An environmental chamber-Fourier transform infrared (FTIR) system was also employed in the kinetic studies. Tropospheric lifetimes of these pollutants were estimated using obtained kinetic

  4. Acid Catalyzed Condensation of Phenylethanal Enol or Thiol Enol Ether to 2-Phenylnaphthalene

    Institute of Scientific and Technical Information of China (English)

    CHANG, Yu-An

    2007-01-01

    Treatment of enol ether or thiol enol ether of phenylethanals with sulfuric or polyphosphoric acid in toluene or xylene gave 2-phenylnaphthalene in good yield. More importantly, a one-pot reaction has been developed.

  5. AFM-assisted fabrication of thiol SAM pattern with alternating quantified surface potential

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    Simons Janet

    2011-01-01

    Full Text Available Abstract Thiol self-assembled monolayers (SAMs are widely used in many nano- and bio-technology applications. We report a new approach to create and characterize a thiol SAMs micropattern with alternating charges on a flat gold-coated substrate using atomic force microscopy (AFM and Kelvin probe force microscopy (KPFM. We produced SAMs-patterns made of alternating positively charged, negatively charged, and hydrophobic-terminated thiols by an automated AFM-assisted manipulation, or nanografting. We show that these thiol patterns possess only small topographical differences as revealed by AFM, and distinguished differences in surface potential (20-50 mV, revealed by KPFM. The pattern can be helpful in the development of biosensor technologies, specifically for selective binding of biomolecules based on charge and hydrophobicity, and serve as a model for creating surfaces with quantified alternating surface potential distribution.

  6. Flavin-catalyzed aerobic oxidation of sulfides and thiols with formic acid/triethylamine.

    Science.gov (United States)

    Murahashi, Shun-Ichi; Zhang, Dazhi; Iida, Hiroki; Miyawaki, Toshio; Uenaka, Masaaki; Murano, Kenji; Meguro, Kanji

    2014-09-14

    An efficient and practical catalytic method for the aerobic oxidative transformation of sulfides into sulfoxides, and thiols into disulfides with formic acid/TEA in the presence of a new, readily available, and stable flavin catalyst 5d is described.

  7. XAFS study on thiol etching of diphosphine-stabilized gold nanoclusters

    Science.gov (United States)

    Bao, Jie; Yang, Lina; Huang, Ting; Sun, Zhihu; Yao, Tao; Jiang, Yong; Wei, Shiqiang

    2017-08-01

    Thiol-etching triphenylphosphine (PPh3)-protected Au nanoclusters has been widely used to synthesize thiolated Au nanoclusters, while few studies have been reported on the thiol-etching reaction starting from diphosphine-protected Au clusters. Here the thiol-etching reaction in chloroform (CHCl3) for 1,5-Bis(diphenylphosphino) pentane (L5) protected Au11 nanoclusters is presented, and synchrotron radiation X-ray absorption fine structure, UV-vis absorption and mass spectra are combined to identify the reaction products. It is revealed that a gold(I)-thiolate complex Au2L5(RS) is produced, contrary to the case of thiol-etching PPh3-protected Au clusters where formation of thermodynamically stable Au25 or Au11 clusters is achieved.

  8. Preparation of reactive three-dimensional microstructures via direct laser writing and thiol-ene chemistry.

    Science.gov (United States)

    Quick, Alexander S; Fischer, Joachim; Richter, Benjamin; Pauloehrl, Thomas; Trouillet, Vanessa; Wegener, Martin; Barner-Kowollik, Christopher

    2013-02-25

    Three-dimensional microstructures are fabricated employing the direct laser writing process and radical thiol-ene polymerization. The resin system consists of a two-photon photoinitiator and multifunctional thiols and olefins. Woodpile photonic crystals with 22 layers and a rod distance of 2 μm are fabricated. The structures are characterized via scanning electron microscopy and focused ion beam milling. The thiol-ene polymerization during fabrication is verified via infrared spectroscopy. The structures are grafted in a subsequent thiol-Michael addition reaction with different functional maleimides. The success of the grafting reaction is evaluated via laser scanning microscopy and X-ray photoelectron spectroscopy. The grafting density is calculated to be close to 200 molecules μm(-2) .

  9. Controlling Topological Entanglement in Engineered Protein Hydrogels with a Variety of Thiol Coupling Chemistries

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    Shengchang eTang

    2014-05-01

    Full Text Available Topological entanglements between polymer chains are achieved in associating protein hydrogels through the synthesis of high molecular weight proteins via chain extension using a variety of thiol coupling chemistries, including disulfide formation, thiol-maleimide, thiol-bromomaleimide and thiol-ene. Coupling of cysteines via disulfide formation results in the most pronounced entanglement effect in hydrogels, while other chemistries provide versatile means of changing the extent of entanglement, achieving faster chain extension, and providing a facile method of controlling the network hierarchy and incorporating stimuli responsivities. The addition of trifunctional coupling agents causes incomplete crosslinking and introduces branching architecture to the protein molecules. The high-frequency plateau modulus and the entanglement plateau modulus can be tuned by changing the ratio of difunctional chain extender to the trifunctional branching unit. Therefore, these chain extension reactions show promise in delicately controlling the relaxation and mechanical properties of engineered protein hydrogels in ways that complement their design through genetic engineering.

  10. Novel pyrazoline-based fluorescent probe for detecting thiols and its application in cells

    Science.gov (United States)

    Zhang, Rong-Rong; Zhang, Jin-Feng; Wang, Sheng-Qing; Cheng, Yan-Long; Miao, Jun-Ying; Zhao, Bao-Xiang

    2015-02-01

    A new compound, N-(4-(1,5-diphenyl-4,5-dihydro-1H-pyrazol-3-yl)phenyl)-acrylamide (probe L), was designed and synthesized as a highly sensitive and selective fluorescent probe for recognizing and detecting thiol from other amino acids. On being mixed with thiol in buffered DMSO:HEPES = 1:1 solution at pH 7.4, the probe exhibited the blue emission at 474 nm. This probe is very sensitive and displayed a linear fluorescence off-on response to thiol. The fluorescence emission of the probe is pH independent in the physiological pH range. Living cell imaging of HeLa cells confirmed its cell permeability and its ability to selectively detect thiol in cells. The structure of the probe was characterized by IR, NMR and HRMS spectroscopy analysis.

  11. Interaction of arylsulfatase A with UDP-N-acetylglucosamine:Lysosomal enzyme-N-acetylglucosamine-1-phosphotransferase.

    Science.gov (United States)

    Schierau, A; Dietz, F; Lange, H; Schestag, F; Parastar, A; Gieselmann, V

    1999-02-05

    The critical step in lysosomal targeting of soluble lysosomal enzymes is the recognition by an UDP-N-acetylglucosamine:lysosomal enzyme-N-acetylglucosamine-1-phosphotransferase. The structure of the determinant common to all lysosomal enzymes for proper recognition by the phosphotransferase is not completely understood. Our current knowledge is largely based on the introduction of targeted amino acid substitutions into lysosomal enzymes and analysis of their effects on phosphotransferase recognition. We have investigated the effect of eight anti-arylsulfatase A monoclonal antibodies on the interaction of arylsulfatase A with the lysosomal enzyme phosphotransferase in vitro. We also show that a lysine-rich surface area of arylsulfatases A and B is essential for proper recognition by the phosphotransferase. Monoclonal antibodies bind to at least six different epitopes at different locations on the surface of arylsulfatase A. All antibodies bind outside the lysine-rich recognition area, but nevertheless Fab fragments of these antibodies prevent interaction of arylsulfatase A with the phosphotransferase. Our data support a model in which binding of arylsulfatase A to the phosphotransferase is not restricted to a limited surface area but involves the simultaneous recognition of large parts of arylsulfatase A.

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

    Science.gov (United States)

    Weiss, Norbert

    2012-03-01

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

  13. TMEM175 deficiency impairs lysosomal and mitochondrial function and increases α-synuclein aggregation

    Science.gov (United States)

    Jinn, Sarah; Drolet, Robert E.; Cramer, Paige E.; Wong, Andus Hon-Kit; Toolan, Dawn M.; Gretzula, Cheryl A.; Voleti, Bhavya; Vassileva, Galya; Disa, Jyoti; Tadin-Strapps, Marija; Stone, David J.

    2017-01-01

    Parkinson disease (PD) is a neurodegenerative disorder pathologically characterized by nigrostriatal dopamine neuron loss and the postmortem presence of Lewy bodies, depositions of insoluble α-synuclein, and other proteins that likely contribute to cellular toxicity and death during the disease. Genetic and biochemical studies have implicated impaired lysosomal and mitochondrial function in the pathogenesis of PD. Transmembrane protein 175 (TMEM175), the lysosomal K+ channel, is centered under a major genome-wide association studies peak for PD, making it a potential candidate risk factor for the disease. To address the possibility that variation in TMEM175 could play a role in PD pathogenesis, TMEM175 function was investigated in a neuronal model system. Studies confirmed that TMEM175 deficiency results in unstable lysosomal pH, which led to decreased lysosomal catalytic activity, decreased glucocerebrosidase activity, impaired autophagosome clearance by the lysosome, and decreased mitochondrial respiration. Moreover, TMEM175 deficiency in rat primary neurons resulted in increased susceptibility to exogenous α-synuclein fibrils. Following α-synuclein fibril treatment, neurons deficient in TMEM175 were found to have increased phosphorylated and detergent-insoluble α-synuclein deposits. Taken together, data from these studies suggest that TMEM175 plays a direct and critical role in lysosomal and mitochondrial function and PD pathogenesis and highlight this ion channel as a potential therapeutic target for treating PD. PMID:28193887

  14. Reduction of mutant huntingtin accumulation and toxicity by lysosomal cathepsins D and B in neurons

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    Ouyang Xiaosen

    2011-06-01

    Full Text Available Abstract Background Huntington's disease is caused by aggregation of mutant huntingtin (mHtt protein containing more than a 36 polyQ repeat. Upregulation of macroautophagy was suggested as a neuroprotective strategy to degrade mutant huntingtin. However, macroautophagy initiation has been shown to be highly efficient in neurons whereas lysosomal activities are rate limiting. The role of the lysosomal and other proteases in Huntington is not clear. Some studies suggest that certain protease activities may contribute to toxicity whereas others are consistent with protection. These discrepancies may be due to a number of mechanisms including distinct effects of the specific intermediate digestion products of mutant huntingtin generated by different proteases. These observations suggested a critical need to investigate the consequence of upregulation of individual lysosomal enzyme in mutant huntingtin accumulation and toxicity. Results In this study, we used molecular approaches to enhance lysosomal protease activities and examined their effects on mutant huntingtin level and toxicity. We found that enhanced expression of lysosomal cathepsins D and B resulted in their increased enzymatic activities and reduced both full-length and fragmented huntingtin in transfected HEK cells. Furthermore, enhanced expression of cathepsin D or B protected against mutant huntingtin toxicity in primary neurons, and their neuroprotection is dependent on macroautophagy. Conclusions These observations demonstrate a neuroprotective effect of enhancing lysosomal cathepsins in reducing mutant huntingtin level and toxicity in transfected cells. They highlight the potential importance of neuroprotection mediated by cathepsin D or B through macroautophagy.

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

    Science.gov (United States)

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

    2015-11-01

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

  16. Lysosomal interaction of Akt with Phafin2: a critical step in the induction of autophagy.

    Directory of Open Access Journals (Sweden)

    Mami Matsuda-Lennikov

    Full Text Available Autophagy is an evolutionarily conserved mechanism for the gross disposal of intracellular proteins in mammalian cells and dysfunction in this pathway has been associated with human disease. Although the serine threonine kinase Akt is suggested to play a role in this process, little is known about the molecular mechanisms by which Akt induces autophagy. Using a yeast two-hybrid screen, Phafin2 (EAPF or PLEKHF2, a lysosomal protein with a unique structure of N-terminal PH (pleckstrin homology domain and C-terminal FYVE (Fab 1, YOTB, Vac 1, and EEA1 domain was found to interact with Akt. A sucrose gradient fractionation experiment revealed that both Akt and Phafin2 co-existed in the same lysosome enriched fraction after autophagy induction. Confocal microscopic analysis and BiFC analysis demonstrated that both Akt and Phafin2 accumulate in the lysosome after induction of autophagy. BiFC analysis using PtdIns (3P interaction defective mutant of Phafin2 demonstrated that lysosomal accumulation of the Akt-Phafin2 complex and subsequent induction of autophagy were lysosomal PtdIns (3P dependent events. Furthermore, in murine macrophages, both Akt and Phafin2 were required for digestion of fluorescent bacteria and/or LPS-induced autophagy. Taken together, these findings establish that lysosomal accumulation of Akt and Phafin2 is a critical step in the induction of autophagy via an interaction with PtdIns (3P.

  17. Activation of the transcription factor EB rescues lysosomal abnormalities in cystinotic kidney cells.

    Science.gov (United States)

    Rega, Laura R; Polishchuk, Elena; Montefusco, Sandro; Napolitano, Gennaro; Tozzi, Giulia; Zhang, Jinzhong; Bellomo, Francesco; Taranta, Anna; Pastore, Anna; Polishchuk, Roman; Piemonte, Fiorella; Medina, Diego L; Catz, Sergio D; Ballabio, Andrea; Emma, Francesco

    2016-04-01

    Nephropathic cystinosis is a rare autosomal recessive lysosomal storage disease characterized by accumulation of cystine into lysosomes secondary to mutations in the cystine lysosomal transporter, cystinosin. The defect initially causes proximal tubular dysfunction (Fanconi syndrome) which in time progresses to end-stage renal disease. Cystinotic patients treated with the cystine-depleting agent, cysteamine, have improved life expectancy, delayed progression to chronic renal failure, but persistence of Fanconi syndrome. Here, we have investigated the role of the transcription factor EB (TFEB), a master regulator of the autophagy-lysosomal pathway, in conditionally immortalized proximal tubular epithelial cells derived from the urine of a healthy volunteer or a cystinotic patient. Lack of cystinosin reduced TFEB expression and induced TFEB nuclear translocation. Stimulation of endogenous TFEB activity by genistein, or overexpression of exogenous TFEB lowered cystine levels within 24 hours in cystinotic cells. Overexpression of TFEB also stimulated delayed endocytic cargo processing within 24 hours. Rescue of other abnormalities of the lysosomal compartment was observed but required prolonged expression of TFEB. These abnormalities could not be corrected with cysteamine. Thus, these data show that the consequences of cystinosin deficiency are not restricted to cystine accumulation and support the role of TFEB as a therapeutic target for the treatment of lysosomal storage diseases, in particular of cystinosis. Copyright © 2016 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

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

    Science.gov (United States)

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

    1989-02-01

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

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

    Science.gov (United States)

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

    2017-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2013-10-18

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

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

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    Bárbara Hissa

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

  2. The role of the thiol group in protein modification with methylglyoxal

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    JELENA M. AĆIMOVIĆ

    2009-08-01

    Full Text Available Methylglyoxal is a highly reactive α-oxoaldehyde with elevated production in hyperglycemia. It reacts with nucleophilic Lys and Arg side-chains and N-terminal amino groups causing protein modification. In the present study, the importance of the reaction of the Cys thiol group with methylglyoxal in protein modification, the competitiveness of this reaction with those of amino and guanidine groups, the time course of these reactions and their role and contribution to protein cross-linking were investigated. Human and bovine serum albumins were used as model systems. It was found that despite the very low levels of thiol groups on the surface of the examined protein molecules (approx. 80 times lower than those of amino and guanidino groups, a very high percentage of it reacts (25–85 %. The amount of reacted thiol groups and the rate of the reaction, the time for the reaction to reach equilibrium, the formation of a stable product and the contribution of thiol groups to protein cross-linking depend on the methylglyoxal concentration. The product formed in the reaction of thiol and an insufficient quantity of methylglyoxal (compared to the concentrations of the groups accessible for modification participates to a significant extent (4 % to protein cross-linking. Metformin applied in equimolar concentration with methylglyoxal prevents its reaction with amino and guanidino groups but, however, not with thiol groups.

  3. Direct observation of pentacene-thiol interaction using x-ray spectroscopy

    Science.gov (United States)

    Jia, Zhang; Lee, Vincent; Floreano, Luca; Verdini, Alberto; Cossaro, Albano; Morgante, Alberto; Kymissis, Ioannis

    2010-03-01

    There has been an intense interest in the surface modification of the source-drain electrodes for organic field effect transistors (OFETs) to improve their performance. A number of thiol based self assembled monolayers demonstrated improvements to the contact resistance and channel performance. Morphological improvements at the contacts, a change in the effective work function, and charge transfer between the thiols and the semiconductor have all been credited with the observed performance improvements. Using in-situ semiconductor deposition together with x-ray photoelectron spectroscopy and near-edge x-ray absorption fine structure, we are able to directly probe two technologically relevant OFET stacks. This work directly measures the interaction between pentacene and two thiols which have been associated to contact improvement: an electroneutral thiol (1-hexadecanethiol) and an electronegative thiol (pentafluorobenzenethiol). Based on our results we observe no chemical interaction between pentacene and the thiol. The electrical improvements to transistor performance, based on these systems, can be attributed to work function shifts of the contacts and morphological improvements of the organic semiconductor.

  4. Intercalation of gaseous thiols and sulfides into Ag+ ion-exchanged aluminum dihydrogen triphosphate.

    Science.gov (United States)

    Hayashi, Aki; Saimen, Hiroki; Watanabe, Nobuaki; Kimura, Hitomi; Kobayashi, Ayumi; Nakayama, Hirokazu; Tsuhako, Mitsutomo

    2005-08-02

    Ag(+) ion-exchanged layered aluminum dihydrogen triphosphate (AlP) with the interlayer distance of 0.85 nm was synthesized by the ion-exchange of proton in triphosphate with Ag(+) ion. The amount of exchanged Ag(+) ion depended on the concentration of AgNO(3) aqueous solution. Ag(+) ion-exchanged AlP adsorbed gaseous thiols and sulfides into the interlayer region. The adsorption amounts of thiols were more than those of sulfides, thiols with one mercapto group > thiol with two mercapto groups > sulfides, and depended on the amount of exchanged Ag(+) ion in the interlayer region. The thiols with one mercapto group were intercalated to expand the interlayer distance of Ag(+) ion-exchanged AlP, whereas there was no expansion in the adsorption of sulfide. In the case of thiol with two mercapto groups, there was observed contraction of the interlayer distance through the bridging with Ag(+) ions of the upper and lower sides of the interlayer region.

