Sample records for lacking ganglioside gm3

  1. Synthesis and characterization of N-parinaroyl analogs of ganglioside GM3 and de-N-acetyl GM3. Interactions with the EGF receptor kinase

    NASA Technical Reports Server (NTRS)

    Song, W.; Welti, R.; Hafner-Strauss, S.; Rintoul, D. A.; Spooner, B. S. (Principal Investigator)

    1993-01-01

    A specific plasma membrane glycosphingolipid, known as ganglioside GM3, can regulate the intrinsic tyrosyl kinase activity of the epidermal growth factor (EGF) receptor; this modulation is not associated with alterations in hormone binding to the receptor. GM3 inhibits EGF receptor tyrosyl kinase activity in detergent micelles, in plasma membrane vesicles, and in whole cells. In addition, immunoaffinity-purified EGF receptor preparations contain ganglioside GM3 (Hanai et al. (1988) J. Biol. Chem. 263, 10915-10921), implying that the glycosphingolipid is intimately associated with the receptor kinase in cell membranes. Both the nature of this association and the molecular mechanism of kinase inhibition remain to be elucidated. In this report, we describe the synthesis of a fluorescent analog of ganglioside GM3, in which the native fatty acid was replaced with trans-parinaric acid. This glycosphingolipid inhibited the receptor kinase activity in a manner similar to that of the native ganglioside. A modified fluorescent glycosphingolipid, N-trans-parinaroyl de-N-acetyl ganglioside GM3, was also prepared. This analog, like the nonfluorescent de-N-acetyl ganglioside GM3, had no effect on receptor kinase activity. Results from tryptophan fluorescence quenching and steady-state anisotropy measurements in membranes containing these fluorescent probes and the human EGF receptor were consistent with the notion that GM3, but not de-N-acetyl GM3, interacts specifically with the receptor in intact membranes.

  2. GM1 and GM2 gangliosides: recent developments.

    PubMed

    Bisel, Blaine; Pavone, Francesco S; Calamai, Martino

    2014-03-01

    GM1 and GM2 gangliosides are important components of the cell membrane and play an integral role in cell signaling and metabolism. In this conceptual overview, we discuss recent developments in our understanding of the basic biological functions of GM1 and GM2 and their involvement in several diseases. In addition to a well-established spectrum of disorders known as gangliosidoses, such as Tay-Sachs disease, more and more evidence points at an involvement of GM1 in Alzheimer's and Parkinson's diseases. New emerging methodologies spanning from single-molecule imaging in vivo to simulations in silico have complemented standard studies based on ganglioside extraction.

  3. Sialidase activities of cultured human fibroblasts and the metabolism of GM3 ganglioside

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Usuki, S.; Lyu, S.C.; Sweeley, C.C.

    1988-05-15

    Free sialic acid has been found in the cell-conditioned medium of human foreskin fibroblasts. It is proposed that the accumulation of extracellular sialic acid may result from the hydrolysis of GM3 ganglioside on the cell surface of these fibroblasts. Sialidase activities with GM3 ganglioside and sialyllactitol as substrates were demonstrated in cell-conditioned medium, and the levels of their activities correlated positively with cell density. The GM3 sialidase activity at pH 4.5 was 4.1 and 38 pmol/h/ml of medium at sparse and confluent densities, respectively; the corresponding activities with sialyllactitol as the substrate were 12 and 75 pmol/h/ml of medium (pHmore » 4.5). The pH versus activity profiles with GM3 as the substrate suggested the presence of a second sialidase with an optimal activity at pH 6.5 in the conditioned medium of preconfluent cells. This activity was virtually absent in the medium of contact-inhibited cells and could not be assayed with sialyllactitol as the substrate. The turnover of cell surface GM3 was assessed by pulse labeling human foreskin fibroblasts with a radioactive precursor of sialic acid ((1-14C)N-acetylmannosamine) and a radioactive precursor of ceramide ((3,3-3H2)serine). During a chase period of 24 h turnover of the doubly labeled cellular GM3 was observed; there was a loss of about 35% of the 14C-labeled sialic acid without any measureable loss of 3H-labeled ceramide from GM3. We have speculated that the enzyme-catalyzed removal of sialic acid from the GM3 ganglioside on the extracellular aspect of the plasma membrane may be a necessary event involved in the modulation of cell growth.« less

  4. Altered Expression of Ganglioside Metabolizing Enzymes Results in GM3 Ganglioside Accumulation in Cerebellar Cells of a Mouse Model of Juvenile Neuronal Ceroid Lipofuscinosis.

    PubMed

    Somogyi, Aleksandra; Petcherski, Anton; Beckert, Benedikt; Huebecker, Mylene; Priestman, David A; Banning, Antje; Cotman, Susan L; Platt, Frances M; Ruonala, Mika O; Tikkanen, Ritva

    2018-02-22

    Juvenile neuronal ceroid lipofuscinosis (JNCL) is caused by mutations in the CLN3 gene. Most JNCL patients exhibit a 1.02 kb genomic deletion removing exons 7 and 8 of this gene, which results in a truncated CLN3 protein carrying an aberrant C-terminus. A genetically accurate mouse model ( Cln3 Δex7/8 mice) for this deletion has been generated. Using cerebellar precursor cell lines generated from wildtype and Cln3 Δex7/8 mice, we have here analyzed the consequences of the CLN3 deletion on levels of cellular gangliosides, particularly GM3, GM2, GM1a and GD1a. The levels of GM1a and GD1a were found to be significantly reduced by both biochemical and cytochemical methods. However, quantitative high-performance liquid chromatography analysis revealed a highly significant increase in GM3, suggesting a metabolic blockade in the conversion of GM3 to more complex gangliosides. Quantitative real-time PCR analysis revealed a significant reduction in the transcripts of the interconverting enzymes, especially of β-1,4- N -acetyl-galactosaminyl transferase 1 (GM2 synthase), which is the enzyme converting GM3 to GM2. Thus, our data suggest that the complex a-series gangliosides are reduced in Cln3 Δex7/8 mouse cerebellar precursor cells due to impaired transcription of the genes responsible for their synthesis.

  5. Altered Expression of Ganglioside Metabolizing Enzymes Results in GM3 Ganglioside Accumulation in Cerebellar Cells of a Mouse Model of Juvenile Neuronal Ceroid Lipofuscinosis

    PubMed Central

    Somogyi, Aleksandra; Petcherski, Anton; Beckert, Benedikt; Huebecker, Mylene; Priestman, David A.; Banning, Antje; Cotman, Susan L.; Platt, Frances M.; Ruonala, Mika O.

    2018-01-01

    Juvenile neuronal ceroid lipofuscinosis (JNCL) is caused by mutations in the CLN3 gene. Most JNCL patients exhibit a 1.02 kb genomic deletion removing exons 7 and 8 of this gene, which results in a truncated CLN3 protein carrying an aberrant C-terminus. A genetically accurate mouse model (Cln3Δex7/8 mice) for this deletion has been generated. Using cerebellar precursor cell lines generated from wildtype and Cln3Δex7/8 mice, we have here analyzed the consequences of the CLN3 deletion on levels of cellular gangliosides, particularly GM3, GM2, GM1a and GD1a. The levels of GM1a and GD1a were found to be significantly reduced by both biochemical and cytochemical methods. However, quantitative high-performance liquid chromatography analysis revealed a highly significant increase in GM3, suggesting a metabolic blockade in the conversion of GM3 to more complex gangliosides. Quantitative real-time PCR analysis revealed a significant reduction in the transcripts of the interconverting enzymes, especially of β-1,4-N-acetyl-galactosaminyl transferase 1 (GM2 synthase), which is the enzyme converting GM3 to GM2. Thus, our data suggest that the complex a-series gangliosides are reduced in Cln3Δex7/8 mouse cerebellar precursor cells due to impaired transcription of the genes responsible for their synthesis. PMID:29470438

  6. Early growth and development impairments in patients with ganglioside GM3 synthase deficiency.

    PubMed

    Wang, H; Wang, A; Wang, D; Bright, A; Sency, V; Zhou, A; Xin, B

    2016-05-01

    Ganglioside GM3 synthase is a key enzyme involved in the biosynthesis of gangliosides. GM3 synthase deficiency (GSD) causes a complete absence of GM3 and all downstream biosynthetic derivatives. The individuals affected by this disorder manifest severe irritability, intractable seizures and profound intellectual disability. However, we have found that most newborns seem symptom-free for a period of time after birth. In order to further understand the onset of the disease, we investigated the early growth and development of patients with this condition through this study. We compared 37 affected individuals with their normal siblings and revealed that all children with GSD had relatively normal intrauterine growth and development, as their weight, length and head circumference were similar to their normal siblings at birth. However, the disease progresses quickly after birth and causes significant constitutional impairments of growth and development by 6 months of age. Neither breastfeeding nor gastrostomy tube placement made significant difference on growth and development as all groups of patients showed the similar pattern. We conclude that GSD causes significant postnatal growth and developmental impairments and the amount of gangliosides in breast milk and general nutritional intervention do not seem to alter these outcomes. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  7. Requirement of GM2 ganglioside activator for phospholipase D activation

    PubMed Central

    Nakamura, Shun-ichi; Akisue, Toshihiro; Jinnai, Hitoshi; Hitomi, Tomohiro; Sarkar, Sukumar; Miwa, Noriko; Okada, Taro; Yoshida, Kimihisa; Kuroda, Shun’ichi; Kikkawa, Ushio; Nishizuka, Yasutomi

    1998-01-01

    Sequence analysis of a heat-stable protein necessary for the activation of ADP ribosylation factor-dependent phospholipase D (PLD) reveals that this protein has a structure highly homologous to the previously known GM2 ganglioside activator whose deficiency results in the AB-variant of GM2 gangliosidosis. The heat-stable activator protein indeed has the capacity to enhance enzymatic conversion of GM2 to GM3 ganglioside that is catalyzed by β-hexosaminidase A. Inversely, GM2 ganglioside activator purified separately from tissues as described earlier [Conzelmann, E. & Sandhoff, K. (1987) Methods Enzymol. 138, 792–815] stimulates ADP ribosylation factor-dependent PLD in a dose-dependent manner. At higher concentrations of ammonium sulfate, the PLD activator protein apparently substitutes for protein kinase C and phosphatidylinositol 4,5-bisphosphate, both of which are known as effective stimulators of the PLD reaction. The mechanism of action of the heat-stable PLD activator protein remains unknown. PMID:9770472

  8. New Insights on Non-Enzymatic Oxidation of Ganglioside GM1 Using Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Couto, Daniela; Melo, Tânia; Maciel, Elisabete; Campos, Ana; Alves, Eliana; Guedes, Sofia; Domingues, M. Rosário M.; Domingues, Pedro

    2016-12-01

    Gangliosides are acidic glycosphingolipids that are present in cell membranes and lipid raft domains, being particularly abundant in central nervous systems. They participate in modulating cell membrane properties, cell-cell recognition, cell regulation, and signaling. Disturbance in ganglioside metabolism has been correlated with the development of diseases, such as neurodegenerative diseases, and in inflammation. Both conditions are associated with an increased production of reactive oxidation species (ROS) that can induce changes in the structure of biomolecules, including lipids, leading to the loss or modification of their function. Oxidized phospholipids are usually involved in chronic diseases and inflammation. However, knowledge regarding oxidation of gangliosides is scarce. In order to evaluate the effect of ROS in gangliosides, an in vitro biomimetic model system was used to study the susceptibility of GM1 (Neu5Ac α2-3(Gal β1-3GalNAc β1-4)Gal β1-4Glc β1Cer) to undergo oxidative modifications. Oxidation of GM1 under Fenton reaction conditions was monitored using high resolution electrospray ionization-mass spectrometry (ESI-MS) and tandem mass spectrometry (ESI-MS/MS). Upon oxidation, GM1 underwent oxidative cleavages in the carbohydrate chain, leading to the formation of other gangliosides GM2 (GalNAcβ1-4Gal(Neu5Acα2-3)1-4Glcβ1Cer), GM3 (Neu5Acα2-3Galβ1-4Glcβ1Cer), asialo-GM1 (Gal β1-3GalNAc β1-4Gal β1-4Glc β1Cer), asialo-GM2 (GalNAc β1-4Gal β1-4Glc β1Cer), of the small glycolipids lactosylceramide (LacCer), glucosylceramide (GlcCer), and of ceramide (Cer). In addition, oxygenated GM1 and GM2 (as keto and hydroxy derivatives), glycans, oxidized glycans, and oxidized ceramides were also identified. Nonenzymatic oxidation of GM1 under oxidative stress contributes to the generation of other gangliosides that may participate in the imbalance of gangliosides metabolism in vivo, through uncontrolled enzymatic pathways and, consequently, play

  9. Mutations in B4GALNT1 (GM2 synthase) underlie a new disorder of ganglioside biosynthesis.

    PubMed

    Harlalka, Gaurav V; Lehman, Anna; Chioza, Barry; Baple, Emma L; Maroofian, Reza; Cross, Harold; Sreekantan-Nair, Ajith; Priestman, David A; Al-Turki, Saeed; McEntagart, Meriel E; Proukakis, Christos; Royle, Louise; Kozak, Radoslaw P; Bastaki, Laila; Patton, Michael; Wagner, Karin; Coblentz, Roselyn; Price, Joy; Mezei, Michelle; Schlade-Bartusiak, Kamilla; Platt, Frances M; Hurles, Matthew E; Crosby, Andrew H

    2013-12-01

    Glycosphingolipids are ubiquitous constituents of eukaryotic plasma membranes, and their sialylated derivatives, gangliosides, are the major class of glycoconjugates expressed by neurons. Deficiencies in their catabolic pathways give rise to a large and well-studied group of inherited disorders, the lysosomal storage diseases. Although many glycosphingolipid catabolic defects have been defined, only one proven inherited disease arising from a defect in ganglioside biosynthesis is known. This disease, because of defects in the first step of ganglioside biosynthesis (GM3 synthase), results in a severe epileptic disorder found at high frequency amongst the Old Order Amish. Here we investigated an unusual neurodegenerative phenotype, most commonly classified as a complex form of hereditary spastic paraplegia, present in families from Kuwait, Italy and the Old Order Amish. Our genetic studies identified mutations in B4GALNT1 (GM2 synthase), encoding the enzyme that catalyzes the second step in complex ganglioside biosynthesis, as the cause of this neurodegenerative phenotype. Biochemical profiling of glycosphingolipid biosynthesis confirmed a lack of GM2 in affected subjects in association with a predictable increase in levels of its precursor, GM3, a finding that will greatly facilitate diagnosis of this condition. With the description of two neurological human diseases involving defects in two sequentially acting enzymes in ganglioside biosynthesis, there is the real possibility that a previously unidentified family of ganglioside deficiency diseases exist. The study of patients and animal models of these disorders will pave the way for a greater understanding of the role gangliosides play in neuronal structure and function and provide insights into the development of effective treatment therapies.

  10. The effect of exogenous GM1 ganglioside on kindled-amygdaloid seizures.

    PubMed

    Albertson, T E; Walby, W F

    1987-01-01

    The effects of 12 daily doses of 30 mg/kg GM1 ganglioside i.p. on the acquisition of kindled-amygdaloid seizures in the rat was studied. No modification in the rate of kindling or the expression of the elicited seizures was noted during the acquisition phase. Further studies with additional fully amygdaloid kindled rats failed to show significant modification of suprathreshold or threshold elicited seizures after single 30-60 mg/kg i.p. doses of GM1 ganglioside. Despite previous studies which have shown antibodies to GM1 ganglioside to be convulsive, no anticonvulsant activity was demonstrated in this study with exogenous GM1 ganglioside using a battery of kindled-amygdaloid seizure tests in the rat.

  11. Therapeutic evaluation of GM2 gangliosidoses by ELISA using anti-GM2 ganglioside antibodies.

    PubMed

    Tsuji, Daisuke; Higashine, Yukari; Matsuoka, Kazuhiko; Sakuraba, Hitoshi; Itoh, Kohji

    2007-03-01

    GM2 gangliosidoses, including Tay-Sachs disease, Sandhoff disease and the AB variant, comprise deficiencies of beta-hexosaminidase isozymes and GM2 ganglioside activator protein associated with accumulation of GM2 ganglioside (GM2) in lysosomes and neurosomatic clinical manifestations. A simple assay system for intracellular quantification of GM2 is required to evaluate the therapeutic effects on GM2-gangliosidoses. We newly established a cell-ELISA system involving anti-GM2 monoclonal antibodies for measuring GM2 storage in fibroblasts from Tay-Sachs and Sandhoff disease patients. We succeeded in detecting the corrective effect of enzyme replacement on elimination of GM2 in the cells with this ELISA system. This simple and sensitive system should be useful as additional diagnosis tool as well as therapeutic evaluation of GM2 gangliosidoses.

  12. Altered distribution of the gangliosides GM1 and GM2 in Alzheimer's disease.

    PubMed

    Pernber, Z; Blennow, K; Bogdanovic, N; Månsson, J-E; Blomqvist, M

    2012-01-01

    Alzheimer's disease (AD) is a neurodegenerative disorder where β-amyloid tends to aggregate and form plaques. Lipid raft-associated ganglioside GM1 has been suggested to facilitate β-amyloid aggregation; furthermore, GM1 and GM2 are increased in lipid rafts isolated from cerebral cortex of AD cases. The distribution of GM1 and GM2 was studied by immunohistochemistry in the frontal and temporal cortex of AD cases. Frontotemporal dementia (FTD) was included as a contrast group. The distribution of GM1 and GM2 changes during the process of AD (n = 5) and FTD (n = 3) compared to controls (n = 5). Altered location of the GM1-positive small circular structures seems to be associated with myelin degradation. In the grey matter, the staining of GM1-positive plasma membranes might reflect neuronal loss in the AD/FTD tissue. The GM1-positive compact bundles were only visible in cells located in the AD frontal grey matter, possibly reflecting raft formation of GM1 and thus a pathological connection. Furthermore, our results suggest GM2 to be enriched within vesicles of pyramidal neurons of the AD/FTD brain. Our study supports the biochemical finding of ganglioside accumulation in cellular membranes of AD patients and shows a redistribution of these molecules. Copyright © 2012 S. Karger AG, Basel.

  13. Ganglioside-Lipid and Ganglioside-Protein Interactions Revealed by Coarse-Grained and Atomistic Molecular Dynamics Simulations.

    PubMed

    Gu, Ruo-Xu; Ingólfsson, Helgi I; de Vries, Alex H; Marrink, Siewert J; Tieleman, D Peter

    2017-04-20

    Gangliosides are glycolipids in which an oligosaccharide headgroup containing one or more sialic acids is connected to a ceramide. Gangliosides reside in the outer leaflet of the plasma membrane and play a crucial role in various physiological processes such as cell signal transduction and neuronal differentiation by modulating structures and functions of membrane proteins. Because the detailed behavior of gangliosides and protein-ganglioside interactions are poorly known, we investigated the interactions between the gangliosides GM1 and GM3 and the proteins aquaporin (AQP1) and WALP23 using equilibrium molecular dynamics simulations and potential of mean force calculations at both coarse-grained (CG) and atomistic levels. In atomistic simulations, on the basis of the GROMOS force field, ganglioside aggregation appears to be a result of the balance between hydrogen bond interactions and steric hindrance of the headgroups. GM3 clusters are slightly larger and more ordered than GM1 clusters due to the smaller headgroup of GM3. The different structures of GM1 and GM3 clusters from atomistic simulations are not observed at the CG level based on the Martini model, implying a difference in driving forces for ganglioside interactions in atomistic and CG simulations. For protein-ganglioside interactions, in the atomistic simulations, GM1 lipids bind to specific sites on the AQP1 surface, whereas they are depleted from WALP23. In the CG simulations, the ganglioside binding sites on the AQP1 surface are similar, but ganglioside aggregation and protein-ganglioside interactions are more prevalent than in the atomistic simulations. Using the polarizable Martini water model, results were closer to the atomistic simulations. Although experimental data for validation is lacking, we proposed modified Martini parameters for gangliosides to more closely mimic the sizes and structures of ganglioside clusters observed at the atomistic level.

  14. Ganglioside-Lipid and Ganglioside-Protein Interactions Revealed by Coarse-Grained and Atomistic Molecular Dynamics Simulations

    PubMed Central

    2016-01-01

    Gangliosides are glycolipids in which an oligosaccharide headgroup containing one or more sialic acids is connected to a ceramide. Gangliosides reside in the outer leaflet of the plasma membrane and play a crucial role in various physiological processes such as cell signal transduction and neuronal differentiation by modulating structures and functions of membrane proteins. Because the detailed behavior of gangliosides and protein-ganglioside interactions are poorly known, we investigated the interactions between the gangliosides GM1 and GM3 and the proteins aquaporin (AQP1) and WALP23 using equilibrium molecular dynamics simulations and potential of mean force calculations at both coarse-grained (CG) and atomistic levels. In atomistic simulations, on the basis of the GROMOS force field, ganglioside aggregation appears to be a result of the balance between hydrogen bond interactions and steric hindrance of the headgroups. GM3 clusters are slightly larger and more ordered than GM1 clusters due to the smaller headgroup of GM3. The different structures of GM1 and GM3 clusters from atomistic simulations are not observed at the CG level based on the Martini model, implying a difference in driving forces for ganglioside interactions in atomistic and CG simulations. For protein-ganglioside interactions, in the atomistic simulations, GM1 lipids bind to specific sites on the AQP1 surface, whereas they are depleted from WALP23. In the CG simulations, the ganglioside binding sites on the AQP1 surface are similar, but ganglioside aggregation and protein-ganglioside interactions are more prevalent than in the atomistic simulations. Using the polarizable Martini water model, results were closer to the atomistic simulations. Although experimental data for validation is lacking, we proposed modified Martini parameters for gangliosides to more closely mimic the sizes and structures of ganglioside clusters observed at the atomistic level. PMID:27610460

  15. Simultaneous quantification of GM1 and GM2 gangliosides by isotope dilution tandem mass spectrometry.

    PubMed

    Gu, Jianghong; Tifft, Cynthia J; Soldin, Steven J

    2008-04-01

    Gangliosides (GGs) are considered as diagnostic biomarkers and therapeutic targets and agents. The goal of this study was to develop a tandem mass spectrometry (MS/MS) method for the simultaneous measurement of both GM1 and GM2 gangliosides in human cerebrospinal fluid (CSF) samples in order to be able to determine their concentrations in patients with Tay-Sachs and Sandhoff disease and assess whether drugs or transplantation affect their concentrations. An API-4000 tandem mass spectrometer equipped with TurboIonSpray source and Shimadzu HPLC system was employed to perform the analysis using isotope dilution with deuterium labeled internal standards. To a 1.5 mL conical plastic Eppendorf centrifuge tube, 40 microL of human CSF sample was added and mixed with 400 microL of internal standard solution for deproteinization. After centrifugation, 100 microL of supernatant was injected onto a C-18 column. After a 2.5 min wash, the switching valve was activated and the analytes were eluted from the column with a water/methanol gradient into the MS/MS system. Quantification by multiple reaction-monitoring (MRM) analysis was performed in the negative mode. The within-day coefficients of variation were <3% for GM1 and <2% for GM2 and the between-day coefficients of variation were <5% for both GM1 and GM2 at all concentrations tested. Accuracy ranged between 98% and 102% for both analytes. Good linearity was also obtained within the concentration range of 10-200 ng/mL (6.5-129.3 nmol/L) for GM1 and 5-100 ng/mL (3.6-72.3 nmol/L) for GM2 (r> or =0.995). A new simple, accurate, and fast isotope dilution tandem mass spectrometry method was developed for the simultaneous quantification of GM1 and GM2 gangliosides in a small amount of human CSF. Concentrations were measured in "normal" CSF and in CSF from patients with Tay-Sachs disease.

  16. Water response to ganglioside GM1 surface remodelling.

    PubMed

    Brocca, P; Rondelli, V; Mallamace, F; Di Bari, M T; Deriu, A; Lohstroh, W; Del Favero, E; Corti, M; Cantu', L

    2017-01-01

    Gangliosides are biological glycolipids participating in rafts, structural and functional domains of cell membranes. Their headgroups are able to assume different conformations when packed on the surface of an aggregate, more lying or standing. Switching between different conformations is possible, and is a collective event. Switching can be induced, in model systems, by concentration or temperature increase, then possibly involving ganglioside-water interaction. In the present paper, the effect of GM1 ganglioside headgroup conformation on the water structuring and interactions is addressed. Depolarized Rayleigh Scattering, Raman Scattering, Quasielastic Neutron Scattering and NMR measurements were performed on GM1 ganglioside solutions, focusing on solvent properties. All used techniques agree in evidencing differences in the structure and dynamics of solvent water on different time-and-length scales in the presence of either GM1 headgroup conformations. In general, all results indicate that both the structural properties of solvent water and its interactions with the sugar headgroups of GM1 respond to surface remodelling. The extent of this modification is much higher than expected and, interestingly, ganglioside headgroups seem to turn from cosmotropes to chaotropes upon collective rearrangement from the standing- to the lying-conformation. In a biological perspective, water structure modulation could be one of the physico-chemical elements contributing to the raft strategy, both for rafts formation and persistence and for their functional aspects. In particular, the interaction with approaching bodies could be favoured or inhibited or triggered by complex-sugar-sequence conformational switch. This article is part of a Special Issue entitled "Science for Life" Guest Editor: Dr. Austen Angell, Dr. Salvatore Magazù and Dr. Federica Migliardo. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. High-throughput imaging method for direct assessment of GM1 ganglioside levels in mammalian cells.

    PubMed

    Acosta, Walter; Martin, Reid; Radin, David N; Cramer, Carole L

    2016-03-01

    GM1-gangliosidosis is an inherited autosomal recessive disorder caused by mutations in the gene GLB1, which encodes acid β-galactosidase (β-gal). The lack of activity in this lysosomal enzyme leads to accumulation of GM1 gangliosides (GM1) in cells. We have developed a high-content-imaging method to assess GM1 levels in fibroblasts that can be used to evaluate substrate reduction in treated GLB1(-/-) cells [1]. This assay allows fluorescent quantification in a multi-well system which generates unbiased and statistically significant data. Fluorescently labeled Cholera Toxin B subunit (CTXB), which specifically binds to GM1 gangliosides, was used to detect in situ GM1 levels in a fixed monolayer of fibroblasts. This sensitive, rapid, and inexpensive method facilitates in vitro drug screening in a format that allows a high number of replicates using low working volumes.

  18. Increased Expression of Simple Ganglioside Species GM2 and GM3 Detected by MALDI Imaging Mass Spectrometry in a Combined Rat Model of Aβ Toxicity and Stroke

    PubMed Central

    Caughlin, Sarah; Hepburn, Jeffrey D.; Park, Dae Hee; Jurcic, Kristina; Yeung, Ken K.-C.; Cechetto, David F.; Whitehead, Shawn N.

    2015-01-01

    The aging brain is often characterized by the presence of multiple comorbidities resulting in synergistic damaging effects in the brain as demonstrated through the interaction of Alzheimer’s disease (AD) and stroke. Gangliosides, a family of membrane lipids enriched in the central nervous system, may have a mechanistic role in mediating the brain’s response to injury as their expression is altered in a number of disease and injury states. Matrix-Assisted Laser Desorption Ionization (MALDI) Imaging Mass Spectrometry (IMS) was used to study the expression of A-series ganglioside species GD1a, GM1, GM2, and GM3 to determine alteration of their expression profiles in the presence of beta-amyloid (Aβ) toxicity in addition to ischemic injury. To model a stroke, rats received a unilateral striatal injection of endothelin-1 (ET-1) (stroke alone group). To model Aβ toxicity, rats received intracerebralventricular (icv) injections of the toxic 25-35 fragment of the Aβ peptide (Aβ alone group). To model the combination of Aβ toxicity with stroke, rats received both the unilateral ET-1 injection and the bilateral icv injections of Aβ₂₅₋₃₅ (combined Aβ/ET-1 group). By 3 d, a significant increase in the simple ganglioside species GM2 was observed in the ischemic brain region of rats who received a stroke (ET-1), with or without Aβ. By 21 d, GM2 levels only remained elevated in the combined Aβ/ET-1 group. GM3 levels however demonstrated a different pattern of expression. By 3 d GM3 was elevated in the ischemic brain region only in the combined Aβ/ET-1 group. By 21 d, GM3 was elevated in the ischemic brain region in both stroke alone and Aβ/ET-1 groups. Overall, results indicate that the accumulation of simple ganglioside species GM2 and GM3 may be indicative of a mechanism of interaction between AD and stroke. PMID:26086081

  19. Increased Expression of Simple Ganglioside Species GM2 and GM3 Detected by MALDI Imaging Mass Spectrometry in a Combined Rat Model of Aβ Toxicity and Stroke.

    PubMed

    Caughlin, Sarah; Hepburn, Jeffrey D; Park, Dae Hee; Jurcic, Kristina; Yeung, Ken K-C; Cechetto, David F; Whitehead, Shawn N

    2015-01-01

    The aging brain is often characterized by the presence of multiple comorbidities resulting in synergistic damaging effects in the brain as demonstrated through the interaction of Alzheimer's disease (AD) and stroke. Gangliosides, a family of membrane lipids enriched in the central nervous system, may have a mechanistic role in mediating the brain's response to injury as their expression is altered in a number of disease and injury states. Matrix-Assisted Laser Desorption Ionization (MALDI) Imaging Mass Spectrometry (IMS) was used to study the expression of A-series ganglioside species GD1a, GM1, GM2, and GM3 to determine alteration of their expression profiles in the presence of beta-amyloid (Aβ) toxicity in addition to ischemic injury. To model a stroke, rats received a unilateral striatal injection of endothelin-1 (ET-1) (stroke alone group). To model Aβ toxicity, rats received intracerebralventricular (i.c.v.) injections of the toxic 25-35 fragment of the Aβ peptide (Aβ alone group). To model the combination of Aβ toxicity with stroke, rats received both the unilateral ET-1 injection and the bilateral icv injections of Aβ25-35 (combined Aβ/ET-1 group). By 3 d, a significant increase in the simple ganglioside species GM2 was observed in the ischemic brain region of rats who received a stroke (ET-1), with or without Aβ. By 21 d, GM2 levels only remained elevated in the combined Aβ/ET-1 group. GM3 levels however demonstrated a different pattern of expression. By 3 d GM3 was elevated in the ischemic brain region only in the combined Aβ/ET-1 group. By 21 d, GM3 was elevated in the ischemic brain region in both stroke alone and Aβ/ET-1 groups. Overall, results indicate that the accumulation of simple ganglioside species GM2 and GM3 may be indicative of a mechanism of interaction between AD and stroke.

  20. Promotion of neuritogenesis in mouse neuroblastoma cells by ganglioside GM3: Involvement of three signal pathways

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Edwards, A.H.

    1989-01-01

    Ganglioside GM3 was extracted from human placentae and tested for neuritogenic properties towards the mouse neuroblastoma cell line Neuro-2A. GM3 (2.5 {mu}M) was found to inhibit cell growth when added exogenously to the cell culture. ({sup 3}H)Thymidine incorporation was inhibited by 49% within 6 hr. Neuritogenesis was evident within 24 hr evidenced by an increase in the number and length of neurites produced compared to control cells. An enzymatic assay for protein kinase C activity was employed to study effects of GM3 on the subcellular localization of the enzyme. Ganglioside GM3 was found to alter the subcellular localization of themore » phospholipid- and calcium-dependent protein kinase C. These results were confirmed using a binding assay employing the labeled phorbol ester ({sup 3}H)phorbol-12,13-dibutyrate. Finally, GM3-modulation of IP{sub 3} formation and cytosolic calcium in the Neuro-2A cells was investigated. GM3 did not alter the phosphoinositol metabolism as evidenced by IP{sub 3} formation in these cells. However, the addition of GM3 (16 {mu}M) to cells loaded with the photoprotein, aequorin, induced an increase in the intracellular calcium concentration within 2 min, which was sustained for 10 min. Removal of external calcium by chelation did not abrogate the response to GM3, indicating that calcium was being released from internal stores. The calcium influx was temporally correlated with the translocation of protein kinase C, providing a rationale whereby GM3 may induce the enzyme to translocate.« less

  1. Enhanced expression of unique gangliosides with GM2-determinant in human uterine cervical carcinoma-derived cell lines.

    PubMed

    Tanaka, Kyoko; Miyazawa, Masaki; Mikami, Mikio; Aoki, Daisuke; Kiguchi, Kazushige; Iwamori, Masao

    2016-10-01

    Monoclonal antibody YHD-06 generated by immunization with GM2 reacted with gangliosides with GM2-determinant, i.e., GM2, GalNAc-GM1b and GalNAc-GD1a, among which GalNAc-GD1a was characterized as an antigen of autoimmune peripheral neuropathies including Guillain-Barré syndrome. When glycolipids were examined by TLC-immunostaining with YHD-06 in seven human cervical carcinoma-derived cell lines, GM2 was found in all cell lines, amounting to 15.5 % to 57.5 % of total gangliosides. Whereas GalNAc-GD1a was present in three cell lines, amounting to 5.4-17.5 % of total gangliosides, and GalNAc-GM1b in four cell lines in amounts of less than 2 %. The elevated amounts of gangliosides with GM2 determinant were closely correlated with the relative intensities of gene expression of GalNAc transferase, this being characteristic of cervical carcinoma-derived cells. However, in tissues from patients with several histological types of cervical carcinomas, GM3 was ubiquitously expressed in amounts of more than 66 % of total gangliosides, GM2 was expressed in only five of 15 tissues, and both GalNAc-GM1b and GalNAc-GD1a were not even detected in trace amounts. Since GM1 was detected in all tissues in amounts of less than 0.06 μg/mg dried tissue, all cervical carcinoma tissues were revealed to exhibit GM2 synthesis, indicating that enhanced synthesis of gangliosides with GM2 determinant is a characteristic of cultivated cells in vitro. Similarly, although I(3)SO3-GalCer was not present in the squamous cell carcinoma (SCC) tissues, SCC-derived cells selectively expressed II(3)SO3-LacCer. Since enhanced synthesis of GM2 has been reported in SV-40 virus-transfected fibroblasts, papilloma virus might be involved in the expression of GM2 in cervical carcinoma-derived cells.

  2. High-throughput imaging method for direct assessment of GM1 ganglioside levels in mammalian cells

    PubMed Central

    Acosta, Walter; Martin, Reid; Radin, David N.; Cramer, Carole L.

    2016-01-01

    GM1-gangliosidosis is an inherited autosomal recessive disorder caused by mutations in the gene GLB1, which encodes acid β-galactosidase (β-gal). The lack of activity in this lysosomal enzyme leads to accumulation of GM1 gangliosides (GM1) in cells. We have developed a high-content-imaging method to assess GM1 levels in fibroblasts that can be used to evaluate substrate reduction in treated GLB1−/− cells [1]. This assay allows fluorescent quantification in a multi-well system which generates unbiased and statistically significant data. Fluorescently labeled Cholera Toxin B subunit (CTXB), which specifically binds to GM1 gangliosides, was used to detect in situ GM1 levels in a fixed monolayer of fibroblasts. This sensitive, rapid, and inexpensive method facilitates in vitro drug screening in a format that allows a high number of replicates using low working volumes. PMID:26958633

  3. GD3- and O-acetylated GD3-gangliosides in the GM2 synthase-deficient mouse brain and their immunohistochemical localization

    PubMed Central

    Matsuda, Junko; Vanier, Marie T.; Popa, Iuliana; Portoukalian, Jacques; Suzuki, Kunihiko

    2006-01-01

    Gangliosides in the brain of the knockout mouse deficient in the activity of β1,4 N-acetylgalactosaminyl transferase (β1,4 GalNAc-T)(GM2 synthase) consisted of nearly exclusively of GM3- and GD3-gangliosides as expected from the known substrate specificity of the enzyme and in confirmation of the initial reports from two laboratories that generated the mutant mouse experimentally. The total molar amount of gangliosides was approximately 30% higher in the mutant mouse brain than that in the wild-type brain. However, contrary to the initial reports, one-fourth of total GD3-ganglioside was O-acetylated. It reacted positively with an anti-O-acetylated GD3 monoclonal antibody and disappeared with a corresponding increase in GD3-ganglioside after mild alkaline treatment. The absence of O-acetylated GD3 in the initial reports can be explained by the saponification step included in their analytical procedures. Although quantitatively much less and identification tentative, we also detected GT3 and O-acetylated GT3. Anti-GD3 and anti-O-acetylated GD3 monoclonal antibodies gave positive reactions in the brain of mutant mouse as expected from the analytical results. Either antibody barely stained wild-type brain except for immunoreactivity of GD3 in the cerebellar Purkinje cells. The distributions of GD3 and O-acetylated GD3 in the brain of mutant mouse were similar but differential localization was noted in the cerebellar Purkinje cells and cerebral cortex. PMID:25792782

  4. In situ detection of GM1 and GM2 gangliosides using immunohistochemical and immunofluorescent techniques for auxiliary diagnosis of canine and feline gangliosidoses.

    PubMed

    Kohyama, Moeko; Yabuki, Akira; Ochiai, Kenji; Nakamoto, Yuya; Uchida, Kazuyuki; Hasegawa, Daisuke; Takahashi, Kimimasa; Kawaguchi, Hiroaki; Tsuboi, Masaya; Yamato, Osamu

    2016-03-31

    GM1 and GM2 gangliosidoses are progressive neurodegenerative lysosomal storage diseases resulting from the excessive accumulation of GM1 and GM2 gangliosides in the lysosomes, respectively. The diagnosis of gangliosidosis is carried out based on comprehensive findings using various types of specimens for histological, ultrastructural, biochemical and genetic analyses. Therefore, the partial absence or lack of specimens might have resulted in many undiagnosed cases. The aim of the present study was to establish immunohistochemical and immunofluorescent techniques for the auxiliary diagnosis of canine and feline gangliosidoses, using paraffin-embedded brain specimens stored for a long period. Using hematoxylin and eosin staining, cytoplasmic accumulation of pale to eosinophilic granular materials in swollen neurons was observed in animals previously diagnosed with GM1 or GM2 gangliosidosis. The immunohistochemical and immunofluorescent techniques developed in this study clearly demonstrated the accumulated material to be either GM1 or GM2 ganglioside. Immunohistochemical and immunofluorescent techniques using stored paraffin-embedded brain specimens are useful for the retrospective diagnosis of GM1 and GM2 gangliosidoses in dogs and cats.

  5. Association to HeLa cells and surface behavior of exogenous gangliosides studied with a fluorescent derivative of GM1

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Masserini, M.; Giuliani, A.; Palestini, P.

    1990-01-23

    Cultured HeLa cells were incubated with pyrene-GM1/{sup 3}H-radiolabeled GM1 ganglioside (1:4 M/M) mixtures for various times. The process of association of pyrene-GM1 with cells was qualitatively and quantitatively the same as that of {sup 3}H-GM1. The pyrene-GM1 and {sup 3}H-GM1 proportions in the various forms of association with cells were similar to that of the starting ganglioside mixture. After 2-h incubation, the association of ganglioside with cells was well established whereas almost no metabolic processing had occurred. During a 24-h incubation, pyrene- and {sup 3}H-GM1 underwent similar metabolic processing and gave rise to catabolic (GM2 and GM3) and anabolic (GDla)more » derivatives. Fluorescence spectroscopy experiments carried out with the excimer formation technique on subcellular fractions containing plasma membranes showed that exogenous ganglioside was, in part, associated with the cells in a micellar form removable by trypsin treatment, and in part inserted in a seemingly molecular dispersion. Addition of Ca{sup 2+} salts caused aggregation of the ganglioside, as indicated by the increase of the excimer:monomer fluorescence ratio. The phenomenon was Ca{sup 2+} concentration dependent (maximum at 10 mM), and subsequent addition of EDTA has no effect. The saccharide portion of exogenously incorporated pyrene-GM1 was available to interact with external ligands, as shown by its ability to bind cholera toxin whose addition reduced the collision rate among the ganglioside lipid moieties.« less

  6. GM2-ganglioside metabolism in hexosaminidase A deficiency states: determination in situ using labeled GM2 added to fibroblast cultures

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Raghavan, S.S.; Krusell, A.; Krusell, J.

    1985-11-01

    To clarify the relationship between hexosaminidase A (HEX A) activity and GM2-ganglioside hydrolysis in atypical clinical situations of HEX A deficiency, we have developed a simple method to assess GM2-ganglioside metabolism in cultured fibroblasts utilizing GM2 labeled with tritium in the sphingosine portion of the molecule. The radioactive lipid is added to the media of cultured skin fibroblasts, and after 10 days the cells are thoroughly washed, then harvested, and their lipid composition analyzed by HPLC. The degree of hydrolysis of the ingested GM2 is determined by comparing the amount of radioactive counts recovered in undegraded substrate with total cellularmore » radioactivity. A deficiency in GM2-ganglioside hydrolysis was demonstrated in seven HEX A-deficient adults with neurological signs and in two healthy-appearing adolescents with older affected siblings. In each case, an analysis of endogenous monosialoganglioside composition revealed an increase in GM2-ganglioside, confirming the presence of a block in the metabolism of GM2. No defect in GM2-catabolism was found in four other healthy individuals with HEX A deficiency. This method of assay is especially helpful in the evaluation of atypical cases of HEX A deficiency for the definitive diagnosis of GM2-gangliosidosis.« less

  7. In cellulo examination of a beta-alpha hybrid construct of beta-hexosaminidase A subunits, reported to interact with the GM2 activator protein and hydrolyze GM2 ganglioside.

    PubMed

    Sinici, Incilay; Yonekawa, Sayuri; Tkachyova, Ilona; Gray, Steven J; Samulski, R Jude; Wakarchuk, Warren; Mark, Brian L; Mahuran, Don J

    2013-01-01

    The hydrolysis in lysosomes of GM2 ganglioside to GM3 ganglioside requires the correct synthesis, intracellular assembly and transport of three separate gene products; i.e., the alpha and beta subunits of heterodimeric beta-hexosaminidase A, E.C. # 3.2.1.52 (encoded by the HEXA and HEXB genes, respectively), and the GM2-activator protein (GM2AP, encoded by the GM2A gene). Mutations in any one of these genes can result in one of three neurodegenerative diseases collectively known as GM2 gangliosidosis (HEXA, Tay-Sachs disease, MIM # 272800; HEXB, Sandhoff disease, MIM # 268800; and GM2A, AB-variant form, MIM # 272750). Elements of both of the hexosaminidase A subunits are needed to productively interact with the GM2 ganglioside-GM2AP complex in the lysosome. Some of these elements have been predicted from the crystal structures of hexosaminidase and the activator. Recently a hybrid of the two subunits has been constructed and reported to be capable of forming homodimers that can perform this reaction in vivo, which could greatly simplify vector-mediated gene transfer approaches for Tay-Sachs or Sandhoff diseases. A cDNA encoding a hybrid hexosaminidase subunit capable of dimerizing and hydrolyzing GM2 ganglioside could be incorporated into a single vector, whereas packaging both subunits of hexosaminidase A into vectors, such as adeno-associated virus, would be impractical due to size constraints. In this report we examine the previously published hybrid construct (H1) and a new more extensive hybrid (H2), with our documented in cellulo (live cell- based) assay utilizing a fluorescent GM2 ganglioside derivative. Unfortunately when Tay-Sachs cells were transfected with either the H1 or H2 hybrid construct and then were fed the GM2 derivative, no significant increase in its turnover was detected. In vitro assays with the isolated H1 or H2 homodimers confirmed that neither was capable of human GM2AP-dependent hydrolysis of GM2 ganglioside.

  8. In Cellulo Examination of a Beta-Alpha Hybrid Construct of Beta-Hexosaminidase A Subunits, Reported to Interact with the GM2 Activator Protein and Hydrolyze GM2 Ganglioside

    PubMed Central

    Sinici, Incilay; Yonekawa, Sayuri; Tkachyova, Ilona; Gray, Steven J.; Samulski, R. Jude; Wakarchuk, Warren; Mark, Brian L.; Mahuran, Don J.

    2013-01-01

    The hydrolysis in lysosomes of GM2 ganglioside to GM3 ganglioside requires the correct synthesis, intracellular assembly and transport of three separate gene products; i.e., the alpha and beta subunits of heterodimeric beta-hexosaminidase A, E.C. # 3.2.1.52 (encoded by the HEXA and HEXB genes, respectively), and the GM2-activator protein (GM2AP, encoded by the GM2A gene). Mutations in any one of these genes can result in one of three neurodegenerative diseases collectively known as GM2 gangliosidosis (HEXA, Tay-Sachs disease, MIM # 272800; HEXB, Sandhoff disease, MIM # 268800; and GM2A, AB-variant form, MIM # 272750). Elements of both of the hexosaminidase A subunits are needed to productively interact with the GM2 ganglioside-GM2AP complex in the lysosome. Some of these elements have been predicted from the crystal structures of hexosaminidase and the activator. Recently a hybrid of the two subunits has been constructed and reported to be capable of forming homodimers that can perform this reaction in vivo, which could greatly simplify vector-mediated gene transfer approaches for Tay-Sachs or Sandhoff diseases. A cDNA encoding a hybrid hexosaminidase subunit capable of dimerizing and hydrolyzing GM2 ganglioside could be incorporated into a single vector, whereas packaging both subunits of hexosaminidase A into vectors, such as adeno-associated virus, would be impractical due to size constraints. In this report we examine the previously published hybrid construct (H1) and a new more extensive hybrid (H2), with our documented in cellulo (live cell- based) assay utilizing a fluorescent GM2 ganglioside derivative. Unfortunately when Tay-Sachs cells were transfected with either the H1 or H2 hybrid construct and then were fed the GM2 derivative, no significant increase in its turnover was detected. In vitro assays with the isolated H1 or H2 homodimers confirmed that neither was capable of human GM2AP-dependent hydrolysis of GM2 ganglioside. PMID:23483939

  9. The biologic role of ganglioside in neuronal differentiation--effects of GM1 ganglioside on human neuroblastoma SH-SY5Y cells.

    PubMed Central

    Lee, M. C.; Lee, W. S.; Park, C. S.; Juhng, S. W.

    1994-01-01

    Human neuroblastoma SH-SY5Y cell is a cloned cell line which has many attractive features for the study of neuronal proliferation and neurite outgrowth, because it has receptors for insulin, IGF-I and PDGF. Gangliosides are sialic acid containing glycosphingolipids which form an integral part of the plasma membrane of many mammalian cells. They inhibit cell growth mediated by tyrosine kinase receptors and ligand-stimulated tyrosine kinase activity, and autophosphorylation of EGF(epidermal growth factor) and PDGF receptors. The experiment was designed to study the effects of GM1 ganglioside on growth of human neuroblastoma SH-SY5Y cells stimulated with trophic factor in vitro. The cells were plated in Eagle's minimum essential medium without serum. The number and morphologic change of SH-SY5Y cells were evaluated in the serum free medium added GM1 ganglioside with insulin or PDGF. SH-SY5Y cells were maintained for six days in serum-free medium, and then cultured for over two weeks in serum-free medium containing either insulin or PDGF. The effect of insulin on cell proliferation developed earlier and was more potent than that of PDGF. These proliferative effects were inhibited by GM1 ganglioside, and the cells showed prominent neurites outgrowth. These findings suggest that GM1 ganglioside inhibits the cell proliferation mediated by tyrosine kinase receptors and directly induces neuritogenesis as one of the neurotrophic factors. PMID:7986393

  10. Endosomal/Lysosomal Processing of Gangliosides Affects Neuronal Cholesterol Sequestration in Niemann-Pick Disease Type C

    PubMed Central

    Zhou, Sharon; Davidson, Cristin; McGlynn, Robert; Stephney, Gloria; Dobrenis, Kostantin; Vanier, Marie T.; Walkley, Steven U.

    2011-01-01

    Niemann-Pick disease type C (NPC) is a severe neurovisceral lysosomal storage disorder caused by defects in NPC1 or NPC2 proteins. Although numerous studies support the primacy of cholesterol storage, neurons of double-mutant mice lacking both NPC1 and an enzyme required for synthesis of all complex gangliosides (β1,4GalNAc transferase) have been reported to exhibit dramatically reduced cholesterol sequestration. Here we show that NPC2-deficient mice lacking this enzyme also exhibit reduced cholesterol, but that genetically restricting synthesis to only a-series gangliosides fully restores neuronal cholesterol storage to typical disease levels. Examining the subcellular locations of sequestered compounds in neurons lacking NPC1 or NPC2 by confocal microscopy revealed that cholesterol and the two principal storage gangliosides (GM2 and GM3) were not consistently co-localized within the same intracellular vesicles. To determine whether the lack of GM2 and GM3 co-localization was due to differences in synthetic versus degradative pathway expression, we generated mice lacking both NPC1 and lysosomal β-galactosidase, and therefore unable to generate GM2 and GM3 in lysosomes. Double mutants lacked both gangliosides, indicating that each is the product of endosomal/lysosomal processing. Unexpectedly, GM1 accumulation in double mutants increased compared to single mutants consistent with a direct role for NPC1 in ganglioside salvage. These studies provide further evidence that NPC1 and NPC2 proteins participate in endosomal/lysosomal processing of both sphingolipids and cholesterol. PMID:21708114

  11. Selective Intracellular Delivery of Ganglioside GM3-Binding Peptide through Caveolae/Raft-Mediated Endocytosis.

    PubMed

    Matsubara, Teruhiko; Otani, Ryohei; Yamashita, Miki; Maeno, Haruka; Nodono, Hanae; Sato, Toshinori

    2017-02-13

    Glycosphingolipids are major components of the membrane raft, and several kinds of viruses and bacterial toxins are known to bind to glycosphingolipids in the membrane raft. Since the viral genes and pathogenic proteins that are taken into cells are directly delivered to their target organelles, caveolae/raft-mediated endocytosis represents a promising pathway for specific delivery. In the present study, we demonstrated the ability of an artificial pentadecapeptide, which binds to ganglioside GM3, to deliver protein into cells by caveolae/raft-mediated endocytosis. The cellular uptake of a biotinylated GM3-binding peptide (GM3BP)-avidin complex into HeLa cells was observed, and the cellular uptake of this complex was inhibited by an incubation with sialic acid or endocytic inhibitors such as methyl-ß-cyclodextrin, and also by an incubation at 4 °C. These results indicate that the GM3BP-avidin complex bind to GM3 in membrane raft, and are taken into cell through caveolae/raft-mediated endocytosis. The GM3BP-avidin complex was transported into cells and localized around the nucleus more slowly than a human immunodeficiency virus type 1 TAT peptide. Furthermore, the uptake of a green fluorescent protein (GFP) linked with GM3BP into HeLa cells was similar to that of the GM3BP-avidin complex, and the localization of the GM3BP-GFP fusion protein was markedly different with that of the TAT-GFP fusion protein. The uptake and trafficking of GM3BP were distinguished from conventional cell-penetrating peptides. GM3BP has potential as a novel peptide for the selective delivery of therapeutic proteins and materials into cells in addition to being a cell-penetrating peptide.

  12. Mice lacking major brain gangliosides develop parkinsonism.

    PubMed

    Wu, Gusheng; Lu, Zi-Hua; Kulkarni, Neil; Amin, Ruchi; Ledeen, Robert W

    2011-09-01

    Parkinson's disease (PD) is the second most prevalent late-onset neurodegenerative disorder that affects nearly 1% of the global population aged 65 and older. Whereas palliative treatments are in use, the goal of blocking progression of motor and cognitive disability remains unfulfilled. A better understanding of the basic pathophysiological mechanisms underlying PD would help to advance that goal. The present study provides evidence that brain ganglioside abnormality, in particular GM1, may be involved. This is based on use of the genetically altered mice with disrupted gene Galgt1 for GM2/GD2 synthase which depletes GM2/GD2 and all the gangliotetraose gangliosides that constitute the major molecular species of brain. These knockout mice show overt motor disability on aging and clear indications of motor impairment with appropriate testing at an earlier age. This disability was rectified by L-dopa administration. These mice show other characteristic symptoms of PD, including depletion of striatal dopamine (DA), loss of DA neurons of the substantia nigra pars compacta, and aggregation of alpha synuclein. These manifestations of parkinsonism were largely attenuated by administration of LIGA-20, a membrane permeable analog of GM1 that penetrates the blood brain barrier and enters living neurons. These results suggest that perturbation of intracellular mechanisms mediated by intracellular GM1 may be a contributing factor to PD.

  13. NEU3 inhibitory effect of naringin suppresses cancer cell growth by attenuation of EGFR signaling through GM3 ganglioside accumulation.

    PubMed

    Yoshinaga, Ayana; Kajiya, Natsuki; Oishi, Kazuki; Kamada, Yuko; Ikeda, Asami; Chigwechokha, Petros Kingstone; Kibe, Toshiro; Kishida, Michiko; Kishida, Shosei; Komatsu, Masaharu; Shiozaki, Kazuhiro

    2016-07-05

    Naringin, which is one of the flavonoids contained in citrus fruits, is well known to possess various healthy functions to humans. It has been reported that naringin suppresses cancer cell growth in vitro and in vivo, although the underlying mechanisms are not fully understood. Recently, the roles of glycoconjugates, such as gangliosides, in cancer cells have been focused because of their regulatory effects of malignant phenotypes. Here, to clarify the roles of naringin in the negative-regulation of cancer cell growth, the alteration of glycoconjugates induced by naringin exposure and its significance on cell signaling were investigated. Human cancer cells, HeLa and A549, were exposed to various concentrations of naringin. Naringin treatment induced the suppression of cell growth toward HeLa and A549 cells accompanied with an increase of apoptotic cells. In naringin-exposed cells, GM3 ganglioside was drastically increased compared to the GM3 content prior to the treatment. Furthermore, naringin inhibited NEU3 sialidase, a GM3 degrading glycosidase. Similarly, NEU3 inhibition activities were also detected by other flavanone, such as hesperidin and neohesperidin dihydrocalcone, but their aglycones showed less inhibitions. Naringin-treated cancer cells showed suppressed EGFR and ERK phosphorylation levels. These results suggest a novel mechanism of naringin in the suppression of cancer cell growth through the alteration of glycolipids. NEU3 inhibitory effect of naringin induced GM3 accumulation in HeLa and A549 cells, leading the attenuation of EGFR/ERK signaling accompanied with a decrease in cell growth. Copyright © 2016 Elsevier B.V. All rights reserved.

  14. Elevation of GM2 ganglioside during ethanol-induced apoptotic neurodegeneration in the developing mouse brain

    PubMed Central

    Saito, Mitsuo; Chakraborty, Goutam; Shah, Relish; Mao, Rui-Fen; Kumar, Asok; Yang, Dun-Sheng; Dobrenis, Kostantin; Saito, Mariko

    2012-01-01

    GM2 ganglioside in the brain increased during ethanol-induced acute apoptotic neurodegeneration in 7-day-old mice. A small but a significant increase observed 2 h after ethanol exposure was followed by a marked increase around 24 h. Subcellular fractionation of the brain 24 h after ethanol treatment indicated that GM2 increased in synaptic and non-synaptic mitochondrial fractions as well as in a lysosome-enriched fraction characteristic to the ethanol-exposed brain. Immunohistochemical staining of GM2 in the ethanol-treated brain showed strong punctate staining mainly in activated microglia, in which it partially overlapped with staining for LAMP1, a late endosomal/lysosomal marker. Also, there was weaker neuronal staining, which partially co-localized with complex IV, a mitochondrial marker, and was augmented in cleaved caspase-3-positive neurons. In contrast, the control brain showed only faint and diffuse GM2 staining in neurons. Incubation of isolated brain mitochondria with GM2 in vitro induced cytochrome c release in a manner similar to that of GD3 ganglioside. Because ethanol is known to trigger mitochondria-mediated apoptosis with cytochrome c release and caspase-3 activation in the 7-day–old mouse brain, the GM2 elevation in mitochondria may be relevant to neuroapoptosis. Subsequently, activated microglia accumulated GM2, indicating a close relationship between GM2 and ethanol-induced neurodegeneration. PMID:22372857

  15. Ganglioside GM2/GM3 complex affixed on silica nanospheres strongly inhibits cell motility through CD82/cMet-mediated pathway

    PubMed Central

    Todeschini, Adriane Regina; Dos Santos, Jose Nilson; Handa, Kazuko; Hakomori, Sen-itiroh

    2008-01-01

    Ganglioside GM2 complexed with tetraspanin CD82 in glycosynaptic microdomain of HCV29 and other epithelial cells inhibits hepatocyte growth factor-induced cMet tyrosine kinase. In addition, adhesion of HCV29 cells to extracellular matrix proteins also activates cMet kinase through “cross-talk” of integrins with cMet, leading to inhibition of cell motility and growth. Present studies indicate that cell motility and growth are greatly influenced by expression of GM2, GM3, or GM2/GM3 complexes, which affect cMet kinase activity of various types of cells, based on the following series of observations: (i) Cells expressing CD82, cultured with GM2 and GM3 cocoated on silica nanospheres, displayed stronger and more consistent motility inhibition than those cultured with GM2 or GM3 alone or with other glycosphingolipids. (ii) GM2-GM3, in the presence of Ca2+ form a heterodimer, as evidenced by electrospray ionization (ESI) mass spectrometry and by specific reactivity with mAb 8E11, directed to GM2/GM3 dimer structure. (iii) Cells expressing cMet and CD82 were characterized by enhanced motility associated with HGF-induced cMet activation. Both cMet and motility were strongly inhibited by culturing cells with GM2/GM3 dimer coated on nanospheres. (iv) Adhesion of HCV29 or YTS-1/CD82 cells to laminin-5-coated plate activated cMet kinase in the absence of HGF, whereas GM2/GM3 dimer inhibited adhesion-induced cMet kinase activity and inhibited cell motility. (v) Inhibited cell motility as in i, iii, and iv was restored to normal level by addition of mAb 8E11, which blocks interaction of GM2/GM3 dimer with CD82. Signaling through Src and MAP kinases is activated or inhibited in close association with cMet kinase, in response to GM2/GM3 dimer interaction with CD82. Thus, a previously uncharacterized GM2/GM3 heterodimer complexed with CD82 inhibits cell motility through CD82-cMet or integrin-cMet pathway. PMID:18272501

  16. Ganglioside GM2/GM3 complex affixed on silica nanospheres strongly inhibits cell motility through CD82/cMet-mediated pathway.

    PubMed

    Todeschini, Adriane Regina; Dos Santos, Jose Nilson; Handa, Kazuko; Hakomori, Sen-itiroh

    2008-02-12

    Ganglioside GM2 complexed with tetraspanin CD82 in glycosynaptic microdomain of HCV29 and other epithelial cells inhibits hepatocyte growth factor-induced cMet tyrosine kinase. In addition, adhesion of HCV29 cells to extracellular matrix proteins also activates cMet kinase through "cross-talk" of integrins with cMet, leading to inhibition of cell motility and growth. Present studies indicate that cell motility and growth are greatly influenced by expression of GM2, GM3, or GM2/GM3 complexes, which affect cMet kinase activity of various types of cells, based on the following series of observations: (i) Cells expressing CD82, cultured with GM2 and GM3 cocoated on silica nanospheres, displayed stronger and more consistent motility inhibition than those cultured with GM2 or GM3 alone or with other glycosphingolipids. (ii) GM2-GM3, in the presence of Ca2+ form a heterodimer, as evidenced by electrospray ionization (ESI) mass spectrometry and by specific reactivity with mAb 8E11, directed to GM2/GM3 dimer structure. (iii) Cells expressing cMet and CD82 were characterized by enhanced motility associated with HGF-induced cMet activation. Both cMet and motility were strongly inhibited by culturing cells with GM2/GM3 dimer coated on nanospheres. (iv) Adhesion of HCV29 or YTS-1/CD82 cells to laminin-5-coated plate activated cMet kinase in the absence of HGF, whereas GM2/GM3 dimer inhibited adhesion-induced cMet kinase activity and inhibited cell motility. (v) Inhibited cell motility as in i, iii, and iv was restored to normal level by addition of mAb 8E11, which blocks interaction of GM2/GM3 dimer with CD82. Signaling through Src and MAP kinases is activated or inhibited in close association with cMet kinase, in response to GM2/GM3 dimer interaction with CD82. Thus, a previously uncharacterized GM2/GM3 heterodimer complexed with CD82 inhibits cell motility through CD82-cMet or integrin-cMet pathway.

  17. A photoreactive derivative of radiolabeled GM1 ganglioside: Preparation and use to establish the involvement of specific proteins in GM1 uptake by human fibroblasts in culture

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Sonnino, S.; Chigorno, V.; Acquotti, D.

    1989-01-10

    A new procedure was used to synthesize a derivative of ganglioside GM1 containing a photoreactive nitrophenyl azide group at the end of the fatty acyl moiety, using deAc-deAcyl-GM1 obtained by deacetylation of the sialic acid and deacylation of the ceramide portion of GM1. This deAc-deAcyl-GM1 was first acylated at the long chain base amino group with 12-aminododecanoic acid, which has the amino group protected by a fluorenyl residue, and tritium labeled at the sialic acid amino group with ({sup 3}H)acetic anhydride of very high specific radioactivity. Cultured human fibroblasts were exposed to mixtures of radioactive photolabeled GM1 for different timesmore » and then illuminated and the radioactive protein patterns studied by SDS-PAGE. After 2 h of exposure, the photolabeled GM1 was stably associated to the cells and underwent almost no metabolic processing, behaving exactly as the underivatized natural GM1. Under these conditions very few proteins became radioactive. Thus, it is evident that the ganglioside binding to fibroblasts and insertion into the outer layer of the plasma membrane involve few individual proteins. When the incubation was prolonged to 24 h, photolabeled GM1 underwent extensive metabolic processing and gave origins to the corresponding ganglioside derivatives of GM2, GM3, and GD1a. Under these conditions many proteins became radioactive, a consequence of GM1 transfer from the surface to the interior or the cell and of the ready availability of interaction of GM1 and its metabolites.« less

  18. Liquid chromatography/electrospray ionisation-tandem mass spectrometry quantification of GM2 gangliosides in human peripheral cells and plasma.

    PubMed

    Fuller, Maria; Duplock, Stephen; Hein, Leanne K; Rigat, Brigitte A; Mahuran, Don J

    2014-08-01

    GM2 gangliosidosis is a group of inherited neurodegenerative disorders resulting primarily from the excessive accumulation of GM2 gangliosides (GM2) in neuronal cells. As biomarkers for categorising patients and monitoring the effectiveness of developing therapies are lacking for this group of disorders, we sought to develop methodology to quantify GM2 levels in more readily attainable patient samples such as plasma, leukocytes, and cultured skin fibroblasts. Following organic extraction, gangliosides were partitioned into the aqueous phase and isolated using C18 solid-phase extraction columns. Relative quantification of three species of GM2 was achieved using LC/ESI-MS/MS with d35GM1 18:1/18:0 as an internal standard. The assay was linear over the biological range, and all GM2 gangliosidosis patients were demarcated from controls by elevated GM2 in cultured skin fibroblast extracts. However, in leukocytes only some molecular species could be used for differentiation and in plasma only one was informative. A reduction in GM2 was easily detected in patient skin fibroblasts after a short treatment with media from normal cells enriched in secreted β-hexosaminidase. This method may show promise for measuring the effectiveness of experimental therapies for GM2 gangliosidosis by allowing quantification of a reduction in the primary storage burden. Copyright © 2014 Elsevier Inc. All rights reserved.

  19. Development of an Immunoassay for Rapid Detection of Ganglioside GM1 Mimicry in Campylobacter jejuni Strains

    PubMed Central

    Prendergast, Martina M.; Kosunen, Timo U.; Moran, Anthony P.

    2001-01-01

    Mimicry of peripheral nerve gangliosides by Campylobacter jejuni lipopolysaccharides (LPSs) has been proposed to induce cross-reacting antiganglioside antibodies in Guillain-Barré syndrome (GBS). Because current methods for LPS characterization are labor-intensive and inhibit the screening of large numbers of strains, a rapid GM1 epitope screening assay was developed. Biomass from two agar plates of confluent growth yielded sufficient LPS using a novel phenol-water and ether extraction procedure. Extracts of LPS were reacted with cholera toxin (GM1 ligand), peanut agglutinin (Galβ1→3GalNAc ligand), and anti-GM1 antibodies. After the assay was validated, 12 of 59 (20%) C. jejuni serostrains, including four serotypes that have not previously been associated with GBS, reacted with two or more anti-GM1 ganglioside reagents. Subsequently, LPS extracts from 5 of 7 (71%) C. jejuni isolates and 2 of 3 (67%) C. jejuni culture collection strains bore GM1 structures. Overall, the assay system was reliable, efficient, and reproducible and may be adapted for large-scale epidemiological studies. PMID:11283076

  20. Elevation of GM2 ganglioside during ethanol-induced apoptotic neurodegeneration in the developing mouse brain.

    PubMed

    Saito, Mitsuo; Chakraborty, Goutam; Shah, Relish; Mao, Rui-Fen; Kumar, Asok; Yang, Dun-Sheng; Dobrenis, Kostantin; Saito, Mariko

    2012-05-01

    GM2 ganglioside in the brain increased during ethanol-induced acute apoptotic neurodegeneration in 7-day-old mice. A small but a significant increase observed 2 h after ethanol exposure was followed by a marked increase around 24 h. Subcellular fractionation of the brain 24 h after ethanol treatment indicated that GM2 increased in synaptic and non-synaptic mitochondrial fractions as well as in a lysosome-enriched fraction characteristic to the ethanol-exposed brain. Immunohistochemical staining of GM2 in the ethanol-treated brain showed strong punctate staining mainly in activated microglia, in which it partially overlapped with staining for LAMP1, a late endosomal/lysosomal marker. Also, there was weaker neuronal staining, which partially co-localized with complex IV, a mitochondrial marker, and was augmented in cleaved caspase 3-positive neurons. In contrast, the control brain showed only faint and diffuse GM2 staining in neurons. Incubation of isolated brain mitochondria with GM2 in vitro induced cytochrome c release in a manner similar to that of GD3 ganglioside. Because ethanol is known to trigger mitochondria-mediated apoptosis with cytochrome c release and caspase 3 activation in the 7-day-old mouse brain, the GM2 elevation in mitochondria may be relevant to neuroapoptosis. Subsequently, activated microglia accumulated GM2, indicating a close relationship between GM2 and ethanol-induced neurodegeneration. © 2012 The Authors. Journal of Neurochemistry © 2012 International Society for Neurochemistry.

  1. Synthesis of gangliosides by cultured oligodendrocytes

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Mack, S.R.; Szuchet, S.; Dawson, G.

    1981-01-01

    Gangliosides are enriched in the nervous system compared to other tissues. The synthesis of gangliosides by monolayer cultures of isolated oligodendrocytes has not previously been investigated. Cells were labeled with (3H) galactose at preselected times and gangliosides isolated by phase partition, purified, and identified by chromatography. Cultured oligodendrocytes showed selectivity in their synthesis of gangliosides, which was expressed in the type of ganglioside synthesized as well as in the change of incorporation over time in culture. For the first ten days, there was very little incorporation of (3H) galactose in gangliosides, but this was followed by a stimulation of uptakemore » for GM3, GM1/GD3, and GD1 gangliosides, reaching a maximum after approximately 25-30 days in vitro. There was little incorporation into GM2 or trisialogangliosides throughout the life of the cultures. Since oligodendrocytes synthesize extensive membranes during this period, one may speculate that the de novo-synthesized gangliosides are used for membranes.« less

  2. IgM ganglioside GM1 antibodies in patients with autoimmune disease or neuropathy, and controls.

    PubMed Central

    Bansal, A S; Abdul-Karim, B; Malik, R A; Goulding, P; Pumphrey, R S; Boulton, A J; Holt, P L; Wilson, P B

    1994-01-01

    AIMS--To compare the titre of anti-ganglioside antibodies (AGA) to GM1 ganglioside in patients with central and peripheral neurological disease and pure motor and sensorimotor neuropathy, in patients with classic autoimmune diseases, and controls. METHODS--AGA to GM1 were measured using an enzyme linked immunosorbent assay (ELISA) technique, highly purified bovine GM1 ganglioside, and sequential dilution of control and test sera. Antibody titre was calculated using the optical density readings of three consecutive serum dilutions multiplied by the dilution factor. RESULTS--A considerable overlap was evident in the titre of AGA to GM1 in control and test sera. High antibody titres were most frequent in patients with multifocal motor neuropathy with conduction block (MMNCB). Low AGA titre were observed in several patient groups. Compared with the controls, the median titre of AGA to GM1 was significantly higher in patients with multiple sclerosis, rheumatoid arthritis, primary Sjögren's syndrome and systemic lupus erythematosus. In contrast, the median titre in patients with diabetic peripheral neuropathy, motor neurone disease, sensorimotor neuropathy and chronic inflammatory demyelinating polyneuropathy was no different from that in normal control subjects. CONCLUSIONS--Estimation of AGA to GM1 may be helpful in the diagnosis of MMNCB in patients with a pure motor neuropathy but in few other conditions. Low titre AGA to GM1 are evident in several autoimmune conditions. The pathogenetic importance of AGA to GM1 in patients with neuropathy is not clear. PMID:8027366

  3. Studies on the biosynthesis and intracellular transport of gangliosides

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Farrer, R.G.

    1987-01-01

    Ganglioside biosynthesis and transport to myelin was studied in brainstem of 17-21 day old rats. Brainstem slices were incubated for up to 2 hours with (/sup 3/H)glucosamine, and gangliosides were isolated by column chromatography and HPTLC. Results from these experiments showed that: (a) ganglioside synthesis was decreased in the slices compared to in vivo, and this decrease was greater in the more complex gangliosides than in the simpler ones; (b) label incorporation into gangliosides GM3 and GM2 increased in a linear fashion, whereas the rate of incorporation continuously increased over the 2 hour period for the more complex gangliosides; (c)more » label incorporated into gangliosides, which showed almost no effect of chase after 30 minutes; (d) monensin at 0.1 uM inhibited the synthesis of all gangliosides except GM3, GM2 and GD3. Compartmentation of ganglioside biosynthesis was examined by analyzing the subcellular location of two ganglioside synthesizing enzymes, lactosylceramide sialosyltransferase (LCST) and GDlb sialosyltransferase (GDlbST), acting early and late in the ganglioside pathway, respectively.« less

  4. Membrane lipids regulate ganglioside GM2 catabolism and GM2 activator protein activity.

    PubMed

    Anheuser, Susi; Breiden, Bernadette; Schwarzmann, Günter; Sandhoff, Konrad

    2015-09-01

    Ganglioside GM2 is the major lysosomal storage compound of Tay-Sachs disease. It also accumulates in Niemann-Pick disease types A and B with primary storage of SM and with cholesterol in type C. Reconstitution of GM2 catabolism with β-hexosaminidase A and GM2 activator protein (GM2AP) at uncharged liposomal surfaces carrying GM2 as substrate generated only a physiologically irrelevant catabolic rate, even at pH 4.2. However, incorporation of anionic phospholipids into the GM2 carrying liposomes stimulated GM2 hydrolysis more than 10-fold, while the incorporation of plasma membrane stabilizing lipids (SM and cholesterol) generated a strong inhibition of GM2 hydrolysis, even in the presence of anionic phospholipids. Mobilization of membrane lipids by GM2AP was also inhibited in the presence of cholesterol or SM, as revealed by surface plasmon resonance studies. These lipids also reduced the interliposomal transfer rate of 2-NBD-GM1 by GM2AP, as observed in assays using Förster resonance energy transfer. Our data raise major concerns about the usage of recombinant His-tagged GM2AP compared with untagged protein. The former binds more strongly to anionic GM2-carrying liposomal surfaces, increases GM2 hydrolysis, and accelerates intermembrane transfer of 2-NBD-GM1, but does not mobilize membrane lipids. Copyright © 2015 by the American Society for Biochemistry and Molecular Biology, Inc.

  5. Approaches in the study of ganglioside metabolism

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Tettamanti, G.; Ghidoni, R.; Sonnino, S.

    1984-01-01

    Ganglioside GM1, /sup 3/H-labeled in the sphingosine or terminal galactose moiety was injected into mice and its metabolic fate in the liver was followed. After administration of sphingosine-labeled GM1 all major liver gangliosides (GM3, GM2, GM1, GD1a-NeuAc, NeuG1) became radioactive, the radioactivity residing in all cases on the sphingosine moiety. The specific radioactivity was highest on GM1, followed by GM2, GM3 and GD1a-NeuAc, NeuG1. Several neutral glycosphingolipids and sphingomyelin were also formed. After administration of galactose-labelled GM1 the only radioactive gangliosides present in the liver were GM1 and GD1a-NeuAc, NeuG1, both carrying the radioactivity on the terminal galactose residue, withmore » no formation of labelled neutral glycosphingolipids. Subcellular studies gave clear evidence that GM1, after being taken up by the liver, was mainly degraded to GM2, GM3 and neutral glycosphingolipids at the level of lysosomes. A part of it was sialylated to more complex gangliosides and some of its metabolic by-products were used for the biosynthesis of other sphingolipid species, likely at the level of the Golgi apparatus. All this suggests that exogenous GM1 is introduced in the metabolic routes of endogenous gangliosides and of other sphingolipids, which are operating in the liver.« less

  6. The Ganglioside GM-1 Inhibits Bupivacaine-Induced Neurotoxicity in Mouse Neuroblastoma Neuro2a Cells.

    PubMed

    Liang, Yujie; Ji, Jiemei; Lin, Yunan; He, Yajun; Liu, Jingchen

    2016-08-01

    Studies indicate that bupivacaine-induced neurotoxicity results from apoptosis. Gangliosides have been shown to promote neuronal repair and recovery of neurological function after spinal cord injury. Previously, we confirmed that in vivo administration of the ganglioside GM-1 attenuated bupivacaine-induced neurotoxicity in various animal models; however, the underlying mechanism remains unclear. Cells of the neuroblastoma line N2a (Neuro2a cells) were divided into three experimental groups: control, bupivacaine-treated, and bupivacaine-treated with GM-1 pretreatment. Cell viability and apoptosis were assessed through CCK-8 assays, Hoechst staining, and flow cytometry analysis of Annexin-V/propidium iodide double labeling. Real-time polymerase chain reaction and western blotting assessed the expression of caspase-3, caspase-8, and caspase-9. Bupivacaine-induced apoptosis worsened with increasing dose and exposure time. Bupivacaine induced increased expression of caspase-3 and caspase-9, but not caspase-8, indicating that the mitochondrial pathway but not the death receptor apoptosis pathway was activated. GM-1 pretreatment inhibited bupivacaine-induced apoptosis and the expression of caspase-3 and caspase-9 in a dose-dependent manner. Bupivacaine induced neurotoxicity by activating apoptosis via the mitochondrial pathway, and this was inhibited by GM-1 pretreatment. Copyright © 2016 John Wiley & Sons, Ltd.

  7. Membrane lipids regulate ganglioside GM2 catabolism and GM2 activator protein activity[S

    PubMed Central

    Anheuser, Susi; Breiden, Bernadette; Schwarzmann, Günter; Sandhoff, Konrad

    2015-01-01

    Ganglioside GM2 is the major lysosomal storage compound of Tay-Sachs disease. It also accumulates in Niemann-Pick disease types A and B with primary storage of SM and with cholesterol in type C. Reconstitution of GM2 catabolism with β-hexosaminidase A and GM2 activator protein (GM2AP) at uncharged liposomal surfaces carrying GM2 as substrate generated only a physiologically irrelevant catabolic rate, even at pH 4.2. However, incorporation of anionic phospholipids into the GM2 carrying liposomes stimulated GM2 hydrolysis more than 10-fold, while the incorporation of plasma membrane stabilizing lipids (SM and cholesterol) generated a strong inhibition of GM2 hydrolysis, even in the presence of anionic phospholipids. Mobilization of membrane lipids by GM2AP was also inhibited in the presence of cholesterol or SM, as revealed by surface plasmon resonance studies. These lipids also reduced the interliposomal transfer rate of 2-NBD-GM1 by GM2AP, as observed in assays using Förster resonance energy transfer. Our data raise major concerns about the usage of recombinant His-tagged GM2AP compared with untagged protein. The former binds more strongly to anionic GM2-carrying liposomal surfaces, increases GM2 hydrolysis, and accelerates intermembrane transfer of 2-NBD-GM1, but does not mobilize membrane lipids. PMID:26175473

  8. Human monoclonal IgM with autoantibody activity against two gangliosides (GM1 and GD1b) in a patient with motor neuron syndrome.

