Loss of the interferon-γ-inducible regulatory immunity-related GTPase (IRG), Irgm1, causes activation of effector IRG proteins on lysosomes, damaging lysosomal function and predicting the dramatic susceptibility of Irgm1-deficient mice to infection.

Science.gov (United States)

Maric-Biresev, Jelena; Hunn, Julia P; Krut, Oleg; Helms, J Bernd; Martens, Sascha; Howard, Jonathan C

2016-04-20

The interferon-γ (IFN-γ)-inducible immunity-related GTPase (IRG), Irgm1, plays an essential role in restraining activation of the IRG pathogen resistance system. However, the loss of Irgm1 in mice also causes a dramatic but unexplained susceptibility phenotype upon infection with a variety of pathogens, including many not normally controlled by the IRG system. This phenotype is associated with lymphopenia, hemopoietic collapse, and death of the mouse. We show that the three regulatory IRG proteins (GMS sub-family), including Irgm1, each of which localizes to distinct sets of endocellular membranes, play an important role during the cellular response to IFN-γ, each protecting specific membranes from off-target activation of effector IRG proteins (GKS sub-family). In the absence of Irgm1, which is localized mainly at lysosomal and Golgi membranes, activated GKS proteins load onto lysosomes, and are associated with reduced lysosomal acidity and failure to process autophagosomes. Another GMS protein, Irgm3, is localized to endoplasmic reticulum (ER) membranes; in the Irgm3-deficient mouse, activated GKS proteins are found at the ER. The Irgm3-deficient mouse does not show the drastic phenotype of the Irgm1 mouse. In the Irgm1/Irgm3 double knock-out mouse, activated GKS proteins associate with lipid droplets, but not with lysosomes, and the Irgm1/Irgm3(-/-) does not have the generalized immunodeficiency phenotype expected from its Irgm1 deficiency. The membrane targeting properties of the three GMS proteins to specific endocellular membranes prevent accumulation of activated GKS protein effectors on the corresponding membranes and thus enable GKS proteins to distinguish organellar cellular membranes from the membranes of pathogen vacuoles. Our data suggest that the generalized lymphomyeloid collapse that occurs in Irgm1(-/-) mice upon infection with a variety of pathogens may be due to lysosomal damage caused by off-target activation of GKS proteins on lysosomal

Distinguishing normal cells from cancer cells via lysosome-targetable pH biomarkers with benzo[a]phenoxazine skeleton

Energy Technology Data Exchange (ETDEWEB)

Zhan, Yan-Hua [College of Chemistry, Chemical Engineering and Material Science, State and Local Joint Engineering Laboratory for Novel Functional Polymeric Materials, Soochow University, 199 Ren’Ai Road, Suzhou, 215123 (China); Li, Xiao-Jun [School of Radiation Medicine and Protection, Medicine College of Soochow University, Suzhou, 215123 (China); Sun, Ru, E-mail: sunru924@hotmail.com [College of Chemistry, Chemical Engineering and Material Science, State and Local Joint Engineering Laboratory for Novel Functional Polymeric Materials, Soochow University, 199 Ren’Ai Road, Suzhou, 215123 (China); Xu, Yu-Jie [School of Radiation Medicine and Protection, Medicine College of Soochow University, Suzhou, 215123 (China); Ge, Jian-Feng, E-mail: ge_jianfeng@hotmail.com [College of Chemistry, Chemical Engineering and Material Science, State and Local Joint Engineering Laboratory for Novel Functional Polymeric Materials, Soochow University, 199 Ren’Ai Road, Suzhou, 215123 (China); Jiangsu Key Laboratory of Medical Optics, Suzhou Institute of Biomedical Engineering and Technology, Chinese Academy of Sciences, Suzhou, 215163 (China)

2016-08-24

In this paper, the design of a lysosome-targetable pH probe that has a fluorescent OFF (pH = 4) to ON (pH = 5–6) response is described to identify lysosomes in normal cells. The mechanism of photoinduced electron transfer with a fluorophore-based reaction (FBR-PET) was proposed. Benzo[a]phenoxazines with electro-donating aryl groups were selected, its (2,5-dimethoxyphenyl)imino-, (2-hydroxyphenyl)imino- and (2-hydroxy-5-methoxyphenyl)- imino-derivatives (probes 1a−c) were prepared and their optical responses towards pH were evaluated; their fluorescence pH titration experiments gave regularly changes with the increasing electro-donating abilities at the linked aryl groups, the (2-hydroxy-5-methoxyphenyl)iminobenzo[a]phenoxazine (probe 1c) exhibited a nearly OFF−ON response at 580–800 nm. All probes were reversible, and they showed excellent selectivity toward the proton over other competitive species. Fluorescence confocal images were performed with HeLa, KB cancer cells and V79 normal cells, probes 1a−c are all lysosome-targetable pH probes, and benzo[a]phenoxazine with (2-hydroxy-5-methoxyphenyl)imino-group (probe 1c) has potential applications in selective differentiation of normal cells from cancer cells. - Highlights: • pH probes for lysosome detection in normal cells. • Differentiation of normal cells from cancer cells by lysosome-biomarker. • The PET mechanism promoted by fluorophore based reactions (FBR-PET).

Distinguishing normal cells from cancer cells via lysosome-targetable pH biomarkers with benzo[a]phenoxazine skeleton

International Nuclear Information System (INIS)

Zhan, Yan-Hua; Li, Xiao-Jun; Sun, Ru; Xu, Yu-Jie; Ge, Jian-Feng

2016-01-01

In this paper, the design of a lysosome-targetable pH probe that has a fluorescent OFF (pH = 4) to ON (pH = 5–6) response is described to identify lysosomes in normal cells. The mechanism of photoinduced electron transfer with a fluorophore-based reaction (FBR-PET) was proposed. Benzo[a]phenoxazines with electro-donating aryl groups were selected, its (2,5-dimethoxyphenyl)imino-, (2-hydroxyphenyl)imino- and (2-hydroxy-5-methoxyphenyl)- imino-derivatives (probes 1a−c) were prepared and their optical responses towards pH were evaluated; their fluorescence pH titration experiments gave regularly changes with the increasing electro-donating abilities at the linked aryl groups, the (2-hydroxy-5-methoxyphenyl)iminobenzo[a]phenoxazine (probe 1c) exhibited a nearly OFF−ON response at 580–800 nm. All probes were reversible, and they showed excellent selectivity toward the proton over other competitive species. Fluorescence confocal images were performed with HeLa, KB cancer cells and V79 normal cells, probes 1a−c are all lysosome-targetable pH probes, and benzo[a]phenoxazine with (2-hydroxy-5-methoxyphenyl)imino-group (probe 1c) has potential applications in selective differentiation of normal cells from cancer cells. - Highlights: • pH probes for lysosome detection in normal cells. • Differentiation of normal cells from cancer cells by lysosome-biomarker. • The PET mechanism promoted by fluorophore based reactions (FBR-PET).

Podocytes degrade endocytosed albumin primarily in lysosomes.

Science.gov (United States)

Carson, John M; Okamura, Kayo; Wakashin, Hidefumi; McFann, Kim; Dobrinskikh, Evgenia; Kopp, Jeffrey B; Blaine, Judith

2014-01-01

Albuminuria is a strong, independent predictor of chronic kidney disease progression. We hypothesize that podocyte processing of albumin via the lysosome may be an important determinant of podocyte injury and loss. A human urine derived podocyte-like epithelial cell (HUPEC) line was used for in vitro experiments. Albumin uptake was quantified by Western blot after loading HUPECs with fluorescein-labeled (FITC) albumin. Co-localization of albumin with lysosomes was determined by confocal microscopy. Albumin degradation was measured by quantifying FITC-albumin abundance in HUPEC lysates by Western blot. Degradation experiments were repeated using HUPECs treated with chloroquine, a lysosome inhibitor, or MG-132, a proteasome inhibitor. Lysosome activity was measured by fluorescence recovery after photo bleaching (FRAP). Cytokine production was measured by ELISA. Cell death was determined by trypan blue staining. In vivo, staining with lysosome-associated membrane protein-1 (LAMP-1) was performed on tissue from a Denys-Drash trangenic mouse model of nephrotic syndrome. HUPECs endocytosed albumin, which co-localized with lysosomes. Choloroquine, but not MG-132, inhibited albumin degradation, indicating that degradation occurs in lysosomes. Cathepsin B activity, measured by FRAP, significantly decreased in HUPECs exposed to albumin (12.5% of activity in controls) and chloroquine (12.8%), and declined further with exposure to albumin plus chloroquine (8.2%, plysosomes are involved in the processing of endocytosed albumin in podocytes, and lysosomal dysfunction may contribute to podocyte injury and glomerulosclerosis in albuminuric diseases. Modifiers of lysosomal activity may have therapeutic potential in slowing the progression of glomerulosclerosis by enhancing the ability of podocytes to process and degrade albumin.

Effect of whole-body X-irradiation on lysosomal enzymes

Energy Technology Data Exchange (ETDEWEB)

D' souza, D W; Vakil, U K; Srinivasan, A [Bhabha Atomic Research Centre, Bombay (India). Biochemistry and Food Technology Div.

1974-06-01

Effects of whole-body x irradiation with sublethal dose (400 rad) on three intestinal lysosomal enzymes, namely, arylsulphatase, cathepsin and acid phosphatases, have been studied. They are almost equally distributed throughout the entire small intestine region. X irradiation adversely affects the integrity of lysosomal membranes. ''Free'' and ''total'' lysosomal enzyme activities exhibit maxima on 6th day. These activities return to normal level on 14th day when there is rapid generation of villi, indicating that lysosomal activities correlate with the progression of injury and of repair mechanism after sublethal dose of x irradiation. The increase in total lysosomal activity may be due to its decreased breakdown, since the rate of protein synthesis in intestinal mucosa is reduced. This is evidenced by reduced incorporation of orally fed /sup 14/C leucine into acid insoluble proteins. (auth)

Acidic nanoparticles are trafficked to lysosomes and restore an acidic lysosomal pH and degradative function to compromised ARPE-19 cells.

Directory of Open Access Journals (Sweden)

Gabriel C Baltazar

Full Text Available Lysosomal enzymes function optimally in acidic environments, and elevation of lysosomal pH can impede their ability to degrade material delivered to lysosomes through autophagy or phagocytosis. We hypothesize that abnormal lysosomal pH is a key aspect in diseases of accumulation and that restoring lysosomal pH will improve cell function. The propensity of nanoparticles to end up in the lysosome makes them an ideal method of delivering drugs to lysosomes. This study asked whether acidic nanoparticles could traffic to lysosomes, lower lysosomal pH and enhance lysosomal degradation by the cultured human retinal pigmented epithelial cell line ARPE-19. Acidic nanoparticles composed of poly (DL-lactide-co-glycolide (PLGA 502 H, PLGA 503 H and poly (DL-lactide (PLA colocalized to lysosomes of ARPE-19 cells within 60 min. PLGA 503 H and PLA lowered lysosomal pH in cells compromised by the alkalinizing agent chloroquine when measured 1 hr. after treatment, with acidification still observed 12 days later. PLA enhanced binding of Bodipy-pepstatin-A to the active site of cathepsin D in compromised cells. PLA also reduced the cellular levels of opsin and the lipofuscin-like autofluorescence associated with photoreceptor outer segments. These observations suggest the acidification produced by the nanoparticles was functionally effective. In summary, acid nanoparticles lead to a rapid and sustained lowering of lysosomal pH and improved degradative activity.

Cocaine induces a mixed lysosomal lipidosis in cultured fibroblasts, by inactivation of acid sphingomyelinase and inhibition of phospholipase A1

International Nuclear Information System (INIS)

Nassogne, Marie-Cecile; Lizarraga, Chantal; N'Kuli, Francisca; Van Bambeke, Francoise; Van Binst, Roger; Wallemacq, Pierre; Tulkens, Paul M.; Mingeot-Leclercq, Marie-Paule; Levade, Thierry; Courtoy, Pierre J.

2004-01-01

This paper reports that cocaine may induce a lysosomal storage disorder. Indeed, culture of Rat-1 fibroblasts with 250-500 μM cocaine induced after 2-3 days a major accumulation in lysosomes of electron-dense lamellar structures. By subcellular fractionation, this was reflected by a selective decrease of the buoyant density of several lysosomal enzymes, indicating lysosomal lipid overload. Biochemical analysis confirmed an increased cellular content of major phospholipids and sphingomyelin, but not of cholesterol. Cocaine, a membrane-permeant weak base, is concentrated by acidotropic sequestration, because its accumulation was abrogated by the proton ionophore, monensin and the vacuolar ATPase inhibitor, bafilomycin A 1 . At its estimated lysosomal concentration, cocaine almost completely inhibited phospholipase A 1 activity on liposomes. Cell incubation with cocaine, but not with its inactive metabolite, benzoylecgonine, rapidly inactivated acid sphingomyelinase, as reflected by a 10-fold decrease in V max with identical K m . Acid sphingomyelinase inactivation was fully prevented by the thiol proteinases inhibitors, leupeptin and E64, indicating that cocaine induces selective sphingomyelinase proteolysis. Upon cocaine removal, acid sphingomyelinase activity was rapidly restored, pointing to its fast turnover. In contrast, the cellular content of several other lysosomal hydrolases was increased up to 2-fold. Together, these data show that acidotropic accumulation of cocaine in lysosomes rapidly inhibits acid phospholipase A 1 and inactivates acid sphingomyelinase, which can explain induction of a mixed lysosomal lipidosis

Functional analysis of lysosomes during mouse preimplantation embryo development.

Science.gov (United States)

Tsukamoto, Satoshi; Hara, Taichi; Yamamoto, Atsushi; Ohta, Yuki; Wada, Ayako; Ishida, Yuka; Kito, Seiji; Nishikawa, Tetsu; Minami, Naojiro; Sato, Ken; Kokubo, Toshiaki

2013-01-01

Lysosomes are acidic and highly dynamic organelles that are essential for macromolecule degradation and many other cellular functions. However, little is known about lysosomal function during early embryogenesis. Here, we found that the number of lysosomes increased after fertilization. Lysosomes were abundant during mouse preimplantation development until the morula stage, but their numbers decreased slightly in blastocysts. Consistently, the protein expression level of mature cathepsins B and D was high from the one-cell to morula stages but low in the blastocyst stage. One-cell embryos injected with siRNAs targeted to both lysosome-associated membrane protein 1 and 2 (LAMP1 and LAMP2) were developmentally arrested at the two-cell stage. Pharmacological inhibition of lysosomes also caused developmental retardation, resulting in accumulation of lipofuscin. Our findings highlight the functional changes in lysosomes in mouse preimplantation embryos.

Ten-year technical and clinical outcomes in TransAtlantic Inter-Society Consensus II infrainguinal C/D lesions using duplex ultrasound arterial mapping as the sole imaging modality for critical lower limb ischemia.

LENUS (Irish Health Repository)

Sultan, Sherif

2013-04-01

The aim of this study was to evaluate duplex ultrasound arterial mapping (DUAM) as the sole imaging modality when planning for bypass surgery (BS) and endovascular revascularization (EvR) in patients with critical limb ischemia for TransAtlantic Inter-Society Consensus (TASC) II C\\/D infrainguinal lesions.

Autophagic dysfunction in a lysosomal storage disorder due to impaired proteolysis.

Science.gov (United States)

Elrick, Matthew J; Lieberman, Andrew P

2013-02-01

Alterations in macroautophagy (hereafter referred to as "autophagy") are a common feature of lysosomal storage disorders, and have been hypothesized to play a major role in the pathogenesis of these diseases. We have recently reported multiple defects in autophagy contributing to the lysosomal storage disorder Niemann-Pick type C (NPC). These include increased formation of autophagosomes, slowed turnover of autophagosomes secondary to impaired lysosomal proteolysis, and delivery of stored lipids to the lysosome via autophagy. The study summarized here describes novel methods for the interrogation of individual stages of the autophagic pathway, and suggests mechanisms by which lipid storage may result in broader lysosomal dysfunction.

Lysosomes shape Ins(1,4,5)P3-evoked Ca2+ signals by selectively sequestering Ca2+ released from the endoplasmic reticulum

Science.gov (United States)

López-Sanjurjo, Cristina I.; Tovey, Stephen C.; Prole, David L.; Taylor, Colin W.

2013-01-01

Summary Most intracellular Ca2+ signals result from opening of Ca2+ channels in the plasma membrane or endoplasmic reticulum (ER), and they are reversed by active transport across these membranes or by shuttling Ca2+ into mitochondria. Ca2+ channels in lysosomes contribute to endo-lysosomal trafficking and Ca2+ signalling, but the role of lysosomal Ca2+ uptake in Ca2+ signalling is unexplored. Inhibition of lysosomal Ca2+ uptake by dissipating the H+ gradient (using bafilomycin A1), perforating lysosomal membranes (using glycyl-L-phenylalanine 2-naphthylamide) or lysosome fusion (using vacuolin) increased the Ca2+ signals evoked by receptors that stimulate inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] formation. Bafilomycin A1 amplified the Ca2+ signals evoked by photolysis of caged Ins(1,4,5)P3 or by inhibition of ER Ca2+ pumps, and it slowed recovery from them. Ca2+ signals evoked by store-operated Ca2+ entry were unaffected by bafilomycin A1. Video-imaging with total internal reflection fluorescence microscopy revealed that lysosomes were motile and remained intimately associated with the ER. Close association of lysosomes with the ER allows them selectively to accumulate Ca2+ released by Ins(1,4,5)P3 receptors. PMID:23097044

Conversion of diphosphatidylglycerol to bis(monoacylglyceryl)phosphate by lysosomes

NARCIS (Netherlands)

Poorthuis, B. J.; Hostetler, K. Y.

1978-01-01

Diphosphatidyl[1',2',3'-14C]glycerol (cardiolipin) is converted to bis(monoacylglyceryl)phosphate when incubated in vitro with rat lysosomes at pH 4.4. The stereochemical configuration of the product is unknown. This reaction probably takes place via lysophosphatidylglycerol, one of the major

Basophil sensitivity through CD63 or CD203c is a functional measure for specific immunotherapy

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Dahl Ronald

2010-02-01

Full Text Available Abstract Background Subcutaneous Immunotherapy (SCIT modifies the allergic response and relieves allergic symptoms. SCIT is the only and a very effective treatment for insect venom allergy. We hypothesized that basophil sensitivity, measured through the basophil activation test, would decrease during SCIT up dosing. Expression of CD203c was compared to CD63 as marker for basophil activation, using a Bland Altman plot and ROC curves. Methods Patients (n = 18 starting subcutaneous SCIT for wasp allergy with an up dosing scheme of 7 to 11 weeks were enrolled. Heparinised blood samples were drawn at weeks 1-4, 7 and at the first maintenance visit. Basophils were stimulated at 7 log dilutions of V. vespula allergen for 15 min, and were stained with CD203c and CD63. Basophils were identified as CD203c+ leukocytes, and the proportion of CD63+ and CD203c+ cells were plotted against allergen concentration. A sigmoid curve was fitted to the points, and the allergen concentration at which half of the maximal activation was achieved, LC50, was calculated. In another series of experiments, LC50 calculated in whole blood (AP was subtracted from LC50 calculated with basophils suspended in plasma from a nonatopic donor (HS to determine the protective effect of soluble factors in blood of patients treated with SCIT. Results Heparin blood basophil activation was similar through CD63 and CD203c. Basophils were significantly more sensitized three weeks after initiation of SCIT compared to baseline (p Conclusion Basophil activation is a versatile and sensitive tool that measures changes in the humoral immune response to allergen during SCIT.

Lysosomal Re-acidification Prevents Lysosphingolipid-Induced Lysosomal Impairment and Cellular Toxicity.

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Christopher J Folts

2016-12-01

Full Text Available Neurodegenerative lysosomal storage disorders (LSDs are severe and untreatable, and mechanisms underlying cellular dysfunction are poorly understood. We found that toxic lipids relevant to three different LSDs disrupt multiple lysosomal and other cellular functions. Unbiased drug discovery revealed several structurally distinct protective compounds, approved for other uses, that prevent lysosomal and cellular toxicities of these lipids. Toxic lipids and protective agents show unexpected convergence on control of lysosomal pH and re-acidification as a critical component of toxicity and protection. In twitcher mice (a model of Krabbe disease [KD], a central nervous system (CNS-penetrant protective agent rescued myelin and oligodendrocyte (OL progenitors, improved motor behavior, and extended lifespan. Our studies reveal shared principles relevant to several LSDs, in which diverse cellular and biochemical disruptions appear to be secondary to disruption of lysosomal pH regulation by specific lipids. These studies also provide novel protective strategies that confer therapeutic benefits in a mouse model of a severe LSD.

H-1, C-13 and N-15 resonance assignments of human DCL-1 (CD302) extracellular domain

Czech Academy of Sciences Publication Activity Database

Pospíšilová, Eliška; Kavan, Daniel; Novák, Petr; Chmelík, Josef

2016-01-01

Roč. 10, č. 1 (2016), s. 189-192 ISSN 1874-2718 R&D Projects: GA MŠk(CZ) LO1509; GA MŠk(CZ) EE2.3.20.0055; GA MŠk(CZ) EE2.3.30.0003; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:61388971 Keywords : DCL-1 (CD302) * C-type lectin like receptor * Protein NMR Subject RIV: EE - Microbiology, Virology Impact factor: 0.459, year: 2016

Lipidomic and Transcriptomic Basis of Lysosomal Dysfunction in Progranulin Deficiency

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Bret M. Evers

2017-09-01

Full Text Available Defective lysosomal function defines many neurodegenerative diseases, such as neuronal ceroid lipofuscinoses (NCL and Niemann-Pick type C (NPC, and is implicated in Alzheimer’s disease (AD and frontotemporal lobar degeneration (FTLD-TDP with progranulin (PGRN deficiency. Here, we show that PGRN is involved in lysosomal homeostasis and lipid metabolism. PGRN deficiency alters lysosome abundance and morphology in mouse neurons. Using an unbiased lipidomic approach, we found that brain lipid composition in humans and mice with PGRN deficiency shows disease-specific differences that distinguish them from normal and other pathologic groups. PGRN loss leads to an accumulation of polyunsaturated triacylglycerides, as well as a reduction of diacylglycerides and phosphatidylserines in fibroblast and enriched lysosome lipidomes. Transcriptomic analysis of PGRN-deficient mouse brains revealed distinct expression patterns of lysosomal, immune-related, and lipid metabolic genes. These findings have implications for the pathogenesis of FTLD-TDP due to PGRN deficiency and suggest lysosomal dysfunction as an underlying mechanism.

Drosophila Vps16A is required for trafficking to lysosomes and biogenesis of pigment granules.

Science.gov (United States)

Pulipparacharuvil, Suprabha; Akbar, Mohammed Ali; Ray, Sanchali; Sevrioukov, Evgueny A; Haberman, Adam S; Rohrer, Jack; Krämer, Helmut

2005-08-15

Mutations that disrupt trafficking to lysosomes and lysosome-related organelles cause multiple diseases, including Hermansky-Pudlak syndrome. The Drosophila eye is a model system for analyzing such mutations. The eye-color genes carnation and deep orange encode two subunits of the Vps-C protein complex required for endosomal trafficking and pigment-granule biogenesis. Here we demonstrate that dVps16A (CG8454) encodes another Vps-C subunit. Biochemical experiments revealed a specific interaction between the dVps16A C-terminus and the Sec1/Munc18 homolog Carnation but not its closest homolog, dVps33B. Instead, dVps33B interacted with a related protein, dVps16B (CG18112). Deep orange bound both Vps16 homologs. Like a deep orange null mutation, eye-specific RNAi-induced knockdown of dVps16A inhibited lysosomal delivery of internalized ligands and interfered with biogenesis of pigment granules. Ubiquitous knockdown of dVps16A was lethal. Together, these findings demonstrate that Drosophila Vps16A is essential for lysosomal trafficking. Furthermore, metazoans have two types of Vps-C complexes with non-redundant functions.

Preparation of Proper Immunogen by Cloning and Stable Expression of cDNA coding for Human Hematopoietic Stem Cell Marker CD34 in NIH-3T3 Mouse Fibroblast Cell Line

Science.gov (United States)

Shafaghat, Farzaneh; Abbasi-Kenarsari, Hajar; Majidi, Jafar; Movassaghpour, Ali Akbar; Shanehbandi, Dariush; Kazemi, Tohid

2015-01-01

Purpose: Transmembrane CD34 glycoprotein is the most important marker for identification, isolation and enumeration of hematopoietic stem cells (HSCs). We aimed in this study to clone the cDNA coding for human CD34 from KG1a cell line and stably express in mouse fibroblast cell line NIH-3T3. Such artificial cell line could be useful as proper immunogen for production of mouse monoclonal antibodies. Methods: CD34 cDNA was cloned from KG1a cell line after total RNA extraction and cDNA synthesis. Pfu DNA polymerase-amplified specific band was ligated to pGEMT-easy TA-cloning vector and sub-cloned in pCMV6-Neo expression vector. After transfection of NIH-3T3 cells using 3 μg of recombinant construct and 6 μl of JetPEI transfection reagent, stable expression was obtained by selection of cells by G418 antibiotic and confirmed by surface flow cytometry. Results: 1158 bp specific band was aligned completely to reference sequence in NCBI database corresponding to long isoform of human CD34. Transient and stable expression of human CD34 on transfected NIH-3T3 mouse fibroblast cells was achieved (25% and 95%, respectively) as shown by flow cytometry. Conclusion: Cloning and stable expression of human CD34 cDNA was successfully performed and validated by standard flow cytometric analysis. Due to murine origin of NIH-3T3 cell line, CD34-expressing NIH-3T3 cells could be useful as immunogen in production of diagnostic monoclonal antibodies against human CD34. This approach could bypass the need for purification of recombinant proteins produced in eukaryotic expression systems. PMID:25789221

Bypass of a 5',8-cyclopurine-2'-deoxynucleoside by DNA polymerase β during DNA replication and base excision repair leads to nucleotide misinsertions and DNA strand breaks.

Science.gov (United States)

Jiang, Zhongliang; Xu, Meng; Lai, Yanhao; Laverde, Eduardo E; Terzidis, Michael A; Masi, Annalisa; Chatgilialoglu, Chryssostomos; Liu, Yuan

2015-09-01

5',8-Cyclopurine-2'-deoxynucleosides including 5',8-cyclo-dA (cdA) and 5',8-cyclo-dG (cdG) are induced by hydroxyl radicals resulting from oxidative stress such as ionizing radiation. 5',8-cyclopurine-2'-deoxynucleoside lesions are repaired by nucleotide excision repair with low efficiency, thereby leading to their accumulation in the human genome and lesion bypass by DNA polymerases during DNA replication and base excision repair (BER). In this study, for the first time, we discovered that DNA polymerase β (pol β) efficiently bypassed a 5'R-cdA, but inefficiently bypassed a 5'S-cdA during DNA replication and BER. We found that cell extracts from pol β wild-type mouse embryonic fibroblasts exhibited significant DNA synthesis activity in bypassing a cdA lesion located in replication and BER intermediates. However, pol β knock-out cell extracts exhibited little DNA synthesis to bypass the lesion. This indicates that pol β plays an important role in bypassing a cdA lesion during DNA replication and BER. Furthermore, we demonstrated that pol β inserted both a correct and incorrect nucleotide to bypass a cdA at a low concentration. Nucleotide misinsertion was significantly stimulated by a high concentration of pol β, indicating a mutagenic effect induced by pol β lesion bypass synthesis of a 5',8-cyclopurine-2'-deoxynucleoside. Moreover, we found that bypass of a 5'S-cdA by pol β generated an intermediate that failed to be extended by pol β, resulting in accumulation of single-strand DNA breaks. Our study provides the first evidence that pol β plays an important role in bypassing a 5',8-cyclo-dA during DNA replication and repair, as well as new insight into mutagenic effects and genome instability resulting from pol β bypassing of a cdA lesion. Copyright © 2015 Elsevier B.V. All rights reserved.

5-FU resistant EMT-like pancreatic cancer cells are hypersensitive to photochemical internalization of the novel endoglin-targeting immunotoxin CD105-saporin.

Science.gov (United States)

Lund, Kaja; Olsen, Cathrine Elisabeth; Wong, Judith Jing Wen; Olsen, Petter Angell; Solberg, Nina Therese; Høgset, Anders; Krauss, Stefan; Selbo, Pål Kristian

2017-12-19

Development of resistance to 5-fluorouracil (5-FU) is a major problem in treatment of various cancers including pancreatic cancer. In this study, we reveal important resistance mechanisms and photochemical strategies to overcome 5-FU resistance in pancreatic adenocarcinoma. 5-FU resistant (5-FUR), epithelial-to-mesenchymal-like sub-clones of the wild type pancreatic cancer cell line Panc03.27 were previously generated in our lab. We investigated the cytotoxic effect of the endosomal/lysosomal-localizing photosensitizer TPCS 2a (fimaporfin) combined with light (photochemical treatment, PCT) using MTS viability assay, and used fluorescence microscopy to show localization of TPCS 2a and to investigate the effect of photodamage of lysosomes. Flow cytometric analysis was performed to investigate uptake of photosensitizer and to assess intracellular ROS levels. Expression and localization of LAMP1 was assessed using RT-qPCR, western blotting, and structured illumination microscopy. MTS viability assay was used to assess the effect of combinations of 5-FU, chloroquine (CQ), and photochemical treatment. Expression of CD105 was investigated using RT-qPCR, western blotting, flow cytometry, and fluorescence microscopy, and co-localization of TPCS 2a and anti-CD105-saporin was assessed using microscopy. Lastly, the MTS assay was used to investigate cytotoxic effects of photochemical internalization (PCI) of the anti-CD105-immunotoxin. The 5-FUR cell lines display hypersensitivity to PCT, which was linked to increased uptake of TPCS 2a , altered lysosomal distribution, lysosomal photodamage and increased expression of the lysosomal marker LAMP-1 in the 5-FUR cells. We show that inhibition of autophagy induced by either chloroquine or lysosomal photodamage increases the sensitivity to 5-FU in the resistant cells. The three 5-FUR sub-clones overexpress Endoglin (CD105). Treatment with the immunotoxin anti-CD105-saporin alone significantly reduced the viability of the CD105

Autophagic dysfunction in a lysosomal storage disorder due to impaired proteolysis

OpenAIRE

Elrick, Matthew J.; Lieberman, Andrew P.