  5. Revisiting the reactions of superoxide with glutathione and other thiols.

    Science.gov (United States)

    Winterbourn, Christine C

    2016-04-01

    The reaction between GSH and superoxide has long been of interest in the free radical biology. Early studies were confusing, as some reports suggested that the reaction could be a major pathway for superoxide removal whereas others questioned whether it happened at all. Further research by several investigators, including Helmut Sies, was required to clarify this complex reaction. We now know that superoxide does react with GSH, but the reaction is relatively slow and occurs mostly by a chain reaction that consumes oxygen and regenerates superoxide. Most of the GSH is converted to GSSG, with a small amount of sulfonic acid. As shown by Sies and colleagues, singlet oxygen is a by-product. Although removal of superoxide by GSH may be a minor pathway, GSH and superoxide have a strong physiological connection. GSH is an efficient free radical scavenger, and when it does so, thiyl radicals are generated. These further react to generate superoxide. Therefore, radical scavenging by GSH and other thiols is a source of superoxide and hydrogen peroxide, and to be an antioxidant pathway, there must be efficient removal of these species.

  6. Thiol passivation of MWIR type II superlattice photodetectors

    Science.gov (United States)

    Salihoglu, O.; Muti, A.; Aydinli, A.

    2013-06-01

    Poor passivation on photodetectors can result in catastrophic failure of the device. Abrupt termination of mesa side walls during pixel definition generates dangling bonds that lead to inversion layers and surface traps leading to surface leakage currents that short circuit diode action. Good passivation, therefore, is critical in the fabrication of high performance devices. Silicondioxide has been the main stay of passivation for commercial photodetectors, deposited at high temperatures and high RF powers using plasma deposition techniques. In photodetectors based on III-V compounds, sulphur passivation has been shown to replace oxygen and saturate the dangling bonds. Despite its effectiveness, it degrades over time. More effort is required to create passivation layers which eliminate surface leakage current. In this work, we propose the use of sulphur based octadecanethiol (ODT), CH3(CH2)17SH, as a passivation layer for the InAs/GaSb superlattice photodetectors that acts as a self assembled monolayer (SAM). ODT SAMs consist of a chain of 18 carbon atoms with a sulphur atom at its head. ODT Thiol coating is a simple process that consist of dipping the sample into the solution for a prescribed time. Excellent electrical performance of diodes tested confirm the effectiveness of the sulphur head stabilized by the intermolecular interaction due to van der Walls forces between the long chains of ODT SAM which results in highly stable ultrathin hydrocarbon layers without long term degradation.

  7. Cytokinin Determines Thiol-Mediated Arsenic Tolerance and Accumulation.

    Science.gov (United States)

    Mohan, Thotegowdanapalya C; Castrillo, Gabriel; Navarro, Cristina; Zarco-Fernández, Sonia; Ramireddy, Eswarayya; Mateo, Cristian; Zamarreño, Angel M; Paz-Ares, Javier; Muñoz, Riansares; García-Mina, Jose M; Hernández, Luis E; Schmülling, Thomas; Leyva, Antonio

    2016-06-01

    The presence of arsenic in soil and water is a constant threat to plant growth in many regions of the world. Phytohormones act in the integration of growth control and stress response, but their role in plant responses to arsenic remains to be elucidated. Here, we show that arsenate [As(V)], the most prevalent arsenic chemical species in nature, causes severe depletion of endogenous cytokinins (CKs) in the model plant Arabidopsis (Arabidopsis thaliana). We found that CK signaling mutants and transgenic plants with reduced endogenous CK levels showed an As(V)-tolerant phenotype. Our data indicate that in CK-depleted plants exposed to As(V), transcript levels of As(V)/phosphate-transporters were similar or even higher than in wild-type plants. In contrast, CK depletion provoked the coordinated activation of As(V) tolerance mechanisms, leading to the accumulation of thiol compounds such as phytochelatins and glutathione, which are essential for arsenic sequestration. Transgenic CK-deficient Arabidopsis and tobacco lines show a marked increase in arsenic accumulation. Our findings indicate that CK is an important regulatory factor in plant adaptation to arsenic stress.

  8. Processing and targeting of the thiol protease, aleurain

    Energy Technology Data Exchange (ETDEWEB)

    Rogers, J.C.

    1989-01-01

    We have identified a cDNA clone from barley aleurone mRNA that encodes a protein with unusual homologies: the C-terminal portion, about 270 amino acids, is 65% identical to the mammalian thiol protease, cathepsin H. This degree of sequence conservation indicates that the enzyme must have some specific function in both plants and mammals that cannot tolerate further divergence. The N-terminal 1/3 of the protein, about 140 amino acids, has no detectable homologies to other known protein sequences; its function is unknown. In aleurone tissue, the mRNA level is increased by gibberellic acid and decreased by abscisic acid, but is expressed apparently constitutively at high levels in leaf and root tissues. The amino acid sequence and cathepsin H homology suggest that the protein will be both secreted into the endoplasmic reticulum and glycosylated. Using our cDNA clone in a bacterial expression system, we have made a fusion protein containing the protease domain of aleurain, and have used it to raise specific antisera in rabbits. These antibodies identify a 32 kd protein in extracts of aleurone layers that is induced with GA treatment but not secreted; a similarly sized protein is specifically identified in extracts of leaf tissue. Experiments are underway to characterize the pattern of expression in different tissues, to identify the subcellular locations of the protein, to characterize processing of the precursor to the 32 kd mature form, and to purify the enzyme from barley. 2 figs.

  9. (Processing and targeting of the thiol protease aleurain)

    Energy Technology Data Exchange (ETDEWEB)

    Rogers, J.C.

    1990-01-01

    Our goal for work during the past two years under this Grant was to characterize the barley thiol protease, aleurain, to determine if it is secreted or retained intracellularly in aleurone cells, and to begin to elucidate structural features that might control targeting of the protein to its final destination. We have shown that aleurain is synthesized as a proenzyme with two N-linked oligosaccharide chains, one high mannose-type and one complex-type. Aleurain undergoes processing to mature form by removal of an Nterminal prosegment, and is retained intracellularly; it cannot be detected among proteins secreted from aleurone cells. Treatment of aleurone cells with tunicamycin to prevent glycosylation of aleurain does not prevent processing of the unglycosylated form. The N-terminal portion of aleurain's prosegment is homologous to the comparable region in two yeast vacuolar proteases, where that region is known to contain the signal necessary for targeting the proteases to the vacuole. 18 refs., 7 figs.

  10. Tip-enhanced Raman spectroscopic imaging of patterned thiol monolayers

    Directory of Open Access Journals (Sweden)

    Johannes Stadler

    2011-08-01

    Full Text Available Full spectroscopic imaging by means of tip-enhanced Raman spectroscopy (TERS was used to measure the distribution of two isomeric thiols (2-mercaptopyridine (2-PySH and 4-mercaptopyridine (4-PySH in a self-assembled monolayer (SAM on a gold surface. From a patterned sample created by microcontact printing, an image with full spectral information in every pixel was acquired. The spectroscopic data is in good agreement with the expected molecular distribution on the sample surface due to the microcontact printing process. Using specific marker bands at 1000 cm−1 for 2-PySH and 1100 cm−1 for 4-PySH, both isomers could be localized on the surface and semi-quantitative information was deduced from the band intensities. Even though nanometer size resolution information was not required, the large signal enhancement of TERS was employed here to detect a monolayer coverage of weakly scattering analytes that were not detectable with normal Raman spectroscopy, emphasizing the usefulness of TERS.

  11. Oligomerization of Indole Derivatives with Incorporation of Thiols

    Directory of Open Access Journals (Sweden)

    Jarl E.S. Wikberg

    2008-08-01

    Full Text Available Abstract: Two molecules of indole derivative, e.g. indole-5-carboxylic acid, reacted with one molecule of thiol, e.g. 1,2-ethanedithiol, in the presence of trifluoroacetic acid to yield adducts such as 3-[2-(2-amino-5-carboxyphenyl-1-(2-mercaptoethylthioethyl]-1Hindole-5-carboxylic acid. Parallel formation of dimers, such as 2,3-dihydro-1H,1'H-2,3'-biindole-5,5'-dicarboxylic acid and trimers, such as 3,3'-[2-(2-amino-5-carboxyphenyl ethane-1,1-diyl]bis(1H-indole-5-carboxylic acid of the indole derivatives was also observed. Reaction of a mixture of indole and indole-5-carboxylic acid with 2-phenylethanethiol proceeded in a regioselective way, affording 3-[2-(2-aminophenyl-1-(phenethylthioethyl]-1H-indole-5-carboxylic acid. An additional product of this reaction was 3-[2-(2-aminophenyl-1-(phenethylthioethyl]-2,3-dihydro-1H,1'H-2,3'-biindole-5'-carboxylic acid, which upon standing in DMSO-d6 solution gave 3-[2-(2-aminophenyl-1-(phenethylthioethyl]-1H,1'H-2,3'-biindole-5'-carboxylic acid. Structures of all compounds were elucidated by NMR, and a mechanism for their formation was suggested.

  12. Lysosomal {beta}-mannosidase: cDNA cloning and characterization

    Energy Technology Data Exchange (ETDEWEB)

    Chen, H.; Leipprandt, J.R.; Traviss, C.E. [Michigan State Univ., East Lansing, MI (United States)] [and others

    1994-09-01

    Lysosomal {beta}-mannosidase is an exoglycosidase that cleaves the single {beta}-linked mannose residue from the non-reducing end of all N-linked glycoprotein oligosaccharides. Deficiency of this enzyme results in {beta}-mannosidosis, a severe neurodegenerative disease in goats and cattle. The human cases described have a milder, highly variable presentation. Study of the molecular pathology of this disease in ruminants and humans and development of the animal model for gene therapy studies required cloning of the gene for {beta}-mannosidase has been cloned. {beta}-Mannosidase cDNA were obtained from a bovine thyroid cDNA library by screening with mixed oligonucleotides derived from peptide sequences resulting from microsequencing of bovine {beta}-mannosidase peptides. A total of six independent positive clones were identified from 5 x 10{sup 5} plaques, covering about 80% of the C-terminal region. The missing 5{prime} region was obtained using 5{prime} RACE. The full-length construct contains 3852-bp nucleotides, encoding 879 amino acids. The initiation codon is followed by 17 amino acids containing the characteristics of a typical signal peptide sequence. The deduced amino acid sequence is colinear with all peptide sequences determined by protein microsequencing. Northern blot analysis demonstrated a 4.2 kb single transcript in various tissues from both normal and affected goats and calves. The mRNA level was decreased in affected {beta}-mannosidosis animals. The gene encoding {beta}-mannosidase was localized on human chromosome 4 by Southern analysis of rodent/human somatic cell hybrids. The mutation in bovine {beta}-mannosidosis has been identified. This is the first report of cloning of the {beta}-mannosidase gene.

  13. Size-controlled silver nanoparticles stabilized on thiol-functionalized MIL-53(Al) frameworks

    Science.gov (United States)

    Cheng, Xinquan; Liu, Min; Zhang, Anfeng; Hu, Shen; Song, Chunshan; Zhang, Guoliang; Guo, Xinwen

    2015-05-01

    A postsynthetic modification method was used to prepare thiol-functionalized metal-organic frameworks (MOFs) by the amidation of mercaptoacetic acid with the amine group, which is present in the frameworks of NH2-MIL-53(Al). By doing this, the thiol group has been successfully grafted on the MOF, which perfectly combined the highly developed pore structures of the MOF with the strong coordination ability of the thiol group. The resulting thiol-functionalized MIL-53(Al) showed a significantly high adsorption capacity for heavy metal ions like Ag+ (182.8 mg g-1). Even more importantly, these grafted thiol groups can be used as anchoring groups for stabilizing metal nanoparticles (NPs) with controllable sizes. Taking silver as an example, monodispersed Ag NPs encapsulated in the cages of MIL-53(Al) have been prepared by using a two-step procedure. In addition, the particle size of the Ag NPs was adjustable to some extent by controlling the initial loading amount. The average size of the smallest Ag NPs is 3.9 +/- 0.9 nm, which is hard to obtain for Ag NPs because of their strong tendency to aggregate.A postsynthetic modification method was used to prepare thiol-functionalized metal-organic frameworks (MOFs) by the amidation of mercaptoacetic acid with the amine group, which is present in the frameworks of NH2-MIL-53(Al). By doing this, the thiol group has been successfully grafted on the MOF, which perfectly combined the highly developed pore structures of the MOF with the strong coordination ability of the thiol group. The resulting thiol-functionalized MIL-53(Al) showed a significantly high adsorption capacity for heavy metal ions like Ag+ (182.8 mg g-1). Even more importantly, these grafted thiol groups can be used as anchoring groups for stabilizing metal nanoparticles (NPs) with controllable sizes. Taking silver as an example, monodispersed Ag NPs encapsulated in the cages of MIL-53(Al) have been prepared by using a two-step procedure. In addition, the particle

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

    Science.gov (United States)

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

    2013-01-01

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

  15. DRAM1 regulates apoptosis through increasing protein levels and lysosomal localization of BAX

    Science.gov (United States)

    Guan, J-J; Zhang, X-D; Sun, W; Qi, L; Wu, J-C; Qin, Z-H

    2015-01-01

    DRAM1 (DNA damage-regulated autophagy modulator 1) is a TP53 target gene that modulates autophagy and apoptosis. We previously found that DRAM1 increased autophagy flux by promoting lysosomal acidification and protease activation. However, the molecular mechanisms by which DRAM1 regulates apoptosis are not clearly defined. Here we report a novel pathway by which DRAM1 regulates apoptosis involving BAX and lysosomes. A549 or HeLa cells were treated with the mitochondrial complex II inhibitor, 3-nitropropionic acid (3NP), or an anticancer drug, doxorubicin. Changes in the protein and mRNA levels of BAX and DRAM1 and the role of DRAM1 in BAX induction were determined. The interaction between DRAM1 and BAX and its effect on BAX degradation, BAX lysosomal localization, the release of cathepsin B and cytochrome c by BAX and the role of BAX in 3NP- or doxorubicin-induced cell death were studied. The results showed that BAX, a proapoptotic protein, was induced by DRAM1 in a transcription-independent manner. BAX was degraded by autophagy under basal conditions; however, its degradation was inhibited when DRAM1 expression was induced. There was a protein interaction between DRAM1 and BAX and this interaction prolonged the half-life of BAX. Furthermore, upregulated DRAM1 recruited BAX to lysosomes, leading to the release of lysosomal cathepsin B and cleavage of BID (BH3-interacting domain death agonist). BAX mediated the release of mitochondrial cytochrome c, activation of caspase-3 and cell death partially through the lysosome-cathepsin B-tBid pathway. These results indicate that DRAM1 regulates apoptosis by inhibiting BAX degradation. In addition to mitochondria, lysosomes may also be involved in BAX-initiated apoptosis. PMID:25633293

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

    Science.gov (United States)

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

    2015-09-01

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

  17. Interactions between autophagic and endo-lysosomal markers in endothelial cells.

    Science.gov (United States)

    Oeste, Clara L; Seco, Esther; Patton, Wayne F; Boya, Patricia; Pérez-Sala, Dolores

    2013-05-01

    Autophagic and endo-lysosomal degradative pathways are essential for cell homeostasis. Availability of reliable tools to interrogate these pathways is critical to unveil their involvement in physiology and pathophysiology. Although several probes have been recently developed to monitor autophagic or lysosomal compartments, their specificity has not been validated through co-localization studies with well-known markers. Here, we evaluate the selectivity and interactions between one lysosomal (Lyso-ID) and one autophagosomal (Cyto-ID) probe under conditions modulating autophagy and/or endo-lysosomal function in live cells. The probe for acidic compartments Lyso-ID was fully localized inside vesicles positive for markers of late endosome-lysosomes, including Lamp1-GFP and GFP-CINCCKVL. Induction of autophagy by amino acid deprivation in bovine aortic endothelial cells caused an early and potent increase in the fluorescence of the proposed autophagy dye Cyto-ID. Cyto-ID-positive compartments extensively co-localized with the autophagosomal fluorescent reporter RFP-LC3, although the time and/or threshold for organelle detection was different for each probe. Interestingly, use of Cyto-ID in combination with Lysotracker Red or Lyso-ID allowed the observation of structures labeled with either one or both probes, the extent of co-localization increasing upon treatment with protease inhibitors. Inhibition of the endo-lysosomal pathway with chloroquine or U18666A resulted in the formation of large Cyto-ID and Lyso-ID-positive compartments. These results constitute the first assessment of the selectivity of Cyto-ID and Lyso-ID as probes for the autophagic and lysosomal pathways, respectively. Our observations show that these probes can be used in combination with protein-based markers for monitoring the interactions of both pathways in live cells.

  18. Thiol-ene immobilisation of carbohydrates onto glass slides as a simple alternative to gold-thiol monolayers, amines or lipid binding.

    Science.gov (United States)

    Biggs, Caroline I; Edmondson, Steve; Gibson, Matthew I

    2015-01-01

    Carbohydrate arrays are a vital tool in studying infection, probing the mechanisms of bacterial, viral and toxin adhesion and the development of new treatments, by mimicking the structure of the glycocalyx. Current methods rely on the formation of monolayers of carbohydrates that have been chemically modified with a linker to enable interaction with a functionalised surface. This includes amines, biotin, lipids or thiols. Thiol-addition to gold to form self-assembled monolayers is perhaps the simplest method for immobilisation as thiolated glycans are readily accessible from reducing carbohydrates in a single step, but are limited to gold surfaces. Here we have developed a quick and versatile methodology which enables the use of thiolated carbohydrates to be immobilised as monolayers directly onto acrylate-functional glass slides via a 'thiol-ene'/Michael-type reaction. By combining the ease of thiol chemistry with glass slides, which are compatible with microarray scanners this offers a cost effective, but also useful method to assemble arrays.

  19. A Study of Functional Polymer Colloids Prepared Using Thiol-Ene/Yne Click Chemistry

    Science.gov (United States)

    Durham, Olivia Z.