    PubMed Central

    Jauberteau, M O; Gualde, N; Preud'Homme, J L; Rigaud, M; Gil, R; Vallat, J M; Baumann, N

    1990-01-01

    Small amounts of oligoclonal immunoglobulins were detected by Western blotting in the serum from a patient with motor neuron syndrome. The prominent one, a monoclonal IgM lambda, reacted strongly with the gangliosides GM1 and GD1b and more weakly with asialo GM1, as shown by immunoenzymatic staining of thin-layer chromatograms of gangliosides, ELISA on purified glycolipid coats and immunoadsorption with purified GM1. Affinity-chromatography with purified GM1 resulted in the purification of monoclonal IgM lambda. This purified IgM and its Fab fragments showed the same pattern of reactivity with gangliosides as that observed with whole serum. Such monoclonal IgM could be responsible for motor neuron diseases in some patients with overt or barely detectable monoclonal gammopathies. Images Fig. 2 Fig. 3 PMID:2357844

  9. Influence of Ganglioside GM1 Concentration on Lipid Clustering and Membrane Properties and Curvature.

    PubMed

    Patel, Dhilon S; Park, Soohyung; Wu, Emilia L; Yeom, Min Sun; Widmalm, Göran; Klauda, Jeffery B; Im, Wonpil

    2016-11-01

    Gangliosides are a class of glycosphingolipids (GSLs) with amphiphilic character that are found at the outer leaflet of the cell membranes, where their ability to organize into special domains makes them vital cell membrane components. However, a molecular understanding of GSL-rich membranes in terms of their clustered organization, stability, and dynamics is still elusive. To gain molecular insight into the organization and dynamics of GSL-rich membranes, we performed all-atom molecular-dynamics simulations of bicomponent ganglioside GM1 in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) phospholipid bilayers with varying concentrations of GM1 (10%, 20%, and 30%). Overall, the simulations show very good agreement with available experimental data, including x-ray electron density profiles along the membrane normal, NMR carbohydrate proton-proton distances, and x-ray crystal structures. This validates the quality of our model systems for investigating GM1 clustering through an ordered-lipid-cluster analysis. The increase in GM1 concentration induces tighter lipid packing, driven mainly by inter-GM1 carbohydrate-carbohydrate interactions, leading to a greater preference for the positive curvature of GM1-containing membranes and larger cluster sizes of ordered-lipid clusters (with a composite of GM1 and POPC). These clusters tend to segregate and form a large percolated cluster at a 30% GM1 concentration at 293 K. At a higher temperature of 330 K, however, the segregation is not maintained. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  10. Specific tritium labeling of gangliosides at the 3-position of sphingosines.

    PubMed

    Ghidoni, R; Sonnino, S; Masserini, M; Orlando, P; Tettamanti, G

    1981-11-01

    GM1 and GD1a gangliosides, treated with 2,3-dichloro-5,6-dicyano benzoquinone (DDQ) in the presence of Triton X-100 and in a toluene medium were specifically oxidized at the 3-position of sphingosine. The maximum reaction yield (65%) was obtained after 40 hours at 37 degrees C with the following molar ratio of reactants: ganglioside-Triton X-100-DDQ 1:70:125. The formation of the 3-keto derivatives of GM1 and GD1a was demonstrated by: a) the appearance of a sharp peak at 1700 cm-1 and of a broad band at 1250 cm-1 (typical of allylic ketones and of carbonyl groups, respectively) in the infra-red spectrum; b) the appearance of an absorption maximum at 230 nm, identical to that featured by 3-keto-cerebrosides, in the ultraviolet spectrum; c) the degradation of long chain bases during the process of release from gangliosides and derivatization for analysis by gas-liquid chromatography (expected for long chain bases carrying a keto group in the 3-position); and d) the quantitative transformation of 3-keto-GM1 and 3-keto-GD1a to GM1 and GD1a, respectively, upon NaBH4 reduction. Reduction of 3-keto-GM1 and 3-keto-GD1a with [3H]-NaBH4 produced 3H-labeled GM1 and GD1a. [3H]GM1 and [3H]GD1a maintained the same carbohydrate and fatty acid composition of the original GM1 and GD1a, and did not contain any saturated long chain bases. Direct proof that the label was at C-3 of long chain bases was given by reoxidation with DDQ, which completely removed the label, and by ozonolysis, after which label was retained on the oligosaccharide-containing fragment. More than 99% of incorporated radioactivity was carried by the long chain bases. The radiochemical purity of labeled gangliosides was greater than 95% and the specific radioactivity was 1.25 and 1.28 Ci/m mol for [3H]GM1 and [3H]GD1a, respectively.

  11. Auditory brainstem responses of CBA/J mice with neonatal conductive hearing losses and treatment with GM1 ganglioside.

    PubMed

    Money, M K; Pippin, G W; Weaver, K E; Kirsch, J P; Webster, D B

    1995-07-01

    Exogenous administration of GM1 ganglioside to CBA/J mice with a neonatal conductive hearing loss ameliorates the atrophy of spiral ganglion neurons, ventral cochlear nucleus neurons, and ventral cochlear nucleus volume. The present investigation demonstrates the extent of a conductive loss caused by atresia and tests the hypothesis that GM1 ganglioside treatment will ameliorate the conductive hearing loss. Auditory brainstem responses were recorded from four groups of seven mice each: two groups received daily subcutaneous injections of saline (one group had normal hearing; the other had a conductive hearing loss); the other two groups received daily subcutaneous injections of GM1 ganglioside (one group had normal hearing; the other had a conductive hearing loss). In mice with a conductive loss, decreases in hearing sensitivity were greatest at high frequencies. The decreases were determined by comparing mean ABR thresholds of the conductive loss mice with those of normal hearing mice. The conductive hearing loss induced in the mice in this study was similar to that seen in humans with congenital aural atresias. GM1 ganglioside treatment had no significant effect on ABR wave I thresholds or latencies in either group.

  12. Specific ganglioside binding to receptor sites on T lymphocytes that couple to ganglioside-induced decrease of CD4 expression

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Morrison, W.J.; Offner, H.; Vandenbark, A.A.

    1989-01-01

    The binding of different gangliosides to rat T-helper lymphocytes was characterized under conditions that decrease CD4 expression on different mammalian T-helper lymphoctyes. Saturation binding by monosialylated ({sub 3}H)-GM{sub 1} to rat T-lymphocytes was time- and temperature-dependent, had a dissociation constant (K{sub D}) of 2.2 {plus minus} 1.4 {mu}M and a binding capacity near 2 fmoles/cell. Competitive inhibition of ({sup 3}H)- GM{sub 1} binding demonstrated a structural-activity related to the number of unconstrained sialic acid moieties on GM{sub 1}-congeneric gangliosides. A comparison between the results of these binding studies and gangliosides-induced decrease of CD4 expression demonstrated that every aspect of ({supmore » 3}H)-GM{sub 1} binding concurs with ganglioside modulation of CD4 expression. It is concluded that the specific decrease of CD4 expression induced by pretreatment with gangliosides involves the initial process of gangliosides binding to specific sites on CD4{sup {double dagger}} T-helper lymphocytes.« less

  13. Ganglioside GM3 content in skeletal muscles is increased in type 2 but decreased in type 1 diabetes rat models: Implications of glycosphingolipid metabolism in pathophysiology of diabetes.

    PubMed

    Bozic, Josko; Markotic, Anita; Cikes-Culic, Vedrana; Novak, Anela; Borovac, Josip A; Vucemilovic, Hrvoje; Trgo, Gorana; Ticinovic Kurir, Tina

    2018-02-01

    Ganglioside GM3 is found in the plasma membrane, where its accumulation attenuates insulin receptor signaling. Considering the role of skeletal muscles in insulin-stimulated glucose uptake, the aim of the present study was to determine the expression of GM3 and its precursors in skeletal muscles of rat models of type 1 and type 2 diabetes mellitus (T1DM and T2DM, respectively). Diabetes was induced in male Sprague-Dawley rats by streptozotocin injection (55 mg/kg, i.p., for T1DM induction; 35 mg/kg, i.p., for T2DM induction), followed by feeding of rats with either a normal pellet diet (T1DM) or a high-fat diet (T2DM). Rats were killed 2 weeks after diabetes induction and samples of skeletal muscle were collected. Frozen quadriceps muscle sections were stained with a primary antibody against GM3 (Neu5Ac) and visualized using a secondary antibody coupled with Texas Red. The muscle content of ganglioside GM3 and its precursors was analyzed by high-performance thin-layer chromatography (HPTLC) followed by GM3 immunostaining. Muscle GM3 content was significantly higher in T2DM compared with control rats (P < 0.001). Furthermore, levels of the GM3 precursors ceramide, glucosylceramide, and lactosylceramide were significantly higher in T2DM compared with control rats (P < 0.05), whereas ceramide content was significantly lower in T1DM rats (P < 0.05). The intensity of the GM3 band on HPTLC was significantly higher in T2DM rats (P < 0.001) and significantly lower in T1DM rats (P < 0.05) compared with control. The expression patterns of GM3 ganglioside and its precursors in diabetic rats suggest that the role of glycosphingolipid metabolism may differ between T2DM and T1DM. © 2017 Ruijin Hospital, Shanghai Jiaotong University School of Medicine and John Wiley & Sons Australia, Ltd.

  14. Amyloid Precursor Protein (APP) Mediated Regulation of Ganglioside Homeostasis Linking Alzheimer's Disease Pathology with Ganglioside Metabolism

    PubMed Central

    Grimm, Marcus O. W.; Zinser, Eva G.; Grösgen, Sven; Hundsdörfer, Benjamin; Rothhaar, Tatjana L.; Burg, Verena K.; Kaestner, Lars; Bayer, Thomas A.; Lipp, Peter; Müller, Ulrike; Grimm, Heike S.; Hartmann, Tobias

    2012-01-01

    Gangliosides are important players for controlling neuronal function and are directly involved in AD pathology. They are among the most potent stimulators of Aβ production, are enriched in amyloid plaques and bind amyloid beta (Aβ). However, the molecular mechanisms linking gangliosides with AD are unknown. Here we identified the previously unknown function of the amyloid precursor protein (APP), specifically its cleavage products Aβ and the APP intracellular domain (AICD), of regulating GD3-synthase (GD3S). Since GD3S is the key enzyme converting a- to b-series gangliosides, it therefore plays a major role in controlling the levels of major brain gangliosides. This regulation occurs by two separate and additive mechanisms. The first mechanism directly targets the enzymatic activity of GD3S: Upon binding of Aβ to the ganglioside GM3, the immediate substrate of the GD3S, enzymatic turnover of GM3 by GD3S was strongly reduced. The second mechanism targets GD3S expression. APP cleavage results, in addition to Aβ release, in the release of AICD, a known candidate for gene transcriptional regulation. AICD strongly down regulated GD3S transcription and knock-in of an AICD deletion mutant of APP in vivo, or knock-down of Fe65 in neuroblastoma cells, was sufficient to abrogate normal GD3S functionality. Equally, knock-out of the presenilin genes, presenilin 1 and presenilin 2, essential for Aβ and AICD production, or of APP itself, increased GD3S activity and expression and consequently resulted in a major shift of a- to b-series gangliosides. In addition to GD3S regulation by APP processing, gangliosides in turn altered APP cleavage. GM3 decreased, whereas the ganglioside GD3, the GD3S product, increased Aβ production, resulting in a regulatory feedback cycle, directly linking ganglioside metabolism with APP processing and Aβ generation. A central aspect of this homeostatic control is the reduction of GD3S activity via an Aβ-GM3 complex and AICD

  15. Genetically engineered humanized anti-ganglioside GM2 antibody against multiple organ metastasis produced by GM2-expressing small-cell lung cancer cells.

    PubMed

    Yamada, Tadaaki; Bando, Hideaki; Takeuchi, Shinji; Kita, Kenji; Li, Qi; Wang, Wei; Akinaga, Shiro; Nishioka, Yasuhiko; Sone, Saburo; Yano, Seiji

    2011-12-01

    Small-cell lung cancer (SCLC) grows rapidly and metastasizes to multiple organs. We examined the antimetastatic effects of the humanized anti-ganglioside GM2 (GM2) antibodies, BIW-8962 and KM8927, compared with the chimeric antibody KM966, in a SCID mouse model of multiple organ metastases induced by GM2-expressing SCLC cells. BIW-8962 and KM8927 induced higher antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity than KM966 against the GM2-expressing SCLC cell line SBC-3 in vitro. These humanized antibodies inhibited the production of multiple organ metastases, increased the number of apoptotic cells, and prolonged the survival of the SCID mice. Histological analyses using clinical specimens showed that SCLC cells expressed GM2. These findings suggest that humanized anti-GM2 antibodies could be therapeutically useful for controlling multiple organ metastases of GM2-expressing SCLC. © 2011 Japanese Cancer Association.

  16. Ganglioside GM2 mediates migration of tumor cells by interacting with integrin and modulating the downstream signaling pathway.

    PubMed

    Kundu, Manjari; Mahata, Barun; Banerjee, Avisek; Chakraborty, Sohini; Debnath, Shibjyoti; Ray, Sougata Sinha; Ghosh, Zhumur; Biswas, Kaushik

    2016-07-01

    The definitive role of ganglioside GM2 in mediating tumor-induced growth and progression is still unknown. Here we report a novel role of ganglioside GM2 in mediating tumor cell migration and uncovered its mechanism. Data shows differential expression levels of GM2-synthase as well as GM2 in different human cancer cells. siRNA mediated knockdown of GM2-synthase in CCF52, A549 and SK-RC-26B cells resulted in significant inhibition of tumor cell migration as well as invasion in vitro without affecting cellular proliferation. Over-expression of GM2-synthase in low-GM2 expressing SK-RC-45 cells resulted in a consequent increase in migration thus confirming the potential role GM2 and its downstream partners play in tumor cell migration and motility. Further, treatment of SK-RC-45 cells with exogenous GM2 resulted in a dramatic increase in migratory and invasive capacity with no change in proliferative capacity, thereby confirming the role of GM2 in tumorigenesis specifically by mediating tumor migration and invasion. Gene expression profiling of GM2-synthase silenced cells revealed altered expression of several genes involved in cell migration primarily those controlling the integrin mediated signaling. GM2-synthase knockdown resulted in decreased phosphorylation of FAK, Src as well as Erk, while over-expression and/or exogenous GM2 treatment caused increased FAK and Erk phosphorylation respectively. Again, GM2 mediated invasion and Erk phosphorylation is blocked in integrin knockdown SK-RC-45 cells, thus confirming that GM2 mediated migration and phosphorylation of Erk is integrin dependent. Finally, confocal microscopy suggested co-localization while co-immunoprecipitation and surface plasmon resonance (SPR) confirmed direct interaction of membrane bound ganglioside, GM2 with the integrin receptor. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. Construction of a hybrid β-hexosaminidase subunit capable of forming stable homodimers that hydrolyze GM2 ganglioside in vivo.

    PubMed

    Tropak, Michael B; Yonekawa, Sayuri; Karumuthil-Melethil, Subha; Thompson, Patrick; Wakarchuk, Warren; Gray, Steven J; Walia, Jagdeep S; Mark, Brian L; Mahuran, Don

    2016-01-01

    Tay-Sachs or Sandhoff disease result from mutations in either the evolutionarily related HEXA or HEXB genes encoding respectively, the α- or β-subunits of β-hexosaminidase A (HexA). Of the three Hex isozymes, only HexA can interact with its cofactor, the GM2 activator protein (GM2AP), and hydrolyze GM2 ganglioside. A major impediment to establishing gene or enzyme replacement therapy based on HexA is the need to synthesize both subunits. Thus, we combined the critical features of both α- and β-subunits into a single hybrid µ-subunit that contains the α-subunit active site, the stable β-subunit interface and unique areas in each subunit needed to interact with GM2AP. To facilitate intracellular analysis and the purification of the µ-homodimer (HexM), CRISPR-based genome editing was used to disrupt the HEXA and HEXB genes in a Human Embryonic Kidney 293 cell line stably expressing the µ-subunit. In association with GM2AP, HexM was shown to hydrolyze a fluorescent GM2 ganglioside derivative both in cellulo and in vitro. Gene transfer studies in both Tay-Sachs and Sandhoff mouse models demonstrated that HexM expression reduced brain GM2 ganglioside levels.

  18. Construction of a hybrid β-hexosaminidase subunit capable of forming stable homodimers that hydrolyze GM2 ganglioside in vivo

    PubMed Central

    Tropak, Michael B; Yonekawa, Sayuri; Karumuthil-Melethil, Subha; Thompson, Patrick; Wakarchuk, Warren; Gray, Steven J; Walia, Jagdeep S; Mark, Brian L; Mahuran, Don

    2016-01-01

    Tay-Sachs or Sandhoff disease result from mutations in either the evolutionarily related HEXA or HEXB genes encoding respectively, the α- or β-subunits of β-hexosaminidase A (HexA). Of the three Hex isozymes, only HexA can interact with its cofactor, the GM2 activator protein (GM2AP), and hydrolyze GM2 ganglioside. A major impediment to establishing gene or enzyme replacement therapy based on HexA is the need to synthesize both subunits. Thus, we combined the critical features of both α- and β-subunits into a single hybrid µ-subunit that contains the α-subunit active site, the stable β-subunit interface and unique areas in each subunit needed to interact with GM2AP. To facilitate intracellular analysis and the purification of the µ-homodimer (HexM), CRISPR-based genome editing was used to disrupt the HEXA and HEXB genes in a Human Embryonic Kidney 293 cell line stably expressing the µ-subunit. In association with GM2AP, HexM was shown to hydrolyze a fluorescent GM2 ganglioside derivative both in cellulo and in vitro. Gene transfer studies in both Tay-Sachs and Sandhoff mouse models demonstrated that HexM expression reduced brain GM2 ganglioside levels. PMID:26966698

  19. Histochemical detection of GM1 ganglioside using cholera toxin-B subunit. Evaluation of critical factors optimal for in situ detection with special emphasis to acetone pre-extraction

    PubMed Central

    Petr, T.; Šmíd, V.; Šmídová, J.; Hůlková, H.; Jirkovská, M.; Elleder, M.; Muchová, L.; Vítek, L.; Šmíd, F.

    2010-01-01

    A comparison of histochemical detection of GM1 ganglioside in cryostat sections using cholera toxin B-subunit after fixation with 4% formaldehyde and dry acetone gave tissue-dependent results. In the liver no pre-treatment showed detectable differences related to GM1 reaction products, while studies in the brain showed the superiority of acetone pre-extraction (followed by formaldehyde), which yielded sharper images compared with the diffuse, blurred staining pattern associated with formaldehyde. Therefore, the aim of our study was to define the optimal conditions for the GM1 detection using cholera toxin B-subunit. Ganglioside extractability with acetone, the ever neglected topic, was tested comparing anhydrous acetone with acetone containing admixture of water. TLC analysis of acetone extractable GM1 ganglioside from liver sections did not exceed 2% of the total GM1 ganglioside content using anhydrous acetone at −20°C, and 4% at room temperature. The loss increased to 30.5% using 9:1 acetone/water. Similarly, photometric analysis of lipid sialic acid, extracted from dried liver homogenates with anhydrous acetone, showed the loss of gangliosides into acetone 3.0±0.3% only. The loss from dried brain homogenate was 9.5±1.1%. Thus, anhydrous conditions (dry tissue samples and anhydrous acetone) are crucial factors for optimal in situ ganglioside detection using acetone pre-treatment. This ensures effective physical fixation, especially in tissues rich in polar lipids (precipitation, prevention of in situ diffusion), and removal of cholesterol, which can act as a hydrophobic blocking barrier. PMID:20558344

  20. Crystallographic structure of human beta-hexosaminidase A: interpretation of Tay-Sachs mutations and loss of GM2 ganglioside hydrolysis.

    PubMed

    Lemieux, M Joanne; Mark, Brian L; Cherney, Maia M; Withers, Stephen G; Mahuran, Don J; James, Michael N G

    2006-06-16

    Lysosomal beta-hexosaminidase A (Hex A) is essential for the degradation of GM2 gangliosides in the central and peripheral nervous system. Accumulation of GM2 leads to severely debilitating neurodegeneration associated with Tay-Sachs disease (TSD), Sandoff disease (SD) and AB variant. Here, we present the X-ray crystallographic structure of Hex A to 2.8 A resolution and the structure of Hex A in complex with NAG-thiazoline, (NGT) to 3.25 A resolution. NGT, a mechanism-based inhibitor, has been shown to act as a chemical chaperone that, to some extent, prevents misfolding of a Hex A mutant associated with adult onset Tay Sachs disease and, as a result, increases the residual activity of Hex A to a level above the critical threshold for disease. The crystal structure of Hex A reveals an alphabeta heterodimer, with each subunit having a functional active site. Only the alpha-subunit active site can hydrolyze GM2 gangliosides due to a flexible loop structure that is removed post-translationally from beta, and to the presence of alphaAsn423 and alphaArg424. The loop structure is involved in binding the GM2 activator protein, while alphaArg424 is critical for binding the carboxylate group of the N-acetyl-neuraminic acid residue of GM2. The beta-subunit lacks these key residues and has betaAsp452 and betaLeu453 in their place; the beta-subunit therefore cleaves only neutral substrates efficiently. Mutations in the alpha-subunit, associated with TSD, and those in the beta-subunit, associated with SD are discussed. The effect of NGT binding in the active site of a mutant Hex A and its effect on protein function is discussed.

  1. Human monoclonal IgM with autoantibody activity against two gangliosides (GM1 and GD1b) in a patient with motor neuron syndrome.

    PubMed

    Jauberteau, M O; Gualde, N; Preud'Homme, J L; Rigaud, M; Gil, R; Vallat, J M; Baumann, N

    1990-05-01

    Small amounts of oligoclonal immunoglobulins were detected by Western blotting in the serum from a patient with motor neuron syndrome. The prominent one, a monoclonal IgM lambda, reacted strongly with the gangliosides GM1 and GD1b and more weakly with asialo GM1, as shown by immunoenzymatic staining of thin-layer chromatograms of gangliosides, ELISA on purified glycolipid coats and immunoadsorption with purified GM1. Affinity-chromatography with purified GM1 resulted in the purification of monoclonal IgM lambda. This purified IgM and its Fab fragments showed the same pattern of reactivity with gangliosides as that observed with whole serum. Such monoclonal IgM could be responsible for motor neuron diseases in some patients with overt or barely detectable monoclonal gammopathies.

  2. Accumulation of cholesterol and GM2 ganglioside in cells cultured in the presence of progesterone: an implication for the basic defect in Niemann-Pick disease type C.

    PubMed

    Sato, M; Akaboshi, S; Katsumoto, T; Taniguchi, M; Higaki, K; Tai, T; Sakuraba, H; Ohno, K

    1998-01-01

    Cultured fibroblasts from patients with Niemann-Pick disease type C (NP-C) are characterized by lysosomal accumulation of unesterified cholesterol and a defect in intracellular trafficking of cholesterol. We have found the accumulation of GM2 ganglioside in NP-C fibroblasts [Yano T, Taniguchi M, Akaboshi S, Vanier MT, Tai T, Sakuraba H, et al. Proc Japan Acad 1996;72B:214-219]. In this communication we show that several inhibitors known to inhibit intracellular cholesterol transport, progesterone, imipramine and KN-62, elicit accumulation of not only unesterified cholesterol but also GM2 ganglioside. This finding suggests that intracellular transport of cholesterol may be coupled with that of GM2 ganglioside. The accumulation of free cholesterol and GM2 ganglioside may be a clue for understanding the basic defect of NP-C. Recently NPC1 gene is found by the positional cloning. The mechanism of accumulating of GM2 ganglioside should be further investigated by studying of the functions of NPC1 gene.

  3. Synthesis of novel ganglioside GM4 analogues containing N-deacetylated and lactamized sialic acid: probes for searching new ligand structures for human L-selectin.

    PubMed

    Otsubo, N; Ishida, H; Kiso, M

    2001-01-15

    Novel ganglioside GM4 analogues, which contain N-deacetylated or lactamized sialic acid instead of usual N-acetylneuraminic acid, were synthesized in a highly efficient manner. (Methyl 4,7,8,9-tetra-O-acetyl-3,5-dideoxy-5-trifluoroacetamido-D-glycero-alpha-D-galacto-2-nonulopyranosylonate)-(2-->3)-4,6-di-O-acetyl-2-O-benzoyl-D-galactopyranosyl trichloroacetimidate was coupled with 2-(tetradecyl)hexadecanol to give the desired beta-glycoside in high yield. Successive O- and N-deacylation, and saponification of the methyl ester group afforded the N-deacetylated sialyl derivative that was converted by treatment with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride in Me2SO into the lactamized sialic acid-containing ganglioside GM4 analogue.

  4. Ganglioside accumulation in activated glia in the developing brain: comparison between WT and GalNAcT KO mice

    PubMed Central

    Saito, Mariko; Wu, Gusheng; Hui, Maria; Masiello, Kurt; Dobrenis, Kostantin; Ledeen, Robert W.; Saito, Mitsuo

    2015-01-01

    Our previous studies have shown accumulation of GM2 ganglioside during ethanol-induced neurodegeneration in the developing brain, and GM2 elevation has also been reported in other brain injuries and neurodegenerative diseases. Using GM2/GD2 synthase KO mice lacking GM2/GD2 and downstream gangliosides, the current study explored the significance of GM2 elevation in WT mice. Immunohistochemical studies indicated that ethanol-induced acute neurodegeneration in postnatal day 7 (P7) WT mice was associated with GM2 accumulation in the late endosomes/lysosomes of both phagocytic microglia and increased glial fibrillary acidic protein (GFAP)-positive astrocytes. However, in KO mice, although ethanol induced robust neurodegeneration and accumulation of GD3 and GM3 in the late endosomes/lysosomes of phagocytic microglia, it did not increase the number of GFAP-positive astrocytes, and the accumulation of GD3/GM3 in astrocytes was minimal. Not only ethanol, but also DMSO, induced GM2 elevation in activated microglia and astrocytes along with neurodegeneration in P7 WT mice, while lipopolysaccharide, which did not induce significant neurodegeneration, caused GM2 accumulation mainly in lysosomes of activated astrocytes. Thus, GM2 elevation is associated with activation of microglia and astrocytes in the injured developing brain, and GM2, GD2, or other downstream gangliosides may regulate astroglial responses in ethanol-induced neurodegeneration. PMID:26063460

  5. Direct evidence that ganglioside is an integral component of the thyrotropin receptor

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kielczynski, W.; Harrison, L.C.; Leedman, P.J.

    1991-03-01

    Gangliosides were extracted from purified human and porcine thyrotropin (TSH) receptors (TSH-R) and were detected by probing with an {sup 125}I-labeled sialic acid-specific lectin, Limax flavus agglutinin. Gangliosides copurified with human and porcine TSH-R migrated between monosialoganglioside GM1 and disialoganglioside GD1a. Ceramide glycanase digestion of the purified human TSH-R-associated glycolipid confirmed its ganglioside nature. It was resistant to Vibrio cholerae sialidase, which digest all gangliosides except GM1, but was sensitive to Arthrobacter ureafaciens sialidase, which digests all gangliosides including GM1. These findings indicate that the human TSH-R contains ganglioside that belongs to the galactosyl({beta}1{r arrow} 3)-N-acetylgalactosaminyl({beta}1{r arrow} 4)-(N-acetylneuraminyl({alpha}2{r arrow} 3))galactosyl({beta}1more » {r arrow} 4)glucosyl({beta}1 {r arrow} 1)ceramide (GM1) family. Its intimate association with receptor protein implies a key role for ganglioside in the structure and function of the TSH-R.« less

  6. Lyso-GM2 ganglioside: a possible biomarker of Tay-Sachs disease and Sandhoff disease.

    PubMed

    Kodama, Takashi; Togawa, Tadayasu; Tsukimura, Takahiro; Kawashima, Ikuo; Matsuoka, Kazuhiko; Kitakaze, Keisuke; Tsuji, Daisuke; Itoh, Kohji; Ishida, Yo-Ichi; Suzuki, Minoru; Suzuki, Toshihiro; Sakuraba, Hitoshi

    2011-01-01

    To find a new biomarker of Tay-Sachs disease and Sandhoff disease. The lyso-GM2 ganglioside (lyso-GM2) levels in the brain and plasma in Sandhoff mice were measured by means of high performance liquid chromatography and the effect of a modified hexosaminidase (Hex) B exhibiting Hex A-like activity was examined. Then, the lyso-GM2 concentrations in human plasma samples were determined. The lyso-GM2 levels in the brain and plasma in Sandhoff mice were apparently increased compared with those in wild-type mice, and they decreased on intracerebroventricular administration of the modified Hex B. The lyso-GM2 levels in plasma of patients with Tay-Sachs disease and Sandhoff disease were increased, and the increase in lyso-GM2 was associated with a decrease in Hex A activity. Lyso-GM2 is expected to be a potential biomarker of Tay-Sachs disease and Sandhoff disease.

  7. Anchored and soluble gangliosides contribute to myelosupportivity of stromal cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ziulkoski, Ana L.; Departamento de Bioquimica, ICBS, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS; Instituto de Ciencias da Saude, Centro Universitario Feevale, Novo Hamburgo, RS

    2009-10-09

    Stroma-mediated myelopoiesis depends upon growth factors and an appropriate intercellular microenvironment. Previous studies have demonstrated that gangliosides, produced by hepatic stromal cell types, are required for optimal myelosupportive function. Here, we compared the mielossuportive functions of a bone marrow stroma (S17) and skin fibroblasts (SF) regarding their ganglioside pattern of synthesis and shedding. The survival and proliferation of a myeloid precursor cell (FDC-P1) were used as reporter. Although the ganglioside synthesis of the two stromal cells was similar, their relative content and shedding were distinct. The ganglioside requirement for mielossuportive function was confirmed by the decreased proliferation of FDC-P1 cellsmore » in ganglioside synthesis-inhibited cultures and in presence of an antibody to GM3 ganglioside. The distinct mielossuportive activities of the S17 and SF stromata may be related to differences on plasma membrane ganglioside concentrations or to differences on the gangliosides shed and their subsequent uptake by myeloid cells, specially, GM3 ganglioside.« less

  8. Multimodal detection of GM2 and GM3 lipid species in the brain of mucopolysaccharidosis type II mouse by serial imaging mass spectrometry and immunohistochemistry.

    PubMed

    Dufresne, Martin; Guneysu, Daniel; Patterson, Nathan Heath; Marcinkiewicz, Mieczyslaw Martin; Regina, Anthony; Demeule, Michel; Chaurand, Pierre

    2017-02-01

    Mucopolysaccharidosis type II (Hunter's disease) mouse model (IdS-KO) was investigated by both imaging mass spectrometry (IMS) and immunohistochemistry (IHC) performed on the same tissue sections. For this purpose, IdS-KO mice brain sections were coated with sublimated 1,5-diaminonaphtalene and analyzed by high spatial resolution IMS (5 μm) and anti-GM3 IHC on the same tissue sections to characterize the ganglioside monosialated ganglioside (GM) deposits found in Hunter's disease. IMS analysis have found that two species of GM3 and GM2 that are only different due to the length of their fatty acid residue (stearic or arachidic residue) were overexpressed in the IdS-KO mice compared to a control mouse. GM3 and GM2 were characterized by on-tissue exact mass and MS/MS compared to a GM3 standard. Realignment of both IMS and IHC data sets further confirmed the observed regioselective signal previously detected by providing direct correlation of the IMS image for the two GM3 overly expressed MS signals with the anti-GM3 IHC image. Furthermore, these regioselective GM MS signals were also found to have highly heterogeneous distributions within the GM3-IHC staining. Some deposits showed high content in GM3 and GM2 stearic species (r = 0.74) and others had more abundant GM3 and GM2 arachidic species (r = 0.76). Same-section analysis of Hunter's disease mouse model by both high spatial resolution IMS and IHC provides a more in-depth analysis of the composition of the GM aggregates while providing spatial distribution of the observed molecular species. Graphical Abstract Ganglioside imaging mass spectrometry followed by immunohistochemistry performed on the same tissue section.

  9. Lyso-GM2 Ganglioside: A Possible Biomarker of Tay-Sachs Disease and Sandhoff Disease

    PubMed Central

    Kodama, Takashi; Togawa, Tadayasu; Tsukimura, Takahiro; Kawashima, Ikuo; Matsuoka, Kazuhiko; Kitakaze, Keisuke; Tsuji, Daisuke; Itoh, Kohji; Ishida, Yo-ichi; Suzuki, Minoru; Suzuki, Toshihiro; Sakuraba, Hitoshi

    2011-01-01

    To find a new biomarker of Tay-Sachs disease and Sandhoff disease. The lyso-GM2 ganglioside (lyso-GM2) levels in the brain and plasma in Sandhoff mice were measured by means of high performance liquid chromatography and the effect of a modified hexosaminidase (Hex) B exhibiting Hex A-like activity was examined. Then, the lyso-GM2 concentrations in human plasma samples were determined. The lyso-GM2 levels in the brain and plasma in Sandhoff mice were apparently increased compared with those in wild-type mice, and they decreased on intracerebroventricular administration of the modified Hex B. The lyso-GM2 levels in plasma of patients with Tay-Sachs disease and Sandhoff disease were increased, and the increase in lyso-GM2 was associated with a decrease in Hex A activity. Lyso-GM2 is expected to be a potential biomarker of Tay-Sachs disease and Sandhoff disease. PMID:22205997

  10. Studies on the turnover and subcellular localization of membrane gangliosides in cultured neuroblastoma cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Clarke, J.T.; Cook, H.W.; Spence, M.W.

    1985-03-01

    To compare the subcellular distribution of endogenously synthesized and exogenous gangliosides, cultured murine neuroblastoma cells (N1E-115) were incubated in suspension for 22 h in the presence of D-(1-/sup 3/H)galactose or (/sup 3/H)GM1 ganglioside, transferred to culture medium containing no radioisotope for periods of up to 72 hr, and then subjected to subcellular fractionation and analysis of lipid-sialic acid and radiolabeled ganglioside levels. The results indicated that GM2 and GM3 were the principal gangliosides in the cells with only traces of GM1 and small amounts of disialogangliosides present. About 50% of the endogenously synthesized radiolabelled ganglioside in the four major subcellularmore » membrane fractions studied was recovered from plasma membrane and only 10-15% from the crude mitochondrial membrane fraction. In contrast, 45% of the exogenous (/sup 3/H)GM1 taken up into the same subcellular membrane fractions was recovered from the crude mitochondrial fraction; less than 15% was localized in the plasma membrane fraction. The results are similar to those obtained from previously reported studies on membrane phospholipid turnover. They suggest that exogenous GM1 ganglioside, like exogenous phosphatidylcholine, does not intermix freely with any quantitatively major pool of endogenous membrane lipid.« less

  11. Partial synthesis of ganglioside and lysoganglioside lipoforms as internal standards for MS quantification.

    PubMed

    Gantner, Martin; Schwarzmann, Günter; Sandhoff, Konrad; Kolter, Thomas

    2014-12-01

    Within recent years, ganglioside patterns have been increasingly analyzed by MS. However, internal standards for calibration are only available for gangliosides GM1, GM2, and GM3. For this reason, we prepared homologous internal standards bearing nonnatural fatty acids of the major mammalian brain gangliosides GM1, GD1a, GD1b, GT1b, and GQ1b, and of the tumor-associated gangliosides GM2 and GD2. The fatty acid moieties were incorporated after selective chemical or enzymatic deacylation of bovine brain gangliosides. For modification of the sphingoid bases, we developed a new synthetic method based on olefin cross metathesis. This method was used for the preparation of a lyso-GM1 and a lyso-GM2 standard. The total yield of this method was 8.7% for the synthesis of d17:1-lyso-GM1 from d20:1/18:0-GM1 in four steps. The title compounds are currently used as calibration substances for MS quantification and are also suitable for functional studies. Copyright © 2014 by the American Society for Biochemistry and Molecular Biology, Inc.

  12. The total chemical synthesis of the monoglycosylated GM2 ganglioside activator using a novel cysteine surrogate.

    PubMed

    Sato, Kohei; Kitakaze, Keisuke; Nakamura, Takahiro; Naruse, Naoto; Aihara, Keisuke; Shigenaga, Akira; Inokuma, Tsubasa; Tsuji, Daisuke; Itoh, Kohji; Otaka, Akira

    2015-06-21

    We describe a novel peptide ligation/desulfurization strategy using a β-mercapto-N-glycosylated asparagine derivative. The newly developed procedure was successfully applied to the total chemical synthesis of the GM2 ganglioside activator protein bearing a monosaccharide on the native glycosylation site.

  13. Cholesterol accelerates the binding of Alzheimer's β-amyloid peptide to ganglioside GM1 through a universal hydrogen-bond-dependent sterol tuning of glycolipid conformation

    PubMed Central

    Fantini, Jacques; Yahi, Nouara; Garmy, Nicolas

    2013-01-01

    Age-related alterations of membrane lipids in brain cell membranes together with high blood cholesterol are considered as major risk factors for Alzheimer's disease. Yet the molecular mechanisms by which these factors increase Alzheimer's risk are mostly unknown. In lipid raft domains of the plasma membrane, neurotoxic Alzheimer's beta-amyloid (Abeta) peptides interact with both cholesterol and ganglioside GM1. Recent data also suggested that cholesterol could stimulate the binding of Abeta to GM1 through conformational modulation of the ganglioside headgroup. Here we used a combination of physicochemical and molecular modeling approaches to decipher the mechanisms of cholesterol-assisted binding of Abeta to GM1. With the aim of decoupling the effect of cholesterol on GM1 from direct Abeta-cholesterol interactions, we designed a minimal peptide (Abeta5-16) containing the GM1-binding domain but lacking the amino acid residues involved in cholesterol recognition. Using the Langmuir technique, we showed that cholesterol (but not phosphatidylcholine or sphingomyelin) significantly accelerates the interaction of Abeta5-16 with GM1. Molecular dynamics simulations suggested that Abeta5-16 interacts with a cholesterol-stabilized dimer of GM1. The main structural effect of cholesterol is to establish a hydrogen-bond between its own OH group and the glycosidic-bond linking ceramide to the glycone part of GM1, thereby inducing a tilt in the glycolipid headgroup. This fine conformational tuning stabilizes the active conformation of the GM1 dimer whose headgroups, oriented in two opposite directions, form a chalice-shaped receptacle for Abeta. These data give new mechanistic insights into the stimulatory effect of cholesterol on Abeta/GM1 interactions. They also support the emerging concept that cholesterol is a universal modulator of protein-glycolipid interactions in the broader context of membrane recognition processes. PMID:23772214

  14. The function of cancer-shed gangliosides in macrophage phenotype: involvement with angiogenesis.

    PubMed

    Chung, Tae-Wook; Choi, Hee-Jung; Park, Mi-Ju; Choi, Hee-Jin; Lee, Syng-Ook; Kim, Keuk-Jun; Kim, Cheorl-Ho; Hong, Changwan; Kim, Kyun-Ha; Joo, Myungsoo; Ha, Ki-Tae

    2017-01-17

    Tumor-derived gangliosides in the tumor microenvironment are involved in the malignant progression of cancer. However, the molecular mechanisms underlying the effects of gangliosides shed from tumors on macrophage phenotype remain unknown. Here, we showed that ganglioside GM1 highly induced the activity and expression of arginase-1 (Arg-1), a major M2 macrophage marker, compared to various gangliosides in bone marrow-derived macrophages (BMDM), peritoneal macrophages and Raw264.7 macrophage cells. We found that GM1 bound to macrophage mannose receptor (MMR/CD206) and common gamma chain (γc). In addition, GM1 increased Arg-1 expression through CD206 and γc-mediated activation of Janus kinase 3 (JAK3) and signal transducer and activator of transcription- 6 (STAT-6). Interestingly, GM1-stimulated macrophages secreted monocyte chemoattractant protein-1 (MCP-1/CCL2) through a CD206/γc/STAT6-mediated signaling pathway and induced angiogenesis. Moreover, the angiogenic effect of GM1-treated macrophages was diminished by RS102895, an MCP-1 receptor (CCR2) antagonist. From these results we suggest that tumor-shed ganglioside is a secretory factor regulating the phenotype of macrophages and consequently enhancing angiogenesis.

  15. On the minor gangliosides of erythrocyte membranes of Japanese cats.

    PubMed

    Ando, N; Yamakawa, T

    1982-03-01

    Seven ganglioside species were isolated and purified from erythrocyte membranes of Japanese cats by DEAE-Sephadex and Iatrobeads column chromatographies. The structures of these gangliosides were determined as Gmi(NeuGc), Gm3(NeuAc), GM3(NeuGc), GD3(NeuGc), GD3(NeuGc comes from NeuAc), GT3(NeuGc), and another GM3 containing a sialic acid of unidentified nature. The occurrence of GT3 suggested the probable presence of a biosynthetic pathway of GM3 leads to GD3 leads to GT3 in erythropoietic cells of Japanese cats. The presence of GD3 having one penultimate N-glycolylneuraminic acid and one terminal N-acetylneuraminic acid, GD3(NeuGc comes from NeuAc) would indicate that this GD3 acts as an intermediate in a possible pathway from GM3(NeuGc) to GD3(NeuGc). Thin layer chromatographic patterns of total erythrocyte membrane gangliosides were compared among Japanese cats (n = 3), lions (n = 3), a serval and a racoon dog. The three species of felid showed similar patterns to each other and contained N-glycolylneuraminic acid as the major sialic acid. On the other hand, erythrocytes of racoon dog, a member of canidae, contained neither GD3 nor GT3, but only GM3.

  16. Botulinum neurotoxin serotype D attacks neurons via two carbohydrate-binding sites in a ganglioside-dependent manner.

    PubMed

    Strotmeier, Jasmin; Lee, Kwangkook; Völker, Anne K; Mahrhold, Stefan; Zong, Yinong; Zeiser, Johannes; Zhou, Jie; Pich, Andreas; Bigalke, Hans; Binz, Thomas; Rummel, Andreas; Jin, Rongsheng

    2010-10-15

    The extraordinarily high toxicity of botulinum neurotoxins primarily results from their specific binding and uptake into neurons. At motor neurons, the seven BoNT (botulinum neurotoxin) serotypes A-G inhibit acetylcholine release leading to flaccid paralysis. Uptake of BoNT/A, B, E, F and G requires a dual interaction with gangliosides and the synaptic vesicle proteins synaptotagmin or SV2 (synaptic vesicle glycoprotein 2), whereas little is known about the cell entry mechanisms of the serotypes C and D, which display the lowest amino acid sequence identity compared with the other five serotypes. In the present study we demonstrate that the neurotoxicity of BoNT/D depends on the presence of gangliosides by employing phrenic nerve hemidiaphragm preparations derived from mice expressing the gangliosides GM3, GM2, GM1 and GD1a, or only GM3 [a description of our use of ganglioside nomenclature is given in Svennerholm (1994) Prog. Brain Res. 101, XI-XIV]. High-resolution crystal structures of the 50 kDa cell-binding domain of BoNT/D alone and in complex with sialic acid, as well as biological analyses of single-site BoNT/D mutants identified two carbohydrate-binding sites. One site is located at a position previously identified in BoNT/A, B, E, F and G, but is lacking the conserved SXWY motif. The other site, co-ordinating one molecule of sialic acid, resembles the second ganglioside-binding pocket (the sialic-acid-binding site) of TeNT (tetanus neurotoxin).

  17. Damaging effects of Clostridium perfringens delta toxin on blood platelets and their relevance to ganglioside GM2.

    PubMed

    Jolivet-Reynaud, C; Launay, J M; Alouf, J E

    1988-04-01

    The lytic effect of Clostridium perfringens delta toxin was investigated on goat, human, rabbit, and guinea pig platelets. In contrast to erythrocytes from the latter three species, which are insensitive to the toxin, the platelets were equally lysed by the same amount of toxin. These results suggest the presence of GM2 or GM2-like ganglioside(s) as a specific recognition site of the toxin on platelet plasmic membrane as previously established for sensitive erythrocytes. Plasmic membrane damage of human platelets was evidenced by the release of entrapped alpha-[14C]aminoisobutyric acid used as a cytoplasmic marker. The specific binding of hemolytically active 125I-delta toxin by human and rabbit platelets was practically identical, dose dependent, and inhibitable by GM2. Labeled toxin was also bound by various subcellular organelles separated from rabbit platelets except the 5-hydroxytryptamine (5-HT)-containing dense bodies, suggesting the absence or inaccessibility of GM2 on the surface of the latter organelles. This result correlates with the low amounts of 5-[3H]HT liberated after platelet challenge with delta toxin whereas this mediator was massively liberated upon lysis by the sulfhydryl-activated toxin alveolysin. The levels of M and P forms of phenol sulfotransferase (PST), involved in 5-HT catabolism, were determined in human platelet lysates after challenge with delta toxin, alveolysin, and other disruptive treatments. The low PST-M activities detected after lysis by delta toxin suggest that this isoenzyme is very likely associated to dense bodies in contrast to PST-P which is cytoplasmic. Platelet lysis by the toxin allows easy separation of these organelles.

  18. A SENSITIVE FLUORESCENCE-BASED ASSAY FOR MONITORING GM2 GANGLIOSIDE HYDROLYSIS IN LIVE PATIENT CELLS AND THEIR LYSATES

    PubMed Central

    Tropak, Michael B.; Bukovac, Scott W.; Rigat, Brigitte A.; Yonekawa, Sayuri; Wakarchuk, Warren; Mahuran, Don J.

    2010-01-01

    Enzyme enhancement therapy, utilizing small molecules as pharmacological chaperones, is anattractive approach for the treatment of lysosomal storage diseases that are associated with protein misfolding. However, pharmacological chaperones are alsoinhibitors of their target enzyme. Thus, a major concern with this approach is that, despite enhancing protein folding within, and intracellular transport of the functional mutant enzyme out of the endoplasmic reticulum, the chaperone will continue to inhibit the enzyme in the lysosome, preventing substrate clearance. Herewe demonstrate that the in vitro hydrolysis of a fluorescent derivative of lyso-GM2 ganglioside, like natural GM2 ganglioside, is specifically carried out by the β-hexosaminidase A isozyme, requires the GM2 activator protein as a co-factor, increases when the derivative is incorporated into anionic liposomes and follows similar Michaelis-Menten kinetics. This substrate can also be used to differentiate between lysates from normal and GM2 activator-deficient cells. When added to the growth medium of cells, the substrate is internalized and primarily incorporated into lysosomes. Utilizing adult Tay-Sachs fibroblasts that have been pre-treated with the pharmacological chaperone Pyrimethamine and subsequently loaded with this substrate, we demonstrate an increase in both the levels of mutant β-hexosaminidase A and substrate-hydrolysis as compared to mock treated cells. PMID:19917668

  19. A sensitive fluorescence-based assay for monitoring GM2 ganglioside hydrolysis in live patient cells and their lysates.

    PubMed

    Tropak, Michael B; Bukovac, Scott W; Rigat, Brigitte A; Yonekawa, Sayuri; Wakarchuk, Warren; Mahuran, Don J

    2010-03-01

    Enzyme enhancement therapy, utilizing small molecules as pharmacological chaperones, is an attractive approach for the treatment of lysosomal storage diseases that are associated with protein misfolding. However, pharmacological chaperones are also inhibitors of their target enzyme. Thus, a major concern with this approach is that, despite enhancing protein folding within, and intracellular transport of the functional mutant enzyme out of the endoplasmic reticulum, the chaperone will continue to inhibit the enzyme in the lysosome, preventing substrate clearance. Here we demonstrate that the in vitro hydrolysis of a fluorescent derivative of lyso-GM2 ganglioside, like natural GM2 ganglioside, is specifically carried out by the beta-hexosaminidase A isozyme, requires the GM2 activator protein as a co-factor, increases when the derivative is incorporated into anionic liposomes and follows similar Michaelis-Menten kinetics. This substrate can also be used to differentiate between lysates from normal and GM2 activator-deficient cells. When added to the growth medium of cells, the substrate is internalized and primarily incorporated into lysosomes. Utilizing adult Tay-Sachs fibroblasts that have been pre-treated with the pharmacological chaperone Pyrimethamine and subsequently loaded with this substrate, we demonstrate an increase in both the levels of mutant beta-hexosaminidase A and substrate-hydrolysis as compared to mock-treated cells.

  20. Oriented 1,2-dimyristoyl-sn-glycero-3-phosphorylcholine/ganglioside membranes: a Fourier transform infrared attenuated total reflection spectroscopic study. Band assignments; orientational, hydrational, and phase behavior; and effects of Ca2+ binding.

    PubMed

    Müller, E; Giehl, A; Schwarzmann, G; Sandhoff, K; Blume, A

    1996-09-01

    Fourier transform infrared (FTIR) attenuated total reflection (ATR) spectroscopy was used to elucidate the hydration behavior and molecular order of phospholipid/ganglioside bilayers. We examined dry and hydrated films of the gangliosides GM1, deacetyl-GM1, lyso-GM1, deacetyllyso-GM1, and GM3 and oriented mixed films of these gangliosides with 1,2-dimyristoyl-sn-glycero-3-phosphorylcholine (DMPC) using polarized light. Analysis of the amide I frequencies reveals that the amide groups are involved in intermolecular interactions via hydrogen bonds of varying strengths. The tilt angle of the acyl chains of the lipids in mixed films was determined as a function of ganglioside structure. Deacetylation of the sialic acid in the headgroup has a stronger influence on the tilt angle than the removal of the ganglioside fatty acid. The phase behavior was examined by FTIR ATR spectroscopy and by differential scanning calorimetry (DSC) measurements on lipid suspensions. At the same molar concentration, lyso-gangliosides have less effect on changes of transition temperature compared to the double-chain analogs. Distinct differences in the amide band shapes were observed between mixtures with lyso-gangliosides and normal double-chain gangliosides. Determined from the dicroic ratio RATR, the orientation of the COO- group in all DMPC/ganglioside mixtures was found to be relatively fixed with respect to the membrane normal. In 4:1 mixtures of DMPC with GM1 and deacetyl-GM1, the binding of Ca2+ leads to a slight decrease in chain tilt in the gel phase, probably caused by a dehydration of the membrane-water interface. In mixtures of DMPC with GM3 and deacetyl-lyso-GM1, a slight increase in chain tilt is observed. The chain tilt in DMPC/lyso-GM1 mixtures is unchanged. Analysis of the COO- band reveals that Ca2+ does not bind to the carboxylate group of the sialic acid of GM1 and deacetyl-GM1, the mixtures in which a decrease in chain tilt was observed. Binding to the sialic acid was

  1. Anti-GM2 ganglioside antibodies are a biomarker for acute canine polyradiculoneuritis

    PubMed Central

    Bianchi, Ezio; Dondi, Maurizio; Penderis, Jacques; Cappell, Joanna; Burgess, Karl; Matiasek, Kaspar; McGonigal, Rhona; Willison, Hugh J.

    2016-01-01

    Acute canine polyradiculoneuritis (ACP) is considered to be the canine equivalent of the human peripheral nerve disorder Guillain-Barré syndrome (GBS); an aetiological relationship, however, remains to be demonstrated. In GBS, anti-glycolipid antibodies (Abs) are considered as important disease mediators. To address the possibility of common Ab biomarkers, the sera of 25 ACP dogs, 19 non-neurological, and 15 epileptic control dogs were screened for IgG Abs to 10 glycolipids and their 1 : 1 heteromeric complexes using combinatorial glycoarrays. Anti-GM2 ganglioside Abs were detected in 14/25 ACP dogs, and anti-GA1 Abs in one further dog. All controls except for one were negative for anti-glycolipid Abs. In this cohort of cases and controls, the glycoarray screen reached a diagnostic sensitivity of 60% and a specificity of 97%; a lower sensitivity (32%) was reported using a conventional glycolipid ELISA. To address the possible pathogenic role for anti-GM2 Abs in ACP, we identified GM2 in canine sciatic nerve by both mass spectrometry and thin layer chromatography overlay. In immunohistological studies, GM2 was localized predominantly to the abaxonal Schwann cell membrane. The presence of anti-GM2 Abs in ACP suggests that it may share a similar pathophysiology with GBS, for which it could thus be considered a naturally occurring animal model. PMID:23521648

  2. Enhanced Autophagy Contributes to Protective Effects of GM1 Ganglioside Against Aβ1-42-Induced Neurotoxicity and Cognitive Deficits.

    PubMed

    Dai, Ruwei; Zhang, Shijie; Duan, Wenjun; Wei, Renrong; Chen, Huifang; Cai, Weibin; Yang, Lei; Wang, Qi

    2017-08-01

    Alzheimer's disease (AD) is a progressive neurodegenerative disorder. The aggregation of Aβ peptides, Aβ1-42 in particular, is thought to be a fundamental pathogenic mechanism leading to the neuronal damage in AD. Recently, monosialoganglioside GM1 is reported to possess pivotal neuroprotection in neurodegenerative diseases. Previous studies have focused on the conformational dynamics and the biochemical interaction of the amyloid-peptide with the GM1 ganglioside, as well as the protective effect of GM1 on cognition. However, the phenomenon of autophagy with regard to neuronal dysfunction in AD is less investigated. In the present study, GM1 treatment were investigated in an AD mouse model and cultured PC12 dells to examine cognition-protective and neuroprotective effects of GM1. Furthermore, GM1 was found to induce autophagy via testing light chain 3 (LC3), Beclin1, neighbor of BRCA1 gene 1 protein and p62 (a substrate of LC3). Chloroquine, an inhibitor of lysosomal, was used to exclude the interference of lysosome, which could fuse with autophagosome and then clear it. In the presence of the inhibitor of autophagy (3-methyladenine; 3-MA), the protective effect of GM1 on PC12 cells in Aβ (1-42) induced toxic conditions was diminished. Interestingly, the expression of histone deacetylase 1 was increased in PC12 cells when treated with GM1, indicating that autophagy might be activated by GM1 through a pathway integrates protein acetylation. This study provides a novel insight into the protective role of GM1 against Aβ (1-42)-induced neurotoxicity via enhancing autophagy.

  3. Anti-GM1 ganglioside antibodies modulate membrane-associated sphingomyelin metabolism by altering neutral sphingomyelinase activity.

    PubMed

    Ueda, Akihiro; Shima, Sayuri; Murate, Kenitiroh; Kikuchi, Kouichi; Nagao, Ryunosuke; Maeda, Toshiki; Muto, Eri; Niimi, Yoshiki; Mizutani, Yasuaki; Mutoh, Tatsuro

    2018-06-01

    Previous studies have shown that patients with Guillain-Barré syndrome express autoantibodies against ganglioside GM1 (GM1), although its pathogenic significance for the development of the disease remains to be elucidated. nSMase2 is the best characterized neutral sphingomyelinase (nSMase) found in neuronal cells. Activation of this enzyme leads to ceramide production, which is a known second messenger of the cell-death program in neuronal cells. We have explored the effects of anti-GM1 antibodies on sphingomyelin metabolism of PC12 cells stably transfected with human trk cDNA (PCtrk cells) by determining their effects on nSMase2 activity. The data we present here strongly suggest that anti-GM1 caused a significant change in sphingomyelin content of the membrane fraction in PCtrk cells. Both nSMase2 activity and the level of nSMase2 protein were significantly decreased by anti-GM1 treatment of PCtrk cells, while acidic SMase activities remained unchanged. Our results indicate, for the first time, that anti-GM1 may produce profound impacts on lipid metabolism in neuronal cell membranes. Copyright © 2018 Elsevier Inc. All rights reserved.

  4. UVB-irradiated keratinocytes induce melanoma-associated ganglioside GD3 synthase gene in melanocytes via secretion of tumor necrosis factor α and interleukin 6

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Miyata, Maiko; Department of Biochemistry II, Nagoya University Graduate School of Medicine, 65 Tsurumai, Showa-ku, Nagoya 466-0065; Ichihara, Masatoshi

    Highlights: • Melanocytes showed low ST8SIA1 and high B3GALT4 levels in contrast with melanomas. • Direct UVB irradiation of melanocytes did not induce ganglioside synthase genes. • Culture supernatants of UVB-irradiated keratinocytes induced ST8SIA1 in melanocytes. • TNFα and IL-6 secreted from keratinocytes enhanced ST8SIA1 expression in melanocytes. • Inflammatory cytokines induced melanoma-related ST8SIA1 in melanocytes. - Abstract: Although expression of gangliosides and their synthetic enzyme genes in malignant melanomas has been well studied, that in normal melanocytes has been scarcely analyzed. In particular, changes in expression levels of glycosyltransferase genes responsible for ganglioside synthesis during evolution of melanomas frommore » melanocytes are very important to understand roles of gangliosides in melanomas. Here, expression of glycosyltransferase genes related to the ganglioside synthesis was analyzed using RNAs from cultured melanocytes and melanoma cell lines. Quantitative RT-PCR revealed that melanomas expressed high levels of mRNA of GD3 synthase and GM2/GD2 synthase genes and low levels of GM1/GD1b synthase genes compared with melanocytes. As a representative exogenous stimulation, effects of ultraviolet B (UVB) on the expression levels of 3 major ganglioside synthase genes in melanocytes were analyzed. Although direct UVB irradiation of melanocytes caused no marked changes, culture supernatants of UVB-irradiated keratinocytes (HaCaT cells) induced definite up-regulation of GD3 synthase and GM2/GD2 synthase genes. Detailed examination of the supernatants revealed that inflammatory cytokines such as TNFα and IL-6 enhanced GD3 synthase gene expression. These results suggest that inflammatory cytokines secreted from UVB-irradiated keratinocytes induced melanoma-associated ganglioside synthase genes, proposing roles of skin microenvironment in the promotion of melanoma-like ganglioside profiles in melanocytes.« less

  5. Size and Shape of Amyloid Fibrils Induced by Ganglioside Nanoclusters: Role of Sialyl Oligosaccharide in Fibril Formation.

    PubMed

    Matsubara, Teruhiko; Nishihara, Masaya; Yasumori, Hanaki; Nakai, Mako; Yanagisawa, Katsuhiko; Sato, Toshinori

    2017-12-05

    Ganglioside-enriched microdomains in the presynaptic neuronal membrane play a key role in the initiation of amyloid ß-protein (Aß) assembly related to Alzheimer's disease. We previously isolated lipids from a detergent-resistant membrane microdomain fraction of synaptosomes prepared from aged mouse brain and found that spherical Aß assemblies were formed on Aß-sensitive ganglioside nanoclusters (ASIGN) of reconstituted lipid bilayers in the synaptosomal fraction. In the present study, we investigated the role of oligosaccharides in Aß fibril formation induced by ganglioside-containing mixed lipid membranes that mimic the features of ASIGN. Ganglioside nanoclusters were constructed as ternary mixed lipid bilayers composed of ganglioside (GM1, GM2, GM3, GD1a, or GT1b), sphingomyelin, and cholesterol, and their surface topography was visualized by atomic force microscopy. Aß fibril formation on the nanocluster was strongly induced in the presence of 10 mol % ganglioside, and Aß-sensitive features were observed at cholesterol contents of 35-55 mol %. GM1-, GD1a-, and GT1b-containing membranes induced longer fibrils than those containing GD1b and GM2, indicating that the terminal galactose of GM1 along with N-acetylneuraminic acid accelerates protofibril elongation. These results demonstrate that Aß fibril formation is induced by ASIGN that are highly enriched ganglioside nanoclusters with a limited number of components and that the generation and elongation of Aß protofibrils are regulated by the oligosaccharide structure of gangliosides.

  6. Anti-GM2 ganglioside antibodies are a biomarker for acute canine polyradiculoneuritis.

    PubMed

    Rupp, Angie; Galban-Horcajo, Francesc; Bianchi, Ezio; Dondi, Maurizio; Penderis, Jacques; Cappell, Joanna; Burgess, Karl; Matiasek, Kaspar; McGonigal, Rhona; Willison, Hugh J

    2013-03-01

    Acute canine polyradiculoneuritis (ACP) is considered to be the canine equivalent of the human peripheral nerve disorder Guillain-Barré syndrome (GBS); an aetiological relationship, however, remains to be demonstrated. In GBS, anti-glycolipid antibodies (Abs) are considered as important disease mediators. To address the possibility of common Ab biomarkers, the sera of 25 ACP dogs, 19 non-neurological, and 15 epileptic control dogs were screened for IgG Abs to 10 glycolipids and their 1 : 1 heteromeric complexes using combinatorial glycoarrays. Anti-GM2 ganglioside Abs were detected in 14/25 ACP dogs, and anti-GA1 Abs in one further dog. All controls except for one were negative for anti-glycolipid Abs. In this cohort of cases and controls, the glycoarray screen reached a diagnostic sensitivity of 60% and a specificity of 97%; a lower sensitivity (32%) was reported using a conventional glycolipid ELISA. To address the possible pathogenic role for anti-GM2 Abs in ACP, we identified GM2 in canine sciatic nerve by both mass spectrometry and thin layer chromatography overlay. In immunohistological studies, GM2 was localized predominantly to the abaxonal Schwann cell membrane. The presence of anti-GM2 Abs in ACP suggests that it may share a similar pathophysiology with GBS, for which it could thus be considered a naturally occurring animal model. © 2013 Peripheral Nerve Society.

  7. Murine Sialidase Neu3 facilitates GM2 degradation and bypass in mouse model of Tay-Sachs disease.

    PubMed

    Seyrantepe, Volkan; Demir, Secil Akyildiz; Timur, Zehra Kevser; Von Gerichten, Johanna; Marsching, Christian; Erdemli, Esra; Oztas, Emin; Takahashi, Kohta; Yamaguchi, Kazunori; Ates, Nurselin; Dönmez Demir, Buket; Dalkara, Turgay; Erich, Katrin; Hopf, Carsten; Sandhoff, Roger; Miyagi, Taeko

    2018-01-01

    Tay-Sachs disease is a severe lysosomal storage disorder caused by mutations in Hexa, the gene that encodes for the α subunit of lysosomal β-hexosaminidase A (HEXA), which converts GM2 to GM3 ganglioside. Unexpectedly, Hexa -/- mice have a normal lifespan and show no obvious neurological impairment until at least one year of age. These mice catabolize stored GM2 ganglioside using sialidase(s) to remove sialic acid and form the glycolipid GA2, which is further processed by β-hexosaminidase B. Therefore, the presence of the sialidase (s) allows the consequences of the Hexa defect to be bypassed. To determine if the sialidase NEU3 contributes to GM2 ganglioside degradation, we generated a mouse model with combined deficiencies of HEXA and NEU3. The Hexa -/- Neu3 -/- mice were healthy at birth, but died at 1.5 to 4.5months of age. Thin-layer chromatography and mass spectrometric analysis of the brains of Hexa -/- Neu3 -/- mice revealed the abnormal accumulation of GM2 ganglioside. Histological and immunohistochemical analysis demonstrated cytoplasmic vacuolation in the neurons. Electron microscopic examination of the brain, kidneys and testes revealed pleomorphic inclusions of many small vesicles and complex lamellar structures. The Hexa -/- Neu3 -/- mice exhibited progressive neurodegeneration with neuronal loss, Purkinje cell depletion, and astrogliosis. Slow movement, ataxia, and tremors were the prominent neurological abnormalities observed in these mice. Furthermore, radiographs revealed abnormalities in the skeletal bones of the Hexa -/- Neu3 -/- mice. Thus, the Hexa -/- Neu3 -/- mice mimic the neuropathological and clinical abnormalities of the classical early-onset Tay-Sachs patients, and provide a suitable model for the future pre-clinical testing of potential treatments for this condition. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. Interaction of Clostridium perfringens delta toxin with erythrocyte and liposome membranes and relation with the specific binding to the ganglioside GM2.

    PubMed

    Jolivet-Reynaud, C; Hauttecoeur, B; Alouf, J E

    1989-01-01

    The specific interaction of the cytolytic Clostridium perfringens delta toxin with membrane GM2 was indicated by: (i) characterization of this glycolipid in the membrane of sheep and goat erythrocytes, which are lysed by the toxin, whereas GM2 was undetectable in insensitive rabbit erythrocytes, (ii) demonstration of 125I-toxin binding to GM2, by autoradiography, following incubation with thin-layer chromatograms containing separated neuroblastoma gangliosides, and (iii) toxin fixation by phospholipid-cholesterol unilamellar vesicles containing either sheep gangliosides or GM2. In order to investigate the intramembrane events leading to membrane disruption following toxin binding, the photoreactive probe 12(4-azido-2-nitrophenoxy)stearoyl 1-14C glucosamine, which inserts into the outer layer and labels integral membrane proteins, was used to establish whether delta toxin penetrates into target cell membrane. No toxin labeling was found, suggesting that toxin action takes place at the membrane surface. This contention is supported by the observation that despite toxin binding, GM2 liposomes did not release entrapped 14C-glucose. Treatment of toxin with carboxypeptidases, but not aminopeptidases, abolished both toxin binding capacity onto erythrocytes and its combination with antitoxin neutralizing antibodies, suggesting that the carboxy terminal end of the toxin is critical for binding to cell membrane.

  9. Cancer vaccines: an update with special focus on ganglioside antigens.

    PubMed

    Bitton, Roberto J; Guthmann, Marcel D; Gabri, Mariano R; Carnero, Ariel J L; Alonso, Daniel F; Fainboim, Leonardo; Gomez, Daniel E

    2002-01-01

    the (CIM) from La Havana, Cuba, to developed new strategies for specific active immunotherapy. The project included two ganglioside based vaccines and one anti-idiotypic vaccine. We focused on two antigens: first GM3, an ubiquitous antigen which is over-expressed in several epithelial tumor types; and a second one, N-Glycolyl-GM3 a more molecule, not being expressed in normal tissues and recently found in several neoplastic cells, in particular breast, melanoma and neuroectodermal cancer cells. We developed two vaccines, one with each antigen, both using proteins derived from the outer membrane proteins (OMP) of Neisseria Meningitidis B, as carriers. We developed also the 1E10 vaccine; an anti-idiotype vaccine designed to mimic the N-Glycolyl-GM3 gangliosides. This monoclonal antibody is an Ab2-type-antibody which recognizes the Ab1 antibody called P3, the latter is a monoclonal antibody that specifically recognizes gangliosides as antigens. Since 1998 we initiated a clinical development program for these three compounds. Results of the phase I clinical trials proved that the three vaccines were safe and able to elicit specific antibody responses. In addition we were able to demonstrate the activation of the cellular arm of the immune response in patients treated with the GM3 vaccine. Although phase I trials are not designed to evaluate antitumor efficacy, it was encouraging to observe tumor shrinkage in some patients treated both with the GM3 and N-Glycolyl-GM3 vaccines. We have already begun a phase II program in several neoplastic diseases, with all three vaccines.

  10. Major and c-series gangliosides in lenticular tissues: mammals to molluscs.

    PubMed

    Saito, M; Sugiyama, K

    2001-10-01

    Gangliosides of eye lenses were examined in mammals (rat, rabbits, pig, cow), bird (chicken), reptile (terrapin), amphibian (bullfrog), bony fish (red sea bream, bluefin tuna, bonito, Pacific mackerel) and molluscs (common squid, Pacific octopus). Besides the fact that GM3 was the common ganglioside species, the composition of major gangliosides in mammalian eye lenses significantly differed from each other. While gangliotetraose gangliosides were abundant in rat eye lens, they did not constitute major components in porcine and bovine tissues. The c-series ganglioside GT3 was expressed in rat eye lenses but were practically absent in other mammalian tissues. The composition of major gangliosides in eye lenses of lower animals varied from species to species, whereas c-series gangliosides were consistently expressed, showing similar compositional profiles. Our results demonstrate the species-specific compositions of lenticular gangliosides. Evidence was also provided suggesting that eye lenses of common squid (Todarodes pacificus) and Pacific octopus (Octopus vulgaris) express gangliosides including gangliotetraose species and c-series gangliosides.

  11. Unique Ganglioside Recognition Strategies for Clostridial Neurotoxins

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Benson, Marc A.; Fu, Zhuji; Kim, Jung-Ja P.

    2012-03-15

    Botulinum neurotoxins (BoNTs) and tetanus neurotoxin are the causative agents of the paralytic diseases botulism and tetanus, respectively. The potency of the clostridial neurotoxins (CNTs) relies primarily on their highly specific binding to nerve terminals and cleavage of SNARE proteins. Although individual CNTs utilize distinct proteins for entry, they share common ganglioside co-receptors. Here, we report the crystal structure of the BoNT/F receptor-binding domain in complex with the sugar moiety of ganglioside GD1a. GD1a binds in a shallow groove formed by the conserved peptide motif E ... H ... SXWY ... G, with additional stabilizing interactions provided by two argininemore » residues. Comparative analysis of BoNT/F with other CNTs revealed several differences in the interactions of each toxin with ganglioside. Notably, exchange of BoNT/F His-1241 with the corresponding lysine residue of BoNT/E resulted in increased affinity for GD1a and conferred the ability to bind ganglioside GM1a. Conversely, BoNT/E was not able to bind GM1a, demonstrating a discrete mechanism of ganglioside recognition. These findings provide a structural basis for ganglioside binding among the CNTs and show that individual toxins utilize unique ganglioside recognition strategies.« less

  12. Bis(monoacylglycero)phosphate and ganglioside GM1 spontaneously form small homogeneous vesicles at specific concentrations

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Chebukati, Janetricks N.; Goff, Philip C.; Frederick, Thomas E.

    2010-04-09

    The morphology and size of hydrated lipid dispersions of bis(monoacylglycero)phosphate (BMP) mixed with varying mole percentages of the ganglioside GM1 were investigated by dynamic light scattering (DLS) and transmission electron microscopy (TEM). Electron paramagnetic resonance (EPR) spectroscopy of these same mixtures, doped at 0.5 mol% with doxyl labeled lipids, was used to investigate acyl-chain packing. Results show that for 20-30% GM1, hydrated BMP:GM1 mixtures spontaneously form small spherical vesicles with diameters {approx}100 nm and a narrow size distribution profile. For other concentrations of GM1, hydrated dispersions with BMP have non-spherical shapes and heterogeneous size profiles, with average vesicle diameters >400more » nm. All samples were prepared at pH 5.5 to mimic the lumen acidity of the late endosome where BMP is an essential component of intraendosomal vesicle budding, lipid sorting and trafficking. These findings indicate that GM1 and BMP under a limited concentration range spontaneously form small vesicles of homogeneous size in an energy independent manner without the need of protein templating. Because BMP is essential for intraendosomal vesicle formation, these results imply that lipid-lipid interactions may play a critical role in the endosomal process of lipid sorting and trafficking.« less

  13. Revealing the Raft Domain Organization in the Plasma Membrane by Single-Molecule Imaging of Fluorescent Ganglioside Analogs.

    PubMed

    Suzuki, Kenichi G N; Ando, Hiromune; Komura, Naoko; Konishi, Miku; Imamura, Akihiro; Ishida, Hideharu; Kiso, Makoto; Fujiwara, Takahiro K; Kusumi, Akihiro

    2018-01-01

    Gangliosides have been implicated in a variety of physiological processes, particularly in the formation and function of raft domains in the plasma membrane. However, the scarcity of suitable fluorescent ganglioside analogs had long prevented us from determining exactly how gangliosides perform their functions in the live-cell plasma membrane. With the development of new fluorescent ganglioside analogs, as described by Komura et al. (2017), this barrier has been broken. We can now address the dynamic behaviors of gangliosides in the live-cell plasma membrane, using fluorescence microscopy, particularly by single-fluorescent molecule imaging and tracking. Single-molecule tracking of fluorescent GM1 and GM3 revealed that these molecules are transiently and dynamically recruited to monomers (monomer-associated rafts) and homodimer rafts of the raftophilic GPI-anchored protein CD59 in quiescent cells, with exponential residency times of 12 and 40ms, respectively, in a manner dependent on raft-lipid interactions. Upon CD59 stimulation, which induces CD59-cluster signaling rafts, the fluorescent GM1 and GM3 analogs were recruited to the signaling rafts, with a lifetime of 48ms. These results represent the first direct evidence that GPI-anchored receptors and gangliosides interact in a cholesterol-dependent manner. Furthermore, they show that gangliosides continually move in and out of rafts that contain CD59 in an extremely dynamic manner, with much higher frequency than expected previously. Such studies would not have been possible without fluorescent ganglioside probes, which exhibit native-like behavior and single-molecule tracking. In this chapter, we review the methods for single-molecule tracking of fluorescent ganglioside analogs and the results obtained by applying these methods. © 2018 Elsevier Inc. All rights reserved.

  14. GM1 ganglioside in Parkinson's disease: Pilot study of effects on dopamine transporter binding.

    PubMed

    Schneider, Jay S; Cambi, Franca; Gollomp, Stephen M; Kuwabara, Hiroto; Brašić, James R; Leiby, Benjamin; Sendek, Stephanie; Wong, Dean F

    2015-09-15

    GM1 ganglioside has been suggested as a treatment for Parkinson's disease (PD), potentially having symptomatic and disease modifying effects. The current pilot imaging study was performed to examine effects of GM1 on dopamine transporter binding, as a surrogate measure of disease progression, studied longitudinally. Positron emission tomography (PET) imaging data were obtained from a subset of subjects enrolled in a delayed start clinical trial of GM1 in PD [1]: 15 Early-start (ES) subjects, 14 Delayed-start (DS) subjects, and 11 Comparison (standard-of-care) subjects. Treatment subjects were studied over a 2.5 year period while Comparison subjects were studied over 2 years. Dynamic PET scans were performed over 90 min following injection of [(11)C]methylphenidate. Regional values of binding potential (BPND) were analyzed for several striatal volumes of interest. Clinical results for this subset of subjects were similar to those previously reported for the larger study group. ES subjects showed early symptomatic improvement and slow symptom progression over the study period. DS and Comparison subjects were initially on the same symptom progression trajectory but diverged once DS subjects received GM1 treatment. Imaging results showed significant slowing of BPND loss in several striatal regions in GM1-treated subjects and in some cases, an increased BPND in some striatal regions was detected after GM1 use. Results of this pilot imaging study provide additional data to suggest a potential disease modifying effect of GM1 on PD. These results need to be confirmed in a larger number of subjects. Copyright © 2015 Elsevier B.V. All rights reserved.

  15. Membrane-lipid homeostasis in a prodromal rat model of Alzheimer's disease: Characteristic profiles in ganglioside distributions during aging detected using MALDI imaging mass spectrometry.

    PubMed

    Caughlin, Sarah; Maheshwari, Shikhar; Agca, Yuksel; Agca, Cansu; Harris, Aaron J; Jurcic, Kristina; Yeung, Ken K-C; Cechetto, David F; Whitehead, Shawn N

    2018-06-01

    Accumulation of simple gangliosides GM2 and GM3, and gangliosides with longer long-chain bases (d20:1) have been linked to toxicity and the pathogenesis of Alzheimer's disease (AD). Conversely, complex gangliosides, such as GM1, have been shown to be neuroprotective. Recent evidence using matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS) has demonstrated that a-series gangliosides are differentially altered during normal aging, yet it remains unclear how simple species are shifting relative to complex gangliosides in the prodromal stages of AD. Ganglioside profiles in wild-type (Wt) and transgenic APP21 Fischer rats were detected and quantified using MALDI-IMS at P0 (birth), 3, 12, and 20 months of age and each species quantified to allow for individual species comparisons. Tg APP21 rats were found to have a decreased level of complex gangliosides in a number of brain regions as compared to Wt rats and showed higher levels of simple gangliosides. A unique pattern of expression was observed in the white matter as compared to gray matter regions, with an age-dependent decrease in GD1 d18:1 species observed and significantly elevated levels of GM3 in Tg APP21 rats. These results are indicative of a pathological shift in ganglioside homeostasis during aging that is exacerbated in Tg APP21 rats. Ganglioside dysregulation may occur in the prodromal stages of neurodegenerative diseases like AD. Copyright © 2018 Elsevier B.V. All rights reserved.

  16. Effect of structural modifications of ganglioside GM2 on intra-molecular carbohydrate-to-carbohydrate interaction and enzymatic susceptibility

    PubMed Central

    Li, Yu-Teh; Li, Su-Chen; Kiso, Makoto; Ishida, Hideharu; Mauri, Laura; Raimondi, Laura; Bernardi, Anna; Sonnino, Sandro

    2008-01-01

    Summary The effect of inter-molecular carbohydrate-to-carbohydrate interaction on basic cell biological processes has been well documented and appreciated. In contrast, very little is known about the intra-molecular carbohydrate-to-carbohydrate interaction. The presence of an interaction between the GalNAc and the Neu5Ac in GM2 detected by NMR spectroscopy represents a well-defined intra-molecular carbohydrate-to-carbohydrate interaction. This intriguing interaction is responsible for the GM2-epitope, GalNAcβ1Π4(Neu5Acα2Π3)Gal-, to exhibit a rigid and compact conformation. We hypothesized that this compact conformation may be the cause for both the GalNAc and the Neu5Ac in GM2 to be refractory to enzymatic hydrolysis and the GM2 activator protein is able to interact with the compact trisaccharide GM2-epitope, rendering the GalNAc and the Neu5Ac accessible to β-hexosaminidase A and sialidase. We have used a series of structurally modified GM2 to study the effect of modifications of sugar chains on the conformation and enzymatic susceptibility of this ganglioside. Our hypothesis was borne out by the fact that when the GalNAcβ1Π4Gal linkage in GM2 was converted to the GalNAcβ1Π6Gal, both the GalNAc and the Neu5Ac became susceptible to β-hexosaminidase A and sialidase, respectively, without GM2 activator protein. We hope our work will engender interest in identifying other intra-molecular carbohydrate-to-carbohydrate interactions in glycoconjugates. PMID:17967427

  17. Effect of structural modifications of ganglioside GM2 on intra-molecular carbohydrate-to-carbohydrate interaction and enzymatic susceptibility.