2013-01-01

Alterations in macroautophagy (hereafter referred to as “autophagy”) are a common feature of lysosomal storage disorders, and have been hypothesized to play a major role in the pathogenesis of these diseases. We have recently reported multiple defects in autophagy contributing to the lysosomal storage disorder Niemann-Pick type C (NPC). These include increased formation of autophagosomes, slowed turnover of autophagosomes secondary to impaired lysosomal proteolysis, and delivery of stored lip...

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....

Pathogenic mechanisms in lysosomal disease: a reappraisal of the role of the lysosome.

Science.gov (United States)

Walkley, Steven U

2007-04-01

The view that lysosomes simply represent end organelles in the serial degradation of polymeric molecules derived from the cell surface and its interior has led to major misconceptions about the nature of lysosomal storage diseases and the pathogenic cascades that characterize them. Accordingly, lysosomal storage bodies are often considered 'inert', inducing cell dysfunction and death primarily through mechanical overcrowding of normal organelles or by other non-specific means leading to generalized cytotoxicity. However, modern studies of lysosomes and their component proteins provide evidence to support a far greater role for these organelles in cell metabolism. In intimate association with endosomal, autophagosomal and related vesicular systems, the greater lysosomal system can be conceptualized as a vital recycling centre that serves as a central metabolic coordinator, influencing literally every aspect of the cell, from signal transduction to regulation of gene expression. This broader view of the role of lysosomes in cells not only provides insight into how single gene defects impacting on lysosomal function can result in the plethora of complex cellular transformations characteristic of these diseases, but also suggests new and innovative therapies that may hold considerable promise for ameliorating disease progression.

Profiling MHC II immunopeptidome of blood-stage malaria reveals that cDC1 control the functionality of parasite-specific CD4 T cells.

Science.gov (United States)

Draheim, Marion; Wlodarczyk, Myriam F; Crozat, Karine; Saliou, Jean-Michel; Alayi, Tchilabalo Dilezitoko; Tomavo, Stanislas; Hassan, Ali; Salvioni, Anna; Demarta-Gatsi, Claudia; Sidney, John; Sette, Alessandro; Dalod, Marc; Berry, Antoine; Silvie, Olivier; Blanchard, Nicolas

2017-11-01

In malaria, CD4 Th1 and T follicular helper (T FH ) cells are important for controlling parasite growth, but Th1 cells also contribute to immunopathology. Moreover, various regulatory CD4 T-cell subsets are critical to hamper pathology. Yet the antigen-presenting cells controlling Th functionality, as well as the antigens recognized by CD4 T cells, are largely unknown. Here, we characterize the MHC II immunopeptidome presented by DC during blood-stage malaria in mice. We establish the immunodominance hierarchy of 14 MHC II ligands derived from conserved parasite proteins. Immunodominance is shaped differently whether blood stage is preceded or not by liver stage, but the same ETRAMP-specific dominant response develops in both contexts. In naïve mice and at the onset of cerebral malaria, CD8α + dendritic cells (cDC1) are superior to other DC subsets for MHC II presentation of the ETRAMP epitope. Using in vivo depletion of cDC1, we show that cDC1 promote parasite-specific Th1 cells and inhibit the development of IL-10 + CD4 T cells. This work profiles the P. berghei blood-stage MHC II immunopeptidome, highlights the potency of cDC1 to present malaria antigens on MHC II, and reveals a major role for cDC1 in regulating malaria-specific CD4 T-cell responses. © 2017 The Authors. Published under the terms of the CC BY 4.0 license.

Lysosomal metabolomics reveals V-ATPase- and mTOR-dependent regulation of amino acid efflux from lysosomes.

Science.gov (United States)

Abu-Remaileh, Monther; Wyant, Gregory A; Kim, Choah; Laqtom, Nouf N; Abbasi, Maria; Chan, Sze Ham; Freinkman, Elizaveta; Sabatini, David M

2017-11-10

The lysosome degrades and recycles macromolecules, signals to the cytosol and nucleus, and is implicated in many diseases. Here, we describe a method for the rapid isolation of mammalian lysosomes and use it to quantitatively profile lysosomal metabolites under various cell states. Under nutrient-replete conditions, many lysosomal amino acids are in rapid exchange with those in the cytosol. Loss of lysosomal acidification through inhibition of the vacuolar H + -adenosine triphosphatase (V-ATPase) increased the luminal concentrations of most metabolites but had no effect on those of the majority of essential amino acids. Instead, nutrient starvation regulates the lysosomal concentrations of these amino acids, an effect we traced to regulation of the mechanistic target of rapamycin (mTOR) pathway. Inhibition of mTOR strongly reduced the lysosomal efflux of most essential amino acids, converting the lysosome into a cellular depot for them. These results reveal the dynamic nature of lysosomal metabolites and that V-ATPase- and mTOR-dependent mechanisms exist for controlling lysosomal amino acid efflux. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Tubular lysosome morphology and distribution within macrophages depend on the integrity of cytoplasmic microtubules

International Nuclear Information System (INIS)

Swanson, J.; Bushnell, A.; Silverstein, S.C.

1987-01-01

Pinocytosis of the fluorescent dye lucifer yellow labels elongated, membrane-bound tubular organelles in several cell types, including cultured human monocytes, thioglycolate-elicited mouse peritoneal macrophages, and the macrophage-like cell line J774.2. These tubular structures can be identified as lysosomes by acid phosphatase histochemistry and immunofluorescence localization of cathepsin L. The abundance of tubular lysosomes is markedly increased by treatment with phorbol 12-myristate 13-acetate. When labeled by pinocytosis of microperoxidase and examined by electron microscopic histochemistry, the tubular lysosomes have an outside diameter of ≅ 75 nm and a length of several micrometers; they radiate from the cell's centrosphere in alignment with cytoplasmic microtubules and intermediate filaments. Incubation of phorbol myristate acetate-treated macrophages at 4 0 C or in medium containing 5 μM colchicine or nocodazole at 37 0 C leads to disassembly of microtubules and fragmentation of the tubular lysosomes. Return of the cultures to 37 0 C or removal of nocodazole from the medium leads to reassembly of microtubules and the reappearance of tubular lysosomes within 10-20 min. The authors conclude that microtubules are essential for the maintenance of tubular lysosome morphology and that, in macrophages, a significant proportion of the lysosomal compartment is contained within these tubular structures

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....

Extracellular ATP reduces HIV-1 transfer from immature dendritic cells to CD4+ T lymphocytes

Directory of Open Access Journals (Sweden)

Barat Corinne

2008-03-01

Full Text Available Abstract Background Dendritic cells (DCs are considered as key mediators of the early events in human immunodeficiency virus type 1 (HIV-1 infection at mucosal sites. Previous studies have shown that surface-bound virions and/or internalized viruses found in endocytic vacuoles of DCs are efficiently transferred to CD4+ T cells. Extracellular adenosine triphosphate (ATP either secreted or released from necrotic cells induces a distorted maturation of DCs, transiently increases their endocytic capacity and affects their migratory capacity. Knowing that high extracellular ATP concentrations are present in situations of tissue injury and inflammation, we investigated the effect of ATP on HIV-1 transmission from DCs to CD4+ T lymphocytes. Results In this study, we show that extracellular ATP reduces HIV-1 transfer from immature monocyte-derived DCs (iDCs to autologous CD4+ T cells. This observed decrease in viral replication was related to a lower proportion of infected CD4+ T cells following transfer, and was seen with both X4- and R5-tropic isolates of HIV-1. Extracellular ATP had no effect on direct CD4+ T cell infection as well as on productive HIV-1 infection of iDCs. These observations indicate that extracellular ATP affects HIV-1 infection of CD4+ T cells in trans with no effect on de novo virus production by iDCs. Additional experiments suggest that extracellular ATP might modulate the trafficking pathway of internalized virions within iDCs leading to an increased lysosomal degradation, which could be partly responsible for the decreased HIV-1 transmission. Conclusion These results suggest that extracellular ATP can act as a factor controlling HIV-1 propagation.

The 2.5 Å Structure of CD1c in Complex with a Mycobacterial Lipid Reveals an Open Groove Ideally Suited for Diverse Antigen Presentation

Energy Technology Data Exchange (ETDEWEB)

Scharf, Louise; Li, Nan-Sheng; Hawk, Andrew J.; Garzón, Diana; Zhang, Tejia; Fox, Lisa M.; Kazen, Allison R.; Shah, Sneha; Haddadian, Esmael J.; Gumperz, Jenny E.; Saghatelian, Alan; Faraldo-Gómez, José D.; Meredith, Stephen C.; Piccirilli, Joseph A.; Adams, Erin J. (Harvard); (UC); (MXPL-G); (UW-MED)

2011-08-24

CD1 molecules function to present lipid-based antigens to T cells. Here we present the crystal structure of CD1c at 2.5 {angstrom} resolution, in complex with the pathogenic Mycobacterium tuberculosis antigen mannosyl-{beta}1-phosphomycoketide (MPM). CD1c accommodated MPM's methylated alkyl chain exclusively in the A pocket, aided by a unique exit portal underneath the {alpha}1 helix. Most striking was an open F pocket architecture lacking the closed cavity structure of other CD1 molecules, reminiscent of peptide binding grooves of classical major histocompatibility complex molecules. This feature, combined with tryptophan-fluorescence quenching during loading of a dodecameric lipopeptide antigen, provides a compelling model by which both the lipid and peptide moieties of the lipopeptide are involved in CD1c presentation of lipopeptides.

NQR and X-ray crystal structure studies of cadmium halide complexes: [C(NH2)3]CdI3 and [4-ClC6H5NH3]3CdBr5

International Nuclear Information System (INIS)

Gesing, Thorsten M.; Lork, Enno; Terao, Hiromitsu; Ishihara, Hideta

2016-01-01

The crystal structures of [C(NH 2 ) 3 ]CdI 3 (1) and [4-ClC 6 H 5 NH 3 ] 3 CdBr 5 (2) have been determined at 100 K: monoclinic, Cc, a = 828.75(3) pm, b = 1615.31(5) pm, c = 810.64(3) pm, and β = 106.5820(10) for 1; monoclinic, P2 1 /c, a = 1486.93(5) pm, b = 794.31(3) pm, c = 2290.59(7) pm, and β = 99.6830(10) for 2. The structure of 1 has an infinite chain of anions consisting of [CdI 4 ] tetrahedra sharing two corners. The structure of 2 has an infinite chain of anions consisting of [CdBr 6 ] octahedra sharing two corners in cis positions. In both structures, isolated cations are connected to the anion chains through weak hydrogen bonds Cd-X..H to result in three-dimensional network structures. In accordance with the crystal structures, three 127 I (m = ±1/2 <-> m = ±3/2), five 81 Br, and three 35 Cl nuclear quadrupole resonance (NQR) lines were observed for 1 and 2. The NQR spectra reflect the anion chain structures and their weak hydrogen bonds. The MO calculations of the models [Cd 5 I 16 ] 6- for 1 and [Cd 3 Br 16 ] 10- for 2 estimate only about half the values for the NQR frequencies but give accurate electric field gradient directions.

Lysosomal exocytosis and lipid storage disorders.

Science.gov (United States)

Samie, Mohammad Ali; Xu, Haoxing

2014-06-01

Lysosomes are acidic compartments in mammalian cells that are primarily responsible for the breakdown of endocytic and autophagic substrates such as membranes, proteins, and lipids into their basic building blocks. Lysosomal storage diseases (LSDs) are a group of metabolic disorders caused by genetic mutations in lysosomal hydrolases required for catabolic degradation, mutations in lysosomal membrane proteins important for catabolite export or membrane trafficking, or mutations in nonlysosomal proteins indirectly affecting these lysosomal functions. A hallmark feature of LSDs is the primary and secondary excessive accumulation of undigested lipids in the lysosome, which causes lysosomal dysfunction and cell death, and subsequently pathological symptoms in various tissues and organs. There are more than 60 types of LSDs, but an effective therapeutic strategy is still lacking for most of them. Several recent in vitro and in vivo studies suggest that induction of lysosomal exocytosis could effectively reduce the accumulation of the storage materials. Meanwhile, the molecular machinery and regulatory mechanisms for lysosomal exocytosis are beginning to be revealed. In this paper, we first discuss these recent developments with the focus on the functional interactions between lipid storage and lysosomal exocytosis. We then discuss whether lysosomal exocytosis can be manipulated to correct lysosomal and cellular dysfunction caused by excessive lipid storage, providing a potentially general therapeutic approach for LSDs. Copyright © 2014 by the American Society for Biochemistry and Molecular Biology, Inc.

Lysosomal exocytosis and lipid storage disorders

Science.gov (United States)

Samie, Mohammad Ali; Xu, Haoxing

2014-01-01

Lysosomes are acidic compartments in mammalian cells that are primarily responsible for the breakdown of endocytic and autophagic substrates such as membranes, proteins, and lipids into their basic building blocks. Lysosomal storage diseases (LSDs) are a group of metabolic disorders caused by genetic mutations in lysosomal hydrolases required for catabolic degradation, mutations in lysosomal membrane proteins important for catabolite export or membrane trafficking, or mutations in nonlysosomal proteins indirectly affecting these lysosomal functions. A hallmark feature of LSDs is the primary and secondary excessive accumulation of undigested lipids in the lysosome, which causes lysosomal dysfunction and cell death, and subsequently pathological symptoms in various tissues and organs. There are more than 60 types of LSDs, but an effective therapeutic strategy is still lacking for most of them. Several recent in vitro and in vivo studies suggest that induction of lysosomal exocytosis could effectively reduce the accumulation of the storage materials. Meanwhile, the molecular machinery and regulatory mechanisms for lysosomal exocytosis are beginning to be revealed. In this paper, we first discuss these recent developments with the focus on the functional interactions between lipid storage and lysosomal exocytosis. We then discuss whether lysosomal exocytosis can be manipulated to correct lysosomal and cellular dysfunction caused by excessive lipid storage, providing a potentially general therapeutic approach for LSDs. PMID:24668941

A novel transgenic mouse model of lysosomal storage disorder

OpenAIRE

Ortiz-Miranda, Sonia; Ji, Rui; Jurczyk, Agata; Aryee, Ken-Edwin; Mo, Shunyan; Fletcher, Terry; Shaffer, Scott A.; Greiner, Dale L.; Bortell, Rita; Gregg, Ronald G.; Cheng, Alan; Hennings, Leah J.; Rittenhouse, Ann R.

2016-01-01

We provide an explanation for striking pathology found in a subset of genetically engineered mice homozygous for a rat CaVβ2a transgene (Tg+/+). Multiple transgene (Tg) copies inserted into chromosome 19; at this same site a large deletion occurred, ablating cholesterol 25-hydroxylase and partially deleting lysosomal acid lipase and CD95. Their loss of function can account for lipid build up and immune system hypertrophy, which defines this phenotype and serendipitously provides a novel model...

Density of liquid Hg(1-x)Cd(x)Te

Science.gov (United States)

Chandra, D.; Holland, L. R.

1983-01-01

Negative thermal expansion has been established in liquid Hg(1-x)Cd(x)Te for x less than 0.2 employing a pycnometric method. Pure HgTe increases in density from its melting point at 670 C to a maximum value at 750 C, where normal thermal expansion progressively resumes. The dependence of density on temperature for liquid Hg(1-x)Cd(x)Te arises almost exclusively from the HgTe portion of the melt, while CdTe acts as a diluent. The temperature corresponding to the maximum density changes slightly with composition, increasing by about 5 C for x = 0.1.

Fullerene (C{sub 60})/CdS nanocomposite with enhanced photocatalytic activity and stability

Energy Technology Data Exchange (ETDEWEB)

Cai, Qiang [Key Laboratory of Advanced Ceramics and Machining Technology, Ministry of Education, School of Materials Science and Engineering, Tianjin University, Tianjin 300072 (China); Hu, Zhuofeng, E-mail: st04hzhf@gmail.com [Department of Chemistry, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong (China); Zhang, Qian; Li, Boyuan [Key Laboratory of Advanced Ceramics and Machining Technology, Ministry of Education, School of Materials Science and Engineering, Tianjin University, Tianjin 300072 (China); Shen, Zhurui, E-mail: shenzhurui@tju.edu.cn [Key Laboratory of Advanced Ceramics and Machining Technology, Ministry of Education, School of Materials Science and Engineering, Tianjin University, Tianjin 300072 (China)

2017-05-01

Highlights: • C{sub 60}/CdS nanocomposite has been fabricated as a novel visible-light-driven photocatalyst. • It exhibits enhanced photocatalytic activity and photostability than that of pure CdS reference. • The C{sub 60} improved the charge separation and transfer of nanocomposite due to its high electron affinity. - Abstract: Herein, the fullerene (C{sub 60})/CdS nanocomposite has been fabricated by a facile one-pot hydrothermal method. Its photocatatlytic hydrogen (H{sub 2}) evolution rate and degradation efficiency of Rhodamine B (Rh B) are evaluated under visible light irradiation (λ ≥ 420 nm). The content of C{sub 60} has been changed from 0.4 wt% to 8 wt%, and the optimal value for photocatalytic activity is determined to be 0.4 wt%. The H{sub 2} evolution rate over this optimal sample reaches 1.73 mmol h{sup −1} g{sup −1} and its apparent degradation rate of Rh B is 0.089 min{sup −1} (degradation efficiency of 97% within 40 min), which is 2.3 times and 1.5 times compared to that of pure CdS reference. Moreover, the photocorrosion of CdS in composite is effectively suppressed, and its photocatalytic activity can be well maintained after three recycles (97.8% retaining for composite vs. 84.4% retaining for CdS). Then, the enhanced photocatalytic activity and stability of C{sub 60}/CdS nanocomposite are further studied by spectroscopic and electrochemical methods. Results show that the C{sub 60} species covering on the surface of CdS can efficiently accelerate the separation and transfer of photoexcited charge carriers, which can improve its activity, and reduce the photocorrosion of CdS.

Regulation of CD93 cell surface expression by protein kinase C isoenzymes.

Science.gov (United States)

Ikewaki, Nobunao; Kulski, Jerzy K; Inoko, Hidetoshi

2006-01-01

Human CD93, also known as complement protein 1, q subcomponent, receptor (C1qRp), is selectively expressed by cells with a myeloid lineage, endothelial cells, platelets, and microglia and was originally reported to be involved in the complement protein 1, q subcomponent (C1q)-mediated enhancement of phagocytosis. The intracellular molecular events responsible for the regulation of its expression on the cell surface, however, have not been determined. In this study, the effect of protein kinases in the regulation of CD93 expression on the cell surface of a human monocyte-like cell line (U937), a human NK-like cell line (KHYG-1), and a human umbilical vein endothelial cell line (HUV-EC-C) was investigated using four types of protein kinase inhibitors, the classical protein kinase C (cPKC) inhibitor Go6976, the novel PKC (nPKC) inhibitor Rottlerin, the protein kinase A (PKA) inhibitor H-89 and the protein tyrosine kinase (PTK) inhibitor herbimycin A at their optimum concentrations for 24 hr. CD93 expression was analyzed using flow cytometry and glutaraldehyde-fixed cellular enzyme-linked immunoassay (EIA) techniques utilizing a CD93 monoclonal antibody (mAb), mNI-11, that was originally established in our laboratory as a CD93 detection probe. The nPKC inhibitor Rottlerin strongly down-regulated CD93 expression on the U937 cells in a dose-dependent manner, whereas the other inhibitors had little or no effect. CD93 expression was down-regulated by Go6976, but not by Rottlerin, in the KHYG-1 cells and by both Rottlerin and Go6976 in the HUV-EC-C cells. The PKC stimulator, phorbol myristate acetate (PMA), strongly up-regulated CD93 expression on the cell surface of all three cell-lines and induced interleukin-8 (IL-8) production by the U937 cells and interferon-gamma (IFN-gamma) production by the KHYG-1 cells. In addition, both Go6976 and Rottlerin inhibited the up-regulation of CD93 expression induced by PMA and IL-8 or IFN-gamma production in the respective cell

Generation and analysis of cDNA library from lipopolysaccharide ...

African Journals Online (AJOL)

These immune-related genes include cytidine deaminase, ferritin, nonmuscle myosin essential light chain, cytochrome c oxidase subunit I, CD63 antigen-like protein and lysosomal-associated transmembrane protein. This study may contribute to the understanding of the immune mechanism of gastropod abalone Haliotis ...

C/EBPα is dispensable for the ontogeny of PD-1+ CD4+ memory T cells but restricts their expansion in an age-dependent manner.

Science.gov (United States)

Norrie, Ida Christine; Ohlsson, Ewa; Nielsen, Olaf; Hasemann, Marie Sigurd; Porse, Bo T

2014-01-01

Ageing and cancer is often associated with altered T cell distributions and this phenomenon has been suggested to be the main driver in the development of immunosenescence. Memory phenotype PD-1+ CD4+ T cells accumulate with age and during leukemic development, and they might account for the attenuated T cell response in elderly or diseased individuals. The transcription factor C/EBPα has been suggested to be responsible for the accumulation as well as for the senescent features of these cells including impaired TCR signaling and decreased proliferation. Thus modulating the activity of C/EBPα could potentially target PD-1+ CD4+ T cells and consequently, impede the development of immunosenescence. To exploit this possibility we tested the importance of C/EBPα for the development of age-dependent PD-1+ CD4+ T cells as well as its role in the accumulation of PD-1+ CD4+ T cells during leukemic progression. In contrast to earlier suggestions, we find that loss of C/EBPα expression in the lymphoid compartment led to an increase of PD-1+ CD4+ T cells specifically in old mice, suggesting that C/EBPα repress the accumulation of these cells in elderly by inhibiting their proliferation. Furthermore, C/EBPα-deficiency in the lymphoid compartment had no effect on leukemic development and did not affect the accumulation of PD-1+ CD4+ T cells. Thus, in addition to contradict earlier suggestions of a role for C/EBPα in immunosenescence, these findings efficiently discard the potential of using C/EBPα as a target for the alleviation of ageing/cancer-associated immunosenescence.

Bypassing non-adherence via PEG in a critically ill HIV-1-infected patient.

Science.gov (United States)

Leipe, J; Hueber, A J; Rech, J; Harrer, T

2008-08-01

This case study describes a 44-year-old, chronically non-adherent, HIV-infected male with relapsing, life threatening toxoplasmic encephalitis (TE) and other recurring opportunistic infections. Non-adherence resulted in critical illness, suppressed CD4 lymphocyte count and elevated viral load. In order to bypass the patient's complete psychological aversion to taking medication, and after exhausting various psychological interventions, a percutaneous endoscopic gastronomy (PEG) tube was inserted for delivery of indispensable medication. During the 15-month follow-up the patient was adherent, exhibiting a consistently undetectable viral load, high CD4 count and a remission of the opportunistic infections. This is an interesting case study demonstrating life-saving and long-term benefit of PEG in an exceptional setting, which has implications for future research and treatment of non-adherent HIV-infected patients.

Cryo-EM structures of the mammalian endo-lysosomal TRPML1 channel elucidate the combined regulation mechanism

Directory of Open Access Journals (Sweden)

Sensen Zhang

2017-09-01

Full Text Available Abstract TRPML1 channel is a non-selective group-2 transient receptor potential (TRP channel with Ca2+ permeability. Located mainly in late endosome and lysosome of all mammalian cell types, TRPML1 is indispensable in the processes of endocytosis, membrane trafficking, and lysosome biogenesis. Mutations of TRPML1 cause a severe lysosomal storage disorder called mucolipidosis type IV (MLIV. In the present study, we determined the cryo-electron microscopy (cryo-EM structures of Mus musculus TRPML1 (mTRPML1 in lipid nanodiscs and Amphipols. Two distinct states of mTRPML1 in Amphipols are added to the closed state, on which could represent two different confirmations upon activation and regulation. The polycystin-mucolipin domain (PMD may sense the luminal/extracellular stimuli and undergo a “move upward” motion during endocytosis, thus triggering the overall conformational change in TRPML1. Based on the structural comparisons, we propose TRPML1 is regulated by pH, Ca2+, and phosphoinositides in a combined manner so as to accommodate the dynamic endocytosis process.

CD11c(hi) Dendritic Cells Regulate Ly-6C(hi) Monocyte Differentiation to Preserve Immune-privileged CNS in Lethal Neuroinflammation.

Science.gov (United States)

Kim, Jin Hyoung; Choi, Jin Young; Kim, Seong Bum; Uyangaa, Erdenebelig; Patil, Ajit Mahadev; Han, Young Woo; Park, Sang-Youel; Lee, John Hwa; Kim, Koanhoi; Eo, Seong Kug

2015-12-02

Although the roles of dendritic cells (DCs) in adaptive defense have been defined well, the contribution of DCs to T cell-independent innate defense and subsequent neuroimmunopathology in immune-privileged CNS upon infection with neurotropic viruses has not been completely defined. Notably, DC roles in regulating innate CD11b(+)Ly-6C(hi) monocyte functions during neuroinflammation have not yet been addressed. Using selective ablation of CD11c(hi)PDCA-1(int/lo) DCs without alteration in CD11c(int)PDCA-1(hi) plasmacytoid DC number, we found that CD11c(hi) DCs are essential to control neuroinflammation caused by infection with neurotropic Japanese encephalitis virus, through early and increased infiltration of CD11b(+)Ly-6C(hi) monocytes and higher expression of CC chemokines. More interestingly, selective CD11c(hi) DC ablation provided altered differentiation and function of infiltrated CD11b(+)Ly-6C(hi) monocytes in the CNS through Flt3-L and GM-CSF, which was closely associated with severely enhanced neuroinflammation. Furthermore, CD11b(+)Ly-6C(hi) monocytes generated in CD11c(hi) DC-ablated environment had a deleterious rather than protective role during neuroinflammation, and were more quickly recruited into inflamed CNS, depending on CCR2, thereby exacerbating neuroinflammation via enhanced supply of virus from the periphery. Therefore, our data demonstrate that CD11c(hi) DCs provide a critical and unexpected role to preserve the immune-privileged CNS in lethal neuroinflammation via regulating the differentiation, function, and trafficking of CD11b(+)Ly-6C(hi) monocytes.

The diabetogenic VPS13C/C2CD4A/C2CD4B rs7172432 variant impairs glucose-stimulated insulin response in 5,722 non-diabetic Danish individuals

DEFF Research Database (Denmark)

Grarup, N; Overvad, M; Sparsø, T

2011-01-01

A genome-wide association study in the Japanese population reported two genome-wide significant loci associated with type 2 diabetes of which the VPS13C/C2CD4A/C2CD4B locus was replicated in Europeans. We looked for potential associations between the diabetogenic VPS13C/C2CD4A/C2CD4B rs7172432 va...

Lysosomes and radiation injury

International Nuclear Information System (INIS)

Watkins, D.K.

1975-01-01

Changes in activities of lysosomal enzymes following whole-body treatment with ionizing radiation have long been recognized (e.g., Douglass and Day 1955, Okada et al., 1957). Attempts to explain nuclear damage by cytoplasmic enzyme attack, concentrated most of the earlier work on DNASE II and acid RNASE. Lysosomal enzymes have subsequently been studied in many tissues following whole-body irradiation. The observations coupled with in vitro results from isolated lysosomes, and u.v. and visible light studies on cells in culture, have led to the presentation of tentative mechanisms of action. General methods of detecting lysosomal damage have utilized the consequent activation or leakage of acid hydrolases. As this is of a temporal nature following irradiation, direct damage to the lysosomal membrane has not as yet been measured and the primary lesion either in the membrane itself or at the hypothetical site of acid hydrolase-membrane attachment has still to be discovered. Despite the accumulating evidence of lysosome disruption subsequent to treatment with radiation of various qualities, the role (if any) of these organelles in cell killing remains obscure. In the following pages a review of the many aspects of radiation damage will be presented and an attempt will be made to correlate the results and to draw general conclusions where possible. A final short section will deal with thecontribution that lysosomal damage may make in cell death and tissue injury and possible implications in radiotherapy

C/EBPα is dispensable for the ontogeny of PD-1+ CD4+ memory T cells but restricts their expansion in an age-dependent manner.