    This project demonstrates the first instance of thiol-ene chemistry as the polymerization method for the production of polymer colloids in two-phase heterogeneous suspensions, miniemulsions, and emulsions. This work was also expanded to thiol-yne chemistry for the production of polymer particles containing increased crosslinking density. The utility of thiol-ene and thiol-yne chemistries for polymerization and polymer modification is well established in bulk systems. These reactions are considered 'click' reactions, which can be defined as processes that are both facile and simple, offering high yields with nearly 100% conversion, no side products, easy product separation, compatibility with a diverse variety of commercially available starting materials, and orthogonality with other chemistries. In addition, thiol-ene and thiol-yne chemistry follow a step-growth mechanism for the development of highly uniform polymer networks, where polymer growth is dependent on the coupling of functional groups. These step-growth polymerization systems are in stark contrast to the chain-growth mechanisms of acrylic and styrenic monomers that have dominated the field of conventional heterogeneous polymerizations. Preliminary studies evaluated the mechanism of particle production in suspension and miniemulsion systems. Monomer droplets were compared to the final polymer particles to confirm that particle growth occurred through the polymerization of monomer droplets. Additional parameters examined include homogenization energy (mechanical mixing), diluent species and concentration, and monomer content. These reactions were conducted using photoinitiation to yield particles in a matter of minutes with diameters in the size range of several microns to hundreds of microns in suspensions or submicron particles in miniemulsions. Improved control over the particle size and size distribution was examined through variation of reaction parameters. In addition, a method of seeded suspension

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

    Science.gov (United States)

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

    2011-01-01

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

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

    DEFF Research Database (Denmark)

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

    2010-01-01

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

  2. Plastid-Localized Glutathione Reductase2–Regulated Glutathione Redox Status Is Essential for Arabidopsis Root Apical Meristem Maintenance[C][W

    Science.gov (United States)

    Yu, Xin; Pasternak, Taras; Eiblmeier, Monika; Ditengou, Franck; Kochersperger, Philip; Sun, Jiaqiang; Wang, Hui; Rennenberg, Heinz; Teale, William; Paponov, Ivan; Zhou, Wenkun; Li, Chuanyou; Li, Xugang; Palme, Klaus

    2013-01-01

    Glutathione is involved in thiol redox signaling and acts as a major redox buffer against reactive oxygen species, helping to maintain a reducing environment in vivo. Glutathione reductase (GR) catalyzes the reduction of glutathione disulfide (GSSG) into reduced glutathione (GSH). The Arabidopsis thaliana genome encodes two GRs: GR1 and GR2. Whereas the cytosolic/peroxisomal GR1 is not crucial for plant development, we show here that the plastid-localized GR2 is essential for root growth and root apical meristem (RAM) maintenance. We identify a GR2 mutant, miao, that displays strong inhibition of root growth and severe defects in the RAM, with GR activity being reduced to ∼50%. miao accumulates high levels of GSSG and exhibits increased glutathione oxidation. The exogenous application of GSH or the thiol-reducing agent DTT can rescue the root phenotype of miao, demonstrating that the RAM defects in miao are triggered by glutathione oxidation. Our in silico analysis of public microarray data shows that auxin and glutathione redox signaling generally act independently at the transcriptional level. We propose that glutathione redox status is essential for RAM maintenance through both auxin/PLETHORA (PLT)-dependent and auxin/PLT-independent redox signaling pathways. PMID:24249834

  3. Regulation of ribonucleotide reductase by Spd1 involves multiple mechanisms

    DEFF Research Database (Denmark)

    Nestoras, Konstantinos; Mohammed, Asma Hadi; Schreurs, Ann-Sofie

    2010-01-01

    The correct levels of deoxyribonucleotide triphosphates and their relative abundance are important to maintain genomic integrity. Ribonucleotide reductase (RNR) regulation is complex and multifaceted. RNR is regulated allosterically by two nucleotide-binding sites, by transcriptional control, and...

  4. Cyclic Voltammetric Responses of Nitrate Reductase on Chemical Modified Electrodes

    Institute of Scientific and Technical Information of China (English)

    YaRuSONG; HuiBoSHAO; 等

    2002-01-01

    Electrochemistry of nitrate reductases (NR) incorporated into 2-aminoethanethiol self-assembled on the gold electrode and polyacrylamide cast on the pyrolytic graphite electrode was examined. NR on chemical modified electrode showed electrochemical cyclic voltammetric responses in phosphate buffers.

  5. Enantioselective imine reduction catalyzed by imine reductases and artificial metalloenzymes.

    Science.gov (United States)

    Gamenara, Daniela; Domínguez de María, Pablo

    2014-05-21

    Adding value to organic synthesis. Novel imine reductases enable the enantioselective reduction of imines to afford optically active amines. Likewise, novel bioinspired artificial metalloenzymes can perform the same reaction as well. Emerging proof-of-concepts are herein discussed.

  6. Uptake of mercury by thiol-grafted chitosan gel beads.

    Science.gov (United States)

    Merrifield, John D; Davids, William G; MacRae, Jean D; Amirbahman, Aria

    2004-07-01

    This study describes the synthesis and characterization of thiol-grafted chitosan beads for use as mercury (Hg) adsorbents. Chitosan flakes were dissolved and formed into spherical beads using a phase inversion technique, then crosslinked to improve their porosity and chemical stability. Cysteine was grafted onto the beads in order to improve the adsorption affinity of Hg to the beads. The beads possessed an average diameter of 3.2 mm, porosity of 0.9, specific surface area of approximately 100 m2/g, average pore size of approximately 120 angstroms, and specific gravity of 2.0. Equilibrium and kinetic uptake experiments were conducted to study the uptake of Hg by the beads. The adsorption capacity was approximately 8.0 mmol-Hg/g-dry beads at pH 7, and decreased with decreasing pH. Hg adsorption kinetics was modeled as radial pore diffusion into a spherical bead with nonlinear adsorption. Use of the nonlinear Freundlich isotherm in the diffusion equation allowed modeling of the uptake kinetics with a single tortuosity factor of 1.5 +/- 0.3 as the fitting parameter for all initial Hg concentrations, chitosan loadings, and agitation rates. At agitation rates of 50 and 75 rpm, where uptake rate was reduced significantly due to the boundary layer effect, the mass transfer coefficient at the outside boundary was also used as a fitting parameter to model the kinetic data. At agitation rates higher than 150 rpm, pore diffusion was the rate-limiting step. The beads exhibited a high initial uptake rate followed by a slower uptake rate suggesting pore diffusion as the rate-determining step especially at high agitation rates. Higher uptake rates observed in this study compared to those in a previous study of chitosan-based crab shells indicate that dissolution and gel formation increase the porosity and pore accessibility of chitosan.

  7. Infection free titanium alloys by stabile thiol based nanocoating.

    Science.gov (United States)

    Cökeliler, Dilek; Göktaş, Hilal; Tosun, Pinar Deniz; Mutlu, Selma

    2010-04-01

    As biomedical materials, titanium and titanium alloys (Ti-6Al-4V) are superior to many materials in terms of mechanical properties and biocompatibility. However, they are still not sufficient for prolonged clinical use because the biocompatibility of these materials must be improved. In this study, the prevention of the attachment of test microorganism on the Ti alloy surfaces by thiol (-SH) and hydroxyl (-OH) functional group containing monomer in plasma based electron beam generator was reported in order to prepare anti-fouling surfaces. The precursor, 11-mercaptoundecanoic acid is used as plasma source to create nano-film with 30-60 nm approximately. The surface chemistry and topology of uncoated and coated samples are characterized by Fourier Transform Infrared Spectroscopy (FTIR) and Atomic Force Microscopy (AFM). Static contact angle measurements are performed to state the change of surface hydrophilicity. All coated samples are tested in-vitro environment with Staphylococcus epidermidis that is chosen as the test bacteria strain in view of its significance for the pathogenesis of medical-device-related infections. This test is repeated after certain period of times and samples are waited in dynamic fluid media in order to investigate the stability of nano-coating. Plasma polymerized 11-mercaptoundecanoic acid film (PP MUA) with 42 +/- 4 nm is found alternative, stabile and simple method to create bacterial anti-fouling surfaces. The static contact angle of the coated surface is 34 +/- 80 whereas the uncoated surface is 57 +/- 50. For the coated surface, the presence of C-OH and C==O groups in infrared spectra defining the PP MUA is achieved by the plasma polymerization. The attachment of the model microorganism on the biomaterial surface prepared by PP MUA is reduced 85.3% if compared to unmodified control surface.

  8. Comparative evaluation of antioxidant capacities of thiol-based antioxidants measured by different in vitro methods.

    Science.gov (United States)

    Güngör, Nilay; Ozyürek, Mustafa; Güçlü, Kubilay; Cekiç, Sema Demirci; Apak, Reşat

    2011-02-15

    Thiol-type compounds are an important class of strong antioxidants and main determinants of total antioxidant capacity (TAC) of cellular homogenates. The TAC of thiol mixtures and the corresponding TEAC (trolox equivalent antioxidant capacity) values of individual thiols were determined by the CUPRAC (CUPric Reducing Antioxidant Capacity) method, and the results were compared with those found by reference assays for method validation. Synthetic mixtures of thiols were prepared, and the expected and found TAC values (in mM trolox (TR) equivalents) of these mixtures showed a good agreement. The technique of standard additions was performed for thiol mixtures and human serum, and the absorbance results confirmed that apparent chemical deviations from Beer's law were absent in the system. The CUPRAC results were compared with those of reference methods, namely 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS)/persulphate and Ferric Reducing Antioxidant Power (FRAP). As being a most important thiol (-SH) peptide at in vivo conditions, glutathione (GSH) showed a TEAC value of 0.57 in the CUPRAC method, as opposed to the corresponding value (1.51) in the ABTS/persulphate method. The ABTS/persulphate result was not in accordance with the reversible 1-e oxidation of GSH to the corresponding disulfide that is expected to occur under physiological conditions. FRAP did not give consistent results, and even at relatively high concentrations of GSH, the TEAC(FRAP) value was only 0.07. The thiol-type antioxidant-bearing pharmaceuticals of Brunac eye drop, Trom and Mentopin effervescent tablets containing N-acetyl-L-cysteine (NAC) were assayed with HPLC for comparison, and the obtained results for NAC were in accordance with those found with CUPRAC.

  9. Electrochemistry behavior of endogenous thiols on fluorine doped tin oxide electrodes

    Energy Technology Data Exchange (ETDEWEB)

    Rojas, Luciana; Molero, Leonard; Tapia, Ricardo A.; Rio, Rodrigo del; Valle, M. Angelica del; Antilen, Monica [Departamento de Quimica Inorganica, Facultad de Quimica, Pontificia Universidad Catolica de Chile, Av Vicuna Mackenna 4860, Casilla 306, Correo 22, Macul, Santiago (Chile); Armijo, Francisco, E-mail: jarmijom@uc.cl [Departamento de Quimica Inorganica, Facultad de Quimica, Pontificia Universidad Catolica de Chile, Av Vicuna Mackenna 4860, Casilla 306, Correo 22, Macul, Santiago (Chile)

    2011-10-01

    Highlights: > The first time that fluorine doped tin oxide electrodes are used for the electrooxidation of endogenous thiols. > Low potentials of electrooxidation were obtained for the different thiols. > The electrochemical behavior of thiols depends on the pH and the ionic electroactive species, the electrooxidation proceeds for a process of adsorption of electroactive species on FTO and high values the heterogeneous electron tranfer rate constant of the reaction were obtained. - Abstract: In this work the electrochemical behavior of different thiols on fluorine doped tin oxide (FTO) electrodes is reported. To this end, the mechanism of electrochemical oxidation of glutathione (GSH), cysteine (Cys), homocysteine (HCys) and acetyl-cysteine (ACys) at different pH was investigated. FTO showed electroactivity for the oxidation of the first three thiols at pH between 2.0 and 4.0, but under these conditions no acetyl-cysteine oxidation was observed on FTO. Voltammetric studies of the electro-oxidation of GSH, Cys and HCys showed peaks at about 0.35, 0.29, and 0.28 V at optimum pH 2.4, 2.8 and 3.4, respectively. In addition, this study demonstrated that GSH, Cys and HCys oxidation occurs when the zwitterion is the electro-active species that interact by adsorption on FTO electrodes. The overall reaction involves 4e{sup -}/4H{sup +} and 2e{sup -}/2H{sup +}, respectively, for HCys and for GSH and Cys and high heterogeneous electron transfer rate constants. Besides, the use of FTO for the determination of different thiols was evaluated. Experimental square wave voltammetry shows a linear current vs. concentrations response between 0.1 and 1.0 mM was found for HCys and GSH, indicating that these FTO electrodes are promising candidates for the efficient electrochemical determination of these endogenous thiols.

  10. Purification and characterization of assimilatory nitrite reductase from Candida utilis.

    Science.gov (United States)

    Sengupta, S; Shaila, M S; Rao, G R

    1996-07-01

    Nitrate assimilation in many plants, algae, yeasts and bacteria is mediated by two enzymes, nitrate reductase (EC 1.6.6.2) and nitrite reductase (EC 1.7.7.1). They catalyse the stepwise reduction of nitrate to nitrite and nitrite to ammonia respectively. The nitrite reductase from an industrially important yeast, Candida utilis, has been purified to homogeneity. Purified nitrite reductase is a heterodimer and the molecular masses of the two subunits are 58 and 66 kDa. The native enzyme exhibits a molecular mass of 126 kDa as analysed by gel filtration. The identify of the two subunits of nitrite reductase was confirmed by immunoblotting using antibody for Cucurbita pepo leaf nitrite reductase. The presence of two different sized transcripts coding for the two subunits was confirmed by (a) in vitro translation of mRNA from nitrate-induced C. utilis followed by immunoprecipitation of the in vitro translated products with heterologous nitrite reductase antibody and (b) Northern-blot analysis. The 66 kDa subunit is acidic in nature which is probably due to its phosphorylated status. The enzyme is stable over a range of temperatures. Both subunits can catalyse nitrite reduction, and the reconstituted enzyme, at a higher protein concentration, shows an activity similar to that of the purified enzyme. Each of these subunits has been shown to contain a few unique peptides in addition to a large number of common peptides. Reduced Methyl Viologen has been found to be as effective an electron donor as NADPH in the catalytic process, a phenomenon not commonly seen for nitrite reductases from other systems.

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

    Directory of Open Access Journals (Sweden)

    Edward R. Bastow

    2012-02-01

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

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

    Science.gov (United States)

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

    2017-02-23

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

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

    Science.gov (United States)

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

    2016-10-15

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

  14. Induction, adaptation and recovery of lysosomal integrity in green-lipped mussel Perna viridis.

    Science.gov (United States)

    Fang, J K H; Wu, R S S; Zheng, G J; Lam, P K S; Shin, P K S

    2008-01-01

    Biomarkers are generally applied to detect pollution in environmental monitoring. Such biological responses should accurately reflect the stress over time in a quantitative manner. As such, the initial and maximum responses induced by stress, as well as adaptation and recovery of these biomarkers, need to be fully understood or else erroneous false-negative or false-positive may be arrived. However, most of the biomarker studies only provided information on initially induced responses under different concentrations of toxicants, while biological adaptation and recovery were poorly known. In this study, the time required for induction, adaptation and recovery of lysosomal integrity in green-lipped mussel Perna viridis upon exposure to benzo[a]pyrene was investigated over a period of 62 days. Maximum induction occurred on day 6 when lysosomal integrity was significantly reduced by 51%, and no further change or adaptation was detected thereafter. When mussels were depurated in clean seawater after 18 days of exposure to benzo[a]pyrene, a gradual recovery was observed, with lysosomal integrity returning to its background level and showing a complete recovery after 20 days of depuration. Lysosomal integrity was significantly correlated with the body burden concentrations of benzo[a]pyrene and condition index of the mussels. The relatively fast induction (6 days) and recovery (20 days) without apparent adaptation suggested that lysosomal integrity in P. viridis can serve as a good biomarker in biomonitoring, as its response is not likely to generate both false-negative and false-positive results.

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

    Science.gov (United States)

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

    2015-05-01

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

  16. Color reduction of melanin by lysosomal and peroxisomal enzymes isolated from mammalian cells.

    Science.gov (United States)

    Park, Dong Jun; Sekhon, Simranjeet Singh; Yoon, Jihee; Kim, Yang-Hoon; Min, Jiho

    2016-02-01

    Lysosomes and peroxisomes are organelles with many functions in all eukaryotic cells. Lysosomes contain hydrolytic enzymes (lysozyme) that degrade molecules, whereas peroxisomes contain enzymes such as catalase that convert hydrogen peroxide (H2O2) to water and oxygen and neutralize toxicity. In contrast, melanin is known as a helpful element to protect the skin against harmful ultraviolet rays. However, a high quantity of melanin leads to hyperpigmentation or skin cancer in human. New materials have already been discovered to inhibit tyrosinase in melanogenesis; however, melanin reduction does not suggest their preparation. In this study, we report that the color intensity because of melanin decreased by the cellular activation of lysosomes and peroxisomes. An increase in the superficial intensity of lysosome and peroxisome activities of HeLa cells was observed. In addition, a decrease in the amount of melanin has also been observed in mammalian cells without using any other chemical, showing that the process can work in vivo for treating melanin. Therefore, the results of this study indicate that the amount of melanin decreases by the lysosome and peroxisome activity after entering the cells, and functional organelles are effective in color reduction. This mechanism can be used in vivo for treating melanin.

  17. Disruption of lysosome function promotes tumor growth and metastasis in Drosophila.

    Science.gov (United States)

    Chi, Congwu; Zhu, Huanhu; Han, Min; Zhuang, Yuan; Wu, Xiaohui; Xu, Tian

    2010-07-09

    Lysosome function is essential to many physiological processes. It has been suggested that deregulation of lysosome function could contribute to cancer. Through a genetic screen in Drosophila, we have discovered that mutations disrupting lysosomal degradation pathway components contribute to tumor development and progression. Loss-of-function mutations in the Class C vacuolar protein sorting (VPS) gene, deep orange (dor), dramatically promote tumor overgrowth and invasion of the Ras(V12) cells. Knocking down either of the two other components of the Class C VPS complex, carnation (car) and vps16A, also renders Ras(V12) cells capable for uncontrolled growth and metastatic behavior. Finally, chemical disruption of the lysosomal function by feeding animals with antimalarial drugs, chloroquine or monensin, leads to malignant tumor growth of the Ras(V12) cells. Taken together, our data provide evidence for a causative role of lysosome dysfunction in tumor growth and invasion and indicate that members of the Class C VPS complex behave as tumor suppressors.