    PubMed

    Li, Yu-Teh; Li, Su-Chen; Kiso, Makoto; Ishida, Hideharu; Mauri, Laura; Raimondi, Laura; Bernardi, Anna; Sonnino, Sandro

    2008-03-01

    The effect of inter-molecular carbohydrate-to-carbohydrate interaction on basic cell biological processes has been well documented and appreciated. In contrast, very little is known about the intra-molecular carbohydrate-to-carbohydrate interaction. The presence of an interaction between the GalNAc and the Neu5Ac in GM2 detected by NMR spectroscopy represents a well-defined intra-molecular carbohydrate-to-carbohydrate interaction. This intriguing interaction is responsible for the GM2-epitope, GalNAcbeta1-->4(Neu5Acalpha2-->3)Gal-, to exhibit a rigid and compact conformation. We hypothesized that this compact conformation may be the cause for both the GalNAc and the Neu5Ac in GM2 to be refractory to enzymatic hydrolysis and the GM2 activator protein is able to interact with the compact trisaccharide GM2-epitope, rendering the GalNAc and the Neu5Ac accessible to beta-hexosaminidase A and sialidase. We have used a series of structurally modified GM2 to study the effect of modifications of sugar chains on the conformation and enzymatic susceptibility of this ganglioside. Our hypothesis was borne out by the fact that when the GalNAcbeta1-->4Gal linkage in GM2 was converted to the GalNAcbeta1-->6Gal, both the GalNAc and the Neu5Ac became susceptible to beta-hexosaminidase A and sialidase, respectively, without GM2 activator protein. We hope our work will engender interest in identifying other intra-molecular carbohydrate-to-carbohydrate interactions in glycoconjugates.

  18. Cholera toxin binding affinity and specificity for gangliosides determined by surface plasmon resonance

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kuziemko, G.M.; Stroh, M.; Stevens, R.C.

    1996-05-21

    The present study determines the affinity of cholera toxin for the ganglioside series GM1, GM2, GM3, GD1A, GD1B, GT1B, asialo GM1, globotriosyl ceramide, and lactosyl ceramide using real time biospecific interaction analysis (surface plasmon resonance, SPR). SPR shows that cholera toxin preferably binds to gangliosides in the following sequence: GM1 > GM2 > GD1A > GM3 > GT1B > GD1B > asialo-GM1. The measured binding affinity of cholera toxin for the ganglioside sequence ranges from 4.61 {times} 10{sup {minus}12} M for GM1 to 1.88 {times} 10{sup {minus}10} M for asialo GM1. The picomolar values obtained by surface plasmon resonance aremore » similar to K{sub d} values determined with whole-cell binding assays. Both whole-cell assays ans SPR measurements on synthetic membranes are higher than free solution measurements by several orders of magnitude. This difference may be caused by the effects of avidity and charged lipid head-groups, which may play a major role in the binding between cholera toxin, the receptor, and the membrane surface. The primary difference between free solution binding studies and surface plasmon resonance studies is that the latter technique is performed on surfaces resembling the cell membrane. Surface plasmon resonance has the further advantage of measuring apparent kinetic association and dissociation rates in real time, providing direct information about binding events at the membrane surface. 34 refs., 8 figs., 2 tabs.« less

  19. Complement Factor H and Simian Virus 40 bind the GM1 ganglioside in distinct conformations.

    PubMed

    Blaum, Bärbel S; Frank, Martin; Walker, Ross C; Neu, Ursula; Stehle, Thilo

    2016-05-01

    Mammalian cell surfaces are decorated with a variety of glycan chains that orchestrate development and defense and are exploited by pathogens for cellular attachment and entry. While glycosidic linkages are, in principle, flexible, the conformational space that a given glycan can sample is subject to spatial and electrostatic restrictions imposed by its overall chemical structure. Here, we show how the glycan moiety of the GM1 ganglioside, a branched, monosialylated pentasaccharide that serves as a ligand for various proteins, undergoes differential conformational selection in its interactions with different lectins. Using STD NMR and X-ray crystallography, we found that the innate immune regulator complement Factor H (FH) binds a previously not reported GM1 conformation that is not compatible with the GM1-binding sites of other structurally characterized GM1-binding lectins such as the Simian Virus 40 (SV40) capsid. Molecular dynamics simulations of the free glycan in explicit solvent on the 10 μs timescale reveal that the FH-bound conformation nevertheless corresponds to a minimum in the Gibbs free energy plot. In contrast to the GM1 conformation recognized by SV40, the FH-bound GM1 conformation is associated with poor NOE restraints, explaining how it escaped(1)H-(1)H NOE-restrained modeling in the past and highlighting the necessity for ensemble representations of glycan structures. © The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  20. Lactational changes in concentration and distribution of ganglioside molecular species in human breast milk from Chinese mothers.

    PubMed

    Ma, Lin; Liu, Xihong; MacGibbon, Alastair K H; Rowan, Angela; McJarrow, Paul; Fong, Bertram Y

    2015-11-01

    Gangliosides play a critical role in human brain development and function. Human breast milk (HBM) is an important dietary source of gangliosides for the growing infant. In this study, ganglioside concentrations were measured in the breast milk from a cross-sectional sample of Chinese mothers over an 8-month lactation period. The average total ganglioside concentration increased from 13.1 mg/l during the first month to 20.9 mg/l by 8 months of lactation. The average concentration during the typically solely breast-feeding period of 1‒6 months was 18.9 mg/l. This is the first study to report the relative distribution of the individual ganglioside molecular species through lactation for any population group. The ganglioside molecular species are made up of different fatty acid moieties that influence the physical properties of these gangliosides, and hence affect their function. The GM(3) molecular species containing long-chain acyl fatty acids had the most prominent changes, increasing in both concentration and relative distribution. The equivalent long-chain acyl fatty acid GD(3) molecular species typically decreased in concentration and relative distribution. The lactational trends for both concentration and relative distribution for the very long-chain acyl fatty acid molecular species were more varied. The major GM(3) and GD(3) molecular species during lactation were d40:1 and d42:1, respectively. An understanding of ganglioside molecular species distribution in HBM is essential for accurate application of mass spectrometry methods for ganglioside quantification.

  1. Childhood-Onset Multifocal Motor Neuropathy With Immunoglobulin M Antibodies to Gangliosides GM1 and GM2: A Case Report and Review of the Literature.

    PubMed

    Ishigaki, Hidetoshi; Hiraide, Takuya; Miyagi, Yoshifumi; Hayashi, Taiju; Matsubayashi, Tomoko; Shimoda, Ayumi; Kusunoki, Susumu; Fukuda, Tokiko

    2016-09-01

    Multifocal motor neuropathy is a rare immune-mediated neuropathy characterized by progressive asymmetric weakness and atrophy without sensory abnormalities. Although disease onset is usually in adulthood, a few childhood-onset cases have been reported. Here, we report the case of an 8-year-old boy with multifocal motor neuropathy who presented with a slowly progressive left and distal upper limb weakness without sensory loss. The initial high-dose intravenous immunoglobulin treatment significantly improved left upper limb muscle weakness. Continued monthly intravenous immunoglobulin treatment gradually improved muscle strength for several months initially. While the muscle strength decreased slightly after 8 months of therapy, it was better than that before intravenous immunoglobulin treatment. One year and eight months after the initiation of treatment, serum testing for IgM antibodies to gangliosides, GM1 and GM2, was negative. This is the first pediatric report of the serum IgM autoantibodies positive to GM1 and GM2. The clinical course is similar to that of partial intravenous immunoglobulin responders among patients with adulthood-onset multifocal motor neuropathy. Since the symptoms plateaued after the initial intravenous immunoglobulin therapy, prognosis appears to be determined by the patient's initial response to intravenous immunoglobulin treatment. Copyright © 2016 Elsevier Inc. All rights reserved.

  2. Atypical juvenile presentation of GM2 gangliosidosis AB in a patient compound-heterozygote for c.259G > T and c.164C > T mutations in the GM2A gene.

    PubMed

    Martins, Carla; Brunel-Guitton, Catherine; Lortie, Anne; Gauvin, France; Morales, Carlos R; Mitchell, Grant A; Pshezhetsky, Alexey V

    2017-06-01

    G M2 -gangliosidosis, AB variant is an extremely rare autosomal recessive inherited disorder caused by mutations in the GM2A gene that encodes G M2 ganglioside activator protein (GM2AP). GM2AP is necessary for solubilisation of G M2 ganglioside in endolysosomes and its presentation to β-hexosaminidase A. Conversely GM2AP deficiency impairs lysosomal catabolism of G M2 ganglioside, leading to its storage in cells and tissues. We describe a 9-year-old child with an unusual juvenile clinical onset of G M2 -gangliosidosis AB. At the age of 3 years he presented with global developmental delay, progressive epilepsy, intellectual disability, axial hypertonia, spasticity, seizures and ataxia, but without the macular cherry-red spots typical for G M2 gangliosidosis. Brain MRI detected a rapid onset of diffuse atrophy, whereas whole exome sequencing showed that the patient is a compound heterozygote for two mutations in GM2A : a novel nonsense mutation, c.259G > T (p.E87X) and a missense mutation c.164C > T (p.P55L) that was recently identified in homozygosity in patients of a Saudi family with a progressive chorea-dementia syndrome. Western blot analysis showed an absence of GM2AP in cultured fibroblasts from the patient, suggesting that both mutations interfere with the synthesis and/or folding of the protein. Finally, impaired catabolism of G M2 ganglioside in the patient's fibroblasts was demonstrated by metabolic labeling with fluorescently labeled G M1 ganglioside and by immunohistochemistry with anti-G M2 and anti-G M3 antibodies. Our observation expands the molecular and clinical spectrum of molecular defects linked to G M2 -gangliosidosis and suggests novel diagnostic approach by whole exome sequencing and perhaps ganglioside analysis in cultured patient's cells.

  3. Identification of ganglioside GM2 activator playing a role in cancer cell migration through proteomic analysis of breast cancer secretomes.

    PubMed

    Shin, Jihye; Kim, Gamin; Lee, Jong Won; Lee, Ji Eun; Kim, Yoo Seok; Yu, Jong-Han; Lee, Seung-Taek; Ahn, Sei Hyun; Kim, Hoguen; Lee, Cheolju

    2016-06-01

    Cancer cell secretomes are considered a potential source for the discovery of cancer markers. In this study, the secretomes of four breast cancer (BC) cell lines (Hs578T, MCF-7, MDA-MB-231, and SK-BR-3) were profiled with liquid chromatography-tandem mass spectrometry analysis. A total of 1410 proteins were identified with less than 1% false discovery rate, of which approximately 55% (796 proteins) were predicted to be secreted from cells. To find BC-specific proteins among the secreted proteins, data of immunohistochemical staining compiled in the Human Protein Atlas were investigated by comparing the data of BC tissues with those of normal tissues. By applying various criteria, including higher expression level in BC tissues, higher predicted potential of secretion, and sufficient number of tandem mass spectra, 12 biomarker candidate proteins including ganglioside GM2 activator (GM2A) were selected for confirmation. Western blot analysis and ELISA for plasma samples of healthy controls and BC patients revealed elevation of GM2A in BC patients, especially those who were estrogen receptor-negative. Additionally, siRNA-mediated knockdown of GM2A in BC cells decreased migration in vitro, whereas the overexpression of GM2A led to an increase in cell migration. Although GM2A as a diagnostic and prognostic marker in BC should be carefully verified further, this study has established the potential role of GM2A in BC progression. © 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

  4. NEU3 sialidase strictly modulates GM3 levels in skeletal myoblasts C2C12 thus favoring their differentiation and protecting them from apoptosis.

    PubMed

    Anastasia, Luigi; Papini, Nadia; Colazzo, Francesca; Palazzolo, Giacomo; Tringali, Cristina; Dileo, Loredana; Piccoli, Marco; Conforti, Erika; Sitzia, Clementina; Monti, Eugenio; Sampaolesi, Maurilio; Tettamanti, Guido; Venerando, Bruno

    2008-12-26

    Membrane-bound sialidase NEU3, often referred to as the "ganglioside sialidase," has a critical regulatory function on the sialoglycosphingolipid pattern of the cell membrane, with an anti-apoptotic function, especially in cancer cells. Although other sialidases have been shown to be involved in skeletal muscle differentiation, the role of NEU3 had yet to be disclosed. Herein we report that NEU3 plays a key role in skeletal muscle differentiation by strictly modulating the ganglioside content of adjacent cells, with special regard to GM3. Induced down-regulation of NEU3 in murine C2C12 myoblasts, even when partial, totally inhibits their capability to differentiate by increasing the GM3 level above a critical point, which causes epidermal growth factor receptor inhibition (and ultimately its down-regulation) and an higher responsiveness of myoblasts to the apoptotic stimuli.

  5. GM1 ganglioside counteracts cholinergic and behavioral deficits induced in the rat by intracerebral injection of vincristine.

    PubMed

    Di Patre, P L; Abbamondi, A; Bartolini, L; Pepeu, G

    1989-03-14

    The intracerebroventricular injection of 0.5 mg of vincristine sulphate in adult male Wistar rats caused within 11 days the impairment of motor and reflexive behavior, evaluated by the elevated platform and hanging wire tests, a decrease in food consumption and loss of body weight, a 45% decrease in hippocampal choline acetyltransferase (ChAT) activity and a 35% decrease in the rate of high-affinity choline uptake (HACU) in the injected side. The latter effects are due to the death of neurons in the respective hemiseptum. Intrafimbrial injection of vincristine caused the same decrease in ChAT activity without behavioral alterations. Daily i.p. administration of GM1 ganglioside, beginning immediately after the vincristine injection, prevented dose dependently the decrease in ChAT activity and HACU rate. Prevention was complete with the 60 mg/kg dose. The same dose was equally active on ChAT activity when given s.c. but was inactive p.o. The ChAT decrease was also prevented when GM1 treatment began 5 days after vincristine. GM1 60 mg/kg i.p. also reduced the behavioral toxicity of vincristine. The possibility that GM1 might prevent vincristine toxicity by antagonizing its disruption of neurofilaments and axonal flow is discussed.

  6. Specific binding of Haemophilus influenzae to minor gangliosides of human respiratory epithelial cells.

    PubMed Central

    Fakih, M G; Murphy, T F; Pattoli, M A; Berenson, C S

    1997-01-01

    Gangliosides are sialylated glycosphingolipids that serve as receptors for various bacteria. To investigate endogenous gangliosides of human respiratory epithelial cells as potential receptors for Haemophilus influenzae, three strains, including nontypeable H. influenzae (NTHI) 1479, and isogenic fimbriated (f+) and nonfimbriated (f0) H. influenzae type b 770235, were 3H labeled and overlaid on two-dimensional thin-layer chromatography (TLC) plates containing either purified HEp-2 gangliosides or murine brain gangliosides. NTHI 1479 bound exclusively to two distinct minor ganglioside doublets, with mobilities near that of GM1. These minor gangliosides comprised only 14.2 and 9.4% of the total, respectively. NTHI 1479 also bound to a distinct ganglioside of human macrophages whose chromatographic mobilities closely resemble those of one of the NTHI-binding gangliosides of HEp-2 cells. H. influenzae type b 770235 f+ and f0 each bound to a different minor HEp-2 ganglioside doublet, with proportionately weaker affinity for a major ganglioside doublet. Remarkably, none of the three strains bound to any murine brain gangliosides. Moreover, when 80 to 90% of sialic acid residues were enzymatically removed from HEp-2 gangliosides, NTHI 1479 binding was proportionately impaired, compared with untreated controls. Our findings support a role for specific gangliosides of specific cells as receptors for H. influenzae strains. Our findings further demonstrate that individual minor gangliosides possess unique biological properties. PMID:9125549

  7. Synthesis of ganglioside epitopes for oligosaccharide specific immunoadsorption therapy of Guillian-Barré syndrome.

    PubMed

    Andersen, Søren M; Ling, Chang-Chun; Zhang, Ping; Townson, Kate; Willison, Hugh J; Bundle, David R

    2004-04-21

    Guillain-Barré syndrome is a postinfectious, autoimmune neuropathy resulting in neuromuscular paralysis. Auto-antibodies, often induced by bacterial infection, bind to human gangliosides possessing monosialoside and diasialoside epitopes and impair the function of nerve junctions, where these ganglioside structures are highly enriched. Truncated gangliosides representive of GD3, GQ1b and GM2 epitopes have been synthesized as methyl glycosides and as a glycosides of an eleven carbon tether. The synthetic oligosaccharide ligands are structural mimics of these highly complex ganglioside epitopes and via their ability to neutralize or remove auto-antibodies have the potential for therapy, either as soluble blocking ligands administered systemically, or as immuno-affinity ligands for use as extracorporeal immunoadsorbents.

  8. Radiometric assay for ganglioside sialidase applied to the determination of the enzyme subcellular location in culture human fibroblasts

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Chigorno, V.; Cardace, G.; Pitto, M.

    1986-03-01

    A radiometric method for the assay of ganglioside sialidase in cultured human fibroblasts was set up. As substrate, highly radioactive (1.28 Ci/mmol) ganglioside GD/sub 1a/ isotopically tritium-labeled at carbon C-3 of the long chain base was employed; the liberated, and TLC separated (/sup 3/H)GM/sub 1/ was determined by computer-assisted radiochromatoscanning. Under experimental conditions that provided a low and quite acceptable (4-5%) coefficient of variation, the detection limit of the method was 0.1 nmol of liberated GM/sub 1/, using as low as 10 ..mu..g of fibroblast homogenate as protein. The detection limit could be lowered to 0.02-0.03 nmol, adopting conditions that,more » however, carried a higher analytical error (coefficient of variation over 10%). The content of ganglioside sialidase in human fibroblasts cultured in 75-cm/sup 2/ plastic flasks was 5.8 -/+ 2.5 (SD) nmol liberated GM/sub 1/ h/sup -1/ mg protein/sup -1/. Subfractionation studies performed on fibroblast homogenate showed that the ganglioside sialidase was mainly associated with the light membrane subfraction that was rich in plasma and intracellular membranes. This subfraction displayed almost no sialidase activity on the artificial substrate 4-methylumbelliferyl-D-N-acetylneuraminic acid. A small but measurable ganglioside sialidase activity was also present in the lysosome-enriched subfraction, which contained a very high sialidase activity on the above artificial substrate.« less

  9. Molecular analysis of a GM2-activator deficiency in two patients with GM2-gangliosidosis AB variant.

    PubMed Central

    Schepers, U.; Glombitza, G.; Lemm, T.; Hoffmann, A.; Chabas, A.; Ozand, P.; Sandhoff, K.

    1996-01-01

    Lysosomal degradation of ganglioside GM2 by beta-hexosaminidase A (hex A) requires the presence of the GM2 activator protein (GM2AP) as an essential cofactor. A deficiency of the GM2 activator causes the AB variant of GM2 gangliosidosis, a recessively inherited disorder characterized by excessive neuronal accumulation of GM2 and related glycolipids. Two novel mutations in the GM2 activator gene (GM2A) have been identified by the reverse-transcriptase-PCR method--a three-base deletion, AAG262-264, resulting in a deletion of Lys88, and a single-base deletion, A410, that causes a frameshift. The latter results in substitution of 33 amino acids and the loss of another 24 amino acid residues. Both patients are homoallelic for their respective mutations inherited from their parents, who are heteroallelic at the GM2A locus. Although the cultured fibroblasts of both patients produce normal levels of activator mRNA, they lack a lysosomal form of GM2AP. Pulse/chase labeling of cultured fibroblasts of the patients, in presence and absence of brefeldin A, indicates a premature degradation of both--mutant and truncated--GM2APs in the endoplasmic reticulum or Golgi. These results were supported by in vitro translation experiments and expression of the mutated proteins. When the mutated GM2APs were expressed in Escherichia coli, both mature GM2AP forms turned proved to exhibit only residual activities in an in vitro assay. Images Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 PMID:8900233

  10. Localization patterns of the ganglioside GM1 in human sperm are indicative of male fertility and independent of traditional semen measures

    PubMed Central

    Cardona, Cristina; Neri, Queenie V.; Simpson, Alana J.; Moody, Melissa A.; Ostermeier, G. Charles; Seaman, Eric K.; Paniza, Theodore; Rosenwaks, Zev; Palermo, Gianpiero D.

    2017-01-01

    Semen analysis lacks a functional component and best identifies extreme cases of infertility. The ganglioside GM1 is known to have functional roles during capacitation and acrosome exocytosis. Here, we assessed whether GM1 localization patterns (Cap‐Score™) correspond with male fertility in different settings: Study 1 involved couples pursuing assisted reproduction in a tertiary care fertility clinic, while Study 2 involved men with known fertility versus those questioning their fertility at a local urology center. In Study 1, we examined various thresholds versus clinical history for 42 patients; 13 had Cap‐Scores ≥39.5%, with 12 of these (92.3%) achieving clinical pregnancy by natural conception or ≤3 intrauterine insemination cycles. Of the 29 patients scoring <39.5%, only six (20.7%) attained clinical pregnancy by natural conception or ≤3 intrauterine insemination cycles. In Study 2, Cap‐Scores were obtained from 76 fertile men (Cohort 1, pregnant partner or recent father) and compared to 122 men seeking fertility assessment (Cohort 2). Cap‐Score values were normally distributed in Cohort 1, with 13.2% having Cap‐Scores more than one standard deviation below the mean (35.3 ± 7.7%). Significantly, more men in Cohort 2 had Cap‐Scores greater than one standard deviation below the normal mean (33.6%; p = 0.001). Minimal/no relationship was found between Cap‐Score and sperm concentration, morphology, or motility. Together, these data demonstrate that Cap‐Score provides novel, clinically relevant insights into sperm function and male fertility that complement traditional semen analysis. Furthermore, the data provide normal reference ranges for fertile men that can help clinicians counsel couples toward the most appropriate fertility treatment. PMID:28418610

  11. Protective effect of gangliosides on DNA in human spermatozoa exposed to cryopreservation.

    PubMed

    Gavella, Mirjana; Lipovac, Vaskresenija; Garaj-Vrhovac, Verica; Gajski, Goran

    2012-01-01

    Gangliosides, the sialic acid-containing glycosphyngolipids, are amphiphilic compounds which in micellar form affect the properties and functions of a cellular membrane. The aim of this study was to test whether exogenous gangliosides supplied to cryopreservation media before freezing could protect sperm cells from cryopreservation-induced DNA damage assessed by Comet assay. Additionally, to investigate whether gangliosides were also able to reduce membrane integrity damage, malonaldialdehyde as a measure of lipid peroxidation and sperm-specific lactate dehydrogenase-C4 activity as an enzyme marker of sperm membrane leakage were determined. The monosialogangliosides (GM1) and trisialogangliosides (GT1b) were examined at a concentration of 100 μM, which was above their respective critical micellar concentrations. Exogenous gangliosides were not found to protect sperm membrane from lipid peroxidation. However, a freezing-/thawing-induced increase in Comet parameters was equally significantly prevented by the presence of both GM1 and GT1b (P < .05), indicating that the ceramide moiety, rather than the polar groups, is involved in the protective ability of gangliosides. The observed phenomena suggest that ganglioside micelles could modulate hydrophobic properties of the sperm membrane responsible for better tolerance to DNA fragmentation, thus protecting DNA integrity from cryopreservation-induced damage.

  12. Analysis of cholera toxin-ganglioside interactions by flow cytometry.

    PubMed

    Lauer, Sabine; Goldstein, Byron; Nolan, Rhiannon L; Nolan, John P

    2002-02-12

    Cholera toxin entry into mammalian cells is mediated by binding of the pentameric B subunit (CTB) to ganglioside GM(1) in the cell membrane. We used flow cytometry to quantitatively measure in real time the interactions of fluorescently labeled pentameric cholera toxin B-subunit (FITC-CTB) with its ganglioside receptor on microsphere-supported phospholipid membranes. A model that describes the multiple steps of this mode of recognition was developed to guide our flow cytometric experiments and extract relevant equilibrium and kinetic rate constants. In contrast to previous studies, our approach takes into account receptor cross-linking, an important feature for multivalent interactions. From equilibrium measurements, we determined an equilibrium binding constant for a single subunit of FITC-CTB binding monovalently to GM(1) presented in bilayers of approximately 8 x 10(7) M(-1) while that for binding to soluble GM(1)-pentasaccharide was found to be approximately 4 x 10(6) M(-1). From kinetic measurements, we determined the rate constant for dissociation of a single site of FITC-CTB from microsphere-supported bilayers to be (3.21 +/- 0.03) x 10(-3) s(-1), and the rate of association of a site on FITC-CTB in solution to a GM(1) in the bilayer to be (2.8 +/- 0.4) x 10(4) M(-1) s(-1). These values yield a lower estimate for the equilibrium binding constant of approximately 1 x 10(7) M(-1). We determined the equilibrium surface cross-linking constant [(1.1 +/- 0.1) x 10(-12) cm(2)] and from this value and the value for the rate constant for dissociation derived a value of approximately 3.5 x 10(-15) cm(2) s(-1) for the forward rate constant for cross-linking. We also compared the interaction of the receptor binding B-subunit with that of the whole toxin (A- and B-subunits). Our results show that the whole toxin binds with approximately 100-fold higher avidity than the pentameric B-subunit alone which is most likely due to the additional interaction of the A(2)-subunit

  13. TNF-alpha induction of GM2 expression on renal cell carcinomas promotes T cell dysfunction.

    PubMed

    Raval, Gira; Biswas, Soumika; Rayman, Patricia; Biswas, Kaushik; Sa, Gaurisankar; Ghosh, Sankar; Thornton, Mark; Hilston, Cynthia; Das, Tanya; Bukowski, Ronald; Finke, James; Tannenbaum, Charles S

    2007-05-15

    Previous studies from our laboratory demonstrated the role of tumor-derived gangliosides as important mediators of T cell apoptosis, and hence, as one mechanism by which tumors evade immune destruction. In this study, we report that TNF-alpha secreted by infiltrating inflammatory cells and/or genetically modified tumors augments tumor-associated GM2 levels, which leads to T cell death and immune dysfunction. The conversion of weakly apoptogenic renal cell carcinoma (RCC) clones to lines that can induce T cell death requires 3-5 days of TNF-alpha pretreatment, a time frame paralleling that needed for TNF-alpha to stimulate GM2 accumulation by SK-RC-45, SK-RC-54, and SK-RC-13. RCC tumor cell lines permanently transfected with the TNF-alpha transgene are similarly toxic for T lymphocytes, which correlates with their constitutively elevated levels of GM2. TNF-alpha increases GM2 ganglioside expression by enhancing the mRNA levels encoding its synthetic enzyme, GM2 synthase, as demonstrated by both RT-PCR and Southern analysis. The contribution of GM2 gangliosides to tumor-induced T cell death was supported by the finding that anti-GM2 Abs significantly blocked T cell apoptosis mediated by TNF-alpha-treated tumor cells, and by the observation that small interfering RNA directed against TNF-alpha abrogated GM2 synthase expression by TNF-transfected SK-RC-45, diminished its GM2 accumulation, and inhibited its apoptogenicity for T lymphocytes. Our results indicate that TNF-alpha signaling promotes RCC-induced killing of T cells by stimulating the acquisition of a distinct ganglioside assembly in RCC tumor cells.

  14. [GM1-dot-EIA for the detection of toxin-producing Vibrio cholerae strains].

    PubMed

    Markina, O V; Alekseeva, L P; Telesmanich, N R; Chemisova, O S; Akulova, M V; Markin, N V

    2011-05-01

    A new variant of enzyme immunoassay (EIA) has been developed on the basis of GM1 gangliosides to detect the toxin-producing Vibrio cholerae strains--GM1-dot-EIA. Experiments were run using a nitrocellulose membrane to bind GM1 gangliosides and polyclonal antitoxic serum to detect cholerogen. GM1-dot-EIA testing identified cholera toxin in 11 of 13 supernatants of V. cholerae eltor ctx(+) strains isolated from man and in 3 of 7 supernatants of V. cholerae eltor ctx(+) strains isolated from water. These data agree with those obtained in CM1-EIA. There was no reaction with the supernatants of other microorganisms. The sensitivity of the technique was 10 ng/ml. Thus, the simple and specific GM1-dot-EIA may be recommended to detect toxin-producing V cholerae strains isolated from man and water.

  15. Glycosylated SV2 and Gangliosides as Dual Receptors for Botulinum Neurotoxin Serotype F

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Fu, Zhuji; Chen, Chen; Barbieri, Joseph T.

    2010-02-22

    Botulinum neurotoxin causes rapid flaccid paralysis through the inhibition of acetylcholine release at the neuromuscular junction. The seven BoNT serotypes (A-G) have been proposed to bind motor neurons via ganglioside-protein dual receptors. To date, the structure-function properties of BoNT/F host receptor interactions have not been resolved. Here, we report the crystal structures of the receptor binding domains (HCR) of BoNT/A and BoNT/F and the characterization of the dual receptors for BoNT/F. The overall polypeptide fold of HCR/A is essentially identical to the receptor binding domain of the BoNT/A holotoxin, and the structure of HCR/F is very similar to that ofmore » HCR/A, except for two regions implicated in neuronal binding. Solid phase array analysis identified two HCR/F binding glycans: ganglioside GD1a and oligosaccharides containing an N-acetyllactosamine core. Using affinity chromatography, HCR/F bound native synaptic vesicle glycoproteins as part of a protein complex. Deglycosylation of glycoproteins using {alpha}(1-3,4)-fucosidase, endo-{beta}-galactosidase, and PNGase F disrupted the interaction with HCR/F, while the binding of HCR/B to its cognate receptor, synaptotagmin I, was unaffected. These data indicate that the HCR/F binds synaptic vesicle glycoproteins through the keratan sulfate moiety of SV2. The interaction of HCR/F with gangliosides was also investigated. HCR/F bound specifically to gangliosides that contain {alpha}2,3-linked sialic acid on the terminal galactose of a neutral saccharide core (binding order GT1b = GD1a GM3; no binding to GD1b and GM1a). Mutations within the putative ganglioside binding pocket of HCR/F decreased binding to gangliosides, synaptic vesicle protein complexes, and primary rat hippocampal neurons. Thus, BoNT/F neuronal discrimination involves the recognition of ganglioside and protein (glycosylated SV2) carbohydrate moieties, providing a structural basis for the high affinity and specificity of BoNT/F for

  16. Dependence of the form factor of ganglioside micelles on a conformational change with temperature

    NASA Astrophysics Data System (ADS)

    Corti, Mario; Boretta, Marco; Cantù, Laura; Del Favero, Elena; Lesieur, Pierre

    1996-09-01

    The gangliosides GM2, GM1 and GD1b, biological amphiphiles with a double tail hydrophobic part and an oligosaccharide chain headgroup, form micelles in solution. Light scattering experiments have shown that ganglioside micelles which have gone through a temperature cycle have a smaller molecular mass and hydrodynamic radius than those which have been kept at room temperature. This fact has been interpreted with the hypothesis that, with temperature, the ganglioside molecules undergo a conformational change which affects their micellar properties appreciably. Careful small angle X-ray experiments, aimed to confirm the light scattering data and to evidence differences in the micellar internal structure are presented. Ganglioside micelles are quite inhomogeneous particles with respect to X-ray scattering, since there is a large contrast variation between the inner lipid part and the external hydrated sugar layer. Experimental form factors are fitted with a double-shell oblate-ellipsoid model.

  17. Imaging mass spectrometry detection of gangliosides species in the mouse brain following transient focal cerebral ischemia and long-term recovery.

    PubMed

    Whitehead, Shawn N; Chan, Kenneth H N; Gangaraju, Sandhya; Slinn, Jacqueline; Li, Jianjun; Hou, Sheng T

    2011-01-01

    Gangliosides, a member of the glycosphingolipid family, are heterogeneously expressed in biological membranes and are particularly enriched within the central nervous system. Gangliosides consist of mono- or poly-sialylated oligosaccharide chains of variable lengths attached to a ceramide unit and are found to be intimately involved in brain disease development. The purpose of this study is to examine the spatial profile of ganglioside species using matrix-assisted laser desorption/ionization (MALDI) imaging (IMS) following middle cerebral artery occlusion (MCAO) reperfusion injury in the mouse. IMS is a powerful method to not only discriminate gangliosides by their oligosaccharide components, but also by their carbon length within their sphingosine base. Mice were subjected to a 30 min unilateral MCAO followed by long-term survival (up to 28 days of reperfusion). Brain sections were sprayed with the matrix 5-Chloro-2-mercaptobenzothiazole, scanned and analyzed for a series of ganglioside molecules using an Applied Biosystems 4800 MALDI TOF/TOF. Traditional histological and immunofluorescence techniques were performed to assess brain tissue damage and verification of the expression of gangliosides of interest. Results revealed a unique anatomical profile of GM1, GD1 and GT1b (d18:1, d20:1 as well as other members of the glycosphingolipid family). There was marked variability in the ratio of expression between ipsilateral and contralateral cortices for the various detected ganglioside species following MCAO-reperfusion injury. Most interestingly, MCAO resulted in the transient induction of both GM2 and GM3 signals within the ipsilateral hemisphere; at the border of the infarcted tissue. Taken together, the data suggest that brain region specific expression of gangliosides, particularly with respect to hydrocarbon length, may play a role in neuronal responses to injury.

  18. GM1 ganglioside reduces the motor incoordination and loss of righting reflex caused by acute ethanol in C57BL/6J mice

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wallis, C.; Rezazadeh, S.M.; Forster, M.J.

    1992-02-26

    Ethanol produces its intoxicating effects by modifying neuronal membranes. Gangliosides stabilize neuronal membranes and promote their recovery from a variety of insults. In this experiment, the efficacy of GM1(i.p.) to reverse ethanol intoxication was evaluated in male mice trained to run on a constantly accelerating rotorod. When mice were tested 15-min following saline or ethanol GM1 pre-treatment reduced rotorod performance by 15% but was ineffective in modifying the ethanol-impaired performance. However, when mice were tested at 15, 35, 55, 75, and 95 min intervals following ethanol, GM1 pre-treatments dose-dependently reduced the efficacy and duration of ethanol in producing motor incoordination.more » Further, GM1 given prior to ethanol significantly prolonged the time to onset of the loss of righting reflex from 1.4 to 1.9 min, and reduced the duration of the righting-reflex loss from 94 to 77 min. This GM1 effect was seen at 24 h, but not at 48 or 72 h after its administration. The blood ethanol concentration at awakening was significantly higher in 24h GM1-treated animals than in controls suggesting that the GM1 effect was not due to an alteration in ethanol clearance. These findings support the hypothesis that GM1 promotes recovery from ethanol intoxication via a neuroprotective mechanism.« less

  19. The GM1 and GM2 Gangliosidoses: Natural History and Progress toward Therapy.

    PubMed

    Regier, Debra S; Proia, Richard L; D'Azzo, Alessandra; Tifft, Cynthia J

    2016-06-01

    The gangliosidoses are lysosomal storage disorders caused by accumulation of GM1 or GM2 gangliosides. GM1 gangliosidosis has both central nervous system and systemic findings; while, GM2 gangliosidosis is restricted primarily to the central nervous system. Both disorders have autosomal recessive modes of inheritance and a continuum of clinical presentations from a severe infantile form to a milder, chronic adult form. Both are devastating diseases without cure or specific treatment however, with the use of supportive aggressive medical management, the lifespan and quality of life has been extended for both diseases. Naturally occurring and engineered animal models that mimic the human diseases have enhanced our understanding of the pathogenesis of disease progression. Some models have shown significant improvement in symptoms and lifespan with enzyme replacement, substrate reduction, and anti-inflammatory treatments alone or in combination. More recently gene therapy has shown impressive results in large and small animal models. Treatment with FDA-approved glucose analogs to reduce the amount of ganglioside substrate is used as off-label treatments for some patients. Therapies also under clinical development include small molecule chaperones and gene therapy.

  20. In search of a solution to the sphinx-like riddle of GM1.

    PubMed

    Ledeen, Robert W; Wu, Gusheng

    2010-12-01

    Among the many glycoconjugates contributing to the sugar code, gangliosides have drawn special attention owing to their predominance as the major sialoglycoconjugate category within the nervous system. However, their occurrence, albeit at lower levels, appears ubiquitous in vertebrate cells and even some invertebrate tissues. Now that over 100 gangliosides have been structurally characterized, their diverse physiological functions constitute a remaining enigma. This has been especially true of GM1, for which a surprising array of functions has already been revealed. Our current research has focused on two areas of GM1 function: (a) signaling induced in neural and immune cells by cross-linking of GM1 in the plasma membrane that leads to activation of TRPC5 (transient receptor potiential, canonical form 5) channels, a process important in neuritogenesis and autoimmune suppression; (b) activation by GM1 of a sodium-calcium exchanger (NCX) in the inner membrane of the nuclear envelope (NE) with resulting modulation of nuclear and cellular calcium. The latter has a role in maintaining neuronal viability, loss of which renders neurons vulnerable to Ca(2+) overload. Pathological manifestations in mutant mice and their cultured neurons lacking GM1 have shown dramatic rescue with a membrane permeable derivative of GM1 that enters the nucleus and restores NCX activity. Nuclear function of GM1 is related to the presence of neuraminidase in the NE, an enzyme that generates GM1 through hydrolysis of GD1a. A different isoform of this enzyme was found in each of the two membranes of the NE.

  1. Rapid Profiling of Bovine and Human Milk Gangliosides by Matrix-Assisted Laser Desorption/Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

    PubMed Central

    Lee, Hyeyoung; An, Hyun Joo; Lerno, Larry A.; German, J. Bruce; Lebrilla, Carlito B.

    2010-01-01

    Gangliosides are anionic glycosphingolipids widely distributed in vertebrate tissues and fluids. Their structural and quantitative expression patterns depend on phylogeny and are distinct down to the species level. In milk, gangliosides are exclusively associated with the milk fat globule membrane. They may participate in diverse biological processes but more specifically to host-pathogen interactions. However, due to the molecular complexities, the analysis needs extensive sample preparation, chromatographic separation, and even chemical reaction, which makes the process very complex and time-consuming. Here, we describe a rapid profiling method for bovine and human milk gangliosides employing matrix-assisted desorption/ionization (MALDI) Fourier transform ion cyclotron resonance (FTICR) mass spectrometry (MS). Prior to the analyses of biological samples, milk ganglioside standards GM3 and GD3 fractions were first analyzed in order to validate this method. High mass accuracy and high resolution obtained from MALDI FTICR MS allow for the confident assignment of chain length and degree of unsaturation of the ceramide. For the structural elucidation, tandem mass spectrometry (MS/MS), specifically as collision-induced dissociation (CID) and infrared multiphoton dissociation (IRMPD) were employed. Complex ganglioside mixtures from bovine and human milk were further analyzed with this method. The samples were prepared by two consecutive chloroform/methanol extraction and solid phase extraction. We observed a number of differences between bovine milk and human milk. The common gangliosides in bovine and human milk are NeuAc-NeuAc-Hex-Hex-Cer (GD3) and NeuAc-Hex-Hex-Cer (GM3); whereas, the ion intensities of ganglioside species are different between two milk samples. Kendrick mass defect plot yields grouping of ganglioside peaks according to their structural similarities. Gangliosides were further probed by tandem MS to confirm the compositional and structural assignments

  2. Anti-ganglioside antibodies in Guillain-Barré syndrome and chronic inflammatory demyelinating polyneuropathy in Chinese patients.

    PubMed

    Fan, Chenghe; Jin, Haiqiang; Hao, Hongjun; Gao, Feng; Sun, Yongan; Lu, Yuanyuan; Liu, Yuanyuan; Lv, Pu; Cui, Wei; Teng, Yuming; Huang, Yining

    2017-04-01

    In this study we investigated the relationships between anti-ganglioside antibodies and Guillain-Barré syndrome (GBS) and chronic inflammatory demyelinating polyneuropathy (CIDP). Samples from 48 Chinese patients diagnosed with GBS and 18 patients diagnosed with CIDP were retrospectively reviewed. In the GBS patients, 62.5% were classified as having acute inflammatory demyelinating polyneuropathy (AIDP), 27.1% were found to have acute motor axonal neuropathy (AMAN), and 10.4% were unclassified. Serum IgG anti-ganglioside antibodies were detected in 46.2% of the AMAN patients and in 6.7% of the AIDP patients (P < 0.05); 5.6% of the 18 CIDP patients were IgG antibody positive, and 27.8% were IgM antibody positive. Facial palsy and sensory impairment were significantly associated with IgM antibodies. These results suggest that IgG anti-GM1 antibodies are associated with AMAN, but not with AIDP, and that IgM antibodies against GM1, GM2, and GM3 are associated with facial nerve palsy. Muscle Nerve 55: 470-475, 2017. © 2016 Wiley Periodicals, Inc.

  3. Design and efficient synthesis of novel GM2 analogues with respect to the elucidation of the function of GM2 activator.

    PubMed

    Komori, Tatsuya; Ando, Takayuki; Imamura, Akihiro; Li, Yu-Teh; Ishida, Hideharu; Kiso, Makoto

    2008-10-01

    To elucidate the mechanism underlying the hydrolysis of the GalNAcbeta1-->4Gal linkage in ganglioside GM2 [GalNAcbeta1-->4(NeuAcalpha2-->3)Galbeta1-->4Glcbeta1-->1' Cer] by beta-hexosaminidase A (Hex A) with GM2 activator protein, we designed and synthesized two kinds of GM2 linkage analogues-6'-NeuAc-GM2 and alpha-GalNAc-GM2. In this paper, the efficient and systematic synthesis of these GM2 analogues was described. The highlight of our synthesis process is that the key intermediates, newly developed sialyllactose derivatives, were efficiently prepared in sufficient quantities; these derivatives directly served as highly reactive glycosyl acceptors and coupled with GalNTroc donors to furnish the assembly of GM2 tetrasaccharides in large quantities.

  4. Complete localization of disulfide bonds in GM2 activator protein.

    PubMed Central

    Schütte, C. G.; Lemm, T.; Glombitza, G. J.; Sandhoff, K.

    1998-01-01

    Lysosomal degradation of ganglioside GM2 by hexosaminidase A requires the presence of a small, non-enzymatic cofactor, the GM2-activator protein (GM2AP). Lack of functional protein leads to the AB variant of GM2-gangliosidosis, a fatal lysosomal storage disease. Although its possible mode of action and functional domains have been discussed frequently in the past, no structural information about GM2AP is available so far. Here, we determine the complete disulfide bond pattern of the protein. Two of the four disulfide bonds present in the protein were open to classical determination by enzymatic cleavage and mass spectrometry. The direct localization of the remaining two bonds was impeded by the close vicinity of cysteines 136 and 138. We determined the arrangement of these disulfide bonds by MALDI-PSD analysis of disulfide linked peptides and by partial reduction, cyanylation and fragmentation in basic solution, as described recently (Wu F, Watson JT, 1997, Protein Sci 6:391-398). PMID:9568910

  5. The possible contribution of a general glycosphingolipid transporter, GM2 activator protein, to atherosclerosis.

    PubMed

    Yanai, Hidekatsu; Yoshida, Hiroshi; Tomono, Yoshiharu; Tada, Norio; Chiba, Hitoshi

    2006-12-01

    We previously found that oxidized low-density lipoprotein (LDL) elevated the expression of mRNA of GalNAcbeta1-4[NeuNAcalpha2-3]Galbeta1-4Glc-Cer (GM2) ganglioside activator protein, in human monocyte-derived macrophages. Recently, GM2 activator protein has become known as a general glycosphingolipid transporter as well as a specific cofactor for the hydrolysis of GM2 ganglioside by lysosomal beta-hexosaminidase A. Accumulation of glycosphingolipids has been observed in the serum or aorta of atherosclerotic model animals and humans. The proliferation of aortic smooth muscle cells, elevation of LDL uptake by macrophages, interfering LDL clearance by the liver, and enhancement of platelet adhesion to collagen have been proposed as the underlying mechanisms of glycosphingolipid-mediated atherogenesis. The GM2 activator protein can bind, solubilize and transport a broad spectrum of lipid molecules, indicating that GM2 activator protein may function as a general intra- and inter-cellular lipid transport protein. Collectively, elevated levels of GM2 activator protein in the aorta may be another feature of human atherosclerosis.

  6. Molecular analysis of a GM2-activator deficiency in two patients with GM2-gangliosidosis AB variant

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Schepers, U.; Glombitza, G.; Lemm, T.

    1996-11-01

    Lysosomal degradation of ganglioside GM2 by {beta}-hexosaminidase A (hex A) requires the presence of the GM2 activator protein (GM2AP) as an essential cofactor. A deficiency of the GM2 activator causes the AB variant of GM2 gangliosidosis, a recessively inherited disorder characterized by excessive neuronal accumulation of GM2 and related glycolipids. Two novel mutations in the GM2 activator gene (GM2A) have been identified by the reverse-transcriptase-PCR method - a three-base deletion, AAG{sup 262-264}, resulting in a deletion of Lys{sup 88}, and a single-base deletion, A{sup 410}, that causes a frameshift. The latter results in substitution of 33 amino acids and themore » loss of another 24 amino acid residues. Both patients are homoallelic for their respective mutations inherited from their parents, who are heteroallelic at the GM2A locus. Although the cultured fibroblasts of both patients produce normal levels of activator mRNA, they lack a lysosomal form of GM2AP. Pulse/chase labeling of cultured fibroblasts of the patients, in presence and absence of brefeldin A, indicates a premature degradation of both-mutant and truncated-GM2APs in the endoplasmic reticulum or Golgi. These results were supported by in vitro translation experiments and expression of the mutated proteins. When the mutated GM2APs were expressed in Escherichia coli, both mature GM2AP forms turned proved to exhibit only residual activities in an in vitro assay. 26 refs., 7 figs.« less

  7. A new approach to the modification of cell membrane glycosphingolipids: Ganglioside composition of JTC-12 P3 cells altered by feeding with galactose as a sole carbohydrate source in protein- and lipid-free synthetic medium

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kawaguchi, Tatsuya; Takaoka, Toshiko; Yoshida, Eiko

    1988-12-01

    A significant difference in the glycosphingolipid composition of JTC-12 P3 cells established from monkey kidney tissue was observed when cells cultured in a protein- and lipid-free synthetic medium containing glucose (DM-160) as a sole carbohydrate source were transferred and cultured in the same medium containing galactose and pyruvic acid (DM-170) in place of glucose. In particular, the amounts of gangliosides GM3, GM2, and GD3 in the cells cultured in DM-170 were 5.3-, 17.8-, and more than 8-fold those in the cells cultured in DM-160, respectively, indicating that anabolism of gangliosides is greatly enhanced in cells cultured in the presence ofmore » galactose and pyruvic acid, as compared with cells cultured in the presence of glucose. In fact, after cultivation of cells in the medium with N-acetyl-D-({sup 14}C)mannosamine for 96 h, the radioactivity incorporated into the gangliosides of the cells in DM-170 was 10-fold that of the cells in DM-160. Among the gangliosides of the cells in DM-170, highly sialylated molecules such as GD3, GD1a, GD1b, and GT1b were preferentially labeled, indicating that the sialytransferases responsible for the synthesis of gangliosides are significantly more activated in cells cultured in DM-170 than in DM-160. These observations reveal that the glycosphingolipid composition of the plasma membrane can be modified epigenetically under well-defined conditions and provide important clues for clarifying the roles of glycosphingolipids associated with particular cell functions.« less

  8. Composite ganglioside autoantibodies and immune treatment response in MMN and MADSAM.

    PubMed

    Martinez-Thompson, Jennifer M; Snyder, Melissa R; Ettore, Michael; McKeon, Andrew; Pittock, Sean J; Roforth, Matthew M; Mandrekar, Jay; Mauermann, Michelle L; Taylor, Bruce V; Dyck, P James B; Windebank, Anthony J; Klein, Christopher J

    2018-06-01

    Multifocal motor neuropathy (MMN) is a motor only, asymmetric onset neuropathy that is relatively treatment-refractory compared with classic chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) and multifocal acquired demyelinating sensory and motor (MADSAM) neuropathy. We reviewed 35 patients seropositive for GM1 (monosialo-asialo [immunoglobulin M, IgM; immunoglobulin G, IgG]) and/or GD1b (disialo [IgG, IgM]) autoantibodies having MMN, classic CIDP, or MADSAM. Immune-treatment responsiveness and clinical course was compared with antibody negative disease controls. Seventy-nine percent of seropositives with an initial diagnosis of MMN were immunotherapy responsive compared with 46% of seronegatives (P = 0.045). Eight ganglioside antibody positive MMN patients of 19 (42%) developed sensory findings consistent with MADSAM compared with 3 of 41 (7%) seronegative MMN patients (P = 0.003). MMN and MADSAM patients with ganglioside antibody positivity had more sustained treatment responses (P = 0.03). Patients initially diagnosed with MMN seropositive for diverse GM1 autoantibodies appear more likely to have sustained treatment response and evolution to MADSAM. Muscle Nerve 57: 1000-1005, 2018. © 2017 Wiley Periodicals, Inc.

  9. Neuraminidases 3 and 4 regulate neuronal function by catabolizing brain gangliosides.

    PubMed

    Pan, Xuefang; De Aragão, Camila De Britto Pará; Velasco-Martin, Juan P; Priestman, David A; Wu, Harry Y; Takahashi, Kohta; Yamaguchi, Kazunori; Sturiale, Luisella; Garozzo, Domenico; Platt, Frances M; Lamarche-Vane, Nathalie; Morales, Carlos R; Miyagi, Taeko; Pshezhetsky, Alexey V

    2017-08-01

    Gangliosides (sialylated glycolipids) play an essential role in the CNS by regulating recognition and signaling in neurons. Metabolic blocks in processing and catabolism of gangliosides result in the development of severe neurologic disorders, including gangliosidoses manifesting with neurodegeneration and neuroinflammation. We demonstrate that 2 mammalian enzymes, neuraminidases 3 and 4, play important roles in catabolic processing of brain gangliosides by cleaving terminal sialic acid residues in their glycan chains. In neuraminidase 3 and 4 double-knockout mice, G M3 ganglioside is stored in microglia, vascular pericytes, and neurons, causing micro- and astrogliosis, neuroinflammation, accumulation of lipofuscin bodies, and memory loss, whereas their cortical and hippocampal neurons have lower rate of neuritogenesis in vitro Double-knockout mice also have reduced levels of G M1 ganglioside and myelin in neuronal axons. Furthermore, neuraminidase 3 deficiency drastically increased storage of G M2 in the brain tissues of an asymptomatic mouse model of Tay-Sachs disease, a severe human gangliosidosis, indicating that this enzyme is responsible for the metabolic bypass of β-hexosaminidase A deficiency. Together, our results provide the first in vivo evidence that neuraminidases 3 and 4 have important roles in CNS function by catabolizing gangliosides and preventing their storage in lipofuscin bodies.-Pan, X., De Britto Pará De Aragão, C., Velasco-Martin, J. P., Priestman, D. A., Wu, H. Y., Takahashi, K., Yamaguchi, K., Sturiale, L., Garozzo, D., Platt, F. M., Lamarche-Vane, N., Morales, C. R., Miyagi, T., Pshezhetsky, A. V. Neuraminidases 3 and 4 regulate neuronal function by catabolizing brain gangliosides. © FASEB.

  10. Anti-ganglioside antibodies in patients with systemic lupus erythematosus and neurological manifestations.

    PubMed

    Labrador-Horrillo, M; Martinez-Valle, F; Gallardo, E; Rojas-Garcia, R; Ordi-Ros, J; Vilardell, M

    2012-05-01

    Anti-ganglioside antibodies (AGA) have been associated with several peripheral neuropathies, such as Miller-Fisher syndrome, Guillain-Barré syndrome and multifocal motor neuropathy. They have also been studied in patients with systemic lupus erythematosus (SLE), focusing on neuropsychiatric manifestations and peripheral neuropathy, but the results are contradictory. To study the presence of AGA in a large cohort of patients with SLE and neuropsychiatric manifestations. Serum from 65 consecutive patients with SLE and neuropsychiatric manifestations, collected from 1985 to 2009, was tested for the presence of AGA antibodies (GM1, GM2, GM3, asialo-GM1 GD1a, GD1b, GD3, GT1b, GQ1b) using a standard enzyme-linked immunosorbent assay ELISA test (INCAT 1999) and thin layer chromatography (TLC). Positive results for asialo-GM1 (IgM) were found in 10 patients, 6 were positive for asialo-GM1 (IgM and IgG), and 4 were positive for other AGA such as GM1, GM2, GM3, GD1b, GT1b, GD3, (mainly IgM). Clinical and statistical studies showed no correlation between AGA and neuropsychiatric manifestations of SLE. Although some patients showed reactivity to AGA, these antibodies are not a useful marker of neuropsychiatric manifestations in SLE patients.

  11. Inefficiency in GM2 ganglioside elimination by human lysosomal beta-hexosaminidase beta-subunit gene transfer to fibroblastic cell line derived from Sandhoff disease model mice.

    PubMed

    Itakura, Tomohiro; Kuroki, Aya; Ishibashi, Yasuhiro; Tsuji, Daisuke; Kawashita, Eri; Higashine, Yukari; Sakuraba, Hitoshi; Yamanaka, Shoji; Itoh, Kohji

    2006-08-01

    Sandhoff disease (SD) is an autosomal recessive GM2 gangliosidosis caused by the defect of lysosomal beta-hexosaminidase (Hex) beta-subunit gene associated with neurosomatic manifestations. Therapeutic effects of Hex subunit gene transduction have been examined on Sandhoff disease model mice (SD mice) produced by the allelic disruption of Hexb gene encoding the murine beta-subunit. We demonstrate here that elimination of GM2 ganglioside (GM2) accumulated in the fibroblastic cell line derived from SD mice (FSD) did not occur when the HEXB gene only was transfected. In contrast, a significant increase in the HexB (betabeta homodimer) activity toward neutral substrates, including GA2 (asialo-GM2) and oligosaccharides carrying the terminal N-acetylglucosamine residues at their non-reducing ends (GlcNAc-oligosaccharides) was observed. Immunoblotting with anti-human HexA (alphabeta heterodimer) serum after native polyacrylamide gel electrophoresis (Native-PAGE) revealed that the human HEXB gene product could hardly form the chimeric HexA through associating with the murine alpha-subunit. However, co-introduction of the HEXA encoding the human alpha-subunit and HEXB genes caused significant corrective effect on the GM2 degradation by producing the human HexA. These results indicate that the recombinant human HexA could interspeciesly associate with the murine GM2 activator protein to degrade GM2 accumulated in the FSD cells. Thus, therapeutic effects of the recombinant human HexA isozyme but not human HEXB gene product could be evaluated by using the SD mice.

  12. Effects of Gangliosides on the Activity of the Plasma Membrane Ca2+-ATPase

    PubMed Central

    Jiang, Lei; Bechtel, Misty D.; Bean, Jennifer L.; Winefield, Robert; Williams, Todd D.; Zaidi, Asma; Michaelis, Elias K.; Michaelis, Mary L.

    2014-01-01

    Control of intracellular calcium concentrations ([Ca2+]i) is essential for neuronal function, and the plasma membrane Ca2+-ATPase (PMCA) is crucial for the maintenance of low [Ca2+]i. We previously reported on loss of PMCA activity in brain synaptic membranes during aging. Gangliosides are known to modulate Ca2+ homeostasis and signal transduction in neurons. In the present study, we observed age-related changes in the ganglioside composition of synaptic plasma membranes. This led us to hypothesize that alterations in ganglioside species might contribute to the age-associated loss of PMCA activity. To probe the relationship between changes in endogenous ganglioside content or composition and PMCA activity in membranes of cortical neurons, we induced depletion of gangliosides by treating neurons with D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP). This caused a marked decrease in the activity of PMCA, which suggested a direct correlation between ganglioside content and PMCA activity. Neurons treated with neuraminidase exhibited an increase in GM1 content, a loss in poly-sialoganglioside content, and a decrease in PMCA activity that was greater than that produced by D-PDMP treatment. Thus, it appeared that poly-sialogangliosides had a stimulatory effect whereas mono-sialogangliosides had the opposite effect. Our observations add support to previous reports of PMCA regulation by gangliosides by demonstrating that manipulations of endogenous ganglioside content and species affect the activity of PMCA in neuronal membranes. Furthermore, our studies suggest that age-associated loss in PMCA activity may result in part from changes in the lipid environment of this Ca2+ transporter. PMID:24434060

  13. Ganglioside structure dictates signal transduction by cholera toxin and association with caveolae-like membrane domains in polarized epithelia.

    PubMed

    Wolf, A A; Jobling, M G; Wimer-Mackin, S; Ferguson-Maltzman, M; Madara, J L; Holmes, R K; Lencer, W I

    1998-05-18

    In polarized cells, signal transduction by cholera toxin (CT) requires apical endocytosis and retrograde transport into Golgi cisternae and perhaps ER (Lencer, W.I., C. Constable, S. Moe, M. Jobling, H.M. Webb, S. Ruston, J.L. Madara, T. Hirst, and R. Holmes. 1995. J. Cell Biol. 131:951-962). In this study, we tested whether CT's apical membrane receptor ganglioside GM1 acts specifically in toxin action. To do so, we used CT and the related Escherichia coli heat-labile type II enterotoxin LTIIb. CT and LTIIb distinguish between gangliosides GM1 and GD1a at the cell surface by virtue of their dissimilar receptor-binding B subunits. The enzymatically active A subunits, however, are homologous. While both toxins bound specifically to human intestinal T84 cells (Kd approximately 5 nM), only CT elicited a cAMP-dependent Cl- secretory response. LTIIb, however, was more potent than CT in eliciting a cAMP-dependent response from mouse Y1 adrenal cells (toxic dose 10 vs. 300 pg/well). In T84 cells, CT fractionated with caveolae-like detergent-insoluble membranes, but LTIIb did not. To investigate further the relationship between the specificity of ganglioside binding and partitioning into detergent-insoluble membranes and signal transduction, CT and LTIIb chimeric toxins were prepared. Analysis of these chimeric toxins confirmed that toxin-induced signal transduction depended critically on the specificity of ganglioside structure. The mechanism(s) by which ganglioside GM1 functions in signal transduction likely depends on coupling CT with caveolae or caveolae-related membrane domains.

  14. The glycolipid GM1 reshapes asymmetric biomembranes and giant vesicles by curvature generation.

    PubMed

    Dasgupta, Raktim; Miettinen, Markus S; Fricke, Nico; Lipowsky, Reinhard; Dimova, Rumiana

    2018-05-29

    The ganglioside GM1 is present in neuronal membranes at elevated concentrations with an asymmetric spatial distribution. It is known to generate curvature and can be expected to strongly influence the neuron morphology. To elucidate these effects, we prepared giant vesicles with GM1 predominantly present in one leaflet of the membrane, mimicking the asymmetric GM1 distribution in neuronal membranes. Based on pulling inward and outward tubes, we developed a technique that allowed the direct measurement of the membrane spontaneous curvature. Using vesicle electroporation and fluorescence intensity analysis, we were able to quantify the GM1 asymmetry across the membrane and to subsequently estimate the local curvature generated by the molecule in the bilayer. Molecular-dynamics simulations confirm the experimentally determined dependence of the membrane spontaneous curvature as a function of GM1 asymmetry. GM1 plays a crucial role in connection with receptor proteins. Our results on curvature generation of GM1 point to an additional important role of this ganglioside, namely in shaping neuronal membranes. Copyright © 2018 the Author(s). Published by PNAS.

  15. Chemical Synthesis of GM2 Glycans, Bioconjugation with Bacteriophage Qβ, and the Induction of Anticancer Antibodies

    PubMed Central

    Yin, Zhaojun; Dulaney, Steven; McKay, Craig S.; Baniel, Claire; Kaczanowska, Katarzyna; Ramadan, Sherif; Finn, M. G.

    2016-01-01

    The development of carbohydrate-based antitumor vaccines is an attractive approach towards tumor prevention and treatment. Herein, we focused on the ganglioside GM2 tumor-associated carbohydrate antigen (TACA), which is overexpressed in a wide range of tumor cells. GM2 was synthesized chemically and conjugated with a virus-like particle derived from bacteriophage Qβ. Although the copper-catalyzed azide–alkyne cyclo-addition reaction efficiently introduced 237 copies of GM2 per Qβ, this construct failed to induce significant amounts of anti-GM2 antibodies compared to the Qβ control. In contrast, GM2 immobilized on Qβ through a thiourea linker elicited high titers of IgG antibodies that recognized GM2-positive tumor cells and effectively induced cell lysis through complement-mediated cytotoxicity. Thus, bacteriophage Qβ is a suitable platform to boost antibody responses towards GM2, a representative member of an important class of TACA: the ganglioside. PMID:26538065

  16. Induction of a melanoma-specific antibody response by a monovalent, but not a divalent, synthetic GM2 neoglycopeptide.

    PubMed

    Bay, S; Fort, S; Birikaki, L; Ganneau, C; Samain, E; Coïc, Y-M; Bonhomme, F; Dériaud, E; Leclerc, C; Lo-Man, R

    2009-04-01

    The GM2 ganglioside represents an important target for specific anticancer immunotherapy. We designed and synthesized a neoglycopeptide immunogen displaying one or two copies of the GM2 tetrasaccharidic moiety. These glycopeptides were prepared using the Huisgen cycloaddition, which enables the efficient ligation of the alkyne-functionalized biosynthesized GM2 with an azido CD4(+) T cell epitope peptide. It is worth noting that the GM2 can be produced on a gram scale in bacteria, which can be advantageous for a scale-up of the process. We show here for the first time that a fully synthetic glycopeptide, which is based on a ganglioside carbohydrate moiety, can induce human tumor cell-specific antibodies after immunization in mice. Interestingly, the monovalent, but not the divalent, form of GM2 peptide construct induced antimelanoma antibodies. Unlike traditional vaccines, this vaccine is a pure chemically-defined entity, a key quality for consistent studies and safe clinical evaluation. Therefore, such carbohydrate-peptide conjugate represents a promising cancer vaccine strategy for active immunotherapy targeting gangliosides.

  17. Rapid and sensitive MRM-based mass spectrometry approach for systematically exploring ganglioside-protein interactions.

    PubMed

    Tian, Ruijun; Jin, Jing; Taylor, Lorne; Larsen, Brett; Quaggin, Susan E; Pawson, Tony

    2013-04-01

    Gangliosides are ubiquitous components of cell membranes. Their interactions with bacterial toxins and membrane-associated proteins (e.g. receptor tyrosine kinases) have important roles in the regulation of multiple cellular functions. Currently, an effective approach for measuring ganglioside-protein interactions especially in a large-scale fashion is largely missing. To this end, we report a facile MS-based approach to explore gangliosides extracted from cells and measure their interactions with protein of interest globally. We optimized a two-step protocol for extracting total gangliosides from cells within 2 h. Easy-to-use magnetic beads conjugated with a protein of interest were used to capture interacting gangliosides. To measure ganglioside-protein interaction on a global scale, we applied a high-sensitive LC-MS system, containing hydrophilic interaction LC separation and multiple reaction monitoring-based MS for ganglioside detection. Sensitivity for ganglioside GM1 is below 100 pg, and the whole analysis can be done in 20 min with isocratic elution. To measure ganglioside interactions with soluble vascular endothelial growth factor receptor 1 (sFlt1), we extracted and readily detected 36 species of gangliosides from perivascular retinal pigment epithelium cells across eight different classes. Twenty-three ganglioside species have significant interactions with sFlt1 as compared with IgG control based on p value cutoff <0.05. These results show that the described method provides a rapid and high-sensitive approach for systematically measuring ganglioside-protein interactions. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. GD3/proteosome vaccines induce consistent IgM antibodies against the ganglioside GD3.

    PubMed

    Livingston, P O; Calves, M J; Helling, F; Zollinger, W D; Blake, M S; Lowell, G H

    1993-09-01

    The gangliosides of melanoma and other tumours of neuroectodermal origin are suitable targets for immune intervention with tumour vaccines. The optimal vaccines in current use contain ganglioside plus bacillus Calmette-Guérin and induce considerable morbidity. We have screened a variety of new adjuvants in the mouse, and describe one antigen-delivery system, proteosomes, which is especially effective. Highly hydrophobic Neisserial outer membrane proteins (OMP) form multimolecular liposome-like vesicular structures termed proteosomes which can readily incorporate amphiphilic molecules such as GD3 ganglioside. The optimal GD3/proteosome vaccine formulation for induction of GD3 antibodies in the mouse is determined. Interestingly, the use of potent immunological adjuvants in addition to proteosomes augments the IgM and IgG antibody titres against OMP in these vaccines but GD3 antibody titres are unaffected. The application of proteosomes to enhance the immune response to GD3 extends the concept of the proteosome immunopotentiating system from lipopeptides to amphipathic carbohydrate epitopes such as cell-surface gangliosides. The demonstrated safety of meningococcal OMP in humans and the data in mice presented here suggest that proteosome vaccines have potential for augmenting the immunogenicity of amphipathic tumour antigens in humans.

  19. Helicobacter pylori and Complex Gangliosides

    PubMed Central

    Roche, Niamh; Ångström, Jonas; Hurtig, Marina; Larsson, Thomas; Borén, Thomas; Teneberg, Susann

    2004-01-01

    Recognition of sialic acid-containing glycoconjugates by the human gastric pathogen Helicobacter pylori has been repeatedly demonstrated. To investigate the structural requirements for H. pylori binding to complex gangliosides, a large number of gangliosides were isolated and characterized by mass spectrometry and proton nuclear magnetic resonance. Ganglioside binding of sialic acid-recognizing H. pylori strains (strains J99 and CCUG 17874) and knockout mutant strains with the sialic acid binding adhesin SabA or the NeuAcα3Galβ4GlcNAcβ3Galβ4GlcNAcβ-binding neutrophil-activating protein HPNAP deleted was investigated using the thin-layer chromatogram binding assay. The wild-type bacteria bound to N-acetyllactosamine-based gangliosides with terminal α3-linked NeuAc, while gangliosides with terminal NeuGcα3, NeuAcα6, or NeuAcα8NeuAcα3 were not recognized. The factors affecting binding affinity were identified as (i) the length of the N-acetyllactosamine carbohydrate chain, (ii) the branches of the carbohydrate chain, and (iii) fucose substitution of the N-acetyllactosamine core chain. While the J99/NAP− mutant strain displayed a ganglioside binding pattern identical to that of the parent J99 wild-type strain, no ganglioside binding was obtained with the J99/SabA− mutant strain, demonstrating that the SabA adhesin is the sole factor responsible for the binding of H. pylori bacterial cells to gangliosides. PMID:14977958

  20. Chemical Synthesis of GM2 Glycans, Bioconjugation with Bacteriophage Qβ, and the Induction of Anticancer Antibodies.

    PubMed

    Yin, Zhaojun; Dulaney, Steven; McKay, Craig S; Baniel, Claire; Kaczanowska, Katarzyna; Ramadan, Sherif; Finn, M G; Huang, Xuefei

    2016-01-01

    The development of carbohydrate-based antitumor vaccines is an attractive approach towards tumor prevention and treatment. Herein, we focused on the ganglioside GM2 tumor-associated carbohydrate antigen (TACA), which is overexpressed in a wide range of tumor cells. GM2 was synthesized chemically and conjugated with a virus-like particle derived from bacteriophage Qβ. Although the copper-catalyzed azide-alkyne cycloaddition reaction efficiently introduced 237 copies of GM2 per Qβ, this construct failed to induce significant amounts of anti-GM2 antibodies compared to the Qβ control. In contrast, GM2 immobilized on Qβ through a thiourea linker elicited high titers of IgG antibodies that recognized GM2-positive tumor cells and effectively induced cell lysis through complement-mediated cytotoxicity. Thus, bacteriophage Qβ is a suitable platform to boost antibody responses towards GM2, a representative member of an important class of TACA: the ganglioside. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Systemic Gene Transfer of a Hexosaminidase Variant Using an scAAV9.47 Vector Corrects GM2 Gangliosidosis in Sandhoff Mice.

    PubMed

    Osmon, Karlaina J L; Woodley, Evan; Thompson, Patrick; Ong, Katalina; Karumuthil-Melethil, Subha; Keimel, John G; Mark, Brian L; Mahuran, Don; Gray, Steven J; Walia, Jagdeep S

    2016-07-01

    GM2 gangliosidosis is a group of neurodegenerative diseases caused by β-hexosaminidase A (HexA) enzyme deficiency. There is currently no cure. HexA is composed of two similar, nonidentical subunits, α and β, which must interact with the GM2 activator protein (GM2AP), a substrate-specific cofactor, to hydrolyze GM2 ganglioside. Mutations in either subunit or the activator can result in the accumulation of GM2 ganglioside within neurons throughout the central nervous system. The resulting neuronal cell death induces the primary symptoms of the disease: motor impairment, seizures, and sensory impairments. This study assesses the long-term effects of gene transfer in a Sandhoff (β-subunit knockout) mouse model. The study utilized a modified human β-hexosaminidase α-subunit (μ-subunit) that contains critical sequences from the β-subunit that enables formation of a stable homodimer (HexM) and interaction with GM2AP to hydrolyze GM2 ganglioside. We investigated a self-complementary adeno-associated viral (scAAV) vector expressing HexM, through intravenous injections of the neonatal mice. We monitored one cohort for 8 weeks and another cohort long-term for survival benefit, behavioral, biochemical, and molecular analyses. Untreated Sandhoff disease (SD) control mice reached a humane endpoint at approximately 15 weeks, whereas treated mice had a median survival age of 40 weeks, an approximate 2.5-fold survival advantage. On behavioral tests, the treated mice outperformed their knockout age-matched controls and perform similarly to the heterozygous controls. Through the enzymatic and GM2 ganglioside analyses, we observed a significant decrease in the GM2 ganglioside level, even though the enzyme levels were not significantly increased. Molecular analyses revealed a global distribution of the vector between brain and spinal cord regions. In conclusion, the neonatal delivery of a novel viral vector expressing the human HexM enzyme is effective in ameliorating the SD

  2. Radioimmune assay of ganglioside GM/sub 1/ synthase using cholera toxin

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Honke, K.; Taniguchi, N.; Makita, A.

    1986-01-01

    A radioimmune assay for uridine 5'-diphosphate-galactose (UDP-Gal):GM/sub 2/ galactosyltransferase, which synthesizes GM/sub 1/, has been developed utilizing cholera toxin. This assay is more sensitive and simpler than previously used assays. Radioactive nucleotide substrate and GM/sub 2/ were incubated with an enzyme sample, and a radiolabeled product, GM/sub 1/, was reacted with cholera toxin. The GM/sub 1/-cholera toxin complex was further reacted with anti-cholera toxin and Staphylococcus aureus cell suspension. The resulting complex was transferred onto a nitrocellulose membrane and quantitated by liquid scintillation counting. This assay was found to be sensitive for the detection of 100 pmol of the reactionmore » product, GM/sub 1/. With this assay method, some properties of the crude enzyme extracts from rat liver were studied. The enzyme had a pH optimum of 6.5-7.0 and required Mn/sup 2 +/. The K/sub m/ values for UDP-Gal and GM/sub 2/ were 0.12 mM and 6 ..mu..M, respectively.« less

  3. Beta-galactosidase deficiency in a Korat cat: a new form of feline GM1-gangliosidosis.

    PubMed

    De Maria, R; Divari, S; Bo, S; Sonnio, S; Lotti, D; Capucchio, M T; Castagnaro, M

    1998-09-01

    A 7-month-old Korat cat was referred for a slowly progressive neurological disease. Circulating monocytes and lymphocytes showed the presence of single or multiple empty vacuoles and blood leukocytes enzyme assay revealed a very low beta-galactosidase activity level (4.7 nmol/mg per h) as compared to unaffected parents and relatives. Histologically, the cat, euthanized at the owner request at 21 months of age, presented diffuse vacuolization and enlargement of neurons throughout the brain, spinal cord and peripheral ganglia, severe cerebellar neuronal cell loss, and moderate astrocytosis. Stored material was stained with periodic acid-Schiff on frozen sections and with the lectins Ricinus conmmunis agglutinin-I, concanavalin A and wheat germ agglutinin on paraffin-embedded sections. Ultrastructurally, neuronal vacuoles were filled with concentrically whorled lamellae and small membrane-bound vesicles. In the affected cat, beta-galactosidase activity was markedly reduced in brain (18.9%) and liver (33.25%), while total beta-hexosaminidase activity showed a remarkable increase. Quantitation of total gangliosides revealed a 3-fold increase in brain and 1.7-fold in liver of affected cat. High-performance thin layer chromatography (HPTLC) detected a striking increase of GM1-ganglioside. On densitometric analysis of HPTLC bands, the absorption of GM1-ganglioside band was 98.52% of all stained bands (GD1a, GD1b, GT1b). Based on clinical onset, morphological and histochemical features, and biochemical findings, the Korat cat GM1-gangliosidosis is comparable with the human type II (juvenile) form. However, clinical progression, survival time and level of beta-galactosidase deficiency do not completely fit with those of human type II GM1-gangliosidosis. The disease in the Korat cat is also different from other reported forms of feline GM1-gangliosidosis.

  4. Cross-reactive antibodies against GM2 and CMV-infected fibroblasts in Guillain-Barré syndrome.

    PubMed

    Ang, C W; Jacobs, B C; Brandenburg, A H; Laman, J D; van der Meché, F G; Osterhaus, A D; van Doorn, P A

    2000-04-11

    To investigate whether anti-GM2 antibodies in patients with Guillain-Barré syndrome (GBS) are induced by molecular mimicry with cytomegalovirus (CMV). Antibodies against ganglioside GM2 are frequently present in the serum from GBS patients with an antecedent infection with CMV. The authors detected inhibition of anti-GM2 reactivity after incubation of GM2-reactive serum samples with fibroblasts infected with a GBS-associated CMV strain. Control sera consisted of GQ1b-reactive samples, and control antigens included uninfected fibroblasts and fibroblasts that were infected with other herpes viruses. Serum immunoglobulin M reactivity with GM2 was decreased in a dose-dependent manner after incubation with CMV-infected fibroblasts. Incubation of anti-GM2-positive serum samples with uninfected fibroblasts and fibroblasts infected with varicella zoster virus did not inhibit anti-GM2 reactivity, whereas this reactivity was slightly decreased after incubation with herpes simplex virus type 1 in one patient. Antibodies against ganglioside GQ1b did not react with CMV-infected fibroblasts. CMV-infected fibroblasts express gangliosidelike epitopes that recognize specifically anti-GM2 antibodies. These results support the hypothesis that antiganglioside antibodies in CMV-infected GBS patients are induced by molecular mimicry between GM2 and antigens that are induced by a CMV infection.

  5. Ganglioside GM1 mimicry in Campylobacter strains from sporadic infections in the United States.

    PubMed

    Nachamkin, I; Ung, H; Moran, A P; Yoo, D; Prendergast, M M; Nicholson, M A; Sheikh, K; Ho, T; Asbury, A K; McKhann, G M; Griffin, J W

    1999-05-01

    To determine whether GM1-like epitopes in Campylobacter species are specific to O serotypes associated with Guillain-Barré syndrome (GBS) or whether they are frequent among random Campylobacter isolates causing enteritis, 275 random enteritis-associated isolates of Campylobacter jejuni were analyzed. To determine whether GM1-like epitopes in Campylobacter species are specific to O serotypes associated with Guillan-Barre syndrome (GBS) or whether they are frequent among random Campylobacter isolates causing enteritis, 275 enteritis-associated isolates, randomly collected in the United States, were analyzed using a cholera-toxin binding assay [corrected]. Overall, 26.2% of the isolates were positive for the GM1-like epitope. Of the 36 different O serotypes in the sample, 21 (58.3%) contained no strains positive for GM1, whereas in 6 serotypes (16.7%), >50% of isolates were positive for GM1. GBS-associated serotypes were more likely to contain strains positive for GM1 than were non-GBS-associated serotypes (37.8% vs. 15.1%, P=.0116). The results suggest that humans are frequently exposed to strains exhibiting GM1-like mimicry and, while certain serotypes may be more likely to possess GM1-like epitopes, the presence of GM1-like epitopes on Campylobacter strains does not itself trigger GBS.

  6. TALEN mediated targeted editing of GM2/GD2-synthase gene modulates anchorage independent growth by reducing anoikis resistance in mouse tumor cells.