Directory of Open Access Journals (Sweden)

Ida Christine Norrie

Full Text Available Ageing and cancer is often associated with altered T cell distributions and this phenomenon has been suggested to be the main driver in the development of immunosenescence. Memory phenotype PD-1+ CD4+ T cells accumulate with age and during leukemic development, and they might account for the attenuated T cell response in elderly or diseased individuals. The transcription factor C/EBPα has been suggested to be responsible for the accumulation as well as for the senescent features of these cells including impaired TCR signaling and decreased proliferation. Thus modulating the activity of C/EBPα could potentially target PD-1+ CD4+ T cells and consequently, impede the development of immunosenescence. To exploit this possibility we tested the importance of C/EBPα for the development of age-dependent PD-1+ CD4+ T cells as well as its role in the accumulation of PD-1+ CD4+ T cells during leukemic progression. In contrast to earlier suggestions, we find that loss of C/EBPα expression in the lymphoid compartment led to an increase of PD-1+ CD4+ T cells specifically in old mice, suggesting that C/EBPα repress the accumulation of these cells in elderly by inhibiting their proliferation. Furthermore, C/EBPα-deficiency in the lymphoid compartment had no effect on leukemic development and did not affect the accumulation of PD-1+ CD4+ T cells. Thus, in addition to contradict earlier suggestions of a role for C/EBPα in immunosenescence, these findings efficiently discard the potential of using C/EBPα as a target for the alleviation of ageing/cancer-associated immunosenescence.

CD36 and malaria: friends or foes? A decade of data provides some answers.

Science.gov (United States)

Cabrera, Ana; Neculai, Dante; Kain, Kevin C

2014-09-01

The past 10 years have generated new insights into the complex interaction between CD36 (cluster of differentiation 36) and malaria. These range from the crystallization of the CD36 homolog, LIMPII (lysosomal integral membrane protein II), permitting modeling of CD36 and its binding to diverse ligands, to cell biology-based studies of CD36 and large population genetic studies assessing the association of CD36 polymorphisms and malarial disease severity. Collectively these lines of evidence indicate that a receptor other than CD36 is associated with severity. CD36 plays an important role in innate immunity and in the phagocytic uptake of multiple pathogens including malaria. CD36 polymorphisms lack association with severity, and isolates that cause severe disease primarily bind to endothelial protein C receptor (EPCR) rather than to CD36. Crown Copyright © 2014. Published by Elsevier Ltd. All rights reserved.

Lysosomal regulation of cholesterol homeostasis in tuberous sclerosis complex is mediated via NPC1 and LDL-R.

Science.gov (United States)

Filippakis, Harilaos; Alesi, Nicola; Ogorek, Barbara; Nijmeh, Julie; Khabibullin, Damir; Gutierrez, Catherine; Valvezan, Alexander J; Cunningham, James; Priolo, Carmen; Henske, Elizabeth P

2017-06-13

Tuberous sclerosis complex (TSC) is a multisystem disease associated with hyperactive mTORC1. The impact of TSC1/2 deficiency on lysosome-mediated processes is not fully understood. We report here that inhibition of lysosomal function using chloroquine (CQ) upregulates cholesterol homeostasis genes in TSC2-deficient cells. This TSC2-dependent transcriptional signature is associated with increased accumulation and intracellular levels of both total cholesterol and cholesterol esters. Unexpectedly, engaging this CQ-induced cholesterol uptake pathway together with inhibition of de novo cholesterol synthesis allows survival of TSC2-deficient, but not TSC2-expressing cells. The underlying mechanism of TSC2-deficient cell survival is dependent on exogenous cholesterol uptake via LDL-R, and endosomal trafficking mediated by Vps34. Simultaneous inhibition of lysosomal and endosomal trafficking inhibits uptake of esterified cholesterol and cell growth in TSC2-deficient, but not TSC2-expressing cells, highlighting the TSC-dependent lysosome-mediated regulation of cholesterol homeostasis and pointing toward the translational potential of these pathways for the therapy of TSC.

TNFα Post-Translationally Targets ZnT2 to Accumulate Zinc in Lysosomes.

Science.gov (United States)

Hennigar, Stephen R; Kelleher, Shannon L

2015-10-01

Mammary epithelial cells undergo widespread lysosomal-mediated cell death (LCD) during early mammary gland involution. Recently, we demonstrated that tumor necrosis factor-α (TNFα), a cytokine released during early involution, redistributes the zinc (Zn) transporter ZnT2 to accumulate Zn in lysosomes and activate LCD and involution. The objective of this study is to determine how TNFα retargets ZnT2 to lysosomes. We tested the hypothesis that TNFα signaling dephosphorylates ZnT2 to uncover a highly conserved dileucine motif (L294L) in the C-terminus of ZnT2, allowing adaptor protein complex-3 (AP-3) to bind and traffic ZnT2 to lysosomes. Confocal micrographs showed that TNFα redistributed wild-type (WT) ZnT2 from late endosomes (Pearson's coefficient = 0.202 ± 0.05 and 0.097 ± 0.03; Plysosomes (0.292 ± 0.03 and 0.649 ± 0.03; Plysosomal Zn (Plysosomes, increase lysosomal Zn, or activate LCD. Moreover, TNFα increased (Plysosomes and activate LCD. Our findings suggest that women with variation in the C-terminus of ZnT2 may be at risk for inadequate involution and breast disease due the inability to traffic ZnT2 to lysosomes. © 2015 Wiley Periodicals, Inc.

Effect of Phosphodiesterase in Regulating the Activity of Lysosomes in the HeLa Cell Line.

Science.gov (United States)

Hong, Eun-Seon; Kim, Bit-Na; Kim, Yang-Hoon; Min, Jiho

2017-02-28

The transport of lysosomal enzymes into the lysosomes depends on the phosphorylation of their chains and the binding of the phosphorylated residues to mannose-6-phosphate receptors. The efficiency of separation depends more on the phosphodiesterases (PDEs) than on the activity of the phosphorylation of mannose residues and can be determined in vitro. PDEs play important roles in regulation of the activation of lysosomes. The expression of proteins was confirmed by western blotting. All PDE4 series protein expression was reduced in high concentrations of rolipram. As a result of observing the fluorescence intensity after rolipram treatment, the lysosomal enzyme was activated at low concentrations and suppressed at high concentrations. High concentrations of rolipram recovered the original function. Antimicrobial activity was not shown in either 10 or 100 µ concentrations of rolipram in treated HeLa cells in vitro. However, the higher anticancer activity at lower rolipram concentration was shown in lysosomal enzyme treated with 10 µ of rolipram. The anticancer activity was confirmed through cathepsin B and D assay. Tranfection allowed examination of the relationship between PDE4 and lysosomal activity in more detail. Protein expression was confirmed to be reduced. Fluorescence intensity showed decreased activity of lysosomes and ROS in cells transfected with the antisense sequences of PDE4 A, B, C, and D. PDE4A showed anticancer activity, whereas lysosome from cells transfected with the antisense sequences of PDE4 B, C, and D had decreased anticancer activity. These results showed the PDE4 A, B, C, and D are conjunctly related with lysosomal activity.

Lysosome and calcium dysregulation in Alzheimer's disease: partners in crime.

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McBrayer, MaryKate; Nixon, Ralph A

2013-12-01

Early-onset FAD (familial Alzheimer's disease) is caused by mutations of PS1 (presenilin 1), PS2 (presenilin 2) and APP (amyloid precursor protein). Beyond the effects of PS1 mutations on proteolytic functions of the γ-secretase complex, mutant or deficient PS1 disrupts lysosomal function and Ca2+ homoeostasis, both of which are considered strong pathogenic factors in FAD. Loss of PS1 function compromises assembly and proton-pumping activity of the vacuolar-ATPase on lysosomes, leading to defective lysosomal acidification and marked impairment of autophagy. Additional dysregulation of cellular Ca2+ by mutant PS1 in FAD has been ascribed to altered ion channels in the endoplasmic reticulum; however, rich stores of Ca2+ in lysosomes are also abnormally released in PS1-deficient cells secondary to the lysosomal acidification defect. The resultant rise in cytosolic Ca2+ activates Ca2+-dependent enzymes, contributing substantially to calpain overactivation that is a final common pathway leading to neurofibrillary degeneration in all forms of AD (Alzheimer's disease). In the present review, we discuss the close inter-relationships among deficits of lysosomal function, autophagy and Ca2+ homoeostasis as a pathogenic process in PS1-related FAD and their relevance to sporadic AD.

Mechanisms and functions of lysosome positioning

Science.gov (United States)

Pu, Jing; Guardia, Carlos M.; Keren-Kaplan, Tal

2016-01-01

ABSTRACT Lysosomes have been classically considered terminal degradative organelles, but in recent years they have been found to participate in many other cellular processes, including killing of intracellular pathogens, antigen presentation, plasma membrane repair, cell adhesion and migration, tumor invasion and metastasis, apoptotic cell death, metabolic signaling and gene regulation. In addition, lysosome dysfunction has been shown to underlie not only rare lysosome storage disorders but also more common diseases, such as cancer and neurodegeneration. The involvement of lysosomes in most of these processes is now known to depend on the ability of lysosomes to move throughout the cytoplasm. Here, we review recent findings on the mechanisms that mediate the motility and positioning of lysosomes, and the importance of lysosome dynamics for cell physiology and pathology. PMID:27799357

Synthesis and Characterization of CdS/CdSxSe1-x Nanowires

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Agueda Lopez, Kleyser; Wu, Marvin

Semiconductor nanowire heterostructures are of interest for potential applications in solar cells and other advanced optoelectronic devices. We report here on synthesis of CdS/CdSxSe1-x nanowires (NWs) using a dual source vapor = liquid - solid technique, and characterization of these NWs with scanning electron microscopy and optical microscopy. We determine the effect of growth parameters, including source / substrate temperatures and time of exposure, on NW size, shape, and composition. The crystal structure and optical properties individual NWs from selected substrates has been mapped using transmission Kikuchi diffraction and photoluminescence (PL) microscopy. NWs consistently exhibit a hexagonal structure, with growth along the c-axis. Strong PL peaks are observed between the expected bandgap emission from CdS and CdSe, confirming formation of CdSxSe1-x. PL peaks vary significantly with intensity along the long axis of the nanowire, suggesting that the NW surface is not uniformly passivated. These nanowires show promise for future investigation and manipulation of energy band gaps contain in CdS/CdSe. CREST.

The Fab1/PIKfyve Phosphoinositide Phosphate Kinase Is Not Necessary to Maintain the pH of Lysosomes and of the Yeast Vacuole*

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Ho, Cheuk Y.; Choy, Christopher H.; Wattson, Christina A.; Johnson, Danielle E.; Botelho, Roberto J.

2015-01-01

Lysosomes and the yeast vacuole are degradative and acidic organelles. Phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2), a master architect of endolysosome and vacuole identity, is thought to be necessary for vacuolar acidification in yeast. There is also evidence that PtdIns(3,5)P2 may play a role in lysosomal acidification in higher eukaryotes. Nevertheless, these conclusions rely on qualitative assays of lysosome/vacuole pH. For example, quinacrine, an acidotropic fluorescent base, does not accumulate in the vacuoles of fab1Δ yeast. Fab1, along with its mammalian ortholog PIKfyve, is the lipid kinase responsible for synthesizing PtdIns(3,5)P2. In this study, we employed several assays that quantitatively assessed the lysosomal and vacuolar pH in PtdIns(3,5)P2-depleted cells. Using ratiometric imaging, we conclude that lysosomes retain a pH lysosomes. PMID:25713145

Uptake and degradation of cytoplasmic RNA by lysosomes in the perfused rat liver

International Nuclear Information System (INIS)

Heydrick, S.J.; Lardeux, B.; Mortimore, G.E.

1987-01-01

The release of [ 14 C]cytidine has been shown previously to be a valid marker for RNA degradation in rat hepatocytes. The breakdown of RNA measured with this marker in perfused livers prelabeled in vivo with [6- 14 C]orotic acid was found to be regulated acutely by perfusate amino acids over a wide range, from 0.29 to 3.48%/h. This regulation paralleled that of lysosomal proteolysis. Chloroquine inhibited RNA degradation 60-70%. In subsequent cell fractionation studies labelled cytidine was released; the distribution of this release paralleled that of a lysosomal marker enzyme. The release plateaued after two hours, defining a distinct lysosomal pool of RNA. The lysosomal location of the RNA pool was confirmed in experiments where a 22% increase in the apparent pool size was obtained by lowering the homogenate pH from 7.0 to 5.5. The pool size correlated linearly with the rate of RNA degradation measured during perfusion, giving a turnover constant in reasonable agreement with values reported for autophagy. These results indicate that cytoplasmic RNA degradation occurs primarily in the lysosome and is regulated under these conditions by the amino acid control of lysosomal sequestration of cytoplasm

The role of complement receptors type 1 (CR1, CD35) and 2 (CR2, CD21) in promoting C3 fragment deposition and membrane attack complex formation on normal peripheral human B cells

DEFF Research Database (Denmark)

Nielsen, Claus Henrik; Pedersen, Morten Løbner; Marquart, Hanne Vibeke Hansen

2002-01-01

Normal human B lymphocytes are known to activate the alternative pathway (AP) of complement, leading to C3-fragment deposition and membrane attack complex (MAC) formation. The process is mediated via complement receptor type 2 (CR2, CD21), with complement receptor type 1 (CR1, CD35) playing...... a subsidiary role. In this study, we examine the relative contributions of CR1 and CR2 to the deposition of C3 fragments and MAC on B lymphocytes under circumstances where all complement pathways are operational. C3-fragment deposition and MAC formation were assessed on human peripheral B lymphocytes...... in the presence of 30% autologous serum. Blocking the CR2 ligand-binding site with monoclonal antibody (mAb) FE8 resulted in significant reduction (37.9+/-11.9%) in C3-fragment deposition, whereas MAC formation was only marginally affected (12.1+/-22.2% reduction). Blocking the CR1 binding-site resulted...

Activation of lysosomal cathepsins in pregnant bovine leukocytes.

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Talukder, Md Abdus Shabur; Balboula, Ahmed Zaky; Shirozu, Takahiro; Kim, Sung Woo; Kunii, Hiroki; Suzuki, Toshiyuki; Ito, Tsukino; Kimura, Koji; Takahashi, Masashi

2018-06-01

In ruminants, interferon-tau (IFNT) - mediated expression of interferon-stimulated genes in peripheral blood leukocytes (PBLs) can indicate pregnancy. Recently, type 1 IFN-mediated activation of lysosomes and lysosomal cathepsins (CTSs) was observed in immune cells. This study investigated the status of lysosomal CTSs and lysosomes in PBLs collected from pregnant (P) and non-pregnant (NP) dairy cows, and conducted in vitro IFNT stimulation of NP blood leukocytes. Blood samples were collected 0, 7, 14 and 18 days post-artificial insemination, and the peripheral blood mononuclear cells (PBMCs) and polymorphonuclear granulocytes (PMNs) separated. The fluorescent activity of CTSB and CTSK in PMNs significantly increased with the progress of pregnancy, especially on day 18. In vitro supplementation of IFNT significantly increased the activities of CTSB and CTSK in NP PBMCs and PMNs. CTSB expression was significantly higher in PBMCs and PMNs collected from P day-18 cows than from NP cows, whereas there was no difference in CTSK expression. IFNT increased CTSB expression but did not affect CTSK expression. Immunodetection showed an increase of CTSB in P day-18 PBMCs and PMNs. In vitro stimulation of IFNT increased CTSB in NP PBMCs and PMNs. Lysosomal acidification showed a significant increase in P day-18 PBMCs and PMNs. IFNT also stimulated lysosomal acidification. Expressions of lysosome-associated membrane protein (LAMP) 1 and LAMP2 were significantly higher in P day-18 PBMCs and PMNs. The results suggest that pregnancy-specific activation of lysosomal functions by CTS activation in blood leukocytes is highly associated with IFNT during maternal and fetal recognition of pregnancy. © 2018 Society for Reproduction and Fertility.

Effects of mercury on lysosomal protein digestion in the kidney proximal tubule

International Nuclear Information System (INIS)

Madsen, K.M.; Christensen, E.I.

1978-01-01

The effect of mercury on renal lysosomal protein digestion was studied after administration of mercury in vitro and in vivo. Mercuric chloride or methylmercury chloride was added in vitro to lysosomal enzymes isolated from normal rats, and subsequently, digestion experiments were carried out using 125 I-labeled lysozyme or cytochrome c as substrate proteins. Both mercury compounds produced a concentration-dependent inhibition of the degradation of the proteins, mercuric chloride being the strongest inhibitor. Mercuric chloride was also administered to rats in vivo for 5 to 8 months. Renal lysosomal enzymes from these animals also had a decreased ability to digest the two substrate proteins. Furthermore, the digestion of lysozyme intravenously injected into mercury-intoxicated rats was decreased in renal cortical slices incubated in vitro. Electron microscope autoradiography showed that intravenously injected labeled lysozyme was located primarily over lysosomes in proximal tubule cells 1 hour after injection in both control animals and mercury-intoxicated rats. These results suggest a decreased catabolism of low molecular weight proteins in the kidney during chronic mercury intoxication

Effect of radioprotectant WR 2721 on cyclic nucleotides, prostaglandins, and lysosomes

International Nuclear Information System (INIS)

Trocha, P.J.; Catravas, G.N.

1983-01-01

Within 1 hr after ip injection of the radioprotectant WR 2721 into rats, splenic cGMP levels dropped and remained suppressed for 6 hr before returning to normal. However, if rats were exposed to ionizing radiation 30-40 min after WR 2721 treatment, they had higher cGMP levels at 3 hr postirradiation than the nonirradiUted, drug-treated controls, but the cGMP content was still found to be lower than that of the irradiated nondrug-treated controls. Radiation exposure of animals pretreated with WR 2721 also resulted in higher liver and spleen levels of cAMP and additional elevations in spleen prostaglandin content, compared with irradiated controls at 3-6 hr after radiation treatment. The secondary fluctuations of lysosomal enzyme activities, prostaglandin content, and cyclic nucleotide levels were also altered in irradiated rats pretreated with WR 2721 when compared with irradiated controls. Liver and spleen lysosomal β-glucuronidase activities, spleen cAMP and cGMP levels, and spleen prostaglandin concentrations were closer to physiological levels at 3 days postirradiation in rats given WR 2721 before the radiation treatment

The position of lysosomes within the cell determines their luminal pH.

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Johnson, Danielle E; Ostrowski, Philip; Jaumouillé, Valentin; Grinstein, Sergio

2016-03-14

We examined the luminal pH of individual lysosomes using quantitative ratiometric fluorescence microscopy and report an unappreciated heterogeneity: peripheral lysosomes are less acidic than juxtanuclear ones despite their comparable buffering capacity. An increased passive (leak) permeability to protons, together with reduced vacuolar H(+)-adenosine triphosphatase (V-ATPase) activity, accounts for the reduced acidifying ability of peripheral lysosomes. The altered composition of peripheral lysosomes is due, at least in part, to more limited access to material exported by the biosynthetic pathway. The balance between Rab7 and Arl8b determines the subcellular localization of lysosomes; more peripheral lysosomes have reduced Rab7 density. This in turn results in decreased recruitment of Rab-interacting lysosomal protein (RILP), an effector that regulates the recruitment and stability of the V1G1 component of the lysosomal V-ATPase. Deliberate margination of lysosomes is associated with reduced acidification and impaired proteolytic activity. The heterogeneity in lysosomal pH may be an indication of a broader functional versatility. © 2016 Johnson et al.

Cellular Uptake and Delivery of Myeloperoxidase to Lysosomes Promote Lipofuscin Degradation and Lysosomal Stress in Retinal Cells*

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Yogalingam, Gouri; Lee, Amanda R.; Mackenzie, Donald S.; Maures, Travis J.; Rafalko, Agnes; Prill, Heather; Berguig, Geoffrey Y.; Hague, Chuck; Christianson, Terri; Bell, Sean M.; LeBowitz, Jonathan H.

2017-01-01

Neutrophil myeloperoxidase (MPO) catalyzes the H2O2-dependent oxidation of chloride anion to generate hypochlorous acid, a potent antimicrobial agent. Besides its well defined role in innate immunity, aberrant degranulation of neutrophils in several inflammatory diseases leads to redistribution of MPO to the extracellular space, where it can mediate tissue damage by promoting the oxidation of several additional substrates. Here, we demonstrate that mannose 6-phosphate receptor-mediated cellular uptake and delivery of MPO to lysosomes of retinal pigmented epithelial (RPE) cells acts to clear this harmful enzyme from the extracellular space, with lysosomal-delivered MPO exhibiting a half-life of 10 h. Lysosomal-targeted MPO exerts both cell-protective and cytotoxic functions. From a therapeutic standpoint, MPO catalyzes the in vitro degradation of N-retinylidene-N-retinylethanolamine, a toxic form of retinal lipofuscin that accumulates in RPE lysosomes and drives the pathogenesis of Stargardt macular degeneration. Furthermore, chronic cellular uptake and accumulation of MPO in lysosomes coincides with N-retinylidene-N-retinylethanolamine elimination in a cell-based model of macular degeneration. However, lysosomal-delivered MPO also disrupts lysosomal acidification in RPE cells, which coincides with nuclear translocation of the lysosomal stress-sensing transcription factor EB and, eventually, cell death. Based on these findings we predict that under periods of acute exposure, cellular uptake and lysosomal degradation of MPO mediates elimination of this harmful enzyme, whereas chronic exposure results in progressive accumulation of MPO in lysosomes. Lysosomal-accumulated MPO can be both cell-protective, by promoting the degradation of toxic retinal lipofuscin deposits, and cytotoxic, by triggering lysosomal stress and cell death. PMID:28115520

Cellular Uptake and Delivery of Myeloperoxidase to Lysosomes Promote Lipofuscin Degradation and Lysosomal Stress in Retinal Cells.

Science.gov (United States)

Yogalingam, Gouri; Lee, Amanda R; Mackenzie, Donald S; Maures, Travis J; Rafalko, Agnes; Prill, Heather; Berguig, Geoffrey Y; Hague, Chuck; Christianson, Terri; Bell, Sean M; LeBowitz, Jonathan H

2017-03-10

Neutrophil myeloperoxidase (MPO) catalyzes the H 2 O 2 -dependent oxidation of chloride anion to generate hypochlorous acid, a potent antimicrobial agent. Besides its well defined role in innate immunity, aberrant degranulation of neutrophils in several inflammatory diseases leads to redistribution of MPO to the extracellular space, where it can mediate tissue damage by promoting the oxidation of several additional substrates. Here, we demonstrate that mannose 6-phosphate receptor-mediated cellular uptake and delivery of MPO to lysosomes of retinal pigmented epithelial (RPE) cells acts to clear this harmful enzyme from the extracellular space, with lysosomal-delivered MPO exhibiting a half-life of 10 h. Lysosomal-targeted MPO exerts both cell-protective and cytotoxic functions. From a therapeutic standpoint, MPO catalyzes the in vitro degradation of N -retinylidene- N -retinylethanolamine, a toxic form of retinal lipofuscin that accumulates in RPE lysosomes and drives the pathogenesis of Stargardt macular degeneration. Furthermore, chronic cellular uptake and accumulation of MPO in lysosomes coincides with N -retinylidene- N -retinylethanolamine elimination in a cell-based model of macular degeneration. However, lysosomal-delivered MPO also disrupts lysosomal acidification in RPE cells, which coincides with nuclear translocation of the lysosomal stress-sensing transcription factor EB and, eventually, cell death. Based on these findings we predict that under periods of acute exposure, cellular uptake and lysosomal degradation of MPO mediates elimination of this harmful enzyme, whereas chronic exposure results in progressive accumulation of MPO in lysosomes. Lysosomal-accumulated MPO can be both cell-protective, by promoting the degradation of toxic retinal lipofuscin deposits, and cytotoxic, by triggering lysosomal stress and cell death. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Targeting Androgen Receptor by Lysosomal Degradation in Prostate Cancer

Science.gov (United States)

2015-11-01

Preparation of the Lysosomes A673 cells were treated with 100 pM chloroquine for 12 h or left untreated. Lysosomes were prepared using the Lysosome...were treated with 100 JlM chloroquine fur 12 h or left tmtreated, and the luciferase activity was determined using the same arnotmt of protein...TFEB levels or by activating TFEB using mTORC1 kinase inhibitor, torin 1. Additionally, we determined that the same approach can be used to target

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

Directory of Open Access Journals (Sweden)

Chih-Peng Chang

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

Sphingosine 1-Phosphate- and C-C Chemokine Receptor 2-Dependent Activation of CD4+ Plasmacytoid Dendritic Cells in the Bone Marrow Contributes to Signs of Sepsis-Induced Immunosuppression

Science.gov (United States)

Smirnov, Anna; Pohlmann, Stephanie; Nehring, Melanie; Ali, Shafaqat; Mann-Nüttel, Ritu; Scheu, Stefanie; Antoni, Anne-Charlotte; Hansen, Wiebke; Büettner, Manuela; Gardiasch, Miriam J.; Westendorf, Astrid M.; Wirsdörfer, Florian; Pastille, Eva; Dudda, Marcel; Flohé, Stefanie B.

2017-01-01

Sepsis is the dysregulated response of the host to systemic, mostly bacterial infection, and is associated with an enhanced susceptibility to life-threatening opportunistic infections. During polymicrobial sepsis, dendritic cells (DCs) secrete enhanced levels of interleukin (IL) 10 due to an altered differentiation in the bone marrow and contribute to the development of immunosuppression. We investigated the origin of the altered DC differentiation using murine cecal ligation and puncture (CLP), a model for human polymicrobial sepsis. Bone marrow cells (BMC) were isolated after sham or CLP operation, the cellular composition was analyzed, and bone marrow-derived DCs (BMDCs) were generated in vitro. From 24 h on after CLP, BMC gave rise to BMDC that released enhanced levels of IL-10. In parallel, a population of CD11chiMHCII+CD4+ DCs expanded in the bone marrow in a MyD88-dependent manner. Prior depletion of the CD11chiMHCII+CD4+ DCs from BMC in vitro reversed the increased IL-10 secretion of subsequently differentiating BMDC. The expansion of the CD11chiMHCII+CD4+ DC population in the bone marrow after CLP required the function of sphingosine 1-phosphate receptors and C-C chemokine receptor (CCR) 2, the receptor for C-C chemokine ligand (CCL) 2, but was not associated with monocyte mobilization. CD11chiMHCII+CD4+ DCs were identified as plasmacytoid DCs (pDCs) that had acquired an activated phenotype according to their increased expression of MHC class II and CD86. A redistribution of CD4+ pDCs from MHC class II− to MHC class II+ cells concomitant with enhanced expression of CD11c finally led to the rise in the number of CD11chiMHCII+CD4+ DCs. Enhanced levels of CCL2 were found in the bone marrow of septic mice and the inhibition of CCR2 dampened the expression of CD86 on CD4+ pDCs after CLP in vitro. Depletion of pDCs reversed the bias of splenic DCs toward increased IL-10 synthesis after CLP in vivo. Thus, during polymicrobial sepsis, CD4+ pDCs are activated

Sphingosine 1-Phosphate- and C-C Chemokine Receptor 2-Dependent Activation of CD4+ Plasmacytoid Dendritic Cells in the Bone Marrow Contributes to Signs of Sepsis-Induced Immunosuppression

Directory of Open Access Journals (Sweden)

Anna Smirnov

2017-11-01

Full Text Available Sepsis is the dysregulated response of the host to systemic, mostly bacterial infection, and is associated with an enhanced susceptibility to life-threatening opportunistic infections. During polymicrobial sepsis, dendritic cells (DCs secrete enhanced levels of interleukin (IL 10 due to an altered differentiation in the bone marrow and contribute to the development of immunosuppression. We investigated the origin of the altered DC differentiation using murine cecal ligation and puncture (CLP, a model for human polymicrobial sepsis. Bone marrow cells (BMC were isolated after sham or CLP operation, the cellular composition was analyzed, and bone marrow-derived DCs (BMDCs were generated in vitro. From 24 h on after CLP, BMC gave rise to BMDC that released enhanced levels of IL-10. In parallel, a population of CD11chiMHCII+CD4+ DCs expanded in the bone marrow in a MyD88-dependent manner. Prior depletion of the CD11chiMHCII+CD4+ DCs from BMC in vitro reversed the increased IL-10 secretion of subsequently differentiating BMDC. The expansion of the CD11chiMHCII+CD4+ DC population in the bone marrow after CLP required the function of sphingosine 1-phosphate receptors and C-C chemokine receptor (CCR 2, the receptor for C-C chemokine ligand (CCL 2, but was not associated with monocyte mobilization. CD11chiMHCII+CD4+ DCs were identified as plasmacytoid DCs (pDCs that had acquired an activated phenotype according to their increased expression of MHC class II and CD86. A redistribution of CD4+ pDCs from MHC class II− to MHC class II+ cells concomitant with enhanced expression of CD11c finally led to the rise in the number of CD11chiMHCII+CD4+ DCs. Enhanced levels of CCL2 were found in the bone marrow of septic mice and the inhibition of CCR2 dampened the expression of CD86 on CD4+ pDCs after CLP in vitro. Depletion of pDCs reversed the bias of splenic DCs toward increased IL-10 synthesis after CLP in vivo. Thus, during polymicrobial sepsis, CD4+ pDCs are

The Fab1/PIKfyve phosphoinositide phosphate kinase is not necessary to maintain the pH of lysosomes and of the yeast vacuole.