  18. Distribution of Prx-linked hydroperoxide reductase activity among microorganisms.

    Science.gov (United States)

    Takeda, Kouji; Nishiyama, Yoshitaka; Yoda, Koji; Watanabe, Toshihiro; Nimura-Matsune, Kaori; Mura, Kiyoshi; Tokue, Chiyoko; Katoh, Tetzuya; Kawasaki, Shinji; Niimura, Youichi

    2004-01-01

    Peroxiredoxin (Prx) constitutes a large family of enzymes found in microorganisms, animals, and plants, but the detection of the activities of Prx-linked hydroperoxide reductases (peroxiredoxin reductases) in cell extracts, and the purification based on peroxide reductase activity, have only been done in bacteria and Trypanosomatidae. A peroxiredoxin reductase (NADH oxidase) from a bacterium, Amphibacillus, displayed only poor activities in the presence of purified Prx from Saccharomyces or Synechocystis, while it is highly active in the presence of bacterial Prx. These results suggested that an enzyme system different from that in bacteria might exist for the reduction of Prx in yeast and cyanobacteria. Prx-linked hydroperoxide reductase activities were detected in cell extracts of Saccharomyces, Synechocystis, and Chlorella, and the enzyme activities of Saccharomyces and Chlorella were induced under vigorously aerated culture conditions and intensive light exposure conditions, respectively. Partial purification of Prx-linked peroxidase from the induced yeast cells indicated that the Prx-linked peroxidase system consists of two protein components, namely, thioredoxin and thioredoxin reductase. This finding is consistent with the previous report on its purification based on its protein protection activity against oxidation [Chae et al., J. Biol. Chem., 269, 27670-27678 (1994)]. In this study we have confirmed that Prx-linked peroxidase activity are widely distributed, not only in bacteria species and Trypanosomatidae, but also in yeast and photosynthetic microorganisms, and showed reconstitution of the activity from partially purified interspecies components.

  19. Evaluation of Thiol Raman Activities and pKa Values Using Internally Referenced Raman-Based pH Titration.

    Science.gov (United States)

    Suwandaratne, Nuwanthi; Hu, Juan; Siriwardana, Kumudu; Gadogbe, Manuel; Zhang, Dongmao

    2016-04-05

    Thiols, including organothiol and thiol-containing biomolecules, are among the most important classes of chemicals that are used broadly in organic synthesis, biological chemistry, and nanosciences. Thiol pKa values are key indicators of thiol reactivity and functionality. Reported herein is an internally referenced Raman-based pH titration method that enables reliable quantification of thiol pKa values for both mono- and dithiols in water. The degree of thiol ionization is monitored directly using the peak intensity of the S-H stretching feature in the 2600 cm(-1) region relative to an internal reference peak as a function of the titration solution's pH. The thiol pKa values and Raman activity relative to its internal reference were then determined by curve fitting the experimental data with equations derived on the basis of the Henderson-Hasselbalch equation. Using this Raman titration method, we determined for the first time the first and second thiol pKa values for 1,2-benzenedithiol in water. This Raman-based method is convenient to implement, and its underlying theory is easy to follow. It should therefore have broad application for thiol pKa determinations and verification.

  20. Aldose reductase, oxidative stress and diabetic mellitus

    Directory of Open Access Journals (Sweden)

    Waiho eTang

    2012-05-01

    Full Text Available Diabetes mellitus (DM is a complex metabolic disorder arising from lack of insulin production or insulin resistance 1. DM is a leading cause of morbidity and mortality in the developed world, particularly from vascular complications such as atherothrombosis in the coronary vessels. Aldose reductase (AR [ALR2; EC 1.1.1.21], a key enzyme in the polyol pathway, catalyzes NADPH-dependent reduction of glucose to sorbitol, leading to excessive accumulation of intracellular reactive oxygen species (ROS in various tissues of DM including the heart, vasculature, neurons, eyes and kidneys. As an example, hyperglycemia through such polyol pathway induced oxidative stress, may have dual heart actions, on coronary blood vessel (atherothrombosis and myocardium (heart failure leading to severe morbidity and mortality (reviewed in 2. In cells cultured under high glucose conditions, many studies have demonstrated similar AR-dependent increases in ROS production, confirming AR as an important factor for the pathogenesis of many diabetic complications. Moreover, recent studies have shown that AR inhibitors may be able to prevent or delay the onset of cardiovascular complications such as ischemia/reperfusion injury, atherosclerosis and atherothrombosis. In this review, we will focus on describing pivotal roles of AR in the pathogenesis of cardiovascular diseases as well as other diabetic complications, and the potential use of AR inhibitors as an emerging therapeutic strategy in preventing DM complications.

  1. Aldose reductase mediates retinal microglia activation.

    Science.gov (United States)

    Chang, Kun-Che; Shieh, Biehuoy; Petrash, J Mark

    2016-04-29

    Retinal microglia (RMG) are one of the major immune cells in charge of surveillance of inflammatory responses in the eye. In the absence of an inflammatory stimulus, RMG reside predominately in the ganglion layer and inner or outer plexiform layers. However, under stress RMG become activated and migrate into the inner nuclear layer (INL) or outer nuclear layer (ONL). Activated RMG in cell culture secrete pro-inflammatory cytokines in a manner sensitive to downregulation by aldose reductase inhibitors. In this study, we utilized CX3CR1(GFP) mice carrying AR mutant alleles to evaluate the role of AR on RMG activation and migration in vivo. When tested on an AR(WT) background, IP injection of LPS induced RMG activation and migration into the INL and ONL. However, this phenomenon was largely prevented by AR inhibitors or in AR null mice, or was exacerbated in transgenic mice that over-express AR. LPS-induced increases in ocular levels of TNF-α and CX3CL-1 in WT mice were substantially lower in AR null mice or were reduced by AR inhibitor treatment. These studies demonstrate that AR expression in RMG may contribute to the proinflammatory phenotypes common to various eye diseases such as uveitis and diabetic retinopathy.

  2. Aldose reductase mediates retinal microglia activation

    Energy Technology Data Exchange (ETDEWEB)

    Chang, Kun-Che; Shieh, Biehuoy; Petrash, J. Mark, E-mail: mark.petrash@ucdenver.edu

    2016-04-29

    Retinal microglia (RMG) are one of the major immune cells in charge of surveillance of inflammatory responses in the eye. In the absence of an inflammatory stimulus, RMG reside predominately in the ganglion layer and inner or outer plexiform layers. However, under stress RMG become activated and migrate into the inner nuclear layer (INL) or outer nuclear layer (ONL). Activated RMG in cell culture secrete pro-inflammatory cytokines in a manner sensitive to downregulation by aldose reductase inhibitors. In this study, we utilized CX3CR1{sup GFP} mice carrying AR mutant alleles to evaluate the role of AR on RMG activation and migration in vivo. When tested on an AR{sup WT} background, IP injection of LPS induced RMG activation and migration into the INL and ONL. However, this phenomenon was largely prevented by AR inhibitors or in AR null mice, or was exacerbated in transgenic mice that over-express AR. LPS-induced increases in ocular levels of TNF-α and CX3CL-1 in WT mice were substantially lower in AR null mice or were reduced by AR inhibitor treatment. These studies demonstrate that AR expression in RMG may contribute to the proinflammatory phenotypes common to various eye diseases such as uveitis and diabetic retinopathy. - Highlights: • AR inhibition prevents retinal microglial activation. • Endotoxin-induced ocular cytokine production is reduced in AR null mice. • Overexpression of AR spontaneously induces retinal microglial activation.

  3. Methylenetetrahydrofolate Reductase Activity and Folate Metabolism

    Directory of Open Access Journals (Sweden)

    Nursen Keser

    2014-04-01

    Full Text Available Folate is a vital B vitamin which is easily water-soluble. It is a natural source which is found in the herbal and animal foods. Folate has important duties in the human metabolism, one of them is the adjustment of the level of plasma homocysteine. Reduction in MTHFR (methylenetetrahydrofolate reductase,which is in charge of the metabolism of homocysteine activity affects the level of homocysteine. Therefore MTHFR is an important enzyme in folate metabolism. Some of the mutations occurring in the MTHFR gene is a risk factor for various diseases and may be caused the hyperhomocysteinemia or the homocystinuria, and they also may lead to metabolic problems. MTHFR is effective in the important pathways such as DNA synthesis, methylation reactions and synthesis of RNA. C677T and A1298C are the most commonly occurring polymorphisms in the gene of MTHFR. The frequency of these polymorphisms show differences in the populations. MTHFR, folate distribution, metabolism of homocysteine and S-adenosylmethionine, by the MTHFR methylation the genetic defects have the potential of affecting the risk of disease in the negative or positive way.

  4. Thioredoxin-related protein of 14 kDa is an efficient L-cystine reductase and S-denitrosylase.

    Science.gov (United States)

    Pader, Irina; Sengupta, Rajib; Cebula, Marcus; Xu, Jianqiang; Lundberg, Jon O; Holmgren, Arne; Johansson, Katarina; Arnér, Elias S J

    2014-05-13

    Thioredoxin-related protein of 14 kDa (TRP14, also called TXNDC17 for thioredoxin domain containing 17, or TXNL5 for thioredoxin-like 5) is an evolutionarily well-conserved member of the thioredoxin (Trx)-fold protein family that lacks activity with classical Trx1 substrates. However, we discovered here that human TRP14 has a high enzymatic activity in reduction of l-cystine, where the catalytic efficiency (2,217 min(-1)⋅µM(-1)) coupled to Trx reductase 1 (TrxR1) using NADPH was fivefold higher compared with Trx1 (418 min(-1)⋅µM(-1)). Moreover, the l-cystine reduction with TRP14 was in contrast to that of Trx1 fully maintained in the presence of a protein disulfide substrate of Trx1 such as insulin, suggesting that TRP14 is a more dedicated l-cystine reductase compared with Trx1. We also found that TRP14 is an efficient S-denitrosylase with similar efficiency as Trx1 in catalyzing TrxR1-dependent denitrosylation of S-nitrosylated glutathione or of HEK293 cell-derived S-nitrosoproteins. Consequently, nitrosylated and thereby inactivated caspase 3 or cathepsin B could be reactivated through either Trx1- or TRP14-catalyzed denitrosylation reactions. TRP14 was also, in contrast to Trx1, completely resistant to inactivation by high concentrations of hydrogen peroxide. The oxidoreductase activities of TRP14 thereby complement those of Trx1 and must therefore be considered for the full understanding of enzymatic control of cellular thiols and nitrosothiols.

  5. Thiol-yne adsorbates for stable, low-density, self-assembled monolayers on gold.

    Science.gov (United States)

    Stevens, Christopher A; Safazadeh, Leila; Berron, Brad J

    2014-03-04

    We present a novel approach toward carboxylate-terminated, low-density monolayers on gold, which provides exceptional adsorbate stability and conformational freedom of interfacial functional groups. Adsorbates are synthesized through the thiol-yne addition of two thiol-containing head groups to an alkyne-containing tail group. The resulting monolayers have two distinct phases: a highly crystalline head phase adjacent to the gold substrate, and a reduced density tail phase, which is in contact with the environment. The ellipsometric thickness of 27 Å is consistent with the proposed structure, where a densely packed decanedithiol monolayer is capped with an 11 carbon long, second layer at 50% lateral chain density. The Fourier transform infrared peak at 1710 cm(-1) supports the presence of the carbonyl group. Further, the peaks associated with asymmetric and symmetric methylene stretching are shifted toward higher wavenumbers compared to those of well-packed self-assembled monolayers (SAMs), which shows a lower average crystallinity of the thiol-yne monolayers compared to a typical monolayer. Contact angle measurements indicate an intermediate surface energy for the thiol-yne monolayer surface, owing to the contribution of exposed methylene functionality at the surface in addition to the carbonyl terminal group. The conformational freedom at the surface was demonstrated through remodeling the thiol-yne surface under an applied potential. Changes in the receding contact angle in response to an external potential support the capacity for reorientation of the surface presenting groups. Despite the low packing at the solution interface, thiol-yne monolayers are resistant to water and ion transport (R(f) ~ 10(5)), supporting the presence of a densely structured layer at the gold surface. Further, the electrochemical stability of the thiol-yne adsorbates exceeded that of well-packed SAMs, requiring a more reductive potential to desorb the thiol-yne monolayers from the

  6. Nebulized and oral thiol derivatives for pulmonary disease in cystic fibrosis.

    Science.gov (United States)

    Tam, Julian; Nash, Edward F; Ratjen, Felix; Tullis, Elizabeth; Stephenson, Anne

    2013-07-12

    Cystic fibrosis is an inherited condition resulting in thickened, sticky respiratory secretions. Respiratory failure, due to recurrent pulmonary infection and inflammation, is the most common cause of mortality. Muco-active therapies (e.g. dornase alfa and nebulized hypertonic saline) may decrease sputum viscosity, increase airway clearance of sputum, reduce infection and inflammation and improve lung function. Thiol derivatives, either oral or nebulized, have shown benefit in other respiratory diseases. Their mode of action is likely to differ according to the route of administration. There are several thiol derivatives, and it is unclear which of these may be beneficial in cystic fibrosis. To evaluate the efficacy and safety of nebulized and oral thiol derivatives in people with cystic fibrosis. We searched the Cochrane Cystic Fibrosis and Genetic Disorders Group Trials Register, comprising references identified from comprehensive electronic database searches, hand searches of relevant journals, abstract books and conference proceedings.Most recent search: 13 June 2013.We also conducted a PubMed search on 26 February 2013 for relevant published articles. Randomized and quasi-randomized controlled trials comparing nebulized or oral thiol derivatives to placebo or another thiol derivative in people with cystic fibrosis. The authors independently assessed trials for inclusion, analysed risk of bias and extracted data. Searches identified 23 trials; nine trials (255 participants) are included, of these seven trials are more than 10 years old. Three trials of nebulized thiol derivatives were identified (one compared 20% N-acetylcysteine to 2% N-acetylcysteine; another compared sodium-2-mercaptoethane sulphonate to 7% hypertonic saline; and another compared glutathione to 4% hypertonic saline). Although generally well-tolerated with no significant adverse effects, there was no evidence of significant clinical benefit in our primary outcomes in participants receiving

  7. Thiol groups controls on arsenite binding by organic matter: new experimental and modeling evidence.

    Science.gov (United States)

    Catrouillet, Charlotte; Davranche, Mélanie; Dia, Aline; Bouhnik-Le Coz, Martine; Pédrot, Mathieu; Marsac, Rémi; Gruau, Gérard

    2015-12-15

    Although it has been suggested that several mechanisms can describe the direct binding of As(III) to organic matter (OM), more recently, the thiol functional group of humic acid (HA) was shown to be an important potential binding site for As(III). Isotherm experiments on As(III) sorption to HAs, that have either been grafted with thiol or not, were thus conducted to investigate the preferential As(III) binding sites. There was a low level of binding of As(III) to HA, which was strongly dependent on the abundance of the thiols. Experimental datasets were used to develop a new model (the modified PHREEQC-Model VI), which defines HA as a group of discrete carboxylic, phenolic and thiol sites. Protonation/deprotonation constants were determined for each group of sites (pKA=4.28±0.03; ΔpKA=2.13±0.10; pKB=7.11±0.26; ΔpKB=3.52±0.49; pKS=5.82±0.052; ΔpKS=6.12±0.12 for the carboxylic, phenolic and thiols sites, respectively) from HAs that were either grafted with thiol or not. The pKS value corresponds to that of single thiol-containing organic ligands. Two binding models were tested: the Mono model, which considered that As(III) is bound to the HA thiol site as monodentate complexes, and the Tri model, which considered that As(III) is bound as tridentate complexes. A simulation of the available literature datasets was used to validate the Mono model, with logKMS=2.91±0.04, i.e. the monodentate hypothesis. This study highlighted the importance of thiol groups in OM reactivity and, notably, determined the As(III) concentration bound to OM (considering that Fe is lacking or at least negligible) and was used to develop a model that is able to determine the As(III) concentrations bound to OM.

  8. Catalysis by methyl-coenzyme M reductase: a theoretical study for heterodisulfide product formation.

    Science.gov (United States)

    Pelmenschikov, Vladimir; Siegbahn, Per E M

    2003-07-01

    Hybrid density functional theory has been used to investigate the catalytic mechanism of methyl-coenzyme M reductase (MCR), an essential enzyme in methanogenesis. In a previous study of methane formation, a scheme was suggested involving oxidation of Ni(I) in the starting square-planar coordination to the high-spin Ni(II) form in the CoM-S-Ni(II)F(430) octahedral intermediate. The methyl radical, concomitantly released by methyl-coenzyme M (CoM), is rapidly quenched by hydrogen atom transfer from the coenzyme B (CoB) thiol group, yielding methane as the first product of the reaction. The present investigation primarily concerns the second and final step of the reaction: oxidation of CoB and CoM to the CoB-S-S-CoM heterodisulfide product and reduction of nickel back to the Ni(I) square-planar form. The activation energy for the second step is found to be around 10 kcal/mol, implying that the first step of methane formation with an activation energy of 20 kcal/mol should be rate-limiting. An oxygen of the Gln147 residue, occupying the rear axial position in the oxidized Ni(II) state, is shown to stabilize the intermediate by 6 kcal/mol, thereby slightly decreasing the barrier for the preceding rate-limiting transition state. The mechanism suggested is discussed in the context of available experimental data. An analysis of the flexibility of the F(430) cofactor during the reaction cycle is also given.