    PubMed

    Mahata, Barun; Banerjee, Avisek; Kundu, Manjari; Bandyopadhyay, Uday; Biswas, Kaushik

    2015-03-12

    Complex ganglioside expression is highly deregulated in several tumors which is further dependent on specific ganglioside synthase genes. Here, we designed and constructed a pair of highly specific transcription-activator like effector endonuclease (TALENs) to disrupt a particular genomic locus of mouse GM2-synthase, a region conserved in coding sequence of all four transcript variants of mouse GM2-synthase. Our designed TALENs effectively work in different mouse cell lines and TALEN induced mutation rate is over 45%. Clonal selection strategy is undertaken to generate stable GM2-synthase knockout cell line. We have also demonstrated non-homologous end joining (NHEJ) mediated integration of neomycin cassette into the TALEN targeted GM2-synthase locus. Functionally, clonally selected GM2-synthase knockout clones show reduced anchorage-independent growth (AIG), reduction in tumor growth and higher cellular adhesion as compared to wild type Renca-v cells. Insight into the mechanism shows that, reduced AIG is due to loss in anoikis resistance, as both knockout clones show increased sensitivity to detachment induced apoptosis. Therefore, TALEN mediated precise genome editing at GM2-synthase locus not only helps us in understanding the function of GM2-synthase gene and complex gangliosides in tumorigenicity but also holds tremendous potential to use TALENs in translational cancer research and therapeutics.

  7. Analysis of anti-ganglioside antibodies by a line immunoassay in patients with chronic-inflammatory demyelinating polyneuropathies (CIDP).

    PubMed

    Klehmet, Juliane; Märschenz, Stefanie; Ruprecht, Klemens; Wunderlich, Benjamin; Büttner, Thomas; Hiemann, Rico; Roggenbuck, Dirk; Meisel, Andreas

    2018-05-24

    Unlike for acute immune-mediated neuropathies (IN), anti-ganglioside autoantibody (aGAAb) testing has been recommended for only a minority of chronic IN yet. Thus, we used a multiplex semi-quantitative line immunoassay (LIA) to search for aGAAb in chronic-inflammatory demyelinating polyneuropathy (CIDP) and its clinical variants. Anti-GAAb to 11 gangliosides and sulfatide (SF) were investigated by LIA in 61 patients with IN (27 typical CIDP, 12 distal-acquired demyelinating polyneuropathy, 6 multifocal-acquired demyelinating sensory/motor polyneuropathy, 10 sensory CIDP, 1 focal CIDP and 5 multifocal-motoric neuropathy), 40 with other neuromuscular disorders (OND) (15 non-immune polyneuropathies, 25 myasthenia gravis), 29 with multiple sclerosis (MS) and 54 healthy controls (HC). In contrast to IgG, positive anti-GAAB IgM against at least one ganglioside/SF was found in 17/61 (27.9%) IN compared to 2/40 (5%) in OND, 2/29 MS (6.9%) and 4/54 (7.4%) in HC (p=0.001). There was a statistically higher prevalence of anti-sulfatide (aSF) IgM in IN compared to OND (p=0.008). Further, aGM1 IgM was more prevalent in IN compared to OND and HC (p=0.009) as well as GD1b in IN compared to HC (p<0.04). The prevalence of aGM1 IgM in CIDP was lower compared to in multifocal motor neuropathy (MMN) (12% vs. 60%, p=0.027). Patients showing aSF, aGM1 and aGM2 IgM were younger compared to aGAAb negatives (p<0.05). Patients with aSF IgM positivity presented more frequently typical CIDP and MMN phenotypes (p<0.05, respectively). The aGAAb LIA revealed an elevated frequency of at least one aGAAb IgM in CIDP/MMN patients. Anti-SF, aGM1 and aGM2 IgM were associated with younger age and anti-SF with IN phenotypes.

  8. Inhibition of glutamate-induced intensification of free radical reactions by gangliosides: possible role in their protective effect in rat cerebellar granule cells and brain synaptosomes.

    PubMed

    Avrova, N F; Victorov, I V; Tyurin, V A; Zakharova, I O; Sokolova, T V; Andreeva, N A; Stelmaschuk, E V; Tyurina, Y Y; Gonchar, V S

    1998-07-01

    The neurotoxic effect of exposure of rat cerebellar granule cells to glutamate (100 microM) is to a large extent prevented by incubation of neurons not only with micromolar, but even with nanomolar concentrations of gangliosides GM1, GD1b, and GT1b. GM1 was also shown to decrease significantly the per cent of dead neurons in culture after induction of lipid peroxidation. Exposure to glutamate was found to cause a significant decrease of the activity of Na+, K+-ATP-ase in rat brain cortex synaptosomes, but superoxide dismutase, alpha-tocopherol, or 10-100 nM GM1 practically prevented its action. Other data showing the ability of gangliosides to inhibit the intensification of free radical reactions by glutamate (based on the estimation of methemoglobin formation, SH group content, etc.) have been obtained. The results suggest that gangliosides are able to decrease the glutamate-induced activation of free radical reactions in nerve cells. This effect appears to contribute to their protective action against glutamate neurotoxicity.

  9. Anti-GM2 gangliosides IgM paraprotein induces neuromuscular block without neuromuscular damage.

    PubMed

    Santafé, Manel M; Sabaté, M Mar; Garcia, Neus; Ortiz, Nico; Lanuza, M Angel; Tomàs, Josep

    2008-11-15

    We analyzed the effect on the mouse neuromuscular synapses of a human monoclonal IgM, which binds specifically to gangliosides with the common epitope [GalNAc beta 1-4Gal(3-2 alpha NeuAc)beta 1-]. We focused on the role of the complement. Evoked neurotransmission was partially blocked by IgM both acutely (1 h) and chronically (10 days). Transmission electron microscopy shows important nerve terminal growth and retraction remodelling though axonal injury can be ruled out. Synapses did not show mouse C5b-9 immunofluorescence and were only immunolabelled when human complement was added. Therefore, the IgM-induced synaptic changes occur without complement-mediated membrane attack.

  10. Phospatidylserine or ganglioside--which of anionic lipids determines the effect of cationic dextran on lipid membrane?

    PubMed

    Hąc-Wydro, Katarzyna; Wydro, Paweł; Cetnar, Andrzej; Włodarczyk, Grzegorz

    2015-02-01

    In this work the influence of cationic polymer, namely diethylaminoethyl DEAE-dextran on model lipid membranes was investigated. This polymer is of a wide application as a biomaterial and a drug carrier and its cytotoxicity toward various cancer cells was also confirmed. It was suggested that anticancer effect of cationic dextran is connected with the binding of the polymer to the negatively charged sialic acid residues overexpressed in cancer membrane. This fact encouraged us to perform the studies aimed at verifying whether the effect of cationic DEAE-dextran on membrane is determined only by the presence of the negatively charged lipid in the system or the kind of anionic lipid is also important. To reach this goal systematic investigations on the effect of dextran on various one-component lipid monolayers and multicomponent hepatoma cell model membranes differing in the level and the kind of anionic lipids (phosphatidylserine, sialic acid-containing ganglioside GM3 or their mixture) were done. As evidenced the results the effect of DEAE-dextran on the model system is determined by anionic lipid-polymer electrostatic interactions. However, the magnitude of the effect of cationic polymer is strongly dependent on the kind of anionic lipid in the model system. Namely, the packing and ordering of the mixtures containing ganglioside GM3 were more affected by DEAE-dextran than phosphatidylserine-containing monolayers. Although the experiments were done on model systems and therefore further studies are highly needed, the collected data may indicate that ganglioside may be important in the differentiation of the effect of cationic dextran on membranes. Copyright © 2014 Elsevier B.V. All rights reserved.

  11. TALEN mediated targeted editing of GM2/GD2-synthase gene modulates anchorage independent growth by reducing anoikis resistance in mouse tumor cells

    PubMed Central

    Mahata, Barun; Banerjee, Avisek; Kundu, Manjari; Bandyopadhyay, Uday; Biswas, Kaushik

    2015-01-01

    Complex ganglioside expression is highly deregulated in several tumors which is further dependent on specific ganglioside synthase genes. Here, we designed and constructed a pair of highly specific transcription-activator like effector endonuclease (TALENs) to disrupt a particular genomic locus of mouse GM2-synthase, a region conserved in coding sequence of all four transcript variants of mouse GM2-synthase. Our designed TALENs effectively work in different mouse cell lines and TALEN induced mutation rate is over 45%. Clonal selection strategy is undertaken to generate stable GM2-synthase knockout cell line. We have also demonstrated non-homologous end joining (NHEJ) mediated integration of neomycin cassette into the TALEN targeted GM2-synthase locus. Functionally, clonally selected GM2-synthase knockout clones show reduced anchorage-independent growth (AIG), reduction in tumor growth and higher cellular adhesion as compared to wild type Renca-v cells. Insight into the mechanism shows that, reduced AIG is due to loss in anoikis resistance, as both knockout clones show increased sensitivity to detachment induced apoptosis. Therefore, TALEN mediated precise genome editing at GM2-synthase locus not only helps us in understanding the function of GM2-synthase gene and complex gangliosides in tumorigenicity but also holds tremendous potential to use TALENs in translational cancer research and therapeutics. PMID:25762467

  12. Synthesis and enzymatic susceptibility of a series of novel GM2 analogs.

    PubMed

    Fuse, Tomoaki; Ando, Hiromune; Imamura, Akihiro; Sawada, Naoki; Ishida, Hideharu; Kiso, Makoto; Ando, Takayuki; Li, Su-Chen; Li, Yu-Teh

    2006-07-01

    A series of GM2 analogs in which GM2 epitope was coupled to a variety of glycosyl lipids were designed and synthesized to investigate the mechanism of enzymatic hydrolysis of GM2 ganglioside. The coupling of N-Troc-protected sialic acid and p-methoxyphenyl galactoside acceptor gave the crystalline disaccharide, which was further coupled with galactosamine donor to give the desired GM2 epitope trisaccharide. After conversion into the corresponding glycosyl donor, the trisaccharide was coupled with galactose, glucose and artificial ceramide (B30) to give the final compounds. The result on hydrolysis of GM2 analogs indicates that GM2 activator protein requires one spacer sugar between GM2 epitope and the lipid moiety to assist the hydrolysis of the terminal GalNAc residue.

  13. Therapeutic potential of intracerebroventricular replacement of modified human β-hexosaminidase B for GM2 gangliosidosis.

    PubMed

    Matsuoka, Kazuhiko; Tamura, Tomomi; Tsuji, Daisuke; Dohzono, Yukie; Kitakaze, Keisuke; Ohno, Kazuki; Saito, Seiji; Sakuraba, Hitoshi; Itoh, Kohji

    2011-06-01

    To develop a novel enzyme replacement therapy for neurodegenerative Tay-Sachs disease (TSD) and Sandhoff disease (SD), which are caused by deficiency of β-hexosaminidase (Hex) A, we designed a genetically engineered HEXB encoding the chimeric human β-subunit containing partial amino acid sequence of the α-subunit by structure-based homology modeling. We succeeded in producing the modified HexB by a Chinese hamster ovary (CHO) cell line stably expressing the chimeric HEXB, which can degrade artificial anionic substrates and GM2 ganglioside in vitro, and also retain the wild-type (WT) HexB-like thermostability in the presence of plasma. The modified HexB was efficiently incorporated via cation-independent mannose 6-phosphate receptor into fibroblasts derived from Tay-Sachs patients, and reduced the GM2 ganglioside accumulated in the cultured cells. Furthermore, intracerebroventricular administration of the modified HexB to Sandhoff mode mice restored the Hex activity in the brains, and reduced the GM2 ganglioside storage in the parenchyma. These results suggest that the intracerebroventricular enzyme replacement therapy involving the modified HexB should be more effective for Tay-Sachs and Sandhoff than that utilizing the HexA, especially as a low-antigenic enzyme replacement therapy for Tay-Sachs patients who have endogenous WT HexB.

  14. Therapeutic Potential of Intracerebroventricular Replacement of Modified Human β-Hexosaminidase B for GM2 Gangliosidosis

    PubMed Central

    Matsuoka, Kazuhiko; Tamura, Tomomi; Tsuji, Daisuke; Dohzono, Yukie; Kitakaze, Keisuke; Ohno, Kazuki; Saito, Seiji; Sakuraba, Hitoshi; Itoh, Kohji

    2011-01-01

    To develop a novel enzyme replacement therapy for neurodegenerative Tay-Sachs disease (TSD) and Sandhoff disease (SD), which are caused by deficiency of β-hexosaminidase (Hex) A, we designed a genetically engineered HEXB encoding the chimeric human β-subunit containing partial amino acid sequence of the α-subunit by structure-based homology modeling. We succeeded in producing the modified HexB by a Chinese hamster ovary (CHO) cell line stably expressing the chimeric HEXB, which can degrade artificial anionic substrates and GM2 ganglioside in vitro, and also retain the wild-type (WT) HexB-like thermostability in the presence of plasma. The modified HexB was efficiently incorporated via cation-independent mannose 6-phosphate receptor into fibroblasts derived from Tay-Sachs patients, and reduced the GM2 ganglioside accumulated in the cultured cells. Furthermore, intracerebroventricular administration of the modified HexB to Sandhoff mode mice restored the Hex activity in the brains, and reduced the GM2 ganglioside storage in the parenchyma. These results suggest that the intracerebroventricular enzyme replacement therapy involving the modified HexB should be more effective for Tay-Sachs and Sandhoff than that utilizing the HexA, especially as a low-antigenic enzyme replacement therapy for Tay-Sachs patients who have endogenous WT HexB. PMID:21487393

  15. Mice lacking sialyltransferase ST3Gal-II develop late-onset obesity and insulin resistance

    PubMed Central

    Lopez, Pablo HH; Aja, Susan; Aoki, Kazuhiro; Seldin, Marcus M; Lei, Xia; Ronnett, Gabriele V; Wong, G William; Schnaar, Ronald L

    2017-01-01

    Sialyltransferases are a family of 20 gene products in mice and humans that transfer sialic acid from its activated precursor, CMP-sialic acid, to the terminus of glycoprotein and glycolipid acceptors. ST3Gal-II (coded by the St3gal2 gene) transfers sialic acid preferentially to the three positions of galactose on the Galβ1-3GalNAc terminus of gangliosides GM1 and GD1b to synthesize GD1a and GT1b, respectively. Mice with a targeted disruption of St3gal2 unexpectedly displayed late-onset obesity and insulin resistance. At 3 months of age, St3gal2-null mice were the same weight as their wild type (WT) counterparts, but by 13 months on standard chow they were visibly obese, 22% heavier and with 37% greater fat/lean ratio than WT mice. St3gal2-null mice became hyperglycemic and displayed impaired glucose tolerance by 9 months of age. They had sharply reduced insulin responsiveness despite equivalent pancreatic islet morphology. Analyses of insulin receptor (IR) tyrosine kinase substrate IRS-1 and downstream target Akt revealed decreased insulin-induced phosphorylation in adipose tissue but not liver or skeletal muscle of St3gal2-null mice. Thin-layer chromatography and mass spectrometry revealed altered ganglioside profiles in the adipose tissue of St3gal2-null mice compared to WT littermates. Metabolically, St3gal2-null mice display a reduced respiratory exchange ratio compared to WT mice, indicating a preference for lipid oxidation as an energy source. Despite their altered metabolism, St3gal2-null mice were hyperactive. We conclude that altered ganglioside expression in adipose tissue results in diminished IR sensitivity and late-onset obesity. PMID:27683310

  16. An inversion of 25 base pairs causes feline GM2 gangliosidosis variant.

    PubMed

    Martin, Douglas R; Krum, Barbara K; Varadarajan, G S; Hathcock, Terri L; Smith, Bruce F; Baker, Henry J

    2004-05-01

    In G(M2) gangliosidosis variant 0, a defect in the beta-subunit of lysosomal beta-N-acetylhexosaminidase (EC 3.2.1.52) causes abnormal accumulation of G(M2) ganglioside and severe neurodegeneration. Distinct feline models of G(M2) gangliosidosis variant 0 have been described in both domestic shorthair and Korat cats. In this study, we determined that the causative mutation of G(M2) gangliosidosis in the domestic shorthair cat is a 25-base-pair inversion at the extreme 3' end of the beta-subunit (HEXB) coding sequence, which introduces three amino acid substitutions at the carboxyl terminus of the protein and a translational stop that is eight amino acids premature. Cats homozygous for the 25-base-pair inversion express levels of beta-subunit mRNA approximately 190% of normal and protein levels only 10-20% of normal. Because the 25-base-pair inversion is similar to mutations in the terminal exon of human HEXB, the domestic shorthair cat should serve as an appropriate model to study the molecular pathogenesis of human G(M2) gangliosidosis variant 0 (Sandhoff disease).

  17. Phase I Study of Anti-GM2 Ganglioside Monoclonal Antibody BIW-8962 as Monotherapy in Patients with Previously Treated Multiple Myeloma.

    PubMed

    Baz, Rachid C; Zonder, Jeffrey A; Gasparetto, Cristina; Reu, Frederic J; Strout, Vincent

    2016-01-01

    BIW-8962 is a monoclonal antibody to GM2 ganglioside that shows preclinical activity towards multiple myeloma (MM) cell lines and in animal models bearing MM xenografts. The objective of this study was to determine the safety, tolerability, maximum tolerated dose (MTD), pharmacokinetics, potential immunogenicity, and preliminary clinical efficacy of BIW-8962 in patients with heavily pretreated MM. Patients ( n  = 23) received escalating doses of BIW-8962 (0.03-3 mg/kg) intravenously every 2 weeks in phase Ia. The highest anticipated dose (10 mg/kg) was not tested and the study was discontinued without proceeding to phases Ib and II. The MTD of BIW-8962 was not established and BIW-8962 was relatively well tolerated. No pattern of consistent toxicity could be inferred from treatment-related AEs grade ≥3 and only two dose-limiting toxicities were recorded (atrial thrombosis + cardiomyopathy and chest pain, respectively). In the efficacy evaluable population ( n  = 22), no patient had a response (complete or partial) and 16 (72.7%) had a best response of stable disease, which was generally not durable. BIW-8962 did not show evidence of clinical activity. The study was therefore stopped and further development of BIW-8962 in MM was halted. This work was funded by Kyowa Kirin Pharmaceutical Development, Inc. ClinicalTrials.gov identifier, NCT00775502.

  18. Gangliosides and Ceramides Change in a Mouse Model of Blast Induced Traumatic Brain Injury

    PubMed Central

    2013-01-01

    Explosive detonations generate atmospheric pressure changes that produce nonpenetrating blast induced “mild” traumatic brain injury (bTBI). The structural basis for mild bTBI has been extremely controversial. The present study applies matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging to track the distribution of gangliosides in mouse brain tissue that were exposed to very low level of explosive detonations (2.5–5.5 psi peak overpressure). We observed major increases of the ganglioside GM2 in the hippocampus, thalamus, and hypothalamus after a single blast exposure. Moreover, these changes were accompanied by depletion of ceramides. No neurological or brain structural signs of injury could be inferred using standard light microscopic techniques. The first source of variability is generated by the Latency between blast and tissue sampling (peak intensity of the blast wave). These findings suggest that subtle molecular changes in intracellular membranes and plasmalemma compartments may be biomarkers for biological responses to mild bTBI. This is also the first report of a GM2 increase in the brains of mature mice from a nongenetic etiology. PMID:23590251

  19. Adjuvant ganglioside GM2-KLH/QS-21 vaccination versus observation after resection of primary tumor > 1.5 mm in patients with stage II melanoma: results of the EORTC 18961 randomized phase III trial.

    PubMed

    Eggermont, Alexander M M; Suciu, Stefan; Rutkowski, Piotr; Marsden, Jeremy; Santinami, Mario; Corrie, Philippa; Aamdal, Steinar; Ascierto, Paolo A; Patel, Poulam M; Kruit, Wim H; Bastholt, Lars; Borgognoni, Lorenzo; Bernengo, Maria Grazia; Davidson, Neville; Polders, Larissa; Praet, Michel; Spatz, Alan

    2013-10-20

    The GM2 ganglioside is an antigen expressed in the majority of melanomas. The GM2-KLH/QS-21 vaccine induces high immunoglobulin M (IgM) and IgG antibody responses. The EORTC 18961 trial compared the efficacy of GM2-KLH/QS-21 vaccination versus observation. A total of 1,314 patients with a primary tumor > 1.50 mm in thickness (T3-4N0M0; American Joint Committee on Cancer stage II) were randomly assigned to GM2-KLH/QS-21 vaccination (n = 657) or observation (n = 657). Treatment consisted of subcutaneous injections once per week from week 1 to 4, then every 3 months for the first 2 years and every 6 months during the third year. Primary end point was relapse-free survival (RFS). Secondary end points were distant metastasis-free survival (DMFS) and overall survival (OS). Analyses were by intent to treat. After a median follow-up of 1.8 years, the trial was stopped at the second interim analysis for futility regarding RFS (hazard ratio [HR], 1.00; P = .99) and detrimental outcome regarding OS (HR, 1.66; P = .02). After a median follow-up of 4.2 years, we had recorded 400 relapses, nine deaths without relapse, a total of 236 deaths. At 4 years, the vaccination arm showed a decreased RFS rate of 1.2% (HR, 1.03; 95% CI, 0.84 to 1.25) and OS rate of 2.1% (HR, 1.16; 95% CI, 0.90 to 1.51). Toxicity was acceptable, with 4.6% of patients ending study participation because of toxicity. GM2-KLH/QS-21 vaccination does not improve outcome for patients with stage II melanoma.

  20. Diversity of glycosphingolipid GM2 and cholesterol accumulation in NPC1 patient-specific iPSC-derived neurons.

    PubMed

    Trilck, Michaela; Peter, Franziska; Zheng, Chaonan; Frank, Marcus; Dobrenis, Kostantin; Mascher, Hermann; Rolfs, Arndt; Frech, Moritz J

    2017-02-15

    Niemann-Pick disease Type C1 (NPC1) is a rare progressive neurodegenerative disorder caused by mutations in the NPC1 gene. On the cellular level NPC1 mutations lead to an accumulation of cholesterol and gangliosides. As a thorough analysis of the severely affected neuronal cells is unfeasible in NPC1 patients, we recently described the cellular phenotype of neuronal cells derived from NPC1 patient iPSCs carrying the compound heterozygous mutation c.1836A>C/c.1628delC. Here we expanded the analysis to cell lines carrying the prevalent mutation c.3182T>C and the novel mutation c.1180T>C, as well as to the determination of GM2 and GM3 gangliosides in NPC1 patient-specific iPSC-derived neurons and glia cells. Immunocytochemical detection of GM2 revealed punctated staining pattern predominantly localized in neurons. Detection of cholesterol by filipin staining showed a comparable staining pattern, colocalized with GM2, indicating a deposit of GM2 and cholesterol in the same cellular compartments. Accumulations were not only restricted to cell bodies, but were also found in the neuronal extensions. A quantification of the GM2 amount by HPLC-MS/MS confirmed significantly higher amounts in neurons carrying a mutation. Additionally, these cells displayed a lowered activity of the catabolic enzyme Hex A, but not B4GALNT1. Molecular docking simulations indicated binding of cholesterol to Hex A, suggesting cholesterol influences the GM2 degradation pathway and, subsequently, leading to the accumulation of GM2. Taken together, this is the first study showing an accumulation of GM2 in neuronal derivatives of patient-specific iPSCs and thus proving further disease-specific hallmarks in this human in vitro model of NPC1. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. Novel Ganglioside-mediated Entry of Botulinum Neurotoxin Serotype D into Neurons

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kroken, Abby R.; Karalewitz, Andrew P.-A.; Fu, Zhuji

    2012-02-07

    Botulinum Neurotoxins (BoNTs) are organized into seven serotypes, A-G. Although several BoNT serotypes enter neurons through synaptic vesicle cycling utilizing dual receptors (a ganglioside and a synaptic vesicle-associated protein), the entry pathway of BoNT/D is less well understood. Although BoNT/D entry is ganglioside-dependent, alignment and structural studies show that BoNT/D lacks key residues within a conserved ganglioside binding pocket that are present in BoNT serotypes A, B, E, F, and G, which indicate that BoNT/D-ganglioside interactions may be unique. In this study BoNT/D is shown to have a unique association with ganglioside relative to the other BoNT serotypes, utilizing amore » ganglioside binding loop (GBL, residues Tyr-1235-Ala-1245) within the receptor binding domain of BoNT/D (HCR/D) via b-series gangliosides, including GT1b, GD1b, and GD2. HCR/D bound gangliosides and entered neurons dependent upon the aromatic ring of Phe-1240 within the GBL. This is the first BoNT-ganglioside interaction that is mediated by a phenylalanine. In contrast, Trp-1238, located near the N terminus of the ganglioside binding loop, was mostly solvent-inaccessible and appeared to contribute to maintaining the loop structure. BoNT/D entry and intoxication were enhanced by membrane depolarization via synaptic vesicle cycling, where HCR/D colocalized with synaptophysin, a synaptic vesicle marker, but immunoprecipitation experiments did not detect direct association with synaptic vesicle protein 2. Thus, BoNT/D utilizes unique associations with gangliosides and synaptic vesicles to enter neurons, which may facilitate new neurotoxin therapies.« less

  2. Thymic involution and corticosterone level in Sandhoff disease model mice: new aspects the pathogenesis of GM2 gangliosidosis.

    PubMed

    Matsuoka, Kazuhiko; Tsuji, Daisuke; Taki, Takao; Itoh, Kohji

    2011-10-01

    Sandhoff disease (SD) is a lysosomal disease caused by a mutation of the HEXB gene associated with excessive accumulation of GM2 ganglioside (GM2) in lysosomes and neurological manifestations. Production of autoantibodies against the accumulated gangliosides has been reported to be involved in the progressive pathogenesis of GM2 gangliosidosis, although the underlying mechanism has not been fully elucidated. The thymus is the key organ in the acquired immune system including the development of autoantibodies. We showed here that thymic involution and an increase in cell death in the organ occur in SD model mice at a late stage of the pathogenesis. Dramatic increases in the populations of Annexin-V(+) cells and terminal deoxynucletidyl transferase dUTP nick end labeling (TUNEL) (+) cells were observed throughout the thymuses of 15-week old SD mice. Enhanced caspase-3/7 activation, but not that of caspase-1/4, -6 ,-8, or -9, was also demonstrated. Furthermore, the serum level of corticosterone, a potent inducer of apoptosis of thymocytes, was elevated during the same period of apoptosis. Our studies suggested that an increase in endocrine corticosterone may be one of the causes that accelerate the apoptosis of thymocytes leading to thymic involution in GM2 gangliosidosis, and thus can be used as a disease marker for evaluation of the thymic condition and disease progression.

  3. An α-subunit loop structure is required for GM2 activator protein binding by β-hexosaminidase A

    PubMed Central

    Zarghooni, Maryam; Bukovac, Scott; Tropak, Michael; Callahan, John; Mahuran, Don

    2010-01-01

    The α- and/or β-subunits of human β-hexosaminidase A (αβ) and B (ββ) are ~60% identical. In vivo only β-hexosaminidase A can utilize GM2 ganglioside as a substrate, but requires the GM2 activator protein to bind GM2 ganglioside and then interact with the enzyme, placing the terminal GalNAc residue in the active site of the α-subunit. A model for this interaction suggests that two loop structures, present only in the α-subunit, may be critical to this binding. Three amino acids in one of these loops are not encoded in the HEXB gene, while four from the other are removed posttranslationally from the pro-β-subunit. Natural substrate assays with forms of hexosaminidase A containing mutant α-subunits demonstrate that only the site that is removed from the β-subunit during its maturation is critical for the interaction. Our data suggest an unexpected biological role for such proteolytic processing events. PMID:15485660

  4. Combined replacement effects of human modified β-hexosaminidase B and GM2 activator protein on GM2 gangliosidoses fibroblasts.

    PubMed

    Kitakaze, Keisuke; Tasaki, Chikako; Tajima, Youichi; Hirokawa, Takatsugu; Tsuji, Daisuke; Sakuraba, Hitoshi; Itoh, Kohji

    2016-09-01

    GM2 gangliosidoses are autosomal recessive lysosomal storage diseases (LSDs) caused by mutations in the HEXA , HEXB and GM2A genes, which encode the human lysosomal β-hexosaminidase (Hex) α- and β-subunits, and GM2 activator protein (GM2A), respectively. These diseases are associated with excessive accumulation of GM2 ganglioside (GM2) in the brains of patients with neurological symptoms. Here we established a CHO cell line overexpressing human GM2A, and purified GM2A from the conditioned medium, which was taken up by fibroblasts derived from a patient with GM2A deficiency, and had the therapeutic effects of reducing the GM2 accumulated in fibroblasts when added to the culture medium. We also demonstrated for the first time that recombinant GM2A could enhance the replacement effect of human modified HexB (modB) with GM2-degrading activity, which is composed of homodimeric altered β-subunits containing a partial amino acid sequence of the α-subunit, including the GSEP loop necessary for binding to GM2A, on reduction of the GM2 accumulated in fibroblasts derived from a patient with Tay-Sachs disease, a HexA (αβ heterodimer) deficiency, caused by HEXA mutations. We predicted the same manner of binding of GM2A to the GSEP loop located in the modified HexB β-subunit to that in the native HexA α-subunit on the basis of the x-ray crystal structures. These findings suggest the effectiveness of combinational replacement therapy involving the human modified HexB and GM2A for GM2 gangliosidoses.

  5. Guillain–Barré syndrome and anti-ganglioside antibodies: a clinician-scientist’s journey

    PubMed Central

    YUKI, Nobuhiro

    2012-01-01

    Guillain–Barré syndrome (GBS) is the most frequent cause of acute flaccid paralysis. Having seen my first GBS patient in 1989, I have since then dedicated my time in research towards understanding the pathogenesis of GBS. Along with several colleagues, we identified IgG autoantibodies against ganglioside GM1 in two patients with GBS subsequent to Campylobacter jejuni enteritis. We proceeded to demonstrate molecular mimicry between GM1 and bacterial lipo-oligosaccharide of C. jejuni isolated from a patient with GBS. Our group then established a disease model for GBS by sensitization with GM1 or GM1-like lipo-oligosaccharide. With this, a new paradigm that carbohydrate mimicry can cause autoimmune disorders was demonstrated, making GBS the first proof of molecular mimicry in autoimmune disease. Patients with Fisher syndrome, characterized by ophthalmoplegia and ataxia, can develop the disease after an infection by C. jejuni. We showed that the genetic polymorphism of C. jejuni sialyltransferase, an enzyme essential to the biosynthesis of ganglioside-like lipo-oligosaccharides determines whether patients develop GBS or Fisher syndrome. This introduces another paradigm that microbial genetic polymorphism can determine the clinical phenotype of human autoimmune diseases. Similarities between the clinical presentation of Fisher syndrome and Bickerstaff brainstem encephalitis have caused debate as to whether they are in fact the same disease. We demonstrated that IgG anti-GQ1b antibodies were common to both, suggesting that they are part of the same disease spectrum. We followed this work by clarifying the nosological relationship between the various clinical presentations within the anti-GQ1b antibody syndrome. In this review, I wanted to share my journey from being a clinician to a clinician-scientist in the hopes of inspiring younger clinicians to follow a similar path. PMID:22850724

  6. GM2 gangliosidosis associated with a HEXA missense mutation in Japanese Chin dogs: a potential model for Tay Sachs disease.

    PubMed

    Sanders, Douglas N; Zeng, Rong; Wenger, David A; Johnson, Gary S; Johnson, Gayle C; Decker, Jared E; Katz, Martin L; Platt, Simon R; O'Brien, Dennis P

    2013-01-01

    GM2 gangliosidosis is a fatal lysosomal storage disease caused by a deficiency of β-hexosaminidase (EC 3.2.1.52). There are two major isoforms of the enzyme: hexosaminidase A composed of an α and a β subunit (encoded by HEXA and HEXB genes, respectively); and, hexosaminidase B composed of two β subunits. Hexosaminidase A requires an activator protein encoded by GM2A to catabolize GM2 ganglioside, but even in the absence of the activator protein, it can hydrolyze the synthetic substrates commonly used to assess enzyme activity. GM2 gangliosidosis has been reported in Japanese Chin dogs, and we identified the disease in two related Japanese Chin dogs based on clinical signs, histopathology and elevated brain GM2 gangliosides. As in previous reports, we found normal or elevated hexosaminidase activity when measured with the synthetic substrates. This suggested that the canine disease is analogous to human AB variant of G(M2) gangliosidosis, which results from mutations in GM2A. However, only common neutral single nucleotide polymorphisms were found upon sequence analysis of the canine ortholog of GM2A from the affected Japanese Chins. When the same DNA samples were used to sequence HEXA, we identified a homozygous HEXA:c967G>A transition which predicts a p.E323K substitution. The glutamyl moiety at 323 is known to make an essential contribution to the active site of hexosaminidase A, and none of the 128 normal Japanese Chins and 92 normal dogs of other breeds that we tested was homozygous for HEXA:c967A. Thus it appears that the HEXA:c967G>A transition is responsible for the GM2 gangliosidosis in Japanese Chins. Copyright © 2012 Elsevier Inc. All rights reserved.

  7. The Pathogenic Role of Ganglioside Metabolism in Alzheimer's Disease-Cholinergic Neuron-Specific Gangliosides and Neurogenesis.

    PubMed

    Ariga, Toshio

    2017-01-01

    Alzheimer's disease (AD) is the most common type of dementia with clinical symptoms that include deficits in memory, judgment, thinking, and behavior. Gangliosides are present on the outer surface of plasma membranes and are especially abundant in the nervous tissues of vertebrates. Ganglioside metabolism, especially the cholinergic neuron-specific gangliosides, GQ1bα and GT1aα, is altered in mouse model of AD and patients with AD. Thus, alterations in ganglioside metabolism may participate in several events related to the pathogenesis of AD. Increased expressions of GT1aα may reflect cholinergic neurogenesis. Most changes in ganglioside metabolism occur in the specific brain areas and their lipid rafts. Targeting ganglioside metabolism in lipid rafts may represent an underexploited opportunity to design novel therapeutic strategies for AD.

  8. Botulinum neurotoxin serotype C associates with dual ganglioside receptors to facilitate cell entry.

    PubMed

    Karalewitz, Andrew P-A; Fu, Zhuji; Baldwin, Michael R; Kim, Jung-Ja P; Barbieri, Joseph T

    2012-11-23

    How botulinum neurotoxin serotype C (BoNT/C) enters neurons is unclear. BoNT/C utilizes dual gangliosides as host cell receptors. BoNT/C accesses gangliosides on the plasma membrane. Plasma membrane accessibility of the dual ganglioside receptors suggests synaptic vesicle exocytosis may not be necessary to expose BoNT/C receptors. Botulinum neurotoxins (BoNTs) cleave SNARE proteins in motor neurons that inhibits synaptic vesicle (SV) exocytosis, resulting in flaccid paralysis. There are seven BoNT serotypes (A-G). In current models, BoNTs initially bind gangliosides on resting neurons and upon SV exocytosis associate with the luminal domains of SV-associated proteins as a second receptor. The entry of BoNT/C is less clear. Characterizing the heavy chain receptor binding domain (HCR), BoNT/C was shown to utilize gangliosides as dual host receptors. Crystallographic and biochemical studies showed that the two ganglioside binding sites, termed GBP2 and Sia-1, were independent and utilized unique mechanisms to bind complex gangliosides. The GBP2 binding site recognized gangliosides that contained a sia5 sialic acid, whereas the Sia-1 binding site recognized gangliosides that contained a sia7 sialic acid and sugars within the backbone of the ganglioside. Utilizing gangliosides that uniquely recognized the GBP2 and Sia-1 binding sites, HCR/C entry into Neuro-2A cells required both functional ganglioside binding sites. HCR/C entered cells differently than the HCR of tetanus toxin, which also utilizes dual gangliosides as host receptors. A point-mutated HCR/C that lacked GBP2 binding potential retained the ability to bind and enter Neuro-2A cells. This showed that ganglioside binding at the Sia-1 site was accessible on the plasma membrane, suggesting that SV exocytosis may not be required to expose BoNT/C receptors. These studies highlight the utility of BoNT HCRs as probes to study the role of gangliosides in neurotransmission.

  9. GM2-gangliosidosis variant 0 (Sandhoff-like disease) in a family of Japanese domestic cats.

    PubMed

    Yamato, O; Matsunaga, S; Takata, K; Uetsuka, K; Satoh, H; Shoda, T; Baba, Y; Yasoshima, A; Kato, K; Takahashi, K; Yamasaki, M; Nakayama, H; Doi, K; Maede, Y; Ogawa, H

    2004-12-04

    A five-month-old, female Japanese domestic shorthair cat with proportionate dwarfism developed neurological disorders, including ataxia, decreased postural responses and generalised body and head tremors, at between two and five months of age. Leucocytosis due to lymphocytosis with abnormal cytoplasmic vacuolations was observed. The concentration of G(M2)-ganglioside in its cerebrospinal fluid was markedly higher than in normal cats, and the activities of beta-hexosaminidases A and B in its leucocytes were markedly reduced. On the basis of these biochemical data, the cat was diagnosed antemortem with G(M2)-gangliosidosis variant 0 (Sandhoff-like disease). The neurological signs became more severe and the cat died at 10 months of age. Histopathologically, neurons throughout the central nervous system were distended, and an ultrastructural study revealed membranous cytoplasmic bodies in these distended neurons. The compound which accumulated in the brain was identified as G(M2)-ganglioside, confirming G(M2)-gangliosidosis. A family study revealed that there were probable heterozygous carriers in which the activities of leucocyte beta-hexosaminidases A and B were less than half the normal value. The Sandhoff-like disease observed in this family of Japanese domestic cats is the first occurrence reported in Japan.

  10. Ganglioside inhibition of sup 125 I-plasmin binding to colorectal carcinoma cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Liepkalns, V.A.; Burtin, M.C.; Correc, P.

    1990-01-01

    The pre-incubation of human colorectal carcinoma cells SW 1116 with 25 to 100 uM purified gangliosides resulted in 35-60% inhibition of specific {sup 125}I-plasmin binding to the cell surface. After 5 to 6 days in culture, tumor cells were pre-incubated at 4 degrees for 1 to 4 h followed by post-incubation with {sup 125}I-plasmin by techniques previously described. At 25 uM the capacity for inhibition of plasmin binding was GT1b greater than GQ1b greater than or equal to GD1a greater than GM1 less than or equal to GgOse 4Cer. Thus a terminal sialyl moiety appears to be necessary (p lessmore » than 0.05) although exogenous N-acetyl neuraminic acid was ineffective (p greater than 0.05), indicating a role for the lipid portion of the ganglioside. Other (glyco)lipids such as sphingosine, fucolipid H-1 and sulfatide were without significant effect. The inhibition could not be reversed by the presence of 10 mM Ca+2, EDTA, pre-treatment of the cell with carboxypeptidase or pretreatment of plasmin with neuraminidases. The inhibition was however reversed by post-incubation in control medium without exogenous ganglioside. Cell counts determined prior to, and after ganglioside incubation showed that the effect was not due to cell death or detachment from the culture surface. The dissociation constant for {sup 125}I-plasmin binding was 5.6 x 10(-8) M (700,000 sites/cell), but in the presence of trisialoganglioside (GT1b), Scatchard plots suggested diversification of binding sites with 280,000 sites/cell at Kd 2.6 x 10(-8) M and 820,000 sites/cell at Kd 2.1 x 10(-7) M. Another interpretation of the Scatchard plot in the presence of ganglioside was that the glycolipid imposed negative cooperativity on plasmin binding to the cell surface. These results suggest that certain gangliosides can affect tumor cell invasiveness by altering protease binding to the cell surface.« less

  11. Determination of ganglioside composition and structure in human brain hemangioma by chip-based nanoelectrospray ionization tandem mass spectrometry.

    PubMed

    Schiopu, Catalin; Flangea, Corina; Capitan, Florina; Serb, Alina; Vukelić, Zeljka; Kalanj-Bognar, Svjetlana; Sisu, Eugen; Przybylski, Michael; Zamfir, Alina D

    2009-12-01

    We report here on a preliminary investigation of ganglioside composition and structure in human hemangioma, a benign tumor in the frontal cortex (HFC) in comparison to normal frontal cortex (NFC) tissue using for the first time advanced mass spectrometric methods based on fully automated chip-nanoelectrospray (nanoESI) high-capacity ion trap (HCT) and collision-induced dissociation (CID). The high ionization efficiency, sensitivity and reproducibility provided by the chip-nanoESI approach allowed for a reliable MS-based ganglioside comparative assay. Unlike NFC, ganglioside mixture extracted from HFC was found dominated by species of short glycan chains exhibiting lower overall sialic acid content. In HFC, only GT1 (d18:1/20:0), and GT3 (d18:1/25:1) polysialylated species were detected. Interestingly, none of these trisialylated forms was detected in NFC, suggesting that such components might selectively be associated with HFC. Unlike the case of previously investigated high malignancy gliosarcoma, in HFC one modified O-Ac-GD2 and one modified O-Ac-GM4 gangliosides were observed. This aspect suggests that these O-acetylated structures could be associated with cerebral tumors having reduced malignancy grade. Fragmentation analysis by CID in MS(2) mode using as precursors the ions corresponding to GT1 (d18:1/20:0) and GD1 (d18:1/20:0) provided data corroborating for the first time the presence of the common GT1a and GT1b isomers and the incidence of unusual GT1c and GT1d glycoforms in brain hemangioma tumor.

  12. Detecting Protein-Glycolipid Interactions Using Glycomicelles and CaR-ESI-MS

    NASA Astrophysics Data System (ADS)

    Han, Ling; Kitova, Elena N.; Klassen, John S.

    2016-11-01

    This study reports on the use of the catch-and-release electrospray ionization mass spectrometry (CaR-ESI-MS) assay, combined with glycomicelles, as a method for detecting specific interactions between water-soluble proteins and glycolipids (GLs) in aqueous solution. The B subunit homopentamers of cholera toxin (CTB5) and Shiga toxin type 1 B (Stx1B5) and the gangliosides GM1, GM2, GM3, GD1a, GD1b, GT1b, and GD2 served as model systems for this study. The CTB5 exhibits broad specificity for gangliosides and binds to GM1, GM2, GM3, GD1a, GD1b, and GT1b; Stx1B5 does not recognize gangliosides. The CaR-ESI-MS assay was used to analyze solutions of CTB5 or Stx1B5 and individual gangliosides (GM1, GM2, GM3, GD1a, GD1b, GT1b, and GD2) or mixtures thereof. The high affinity interaction of CTB5 with GM1 was successfully detected. However, the apparent affinity, as determined from the mass spectra, is significantly lower than that of the corresponding pentasaccharide or when GM1 is presented in model membranes such as nanodiscs. Interactions between CTB5 and the low affinity gangliosides GD1a, GD1b, and GT1b, as well as GD2, which served as a negative control, were detected; no binding of CTB5 to GM2 or GM3 was observed. The CaR-ESI-MS results obtained for Stx1B5 reveal that nonspecific protein-ganglioside binding can occur during the ESI process, although the extent of binding varies between gangliosides. Consequently, interactions detected for CTB5 with GD1a, GD1b, and GT1b are likely nonspecific in origin. Taken together, these results reveal that the CaR-ESI-MS/glycomicelle approach for detecting protein-GL interactions is prone to false positives and false negatives and must be used with caution.

  13. Genetics and Therapies for GM2 Gangliosidosis.

    PubMed

    Cachon-Gonzalez, María Begona; Zaccariotto, Eva; Cox, Timothy Martin

    2018-04-04

    Tay-Sachs disease, caused by impaired β-N-acetylhexosaminidase activity, was the first GM2 gangliosidosis to be studied and one of the most severe and earliest lysosomal diseases to be described. The condition, associated with the pathological build-up of GM2 ganglioside, has acquired almost iconic status and serves as a paradigm in the study of lysosomal storage diseases. Inherited as a classical autosomal recessive disorder, this global disease of the nervous system induces developmental arrest with regression of attained milestones; neurodegeneration progresses rapidly to cause premature death in young children. There is no effective treatment beyond palliative care, and while the genetic basis of GM2 gangliosidosis is well established, the molecular and cellular events, from disease-causing mutations and glycosphingolipid storage to disease manifestations, remain to be fully delineated. Several therapeutic approaches has been attempted in patients, including enzymatic augmentation, bone marrow transplantation, enzyme enhancement, and substrate reduction therapy. Hitherto, none of these stratagems has materially altered the course of the disease. Authentic animal models of GM2 gangliodidosis have facilitated in-depth evaluation of innovative applications such as gene transfer, which in contrast to other interventions, shows great promise. This review outlines current knowledge pertaining the pathobiology as well as potential innovative treatments for the GM2 gangliosidoses. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  14. The reactivities of human erythrocyte autoantibodies anti-Pr2, anti-Gd, Fl and Sa with gangliosides in a chromatogram binding assay.

    PubMed Central

    Uemura, K; Roelcke, D; Nagai, Y; Feizi, T

    1984-01-01

    The thin layer chromatogram binding assay was used to study the reaction of several natural-monoclonal autoantibodies which recognize sialic acid-dependent antigens of human erythrocytes. Immunostaining of gangliosides derived from human and bovine erythrocytes was achieved with four autoantibodies designated anti-Pr2, anti-Gd, Sa and Fl, each of which has a different haemagglutination pattern with untreated and proteinase-treated erythrocytes and with cells of I and i antigen types. From the chromatogram binding patterns of anti-Pr2 with gangliosides of the neolacto and the ganglio series, it is deduced that this antibody reacts best with N-acetylneuraminic acid when it is alpha 2-3- or alpha 2-6-linked to a terminal Gal(beta 1-4)Glc/GlcNAc GlcNAc sequence and to a lesser extent when it is alpha 2-3-linked to a terminal Gal(beta 1-3)GalNAc sequence or to an internal galactose and when it is alpha 2-8-linked to another, internal N-acetylneuraminic acid residue. The other three antibodies differ from anti-Pr2 in their lack of reaction with glycolipids of the ganglio series. They react with the NeuAc(alpha 2-3)Gal(beta 1-4)Glc/GlcNAc sequence as found in GM3 and in glycolipids of the neolacto series, but show a preference for the latter, longer sequences. Thus all four antibodies react with sialylated oligosaccharides containing i type (linear) and I type (branched) neolacto backbones. Fl antibody differs from the other three in its stronger reaction with branched neolacto sequences in accordance with its stronger agglutination of erythrocytes of I rather than i type. The four antibodies show a specificity for N-acetyl- rather than N-glycolyl-neuraminic acid. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. PMID:6204642

  15. Ganglioside GM1 protects against high altitude cerebral edema in rats by suppressing the oxidative stress and inflammatory response via the PI3K/AKT-Nrf2 pathway.

    PubMed

    Gong, Gu; Yin, Liang; Yuan, Libang; Sui, Daming; Sun, Yangyang; Fu, Haiyu; Chen, Liang; Wang, Xiaowu

    2018-03-01

    High altitude cerebral edema (HACE) is a severe type of acute mountain sickness (AMS) that occurs in response to a high altitude hypobaric hypoxic (HH) environment. GM1 monosialoganglioside can alleviate brain injury under adverse conditions including amyloid-β-peptide, ischemia and trauma. However, its role in HACE-induced brain damage remains poorly elucidated. In this study, GM1 supplementation dose-dependently attenuated increase in rat brain water content (BWC) induced by hypobaric chamber (7600 m) exposurefor 24 h. Compared with the HH-treated group, rats injected with GM1 exhibited less brain vascular leakage, lower aquaporin-4 and higher occludin expression, but they also showed increase in Na+/K+-ATPase pump activities. Importantly, HH-incurred consciousness impairment and coordination loss also were ameliorated following GM1 administration. Furthermore, the increased oxidative stress and decrease in anti-oxidant stress system under the HH condition were also reversely abrogated by GM1 treatment via suppressing accumulation of ROS, MDA and elevating the levels of SOD and GSH. Simultaneously, GM1 administration also counteracted the enhanced inflammation in HH-exposed rats by muting pro-inflammatory cytokines IL-1β, TNF-α, and IL-6 levels in serum and brain tissues. Subsequently, GM1 potentiated the activation of the PI3K/AKT-Nrf2 pathway. Cessation of this pathway by LY294002 reversed GM1-mediated inhibitory effects on oxidative stress and inflammation, and ultimately abrogated the protective role of GM1 in abating brain edema, cognitive and motor dysfunction. Overall, GM1 may afford a protective intervention in HACE by suppressing oxidative stress and inflammatory response via activating the PI3K/AKT-Nrf2 pathway, implying a promising agent for the treatment of HACE. Copyright © 2018 Elsevier Ltd. All rights reserved.

  16. GM2 Gangliosidosis in Shiba Inu Dogs with an In-Frame Deletion in HEXB.

    PubMed

    Kolicheski, A; Johnson, G S; Villani, N A; O'Brien, D P; Mhlanga-Mutangadura, T; Wenger, D A; Mikoloski, K; Eagleson, J S; Taylor, J F; Schnabel, R D; Katz, M L

    2017-09-01

    Consistent with a tentative diagnosis of neuronal ceroid lipofuscinosis (NCL), autofluorescent cytoplasmic storage bodies were found in neurons from the brains of 2 related Shiba Inu dogs with a young-adult onset, progressive neurodegenerative disease. Unexpectedly, no potentially causal NCL-related variants were identified in a whole-genome sequence generated with DNA from 1 of the affected dogs. Instead, the whole-genome sequence contained a homozygous 3 base pair (bp) deletion in a coding region of HEXB. The other affected dog also was homozygous for this 3-bp deletion. Mutations in the human HEXB ortholog cause Sandhoff disease, a type of GM2 gangliosidosis. Thin-layer chromatography confirmed that GM2 ganglioside had accumulated in an affected Shiba Inu brain. Enzymatic analysis confirmed that the GM2 gangliosidosis resulted from a deficiency in the HEXB encoded protein and not from a deficiency in products from HEXA or GM2A, which are known alternative causes of GM2 gangliosidosis. We conclude that the homozygous 3-bp deletion in HEXB is the likely cause of the Shiba Inu neurodegenerative disease and that whole-genome sequencing can lead to the early identification of potentially disease-causing DNA variants thereby refocusing subsequent diagnostic analyses toward confirming or refuting candidate variant causality. Copyright © 2017 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.

  17. Detecting Protein-Glycolipid Interactions Using Glycomicelles and CaR-ESI-MS.

    PubMed

    Han, Ling; Kitova, Elena N; Klassen, John S

    2016-11-01

    This study reports on the use of the catch-and-release electrospray ionization mass spectrometry (CaR-ESI-MS) assay, combined with glycomicelles, as a method for detecting specific interactions between water-soluble proteins and glycolipids (GLs) in aqueous solution. The B subunit homopentamers of cholera toxin (CTB 5 ) and Shiga toxin type 1 B (Stx1B 5 ) and the gangliosides GM1, GM2, GM3, GD1a, GD1b, GT1b, and GD2 served as model systems for this study. The CTB 5 exhibits broad specificity for gangliosides and binds to GM1, GM2, GM3, GD1a, GD1b, and GT1b; Stx1B 5 does not recognize gangliosides. The CaR-ESI-MS assay was used to analyze solutions of CTB 5 or Stx1B 5 and individual gangliosides (GM1, GM2, GM3, GD1a, GD1b, GT1b, and GD2) or mixtures thereof. The high affinity interaction of CTB 5 with GM1 was successfully detected. However, the apparent affinity, as determined from the mass spectra, is significantly lower than that of the corresponding pentasaccharide or when GM1 is presented in model membranes such as nanodiscs. Interactions between CTB 5 and the low affinity gangliosides GD1a, GD1b, and GT1b, as well as GD2, which served as a negative control, were detected; no binding of CTB 5 to GM2 or GM3 was observed. The CaR-ESI-MS results obtained for Stx1B 5 reveal that nonspecific protein-ganglioside binding can occur during the ESI process, although the extent of binding varies between gangliosides. Consequently, interactions detected for CTB 5 with GD1a, GD1b, and GT1b are likely nonspecific in origin. Taken together, these results reveal that the CaR-ESI-MS/glycomicelle approach for detecting protein-GL interactions is prone to false positives and false negatives and must be used with caution. Graphical Abstract .

  18. Structure of ganglioside with CAD blood group antigen activity

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Gillard, B.K.; Blanchard, D.; Cartron, J.P.

    1986-05-01

    The novel erythrocyte ganglioside which carries the blood group Cad determinant has been isolated, and its structure has been determined. The ganglioside contained Glu:Gal:GalNAc:GlcNAc in a molar ratio of 1.00:1.94:0.93:0.95. The ganglioside binds Helix pomatia lectin and its chromatographic mobility is similar to G/sub D3/. After treatment with ..beta..-hexosaminidase (human placenta HexA) the product migrated with sialosylparagloboside (SPG), no longer binds Helix lectin, and binds a human anti-SPG antibody. Treatment of this material with neuraminidase (V. cholera) yielded a product with the mobility of paragloboside that bound monoclonal antibody 1B2. NMR analysis revealed that the terminal GalNAc is linked ..beta..1-4more » to Gal, and confirms the structure proposed previously: GalNAc..beta..1-4(NeuAc..cap alpha..2-3)Gal..beta..1-4GlcNAc..beta..1-3Gal..beta..1-4Glc-Cer. This structure is consistent with the previous demonstration that a compound with the same chromatographic mobility as the Cad ganglioside could be synthesized by enzymatic transfer of GalNAc to sialosylparagloboside.« less

  19. Role of gangliosides in active immunotherapy with melanoma vaccine.

    PubMed

    Ravindranath, M H; Morton, D L

    1991-01-01

    Among various tumor associated cell surface antigens, gangliosides, the glycosphingolipids that contain sialic acids, offer a variety of epitopes, some of which are preferentially expressed on melanoma cells. These surface components of the bilayered lipid membrane of tumor cells are the targets of active immunotherapy with melanoma vaccine. Purified gangliosides in aqueous solution form micelles and, at high density, form lactones. Their antigenic expression (physical conformation and orientation) on the cell surface is governed by the nature of the sphingosine and the fatty acids they contain. Evidence is accruing to show that the nature of the fatty acid moiety of gangliosides differs in normal and neoplastic cells. Gangliosides per se are not immunogenic and require extrinsic adjuvanticity. Preparation of a melanoma cell vaccine for active immunotherapy requires an understanding of the ganglioside profile of melanoma, the ganglioside-associated heterogeneity of melanoma, and the role of shed melanoma gangliosides in the immunosuppression of cell mediated and humoral immunity. In addition, the role of some of the anti-ganglioside antibodies in the elimination of shed gangliosides, the cytotoxic killing of tumor cells, as well as in the down-regulation of lymphocyte functions must be considered in the formulation of vaccine. Different strategies for augmenting the immunogenicity of melanoma associated gangliosides with melanoma vaccine are evaluated.

  20. Evidence for Lipid Packaging in the Crystal Structure of the GM2-Activator Complex with Platelet Activating Factor

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Wright, Christine S.; Mi, Li-Zhi; Rastinejad, Fraydoon

    2010-11-16

    GM2-activator protein (GM2-AP) is a lipid transfer protein that has the ability to stimulate the enzymatic processing of gangliosides as well as T-cell activation through lipid presentation. Our previous X-ray crystallographic studies of GM2-AP have revealed a large lipid binding pocket as the central overall feature of the structure with non-protein electron density within this pocket suggesting bound lipid. To extend these studies, we present here the 2 {angstrom} crystal structure of GM2-AP complexed with platelet activating factor (PAF). PAF is a potent phosphoacylglycerol whose toxic patho-physiological effects can be inhibited by GM2-AP. The structure shows an ordered arrangement ofmore » two bound lipids and a fatty acid molecule. One PAF molecule binds in an extended conformation within the hydrophobic channel that has an open and closed conformation, and was seen to contain bound phospholipid in the low pH apo structure. The second molecule is submerged inside the pocket in a U-shaped conformation with its head group near the single polar residue S141. It was refined as lyso-PAF as it lacks electron density for the sn-2 acetate group. The alkyl chains of PAF interact through van der Waals contacts, while the head groups bind in different environments with their phosphocholine moieties in contact with aromatic rings (Y137, F80). The structure has revealed further insights into the lipid binding properties of GM2-AP, suggesting an unexpected unique mode of lipid packaging that may explain the efficiency of GM2-AP in inhibiting the detrimental biological effects of PAF.« less

  1. The GM2 Glycan Serves as a Functional Coreceptor for Serotype 1 Reovirus

    PubMed Central

    Liu, Yan; Blaum, Bärbel S.; Reiter, Dirk M.; Feizi, Ten; Dermody, Terence S.; Stehle, Thilo

    2012-01-01

    Viral attachment to target cells is the first step in infection and also serves as a determinant of tropism. Like many viruses, mammalian reoviruses bind with low affinity to cell-surface carbohydrate receptors to initiate the infectious process. Reoviruses disseminate with serotype-specific tropism in the host, which may be explained by differential glycan utilization. Although α2,3-linked sialylated oligosaccharides serve as carbohydrate receptors for type 3 reoviruses, neither a specific glycan bound by any reovirus serotype nor the function of glycan binding in type 1 reovirus infection was known. We have identified the oligosaccharide portion of ganglioside GM2 (the GM2 glycan) as a receptor for the attachment protein σ1 of reovirus strain type 1 Lang (T1L) using glycan array screening. The interaction of T1L σ1 with GM2 in solution was confirmed using NMR spectroscopy. We established that GM2 glycan engagement is required for optimal infection of mouse embryonic fibroblasts (MEFs) by T1L. Preincubation with GM2 specifically inhibited type 1 but not type 3 reovirus infection of MEFs. To provide a structural basis for these observations, we defined the mode of receptor recognition by determining the crystal structure of T1L σ1 in complex with the GM2 glycan. GM2 binds in a shallow groove in the globular head domain of T1L σ1. Both terminal sugar moieties of the GM2 glycan, N-acetylneuraminic acid and N-acetylgalactosamine, form contacts with the protein, providing an explanation for the observed specificity for GM2. Viruses with mutations in the glycan-binding domain display diminished hemagglutination capacity, a property dependent on glycan binding, and reduced capacity to infect MEFs. Our results define a novel mode of virus-glycan engagement and provide a mechanistic explanation for the serotype-dependent differences in glycan utilization by reovirus. PMID:23236285

  2. The GM2 glycan serves as a functional coreceptor for serotype 1 reovirus.

    PubMed

    Reiss, Kerstin; Stencel, Jennifer E; Liu, Yan; Blaum, Bärbel S; Reiter, Dirk M; Feizi, Ten; Dermody, Terence S; Stehle, Thilo

    2012-01-01

    Viral attachment to target cells is the first step in infection and also serves as a determinant of tropism. Like many viruses, mammalian reoviruses bind with low affinity to cell-surface carbohydrate receptors to initiate the infectious process. Reoviruses disseminate with serotype-specific tropism in the host, which may be explained by differential glycan utilization. Although α2,3-linked sialylated oligosaccharides serve as carbohydrate receptors for type 3 reoviruses, neither a specific glycan bound by any reovirus serotype nor the function of glycan binding in type 1 reovirus infection was known. We have identified the oligosaccharide portion of ganglioside GM2 (the GM2 glycan) as a receptor for the attachment protein σ1 of reovirus strain type 1 Lang (T1L) using glycan array screening. The interaction of T1L σ1 with GM2 in solution was confirmed using NMR spectroscopy. We established that GM2 glycan engagement is required for optimal infection of mouse embryonic fibroblasts (MEFs) by T1L. Preincubation with GM2 specifically inhibited type 1 but not type 3 reovirus infection of MEFs. To provide a structural basis for these observations, we defined the mode of receptor recognition by determining the crystal structure of T1L σ1 in complex with the GM2 glycan. GM2 binds in a shallow groove in the globular head domain of T1L σ1. Both terminal sugar moieties of the GM2 glycan, N-acetylneuraminic acid and N-acetylgalactosamine, form contacts with the protein, providing an explanation for the observed specificity for GM2. Viruses with mutations in the glycan-binding domain display diminished hemagglutination capacity, a property dependent on glycan binding, and reduced capacity to infect MEFs. Our results define a novel mode of virus-glycan engagement and provide a mechanistic explanation for the serotype-dependent differences in glycan utilization by reovirus.

  3. Gangliosides in the Nervous System: Biosynthesis and Degradation

    NASA Astrophysics Data System (ADS)

    Yu, Robert K.; Ariga, Toshio; Yanagisawa, Makoto; Zeng, Guichao

    Gangliosides, abundant in the nervous system, are known to play crucial modulatory roles in cellular recognition, interaction, adhesion, and signal transduction, particularly during early developmental stages. The expression of gangliosides in the nervous system is developmentally regulated and is closely related to the differentiation state of the cell. Ganglioside biosynthesis occurs in intracellular organelles, from which gangliosides are transported to the plasma membrane. During brain development, the ganglioside composition of the nervous system undergoes remarkable changes and is strictly regulated by the activities of glycosyltransferases, which can occur at different levels of control, including glycosyltransferase gene transcription and posttranslational modification. Genes for glycosyltransferase involved in ganglioside biosynthesis have been cloned and classified into families of glycosyltransferases based on their amino acid sequence similarities. The donor and acceptor substrate specificities are determined by enzymatic analysis of the glycosyltransferase gene products. Cell-type specific regulation of these genes has also been studied. Gangliosides are degraded by lysosomal exoglycosidases. The action of these enzymes occurs frequently in cooperation with activator proteins. Several human diseases are caused by defects of degradative enzymes, resulting in massive accumulation of certain glycolipids, including gangliosides in the lysosomal compartment and other organelles in the brain and visceral organs. Some of the representative lysosomal storage diseases (LSDs) caused by the accumulation of lipids in late endosomes and lysosomes will be discussed.

  4. N-butyldeoxynojirimycin treatment restores the innate fear response and improves learning in mucopolysaccharidosis IIIA mice.

    PubMed

    Kaidonis, Xenia; Byers, Sharon; Ranieri, Enzo; Sharp, Peter; Fletcher, Janice; Derrick-Roberts, Ainslie

    2016-06-01

    Mucopolysaccharidosis IIIA is a heritable neurodegenerative disorder resulting from the dysfunction of the lysosomal hydrolase sulphamidase. This leads to the primary accumulation of the complex carbohydrate heparan sulphate in a wide range of tissues and the secondary neuronal storage of gangliosides GM2 and GM3 in the brain. GM2 storage is associated with CNS deterioration in the GM2 gangliosidosis group of lysosomal storage disorders and may also contribute to MPS CNS disease. N-butyldeoxynojirimycin, an inhibitor of ceramide glucosyltransferase activity and therefore of ganglioside synthesis, was administered to MPS IIIA mice both prior to maximal GM2 and GM3 accumulation (early treatment) and after the maximum level of ganglioside had accumulated in the brain (late treatment) to determine if behaviour was altered by ganglioside level. Ceramide glucosyltransferase activity was decreased in both treatment groups; however, brain ganglioside levels were only decreased in the late treatment group. Learning in the water cross maze was improved in both groups and the innate fear response was also restored in both groups. A reduction in the expression of inflammatory gene Ccl3 was observed in the early treatment group, while IL1β expression was reduced in both treatment groups. Thus, it appears that NB-DNJ elicits a transient decrease in brain ganglioside levels, some modulation of inflammatory cytokines and a functional improvement in behaviour that can be elicited both before and after overt neurological changes manifest. NB-DNJ improves learning and restores the innate fear response in MPS IIIA mice by decreasing ceramide glucosyltransferase activity and transiently reducing ganglioside storage and/or modulating inflammatory signals. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. A novel human model of the neurodegenerative disease GM1 gangliosidosis using induced pluripotent stem cells demonstrates inflammasome activation.

    PubMed

    Son, Mi-Young; Kwak, Jae Eun; Seol, Binna; Lee, Da Yong; Jeon, Hyejin; Cho, Yee Sook

    2015-09-01

    GM1 gangliosidosis (GM1) is an inherited neurodegenerative disorder caused by mutations in the lysosomal β-galactosidase (β-gal) gene. Insufficient β-gal activity leads to abnormal accumulation of GM1 gangliosides in tissues, particularly in the central nervous system, resulting in progressive neurodegeneration. Here, we report an in vitro human GM1 model, based on induced pluripotent stem cell (iPSC) technology. Neural progenitor cells differentiated from GM1 patient-derived iPSCs (GM1-NPCs) recapitulated the biochemical and molecular phenotypes of GM1, including defective β-gal activity and increased lysosomes. Importantly, the characterization of GM1-NPCs established that GM1 is significantly associated with the activation of inflammasomes, which play a critical role in the pathogenesis of various neurodegenerative diseases. Specific inflammasome inhibitors potently alleviated the disease-related phenotypes of GM1-NPCs in vitro and in vivo. Our data demonstrate that GM1-NPCs are a valuable in vitro human GM1 model and suggest that inflammasome activation is a novel target pathway for GM1 drug development. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  6. The role of gangliosides in brain development and the potential benefits of perinatal supplementation.

    PubMed

    Ryan, Jennifer M; Rice, Gregory E; Mitchell, Murray D

    2013-11-01

    The maternal diet provides critical nutrients that can influence fetal and infant brain development and function. This review highlights the potential benefits of maternal dietary ganglioside supplementation on fetal and infant brain development. English-language systematic reviews, preclinical studies, and clinical studies were obtained through searches on PubMed. Reports were selected if they included benefits and harms of maternal ganglioside supplementation during pregnancy or ganglioside-supplemented formula after pregnancy. The potential benefits of ganglioside supplementation were explored by investigating the following: (1) their role in neural development, (2) their therapeutic use in neural injury and disease, (3) their presence in human breast milk, and (4) their use as a dietary supplement during or after pregnancy. Preclinical studies indicate that ganglioside supplementation at high doses (1% of total dietary intake) can significantly increase cognitive development and body weight when given prenatally. However, lower ganglioside supplementation doses have no beneficial cognitive effects, even when given throughout pregnancy and lactation. In human clinical trials, infants given formula supplemented with gangliosides showed increased cognitive development and an increase in ganglioside content. Ganglioside supplementation may promote brain development and function in offspring when administered at the optimum dosage. We propose that prenatal maternal dietary supplementation with gangliosides throughout pregnancy may promote greater long-term effects on brain development and function. Before this concept can be encouraged in preconception clinics, future research and clinical trials are needed to confirm the ability of dietary gangliosides to improve cognitive development, but available results already encourage this area of research. © 2013.

  7. Expression of the GM2 activator protein in mouse testis.

    PubMed

    Li, Yu-Teh; Li, Su-Chen; Chen, I-Li

    2017-12-01

    The GM2-activator protein (GM2-AP), revealed by Li et al. in 1973 in human liver, was initially identified as a protein cofactor that stimulated β-hexosaminidase A to hydrolyze N-acetylgalactosamine from GM2 ganglioside. This cofactor was found to be missing in human variant AB Tay-Sachs disease. Over the years, the GM2-AP has also been shown to be involved in kidney vesicular transport, lipid presentation by CD1 molecule to T-cells, and interaction of human sperm with zona pellucida. Since the expression of the GM2-AP via mRNA detection in mouse tissues was found to be the highest in testis, we became interested in the localization of the GM2-AP at cellular level in mouse testis during spermatogenesis. Using immunohistochemical analysis and electron microscopy, we found that the GM2-AP was predominantly localized in the basal cytoplasm and the attenuated processes of Sertoli cells. The stained structure appeared to be lysosomes. The most interesting finding was the association of the GM2-AP with the acrosomal apparatus in early spermatids. A modest to intense staining was observed in some acrosomal granules and acrosomal caps. The GM2-AP seemed to disappear from acrosomal caps in the later stage of spermatids, in which the nucleus became elongated and condensed. These results suggest that the GM2-AP may be involved in the normal functions of Sertoli cells and play important roles during the development of acrosomal caps in the early spermatids. Copyright © 2017 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

  8. [Molecular pathogenesis and therapeutic approach of GM2 gangliosidosis].

    PubMed

    Tsuji, Daisuke

    2013-01-01

    Tay-Sachs and Sandhoff diseases (GM2 gangliosidoses) are autosomal recessive lysosomal storage diseases caused by gene mutations in HEXA and HEXB, each encoding human lysosomal β-hexosaminidase α-subunits and β-subunits, respectively. In Tay-Sachs disease, excessive accumulation of GM2 ganglioside (GM2), mainly in the central nervous system, is caused by a deficiency of the HexA isozyme (αβ heterodimer), resulting in progressive neurologic disorders. In Sandhoff disease, combined deficiencies of HexA and HexB (ββ homodimer) cause not only the accumulation of GM2 but also of oligosaccharides carrying terminal N-acetylhexosamine residues (GlcNAc-oligosaccharides), resulting in systemic manifestations including hepatosplenomegaly as well as neurologic symptoms. Hence there is little clinically effective treatment for these GM2 gangliosidoses. Recent studies on the molecular pathogenesis in Sandhoff disease patients and disease model mice have shown the involvement of microglial activation and chemokine induction in neuroinflammation and neurodegeneration in this disease. Experimental and therapeutic approaches, including recombinant enzyme replacement, have been performed using Sandhoff disease model mice, suggesting the future application of novel techniques to treat GM2 gangliosidoses (Hex deficiencies), including Sandhoff disease as well as Tay-Sachs disease. In this study, we isolated astrocytes and microglia from the neonatal brain of Sandhoff disease model mice and demonstrated abnormalities of glial cells. Moreover, we demonstrated the therapeutic effect of an intracerebroventricular administration of novel recombinant human HexA carrying a high content of M6P residue in Sandhoff disease model mice.

  9. Tay-Sachs disease

    MedlinePlus

    GM2 gangliosidosis - Tay-Sachs; Lysosomal storage disease - Tay-Sachs disease ... called gangliosides. Without this protein, gangliosides, particularly ganglioside GM2, build up in cells, often nerve cells in ...

  10. Therapeutic activity of glycoengineered anti-GM2 antibodies against malignant pleural mesothelioma

    PubMed Central

    Li, Qi; Wang, Wei; Machino, Yusuke; Yamada, Tadaaki; Kita, Kenji; Oshima, Masanobu; Sekido, Yoshitaka; Tsuchiya, Mami; Suzuki, Yui; Nan-ya, Ken-ichiro; Iida, Shigeru; Nakamura, Kazuyasu; Iwakiri, Shotaro; Itoi, Kazumi; Yano, Seiji

    2015-01-01

    Malignant pleural mesothelioma (MPM) is a rare and highly aggressive neoplasm that arises from the pleural, pericardial, or peritoneal lining. Although surgery, chemotherapy, radiotherapy, and combinations of these therapies are used to treat MPM, the median survival of such patients is dismal. Therefore, there is a compelling need to develop novel therapeutics with different modes of action. Ganglioside GM2 is a glycolipid that has been shown to be overexpressed in various types of cancer. However, there are no published reports regarding the use of GM2 as a potential therapeutic target in cases of MPM. In this study, we evaluated the efficacy of the anti-GM2 antibody BIW-8962 as an anti-MPM therapeutic using in vitro and in vivo assays. Consequently, the GM2 expression in the MPM cell lines was confirmed using flow cytometry. In addition, eight of 11 cell lines were GM2-positive (73%), although the GM2 expression was variable. BIW-8962 showed a significant antibody-dependent cellular cytotoxicity activity against the GM2-expressing MPM cell line MSTO-211H, the effect of which depended on the antibody concentration and effector/target ratio. In an in vivo orthotropic mouse model using MSTO-211H cells, BIW-8962 significantly decreased the incidence and size of tumors. Additionally, the GM2 expression was confirmed in the MPM clinical specimens. Fifty-eight percent of the MPM tumors were positive for GM2, with individual variation in the intensity and frequency of staining. These data suggest that anti-GM2 antibodies may become a therapeutic option for MPM patients. PMID:25421609

  11. Interactions of the GM2 activator protein with phosphatidylcholine bilayers: a site-directed spin-labeling power saturation study.

    PubMed

    Mathias, Jordan D; Ran, Yong; Carter, Jeffery D; Fanucci, Gail E

    2009-09-02

    The GM2 activator protein (GM2AP) is an accessory protein that is an essential component in the catabolism of the ganglioside GM2. A function of GM2AP is to bind and extract GM2 from intralysosomal vesicles, forming a soluble protein-lipid complex, which interacts with the hydrolase Hexosaminidase A, the enzyme that cleaves the terminal sugar group of GM2. Here, we used site-directed spin labeling with power saturation electron paramagnetic resonance to determine the surface-bound orientation of GM2AP upon phosphatidylcholine vesicles. Because GM2AP extracts lipid ligands from the vesicle and is undergoing exchange on and off the vesicle surface, we utilized a nickel-chelating lipid to localize the paramagnetic metal collider to the lipid bilayer-aqueous interface. Spin-labeled sites that collide with the lipid-bound metal relaxing agent provide a means for mapping sites of the protein that interact with the lipid bilayer interface. Results show that GM2AP binds to lipid bilayers such that the residues lining the lipid-binding cavity lie on the vesicle surface. This orientation creates a favorable microenvironment that can allow for the lipid tails to flip out of the bilayer directly into the hydrophobic pocket of GM2AP.

  12. GM2 gangliosidosis in an adult pet rabbit.

    PubMed

    Rickmeyer, T; Schöniger, S; Petermann, A; Harzer, K; Kustermann-Kuhn, B; Fuhrmann, H; Schoon, H-A

    2013-02-01

    A 1.5-year-old neutered male rabbit was presented with chronic nasal discharge and ataxia. Rapid progression of neurological signs was noted subsequent to general anaesthesia and the rabbit was humanely destroyed due to the poor prognosis. At necropsy examination there were no gross changes affecting the brain or spinal cord. Microscopical examination revealed that the perikarya of numerous neurons in the brain and spinal cord were distended by the intracytoplasmic accumulation of pale, finely granular to vacuolar material. Transmission electron microscopy showed this to be composed of concentric membranous cytoplasmic bodies. Thin layer chromatography revealed elevation of GM2 ganglioside in the brain of this rabbit compared with that of an unaffected control rabbit. Enzymatically, there was markedly reduced activity of tissue β-hexosaminidase A in brain and liver tissue from the rabbit. This was a result of an almost complete absence of the enzymatic activity of the α-subunit of that enzyme. These findings are consistent with sphingolipidosis comparable with human GM2 gangliosidosis variant B1. Copyright © 2012 Elsevier Ltd. All rights reserved.

  13. Isoflavone Malonyltransferases GmIMaT1 and GmIMaT3 Differently Modify Isoflavone Glucosides in Soybean (Glycine max) under Various Stresses

    PubMed Central

    Ahmad, Muhammad Z.; Li, Penghui; Wang, Junjie; Rehman, Naveed Ur; Zhao, Jian

    2017-01-01

    Malonylated isoflavones are the major forms of isoflavonoids in soybean plants, the genes responsible for their biosyntheses are not well understood, nor their physiological functions. Here we report a new benzylalcohol O-acetyltransferase, anthocyanin O-hydroxycinnamoyltransferase, anthranilate N-hydroxycinnamoyl/benzoyltransferase, deacetylvindoline 4-O-acetyltransferase (BAHD) family isoflavone glucoside malonyltransferase GmIMaT1, and GmIMaT3, which is allelic to the previously characterized GmMT7 and GmIF7MaT. Biochemical studies showed that recombinant GmIMaT1 and GmIMaT3 enzymes used malonyl-CoA and several isoflavone 7-O-glucosides as substrates. The Km values of GmIMaT1 for glycitin, genistin, and daidzin were 13.11, 23.04, and 36.28 μM, respectively, while these of GmIMaT3 were 12.94, 26.67, and 30.12 μM, respectively. Transgenic hairy roots overexpressing both GmIMaTs had increased levels of malonyldaidzin and malonylgenistin, and contents of daidzin and glycitin increased only in GmIMaT1-overexpression lines. The increased daidzein and genistein contents were detected only in GmIMaT3-overexpression lines. Knockdown of GmIMaT1 and GmIMaT3 reduced malonyldaidzin and malonylgenistin contents, and affected other isoflavonoids differently. GmIMaT1 is primarily localized to the endoplasmic reticulum while GmIMaT3 is primarily in the cytosol. By examining their transcript changes corresponding to the altered isoflavone metabolic profiles under various environmental and hormonal stresses, we probed the possible functions of GmIMaTs. Two GmIMaTs displayed distinct tissue expression patterns and respond differently to various factors in modifying isoflavone 7-O-glucosides under various stresses. PMID:28559900

  14. Ganglioside biochemistry.

    PubMed

    Kolter, Thomas

    2012-01-01

    Gangliosides are sialic acid-containing glycosphingolipids. They occur especially on the cellular surfaces of neuronal cells, where they form a complex pattern, but are also found in many other cell types. The paper provides a general overview on their structures, occurrence, and metabolism. Key functional, biochemical, and pathobiochemical aspects are summarized.