Science.gov (United States)

Ho, Cheuk Y; Choy, Christopher H; Wattson, Christina A; Johnson, Danielle E; Botelho, Roberto J

2015-04-10

Lysosomes and the yeast vacuole are degradative and acidic organelles. Phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2), a master architect of endolysosome and vacuole identity, is thought to be necessary for vacuolar acidification in yeast. There is also evidence that PtdIns(3,5)P2 may play a role in lysosomal acidification in higher eukaryotes. Nevertheless, these conclusions rely on qualitative assays of lysosome/vacuole pH. For example, quinacrine, an acidotropic fluorescent base, does not accumulate in the vacuoles of fab1Δ yeast. Fab1, along with its mammalian ortholog PIKfyve, is the lipid kinase responsible for synthesizing PtdIns(3,5)P2. In this study, we employed several assays that quantitatively assessed the lysosomal and vacuolar pH in PtdIns(3,5)P2-depleted cells. Using ratiometric imaging, we conclude that lysosomes retain a pH lysosomes. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Activation of lysosomal P2X4 by ATP transported into lysosomes via VNUT/SLC17A9 using V‐ATPase generated voltage gradient as the driving force

Science.gov (United States)

Zhong, Xi Zoë; Cao, Qi; Sun, Xue

2016-01-01

Key points SLC17A9 proteins function as a lysosomal ATP transporter responsible for lysosomal ATP accumulation.P2X4 receptors act as lysosomal ion channels activated by luminal ATP.SLC17A9‐mediated ATP transport across the lysosomal membrane is suppressed by Bafilomycin A1, the V‐ATPase inhibitor.SLC17A9 mainly uses voltage gradient but not pH gradient generated by the V‐ATPase as the driving force to transport ATP into the lysosome to activate P2X4. Abstract The lysosome contains abundant ATP which plays important roles in lysosome functions and in cell signalling. Recently, solute carrier family 17 member 9 (SLC17A9, also known as VNUT for vesicular nucleotide transporter) proteins were suggested to function as a lysosomal ATP transporter responsible for lysosomal ATP accumulation, and P2X4 receptors were suggested to be lysosomal ion channels that are activated by luminal ATP. However, the molecular mechanism of SLC17A9 transporting ATP and the regulatory mechanism of lysosomal P2X4 are largely unknown. In this study, we report that SLC17A9‐mediated ATP transport across lysosomal membranes is suppressed by Bafilomycin A1, the V‐ATPase inhibitor. By measuring P2X4 activity, which is indicative of ATP transport across lysosomal membranes, we further demonstrated that SLC17A9 mainly uses voltage gradient but not pH gradient as the driving force to transport ATP into lysosomes. This study provides a molecular mechanism for lysosomal ATP transport mediated by SLC17A9. It also suggests a regulatory mechanism of lysosomal P2X4 by SLC17A9. PMID:27477609

Earthworm responses to Cd and Cu under fluctuating environmental conditions: a comparison with results from laboratory exposures

International Nuclear Information System (INIS)

Spurgeon, David J.; Svendsen, Claus; Lister, Lindsay J.; Hankard, Peter K.; Kille, Peter

2005-01-01

Laboratory toxicity tests are usually conducted under stable ambient conditions, while exposures in ecosystems occur in a fluctuating climate. To assess how climate influences the toxicity of Cu and Cd for the earthworm Lumbricus rubellus, this study compared effects for life-cycle parameters (survival, reproduction), cellular status (lysosomal membrane stability), gene expression (transcript of the metal binding protein metallothionein-2) and tissue metal concentration measured under outdoor conditions, with the same responses under constant conditions as measured by Spurgeon et al. [Spurgeon, D.J., Svendsen, C., Weeks, J.M., Hankard, P.K., Stubberud, H.E., Kammenga, J.E., 2003. Quantifying copper and cadmium impacts on intrinsic rate of population increase in the terrestrial oligochaete Lumbricus rubellus. Environmental Toxicology and Chemistry 22, 1465-1472]. Both metals were found to significantly influence earthworm reproduction, compromise lysosomal membrane stability and induce MT-2 gene expression in the outdoor system. Comparison with physiological and life-cycle responses in the laboratory indicated similar response patterns and effect concentrations for Cu. For Cd, lysosomal membrane stability and MT-2expression showed comparable responses in both exposures. Juvenile production rate, however, gave different dose response relationships, with the EC- 50 in the outdoor test approximately half that in the laboratory test. A difference in Cd accumulation was also seen. Overall, however, the comparison indicated only a marginal effect of environmental fluctuations typical for northern temperate Europe on earthworm sensitivity to the two metals. - Comparative analysis of life-cycle, physiological and molecular responses to Cu and Cd indicate similar responses under static and fluctuating climate regimes

mTORC1 Activator SLC38A9 Is Required to Efflux Essential Amino Acids from Lysosomes and Use Protein as a Nutrient.

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Wyant, Gregory A; Abu-Remaileh, Monther; Wolfson, Rachel L; Chen, Walter W; Freinkman, Elizaveta; Danai, Laura V; Vander Heiden, Matthew G; Sabatini, David M

2017-10-19

The mTORC1 kinase is a master growth regulator that senses many environmental cues, including amino acids. Activation of mTORC1 by arginine requires SLC38A9, a poorly understood lysosomal membrane protein with homology to amino acid transporters. Here, we validate that SLC38A9 is an arginine sensor for the mTORC1 pathway, and we uncover an unexpectedly central role for SLC38A9 in amino acid homeostasis. SLC38A9 mediates the transport, in an arginine-regulated fashion, of many essential amino acids out of lysosomes, including leucine, which mTORC1 senses through the cytosolic Sestrin proteins. SLC38A9 is necessary for leucine generated via lysosomal proteolysis to exit lysosomes and activate mTORC1. Pancreatic cancer cells, which use macropinocytosed protein as a nutrient source, require SLC38A9 to form tumors. Thus, through SLC38A9, arginine serves as a lysosomal messenger that couples mTORC1 activation to the release from lysosomes of the essential amino acids needed to drive cell growth. Copyright © 2017 Elsevier Inc. All rights reserved.

Isolating Lysosomes from Rat Liver.

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Pryor, Paul R

2016-04-01

This protocol describes the generation of a fraction enriched in lysosomes from rat liver. The lysosomes are rapidly isolated using density-gradient centrifugation with gradient media that retain the osmolarity of the lysosomes such that they are functional and can be used in in vitro assays. © 2016 Cold Spring Harbor Laboratory Press.

Drosophila melanogaster cellular repressor of E1A-stimulated genes is a lysosomal protein essential for fly development.

Science.gov (United States)

Kowalewski-Nimmerfall, Elisabeth; Schähs, Philipp; Maresch, Daniel; Rendic, Dubravko; Krämer, Helmut; Mach, Lukas

2014-12-01

Mammalian cellular repressor of E1A-stimulated genes is a lysosomal glycoprotein implicated in cellular growth and differentiation. The genome of the fruit fly Drosophila melanogaster encodes a putative orthologue (dCREG), suggesting evolutionarily conserved physiological functions of this protein. In D. melanogaster S2 cells, dCREG was found to localize in lysosomes. Further studies revealed that intracellular dCREG is subject of proteolytic maturation. Processing and turnover could be substantially reduced by RNAi-mediated silencing of cathepsin L. In contrast to mammalian cells, lysosomal delivery of dCREG does not depend on its carbohydrate moiety. Furthermore, depletion of the putative D. melanogaster lysosomal sorting receptor lysosomal enzyme receptor protein did not compromise cellular retention of dCREG. We also investigated the developmental consequences of dCREG ablation in whole D. melanogaster flies. Ubiquitous depletion of dCREG proved lethal at the late pupal stage once a knock-down efficiency of >95% was achieved. These results demonstrate that dCREG is essential for proper completion of fly development. Copyright © 2014. Published by Elsevier B.V.

Measuring the gypsum content of C&D debris fines.

Science.gov (United States)

Musson, Stephen E; Xu, Qiyong; Townsend, Timothy G

2008-11-01

Construction and demolition (C&D) debris recycling facilities often produce a screened material intended for use as alternative daily cover (ADC) at active landfills or for shaping and grading at closed landfills. This product contains soil and small pieces of wood, concrete, gypsum drywall, shingles and other components of C&D debris. Concerns have been raised over the contribution of gypsum drywall in C&D debris fines to odor problems at landfills where the product is used. To address such concerns, limitations may be placed on the percentage of gypsum (or sulfate) that can occur, and standardized testing procedures are required to permit valid compliance testing. A test procedure was developed for measuring the gypsum content in C&D debris fines. The concentration of sulfate leached in an aqueous solution was used to estimate the initial gypsum content of the sample. The impact of sample size and leaching time were evaluated. Precision and accuracy increased with increasing gypsum content. Results from replicate samples had an average relative standard deviation of 9%. The gypsum content of fines obtained from different facilities in the US varied widely from 1% to over 25%. These variations not only occurred between differing facilities, but within batches produced within a single facility.

Structure and Formation of cD Galaxies: NGC 6166 in ABELL 2199

Science.gov (United States)

Bender, Ralf; Kormendy, John; Cornell, Mark E.; Fisher, David B.

2015-07-01

 Hobby-Eberly Telescope (HET) spectroscopy is used to measure the velocity dispersion profile of the nearest prototypical cD galaxy, NGC 6166 in the cluster Abell 2199. We also present composite surface photometry from many telescopes. We confirm the defining feature of a cD galaxy; i.e., (we suggest), a halo of stars that fills the cluster center and that is controlled dynamically by cluster gravity, not by the central galaxy. Our HET spectroscopy shows that the velocity dispersion of NGC 6166 rises from σ ≃ 300 km s-1 in the inner r˜ 10\\prime\\prime to σ =865+/- 58 km s-1 at r ˜ 100″ in the cD halo. This extends published observations of an outward σ increase and shows for the first time that σ rises all the way to the cluster velocity dispersion of 819 ± 32 km s-1. We also observe that the main body of NGC 6166 moves at +206 ± 39 km s-1 with respect to the cluster mean velocity, but the velocity of the inner cD halo is ˜70 km s-1 closer to the cluster velocity. These results support our picture that cD halos consist of stars that were stripped from individual cluster galaxies by fast tidal encounters.  However, our photometry does not confirm the widespread view that cD halos are identifiable as an extra, low-surface-brightness component that is photometrically distinct from the inner, steep-Sérsic-function main body of an otherwise-normal giant elliptical galaxy. Instead, all of the brightness profile of NGC 6166 outside its core is described to ±0.037 V mag arcsec-2 by a single Sérsic function with index n≃ 8.3. The cD halo is not recognizable from photometry alone. This blurs the distinction between cluster-dominated cD halos and the similarly-large-Sérsic-index halos of giant, core-boxy-nonrotating ellipticals. These halos are believed to be accreted onto compact, high-redshift progenitors (“red nuggets”) by large numbers of minor mergers. They belong dynamically to their central galaxies. Still, cDs and core-boxy-nonrotating Es

Determination of frequencies of alleles, associated with the pseudodeficiency of lysosomal hydrolases, in population of Ukraine

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N. V. Olkhovych

2016-10-01

Full Text Available The pseudodeficiency of lysosomal hydrolases described as a significant reduction in enzyme activi­ty in vitro in clinically healthy individuals, can lead to diagnostic errors in the process of biochemical analysis of lysosomal storage disease in case of its combination with pathology of another origin. Pseudodeficiency is mostly caused by some non-pathogenic changes in the corresponding gene. These changes lead to the in vitro lability of the enzyme molecule, whereas in vivo the enzyme retains its functional activity. To assess the prevalence of the most common lysosomal hydrolases pseudodeficiency alleles in Ukraine, we have determined the frequency of alleles c.1055A>G and c.* 96A>G in the ARSA gene, substitutions c.739C>T (R247W and c.745C>T (R249W in the HEXA gene, c.1726G>A (G576S and c.2065G>A (E689K in the GAA gene, c.937G>T (D313Y in the GLA1 gene and c.898G>A (A300T in the IDUA gene in a group of 117 healthy individuals from different regions of the country and 14 heterozygous carriers of pathogenic mutations in the HEXA gene (parents of children with confirmed diagnosis of Tay-Sachs disease. The total frequency of haplotypes, associated with arylsulfatase A pseudodeficiency, in healthy people in Ukraine (c.1055G/c.*96G and c.1055G/c.*96A haplotypes was 10.3%. The frequency of c.739C>T (R247W allele, associated with hexo­saminidase A pseudodeficiency, among Tay-Sachs carriers from Ukraine was 7.1%. The total frequency of α-glucosidase pseudodeficiency haplotypes in healthy individuals in Ukraine (c.1726A/c.2065A and c.1726G/c.2065A haplotypes was 2.6%. No person among examined individuals with the substitution c.937G>T (D313Y in the GLA1 gene and c.898G>A (A300T in the IDUA gene was found. The differential diagnostics of lysosomal storage diseases requires obligatory determination of the presence of the pseudodeficiency alleles, particularly the ones with high incidence in the total population. Ignoring phenomenon of

Determination of frequencies of alleles, associated with the pseudodeficiency of lysosomal hydrolases, in population of Ukraine.

Science.gov (United States)

Olkhovych, N V; Gorovenko, N G

2016-01-01

The pseudodeficiency of lysosomal hydrolases described as a significant reduction in enzyme activi­ty in vitro in clinically healthy individuals, can lead to diagnostic errors in the process of biochemical analysis of lysosomal storage disease in case of its combination with pathology of another origin. Pseudodeficiency is mostly caused by some non-pathogenic changes in the corresponding gene. These changes lead to the in vitro lability of the enzyme molecule, whereas in vivo the enzyme retains its functional activity. To assess the prevalence of the most common lysosomal hydrolases pseudodeficiency alleles in Ukraine, we have determined the frequency of alleles c.1055A>G and c.* 96A>G in the ARSA gene, substitutions c.739C>T (R247W) and c.745C>T (R249W) in the HEXA gene, c.1726G>A (G576S) and c.2065G>A (E689K) in the GAA gene, c.937G>T (D313Y) in the GLA1 gene and c.898G>A (A300T) in the IDUA gene in a group of 117 healthy individuals from different regions of the country and 14 heterozygous carriers of pathogenic mutations in the HEXA gene (parents of children with confirmed diagnosis of Tay-Sachs disease). The total frequency of haplotypes, associated with arylsulfatase A pseudodeficiency, in healthy people in Ukraine (c.1055G/c.*96G and c.1055G/c.*96A haplotypes) was 10.3%. The frequency of c.739C>T (R247W) allele, associated with hexo­saminidase A pseudodeficiency, among Tay-Sachs carriers from Ukraine was 7.1%. The total frequency of α-glucosidase pseudodeficiency haplotypes in healthy individuals in Ukraine (c.1726A/c.2065A and c.1726G/c.2065A haplotypes) was 2.6%. No person among examined individuals with the substitution c.937G>T (D313Y) in the GLA1 gene and c.898G>A (A300T) in the IDUA gene was found. The differential diagnostics of lysosomal storage diseases requires obligatory determination of the presence of the pseudodeficiency alleles, particularly the ones with high incidence in the total population. Ignoring phenomenon of pseudodeficiency may

A Novel Method of Imaging Lysosomes in Living Human Mammary Epithelial Cells

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Kristine Glunde

2003-01-01

Full Text Available Cancer cells invade by secreting degradative enzymes which, under normal conditions, are sequestered in lysosomal vesicles. The ability to noninvasively label lysosomes and track lysosomal trafficking would be extremely useful to understand the mechanisms by which degradative enzymes are secreted in the presence of pathophysiological environments, such as hypoxia and acidic extracellular pH, which are frequently encountered in solid tumors. In this study, a novel method of introducing a fluorescent label into lysosomes of human mammary epithelial cells (HMECs was evaluated. Highly glycosylated lysosomal membrane proteins were labeled with a newly synthesized compound, 5-dimethylamino-naphthalene-1-sulfonic acid 5-amino-3,4,6-trihydroxy-tetrahydro-pyran-2-ylmethyl ester (6-O-dansyl-GlcNH2. The ability to optically image lysosomes using this new probe was validated by determining the colocalization of the fluorescence from the dansyl group with immunofluorescent staining of two well-established lysosomal marker proteins, LAMP-1 and LAMP-2. The location of the dansyl group in lysosomes was also verified by using an anti-dansyl antibody in Western blots of lysosomes isolated using isopycnic density gradient centrifugation. This novel method of labeling lysosomes biosynthetically was used to image lysosomes in living HMECs perfused in a microscopy-compatible cell perfusion system.

Localization of radioactivity from 2-methoxy[1,2-14C]ethanol in maternal and conceptus compartments of CD-1 mice

International Nuclear Information System (INIS)

Sleet, R.B.; John-Greene, J.A.; Welsch, F.

1986-01-01

2-Methoxyethanol (ME) induces paw malformations in CD-1 mice when given by gavage on gestation day (gd) 11 (vaginal plug + day = gd 0). The distribution of radioactivity originating from 2-methoxy[1,2- 14 C]ethanol ([ 14 C]ME) was examined by liquid scintillation spectrophotometry and whole body autoradiography in pregnant (gd 11) CD-1 mice from 5 min to 48 hr after oral administration. Each dam received either a trace dose of [ 14 C]ME (0.92 mumol; 13 muCi) combined with an unlabeled teratogenic dose (187 mumol). By 5 min after the trace dose was administered, 14 C had distributed throughout the maternal and conceptus compartments. Radioactivity in the maternal compartment was most concentrated in the liver, blood and gastrointestinal tract. Conceptus 14 C was associated with the placenta, yolk sac, and embryonal structures such as limb buds, somites, and neuroepithelium. The concentration of blood 14 C plateaued within 30 min after administration of the trace or combined trace/teratogenic dose. It remained stable for 1.5 hr and then gradually declined, reaching 2 to 10% of the maximal concentration by 48 hr. 14 C content in the maternal liver, conceptuses, and embryos per se was always greater than that of the blood and was inversely related to ME dose at 6 hr but not 48 hr. At 6 hr after administration of the trace dose, 69% of total liver and 33% of embryonal 14 C were acid insoluble. Tissue-specific interaction with [ 14 C]ME was demonstrated by the distribution of acid insoluble radioactivity among various cellular components of the maternal liver and embryo. The findings indicate that the embryo is readily susceptible to blood borne ME and/or its metabolites. In addition, the chemical characteristics of the labeled molecule(s) apparently favored label incorporation into macromolecules by the liver and embryo

HIV Controllers Exhibit Enhanced Frequencies of Major Histocompatibility Complex Class II Tetramer+ Gag-Specific CD4+ T Cells in Chronic Clade C HIV-1 Infection.

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Laher, Faatima; Ranasinghe, Srinika; Porichis, Filippos; Mewalal, Nikoshia; Pretorius, Karyn; Ismail, Nasreen; Buus, Søren; Stryhn, Anette; Carrington, Mary; Walker, Bruce D; Ndung'u, Thumbi; Ndhlovu, Zaza M

2017-04-01

Immune control of viral infections is heavily dependent on helper CD4 + T cell function. However, the understanding of the contribution of HIV-specific CD4 + T cell responses to immune protection against HIV-1, particularly in clade C infection, remains incomplete. Recently, major histocompatibility complex (MHC) class II tetramers have emerged as a powerful tool for interrogating antigen-specific CD4 + T cells without relying on effector functions. Here, we defined the MHC class II alleles for immunodominant Gag CD4 + T cell epitopes in clade C virus infection, constructed MHC class II tetramers, and then used these to define the magnitude, function, and relation to the viral load of HIV-specific CD4 + T cell responses in a cohort of untreated HIV clade C-infected persons. We observed significantly higher frequencies of MHC class II tetramer-positive CD4 + T cells in HIV controllers than progressors ( P = 0.0001), and these expanded Gag-specific CD4 + T cells in HIV controllers showed higher levels of expression of the cytolytic proteins granzymes A and B. Importantly, targeting of the immunodominant Gag41 peptide in the context of HLA class II DRB1*1101 was associated with HIV control ( r = -0.5, P = 0.02). These data identify an association between HIV-specific CD4 + T cell targeting of immunodominant Gag epitopes and immune control, particularly the contribution of a single class II MHC-peptide complex to the immune response against HIV-1 infection. Furthermore, these results highlight the advantage of the use of class II tetramers in evaluating HIV-specific CD4 + T cell responses in natural infections. IMPORTANCE Increasing evidence suggests that virus-specific CD4 + T cells contribute to the immune-mediated control of clade B HIV-1 infection, yet there remains a relative paucity of data regarding the role of HIV-specific CD4 + T cells in shaping adaptive immune responses in individuals infected with clade C, which is responsible for the majority of HIV

Pathogenic cascades in lysosomal disease-Why so complex?

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Walkley, S U

2009-04-01

Lysosomal disease represents a large group of more than 50 clinically recognized conditions resulting from inborn errors of metabolism affecting the organelle known as the lysosome. The lysosome is an integral part of the larger endosomal/lysosomal system, and is closely allied with the ubiquitin-proteosomal and autophagosomal systems, which together comprise essential cell machinery for substrate degradation and recycling, homeostatic control, and signalling. More than two-thirds of lysosomal diseases affect the brain, with neurons appearing particularly vulnerable to lysosomal compromise and showing diverse consequences ranging from specific axonal and dendritic abnormalities to neuron death. While failure of lysosomal function characteristically leads to lysosomal storage, new studies argue that lysosomal diseases may also be appropriately viewed as 'states of deficiency' rather than simply overabundance (storage). Interference with signalling events and salvage processing normally controlled by the endosomal/lysosomal system may represent key mechanisms accounting for the inherent complexity of lysosomal disorders. Analysis of lysosomal disease pathogenesis provides a unique window through which to observe the importance of the greater lysosomal system for normal cell health.

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

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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.

PD-1 expression on peripheral CD8+ TEM/TEMRA subsets closely correlated with HCV viral load in chronic hepatitis C patients

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Zhang Weidong

2010-11-01

Full Text Available Abstract Background Tight correlation between host circulating CD8+ T cell-mediated immune response and control of viral replication is classical characteristic of long-term HCV infection. CD8+ T cell maturation/activation markers are expected to be associated with viral replication and disease progression in chronic HCV infection. The aim of the present study was to explore novel markers on CD8+ T cells with ability to evaluate HCV viral replication and disease progression. Methods PBMCs were isolated from 37 chronic HCV-infected patients and 17 healthy controls. Distributed pattern of CD8+ T cells subsets and expression of PD-1, CD38, HLA-DR and CD127 were analyzed by flow cytometry. The correlation between expression of surface markers and HCV viral load or ALT was studied. Results Declined naïve and increased TEMRA CD8+ T subsets were found in HCV-infected individuals compared with healthy controls. Percentage and MFI of PD-1, CD38 and HLA-DR on all CD8+ T cell subsets were higher in HCV-infected patients than healthy controls. In contrast, CD127 expression on CD8+ TCM showed an opposite trend as PD-1, CD38 and HLA-DR did. In chronic HCV infection, MFI of PD-1 on CD8+ TEM (p Conclusion PD-1 level on peripheral CD8+ TEM/TEMRA was highly correlated with HCV viral load in chronic HCV-infected patients, which made PD-1 a novel indicator to evaluate HCV replication and disease progression in chronic hepatitis C patients.

Performance of single-pass and by-pass multi-step multi-soil-layering systems for low-(C/N)-ratio polluted river water treatment.

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Wei, Cai-Jie; Wu, Wei-Zhong

2018-09-01

Two kinds of hybrid two-step multi-soil-layering (MSL) systems loaded with different filter medias (zeolite-ceramsite MSL-1 and ceramsite-red clay MSL-2) were set-up for the low-(C/N)-ratio polluted river water treatment. A long-term pollutant removal performance of these two kinds of MSL systems was evaluated for 214 days. By-pass was employed in MSL systems to evaluate its effect on nitrogen removal enhancement. Zeolite-ceramsite single-pass MSL-1 system owns outstanding ammonia removal capability (24 g NH 4 + -Nm -2 d -1 ), 3 times higher than MSL-2 without zeolite under low aeration rate condition (0.8 × 10 4  L m -2 .h -1 ). Aeration rate up to 1.6 × 10 4  L m -2 .h -1 well satisfied the requirement of complete nitrification in first unit of both two MSLs. However, weak denitrification in second unit was commonly observed. By-pass of 50% influent into second unit can improve about 20% TN removal rate for both MSL-1 and MSL-2. Complete nitrification and denitrification was achieved in by-pass MSL systems after addition of carbon source with the resulting C/N ratio up to 2.5. The characters of biofilms distributed in different sections inside MSL-1 system well illustrated the nitrogen removal mechanism inside MSL systems. Two kinds of MSLs are both promising as an appealing nitrifying biofilm reactor. Recirculation can be considered further for by-pass MSL-2 system to ensure a complete ammonia removal. Copyright © 2018 Elsevier Ltd. All rights reserved.

AZ91C magnesium alloy modified by Cd

DEFF Research Database (Denmark)

Shabadi, R.; Ambat, Rajan; Dwarakadasa, E.S.

2014-01-01

In the present work, the effect of Cd on the microstructure, mechanical properties and general corrosion behaviour of AZ91C alloys was investigated. Addition of Cd was found not to be efficient in modifying/refining the microstructure or β-phase. A morphology change in β-phase from fine continuous...... precipitates to discontinuous β-phase upon the addition of Cd was observed. A marginal increment in mechanical properties was observed. General corrosion behaviour was followed with weight loss measurements, potentiostatic polarisation studies and surface studies in 3.5% sodium chloride solution and 3.......5% sodium chloride with 2% potassium dichromate solution. Cd addition deteriorated the corrosion behaviour of AZ91C. This behaviour was attributed to the formation of chunks of β-phase upon the addition of Cd. AZ91C with refined β-phase distribution, performed rather better in the NaCl solutions....

Id1 expression promotes peripheral CD4{sup +} T cell proliferation and survival upon TCR activation without co-stimulation

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Liu, Chen; Jin, Rong [Department of Immunology, Peking University Health Science Center, Beijing (China); Wang, Hong-Cheng [Oklahoma Medical Research Foundation, Oklahoma City, OK (United States); Tang, Hui; Liu, Yuan-Feng; Qian, Xiao-Ping; Sun, Xiu-Yuan; Ge, Qing [Department of Immunology, Peking University Health Science Center, Beijing (China); Sun, Xiao-Hong, E-mail: sunx@omrf.org [Oklahoma Medical Research Foundation, Oklahoma City, OK (United States); Zhang, Yu, E-mail: zhangyu007@bjmu.edu.cn [Department of Immunology, Peking University Health Science Center, Beijing (China)

2013-06-21

Highlights: •Id1 expression enables naïve T cell proliferation without anti-CD28 co-stimulation. •Id1 expression facilitates T cells survival when stimulated with anti-CD3. •Elevation of IL-2 production by Id1 contributes increased proliferation and survival. •Id1 potentiates NF-κB activation by anti-CD3 stimulation. -- Abstract: Although the role of E proteins in the thymocyte development is well documented, much less is known about their function in peripheral T cells. Here we demonstrated that CD4 promoter-driven transgenic expression of Id1, a naturally occurring dominant-negative inhibitor of E proteins, can substitute for the co-stimulatory signal delivered by CD28 to facilitate the proliferation and survival of naïve CD4{sup +} cells upon anti-CD3 stimulation. We next discovered that IL-2 production and NF-κB activity after anti-CD3 stimulation were significantly elevated in Id1-expressing cells, which may be, at least in part, responsible for the augmentation of their proliferation and survival. Taken together, results from this study suggest an important role of E and Id proteins in peripheral T cell activation. The ability of Id proteins to by-pass co-stimulatory signals to enable T cell activation has significant implications in regulating T cell immunity.

Cooperative use of cytochrome cd1 nitrite reductase and its redox partner cytochrome c552 to improve the selectivity of nitrite biosensing

International Nuclear Information System (INIS)

Serra, A.S.; Jorge, S.R.; Silveira, C.M.; Moura, J.J.G.; Jubete, E.; Ochoteco, E.; Cabanero, G.; Grande, H.; Almeida, M.G.

2011-01-01

In this work, a novel enzymatic biosensor for determination of nitrites constructed on an electrochemical transducing platform is proposed. The sensor is based on cytochrome-cd 1 (cyt-cd 1 ) nitrite reductase from Marinobacter hydrocarbonoclasticus strain 617 as biological recognition element, and its putative physiological redox partner cytochrome-c 552 (cyt-c 552 ), as electron mediator. The proteins were co-immobilized using a photopolymerizable polyvinyl alcohol (PVA) derivative, onto carbon paste screen printed electrodes (CPSPEs); the optimal modification conditions were 100 μM cyt-cd 1 /100 μM cyt-c 552 and 50% PVA, after a 48 h polymerization time. Electrochemical characterization of the mediator was carried out by cyclic voltammetry. The one-electron exchange between cyt-c 552 and the working electrode is a quasi-reversible process, without mass transport limitations. The formal potential of the mediator is 254 ± 2 mV vs NHE and the intermolecular electron transfer rate constant between cytochromes c 552 and cd 1 is 9.9 x 10 3 M -1 s -1 . The analytical parameters of the biosensor response to nitrite as assessed by amperometric measurements were: linear range from 10 to 200 μM; detection and quantification limits of 7 and 24 μM, respectively; sensitivity of 2.49 ± 0.08 A mol -1 cm 2 μM -1 . Catalytic profiles in the presence of possible interfering species were also investigated. The interference from competitive enzymatic reduction of dissolved oxygen could be overcome by tuning the cyclic voltammograms for faster sweep rates.

Effect of Lactobacillus salivarius on Th1/Th2 cytokines and the number of spleen CD4⁺ CD25⁺ Foxp3⁺ Treg in asthma Balb/c mouse.