  9. Near-Edge X-ray Absorption Fine Structure Spectroscopy of Diamondoid Thiol Monolayers on Gold

    Energy Technology Data Exchange (ETDEWEB)

    Willey, T M; Fabbri, J; Lee, J I; Schreiner, P; Fokin, A A; Tkachenko, B A; Fokina, N A; Dahl, J; Carlson, B; Vance, A L; Yang, W; Terminello, L J; van Buuren, T; Melosh, N

    2007-11-27

    Diamondoids, hydrocarbon molecules with cubic-diamond-cage structures, have unique properties with potential value for nanotechnology. The availability and ability to selectively functionalize this special class of nanodiamond materials opens new possibilities for surface-modification, for high-efficiency field emitters in molecular electronics, as seed crystals for diamond growth, or as robust mechanical coatings. The properties of self-assembled monolayers (SAMs) of diamondoids are thus of fundamental interest for a variety of emerging applications. This paper presents the effects of thiol substitution position and polymantane order on diamondoid SAMs on gold using near-edge X-ray absorption fine structure spectroscopy (NEXAFS) and X-ray photoelectron spectroscopy (XPS). A framework to determine both molecular tilt and twist through NEXAFS is presented and reveals highly ordered diamondoid SAMs, with the molecular orientation controlled by the thiol location. C 1s and S 2p binding energies are lower in adamantane thiol than alkane thiols on gold by 0.67 {+-} 0.05 eV and 0.16 {+-} 0.04 eV respectively. These binding energies vary with diamondoid monolayer structure and thiol substitution position, consistent with different amounts of steric strain and electronic interaction with the substrate. This work demonstrates control over the assembly, in particular the orientational and electronic structure, providing a flexible design of surface properties with this exciting new class of diamond clusters.

  10. Selective Adsorption of Thiols Using Gold Nanoparticles Supported on Metal Oxides.

    Science.gov (United States)

    Sui, Ruohong; Lesage, Kevin L; Carefoot, Sarah K; Fürstenhaupt, Tobias; Rose, Chelsea J; Marriott, Robert A

    2016-09-13

    Selective capture of thiols from a synthetic hydrogen sulfide containing mixture using supported nanogold materials has been explored for the potential removal of thiols from sour gas production fluids. In this research, TiO2-, Al2O3-, SiO2-, and ZnO-supported gold nanoparticles have been studied for their usage as regeneratable adsorbents to capture CH3SH, C2H5SH, and i-C3H7SH. Au/TiO2 and Au/Al2O3 showed promising properties for removing the thiols efficiently from a gas-phase mixture; however, Au/Al2O3 did catalyze some undesirable side reactions, e.g., carbonyl sulfide formation. It was found that a mild temperature of T = 200 °C was sufficient for regeneration of either Au/TiO2 or Au/Al2O3 adsorbent. The metal oxide mesopores played an important role for accommodating gold particles and chemisorption of the thiols, where smaller pore sizes were found to inhibit the agglomeration/growth of gold particles. The nature of thiol adsorption and the impact of multiple adsorption-desorption cycles on the adsorbents have been studied using electron microscopy, XPS, XRD, GC, and physi/chemiadsorption analyses.

  11. Superoxide dismutase and media dependence of far-UV radiation resistance in thiol-treated cells

    Energy Technology Data Exchange (ETDEWEB)

    Claycamp, H.G.; McCormick, M.L.; DeRose, C.M.; Elwell, J.H.; Oberley, L.W. (Iowa Univ., Iowa City, IA (USA). Radiation Research Lab.)

    1990-09-01

    Pretreatment of wild-type Escherichia coli K12 cells with dithiothreitol (DTT) induces far-UV radiation resistance after the thiol is removed (Claycamp 1988). The present study shows that a 1 h treatment of cells with DTT in minimal medium followed by a 0.5 h incubation in buffer (37{sup 0}C) results in a dose reduction factor (DRF) calculated at F{sub 37} of 1.81. When the thio pretreatment was in rich medium, sensitization occurs with DRF = 0.729. This could be reversed to protection by inhibiting extracellular thiol oxidation in rich medium with the chelator, DETAPAC, such that thiol oxidation rate was equivalent to that of DTT in minimal medium. Both thiol-induced resistance and sensitization produced changes predominantly in the shoulders of survival curves. For either protection or sensitization, at least one form of endogenous superoxide dismutase (SOD) was required. These results suggest that different targets are involved in thiol-induced UV protection and sensitization: DNA and extracellular targets (e.g. the membrane), respectively. (author).

  12. Thiol-disulfide exchange in peptides derived from human growth hormone.

    Science.gov (United States)

    Chandrasekhar, Saradha; Epling, Daniel E; Sophocleous, Andreas M; Topp, Elizabeth M

    2014-04-01

    Disulfide bonds stabilize proteins by cross-linking distant regions into a compact three-dimensional structure. They can also participate in hydrolytic and oxidative pathways to form nonnative disulfide bonds and other reactive species. Such covalent modifications can contribute to protein aggregation. Here, we present experimental data for the mechanism of thiol-disulfide exchange in tryptic peptides derived from human growth hormone in aqueous solution. Reaction kinetics was monitored to investigate the effect of pH (6.0-10.0), temperature (4-50°C), oxidation suppressants [ethylenediaminetetraacetic acid (EDTA) and N2 sparging], and peptide secondary structure (amide cyclized vs. open form). The concentrations of free thiol containing peptides, scrambled disulfides, and native disulfide-linked peptides generated via thiol-disulfide exchange and oxidation reactions were determined using reverse-phase HPLC and liquid chromatography-mass spectrometry. Concentration versus time data were fitted to a mathematical model using nonlinear least squares regression analysis. At all pH values, the model was able to fit the data with R(2) ≥ 0.95. Excluding oxidation suppressants (EDTA and N2 sparging) resulted in an increase in the formation of scrambled disulfides via oxidative pathways but did not influence the intrinsic rate of thiol-disulfide exchange. In addition, peptide secondary structure was found to influence the rate of thiol-disulfide exchange.

  13. Heavy metal ion removal by thiol functionalized aluminum oxide hydroxide nanowhiskers

    Science.gov (United States)

    Xia, Zhiyong; Baird, Lance; Zimmerman, Natasha; Yeager, Matthew

    2017-09-01

    In this study, we developed a cost effective method of using thiol functionalized γ-aluminum oxide hydroxide (γ-AlOOH) filters for removing three key heavy metals from water: mercury, lead, and cadmium under non-concomitant conditions. Compared to non-thiol treated γ-AlOOH filters, the introduction of thiol functional groups greatly improved the heavy metal removal efficiency under both static and dynamic filtration conditions. The adsorption kinetics of thiol functionalized γ-AlOOH were investigated using the Lagergren first order and pseudo-second order kinetics models; whereas the isothermal adsorption behavior of these membranes was revealed through the Langmuir and Freundlich models. Heavy metal concentration was quantified by Inductively Coupled Plasma-Mass Spectroscopy, and the thiol level on γ-AlOOH surface was measured by a colorimetric assay using Ellman's reagent. X-ray photoelectron spectroscopy was used to further address the surface sulfur state on the membranes after heavy metal exposure. Mechanisms for heavy metal adsorption were also discussed.

  14. Pore surface engineering in a zirconium metal–organic framework via thiol-ene reaction

    Energy Technology Data Exchange (ETDEWEB)

    Gui, Bo; Hu, Guiping; Zhou, Tailin; Wang, Cheng, E-mail: chengwang@whu.edu.cn

    2015-03-15

    A porous olefin-functionalized Zr(IV)-based metal–organic framework, denoted as UiO-68-allyl, has been constructed. Our results clearly demonstrated that the surface of UiO-68-allyl could be decorated with organic molecule (ethanethiol) via thiol-ene reaction. More importantly, the crystallinity of the framework were maintained during the post-synthetic modification process. However, the microporosity of the framework is retained but the surface area decreased, due to the grafting of ethylthio groups into the pores. From our studies, we can conclude that the strategy of post-synthetic modification of UiO-68-allyl via thiol-ene reaction may be general. Furthermore, we may anchor other desired functional group onto the pore walls in Zr-MOFs via thiol-ene reaction, enabling more potential applications. - graphical abstract: In this manuscript, we reported the post-synthetic modification of an olefin-functionalized Zr(IV)-based metal–organic framework via thiol-ene reaction. - Highlights: • A porous olefin-functionalized Zr(IV)-based metal–organic framework has been constructed. • The surface of olefin-functionalized Zr-MOF could be decorated with organic molecules via thiol-ene reaction. • The crystallinity and permanent porosity of the framework were maintained during the post-synthetic modification process.

  15. Quantification of protein thiols using ThioGlo 1 fluorescent derivatives and HPLC separation.

    Science.gov (United States)

    Hoff, Signe; Larsen, Flemming H; Andersen, Mogens L; Lund, Marianne N

    2013-04-07

    A method for quantification of total soluble protein-derived thiols in beer was developed based on the formation of fluorescent adducts with the maleimide compound ThioGlo 1. The problem of interference from fluorescent adducts of sulfite and ThioGlo 1 was solved by HPLC separation of the adducts followed by fluorescence detection. Using standard addition of GSH, a detection limit of 0.028 μM thiols was achieved. The application and validation of the method was demonstrated for beers with different color intensities, and the application range is in principle for any biological system containing thiols. However, the quantification of cysteine was complicated by a lower fluorescence response of its ThioGlo 1 adducts. Based on the studies of the responses of a series of cysteine-derived thiols and (1)H NMR studies of the structures of ThioGlo 1 adducts with GSH and cysteine, it was concluded that thiols with a neighboring free amino group yield ThioGlo 1 adducts with a reduced fluorescence intensity.

  16. Self-Healing Photocurable Epoxy/thiol-ene Systems Using an Aromatic Epoxy Resin

    Directory of Open Access Journals (Sweden)

    Ricardo Acosta Ortiz

    2016-01-01

    Full Text Available A rapid and efficient method to obtain self-healing epoxy resins is discussed. This method is based on the use of a thiol-disulfide oligomer obtained by partial oxidation of a multifunctional thiol using a hypervalent iodine (III compound as oxidant. The oligomer was characterized by Fourier transform infrared spectroscopy (FTIR, Raman and nuclear magnetic resonance spectroscopies, and gel permeation chromatography (GPC. The oligomer was a joint component of the thiol-ene system along with a tetra-allyl-functionalized curing agent. The kinetics of the photopolymerization of diglycidylether of bisphenol A (DGEBA revealed that conversions of the epoxy groups as high as 80% were achieved in only 15 minutes by increasing the concentration of the thiol-ene system in the formulation. The disulfide bonds introduced in the copolymer using the thiol-disulfide oligomer allowed the repairing of the test specimens in as little as 10 minutes when the specimens were heated at 80°C or for 500 minutes at room temperature. The analysis of the mechanical properties using dynamic mechanical analysis (DMA showed that the specimens displayed a healing efficiency up to 111% compared with the unhealed specimens, depending on the amount of polythioethers present in the copolymer.

  17. A Central Role for Thiols in Plant Tolerance to Abiotic Stress

    Directory of Open Access Journals (Sweden)

    Lyuben Zagorchev

    2013-04-01

    Full Text Available Abiotic stress poses major problems to agriculture and increasing efforts are being made to understand plant stress response and tolerance mechanisms and to develop new tools that underpin successful agriculture. However, the molecular mechanisms of plant stress tolerance are not fully understood, and the data available is incomplete and sometimes contradictory. Here, we review the significance of protein and non-protein thiol compounds in relation to plant tolerance of abiotic stress. First, the roles of the amino acids cysteine and methionine, are discussed, followed by an extensive discussion of the low-molecular-weight tripeptide, thiol glutathione, which plays a central part in plant stress response and oxidative signalling and of glutathione-related enzymes, including those involved in the biosynthesis of non-protein thiol compounds. Special attention is given to the glutathione redox state, to phytochelatins and to the role of glutathione in the regulation of the cell cycle. The protein thiol section focuses on glutaredoxins and thioredoxins, proteins with oxidoreductase activity, which are involved in protein glutathionylation. The review concludes with a brief overview of and future perspectives for the involvement of plant thiols in abiotic stress tolerance.

  18. Facile and Efficient Synthesis of Carbosiloxane Dendrimers via Orthogonal Click Chemistry Between Thiol and Ene.

    Science.gov (United States)

    Zhang, Zhida; Feng, Shengyu; Zhang, Jie

    2016-02-01

    A combination of a thiol-Michael addition reaction and a free radical mediated thiol-ene reaction is employed as a facile and efficient approach to carbosiloxane dendrimer synthesis. For the first time, carbosiloxane dendrimers are constructed rapidly by an orthogonal click strategy without protection/deprotection procedures. The chemoselectivity of these two thiol-ene click reactions leads to a design of a new monomer containing both electron-deficient carbon-carbon double bonds and unconjugated carbon-carbon double bonds. Siloxane bonds are introduced as the linker between these two kinds of carbon-carbon double bonds. Starting from a bifunctional thiol core, the dendrimers are constructed by iterative thiol-ene click reactions under different but both mild reaction conditions. After simple purification steps the fifth dendrimer with 54 peripheral functional groups is obtained with an excellent overall yield in a single day. Furthermore, a strong blue glow is observed when the dendrimer is excited by a UV lamp.

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

    Science.gov (United States)

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

    2011-10-01

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

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

    Institute of Scientific and Technical Information of China (English)

    Qinghua Zhou; Haimin Li; Ding Xue

    2011-01-01

    In mammals,the inheritance of mitochondrion and its DNA (mtDNA) is strictly maternal,despite the fact that a sperm can inject up to 100 functional mitochondria into the oocyte during fertilization.The mechanisms responsible for the elimination of the paternal mitochondria remain largely unknown.We report here that this paternal mitochondrial elimination process is conserved in Caenorhabditis elegans,and that the lysosomal pathway actively participates in this process.Molecular and cell biological analyses indicate that in wild-type animals paternal mitoehondria and mtDNA are destroyed within two hours after fertilization.In animals with compromised lysosomes,paternal mitochondria persist until late embryonic stages.Therefore,the lysosomal pathway plays an important role in degrading paternal mitochondria introduced into the oocyte during fertilization.Our study indicates that C.elegans is an excellent animal model for understanding and dissecting this conserved biological process critical for animal development and reproduction.

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

    Science.gov (United States)

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

    2010-07-01

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

  2. Molecular characterization of aspartylglucosaminidase, a lysosomal hydrolase upregulated during strobilation in the moon jellyfish, Aurelia aurita.

    Science.gov (United States)

    Tsujita, Natsumi; Kuwahara, Hiroyuki; Koyama, Hiroki; Yanaka, Noriyuki; Arakawa, Kenji; Kuniyoshi, Hisato

    2017-05-01

    The life cycle of the moon jellyfish, Aurelia aurita, alternates between a benthic asexual polyp stage and a planktonic sexual medusa (jellyfish) stage. Transition from polyp to medusa is called strobilation. To investigate the molecular mechanisms of strobilation, we screened for genes that are upregulated during strobilation using the differential display method and we identified aspartylglucosaminidase (AGA), which encodes a lysosomal hydrolase. Similar to AGAs from other species, Aurelia AGA possessed an N-terminal signal peptide and potential N-glycosylation sites. The genomic region of Aurelia AGA was approximately 9.8 kb in length and contained 12 exons and 11 introns. Quantitative RT-PCR analysis revealed that AGA expression increased during strobilation, and was then decreased in medusae. To inhibit AGA function, we administered the lysosomal acidification inhibitors, chloroquine or bafilomycin A1, to animals during strobilation. Both inhibitors disturbed medusa morphogenesis at the oral end, suggesting involvement of lysosomal hydrolases in strobilation.

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

    DEFF Research Database (Denmark)

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

    2007-01-01

    The endosomal-lysosomal system (ELS) has been suggested to play a role in the pathogenesis of prion diseases. The purpose of this study was to examine how experimental observations can be translated to human neuropathology and whether alterations of the ELS relate to neuropathologic changes....... Combined with stereologic techniques, we examined components of the ELS in human sporadic Creutzfeldt-Jakob disease brains. We immunostained for the early endosomal marker Rab5 and lysosomal enzymes cathepsin D and B. We determined neuron-specific changes in their expression and correlated......-immunoreactive lysosomes. The intraneuronal distribution of cathepsin D and B diverges between Purkinje cells and frontal cortical neurons in sporadic Creutzfeldt-Jakob disease brains. We demonstrated focal intra- and perineuronal colocalization of cathepsin D and PrP. Our results indicate that effects in the ELS...

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

    Energy Technology Data Exchange (ETDEWEB)

    Zeng, Jibin; Racicott, Jesse [Department of Anatomy and Cell Biology, McGill University, Montreal (Canada); Morales, Carlos R., E-mail: carlos.morales@mcgill.ca [Department of Anatomy and Cell Biology, McGill University, Montreal (Canada)

    2009-11-01

    Lysosomes are intracellular organelles which contain enzymes and activator proteins involved in the digestion and recycling of a variety of cellular and extracellular substances. We have identified a novel sorting receptor, sortilin, which is involved in the lysosomal trafficking of the sphingolipid activator proteins, prosaposin and GM{sub 2}AP, and the soluble hydrolases cathepsin D, cathepsin H, and acid sphingomyelinase. Sortilin belongs to a growing family of receptors with homology to the yeast Vps10 protein, which acts as a lysosomal sorting receptor for carboxypeptidase Y. In this study we examined the effects of the sortilin gene inactivation in mice. The inactivation of this gene did not yield any noticeable lysosomal pathology. To determine the existence of an alternative receptor complementing the sorting function of sortilin, we quantified the concentration of prosaposin in the lysosomes of the nonciliated epithelial cells lining the efferent ducts. These cells were chosen because they express sortilin and have a large number of lysosomes containing prosaposin. In addition, the nonciliated cells are known to endocytose luminal prosaposin that is synthesized and secreted by Sertoli cells into the seminiferous luminal fluids. Consequently, the nonciliated cells are capable of targeting both exogenous and endogenous prosaposin to the lysosomes. Using electron microscope immunogold labeling and quantitative analysis, our results demonstrate that inactivation of the sortilin gene produces a significant decrease of prosaposin in the lysosomes. When luminal prosaposin was excluded from the efferent ducts, the level of prosaposin in lysosomes was even lower in the mutant mice. Nonetheless, a significant amount of prosaposin continues to reach the lysosomal compartment. These results strongly suggest the existence of an alternative receptor that complements the function of sortilin and explains the lack of lysosomal storage disorders in the sortilin

  5. Isolation and characterization of cDNAs encoding leucoanthocyanidin reductase and anthocyanidin reductase from Populus trichocarpa.