  15. Late onset GM2 gangliosidosis presenting with motor neuron disease: an autopsy case.

    PubMed

    Yokoyama, Teruo; Nakamura, Seigo; Horiuchi, Emiko; Ishiyama, Miyako; Kawashima, Rei; Nakamura, Kazuo; Hasegawa, Kazuko; Yagishita, Saburo

    2014-06-01

    Adult-onset GM2 gangliosidosis is very rare and only three autopsy cases have been reported up to now. We report herein an autopsy case of adult-onset GM2 gangliosidosis. The patient developed slowly progressive motor neuron disease-like symptoms after longstanding mood disorder and cognitive dysfunction. He developed gait disturbance and weakness of lower limbs at age 52 years. Because of progressive muscle weakness and atrophy, he became bed-ridden at age 65. At age of 68, he died. His neurological findings presented slight cognitive disturbance, slight manic state, severe muscle weakness, atrophy of four limbs and no extrapyramidal signs and symptoms, and cerebellar ataxia. Neuropathologically, mild neuronal loss and abundant lipid deposits were noted in the neuronal cytoplasm throughout the nervous system, including peripheral autonomic neurons. The most outstanding findings were marked neuronal loss and distended neurons in the anterior horn of the spinal cord, which supports his clinical symptomatology of lower motor neuron disease in this case. The presence of lipofuscin, zebra bodies and membranous cytoplasmic bodies (MCB) and the increase of GM2 ganglioside by biochemistry led to diagnosis of GM2 gangliosidosis. © 2013 Japanese Society of Neuropathology.

  16. Mechanism of abnormal growth in astrocytes derived from a mouse model of GM2 gangliosidosis.

    PubMed

    Kawashima, Nagako; Tsuji, Daisuke; Okuda, Tetsuya; Itoh, Kohji; Nakayama, Ken-ichi

    2009-11-01

    Sandhoff disease is a progressive neurodegenerative disorder caused by mutations in the HEXB gene which encodes the beta-subunit of N-acetyl-beta-hexosaminidase A and B, resulting in the accumulation of the ganglioside GM2. We isolated astrocytes from the neonatal brain of Sandhoff disease model mice in which the N-acetyl-beta-hexosaminidase beta-subunit gene is genetically disrupted (ASD). Glycolipid profiles revealed that GM2/GA2 accumulated in the lysosomes and not on the cell surface of ASD astrocytes. In addition, GM3 was increased on the cell surface. We found remarkable differences in the cell proliferation of ASD astrocytes when compared with cells isolated from wild-type mice, with a faster growth rate of ASD cells. In addition, we observed increased extracellular, signal-regulated kinase (ERK) phosphorylation in ASD cells, but Akt phosphorylation was decreased. Furthermore, the phosphorylation of ERK in ASD cells was not dependent upon extracellular growth factors. Treatment of ASD astrocytes with recombinant N-acetyl-beta-hexosaminidase A resulted in a decrease of their growth rate and ERK phosphorylation. These results indicated that the up-regulation of ERK phosphorylation and the increase in proliferation of ASD astrocytes were dependent upon GM2/GA2 accumulation. These findings may represent a mechanism in linking the nerve cell death and reactive gliosis observed in Sandhoff disease.

  17. Ganglioside Biochemistry

    PubMed Central

    Kolter, Thomas

    2012-01-01

    Gangliosides are sialic acid-containing glycosphingolipids. They occur especially on the cellular surfaces of neuronal cells, where they form a complex pattern, but are also found in many other cell types. The paper provides a general overview on their structures, occurrence, and metabolism. Key functional, biochemical, and pathobiochemical aspects are summarized. PMID:25969757

  18. Antiganglioside antibodies in Guillain-Barré syndrome after a recent cytomegalovirus infection

    PubMed Central

    Khalili-Shirazi, A.; Gregson, N.; Gray, I.; Rees, J.; Winer, J.; Hughes, R.

    1999-01-01

    OBJECTIVE—To study the association between anti-ganglioside antibody responses and Guillan-Barré syndrome (GBS) after a recent cytomegalovirus (CMV) infection.
METHODS—Enzyme linked immunosorbant assay (ELISA) was undertaken on serum samples from 14 patients with GBS with recent cytomegalovirus (CMV) infection (CMV+GBS) and 12 without (CMV-GBS), 17 patients with other neurological diseases (OND), 11 patients with a recent CMV infection but without neurological involvement, 11 patients with recent Epstein-Barr virus (EBV) infection but without neurological involvement, and 20 normal control (NC) subjects.
RESULTS—IgM antibodies were found at 1:100 serum dilution to gangliosides GM2 (six of 14 patients), GM1 (four of 14), GD1a (three of 14) and GD1b (two of 14) in the serum samples of the CMV+GBS patients, but not in those of any of the CMV-GBS patients. IgM antibodies were also found to gangliosides GM1, GD1a, and GD1b in one of 11 OND patients, to ganglioside GM1 in one of 11 non- neurological CMV patients, and to ganglioside GD1b in one of 20NC subjects. Some patients with EBV infection had IgM antibodies to gangliosides GM1 (five of 11), GM2 (three of 11), and GD1a (two of 11). However, the antibodies to ganglioside GM2 had a low titre, none being positive at 1:200 dilution, whereas five of the CMV+GBS serum samples remained positive at this dilution.
CONCLUSION—Antibodies to ganglioside GM2 are often associated with GBS after CMV infection, but their relevance is not known. It is unlikely that CMV infection and anti-ganglioside GM2 antibodies are solely responsible and an additional factor is required to elicit GBS.

 PMID:10084538

  19. Antiganglioside antibodies in Guillain-Barré syndrome after a recent cytomegalovirus infection.

    PubMed

    Khalili-Shirazi, A; Gregson, N; Gray, I; Rees, J; Winer, J; Hughes, R

    1999-03-01

    To study the association between anti-ganglioside antibody responses and Guillan-Barré syndrome (GBS) after a recent cytomegalovirus (CMV) infection. Enzyme linked immunosorbant assay (ELISA) was undertaken on serum samples from 14 patients with GBS with recent cytomegalovirus (CMV) infection (CMV+GBS) and 12 without (CMV-GBS), 17 patients with other neurological diseases (OND), 11 patients with a recent CMV infection but without neurological involvement, 11 patients with recent Epstein-Barr virus (EBV) infection but without neurological involvement, and 20 normal control (NC) subjects. IgM antibodies were found at 1:100 serum dilution to gangliosides GM2 (six of 14 patients), GM1 (four of 14), GD1a (three of 14) and GD1b (two of 14) in the serum samples of the CMV+GBS patients, but not in those of any of the CMV-GBS patients. IgM antibodies were also found to gangliosides GM1, GD1a, and GD1b in one of 11 OND patients, to ganglioside GM1 in one of 11 non- neurological CMV patients, and to ganglioside GD1b in one of 20 NC subjects. Some patients with EBV infection had IgM antibodies to gangliosides GM1 (five of 11), GM2 (three of 11), and GD1a (two of 11). However, the antibodies to ganglioside GM2 had a low titre, none being positive at 1:200 dilution, whereas five of the CMV+GBS serum samples remained positive at this dilution. Antibodies to ganglioside GM2 are often associated with GBS after CMV infection, but their relevance is not known. It is unlikely that CMV infection and anti-ganglioside GM2 antibodies are solely responsible and an additional factor is required to elicit GBS.

  20. Frog brain uridine diphosphate galactose–N-acetylgalactosaminyl-N-acetylneuraminylgalactosylglucosylceramide galactosyltransferase

    PubMed Central

    Yip, Morris C. M.; Dain, Joel A.

    1970-01-01

    1. The enzyme that catalyses the transfer of galactose from UDP-galactose to N-acetylgalactosaminyl-(1→4)-N-acetylneuraminyl-(2→3)-galactosyl-(1→4)-glucosylceramide (GM2) was found mainly in the heavy- and light-microsomal fractions of the adult frog brain. 2. The subcellular distribution of the enzyme, UDP-galactose–GM2 galactosyltransferase, parallels that of gangliosides in adult frog brain. 3. The enzymic activity was first detected at late gastrulation (Shumway stage 11½) and increased until the completion of the operculum (Shumway stage 25) and then decreased in the tadpoles. 4. In adult frog brain, the enzyme exhibited a pH optimum of 7.2–7.3 in both cacodylate and tris buffers. The enzyme required 10mm-Mn2+ for maximal activity and the Km for Mn2+ was determined as 2.2mm. The half-maximal velocity was obtained at a GM2 concentration of 0.18mm. Inhibition of the enzymic reaction was found when the GM2 concentration was greater than 1.38mm. 5. The enzymic activity was also inhibited by the products in the pathway of ganglioside synthesis, i.e. either by a mixture of gangliosides or by individual ganglioside components. The most active inhibitor was disialoganglioside. The degree of inhibition is a function of the individual ganglioside concentration. 6. A product-inhibition mechanism for the regulation of ganglioside biosynthesis is discussed. PMID:5484669

  1. Therapeutic activity of glycoengineered anti-GM2 antibodies against malignant pleural mesothelioma.

    PubMed

    Li, Qi; Wang, Wei; Machino, Yusuke; Yamada, Tadaaki; Kita, Kenji; Oshima, Masanobu; Sekido, Yoshitaka; Tsuchiya, Mami; Suzuki, Yui; Nan-Ya, Ken-ichiro; Iida, Shigeru; Nakamura, Kazuyasu; Iwakiri, Shotaro; Itoi, Kazumi; Yano, Seiji

    2015-01-01

    Malignant pleural mesothelioma (MPM) is a rare and highly aggressive neoplasm that arises from the pleural, pericardial, or peritoneal lining. Although surgery, chemotherapy, radiotherapy, and combinations of these therapies are used to treat MPM, the median survival of such patients is dismal. Therefore, there is a compelling need to develop novel therapeutics with different modes of action. Ganglioside GM2 is a glycolipid that has been shown to be overexpressed in various types of cancer. However, there are no published reports regarding the use of GM2 as a potential therapeutic target in cases of MPM. In this study, we evaluated the efficacy of the anti-GM2 antibody BIW-8962 as an anti-MPM therapeutic using in vitro and in vivo assays. Consequently, the GM2 expression in the MPM cell lines was confirmed using flow cytometry. In addition, eight of 11 cell lines were GM2-positive (73%), although the GM2 expression was variable. BIW-8962 showed a significant antibody-dependent cellular cytotoxicity activity against the GM2-expressing MPM cell line MSTO-211H, the effect of which depended on the antibody concentration and effector/target ratio. In an in vivo orthotropic mouse model using MSTO-211H cells, BIW-8962 significantly decreased the incidence and size of tumors. Additionally, the GM2 expression was confirmed in the MPM clinical specimens. Fifty-eight percent of the MPM tumors were positive for GM2, with individual variation in the intensity and frequency of staining. These data suggest that anti-GM2 antibodies may become a therapeutic option for MPM patients. © 2014 The Authors. Cancer Science published by Wiley Publishing Asia Pty Ltd on behalf of Japanese Cancer Association.

  2. Natural History of Infantile GM2 Gangliosidosis

    PubMed Central

    Bley, Annette E.; Giannikopoulos, Ourania A.; Hayden, Doug; Kubilus, Kim; Tifft, Cynthia J.

    2011-01-01

    OBJECTIVE: GM2 gangliosidoses are caused by an inherited deficiency of lysosomal β-hexosaminidase and result in ganglioside accumulation in the brain. Onset during infancy leads to rapid neurodegeneration and death before 4 years of age. We set out to quantify the rate of functional decline in infantile GM2 gangliosidosis on the basis of patient surveys and a comprehensive review of existing literature. METHODS: Patients with infantile GM2 gangliosidosis (N = 237) were surveyed via questionnaire by the National Tay Sachs & Allied Diseases Association (NTSAD). These data were supplemented by survival data from the NTSAD database and a literature survey. Detailed retrospective surveys from 97 patients were available. Five patients who had received hematopoietic stem cell transplantation were evaluated separately. The mortality rate of the remaining 92 patients was comparable to that of the 103 patients from the NTSAD database and 121 patients reported in the literature. RESULTS: Common symptoms at onset were developmental arrest (83%), startling (65%), and hypotonia (60%). All 55 patients who had learned to sit without support lost that ability within 1 year. Individual functional measures correlated with each other but not with survival. Gastric tube placement was associated with prolonged survival. Tay Sachs and Sandhoff variants did not differ. Hematopoietic stem cell transplantation was not associated with prolonged survival. CONCLUSIONS: We studied the timing of regression in 97 cases of infantile GM2 gangliosidosis and conclude that clinical disease progression does not correlate with survival, likely because of the impact of improved supportive care over time. However, functional measures are quantifiable and can inform power calculations and study design of future interventions. PMID:22025593

  3. Protease-resistant modified human β-hexosaminidase B ameliorates symptoms in GM2 gangliosidosis model.

    PubMed

    Kitakaze, Keisuke; Mizutani, Yasumichi; Sugiyama, Eiji; Tasaki, Chikako; Tsuji, Daisuke; Maita, Nobuo; Hirokawa, Takatsugu; Asanuma, Daisuke; Kamiya, Mako; Sato, Kohei; Setou, Mitsutoshi; Urano, Yasuteru; Togawa, Tadayasu; Otaka, Akira; Sakuraba, Hitoshi; Itoh, Kohji

    2016-05-02

    GM2 gangliosidoses, including Tay-Sachs and Sandhoff diseases, are neurodegenerative lysosomal storage diseases that are caused by deficiency of β-hexosaminidase A, which comprises an αβ heterodimer. There are no effective treatments for these diseases; however, various strategies aimed at restoring β-hexosaminidase A have been explored. Here, we produced a modified human hexosaminidase subunit β (HexB), which we have termed mod2B, composed of homodimeric β subunits that contain amino acid sequences from the α subunit that confer GM2 ganglioside-degrading activity and protease resistance. We also developed fluorescent probes that allow visualization of endocytosis of mod2B via mannose 6-phosphate receptors and delivery of mod2B to lysosomes in GM2 gangliosidosis models. In addition, we applied imaging mass spectrometry to monitor efficacy of this approach in Sandhoff disease model mice. Following i.c.v. administration, mod2B was widely distributed and reduced accumulation of GM2, asialo-GM2, and bis(monoacylglycero)phosphate in brain regions including the hypothalamus, hippocampus, and cerebellum. Moreover, mod2B administration markedly improved motor dysfunction and a prolonged lifespan in Sandhoff disease mice. Together, the results of our study indicate that mod2B has potential for intracerebrospinal fluid enzyme replacement therapy and should be further explored as a gene therapy for GM2 gangliosidoses.

  4. IL-3 specifically inhibits GM-CSF binding to the higher affinity receptor

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Taketazu, F.; Chiba, S.; Shibuya, K.

    1991-02-01

    The inhibition of binding between human granulocyte-macrophage colony-stimulating factor (GM-CSF) and its receptor by human interleukin-3 (IL-3) was observed in myelogenous leukemia cell line KG-1 which bore the receptors both for GM-CSF and IL-3. In contrast, this phenomenon was not observed in histiocytic lymphoma cell line U-937 or in gastric carcinoma cell line KATO III, both of which have apparent GM-CSF receptor but an undetectable IL-3 receptor. In KG-1 cells, the cross-inhibition was preferentially observed when the binding of GM-CSF was performed under the high-affinity binding condition; i.e., a low concentration of 125I-GM-CSF was incubated. Scatchard analysis of 125I-GM-CSF bindingmore » to KG-1 cells in the absence and in the presence of unlabeled IL-3 demonstrated that IL-3 inhibited GM-CSF binding to the higher-affinity component of GM-CSF receptor on KG-1 cells. Moreover, a chemical cross-linking study has revealed that the cross-inhibition of the GM-CSF binding observed in KG-1 cells is specific for the beta-chain, Mr 135,000 binding protein which has been identified as a component forming the high-affinity GM-CSF receptor existing specifically on hemopoietic cells.« less

  5. Animal models of GM2 gangliosidosis: utility and limitations.

    PubMed

    Lawson, Cheryl A; Martin, Douglas R

    2016-01-01

    GM2 gangliosidosis, a subset of lysosomal storage disorders, is caused by a deficiency of the glycohydrolase, β-N-acetylhexosaminidase, and includes the closely related Tay-Sachs and Sandhoff diseases. The enzyme deficiency prevents the normal, stepwise degradation of ganglioside, which accumulates unchecked within the cellular lysosome, particularly in neurons. As a result, individuals with GM2 gangliosidosis experience progressive neurological diseases including motor deficits, progressive weakness and hypotonia, decreased responsiveness, vision deterioration, and seizures. Mice and cats are well-established animal models for Sandhoff disease, whereas Jacob sheep are the only known laboratory animal model of Tay-Sachs disease to exhibit clinical symptoms. Since the human diseases are relatively rare, animal models are indispensable tools for further study of pathogenesis and for development of potential treatments. Though no effective treatments for gangliosidoses currently exist, animal models have been used to test promising experimental therapies. Herein, the utility and limitations of gangliosidosis animal models and how they have contributed to the development of potential new treatments are described.

  6. Animal models of GM2 gangliosidosis: utility and limitations

    PubMed Central

    Lawson, Cheryl A; Martin, Douglas R

    2016-01-01

    GM2 gangliosidosis, a subset of lysosomal storage disorders, is caused by a deficiency of the glycohydrolase, β-N-acetylhexosaminidase, and includes the closely related Tay–Sachs and Sandhoff diseases. The enzyme deficiency prevents the normal, stepwise degradation of ganglioside, which accumulates unchecked within the cellular lysosome, particularly in neurons. As a result, individuals with GM2 gangliosidosis experience progressive neurological diseases including motor deficits, progressive weakness and hypotonia, decreased responsiveness, vision deterioration, and seizures. Mice and cats are well-established animal models for Sandhoff disease, whereas Jacob sheep are the only known laboratory animal model of Tay–Sachs disease to exhibit clinical symptoms. Since the human diseases are relatively rare, animal models are indispensable tools for further study of pathogenesis and for development of potential treatments. Though no effective treatments for gangliosidoses currently exist, animal models have been used to test promising experimental therapies. Herein, the utility and limitations of gangliosidosis animal models and how they have contributed to the development of potential new treatments are described. PMID:27499644

  7. Lo/Ld phase coexistence modulation induced by GM1.

    PubMed

    Puff, Nicolas; Watanabe, Chiho; Seigneuret, Michel; Angelova, Miglena I; Staneva, Galya

    2014-08-01

    Lipid rafts are assumed to undergo biologically important size-modulations from nanorafts to microrafts. Due to the complexity of cellular membranes, model systems become important tools, especially for the investigation of the factors affecting "raft-like" Lo domain size and the search for Lo nanodomains as precursors in Lo microdomain formation. Because lipid compositional change is the primary mechanism by which a cell can alter membrane phase behavior, we studied the effect of the ganglioside GM1 concentration on the Lo/Ld lateral phase separation in PC/SM/Chol/GM1 bilayers. GM1 above 1mol % abolishes the formation of the micrometer-scale Lo domains observed in GUVs. However, the apparently homogeneous phase observed in optical microscopy corresponds in fact, within a certain temperature range, to a Lo/Ld lateral phase separation taking place below the optical resolution. This nanoscale phase separation is revealed by fluorescence spectroscopy, including C12NBD-PC self-quenching and Laurdan GP measurements, and is supported by Gaussian spectral decomposition analysis. The temperature of formation of nanoscale Lo phase domains over an Ld phase is determined, and is shifted to higher values when the GM1 content increases. A "morphological" phase diagram could be made, and it displays three regions corresponding respectively to Lo/Ld micrometric phase separation, Lo/Ld nanometric phase separation, and a homogeneous Ld phase. We therefore show that a lipid only-based mechanism is able to control the existence and the sizes of phase-separated membrane domains. GM1 could act on the line tension, "arresting" domain growth and thereby stabilizing Lo nanodomains. Copyright © 2014 Elsevier B.V. All rights reserved.

  8. Reduction of the ganglioside binding activity of the tetanus toxin HC fragment destroys immunogenicity: implications for development of novel tetanus vaccines.

    PubMed

    Qazi, Omar; Sesardic, Dorothea; Tierney, Robert; Söderbäck, Zahra; Crane, Dennis; Bolgiano, Barbara; Fairweather, Neil

    2006-08-01

    In this study, the immunogenicities of the nontoxic H(C) fragment of tetanus toxin and derivatives lacking ganglioside binding activity were compared with that of tetanus toxoid after subcutaneous immunization of mice. Wild-type H(C) (H(C)WT) protein and tetanus toxoid both elicited strong antibody responses against toxoid and H(C) antigens and provided complete protection against toxin challenge. Mutants of H(C) containing deletions essential for ganglioside binding elicited lower responses than H(C)WT. H(C)M115, containing two amino acid substitutions within the ganglioside binding site, provided reduced protection against tetanus toxin challenge compared with H(C)WT, consistent with lower anti-H(C) and anti-toxoid antibody titers. Circular-dichroism spectroscopy and intrinsic fluorescence spectroscopy showed minimal structural perturbation in H(C)M115. We conclude that the presence of the ganglioside binding site within H(C) may be essential for induction of a fully protective anti-tetanus response comparable to that induced by tetanus toxoid by subcutaneous injection.

  9. Reduction of the Ganglioside Binding Activity of the Tetanus Toxin HC Fragment Destroys Immunogenicity: Implications for Development of Novel Tetanus Vaccines

    PubMed Central

    Qazi, Omar; Sesardic, Dorothea; Tierney, Robert; Söderbäck, Zahra; Crane, Dennis; Bolgiano, Barbara; Fairweather, Neil

    2006-01-01

    In this study, the immunogenicities of the nontoxic HC fragment of tetanus toxin and derivatives lacking ganglioside binding activity were compared with that of tetanus toxoid after subcutaneous immunization of mice. Wild-type HC (HCWT) protein and tetanus toxoid both elicited strong antibody responses against toxoid and HC antigens and provided complete protection against toxin challenge. Mutants of HC containing deletions essential for ganglioside binding elicited lower responses than HCWT. HCM115, containing two amino acid substitutions within the ganglioside binding site, provided reduced protection against tetanus toxin challenge compared with HCWT, consistent with lower anti-HC and anti-toxoid antibody titers. Circular-dichroism spectroscopy and intrinsic fluorescence spectroscopy showed minimal structural perturbation in HCM115. We conclude that the presence of the ganglioside binding site within HC may be essential for induction of a fully protective anti-tetanus response comparable to that induced by tetanus toxoid by subcutaneous injection. PMID:16861677

  10. Isolation and structure of a monomethylated ganglioside possessing neuritogenic activity from the ovary of the sea urchin Diadema setosum.

    PubMed

    Yamada, Koji; Tanabe, Kaoru; Miyamoto, Tomofumi; Kusumoto, Toshihide; Inagaki, Masanori; Higuchi, Ryuichi

    2008-05-01

    A new monomethylated ganglioside, DSG-A (3), was obtained, together with four known gangliosides, compounds (1, 2, 4, 5), from the lipid fraction of the chloroform/methanol extract of the ovary of the sea urchin Diadema setosum. The structures of the new ganglioside was determined on the basis of chemical and spectroscopic evidence to be 1-O-[9-O-methyl-(N-acetyl-alpha-D-neuraminosyl)-(2-->6)-beta-D-glucopyranosyl]-ceramide (3). The ceramide moiety of 3 was composed of C18-phytosphingosine base, and 2-hydroxy and nonhydroxylated fatty acid units. These gangliosides showed neuritogenic activity toward the rat pheochromocytoma cell line PC-12 in the presence of nerve growth factor, in which compound 3 showed the most potent activity.

  11. Molecular recognition and colorimetric detection of cholera toxin by poly(diacetylene) liposomes incorporating G{sub m1} ganglioside

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Pan, J.J.; Charych, D.

    1997-03-19

    Molecular recognition sites on cell membranes serve as the main communication channels between the inside of a cell and its surroundings. Upon receptor binding, cellular messages such as ion channel opening or activation of enzymes are triggered. In this report, we demonstrate that artificial cell membranes made from conjugated lipid polymers (poly(diacetylene)) can, on a simple level, mimic membrane processes of molecular recognition and signal transduction. The ganglioside GM1 was incorporated into poly(diacetylene) liposomes. Molecular recognition of cholera toxin at the interface of the liposome resulted in a change of the membrane color due to conformational charges in the conjugatedmore » (ene-yne) polymer backbone. The `colored liposomes` might be used as simple colorimetric sensors for drug screening or as new tools to study membrane-membrane or membrane-receptor interactions. 21 refs., 3 figs.« less

  12. Vaccination of High-Risk Breast Cancer Patients with Carbohydrate Mimicking Peptides

    DTIC Science & Technology

    2008-05-01

    Spontaneous pyroglutamic acid formation for peptides starting with glutamic acid or glutamine residues is not considered an impurity. Spontaneous...Examples of tumor- associated carbohydrate antigens include the gangliosides GM2, GD2, GD3, and fucosyl GM1, Globo H, polysialic acid , STn and the...directed toward gangliosides, polysialic acid , Globo, Lewis Y (LeY), and the STn antigen. Because TACA are T-cell–independent antigens and self-antigens

  13. The Diagnostic Utility of Determining Anti-GM1: GalC Complex Antibodies in Multifocal Motor Neuropathy: A Validation Study

    PubMed Central

    Galban-Horcajo, Francesc; Vlam, Lotte; Delmont, Emilien; Halstead, Susan K.; van den Berg, Leonard; van der Pol, W-Ludo; Willison, Hugh J.

    2015-01-01

    Abstract Background: Multifocal motor neuropathy (MMN) is associated with IgM antibodies to GM1 ganglioside. The importance of the lipid milieu that might facilitate or inhibit antibody binding to GM1 in immunoassays is well recognised. Existing studies, using a range of different approaches, generally concur that anti-GM1 IgM antibody detection rates are improved by the addition of galactocerebroside (GalC) to the GM1 assay. Objective: The current study sought to formally evaluate the clinical utility of the GM1:GalC complex assay in the diagnosis of MMN. Methods: Anti-GM1 and -GM1:GalC antibodies were examined using ELISA and glycoarray (dot blot) in a fully blinded study design, consisting of 100 MMN patients, 100 ALS cases and 100 healthy controls. Results: The detection of anti-GM1 Abs using glycoarray was 67% sensitive and 85% specific. The addition of GalC to GM1, (1:1 weight to weight ratio), increased the sensitivity to 81% , whilst dropping specificity to 80% . Increasing the GalC content to a 1:5 ratio (or higher) further decreased specificity, and in doing so limited the usefulness of the GM1:GalC assay to the level of GM1 alone. The addition of GalC to the ELISA method also significantly increased sensitivity compared with GM1 alone, albeit with a significant decrease in specificity. Conclusions: This study indicates that the GM1:GalC assay is an advantageous assay adaptation for detecting anti-GM1 antibodies in MMN, using either glycoarray or ELISA, and warrants introduction into clinical diagnostic practice. PMID:27858734

  14. Ganglioside Composition in Beef, Chicken, Pork, and Fish Determined Using Liquid Chromatography-High-Resolution Mass Spectrometry.

    PubMed

    Fong, Bertram Y; Ma, Lin; Khor, Geok Lin; van der Does, Yvonne; Rowan, Angela; McJarrow, Paul; MacGibbon, Alastair K H

    2016-08-17

    Gangliosides (GA) are found in animal tissues and fluids, such as blood and milk. These sialo-glycosphingolipids have bioactivities in neural development, the gastrointestinal tract, and the immune system. In this study, a high-performance liquid chromatography-mass spectrometry (HPLC-MS) method was validated to characterize and quantitate the GA in beef, chicken, pork, and fish species (turbot, snapper, king salmon, and island mackerel). For the first time, we report the concentration of GM3, the dominant GA in these foods, as ranging from 0.35 to 1.1 mg/100 g and 0.70 to 5.86 mg/100 g of meat and fish, respectively. The minor GAs measured were GD3, GD1a, GD1b, and GT1b. Molecular species distribution revealed that the GA contained long- to very-long-chain acyl fatty acids attached to the ceramide moiety. Fish GA contained only N-acetylneuraminic acid (NeuAc) sialic acid, while beef, chicken, and pork contained GD1a/b species that incorporated both NeuAc and N-glycolylneuraminic acid (NeuGc) and hydroxylated fatty acids.

  15. Highly phosphomannosylated enzyme replacement therapy for GM2 gangliosidosis.

    PubMed

    Tsuji, Daisuke; Akeboshi, Hiromi; Matsuoka, Kazuhiko; Yasuoka, Hiroko; Miyasaki, Eri; Kasahara, Yoshiko; Kawashima, Ikuo; Chiba, Yasunori; Jigami, Yoshifumi; Taki, Takao; Sakuraba, Hitoshi; Itoh, Kohji

    2011-04-01

    Novel recombinant human lysosomal β-hexosaminidase A (HexA) was developed for enzyme replacement therapy (ERT) for Tay-Sachs and Sandhoff diseases, ie, autosomal recessive GM2 gangliosidoses, caused by HexA deficiency. A recombinant human HexA (Om4HexA) with a high mannose 6-phosphate (M6P)-type-N-glycan content, which was produced by a methylotrophic yeast strain, Ogataea minuta, overexpressing the OmMNN4 gene, was intracerebroventricularly (ICV) administered to Sandhoff disease model mice (Hexb⁻/⁻ mice) at different doses (0.5-2.5 mg/kg), and then the replacement and therapeutic effects were examined. The Om4HexA was widely distributed across the ependymal cell layer, dose-dependently restored the enzyme activity due to uptake via cell surface cation-independent M6P receptor (CI-M6PR) on neural cells, and reduced substrates, including GM2 ganglioside (GM2), asialo GM2 (GA2), and oligosaccharides with terminal N-acetylglucosamine residues (GlcNAc-oligosaccharides), accumulated in brain parenchyma. A significant inhibition of chemokine macrophage inflammatory protein-1 α (MIP-1α) induction was also revealed, especially in the hindbrain (< 63%). The decrease in central neural storage correlated with an improvement of motor dysfunction as well as prolongation of the lifespan. This lysosome-directed recombinant human enzyme drug derived from methylotrophic yeast has the high therapeutic potential to improve the motor dysfunction and quality of life of the lysosomal storage diseases (LSDs) patients with neurological manifestations. We emphasize the importance of neural cell surface M6P receptor as a delivery target of neural cell-directed enzyme replacement therapy (NCDERT) for neurodegenerative metabolic diseases. Copyright © 2010 American Neurological Association.

  16. Possible role of autoantibodies in the pathophysiology of GM2 gangliosidoses

    PubMed Central

    Yamaguchi, Akira; Katsuyama, Kayoko; Nagahama, Kiyotaka; Takai, Toshiyuki; Aoki, Ichiro; Yamanaka, Shoji

    2004-01-01

    Mice containing a disruption of the Hexb gene have provided a useful model system for the study of the human lysosomal storage disorder known as Sandhoff disease (SD). Hexb–/– mice rapidly develop a progressive neurologic disease of ganglioside GM2 and GA2 storage. Our study revealed that the disease states in this model are associated with the appearance of antiganglioside autoantibodies. Both elevation of serum antiganglioside autoantibodies and IgG deposition to CNS neurons were found in the advanced stages of the disease in Hexb–/– mice; serum transfer from these mice showed IgG binding to neurons. To determine the role of these autoantibodies, the Fc receptor γ gene (FcRγ) was additionally disrupted in Hexb–/– mice, as it plays a key role in immune complex–mediated autoimmune diseases. Clinical symptoms were improved and life spans were extended in the Hexb–/–FcRγ–/– mice; the number of apoptotic cells was also decreased. The level of ganglioside accumulation, however, did not change. IgG deposition was also confirmed in the brain of an autopsied SD patient. Taken together, these findings suggest that the production of autoantibodies plays an important role in the pathogenesis of neuropathy in SD and therefore provides a target for novel therapies. PMID:14722612

  17. Wisp2/CCN5 up-regulated in the central nervous system of GM3-only mice facilitates neurite formation in Neuro2a cells via integrin-Akt signaling

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ohkawa, Yuki, E-mail: tomilbio@med.nagoya-u.ac.jp; Ohmi, Yuhsuke, E-mail: ooumi82@med.nagoya-u.ac.jp; Tajima, Orie, E-mail: oriet@isc.chubu.ac.jp

    Highlights: {yields} Wisp2/CCN5 was up-regulated in nervous tissues of GM3-only mutant mice. {yields} Wisp2/CCN5 was found in neurons more strongly in the mutant mice. {yields} Wisp2/CCN5 induces Akt phosphorylation via integrins and facilitates neurite formation. {yields} Wisp2/CCN5 conferred resistance to H{sub 2}O{sub 2}-induced apoptosis. {yields} Up-regulation of Wisp2/CCN5 in GM3-only mice seemed for protection of brains from neurodegeneration. -- Abstract: Wisp2/CCN5 belongs to CCN family proteins which are involved in cell proliferation, angiogenesis, tumorigenesis and wound healing. Although a number of studies on the roles of Wisp2/CCN5 in cancers have been reported, no study on the expression and function ofmore » Wisp2/CCN5 in the central nervous system has been reported. In this study, we focused on Wisp2/CCN5 that was up-regulated in nervous tissues in GM3-only mice. Over-expression of Wisp2/CCN5 enhanced neurite outgrowth potently after serum withdrawal with increased phosphorylation levels of Akt and ERKs. When cells were cultured with recombinant Wisp2/CCN5 proteins, more and longer neurites were formed than in the controls. Thus, we demonstrated for the first time that Wisp2/CCN5 facilitates neurite formation in a mouse neuroblastoma cell line, Neuro2a. Akt phosphorylation induced by recombinant Wisp2/CCN5 was suppressed after knockdown of integrin {beta}1. Moreover, Wisp2/CCN5-over-expressing cells were resistant to apoptosis induced by H{sub 2}O{sub 2}. These results suggested that secreted Wisp2/CCN5 induces Akt and ERK phosphorylation via integrins, and consequently facilitates neurite formation and conferred resistance to apoptosis. Up-regulation of Wisp2/CCN5 in GM3-only mice should be, therefore, a reaction to protect nervous tissues from neurodegeneration caused by ganglioside deficiency.« less

  18. Effect of compounds with antibacterial activities in human milk on respiratory syncytial virus and cytomegalovirus in vitro.

    PubMed

    Portelli, J; Gordon, A; May, J T

    1998-11-01

    The effect of some antibacterial compounds present in human milk were tested for antiviral activity against respiratory syncytial virus, Semliki Forest virus and cytomegalovirus. These included the gangliosides GM1, GM2 and GM3, sialyl-lactose, lactoferrin and chondroitin sulphate A, B and C, which were all tested for their ability to inhibit the viruses in cell culture. Of the compounds tested, only the ganglioside GM2, chondroitin sulphate B and lactoferrin inhibited the absorption and growth of respiratory syncytial virus in cell culture, and none inhibited the growth of Semliki Forest virus, indicating that lipid antiviral activity was not associated with any of the gangliosides. While the concentrations of these two compounds required to inhibit respiratory syncytial virus were in excess of those present in human milk, sialyl-lactose concentrations similar to those present in human milk increased the growth of cytomegalovirus. Lactoferrin was confirmed as inhibiting both respiratory syncytial virus and cytomegalovirus growth in culture even when used at lower concentrations than those present in human milk. The antiviral activities of GM2, chondroitin sulphate B and lactoferrin were tested when added to an infant formula. Lactoferrin continued to have antiviral activity against cytomegalovirus, but a lower activity against respiratory syncytial virus; ganglioside GM2 and chondroitin sulphate B still maintained antiviral activity against respiratory syncytial virus.

  19. Distinct Immunoglobulin Class and Immunoglobulin G Subclass Patterns against Ganglioside GQ1b in Miller Fisher Syndrome following Different Types of Infection

    PubMed Central

    Schwerer, Beatrix; Neisser, Andrea; Bernheimer, Hanno

    1999-01-01

    We studied serum antibodies against gangliosides GQ1b and GM1 in 13 patients with Miller Fisher syndrome (MFS) and in 18 patients with Guillain-Barré syndrome (GBS) with cranial nerve involvement. Anti-GQ1b titers were elevated in all patients with MFS cases (immunoglobulin G [IgG] > IgA, IgM), and in 8 of the 18 with GBS. Lower frequencies of increased anti-GM1 titers were observed in MFS patients (3 of 13), as well as in GBS patients (5 of 18). During the course of MFS, anti-GQ1b titers of all Ig classes decreased within 3 weeks after onset. By contrast, anti-GM1 titers (mainly IgM) transiently increased during the course of MFS in five of six patients, suggesting a nonspecific secondary immune response. In patients with MFS following respiratory infections, IgG was the major anti-GQ1b Ig class (six of six patients) and IgG3 was the major subclass (five of six). In contrast, four of five patients with MFS following gastrointestinal infections showed predominance of anti-GQ1b IgA or IgM over IgG and predominance of the IgG2 subclass; anti-GQ1b IgG (IgG3) prevailed in one patient only. These distinct Ig patterns strongly suggest that different infections may trigger different mechanisms of anti-GQ1b production, such as via T-cell-dependent as opposed to T-cell-independent pathways. Thus, the origin of antibodies against GQ1b in MFS may be determined by the type of infectious agent that precipitates the disease. PMID:10225903

  20. Deficits in cognitive function and hippocampal plasticity in GM2/GD2 synthase knockout mice.

    PubMed

    Sha, Sha; Zhou, Libin; Yin, Jun; Takamiya, Koga; Furukawa, Keiko; Furukawa, Koichi; Sokabe, Masahiro; Chen, Ling

    2014-04-01

    In this study, we used GM2/GD2 synthase knockout (GM2/GD2−/−) mice to examine the influence of deficiency in ganglioside “a-pathway” and “b-pathway” on cognitive performances and hippocampal synaptic plasticity. Eight-week-old GM2/GD2−/− male mice showed a longer escape-latency in Morris water maze test and a shorter latency in step-down inhibitory avoidance task than wild-type (WT) mice. Schaffer collateral-CA1 synapses in the hippocampal slices from GM2/GD2−/− mice showed an increase in the slope of EPSPs with reduced paired-pulse facilitation, indicating an enhancement of their presynaptic glutamate release. In GM2/GD2−/− mice, NMDA receptor (NMDAr)-dependent LTP could not be induced by high-frequency (100–200 Hz) tetanus or θ-burst conditioning stimulation (CS), whereas NMDAr-independent LTP was induced by medium-frequency CS (20–50 Hz). The application of mono-sialoganglioside GM1 in the slice from GM2/GD2−/− mice, to specifically recover the a-pathway, prevented the increased presynaptic glutamate release and 20 Hz-LTP induction, whereas it could not rescue the impaired NMDAr-dependent LTP. These findings suggest that b-pathway deficiency impairs cognitive function probably through suppression of NMDAr-dependent LTP, while a-pathway deficiency may facilitate NMDAr-independent LTP through enhancing presynaptic glutamate release. As both of the NMDAr-independent LTP and increased presynaptic glutamate release were sensitive to the blockade of L-type voltage-gated Ca2+ channels (L-VGCC), a-pathway deficiency may affect presynaptic L-VGCC.

  1. Genetics Home Reference: GM3 synthase deficiency

    MedlinePlus

    ... GM3 synthase deficiency is characterized by recurrent seizures (epilepsy) and problems with brain development. Within the first ... Testing (1 link) Genetic Testing Registry: Amish infantile epilepsy syndrome Other Diagnosis and Management Resources (2 links) ...

  2. Identification of New Serum Biomarkers for Early Breast Cancer Diagnosis and Prognosis Using Lipid Microarrays

    DTIC Science & Technology

    2008-09-01

    specific for asialo-GM1 bound specifically to GM1, but not to the closely related gangliosides GM1 or GM2 (Fig. 2). Monoclonal antibodies raised...against GD3 specifically bound GD3, but not to asialo-GM1, GM1 and GM2 (Fig. 2). The secondary antibodies did not show reactivity against lipids (data not...fluorescent intensity on different membranes. Asialo GM1 GM1 GM2 GD3 Fig 2. Lipids on the PVDF membrane can be detected by specific

  3. Identification of New Serum Biomarkers for Early Breast Cancer Diagnosis and Prognosis Using Lipid Microarrays

    DTIC Science & Technology

    2007-09-01

    specific for asialo-GM1 bound specifically to GM1, but not to the closely related gangliosides GM1 or GM2 (Fig. 2). Monoclonal antibodies raised against...GD3 specifically bound GD3, but not to asialo-GM1, GM1 and GM2 (Fig. 2). The secondary antibodies did not show reactivity against lipids (data not...fluorescent intensity on different membranes. Asialo GM1 GM1 GM2 GD3 Fig 2. Lipids on the PVDF membrane can be detected by specific antibodies

  4. STAT3-Activated GM-CSFRα Translocates to the Nucleus and Protects CLL Cells from Apoptosis

    PubMed Central

    Li, Ping; Harris, David; Liu, Zhiming; Rozovski, Uri; Ferrajoli, Alessandra; Wang, Yongtao; Bueso-Ramos, Carlos; Hazan-Halevy, Inbal; Grgurevic, Srdana; Wierda, William; Burger, Jan; O'Brien, Susan; Faderl, Stefan; Keating, Michael; Estrov, Zeev

    2014-01-01

    Here it was determined that Chronic Lymphocytic Leukemia (CLL) cells express the α-subunit but not the β-subunit of the granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR/CSF3R). GM-CSFRα was detected on the surface, in the cytosol, and the nucleus of CLL cells via confocal microscopy, cell fractionation, and GM-CSFRα antibody epitope mapping. Because STAT3 is frequently activated in CLL and the GM-CSFRα promoter harbors putative STAT3 consensus binding sites, MM1 cells were transfected with truncated forms of the GM-CSFRα promoter, then stimulated with IL-6 to activate STAT3 to identify STAT3 binding sites. Chromatin immunoprecipitation (ChIP) and an electoromobility shift assay (EMSA) confirmed STAT3 occupancy to those promoter regions in both IL-6 stimulated MM1 and CLL cells. Transfection of MM1 cells with STAT3 siRNA or CLL cells with STAT3 shRNA significantly down-regulated GM-CSFRα mRNA and protein levels. RNA transcripts, involved in regulating cell-survival pathways, and the proteins KAP1 (TRIM28) and ISG15 co-immunoprecipitated with GM-CSFRα. GM-CSFRα-bound KAP1 enhanced the transcriptional activity of STAT3, whereas ISG15 inhibited the NF-κB pathway. Nevertheless, overexpression of GM-CSFRα protected MM1 cells from dexamethasone-induced apoptosis, and GM-CSFRα knockdown induced apoptosis in CLL cells, suggesting that GM-CSFRα provides a ligand-independent survival advantage. PMID:24836891

  5. Effect of anti-GM2 antibodies on rat sciatic nerve: electrophysiological and morphological study.

    PubMed

    Ortiz, Nicolau; Sabaté, M Mar; Garcia, Neus; Santafe, Manel M; Lanuza, M Angel; Tomàs, Marta; Tomàs, Josep

    2009-03-31

    We found that a monoclonal human IgM anti-GM2 was fixed in rat sciatic axons and Schwann cells and was able to activate human complement. The passive transfer of IgM and complement in sciatic nerves can induce an acute alteration in nerve conduction. When the transfer of IgM plus complement was repeated for 10 days, the compound action motor potential amplitude was very low and the morphological study showed axons and myelin damage. Without human complement, IgM can only slightly disorganize the myelin by separating some layers, probably by interfering with the functional role of gangliosides in the myelin package.

  6. Synthesis and Immunological Properties of N-Modified GM3 Antigens as Therapeutic Cancer Vaccines

    PubMed Central

    Pan, Yanbin; Chefalo, Peter; Nagy, Nancy; Harding, Clifford; Guo, Zhongwu

    2011-01-01

    The problem of immunotolerance to GM3, an important tumor-associated trisaccharide antigen, seriously hinders its usage in cancer vaccine development. To solve this problem, the keyhole limpet hemocyanin (KLH) conjugates of a series of GM3 derivatives were synthesized and screened as therapeutic cancer vaccines. First, the β-linked anomeric azides of differently N-acylated GM3 analogs were prepared by a highly convergent procedure. Next, a pentenoyl group was linked to the reducing end of the carbohydrate antigens following selective reduction of the azido group. The linker was thereafter ozonolyzed to give an aldehyde functionality permitting the conjugation of the antigens to KLH via reductive amination. Finally, the immunological properties of the resultant glycoconjugates were studied in C57BL/6 mice by assessing the titers of specific antibodies induced by the GM3 analogs. While KLH-GM3 elicited low levels of immune response, the KLH conjugates of N-propionyl, N-butanoyl, N-iso-butanoyl and N-phenylacetyl GM3’s induced robust immune reactions with antibodies of multiple isotypes, indicating significantly improved and T-cell dependent immune responses that lead to isotype switching, affinity maturation and the induction of immunological ‘memory’. It was suggested that GM3PhAc-KLH is a promising vaccine candidate for glycoengineered immunotherapy of cancer with GM3 as the primary target. PMID:15689172

  7. Serum antibodies against gangliosides and Campylobacter jejuni lipopolysaccharides in Miller Fisher syndrome.

    PubMed Central

    Neisser, A; Bernheimer, H; Berger, T; Moran, A P; Schwerer, B

    1997-01-01

    Seven patients with Miller Fisher syndrome (MFS), six in the acute phase and one in the recovery phase, were investigated for serum antibodies against gangliosides and purified lipopolysaccharides (LPS) from different strains of Campylobacter jejuni, including the MFS-associated serotypes O:2 and O:23. Immunoglobulin G antibodies against gangliosides GT1a and GQ1b were found in five of six patients in the acute phase of disease. Three of these patients also displayed antibodies to ganglioside GD2, a finding not previously reported for MFS. All anti-GT1a- and anti-GQ1b-seropositive patients showed antibody binding to C. jejuni LPS, predominantly to O:2 and O:23 LPS. Antibody cross-reactivity between gangliosides GT1a and GQ1b and O:2 and O:23 LPS was demonstrated by adsorption studies. This cross-reactivity between gangliosides and C.jejuni LPS, which is obviously due to oligosaccharide homologies, may be an important pathogenetic factor in the development of MFS after C. jejuni infection. PMID:9317004

  8. Dietary lipids containing gangliosides reduce Giardia muris infection in vivo and survival of Giardia lamblia trophozoites in vitro.

    PubMed

    Suh, M; Belosevic, M; Clandinin, M T

    2004-06-01

    We examined whether a ganglioside supplemented diet affected the course of Giardia muris infection in mice and survival of Giardia lamblia trophozoites in vitro. Female CD-1 mice were fed 1 of 5 experimental diets: standard lab chow as a control diet; semi-synthetic diets containing 20% (w/w) triglyceride based on the fat composition of a conventional infant formula; triglyceride diet; triglyceride diet containing a low level of ganglioside (0.1% w/w); and triglyceride diet containing a high level of ganglioside (1.0% w/w of diet). After 2 weeks of feeding, mice were inoculated with G. muris by gastric intubation and fed the experimental diets during the course of the infection. Cysts released in the faeces and trophozoites present in the small intestine were enumerated at various times post-infection. The average cyst output and the number of trophozoites during the course of the infection in mice fed ganglioside-containing diet were found to be significantly lower (3-log10 reduction) compared to animals fed control diets. The results of in vitro growth studies indicated that gangliosides may be directly toxic to the parasites. Thus, gangliosides have a protective effect against G. muris infection in vivo and affect the survival of G. lamblia trophozoites in vitro.

  9. Inhibition of Lysosome Membrane Recycling Causes Accumulation of Gangliosides that Contribute to Neurodegeneration.

    PubMed

    Boutry, Maxime; Branchu, Julien; Lustremant, Céline; Pujol, Claire; Pernelle, Julie; Matusiak, Raphaël; Seyer, Alexandre; Poirel, Marion; Chu-Van, Emeline; Pierga, Alexandre; Dobrenis, Kostantin; Puech, Jean-Philippe; Caillaud, Catherine; Durr, Alexandra; Brice, Alexis; Colsch, Benoit; Mochel, Fanny; El Hachimi, Khalid Hamid; Stevanin, Giovanni; Darios, Frédéric

    2018-06-26

    Lysosome membrane recycling occurs at the end of the autophagic pathway and requires proteins that are mostly encoded by genes mutated in neurodegenerative diseases. However, its implication in neuronal death is still unclear. Here, we show that spatacsin, which is required for lysosome recycling and whose loss of function leads to hereditary spastic paraplegia 11 (SPG11), promotes clearance of gangliosides from lysosomes in mouse and human SPG11 models. We demonstrate that spatacsin acts downstream of clathrin and recruits dynamin to allow lysosome membrane recycling and clearance of gangliosides from lysosomes. Gangliosides contributed to the accumulation of autophagy markers in lysosomes and to neuronal death. In contrast, decreasing ganglioside synthesis prevented neurodegeneration and improved motor phenotype in a SPG11 zebrafish model. Our work reveals how inhibition of lysosome membrane recycling leads to the deleterious accumulation of gangliosides, linking lysosome recycling to neurodegeneration. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

  10. Constituents of ophiuroidea. 1. Isolation and structure of three ganglioside molecular species from the brittle star Ophiocoma scolopendrina.

    PubMed

    Inagaki, M; Shibai, M; Isobe, R; Higuchi, R

    2001-12-01

    Three ganglioside molecular species, OSG-0 (1), OSG-1 (2), and OSG-2 (3) have been obtained from the polar lipid fraction of the chloroform/methanol extract of the brittle star Ophiocoma scolopendrina. The structures of these gangliosides have been determined on the basis of chemical and spectroscopic evidence as 1-O-[(N-glycolyl-alpha-D-neuraminosyl)-(2-->6)-beta-D-glucopyranosyl]-ceramide (1), 1-O-[8-O-sulfo-(N-acetyl-alpha-D-neuraminosyl)-(2-->6)-beta-D-glucopyranosyll-ceramide (2) and 1-O-[(N-glycolyl-alpha-D-neuraminosyl)-(2-->8)-(N-acetyl- and N-glycolyl-alpha-D-neuraminosyl)-(2-->6)-beta-D-glucopyranosyl]-ceramide (3). The ceramide moieties were composed of heterogeneous unsubstituted fatty acid, 2-hydroxy fatty acid and phytosphingosine units. Compounds 2 and 3 represent new ganglioside molecular species.

  11. Detection of Sendai virus receptor, the ganglioside GDla, in target tissue (mouse lung)

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Markwell, M.A.K.; Sato, E.

    1986-05-01

    Previously the authors had shown that the gangliosides GDla, GTlb, and GQlb derived from brain function as receptors for the paramyxovirus Sendai virus by their ability to induce infection when incubated with receptor-deficient cells. Analyses of MDBK, HeLa, and MDCK cells in culture demonstrated that these putative receptors were present in host cells in the quantities required for infection. The primary site of infection for Sendai virus in the whole animal is the respiratory tract, culminating in the lung. Therefore, the ganglioside content of this target organ was analyzed to determine the endogenous receptor population available to Sendai virus. Themore » total ganglioside fraction of lung was resolved into individual species by HPTLC. Gangliosides of the gangliotetraose series were identified by the specific binding of /sup 125/I-labeled tetanus and cholera toxins before and after exposure with sialidase. In this manner one of the major resorcinol-positive bands was identified as GDla. Evidence of the more complex ganglioside receptors for Sendai virus was also seen.« less

  12. Analysis of GD2/GM2 synthase mRNA as a biomarker for small cell lung cancer.

    PubMed

    Chen, Lin-Chi; Brown, Andrew B; Cheung, Irene Y; Cheung, Nai-Kong V; Kris, Mark G; Krug, Lee M

    2010-02-01

    GD2/GM2 synthase is a key enzyme in the synthesis of GD2 and GM2 gangliosides found on the surface of neuroblastoma and small cell lung carcinoma (SCLC) cells. In neuroblastoma, persistent levels of GD2/GM2 synthase RNA in bone marrow (BM) following therapy portend poorer progression-free and overall survival. We conducted this study to determine if GD2/GM2 synthase RNA could be detected in SCLC cell lines and human tissues, and whether mRNA transcript levels corresponded with disease status. Initially, a pilot study enrolled patients with SCLC to determine the rate of GD2 expression at various points in the patients' disease course. Peripheral blood (PB), bone marrow and tumor tissues were used to measure GD2/GM2 synthase levels. In addition, SCLC cell lines were analyzed for GD2/GM2 synthase expression. Based on data from that initial analysis, a prospective trial was developed enrolling patients with newly diagnosed SCLC and following them serially. GD2/GM2 synthase transcript was determined by a sensitive quantitative reverse transcription-PCR (qRT-PCR) assay and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Six SCLC cell lines were assayed for expression of GD2/GM2 synthase, and high expression was detected in all. GD2/GM2 synthase transcript levels were obtained from tumor tissue, BM, or PB of 29 patients in the pilot study. 6/10 (60%) tumor tissues or BM samples were positive (median 332.7 units; range 13-2323 units); 8/19 (42%) untreated patients were GD2/GM2 synthase positive in their PB prior to beginning therapy (median 10.2; range 5.1-32.2); 3/4 (75%) patients who were first tested when they developed recurrent disease were positive in their PB (median 16.1; range 8.5-19.9). The fourth patient had an initial value of 2.0 (negative), which increased to 8.4 (positive) within 1 month without treatment. Seven of 12 patients with baseline positive GD2/GM2 synthase values had post-treatment levels measured, all of which were

  13. Isolation and characterization of GmMYBJ3, an R2R3-MYB transcription factor that affects isoflavonoids biosynthesis in soybean

    PubMed Central

    Zhao, Mingzhu; Wang, Tianliang; Wu, Ping; Guo, Wenyun; Su, Liantai; Wang, Ying; Liu, Yajing; Yan, Fan

    2017-01-01

    Isoflavonoids are secondary metabolites that play a variety of roles in plant-microbe interactions and plant defenses against abiotic stresses. Here we report a new MYB transcription factor (TF) gene, GmMYBJ3, that is involved in the isoflavonoids biosynthesis. The GmMYBJ3 gene is 1,002 bp long and encodes a protein of 333 amino acids. Amino acid sequence analysis showed that GmMYBJ3 is a typical R2R3 MYB TF. Yeast expression experiment demonstrated that GmMYBJ3 has its transcription activity in the nucleus and is transiently expressed in onion epidermal cells. The GmMYBJ3 gene was transformed into soybean and the expression activity of the GmMYBJ3 gene was significantly positively correlated with total isoflavonoid accumulation in soybean. Transient expression assays indicated that GmMYBJ3 can activate CHS8 expression. Furthermore, we analyzed the expressions of several genes known involved in the isoflavonoid biosynthesis, including CHS8, CHI1A, PAL1, IFS2 and F3H, in the GmMYBJ3 transgenic plants. The results showed that the expression levels of CHS8 and CHI1A were significantly increased in the transgenic plants compared to wild-type plants, but those of PAL1, IFS2 and F3H remained similar between the transgenic and wild-type plants. These results suggest that GmMYBJ3 participates in the isoflavonoid biosynthesis through regulation of CHS8 and CHI1A in soybean. PMID:28654660

  14. Binding, Conformational Transition and Dimerization of Amyloid-β Peptide on GM1-Containing Ternary Membrane: Insights from Molecular Dynamics Simulation

    PubMed Central

    Manna, Moutusi; Mukhopadhyay, Chaitali

    2013-01-01

    Interactions of amyloid-β (Aβ) with neuronal membrane are associated with the progression of Alzheimer’s disease (AD). Ganglioside GM1 has been shown to promote the structural conversion of Aβ and increase the rate of peptide aggregation; but the exact nature of interaction driving theses processes remains to be explored. In this work, we have carried out atomistic-scale computer simulations (totaling 2.65 µs) to investigate the behavior of Aβ monomer and dimers in GM1-containing raft-like membrane. The oligosaccharide head-group of GM1 was observed to act as scaffold for Aβ-binding through sugar-specific interactions. Starting from the initial helical peptide conformation, a β-hairpin motif was formed at the C-terminus of the GM1-bound Aβ-monomer; that didn’t appear in absence of GM1 (both in fluid POPC and liquid-ordered cholesterol/POPC bilayers and also in aqueous medium) within the simulation time span. For Aβ-dimers, the β-structure was further enhanced by peptide-peptide interactions, which might influence the propensity of Aβ to aggregate into higher-ordered structures. The salt-bridges and inter-peptide hydrogen bonds were found to account for dimer stability. We observed spontaneous formation of intra-peptide D23-K28 salt-bridge and a turn at V24GSN27 region - long been accepted as characteristic structural-motifs for amyloid self-assembly. Altogether, our results provide atomistic details of Aβ-GM1 and Aβ-Aβ interactions and demonstrate their importance in the early-stages of GM1-mediated Aβ-oligomerisation on membrane surface. PMID:23951128

  15. The biological activity of botulinum neurotoxin type C is dependent upon novel types of ganglioside binding sites.

    PubMed

    Strotmeier, Jasmin; Gu, Shenyan; Jutzi, Stephan; Mahrhold, Stefan; Zhou, Jie; Pich, Andreas; Eichner, Timo; Bigalke, Hans; Rummel, Andreas; Jin, Rongsheng; Binz, Thomas

    2011-07-01

    The seven botulinum neurotoxins (BoNT) cause muscle paralysis by selectively cleaving core components of the vesicular fusion machinery. Their extraordinary activity primarily relies on highly specific entry into neurons. Data on BoNT/A, B, E, F and G suggest that entry follows a dual receptor interaction with complex gangliosides via an established ganglioside binding region and a synaptic vesicle protein. Here, we report high resolution crystal structures of the BoNT/C cell binding fragment alone and in complex with sialic acid. The WY-motif characteristic of the established ganglioside binding region was located on an exposed loop. Sialic acid was co-ordinated at a novel position neighbouring the binding pocket for synaptotagmin in BoNT/B and G and the sialic acid binding site in BoNT/D and TeNT respectively. Employing synaptosomes and immobilized gangliosides binding studies with BoNT/C mutants showed that the ganglioside binding WY-loop, the newly identified sialic acid-co-ordinating pocket and the area corresponding to the established ganglioside binding region of other BoNTs are involved in ganglioside interaction. Phrenic nerve hemidiaphragm activity tests employing ganglioside deficient mice furthermore evidenced that the biological activity of BoNT/C depends on ganglioside interaction with at least two binding sites. These data suggest a unique cell binding and entry mechanism for BoNT/C among clostridial neurotoxins. © 2011 Blackwell Publishing Ltd.

  16. Identification of a Unique Ganglioside Binding Loop within Botulinum Neurotoxins C and D-SA

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Karalewitz, Andrew P.-A.; Kroken, Abby R.; Fu, Zhuji

    2010-09-22

    The botulinum neurotoxins (BoNTs) are the most potent protein toxins for humans. There are seven serotypes of BoNTs (A-G) based on a lack of cross antiserum neutralization. BoNTs utilize gangliosides as components of the host receptors for binding and entry into neurons. Members of BoNT/C and BoNT/D serotypes include mosaic toxins that are organized in D/C and C/D toxins. One D/C mosaic toxin, BoNT/D-South Africa (BoNT/D-SA), was not fully neutralized by immunization with BoNT serotype C or D, which stimulated this study. Here the crystal structures of the receptor binding domains of BoNT/C, BoNT/D, and BoNT/D-SA are presented. Biochemical andmore » cell binding studies show that BoNT/C and BoNT/D-SA possess unique mechanisms for ganglioside binding. These studies provide new information about how the BoNTs can enter host cells as well as a basis for understanding the immunological diversity of these neurotoxins.« less

  17. Effect of gangliosides in the autoimmune response induced by liposome-associated antigens.

    PubMed

    Correa, S G; Rivero, V E; Yranzo-Volonté, N; Romero-Piffiguer, M; Ferro, M E; Riera, C M

    1993-01-01

    A model of autoimmunity to rat male accessory glands (RAG) was recently developed by intraperitoneal administration of three doses of native RAG associated with liposomes. In this work we analysed the effects of gangliosides in the cellular response to RAG when they were intraperitoneally administrated prior to the second dose of liposome-associated RAG. Results show that the ganglioside treatment could modify an established DTH response. Also, gangliosides markedly reduced the number of Ia antigen-positive peritoneal exudated cells (PEC). However, they modified neither the processing of liposomes through PEC nor their viability. Moreover, we obtained cellular response by transferring PEC from immunized donors into naive receptors.

  18. Protective Role of Endogenous Gangliosides for Lysosomal Pathology in a Cellular Model of Synucleinopathies

    PubMed Central

    Wei, Jianshe; Fujita, Masayo; Nakai, Masaaki; Waragai, Masaaki; Sekigawa, Akio; Sugama, Shuei; Takenouchi, Takato; Masliah, Eliezer; Hashimoto, Makoto

    2009-01-01

    Gangliosides may be involved in the pathogenesis of Parkinson’s disease and related disorders, although the precise mechanisms governing this involvement remain unknown. In this study, we determined whether changes in endogenous ganglioside levels affect lysosomal pathology in a cellular model of synucleinopathy. For this purpose, dementia with Lewy body-linked P123H β-synuclein (β-syn) neuroblastoma cells transfected with α-synuclein were used as a model system because these cells were characterized as having extensive formation of lysosomal inclusions bodies. Treatment of these cells with d-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), an inhibitor of glycosyl ceramide synthase, resulted in various features of lysosomal pathology, including compromised lysosomal activity, enhanced lysosomal membrane permeabilization, and increased cytotoxicity. Consistent with these findings, expression levels of lysosomal membrane proteins, ATP13A2 and LAMP-2, were significantly decreased, and electron microscopy demonstrated alterations in the lysosomal membrane structures. Furthermore, the accumulation of both P123H β-syn and α-synuclein proteins was significant in PDMP-treated cells because of the suppressive effect of PDMP on the autophagy pathway. Finally, the detrimental effects of PDMP on lysosomal pathology were significantly ameliorated by the addition of gangliosides to the cultured cells. These data suggest that endogenous gangliosides may play protective roles against the lysosomal pathology of synucleinopathies. PMID:19349362

  19. Altered lipid raft–associated signaling and ganglioside expression in T lymphocytes from patients with systemic lupus erythematosus

    PubMed Central

    Jury, Elizabeth C.; Kabouridis, Panagiotis S.; Flores-Borja, Fabian; Mageed, Rizgar A.; Isenberg, David A.

    2004-01-01

    Systemic lupus erythematosus (SLE) is characterized by abnormalities in T lymphocyte receptor–mediated signal transduction pathways. Our previous studies have established that lymphocyte-specific protein tyrosine kinase (LCK) is reduced in T lymphocytes from patients with SLE and that this reduction is associated with disease activity and parallels an increase in LCK ubiquitination independent of T cell activation. This study investigated the expression of molecules that regulate LCK homeostasis, such as CD45, C-terminal Src kinase (CSK), and c-Cbl, in lipid raft domains from SLE T cells and investigated the localization of these proteins during T cell receptor (TCR) triggering. Our results indicate that the expression of raft-associated ganglioside, GM1, is increased in T cells from SLE patients and LCK may be differentially regulated due to an alteration in the association of CD45 with lipid raft domains. CD45 tyrosine phosphatase, which regulates LCK activity, was differentially expressed and its localization into lipid rafts was increased in T cells from patients with SLE. Furthermore, T cells allowed to “rest” in vitro showed a reversal of the changes in LCK, CD45, and GM1 expression. The results also revealed that alterations in the level of GM1 expression and lipid raft occupancy cannot be induced by serum factors from patients with SLE but indicated that cell-cell contact, activating aberrant proximal signaling pathways, may be important in influencing abnormalities in T cell signaling and, therefore, function in patients with SLE. PMID:15085197

  20. The Protective Effect of Gangliosides on Lead (Pb)-Induced Neurotoxicity Is Mediated by Autophagic Pathways.

    PubMed

    Meng, Hongtao; Wang, Lan; He, Junhong; Wang, Zhufeng

    2016-03-25

    Lead (Pb) is a ubiquitous environmental and industrial pollutant and can affect intelligence development and the learning ability and memory of children. Therefore, necessary measures should be taken to protect the central nervous system (CNS) from Pb toxicity. Gangliosides are sialic acid-containing glycosphingolipids that are constituents of mammalian cell membranes and are more abundantly expressed in the CNS. Studies have shown that gangliosides constitute a useful tool in the attempt to promote functional recovery of CNS and can reverse Pb-induced impairments of synaptic plasticity in rats. However, the detailed mechanisms have yet to be fully understood. In our present study, we tried to investigate the role of gangliosides in Pb-induced injury in hippocampus neurons and to further confirm the detailed mechanism. Our results show that Pb-induced injuries in the spatial reference memory were associated with a reduction of cell viability and cell apoptosis, and treatment with gangliosides markedly ameliorated the Pb-induced injury by inhibition of apoptosis action. Gangliosides further attenuated Pb-induced the abnormal autophagic process by regulation of mTOR pathways. In summary, our study establishes the efficacy of gangliosides as neuroprotective agents and provides a strong rationale for further studies on the underlying mechanisms of their neuroprotective functions.

  1. Efficient Metabolic Engineering of GM3 on Tumor Cells by N-Phenylacetyl-D-mannosamine†

    PubMed Central

    Chefalo, Peter; Pan, Yanbin; Nagy, Nancy; Guo, Zhongwu; Harding, Clifford V.

    2008-01-01

    Abnormal carbohydrates expressed on tumor cells, which are referred to as tumor-associated carbohydrate antigens (TACAs), are potential targets for development of cancer vaccines. However, immune tolerance to TACAs has severely hindered progress in this area. To overcome this problem, we have developed a novel immunotherapeutic strategy based on synthetic cancer vaccines and metabolic engineering of TACAs on tumor cells. One critical step of this new strategy is metabolic engineering of cancer, namely to induce expression of an artificial form of a TACA by supplying tumors with an artificial monosaccharide precursor. To identify the proper precursor for this application, N-propionyl, N-butanoyl, N-iso-butanoyl and N-phenylacetyl derivatives of D-mannosamine were synthesized, and their efficiency as biosynthetic precursors to modify sialic acid and induce expression of modified forms of GM3 antigen on tumor cells was investigated. For this purpose, tumor cells were incubated with different N-acyl-D-mannosamines, and modified forms of GM3 expressed on tumor cells were analyzed by flow cytometry using antigen-specific antisera. N-phenylacetyl-D-mannosamine was efficiently incorporated in a time and dose dependent manner to bioengineer GM3 expression by several tumor cell lines including K562, SKMEL-28 and B16-F0. Moreover, these tumor cell lines also showed ManPAc-dependent sensitivity to cytotoxicity medicated by anti-PAcGM3 immune serum and complement. These results provide an important validation for this novel therapeutic strategy. Because N-phenylacetyl GM3-protein conjugates are particularly immunogenic, the combination of an N-phenylacetyl GM3 conjugate vaccine with systemic N-phenylacetyl-D-mannosamine treatment is a promising immunotherapy for future development and application to melanoma and other GM3-bearing tumors. PMID:16533056

  2. Early Supplementation of Phospholipids and Gangliosides Affects Brain and Cognitive Development in Neonatal Piglets123

    PubMed Central

    Liu, Hongnan; Radlowski, Emily C; Conrad, Matthew S; Li, Yao; Dilger, Ryan N; Johnson, Rodney W

    2014-01-01

    Background: Because human breast milk is a rich source of phospholipids and gangliosides and breastfed infants have improved learning compared with formula-fed infants, the importance of dietary phospholipids and gangliosides for brain development is of interest. Objective: We sought to determine the effects of phospholipids and gangliosides on brain and cognitive development. Methods: Male and female piglets from multiple litters were artificially reared and fed formula containing 0% (control), 0.8%, or 2.5% Lacprodan PL-20 (PL-20; Arla Foods Ingredients), a phospholipid/ganglioside supplement, from postnatal day (PD) 2 to PD28. Beginning on PD14, performance in a spatial T-maze task was assessed. At PD28, brain MRI data were acquired and piglets were killed to obtain hippocampal tissue for metabolic profiling. Results: Diet affected maze performance, with piglets that were fed 0.8% and 2.5% PL-20 making fewer errors than control piglets (80% vs. 75% correct on average; P < 0.05) and taking less time to make a choice (3 vs. 5 s/trial; P < 0.01). Mean brain weight was 5% higher for piglets fed 0.8% and 2.5% PL-20 (P < 0.05) than control piglets, and voxel-based morphometry revealed multiple brain areas with greater volumes and more gray and white matter in piglets fed 0.8% and 2.5% PL-20 than in control piglets. Metabolic profiling of hippocampal tissue revealed that multiple phosphatidylcholine-related metabolites were altered by diet. Conclusion: In summary, dietary phospholipids and gangliosides improved spatial learning and affected brain growth and composition in neonatal piglets. PMID:25411030

  3. Dissecting the Role of Anti-ganglioside Antibodies in Guillain-Barré Syndrome: an Animal Model Approach.

    PubMed

    Asthana, Pallavi; Vong, Joaquim Si Long; Kumar, Gajendra; Chang, Raymond Chuen-Chung; Zhang, Gang; Sheikh, Kazim A; Ma, Chi Him Eddie

    2016-09-01

    Guillain-Barré syndrome (GBS) is an autoimmune polyneuropathy disease affecting the peripheral nervous system (PNS). Most of the GBS patients experienced neurological symptoms such as paresthesia, weakness, pain, and areflexia. There are also combinations of non-neurological symptoms which include upper respiratory tract infection and diarrhea. One of the major causes of GBS is due largely to the autoantibodies against gangliosides located on the peripheral nerves. Gangliosides are sialic acid-bearing glycosphingolipids consisting of a ceramide lipid anchor with one or more sialic acids attached to a neutral sugar backbone. Molecular mimicry between the outer components of oligosaccharide of gangliosides on nerve membrane and lipo-oligosaccharide of microbes is thought to trigger the autoimmunity. Intra-peritoneal implantation of monoclonal ganglioside antibodies secreting hybridoma into animals induced peripheral neuropathy. Recent studies demonstrated that injection of synthesized anti-ganglioside antibodies raised by hybridoma cells into mice initiates immune response against peripheral nerves, and eventually failure in peripheral nerve regeneration. Accumulating evidences indicate that the conjugation of anti-ganglioside monoclonal antibodies to activating FcγRIII present on the circulating macrophages inhibits axonal regeneration. The activation of RhoA signaling pathways is also involved in neurite outgrowth inhibition. However, the link between these two molecular events remains unresolved and requires further investigation. Development of anti-ganglioside antagonists can serve as targeted therapy for the treatment of GBS and will open a new approach of drug development with maximum efficacy and specificity.

  4. Structural and mutational analyses of the receptor binding domain of botulinum D/C mosaic neurotoxin: Insight into the ganglioside binding mechanism

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Nuemket, Nipawan; Tanaka, Yoshikazu; Faculty of Advanced Life Science, Hokkaido University, Sapporo 060-0810

    2011-07-29

    Highlights: {yields} We determined the crystal structure of the receptor binding domain of BoNT in complex with 3'-sialyllactose. {yields} An electron density derived from the 3'-sialyllactose was confirmed at the cleft in the C-terminal subdomain. {yields} Alanine site-directed mutagenesis showed that GBS and GBL are important for ganglioside binding. {yields} A cell binding mechanism, which involves cooperative contribution of two sites, was proposed. -- Abstract: Clostridium botulinum type D strain OFD05, which produces the D/C mosaic neurotoxin, was isolated from cattle killed by the recent botulism outbreak in Japan. The D/C mosaic neurotoxin is the most toxic of the botulinummore » neurotoxins (BoNT) characterized to date. Here, we determined the crystal structure of the receptor binding domain of BoNT from strain OFD05 in complex with 3'-sialyllactose at a resolution of 3.0 A. In the structure, an electron density derived from the 3'-sialyllactose was confirmed at the cleft in the C-terminal subdomain. Alanine site-directed mutagenesis showed the significant contribution of the residues surrounding the cleft to ganglioside recognition. In addition, a loop adjoining the cleft also plays an important role in ganglioside recognition. In contrast, little effect was observed when the residues located around the surface previously identified as the protein receptor binding site in other BoNTs were substituted. The results of cell binding analysis of the mutants were significantly correlated with the ganglioside binding properties. Based on these observations, a cell binding mechanism of BoNT from strain OFD05 is proposed, which involves cooperative contribution of two ganglioside binding sites.« less

  5. GM crops and the rat digestive tract: a critical review.

    PubMed

    Zdziarski, I M; Edwards, J W; Carman, J A; Haynes, J I

    2014-12-01

    The aim of this review is to examine the relationship between genetically modified (GM) crops and health, based on histopathological investigations of the digestive tract in rats. We reviewed published long-term feeding studies of crops containing one or more of three specific traits: herbicide tolerance via the EPSPS gene and insect resistance via cry1Ab or cry3Bb1 genes. These genes are commonly found in commercialised GM crops. Our search found 21 studies for nine (19%) out of the 47 crops approved for human and/or animal consumption. We could find no studies on the other 38 (81%) approved crops. Fourteen out of the 21 studies (67%) were general health assessments of the GM crop on rat health. Most of these studies (76%) were performed after the crop had been approved for human and/or animal consumption, with half of these being published at least nine years after approval. Our review also discovered an inconsistency in methodology and a lack of defined criteria for outcomes that would be considered toxicologically or pathologically significant. In addition, there was a lack of transparency in the methods and results, which made comparisons between the studies difficult. The evidence reviewed here demonstrates an incomplete picture regarding the toxicity (and safety) of GM products consumed by humans and animals. Therefore, each GM product should be assessed on merit, with appropriate studies performed to indicate the level of safety associated with them. Detailed guidelines should be developed which will allow for the generation of comparable and reproducible studies. This will establish a foundation for evidence-based guidelines, to better determine if GM food is safe for human and animal consumption. Copyright © 2014. Published by Elsevier Ltd.

  6. Nitrogen dioxide-induced alterations in ganglioside content and structure of pulmonary artery endothelial cell plasma membranes

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Sekharam, M.; Patel, J.M.; Block, E.R.

    1990-02-26

    Nitrogen dioxide (NO{sub 2}), an environmental oxidant, is known to cause injury to the surface of pulmonary artery endothelial cells (PAEC). Because gangliosides are present in the outer leaflet of plasma membranes, the authors hypothesize that NO{sub 2} exposure may alter the ganglioside content and structure of PAEC plasma membranes. To test this, confluent porcine PAEC were exposed to 5 ppm NO{sub 2} containing 5% CO{sub 2} for 48 hours at 37 C in a CO{sub 2} incubator. Controls were exposed to air containing 5% Co{sub 2} under identical conditions. After exposure: (1) total lipids were extracted and ganglioside basesmore » were separated and estimated by fluorescamine, (2) the sialic acid content of intact cells was measured by the resorcinol method, and (3) freeze-fracture analysis of the intact cell plasma membrane was done by propane jet freezing and shadowing with platinum and carbon to form a replica. The ganglioside and sialic acid/{mu}g protein, respectively. In No{sub 2}-exposed cells, ganglioside content was reduced by 45% and sialic acid content was increased by 30%. Freeze-fracture analysis of the plasma membrane of control cells showed the presence of 160{+-}12 particles/cm area at 45000x. In contrast, the number of particles on the No{sub 2}-exposed plasma membrane was reduced to 68{+-}5 particles/cm at 45000x (p < 0.05). These results demonstrate that NO{sub 2} causes structural changes in the surface of PAEC plasma membranes, and these are temporally associated with a reduction in the number of gagliosides in these cells.« less

  7. Clustering T cell GM1 Lipid Rafts Increases Cellular Resistance to Shear on Fibronectin through Changes in Integrin Affinity and Cytoskeletal Dynamics

    PubMed Central

    Mitchell, Jason S.; Brown, Wells S.; Woodside, Darren G.; Vanderslice, Peter; McIntyre, Bradley W.

    2008-01-01

    Lipid rafts are small laterally mobile microdomains that are highly enriched in lymphocyte signaling molecules. GM1 gangliosides are a common lipid raft component and have been shown to be important in many T cell functions. The aggregation of specific GM1 lipid rafts can control many T cell activation events, including their novel association with T cell integrins. We found that clustering GM1 lipid rafts can regulate β1 integrin function. This was apparent through increased resistance to shear flow dependent detachment of T cells adherent to the α4β1 and α5β1 integrin ligand fibronectin (FN). Adhesion strengthening as a result of clustering GM1 enriched lipid rafts correlated with increased cellular rigidity and morphology through the localization of cortical F-actin, the resistance to shear induced cell stretching, and an increase in the surface area and symmetry of the contact area between the cell surface and adhesive substrate. Furthermore, clustering GM1 lipid rafts could initiate integrin “inside-out” signaling mechanisms. This was seen through increased integrin-cytoskeleton associations and enhanced soluble binding of FN and VCAM-1 suggesting the induction of high affinity integrin conformations. The activation of these adhesion strengthening characteristics appear to be specific for the aggregation of GM1 lipid rafts as the aggregation of the heterogeneous raft associated molecule CD59 failed to activate these functions. These findings indicate a novel mechanism to signal to β1 integrins and to activate adhesion strengthening processes. PMID:19139760

  8. An antibody to the GM1/GalNAc-GD1a complex correlates with development of pure motor Guillain-Barré syndrome with reversible conduction failure.