Science.gov (United States)

Yun, Xiang; Shang, Yunxiao; Li, Miao

2015-01-01

Bronchial asthma is a chronic airway inflammatory disease that involves T lymphocytes. In order to explore the effect of Lactobacillus salivarius on Th1/Th2 cytokines and the number of spleen CD4(+) CD25(+) Foxp3(+) Treg in asthma Balb/c mouse, we constructed acute asthma model with ovalbumin to observe the mouse behavior change in Balb/c mice. The expression of GATA-3 mRNA and T-bet mRNA was measured by real-time PCR. The proportion of CD4(+) CD25(+) Foxp3(+) Treg/CD4(+) was determined by flow cytometry. The results demonstrated that oral gavage with Lactobacillus salivarius before sensitization could alleviate the clinical symptoms, airway hyper-reactivity and airway inflammation in asthma mouse to some extent; Lactobacillus salivarius may improve the imbalance of Th1/Th2 in asthma mouse through increasing the expression of T-bet mRNA at the transcriptional level and inhibiting the expression of GATA-3 mRNA simultaneously. CD4(+) CD25(+) Foxp3(+) Treg cells may be involved in the pathogenesis of bronchial asthma, and may be the upstream regulatory mechanism of the improvement of Th1/Th2 imbalance by Lactobacillus salivarius.

The role of CD154-CD40 versus CD28-B7 costimulatory pathways in regulating allogeneic Th1 and Th2 responses in vivo

DEFF Research Database (Denmark)

Kishimoto, K; Dong, V M; Issazadeh-Navikas, Shohreh

2000-01-01

We used signal transducer and activator of transcription 4 (STAT4) and STAT6 gene knockout (-/-) mice as recipients of fully mismatched cardiac allografts to study the role of T-cell costimulatory pathways in regulating allogeneic T-helper 1 (Th1) versus Th2 responses in vivo. STAT4(-/-) mice have...... impaired Th1 responses, whereas STAT6(-/-) mice do not generate normal Th2 responses. Cardiac allografts from C57BL/6 mice were transplanted into normal wild-type (WT), STAT4(-/-), and STAT6(-/-) BALB/c recipients. STAT4(-/-) and STAT6(-/-) mice rejected their grafts with the same tempo as untreated WT....... Furthermore, there was a similar differential effect of CD28-B7 versus CD154-CD40 blockade in inhibiting immune responses in animals immunized with ovalbumin and complete Freund's adjuvant. These novel data indicate that Th1 and Th2 cells are differentially regulated by CD28-B7 versus CD154-CD40 costimulation...

The Role of Aquaporin 1 Activated by cGMP in Myocardial Edema Caused by Cardiopulmonary Bypass in Sheep

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Fang-bao Ding

2013-11-01

Full Text Available Background/Aims: Most cardiac procedures involve the use of cardiopulmonary bypass (CPB, which pumps oxygenated blood to the body while the heart and lungs are isolated. CPB can cause profound alterations V in the homeostasis of physiological fluids, which often results in myocardial edema. In our study, we used sheep CPB model of in vivo and in vitro to assess the relationship between cGMP and AQP1 during CPB. Methods: ODQ, a specific inhibitor of soluble guanylate cyclase (sGC, was used to treat the CPB animals or cardiomyocytes. Left ventricular function of each group was determined by pressure-volume system. Water content of myocardial tissue was assessed by dry-wet weight, and cardiomyocytes water permeability was also calculated. The concentration of cGMP was determined by Radioimmunoassay (RIA. mRNA and protein expression of AQP1 were detected by real-time PCR and western blot, respectively. Results: The relative expression level of AQP1 mRNA and protein at each time point (0, 6, 12, 24 or 48 h after CPB was significantly increased (1.18-fold at 12 h, 1.77-fold at 24 h and 2.18-fold at 48h compared with each sham group, the protein expression of AQP1 also showed a rising trend after CPB. The degree of myocardial edema (75.1% at 12 h, 79.3% at 24 h and 81.0% at 48h increased following the CPB surgery. The mRNA expression level of AQP1 was significantly decreased by 39.7% (pin vitro experiments showed the same changing trends as in vivo. Conclusion: cGMP pathway controls water channels and then affects water intake during CPB through an AQP1-mediated pathway.

Mitochondrial Dysfunction in Lysosomal Storage Disorders

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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.

Modulation of phenotype and function of human CD4+CD25+ T regulatory lymphocytes mediated by cAMP elevating agents

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Antonella Riccomi

2016-09-01

Full Text Available We have shown that Cholera Toxin (CT and other cyclic AMP (cAMP elevating agents induce up-regulation of the inhibitory molecule CTLA-4 in human resting CD4+ T lymphocytes, which following the treatment acquired suppressive functions. In this study, we evaluated the effect of cAMP elevating agents on human CD4+CD25+ T cells, which include the T regulatory (Treg cells that play a pivotal role in the maintenance of immunological tolerance. We found that cAMP elevating agents induce up-regulation of CTLA-4 in CD4+CD25- and further enhance its expression in CD4+CD25+ T cells. We observed an increase of two isoforms of mRNA coding for the membrane and the soluble CTLA-4 molecules, suggesting that the regulation of CTLA-4 expression by cAMP is at the transcriptional level. In addition, we found that the increase of cAMP in CD4+CD25+ T cells converts the CD4+CD25+Foxp3- T cells in CD4+CD25+Foxp3+ T cells, whereas the increase of cAMP in CD4+CD25- T cells did not up-regulate Foxp3 in the absence of activation stimuli. To investigate the function of these cells, we performed an in vitro suppression assay by culturing CD4+CD25+ T cells untreated or pre-treated with CT with anti-CD3 mAbs-stimulated autologous PBMC. We found that CT enhances the inhibitory function of CD4+CD25+ T cells, CD4+ and CD8+ T cell proliferation and IFNγ production are strongly inhibited by CD4+CD25+ T cells pre-treated with cAMP elevating agents. Furthermore, we found that CD4+CD25+ T lymphocytes pre-treated with cAMP elevating agents induce the up-regulation of CD80 and CD86 co-stimulatory molecules on immature dendritic cells (DCs in the absence of antigenic stimulation, however without leading to full DC maturation. These data show that the increase of intracellular cAMP modulates the phenotype and function of human CD4+CD25+ T cells.

Tracking virus-specific CD4+ T cells during and after acute hepatitis C virus infection.

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Michaela Lucas

2007-07-01

Full Text Available CD4+ T cell help is critical in maintaining antiviral immune responses and such help has been shown to be sustained in acute resolving hepatitis C. In contrast, in evolving chronic hepatitis C CD4+ T cell helper responses appear to be absent or short-lived, using functional assays.Here we used a novel HLA-DR1 tetramer containing a highly targeted CD4+ T cell epitope from the hepatitis C virus non-structural protein 4 to track number and phenotype of hepatitis C virus specific CD4+ T cells in a cohort of seven HLA-DR1 positive patients with acute hepatitis C in comparison to patients with chronic or resolved hepatitis C. We observed peptide-specific T cells in all seven patients with acute hepatitis C regardless of outcome at frequencies up to 0.65% of CD4+ T cells. Among patients who transiently controlled virus replication we observed loss of function, and/or physical deletion of tetramer+ CD4+ T cells before viral recrudescence. In some patients with chronic hepatitis C very low numbers of tetramer+ cells were detectable in peripheral blood, compared to robust responses detected in spontaneous resolvers. Importantly we did not observe escape mutations in this key CD4+ T cell epitope in patients with evolving chronic hepatitis C.During acute hepatitis C a CD4+ T cell response against this epitope is readily induced in most, if not all, HLA-DR1+ patients. This antiviral T cell population becomes functionally impaired or is deleted early in the course of disease in those where viremia persists.

CD147 reprograms fatty acid metabolism in hepatocellular carcinoma cells through Akt/mTOR/SREBP1c and P38/PPARα pathways.

Science.gov (United States)

Li, Jibin; Huang, Qichao; Long, Xiaoyu; Zhang, Jing; Huang, Xiaojun; Aa, Jiye; Yang, Hushan; Chen, Zhinan; Xing, Jinliang

2015-12-01

CD147 is a transmembrane glycoprotein which is highly expressed in various human cancers including hepatocellular carcinoma (HCC). A drug Licartin developed with (131)Iodine-labeled antibody against CD147 has been approved by the Chinese Food and Drug Administration (FDA) and enters into clinical use for HCC treatment. Increasing lines of evidence indicate that CD147 is implicated in the metabolism of cancer cells, especially glycolysis. However, the molecular mechanism underlying the relationship between CD147 and aberrant tumor lipid metabolism remains elusive. We systematically investigated the role of CD147 in the regulation of lipid metabolism, including de novo lipogenesis and fatty acid β-oxidation, in HCC cells and explored the underlying molecular mechanisms. Bioinformatic analysis and experimental evidence demonstrated that CD147 significantly contributed to the reprogramming of fatty acid metabolism in HCC cells mainly through two mechanisms. On one hand, CD147 upregulated the expression of sterol regulatory element binding protein 1c (SREBP1c) by activating the Akt/mTOR signaling pathway, which in turn directly activated the transcription of major lipogenic genes FASN and ACC1 to promote de novo lipogenesis. On the other hand, CD147 downregulated peroxisome proliferator-activated receptor alpha (PPARα) and its transcriptional target genes CPT1A and ACOX1 by activating the p38 MAPK signaling pathway to inhibit fatty acid β-oxidation. Moreover, in vitro and in vivo assays indicated that the CD147-mediated reprogramming of fatty acid metabolism played a critical role in the proliferation and metastasis of HCC cells. Our findings demonstrate that CD147 is a critical regulator of fatty acid metabolism, which provides a strong line of evidence for this molecule to be used as a drug target in cancer treatment. Copyright © 2015 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.

Lysosomes as mediators of drug resistance in cancer.

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Zhitomirsky, Benny; Assaraf, Yehuda G

2016-01-01

Drug resistance remains a leading cause of chemotherapeutic treatment failure and cancer-related mortality. While some mechanisms of anticancer drug resistance have been well characterized, multiple mechanisms remain elusive. In this respect, passive ion trapping-based lysosomal sequestration of multiple hydrophobic weak-base chemotherapeutic agents was found to reduce the accessibility of these drugs to their target sites, resulting in a markedly reduced cytotoxic effect and drug resistance. Recently we have demonstrated that lysosomal sequestration of hydrophobic weak base drugs triggers TFEB-mediated lysosomal biogenesis resulting in an enlarged lysosomal compartment, capable of enhanced drug sequestration. This study further showed that cancer cells with an increased number of drug-accumulating lysosomes are more resistant to lysosome-sequestered drugs, suggesting a model of drug-induced lysosome-mediated chemoresistance. In addition to passive drug sequestration of hydrophobic weak base chemotherapeutics, other mechanisms of lysosome-mediated drug resistance have also been reported; these include active lysosomal drug sequestration mediated by ATP-driven transporters from the ABC superfamily, and a role for lysosomal copper transporters in cancer resistance to platinum-based chemotherapeutics. Furthermore, lysosomal exocytosis was suggested as a mechanism to facilitate the clearance of chemotherapeutics which highly accumulated in lysosomes, thus providing an additional line of resistance, supplementing the organelle entrapment of chemotherapeutics away from their target sites. Along with these mechanisms of lysosome-mediated drug resistance, several approaches were recently developed for the overcoming of drug resistance or exploiting lysosomal drug sequestration, including lysosomal photodestruction and drug-induced lysosomal membrane permeabilization. In this review we explore the current literature addressing the role of lysosomes in mediating cancer drug

Lysosomal storage diseases

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Ferreira, Carlos R.; Gahl, William A.

2016-01-01

Lysosomes are cytoplasmic organelles that contain a variety of different hydrolases. A genetic deficiency in the enzymatic activity of one of these hydrolases will lead to the accumulation of the material meant for lysosomal degradation. Examples include glycogen in the case of Pompe disease, glycosaminoglycans in the case of the mucopolysaccharidoses, glycoproteins in the cases of the oligosaccharidoses, and sphingolipids in the cases of Niemann-Pick disease types A and B, Gaucher disease, Tay-Sachs disease, Krabbe disease, and metachromatic leukodystrophy. Sometimes, the lysosomal storage can be caused not by the enzymatic deficiency of one of the hydrolases, but by the deficiency of an activator protein, as occurs in the AB variant of GM2 gangliosidosis. Still other times, the accumulated lysosomal material results from failed egress of a small molecule as a consequence of a deficient transporter, as in cystinosis or Salla disease. In the last couple of decades, enzyme replacement therapy has become available for a number of lysosomal storage diseases. Examples include imiglucerase, taliglucerase and velaglucerase for Gaucher disease, laronidase for Hurler disease, idursulfase for Hunter disease, elosulfase for Morquio disease, galsulfase for Maroteaux-Lamy disease, alglucosidase alfa for Pompe disease, and agalsidase alfa and beta for Fabry disease. In addition, substrate reduction therapy has been approved for certain disorders, such as eliglustat for Gaucher disease. The advent of treatment options for some of these disorders has led to newborn screening pilot studies, and ultimately to the addition of Pompe disease and Hurler disease to the Recommended Uniform Screening Panel (RUSP) in 2015 and 2016, respectively. PMID:29152458

Autophagy failure in Alzheimer's disease and the role of defective lysosomal acidification.

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Wolfe, Devin M; Lee, Ju-Hyun; Kumar, Asok; Lee, Sooyeon; Orenstein, Samantha J; Nixon, Ralph A

2013-06-01

Autophagy is a lysosomal degradative process which recycles cellular waste and eliminates potentially toxic damaged organelles and protein aggregates. The important cytoprotective functions of autophagy are demonstrated by the diverse pathogenic consequences that may stem from autophagy dysregulation in a growing number of neurodegenerative disorders. In many of the diseases associated with autophagy anomalies, it is the final stage of autophagy-lysosomal degradation that is disrupted. In several disorders, including Alzheimer's disease (AD), defective lysosomal acidification contributes to this proteolytic failure. The complex regulation of lysosomal pH makes this process vulnerable to disruption by many factors, and reliable lysosomal pH measurements have become increasingly important in investigations of disease mechanisms. Although various reagents for pH quantification have been developed over several decades, they are not all equally well suited for measuring the pH of lysosomes. Here, we evaluate the most commonly used pH probes for sensitivity and localisation, and identify LysoSensor yellow/blue-dextran, among currently used probes, as having the optimal profile of properties for measuring lysosomal pH. In addition, we review evidence that lysosomal acidification is defective in AD and extend our original findings, of elevated lysosomal pH in presenilin 1 (PS1)-deficient blastocysts and neurons, to additional cell models of PS1 and PS1/2 deficiency, to fibroblasts from AD patients with PS1 mutations, and to neurons in the PS/APP mouse model of AD. © 2013 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

A coordination polymer of CdII with benzene-1,3-dicarboxylate and 1,4-bis[1-(2-pyridylmethylbenzimidazol-2-yl]butane

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Wei-Ping Zhang

2009-11-01

Full Text Available The title CdII coordination polymer, catena-poly[[{1,4-bis[1-(2-pyridylmethylbenzimidazol-2-yl]butane}cadmium(II]-μ-benzene-1,3-dicarboxylato], [Cd(C8H4O4(C30H28N6]n, was obtained by reaction of CdCO3, benzene-1,3-dicarboxylic acid (H2btc and 1,4-bis[1-(2-pyridylmethylbenzimidazol-2-yl]butane (L. The CdII cation is six-coordinated by an N2O4-donor set. L acts as a bidentate ligand and btc anions link CdII centers into a chain propagating parallel to [010].

The lysosomal enzyme receptor protein (LERP is not essential, but is implicated in lysosomal function in Drosophila melanogaster

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Medina Hasanagic

2015-10-01

Full Text Available The lysosomal enzyme receptor protein (LERP of Drosophila melanogaster is the ortholog of the mammalian cation-independent mannose 6-phosphate (Man 6-P receptor, which mediates trafficking of newly synthesized lysosomal acid hydrolases to lysosomes. However, flies lack the enzymes necessary to make the Man 6-P mark, and the amino acids implicated in Man 6-P binding by the mammalian receptor are not conserved in LERP. Thus, the function of LERP in sorting of lysosomal enzymes to lysosomes in Drosophila is unclear. Here, we analyze the consequence of LERP depletion in S2 cells and intact flies. RNAi-mediated knockdown of LERP in S2 cells had little or no effect on the cellular content or secretion of several lysosomal hydrolases. We generated a novel Lerp null mutation, LerpF6, which abolishes LERP protein expression. Lerp mutants have normal viability and fertility and display no overt phenotypes other than reduced body weight. Lerp mutant flies exhibit a 30–40% decrease in the level of several lysosomal hydrolases, and are hypersensitive to dietary chloroquine and starvation, consistent with impaired lysosome function. Loss of LERP also enhances an eye phenotype associated with defective autophagy. Our findings implicate Lerp in lysosome function and autophagy.

FIG4 regulates lysosome membrane homeostasis independent of phosphatase function.

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

2016-02-15

FIG4 is a phosphoinositide phosphatase that is mutated in several diseases including Charcot-Marie-Tooth Disease 4J (CMT4J) and Yunis-Varon syndrome (YVS). To investigate the mechanism of disease pathogenesis, we generated Drosophila models of FIG4-related diseases. Fig4 null mutant animals are viable but exhibit marked enlargement of the lysosomal compartment in muscle cells and neurons, accompanied by an age-related decline in flight ability. Transgenic animals expressing Drosophila Fig4 missense mutations corresponding to human pathogenic mutations can partially rescue lysosomal expansion phenotypes, consistent with these mutations causing decreased FIG4 function. Interestingly, Fig4 mutations predicted to inactivate FIG4 phosphatase activity rescue lysosome expansion phenotypes, and mutations in the phosphoinositide (3) phosphate kinase Fab1 that performs the reverse enzymatic reaction also causes a lysosome expansion phenotype. Since FIG4 and FAB1 are present together in the same biochemical complex, these data are consistent with a model in which FIG4 serves a phosphatase-independent biosynthetic function that is essential for lysosomal membrane homeostasis. Lysosomal phenotypes are suppressed by genetic inhibition of Rab7 or the HOPS complex, demonstrating that FIG4 functions after endosome-to-lysosome fusion. Furthermore, disruption of the retromer complex, implicated in recycling from the lysosome to Golgi, does not lead to similar phenotypes as Fig4, suggesting that the lysosomal defects are not due to compromised retromer-mediated recycling of endolysosomal membranes. These data show that FIG4 plays a critical noncatalytic function in maintaining lysosomal membrane homeostasis, and that this function is disrupted by mutations that cause CMT4J and YVS. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Reduced synaptic vesicle protein degradation at lysosomes curbs TBC1D24/sky-induced neurodegeneration.

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Fernandes, Ana Clara; Uytterhoeven, Valerie; Kuenen, Sabine; Wang, Yu-Chun; Slabbaert, Jan R; Swerts, Jef; Kasprowicz, Jaroslaw; Aerts, Stein; Verstreken, Patrik

2014-11-24

Synaptic demise and accumulation of dysfunctional proteins are thought of as common features in neurodegeneration. However, the mechanisms by which synaptic proteins turn over remain elusive. In this paper, we study Drosophila melanogaster lacking active TBC1D24/Skywalker (Sky), a protein that in humans causes severe neurodegeneration, epilepsy, and DOOR (deafness, onychdystrophy, osteodystrophy, and mental retardation) syndrome, and identify endosome-to-lysosome trafficking as a mechanism for degradation of synaptic vesicle-associated proteins. In fly sky mutants, synaptic vesicles traveled excessively to endosomes. Using chimeric fluorescent timers, we show that synaptic vesicle-associated proteins were younger on average, suggesting that older proteins are more efficiently degraded. Using a genetic screen, we find that reducing endosomal-to-lysosomal trafficking, controlled by the homotypic fusion and vacuole protein sorting (HOPS) complex, rescued the neurotransmission and neurodegeneration defects in sky mutants. Consistently, synaptic vesicle proteins were older in HOPS complex mutants, and these mutants also showed reduced neurotransmission. Our findings define a mechanism in which synaptic transmission is facilitated by efficient protein turnover at lysosomes and identify a potential strategy to suppress defects arising from TBC1D24 mutations in humans. © 2014 Fernandes et al.

αvβ5 Integrin/FAK/PGC-1α Pathway Confers Protective Effects on Retinal Pigment Epithelium.

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Murilo F Roggia

Full Text Available To elucidate the mechanism of the induction of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α by photoreceptor outer segments (POS and its effects on retinal pigment epithelium (RPE.PGC-1α upregulation by POS was confirmed in ARPE-19 cells and in RPE ex vivo. To elucidate the mechanism, siRNAs against β5 integrin, CD36, Mer tyrosine kinase (MerTK, and Atg5, blocking antibodies against CD36 and MerTK, and a specific inhibitor for focal adhesion kinase (FAK were used. We examined the effect of POS-induced PGC-1α upregulation on the levels of reactive oxygen species (ROS, mitochondrial biogenesis, senescence-associated β-galactosidase (SA-β-gal after H2O2 treatment, and lysosomal activity. Lysosomal activity was evaluated through transcriptional factor EB and its target genes, and the activity of cathepsin D. Lipid metabolism after POS treatment was assessed using Oil Red O and BODIPY C11. RPE phenotypes of PGC-1α-deficient mice were examined.POS-induced PGC-1α upregulation was suppressed by siRNA against β5 integrin and a FAK inhibitor. siRNAs and blocking antibodies against CD36 and MerTK enhanced the effect of POS on PGC-1α. The upregulation of PGC-1α increased the levels of mRNA for antioxidant enzymes and stimulated mitochondrial biogenesis, decreased ROS levels, and reduced SA-β-gal staining in H2O2-treated ARPE-19 cells. PGC-1α was critical for lysosomal activity and lipid metabolism after POS treatment. PGC-1α-deficient mice demonstrated an accumulation of type 2 lysosomes in RPE, thickening of Bruch's membrane, and poor choriocapillaris vasculature.The binding, but not the internalization of POS confers protective effects on RPE cells through the αvβ5 integrin/FAK/PGC-1α pathway.

GP2 is selectively expressed by small intestinal CD103⁺CD11b⁺ cDC

DEFF Research Database (Denmark)

Müller-Luda, Katarzyna; Ahmadi, Fatemeh; Ohno, Hiroshi

DC in the small intestine. Moreover, GP2 expressing cDC in the small intestine were dramatically reduced in the setting of intestinal inflammation. We have previously shown that mice with an IRF4 deletion in CD11c+ cells (Cd11c-cre.Irf4 fl/fl mice) have reduced numbers of small intestinal CD103+CD11b+ c......DC. Interesting, we found that GP2+ CD103+CD11b+ cDC were dramatically reduced in these mice. Finally, to address the in vivo role of GP2 expression by cDC, we have generated mice with a selective deletion of GP2 in CD103+CD11b+ cDC (huLangerin-cre.gp2 fl/fl  mice). Results from these ongoing studies...

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....

Iowa Mutant Apolipoprotein A-I (ApoA-IIowa) Fibrils Target Lysosomes.

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Kameyama, Hirokazu; Nakajima, Hiroyuki; Nishitsuji, Kazuchika; Mikawa, Shiho; Uchimura, Kenji; Kobayashi, Norihiro; Okuhira, Keiichiro; Saito, Hiroyuki; Sakashita, Naomi

2016-07-28

The single amino acid mutation G26R in human apolipoprotein A-I (apoA-IIowa) is the first mutation that was associated with familial AApoA1 amyloidosis. The N-terminal fragments (amino acid residues 1-83) of apoA-I containing this mutation deposit as amyloid fibrils in patients' tissues and organs, but the mechanisms of cellular degradation and cytotoxicity have not yet been clarified. In this study, we demonstrated degradation of apoA-IIowa fibrils via the autophagy-lysosomal pathway in human embryonic kidney 293 cells. ApoA-IIowa fibrils induced an increase in lysosomal pH and the cytosolic release of the toxic lysosomal protease cathepsin B. The mitochondrial dysfunction caused by apoA-IIowa fibrils depended on cathepsin B and was ameliorated by increasing the degradation of apoA-IIowa fibrils. Thus, although apoA-IIowa fibril transport to lysosomes and fibril degradation in lysosomes may have occurred, the presence of an excess number of apoA-IIowa fibrils, more than the lysosomes could degrade, may be detrimental to cells. Our results thus provide evidence that the target of apoA-IIowa fibrils is lysosomes, and we thereby gained a novel insight into the mechanism of AApoA1 amyloidosis.

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....

Dicty_cDB: Contig-U15201-1 [Dicty_cDB

Lifescience Database Archive (English)

Full Text Available 2e-04 2 ( EX698640 ) GF_AW109651c08 AW1 Schistosoma mansoni cDNA clone... 44 2e-04 2 ( CD147...-0107T-L395-E12-U.B MG1-0107 Schistosoma manso... 44 2e-04 2 ( CD147233 ) ML1-0002T-M131-E11-U.G ML1-0002 Sc

BACE is degraded via the lysosomal pathway.

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Koh, Young Ho; von Arnim, Christine A F; Hyman, Bradley T; Tanzi, Rudolph E; Tesco, Giuseppina

2005-09-16

Amyloid plaques are formed by aggregates of amyloid-beta-peptide, a 37-43-amino acid fragment (primarily Abeta(40) and Abeta(42)) generated by proteolytic processing of the amyloid precursor protein (APP) by beta- and gamma-secretases. A type I transmembrane aspartyl protease, BACE (beta-site APP cleaving enzyme), has been identified to be the beta-secretase. BACE is targeted through the secretory pathway to the plasma membrane where it can be internalized to endosomes. The carboxyl terminus of BACE contains a di-leucine-based signal for sorting of transmembrane proteins to endosomes and lysosomes. In this study, we set out to determine whether BACE is degraded by the lysosomal pathway and whether the di-leucine motif is necessary for targeting BACE to the lysosomes. Here we show that lysosomal inhibitors, chloroquine and NH(4)Cl, lead to accumulation of endogenous and ectopically expressed BACE in a variety of cell types, including primary neurons. Furthermore, the inhibition of lysosomal hydrolases results in the redistribution and accumulation of BACE in the late endosomal/lysosomal compartments (lysosome-associated membrane protein 2 (LAMP2)-positive). In contrast, the BACE-LL/AA mutant, in which Leu(499) and Leu(500) in the COOH-terminal sequence (DDISLLK) were replaced by alanines, only partially co-localized with LAMP2-positive compartments following inhibition of lysosomal hydrolases. Collectively, our data indicate that BACE is transported to the late endosomal/lysosomal compartments where it is degraded via the lysosomal pathway and that the di-leucine motif plays a role in sorting BACE to lysosomes.

Ultraviolet induced lysosome activity in corneal epithelium

International Nuclear Information System (INIS)

Cullen, A.P.

1980-01-01

A 5.000 W Xe-Hg high pressure lamp and a double monochromator were used to produce a 3.3 nm half-bandpass ultraviolet radiation at 295 nm. Pigmented rabbit eyes were irradiated with radiant exposures from 140 Jm -2 to 10.000 Jm -2 and evaluated by slit-lamp biomicroscopy, light and electron microscopy. Corneal threshold (Hsub(c) was 200 Jm -2 and lens threshold (Hsub(L)) was 7.500 Jm -2 . The most repeatable and reliable corneal response to these levels of UV was the development of corneal epithelial granules. Histological changes included a loss of superficial epithelial cells and selective UV induced autolysis of the wing cells. It is suggested that the biomicroscopically observed granules are the clinical manifestation of the secondary lysosomes revealed by light and electron microscopy. It is proposed that UV breaks down the primary lysosome membranes to release hydrolytic enzymes which in turn form the secondary lysosomes during autolysis. Extreme levels of radiant exposure at 295 nm result in indiscriminate destruction of all layers of the corneal epithelium, but the posterior cornea was spared. (orig.) [de

TFEB ameliorates the impairment of the autophagy-lysosome pathway in neurons induced by doxorubicin

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Moruno Manchon, Jose Felix; Uzor, Ndidi-Ese; Kesler, Shelli R.; Wefel, Jeffrey S.; Townley, Debra M.; Nagaraja, Archana Sidalaghatta; Pradeep, Sunila; Mangala, Lingegowda S.; Sood, Anil K.; Tsvetkov, Andrey S.

2016-01-01

Doxorubicin, a commonly used chemotherapy agent, induces severe cardio- and neurotoxicity. Molecular mechanisms of cardiotoxicity have been extensively studied, but mechanisms by which doxorubicin exhibits its neurotoxic properties remain unclear. Here, we show that doxorubicin impairs neuronal autophagy, leading to the accumulation of an autophagy substrate p62. Neurons treated with doxorubicin contained autophagosomes, damaged mitochondria, and lipid droplets. The brains from mice treated with pegylated liposomal doxorubicin exhibited autophagosomes, often with mitochondria, lipofuscin, and lipid droplets. Interestingly, lysosomes were less acidic in doxorubicin-treated neurons. Overexpression of the transcription factor EB (TFEB), which controls the autophagy-lysosome axis, increased survival of doxorubicin-treated neurons. 2-Hydroxypropyl-β-cyclodextrin (HPβCD), an activator of TFEB, also promoted neuronal survival, decreased the levels of p62, and lowered the pH in lysosomes. Taken together, substantial changes induced by doxorubicin contribute to neurotoxicity, cognitive disturbances in cancer patients and survivors, and accelerated brain aging. The TFEB pathway might be a new approach for mitigating damage of neuronal autophagy caused by doxorubicin. PMID:27992857

Hydrogen sulfide flux measurements from construction and demolition debris (C&D) landfills.