    Directory of Open Access Journals (Sweden)

    Lijun Wang

    Full Text Available Proanthocyanidins (PAs contribute to poplar defense mechanisms against biotic and abiotic stresses. Transcripts of PA biosynthetic genes accumulated rapidly in response to infection by the fungus Marssonina brunnea f.sp. multigermtubi, treatments of salicylic acid (SA and wounding, resulting in PA accumulation in poplar leaves. Anthocyanidin reductase (ANR and leucoanthocyanidin reductase (LAR are two key enzymes of the PA biosynthesis that produce the main subunits: (+-catechin and (--epicatechin required for formation of PA polymers. In Populus, ANR and LAR are encoded by at least two and three highly related genes, respectively. In this study, we isolated and functionally characterized genes PtrANR1 and PtrLAR1 from P. trichocarpa. Phylogenetic analysis shows that Populus ANR1 and LAR1 occurr in two distinct phylogenetic lineages, but both genes have little difference in their tissue distribution, preferentially expressed in roots. Overexpression of PtrANR1 in poplar resulted in a significant increase in PA levels but no impact on catechin levels. Antisense down-regulation of PtrANR1 showed reduced PA accumulation in transgenic lines, but increased levels of anthocyanin content. Ectopic expression of PtrLAR1 in poplar positively regulated the biosynthesis of PAs, whereas the accumulation of anthocyanin and flavonol was significantly reduced (P<0.05 in all transgenic plants compared to the control plants. These results suggest that both PtrANR1 and PtrLAR1 contribute to PA biosynthesis in Populus.

  6. Transcripts of Anthocyanidin Reductase and Leucoanthocyanidin Reductase and Measurement of Catechin and Epicatechin in Tartary Buckwheat

    Directory of Open Access Journals (Sweden)

    Yeon Bok Kim

    2014-01-01

    Full Text Available Anthocyanidin reductase (ANR and leucoanthocyanidin reductase (LAR play an important role in the monomeric units biosynthesis of proanthocyanidins (PAs such as catechin and epicatechin in several plants. The aim of this study was to clone ANR and LAR genes involved in PAs biosynthesis and examine the expression of these two genes in different organs under different growth conditions in two tartary buckwheat cultivars, Hokkai T8 and T10. Gene expression was carried out by quantitative real-time RT-PCR, and catechin and epicatechin content was analyzed by high performance liquid chromatography. The expression pattern of ANR and LAR did not match the accumulation pattern of PAs in different organs of two cultivars. Epicatechin content was the highest in the flowers of both cultivars and it was affected by light in only Hokkai T8 sprouts. ANR and LAR levels in tartary buckwheat might be regulated by different mechanisms for catechin and epicatechin biosynthesis under light and dark conditions.

  7. Detection of biological thiols based on a colorimetric method

    Institute of Scientific and Technical Information of China (English)

    Yuan-yuan XU; Yang-yang SUN; Yu-juan ZHANG; Chen-he LU; Jin-feng MIAO‡

    2016-01-01

    Biological thiols (biothiols), an important kind of functional biomolecules, such as cysteine (Cys) and glutathione (GSH), play vital roles in maintaining the stability of the intracellular environment. In past decades, studies have demonstrated that metabolic disorder of biothiols is related to many serious disease processes and wil lead to extreme damage in human and numerous animals. We carried out a series of experiments to detect biothiols in bi-osamples, including bovine plasma and cel lysates of seven different cel lines based on a simple colorimetric method. In a typical test, the color of the test solution could gradualy change from blue to colorless after the addition of biothiols. Based on the color change displayed, experimental results reveal that the percentage of biothiols in the embryonic fibroblast cell line is significantly higher than those in the other six cell lines, which provides the basis for the following biothiols-related study.%中文概要题目:生物巯化物的可视化检测目的:通过简单可靠的可视化检测方法评估牛血清及各细胞系中生物巯化物的含量。创新点:基于银纳米颗粒形成的比色变化过程对牛血清及细胞中生物巯化物进行了检测。方法:将6组不同的细胞系培养后进行裂解,其裂解产物分别与3,3',5,5'-四甲基联苯胺(TMB)和硝酸银(AgNO3)的混合液室温孵育后,用紫外可见分光光度计测量细胞中生物巯化物的含量。结论:通过不同细胞系中生物巯化物含量的比对,证实胚胎成纤维细胞中生物巯化物的含量明显高于其他细胞。

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

    Science.gov (United States)

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

    2014-02-26

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

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

    Directory of Open Access Journals (Sweden)

    Corinna Hiller

    2014-04-01

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

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

    Directory of Open Access Journals (Sweden)

    Anna Stypułkowska

    2010-11-01

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

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

    Science.gov (United States)

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

    2013-11-01

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

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

    Directory of Open Access Journals (Sweden)

    Cristina I López Sanjurjo

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

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

    OpenAIRE

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

    2010-01-01

    Two-pore channels (TPCNs) have been proposed to form lysosomal Ca2+ release channels that are activated by nicotinic acid adenine dinucleotide phosphate. Here, we employ a glass chip-based method to record for the first time nicotinic acid adenine dinucleotide phosphate -dependent currents through a two-pore channel (TPCN2) from intact lysosomes. We show that TPCN2 is a highly selective Ca2+ channel that is regulated by intralysosomal pH. Using site-directed mutagenesis, we identify an amino ...

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

    DEFF Research Database (Denmark)

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

    2015-01-01

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

  15. Effects of the lysosomal destabilizing drug siramesine on glioblastoma in vitro and in vivo

    DEFF Research Database (Denmark)

    Jensen, Stine S.; Asferg Petterson, Stine; Halle, Bo

    2017-01-01

    confirmed by immunohistochemical staining of histological sections of spheroids, spheroids in brain slice cultures and tumors in mice brains. Results: The results showed that siramesine killed standard glioma cell lines in vitro, and loss of acridine orange staining suggested a compromised lysosomal...... cell death and inhibited tumor cell migration. This could not be reproduced in the organotypic three dimensional spheroid-brain slice culture model or in the mice xenograft model. Conclusions: In conclusion the in vitro results obtained with tumor cells and spheroids suggest a potential of lysosomal...

  16. Amyloid-β secretion, generation, and lysosomal sequestration in response to proteasome inhibition

    DEFF Research Database (Denmark)

    Agholme, Lotta; Hallbeck, Martin; Benedikz, Eirikur

    2012-01-01

    that proteasome inhibition resulted in autophagy-dependent accumulation of Aβ in lysosomes, and increased levels of intracellular and secreted Aβ. The enhanced levels of Aβ could not be explained by increased amounts of AβPP. Instead, reduced degradation of the C-terminal fragment of AβPP (C99) by the proteasome....... Furthermore, proteasome inhibition caused a reduction in cellular viability, which was reverted by inhibition of autophagy. Dysfunction of the proteasome could cause lysosomal accumulation of Aβ, as well as increased generation and secretion of Aβ, which is partly facilitated by autophagy. As a decrease...

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

    Science.gov (United States)

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

    2015-05-01

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

  18. Effect of various lysosomes and endotoxin on vascular permeability in frogs and mice.

    Science.gov (United States)

    Csákó, G; Reichel, A; Csernyánszky, H; Reichel, U

    1975-01-01

    Blood-lymph permeability increasing effects of frog liver lysosomes, Escherichia coli 0111 endotoxin, bradykinin and serotonin were demonstrated in frogs with a method developed by the authors. These actions were expressed in a faster dye saturation in the lymph as compared to that of the controls. 2. The method is based on the determinations of concentration of Evans blue transported as protein-bound dye into the lymph. 3. Frog liver and polymorphonuclear leukocyte lysosomes had a capillary permeability increasing action tested by local skin response when injecting Evans blue intravenously in mice. 4. All these phenomena are similar to events described earlier in mammalian systems.

  19. Hybrid thiol-ene network nanocomposites based on multi(meth)acrylate POSS.

    Science.gov (United States)

    Li, Liguo; Liang, Rendong; Li, Yajie; Liu, Hongzhi; Feng, Shengyu

    2013-09-15

    First, multi(meth)acrylate functionalized POSS monomers were synthesized in this paper. Secondly, FTIR was used to evaluate the homopolymerization behaviors of multi(meth)acrylate POSS and their copolymerization behaviors in the thiol-ene reactions with octa(3-mercaptopropyl) POSS in the presence of photoinitiator. Results showed that the photopolymerization rate of multimethacrylate POSS was faster than that of multiacrylate POSS. The FTIR results also showed that the copolymerizations were dominant in the thiol-ene reactions with octa(3-mercaptopropyl) POSS, different from traditional (meth)acrylate-thiol system, in which homopolymerizations were predominant. Finally, the resulted hybrid networks based on POSS were characterized by XRD, FE-SEM, DSC, and TGA. The characterization results showed that hybrid networks based on POSS were homogeneous and exhibited high thermal stability.

  20. Influence of volatile thiols in the development of blackcurrant aroma in red wine.

    Science.gov (United States)

    Rigou, Peggy; Triay, Aurélie; Razungles, Alain

    2014-01-01

    A strong blackcurrant aroma was recently perceived in some red wines originating from the same appellation. Varietal thiols such as 4-mercapto-4-methyl-2-pentanone (4MMP), 3-(mercapto)hexyl acetate (3MHA) and 3-mercapto-1-hexanol (3MH) are compounds potentially responsible for the development of this aroma. In order to demonstrate the correlation between thiols concentrations in red wines and blackcurrant aroma intensity, a multiple variable analysis was realised with thiols concentrations obtained by chemical analysis and blackcurrant aroma intensities obtained by descriptive sensory analysis. The 4MMP concentration was very well correlated to the blackcurrant aroma, and 3MHA and 3MH present at high concentrations act as enhancers of the perception of this aroma. This correlation was further supported after performing a sensory comparison by classification test. The different factors that could impact on the development of blackcurrant aroma in red wine were discussed.

  1. Electrical resistivity of nanoporous gold modified with thiol self-assembled monolayers

    Science.gov (United States)

    Hakamada, Masataka; Kato, Naoki; Mabuchi, Mamoru

    2016-11-01

    The electrical resistivity of nanoporous gold (NPG) modified with thiol self-assembled monolayers (SAMs) has been measured at 298 K using a four-probe method. We found that the adsorption of thiol SAMs increases the electrical resistivity of NPG by up to 22.2%. Dependence of the electrical resistivity on the atmosphere (air or water) was also observed in SAMs-modified NPG, suggesting that the electronic states of the tail groups affect the electrons of the binding sulfur and adjacent surface gold atoms. The present results suggest that adsorption of thiol molecules can influence the behavior of the conducting electrons in NPG and that modification of NPG with SAMs may be useful for environmental sensing.

  2. Mutations in the RAM network confer resistance to the thiol oxidant 4,4'-dipyridyl disulfide

    DEFF Research Database (Denmark)

    López-Mirabal, H Reynaldo; Winther, Jakob R; Thorsen, Michael

    2008-01-01

    might relate to bypass for abnormal septum-associated protein sorting. The broad resistance toward oxidants (DPS, diamide and H(2)O(2)) of the Deltacts1 strain links cell wall function to the resistance to oxidative stress and suggests the existence of targets that are common for these oxidants.......Thiol oxidants are expected to have multiple effects in living cells. Hence, mutations giving resistance to such agents are likely to reveal important targets and/or mechanisms influencing the cellular capacity to withstand thiol oxidation. A screen for mutants resistant to the thiol......-specific oxidant dipyridyl disulfide (DPS) yielded tao3-516, which is impaired in the function of the RAM signaling network protein Tao3/Pag1p. We suggest that the DPS-resistance of the tao3-516 mutant might be due to deficient cell-cycle-regulated production of the chitinase Cts1p, which functions in post...

  3. Thiolated pyrimidine nucleotides may interfere thiol groups concentrated at lipid rafts of HIV-1 infected cells.

    Science.gov (United States)

    Kanizsai, Szilvia; Ongrádi, Joseph; Aradi, János; Nagy, Károly

    2014-12-01

    Upon HIV infection, cells become activated and cell surface thiols are present in increased number. Earlier we demonstrated in vitro anti-HIV effect of thiolated pyrimidine nucleotide UD29, which interferes thiol function. To further analyse the redox processes required for HIV-1 entry and infection, toxicity assays were performed using HIV-1 infected monolayer HeLaCD4-LTR/ β-gal cells and suspension H9 T cells treated with several thiolated nucleotide derivatives of UD29. Selective cytotoxicity of thiolated pyrimidines on HIV-1 infected cells were observed. Results indicate that thiolated pyrimidine derivates may interfere with -SH (thiol) groups concentrated in lipid rafts of cell membrane and interacts HIV-1 infected (activated) cells resulting in a selective cytotoxicity of HIV-1 infected cells, and reducing HIV-1 entry.

  4. Strategies for creating antifouling surfaces using selfassembled poly(ethylene glycol) thiol molecules

    DEFF Research Database (Denmark)

    Lokanathan, Arcot R.

    2011-01-01

    conditions for the reversible, initial attachment of microbial cells. This effect can be obtained by grafting hydrophilic polymeric chains onto surfaces and thereby provide a steric barrier between the substrate surface and the microbial cell. Poly (ethylene glycol) (PEG) is one of the most widely used....... The work focuses on novel strategies to self assemble PEG thiol monolayers with high graft density. One of the strategies investigated involved backfilling a self assembled layer of 2000 Da PEG thiol with shorter oligo (ethylene glycol) (OEG) thiol molecules to form a mixed monolayer. Detailed quantitative......Microorganisms are one of the most important parts of our ecosystem influencing the sustenance of human society. The beneficial microbes are of high relevance to food industry, development of antibiotics and processing of many raw materials. Mankind has indeed benefitted a lot from large number...

  5. Diamond surface functionalization with biomimicry – Amine surface tether and thiol moiety for electrochemical sensors

    Energy Technology Data Exchange (ETDEWEB)

    Sund, James B., E-mail: jim@jamessund.com [Department of Electrical and Computer Engineering, Duke University, Durham, NC (United States); Causey, Corey P. [Departments of Chemistry and Biochemistry, Duke University, Durham, NC (United States); Wolter, Scott D. [Department of Physics, Elon University, Elon, NC 27244 (United States); Parker, Charles B., E-mail: charles.parker@duke.edu [Department of Electrical and Computer Engineering, Duke University, Durham, NC (United States); Stoner, Brian R. [Department of Electrical and Computer Engineering, Duke University, Durham, NC (United States); Research Triangle Institute (RTI) International, Research Triangle Park, NC (United States); Toone, Eric J. [Departments of Chemistry and Biochemistry, Duke University, Durham, NC (United States); Glass, Jeffrey T. [Department of Electrical and Computer Engineering, Duke University, Durham, NC (United States)

    2014-05-01

    Highlights: • Diamond surfaces were functionalized with organic molecules using a novel approach. • Used biomimicry to select a molecule to bind NO, similar to the human body. • Molecular orbital theory predicted the molecule-analyte oxidation behavior. • A thiol moiety was attached to an amine surface tether on the diamond surface. • XPS analysis verified each surface functionalization step. - Abstract: The surface of conducting diamond was functionalized with a terminal thiol group that is capable of binding and detecting nitrogen–oxygen species. The functionalization process employed multiple steps starting with doped diamond films grown by plasma enhanced chemical vapor deposition followed by hydrogen termination and photochemical attachment of a chemically protected amine alkene. The surface tether was deprotected to reveal the amine functionality, which enabled the tether to be extended with surface chemistry to add a terminal thiol moiety for electrochemical sensing applications. Each step of the process was validated using X-ray photoelectron spectroscopy analysis.

  6. Serum paraoxonase activity, total thiols levels, and oxidative status in patients with acute brucellosis.

    Science.gov (United States)

    Esen, Ramazan; Aslan, Mehmet; Kucukoglu, Mehmet Emin; Cıkman, Aytekin; Yakan, Umit; Sunnetcioglu, Mahmut; Selek, Sahbettin

    2015-06-01

    It is well known that paraoxonase-1 (PON1) activity may decrease during the course of infection and inflammation. The aim of this study was to investigate serum PON1 activity, oxidative status, and thiols levels in patients with acute brucellosis. In addition, we investigated the PON1 phenotype in patients with acute brucellosis. Thirty patients with acute brucellosis and 35 healthy controls were enrolled. Serum paraoxonase and arylesterase activities, thiols levels, lipid hydroperoxide levels, total antioxidant capacity (TAC), total oxidant status (TOS) and oxidative stress index (OSI) were determined. Serum basal and salt-stimulated paraoxonase-arylesterase activities, TAC levels and thiols levels were significantly lower in patients with acute brucellosis than controls (for all, p brucellosis. These results indicate that lower PON1 activity is associated with oxidant-antioxidant imbalance.

  7. Thiol peptides induction in the seagrass Thalassia testudinum (Banks ex Koenig) in response to cadmium exposure

    Energy Technology Data Exchange (ETDEWEB)

    Alvarez-Legorreta, Teresa [Departamento de Recursos del Mar, CINVESTAV-IPN, Unidad Merida, Apdo. Postal 73-Cordemex, Merida, Yucatan 97310 (Mexico); Mendoza-Cozatl, David; Moreno-Sanchez, Rafael [Departamento de Bioquimica, Instituto Nacional de Cardiologia, Mexico D.F. 14080 (Mexico); Gold-Bouchot, Gerardo [Departamento de Recursos del Mar, CINVESTAV-IPN, Unidad Merida, Apdo. Postal 73-Cordemex, Merida, Yucatan 97310 (Mexico)], E-mail: gold@mda.cinvestav.mx

    2008-01-20

    Trace metal accumulation and thiol compounds synthesis as induced by cadmium exposure was studied in the seagrass Thalassia testudinum. Shoots were exposed for 24, 48, 96 and 144 h to several CdCl{sub 2} concentrations (0, 30, 50 and 70 {mu}M). Levels of cadmium, cysteine, glutathione (GSH), {gamma}-glutamylcysteine ({gamma}-EC), and phytochelatin-like peptides were determined in green blades, live sheaths and root/rhizomes tissues. Metal accumulation was dependent on Cd concentration and type of tissue, with green blades showing the highest content followed by live sheaths and root/rhizomes. All tissues experienced an increase in thiol-containing compounds as a response to cadmium exposure. Live sheaths showed the highest levels of cysteine, GSH and {gamma}-EC. This is the first report of induction of thiol peptides, presumably phytochelatins, by a trace metal in a sea grass species.