    PubMed

    Ogawa, Go; Kaida, Ken-ichi; Kuwahara, Motoi; Kimura, Fumihiko; Kamakura, Keiko; Kusunoki, Susumu

    2013-01-15

    Antibodies to a ganglioside complex consisting of GM1 and GalNAc-GD1a (GM1/GalNAc-GD1a) are found in sera from patients with Guillain-Barré syndrome (GBS). To elucidate the clinical significance of anti-GM1/GalNAc-GD1a antibodies in GBS, clinical features of 58 GBS patients with IgG anti-GM1/GalNAc-GD1a antibodies confirmed by enzyme-linked immunosorbent assay and thin layer chromatography immunostaining were analyzed. Compared to GBS patients without anti-GM1/GalNAc-GD1a antibodies, anti-GM1/GalNAc-GD1a-positive patients more frequently had a preceding respiratory infection (n=38, 66%, p<0.01) and were characterized by infrequency of cranial nerve deficits (n=9, 16%, p<0.01) and sensory disturbances (n=26, 45%, p<0.01). Of the 28 anti-GM1/GalNAc-GD1a-positive patients for whom electrophysiological data were available, 14 had conduction blocks (CBs) at intermediate segments of motor nerves, which were not followed by evident remyelination. Eight of 10 bedridden cases were able to walk independently within one month after the nadir. These results show that the presence of anti-GM1/GalNAc-GD1a antibodies correlated with pure motor GBS characterized by antecedent respiratory infection, fewer cranial nerve deficits, and CBs at intermediate sites of motor nerves. The CB may be generated through alteration of the regulatory function of sodium channels in the nodal axolemma. Copyright © 2012 Elsevier B.V. All rights reserved.

  9. Broad neutralization of calcium-permeable amyloid pore channels with a chimeric Alzheimer/Parkinson peptide targeting brain gangliosides.

    PubMed

    Di Scala, Coralie; Yahi, Nouara; Flores, Alessandra; Boutemeur, Sonia; Kourdougli, Nazim; Chahinian, Henri; Fantini, Jacques

    2016-02-01

    Growing evidence supports a role for brain gangliosides in the pathogenesis of neurodegenerative diseases including Alzheimer's and Parkinson's. Recently we deciphered the ganglioside-recognition code controlling specific ganglioside binding to Alzheimer's β-amyloid (Aβ1-42) peptide and Parkinson's disease-associated protein α-synuclein. Cracking this code allowed us to engineer a short chimeric Aβ/α-synuclein peptide that recognizes all brain gangliosides. Here we show that ganglioside-deprived neural cells do no longer sustain the formation of zinc-sensitive amyloid pore channels induced by either Aβ1-42 or α-synuclein, as assessed by single-cell Ca(2+) fluorescence microscopy. Thus, amyloid channel formation, now considered a key step in neurodegeneration, is a ganglioside-dependent process. Nanomolar concentrations of chimeric peptide competitively inhibited amyloid pore formation induced by Aβ1-42 or α-synuclein in cultured neural cells. Moreover, this peptide abrogated the intracellular calcium increases induced by Parkinson's-associated mutant forms of α-synuclein (A30P, E46K and A53T). The chimeric peptide also prevented the deleterious effects of Aβ1-42 on synaptic vesicle trafficking and decreased the Aβ1-42-induced impairment of spontaneous activity in rat hippocampal slices. Taken together, these data show that the chimeric peptide has broad anti-amyloid pore activity, suggesting that a common therapeutic strategy based on the prevention of amyloid-ganglioside interactions is a reachable goal for both Alzheimer's and Parkinson's diseases. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. GmPGIP3 enhanced resistance to both take-all and common root rot diseases in transgenic wheat.

    PubMed

    Wang, Aiyun; Wei, Xuening; Rong, Wei; Dang, Liang; Du, Li-Pu; Qi, Lin; Xu, Hui-Jun; Shao, Yanjun; Zhang, Zengyan

    2015-05-01

    Take-all (caused by the fungal pathogen Gaeumannomyces graminis var. tritici, Ggt) and common root rot (caused by Bipolaris sorokiniana) are devastating root diseases of wheat (Triticum aestivum L.). Development of resistant wheat cultivars has been a challenge since no resistant wheat accession is available. GmPGIP3, one member of polygalacturonase-inhibiting protein (PGIP) family in soybean (Glycine max), exhibited inhibition activity against fungal endopolygalacturonases (PGs) in vitro. In this study, the GmPGIP3 transgenic wheat plants were generated and used to assess the effectiveness of GmPGIP3 in protecting wheat from the infection of Ggt and B. sorokiniana. Four independent transgenic lines were identified by genomic PCR, Southern blot, and reverse transcription PCR (RT-PCR). The introduced GmPGIP3 was integrated into the genomes of these transgenic lines and could be expressed. The expressing GmPGIP3 protein in these transgenic wheat lines could inhibit the PGs produced by Ggt and B. sorokiniana. The disease response assessments postinoculation showed that the GmPGIP3-expressing transgenic wheat lines displayed significantly enhanced resistance to both take-all and common root rot diseases caused by the infection of Ggt and B. sorokiniana. These data suggested that GmPGIP3 is an attractive gene resource in improving resistance to both take-all and common root rot diseases in wheat.

  11. The assembly of GM1 glycolipid- and cholesterol-enriched raft-like membrane microdomains is important for giardial encystation.

    PubMed

    De Chatterjee, Atasi; Mendez, Tavis L; Roychowdhury, Sukla; Das, Siddhartha

    2015-05-01

    Although encystation (or cyst formation) is an important step of the life cycle of Giardia, the cellular events that trigger encystation are poorly understood. Because membrane microdomains are involved in inducing growth and differentiation in many eukaryotes, we wondered if these raft-like domains are assembled by this parasite and participate in the encystation process. Since the GM1 ganglioside is a major constituent of mammalian lipid rafts (LRs) and known to react with cholera toxin B (CTXB), we used Alexa Fluor-conjugated CTXB and GM1 antibodies to detect giardial LRs. Raft-like structures in trophozoites are located in the plasma membranes and on the periphery of ventral discs. In cysts, however, they are localized in the membranes beneath the cyst wall. Nystatin and filipin III, two cholesterol-binding agents, and oseltamivir (Tamiflu), a viral neuraminidase inhibitor, disassembled the microdomains, as evidenced by reduced staining of trophozoites with CTXB and GM1 antibodies. GM1- and cholesterol-enriched LRs were isolated from Giardia by density gradient centrifugation and found to be sensitive to nystatin and oseltamivir. The involvement of LRs in encystation could be supported by the observation that raft inhibitors interrupted the biogenesis of encystation-specific vesicles and cyst production. Furthermore, culturing of trophozoites in dialyzed medium containing fetal bovine serum (which is low in cholesterol) reduced raft assembly and encystation, which could be rescued by adding cholesterol from the outside. Our results suggest that Giardia is able to form GM1- and cholesterol-enriched lipid rafts and these raft domains are important for encystation. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  12. Will GM animals follow the GM plant fate?

    PubMed

    Vàzquez-Salat, Núria; Houdebine, Louis-Marie

    2013-02-01

    Despite being both Genetically Modified Organisms (GMOs), GM plants and GM animals share few similarities outside the laboratory premises. Whilst GM plants were soon embraced by industry and became a commercial success, only recently have GM animals reached the market. However, an area where GM animals are likely to follow the GM plant path is on their potential to cause social unrest. One of the major flaws of the 90s GMO crisis was the underestimation of the influence that different players can have in the adoption of new biotechnological applications. In this article we describe the unique evolution of GM animals in two of the most important fields: the pharmaceutical and the breeding sectors. For our analysis, we have subdivided the production chain into three governance domains: Science, Market and Public. We describe the influence and interaction of each of these domains as a vehicle for predicting the future adoptability of GM animals and to highlight conflicting areas.

  13. Synthesis and biological evaluation of several dephosphonated analogues of CMP-Neu5Ac as inhibitors of GM3-synthase.

    PubMed

    Rota, Paola; Cirillo, Federica; Piccoli, Marco; Gregorio, Antonio; Tettamanti, Guido; Allevi, Pietro; Anastasia, Luigi

    2015-10-05

    Previous studies demonstrated that reducing the GM3 content in myoblasts increased the cell resistance to hypoxic stress, suggesting that a pharmacological inhibition of the GM3 synthesis could be instrumental for the development of new treatments for ischemic diseases. Herein, the synthesis of several dephosphonated CMP-Neu5Ac congeners and their anti-GM3-synthase activity is reported. Biological activity testes revealed that some inhibitors almost completely blocked the GM3-synthase activity in vitro and reduced the GM3 content in living embryonic kidney 293A cells, eventually activating the epidermal growth factor receptor (EGFR) signaling cascade. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. HETEROCLITIC POLYCLONAL AND MONOCLONAL ANTI-Gm(a) AND ANTI-Gm(g) HUMAN RHEUMATOID FACTORS REACT WITH EPITOPES INDUCED IN Gm(a−) Gm(g−) IgG BY INTERACTION WITH ANTIGEN OR BY NONSPECIFIC AGGREGATION

    PubMed Central

    WILLIAMS, RALPH C.; MALONE, CHRISTINE C.; CASALI, PAOLO

    2015-01-01

    vitro-stimulated Gm(a−) normals or rheumatoid patients’ PBMC provided no evidence for Gm(a) nucleotide sequences. The present data suggest that acquisition of the Gm(a) determinant by Gm(a−) IgG may result from subtle changes in the CH2-CH3 RF-binding region. Such changes would occur when Gm(a) IgG are complexed with Ag or nonspecifically altered, thereby providing a possible explanation for the induction of heteroclitic RF in Gm(a−) rheumatoid arthritis patients. PMID:1380541

  15. A Drosophila protein family implicated in pheromone perception is related to Tay-Sachs GM2-activator protein.

    PubMed

    Starostina, Elena; Xu, Aiguo; Lin, Heping; Pikielny, Claudio W

    2009-01-02

    Low volatility, lipid-like cuticular hydrocarbon pheromones produced by Drosophila melanogaster females play an essential role in triggering and modulating mating behavior, but the chemosensory mechanisms involved remain poorly understood. Recently, we showed that the CheB42a protein, which is expressed in only 10 pheromone-sensing taste hairs on the front legs of males, modulates progression to late stages of male courtship behavior in response to female-specific cuticular hydrocarbons. Here we report that expression of all 12 genes in the CheB gene family is predominantly or exclusively gustatory-specific, and occurs in many different, often non-overlapping patterns. Only the Gr family of gustatory receptor genes displays a comparable variety of gustatory-specific expression patterns. Unlike Grs, however, expression of all but one CheB gene is sexually dimorphic. Like CheB42a, other CheBs may therefore function specifically in gustatory perception of pheromones. We also show that CheBs belong to the ML superfamily of lipid-binding proteins, and are most similar to human GM2-activator protein (GM2-AP). In particular, GM2-AP residues involved in ligand binding are conserved in CheBs but not in other ML proteins. Finally, CheB42a is specifically secreted into the inner lumen of pheromone-sensing taste hairs, where pheromones interact with membrane-bound receptors. We propose that CheB proteins interact directly with lipid-like Drosophila pheromones and modulate their detection by the gustatory signal transduction machinery. Furthermore, as loss of GM2-AP in Tay-Sachs disease prevents degradation of GM2 gangliosides and results in neurodegeneration, the function of CheBs in pheromone response may involve biochemical mechanisms critical for lipid metabolism in human neurons.

  16. Effects of Ganglioside on Working Memory and the Default Mode Network in Individuals with Subjective Cognitive Impairment: A Randomized Controlled Trial.

    PubMed

    Jeon, Yujin; Kim, Binna; Kim, Jieun E; Kim, Bori R; Ban, Soonhyun; Jeong, Jee Hyang; Kwon, Oran; Rhie, Sandy Jeong; Ahn, Chang-Won; Kim, Jong-Hoon; Jung, Sung Ug; Park, Soo-Hyun; Lyoo, In Kyoon; Yoon, Sujung

    2016-01-01

    This randomized, double-blind, placebo-controlled trial examined whether the administration of ganglioside, an active ingredient of deer bone extract, can improve working memory performance by increasing gray matter volume and functional connectivity in the default mode network (DMN) in individuals with subjective cognitive impairment. Seventy-five individuals with subjective cognitive impairment were chosen to receive either ganglioside (330[Formula: see text][Formula: see text]g/day or 660[Formula: see text][Formula: see text]g/day) or a placebo for 8 weeks. Changes in working memory performance with treatment of either ganglioside or placebo were assessed as cognitive outcome measures. Using voxel-based morphometry and functional connectivity analyses, changes in gray matter volume and functional connectivity in the DMN were also assessed as brain outcome measures. Improvement in working memory performance was greater in the ganglioside group than in the placebo group. The ganglioside group, relative to the placebo group, showed greater increases in gray matter volume and functional connectivity in the DMN. A significant relationship between increased functional connectivity of the precuneus and improved working memory performance was observed in the ganglioside group. The current findings suggest that ganglioside has cognitive-enhancing effects in individuals with subjective cognitive impairment. Ganglioside-induced increases in gray matter volume and functional connectivity in the DMN may partly be responsible for the potential nootropic effects of ganglioside. The clinical trial was registered with ClinicalTrials.gov (identifier: NCT02379481).

  17. AP-MALDI Mass Spectrometry Imaging of Gangliosides Using 2,6-Dihydroxyacetophenone

    NASA Astrophysics Data System (ADS)

    Jackson, Shelley N.; Muller, Ludovic; Roux, Aurelie; Oktem, Berk; Moskovets, Eugene; Doroshenko, Vladimir M.; Woods, Amina S.

    2018-03-01

    Matrix-assisted laser/desorption ionization (MALDI) mass spectrometry imaging (MSI) is widely used as a unique tool to record the distribution of a large range of biomolecules in tissues. 2,6-Dihydroxyacetophenone (DHA) matrix has been shown to provide efficient ionization of lipids, especially gangliosides. The major drawback for DHA as it applies to MS imaging is that it sublimes under vacuum (low pressure) at the extended time necessary to complete both high spatial and mass resolution MSI studies of whole organs. To overcome the problem of sublimation, we used an atmospheric pressure (AP)-MALDI source to obtain high spatial resolution images of lipids in the brain using a high mass resolution mass spectrometer. Additionally, the advantages of atmospheric pressure and DHA for imaging gangliosides are highlighted. The imaging of [M-H]- and [M-H2O-H]- mass peaks for GD1 gangliosides showed different distribution, most likely reflecting the different spatial distribution of GD1a and GD1b species in the brain. [Figure not available: see fulltext.

  18. Characterization of a new model of GM2-gangliosidosis (Sandhoff's disease) in Korat cats.

    PubMed Central

    Neuwelt, E A; Johnson, W G; Blank, N K; Pagel, M A; Maslen-McClure, C; McClure, M J; Wu, P M

    1985-01-01

    We have detected a disorder in Korat cats (initially imported from Thailand) that is analogous to human Sandhoff's disease. Pedigree analysis indicates that this disease in an autosomal recessive disorder in the American Korat. Postmortem studies on one affected cat showed hepatomegaly that was not reported in the only other known feline model of GM2-gangliosidosis type II. Histologic and ultra-structural evaluation revealed typical storage vacuoles. There was a marked deficiency in the activity of hexosaminidase (HEX) A and B in affected brain and liver as compared to controls. Electrophoresis of a liver extract revealed a deficiency of normal HEX A and B in the affected animals. The blocking primary enzyme immunoassay verified the presence of antigenically reactive HEX present in affected cat livers in quantities slightly elevated with respect to the normal HEX concentration in control cats. In leukocytes, obligate heterozygotes had intermediate levels of total HEX activity with a slight increase in the percent activity due to HEX A. Indeed, 4 of 11 phenotypically normal animals in addition to four obligate heterozygotes appear to be carriers using this assay. Affected brain and liver compared with control brain and liver contained a great excess of bound N-acetylneuraminic acid in the Folch upper-phase solids; thin-layer chromatography showed a marked increase in GM2-ganglioside. In summary, we have characterized the pedigree, pathology, and biochemistry of a new feline model of GM2-gangliosidosis which is similar to but different from the only other known feline model. Images PMID:4040927

  19. Characterization of a new model of GM2-gangliosidosis (Sandhoff's disease) in Korat cats.

    PubMed

    Neuwelt, E A; Johnson, W G; Blank, N K; Pagel, M A; Maslen-McClure, C; McClure, M J; Wu, P M

    1985-08-01

    We have detected a disorder in Korat cats (initially imported from Thailand) that is analogous to human Sandhoff's disease. Pedigree analysis indicates that this disease in an autosomal recessive disorder in the American Korat. Postmortem studies on one affected cat showed hepatomegaly that was not reported in the only other known feline model of GM2-gangliosidosis type II. Histologic and ultra-structural evaluation revealed typical storage vacuoles. There was a marked deficiency in the activity of hexosaminidase (HEX) A and B in affected brain and liver as compared to controls. Electrophoresis of a liver extract revealed a deficiency of normal HEX A and B in the affected animals. The blocking primary enzyme immunoassay verified the presence of antigenically reactive HEX present in affected cat livers in quantities slightly elevated with respect to the normal HEX concentration in control cats. In leukocytes, obligate heterozygotes had intermediate levels of total HEX activity with a slight increase in the percent activity due to HEX A. Indeed, 4 of 11 phenotypically normal animals in addition to four obligate heterozygotes appear to be carriers using this assay. Affected brain and liver compared with control brain and liver contained a great excess of bound N-acetylneuraminic acid in the Folch upper-phase solids; thin-layer chromatography showed a marked increase in GM2-ganglioside. In summary, we have characterized the pedigree, pathology, and biochemistry of a new feline model of GM2-gangliosidosis which is similar to but different from the only other known feline model.

  20. Scientific literature on monosialoganglioside in the Science Citation Index-Expanded: A bibliometric analysis of articles from 1942 to 2011 by each decade.

    PubMed

    Xu, Yanli; Li, Miaojing; Liu, Zhijun; Liu, Ruichun; Zhang, Jianzhong

    2012-01-05

    The monosialoganglioside (GM1) is a popular topic of research but the bibliometric analysis of GM1 over the decades in Science Citation Index-Expanded (SCI-E) remains poorly understood. To identify the global research and to improve the understanding of research trends in the GM1 field from 1942 to 2011. A bibliometric study. We performed a bibliometric analysis based on the SCI-E published by the Institute of Scientific Information. Articles closely related to GM1 were included. Exclusive criteria: (1) Articles related to gangliosidosis, disialo-ganglioside, trisialo-ganglioside or ganglioside GQIb. (2) Document types such as meeting abstracts, reviews, proceedings papers, notes, and letters. (1) Type of publication output; (2) number of author outputs; (3) distribution of output in subject categories; (4) publication distribution of countries; (5) distribution of output in journals, and (6) distribution of citations in each decade. During 1942 to 2011, there were 10 126 papers on GM1 that were added to the SCI. Articles (8 004) were the most frequently used document type comprising 79.0%, followed by meeting abstracts, reviews and proceedings papers. Research on GM1 could be found in the SCI from 1942, it was developed in the 1970s, greatly increased in the 1980s, and reached a peak in the 1990s, and it was slightly decreased in 2000. The distribution of subject categories showed that GM1 research covered both clinical and basic science research. The USA, Japan, and Germany were the three most productive countries, and the publication numbers in the USA were highest in all decades. The Journal of Biological Chemistry, Journal of Neurochemistry and Biochemistry were core subject journals in GM1 studies in each decade. This study highlights the topics in GM1 research that are being published around the world.

  1. Tetanus toxin interaction with human erythrocytes. II. Kinetic properties of toxin association and evidence for a ganglioside-toxin macromolecular complex formation.

    PubMed

    Lazarovici, P; Yavin, E

    1985-01-25

    The properties of tetanus toxin interaction with human erythrocytes supplemented with disialo- and trisialo-gangliosides have been investigated. Binding of toxin is linear with time for 1 h and is 3-4-fold higher at 37 degrees C than at 4 degrees C during incubation of long duration. It exhibits saturation at toxin concentrations between 0.1 and 1 microgram/ml; however, it is nonsaturable between 1 and up to 50 micrograms/ml. It is effectively prevented by free gangliosides and antibodies or by pretreatment with sialidase but is unaffected by a number of closely related ligands including toxoid and toxin fragments. NaCl (1 M) removes a great portion (86%) of cell-associated toxin while Triton X-100 extracts an additional fraction (30%) of the salt-resistant cell-bound toxin. The residual sequestred toxin after detergent extraction is sensitive to proteolytic degradation. The trypsin-stable fraction (1.5%) is biotoxic and may be indicative of internalization of toxin. A macromolecular complex of about 700 kDa containing toxin and gangliosides has been isolated and characterized by Sephacryl S-300 gel permeation chromatography, SDS-gel electrophoresis, immunoprecipitability and biotoxicity. This complex is obtained only in ganglioside-supplemented cells and not when free 3H-labeled GD1b is reacted with 125I-labeled toxin in solution in the absence of cells. The hydrophobicity properties acquired as a result of ganglioside-toxin interaction, presumably at the cell surface, suggest a conformational change of the toxin which may enable its penetration into the bilayer.

  2. Epidermal growth factor-induced mobilization of a ganglioside-specific sialidase (NEU3) to membrane ruffles

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Yamaguchi, Kazunori; Hata, Keiko; Wada, Tadashi

    2006-07-28

    Human ganglioside-specific sialidase, NEU3, localized at cell membranes is thought to regulate various biological processes at cell surfaces. We here explored functional subcellular localization of the sialidase by immunofluorescence and found accumulation at leading edges of cell membranes in the presence of serum in culture. In response to EGF, the sialidase redistributed rapidly to ruffling cell membranes of squamous carcinoma A431 cells and co-localized with Rac-1. NEU3 overexpression enhanced Rac-1 activation and cell migration as compared with controls in HeLa cells as well as in A431 cells. Consistent with co-localization with Rac-1 by immunofluorescence, NEU3 was found to co-precipitate withmore » activated Rac bound to GST-PAK-1 fusion protein. NEU3 silencing by siRNA, in contrast, resulted in inhibition of Rac-1 activation. These results indicate that NEU3 is able to mobilize to membrane ruffles in response to growth stimuli and activate the Rac-1 signaling by co-localization with Rac-1, leading to increased cell motility.« less

  3. Novel insights into the lipidome of glioblastoma cells based on a combined PLSR and DD-HDS computational analysis

    NASA Astrophysics Data System (ADS)

    Lespinats, S.; Meyer-Bäse, Anke; He, Huan; Marshall, Alan G.; Conrad, Charles A.; Emmett, Mark R.

    2009-05-01

    Partial Least Square Regression (PLSR) and Data-Driven High Dimensional Scaling (DD-HDS) are employed for the prediction and the visualization of changes in polar lipid expression induced by different combinations of wild-type (wt) p53 gene therapy and SN38 chemotherapy of U87 MG glioblastoma cells. A very detailed analysis of the gangliosides reveals that certain gangliosides of GM3 or GD1-type have unique properties not shared by the others. In summary, this preliminary work shows that data mining techniques are able to determine the modulation of gangliosides by different treatment combinations.

  4. Over-expression of mammalian sialidase NEU3 reduces Newcastle disease virus entry and propagation in COS7 cells.

    PubMed

    Anastasia, Luigi; Holguera, Javier; Bianchi, Anna; D'Avila, Francesca; Papini, Nadia; Tringali, Cristina; Monti, Eugenio; Villar, Enrique; Venerando, Bruno; Muñoz-Barroso, Isabel; Tettamanti, Guido

    2008-03-01

    The paramyxovirus Newcastle Disease Virus (NDV) binds to sialic acid-containing glycoconjugates, sialoglycoproteins and sialoglycolipids (gangliosides) of host cell plasma membrane through its hemagglutinin-neuraminidase (sialidase) HN glycoprotein. We hypothesized that the modifications of the cell surface ganglioside pattern determined by over-expression of the mammalian plasma-membrane associated, ganglioside specific, sialidase NEU3 would affect the virus-host cell interactions. Using COS7 cells as a model system, we observed that over-expression of the murine MmNEU3 did not affect NDV binding but caused a marked reduction in NDV infection and virus propagation through cell-cell fusion. Moreover, since GD1a was greatly reduced in COS7 cells following NEU3-over-expression, we added [(3)H]-labelled GD1a to COS7 cells under conditions that block intralysosomal metabolic processing, and we observed a marked increase of GD1a cleavage to GM1 during NDV infection, indicating a direct involvement of the virus sialidase and host cell GD1a in NDV infectivity. Therefore, the decrease of GD1a in COS7 cell membrane upon MmNEU3 over-expression is likely to be instrumental to NDV reduced infection. Evidence was also provided for the preferential association of NDV-HN at 4 degrees C to detergent resistant microdomains (DRMs) of COS7 cells plasma membranes.

  5. Long-Term Correction of Sandhoff Disease Following Intravenous Delivery of rAAV9 to Mouse Neonates

    PubMed Central

    Walia, Jagdeep S; Altaleb, Naderah; Bello, Alexander; Kruck, Christa; LaFave, Matthew C; Varshney, Gaurav K; Burgess, Shawn M; Chowdhury, Biswajit; Hurlbut, David; Hemming, Richard; Kobinger, Gary P; Triggs-Raine, Barbara

    2015-01-01

    GM2 gangliosidoses are severe neurodegenerative disorders resulting from a deficiency in β-hexosaminidase A activity and lacking effective therapies. Using a Sandhoff disease (SD) mouse model (Hexb−/−) of the GM2 gangliosidoses, we tested the potential of systemically delivered adeno-associated virus 9 (AAV9) expressing Hexb cDNA to correct the neurological phenotype. Neonatal or adult SD and normal mice were intravenously injected with AAV9-HexB or –LacZ and monitored for serum β-hexosaminidase activity, motor function, and survival. Brain GM2 ganglioside, β-hexosaminidase activity, and inflammation were assessed at experimental week 43, or an earlier humane end point. SD mice injected with AAV9-LacZ died by 17 weeks of age, whereas all neonatal AAV9-HexB–treated SD mice survived until 43 weeks (P < 0.0001) with only three exhibiting neurological dysfunction. SD mice treated as adults with AAV9-HexB died between 17 and 35 weeks. Neonatal SD-HexB–treated mice had a significant increase in brain β-hexosaminidase activity, and a reduction in GM2 ganglioside storage and neuroinflammation compared to adult SD-HexB– and SD-LacZ–treated groups. However, at 43 weeks, 8 of 10 neonatal-HexB injected control and SD mice exhibited liver or lung tumors. This study demonstrates the potential for long-term correction of SD and other GM2 gangliosidoses through early rAAV9 based systemic gene therapy. PMID:25515709

  6. Canine GM2-Gangliosidosis Sandhoff Disease Associated with a 3-Base Pair Deletion in the HEXB Gene.

    PubMed

    Wang, P; Henthorn, P S; Galban, E; Lin, G; Takedai, T; Casal, M

    2018-01-01

    GM2-gangliosidosis is a fatal neurodegenerative lysosomal storage disease (LSD) caused by deficiency of either β-hexosaminidase A (Hex-A) and β-hexosaminidase B (Hex-B) together, or the GM2 activator protein. Clinical signs can be variable and are not pathognomonic for the specific, causal deficiency. To characterize the phenotype and genotype of GM2-gangliosidosis disease in an affected dog. One affected Shiba Inu and a clinically healthy dog. Clinical and neurologic evaluation, brain magnetic resonance imaging (MRI), assays of lysosomal enzyme activities, and sequencing of all coding regions of HEXA, HEXB, and GM2A genes. A 14-month-old, female Shiba Inu presented with clinical signs resembling GM2-gangliosidosis in humans and GM1-gangliosidosis in the Shiba Inu. Magnetic resonance imaging (MRI) of the dog's brain indicated neurodegenerative disease, and evaluation of cerebrospinal fluid (CSF) identified storage granules in leukocytes. Lysosomal enzyme assays of plasma and leukocytes showed deficiencies of Hex-A and Hex-B activities in both tissues. Genetic analysis identified a homozygous, 3-base pair deletion in the HEXB gene (c.618-620delCCT). Clinical, biochemical, and molecular features are characterized in a Shiba Inu with GM2-gangliosidosis. The deletion of 3 adjacent base pairs in HEXB predicts the loss of a leucine residue at amino acid position 207 (p.Leu207del) supporting the hypothesis that GM2-gangliosidosis seen in this dog is the Sandhoff type. Because GM1-gangliosidosis also exists in this breed with almost identical clinical signs, genetic testing for both GM1- and GM2-gangliosidosis should be considered to make a definitive diagnosis. Copyright © 2017 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.

  7. Measuring the diffusion coefficient of ganglioside on cell membrane by fluorescence correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Dong, Shiqing; You, Minghai; Chen, Jianling; Zhou, Jie; Xie, Shusen; Yang, Hongqin

    2017-06-01

    The fluidity of proteins and lipids on cell membrane plays an important role in cell’s physiological functions. Fluorescence correlation spectroscopy (FCS) is an effective technique to detect the rapid dynamic behaviors of proteins and/or lipids in living cells. In this study, we used the rhodamine6G solution to optimize the FCS system. And, cholera toxin B subunit (CT-B) was used to label ganglioside on living Hela cell membranes. The diffusion time and coefficients of ganglioside can be obtained through fitting the autocorrelation curve based on the model of two-dimensional cell membrane. The results showed that the diffusion coefficients of ganglioside distributed within a wide range. It revealed the lateral diffusion of lipids on cell membrane was inhomogeneous, which was due to different microstructures of cytoplasmic membrane. The study provides a helpful method for further studying the dynamic characteristics of proteins and lipids molecules on living cell membrane.

  8. Extensive Analysis of GmFTL and GmCOL Expression in Northern Soybean Cultivars in Field Conditions

    PubMed Central

    Zhu, Jinlong; Lu, Mingyang; Chen, Fulu; Liu, Linpo; Xi, Zhang-Ying; Bachmair, Andreas; Chen, Qingshan; Fu, Yong-Fu

    2015-01-01

    The FLOWERING LOCUS T (FT) gene is a highly conserved florigen gene among flowering plants. Soybean genome encodes six homologs of FT, which display flowering activity in Arabidopsis thaliana. However, their contributions to flowering time in different soybean cultivars, especially in field conditions, are unclear. We employed six soybean cultivars with different maturities to extensively investigate expression patterns of GmFTLs (Glycine max FT-like) and GmCOLs (Glycine max CO-like) in the field conditions. The results show that GmFTL3 is an FT homolog with the highest transcript abundance in soybean, but other GmFTLs may also contribute to flower induction with different extents, because they have more or less similar expression patterns in developmental-, leaf-, and circadian-specific modes. And four GmCOL genes (GmCOL1/2/5/13) may confer to the expression of GmFTL genes. Artificial manipulation of GmFTL expression by transgenic strategy (overexpression and RNAi) results in a distinct change in soybean flowering time, indicating that GmFTLs not only impact on the control of flowering time, but have potential applications in the manipulation of photoperiodic adaptation in soybean. Additionally, transgenic plants show that GmFTLs play a role in formation of the first flowers and in vegetative growth. PMID:26371882

  9. Extensive Analysis of GmFTL and GmCOL Expression in Northern Soybean Cultivars in Field Conditions.

    PubMed

    Guo, Guangyu; Xu, Kun; Zhang, Xiaomei; Zhu, Jinlong; Lu, Mingyang; Chen, Fulu; Liu, Linpo; Xi, Zhang-Ying; Bachmair, Andreas; Chen, Qingshan; Fu, Yong-Fu

    2015-01-01

    The FLOWERING LOCUS T (FT) gene is a highly conserved florigen gene among flowering plants. Soybean genome encodes six homologs of FT, which display flowering activity in Arabidopsis thaliana. However, their contributions to flowering time in different soybean cultivars, especially in field conditions, are unclear. We employed six soybean cultivars with different maturities to extensively investigate expression patterns of GmFTLs (Glycine max FT-like) and GmCOLs (Glycine max CO-like) in the field conditions. The results show that GmFTL3 is an FT homolog with the highest transcript abundance in soybean, but other GmFTLs may also contribute to flower induction with different extents, because they have more or less similar expression patterns in developmental-, leaf-, and circadian-specific modes. And four GmCOL genes (GmCOL1/2/5/13) may confer to the expression of GmFTL genes. Artificial manipulation of GmFTL expression by transgenic strategy (overexpression and RNAi) results in a distinct change in soybean flowering time, indicating that GmFTLs not only impact on the control of flowering time, but have potential applications in the manipulation of photoperiodic adaptation in soybean. Additionally, transgenic plants show that GmFTLs play a role in formation of the first flowers and in vegetative growth.

  10. FURTHER STUDIES ON THE γG-HEAVY CHAIN GENE COMPLEXES, WITH PARTICULAR REFERENCE TO THE GENETIC MARKERS Gm(g) AND Gm(n)

    PubMed Central

    Natvig, J. B.; Kunkel, H. G.; Yount, W. J.; Nielsen, J. C.

    1968-01-01

    The recently described Gm (g) and Gm (n) genetic markers of the γG3- and γG2-subgroups of γ-globulin were characterized in detail primarily through studies of myeloma proteins, their polypeptide chains and fragments. Antisera derived from rabbits, non-human primates and rheumatoid arthritis patients gave identical results. This contrasted with the Gm (b) system where the rabbit antisera react with a different genetic determinant (b0) than the sera from rheumatoid arthritis patients (b). The Gm (g) and Gm (n) antigens were detected both by precipitin analysis and by hemagglutination inhibition. The Gm (g) antigen was not associated with any of the other genetic antigens of the γG3-proteins which all belonged in the Gm (b) class. The genes for the latter were always allelic to the gene coding for Gm (g), with that for Gm (b0) constantly present when that for Gm (g) was absent. The Gm (g) and Gm (n) markers were of particular value in tracing the various gene complexes made up of the closely linked subgroup genes. Further support was gained for the concept that the different gene complexes of various population groups arose primarily through crossing-over. The Gm g and Gm b genes for the γG3-subgroup were extremely closely linked to those for the γG1-subgroup. However the Gm (n) marker indicated that the γG2-subgroup genes were probably further separated on the chromosome. Additional evidence was obtained for the γG2-γG3-γG1-order of the subgroup cistrons. Among the wide range of gene complexes a new type (γG2,—,γ/G1) was described. This complex appeared to have a deletion of the γG3-cistron. Lower levels of γG3-globulin were found in the sera of the individuals with this gene in the heterozygous state. The possibility that this unusual complex arose through an unequal nonhomologous crossing-over is discussed. PMID:19867305

  11. The soybean R2R3 MYB transcription factor GmMYB100 negatively regulates plant flavonoid biosynthesis.

    PubMed

    Yan, Junhui; Wang, Biao; Zhong, Yunpeng; Yao, Luming; Cheng, Linjing; Wu, Tianlong

    2015-09-01

    Soybean flavonoids, a group of important signaling molecules in plant-environment interaction, ubiquitously exist in soybean and are tightly regulated by many genes. Here we reported that GmMYB100, a gene encoding a R2R3 MYB transcription factor, is involved in soybean flavonoid biosynthesis. GmMYB100 is mainly expressed in flowers, leaves and immature embryo, and its level is decreased after pod ripening. Subcellular localization assay indicates that GmMYB100 is a nuclear protein. GmMYB100 has transactivation ability revealed by a yeast functional assay; whereas bioinformatic analysis suggests that GmMYB100 has a negative function in flavonoid biosynthesis. GmMYB100-overexpression represses the transcript levels of flavonoid-related genes in transgenic soybean hairy roots and Arabidopsis, and inhibits isoflavonoid (soybean) and flavonol (Arabidopsis) production in transgenic plants. Furthermore, the transcript levels of six flavonoid-related genes and flavonoid (isoflavonoid and flavone aglycones) accumulation are elevated in the GmMYB100-RNAi transgenic hairy roots. We also demonstrate that GmMYB100 protein depresses the promoter activities of soybean chalcone synthase and chalcone isomerase. These findings indicate that GmMYB100 is a negative regulator in soybean flavonoid biosynthesis pathway.

  12. GM2 gangliosidosis variant 0 (Sandhoff-like disease) in a family of toy poodles.

    PubMed

    Tamura, S; Tamura, Y; Uchida, K; Nibe, K; Nakaichi, M; Hossain, M A; Chang, H S; Rahman, M M; Yabuki, A; Yamato, O

    2010-01-01

    GM2 gangliosidosis variant 0 (human Sandhoff disease) is a lysosomal storage disorder caused by deficiencies of acid β-hexosaminidase (Hex) A and Hex B because of an abnormality of the β-subunit, a common component in these enzyme molecules, which is coded by the HEXB gene. To describe the clinical, pathological, biochemical, and magnetic resonance imaging (MRI) findings of Sandhoff-like disease identified in a family of Toy Poodles. Three red-haired Toy Poodles demonstrated clinical signs including motor disorders and tremor starting between 9 and 12 months of age. The animals finally died of neurological deterioration between 18 and 23 months of age. There were some lymphocytes with abnormal cytoplasmic vacuoles detected. Observational case study. The common MRI finding was diffuse T2-hyperintensity of the subcortical white matter in the cerebrum. Bilateral T2-hyperintensity and T1-hypointensity in the nucleus caudatus, and atrophic findings of the cerebrum and cerebellum, were observed in a dog in the late stage. Histopathologically, swollen neurons with pale to eosinophilic granular materials in the cytoplasm were observed throughout the central nervous system. Biochemically, GM2 ganglioside had accumulated in the brain, and Hex A and Hex B were deficient in the brain and liver. Pedigree analysis demonstrated that the 3 affected dogs were from the same family line. The Sandhoff-like disease observed in this family of Toy Poodles is the 2nd occurrence of the canine form of this disease and the 1st report of its identification in a family of dogs. Copyright © 2010 by the American College of Veterinary Internal Medicine.

  13. Human IL-3/GM-CSF knock-in mice support human alveolar macrophage development and human immune responses in the lung

    PubMed Central

    Willinger, Tim; Rongvaux, Anthony; Takizawa, Hitoshi; Yancopoulos, George D.; Valenzuela, David M.; Murphy, Andrew J.; Auerbach, Wojtek; Eynon, Elizabeth E.; Stevens, Sean; Manz, Markus G.; Flavell, Richard A.

    2011-01-01

    Mice with a functional human immune system have the potential to allow in vivo studies of human infectious diseases and to enable vaccine testing. To this end, mice need to fully support the development of human immune cells, allow infection with human pathogens, and be capable of mounting effective human immune responses. A major limitation of humanized mice is the poor development and function of human myeloid cells and the absence of human immune responses at mucosal surfaces, such as the lung. To overcome this, we generated human IL-3/GM-CSF knock-in (hIL-3/GM-CSF KI) mice. These mice faithfully expressed human GM-CSF and IL-3 and developed pulmonary alveolar proteinosis because of elimination of mouse GM-CSF. We demonstrate that hIL-3/GM-CSF KI mice engrafted with human CD34+ hematopoietic cells had improved human myeloid cell reconstitution in the lung. In particular, hIL-3/GM-CSF KI mice supported the development of human alveolar macrophages that partially rescued the pulmonary alveolar proteinosis syndrome. Moreover, human alveolar macrophages mounted correlates of a human innate immune response against influenza virus. The hIL-3/GM-CSF KI mice represent a unique mouse model that permits the study of human mucosal immune responses to lung pathogens. PMID:21262803

  14. Biochemical characterization of the interactions between doxorubicin and lipidic GM1 micelles with or without paclitaxel loading

    PubMed Central

    Leonhard, Victoria; Alasino, Roxana V; Bianco, Ismael D; Garro, Ariel G; Heredia, Valeria; Beltramo, Dante M

    2015-01-01

    Doxorubicin (Dox) is an anthracycline anticancer drug with high water solubility, whose use is limited primarily due to significant side effects. In this study it is shown that Dox interacts with monosialoglycosphingolipid (GM1) ganglioside micelles primarily through hydrophobic interactions independent of pH and ionic strength. In addition, Dox can be incorporated even into GM1 micelles already containing highly hydrophobic paclitaxel (Ptx). However, it was not possible to incorporate Ptx into Dox-containing GM1 micelles, suggesting that Dox could be occupying a more external position in the micelles. This result is in agreement with a higher hydrolysis of Dox than of Ptx when micelles were incubated at alkaline pH. The loading of Dox into GM1 micelles was observed over a broad range of temperature (4°C–55°C). Furthermore, Dox-loaded micelles were stable in aqueous solutions exhibiting no aggregation or precipitation for up to 2 months when kept at 4°C–25°C and even after freeze–thawing cycles. Upon exposure to blood components, Dox-containing micelles were observed to interact with human serum albumin. However, the amount of human serum albumin that ended up being associated to the micelles was inversely related to the amount of Dox, suggesting that both could share their binding sites. In vitro studies on Hep2 cells showed that the cellular uptake and cytotoxic activity of Dox and Ptx from the micellar complexes were similar to those of the free form of these drugs, even when the micelle was covered with albumin. These results support the idea of the existence of different nano-domains in a single micelle and the fact that this micellar model could be used as a platform for loading and delivering hydrophobic and hydrophilic active pharmaceutical ingredients. PMID:26005348

  15. Novel GM animal technologies and their governance.

    PubMed

    Bruce, Ann; Castle, David; Gibbs, Corrina; Tait, Joyce; Whitelaw, C Bruce A

    2013-08-01

    Scientific advances in methods of producing genetically modified (GM) animals continue, yet few such animals have reached commercial production. Existing regulations designed for early techniques of genetic modification pose formidable barriers to commercial applications. Radically improved techniques for producing GM animals invite a re-examination of current regulatory regimes. We critically examine current GM animal regulations, with a particular focus on the European Union, through a framework that recognises the importance of interactions among regulatory regimes, innovation outcomes and industry sectors. The current focus on the regulation of risk is necessary but is unable to discriminate among applications and tends to close down broad areas of application rather than facilitate innovation and positive industry interactions. Furthermore, the fields of innovative animal biosciences appear to lack networks of organisations with co-ordinated future oriented actions. Such networks could drive coherent programmes of innovation towards particular visions and contribute actively to the development of regulatory systems for GM animals. The analysis presented makes the case for regulatory consideration of each animal bioscience related innovation on the basis of the nature of the product itself and not the process by which it was developed.

  16. Campylobacter jejuni chromosomal sequences that hybridize to Vibrio cholerae and Escherichia coli LT enterotoxin genes.

    PubMed

    Calva, E; Torres, J; Vázquez, M; Angeles, V; de la Vega, H; Ruíz-Palacios, G M

    1989-02-20

    Campylobacter jejuni is one of the main etiologic agents of gastrointestinal illness in developing and developed areas throughout the world. Isolation of enterotoxin-producing C. jejuni has been associated with clinical symptoms of a watery-secretory type of diarrhea. Although physiological and immunological relatedness has been demonstrated between the C. jejuni enterotoxin (CJT), the Vibrio cholerae enterotoxin (CT), and the heat-labile cholera-like Escherichia coli enterotoxin (LT), nucleotide sequence similarity between C. jejuni DNA and either the toxA, toxB, eltA or eltB genes remained to be shown. We found that binding to ganglioside GM1 prevented recognition of CJT by monoclonal antibodies directed to either CT or LT. This indicates antigenic similarity between the three enterotoxins in the ganglioside GM1-binding site. Therefore we searched for corresponding similarities at the DNA level and found, by oligodeoxynucleotide hybridization, C. jejuni chromosomal nucleotide sequences similar to the coding region for a postulated ganglioside GM1-binding site on toxB and eltB.

  17. Genetically Modified (GM) Foods and Ethical Eating.

    PubMed

    Dizon, Francis; Costa, Sarah; Rock, Cheryl; Harris, Amanda; Husk, Cierra; Mei, Jenny

    2016-02-01

    The ability to manipulate and customize the genetic code of living organisms has brought forth the production of genetically modified organisms (GMOs) and consumption of genetically modified (GM) foods. The potential for GM foods to improve the efficiency of food production, increase customer satisfaction, and provide potential health benefits has contributed to the rapid incorporation of GM foods into the American diet. However, GM foods and GMOs are also a topic of ethical debate. The use of GM foods and GM technology is surrounded by ethical concerns and situational judgment, and should ideally adhere to the ethical standards placed upon food and nutrition professionals, such as: beneficence, nonmaleficence, justice and autonomy. The future of GM foods involves many aspects and trends, including enhanced nutritional value in foods, strict labeling laws, and potential beneficial economic conditions in developing nations. This paper briefly reviews the origin and background of GM foods, while delving thoroughly into 3 areas: (1) GMO labeling, (2) ethical concerns, and (3) health and industry applications. This paper also examines the relationship between the various applications of GM foods and their corresponding ethical issues. Ethical concerns were evaluated in the context of the code of ethics developed by the Academy of Nutrition and Dietetics (AND) that govern the work of food and nutrition professionals. Overall, there is a need to stay vigilant about the many ethical implications of producing and consuming GM foods and GMOs. © 2015 Institute of Food Technologists®

  18. GmWRKY31 and GmHDL56 Enhances Resistance to Phytophthora sojae by Regulating Defense-Related Gene Expression in Soybean.

    PubMed

    Fan, Sujie; Dong, Lidong; Han, Dan; Zhang, Feng; Wu, Junjiang; Jiang, Liangyu; Cheng, Qun; Li, Rongpeng; Lu, Wencheng; Meng, Fanshan; Zhang, Shuzhen; Xu, Pengfei

    2017-01-01

    Phytophthora root and stem rot of soybean [ Glycine max (L.) Merr.] caused by the oomycete Phytophthora sojae , is a destructive disease worldwide. The molecular mechanism of the soybean response to P. sojae is largely unclear. We report a novel WRKY transcription factor (TF) in soybean, GmWRKY31, in the host response to P. sojae . Overexpression and RNA interference analysis demonstrated that GmWRKY31 enhanced resistance to P. sojae in transgenic soybean plants. GmWRKY31 was targeted to the nucleus, where it bound to the W-box and acted as an activator of gene transcription. Moreover, we determined that GmWRKY31 physically interacted with GmHDL56, which improved resistance to P. sojae in transgenic soybean roots. GmWRKY31 and GmHDL56 shared a common target GmNPR1 which was induced by P. sojae . Overexpression and RNA interference analysis demonstrated that GmNPR1 enhanced resistance to P. sojae in transgenic soybean plants. Several pathogenesis-related ( PR ) genes were constitutively activated, including GmPR1a , GmPR2 , GmPR3 , GmPR4 , GmPR5a , and GmPR10 , in soybean plants overexpressing GmNPR1 transcripts. By contrast, the induction of PR genes was compromised in transgenic GmNPR1 -RNAi lines. Taken together, these findings suggested that the interaction between GmWRKY31 and GmHDL56 enhances resistance to P. sojae by regulating defense-related gene expression in soybean.

  19. GmWRKY31 and GmHDL56 Enhances Resistance to Phytophthora sojae by Regulating Defense-Related Gene Expression in Soybean

    PubMed Central

    Fan, Sujie; Dong, Lidong; Han, Dan; Zhang, Feng; Wu, Junjiang; Jiang, Liangyu; Cheng, Qun; Li, Rongpeng; Lu, Wencheng; Meng, Fanshan; Zhang, Shuzhen; Xu, Pengfei

    2017-01-01

    Phytophthora root and stem rot of soybean [Glycine max (L.) Merr.] caused by the oomycete Phytophthora sojae, is a destructive disease worldwide. The molecular mechanism of the soybean response to P. sojae is largely unclear. We report a novel WRKY transcription factor (TF) in soybean, GmWRKY31, in the host response to P. sojae. Overexpression and RNA interference analysis demonstrated that GmWRKY31 enhanced resistance to P. sojae in transgenic soybean plants. GmWRKY31 was targeted to the nucleus, where it bound to the W-box and acted as an activator of gene transcription. Moreover, we determined that GmWRKY31 physically interacted with GmHDL56, which improved resistance to P. sojae in transgenic soybean roots. GmWRKY31 and GmHDL56 shared a common target GmNPR1 which was induced by P. sojae. Overexpression and RNA interference analysis demonstrated that GmNPR1 enhanced resistance to P. sojae in transgenic soybean plants. Several pathogenesis-related (PR) genes were constitutively activated, including GmPR1a, GmPR2, GmPR3, GmPR4, GmPR5a, and GmPR10, in soybean plants overexpressing GmNPR1 transcripts. By contrast, the induction of PR genes was compromised in transgenic GmNPR1-RNAi lines. Taken together, these findings suggested that the interaction between GmWRKY31 and GmHDL56 enhances resistance to P. sojae by regulating defense-related gene expression in soybean. PMID:28553307

  20. Gangliosides as a potential new class of stem cell markers: the case of GD1a in human bone marrow mesenchymal stem cells.

    PubMed

    Bergante, Sonia; Torretta, Enrica; Creo, Pasquale; Sessarego, Nadia; Papini, Nadia; Piccoli, Marco; Fania, Chiara; Cirillo, Federica; Conforti, Erika; Ghiroldi, Andrea; Tringali, Cristina; Venerando, Bruno; Ibatici, Adalberto; Gelfi, Cecilia; Tettamanti, Guido; Anastasia, Luigi

    2014-03-01

    Owing to their exposure on the cell surface and the possibility of being directly recognized with specific antibodies, glycosphingolipids have aroused great interest in the field of stem cell biology. In the search for specific markers of the differentiation of human bone marrow mesenchymal stem cells (hBMSCs) toward osteoblasts, we studied their glycosphingolipid pattern, with particular attention to gangliosides. After lipid extraction and fractionation, gangliosides, metabolically (3)H-labeled in the sphingosine moiety, were separated by high-performance TLC and chemically characterized by MALDI MS. Upon induction of osteogenic differentiation, a 3-fold increase of ganglioside GD1a was observed. Therefore, the hypothesis of GD1a involvement in hBMSCs commitment toward the osteogenic phenotype was tested by comparison of the osteogenic propensity of GD1a-highly expressing versus GD1a-low expressing hBMSCs and direct addition of GD1a in the differentiation medium. It was found that either the high expression of GD1a in hBMSCs or the addition of GD1a in the differentiation medium favored osteogenesis, providing a remarkable increase of alkaline phosphatase. It was also observed that ganglioside GD2, although detectable in hBMSCs by immunohistochemistry with an anti-GD2 antibody, could not be recognized by chemical analysis, likely reflecting a case, not uncommon, of molecular mimicry.

  1. Ganglioside GT1b protects human spermatozoa from hydrogen peroxide-induced DNA and membrane damage.

    PubMed

    Gavella, Mirjana; Garaj-Vrhovac, Verica; Lipovac, Vaskresenija; Antica, Mariastefania; Gajski, Goran; Car, Nikica

    2010-06-01

    We have reported previously that various gangliosides, the sialic acid containing glycosphingolipids, provide protection against sperm injury caused by reactive oxygen species (ROS). In this study, we investigated the effect of treatment of human spermatozoa with ganglioside GT1b on hydrogen peroxide (H(2)O(2))-induced DNA fragmentation and plasma membrane damage. Single-cell gel electrophoresis (Comet assay) used in the assessment of sperm DNA integrity showed that in vitro supplemented GT1b (100 microm) significantly reduced DNA damage induced by H(2)O(2) (200 microm) (p < 0.05). Measurements of Annexin V binding in combination with the propidium iodide vital dye labelling demonstrated that the spermatozoa pre-treated with GT1b exhibited a significant increase (p < 0.05) in the percentage of live cells with intact membrane and decreased phosphatidylserine translocation after exposure to H(2)O(2). Flow cytometry using the intracellular ROS-sensitive fluorescence dichlorodihydrofluorescein diacetate dye employed to investigate the transport of the extracellularly supplied H(2)O(2) into the cell interior revealed that ganglioside GT1b completely inhibited the passage of H(2)O(2) through the sperm membrane. These results suggest that ganglioside GT1b may protect human spermatozoa from H(2)O(2)-induced damage by rendering sperm membrane more hydrophobic, thus inhibiting the diffusion of H(2)O(2) across the membrane.

  2. Assignment by Negative-Ion Electrospray Tandem Mass Spectrometry of the Tetrasaccharide Backbones of Monosialylated Glycans Released from Bovine Brain Gangliosides

    NASA Astrophysics Data System (ADS)

    Chai, Wengang; Zhang, Yibing; Mauri, Laura; Ciampa, Maria G.; Mulloy, Barbara; Sonnino, Sandro; Feizi, Ten

    2018-05-01

    Gangliosides, as plasma membrane-associated sialylated glycolipids, are antigenic structures and they serve as ligands for adhesion proteins of pathogens, for toxins of bacteria, and for endogenous proteins of the host. The detectability by carbohydrate-binding proteins of glycan antigens and ligands on glycolipids can be influenced by the differing lipid moieties. To investigate glycan sequences of gangliosides as recognition structures, we have underway a program of work to develop a "gangliome" microarray consisting of isolated natural gangliosides and neoglycolipids (NGLs) derived from glycans released from them, and each linked to the same lipid molecule for arraying and comparative microarray binding analyses. Here, in the first phase of our studies, we describe a strategy for high-sensitivity assignment of the tetrasaccharide backbones and application to identification of eight of monosialylated glycans released from bovine brain gangliosides. This approach is based on negative-ion electrospray mass spectrometry with collision-induced dissociation (ESI-CID-MS/MS) of the desialylated glycans. Using this strategy, we have the data on backbone regions of four minor components among the monosialo-ganglioside-derived glycans; these are of the ganglio-, lacto-, and neolacto-series.

  3. Gangliosides as a potential new class of stem cell markers: the case of GD1a in human bone marrow mesenchymal stem cells[S

    PubMed Central

    Bergante, Sonia; Torretta, Enrica; Creo, Pasquale; Sessarego, Nadia; Papini, Nadia; Piccoli, Marco; Fania, Chiara; Cirillo, Federica; Conforti, Erika; Ghiroldi, Andrea; Tringali, Cristina; Venerando, Bruno; Ibatici, Adalberto; Gelfi, Cecilia; Tettamanti, Guido; Anastasia, Luigi

    2014-01-01

    Owing to their exposure on the cell surface and the possibility of being directly recognized with specific antibodies, glycosphingolipids have aroused great interest in the field of stem cell biology. In the search for specific markers of the differentiation of human bone marrow mesenchymal stem cells (hBMSCs) toward osteoblasts, we studied their glycosphingolipid pattern, with particular attention to gangliosides. After lipid extraction and fractionation, gangliosides, metabolically 3H-labeled in the sphingosine moiety, were separated by high-performance TLC and chemically characterized by MALDI MS. Upon induction of osteogenic differentiation, a 3-fold increase of ganglioside GD1a was observed. Therefore, the hypothesis of GD1a involvement in hBMSCs commitment toward the osteogenic phenotype was tested by comparison of the osteogenic propensity of GD1a-highly expressing versus GD1a-low expressing hBMSCs and direct addition of GD1a in the differentiation medium. It was found that either the high expression of GD1a in hBMSCs or the addition of GD1a in the differentiation medium favored osteogenesis, providing a remarkable increase of alkaline phosphatase. It was also observed that ganglioside GD2, although detectable in hBMSCs by immunohistochemistry with an anti-GD2 antibody, could not be recognized by chemical analysis, likely reflecting a case, not uncommon, of molecular mimicry. PMID:24449473

  4. Efficient glycoengineering of GM3 on melanoma cell and monoclonal antibody-mediated selective killing of the glycoengineered cancer cell

    PubMed Central

    Wang, Qianli; Zhang, Junping; Guo, Zhongwu

    2007-01-01

    To verify the principal of a new immunotherapeutic strategy for cancer, a monoclonal antibody 2H3 against N-phenylacetyl GM3, an unnatural form of the tumor-associated antigen GM3, was prepared and employed to demonstrate that murine melanoma cell B16F0 could be effectively glycoengineered by N-phenylacetyl-d-mannosamine to express N-phenylacetyl GM3 and that 2H3 was highly cytotoxic to the glycoengineered B16F0 cell in the presence of complements. It was further demonstrated that B16F0 cell could be glycoengineered 4-5 times more effectively than 3T3 A31 cell, a normal murine embryo fibroblast cell, and that the antibody and complement mediated cytotoxicity was at least 200 times more potent to the glycoengineered B16F0 cell than to the N-phenylacetyl-d-mannosamine-treated 3T3 A31 cell. These results show the promise for developing useful melanoma immunotherapies based on vaccination against N-phenylacetyl GM3 followed by treatment with N-phenylacetyl-d-mannosamine. PMID:17892942

  5. Efficient glycoengineering of GM3 on melanoma cell and monoclonal antibody-mediated selective killing of the glycoengineered cancer cell.

    PubMed

    Wang, Qianli; Zhang, Junping; Guo, Zhongwu

    2007-12-15

    To verify the principal of a new immunotherapeutic strategy for cancer, a monoclonal antibody 2H3 against N-phenylacetyl GM3, an unnatural form of the tumor-associated antigen GM3, was prepared and employed to demonstrate that murine melanoma cell B16F0 could be effectively glycoengineered by N-phenylacetyl-d-mannosamine to express N-phenylacetyl GM3 and that 2H3 was highly cytotoxic to the glycoengineered B16F0 cell in the presence of complements. It was further demonstrated that B16F0 cell could be glycoengineered 4-5 times more effectively than 3T3 A31 cell, a normal murine embryo fibroblast cell, and that the antibody and complement mediated cytotoxicity was at least 200 times more potent to the glycoengineered B16F0 cell than to the N-phenylacetyl-d-mannosamine-treated 3T3 A31 cell. These results show the promise for developing useful melanoma immunotherapies based on vaccination against N-phenylacetyl GM3 followed by treatment with N-phenylacetyl-d-mannosamine.

  6. GmDREB1 overexpression affects the expression of microRNAs in GM wheat seeds.

    PubMed

    Jiang, Qiyan; Sun, Xianjun; Niu, Fengjuan; Hu, Zheng; Chen, Rui; Zhang, Hui

    2017-01-01

    MicroRNAs (miRNAs) are small regulators of gene expression that act on many different molecular and biochemical processes in eukaryotes. To date, miRNAs have not been considered in the current evaluation system for GM crops. In this study, small RNAs from the dry seeds of a GM wheat line overexpressing GmDREB1 and non-GM wheat cultivars were investigated using deep sequencing technology and bioinformatic approaches. As a result, 23 differentially expressed miRNAs in dry seeds were identified and confirmed between GM wheat and a non-GM acceptor. Notably, more differentially expressed tae-miRNAs between non-GM wheat varieties were found, indicating that the degree of variance between non-GM cultivars was considerably higher than that induced by the transgenic event. Most of the target genes of these differentially expressed miRNAs between GM wheat and a non-GM acceptor were associated with abiotic stress, in accordance with the product concept of GM wheat in improving drought and salt tolerance. Our data provided useful information and insights into the evaluation of miRNA expression in edible GM crops.

  7. GM-CSF and IL-3 Modulate Human Monocyte TNF-α Production and Renewal in In Vitro Models of Trained Immunity.

    PubMed

    Borriello, Francesco; Iannone, Raffaella; Di Somma, Sarah; Loffredo, Stefania; Scamardella, Eloise; Galdiero, Maria Rosaria; Varricchi, Gilda; Granata, Francescopaolo; Portella, Giuseppe; Marone, Gianni

    2016-01-01

    GM-CSF and IL-3 are hematopoietic cytokines that also modulate the effector functions of several immune cell subsets. In particular, GM-CSF and IL-3 exert a significant control on monocyte and macrophage effector functions, as assessed in experimental models of inflammatory and autoimmune diseases and also in human studies. Here, we sought to investigate the mechanisms and the extent to which GM-CSF and IL-3 modulate the pro-inflammatory, LPS-mediated, activation of human CD14 + monocytes taking into account the new concept of trained immunity (i.e., the priming stimulus modulates the response to subsequent stimuli mainly by inducing chromatin remodeling and increased transcription at relevant genetic loci). We demonstrate that GM-CSF and IL-3 priming enhances TNF-α production upon subsequent LPS stimulation (short-term model of trained immunity) in a p38- and SIRT2-dependent manner without increasing TNF primary transcript levels (a more direct measure of transcription), thus supporting a posttranscriptional regulation of TNF-α in primed monocytes. GM-CSF and IL-3 priming followed by 6 days of resting also results in increased TNF-α production upon LPS stimulation (long-term model of trained immunity). In this case, however, GM-CSF and IL-3 priming induces a c-Myc-dependent monocyte renewal and increase in cell number that is in turn responsible for heightened TNF-α production. Overall, our results provide insights to understand the biology of monocytes in health and disease conditions in which the hematopoietic cytokines GM-CSF and IL-3 play a role and also extend our knowledge of the cellular and molecular mechanisms of trained immunity.

  8. Activation of human naïve Th cells increases surface expression of GD3 and induces neoexpression of GD2 that colocalize with TCR clusters.

    PubMed

    Villanueva-Cabello, Tania M; Mollicone, Rosella; Cruz-Muñoz, Mario E; López-Guerrero, Delia V; Martínez-Duncker, Iván

    2015-12-01

    CD4+ T helper lymphocytes (Th) orchestrate the immune response after their activation by antigen-presenting cells. Activation of naïve Th cells is reported to generate the reduction in surface epitopes of sialic acid (Sia) in α2,3 and α2,6 linkages. In this work, we report that in spite of this glycophenotype, anti-CD3/anti-CD28-activated purified human naïve Th cells show a significant increase in surface Sia, as assessed by metabolic labeling, compared with resting naïve Th cells, suggesting an increased flux of Sia toward Siaα2,8 glycoconjugates. To understand this increase as a result of ganglioside up-regulation, we observed that very early after activation, human naïve Th cells show an increased expression in surface GD3 and neoexpression of surface GD2 gangliosides, the latter clustering with the T cell receptor (TCR). Also, we report that in contrast to GM2/GD2 synthase null mice, lentiviral vector-mediated silencing of the GM2/GD2 synthase in activated human naïve Th cells reduced efficient TCR clustering and downstream signaling, as assessed by proliferation assays and IL-2 and IL-2R expression, pointing to an important role of this enzyme in activation of human naive Th cells. © The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  9. Curative effects of GM1 in the treatment of severe ischemic brain injury and its effects on serum TNF-α and NDS.

    PubMed

    Liu, Fang; Sun, Xiaoling; Zhang, Yunxia; Zhao, Guoliang; Liu, Yancang; Zhang, Yiwu

    2018-06-01

    The curative effects of monosialotetrahexosyl ganglioside (GM1) in the treatment of severe ischemic brain injury and its effects on tumor necrosis factor-α (TNF-α) and neuropathy disability score (NDS). Sixty patients with severe ischemic brain injury admitted to The First People's Hospital of Jining (Jining, China) from June 2014 to March 2016 were selected. They were randomly divided into the control group (n=30) and the experimental group (n=30). The patients in the control group were treated with routine therapy while those in the experimental group were treated with GM1. The level of TNF-α in the serum was measured by the enzyme-linked immunosorbent assay. The NDS was used to grade the two groups; Pearson's correlation coefficient was applied to analyze the correlation between the content of TNF-α and NDS; the content of superoxide dismutase (SOD) was detected using xanthine oxidase assay, and the content of malondialdehyde (MDA) was detected by thiobarbituric acid method. The clinical recovery time of two groups of patients was recorded. At 14 days after GM1 treatment, the serum TNF-α content and the NDS in the experimental group were significantly lower than those in the control group (P<0.05). The content of TNF-α in the patients was positively correlated with the NDS. After treatment, the serum MDA content of patients in the experimental group was lower, while the SOD content was significantly higher than that in the control group (P<0.05). After GM1 treatment, hemodynamic parameters of patients in the experimental group were significantly improved compared with those in the control group. The total effective rate of GM1 treatment in the experimental group was higher than that in the control group (P<0.05). GM1 has a good clinical significance in the treatment of patients with severe ischemic brain injury and is worthy of clinical promotion and application.

  10. GmDREB1 overexpression affects the expression of microRNAs in GM wheat seeds

    PubMed Central

    Niu, Fengjuan; Hu, Zheng; Chen, Rui; Zhang, Hui

    2017-01-01

    MicroRNAs (miRNAs) are small regulators of gene expression that act on many different molecular and biochemical processes in eukaryotes. To date, miRNAs have not been considered in the current evaluation system for GM crops. In this study, small RNAs from the dry seeds of a GM wheat line overexpressing GmDREB1 and non-GM wheat cultivars were investigated using deep sequencing technology and bioinformatic approaches. As a result, 23 differentially expressed miRNAs in dry seeds were identified and confirmed between GM wheat and a non-GM acceptor. Notably, more differentially expressed tae-miRNAs between non-GM wheat varieties were found, indicating that the degree of variance between non-GM cultivars was considerably higher than that induced by the transgenic event. Most of the target genes of these differentially expressed miRNAs between GM wheat and a non-GM acceptor were associated with abiotic stress, in accordance with the product concept of GM wheat in improving drought and salt tolerance. Our data provided useful information and insights into the evaluation of miRNA expression in edible GM crops. PMID:28459812

  11. Lysosomal Accumulation of SCMAS (Subunit c of Mitochondrial ATP Synthase) in Neurons of the Mouse Model of Mucopolysaccharidosis III B

    PubMed Central

    Ryazantsev, Sergey; Yu, Wei-Hong; Zhao, Hui-Zhi; Neufeld, Elizabeth F.; Ohmi, Kazuhiro

    2007-01-01

    The neurodegenerative disease MPS III B (Sanfilippo syndrome type B) is caused by mutations in the gene encoding the lysosomal enzyme α-N-acetylglucosaminidase, with a resulting block in heparan sulfate degradation. A mouse model with disruption of the Naglu gene allows detailed study of brain pathology. In contrast to somatic cells, which accumulate primarily heparan sulfate, neurons accumulate a number of apparently unrelated metabolites, including subunit c of mitochondrial ATP synthase (SCMAS). SCMAS accumulated from 1 month of age, primarily in the medial entorhinal cortex and layer V of the somatosensory cortex. Its accumulation was not due to the absence of specific proteases. Light microscopy of brain sections of 6 months-old mice showed SCMAS to accumulate in the same areas as glycosaminoglycan and unesterified cholesterol, in the same cells as ubiquitin and GM3 ganglioside, and in the same organelles as Lamp 1 and Lamp 2. Cryo-immuno electron microscopy showed SCMAS to be present in Lamp positive vesicles bounded by a single membrane (lysosomes), in fingerprint-like layered arrays. GM3 ganglioside was found in the same lysosomes, but was not associated with the SCMAS arrays. GM3 ganglioside was also seen in lysosomes of microglia, suggesting phagocytosis of neuronal membranes. Samples used for cryo-EM and further processed by standard EM procedures (osmium tetroxide fixation and plastic embedding) showed the disappearance of the SCMAS fingerprint arrays and appearance in the same location of “zebra bodies”, well known but little understood inclusions in the brain of patients with mucopolysaccharidoses. PMID:17185018

  12. Characterization of a new GmFAD3A allele in Brazilian CS303TNKCA soybean cultivar.

    PubMed

    Silva, Luiz Claudio Costa; Bueno, Rafael Delmond; da Matta, Loreta Buuda; Pereira, Pedro Henrique Scarpelli; Mayrink, Danyelle Barbosa; Piovesan, Newton Deniz; Sediyama, Carlos Sigueyuki; Fontes, Elizabeth Pacheco Batista; Cardinal, Andrea J; Dal-Bianco, Maximiller

    2018-05-01

    We molecularly characterized a new mutation in the GmFAD3A gene associated with low linolenic content in the Brazilian soybean cultivar CS303TNKCA and developed a molecular marker to select this mutation. Soybean is one of the most important crops cultivated worldwide. Soybean oil has 13% palmitic acid, 4% stearic acid, 20% oleic acid, 55% linoleic acid and 8% linolenic acid. Breeding programs are developing varieties with high oleic and low polyunsaturated fatty acids (linoleic and linolenic) to improve the oil oxidative stability and make the varieties more attractive for the soy industry. The main goal of this study was to characterize the low linoleic acid trait in CS303TNKCA cultivar. We sequenced CS303TNKCA GmFAD3A, GmFAD3B and GmFAD3C genes and identified an adenine point deletion in the GmFAD3A exon 5 (delA). This alteration creates a premature stop codon, leading to a truncated protein with just 207 residues that result in a non-functional enzyme. Analysis of enzymatic activity by heterologous expression in yeast support delA as the cause of low linolenic acid content in CS303TNKCA. Thus, we developed a TaqMan genotyping assay to associate delA with low linolenic acid content in segregating populations. Lines homozygous for delA had a linolenic acid content of 3.3 to 4.4%, and the variation at this locus accounted for 50.83 to 73.70% of the phenotypic variation. This molecular marker is a new tool to introgress the low linolenic acid trait into elite soybean cultivars and can be used to combine with high oleic trait markers to produce soybean with enhanced economic value. The advantage of using CS303TNKCA compared to other lines available in the literature is that this cultivar has good agronomic characteristics and is adapted to Brazilian conditions.

  13. The 3GM Radio Science Experiment on board the ESA JUICE Mission

    NASA Astrophysics Data System (ADS)

    Di Benedetto, M.; Ciarcia, S., Sr.; Iess, L.; Kaspi, Y.; Mann, R., Sr.; Shapira, A., Sr.

    2017-12-01

    The JUpiter ICy moons Explorer (JUICE) is a mission of the European Space Agency devoted to the exploration of the Jupiter system. The main mission themes are the study of Jupiter as an archetype for gas giants and exoplanetary systems, and the characterisation of the potentially habitable worlds Europa, Ganymede and Callisto. During the Jovian tour, JUICE will encounter multiple times the moons Europa and Callisto, while its final target, Ganymede, will be thoroughly investigated in the last phase of the mission, when JUICE will enter orbiting around it. The spacecraft payload consists of a suite of ten instruments, comprising a remote sensing, a geophysical, and a plasma and waves in situ package, plus a radio science instrument, named 3GM (Gravity and Geophysics of Jupiter and the Galilean Moons). An additional experiment, having no dedicated onboard hardware, will be carried out using ground-based VLBI measurements. The 3GM instrument is made up of two separate and independent units incorporated in the spacecraft TT&C subsystem: a Ka band Transponder (KaT) and an Ultra Stable Oscillator (USO). The KaT will enable two-way range and range-rate measurements at Ka band (34.5-32.2 GHz), accurate respectively to 20 cm and 3 μm/s (at 1000 s integration time). The gravity experiment relies on the Ka band radio link to help unveiling the interior structure of Ganymede, inferring the extent of the subsurface global ocean through measurements of the moon's gravity field and tidal response. For Europa and Callisto, only the low degree gravity coefficients can be measured during flybys. 3GM will refine the moment of inertia of the two moons and will determine departures from the hydrostatic equilibrium. The USO will be used for one-way downlink occultation experiments. It will generate on board a highly stable 4.79 MHz reference signal with an Allan deviation of few parts of 10-13 at 1-1000 s integration time. Then the signal will be routed to the DST after an internal x12

  14. Cerebral organoids derived from Sandhoff disease-induced pluripotent stem cells exhibit impaired neurodifferentiation.

    PubMed

    Allende, Maria L; Cook, Emily K; Larman, Bridget C; Nugent, Adrienne; Brady, Jacqueline M; Golebiowski, Diane; Sena-Esteves, Miguel; Tifft, Cynthia J; Proia, Richard L

    2018-03-01

    Sandhoff disease, one of the GM2 gangliosidoses, is a lysosomal storage disorder characterized by the absence of β-hexosaminidase A and B activity and the concomitant lysosomal accumulation of its substrate, GM2 ganglioside. It features catastrophic neurodegeneration and death in early childhood. How the lysosomal accumulation of ganglioside might affect the early development of the nervous system is not understood. Recently, cerebral organoids derived from induced pluripotent stem (iPS) cells have illuminated early developmental events altered by disease processes. To develop an early neurodevelopmental model of Sandhoff disease, we first generated iPS cells from the fibroblasts of an infantile Sandhoff disease patient, then corrected one of the mutant HEXB alleles in those iPS cells using CRISPR/Cas9 genome-editing technology, thereby creating isogenic controls. Next, we used the parental Sandhoff disease iPS cells and isogenic HEXB -corrected iPS cell clones to generate cerebral organoids that modeled the first trimester of neurodevelopment. The Sandhoff disease organoids, but not the HEXB -corrected organoids, accumulated GM2 ganglioside and exhibited increased size and cellular proliferation compared with the HEXB -corrected organoids. Whole-transcriptome analysis demonstrated that development was impaired in the Sandhoff disease organoids, suggesting that alterations in neuronal differentiation may occur during early development in the GM2 gangliosidoses.

  15. LincRNA-Gm4419 knockdown ameliorates NF-κB/NLRP3 inflammasome-mediated inflammation in diabetic nephropathy.

    PubMed

    Yi, Hong; Peng, Rui; Zhang, Lu-Yu; Sun, Yan; Peng, Hui-Min; Liu, Han-Deng; Yu, Li-Juan; Li, Ai-Ling; Zhang, Ya-Juan; Jiang, Wen-Hao; Zhang, Zheng

    2017-02-02

    Diabetic nephropathy (DN) as the primary cause of end-stage kidney disease is a common complication of diabetes. Recent researches have shown the activation of nuclear factor kappa light-chain enhancer of activated B cells (NF-κB) and NACHT, LRR and PYD domain-containing protein 3 (NLRP3) inflammasome are associated with inflammation in the progression of DN, but the exact mechanism is unclear. Long noncoding RNAs (lncRNAs) have roles in the development of many diseases including DN. However, the relationship between lncRNAs and inflammation in DN remains largely unknown. Our previous study has revealed that 14 lncRNAs are abnormally expressed in DN by RNA sequencing and real-time quantitative PCR (qRT-PCR) in the renal tissues of db/db DN mice. In this study, these lncRNAs were verified their expressions by qRT-PCR in mesangial cells (MCs) cultured under high- and low-glucose conditions. Twelve lncRNAs displayed the same expressional tendencies in both renal tissues and MCs. In particular, long intergenic noncoding RNA (lincRNA)-Gm4419 was the only one associating with NF-κB among these 12 lncRNAs by bioinformatics methods. Moreover, Gm4419 knockdown could obviously inhibit the expressions of pro-inflammatory cytokines and renal fibrosis biomarkers, and reduce cell proliferation in MCs under high-glucose condition, whereas overexpression of Gm4419 could increase the inflammation, fibrosis and cell proliferation in MCs under low-glucose condition. Interestingly, our results showed that Gm4419 could activate the NF-κB pathway by directly interacting with p50, the subunit of NF-κB. In addition, we found that p50 could interact with NLRP3 inflammasome in MCs. In conclusion, our findings suggest lincRNA-Gm4419 may participate in the inflammation, fibrosis and proliferation in MCs under high-glucose condition through NF-κB/NLRP3 inflammasome signaling pathway, and may provide new insights into the regulation of Gm4419 during the progression of DN.

  16. The 2.3 {angstrom} crystal structure of cholera toxin B subunit pentamer: Choleragenoid

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Zhang, Rong-Guang; Westbrook, M.L.; Maulik, P.R.

    1996-02-01

    Cholera toxin, a heterohexameric AB{sub 5} enterotoxin released by Vibrio cholera, induces a profuse secretory diarrhea in susceptible hosts. Choleragenoid, the B subunit pentamer of cholera toxin, directs the enzymatic A subunit to its target by binding to GM{sub 1} gangliosides exposed on the luminal surface of intestinal epithelial cells. We have solved the crystal structure of choleragenoid at 2.3 {Angstrom} resolution by combining single isomorphous replacement with non-crystallographic symmetry averaging. The structure of the B subunits, and their pentameric arrangement, closely resembles that reported for the intact holotoxin (choleragen), the heat-labile enterotoxin from E. coli, and for a choleragenoid-GM{submore » 1} pentasaccharide complex. In the absence of the A subunit the central cavity of the B pentamer is a highly solvated channel. The binding of the A subunit or the receptor pentasaccharide to choleragenoid has only a modest effect on the local stereochemistry and does not perceptibly alter the subunit interface.« less

  17. Conflicts of Interest in GM Bt Crop Efficacy and Durability Studies

    PubMed Central

    Lombaert, Eric

    2016-01-01

    Public confidence in genetically modified (GM) crop studies is tenuous at best in many countries, including those of the European Union in particular. A lack of information about the effects of ties between academic research and industry might stretch this confidence to the breaking point. We therefore performed an analysis on a large set of research articles (n = 672) focusing on the efficacy or durability of GM Bt crops and ties between the researchers carrying out these studies and the GM crop industry. We found that ties between researchers and the GM crop industry were common, with 40% of the articles considered displaying conflicts of interest (COI). In particular, we found that, compared to the absence of COI, the presence of a COI was associated with a 50% higher frequency of outcomes favorable to the interests of the GM crop company. Using our large dataset, we were able to propose possible direct and indirect mechanisms behind this statistical association. They might notably include changes of authorship or funding statements after the results of a study have been obtained and a choice in the topics studied driven by industrial priorities. PMID:27977705

  18. CSF N-glycan profile reveals sialylation deficiency in a patient with GM2 gangliosidosis presenting as childhood disintegrative disorder.