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Eun, Sangho; Reinhart, Debra R; Cooper, C David; Townsend, Timothy G; Faour, Ayman

2007-01-01

Hydrogen sulfide (H2S) has been identified as a principal odorous component of gaseous emissions from construction and demolition debris (C&D) landfills. Although several studies have reported the ambient concentrations of H2S near C&D landfills, few studies have quantified emission rates of H2S. One of the most widely used techniques for measuring surface gas emission rates from landfills is the flux chamber method. Flux measurements using the flux chamber were performed at five different C&D landfills from April to August, 2003. The flux rates of H2S measured in this research were between 0.192 and 1.76 mg/(m2-d).

Dysregulated CD46 shedding interferes with Th1-contraction in systemic lupus erythematosus.

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Ellinghaus, Ursula; Cortini, Andrea; Pinder, Christopher L; Le Friec, Gaelle; Kemper, Claudia; Vyse, Timothy J

2017-07-01

IFN-γ-producing T helper 1 (Th1) cell responses mediate protection against infections but uncontrolled Th1 activity also contributes to a broad range of autoimmune diseases. Autocrine complement activation has recently emerged as key in the induction and contraction of human Th1 immunity: activation of the complement regulator CD46 and the C3aR expressed by CD4 + T cells via autocrine generated ligands C3b and C3a, respectively, are critical to IFN-γ production. Further, CD46-mediated signals also induce co-expression of immunosuppressive IL-10 in Th1 cells and transition into a (self)-regulating and contracting phase. In consequence, C3 or CD46-deficient patients suffer from recurrent infections while dysregulation of CD46 signaling contributes to Th1 hyperactivity in rheumatoid arthritis and multiple sclerosis. Here, we report a defect in CD46-regulated Th1 contraction in patients with systemic lupus erythematosus (SLE). We observed that MMP-9-mediated increased shedding of soluble CD46 by Th1 cells was associated with this defect and that inhibition of MMP-9 activity normalized release of soluble CD46 and restored Th1 contraction in patients' T cells. These data may deliver the first mechanistic explanation for the increased serum CD46 levels observed in SLE patients and indicate that targeting CD46-cleaving proteases could be a novel avenue to modulate Th1 responses. © 2017 The Authors. European Journal of Immunology published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A novel kinesin-like protein, KIF1Bbeta3 is involved in the movement of lysosomes to the cell periphery in non-neuronal cells.

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Matsushita, Masafumi; Tanaka, Shingo; Nakamura, Norihiro; Inoue, Hiroki; Kanazawa, Hiroshi

2004-03-01

The kinesin superfamily protein, KIF1Bbeta, a splice variant of KIF1B, is involved in the transport of synaptic vesicles in neuronal cells, and is also expressed in various non-neuronal tissues. To elucidate the functions of KIF1Bbeta in non-neuronal cells, we analyzed the intracellular localization of KIF1Bbeta and characterized its isoform expression profile. In COS-7 cells, KIF1B colocalized with lysosomal markers and expression of a mutant form of KIF1Bbeta, lacking the motor domain, impaired the intracellular distribution of lysosomes. A novel isoform of the kinesin-like protein, KIF1Bbeta3, was identified in rat and simian kidney. It lacks the 5th exon of the KIF1Bbeta-specific tail region. Overexpression of KIF1Bbeta3 induced the translocation of lysosomes to the cell periphery. However, overexpression of KIF1Bbeta3-Q98L, which harbors a pathogenic mutation associated with a familial neuropathy, Charcot-Marie-Tooth disease type 2 A, resulted in the abnormal perinuclear clustering of lysosomes. These results indicate that KIF1Bbeta3 is involved in the translocation of lysosomes from perinuclear regions to the cell periphery.

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

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Yanyan Li

2016-01-01

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

Whole-gene analysis of two groups of hepatitis B virus C/D inter-genotype recombinant strains isolated in Tibet, China.

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Tiezhu Liu

Full Text Available Tibet is a highly hepatitis B virus (HBV endemic area. Two types of C/D recombinant HBV are commonly isolated in Tibet and have been previously described. In an effort to better understand the molecular characteristic of these C/D recombinant strains from Tibet, we undertook a multistage random sampling project to collect HBsAg positive samples. Molecular epidemiological and bio-informational technologies were used to analyze the characteristics of the sequences found in this study. There were 60 samples enrolled in the survey, and we obtained 19 whole-genome sequences. 19 samples were all C/D recombinant, and could be divided into two sub-types named C/D1 and C/D2 according to the differences in the location of the recombinant breakpoint. The recombination breakpoint of the 10 strains belonging to the C/D1 sub-type was located at nt750, while the 9 stains belonging to C/D2 had their recombination break point at nt1530. According to whole-genome sequence analysis, the 19 identified strains belong to genotype C, but the nucleotide distance was more than 5% between the 19 strains and sub-genotypes C1 to C15. The distance between C/D1with C2 was 5.8±2.1%, while the distance between C/D2 with C2 was 6.4±2.1%. The parental strain was most likely sub-genotype C2. C/D1 strains were all collected in the middle and northern areas of Tibet including Lhasa, Linzhi and Ali, while C/D2 was predominant in Shannan in southern Tibet. This indicates that the two recombinant genotypes are regionally distributed in Tibet. These results provide important information for the study of special HBV recombination events, gene features, virus evolution, and the control and prevention policy of HBV in Tibet.

Cancer-associated lysosomal changes: friends or foes?

Science.gov (United States)

Kallunki, T; Olsen, O D; Jäättelä, M

2013-04-18

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 changes in the lysosomal compartment can be regarded as friends or foes. Most of them are clearly transforming as they promote invasive growth, angiogenesis and drug resistance. The same changes can, however, strongly sensitize cells to lysosomal membrane permeabilization and thereby to lysosome-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.

Purification and primary structure determination of human lysosomal dipeptidase.

Science.gov (United States)

Dolenc, Iztok; Mihelic, Marko

2003-02-01

The lysosomal metallopeptidase is an enzyme that acts preferentially on dipeptides with unsubstituted N- and C-termini. Its activity is highest in slightly acidic pH. Here we describe the isolation and characterization of lysosomal dipeptidase from human kidney. The isolated enzyme has the amino-terminal sequence DVAKAIINLAVY and is a homodimer with a molecular mass of 100 kDa. So far no amino acid sequence has been determined for this metallopeptidase. The complete primary structure as deduced from the nucleotide sequence revealed that the isolated dipeptidase is similar to blood plasma glutamate carboxypeptidase.

Glucose Modulation Induces Lysosome Formation and Increases Lysosomotropic Drug Sequestration via the P-Glycoprotein Drug Transporter.

Science.gov (United States)

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

2016-02-19

Pgp is functional on the plasma membrane and lysosomal membrane. Lysosomal-Pgp can pump substrates into the organelle, thereby trapping certain chemotherapeutics (e.g. doxorubicin; DOX). This mechanism serves as a "safe house" to protect cells against cytotoxic drugs. Interestingly, in contrast to DOX, lysosomal sequestration of the novel anti-tumor agent and P-glycoprotein (Pgp) substrate, di-2-pyridylketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT), induces lysosomal membrane permeabilization. This mechanism of lysosomal-Pgp utilization enhances cytotoxicity to multidrug-resistant cells. Consequently, Dp44mT has greater anti-tumor activity in drug-resistant relative to non-Pgp-expressing tumors. Interestingly, stressors in the tumor microenvironment trigger endocytosis for cell signaling to assist cell survival. Hence, this investigation examined how glucose variation-induced stress regulated early endosome and lysosome formation via endocytosis of the plasma membrane. Furthermore, the impact of glucose variation-induced stress on resistance to DOX was compared with Dp44mT and its structurally related analogue, di-2-pyridylketone 4-cyclohexyl-4-methyl-3-thiosemicarbazone (DpC). These studies showed that glucose variation-induced stress-stimulated formation of early endosomes and lysosomes. In fact, through the process of fluid-phase endocytosis, Pgp was redistributed from the plasma membrane to the lysosomal membrane via early endosome formation. This lysosomal-Pgp actively transported the Pgp substrate, DOX, into the lysosome where it became trapped as a result of protonation at pH 5. Due to increased lysosomal DOX trapping, Pgp-expressing cells became more resistant to DOX. In contrast, cytotoxicity of Dp44mT and DpC was potentiated due to more lysosomes containing functional Pgp under glucose-induced stress. These thiosemicarbazones increased lysosomal membrane permeabilization and cell death. This mechanism has critical implications for drug-targeting in

CD68/macrosialin: not just a histochemical marker.

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Chistiakov, Dimitry A; Killingsworth, Murry C; Myasoedova, Veronika A; Orekhov, Alexander N; Bobryshev, Yuri V

2017-01-01

CD68 is a heavily glycosylated glycoprotein that is highly expressed in macrophages and other mononuclear phagocytes. Traditionally, CD68 is exploited as a valuable cytochemical marker to immunostain monocyte/macrophages in the histochemical analysis of inflamed tissues, tumor tissues, and other immunohistopathological applications. CD68 alone or in combination with other cell markers of tumor-associated macrophages showed a good predictive value as a prognostic marker of survival in cancer patients. Lowression of CD68 was found in the lymphoid cells, non-hematopoietic cells (fibroblasts, endothelial cells, etc), and tumor cells. Cell-specific CD68 expression and differentiated expression levels are determined by the complex interplay between transcription factors, regulatory transcriptional elements, and epigenetic factors. Human CD68 and its mouse ortholog macrosialin belong to the family of LAMP proteins located in the lysosomal membrane and share many structural similarities such as the presence of the LAMP-like domain. Except for a second LAMP-like domain present in LAMPs, CD68/microsialin has a highly glycosylated mucin-like domain involved in ligand binding. CD68 has been shown to bind oxLDL, phosphatidylserine, apoptotic cells and serve as a receptor for malaria sporozoite in liver infection. CD68 is mainly located in the endosomal/lysosomal compartment but can rapidly shuttle to the cell surface. However, the role of CD68 as a scavenger receptor remains to be confirmed. It seems that CD68 is not involved in binding bacterial/viral pathogens, innate, inflammatory or humoral immune responses, although it may potentially be involved in antigen processing/presentation. CD68 could be functionally important in osteoclasts since its deletion leads to reduced bone resorption capacity. The role of CD68 in atherosclerosis is contradictory.

Adoptive immunotherapy mediated by ex vivo expanded natural killer T cells against CD1d-expressing lymphoid neoplasms.

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Bagnara, Davide; Ibatici, Adalberto; Corselli, Mirko; Sessarego, Nadia; Tenca, Claudya; De Santanna, Amleto; Mazzarello, Andrea; Daga, Antonio; Corvò, Renzo; De Rossi, Giulio; Frassoni, Francesco; Ciccone, Ermanno; Fais, Franco

2009-07-01

CD1d is a monomorphic antigen presentation molecule expressed in several hematologic malignancies. Alpha-galactosylceramide (alpha-GalCer) is a glycolipid that can be presented to cytotoxic CD1d-restricted T cells. These reagents represent a potentially powerful tool for cell mediated immunotherapy. We set up an experimental model to evaluate the use of adoptively transferred cytotoxic CD1d-restricted T cells and alpha-GalCer in the treatment of mice engrafted with CD1d(+) lymphoid neoplastic cells. To this end the C1R cell line was transfected with CD1c or CD1d molecules. In addition, upon retroviral infection firefly luciferase was expressed on C1R transfected cell lines allowing the evaluation of tumor growth in xenografted immunodeficient NOD/SCID mice. The C1R-CD1d cell line was highly susceptible to specific CD1d-restricted T cell cytotoxicity in the presence alpha-GalCer in vitro. After adoptive transfer of CD1d-restricted T cells and alpha-GalCer to mice engrafted with both C1R-CD1c and C1R-CD1d, a reduction in tumor growth was observed only in CD1d(+) masses. In addition, CD1d-restricted T-cell treatment plus alpha-GalCer eradicated small C1R-CD1d(+) nodules. Immunohistochemical analysis revealed that infiltrating NKT cells were mainly observed in CD1d nodules. Our results indicate that ex vivo expanded cytotoxic CD1d-restricted T cells and alpha-GalCer may represent a new immunotherapeutic tool for treatment of CD1d(+) hematologic malignancies.

Activation of lysosomal enzymes and tumour regression caused by irradiation and steroid hormones

International Nuclear Information System (INIS)

Ball, A.; Barratt, G.M.; Wills, E.D.

1982-01-01

The lysosomal enzyme activity and membrane permeability of mouse C3H mammary tumours has been studied using quantitative cytochemical methods following irradiation of the tumours with doses of 1500, 3500 or 6000 rad ν rays. No change in the lysosomal enzyme activity was observed immediately after irradiation, but increased enzyme activity and increased membrane permeability were observed 24 hr after irradiation with doses of 3500 or 6000 rad. Twenty-four hours after injection of prednisolone there was a marked increase of lysosomal membrane permeability and enzyme activity, and injection of prednisolone soon after irradiation enhanced the effect of irradiation. After a dose of 6000 rad and prednisolone, the lysosomal membrane permeability increased to 191% of the control and the enzyme activity to 326% of the value of the control tumours. Measurement of tumour size after irradiation or after a combined treatment with irradiation and prednisolone showed that a close correlation exists between tumour regression and lysosomal enzyme activity. The experiments support the view that lysosomal enzymes play an important role in tumour regression following irradiation. (author)

In utero exposure to benzene increases embryonic c-Myb and Pim-1 protein levels in CD-1 mice

International Nuclear Information System (INIS)

Wan, Joanne; Winn, Louise M.

2008-01-01

Benzene is a known human leukemogen, but its role as an in utero leukemogen remains controversial. Epidemiological studies have correlated parental exposure to benzene with an increased incidence of childhood leukemias. We hypothesize that in utero exposure to benzene may cause leukemogenesis by affecting the embryonic c-Myb/Pim-1 signaling pathway and that this is mediated by oxidative stress. To investigate this hypothesis, pregnant CD-1 mice were treated with either 800 mg/kg of benzene or corn oil (i.p.) on days 10 and 11 of gestation and in some cases pretreated with 25 kU/kg of PEG-catalase. Phosphorylated and total embryonic c-Myb and Pim-1 protein levels were assessed using Western blotting and maternal and embryonic oxidative stress were assessed by measuring reduced to oxidized glutathione ratios. Our results show increased oxidative stress at 4 and 24 h after exposure, increased phosphorylated Pim-1 protein levels 4 h after benzene exposure, and increased Pim-1 levels at 24 and 48 h after benzene exposure. Embryonic c-Myb levels were elevated at 24 h after exposure. PEG-catalase pretreatment prevented benzene-mediated increases in embryonic c-Myb and Pim-1 protein levels, and benzene-induced oxidative stress. These results support a role for ROS in c-Myb and Pim-1 alterations after in utero benzene exposure

TOM1L Is Involved in a Novel Signaling Pathway Important for the IL-2 Production in Jurkat T Cells Stimulated by CD3/CD28 CoLigation

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Ahmed Elmarghani

2009-01-01

Full Text Available TOM1L (target of Myb-1 Like was identified as a binding partner for the full length and catalytically-active Lck in a yeast 2-hybrid screening assay. Here we show that in Jurkat T cells stimulated by CD3/CD28 coligation where the expression of TOM1L is reduced by lenti virus mediated-siRNA results in a dramatically lower IL-2 production. The production of IL-2 in siRNA treated cells stimulated with PMA/ionomycin was not affected indicating an involvement of TOM1L in a pathway proximal of TCR and CD28. The coexpression of Fyn with TOM1L increased the level of the phosphorylated form of Fyn indicating that TOM1L has the ability to activate Fyn. The ability of TOM1L to activate Fyn was further shown in a kinase assay using angiotensin II as a substrate. By confocal microscopy, we show that the expression of TOM1L in non-treated HeLa and SK-N-SH cells colocalizes with the mitochondrial membrane but not with lysosomal compartments or the trans-Golgi network. Furthermore, we show that the over-expression of TOM1L in Jurkat cells causes an increase of the STAT3 expression . Based on our results, we here propose that TOM1L is involved in a novel signaling pathway that is important for the IL-2 production in T cells.

Progranulin, lysosomal regulation and neurodegenerative disease.

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Kao, Aimee W; McKay, Andrew; Singh, Param Priya; Brunet, Anne; Huang, Eric J

2017-06-01

The discovery that heterozygous and homozygous mutations in the gene encoding progranulin are causally linked to frontotemporal dementia and lysosomal storage disease, respectively, reveals previously unrecognized roles of the progranulin protein in regulating lysosome biogenesis and function. Given the importance of lysosomes in cellular homeostasis, it is not surprising that progranulin deficiency has pleiotropic effects on neural circuit development and maintenance, stress response, innate immunity and ageing. This Progress article reviews recent advances in progranulin biology emphasizing its roles in lysosomal function and brain innate immunity, and outlines future avenues of investigation that may lead to new therapeutic approaches for neurodegeneration.

THE RESPIRATORY SUBSTRATE RHODOQUINOL INDUCES Q-CYCLE BYPASS REACTIONS IN THE YEAST CYTOCHROME bc1 COMPLEX - MECHANISTIC AND PHYSIOLOGICAL IMPLICATIONS

International Nuclear Information System (INIS)

Cape, Jonathan L.; Strahan, Jeff R.; Lenaeus, Michael J.; Yuknis, Brook A.; Le, Trieu T.; Shepherd, Jennifer; Bowman, Michael K.; Kramer, David M.

2005-01-01

The mitochondrial cytochrome bc1 complex catalyzes the transfer of electrons from ubiquinol to cyt c, while generating a proton motive force for ATP synthesis, via the ''Qcycle'' mechanism. Under certain conditions, electron flow through the Q-cycle is blocked at the level of a reactive intermediate in the quinol oxidase site of the enzyme, resulting in ''bypass reactions'', some of which lead to superoxide production. Using analogs of the respiratory substrates, ubiquinol-3 and rhodoquinol-3, we show that the relative rates of Q-cycle bypass reactions in the Saccharomyces cerevisiae cyt bc1 complex are highly dependent, by a factor of up to one hundred-fold, on the properties of the substrate quinol. Our results suggest that the rate of Q-cycle bypass reactions is dependent on the steady state concentration of reactive intermediates produced at the quinol oxidase site of the enzyme. We conclude that normal operation of the Q-cycle requires a fairly narrow window of redox potentials, with respect to the quinol substrate, to allow normal turnover of the complex while preventing potentially damaging bypass reactions

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...

Clinical Trials Using Anti-CD19/CD28/CD3zeta CAR Gammaretroviral Vector-transduced Autologous T Lymphocytes KTE-C19

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NCI supports clinical trials that test new and more effective ways to treat cancer. Find clinical trials studying anti-cd19/cd28/cd3zeta car gammaretroviral vector-transduced autologous t lymphocytes kte-c19.

Regulation of HIV-Gag Expression and Targeting to the Endolysosomal/Secretory Pathway by the Luminal Domain of Lysosomal-Associated Membrane Protein (LAMP-1) Enhance Gag-Specific Immune Response

Science.gov (United States)

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

2014-01-01

We have previously demonstrated that a DNA vaccine encoding HIV-p55gag in association with the lysosomal associated membrane protein-1 (LAMP-1) elicited a greater Gag-specific immune response, in comparison to a DNA encoding the native gag. In vitro studies have also demonstrated that LAMP/Gag was highly expressed and was present in MHCII containing compartments in transfected cells. In this study, the mechanisms involved in these processes and the relative contributions of the increased expression and altered traffic for the enhanced immune response were addressed. Cells transfected with plasmid DNA constructs containing p55gag attached to truncated sequences of LAMP-1 showed that the increased expression of gag mRNA required p55gag in frame with at least 741 bp of the LAMP-1 luminal domain. LAMP luminal domain also showed to be essential for Gag traffic through lysosomes and, in this case, the whole sequence was required. Further analysis of the trafficking pathway of the intact LAMP/Gag chimera demonstrated that it was secreted, at least in part, associated with exosome-like vesicles. Immunization of mice with LAMP/gag chimeric plasmids demonstrated that high expression level alone can induce a substantial transient antibody response, but targeting of the antigen to the endolysosomal/secretory pathways was required for establishment of cellular and memory response. The intact LAMP/gag construct induced polyfunctional CD4+ T cell response, which presence at the time of immunization was required for CD8+ T cell priming. LAMP-mediated targeting to endolysosomal/secretory pathway is an important new mechanistic element in LAMP-mediated enhanced immunity with applications to the development of novel anti-HIV vaccines and to general vaccinology field. PMID:24932692

[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.

CD80 and CD86 IgC domains are important for quaternary structure, receptor binding and co-signaling function.

Science.gov (United States)

Girard, Tanya; Gaucher, Denis; El-Far, Mohamed; Breton, Gaëlle; Sékaly, Rafick-Pierre

2014-09-01

CD86 and CD80, the ligands for the co-stimulatory molecules CD28 and CTLA-4, are members of the Ig superfamily. Their structure includes Ig variable-like (IgV) domains, Ig constant-like (IgC) domains and intracellular domains. Although crystallographic studies have clearly identified the IgV domain to be responsible for receptor interactions, earlier studies suggested that both Ig domains are required for full co-signaling function. Herein, we have used deletion and chimeric human CD80 and CD86 molecules in co-stimulation assays to study the impact of the multimeric state of IgV and IgC domains on receptor binding properties and on co-stimulatory function in a peptide-specific T cell activation model. We report for the first time the presence of CD80 dimers and CD86 monomers in living cells. Moreover, we show that the IgC domain of both molecules inhibits multimer formation and greatly affects binding to the co-receptors CD28 and CTLA-4. Finally, both IgC and intracellular domains are required for full co-signaling function. These findings reveal the distinct but complementary roles of CD80 and CD86 IgV and IgC domains in T cell activation. Copyright © 2014 Elsevier B.V. All rights reserved.

Drosophila melanogaster cellular repressor of E1A-stimulated genes is a lysosomal protein essential for fly development

OpenAIRE

Kowalewski-Nimmerfall, Elisabeth; Sch?hs, Philipp; Maresch, Daniel; Rendic, Dubravko; Kr?mer, Helmut; Mach, Lukas

2014-01-01

Mammalian cellular repressor of E1A-stimulated genes is a lysosomal glycoprotein implicated in cellular growth and differentiation. The genome of the fruit fly Drosophila melanogaster encodes a putative orthologue (dCREG), suggesting evolutionarily conserved physiological functions of this protein. In D. melanogaster S2 cells, dCREG was found to localize in lysosomes. Further studies revealed that intracellular dCREG is subject of proteolytic maturation. Processing and turnover could be subst...

PDMP, a ceramide analogue, acts as an inhibitor of mTORC1 by inducing its translocation from lysosome to endoplasmic reticulum

Energy Technology Data Exchange (ETDEWEB)

Ode, Takashi [Department of Periodontology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (TMDU), 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510 (Japan); Research Fellow of the Japan Society for the Promotion of Science (JSPS), 5-3-1 Kojimachi, Chiyoda-ku, Tokyo 102-0083 (Japan); Podyma-Inoue, Katarzyna A.; Terasawa, Kazue [Department of Biochemistry, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (TMDU), 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510 (Japan); Inokuchi, Jin-ichi [Division of Glycopathology, Institute of Molecular Biomembrane and Glycobiology, Tohoku Medical and Pharmaceutical University, 4-4-1, Komatsushima, Aoba-ku, Sendai, Miyagi 981-8558 (Japan); Kobayashi, Toshihide [Lipid Biology Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198 (Japan); CNRS, UMR 7213, University of Strasbourg, 67401 Illkirch (France); Watabe, Tetsuro [Department of Biochemistry, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (TMDU), 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510 (Japan); Izumi, Yuichi [Department of Periodontology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (TMDU), 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510 (Japan); Hara-Yokoyama, Miki, E-mail: m.yokoyama.bch@tmd.ac.jp [Department of Biochemistry, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University (TMDU), 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510 (Japan)

2017-01-01

Mammalian or mechanistic target of rapamycin complex 1 (mTORC1) is a master regulator of cell growth, metabolism, and cell differentiation. Recent studies have revealed that the recruitment of mTORC1 to lysosomes is essential for its activation. The ceramide analogue 1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), a well known glycosphingolipid synthesis inhibitor, also affects the structures and functions of various organelles, including lysosomes and endoplasmic reticulum (ER). We investigated whether PDMP regulates the mTORC1 activity through its effects on organellar behavior. PDMP induced the translocation of mTORC1 from late endosomes/lysosomes, leading to the dissociation of mTORC1 from its activator Rheb in MC3T3-E1 cells. Surprisingly, we found mTORC1 translocation to the ER upon PDMP treatment. This effect of PDMP was independent of its action as the inhibitor, since two stereoisomers of PDMP, with and without the inhibitor activity, showed essentially the same effect. We confirmed that PDMP inhibits the mTORC1 activity based on the decrease in the phosphorylation of ribosomal S6 kinase, a downstream target of mTORC1, and the increase in LC3 puncta, reflecting autophagosome formation. Furthermore, PDMP inhibited the mTORC1-dependent osteoblastic cell proliferation and differentiation of MC3T3-E1 cells. Accordingly, the present results reveal a novel mechanism of PDMP, which inhibits the mTORC1 activity by inducing the translocation of mTOR from lysosomes to the ER. - Highlights: • The ceramide analogue, PDMP, suppressed the activation of mTORC1. • PDMP induced the translocation of mTOR from lysosomes to ER. • PDMP led to the dissociation of mTOR from its activator Rheb. • PDMP inhibited the mTORC1-dependent osteoblastic cell proliferation.

Lysosomal multienzyme complex: pros and cons of working together.

Science.gov (United States)

Bonten, Erik J; Annunziata, Ida; d'Azzo, Alessandra

2014-06-01

The ubiquitous distribution of lysosomes and their heterogeneous protein composition reflects the versatility of these organelles in maintaining cell homeostasis and their importance in tissue differentiation and remodeling. In lysosomes, the degradation of complex, macromolecular substrates requires the synergistic action of multiple hydrolases that usually work in a stepwise fashion. This catalytic machinery explains the existence of lysosomal enzyme complexes that can be dynamically assembled and disassembled to efficiently and quickly adapt to the pool of substrates to be processed or degraded, adding extra tiers to the regulation of the individual protein components. An example of such a complex is the one composed of three hydrolases that are ubiquitously but differentially expressed: the serine carboxypeptidase, protective protein/cathepsin A (PPCA), the sialidase, neuraminidase-1 (NEU1), and the glycosidase β-galactosidase (β-GAL). Next to this 'core' complex, the existence of sub-complexes, which may contain additional components, and function at the cell surface or extracellularly, suggests as yet unexplored functions of these enzymes. Here we review how studies of basic biological processes in the mouse models of three lysosomal storage disorders, galactosialidosis, sialidosis, and GM1-gangliosidosis, revealed new and unexpected roles for the three respective affected enzymes, Ppca, Neu1, and β-Gal, that go beyond their canonical degradative activities. These findings have broadened our perspective on their functions and may pave the way for the development of new therapies for these lysosomal storage disorders.

Reactive glia promote development of CD103+ CD69+ CD8+ T-cells through programmed cell death-ligand 1 (PD-L1).

Science.gov (United States)

Prasad, Sujata; Hu, Shuxian; Sheng, Wen S; Chauhan, Priyanka; Lokensgard, James R

2018-06-01

Previous work from our laboratory has demonstrated in vivo persistence of CD103 + CD69 + brain resident memory CD8 + T-cells (bT RM ) following viral infection, and that the PD-1: PD-L1 pathway promotes development of these T RM cells within the brain. Although glial cells express low basal levels of PD-L1, its expression is upregulated upon IFN-γ-treatment, and they have been shown to modulate antiviral T-cell effector responses through the PD-1: PD-L1 pathway. We performed flow cytometric analysis of cells from co-cultures of mixed glia and CD8 + T-cells obtained from wild type mice to investigate the role of glial cells in the development of bT RM . In this study, we show that interactions between reactive glia and anti-CD3 Ab-stimulated CD8 + T-cells promote development of CD103 + CD69 + CD8 + T-cells through engagement of the PD-1: PD-L1 pathway. These studies used co-cultures of primary murine glial cells obtained from WT animals along with CD8 + T-cells obtained from either WT or PD-1 KO mice. We found that αCD3 Ab-stimulated CD8 + T-cells from WT animals increased expression of CD103 and CD69 when co-cultured with primary murine glial cells. In contrast, significantly reduced expression of CD103 and CD69 was observed using CD8 + T-cells from PD-1 KO mice. We also observed that reactive glia promoted high levels of CD127, a marker of memory precursor effector cells (MPEC), on CD69 + CD8 + T-cells, which promotes development of T RM cells. Interestingly, results obtained using T-cells from PD-1 KO animals showed significantly reduced expression of CD127 on CD69 + CD8 + cells. Additionally, blocking of glial PD-L1 resulted in decreased expression of CD103, along with reduced CD127 on CD69 + CD8 + T-cells. Taken together, these results demonstrate a role for activated glia in promoting development of bT RM through the PD-1: PD-L1 pathway. © 2018 The Authors. Immunity, Inflammation and Disease Published by John Wiley & Sons Ltd.

Quantitative proteomic profiling for clarification of the crucial roles of lysosomes in microbial infections.