  8. Enhancement in the Glass Transition Temperature in Latent Thiol-Epoxy Click Cured Thermosets

    Directory of Open Access Journals (Sweden)

    Dailyn Guzmán

    2015-04-01

    Full Text Available Tri and tetrafunctional thiol were used as curing agent for diglycidyl ether of bisphenol A (DGEBA catalyzed by a commercially available amine precursor, LC-80. Triglycidyl isocianurate (TGIC was added in different proportions to the mixture to increase rigidity and glass transition temperature (Tg. The cooperative effect of increasing functionality of thiol and the presence of TGIC in the formulation leads to an increased Tg without affecting thermal stability. The kinetics of the curing of mixtures was studied by calorimetry under isothermal and non-isothermal conditions. The latent characteristics of the formulations containing amine precursors were investigated by rheometry and calorimetry. The increase in the functionality of the thiol produces a slight decrease in the storage lifetime of the mixture. The materials obtained with tetrathiol as curing agent showed the highest values of Young’s modulus and Tg.

  9. Janus Gold Nanoparticles from Nanodroplets of Alkyl Thiols: A Molecular Dynamics Study.

    Science.gov (United States)

    Bhandary, Debdip; Valechi, Vasumathi; Cordeiro, Maria Natália D S; Singh, Jayant K

    2017-03-28

    Janus particles provide an asymmetry in structure, which can impart diverse functionalities leading to immense importance in various applications, ranging from targeted delivery to interfacial phenomena, including catalysis, electronics, and optics. In this work, we present results of a molecular dynamics study of the growth mechanism of coating on gold nanoparticles (AuNPs) from droplets of n-alkyl thiols with different chain lengths (C5 and C11) and terminal groups (CH3 and COOH). The effect of chain lengths and functional groups on the formation of a monolayer of alkyl thiols on AuNPs is investigated. A two-step mechanism, initiated by the binding of the droplet to the nanoparticle surface with a time constant on the order of ∼1 ns, followed by the diffusion-driven growth with a larger time constant (on the order of 100 ns), is shown to capture the growth dynamics of the monolayer. It is observed that the time required for complete wetting increases with an increase in the chain length. Moreover, the monolayer formation is slowed down in the presence of carboxyl groups because of strong hydrogen bonding. The kinetics of the n-alkyl thiols coating on the nanoparticles is found to be independent of the droplet size but carboxyl-terminated thiols spread more with increasing droplet size. Furthermore, different time constants for different chains and functional groups yield Janus coating when two droplets of alkyl thiols with different terminal groups are allowed to form monolayers on the nanoparticle. The Janus balance (β) for different combinations of alkyl thiols and nanoparticle sizes varies in the range of 0.42-0.71.

  10. Stretching of BDT-gold molecular junctions: Thiol or thiolate termination?

    KAUST Repository

    Souza, Amaury De Melo

    2014-01-01

    It is often assumed that the hydrogen atoms in the thiol groups of a benzene-1,4-dithiol dissociate when Au-benzene-1,4-dithiol-Au junctions are formed. We demonstrate, by stability and transport property calculations, that this assumption cannot be made. We show that the dissociative adsorption of methanethiol and benzene-1,4-dithiol molecules on a flat Au(111) surface is energetically unfavorable and that the activation barrier for this reaction is as high as 1 eV. For the molecule in the junction, our results show, for all electrode geometries studied, that the thiol junctions are energetically more stable than their thiolate counterparts. Due to the fact that density functional theory (DFT) within the local density approximation (LDA) underestimates the energy difference between the lowest unoccupied molecular orbital and the highest occupied molecular orbital by several electron-volts, and that it does not capture the renormalization of the energy levels due to the image charge effect, the conductance of the Au-benzene-1,4-dithiol-Au junctions is overestimated. After taking into account corrections due to image charge effects by means of constrained-DFT calculations and electrostatic classical models, we apply a scissor operator to correct the DFT energy level positions, and calculate the transport properties of the thiol and thiolate molecular junctions as a function of the electrode separation. For the thiol junctions, we show that the conductance decreases as the electrode separation increases, whereas the opposite trend is found for the thiolate junctions. Both behaviors have been observed in experiments, therefore pointing to the possible coexistence of both thiol and thiolate junctions. Moreover, the corrected conductance values, for both thiol and thiolate, are up to two orders of magnitude smaller than those calculated with DFT-LDA. This brings the theoretical results in quantitatively good agreement with experimental data.

  11. Production of Thiol Species From An Exponential Growth Diatom Under Copper Exposure

    Science.gov (United States)

    Tang, D.; Shafer, M. M.; Karner, D. A.; Armstrong, D. E.; Schauer, J.

    2003-12-01

    The intracellar induction of phytochelatins is a well documented response of eukaryotic microorganisms to aqueous metal exposure. The extracellular release of thiolic compounds from algal species has been observed; and in some cases, this release can contribute a significant fraction of the uncharacterized metal-complexing ligands. Glutathione (GSH) or cysteine is among the detectable thiols excreted. A quantitative assessment of the excretion of thiols from algae cells into growth media is needed to assess the significance of biogenic-thiols as a source of strong ligands in natural waters and as a "forgotten" route in sulfur biogeochemical cycle. Unbuffered growth media (e.g., without adding complexing ligand such as EDTA) have only rarely been used to study the possible release of metal-complexing ligands from algal species, and the ligand titration techniques used varied considerably. The majority of culture studies have applied metal-buffered media. A direct comparison of released ligands under buffered and unbuffered conditions is lacking, partially due to the inherent difficulties of the titration methods applied. Using HPLC with fluorescence detection of thiol-monobromobimane derivatives, we were able to follow the dynamic change of GSH released in both media types during algal growth: (1) the cell quotas for thiols and pigments varied (mostly decreases) with growth time. Therefore, pigment-normalized cellular thiol concentrations were more or less conservative. (2) GSH was released into both the EDTA-buffered and -unbuffered growth media at similar concentrations. (3) at similar available Cu concentrations, EDTA possibly enhanced, rather than hindered, the release of GSH.

  12. Surface modification of cyclomatrix polyphosphazene microsphere by thiol-ene chemistry and lectin recognition

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Chen; Zhu, Xue-yan; Gao, Qiao-ling; Fang, Fei; Huang, Xiao-jun, E-mail: hxjzxh@zju.edu.cn

    2016-11-30

    Graphical abstract: A new synthetic route leading to polyphosphazene cyclomatrix microsphere with various functional groups has achieved via thiol-ene click modification. Herein, hexacholorocyclophosphazene (HCCP) crosslinked with bisphenol-S and 4,4′-diallyl bisphenol-S to generate broadly dispersed microspheres. Thiol-ene modification under UV irradiation not only presented high efficiency and flexibility for post-functionalization, but also imposed no harm on global morphology and crosslinked skeleton of such microspheres. - Highlights: • Functional polyphosphazene microspheres with high chemical flexibility were synthesized by thiol-ene modification. • Polyphosphazene microspheres possessed high thermal stability. • Glycosylated polyphosphazene microspheres showed affinity to lectin Con-A, which inferred potential application in biomedicine. - Abstract: A new synthetic route leading to functional polyphosphazene cyclomatrix microsphere has been developed via thiol-ene click modification. Hexacholorocyclophosphazene (HCCP) was crosslinked with both bisphenol-S and 4,4′-diallyl bisphenol-S to obtain vinyl polyphosphazene microspheres (VPZM) in order to ensure high crosslinking degree and introduce vinyl moieties. Compared to the microspheres obtained by HCCP and bisphenol-S, the size of VPZM was broadly dispersed from 400 nm to 1.40 μm. Thiol-ene click reactions were carried out to attach functional groups, such as glucosyl, carboxyl, ester and dodecyl groups onto polyphosphazene microspheres, which demonstrated no change in morphology and size after modification. Solid state NMR (SSNMR) and Fourier transform infrared spectoscopy (FT-IR) results showed that the vinyl moieties were introduced in the period of crosslinking and functionalization was also successful via click reactions. Moreover, the microspheres presented a little difference in thermal properties after modification. Concanavalin A (Con-A) fluorescent adsorption was also observed for

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

    DEFF Research Database (Denmark)

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

    2013-01-01

    " hypothesis. Our measurements show that PEI does not induce change in lysosomal pH as previously suggested and quantification of PEI concentrations in lysosomes makes it uncertain that the "proton sponge " effect is the dominant mechanism of polyplex escape.Molecular Therapy (2012); doi:10.1038/mt.2012.185....

  14. Disorders of lysosomal acidification-The emerging role of v-ATPase in aging and neurodegenerative disease.

    Science.gov (United States)

    Colacurcio, Daniel J; Nixon, Ralph A

    2016-12-01

    Autophagy and endocytosis deliver unneeded cellular materials to lysosomes for degradation. Beyond processing cellular waste, lysosomes release metabolites and ions that serve signaling and nutrient sensing roles, linking the functions of the lysosome to various pathways for intracellular metabolism and nutrient homeostasis. Each of these lysosomal behaviors is influenced by the intraluminal pH of the lysosome, which is maintained in the low acidic range by a proton pump, the vacuolar ATPase (v-ATPase). New reports implicate altered v-ATPase activity and lysosomal pH dysregulation in cellular aging, longevity, and adult-onset neurodegenerative diseases, including forms of Parkinson disease and Alzheimer disease. Genetic defects of subunits composing the v-ATPase or v-ATPase-related proteins occur in an increasingly recognized group of familial neurodegenerative diseases. Here, we review the expanding roles of the v-ATPase complex as a platform regulating lysosomal hydrolysis and cellular homeostasis. We discuss the unique vulnerability of neurons to persistent low level lysosomal dysfunction and review recent clinical and experimental studies that link dysfunction of the v-ATPase complex to neurodegenerative diseases across the age spectrum. Copyright © 2016 Elsevier B.V. All rights reserved.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1988-03-01

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

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

    Science.gov (United States)

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

    2016-03-18

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

  17. Therapeutic effects of remediating autophagy failure in a mouse model of Alzheimer disease by enhancing lysosomal proteolysis.

    Science.gov (United States)

    Yang, Dun-Sheng; Stavrides, Philip; Mohan, Panaiyur S; Kaushik, Susmita; Kumar, Asok; Ohno, Masuo; Schmidt, Stephen D; Wesson, Daniel W; Bandyopadhyay, Urmi; Jiang, Ying; Pawlik, Monika; Peterhoff, Corrinne M; Yang, Austin J; Wilson, Donald A; St George-Hyslop, Peter; Westaway, David; Mathews, Paul M; Levy, Efrat; Cuervo, Ana M; Nixon, Ralph A

    2011-07-01

    The extensive autophagic-lysosomal pathology in Alzheimer disease (AD) brain has revealed a major defect: in the proteolytic clearance of autophagy substrates. Autophagy failure contributes on several levels to AD pathogenesis and has become an important therapeutic target for AD and other neurodegenerative diseases. We recently observed broad therapeutic effects of stimulating autophagic-lysosomal proteolysis in the TgCRND8 mouse model of AD that exhibits defective proteolytic clearance of autophagic substrates, robust intralysosomal amyloid-β peptide (Aβ) accumulation, extracellular β-amyloid deposition and cognitive deficits. By genetically deleting the lysosomal cysteine protease inhibitor, cystatin B (CstB), to selectively restore depressed cathepsin activities, we substantially cleared Aβ, ubiquitinated proteins and other autophagic substrates from autolysosomes/lysosomes and rescued autophagic-lysosomal pathology, as well as reduced total Aβ40/42 levels and extracellular amyloid deposition, highlighting the underappreciated importance of the lysosomal system for Aβ clearance. Most importantly, lysosomal remediation prevented the marked learning and memory deficits in TgCRND8 mice. Our findings underscore the pathogenic significance of autophagic-lysosomal dysfunction in AD and demonstrate the value of reversing this dysfunction as an innovative therapeautic strategy for AD.

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

    Science.gov (United States)

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

    2017-03-01

    Parkinson's disease is characterized by accumulation of intraneuronal cytoplasmic inclusions, Lewy bodies, which mainly consist of aggregated α-synuclein. Controversies exist as to whether high blood cholesterol is a risk factor for the development of the disease and whether statin treatment could have a protective effect. Using a model system of BE(2)-M17 neuroblastoma cells treated with the neurotoxin 1-methyl-4-phenylpyridinium (MPP(+)), we found that MPP(+)-induced cell death was accompanied by cholesterol accumulation in a lysosomal-like pattern in pre-apoptotic cells. To study the effects of lysosomal cholesterol accumulation, we increased lysosomal cholesterol through pre-treatment with U18666A and found delayed leakage of lysosomal contents into the cytosol, which reduced cell death. This suggests that increased lysosomal cholesterol is a stress response mechanism to protect lysosomal membrane integrity in response to early apoptotic stress. However, high cholesterol also stimulated the accumulation of α-synuclein. Treatment with the cholesterol-lowering drug lovastatin reduced MPP(+)-induced cell death by inhibiting the production of reactive oxygen species, but did not prevent lysosomal cholesterol increase nor affect α-synuclein accumulation. Our study indicates a dual role of high cholesterol in Parkinson's disease, in which it acts both as a protector against lysosomal membrane permeabilization and as a stimulator of α-synuclein accumulation. Copyright © 2017 Elsevier GmbH. All rights reserved.

  19. Thiol-catalyzed formation of lactate and glycerate from glyceraldehyde. [significance in molecular evolution

    Science.gov (United States)

    Weber, A. L.

    1983-01-01

    The rate of lactate formation from glyceraldehyde, catalyzed by N-acetyl-cysteine at ambient temperature in aqueous sodium phosphate (pH 7.0), is more rapid at higher sodium phosphate concentrations and remains essentially the same in the presence and absence of oxygen. The dramatic increase in the rate of glycerate formation that is brought about by this thiol, N-acetylcysteine, is accompanied by commensurate decreases in the rates of glycolate and formate production. It is suggested that the thiol-dependent formation of lactate and glycerate occurs by way of their respective thioesters. Attention is given to the significance of these reactions in the context of molecular evolution.

  20. Characterization of self-assembled monolayers of porphyrins bearing multiple-thiol and photoelectric response

    Institute of Scientific and Technical Information of China (English)

    Jian Dong Yang; Xiao Quan Lu

    2012-01-01

    Self-assembled monolayers (SAMs) of thiol-derivatized porphyrin molecules on Au substrate have attracted extensively interest for use in sensing,optoelectronic devices and molecular electronics.In this paper,tetra-[p-(3-mercaptopropyloxy)-phenyl]-porphyrin was synthesized and self-assembled with thiol on Au substrate for porphyrin SAMs (PPS4).The electrochemical results demonstrated that PPS4 could form excellent SAMs on gold surface.Self-assembled nanojunctions of PPS4 were fabricated by using gold nanogap electrodes (gap width:ca.100 nm).With the light on/off,the nanojunctions showed current high/low as nanometer scaled photo switch.

  1. Endothelium-dependent vasorelaxation in inhibited by in vivo depletion of vascular thiol levels

    DEFF Research Database (Denmark)

    Laursen, J B; Boesgaard, S; Trautner, S;

    2001-01-01

    Thiols like glutathione may serve as reducing cofactors in the production of nitric oxide (NO) and protect NO from inactivation by radical oxygen species. Depletion of thiol compounds reduces NO-mediated vascular effects in vitro and in vivo. The mechanisms underlying these actions are not clear......, but may involve decreased synthesis of NO and/or increased degradation of NO. This study investigates the effect of glutathione depletion on the response to NO-mediated vasodilation induced by acetylcholine (Ach, 10 micrograms/kg), endothelial NO synthase (eNOS) activity and potential markers of vascular...

  2. Diamond surface functionalization with biomimicry - Amine surface tether and thiol moiety for electrochemical sensors

    Science.gov (United States)

    Sund, James B.; Causey, Corey P.; Wolter, Scott D.; Parker, Charles B.; Stoner, Brian R.; Toone, Eric J.; Glass, Jeffrey T.

    2014-05-01

    The surface of conducting diamond was functionalized with a terminal thiol group that is capable of binding and detecting nitrogen-oxygen species. The functionalization process employed multiple steps starting with doped diamond films grown by plasma enhanced chemical vapor deposition followed by hydrogen termination and photochemical attachment of a chemically protected amine alkene. The surface tether was deprotected to reveal the amine functionality, which enabled the tether to be extended with surface chemistry to add a terminal thiol moiety for electrochemical sensing applications. Each step of the process was validated using X-ray photoelectron spectroscopy analysis.

  3. Mutagenesis of the redox-active disulfide in mercuric ion reductase: Catalysis by mutant enzymes restricted to flavin redox chemistry

    Energy Technology Data Exchange (ETDEWEB)

    Distefano, M.D.; Au, K.G.; Walsh, C.T. (Harvard Medical School, Boston, MA (USA))

    1989-02-07

    Mercuric reductase, a flavoenzyme that possesses a redox-active cystine, Cys{sub 135}Cys{sub 140}, catalyzes the reduction of Hg(II) to Hg(0) by NADPH. As a probe of mechanism, the authors have constructed mutants lacking a redox-active disulfide by eliminating Cys{sub 135} (Ala{sub 135}Cys{sub 140}), Cys{sub 14} (Cys{sub 135}Ala{sub 140}), or both (Ala{sub 135}Ala{sub 140}). Additionally, they have made double mutants that lack Cys{sub 135} (Ala{sub 135}Cys{sub 139}Cys{sub 140}) or Cys{sub 140} (Cys{sub 135}Cys{sub 139}Ala{sub 140}) but introduce a new Cys in place of Gly{sub 139} with the aim of constructing dithiol pairs in the active site that do not form a redox-active disulfide. The resulting mutant enzymes all lack redox-active disulfides and are hence restricted to FAD/FADH{sub 2} redox chemistry. Each mutant enzyme possesses unique physical and spectroscopic properties that reflect subtle differences in the FAD microenvironment. Preliminary evidence for the Ala{sub 135}Cys{sub 139}Cys{sub 14} mutant enzyme suggests that this protein forms a disulfide between the two adjacent Cys residues. Hg(II) titration experiments that correlate the extent of charge-transfer quenching with Hg(II) binding indicate that the Ala{sub 135}Cys{sub 140} protein binds Hg(II) with substantially less avidity than does the wild-type enzyme. All mutant mercuric reductases catalyze transhydrogenation and oxygen reduction reactions through obligatory reduced flavin intermediates at rates comparable to or greater than that of the wild-type enzyme. In multiple-turnover assays which monitored the production of Hg(0), two of the mutant enzymes were observed to proceed through at least 30 turnovers at rates ca. 1000-fold slower than that of wild-type mercuric reductase. They conclude that the Cys{sub 135} and Cys{sub 140} thiols serve as Hg(II) ligands that orient the Hg(II) for subsequent reduction by a reduced flavin intermediate.