    PubMed

    Barone, Rita; Sturiale, Luisella; Fiumara, Agata; Palmigiano, Angelo; Bua, Rosaria O; Rizzo, Renata; Zappia, Mario; Garozzo, Domenico

    2016-04-01

    Protein N-glycosylation consists in the synthesis and processing of the oligosaccharide moiety (N-glycan) linked to a protein and it serves several functions for the proper central nervous system (CNS) development and function. Previous experimental and clinical studies have shown the importance of proper glycoprotein sialylation for the synaptic function and the occurrence of autism spectrum disorders (ASD) in the presence of sialylation deficiency in the CNS. Late-onset Tay Sachs disease (LOTSD) is a lysosomal disorder caused by mutations in the HEXA gene resulting in GM2-ganglioside storage in the CNS. It is characterized by progressive neurological impairment and high co-occurrence of psychiatric disturbances. We studied the N-glycome profile of the cerebrospinal fluid (CSF) in a 14 year-old patient with GM2-gangliosidosis (LOTSD). At the age of 4, the patient presented regressive autism fulfilling criteria for childhood disintegrative disorder (CDD). A CSF sample was obtained in the course of diagnostic work-up for the suspicion of an underlying neurodegenerative disorder. We found definite changes of CSF N-glycans due to a dramatic decrease of sialylated biantennary and triantennary structures and an increase of asialo-core fucosylated bisected N-glycans. No changes of total plasma N-glycans were found. Herein findings highlight possible relationships between the early onset psychiatric disturbance featuring CDD in the patient and defective protein sialylation in the CNS. In conclusion, the study first shows aberrant N-glycan structures of CSF proteins in LOTSD; unveils possible pathomechanisms of GM2-gangliosidosis; supports existing relationships between neuropsychiatric disorders and unproper protein glycosylation in the CNS. © 2015 International Society for Autism Research, Wiley Periodicals, Inc.

  19. The world of "GM-free".

    PubMed

    Moses, Vivian; Brookes, Graham

    2013-01-01

    The rapid global development of agricultural production systems using seeds derived from genetic modification (GM) has been paralleled by the growth of attempts to keep at least a part of the world's agriculture and food as free from GM-crops and their products as possible. The ideal for some proponents of such "GM-free" activity would be a total absence, usually styled "zero content"; others, perhaps more realistically, opt for a definition usually not precisely defined that allows for minimal trace levels of GM material. The reasons for wanting "GM-free" agriculture and its products are varied; they include philosophical and religious beliefs, concern for human (and animal) health--and for "the environment"-as well as commercial and political interests. With such a variety of motivations, and in the absence of legal rulings, the definitions of "GM-free" vary according to inclination and circumstances. Whatever the precise meaning, the maintenance of a "GM-free" product stream in a world where GM crop production is widespread requires the establishment of identity preservation and segregation systems in which traceability and testing are cornerstones. Inevitably these have cost implications for the supply chain and/or the ultimate consumer. In a number of countries different forms of "GM-free" labels exist for some products; the style of such labels is variable with schemes and labels typically voluntary or privately organized. In more recent years, some governments have begun to regularize the definition and meaning of "GM-free." We conclude our analysis by exploring consumer reactions both to "GM-free" and to "GM-free" labels, and ask who ultimately benefits from preserving a product stream substantially or entirely devoid of GM-content.

  20. An R2R3-type MYB transcription factor, GmMYB29, regulates isoflavone biosynthesis in soybean

    PubMed Central

    Liu, Shulin; Zhou, Xiaoqiong; Zhang, Huairen; Wang, Chun-e; Yang, Wenming; Tian, Zhixi; Cheng, Hao; Yu, Deyue

    2017-01-01

    Isoflavones comprise a group of secondary metabolites produced almost exclusively by plants in the legume family, including soybean [Glycine max (L.) Merr.]. They play vital roles in plant defense and have many beneficial effects on human health. Isoflavone content is a complex quantitative trait controlled by multiple genes, and the genetic mechanisms underlying isoflavone biosynthesis remain largely unknown. Via a genome-wide association study (GWAS), we identified 28 single nucleotide polymorphisms (SNPs) that are significantly associated with isoflavone concentrations in soybean. One of these 28 SNPs was located in the 5’-untranslated region (5’-UTR) of an R2R3-type MYB transcription factor, GmMYB29, and this gene was thus selected as a candidate gene for further analyses. A subcellular localization study confirmed that GmMYB29 was located in the nucleus. Transient reporter gene assays demonstrated that GmMYB29 activated the IFS2 (isoflavone synthase 2) and CHS8 (chalcone synthase 8) gene promoters. Overexpression and RNAi-mediated silencing of GmMYB29 in soybean hairy roots resulted in increased and decreased isoflavone content, respectively. Moreover, a candidate-gene association analysis revealed that 11 natural GmMYB29 polymorphisms were significantly associated with isoflavone contents, and regulation of GmMYB29 expression could partially contribute to the observed phenotypic variation. Taken together, these results provide important genetic insights into the molecular mechanisms underlying isoflavone biosynthesis in soybean. PMID:28489859

  1. Cerebral organoids derived from Sandhoff disease-induced pluripotent stem cells exhibit impaired neurodifferentiation[S

    PubMed Central

    Allende, Maria L.; Cook, Emily K.; Larman, Bridget C.; Nugent, Adrienne; Brady, Jacqueline M.; Golebiowski, Diane; Sena-Esteves, Miguel; Tifft, Cynthia J.

    2018-01-01

    Sandhoff disease, one of the GM2 gangliosidoses, is a lysosomal storage disorder characterized by the absence of β-hexosaminidase A and B activity and the concomitant lysosomal accumulation of its substrate, GM2 ganglioside. It features catastrophic neurodegeneration and death in early childhood. How the lysosomal accumulation of ganglioside might affect the early development of the nervous system is not understood. Recently, cerebral organoids derived from induced pluripotent stem (iPS) cells have illuminated early developmental events altered by disease processes. To develop an early neurodevelopmental model of Sandhoff disease, we first generated iPS cells from the fibroblasts of an infantile Sandhoff disease patient, then corrected one of the mutant HEXB alleles in those iPS cells using CRISPR/Cas9 genome-editing technology, thereby creating isogenic controls. Next, we used the parental Sandhoff disease iPS cells and isogenic HEXB-corrected iPS cell clones to generate cerebral organoids that modeled the first trimester of neurodevelopment. The Sandhoff disease organoids, but not the HEXB-corrected organoids, accumulated GM2 ganglioside and exhibited increased size and cellular proliferation compared with the HEXB-corrected organoids. Whole-transcriptome analysis demonstrated that development was impaired in the Sandhoff disease organoids, suggesting that alterations in neuronal differentiation may occur during early development in the GM2 gangliosidoses. PMID:29358305

  2. Binding to Gangliosides Containing N-Acetylneuraminic Acid Is Sufficient To Mediate the Immunomodulatory Properties of the Nontoxic Mucosal Adjuvant LT-IIb(T13I) ▿

    PubMed Central

    Nawar, Hesham F.; Berenson, Charles S.; Hajishengallis, George; Takematsu, Hiromu; Mandell, Lorrie; Clare, Ragina L.; Connell, Terry D.

    2010-01-01

    By use of a mouse mucosal immunization model, LT-IIb(T13I), a nontoxic mutant type II heat-labile enterotoxin, was shown to have potent mucosal and systemic adjuvant properties. In contrast to LT-IIb, which binds strongly to ganglioside receptors decorated with either N-acetylneuraminic acid (NeuAc) or N-glycolylneuraminic acid (NeuGc), LT-IIb(T13I) binds NeuAc gangliosides much less well. Rather, LT-IIb(T13I) binds preferentially to NeuGc gangliosides. To determine if the adjuvant properties of LT-IIb(T13I) are altered in the absence of NeuGc ganglioside receptors, experiments were conducted using a Cmah-null mouse line which is deficient in the synthesis of NeuGc gangliosides. Several immunomodulatory properties of LT-IIb(T13I) were shown to be dependent on NeuGc gangliosides. LT-IIb(T13I) had reduced binding activity for NeuGc-deficient B cells and macrophages; binding to NeuGc-deficient T cells and dendritic cells (DC) was essentially undetectable. Treatment of Cmah-null macrophages with LT-IIb(T13I), however, upregulated the transcription of interleukin-4 (IL-4), IL-6, IL-17, and gamma interferon (IFN-γ), four cytokines important for promoting immune responses. The production of mucosal IgA and serum IgG against an immunizing antigen was augmented in NeuGc-deficient mice administered LT-IIb(T13I) as a mucosal adjuvant. Notably, NeuGc gangliosides are not expressed in humans. Still, treatment of human monocytes with LT-IIb(T13I) induced the secretion of IL-6, an inflammatory cytokine that mediates differential control of leukocyte activation. These results suggested that NeuAc gangliosides are sufficient to mediate the immunomodulatory properties of LT-IIb(T13I) in mice and in human cells. The nontoxic mutant enterotoxin LT-IIb(T13I), therefore, is potentially a new and safe human mucosal adjuvant. PMID:20392887

  3. Imbalance in Fatty-Acid-Chain Length of Gangliosides Triggers Alzheimer Amyloid Deposition in the Precuneus

    PubMed Central

    Oikawa, Naoto; Matsubara, Teruhiko; Fukuda, Ryoto; Yasumori, Hanaki; Hatsuta, Hiroyuki; Murayama, Shigeo; Sato, Toshinori; Suzuki, Akemi; Yanagisawa, Katsuhiko

    2015-01-01

    Amyloid deposition, a crucial event of Alzheimer’s disease (AD), emerges in distinct brain regions. A key question is what triggers the assembly of the monomeric amyloid ß-protein (Aß) into fibrils in the regions. On the basis of our previous findings that gangliosides facilitate the initiation of Aß assembly at presynaptic neuritic terminals, we investigated how lipids, including gangliosides, cholesterol and sphingomyelin, extracted from synaptic plasma membranes (SPMs) isolated from autopsy brains were involved in the Aß assembly. We focused on two regions of the cerebral cortex; precuneus and calcarine cortex, one of the most vulnerable and one of the most resistant regions to amyloid deposition, respectively. Here, we show that lipids extracted from SPMs isolated from the amyloid-bearing precuneus, but neither the amyloid-free precuneus nor the calcarine cortex, markedly accelerate the Aß assembly in vitro. Through liquid chromatography-mass spectrometry of the lipids, we identified an increase in the ratio of the level of GD1b-ganglioside containing C20:0 fatty acid to that containing C18:0 as a cause of the enhanced Aß assembly in the precuneus. Our results suggest that the local glycolipid environment play a critical role in the initiation of Alzheimer amyloid deposition. PMID:25798597

  4. LONG-TERM LIGHT CURVE OF HIGHLY VARIABLE PROTOSTELLAR STAR GM CEP

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Xiao Limin; Kroll, Peter; Henden, Arne A.

    2010-04-15

    We present data from the archival plates at Harvard College Observatory and Sonneberg Observatory showing the field of the solar-type pre-main-sequence star GM Cep. A total of 186 magnitudes of GM Cep have been measured on these archival plates, with 176 in blue sensitivity, six in visible, and four in red. We combine our data with data from the literature and from the American Association of Variable Star Observers to depict the long-term light curves of GM Cep in both B and V wavelengths. The light curves span from 1895 until now, with two densely sampled regions (1935-1945 in themore » B band, and 2006 until now in the V band). The long-term light curves do not show any fast rise behavior as predicted by an accretion mechanism. Both the light curves and the magnitude histograms confirm the conclusion that the light curves are dominated by dips (possibly from extinction) superposed on some quiescence state, instead of outbursts caused by accretion flares. Our result excludes the possibility of GM Cep being a FUor, EXor, or McNeil's Nebula-type star. Several special cases of T Tauri stars were checked, but none of these light curves were compatible with that of GM Cep. The lack of periodicity in the light curve excludes the possibility of GM Cep being a KH 15D system.« less

  5. Chemoenzymatic Syntheses of Sialylated Oligosaccharides Containing C5-Modified Neuraminic Acids for Dual Inhibition of Hemagglutinins and Neuraminidases.

    PubMed

    Birikaki, Lémonia; Pradeau, Stéphanie; Armand, Sylvie; Priem, Bernard; Márquez-Domínguez, Luis; Reyes-Leyva, Julio; Santos-López, Gerardo; Samain, Eric; Driguez, Hugues; Fort, Sébastien

    2015-07-20

    A fast chemoenzymatic synthesis of sialylated oligosaccharides containing C5-modified neuraminic acids is reported. Analogues of GM3 and GM2 ganglioside saccharidic portions where the acetyl group of NeuNAc has been replaced by a phenylacetyl (PhAc) or a propanoyl (Prop) moiety have been efficiently prepared with metabolically engineered E. coli bacteria. GM3 analogues were either obtained by chemoselective modification of biosynthetic N-acetyl-sialyllactoside (GM3 NAc) or by direct bacterial synthesis using C5-modified neuraminic acid precursors. The latter strategy proved to be very versatile as it led to an efficient synthesis of GM2 analogues. These glycomimetics were assessed against hemagglutinins and sialidases. In particular, the GM3 NPhAc displayed a binding affinity for Maackia amurensis agglutinin (MAA) similar to that of GM3 NAc, while being resistant to hydrolysis by Vibrio cholerae (VC) neuraminidase. A preliminary study with influenza viruses also confirmed a selective inhibition of N1 neuraminidase by GM3 NPhAc, suggesting potential developments for the detection of flu viruses and for fighting them. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. A 3-year-old boy with Guillain-Barré syndrome and encephalitis associated with Mycoplasma pneumoniae infection.

    PubMed

    Hanzawa, Fumie; Fuchigami, Tatsuo; Ishii, Wakako; Nakajima, Sonoko; Kawamura, Yuki; Endo, Ayumi; Arakawa, Chikako; Kohira, Ryutaro; Fujita, Yukihiko; Takahashi, Shori

    2014-02-01

    Mycoplasma pneumoniae is a common cause of respiratory tract illness in children. Among the most common extrapulmonary manifestations are disorders of the central nervous system, including meningitis, meningoencephalitis, cerebellitis, polyneuropathy, acute disseminated encephalomyelitis, and Guillain-Barré syndrome. Guillain-Barré syndrome, also known as acute inflammatory demyelinating polyradiculoneuropathy, is an acute-onset, immune-mediated disorder of the peripheral nervous system. The central nervous system is usually intact in patients with Guillain-Barré syndrome. However, there have been some reports of an association of Guillain-Barré syndrome with central nervous system involvement in children. We report a 3-year-old boy with M. pneumoniae infection associated with Guillain-Barré syndrome and encephalitis. Both serum anti-GM1 ganglioside (IgG and IgM) and anti-galactocerebroside IgG antibodies were detected in our patient: the former in the earlier stage of the disease, and the latter in the later stage. We speculate that anti-GM1 ganglioside was associated more with encephalitis, and anti-galactocerebroside antibody was associated more with GBS in our case. Our patient is the youngest report of Guillain-Barré syndrome with central nervous system involvement, and the first report of a pediatric patient with associated M. pneumoniae infection. Such cases are rarely reported, but highlight the need for awareness of the association of the infection with Guillain-Barré syndrome with central nervous system involvement. Copyright © 2013 Japanese Society of Chemotherapy and The Japanese Association for Infectious Disease. Published by Elsevier Ltd. All rights reserved.

  7. Thymic alterations in GM2 gangliosidoses model mice.

    PubMed

    Kanzaki, Seiichi; Yamaguchi, Akira; Yamaguchi, Kayoko; Kojima, Yoshitsugu; Suzuki, Kyoko; Koumitsu, Noriko; Nagashima, Yoji; Nagahama, Kiyotaka; Ehara, Michiko; Hirayasu, Yoshio; Ryo, Akihide; Aoki, Ichiro; Yamanaka, Shoji

    2010-08-10

    Sandhoff disease is a lysosomal storage disorder characterized by the absence of β-hexosaminidase and storage of GM2 ganglioside and related glycolipids. We have previously found that the progressive neurologic disease induced in Hexb(-/-) mice, an animal model for Sandhoff disease, is associated with the production of pathogenic anti-glycolipid autoantibodies. In our current study, we report on the alterations in the thymus during the development of mild to severe progressive neurologic disease. The thymus from Hexb(-/-) mice of greater than 15 weeks of age showed a marked decrease in the percentage of immature CD4(+)/CD8(+) T cells and a significantly increased number of CD4(+)/CD8(-) T cells. During involution, the levels of both apoptotic thymic cells and IgG deposits to T cells were found to have increased, whilst swollen macrophages were prominently observed, particularly in the cortex. We employed cDNA microarray analysis to monitor gene expression during the involution process and found that genes associated with the immune responses were upregulated, particularly those expressed in macrophages. CXCL13 was one of these upregulated genes and is expressed specifically in the thymus. B1 cells were also found to have increased in the thy mus. It is significant that these alterations in the thymus were reduced in FcRγ additionally disrupted Hexb(-/-) mice. These results suggest that the FcRγ chain may render the usually poorly immunogenic thymus into an organ prone to autoimmune responses, including the chemotaxis of B1 cells toward CXCL13.

  8. Soybean GmMYB73 promotes lipid accumulation in transgenic plants

    PubMed Central

    2014-01-01

    Background Soybean is one of the most important oil crops. The regulatory genes involved in oil accumulation are largely unclear. We initiated studies to identify genes that regulate this process. Results One MYB-type gene GmMYB73 was found to display differential expression in soybean seeds of different developing stages by microarray analysis and was further investigated for its functions in lipid accumulation. GmMYB73 is a small protein with single MYB repeat and has similarity to CPC-like MYB proteins from Arabidopsis. GmMYB73 interacted with GL3 and EGL3, and then suppressed GL2, a negative regulator of oil accumulation. GmMYB73 overexpression enhanced lipid contents in both seeds and leaves of transgenic Arabidopsis plants. Seed length and thousand-seed weight were also promoted. GmMYB73 introduction into the Arabidopsis try cpc double mutant rescued the total lipids, seed size and thousand-seed weight. GmMYB73 also elevated lipid levels in seeds and leaves of transgenic Lotus, and in transgenic hairy roots of soybean plants. GmMYB73 promoted PLDα1 expression, whose promoter can be bound and inhibited by GL2. PLDα1 mutation reduced triacylglycerol levels mildly in seeds but significantly in leaves of Arabidopsis plants. Conclusions GmMYB73 may reduce GL2, and then release GL2-inhibited PLDα1 expression for lipid accumulation. Manipulation of GmMYB73 may potentially improve oil production in legume crop plants. PMID:24655684

  9. Detecting Protein-Glycolipid Interactions Using CaR-ESI-MS and Model Membranes: Comparison of Pre-loaded and Passively Loaded Picodiscs.

    PubMed

    Li, Jun; Han, Ling; Li, Jianing; Kitova, Elena N; Xiong, Zi Jian; Privé, Gilbert G; Klassen, John S

    2018-04-13

    Catch-and-release electrospray ionization mass spectrometry (CaR-ESI-MS), implemented using model membranes (MMs), is a promising approach for the discovery of glycolipid ligands of glycan-binding proteins (GBPs). Picodiscs (PDs), which are lipid-transporting complexes composed of the human sphingolipid activator protein saposin A and phospholipids, have proven to be useful MMs for such studies. The present work compares the use of conventional (pre-loaded) PDs with passively loaded PDs ( PL PDs) for CaR-ESI-MS screening of glycolipids against cholera toxin B subunit homopentamer (CTB 5 ). The pre-loaded PDs were prepared from a mixture of purified glycolipid and phospholipid or a mixture of lipids extracted from tissue, while the PL PDs were prepared by incubating PDs containing only phospholipid with glycolipid-containing lipid mixtures in aqueous solution. Time-dependent changes in the composition of the PL PDs produced by incubation with glycomicelles of the ganglioside GM1 were monitored using collision-induced dissociation of the gaseous PD ions and from the extent of ganglioside binding to CTB 5 measured by ESI-MS. GM1 incorporation into PDs was evident within a few hours of incubation. At incubation times ≥ 10 days, GM1 binding to CTB 5 was indistinguishable from that observed with pre-loaded PDs produced directly from GM1 at the same concentration. Comparison of ganglioside binding to CTB 5 measured for pre-loaded PDs and PL PDs prepared from glycolipids extracted from pig and mouse brain revealed that the PL PDs allow for the detection of a greater number of ganglioside ligands. Together, the results of this study suggest PL PDs may have advantages over conventionally prepared PDs for screening glycolipids against GBPs using CaR-ESI-MS. Graphical Abstract ᅟ.

  10. Detecting Protein-Glycolipid Interactions Using CaR-ESI-MS and Model Membranes: Comparison of Pre-loaded and Passively Loaded Picodiscs

    NASA Astrophysics Data System (ADS)

    Li, Jun; Han, Ling; Li, Jianing; Kitova, Elena N.; Xiong, Zi Jian; Privé, Gilbert G.; Klassen, John S.

    2018-04-01

    Catch-and-release electrospray ionization mass spectrometry (CaR-ESI-MS), implemented using model membranes (MMs), is a promising approach for the discovery of glycolipid ligands of glycan-binding proteins (GBPs). Picodiscs (PDs), which are lipid-transporting complexes composed of the human sphingolipid activator protein saposin A and phospholipids, have proven to be useful MMs for such studies. The present work compares the use of conventional (pre-loaded) PDs with passively loaded PDs (PLPDs) for CaR-ESI-MS screening of glycolipids against cholera toxin B subunit homopentamer (CTB5). The pre-loaded PDs were prepared from a mixture of purified glycolipid and phospholipid or a mixture of lipids extracted from tissue, while the PLPDs were prepared by incubating PDs containing only phospholipid with glycolipid-containing lipid mixtures in aqueous solution. Time-dependent changes in the composition of the PLPDs produced by incubation with glycomicelles of the ganglioside GM1 were monitored using collision-induced dissociation of the gaseous PD ions and from the extent of ganglioside binding to CTB5 measured by ESI-MS. GM1 incorporation into PDs was evident within a few hours of incubation. At incubation times ≥ 10 days, GM1 binding to CTB5 was indistinguishable from that observed with pre-loaded PDs produced directly from GM1 at the same concentration. Comparison of ganglioside binding to CTB5 measured for pre-loaded PDs and PLPDs prepared from glycolipids extracted from pig and mouse brain revealed that the PLPDs allow for the detection of a greater number of ganglioside ligands. Together, the results of this study suggest PLPDs may have advantages over conventionally prepared PDs for screening glycolipids against GBPs using CaR-ESI-MS. [Figure not available: see fulltext.

  11. Expression of non-toxic mutant of Escherichia coli heat-labile enterotoxin in tobacco chloroplasts.

    PubMed

    Kang, Tae-Jin; Han, So-Chon; Kim, Mi-Young; Kim, Young-Sook; Yang, Moon-Sik

    2004-11-01

    Chloroplast transformation systems offer unique advantages in biotechnology, including high level of foreign gene expression, maternal inheritance, and polycistronic expression. We studied chloroplast expression of LTK63 (change Ser-->Lys at position 63 in the A subunit) which is the mutant of Escherichia coli heat-labile toxin. LTK63 is devoid of any toxic activity, but still retains its mucosal adjuvanticity. The LTK63 was cloned into chloroplast targeting vector and transformed to tobacco chloroplasts by particle bombardment. PCR and Southern blot analyses confirmed stable homologous recombination of the LTK63 gene into the chloroplast genome. The amount of LTK63 protein detected in tobacco chloroplasts was approximately 3.7% of the total soluble protein. The GM1-ganglioside binding assay confirmed that chloroplast-synthesized LTB of LTK63 binds to the intestinal membrane GM1-ganglioside receptor. Thus, the expression of LTK63 in chloroplasts provides a potential route toward the development of a plant-based edible vaccine for high expression system and environmentally friendly approach.

  12. Lag phase and biomass determination of Rhodococcus pyridinivorans GM3 for degradation of phenol

    NASA Astrophysics Data System (ADS)

    Al-Defiery, M. E. J.; Reddy, G.

    2018-05-01

    Among various techniques available for removal of phenol, biodegradation is an eco-friendly and cost effective method. Thus, it is required to understand the process of biodegradation of phenol, such as investigate on lag phase and biomass concentration. Phenol degrading bacteria were isolated from soil samples of industrial sites in enriched mineral salts medium (MSM) with phenol as a sole source of energy and carbon. One isolate of potential phenol degradation from consortium for phenol degrading studies was identified as Rhodococcus pyridinivorans GM3. Lag phase and biomass determination of R. pyridinivorans GM3 was studied with different phenol concentrations under pH 8.5 at temperature 32 Co and 200 rpm. Microbial biomass was directly proportional to increasing phenol concentration between 1.0 to 2.0 g/L with a maximum dry biomass of 1.745 g/L was noted after complete degradation of 2.0 g/L phenol in 48 hours.

  13. Isotopic labeling of milk disialogangliosides (GD3).

    PubMed

    Reis, Mariza Gomes; Bibiloni, Rodrigo; McJarrow, Paul; MacGibbon, Alastair; Fong, Bertram; Bassett, Shalome; Roy, Nicole; Dos Reis, Marlon Martins

    2016-10-01

    The most abundant ganglioside group in both human milk and bovine milk during the first postnatal week is ganglioside GD3. This group of disialogangliosides forms up to 80% of the total ganglioside content of colostrum. Although dietary gangliosides have shown biological activity such as improvement of cognitive development, gastrointestinal health, and immune function, there is still a gap in our understanding of the molecular mechanisms governing its uptake and the metabolic processes affecting its bioavailability. The use of isotopically labeled ganglioside to track the bioavailability, absorption, distribution, and metabolism of gangliosides may provide key information to bridge this gap. However, isotope labeled GD3 is not commercially available and its preparation has not been described. We report for the first time the preparation of labeled GD3 with stable isotopes. Using alkaline hydrolysis, we were able to selectively remove both acetyl groups from the tetrasaccharide portion of GD3 without promoting significant hydrolysis of the ceramide portion of the molecule to generate N-deacetyl-GD3 (Neu5α2-8Neu5-GD3). The N-deacetyl-GD3 was then chemoselectively re-acetylated in aqueous medium using deuterated acetic anhydride in the presence of Triton X 100 to produce 2 H 6 -GD3 {GD3[(Neu5Ac-11- 2 H 3 )-(Neu5Ac-11- 2 H 3 )]}. This method provided 2 H 6 -GD3 with approximately 60% yield. This compound was characterized by proton nuclear magnetic resonance ( 1 H NMR) and liquid chromatography mass spectrometry (LC-MS). The oral absorption of the 2 H 6 -GD3 was demonstrated using a Sprague-Dawley weaning rats. Our results indicate that some ingested labeled milk gangliosides are absorbed and transported into the bloodstream without modification. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  14. Functional analysis of structurally related soybean GmWRKY58 and GmWRKY76 in plant growth and development.

    PubMed

    Yang, Yan; Chi, Yingjun; Wang, Ze; Zhou, Yuan; Fan, Baofang; Chen, Zhixiang

    2016-08-01

    WRKY transcription factors constitute a large protein superfamily with a predominant role in plant stress responses. In this study we report that two structurally related soybean WRKY proteins, GmWRKY58 and GmWRKY76, play a critical role in plant growth and flowering. GmWRKY58 and GmWRKY76 are both Group III WRKY proteins with a C2HC zinc finger domain and are close homologs of AtWRKY70 and AtWRKY54, two well-characterized Arabidopsis WRKY proteins with an important role in plant responses to biotic and abiotic stresses. GmWRKY58 and GmWRKY76 are both localized to the nucleus, recognize the TTGACC W-box sequence with a high specificity, and function as transcriptional activators in both yeast and plant cells. Expression of GmWRKY58 and GmWRKY76 was detected at low levels in roots, stem, leaves, flowers, and pods. Expression of the two genes in leaves increased substantially during the first 4 weeks after germination but steadily declined thereafter with increased age. To determine their biological functions, transgenic Arabidopsis plants were generated overexpressing GmWRKY58 or GmWRKY76 Unlike AtWRKY70 and AtWRKY54, overexpression of GmWRKY58 or GmWRKY76 had no effect on disease resistance and only small effects on abiotic stress tolerance of the transgenic plants. Significantly, transgenic Arabidopsis plants overexpressing GmWRKY58 or GmWRKY76 flowered substantially earlier than control plants and this early flowering phenotype was associated with increased expression of several flowering-promoting genes, some of which are enriched in W-box sequences in their promoters recognized by GmWRKY58 and GmWRKY76. In addition, virus-induced silencing of GmWRKY58 and GmWRKY76 in soybean resulted in stunted plants with reduced leaf expansion and terminated stem growth. These results provide strong evidence for functional divergence among close structural homologs of WRKY proteins from different plant species. © The Author 2016. Published by Oxford University Press on

  15. Functional analysis of structurally related soybean GmWRKY58 and GmWRKY76 in plant growth and development

    PubMed Central

    Yang, Yan; Chi, Yingjun; Wang, Ze; Zhou, Yuan; Fan, Baofang; Chen, Zhixiang

    2016-01-01

    WRKY transcription factors constitute a large protein superfamily with a predominant role in plant stress responses. In this study we report that two structurally related soybean WRKY proteins, GmWRKY58 and GmWRKY76, play a critical role in plant growth and flowering. GmWRKY58 and GmWRKY76 are both Group III WRKY proteins with a C2HC zinc finger domain and are close homologs of AtWRKY70 and AtWRKY54, two well-characterized Arabidopsis WRKY proteins with an important role in plant responses to biotic and abiotic stresses. GmWRKY58 and GmWRKY76 are both localized to the nucleus, recognize the TTGACC W-box sequence with a high specificity, and function as transcriptional activators in both yeast and plant cells. Expression of GmWRKY58 and GmWRKY76 was detected at low levels in roots, stem, leaves, flowers, and pods. Expression of the two genes in leaves increased substantially during the first 4 weeks after germination but steadily declined thereafter with increased age. To determine their biological functions, transgenic Arabidopsis plants were generated overexpressing GmWRKY58 or GmWRKY76. Unlike AtWRKY70 and AtWRKY54, overexpression of GmWRKY58 or GmWRKY76 had no effect on disease resistance and only small effects on abiotic stress tolerance of the transgenic plants. Significantly, transgenic Arabidopsis plants overexpressing GmWRKY58 or GmWRKY76 flowered substantially earlier than control plants and this early flowering phenotype was associated with increased expression of several flowering-promoting genes, some of which are enriched in W-box sequences in their promoters recognized by GmWRKY58 and GmWRKY76. In addition, virus-induced silencing of GmWRKY58 and GmWRKY76 in soybean resulted in stunted plants with reduced leaf expansion and terminated stem growth. These results provide strong evidence for functional divergence among close structural homologs of WRKY proteins from different plant species. PMID:27335454

  16. Immunoglobulin GM and KM genes and measles vaccine-induced humoral immunity.

    PubMed

    Ovsyannikova, Inna G; Larrabee, Beth R; Schaid, Daniel J; Poland, Gregory A

    2017-10-04

    Identifying genetic polymorphisms that explain variations in humoral immunity to live measles virus vaccine is of great interest. Immunoglobulin GM (heavy chain) and KM (light chain) allotypes are genetic markers known to be associated with susceptibility to several infectious diseases. We assessed associations between GM and KM genotypes and measles vaccine humoral immunity (neutralizing antibody titers) in a combined cohort (n=1796) of racially diverse healthy individuals (age 18-41years). We did not discover any significant associations between GM and/or KM genotypes and measles vaccine-induced neutralizing antibody titers. African-American subjects had higher neutralizing antibody titers than Caucasians (1260mIU/mL vs. 740mIU/mL, p=7.10×10 -13 ), and those titers remained statistically significant (p=1.68×10 -09 ) after adjusting for age at enrollment and time since last vaccination. There were no statistically significant sex-specific differences in measles-induced neutralizing antibody titers in our study (p=0.375). Our data indicate a surprising lack of evidence for an association between GM and KM genotypes and measles-specific neutralizing antibody titers, despite the importance of these immune response genes. Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. Can GM sorghum impact Africa?

    PubMed

    Botha, Gerda M; Viljoen, Christopher D

    2008-02-01

    It is said that genetic modification (GM) of grain sorghum has the potential to alleviate hunger in Africa. To this end, millions of dollars have been committed to developing GM sorghum. Current developments in the genetic engineering of sorghum are similar to efforts to improve cassava and other traditional African crops, as well as rice in Asia. On closer analysis, GM sorghum is faced with the same limitations as 'Golden Rice' (GM rice) in the context of combating vitamin A deficiency (VAD) efficiently and sustainably. Thus, it is questionable whether the cost of developing GM sorghum can be justified when compared to the cost of investing in sustainable agricultural practice in Africa.

  18. Investigating GM Risk Perceptions: A Survey of Anti-GM and Environmental Campaign Group Members

    ERIC Educational Resources Information Center

    Hall, Clare; Moran, Dominic

    2006-01-01

    This study investigates how members of anti-GM campaign groups and environment groups perceive the risks and benefits of genetically modified (GM) technology in food and agriculture. The study targeted these groups as the most risk-averse sector of society when considering GM technology. Survey respondents were asked to rank the current and future…

  19. Neuroprotective Ganglioside Derivatives

    DTIC Science & Technology

    2006-09-01

    SH - SY5Y human neuroblastoma cell line . Derivatives determined to have therapeutic potential are tested in vitro for their...to be cytoprotective in in vitro models using the dopaminergic neurotoxin, 1-methyl-4-phenylpyridinium (MPP+) and the SH - SY5Y cell line . Derivatives...action of these two compounds remains unknown. The ability of GM1 (no preincubation) to protect RA- differentiated SH - SY5Y cells from MPP+

  20. GM2 Activator Deficiency Caused by a Homozygous Exon 2 Deletion in GM2A.

    PubMed

    Hall, Patricia L; Laine, Regina; Alexander, John J; Ankala, Arunkanth; Teot, Lisa A; Lidov, Hart G W; Anselm, Irina

    2018-01-01

    GM2 activator (GM2A) deficiency (OMIM 613109) is a rare lysosomal storage disorder, with onset typically in infancy or early childhood. Clinically, it is almost indistinguishable from Tay-Sachs disease (OMIM 272800) or Sandhoff disease (OMIM 268800); however, traditionally available biochemical screening tests will most likely reveal normal results. We report a 2-year-old male with initially normal development until the age of 9 months, when he presented with developmental delay and regression. Workup at that time was unrevealing; at 15 months, he had abnormal brain MRI findings and a cherry red spot on ophthalmological examination. Family history and all laboratory studies were uninformative. The combination of a cherry red spot and developmental regression was strongly suggestive of a lysosomal storage disorder. Sequence analysis of GM2A did not reveal any pathogenic variants; however, exon 2 of GM2A could not be amplified by PCR, raising suspicion for a large, homozygous deletion. Subsequent copy number analysis confirmed a homozygous deletion of exon 2 in GM2A. This is the first reported case of GM2A deficiency being caused by a whole exon deletion. We describe previously unreported electron microscopy findings in this disease, thus expanding the clinical and variant spectrum for GM2 activator deficiency. These findings demonstrate the increased degree of suspicion required for diagnosis of this rare disorder. Brief Summary: This case of GM2 activator deficiency was caused by a homozygous deletion in GM2A, demonstrating the need to include exon level copy number analysis in any workup to fully exclude this disorder.

  1. Replication of human immunodeficiency virus in monocytes. Granulocyte/macrophage colony-stimulating factor (GM-CSF) potentiates viral production yet enhances the antiviral effect mediated by 3'-azido- 2'3'-dideoxythymidine (AZT) and other dideoxynucleoside congeners of thymidine

    PubMed Central

    1989-01-01

    We have investigated the influence of granulocyte-macrophage CSF (GM- CSF) on the replication of HIV-1 in cells of monocyte/macrophage (M/M) lineage, and its effect on the anti-HIV activity of several 2'3'- dideoxynucleoside congeners of thymidine in these cells in vitro. We found that replication of both HTLV-IIIBa-L (a monocytotropic strain of HIV-1) and HTLV-IIIB (a lymphocytotropic strain) is markedly enhanced in M/M, but not in lymphocytes exposed to GM-CSF in culture. Moreover, GM-CSF reduced the dose of HIV required to obtain productive infection in M/M. Even in the face of this increased infection, GM-CSF also enhanced the net anti-HIV activity of 3'-azido-2'3'-dideoxythymidine (AZT) and several related congeners: 2'3'-dideoxythymidine (ddT), 2'3'- dideoxy-2'3'-didehydrothymidine (D4T), and 3'-azido-2'3'-dideoxyuridine (AZddU). Inhibition of viral replication in GM-CSF-exposed M/M was achieved with concentrations of AZT and related drugs, which were 10- 100 times lower than those inhibitory for HIV-1 in monocytes in the absence of GM-CSF. Other dideoxynucleosides not related to AZT showed unchanged or decreased anti-HIV activity in GM-CSF-exposed M/M. To investigate the possible biochemical basis for these effects, we evaluated the metabolism of several drugs in M/M exposed to GM-CSF. We observed in these cells markedly increased levels of both parent and mono-, di-, and triphosphate anabolites of AZT and D4T compared with M/M not exposed to GM-CSF. By contrast, only limited increases of endogenous competing 2'-deoxynucleoside-5'-triphosphate pools were observed after GM-CSF exposure. Thus, the ratio of AZT-5'- triphosphate/2'-deoxythymidine-5'-triphosphate and 2'3'-dideoxy-2'3'- didehydrothymidine-5'-triphosphate/2'-deoxythymi dine- 5'-triphosphate is several-fold higher in GM-CSF-exposed M/M, and this may account for the enhanced activity of such drugs in these cells. Taken together, these findings suggest that GM-CSF increases HIV-1 replication in M

  2. The Src-like adaptor protein regulates GM-CSFR signaling and monocytic dendritic cell maturation.

    PubMed

    Liontos, Larissa M; Dissanayake, Dilan; Ohashi, Pamela S; Weiss, Arthur; Dragone, Leonard L; McGlade, C Jane

    2011-02-15

    GM-CSF is an important cytokine involved in myeloid differentiation and inflammatory processes. Signaling through the GM-CSFR also plays a critical role in the generation of monocyte-derived dendritic cells (DC). In this article, we report that the Src-like adaptor protein (SLAP) functions as a negative regulator of the GM-CSFR. In bone marrow-derived DC (BM-DC) lacking SLAP and the closely related SLAP2, downregulation of GM-CSFRβ is impaired, leading to enhanced phosphorylation of Jak2 and prolonged activation of Akt and Erk1/2 in response to GM-CSF stimulation. Compared with wild-type bone marrow, SLAP/SLAP2(-/-) bone marrow gave rise to similar numbers of CD11c(+) and CD11b(+) DC, but SLAP/SLAP2(-/-) BM-DC failed to acquire high levels of MHC class II, CD80, and CD86, indicating an impairment in maturation. Furthermore, MHC class II expression in SLAP/SLAP2(-/-) BM-DC was rescued by decreasing GM-CSF concentration, suggesting that enhanced GM-CSF signaling mediates the block in maturation. In addition, SLAP/SLAP2(-/-) BM-DC produced less IL-12 and TNF-α in response to LPS compared with controls and failed to stimulate T cells in an MLR. Ag-specific T cell activation assays showed that SLAP/SLAP2(-/-) BM-DC were less robust at inducing IFN-γ secretion by DO11.10 T cells. These results indicated that SLAP-mediated GM-CSFR regulation is important for the generation of functionally mature monocytic DC.

  3. Sialic Acids in the Brain: Gangliosides and Polysialic Acid in Nervous System Development, Stability, Disease, and Regeneration

    PubMed Central

    Gerardy-Schahn, Rita; Hildebrandt, Herbert

    2014-01-01

    Every cell in nature carries a rich surface coat of glycans, its glycocalyx, which constitutes the cell's interface with its environment. In eukaryotes, the glycocalyx is composed of glycolipids, glycoproteins, and proteoglycans, the compositions of which vary among different tissues and cell types. Many of the linear and branched glycans on cell surface glycoproteins and glycolipids of vertebrates are terminated with sialic acids, nine-carbon sugars with a carboxylic acid, a glycerol side-chain, and an N-acyl group that, along with their display at the outmost end of cell surface glycans, provide for varied molecular interactions. Among their functions, sialic acids regulate cell-cell interactions, modulate the activities of their glycoprotein and glycolipid scaffolds as well as other cell surface molecules, and are receptors for pathogens and toxins. In the brain, two families of sialoglycans are of particular interest: gangliosides and polysialic acid. Gangliosides, sialylated glycosphingolipids, are the most abundant sialoglycans of nerve cells. Mouse genetic studies and human disorders of ganglioside metabolism implicate gangliosides in axon-myelin interactions, axon stability, axon regeneration, and the modulation of nerve cell excitability. Polysialic acid is a unique homopolymer that reaches >90 sialic acid residues attached to select glycoproteins, especially the neural cell adhesion molecule in the brain. Molecular, cellular, and genetic studies implicate polysialic acid in the control of cell-cell and cell-matrix interactions, intermolecular interactions at cell surfaces, and interactions with other molecules in the cellular environment. Polysialic acid is essential for appropriate brain development, and polymorphisms in the human genes responsible for polysialic acid biosynthesis are associated with psychiatric disorders including schizophrenia, autism, and bipolar disorder. Polysialic acid also appears to play a role in adult brain plasticity

  4. Strategies for coexistence of GM and non-GM soy from import to feed processing.

    PubMed

    Gryson, Nicolas; Eeckhout, Mia; Trouillier, Aurélie; Le Bail, Marianne; Soler, Louis-Georges

    2009-01-01

    Regulations 1829/2003/CE and 1830/2003/CE have allowed the placing on the European market of GM products in food and feed chains, and have defined their rules of traceability and labeling. For some supply chains, like for soy and its derived products that are used in the production of feed, manufacturers have to face both non-GM and GM production, although there are no labeling requirements for animal products derived from animals fed with GMOs. This study presents the strategies of stakeholders involved in the feed production chain to maintain concurrent production of compound feed with GM and non-GM soy products, by dealing with the coexistence between those two crops. The stakeholders include importers, traders, soy processors, feed processors and retailers. The study shows that many tools are in place to ensure and maintain the current coexistence. However, a profound harmonization of procedures and methods at a European level should be encouraged.

  5. Defective macroautophagic turnover of brain lipids in the TgCRND8 Alzheimer mouse model: prevention by correcting lysosomal proteolytic deficits

    PubMed Central

    Stavrides, Philip; Saito, Mitsuo; Kumar, Asok; Rodriguez-Navarro, Jose A.; Pawlik, Monika; Huo, Chunfeng; Walkley, Steven U.; Saito, Mariko; Cuervo, Ana M.

    2014-01-01

    Autophagy, the major lysosomal pathway for the turnover of intracellular organelles is markedly impaired in neurons in Alzheimer’s disease and Alzheimer mouse models. We have previously reported that severe lysosomal and amyloid neuropathology and associated cognitive deficits in the TgCRND8 Alzheimer mouse model can be ameliorated by restoring lysosomal proteolytic capacity and autophagy flux via genetic deletion of the lysosomal protease inhibitor, cystatin B. Here we present evidence that macroautophagy is a significant pathway for lipid turnover, which is defective in TgCRND8 brain where lipids accumulate as membranous structures and lipid droplets within giant neuronal autolysosomes. Levels of multiple lipid species including several sphingolipids (ceramide, ganglioside GM3, GM2, GM1, GD3 and GD1a), cardiolipin, cholesterol and cholesteryl esters are elevated in autophagic vacuole fractions and lysosomes isolated from TgCRND8 brain. Lipids are localized in autophagosomes and autolysosomes by double immunofluorescence analyses in wild-type mice and colocalization is increased in TgCRND8 mice where abnormally abundant GM2 ganglioside-positive granules are detected in neuronal lysosomes. Cystatin B deletion in TgCRND8 significantly reduces the number of GM2-positive granules and lowers the levels of GM2 and GM3 in lysosomes, decreases lipofuscin-related autofluorescence, and eliminates giant lipid-containing autolysosomes while increasing numbers of normal-sized autolysosomes/lysosomes with reduced content of undigested components. These findings have identified macroautophagy as a previously unappreciated route for delivering membrane lipids to lysosomes for turnover, a function that has so far been considered to be mediated exclusively through the endocytic pathway, and revealed that autophagic-lysosomal dysfunction in TgCRND8 brain impedes lysosomal turnover of lipids as well as proteins. The amelioration of lipid accumulation in TgCRND8 by removing cystatin

  6. Acceptability of GM foods among Pakistani consumers.

    PubMed

    Ali, Akhter; Rahut, Dil Bahadur; Imtiaz, Muhammad

    2016-04-02

    In Pakistan majority of the consumers do not have information about genetically modified (GM) foods. In developing countries particularly in Pakistan few studies have focused on consumers' acceptability about GM foods. Using comprehensive primary dataset collected from 320 consumers in 2013 from Pakistan, this study analyzes the determinants of consumers' acceptability of GM foods. The data was analyzed by employing the bivariate probit model and censored least absolute deviation (CLAD) models. The empirical results indicated that urban consumers are more aware of GM foods compared to rural consumers. The acceptance of GM foods was more among females' consumers as compared to male consumers. In addition, the older consumers were more willing to accept GM food compared to young consumers. The acceptability of GM foods was also higher among wealthier households. Low price is the key factor leading to the acceptability of GM foods. The acceptability of the GM foods also reduces the risks among Pakistani consumers.

  7. Acceptability of GM foods among Pakistani consumers

    PubMed Central

    Ali, Akhter; Rahut, Dil Bahadur; Imtiaz, Muhammad

    2016-01-01

    ABSTRACT In Pakistan majority of the consumers do not have information about genetically modified (GM) foods. In developing countries particularly in Pakistan few studies have focused on consumers' acceptability about GM foods. Using comprehensive primary dataset collected from 320 consumers in 2013 from Pakistan, this study analyzes the determinants of consumers' acceptability of GM foods. The data was analyzed by employing the bivariate probit model and censored least absolute deviation (CLAD) models. The empirical results indicated that urban consumers are more aware of GM foods compared to rural consumers. The acceptance of GM foods was more among females' consumers as compared to male consumers. In addition, the older consumers were more willing to accept GM food compared to young consumers. The acceptability of GM foods was also higher among wealthier households. Low price is the key factor leading to the acceptability of GM foods. The acceptability of the GM foods also reduces the risks among Pakistani consumers. PMID:27494790

  8. Ex-vivo expansion of CFU-GM and BFU-E in unselected PBMC cultures with Flt3L is enhanced by autologous plasma.

    PubMed

    Guo, M; Miller, W M; Papoutsakis, E T; Patel, S; James, C; Goolsby, C; Winter, J N

    1999-01-01

    Previous ex-vivo expansion studies in our laboratory, comparing unselected and CD34(+)-selected PBMC, have shown no advantage for CD34(+) cell selection, in terms of the expansion achieved. Our goal was to develop procedures for consistent generation of large numbers of hematopoietic progenitor and post-progenitor cells from unselected PBMC. Unselected PBMC, collected from cancer patients undergoing apheresis prior to high-dose chemotherapy and autologous stem cell rescue, were expanded ex vivo in static cultures, without a stromal layer, in the presence of Flt3 ligand (Flt3L), a recombinant GM-CSF/IL-3 fusion protein (PIXY321), G-CSF and GM-CSF for 10 days. The addition of 2% autologous plasma to this cytokine combination enhanced expansion of total cell numbers (3.2 fold versus 1.9 fold; p < 0.01), colony-forming units granulocyte-macrophage (CFU-GM) (22.0 fold versus 8.1 fold, p < 0.01) and burst-forming units erythroid (BFU-E) (17.6 fold versus 7.0 fold, 0.01 < p < 0.02). The optimal seeding density for a given specimen was inversely related to the frequency of CD34(+) cells in the sample. CFU-GM expansion with the Flt3L-containing cytokine cocktail was equivalent to that obtained with IL-3, IL-6, G-CSF and SCF, whether or not the cultures were supplemented with autologous plasma. In plasma-free cultures, BFU-E expansion was significantly higher with IL-3, IL-6, G-CSF and SCF than with Flt3L, PIXY321, G-CSF and GM-CSF. In the presence of autologous plasma, however BFU-E expansion was higher in the Flt3L-containing media. In comparison studies, autologous plasma suppressed BFU-E expansion in SCF-containing cultures. Consistent with our colony assay results, dual-parameter flow cytometric analysis of the expanded cell population revealed that supplementation with autologous plasma yielded a significant increase in the numbers of myeloid progenitors in Flt3L-containing cultures. Unselected PBMC from cancer patients can be effectively expanded ex vivo in Flt3L

  9. Recent progress in GM-CSF-based cancer immunotherapy.

    PubMed

    Yan, Wan-Lun; Shen, Kuan-Yin; Tien, Chun-Yuan; Chen, Yu-An; Liu, Shih-Jen

    2017-03-01

    Cancer immunotherapy is a growing field. GM-CSF, a potent cytokine promoting the differentiation of myeloid cells, can also be used as an immunostimulatory adjuvant to elicit antitumor immunity. Additionally, GM-CSF is essential for the differentiation of dendritic cells, which are responsible for processing and presenting tumor antigens for the priming of antitumor cytotoxic T lymphocytes. Some strategies have been developed for GM-CSF-based cancer immunotherapy in clinical practice: GM-CSF monotherapy, GM-CSF-secreting cancer cell vaccines, GM-CSF-fused tumor-associated antigen protein-based vaccines, GM-CSF-based DNA vaccines and GM-CSF combination therapy. GM-CSF also contributes to the regulation of immunosuppression in the tumor microenvironment. This review provides recommendations regarding GM-CSF-based cancer immunotherapy.

  10. GM Risk Assessment

    NASA Astrophysics Data System (ADS)

    Sparrow, Penny A. C.

    GM risk assessments play an important role in the decision-making process surrounding the regulation, notification and permission to handle Genetically Modified Organisms (GMOs). Ultimately the role of a GM risk assessment will be to ensure the safe handling and containment of the GMO; and to assess any potential impacts on the environment and human health. A risk assessment should answer all ‘what if’ scenarios, based on scientific evidence.

  11. GM biofortified crops: potential effects on targeting the micronutrient intake gap in human populations.

    PubMed

    De Steur, Hans; Mehta, Saurabh; Gellynck, Xavier; Finkelstein, Julia L

    2017-04-01

    Genetic engineering has been successfully applied to increase micronutrient content in staple crops. Nutrition evidence is key to ensure scale-up and successful implementation. Unlike conventional plant breeding efforts, research on the efficacy or effectiveness of GM biofortified crops on nutritional status in human populations is lacking. This review reports on the potential role of GM biofortified crops in closing the micronutrient gap - increasing the dietary intake of micronutrients in human populations. To date, one clinical trial in the United States reported a high bio-conversion rate of β-carotene in Golden Rice, and potential effects of GM biofortified crop consumption on dietary intake and nutritional outcomes are promising. However, further research needs to confirm the ex ante assessments in target regions. Copyright © 2017. Published by Elsevier Ltd.

  12. Epithelial GM-CSF induction by Candida glabrata.

    PubMed

    Li, L; Dongari-Bagtzoglou, A

    2009-08-01

    The main cytokine induced by the interaction of oral epithelial cells with C. glabrata is granulocyte monocyte colony-stimulating factor (GM-CSF); however, the mechanisms regulating this response are unknown. Based on previously published information on the interactions of C. albicans with oral epithelial cells, we hypothesized that interaction with viable C. glabrata triggers GM-CSF synthesis via NF-kappaB activation. We found that C. glabrata-induced GM-CSF synthesis was adhesion-dependent, enhanced by endocytosis, and required fungal viability. NF-kappaB activation was noted during interaction of epithelial cells with C. glabrata, and pre-treatment with an NF-kappaB inhibitor partly inhibited GM-CSF synthesis. Blocking TLR4 with anti-TLR4 antibody did not inhibit GM-CSF production. In contrast, an anti-CDw17 antibody triggered significant inhibition of NF-kappaB activation and GM-CSF synthesis. beta-glucans did not stimulate GM-CSF synthesis, suggesting that the CDw17/NF-kappaB/GM-CSF pathway may be beta-glucan-independent. This study provides new insights into the mechanism of GM-CSF induction by C. glabrata.

  13. Granulocyte-macrophage colony-stimulating factor (GM-CSF) regulates cytokine induction by 1,3-beta-D-glucan SCG in DBA/2 mice in vitro.

    PubMed

    Harada, Toshie; Miura, Noriko N; Adachi, Yoshiyuki; Nakajima, Mitsuhiro; Yadomae, Toshiro; Ohno, Naohito

    2004-08-01

    Sparassis crispa Fr. is an edible/medicinal mushroom that recently became cultivable in Japan. SCG is a major 6-branched 1,3-beta-D-glucan in S. crispa showing antitumor activity. We recently found that the splenocytes from naive DBA/1 and DBA/2 mice strongly react with SCG to produce interferon-gamma (IFN-gamma). In this study, cytokines induced by SCG were screened and found to be IFN-gamma, tumor necrosis factor-alpha (TNF-alpha), granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin-12 (IL-12p70). The addition of recombinant murine GM-CSF (rMuGM-CSF) to spleen cell cultures from various strains of mice synergistically enhanced IFN-gamma, TNF-alpha and IL-12p70 in the presence of SCG. In contrast, neutralizing GM-CSF using anti-GM-CSF monoclonal antibody (mAb) significantly inhibited IFN-gamma, TNF-alpha, and IL-12p70 elicited by SCG. We conclude that GM-CSF is a key molecule for cytokine induction by beta-glucan, and GM-CSF induction by SCG is the specific step in DBA/2 mice in vitro.

  14. Novel adapter proteins that link the human GM-CSF receptor to the phosphatidylino-sitol 3-kinase and Shc/Grb2/ras signaling pathways.

    PubMed

    Jücker, M; Feldman, R A

    1996-01-01

    We have used a human GM-CSF-dependent hematopoietic cell line that responds to physiological concentrations of hGM-CSF to analyze a set of signaling events that occur in normal myelopoiesis and whose deregulation may lead to leukemogenesis. Stimulation of these cells with hGM-CSF induced the assembly of multimeric complexes that contained known and novel phosphotyrosyl proteins. One of the new proteins was a major phosphotyrosyl substrate of 76-85 kDa (p80) that was directly associated with the p85 subunit of phosphatidylinositol (PI) 3-kinase through the SH2 domains of p85. p80 also associated with the beta subunit of the activated hGM-CSF receptor, and assembly of this complex correlated with activation of PI 3-kinase. A second phosphotyrosyl protein we identified, p140, associated with the Shc and Grb2 adapter proteins by direct binding to a novel phosphotyrosine-interacting domain located at the N-terminus of Shc. and to the SH3 domains of Grb2, respectively. The Shc/p140/Grb2 complex was found to be constitutively activated in acute myeloid leukemia cells, indicating that activation of this pathway may be a necessary step in the development of some leukemias. The p80/p85/PI 3-kinase and the Shc/Grb2/p140 complexes were tightly associated with Src family kinases, which were prime candidates for phosphorylation of Shc, p80, p140 and other phosphotyrosyl substrates present in these complexes. Our studies suggest that p80 and p140 may link the hGM-CSF receptor to the PI 3-kinase and Shc/Grb2/ras signaling pathways, respectively, and that abnormal activation of hGM-CSF-dependent targets may play a role in leukemogenesis.

  15. [Application of DNA extraction kit, 'GM quicker' for detection of genetically modified soybeans].

    PubMed

    Sato, Noriko; Sugiura, Yoshitsugu; Tanaka, Toshitsugu

    2012-01-01

    Several DNA extraction methods have been officially introduced to detect genetically modified soybeans, but the choice of DNA extraction kits depend on the nature of the samples, such as grains or processed foods. To overcome this disadvantage, we examined whether the GM quicker kit is available for both grains and processed foods. We compared GM quicker with four approved DNA extraction kits in respect of DNA purity, copy numbers of lectin gene, and working time. We found that the DNA quality of GM quicker was superior to that of the other kits for grains, and the procedure was faster. However, in the case of processed foods, GM quicker was not superior to the other kits. We therefore investigated an unapproved GM quicker 3 kit, which is available for DNA extraction from processed foods, such as tofu and boiled soybeans. The GM quicker 3 kit provided good DNA quality from both grains and processed foods, so we made a minor modification of the GM quicker-based protocol that was suitable for processed foods, using GM quicker and its reagents. The modified method enhanced the performance of GM quicker with processed foods. We believe that GM quicker with the modified protocol is an excellent tool to obtain high-quality DNA from grains and processed foods for detection of genetically modified soybeans.

  16. Enzyme replacement for GM1-gangliosidosis: Uptake, lysosomal activation, and cellular disease correction using a novel β-galactosidase:RTB lectin fusion.

    PubMed

    Condori, Jose; Acosta, Walter; Ayala, Jorge; Katta, Varun; Flory, Ashley; Martin, Reid; Radin, Jonathan; Cramer, Carole L; Radin, David N

    2016-02-01

    New enzyme delivery technologies are required for treatment of lysosomal storage disorders with significant pathologies associated with the so-called "hard-to-treat" tissues and organs. Genetic deficiencies in the GLB1 gene encoding acid β-galactosidase lead to GM1-gangliosidosis or Morquio B, lysosomal diseases with predominant disease manifestation associated with the central nervous system or skeletal system, respectively. Current lysosomal ERTs are delivered into cells based on receptor-mediated endocytosis and do not effectively address several hard-to-treat organs including those critical for GM1-gangliosidosis patients. Lectins provide alternative cell-uptake mechanisms based on adsorptive-mediated endocytosis and thus may provide unique biodistribution for lysosomal disease therapeutics. In the current study, genetic fusions of the plant galactose/galactosamine-binding lectin, RTB, and the human acid β-galactosidase enzyme were produced using a plant-based bioproduction platform. β-gal:RTB and RTB:β-gal fusion products retained both lectin activity and β-galactosidase activity. Purified proteins representing both fusion orientations were efficiently taken up into GM1 patient fibroblasts and mediated the reduction of GM1 ganglioside substrate with activities matching mammalian cell-derived β-galactosidase. In contrast, plant-derived β-gal alone was enzymatically active but did not mediate uptake or correction indicating the need for either lectin-based (plant product) or mannose-6-phosphate-based (mammalian product) delivery. Native β-galactosidase undergoes catalytic activation (cleavage within the C-terminal region) in lysosomes and is stabilized by association with protective protein/cathepsin A. Enzymatic activity and lysosomal protein processing of the RTB fusions were assessed following internalization into GM1 fibroblasts. Within 1-4h, both β-gal:RTB and RTB:β-gal were processed to the ~64kDa "activated" β-gal form; the RTB lectin was

  17. Brain Structural Differences between Normal and Obese Adults and their Links with Lack of Perseverance, Negative Urgency, and Sensation Seeking.

    PubMed

    Wang, Haifeng; Wen, Baohong; Cheng, Jingliang; Li, Hongpeng

    2017-01-16

    In order to examine the difference in brain structure between obese and normal weight individuals, and to explore the relationship between the neuroanatomical changes and impulsivity traits, this study used a voxel-based morphometry method to examine gray matter (GM) volume alterations related to impulsive personality traits in obese individuals relative to normal weight. Eighty adults that completed the UPPS-P Impulsive Behavior Scale were analyzed. Possible GM volume alterations were first analyzed at the whole brain level, and then the relationship between regional GM volume differences and UPPS-P scores were examined in selected regions of interest. Reduced GM volumes were found in the frontal and limbic regions in the obese group compared to normal weight individuals. In the normal weight group, lack of perseverance was negatively correlated with GM volume in the anterior cingulate cortex, and negative urgency was negatively correlated with GM volume in the insula. In the obese group, sensation seeking was negatively correlated with GM volume in the left amygdala and right pallidum. These findings might improve our understanding of the relationship between lack of perseverance, negative urgency, and sensation seeking and body weight fluctuations.

  18. Brain Structural Differences between Normal and Obese Adults and their Links with Lack of Perseverance, Negative Urgency, and Sensation Seeking

    PubMed Central

    Wang, Haifeng; Wen, Baohong; Cheng, Jingliang; Li, Hongpeng

    2017-01-01

    In order to examine the difference in brain structure between obese and normal weight individuals, and to explore the relationship between the neuroanatomical changes and impulsivity traits, this study used a voxel-based morphometry method to examine gray matter (GM) volume alterations related to impulsive personality traits in obese individuals relative to normal weight. Eighty adults that completed the UPPS-P Impulsive Behavior Scale were analyzed. Possible GM volume alterations were first analyzed at the whole brain level, and then the relationship between regional GM volume differences and UPPS-P scores were examined in selected regions of interest. Reduced GM volumes were found in the frontal and limbic regions in the obese group compared to normal weight individuals. In the normal weight group, lack of perseverance was negatively correlated with GM volume in the anterior cingulate cortex, and negative urgency was negatively correlated with GM volume in the insula. In the obese group, sensation seeking was negatively correlated with GM volume in the left amygdala and right pallidum. These findings might improve our understanding of the relationship between lack of perseverance, negative urgency, and sensation seeking and body weight fluctuations. PMID:28091559

  19. The natural history of juvenile or subacute GM2 gangliosidosis: 21 new cases and literature review of 134 previously reported.

    PubMed

    Maegawa, Gustavo H B; Stockley, Tracy; Tropak, Michael; Banwell, Brenda; Blaser, Susan; Kok, Fernando; Giugliani, Roberto; Mahuran, Don; Clarke, Joe T R

    2006-11-01

    Juvenile GM2 gangliosidosis is a group of inherited neurodegenerative diseases caused by deficiency of lysosomal beta-hexosaminidase resulting in GM2 ganglioside accumulation in brain. The purpose of this study was to delineate the natural history of the condition and identify genotype-phenotype correlations that might be helpful in predicting the course of the disease in individual patients. A cohort of 21 patients with juvenile GM2 gangliosidosis, 15 with the Tay-Sachs variant and 6 with the Sandhoff variant, was studied prospectively in 2 centers. Our experience was compared with previously published reports on 134 patients. Information about clinical features, beta-hexosaminidase enzyme activity, and mutation analysis was collected. In our cohort of patients, the mean (+/-SD) age of onset of symptoms was 5.3 +/- 4.1 years, with a mean follow-up time of 8.4 years. The most common symptoms at onset were gait disturbances (66.7%), incoordination (52.4%), speech problems (28.6%), and developmental delay (28.6%). The age of onset of gait disturbances was 7.1 +/- 5.6 years. The mean time for progression to becoming wheelchair-bound was 6.2 +/- 5.5 years. The mean age of onset of speech problems was 7.0 +/- 5.6 years, with a mean time of progression to anarthria of 5.6 +/- 5.3 years. Muscle wasting (10.6 +/- 7.4 years), proximal weakness (11.1 +/- 7.7 years), and incontinence of sphincters (14.6 +/- 9.7 years) appeared later in the course of the disease. Psychiatric disturbances and neuropathy were more prevalent in patients with the Sandhoff variant than in those with the Tay-Sachs variant. However, dysphagia, sphincter incontinence, and sleep problems occurred earlier in those with the Tay-Sachs variant. Cerebellar atrophy was the most common finding on brain MRI (52.9%). The median survival time among the studied and reviewed patients was 14.5 years. The genotype-phenotype correlation revealed that in patients with the Tay-Sachs variant, the presence of R178H and R

  20. Characterization of the soybean GmALMT family genes and the function of GmALMT5 in response to phosphate starvation.

    PubMed

    Peng, Wenting; Wu, Weiwei; Peng, Junchu; Li, Jiaojiao; Lin, Yan; Wang, Yanan; Tian, Jiang; Sun, Lili; Liang, Cuiyue; Liao, Hong

    2018-03-01

    A potential mechanism to enhance utilization of sparingly soluble forms of phosphorus (P) is the root secretion of malate, which is mainly mediated by the ALMT gene family in plants. In this study, a total of 34 GmALMT genes were identified in the soybean genome. Expression patterns diverged considerably among GmALMTs in response to phosphate (Pi) starvation in leaves, roots and flowers, with expression altered by P availability in 26 of the 34 GmALMTs. One root-specific GmALMT whose expression was significantly enhanced by Pi-starvation, GmALMT5, was studied in more detail to determine its possible role in soybean P nutrition. Analysis of GmALMT5 tissue expression patterns, subcellular localization, and malate exudation from transgenic soybean hairy roots overexpressing GmALMT5, demonstrated that GmALMT5 is a plasma membrane protein that mediates malate efflux from roots. Furthermore, both growth and P content of transgenic Arabidopsis overexpressing GmALMT5 were significantly increased when sparingly soluble Ca-P was used as the external P source. Taken together, these results indicate that members of the soybean GmALMT gene family exhibit diverse responses to Pi starvation. One member of this family, GmALMT5, might contribute to soybean P efficiency by enhancing utilization of sparingly soluble P sources under P limited conditions. © 2017 Institute of Botany, Chinese Academy of Sciences.

  1. The economic and environmental cost of delayed GM crop adoption: The case of Australia's GM canola moratorium.

    PubMed

    Biden, Scott; Smyth, Stuart J; Hudson, David

    2018-01-02

    Incorporating socio-economic considerations (SECs) into national biosafety regulations regarding genetically modified (GM) crops have opportunity costs. Australia approved the cultivation of GM canola through a science-based risk assessment in 2003, but allowed state moratoria to be instituted based on potential trade impacts over the period 2004 to 2008 and 2010 in the main canola growing states. This analysis constructs a counterfactual assessment using Canadian GM canola adoption data to create an S-Curve of adoption in Australia to measure the environmental and economic opportunity costs of Australia's SEC-based moratoria between 2004 and 2014. The environmental impacts are measured through the amount of chemical active ingredients applied during pest management, the Environmental Impact Quotient indicator, and greenhouse gas emissions. The economic impacts are measured through the variable costs of the weed control programs, yield and the contribution margin. The environmental opportunity costs from delaying the adoption of GM canola in Australia include an additional 6.5 million kilograms of active ingredients applied to canola land; a 14.3% increase in environmental impact to farmers, consumers and the ecology; 8.7 million litres of diesel fuel burned; and an additional 24.2 million kilograms of greenhouse gas (GHG) and compound emissions released. The economic opportunity costs of the SEC-based moratoria resulted in foregone output of 1.1 million metric tonnes of canola and a net economic loss to canola farmers' of AU$485.6 million. The paper provides some of the first quantified, post-adoption evidence on the opportunity cost and environmental impacts of incorporating SECs into GM crop regulation.

  2. Optimization of GM(1,1) power model

    NASA Astrophysics Data System (ADS)

    Luo, Dang; Sun, Yu-ling; Song, Bo

    2013-10-01

    GM (1,1) power model is the expansion of traditional GM (1,1) model and Grey Verhulst model. Compared with the traditional models, GM (1,1) power model has the following advantage: The power exponent in the model which best matches the actual data values can be found by certain technology. So, GM (1,1) power model can reflect nonlinear features of the data, simulate and forecast with high accuracy. It's very important to determine the best power exponent during the modeling process. In this paper, according to the GM(1,1) power model of albino equation is Bernoulli equation, through variable substitution, turning it into the GM(1,1) model of the linear albino equation form, and then through the grey differential equation properly built, established GM(1,1) power model, and parameters with pattern search method solution. Finally, we illustrate the effectiveness of the new methods with the example of simulating and forecasting the promotion rates from senior secondary schools to higher education in China.

  3. Pathology of GM2 gangliosidosis in Jacob sheep.

    PubMed

    Porter, B F; Lewis, B C; Edwards, J F; Alroy, J; Zeng, B J; Torres, P A; Bretzlaff, K N; Kolodny, E H

    2011-07-01

    The G(M2) gangliosidoses are a group of lysosomal storage diseases caused by defects in the genes coding for the enzyme hexosaminidase or the G(M2) activator protein. Four Jacob sheep from the same farm were examined over a 3-year period for a progressive neurologic disease. Two lambs were 6-month-old intact males and 2 were 8-month-old females. Clinical findings included ataxia in all 4 limbs, proprioceptive deficits, and cortical blindness. At necropsy, the nervous system appeared grossly normal. Histologically, most neurons within the brain, spinal cord, and peripheral ganglia were enlarged, and the cytoplasm was distended by foamy to granular material that stained positively with Luxol fast blue and Sudan black B stains. Other neuropathologic findings included widespread astrocytosis, microgliosis, and scattered spheroids. Electron microscopy revealed membranous cytoplasmic bodies within the cytoplasm of neurons. Biochemical and molecular genetic studies confirmed the diagnosis of G(M2) gangliosidosis. This form of G(M2) gangliosidosis in Jacob sheep is very similar to human Tay-Sachs disease and is potentially a useful animal model. © The Authors 2011

  4. Aβ1-25-Derived Sphingolipid-Domain Tracer Peptide SBD Interacts with Membrane Ganglioside Clusters via a Coil-Helix-Coil Motif

    PubMed Central

    Wang, Yaofeng; Kraut, Rachel; Mu, Yuguang

    2015-01-01

    The Amyloid-β (Aβ)-derived, sphingolipid binding domain (SBD) peptide is a fluorescently tagged probe used to trace the diffusion behavior of sphingolipid-containing microdomains in cell membranes through binding to a constellation of glycosphingolipids, sphingomyelin, and cholesterol. However, the molecular details of the binding mechanism between SBD and plasma membrane domains remain unclear. Here, to investigate how the peptide recognizes the lipid surface at an atomically detailed level, SBD peptides in the environment of raft-like bilayers were examined in micro-seconds-long molecular dynamics simulations. We found that SBD adopted a coil-helix-coil structural motif, which binds to multiple GT1b gangliosides via salt bridges and CH–π interactions. Our simulation results demonstrate that the CH–π and electrostatic forces between SBD monomers and GT1b gangliosides clusters are the main driving forces in the binding process. The presence of the fluorescent dye and linker molecules do not change the binding mechanism of SBD probes with gangliosides, which involves the helix-turn-helix structural motif that was suggested to constitute a glycolipid binding domain common to some sphingolipid interacting proteins, including HIV gp120, prion, and Aβ. PMID:26540054

  5. Epstein–Barr virus glycoprotein gM can interact with the cellular protein p32 and knockdown of p32 impairs virus

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Changotra, Harish; Turk, Susan M.; Artigues, Antonio

    The Epstein–Barr virus glycoprotein complex gMgN has been implicated in assembly and release of fully enveloped virus, although the precise role that it plays has not been elucidated. We report here that the long predicted cytoplasmic tail of gM is not required for complex formation and that it interacts with the cellular protein p32, which has been reported to be involved in nuclear egress of human cytomegalovirus and herpes simplex virus. Although redistribution of p32 and colocalization with gM was not observed in virus infected cells, knockdown of p32 expression by siRNA or lentivirus-delivered shRNA recapitulated the phenotype of amore » virus lacking expression of gNgM. A proportion of virus released from cells sedimented with characteristics of virus lacking an intact envelope and there was an increase in virus trapped in nuclear condensed chromatin. The observations suggest the possibility that p32 may also be involved in nuclear egress of Epstein–Barr virus. - Highlights: • The predicted cytoplasmic tail of gM is not required to complex with gN. • Cellular p32 can interact with the predicted cytoplasmic tail of EBV gM. • Knockdown of p32 recapitulates the phenotype of virus lacking the gNgM complex.« less

  6. Risk Management of GM Crops

    EPA Science Inventory

    Driven by biofuel demand, a significant increase in GM corn acreage is anticipated for the 2007 growing season with future planted GM corn acreage approaching 80% of the corn crop by 2009. As demand increases, grower non-compliance with mandated planting requirements is likely to...

  7. Tanzanian farmers' knowledge and attitudes to GM biotechnology and the potential use of GM crops to provide improved levels of food security. A Qualitative Study.

    PubMed

    Lewis, Christopher P; Newell, James N; Herron, Caroline M; Nawabu, Haidari

    2010-07-12

    Genetically Modified (GM) crops have been championed as one possible method to improve food security and individual nutritional status in sub Saharan Africa. Understanding and acceptability of GM crop technology to farmers and consumers have not been assessed. We developed a qualitative research study involving farmers as both producers and consumers to gauge the understanding of GM crop technology, its acceptability, and identifying issues of concern. Nineteen individual interviews (10 male and 9 female) and five mixed gender focus group discussions with local farmers were conducted in 3 regions in Tanzania. Analysis took place concurrently with data collection. Following initial interviews, subsequent questions were adjusted based on emerging themes. Understanding, awareness and knowledge of GM crop technology and terminology and its potential risks and benefits was very poor in all regions. Receptivity to the potential use of GM crops was, however, high. Respondents focused on the potential benefits of GM crops rather than any potential longer term health risks. A number of factors, most significantly field trial data, would influence farmers' decisions regarding the introduction of GM crop varieties into their farming practice. Understanding of the potential improved health provision possible by changes in agricultural practice and food-related decision making, and the health benefits of a diet containing essential vitamins, minerals and micronutrients is also poor in these communities. This study forms a basis from which further research work can be undertaken. It is important to continue to assess opinions and attitudes of farmers and consumers in sub Saharan Africa towards potential use of GM technologies whilst highlighting the importance of the relationship between agriculture, health and development. This will allow people in the region to make accurate, informed decisions about whether they believe use of GM biotechnology is an appropriate way in which

  8. Unique transcriptome signatures and GM-CSF expression in lymphocytes from patients with spondyloarthritis.

    PubMed

    Al-Mossawi, M H; Chen, L; Fang, H; Ridley, A; de Wit, J; Yager, N; Hammitzsch, A; Pulyakhina, I; Fairfax, B P; Simone, D; Yi, Yao; Bandyopadhyay, S; Doig, K; Gundle, R; Kendrick, B; Powrie, F; Knight, J C; Bowness, P

    2017-11-15

    Spondyloarthritis encompasses a group of common inflammatory diseases thought to be driven by IL-17A-secreting type-17 lymphocytes. Here we show increased numbers of GM-CSF-producing CD4 and CD8 lymphocytes in the blood and joints of patients with spondyloarthritis, and increased numbers of IL-17A + GM-CSF + double-producing CD4, CD8, γδ and NK cells. GM-CSF production in CD4 T cells occurs both independently and in combination with classical Th1 and Th17 cytokines. Type 3 innate lymphoid cells producing predominantly GM-CSF are expanded in synovial tissues from patients with spondyloarthritis. GM-CSF + CD4 + cells, isolated using a triple cytokine capture approach, have a specific transcriptional signature. Both GM-CSF + and IL-17A + GM-CSF + double-producing CD4 T cells express increased levels of GPR65, a proton-sensing receptor associated with spondyloarthritis in genome-wide association studies and pathogenicity in murine inflammatory disease models. Silencing GPR65 in primary CD4 T cells reduces GM-CSF production. GM-CSF and GPR65 may thus serve as targets for therapeutic intervention of spondyloarthritis.

  9. Essential mechanisms of differential activation of eosinophils by IL-3 compared to GM-CSF and IL-5

    PubMed Central

    Esnault, Stephane; Kelly, Elizabeth A.

    2017-01-01

    There is compelling evidence that the eosinophils bring negative biological outcomes in several diseases, including eosinophilic asthma and hypereosinophilic syndromes. Eosinophils produce and store a broad range of toxic proteins and other mediators that enhance the inflammatory response and lead to tissue damage. For instance, in asthma, there is a close relationship between increased lung eosinophilia, asthma exacerbation, and loss of lung function. The use of an anti-IL-5 therapy in severe eosinophilic asthmatic patients is efficient to reduce exacerbations. However, anti-IL-5-treated patients still display a relatively high amount of functional lung tissue eosinophils, indicating that supplemental therapies are required to damper the eosinophil functions. Our recent published works, suggest that compared to IL-5, IL-3 can more strongly and differentially affect eosinophil functions. In this review, we will summarize our and other investigations that have compared the effects of the three β-chain receptor cytokines (IL-5, GM-CSF and IL-3) on eosinophil biology. We will focus on how IL-3 differentially activates eosinophils compared to IL-5 or GM-CSF. PMID:28605348

  10. Defective macroautophagic turnover of brain lipids in the TgCRND8 Alzheimer mouse model: prevention by correcting lysosomal proteolytic deficits.