Science.gov (United States)

Xu, Benhong; Gao, Yanpan; Zhan, Shaohua; Ge, Wei

2017-07-01

Lysosomes play vital roles in both innate and adaptive immunity. It is widely accepted that lysosomes do not function exclusively as a digestive organelle. It is also involved in the process of immune cells against pathogens. However, the changes in the lysosomal proteome caused by infection with various microbes are still largely unknown, and our understanding of the proteome of the purified lysosome is another obstacle that needs to be resolved. Here, we performed a proteomic study on lysosomes enriched from THP1 cells after infection with Listeria monocytogenes (L.m), Herpes Simplex Virus 1 (HSV-1) and Vesicular Stomatitis Virus (VSV). In combination with the gene ontology (GO) analysis, we identified 284 lysosomal-related proteins from a total of 4560 proteins. We also constructed the protein-protein interaction networks for the differentially expressed proteins and revealed the core lysosomal proteins, including SRC in the L. m treated group, SRC, GLB1, HEXA and HEXB in the HSV-1 treated group and GLB1, CTSA, CTSB, HEXA and HEXB in the VSV treated group, which are involved in responding to diverse microbial infections. This study not only reveals variable lysosome responses depending on the bacterial or virus infection, but also provides the evidence based on which we propose a novel approach to proteome research for investigation of the function of the enriched organelles. Copyright © 2017 Elsevier Ltd. All rights reserved.

CD52 expression on CD4+ T cells in HIV-positive individuals on cART

DEFF Research Database (Denmark)

Vojdeman, Fie Juhl; Gaardbo, Julie Christine; Hartling, Hans Jakob

2018-01-01

BACKGROUND: Human immune defect virus (HIV) persists in a latent state in quiescent CD4+ T cells preventing eradication of HIV. CD52 is a surface molecule modulated by HIV. We aimed at examining factors related to CD52 expression on CD4+ T cells in HIV-positive individuals and the impact...... of initiation of combination antiretroviral therapy (cART). METHODS: Peripheral blood mononuclear cells (PBMC) from 18 HIV-positive individuals and 10 uninfected age and gender matched controls were examined by flow cytometry for CD38 and CD52 expression on CD4+ T cells. Stimulation assays were performed on 8...... healthy blood donors to determine a cut-off for CD52 expression. RESULTS: All examined CD4+ T cells expressed CD52. However, both CD4+ T cells with higher (CD52++) and with lower CD52 expression (CD52dim) were found in HIV-positive individuals compared to uninfected controls. Two % CD52dim cells defined...

A lysosome-locating and acidic pH-activatable fluorescent probe for visualizing endogenous H2O2 in lysosomes.

Science.gov (United States)

Liu, Jun; Zhou, Shunqing; Ren, Jing; Wu, Chuanliu; Zhao, Yibing

2017-11-20

There is increasing evidence indicating that lysosomal H 2 O 2 is closely related to autophagy and apoptotic pathways under both physiological and pathological conditions. Therefore, fluorescent probes that can be exploited to visualize H 2 O 2 in lysosomes are potential tools for exploring diverse roles of H 2 O 2 in cells. However, functional exploration of lysosomal H 2 O 2 is limited by the lack of fluorescent probes capable of compatibly sensing H 2 O 2 under weak acidic conditions (pH = 4.5) of lysosomes. Lower spatial resolution of the fluorescent visualization of lysosomal H 2 O 2 might be caused by the interference of signals from cytosolic and mitochondrial H 2 O 2 , as well as the non-specific distribution of the probes in cells. In this work, we developed a lysosome-locating and acidic-pH-activatable fluorescent probe for the detection and visualization of H 2 O 2 in lysosomes, which consists of a H 2 O 2 -responsive boronate unit, a lysosome-locating morpholine group, and a pH-activatable benzorhodol fluorophore. The response of the fluorescent probe to H 2 O 2 is significantly more pronounced under acidic pH conditions than that under neutral pH conditions. Notably, the present probe enables the fluorescence sensing of endogenous lysosomal H 2 O 2 in living cells without external stimulations, with signal interference from the cytoplasm and other intracellular organelles being negligible.

Ultraviolet induced lysosome activity in corneal epithelium

Energy Technology Data Exchange (ETDEWEB)

Cullen, A.P.

1980-01-01

A 5.000 W Xe-Hg high pressure lamp and a double monochromator were used to produce a 3.3 nm half-bandpass ultraviolet radiation at 295 nm. Pigmented rabbit eyes were irradiated with radiant exposures from 140 Jm/sup -2/ to 10.000 Jm/sup -2/ and evaluated by slit-lamp biomicroscopy, light and electron microscopy. Corneal threshold (Hsub(c) was 200 Jm/sup -2/ and lens threshold (Hsub(L)) was 7.500 Jm/sup -2/. The most repeatable and reliable corneal response to these levels of UV was the development of corneal epithelial granules. Histological changes included a loss of superficial epithelial cells and selective UV induced autolysis of the wing cells. It is suggested that the biomicroscopically observed granules are the clinical manifestation of the secondary lysosomes revealed by light and electron microscopy. It is proposed that UV breaks down the primary lysosome membranes to release hydrolytic enzymes which in turn form the secondary lysosomes during autolysis. Extreme levels of radiant exposure at 295 nm result in indiscriminate destruction of all layers of the corneal epithelium, but the posterior cornea was spared.

Radionuclide angiographical assessment of the effects of aorto-coronary bypass grafting on ventricular function during rest and exercise

International Nuclear Information System (INIS)

Kito, Yoshitsugu; Fujita, Tsuyoshi; Ohara, Kuniyoshi

1983-01-01

Ventricular function was measured by radionuclide angiography during rest and exercise in 27 patients before and after aorto-coronary bypass grafting (AC bypass). The patients were divided in three groups: 6 patients in complete revascularisation (group A), 8 patients in surgical complete revascularisation (group B), and 13 patients in incomplete revascularisation (group C). Heart rate increased by exercise from 58 +- 5 bpm to 87 +- 5 bpm, from 60 +- 5 bpm to 90 +- 15 bpm, and from 61 +- 8 bpm to 84 +- 7 bpm before AC bypass, and increased from 81 +- 7 bpm to 116 +- 11 bpm, from 86 +- 13 bpm to 114 +- 12 bpm, and from 80 +- 11 bpm to 106 +- 13 bpm after AC bypass in group A, B and C, respecitively. EF decreased by exercise from 61 +- 7 % to 58 +- 12 %, from 53 +- 10 % to 49 +- 7 %, and from 54 +- 8 % to 52 +- 8 % before AC bypass, and increased by exercise from 66 +- 7 % to 77 +- 6 %, from 49 +- 11 % to 56 +- 13 %, and from 54 +- 8 % to 59 +- 10 % after AC bypass in group A,FB and C, respectively. dV/dt(Vmax/sec) increased by exercise from 4.0 +- 1.0 to 4.4 +- 1.0, from 2.5 +- 1.0 to 3.4 +- 1.5 and from 3.5 +- 0.5 to 3.4 +- 0.6 before AC bypass, and increased from 3.9 +- 0.5 to 6.2 +- 0.7, from 3.5 +- 0.9 to 5.0 +- 0.7, and from 3.6 +- 1.0 to 5.4 +- 1.5 after AC bypass in group A,B and C, respectively. In conclusion, left ventricular performance during exercise was significantly increased after AC bypass in all patients. However, the rate of increase in left ventricular performance during exercise after AC bypass was more significant in the group of complete revascularisation than in the group of others. Serial application of radionuclide angiography to patients after AC bypass appears promising in evaluating the long-term effects of operation on ventricular function. (author)

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.

Expression of KAI1/CD82 and MRP-1/CD9 in transitional cell carcinoma of bladder.

Science.gov (United States)

Ai, Xing; Zhang, Xu; Wu, Zhun; Ma, Xin; Ju, Zhenghua; Wang, Baojun; Shi, Taoping

2007-02-01

The expression of KAI1/CD82 and MRP-1/CD9 in transitional cell carcinoma of bladder (TCCB) and its clinical significance were investigated. Immunohistochemistry was used to detect KAI1/CD82 and MRP-1/CD9 protein expression in 52 TCCB specimens. Correlation between the expression of KAI1/CD82 and MRP-1/CD9 to clinicopathologic factors was statistically analyzed. The results showed that the positive rate of KAI1/CD82 and MRP-1/CD9 in TCCB was 50% and 61.5%, respectively. The MRP-1/CD9 and KAI1/CD82 expression was significantly associated with grade of TCCB (PMRP-1/CD9 or KAI1/CD82 expression and clinical stage of TCCB (P>0.05). The expression level of MRP-1/CD9 and KAI1/CD82 in recurrent TCCB samples was lower than that in non-recurrent samples (PMRP-1/CD9 expression was statistically significant (r=0.316, PMRP-1/CD9 expression may be important prognostic indicators and potentially useful for assessing the biological behavior of TCCB.

Redox-mediated bypass of restriction point via skipping of G1pm

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Greene James J

2006-07-01

Full Text Available Abstract Background It is well known that cancer cells bypass the restriction point, R, and undergo uncontrolled cell proliferation. Hypothesis and evidence We suggest here that fibrosarcoma cells enter G1ps directly from M, skipping G1pm, hence bypassing R, in response to redox modulation. Evidence is presented from the published literature that demonstrate a shortening of the cycle period of transformed fibroblasts (SV-3T3 compared to the nontransformed 3T3 fibroblasts, corresponding to the duration of G1pm in the 3T3 fibroblasts. Evidence is also presented that demonstrate that redox modulation can induce the CUA-4 fibroblasts to bypass R, resulting in a cycle period closely corresponding to the cycle period of fibrosarcoma cells (HT1080. Conclusion The evidence supports our hypothesis that a low internal redox potential can cause fibrosarcoma cells to skip the G1pm phase of the cell cycle.

Changes in adhesion molecule expression and oxidative burst activity of granulocytes and monocytes during open-heart surgery with cardiopulmonary bypass compared with abdominal surgery

DEFF Research Database (Denmark)

Toft, P; Nielsen, C H; Tønnesen, Else Kirstine

1998-01-01

Cardiac and major abdominal surgery are associated with granulocytosis in peripheral blood. The purpose of the present study was to describe the granulocyte and monocyte oxidative burst and the expression of adhesion molecules following cardiac surgery with cardiopulmonary bypass and abdominal...... during cardiopulmonary bypass was observed. The percentage of CD11a-positive granulocytes increased from 30% pre-operatively to 75% following cardiopulmonary bypass, while CD44-positive granulocytes increased from 5% to 13%. Despite the extent of the changes, these were not significant. The oxidative...... to an increased per-operative oxidative burst activity, and the induction of adhesion molecules on granulocytes associated with the cardiopulmonary bypass and surgery. In conclusion, open-heart surgery with cardiopulmonary bypass was associated with a rapid and pronounced activation of leukocytes which may play...

Delivery of Cargo to Lysosomes Using GNeosomes.

Science.gov (United States)

Hamill, Kristina M; Wexselblatt, Ezequiel; Tong, Wenyong; Esko, Jeffrey D; Tor, Yitzhak

2017-01-01

Liposomes have been used to improve the intracellular delivery of a variety of cargos. Encapsulation of cargos in liposomes leads to improved plasma half-lives and minimized degradation. Here, we present a method for improving the selective delivery of liposomes to the lysosomes using a guanidinylated neomycin (GNeo) transporter. The method for synthesizing GNeo-lipids, incorporating them into liposomes, and the enhanced lysosomal delivery of encapsulated cargo are presented. GNeo-liposomes, termed GNeosomes, are capable of delivering a fluorescent dye to the lysosomes of Chinese hamster ovary cells as shown using confocal microscopy. GNeosomes can also be used to deliver therapeutic quantities of lysosomal enzymes to fibroblasts isolated from patients with a lysosomal storage disorder.

Detection of high CD44 expression in oral cancers using the novel monoclonal antibody, C44Mab-5

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Shinji Yamada

2018-07-01

Full Text Available CD44 is a transmembrane glycoprotein that regulates a variety of genes related to cell-adhesion, migration, proliferation, differentiation, and survival. A large number of alternative splicing isoforms of CD44, containing various combinations of alternative exons, have been reported. CD44 standard (CD44s, which lacks variant exons, is widely expressed on the surface of most tissues and all hematopoietic cells. In contrast, CD44 variant isoforms show tissue-specific expression patterns and have been extensively studied as both prognostic markers and therapeutic targets in cancer and other diseases. In this study, we immunized mice with CHO-K1 cell lines overexpressing CD44v3-10 to obtain novel anti-CD44 mAbs. One of the clones, C44Mab-5 (IgG1, kappa, recognized both CD44s and CD44v3-10. C44Mab-5 also reacted with oral cancer cells such as Ca9-22, HO-1-u-1, SAS, HSC-2, HSC-3, and HSC-4 using flow cytometry. Moreover, immunohistochemical analysis revealed that C44Mab-5 detected 166/182 (91.2% of oral cancers. These results suggest that the C44Mab-5 antibody may be useful for investigating the expression and function of CD44 in various cancers.

Detection of high CD44 expression in oral cancers using the novel monoclonal antibody, C44Mab-5.

Science.gov (United States)

Yamada, Shinji; Itai, Shunsuke; Nakamura, Takuro; Yanaka, Miyuki; Kaneko, Mika K; Kato, Yukinari

2018-07-01

CD44 is a transmembrane glycoprotein that regulates a variety of genes related to cell-adhesion, migration, proliferation, differentiation, and survival. A large number of alternative splicing isoforms of CD44, containing various combinations of alternative exons, have been reported. CD44 standard (CD44s), which lacks variant exons, is widely expressed on the surface of most tissues and all hematopoietic cells. In contrast, CD44 variant isoforms show tissue-specific expression patterns and have been extensively studied as both prognostic markers and therapeutic targets in cancer and other diseases. In this study, we immunized mice with CHO-K1 cell lines overexpressing CD44v3-10 to obtain novel anti-CD44 mAbs. One of the clones, C 44 Mab-5 (IgG 1 , kappa), recognized both CD44s and CD44v3-10. C 44 Mab-5 also reacted with oral cancer cells such as Ca9-22, HO-1-u-1, SAS, HSC-2, HSC-3, and HSC-4 using flow cytometry. Moreover, immunohistochemical analysis revealed that C 44 Mab-5 detected 166/182 (91.2%) of oral cancers. These results suggest that the C 44 Mab-5 antibody may be useful for investigating the expression and function of CD44 in various cancers.

Antibody Therapy Targeting CD47 and CD271 Effectively Suppresses Melanoma Metastasis in Patient-Derived Xenografts

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Michael Ngo

2016-08-01

Full Text Available The high rate of metastasis and recurrence among melanoma patients indicates the existence of cells within melanoma that have the ability to both initiate metastatic programs and bypass immune recognition. Here, we identify CD47 as a regulator of melanoma tumor metastasis and immune evasion. Protein and gene expression analysis of clinical melanoma samples reveals that CD47, an anti-phagocytic signal, correlates with melanoma metastasis. Antibody-mediated blockade of CD47 coupled with targeting of CD271+ melanoma cells strongly inhibits tumor metastasis in patient-derived xenografts. This therapeutic effect is mediated by drastic changes in the tumor and metastatic site immune microenvironments, both of whichwhich exhibit greatly increased density of differentiated macrophages and significantly fewer inflammatory monocytes, pro-metastatic macrophages (CCR2+/VEGFR1+, and neutrophils, all of which are associated with disease progression. Thus, antibody therapy that activates the innate immune response in combination with selective targeting of CD271+ melanoma cells represents a powerful therapeutic approach against metastatic melanoma.

Riccardin D-N induces lysosomal membrane permeabilization by inhibiting acid sphingomyelinase and interfering with sphingomyelin metabolism in vivo

Energy Technology Data Exchange (ETDEWEB)

Li, Lin [Department of Natural Product Chemistry, Key Lab of Chemical Biology of MOE (Ministry of Education), Shandong University, Jinan 250012 (China); Niu, Huanmin [Department of Biochemistry and Molecular Biology, School of Medicine, Shandong University, Jinan 250012 (China); Sun, Bin [Department of Natural Product Chemistry, Key Lab of Chemical Biology of MOE (Ministry of Education), Shandong University, Jinan 250012 (China); Xiao, Yanan [School of Pharmaceutical Science, Shandong University, Jinan 250012 (China); Li, Wei [Department of Natural Product Chemistry, Key Lab of Chemical Biology of MOE (Ministry of Education), Shandong University, Jinan 250012 (China); Yuan, Huiqing [Department of Biochemistry and Molecular Biology, School of Medicine, Shandong University, Jinan 250012 (China); Lou, Hongxiang, E-mail: louhongxiang@sdu.edu.cn [Department of Natural Product Chemistry, Key Lab of Chemical Biology of MOE (Ministry of Education), Shandong University, Jinan 250012 (China)

2016-11-01

Lysosomes are important targets for anticancer drug discovery. Our previous study showed that Riccardin D-N (RD-N), a natural macrocylic bisbibenzyl derivative produced by Mannich reaction, induced cell death by accumulating in lysosomes. Experiments were performed on human lung squamous cell carcinoma tissue from left inferior lobar bronchus of patient xenografts and H460 cells. RD-N was administrated for 25 days. The specimens of xenografts in Balb/c athymic (nu +/nu +) male mice were removed for immunohistochemistry, subcellular fractionation, enzyme activities and Western blotting analysis. mRFP-GFP-LC3 reporter was used to examine autophagy in H460 cells. Sphingomyelin assay was evaluated by thin-layer chromatography and assay kit. Lysosomal membrane permeabilization (LMP) caused by acid sphingomyelinase (ASM) inhibition and subsequent changes of sphingomyelin (SM) metabolism selectively destabilized the cancer cell lysosomes in RD-N-treated H460 cells in vitro and tumor xenograft model in vivo. The destabilized lysosomes induced the release of cathepsins from the lysosomes into the cytosol and further triggered cell death. These results explain the underlying mechanism of RD-N induced LMP. It can be concluded that a more lysosomotropic derivative was synthesized by introduction of an amine group, which could have more potential applications in cancer therapy. - Highlights: • Riccardin D-N (RD-N) significantly downregulated LAMP1 expressions. • RD-N inhibited the acid sphingomyelinase activity. • RD-N induced lysosomal membrane permeabilization in vivo. • RD-N induced SM accumulation in the lysosomal membranes. • RD-N also induced the release of cathepsins from destabilized lysosomes.

Riccardin D-N induces lysosomal membrane permeabilization by inhibiting acid sphingomyelinase and interfering with sphingomyelin metabolism in vivo

International Nuclear Information System (INIS)

Li, Lin; Niu, Huanmin; Sun, Bin; Xiao, Yanan; Li, Wei; Yuan, Huiqing; Lou, Hongxiang

2016-01-01

Lysosomes are important targets for anticancer drug discovery. Our previous study showed that Riccardin D-N (RD-N), a natural macrocylic bisbibenzyl derivative produced by Mannich reaction, induced cell death by accumulating in lysosomes. Experiments were performed on human lung squamous cell carcinoma tissue from left inferior lobar bronchus of patient xenografts and H460 cells. RD-N was administrated for 25 days. The specimens of xenografts in Balb/c athymic (nu +/nu +) male mice were removed for immunohistochemistry, subcellular fractionation, enzyme activities and Western blotting analysis. mRFP-GFP-LC3 reporter was used to examine autophagy in H460 cells. Sphingomyelin assay was evaluated by thin-layer chromatography and assay kit. Lysosomal membrane permeabilization (LMP) caused by acid sphingomyelinase (ASM) inhibition and subsequent changes of sphingomyelin (SM) metabolism selectively destabilized the cancer cell lysosomes in RD-N-treated H460 cells in vitro and tumor xenograft model in vivo. The destabilized lysosomes induced the release of cathepsins from the lysosomes into the cytosol and further triggered cell death. These results explain the underlying mechanism of RD-N induced LMP. It can be concluded that a more lysosomotropic derivative was synthesized by introduction of an amine group, which could have more potential applications in cancer therapy. - Highlights: • Riccardin D-N (RD-N) significantly downregulated LAMP1 expressions. • RD-N inhibited the acid sphingomyelinase activity. • RD-N induced lysosomal membrane permeabilization in vivo. • RD-N induced SM accumulation in the lysosomal membranes. • RD-N also induced the release of cathepsins from destabilized lysosomes.

Group theoretical treatment of the low-temperature phase transition of the Cd6Ca 1/1-cubic approximant

International Nuclear Information System (INIS)

Tamura, R.; Shibata, K.; Nishimoto, K.; Takeuchi, S.; Edagawa, K.; Saitoh, K.; Isobe, M.; Ueda, Y.

2005-01-01

An antiparallel orientational transition is reported for an intermetallic compound, i.e., Cd 6 Ca crystal, which is a 1/1-1/1-1/1 crystalline approximant to the icosahedral quasicrystal Cd 5.7 Ca. A group theoretical analysis based on the Landau theory predicts that the space group of the low-temperature phase is either C2/c or C2/m, in good agreement with the observations. Accordingly, two types of orientational orderings of Cd 4 tetrahedra, which are located in the center of icosahedral clusters, may occur below 100 K: In both cases, the Cd 4 tetrahedra are orientationally ordered in an antiparallel fashion along the [110] direction of the high temperature body-centered-cubic phase. Such a transition in a metal is reminiscent of orientational transitions in molecular solids

Effect of irradiation on lysosomal enzyme activation in cultured macrophages

International Nuclear Information System (INIS)

Clarke, C.; Wills, E.D.

1980-01-01

The effect of γrays on lysosomal enzyme activity of normal and immune macrophages of DBA/2 mice cultured in vitro has been studied. A dose of 500 rad did not significantly affect lysosomal enzyme activity 3 hours after irradiation but caused the activity to increase to 1.4 times the control value 22.5 hours after irradiation. 22.5 hours after a dose of 3000 rad the enzyme activity increased to 2.5 times the control. Lysosomal enzyme activity of the macrophages was also markedly increased by immunization of the mice with D lymphoma cells, before culture in vitro, but irradiation of these cells with a dose of 500 rad caused a further increase in lysosomal enzyme activity. The results indicate that immunization and irradiation both cause stimulation of lysosomal enzyme activity in macrophages but that the mechanisms of activation are unlikely to be identical. (author)

Solution NMR investigation of the CD95/FADD homotypic death domain complex suggests lack of engagement of the CD95 C terminus.

Science.gov (United States)

Esposito, Diego; Sankar, Andrew; Morgner, Nina; Robinson, Carol V; Rittinger, Katrin; Driscoll, Paul C

2010-10-13

We have addressed complex formation between the death domain (DD) of the death receptor CD95 (Fas/APO-1) with the DD of immediate adaptor protein FADD using nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry, and size-exclusion chromatography with in-line light scattering. We find complexation to be independent of the C-terminal 12 residues of CD95 and insensitive to mutation of residues that engage in the high-order clustering of CD95-DD molecules in a recently reported crystal structure obtained at pH 4. Differential NMR linewidths indicate that the C-terminal region of the CD95 chains remains in a disordered state and (13)C-methyl TROSY data are consistent with a lack of high degree of symmetry for the complex. The overall molecular mass of the complex is inconsistent with that in the crystal structure, and the complex dissociates at pH 4. We discuss these findings using sequence analysis of CD95 orthologs and the effect of FADD mutations on the interaction with CD95. Copyright © 2010 Elsevier Ltd. All rights reserved.

Cellular repressor of E1A-stimulated genes is a bona fide lysosomal protein which undergoes proteolytic maturation during its biosynthesis

International Nuclear Information System (INIS)

Schaehs, Philipp; Weidinger, Petra; Probst, Olivia C.; Svoboda, Barbara; Stadlmann, Johannes; Beug, Hartmut; Waerner, Thomas; Mach, Lukas

2008-01-01

Cellular repressor of E1A-stimulated genes (CREG) has been reported to be a secretory glycoprotein implicated in cellular growth and differentiation. We now show that CREG is predominantly localized within intracellular compartments. Intracellular CREG was found to lack an N-terminal peptide present in the secreted form of the protein. In contrast to normal cells, CREG is largely secreted by fibroblasts missing both mannose 6-phosphate receptors. This is not observed in cells lacking only one of them. Mass spectrometric analysis of recombinant CREG revealed that the protein contains phosphorylated oligosaccharides at either of its two N-glycosylation sites. Cellular CREG was found to cosediment with lysosomal markers upon subcellular fractionation by density-gradient centrifugation. In fibroblasts expressing a CREG-GFP fusion construct, the heterologous protein was detected in compartments containing lysosomal proteins. Immunolocalization of endogenous CREG confirmed that intracellular CREG is localized in lysosomes. Proteolytic processing of intracellular CREG involves the action of lysosomal cysteine proteinases. These results establish that CREG is a lysosomal protein that undergoes proteolytic maturation in the course of its biosynthesis, carries the mannose 6-phosphate recognition marker and depends on the interaction with mannose 6-phosphate receptors for efficient delivery to lysosomes

Synthesis of the C1-C28 Portion of Spongistatin 1 (Altohyrtin A).

Science.gov (United States)

Claffey, Michelle M.; Hayes, Christopher J.; Heathcock, Clayton H.

1999-10-29

A synthetic approach was developed to the C1-C28 subunit of spongistatin 1 (altohyrtin A, 65). The key step was the coupling of the AB and CD spiroketal moieties via an anti-aldol reaction of aldehyde 62 and ethyl ketone 57. The development of a method for the construction of the AB spiroketal fragment is described and included the desymmetrization of C(2)-symmetric diketone 10 and the differentiation of the two primary alcohols of 16. Further elaboration of this advanced intermediate to the desired aldehyde 62 included an Evans' syn-aldol reaction and Tebbe olefination. The synthesis of the CD spiroketal fragment 56 involved the ketalization of a triol-dione, generated in situ by deprotection of 45, to provide a favorable ratio (6-7:1) of spiroketal isomers 46 and 47, respectively. The overall protecting group strategy, involving many selective manipulations of silyl protecting groups, was successfully developed to provide the desired C1-C28 subunit of spongistatin 1 (altohyrtin A) (65).

Cooperative use of cytochrome cd{sub 1} nitrite reductase and its redox partner cytochrome c{sub 552} to improve the selectivity of nitrite biosensing

Energy Technology Data Exchange (ETDEWEB)

Serra, A.S.; Jorge, S.R.; Silveira, C.M.; Moura, J.J.G. [REQUIMTE - Dept. de Quimica, CQFB, Faculdade de Ciencias e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica (Portugal); Jubete, E.; Ochoteco, E.; Cabanero, G.; Grande, H. [CIDETEC - Centro de Tecnologias Electroquimicas, Parque Tecnologico de San Sebastian, Po Miramon, 196, 20009 Donostia - San Sebastian (Spain); Almeida, M.G., E-mail: mga@dq.fct.unl.pt [REQUIMTE - Dept. de Quimica, CQFB, Faculdade de Ciencias e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica (Portugal); Escola Superior de Saude Egas Moniz, Monte de Caparica, 2829-511 Caparica (Portugal)

2011-05-05

In this work, a novel enzymatic biosensor for determination of nitrites constructed on an electrochemical transducing platform is proposed. The sensor is based on cytochrome-cd{sub 1} (cyt-cd{sub 1}) nitrite reductase from Marinobacter hydrocarbonoclasticus strain 617 as biological recognition element, and its putative physiological redox partner cytochrome-c{sub 552} (cyt-c{sub 552}), as electron mediator. The proteins were co-immobilized using a photopolymerizable polyvinyl alcohol (PVA) derivative, onto carbon paste screen printed electrodes (CPSPEs); the optimal modification conditions were 100 {mu}M cyt-cd{sub 1}/100 {mu}M cyt-c{sub 552} and 50% PVA, after a 48 h polymerization time. Electrochemical characterization of the mediator was carried out by cyclic voltammetry. The one-electron exchange between cyt-c{sub 552} and the working electrode is a quasi-reversible process, without mass transport limitations. The formal potential of the mediator is 254 {+-} 2 mV vs NHE and the intermolecular electron transfer rate constant between cytochromes c{sub 552} and cd{sub 1} is 9.9 x 10{sup 3} M{sup -1} s{sup -1}. The analytical parameters of the biosensor response to nitrite as assessed by amperometric measurements were: linear range from 10 to 200 {mu}M; detection and quantification limits of 7 and 24 {mu}M, respectively; sensitivity of 2.49 {+-} 0.08 A mol{sup -1} cm{sup 2} {mu}M{sup -1}. Catalytic profiles in the presence of possible interfering species were also investigated. The interference from competitive enzymatic reduction of dissolved oxygen could be overcome by tuning the cyclic voltammograms for faster sweep rates.

Identification of a lysosome membrane protein which could mediate ATP-dependent stable association of lysosomes to microtubules

International Nuclear Information System (INIS)

Mithieux, G.; Rousset, B.

1989-01-01

We have previously reported that purified thyroid lysosomes bind to reconstituted microtubules to form stable complexes, a process which is inhibited by ATP. Among detergent-solubilized lysosomal membrane protein, we identified a 50-kDa molecular component which binds to preassembled microtubules. The binding of this polypeptide to microtubules was decreased in the presence of ATP. We purified this 50-kDa protein by affinity chromatography on immobilized ATP. The 50-kDa protein bound to the ATP column was eluted by 1 mM ATP. The purified protein, labeled with 125I, exhibited the ability of interacting with microtubules. The binding process was inhibited by increasing concentrations of ATP, the half-maximal inhibitory effect being obtained at an ATP concentration of 0.35 mM. The interaction of the 50-kDa protein with microtubules is a saturable phenomenon since the binding of the 125I-labeled 50-kDa protein was inhibited by unlabeled solubilized lysosomal membrane protein containing the 50-kDa polypeptide but not by the same protein fraction from which the 50-kDa polypeptide had been removed by the ATP affinity chromatography procedure. The 50-kDa protein has the property to bind to pure tubulin coupled to an insoluble matrix. The 50-kDa protein was eluted from the tubulin affinity column by ATP. These findings support the conclusion that a protein inserted into the lysosomal membrane is able to bind directly to microtubules in a process which can be regulated by ATP. We propose that this protein could account for the association of lysosomes to microtubules demonstrated both in vitro and in intact cells

Clonal expansion of genome-intact HIV-1 in functionally polarized Th1 CD4+ T cells.