  4. Rapid and simple preparation of thiol-ene emulsion-templated monoliths and their application as enzymatic microreactors

    DEFF Research Database (Denmark)

    Lafleur, Josiane P; Senkbeil, Silja; Novotny, Jakub;

    2015-01-01

    electron microscopy showed that the methanol-based emulsion templating process resulted in a network of highly interconnected and regular thiol-ene beads anchored solidly inside thiol-ene microchannels. Surface area measurements indicate that the monoliths are macroporous, with no or little micro...

  5. Cytoplasmic glutathione redox status determines survival upon exposure to the thiol-oxidant 4,4'-dipyridyl disulfide

    DEFF Research Database (Denmark)

    López-Mirabal, H Reynaldo; Thorsen, Michael; Kielland-Brandt, Morten C

    2007-01-01

    Dipyridyl disulfide (DPS) is a highly reactive thiol oxidant that functions as electron acceptor in thiol-disulfide exchange reactions. DPS is very toxic to yeasts, impairing growth at low micromolar concentrations. The genes TRX2 (thioredoxin), SOD1 (superoxide dismutase), GSH1 (gamma-glutamyl-c...

  6. Functionalization of embedded thiol-ene waveguides for evanescent wave-induced fluorescence detection in a microfluidic device

    DEFF Research Database (Denmark)

    Feidenhans, Nikolaj A.; Jensen, Thomas Glasdam; Lafleur, Josiane P.;

    2013-01-01

    We demonstrate the use of functional surface groups inherently present on off-stoichiometric thiol-ene polymers, for site-specific immobilization of biomolecules and detection by evanescent wave-induced fluorescence. An optofluidic chip featuring an embedded thiol-ene waveguide was selectively fu...

  7. Methylphosphonic Dichloride as Reagent for the Determination of the Enantiomeric Excess of Chiral Thiols. Scope and Limitations

    NARCIS (Netherlands)

    Strijtveen, Bert; Kellogg, Richard M.; Feringa, Bernard

    1987-01-01

    Methylphosphonic dichloride, CH3P(=O)Cl2, reacts cleanly and quantitatively with thiols to form dialkylthiophosphonates, CH3P(=O)(SR)2. From the ratio of the integrations of the 31P absorptions in the NMR spectra, the enantiomeric excesses of the thiols can be obtained for the cases that R is chiral

  8. Role of lysosomal enzymes released by alveolar macrophages in the pathogenesis of the acute phase of hypersensitivity pneumonitis

    Directory of Open Access Journals (Sweden)

    J. L. Pérez-Arellano

    1995-01-01

    Full Text Available Hydrolytic enzymes are the major constituents of alveolar macrophages (AM and have been shown to be involved in many aspects of the inflammatory pulmonary response. The aim of this study was to evaluate the role of lysosomal enzymes in the acute phase of hypersensitivity pneumonitis (HPs. An experimental study on AM lysosomal enzymes of an HP-guinea-pig model was performed. The results obtained both in vivo and in vitro suggest that intracellular enzymatic activity decrease is, at least partly, due to release of lysosomal enzymes into the medium. A positive but slight correlation was found between extracellular lysosomal activity and four parameters of lung lesion (lung index, bronchoalveolar fluid total (BALF protein concentration, BALF LDH and BALF alkaline phosphatase activities. All the above findings suggest that the AM release of lysosomal enzymes during HP is a factor involved, although possibly not the only one, in the pulmonary lesions appearing in this disease.

  9. In Vivo Evidence for Lysosome Depletion and Impaired Autophagic Clearance in Hereditary Spastic Paraplegia Type SPG11.

    Science.gov (United States)

    Varga, Rita-Eva; Khundadze, Mukhran; Damme, Markus; Nietzsche, Sandor; Hoffmann, Birgit; Stauber, Tobias; Koch, Nicole; Hennings, J Christopher; Franzka, Patricia; Huebner, Antje K; Kessels, Michael M; Biskup, Christoph; Jentsch, Thomas J; Qualmann, Britta; Braulke, Thomas; Kurth, Ingo; Beetz, Christian; Hübner, Christian A

    2015-08-01

    Hereditary spastic paraplegia (HSP) is characterized by a dying back degeneration of corticospinal axons which leads to progressive weakness and spasticity of the legs. SPG11 is the most common autosomal-recessive form of HSPs and is caused by mutations in SPG11. A recent in vitro study suggested that Spatacsin, the respective gene product, is needed for the recycling of lysosomes from autolysosomes, a process known as autophagic lysosome reformation. The relevance of this observation for hereditary spastic paraplegia, however, has remained unclear. Here, we report that disruption of Spatacsin in mice indeed causes hereditary spastic paraplegia-like phenotypes with loss of cortical neurons and Purkinje cells. Degenerating neurons accumulate autofluorescent material, which stains for the lysosomal protein Lamp1 and for p62, a marker of substrate destined to be degraded by autophagy, and hence appears to be related to autolysosomes. Supporting a more generalized defect of autophagy, levels of lipidated LC3 are increased in Spatacsin knockout mouse embryonic fibrobasts (MEFs). Though distinct parameters of lysosomal function like processing of cathepsin D and lysosomal pH are preserved, lysosome numbers are reduced in knockout MEFs and the recovery of lysosomes during sustained starvation impaired consistent with a defect of autophagic lysosome reformation. Because lysosomes are reduced in cortical neurons and Purkinje cells in vivo, we propose that the decreased number of lysosomes available for fusion with autophagosomes impairs autolysosomal clearance, results in the accumulation of undegraded material and finally causes death of particularly sensitive neurons like cortical motoneurons and Purkinje cells in knockout mice.

  10. In Vivo Evidence for Lysosome Depletion and Impaired Autophagic Clearance in Hereditary Spastic Paraplegia Type SPG11.

    Directory of Open Access Journals (Sweden)

    Rita-Eva Varga

    2015-08-01

    Full Text Available Hereditary spastic paraplegia (HSP is characterized by a dying back degeneration of corticospinal axons which leads to progressive weakness and spasticity of the legs. SPG11 is the most common autosomal-recessive form of HSPs and is caused by mutations in SPG11. A recent in vitro study suggested that Spatacsin, the respective gene product, is needed for the recycling of lysosomes from autolysosomes, a process known as autophagic lysosome reformation. The relevance of this observation for hereditary spastic paraplegia, however, has remained unclear. Here, we report that disruption of Spatacsin in mice indeed causes hereditary spastic paraplegia-like phenotypes with loss of cortical neurons and Purkinje cells. Degenerating neurons accumulate autofluorescent material, which stains for the lysosomal protein Lamp1 and for p62, a marker of substrate destined to be degraded by autophagy, and hence appears to be related to autolysosomes. Supporting a more generalized defect of autophagy, levels of lipidated LC3 are increased in Spatacsin knockout mouse embryonic fibrobasts (MEFs. Though distinct parameters of lysosomal function like processing of cathepsin D and lysosomal pH are preserved, lysosome numbers are reduced in knockout MEFs and the recovery of lysosomes during sustained starvation impaired consistent with a defect of autophagic lysosome reformation. Because lysosomes are reduced in cortical neurons and Purkinje cells in vivo, we propose that the decreased number of lysosomes available for fusion with autophagosomes impairs autolysosomal clearance, results in the accumulation of undegraded material and finally causes death of particularly sensitive neurons like cortical motoneurons and Purkinje cells in knockout mice.

  11. In Vivo Evidence for Lysosome Depletion and Impaired Autophagic Clearance in Hereditary Spastic Paraplegia Type SPG11.

    Directory of Open Access Journals (Sweden)

    Rita-Eva Varga

    2015-08-01

    Full Text Available Hereditary spastic paraplegia (HSP is characterized by a dying back degeneration of corticospinal axons which leads to progressive weakness and spasticity of the legs. SPG11 is the most common autosomal-recessive form of HSPs and is caused by mutations in SPG11. A recent in vitro study suggested that Spatacsin, the respective gene product, is needed for the recycling of lysosomes from autolysosomes, a process known as autophagic lysosome reformation. The relevance of this observation for hereditary spastic paraplegia, however, has remained unclear. Here, we report that disruption of Spatacsin in mice indeed causes hereditary spastic paraplegia-like phenotypes with loss of cortical neurons and Purkinje cells. Degenerating neurons accumulate autofluorescent material, which stains for the lysosomal protein Lamp1 and for p62, a marker of substrate destined to be degraded by autophagy, and hence appears to be related to autolysosomes. Supporting a more generalized defect of autophagy, levels of lipidated LC3 are increased in Spatacsin knockout mouse embryonic fibrobasts (MEFs. Though distinct parameters of lysosomal function like processing of cathepsin D and lysosomal pH are preserved, lysosome numbers are reduced in knockout MEFs and the recovery of lysosomes during sustained starvation impaired consistent with a defect of autophagic lysosome reformation. Because lysosomes are reduced in cortical neurons and Purkinje cells in vivo, we propose that the decreased number of lysosomes available for fusion with autophagosomes impairs autolysosomal clearance, results in the accumulation of undegraded material and finally causes death of particularly sensitive neurons like cortical motoneurons and Purkinje cells in knockout mice.

  12. In Vivo Evidence for Lysosome Depletion and Impaired Autophagic Clearance in Hereditary Spastic Paraplegia Type SPG11

    Science.gov (United States)

    Varga, Rita-Eva; Khundadze, Mukhran; Damme, Markus; Nietzsche, Sandor; Hoffmann, Birgit; Stauber, Tobias; Koch, Nicole; Hennings, J. Christopher; Franzka, Patricia; Huebner, Antje K.; Kessels, Michael M.; Biskup, Christoph; Jentsch, Thomas J.; Qualmann, Britta; Braulke, Thomas; Kurth, Ingo; Beetz, Christian; Hübner, Christian A.

    2015-01-01

    Hereditary spastic paraplegia (HSP) is characterized by a dying back degeneration of corticospinal axons which leads to progressive weakness and spasticity of the legs. SPG11 is the most common autosomal-recessive form of HSPs and is caused by mutations in SPG11. A recent in vitro study suggested that Spatacsin, the respective gene product, is needed for the recycling of lysosomes from autolysosomes, a process known as autophagic lysosome reformation. The relevance of this observation for hereditary spastic paraplegia, however, has remained unclear. Here, we report that disruption of Spatacsin in mice indeed causes hereditary spastic paraplegia-like phenotypes with loss of cortical neurons and Purkinje cells. Degenerating neurons accumulate autofluorescent material, which stains for the lysosomal protein Lamp1 and for p62, a marker of substrate destined to be degraded by autophagy, and hence appears to be related to autolysosomes. Supporting a more generalized defect of autophagy, levels of lipidated LC3 are increased in Spatacsin knockout mouse embryonic fibrobasts (MEFs). Though distinct parameters of lysosomal function like processing of cathepsin D and lysosomal pH are preserved, lysosome numbers are reduced in knockout MEFs and the recovery of lysosomes during sustained starvation impaired consistent with a defect of autophagic lysosome reformation. Because lysosomes are reduced in cortical neurons and Purkinje cells in vivo, we propose that the decreased number of lysosomes available for fusion with autophagosomes impairs autolysosomal clearance, results in the accumulation of undegraded material and finally causes death of particularly sensitive neurons like cortical motoneurons and Purkinje cells in knockout mice. PMID:26284655

  13. Selective chromogenic detection of thiol-containing biomolecules using carbonaceous nanospheres loaded with silver nanoparticles as carrier.

    Science.gov (United States)

    Hu, Bo; Zhao, Yang; Zhu, Hai-Zhou; Yu, Shu-Hong

    2011-04-26

    Thiol-containing biomolecules show strong affinity with noble metal nanostructures and could not only stably protect them but also control the self-assembly process of these special nanostructures. A highly selective and sensitive chromogenic detection method has been designed for the low and high molecular weight thiol-containing biomolecules, including cysteine, glutathione, dithiothreitol, and bovine serum albumin, using a new type of carbonaceous nanospheres loaded with silver nanoparticles (Ag NPs) as carrier. This strategy relies upon the place-exchange process between the reporter dyes on the surface of Ag NPs and the thiol groups of thiol-containing biomolecules. The concentration of biomolecules can be determined by monitoring with the fluorescence intensity of reporter dyes dispersed in solution. This new chromogenic assay method could selectively detect these biomolecules in the presence of various other amino acids and monosaccharides and even sensitively detect the thiol-containing biomolecules with different molecular weight, even including proteins.

  14. Preparation of a novel carboxyl stationary phase by "thiol-ene" click chemistry for hydrophilic interaction chromatography.

    Science.gov (United States)

    Peng, Xi-Tian; Liu, Tao; Ji, Shu-Xian; Feng, Yu-Qi

    2013-08-01

    A novel carboxyl-bonded silica stationary phase was prepared by "thiol-ene" click chemistry. The resultant Thiol-Click-COOH phase was evaluated under hydrophilic interaction liquid chromatography (HILIC) mobile phase conditions. A comparison of the chromatographic performance of Thiol-Click-COOH and pure silica columns was performed according to the retention behaviors of analytes and the charged state of the stationary phases. The results indicated that the newly developed Thiol-Click-COOH column has a higher surface charge and stronger hydrophilicity than the pure silica column. Furthermore, the chromatographic behaviors of five nucleosides on the Thiol-Click-COOH phase were investigated in detail. Finally, a good separation of 13 nucleosides and bases, and four water-soluble vitamins was achieved.

  15. The Nox/Ferric reductase/Ferric reductase-like families of Eumycetes.

    Science.gov (United States)

    Grissa, Ibtissem; Bidard, Frédérique; Grognet, Pierre; Grossetete, Sandrine; Silar, Philippe

    2010-09-01

    Reactive Oxygen Species (ROS) are involved in plant biomass degradation by fungi and development of fungal structures. While the ROS-generating NADPH oxidases from filamentous fungi are under strong scrutiny, much less is known about the related integral Membrane (or Ferric) Reductases (IMRs). Here, we present a survey of these enzymes in 29 fungal genomes covering the entire available range of fungal diversity. IMRs are present in all fungal genomes. They can be classified into at least 24 families, underscoring the high diversity of these enzymes. Some are differentially regulated during colony or fruiting body development, as well as by the nature of the carbon source of the growth medium. Importantly, functional characterization of IMRs has been made on proteins belonging to only two families, while nothing or very little is known about the proteins of the other 22 families.

  16. TPC1 Has Two Variant Isoforms, and Their Removal Has Different Effects on Endo-Lysosomal Functions Compared to Loss of TPC2

    OpenAIRE

    Ruas, Margarida; Chuang, Kai-Ting; Davis, Lianne C.; Al-Douri, Areej; Tynan, Patricia W.; Tunn, Ruth; Teboul, Lydia; Galione, Antony; Parrington, John

    2014-01-01

    Organelle ion homeostasis within the endo-lysosomal system is critical for physiological functions. Two-pore channels (TPCs) are cation channels that reside in endo-lysosomal organelles, and overexpression results in endo-lysosomal trafficking defects. However, the impact of a lack of TPC expression on endo-lysosomal trafficking is unknown. Here, we characterize Tpcn1 expression in two transgenic mouse lines (Tpcn1 XG716 and Tpcn1 T159) and show expression of a novel evolutionarily conserved ...

  17. Involvement of lysosomal dysfunction in autophagosome accumulation and early pathologies in adipose tissue of obese mice.

    Science.gov (United States)

    Mizunoe, Yuhei; Sudo, Yuka; Okita, Naoyuki; Hiraoka, Hidenori; Mikami, Kentaro; Narahara, Tomohiro; Negishi, Arisa; Yoshida, Miki; Higashibata, Rikako; Watanabe, Shukoh; Kaneko, Hiroki; Natori, Daiki; Furuichi, Takuma; Yasukawa, Hiromine; Kobayashi, Masaki; Higami, Yoshikazu

    2017-04-03

    Whether obesity accelerates or suppresses autophagy in adipose tissue is still debatable. To clarify dysregulation of autophagy and its role in pathologies of obese adipose tissue, we focused on lysosomal function, protease maturation and activity, both in vivo and in vitro. First, we showed that autophagosome formation was accelerated, but autophagic clearance was impaired in obese adipose tissue. We also found protein and activity levels of CTSL (cathepsin L) were suppressed in obese adipose tissue, while the activity of CTSB (cathepsin B) was significantly enhanced. Moreover, cellular senescence and inflammasomes were activated in obese adipose tissue. In 3T3L1 adipocytes, downregulation of CTSL deteriorated autophagic clearance, upregulated expression of CTSB, promoted cellular senescence and activated inflammasomes. Upregulation of CTSB promoted additional activation of inflammasomes. Therefore, we suggest lysosomal dysfunction observed in obese adipose tissue leads to lower autophagic clearance, resulting in autophagosome accumulation. Simultaneously, lysosomal abnormalities, including deteriorated CTSL function and compensatory activation of CTSB, caused cellular senescence and inflammasome activation. Our findings strongly suggest lysosomal dysfunction is involved in early pathologies of obese adipose tissue.

  18. Structural and functional analysis of lysosomal ss-galactosidase and its relation to the protective protein.

    NARCIS (Netherlands)

    H. Morreau (Hans)

    1992-01-01

    textabstractLysosomal B-galactosidase is the glycosidase, that cleaves B-linked galactosyl mmenes from a variety of natural and synthetic substrates. In normal tissues of various species this enzyme appears to associate with two other hydrolases, N-acetyl-o:-neuraminidase and the protective protein.

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

    Directory of Open Access Journals (Sweden)

    Viorica Ivan

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

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

    Science.gov (United States)

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

    2001-01-01

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