Science.gov (United States)

Lee, Guinevere Q; Orlova-Fink, Nina; Einkauf, Kevin; Chowdhury, Fatema Z; Sun, Xiaoming; Harrington, Sean; Kuo, Hsiao-Hsuan; Hua, Stephane; Chen, Hsiao-Rong; Ouyang, Zhengyu; Reddy, Kavidha; Dong, Krista; Ndung'u, Thumbi; Walker, Bruce D; Rosenberg, Eric S; Yu, Xu G; Lichterfeld, Mathias

2017-06-30

HIV-1 causes a chronic, incurable disease due to its persistence in CD4+ T cells that contain replication-competent provirus, but exhibit little or no active viral gene expression and effectively resist combination antiretroviral therapy (cART). These latently infected T cells represent an extremely small proportion of all circulating CD4+ T cells but possess a remarkable long-term stability and typically persist throughout life, for reasons that are not fully understood. Here we performed massive single-genome, near-full-length next-generation sequencing of HIV-1 DNA derived from unfractionated peripheral blood mononuclear cells, ex vivo-isolated CD4+ T cells, and subsets of functionally polarized memory CD4+ T cells. This approach identified multiple sets of independent, near-full-length proviral sequences from cART-treated individuals that were completely identical, consistent with clonal expansion of CD4+ T cells harboring intact HIV-1. Intact, near-full-genome HIV-1 DNA sequences that were derived from such clonally expanded CD4+ T cells constituted 62% of all analyzed genome-intact sequences in memory CD4 T cells, were preferentially observed in Th1-polarized cells, were longitudinally detected over a duration of up to 5 years, and were fully replication- and infection-competent. Together, these data suggest that clonal proliferation of Th1-polarized CD4+ T cells encoding for intact HIV-1 represents a driving force for stabilizing the pool of latently infected CD4+ T cells.

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

Science.gov (United States)

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

2009-03-01

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

Soluble CD40 ligand stimulates CD40-dependent activation of the β2 integrin Mac-1 and protein kinase C zeda (PKCζ in neutrophils: implications for neutrophil-platelet interactions and neutrophil oxidative burst.

Directory of Open Access Journals (Sweden)

Rong Jin

Full Text Available Recent work has revealed an essential involvement of soluble CD40L (sCD40L in inflammation and vascular disease. Activated platelets are the major source of sCD40L, which has been implicated in platelet and leukocyte activation, although its exact functional impact on leukocyte-platelet interactions and the underlying mechanisms remain undefined. We aimed to determine the impact and the mechanisms of sCD40L on neutrophils. We studied neutrophil interactions with activated, surface-adherent platelets as a model for leukocyte recruitment to the sites of injury. Our data show that CD40L contributes to neutrophil firm adhesion to and transmigration across activated surface-adherent platelets, possibly through two potential mechanisms. One involves the direct interaction of ligand-receptor (CD40L-CD40, i.e., platelet surface CD40L interaction with neutrophil CD40; another involves an indirect mechanism, i.e. soluble CD40L stimulates activation of the leukocyte-specific β2 integrin Mac-1 in neutrophils and thereby further promotes neutrophil adhesion and migration. Activation of the integrin Mac-1 is known to be critical for mediating neutrophil adhesion and migration. sCD40L activated Mac-1 in neutrophils and enhanced neutrophil-platelet interactions in wild-type neutrophils, but failed to elicit such responses in CD40-deficient neutrophils. Furthermore, our data show that the protein kinase C zeta (PKCζ is critically required for sCD40L-induced Mac-1 activation and neutrophil adhesive function. sCD40L strongly stimulated the focal clustering of Mac-1 (CD11b and the colocalization of Mac-1 with PKCζ in wild-type neutrophils, but had minimal effect in CD40-deficient neutrophils. Blocking PKCζ completely inhibited sCD40L-induced neutrophil firm adhesion. Moreover, sCD40L strongly stimulates neutrophil oxidative burst via CD40-dependent activation of PI3K/NF-KB, but independent of Mac-1 and PKCζ. These findings may contribute to a better

Immunohistochemistry of a choroidal melanoma: nestin, CD34 and CD117÷c-kit labeling.

Science.gov (United States)

Vrapciu, Alexandra Diana; Rusu, Mugurel Constantin; Voinea, Liliana Mary

2014-01-01

In a case of choroidal melanoma (CM) in a 70-year-old male patient, was firstly aimed at studying the processes of angiogenesis by use of nestin and CD34 antibodies. Anti-CD117÷c-kit antibodies were further considered for their progenitor cells specificity. Choroidal melanoma was histopathologically confirmed. Nestin-positive endothelia were found in the CM and the adjacent retina, but not in endothelia elsewhere in that eye. Nestin-positive non-pigmentary cells were found within the CM. Filopodia-projecting endothelial tip cells (ETCs), nestin- and CD34-positive were found in the CM. CD34-positive ETCs were also found in the iridial stroma. There were found two different immune patterns of the retinal Müller cells (MCs). They were nestin-positive in the retina adjacent to the tumor, but negative in any other part of retina. On the other hand, CD117÷c-kit antibodies labeled MCs as follows: (a) discontinuously, or continuously, in the retina adjacent to the CM; (b) only the inner segments of the MCs were labeled in the retina unrelated to the CM. While nestin could be a reliable marker for retinal damage, the CD117÷c-kit phenotype of MCs still needs further investigations. Antiangiogenic therapy appears as a good choice for tumor therapy.

Triptolide induces lysosomal-mediated programmed cell death in MCF-7 breast cancer cells

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Owa C

2013-09-01

Full Text Available Chie Owa, Michael E Messina Jr, Reginald HalabyDepartment of Biology, Montclair State University, Montclair, NJ, USABackground: Breast cancer is a major cause of death; in fact, it is the most common type, in order of the number of global deaths, of cancer in women worldwide. This research seeks to investigate how triptolide, an extract from the Chinese herb Tripterygium wilfordii Hook F, induces apoptosis in MCF-7 human breast cancer cells. Accumulating evidence suggests a role for lysosomal proteases in the activation of apoptosis. However, there is also some controversy regarding the direct participation of lysosomal proteases in activation of key apoptosis-related caspases and release of mitochondrial cytochrome c. In the present study, we demonstrate that triptolide induces an atypical, lysosomal-mediated apoptotic cell death in MCF-7 cells because they lack caspase-3.Methods: MCF-7 cell death was characterized via cellular morphology, chromatin condensation, 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide colorimetric cell growth inhibition assay and the expression levels of proapoptotic proteins. Acridine orange and LysoTracker® staining were performed to visualize lysosomes. Lysosomal enzymatic activity was monitored using an acid phosphatase assay and western blotting of cathepsin B protein levels in the cytosolic fraction, which showed increased enzymatic activity in drug-treated cells.Results: These experiments suggest that triptolide-treated MCF-7 cells undergo atypical apoptosis and that, during the early stages, lysosomal enzymes leak into the cytosol, indicating lysosomal membrane permeability.Conclusion: Our results suggest that further studies are warranted to investigate triptolide's potential as an anticancer therapeutic agent.Keywords: triptolide, MCF-7 breast cancer cells, apoptosis, lysosomes, lysosomal membrane permeabilization (LMP

Vitamin C in prevention of atrial fibrillation after coronary artery bypass graft: double blind randomized clinical trial

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Mahmoodreza Sarzaeem

2014-03-01

Conclusion: Vitamin C is relatively safe, inexpensive, well tolerated and has a low complication. According to the 44% reduction in the incidence of atrial fibrillation in vitamin C patients undergoing coronary artery bypass grafting surgery, this drug can be prescribed as a prophylaxis for prevention of post-CABG atrial fibrillation.

Genetic localization of Cd63, a member of the transmembrane 4 superfamily, reveals two distinct loci in the mouse genome

Energy Technology Data Exchange (ETDEWEB)

Gwynn, B.; Eicher, E.M.; Peters, L.L. [Jackson Lab., Bar Harbor, ME (United States)

1996-07-15

The membrane protein CD63, a molecular marker for early stages of melanoma progression, has been associated with platelet storage pool deficiency disorders (SPD). CD63 localizes to the membranes of platelets, lysosomes, and melanosomes, all of which are affected in a specific subgroup of SPD. The cDNA encoding CD63 detects two closely related sequences that map to different regions of the mouse genome. One locus maps to mouse Chromosome (Chr) 10 in a region that shares linkage homology with the human chromosome encoding human CD63. The second locus maps to mouse Chr 18 in a region that bears no known human CD63-related genes. No SPD has been localized to these regions of either the mouse or the human chromosomes. 15 refs., 2 figs.

The emerging role of lysosomes in copper homeostasis.

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Polishchuk, Elena V; Polishchuk, Roman S

2016-09-01

The lysosomal system operates as a focal point where a number of important physiological processes such as endocytosis, autophagy and nutrient sensing converge. One of the key functions of lysosomes consists of regulating the metabolism/homeostasis of metals. Metal-containing components are carried to the lysosome through incoming membrane flows, while numerous transporters allow metal ions to move across the lysosome membrane. These properties enable lysosomes to direct metal fluxes to the sites where metal ions are either used by cellular components or sequestered. Copper belongs to a group of metals that are essential for the activity of vitally important enzymes, although it is toxic when in excess. Thus, copper uptake, supply and intracellular compartmentalization have to be tightly regulated. An increasing number of publications have indicated that these processes involve lysosomes. Here we review studies that reveal the expanding role of the lysosomal system as a hub for the control of Cu homeostasis and for the regulation of key Cu-dependent processes in health and disease.

75 FR 71145 - San Joaquin River Restoration Program: Reach 4B, Eastside Bypass, and Mariposa Bypass Channel and...

Science.gov (United States)

2010-11-22

..., Eastside Bypass, and Mariposa Bypass Channel and Structural Improvements Project, Merced County, CA AGENCY... on the effects of the proposed Reach 4B, Eastside Bypass, and Mariposa Bypass Channel and Structural... Mariposa Bypass with the San Joaquin River (generally referred to as Reach 4B1). The improvements will...

CD38/cADPR Signaling Pathway in Airway Disease: Regulatory Mechanisms

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Deepak A. Deshpande

2018-01-01

Full Text Available Asthma is an inflammatory disease in which proinflammatory cytokines have a role in inducing abnormalities of airway smooth muscle function and in the development of airway hyperresponsiveness. Inflammatory cytokines alter calcium (Ca2+ signaling and contractility of airway smooth muscle, which results in nonspecific airway hyperresponsiveness to agonists. In this context, Ca2+ regulatory mechanisms in airway smooth muscle and changes in these regulatory mechanisms encompass a major component of airway hyperresponsiveness. Although dynamic Ca2+ regulation is complex, phospholipase C/inositol tris-phosphate (PLC/IP3 and CD38-cyclic ADP-ribose (CD38/cADPR are two major pathways mediating agonist-induced Ca2+ regulation in airway smooth muscle. Altered CD38 expression or enhanced cyclic ADP-ribosyl cyclase activity associated with CD38 contributes to human pathologies such as asthma, neoplasia, and neuroimmune diseases. This review is focused on investigations on the role of CD38-cyclic ADP-ribose signaling in airway smooth muscle in the context of transcriptional and posttranscriptional regulation of CD38 expression. The specific roles of transcription factors NF-kB and AP-1 in the transcriptional regulation of CD38 expression and of miRNAs miR-140-3p and miR-708 in the posttranscriptional regulation and the underlying mechanisms of such regulation are discussed.

CD38/cADPR Signaling Pathway in Airway Disease: Regulatory Mechanisms

Science.gov (United States)

Deshpande, Deepak A.; Guedes, Alonso G. P.; Graeff, Richard; Dogan, Soner; Subramanian, Subbaya; Walseth, Timothy F.

2018-01-01

Asthma is an inflammatory disease in which proinflammatory cytokines have a role in inducing abnormalities of airway smooth muscle function and in the development of airway hyperresponsiveness. Inflammatory cytokines alter calcium (Ca2+) signaling and contractility of airway smooth muscle, which results in nonspecific airway hyperresponsiveness to agonists. In this context, Ca2+ regulatory mechanisms in airway smooth muscle and changes in these regulatory mechanisms encompass a major component of airway hyperresponsiveness. Although dynamic Ca2+ regulation is complex, phospholipase C/inositol tris-phosphate (PLC/IP3) and CD38-cyclic ADP-ribose (CD38/cADPR) are two major pathways mediating agonist-induced Ca2+ regulation in airway smooth muscle. Altered CD38 expression or enhanced cyclic ADP-ribosyl cyclase activity associated with CD38 contributes to human pathologies such as asthma, neoplasia, and neuroimmune diseases. This review is focused on investigations on the role of CD38-cyclic ADP-ribose signaling in airway smooth muscle in the context of transcriptional and posttranscriptional regulation of CD38 expression. The specific roles of transcription factors NF-kB and AP-1 in the transcriptional regulation of CD38 expression and of miRNAs miR-140-3p and miR-708 in the posttranscriptional regulation and the underlying mechanisms of such regulation are discussed. PMID:29576747

Mechanisms of communication between mitochondria and lysosomes.

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Raimundo, Nuno; Fernández-Mosquera, Lorena; Yambire, King Faisal; Diogo, Cátia V

2016-10-01

Mitochondria and lysosomes have long been studied in the context of their classic functions: energy factory and recycle bin, respectively. In the last twenty years, it became evident that these organelles are much more than simple industrial units, and are indeed in charge of many of cellular processes. Both mitochondria and lysosomes are now recognized as far-reaching signaling platforms, regulating many key aspects of cell and tissue physiology. It has furthermore become clear that mitochondria and lysosomes impact each other. The mechanisms underlying the cross-talk between these organelles are only now starting to be addressed. In this review, we briefly summarize how mitochondria, lysosomes and the lysosome-related process of autophagy affect each other in physiology and pathology. Copyright © 2016 Elsevier Ltd. All rights reserved.

A novel transgenic mouse model of lysosomal storage disorder.

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Ortiz-Miranda, Sonia; Ji, Rui; Jurczyk, Agata; Aryee, Ken-Edwin; Mo, Shunyan; Fletcher, Terry; Shaffer, Scott A; Greiner, Dale L; Bortell, Rita; Gregg, Ronald G; Cheng, Alan; Hennings, Leah J; Rittenhouse, Ann R

2016-11-01

Knockout technology has proven useful for delineating functional roles of specific genes. Here we describe and provide an explanation for striking pathology that occurs in a subset of genetically engineered mice expressing a rat Ca V β2a transgene under control of the cardiac α-myosin heavy chain promoter. Lesions were limited to mice homozygous for transgene and independent of native Cacnb2 genomic copy number. Gross findings included an atrophied pancreas; decreased adipose tissue; thickened, orange intestines; and enlarged liver, spleen, and abdominal lymph nodes. Immune cell infiltration and cell engulfment by macrophages were associated with loss of pancreatic acinar cells. Foamy macrophages diffusely infiltrated the small intestine's lamina propria, while similar macrophage aggregates packed liver and splenic red pulp sinusoids. Periodic acid-Schiff-positive, diastase-resistant, iron-negative, Oil Red O-positive, and autofluorescent cytoplasm was indicative of a lipid storage disorder. Electron microscopic analysis revealed liver sinusoids distended by clusters of macrophages containing intracellular myelin "swirls" and hepatocytes with enlarged lysosomes. Additionally, build up of cholesterol, cholesterol esters, and triglycerides, along with changes in liver metabolic enzyme levels, were consistent with a lipid processing defect. Because of this complex pathology, we examined the transgene insertion site. Multiple transgene copies inserted into chromosome 19; at this same site, an approximate 180,000 base pair deletion occurred, ablating cholesterol 25-hydroxylase and partially deleting lysosomal acid lipase and CD95 Loss of gene function can account for the altered lipid processing, along with hypertrophy of the immune system, which define this phenotype, and serendipitously provides a novel mouse model of lysosomal storage disorder. Copyright © 2016 the American Physiological Society.

Characterization of recombinant human lysosomal beta-hexosaminidases produced in the methylotrophic yeast Pichia pastoris

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Angela Johana Espejo Mojica

2016-08-01

Full Text Available β-hexosaminidases (Hex are dimeric enzymes involved in the lysosomal degradation of glycolipids and glycans. They are formed by α- and/or β-subunits encoded byHEXA and HEXB genes, respectively. Mutations in these genes lead to Tay Sachs or Sandhoff diseases, which are neurodegenerative disorders caused by the accumulation of non-degraded glycolipids. Although tissue-derived Hex have been widely characterized, limited information is available for recombinant β-hexosaminidases. In this study, human lysosomal recombinant Hex (rhHex-A, rhHex-B, and rhHex-S were produced in the methylotrophic yeast Pichia pastoris GS115. The highest specific enzyme activities were 13,124 for rhHexA; 12,779 for rhHex-B; and 14,606 U .mg-1 for rhHex-S. These results were 25- to 50-fold higher than those obtained from normal human leukocytes. Proteins were purified and characterized at different pH and temperature conditions. All proteins were stable at acidic pH, and at 4 °C and 37 °C. At 45 °C rhHex-S was completely inactivated, while rhHex-A and rhHex-B showed high stability. This study demonstrates P. pastoris GS115 potential for polymeric lysosomal enzyme production, and describes the characterization of recombinant β-hexosaminidases produced within the same host.

HIV-1 Nef down-modulates C-C and C-X-C chemokine receptors via ubiquitin and ubiquitin-independent mechanism.

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Prabha Chandrasekaran

Full Text Available Human and Simian Immunodeficiency virus (HIV-1, HIV-2, and SIV encode an accessory protein, Nef, which is a pathogenesis and virulence factor. Nef is a multivalent adapter that dysregulates the trafficking of many immune cell receptors, including chemokine receptors (CKRs. Physiological endocytic itinerary of agonist occupied CXCR4 involves ubiquitinylation of the phosphorylated receptor at three critical lysine residues and dynamin-dependent trafficking through the ESCRT pathway into lysosomes for degradation. Likewise, Nef induced CXCR4 degradation was critically dependent on the three lysines in the C-terminal -SSLKILSKGK- motif. Nef directly recruits the HECT domain E3 ligases AIP4 or NEDD4 to CXCR4 in the resting state. This mechanism was confirmed by ternary interactions of Nef, CXCR4 and AIP4 or NEDD4; by reversal of Nef effect by expression of catalytically inactive AIP4-C830A mutant; and siRNA knockdown of AIP4, NEDD4 or some ESCRT-0 adapters. However, ubiquitinylation dependent lysosomal degradation was not the only mechanism by which Nef downregulated CKRs. Agonist and Nef mediated CXCR2 (and CXCR1 degradation was ubiquitinylation independent. Nef also profoundly downregulated the naturally truncated CXCR4 associated with WHIM syndrome and engineered variants of CXCR4 that resist CXCL12 induced internalization via an ubiquitinylation independent mechanism.

NQR and X-ray crystal structure studies of cadmium halide complexes: [C(NH{sub 2}){sub 3}]CdI{sub 3} and [4-ClC{sub 6}H{sub 5}NH{sub 3}]{sub 3}CdBr{sub 5}

Energy Technology Data Exchange (ETDEWEB)

Gesing, Thorsten M.; Lork, Enno [Bremen Univ. (Germany). MAPEX Center for Material and Processes; Terao, Hiromitsu [Tokushima Univ. (Japan). Faculty of Integrated Arts and Sciences; Ishihara, Hideta [Saga Univ. (Japan). Faculty of Culture and Education

2016-05-01

The crystal structures of [C(NH{sub 2}){sub 3}]CdI{sub 3} (1) and [4-ClC{sub 6}H{sub 5}NH{sub 3}]{sub 3}CdBr{sub 5} (2) have been determined at 100 K: monoclinic, Cc, a = 828.75(3) pm, b = 1615.31(5) pm, c = 810.64(3) pm, and β = 106.5820(10) for 1; monoclinic, P2{sub 1}/c, a = 1486.93(5) pm, b = 794.31(3) pm, c = 2290.59(7) pm, and β = 99.6830(10) for 2. The structure of 1 has an infinite chain of anions consisting of [CdI{sub 4}] tetrahedra sharing two corners. The structure of 2 has an infinite chain of anions consisting of [CdBr{sub 6}] octahedra sharing two corners in cis positions. In both structures, isolated cations are connected to the anion chains through weak hydrogen bonds Cd-X..H to result in three-dimensional network structures. In accordance with the crystal structures, three {sup 127}I (m = ±1/2 <-> m = ±3/2), five {sup 81}Br, and three {sup 35}Cl nuclear quadrupole resonance (NQR) lines were observed for 1 and 2. The NQR spectra reflect the anion chain structures and their weak hydrogen bonds. The MO calculations of the models [Cd{sub 5}I{sub 16}]{sup 6-} for 1 and [Cd{sub 3}Br{sub 16}]{sup 10-} for 2 estimate only about half the values for the NQR frequencies but give accurate electric field gradient directions.

C5a receptor (CD88) blockade protects against MPO-ANCA GN.

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Xiao, Hong; Dairaghi, Daniel J; Powers, Jay P; Ertl, Linda S; Baumgart, Trageen; Wang, Yu; Seitz, Lisa C; Penfold, Mark E T; Gan, Lin; Hu, Peiqi; Lu, Bao; Gerard, Norma P; Gerard, Craig; Schall, Thomas J; Jaen, Juan C; Falk, Ronald J; Jennette, J Charles

2014-02-01

Necrotizing and crescentic GN (NCGN) with a paucity of glomerular immunoglobulin deposits is associated with ANCA. The most common ANCA target antigens are myeloperoxidase (MPO) and proteinase 3. In a manner that requires activation of the alternative complement pathway, passive transfer of antibodies to mouse MPO (anti-MPO) induces a mouse model of ANCA NCGN that closely mimics human disease. Here, we confirm the importance of C5aR/CD88 in the mediation of anti-MPO-induced NCGN and report that C6 is not required. We further demonstrate that deficiency of C5a-like receptor (C5L2) has the reverse effect of C5aR/CD88 deficiency and results in more severe disease, indicating that C5aR/CD88 engagement enhances inflammation and C5L2 engagement suppresses inflammation. Oral administration of CCX168, a small molecule antagonist of human C5aR/CD88, ameliorated anti-MPO-induced NCGN in mice expressing human C5aR/CD88. These observations suggest that blockade of C5aR/CD88 might have therapeutic benefit in patients with ANCA-associated vasculitis and GN.

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

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

2016-03-18

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.

Limited and selective transfer of plasma membrane glycoproteins to membrane of secondary lysosomes

International Nuclear Information System (INIS)

Haylett, T.; Thilo, L.

1986-01-01

Radioactive galactose, covalently bound to cell surface glycoconjugates on mouse macrophage cells, P388D 1 , was used as a membrane marker to study the composition, and the kinetics of exchange, of plasma membrane-derived constituents in the membrane of secondary lysosomes. Secondary lysosomes were separated from endosomes and plasma membrane by self-forming Percoll density gradients. Horseradish peroxidase, taken up by fluid-phase pinocytosis, served as a vesicle contents marker to monitor transfer of endosomal contents into secondary lysosomes. Concurrently, the fraction of plasma membrane-derived label of secondary lysosomes increased by first order kinetics from 4 PAGE, labeled molecules of M/sub r/ 160-190 kD were depleted and of the M/sub r/ 100-120 kD were enriched in lysosome membrane compared with the relative composition of label on the cell surface. No corresponding selectivity was observed for the degradation of label, with all M/sub r/ classes being affected to the same relative extent. The results indicate that endocytosis-derived transfer of plasma membrane constitutents to secondary lysosomes is a limited and selective process, and that only ∼1% of internalized membrane is recycled via a membrane pool of secondary lysosomes

Occupational Analysis Products: Space Systems Operations - AFSC 1C6X1 (CD-ROM)

National Research Council Canada - National Science Library

Boerstler, Robert E

2004-01-01

...: 1 CD-ROM; 4 3/4 in.; 30.4 MB. SYSTEMS DETAIL NOTE: ABSTRACT: The Space Systems Operations career ladder was surveyed to obtain current task and equipment data for use in evaluating current training programs...

Allorecognition of HLA-C mismatches by CD8+ T cells in hematopoietic stem cell transplantation is a complex interplay between mismatched peptide binding region residues, HLA-C expression and HLA-DPB1 disparities

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Florence Bettens

2016-12-01

Full Text Available HLA-C locus mismatches are the most frequent class I disparities in unrelated hematopoietic stem cell transplantation (HSCT and have a detrimental impact on clinical outcome. Recently, a few retrospective clinical studies have reported some variability in the immunogenicity of HLA-C incompatibilities. To get better insight into presumably permissive HLA-C mismatches we have developed a one-way in vitro mixed lymphocyte reaction (MLR assay allowing to quantify activated CD56-CD137+CD8+ lymphocytes in HLA-C incompatible combinations. T cell-mediated alloresponses were correlated with genetic markers such as HLA-C mRNA expression and the number of amino acid mismatches in the α1/α2 domains (peptide binding region. Because of the high rate of HLA-DPB1 incompatibilities in HLA-A, B, C, DRB1 and DQB1 matched unrelated HSCT patient/donor pairs, the impact of HLA-DPB1 mismatching, a potential bystander of CD4+ T cell activation, was also considered. Heterogeneous alloresponses were measured in 63 HLA-C mismatched pairs with a positive assay in 52% of the combinations (2.3-18.6% activated CTLs, representing 24 different HLA-A~B~DRB1~DQB1 haplotypes. There was no correlation between measured alloresponses and mRNA expression of the mismatched HLA-C alleles. The HLA-C*03:03/03:04 mismatch did not induce any positive alloresponse in 5 MLRs. We also identified HLA-C*02:02 and HLA-C*06:02 as mismatched alleles with lower immunogenicity, and HLA-C*14:02 as a more immunogenic mismatch. A difference of at least 10 amino acid residues known to impact peptide/TCR binding and a bystander HLA-DPB1 incompatibility had a significant impact on CTL alloreactivity (p=0.021. The same HLA-C mismatch, when recognized by two different responders with the same HLA haplotypes, was recognized differently, emphasizing the role of the T-cell repertoire of responding cells. In conclusion, mismatched HLA-C alleles differing by10 or more amino acids in the peptide/TCR binding

Optical, Electrical and Photocatalytic Properties of the Ternary Semiconductors ZnxCd1-xS, CuxCd1-xS and CuxZn1-xS

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Sandra Andrea Mayén-Hernández

2014-01-01

Full Text Available The effects of vacuum annealing at different temperatures on the optical, electrical and photocatalytic properties of polycrystalline and amorphous thin films of the ternary semiconductor alloys ZnxCd1-xS, CuxCd1-xS and CuxZn1-xS were investigated in stacks of binary semiconductors obtained by chemical bath deposition. The electrical properties were measured at room temperature using a four-contact probe in the Van der Pauw configuration. The energy band gap of the films varied from 2.30 to 2.85 eV. The photocatalytic activity of the semiconductor thin films was evaluated by the degradation of an aqueous methylene blue solution. The thin film of ZnxCd1-xS annealed under vacuum at 300°C exhibited the highest photocatalytic activity.

The gene expression profile of CD11c+ CD8α- dendritic cells in the pre-diabetic pancreas of the NOD mouse.

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Wouter Beumer

Full Text Available Two major dendritic cell (DC subsets have been described in the pancreas of mice: The CD11c+ CD8α- DCs (strong CD4+ T cell proliferation inducers and the CD8α+ CD103+ DCs (T cell apoptosis inducers. Here we analyzed the larger subset of CD11c+ CD8α- DCs isolated from the pancreas of pre-diabetic NOD mice for genome-wide gene expression (validated by Q-PCR to elucidate abnormalities in underlying gene expression networks. CD11c+ CD8α- DCs were isolated from 5 week old NOD and control C57BL/6 pancreas. The steady state pancreatic NOD CD11c+ CD8α- DCs showed a reduced expression of several gene networks important for the prime functions of these cells, i.e. for cell renewal, immune tolerance induction, migration and for the provision of growth factors including those for beta cell regeneration. A functional in vivo BrdU incorporation test showed the reduced proliferation of steady state pancreatic DC. The reduced expression of tolerance induction genes (CD200R, CCR5 and CD24 was supported on the protein level by flow cytometry. Also previously published functional tests on maturation, immune stimulation and migration confirm the molecular deficits of NOD steady state DC. Despite these deficiencies NOD pancreas CD11c+ CD8α- DCs showed a hyperreactivity to LPS, which resulted in an enhanced pro-inflammatory state characterized by a gene profile of an enhanced expression of a number of classical inflammatory cytokines. The enhanced up-regulation of inflammatory genes was supported by the in vitro cytokine production profile of the DCs. In conclusion, our data show that NOD pancreatic CD11c+ CD8α- DCs show various deficiencies in steady state, while hyperreactive when encountering a danger signal such as LPS.