    PubMed

    Yang, Dun-Sheng; Stavrides, Philip; Saito, Mitsuo; Kumar, Asok; Rodriguez-Navarro, Jose A; Pawlik, Monika; Huo, Chunfeng; Walkley, Steven U; Saito, Mariko; Cuervo, Ana M; Nixon, Ralph A

    2014-12-01

    Autophagy, the major lysosomal pathway for the turnover of intracellular organelles is markedly impaired in neurons in Alzheimer's disease and Alzheimer mouse models. We have previously reported that severe lysosomal and amyloid neuropathology and associated cognitive deficits in the TgCRND8 Alzheimer mouse model can be ameliorated by restoring lysosomal proteolytic capacity and autophagy flux via genetic deletion of the lysosomal protease inhibitor, cystatin B. Here we present evidence that macroautophagy is a significant pathway for lipid turnover, which is defective in TgCRND8 brain where lipids accumulate as membranous structures and lipid droplets within giant neuronal autolysosomes. Levels of multiple lipid species including several sphingolipids (ceramide, ganglioside GM3, GM2, GM1, GD3 and GD1a), cardiolipin, cholesterol and cholesteryl esters are elevated in autophagic vacuole fractions and lysosomes isolated from TgCRND8 brain. Lipids are localized in autophagosomes and autolysosomes by double immunofluorescence analyses in wild-type mice and colocalization is increased in TgCRND8 mice where abnormally abundant GM2 ganglioside-positive granules are detected in neuronal lysosomes. Cystatin B deletion in TgCRND8 significantly reduces the number of GM2-positive granules and lowers the levels of GM2 and GM3 in lysosomes, decreases lipofuscin-related autofluorescence, and eliminates giant lipid-containing autolysosomes while increasing numbers of normal-sized autolysosomes/lysosomes with reduced content of undigested components. These findings have identified macroautophagy as a previously unappreciated route for delivering membrane lipids to lysosomes for turnover, a function that has so far been considered to be mediated exclusively through the endocytic pathway, and revealed that autophagic-lysosomal dysfunction in TgCRND8 brain impedes lysosomal turnover of lipids as well as proteins. The amelioration of lipid accumulation in TgCRND8 by removing cystatin B

  11. The effect of recombinant GM-CSF on the recovery of monkeys transplanted with autologous bone marrow.

    PubMed

    Monroy, R L; Skelly, R R; MacVittie, T J; Davis, T A; Sauber, J J; Clark, S C; Donahue, R E

    1987-11-01

    The regulatory function of recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF) on granulocyte production in vivo was evaluated in an autologous bone marrow transplantation model using rhesus monkeys. Monkeys were exposed to 9.0 Gy total body irradiation and then transplanted with 5.0 x 10(7) low-density bone marrow cells/kg. Alzet miniosmotic pumps were subcutaneously implanted to deliver rhGM-CSF at a rate of 50,400 U/kg/d. Minipumps, containing either rhGM-CSF or saline, were implanted between zero and five days after transplantation for seven days. Kinetic recoveries of peripheral blood cells after either saline or rhGM-CSF treatment were compared. Treatment with rhGM-CSF accelerated the recovery of neutrophils. Neutrophils in rhGM-CSF-treated animals recovered to 80% (3.4 x 10(3)/mm3) pre-irradiation control levels by day 20, in comparison with only 33% (0.9 x 10(3)/mm3) recovery for saline control monkeys. In addition, the recovery of neutrophils was enhanced over that of the controls, reaching 140% v 70% on day 30. Another prominent feature of rhGM-CSF-treated monkeys was the accelerated recovery of platelets, reaching near 50% normal levels by day 24 in comparison with 20% of normal levels for controls. The infusion of rhGM-CSF was shown to be an effective regulator of early hematopoietic regeneration, leading to the accelerated recovery of both neutrophils and platelets and then providing a consistent sustained increase of neutrophils even in the absence of rhGM-CSF.

  12. FAS promoter polymorphisms and serum sFas level are associated with increased risk of nerve damage in Bangladeshi patients with Guillain-Barré syndrome.

    PubMed

    Islam, Zhahirul; Jahan, Israt; Ahammad, Rijwan U; Shahnaij, Mohammad; Nahar, Shamsun; Mohammad, Quazi D

    2018-01-01

    Guillain-Barré syndrome (GBS) is an autoimmune disorder of the peripheral nervous system triggered by molecular mimicry between pathogen lipopolysaccharides and host nerve gangliosides. Polymorphisms in the Fas receptor (FAS) and Fas ligand (FASL) genes may potentially alter the elimination of autoreactive immune cells and affect disease susceptibility or disease severity in GBS. We detected single nucleotide polymorphisms (SNPs) in FAS (-1377G/A and -670A/G) and FASL (-843C/T) in a prospective cohort of 300 patients with GBS and 300 healthy controls from the Bangladeshi population. Genotype distributions were not significantly different between patients with GBS and healthy controls. The FAS -670 AG heterozygous (P = 0.0005, OR = 2.5, 95% CI = 1.5-4.2) and GG homozygous (P = 0.0048, OR = 2.6, 95% CI = 1.3-5.0) genotypes were more common in patients with anti-GM1 antibodies than patients without anti-GM1 antibodies. The FAS -670 G allele was more prevalent in anti-GM1 antibody-positive than -negative patients (P = 0.0002, OR = 1.9, 95% CI = 1.4-2.7) and also in patients with the axonal subtype than demyelinating subtype (P < 0.0001, OR = 4.8, 95% CI = 2.3-10.1). The 1377G/-670G GG haplotype was significantly associated with the axonal subtype (P < 0.0001) and anti-ganglioside antibody-positivity (P = 0.0008) in GBS. Serum sFas (237.5 pg/mL vs. 159.5 pg/mL; P < 0.0001) and sFasL (225.1 pg/mL vs. 183.4 pg/mL; P = 0.0069) were elevated in patients with GBS compared to healthy controls, and among patients with high serum sFas was associated with severe GBS (P = 0.0406). In conclusion, this study indicates FAS-FASL promoter SNPs may promote the production of cross-reactive anti-ganglioside antibodies in GBS.

  13. Temperature oscillation suppression of GM cryocooler

    NASA Astrophysics Data System (ADS)

    Okidono, K.; Oota, T.; Kurihara, H.; Sumida, T.; Nishioka, T.; Kato, H.; Matsumura, M.; Sasaki, O.

    2012-12-01

    GM cryocooler is a convenient refrigerator to achieve low temperatures about 4 K, while it is not suitable for precise measurements because of the large temperature oscillation of typically about 0.3 K. To resolve this problem, we have developed an adapter (He-pot) with a simple structure as possible. From the thermodynamic consideration, both heat capacity and thermal conductance should be large in order to reduce the temperature oscillation without compromising cooling power. Optimal structure of the He-pot is a copper cylinder filled with high pressure He-gas at room temperature. This can reduce the temperature oscillation to less than 10 mK below a certain temperature TH without compromising cooling power. TH are 3.8 and 4.5 for filled He-gas pressures of 90 and 60 atm, respectively. By using this He-pot, GM cryocooler can be applied to such as precise physical property measurements and THz detection.

  14. The Natural History of Juvenile or Subacute GM2 Gangliosidosis: 21 New Cases and Literature Review of 134 Previously Reported

    PubMed Central

    Maegawa, Gustavo H. B.; Stockley, Tracy; Tropak, Michael; Banwell, Brenda; Blaser, Susan; Kok, Fernando; Giugliani, Roberto; Mahuran, Don; Clarke, Joe T. R.

    2010-01-01

    OBJECTIVE Juvenile GM2 gangliosidosis is a group of inherited neurodegenerative diseases caused by deficiency of lysosomal β-hexosaminidase resulting in GM2 ganglioside accumulation in brain. The purpose of this study was to delineate the natural history of the condition and identify genotype-phenotype correlations that might be helpful in predicting the course of the disease in individual patients. METHODS A cohort of 21 patients with juvenile GM2 gangliosidosis, 15 with the Tay-Sachs variant and 6 with the Sandhoff variant, was studied prospectively in 2 centers. Our experience was compared with previously published reports on 134 patients. Information about clinical features, β-hexosaminidase enzyme activity, and mutation analysis was collected. RESULTS In our cohort of patients, the mean (±SD) age of onset of symptoms was 5.3 ± 4.1 years, with a mean follow-up time of 8.4 years. The most common symptoms at onset were gait disturbances (66.7%), incoordination (52.4%), speech problems (28.6%), and developmental delay (28.6%). The age of onset of gait disturbances was 7.1 ± 5.6 years. The mean time for progression to becoming wheelchair-bound was 6.2 ± 5.5 years. The mean age of onset of speech problems was 7.0 ± 5.6 years, with a mean time of progression to anarthria of 5.6 ± 5.3 years. Muscle wasting (10.6 ± 7.4 years), proximal weakness (11.1 ± 7.7 years), and incontinence of sphincters (14.6 ± 9.7 years) appeared later in the course of the disease. Psychiatric disturbances and neuropathy were more prevalent in patients with the Sandhoff variant than in those with the Tay-Sachs variant. However, dysphagia, sphincter incontinence, and sleep problems occurred earlier in those with the Tay-Sachs variant. Cerebellar atrophy was the most common finding on brain MRI (52.9%). The median survival time among the studied and reviewed patients was 14.5 years. The genotype-phenotype correlation revealed that in patients with the Tay-Sachs variant, the presence

  15. Penetration of blood-brain barrier and antitumor activity and nerve repair in glioma by doxorubicin-loaded monosialoganglioside micelles system.

    PubMed

    Zou, Dan; Wang, Wei; Lei, Daoxi; Yin, Ying; Ren, Peng; Chen, Jinju; Yin, Tieying; Wang, Bochu; Wang, Guixue; Wang, Yazhou

    2017-01-01

    For the treatment of glioma and other central nervous system diseases, one of the biggest challenges is that most therapeutic drugs cannot be delivered to the brain tumor tissue due to the blood-brain barrier (BBB). The goal of this study was to construct a nanodelivery vehicle system with capabilities to overcome the BBB for central nervous system administration. Doxorubicin as a model drug encapsulated in ganglioside GM1 micelles was able to achieve up to 9.33% loading efficiency and 97.05% encapsulation efficiency by orthogonal experimental design. The in vitro study demonstrated a slow and sustainable drug release in physiological conditions. In the cellular uptake studies, mixed micelles could effectively transport into both human umbilical vein endothelial cells and C6 cells. Furthermore, biodistribution imaging of mice showed that the DiR/GM1 mixed micelles were accumulated sustainably and distributed centrally in the brain. Experiments on zebrafish confirmed that drug-loaded GM1 micelles can overcome the BBB and enter the brain. Among all the treatment groups, the median survival time of C6-bearing rats after administering DOX/GM1 micelles was significantly prolonged. In conclusion, the ganglioside nanomicelles developed in this work can not only penetrate BBB effectively but also repair nerves and kill tumor cells at the same time.

  16. GM organisms and the EU regulatory environment: allergenicity as a risk component.

    PubMed

    Davies, Howard V

    2005-11-01

    The European Food Safety Authority, following a request from the European Commission, has published a guidance document for the risk assessment of GM plants and derived food and feed to assist in the implementation of provisions of Regulation (EC) 1829/2003 of the European Parliament and Council on GM food and feed. This regulation has applied since 18 April 2004. In principle, hazard identification and characterisation of GM crops is conducted in four steps: characterisation of the parent crop and any hazards associated with it; characterisation of the transformation process and of the inserted recombinant DNA, including an assessment of the possible production of new fusion proteins or allergens; assessment of the introduced proteins (toxicity, allergenicity) and metabolites; identification of any other targetted and unexpected alterations in the GM crop, including changes in the plant metabolism resulting in compositional changes and assessment of their toxicological, allergenic or nutritional impact. In relation to allergenicity specifically, it is clear that this property of a given protein is not intrinsic and fully predictable but is a biological activity requiring an interaction with individuals with a predisposed genetic background. Allergenicity, therefore, depends on the genetic diversity and variability in atopic human subjects. Given this lack of complete predictability it is necessary to obtain, from several steps in the risk-assessment process, a cumulative body of evidence that minimises any uncertainty about the protein(s) in question.

  17. Chitosan solution enhances the immunoadjuvant properties of GM-CSF

    PubMed Central

    Zaharoff, David A.; Rogers, Connie J.; Hance, Kenneth W.; Schlom, Jeffrey; Greiner, John W.

    2008-01-01

    Sustained, local delivery of immunomodulatory cytokines is under investigation for its ability to enhance vaccine and anti-tumor responses both clinically and preclinically. This study evaluates the ability of chitosan, a biocompatible polysaccharide, to (1) control the dissemination of a cytokine, GM-CSF, and (2) enhance the immunoadjuvant properties of GM-CSF. While cytokines have previously been delivered in lipid-based adjuvants and other vehicles, these do not have the clinical safety profile or unique properties of chitosan. We found that chitosan solution maintained a measurable depot of recombinant GM-CSF (rGM-CSF) at a subcutaneous injection site for up to 9 days. In contrast, when delivered in a saline vehicle, rGM-CSF was undetectable in 12 to 24 hours. Furthermore, a single s.c. injection of 20μg rGM-CSF in chitosan solution (chitosan/rGM-CSF(20μg)) transiently expanded lymph nodes up to 4.6-fold and increased the number of MHC class II expressing cells and dendritic cells by 7.4-fold and 6.8-fold, respectively. These increases were significantly greater than those measured when rGM-CSF was administered in saline at the standard preclinical dose and schedule, i.e. 4 daily s.c. injections of 20μg. Furthermore, lymph node cells from mice injected with chitosan/rGM-CSF(20μg) induced greater allogeneic T cell proliferation, indicating enhanced antigen presenting capability, than lymph node cells from mice injected with rGM-CSF alone. Finally, in vaccination experiments, chitosan/rGM-CSF was superior to either chitosan or rGM-CSF alone in enhancing the induction of antigen-specific CD4+ proliferation, peptide-specific CD8+ pentamer staining and cytotoxic T cell lysis. Altogether, chitosan/rGM-CSF outperformed standard rGM-CSF administrations in dendritic cell recruitment, antigen presentation and vaccine enhancement. We conclude that chitosan solution is a promising delivery platform for the sustained, local delivery of rGM-CSF. PMID:18037196

  18. Dynamics and Size of Cross-Linking-Induced Lipid Nanodomains in Model Membranes

    PubMed Central

    Štefl, Martin; Šachl, Radek; Humpolíčková, Jana; Cebecauer, Marek; Macháň, Radek; Kolářová, Marie; Johansson, Lennart B.-Å.; Hof, Martin

    2012-01-01

    Changes of membrane organization upon cross-linking of its components trigger cell signaling response to various exogenous factors. Cross-linking of raft gangliosides GM1 with cholera toxin (CTxB) was shown to cause microscopic phase separation in model membranes, and the CTxB-GM1 complexes forming a minimal lipid raft unit are the subject of ongoing cell membrane research. Yet, those subdiffraction sized rafts have never been described in terms of size and dynamics. By means of two-color z-scan fluorescence correlation spectroscopy, we show that the nanosized domains are formed in model membranes at lower sphingomyelin (Sph) content than needed for the large-scale phase separation and that the CTxB-GM1 complexes are confined in the domains poorly stabilized with Sph. Förster resonance energy transfer together with Monte Carlo modeling of the donor decay response reveal the domain radius of ∼8 nm, which increases at higher Sph content. We observed two types of domains behaving differently, which suggests a dual role of the cross-linker: first, local transient condensation of the GM1 molecules compensating for a lack of Sph and second, coalescence of existing nanodomains ending in large-scale phase separation. PMID:22824274

  19. Tanzanian farmers' knowledge and attitudes to GM biotechnology and the potential use of GM crops to provide improved levels of food security. A Qualitative Study

    PubMed Central

    2010-01-01

    Background Genetically Modified (GM) crops have been championed as one possible method to improve food security and individual nutritional status in sub Saharan Africa. Understanding and acceptability of GM crop technology to farmers and consumers have not been assessed. We developed a qualitative research study involving farmers as both producers and consumers to gauge the understanding of GM crop technology, its acceptability, and identifying issues of concern. Methods Nineteen individual interviews (10 male and 9 female) and five mixed gender focus group discussions with local farmers were conducted in 3 regions in Tanzania. Analysis took place concurrently with data collection. Following initial interviews, subsequent questions were adjusted based on emerging themes. Results Understanding, awareness and knowledge of GM crop technology and terminology and its potential risks and benefits was very poor in all regions. Receptivity to the potential use of GM crops was, however, high. Respondents focused on the potential benefits of GM crops rather than any potential longer term health risks. A number of factors, most significantly field trial data, would influence farmers' decisions regarding the introduction of GM crop varieties into their farming practice. Understanding of the potential improved health provision possible by changes in agricultural practice and food-related decision making, and the health benefits of a diet containing essential vitamins, minerals and micronutrients is also poor in these communities. Conclusion This study forms a basis from which further research work can be undertaken. It is important to continue to assess opinions and attitudes of farmers and consumers in sub Saharan Africa towards potential use of GM technologies whilst highlighting the importance of the relationship between agriculture, health and development. This will allow people in the region to make accurate, informed decisions about whether they believe use of GM

  20. Experiences with a PDS 2020 GM

    NASA Technical Reports Server (NTRS)

    Teuber, D.

    1984-01-01

    Various perturbations which occurred during the operation of a PDS 2020 GM microdensitometer are reviewed. The effects of elevated temperature, vibration, transient electrical noise, and photometric perturbations are briefly discussed. Sample astronomical results from the PDS 2020 GM unit are presented.

  1. [The therapeutic effect of HSV1-hGM-CSF combined with doxorubicin on the mouse breast cancer model].

    PubMed

    Zhuang, X F; Zhang, S R; Liu, B L; Wu, J L; Li, X Q; Gu, H G; Shu, Y

    2018-03-23

    Objective: To evaluate the oncolytic effect of herpes simplex virus type 1 which carried recombined human granulocyte-macrophage colony-stimulating factor (HSV1-hGM-CSF) on the mouse breast cancer cell line 4T1 and compare the anticancer effects of HSV1-hGM-CSF, doxorubicin alone or combination on the breast cancer in mice. Methods: We investigated the cytotoxic effect on 4T1 cells in vitro, the cell growth, cell apoptosis and cell cycle of 4T1 cells treated with oncolytic HSV1-hGM-CSF at different MOIs (0, 0.5, 1 and 2) and doxorubicin at different concentrations (0, 2, 4 and 8 μg/ml). The effects of oncolytic HSV1-hGM-CSF and doxorubicin on the tumor growth, survival time and their side effects on the mouse breast cancer model were observed. Results: Both oncolytic HSV1-hGM-CSF and doxorubicin significantly inhibited the proliferation of 4T1 cells in vitro . Doxorubicin induced the G(2)/M phase arrest of 4T1 cells, while the cytotoxicity of oncolytic HSV1-hGM-CSF was no cell cycle-dependent.At day 16 after treatment with doxorubicin and HSV1-hGM-CSF, the tumor volume of 4T1 tumor bearing mice were (144.40±27.68)mm(3,) (216.80±57.18)mm(3,) (246.10±21.90)mm(3,) (327.50±44.24)mm(3,) (213.30±32.31)mm(3) and (495.80±75.87)mm(3) in the groups of doxorubicin combined with high dose HSV1-hGM-CSF, doxorubicin combined with low dose HSV1-hGM-CSF, doxorubicin alone, high dose HSV1-hGM-CSF alone, low dose HSV1-hGM-CSF alone and control, respectively.Compared with the control group, both doxorubicin and HSV1-hGM-CSF treatment exhibited significant reduction of primary tumor volume in vivo ( P <0.001). The median survival times were 48, 50, 40, 42, 43 and 37 days in the six groups mentioned above, respectively. The median survival period of doxorubicin alone, high dose HSV1-hGM-CSF alone and low dose HSV1-hGM-CSF alone were significantly longer than that of control ( P <0.05). Conclusion: Synergistic effect of sequential treatment with doxorubicin and oncolytic HSV1-hGM

  2. Granulocyte-macrophage colony stimulating factor (GM-CSF) enhances cumulus cell expansion in bovine oocytes

    PubMed Central

    2013-01-01

    Background The objectives of the study were to characterize the expression of the α- and β-subunits of granulocyte-macrophage colony stimulating factor (GM-CSF) receptor in bovine cumulus cells and oocytes and to determine the effect of exogenous GM-CSF on cumulus cells expansion, oocyte maturation, IGF-2 transcript expression and subsequent competence for embryonic development. Methods Cumulus-oocyte complexes (COC) were obtained by aspirating follicles 3- to 8-mm in diameter with an 18 G needle connected to a vacuum pump at −50 mmHg. Samples of cumulus cells and oocytes were used to detect GM- CSF receptor by immunofluorescence. A dose–response experiment was performed to estimate the effect of GM-CSF on cumulus cell expansion and nuclear/cytoplasmic maturation. Also, the effect of GM-CSF on IGF-2 expression was evaluated in oocytes and cumulus cells after in vitro maturation by Q-PCR. Finally, a batch of COC was randomly assigned to in vitro maturation media consisting of: 1) synthetic oviductal fluid (SOF, n = 212); 2) synthetic oviductal fluid supplemented with 100 ng/ml of GM-CSF (SOF + GM-CSF, n = 224) or 3) tissue culture medium (TCM 199, n = 216) and then subsequently in vitro fertilized and cultured for 9 days. Results Immunoreactivity for both α and β GM-CSF receptors was localized in the cytoplasm of both cumulus cells and oocytes. Oocytes in vitro matured either with 10 or 100 ng/ml of GM-CSF presented a higher (P < 0.05) cumulus cells expansion than that of the control group (0 ng/ml of GM-CSF). GM-CSF did not affect the proportion of oocytes in metaphase II, cortical granules dispersion and IGF-2 expression. COC exposed to 100 ng/ml of GM-CSF during maturation did not display significant differences in terms of embryo cleavage rate (50.4% vs. 57.5%), blastocyst development at day 7 (31.9% vs. 28.7%) and at day 9 (17.4% vs. 17.9%) compared to untreated control (SOF alone, P = 0.2). Conclusions GM-CSF enhanced cumulus

  3. Case reports of juvenile GM1 gangliosidosisis type II caused by mutation in GLB1 gene.

    PubMed

    Karimzadeh, Parvaneh; Naderi, Samaneh; Modarresi, Farzaneh; Dastsooz, Hassan; Nemati, Hamid; Farokhashtiani, Tayebeh; Shamsian, Bibi Shahin; Inaloo, Soroor; Faghihi, Mohammad Ali

    2017-07-17

    Type II or juvenile GM1-gangliosidosis is an autosomal recessive lysosomal storage disorder, which is clinically distinct from infantile form of the disease by the lack of characteristic cherry-red spot and hepatosplenomegaly. The disease is characterized by slowly progressive neurodegeneration and mild skeletal changes. Due to the later age of onset and uncharacteristic presentation, diagnosis is frequently puzzled with other ataxic and purely neurological disorders. Up to now, 3-4 types of GM1-gangliosidosis have been reported and among them type I is the most common phenotype with the age of onset around 6 months. Various forms of GM1-gangliosidosis are caused by GLB1 gene mutations but severity of the disease and age of onset are directly related to the position and the nature of deleterious mutations. However, due to its unique genetic cause and overlapping clinical features, some researchers believe that GM1 gangliosidosis represents an overlapped disease spectrum instead of four distinct types. Here, we report a less frequent type of autosomal recessive GM1 gangliosidosis with perplexing clinical presentation in three families in the southwest part of Iran, who are unrelated but all from "Lurs" ethnic background. To identify disease-causing mutations, Whole Exome Sequencing (WES) utilizing next generation sequencing was performed. Four patients from three families were investigated with the age of onset around 3 years old. Clinical presentations were ataxia, gate disturbances and dystonia leading to wheelchair-dependent disability, regression of intellectual abilities, and general developmental regression. They all were born in consanguineous families with no previous documented similar disease in their parents. A homozygote missense mutation in GLB1 gene (c. 601 G > A, p.R201C) was found in all patients. Using Sanger sequencing this identified mutation was confirmed in the proband, their parents, grandparents, and extended family members, confirming

  4. 5 CFR 531.243 - Promotion of a GM employee.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 5 Administrative Personnel 1 2010-01-01 2010-01-01 false Promotion of a GM employee. 531.243... Promotion of a GM employee. (a) Upon promotion, an employee's status as a GM employee ends, as provided in § 531.241(b). (b) When an employee loses status as a GM employee because of a temporary promotion and is...

  5. Science, politics, and the GM debate in Europe.

    PubMed

    Tencalla, Francesca

    2006-02-01

    Europe today stands at a crossroad, facing challenges but also opportunities. In its intent to make Europe a leading technology-based economy by 2010, the European Commission has identified biotechnology and genomics as fields for future growth, crucial for supporting the agricultural and food processing industry. Since first commercialization in 1996, GM crop areas have grown at double-digit rates, making this one of the most rapidly adopted technologies in agriculture. However, in contrast to other world areas and despite European Commission support, Europe has found itself 'bogged-down' in a polemic between opponents and supporters of plant biotechnology. As a result, planted areas have remained small. This stalemate is due to a lack of political leadership, especially at the Member State level, all the more surprising in light of European early development and competitive advantage with crop biotechnology. This situation proves once again that, for cutting-edge innovations, a solid science base alone is not sufficient. Acceptance or rejection of new technologies depends on interlinked political, economic, and societal factors that create a favorable or unfavorable situation at a given time. This article will look at GM crops in Europe and the role science and politics have played in the introduction of crop biotechnology.

  6. Stimulation of a Ca sup 2+ -dependent protein kinase by G sub M1 ganglioside in nerve growth factor-treated PC12 cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Hilbush, B.S.; Levine, J.M.

    1991-07-01

    The authors have investigated the ability of exogenous gangliosides to modulate nerve growth factor (NGF) signal transduction in PC12 cells. The effects of exogenous ganglioside G{sub M1} on multiple protein kinase activities were assayed by analyzing site-specific serine phosphorylation of tyrosine hydroxylase (TyrOHase) by two-dimensional phosphopeptide mapping. In the presence of NGF, exogenous G{sub M1} increased {sup 32}P incorporation into TyrOHase phosphopeptide T2, a Ca{sup 2+}/calmodulin-dependent protein kinase substrate whose phosphorylation is not normally affected by NGF treatment. In the absence of NGF, G{sub M1} treatment had no significant effects on TyrOHase phosphorylation. The removal of extracellular Ca{sup 2+} ormore » blockade of dihydropyridine-sensitive Ca{sup 2+} channels prevented the G{sub M1}-induced increases in {sup 32}P incorporation into phosphopeptide T2. Exogenous G{sub M1} also potentiated K{sup +} depolarization-induced increases in the phosphorylation of TyrOHase. These results suggest that the stimulatory effects of exogenous G{sub M1} ganglioside on NGF actions may be due to its ability to potentiate a Ca{sup 2+}-dependent signaling pathway.« less

  7. GM-CSF produced by non-hematopoietic cells is required for early epithelial cell proliferation and repair of injured colonic mucosa1,2

    PubMed Central

    Egea, Laia; McAllister, Christopher S.; Lakhdari, Omar; Minev, Ivelina; Shenouda, Steve; Kagnoff, Martin F.

    2012-01-01

    GM-CSF is a growth factor that promotes the survival and activation of macrophages and granulocytes, and dendritic cell (DC) differentiation and survival in vitro. The mechanism by which exogenous GM-CSF ameliorates the severity of Crohn’s disease in humans and colitis in murine models has been considered mainly to reflect its activity on myeloid cells. We used GM-CSF deficient (GM-CSF−/−) mice to probe the functional role of endogenous host-produced GM-CSF in a colitis model induced after injury to the colon epithelium. Dextran sodium sulfate (DSS) at doses that resulted in little epithelial damage and mucosal ulceration in wild type (WT) mice resulted in marked colon ulceration and delayed ulcer healing in GM-CSF−/− mice. Colon crypt epithelial cell proliferation in vivo was significantly decreased in GM-CSF−/− mice at early times after DSS injury. This was paralleled by decreased expression of crypt epithelial cell genes involved in cell cycle, proliferation, and wound healing. Decreased crypt cell proliferation and delayed ulcer healing in GM-CSF−/− mice were rescued by exogenous GM-CSF, indicating the lack of a developmental abnormality in the epithelial cell proliferative response in those mice. Non-hematopoietic cells and not myeloid cells produced the GM-CSF important for colon epithelial proliferation after DSS-induced injury as revealed by bone marrow chimera and DC depletion experiments, with colon epithelial cells being the cellular source of GM-CSF. Endogenous epithelial cell produced GM-CSF has a novel non-redundant role in facilitating epithelial cell proliferation and ulcer healing in response to injury of the colon crypt epithelium. PMID:23325885

  8. The regulation of ER export and Golgi retention of ST3Gal5 (GM3/GM4 synthase) and B4GalNAcT1 (GM2/GD2/GA2 synthase) by arginine/lysine-based motif adjacent to the transmembrane domain.

    PubMed

    Uemura, Satoshi; Shishido, Fumi; Kashimura, Madoka; Inokuchi, Jin-ichi

    2015-12-01

    In the Golgi maturation model, the Golgi cisternae dynamically mature along a secretory pathway. In this dynamic process, glycosyltransferases are transported from the endoplasmic reticulum (ER) to the Golgi apparatus where they remain and function. The precise mechanism behind this maturation process remains unclear. We investigated two glycosyltransferases, ST3Gal5 (ST3G5) and B4GalNAcT1 (B4GN1), involved in ganglioside synthesis and examined their signal sequences for ER export and Golgi retention. Reports have suggested that the [R/K](X)[R/K] motif functions as an ER exporting signal; however, this signal sequence is insufficient in stably expressed, full-length ST3G5. Through further analysis, we have clarified that the (2)R(3)R(X)(5) (9)K(X)(3) (13)K sequence in ST3G5 is essential for ER export. We have named the sequence the R/K-based motif. On the other hand, for ER export of B4GN1, the homodimer formation in addition to the R/K-based motif is required for ER export suggesting the importance of unidentified lumenal side interaction. We found that ST3G5 R2A/R3A and K9A/K13A mutants localized not only in Golgi apparatus but also in endosomes. Furthermore, the amounts of mature type asparagine-linked (N)-glycans in ST3G5 R2A/R3A and K9A/K13A mutants were decreased compared with those in wild-type proteins, and the stability of the mutants was lower. These results suggest that the R/K-based motif is necessary for the Golgi retention of ST3G5 and that the retention is involved in the maturation of N-glycans and in stability. Thus, several basic amino acids located on the cytoplasmic tail of ST3G5 play important roles in both ER export and Golgi retention. © The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  9. Expression of cholera toxin B subunit in transgenic tomato plants.

    PubMed

    Jani, Dewal; Meena, Laxman Singh; Rizwan-ul-Haq, Quazi Mohammad; Singh, Yogendra; Sharma, Arun K; Tyagi, Akhilesh K

    2002-10-01

    Cholera toxin, secreted by Vibrio cholerae, consists of A and B subunits. The latter binds to G(M1)-ganglioside receptors as a pentamer (approximately 55 kDa). Tomato plants were transformed with the gene encoding cholera toxin B subunit (ctxB) along with an endoplasmic reticulum retention signal (SEKDEL) under the control of the CaMV 35S promoter via Agrobacterium-mediated transformation. PCR and Southern analysis confirmed the presence of the ctxB gene in transformed tomato plants. Northern analysis showed the presence of the ctxB-specific transcript. Immunoblot assays of the plant-derived protein extract showed the presence of cholera toxin subunit B (CTB) with mobility similar to purified CTB from V. cholerae. Both tomato leaves and fruits expressed CTB at levels up to 0.02 and 0.04% of total soluble protein, respectively. The G(M1)-ELISA showed that the plant-derived CTB bound specifically to G(M1)-ganglioside receptor, suggesting that it retained its native pentameric form. This study forms a basis for exploring the utility of CTB to develop tomato-based edible vaccines against cholera.

  10. Ligand Extraction Properties of the GM2 Activator Protein and Its Interactions with Lipid Vesicles

    PubMed Central

    Ran, Yong; Fanucci, Gail E.

    2009-01-01

    Abstract The GM2 activator protein (GM2AP) is an accessory protein required for the enzymatic conversion of GM2 to GM3 by hydrolases in the lysosomal compartments of cells. Here, GM2AP interactions with lipid vesicles are investigated by sucrose-loaded vesicle sedimentation and gel filtration assays, and the effects of pH and lipid composition on membrane binding and lipid extraction are characterized. The sedimentation experiments allow for facile quantification of the percentage of protein in solution and on the bilayer surface, with detailed analysis of the protein:lipid complex that remains in solution. Optimum binding and ligand extraction is found for pH 4.8 where <15% of the protein remains surface associated regardless of the lipid composition. In addition to extracting GM2, we find that GM2AP readily extracts dansyl-headgroup-labeled lipids as well as other phospholipids from vesicles. The ability of GM2AP to extract dansyl-DHPE from vesicles is altered by pH and the specific ligand GM2. Although the unique endosomal lipid, bis(monoacylglycero)phosphate, is not required for ligand extraction, it does enhance the extraction efficiency of GM2 when cholesterol is present in the vesicles. PMID:19580763

  11. Ligand extraction properties of the GM2 activator protein and its interactions with lipid vesicles.

    PubMed

    Ran, Yong; Fanucci, Gail E

    2009-07-08

    The GM2 activator protein (GM2AP) is an accessory protein required for the enzymatic conversion of GM2 to GM3 by hydrolases in the lysosomal compartments of cells. Here, GM2AP interactions with lipid vesicles are investigated by sucrose-loaded vesicle sedimentation and gel filtration assays, and the effects of pH and lipid composition on membrane binding and lipid extraction are characterized. The sedimentation experiments allow for facile quantification of the percentage of protein in solution and on the bilayer surface, with detailed analysis of the protein:lipid complex that remains in solution. Optimum binding and ligand extraction is found for pH 4.8 where <15% of the protein remains surface associated regardless of the lipid composition. In addition to extracting GM2, we find that GM2AP readily extracts dansyl-headgroup-labeled lipids as well as other phospholipids from vesicles. The ability of GM2AP to extract dansyl-DHPE from vesicles is altered by pH and the specific ligand GM2. Although the unique endosomal lipid, bis(monoacylglycero)phosphate, is not required for ligand extraction, it does enhance the extraction efficiency of GM2 when cholesterol is present in the vesicles.

  12. Low doses of GM-CSF (molgramostim) and G-CSF (filgrastim) after cyclophosphamide (4 g/m2) enhance the peripheral blood progenitor cell harvest: results of two randomized studies including 120 patients

    PubMed Central

    Quittet, Philippe; Ceballos, Patrice; Lopez, Ernesto; Lu, Zhao-Yang; Latry, Pascal; Becht, Catherine; Legouffe, Eric; Fegueux, Nathalie; Exbrayat, Carole; Pouessel, Damien; Rouillé, Valérie; Daures, Jean-Pierre; Klein, Bernard; Rossi, Jean-François

    2006-01-01

    The use of a combination of G-CSF and GM-CSF to G-CSF alone, after cyclophosphamide (4g/m2) was compared in 2 randomized phase III studies, including 120 patients. In study A, 60 patients received 5 × 2 μg/kg/day of G-CSF and GM-CSF compared to 5 μg/kg/day of G-CSF. In study B, 60 patients received 2.5 × 2 μg/kg/day G-CSF and GM-CSF compared to G-CSF alone (5 μg/kg/day). With the aim to collect at least 5 × 106/kg CD34 cells in a maximum of 3 large volume leukapherisis (LK), 123 LK were performed in study A, showing significant higher number of patients reaching 10 × 106/kg CD34 cells (21/29 in G+GM-CSF arm vs 11/27 in G-CSF arm, P= .00006). In study B, 109 LK were performed, with similar results (10/27 vs 15/26, P= .003). In both the study, the total harvest of CD34 cells/kg was 2-fold higher in G-CSF plus GM-CSF group (18.3 × 106 in study A and 15.85 × 106 in study B) than in G-CSF group (9 × 106 in study A and 8.1 × 106 in study B), a difference particularly seen in multiple myeloma, with no significant difference in terms of mobilized myeloma cells between G-CSF and GM-CSF groups. PMID:16883311

  13. [Effect of non-genetically modified (non-GM) soy varieties on the measured value of GM soy by a quantitative PCR method].

    PubMed

    Watanabe, Takahiro; Sekino, Ayako; Shiramasa, Yuko; Matsuda, Rieko; Maitani, Tamio

    2008-08-01

    It is very important to examine the effect of non-genetically modified (non-GM) soy varieties, which constitute the matrix of the testing sample used to quantify GM soy (RRS), on the measured value of RRS by quantitative PCR methods. Therefore, we quantified the amount of RRS in powder-mixed samples containing 1 or 5% RRS prepared by using 10 different varieties of non-GM soy as the matrix. The results revealed that the measured values were not in agreement with the powder-mixing levels and that the extent of the difference depended on the variety of non-GM soy used as the matrix. The yields of DNA extracted differed among the soy varieties. On the other hand, analysis of DNA-mixed samples, that were prepared with the DNAs extracted from RRS and non-GM soy varieties, showed that the measured values of RRS were in agreement with the DNA-mixing levels. These results strongly suggest that the proportions of DNA derived from RRS and non-GM soy were not consistent with the powder-mixing ratio in the case of some non-GM soy varieties used as a matrix, resulting in the discrepancy between the measured values and the powder-mixing levels.

  14. Lipid-Mediated Targeting with Membrane Wrapped Nanoparticles in the Presence of Corona Formation

    PubMed Central

    Xu, Fangda; Reiser, Michael; Yu, Xinwei; Gummuluru, Suryaram; Wetzler, Lee; Reinhard, Björn M.

    2016-01-01

    Membrane wrapped nanoparticles represent a versatile platform for utilizing specific lipid-receptor interactions, such as siallyllactose-mediated binding of the ganglioside GM3 to Siglec1 (CD169), for targeting purposes. The membrane wrap around the nanoparticles does not only serve as a matrix to incorporate GM3 as targeting moiety for antigen presenting cells but also offers unique opportunities for constructing a biomimetic surface from lipids with potentially protein repellent properties. We characterize non-specific protein adsorption (corona formation) to membrane wrapped nanoparticles with core diameters of approx. 35 nm and 80 nm and its effect on the GM3-mediated targeting efficacy as function of surface charge through combined in vitro and in vivo studies. The stability and fate of the membrane wrap around the nanoparticles in a simulated biological fluid and after uptake in CD169 expressing antigen presenting cells is experimentally tested. Finally, we demonstrate in hock immunization studies in mice that GM3 decorated membrane wrapped nanoparticles achieve a selective enrichment in the peripheral regions of popliteal lymph nodes that contain high concentrations of CD169 expressing antigen presenting cells. PMID:26720275

  15. Genetics Home Reference: GM2-gangliosidosis, AB variant

    MedlinePlus

    ... link) National Institute of Neurological Disorders and Stroke: Lipid Storage Diseases Fact Sheet Educational Resources (3 links) ... Chen B, Rigat B, Curry C, Mahuran DJ. Structure of the GM2A gene: identification of an exon ...

  16. A Conformational Change of C Fragment of Tetanus Neurotoxin Reduces Its Ganglioside-Binding Activity but Does Not Destroy Its Immunogenicity ▿

    PubMed Central

    Yu, Rui; Yi, Shaoqiong; Yu, Changming; Fang, Ting; Liu, Shuling; Yu, Ting; Song, Xiaohong; Fu, Ling; Hou, Lihua; Chen, Wei

    2011-01-01

    The C fragment of tetanus neurotoxin (TeNT-Hc) with different conformations was observed due to the four cysteine residues within it which could form different intramolecular disulfide bonds. In this study, we prepared and compared three types of monomeric TeNT-Hc with different conformational components: free sulfhydryls (50 kDa), bound sulfhydryls (44 kDa), and a mixture of the two conformational proteins (half 50 kDa and half 44 kDa). TeNT-Hc with bound sulfhydryls reduced its binding activity to ganglioside GT1b and neuronal PC-12 cells compared to what was seen for TeNT-Hc with free sulfhydryls. However, there was no significant difference among their immunogenicities in mice, including induction of antitetanus toxoid IgG titers, antibody types, and protective capacities against tetanus neurotoxin challenge. Our results showed that the conformational changes of TeNT-Hc resulting from disulfide bond formation reduced its ganglioside-binding activity but did not destroy its immunogenicity, and the protein still retained continuous B cell and T cell epitopes; that is, the presence of the ganglioside-binding site within TeNT-Hc may be not essential for the induction of a fully protective antitetanus response. TeNT-Hc with bound sulfhydryls may be developed into an ideal human vaccine with a lower potential for side effects. PMID:21813664

  17. Comparative Analysis of Two Industries for Validating Green Manufacturing (GM) Framework: An Indian Scenario

    NASA Astrophysics Data System (ADS)

    Rehman, Minhaj Ahemad Abdul; Shrivastava, Rakesh Lakshmikumar; Shrivastava, Rashmi Rakesh

    2017-04-01

    Green Manufacturing (GM) deals with manufacturing practices that reduces or eliminates the adverse environmental impact during any of its phases. It emphasizes the use of processes that do not contaminate the environment or hurt consumers, employees, or other stakeholders. This paper presents the comparative analysis of two Indian industries representing different sectors for validating GM framework. It also highlights the road map of the companies for achieving performance improvement through GM implementation and its impact on organisational performance. The case studies helps in evaluating the companies GM implementation and overall business performance. For this, a developed diagnostic instrument in the form of questionnaire was administered amongst employees in the companies respectively and their responses were analysed. In order to have a better understanding of the impact of GM implementation, the information about overall business performance was obtained over the last 3 years. The diagnostic instrument developed here may be used by manufacturing organisations to prioritise their management efforts to assess and implement GM.

  18. GM2-activator protein: a new biomarker for lung cancer.

    PubMed

    Potprommanee, Laddawan; Ma, Haou-Tzong; Shank, Lalida; Juan, Yi-Hsiu; Liao, Wei-Yu; Chen, Shui-Tein; Yu, Chong-Jen

    2015-01-01

    Effective biomarkers for early diagnosis of lung cancer are needed. A recent study demonstrated that urinary GM2-activator protein (GM2AP) level was increased in lung cancer patients. This study aims to validate the potential application of GM2AP as a biomarker for diagnosis of lung cancer. Serum and urine samples were obtained from 189 participants (133 patients for treatment naive lung cancer, 26 healthy volunteers for urine, and 30 healthy volunteers for serum). GM2AP level was detected by Western blotting and quantified using enzyme-linked immunosorbent assay (ELISA). The GM2AP expression in tumors and nontumor parts of lung tissues from 143 nonsmall cell lung cancers was detected by immunohistochemical stains. There was an 8.11 ± 1.36 folds increase in urine and a 5.41 ± 0.73 folds increase in serum level of GM2AP in lung cancer patients compared with healthy volunteers (p < 0.0001), achieving a 0.89 AUC value in urine and 0.90 AUC value in serum for the receiver-operating characteristic curves. Both serum and urine levels of GM2AP correlated significantly with pathology stages (urine, p = 0.009; serum, p < 0.0001). Using immunohistochemical, positive expression of GM2AP was found at 83.9% of nonsmall cell lung cancers patients and none in normal tissue. The GM2AP expression was significantly correlated with pathology stage (p = 0.0001). Patients with higher GM2AP expression had shorter overall survival (p = 0.045) and disease-free survival (p = 0.049) than lower GM2AP expression. Moreover, the multivariate analysis suggested GM2AP as an independent predictors of disease-free survival and overall survival. Our study demonstrates that GM2AP might serve as potential diagnostic and prognostic biomarkers in patients with lung cancer.

  19. Paediatric Crohn disease patients with stricturing behaviour exhibit ileal granulocyte–macrophage colony-stimulating factor (GM-CSF) autoantibody production and reduced neutrophil bacterial killing and GM-CSF bioactivity

    PubMed Central

    Jurickova, I; Collins, M H; Chalk, C; Seese, A; Bezold, R; Lake, K; Allmen, D; Frischer, J S; Falcone, R A; Trapnell, B C; Denson, L A

    2013-01-01

    Granulocyte–macrophage colony-stimulating factor (GM-CSF) autoantibodies are associated with stricturing behaviour in Crohn disease (CD). We hypothesized that CD ileal lamina propria mononuclear cells (LPMC) would produce GM-CSF autoantibodies and peripheral blood (PB) samples would contain GM-CSF neutralizing capacity (NC). Paediatric CD and control PBMC and ileal biopsies or LPMC were isolated and cultured and GM-CSF, immunoglobulin (Ig)G and GM-CSF autoantibodies production were measured by enzyme-linked immunosorbent assay (ELISA). Basal and GM-CSF-primed neutrophil bacterial killing and signal transducer and activator of transcription 5 (STAT5) tyrosine phosphorylation (pSTAT5) were measured by flow cytometry. GM-CSF autoantibodies were enriched within total IgG for LPMC isolated from CD ileal strictures and proximal margins compared to control ileum. Neutrophil bacterial killing was reduced in CD patients compared to controls. Within CD, neutrophil GM-CSF-dependent STAT5 activation and bacterial killing were reduced as GM-CSF autoantibodies increased. GM-CSF stimulation of pSTAT5 did not vary between controls and CD patients in washed PB granulocytes in which serum was removed. However, GM-CSF stimulation of pSTAT5 was reduced in whole PB samples from CD patients. These data were used to calculate the GM-CSF NC. CD patients with GM-CSF NC greater than 25% exhibited a fourfold higher rate of stricturing behaviour and surgery. The likelihood ratio (95% confidence interval) for stricturing behaviour for patients with elevation in both GM-CSF autoantibodies and GM-CSF NC was equal to 5 (2, 11). GM-CSF autoantibodies are produced by LPMC isolated from CD ileal resection specimens and are associated with reduced neutrophil bacterial killing. CD peripheral blood contains GM-CSF NC, which is associated with increased rates of stricturing behaviour. PMID:23600834

  20. [Construction of a new oncolytic virus oHSV2hGM-CSF and its anti-tumor effects].

    PubMed

    Shi, Gui-Lan; Zhuang, Xiu-Fen; Han, Xiang-Ping; Li, Jie; Zhang, Yu; Zhang, Shu-Ren; Liu, Bin-Lei

    2012-02-01

    The aim of this study was to construct a new oncolytic virus oHSV2hGM-CSF and evaluate its oncolytic activity in vitro and in vivo in parallel with oHSV1hGM-CSF. oHSV2hGM-CSF was a replication-competent, attenuated HSV2 based on the HG52 virus (an HSV2 strain). It was engineered to be specific for cancer by deletion of the viral genes ICP34.5 and ICP47 and insertion of the gene encoding hGM-CSF. To measure the in vitro killing effect of the virus, 15 human tumor cell lines (HeLa, Eca-109, PG, HepG2, SK/FU, CNE-2Z, PC-3, SK-OV3, A-549, 786-0, MCF-7, Hep-2, HT-29, SK-Mel-28, U87-MG) and mouse melanoma (B16R) cell line were seeded into 24-well plates and infected with viruses at MOI = 1 (multiplicity of infection, MOI), or left uninfected. The cells were harvested 24 and 48 hours post infection, and observed under the microscope. For animal studies, the oncolytic viruses were administered intratumorally (at 3-day interval) at a dose of 2.3 x 10(6) PFU (plaque forming unit, PFU) for three times when the tumor volume reached 7-8 mm3. The tumor volume was measured at 3-day intervals and animal survival was recorded. Both oHSV2hCM-CSFand oHSV1hGM-CSF induced widespread cytopathic effects at 24 h after infection. OHSV2hGM-CSF, by contrast, produced more plaques with a syncytial phenotype than oHSV1hGM-CSF. In the in vitro killing experiments for the cell lines HeLa, HepG2, SK-Mel-28, B16R and U87-MG, oHSV2hGM-CSF eradicated significantly more cells than oHSV1hGM-CSF under the same conditions. For the mouse experiments, it was observed that oHSV2hGM-CSF significantly inhibited the tumor growth. At 15 days after B16R tumor cells inoculation, the tumor volumes of the PBS, oHSV1hGCM-CSF and oHSV2hGM-CSF groups were (374.7 +/- 128.24) mm3, (128.23 +/- 45.32) mm3 (P < 0.05, vs. PBS group) or (10.06 +/- 5.1) mm3 (P < 0.01, vs. PBS group), respectively (mean +/- error). The long term therapeutic effect of oHSV2hGM-CSF on the B16R animal model was evaluated by recording animal

  1. Gangliosides inhibit bee venom melittin cytotoxicity but not phospholipase A{sub 2}-induced degranulation in mast cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Nishikawa, Hirofumi; Kitani, Seiichi, E-mail: drkitani@kaiyodai.ac.jp

    Sting accident by honeybee causes severe pain, inflammation and allergic reaction through IgE-mediated anaphylaxis. In addition to this hypersensitivity, an anaphylactoid reaction occurs by toxic effects even in a non-allergic person via cytolysis followed by similar clinical manifestations. Auto-injectable epinephrine might be effective for bee stings, but cannot inhibit mast cell lysis and degranulation by venom toxins. We used connective tissue type canine mast cell line (CM-MC) for finding an effective measure that might inhibit bee venom toxicity. We evaluated degranulation and cytotoxicity by measurement of {beta}-hexosaminidase release and MTT assay. Melittin and crude bee venom induced the degranulation andmore » cytotoxicity, which were strongly inhibited by mono-sialoganglioside (G{sub M1}), di-sialoganglioside (G{sub D1a}) and tri-sialoganglioside (G{sub T1b}). In contrast, honeybee venom-derived phospholipase A{sub 2} induced the net degranulation directly without cytotoxicity, which was not inhibited by G{sub M1}, G{sub D1a} and G{sub T1b}. For analysis of distribution of G{alpha}{sub q} and G{alpha}{sub i} protein by western blotting, lipid rafts were isolated by using discontinuous sucrose gradient centrifuge. Melittin disrupted the localization of G{alpha}{sub q} and G{alpha}{sub i} at lipid raft, but gangliosides stabilized the rafts. As a result from this cell-based study, bee venom-induced anaphylactoid reaction can be explained with melittin cytotoxicity and phospholipase A{sub 2}-induced degranulation. Taken together, gangliosides inhibit the effect of melittin such as degranulation, cytotoxicity and lipid raft disruption but not phospholipase A{sub 2}-induced degranulation in mast cells. Our study shows a potential of gangliosides as a therapeutic tool for anaphylactoid reaction by honeybee sting.« less

  2. Pivotal Roles of GM-CSF in Autoimmunity and Inflammation

    PubMed Central

    Shiomi, Aoi; Usui, Takashi

    2015-01-01

    Granulocyte macrophage-colony stimulating factor (GM-CSF) is a hematopoietic growth factor, which stimulates the proliferation of granulocytes and macrophages from bone marrow precursor cells. In autoimmune and inflammatory diseases, Th17 cells have been considered as strong inducers of tissue inflammation. However, recent evidence indicates that GM-CSF has prominent proinflammatory functions and that this growth factor (not IL-17) is critical for the pathogenicity of CD4+ T cells. Therefore, the mechanism of GM-CSF-producing CD4+ T cell differentiation and the role of GM-CSF in the development of autoimmune and inflammatory diseases are gaining increasing attention. This review summarizes the latest knowledge of GM-CSF and its relationship with autoimmune and inflammatory diseases. The potential therapies targeting GM-CSF as well as their possible side effects have also been addressed in this review. PMID:25838639

  3. Fucosylation and protein glycosylation create functional receptors for cholera toxin

    PubMed Central

    Wands, Amberlyn M; Fujita, Akiko; McCombs, Janet E; Cervin, Jakob; Dedic, Benjamin; Rodriguez, Andrea C; Nischan, Nicole; Bond, Michelle R; Mettlen, Marcel; Trudgian, David C; Lemoff, Andrew; Quiding-Järbrink, Marianne; Gustavsson, Bengt; Steentoft, Catharina; Clausen, Henrik; Mirzaei, Hamid; Teneberg, Susann; Yrlid, Ulf; Kohler, Jennifer J

    2015-01-01

    Cholera toxin (CT) enters and intoxicates host cells after binding cell surface receptors using its B subunit (CTB). The ganglioside (glycolipid) GM1 is thought to be the sole CT receptor; however, the mechanism by which CTB binding to GM1 mediates internalization of CT remains enigmatic. Here we report that CTB binds cell surface glycoproteins. Relative contributions of gangliosides and glycoproteins to CTB binding depend on cell type, and CTB binds primarily to glycoproteins in colonic epithelial cell lines. Using a metabolically incorporated photocrosslinking sugar, we identified one CTB-binding glycoprotein and demonstrated that the glycan portion of the molecule, not the protein, provides the CTB interaction motif. We further show that fucosylated structures promote CTB entry into a colonic epithelial cell line and subsequent host cell intoxication. CTB-binding fucosylated glycoproteins are present in normal human intestinal epithelia and could play a role in cholera. DOI: http://dx.doi.org/10.7554/eLife.09545.001 PMID:26512888

  4. The use of antioxidants to prevent glutamate-induced derangement of calcium ion metabolism in rat cerebral cortex synaptosomes.

    PubMed

    Avrova, N F; Shestak, K I; Zakharova, I O; Sokolova, T V; Tyurina, Y Y; Tyurin, V A

    2000-01-01

    Glutamate is shown to induce increases in intracellular Ca2+ concentrations ([Ca2+]i), increases in 45Ca2+ influx, decreases in the activity of Na+,K+-ATPase activity, and activation of the Na+/Ca2+ exchanger in rat cerebral cortex synaptosomes. NMDA receptor antagonists virtually prevented these effects. Preincubation of synaptosomes with alpha-tocopherol, superoxide dismutase, and ganglioside GM1 normalized [Ca2+]i, 45Ca2+ influx, and Na+,K+-ATPase activity in rat cerebral cortex synaptosomes exposed to glutamate. Glutamate and GM1 activated the Na+/K+ exchanger, and their effects were additive. Calcium ions entering cerebral cortex nerve cells via NMDA receptors during exposure to high glutamate concentrations appeared to be only the trigger for the processes activating free-radical reactions. Activation of these reactions led to increases in Ca2+ influx into cells, decreases in Na+,K+-ATPase activity, and significant increases in [Ca2+]i, though this could be prevented by antioxidants and gangliosides.

  5. Guillain Barré Syndrome is induced in Non-Obese Diabetic (NOD) mice following Campylobacter jejuni infection and is exacerbated by antibiotics.

    PubMed

    St Charles, J L; Bell, J A; Gadsden, B J; Malik, A; Cooke, H; Van de Grift, L K; Kim, H Y; Smith, E J; Mansfield, L S

    2017-02-01

    Campylobacter jejuni is a leading cause of bacterial gastroenteritis linked to several serious autoimmune sequelae such as the peripheral neuropathies Guillain Barré syndrome (GBS) and Miller Fisher syndrome (MFS). We hypothesized that GBS and MFS can result in NOD wild type (WT) mice or their congenic interleukin (IL)-10 or B7-2 knockouts secondary to C. jejuni infection. Mice were gavaged orally with C. jejuni strains HB93-13 and 260.94 from patients with GBS or CF93-6 from a patient with MFS and assessed for clinical neurological signs and phenotypes, anti-ganglioside antibodies, and cellular infiltrates and lesions in gut and peripheral nerve tissues. Significant increases in autoantibodies against single gangliosides (GM1, GQ1b, GD1a) occurred in infected NOD mice of all genotypes, although the isotypes varied (NOD WT had IgG1, IgG3; NOD B7-2 -/- had IgG3; NOD IL-10 -/- had IgG1, IgG3, IgG2a). Infected NOD WT and NOD IL-10 -/- mice also produced anti-ganglioside antibodies of the IgG1 isotype directed against a mixture of GM1/GQ1b gangliosides. Phenotypic tests showed significant differences between treatment groups of all mouse genotypes. Peripheral nerve lesions with macrophage infiltrates were significantly increased in infected mice of NOD WT and IL-10 -/- genotypes compared to sham-inoculated controls, while lesions with T cell infiltrates were significantly increased in infected mice of the NOD B7-2 -/- genotype compared to sham-inoculated controls. In both infected and sham inoculated NOD IL-10 -/- mice, antibiotic treatment exacerbated neurological signs, lesions and the amount and number of different isotypes of antiganglioside autoantibodies produced. Thus, inducible mouse models of post-C. jejuni GBS are feasible and can be characterized based on evaluation of three factors-onset of GBS clinical signs/phenotypes, anti-ganglioside autoantibodies and nerve lesions. Based on these factors we characterized 1) NOD B-7 -/- mice as an acute

  6. A comparative study of human IgE binding to proteins of a genetically modified (GM) soybean and six non-GM soybeans grown in multiple locations.

    PubMed

    Lu, Mei; Jin, Yuan; Ballmer-Weber, Barbara; Goodman, Richard E

    2018-02-01

    Prior to commercialization, genetically modified (GM) crops are evaluated to determine the allergenicity of the newly expressed protein. Some regulators require an evaluation of endogenous allergens in commonly allergenic crops including soybean to determine if genetic transformation increased endogenous allergen concentrations, even asking for IgE testing using sera from individual sensitized subjects. Little is known about the variability of the expression of endogenous allergens among non-GM varieties or under different environmental conditions. We tested IgE binding to endogenous allergenic proteins in an experimental non-commercial GM line, a non-GM near-isoline control, and five non-GM commercial soybean lines replicated at three geographically separated locations. One-dimensional (1D) and two-dimensional (2D) immunoblotting and ELISA were performed using serum or plasma from eleven soybean allergic patients. The results of immunoblots and ELISA showed no significant differences in IgE binding between the GM line and its non-GM near-isoline control. However, some distinct differences in IgE binding patterns were observed among the non-GM commercial soybean lines and between different locations, highlighting the inherent variability in endogenous allergenic proteins. Understanding the potential variability in the levels of endogenous allergens is necessary to establish a standard of acceptance for GM soybeans compared to non-GM soybean events and lines. Copyright © 2018. Published by Elsevier Ltd.

  7. Rare Variant of GM2 Gangliosidosis through Activator-Protein Deficiency.

    PubMed

    Brackmann, Florian; Kehrer, Christiane; Kustermann, Wibke; Böhringer, Judith; Krägeloh-Mann, Ingeborg; Trollmann, Regina

    2017-04-01

    GM2 gangliosidosis, AB variant, is a very rare form of GM2 gangliosidosis due to a deficiency of GM2 activator protein. We report on two patients with typical clinical features suggestive of GM2 gangliosidosis, but normal results for hexosaminidase A and hexosaminidase B as well as their corresponding genes. Genetic analysis of the gene encoding the activator protein, the GM2A gene, elucidated the cause of the disease, adding a novel mutation to the spectrum of GM2 AB variant. This report points out that in typical clinical constellations with normal enzyme results, genetic diagnostic for activator protein defects should be performed. Georg Thieme Verlag KG Stuttgart · New York.

  8. GM2 gangliosidosis AB variant: novel mutation from India - a case report with a review.

    PubMed

    Sheth, Jayesh; Datar, Chaitanya; Mistri, Mehul; Bhavsar, Riddhi; Sheth, Frenny; Shah, Krati

    2016-07-11

    GM2 gangliosidosis-AB variants a rare autosomal recessive neurodegenerative disorder occurring due to deficiency of GM2 activator protein resulting from the mutation in GM2A gene. Only seven mutations in nine cases have been reported from different population except India. Present case is a one year old male born to 3rd degree consanguineous Indian parents from Maharashtra. He was presented with global developmental delay, hypotonia and sensitive to hyperacusis. Horizontal nystagmus and cherry red spot was detected during ophthalmic examination. MRI of brain revealed putaminal hyperintensity and thalamic hypointensity with some unmyelinated white matter in T2/T1 weighted images. Initially he was suspected having Tay-Sachs disease and finally diagnosed as GM2 gangliosidosis, AB variant due to truncated protein caused by nonsense mutation c.472 G > T (p.E158X) in GM2Agene. Children with phenotypic presentation as GM2 gangliosidosis (Tay-Sachs or Sandhoff disease) and normal enzyme activity of β-hexosaminidase-A and -B in leucocytes need to be investigated for GM2 activator protein deficiency.

  9. Tailored Synthesis of 162-Residue S-Monoglycosylated GM2-Activator Protein (GM2AP) Analogues that Allows Facile Access to a Protein Library.

    PubMed

    Nakamura, Takahiro; Sato, Kohei; Naruse, Naoto; Kitakaze, Keisuke; Inokuma, Tsubasa; Hirokawa, Takatsugu; Shigenaga, Akira; Itoh, Kohji; Otaka, Akira

    2016-10-17

    A synthetic protocol for the preparation of 162-residue S-monoglycosylated GM2-activator protein (GM2AP) analogues bearing various amino acid substitutions for Thr69 has been developed. The facile incorporation of the replacements into the protein was achieved by means of a one-pot/N-to-C-directed sequential ligation strategy using readily accessible middle N-sulfanylethylanilide (SEAlide) peptides each consisting of seven amino acid residues. A kinetically controlled ligation protocol was successfully applied to the assembly of three peptide segments covering the GM2AP. The native chemical ligation (NCL) reactivities of the SEAlide peptides can be tuned by the presence or absence of phosphate salts. Furthermore, NCL of the alkyl thioester fragment [GM2AP (1-31)] with the N-terminal cysteinyl prolyl thioester [GM2AP (32-67)] proceeded smoothly to yield the 67-residue prolyl thioester, with the prolyl thioester moiety remaining intact. This newly developed strategy enabled the facile synthesis of GM2AP analogues. Thus, we refer to this synthetic protocol as "tailored synthesis" for the construction of a GM2AP library. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Association between Immunoglobulin GM and KM Genotypes and Placental Malaria in HIV-1 Negative and Positive Women in Western Kenya

    PubMed Central

    Iriemenam, Nnaemeka C.; Pandey, Janardan P.; Williamson, John; Blackstock, Anna J.; Yesupriya, Ajay; Namboodiri, Aryan M.; Rocca, Keith M.; van Eijk, Anna Maria; Ayisi, John; Oteino, Juliana; Lal, Renu B.; ter Kuile, Feiko O.; Steketee, Richard; Nahlen, Bernard; Slutsker, Laurence; Shi, Ya Ping

    2013-01-01

    Immunoglobulin (Ig) GM and KM allotypes, genetic markers of γ and κ chains, are associated with humoral immune responsiveness. Previous studies have shown the relationships between GM6-carrying haplotypes and susceptibility to malaria infection in children and adults; however, the role of the genetic markers in placental malaria (PM) infection and PM with HIV co-infection during pregnancy has not been investigated. We examined the relationship between the gene polymorphisms of Ig GM6 and KM allotypes and the risk of PM infection in pregnant women with known HIV status. DNA samples from 728 pregnant women were genotyped for GM6 and KM alleles using polymerase chain reaction-restriction fragment length polymorphism method. Individual GM6 and KM genotypes and the combined GM6 and KM genotypes were assessed in relation to PM in HIV-1 negative and positive women, respectively. There was no significant effect of individual GM6 and KM genotypes on the risk of PM infection in HIV-1 negative and positive women. However, the combination of homozygosity for GM6(+) and KM3 was associated with decreased risk of PM (adjusted OR, 0.25; 95% CI, 0.08–0.8; P = 0.019) in HIV-1 negative women while in HIV-1 positive women the combination of GM6(+/−) with either KM1-3 or KM1 was associated with increased risk of PM infection (adjusted OR, 2.10; 95% CI, 1.18–3.73; P = 0.011). Hardy-Weinberg Equilibrium (HWE) tests further showed an overall significant positive F(is) (indication of deficit in heterozygotes) for GM6 while there was no deviation for KM genotype frequency from HWE in the same population. These findings suggest that the combination of homozygous GM6(+) and KM3 may protect against PM in HIV-1 negative women while the HIV-1 positive women with heterozygous GM6(+/−) combined with KM1-3 or KM1 may be more susceptible to PM infection. The deficit in heterozygotes for GM6 further suggests that GM6 could be under selection likely by malaria infection. PMID

  11. Compositional differences in soybeans on the market: glyphosate accumulates in Roundup Ready GM soybeans.

    PubMed

    Bøhn, T; Cuhra, M; Traavik, T; Sanden, M; Fagan, J; Primicerio, R

    2014-06-15

    This article describes the nutrient and elemental composition, including residues of herbicides and pesticides, of 31 soybean batches from Iowa, USA. The soy samples were grouped into three different categories: (i) genetically modified, glyphosate-tolerant soy (GM-soy); (ii) unmodified soy cultivated using a conventional "chemical" cultivation regime; and (iii) unmodified soy cultivated using an organic cultivation regime. Organic soybeans showed the healthiest nutritional profile with more sugars, such as glucose, fructose, sucrose and maltose, significantly more total protein, zinc and less fibre than both conventional and GM-soy. Organic soybeans also contained less total saturated fat and total omega-6 fatty acids than both conventional and GM-soy. GM-soy contained high residues of glyphosate and AMPA (mean 3.3 and 5.7 mg/kg, respectively). Conventional and organic soybean batches contained none of these agrochemicals. Using 35 different nutritional and elemental variables to characterise each soy sample, we were able to discriminate GM, conventional and organic soybeans without exception, demonstrating "substantial non-equivalence" in compositional characteristics for 'ready-to-market' soybeans. Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.

  12. GM foods and the misperception of risk perception.

    PubMed

    Gaskell, George; Allum, Nick; Wagner, Wolfgang; Kronberger, Nicole; Torgersen, Helge; Hampel, Juergen; Bardes, Julie

    2004-02-01

    Public opposition to genetically modified (GM) food and crops is widely interpreted as the result of the public's misperception of the risks. With scientific assessment pointing to no unique risks from GM crops and foods, a strategy of accurate risk communication from trusted sources has been advocated. This is based on the assumption that the benefits of GM crops and foods are self-evident. Informed by the interpretation of some qualitative interviews with lay people, we use data from the Eurobarometer survey on biotechnology to explore the hypothesis that it is not so much the perception of risks as the absence of benefits that is the basis of the widespread rejection of GM foods and crops by the European public. Some respondents perceive both risks and benefits, and may be trading off these attributes along the lines of a rational choice model. However, for others, one attribute-benefit-appears to dominate their judgments: the lexicographic heuristic. For these respondents, their perception of risk is of limited importance in the formation of attitudes toward GM food and crops. The implication is that the absence of perceived benefits from GM foods and crops calls into question the relevance of risk communication strategies for bringing about change in public opinion.

  13. Serotype-specific differences in inhibition of reovirus infectivity by human-milk glycans are determined by viral attachment protein σ1.

    PubMed

    Iskarpatyoti, Jason A; Morse, E Ashley; McClung, R Paul; Ikizler, Miné; Wetzel, J Denise; Contractor, Nikhat; Dermody, Terence S

    2012-11-25

    Human milk contains many bioactive components, including secretory IgA, oligosaccharides, and milk-associated proteins. We assessed the antiviral effects of several components of milk against mammalian reoviruses. We found that glucocerebroside (GCB) inhibited the infectivity of reovirus strain type 1 Lang (T1L), whereas gangliosides GD3 and GM3 and 3'-sialyllactose (3SL) inhibited the infectivity of reovirus strain type 3 Dearing (T3D). Agglutination of erythrocytes mediated by T1L and T3D was inhibited by GD3, GM3, and bovine lactoferrin. Additionally, α-sialic acid, 3SL, 6'-sialyllactose, sialic acid, human lactoferrin, osteopontin, and α-lactalbumin inhibited hemagglutination mediated by T3D. Using single-gene reassortant viruses, we found that serotype-specific differences segregate with the gene encoding the viral attachment protein. Furthermore, GD3, GM3, and 3SL inhibit T3D infectivity by blocking binding to host cells, whereas GCB inhibits T1L infectivity post-attachment. These results enhance an understanding of reovirus cell attachment and define a mechanism for the antimicrobial activity of human milk. Copyright © 2012 Elsevier Inc. All rights reserved.

  14. Comprehensive Analysis of the Soybean (Glycine max) GmLAX Auxin Transporter Gene Family

    PubMed Central

    Chai, Chenglin; Wang, Yongqin; Valliyodan, Babu; Nguyen, Henry T.

    2016-01-01

    The phytohormone auxin plays a critical role in regulation of plant growth and development as well as plant responses to abiotic stresses. This is mainly achieved through its uneven distribution in plant via a polar auxin transport process. Auxin transporters are major players in polar auxin transport. The AUXIN RESISTENT 1/LIKE AUX1 (AUX/LAX) auxin influx carriers belong to the amino acid permease family of proton-driven transporters and function in the uptake of indole-3-acetic acid (IAA). In this study, genome-wide comprehensive analysis of the soybean AUX/LAX (GmLAX) gene family, including phylogenic relationships, chromosome localization, and gene structure, was carried out. A total of 15 GmLAX genes, including seven duplicated gene pairs, were identified in the soybean genome. They were distributed on 10 chromosomes. Despite their higher percentage identities at the protein level, GmLAXs exhibited versatile tissue-specific expression patterns, indicating coordinated functioning during plant growth and development. Most GmLAXs were responsive to drought and dehydration stresses and auxin and abscisic acid (ABA) stimuli, in a tissue- and/or time point- sensitive mode. Several GmLAX members were involved in responding to salt stress. Sequence analysis revealed that promoters of GmLAXs contained different combinations of stress-related cis-regulatory elements. These studies suggest that the soybean GmLAXs were under control of a very complex regulatory network, responding to various internal and external signals. This study helps to identity candidate GmLAXs for further analysis of their roles in soybean development and adaption to adverse environments. PMID:27014306

  15. TGF-β Affects the Differentiation of Human GM-CSF+ CD4+ T Cells in an Activation- and Sodium-Dependent Manner.

    PubMed

    Éliás, Szabolcs; Schmidt, Angelika; Kannan, Venkateshan; Andersson, John; Tegnér, Jesper

    2016-01-01

    The cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) is involved in the pathogenesis of chronic inflammatory diseases such as multiple sclerosis. However, the environmental cues promoting differentiation of GM-CSF producing T cells are unclear. Herein, we performed a broad experimental screening of cytokines and data-driven analysis assessing their ability to induce human GM-CSF + CD4 + T cells and their subpopulations. TGF-β was discovered to induce GM-CSF production independently of proliferation and IL-2 signaling including STAT5. In contrast, IL-6 and IL-23 decreased GM-CSF production. On the population level, GM-CSF induction was highly correlated with expression of FOXP3 across cytokine stimulations but not with that of IL-17. However, on single-cell level GM-CSF and IFN-γ expression were most correlated, independently of the cytokine environment. Importantly, under low sodium conditions in the medium or upon stimulation with plate-bound instead of bead-bound anti-CD3 and anti-CD28 antibodies, the effects of TGF-β on GM-CSF, but not on FOXP3, were reversed. Our analysis indicates a novel role for TGF-β in generating GM-CSF + subsets of human CD4 + T cells. These results are important for understanding of autoimmune disease and therapeutic considerations.

  16. TGF-β Affects the Differentiation of Human GM-CSF+ CD4+ T Cells in an Activation- and Sodium-Dependent Manner

    PubMed Central

    Éliás, Szabolcs; Schmidt, Angelika; Kannan, Venkateshan; Andersson, John; Tegnér, Jesper

    2016-01-01

    The cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) is involved in the pathogenesis of chronic inflammatory diseases such as multiple sclerosis. However, the environmental cues promoting differentiation of GM-CSF producing T cells are unclear. Herein, we performed a broad experimental screening of cytokines and data-driven analysis assessing their ability to induce human GM-CSF+ CD4+ T cells and their subpopulations. TGF-β was discovered to induce GM-CSF production independently of proliferation and IL-2 signaling including STAT5. In contrast, IL-6 and IL-23 decreased GM-CSF production. On the population level, GM-CSF induction was highly correlated with expression of FOXP3 across cytokine stimulations but not with that of IL-17. However, on single-cell level GM-CSF and IFN-γ expression were most correlated, independently of the cytokine environment. Importantly, under low sodium conditions in the medium or upon stimulation with plate-bound instead of bead-bound anti-CD3 and anti-CD28 antibodies, the effects of TGF-β on GM-CSF, but not on FOXP3, were reversed. Our analysis indicates a novel role for TGF-β in generating GM-CSF+ subsets of human CD4+ T cells. These results are important for understanding of autoimmune disease and therapeutic considerations. PMID:28066414

  17. Interaction of RNA-binding protein HuR and miR-466i regulates GM-CSF expression.

    PubMed

    Chen, Jing; Adamiak, William; Huang, Ganlei; Atasoy, Ulus; Rostami, Abdolmohamad; Yu, Shiguang

    2017-12-08

    Granulocyte-macrophage colony-stimulating factor (GM-CSF) produced by T helper 17 (Th17) cells plays an essential role in autoimmune diseases. Transcriptional regulation of Th17 cell differentiation has been extensively studied, but post-transcriptional regulation of Th17 cell differentiation has remained less well characterized. The RNA-binding protein HuR functions to promote the stability of target mRNAs via binding the AU-rich elements of the 3' untranslated region (3'UTR) of numerous pro-inflammatory cytokines including IL-4, IL-13, IL-17 and TNF-α. However, whether HuR regulates GM-CSF expression in Th17 cells has not been fully investigated. Here we showed that HuR conditional knockout (KO) Th17 cells have decreased GM-CSF mRNA in comparison with wild-type (WT) Th17 cells, and that HuR binds directly to GM-CSF mRNA 3'UTR. Interestingly, HuR deficiency increased the levels of certain microRNA expression in Th17 cells; for example, miR-466i functioned to mediate GM-CSF and IL-17 mRNA decay, which was confirmed by in vitro luciferase assay. Furthermore, we found that HuR promoted Mxi1 expression to inhibit certain miRNA expression. Taken together, these findings indicate that interaction of HuR and miR-466i orchestrates GM-CSF expression in Th17 cells.

  18. Comparative Proteomic Analysis of Two Varieties of Genetically Modified (GM) Embrapa 5.1 Common Bean (Phaseolus vulgaris L.) and Their Non-GM Counterparts.

    PubMed

    Balsamo, Geisi M; Valentim-Neto, Pedro A; Mello, Carla S; Arisi, Ana C M

    2015-12-09

    The genetically modified (GM) common bean event Embrapa 5.1 was commercially approved in Brazil in 2011; it is resistant to golden mosaic virus infection. In the present work grain proteome profiles of two Embrapa 5.1 common bean varieties, Pérola and Pontal, and their non-GM counterparts were compared by two-dimensional gel electrophoresis (2-DE) followed by mass spectrometry (MS). Analyses detected 23 spots differentially accumulated between GM Pérola and non-GM Pérola and 21 spots between GM Pontal and non-GM Pontal, although they were not the same proteins in Pérola and Pontal varieties, indicating that the variability observed may not be due to the genetic transformation. Among them, eight proteins were identified in Pérola varieties, and four proteins were identified in Pontal. Moreover, we applied principal component analysis (PCA) on 2-DE data, and variation between varieties was explained in the first two principal components. This work provides a first 2-DE-MS/MS-based analysis of Embrapa 5.1 common bean grains.

  19. Examining consumer behavior toward genetically modified (GM) food in Britain.

    PubMed

    Spence, Alexa; Townsend, Ellen

    2006-06-01

    This study examined behavior toward genetically modified (GM) food in a British community-based sample. We used an equivalent gain task in which participants actually received the options they chose to encourage truthful responding. In conjunction with this, theory of planned behavior (TPB) components were evaluated so as to examine the relative importance of behavioral influences in this domain. Here, the TPB was extended to include additional components to measure self-identity, moral norms, and emotional involvement. Results indicated that the monetary amounts participants accepted in preference to GM food were significantly lower than those accepted in preference to non-GM food. However, the vast majority of participants were indifferent between GM and non-GM food options. All TPB components significantly predicted behavioral intentions to try GM food, with attitudes toward GM being the strongest predictor. Self-identity and emotional involvement were also found to be significant predictors of behavioral intentions but moral norms were not. In addition, behavioral intentions significantly predicted behavior; however, PBC did not. An additional measure of participants' propensity to respond in a socially desirable manner indicated that our results were not influenced by self-presentation issues, giving confidence to our findings. Overall, it appears that the majority of participants (74.5%) would purchase GM food at some price.

  20. Glycosphingolipid-functionalized nanoparticles recapitulate CD169-dependent HIV-1 uptake and trafficking in dendritic cells

    NASA Astrophysics Data System (ADS)

    Yu, Xinwei; Feizpour, Amin; Ramirez, Nora-Guadalupe P.; Wu, Linxi; Akiyama, Hisashi; Xu, Fangda; Gummuluru, Suryaram; Reinhard, Björn M.

    2014-06-01

    Ganglioside GM3, a host-derived glycosphingolipid incorporated in the membrane of human immunodeficiency virus-1 (HIV-1) viral particles, mediates interactions between HIV-1 and Siglec1/CD169, a protein expressed on dendritic cells (DCs). Such interactions, which seem to be independent of viral envelope glycoprotein gp120, are poorly understood. Here we develop a model system consisting of self-assembled artificial virus nanoparticles (AVNs) that are free of viral glycoproteins or other host-derived glycolipids and glycoproteins. These plasmonic AVNs contain a membrane of defined composition wrapped around a solid metal core. GM3-containing AVNs are captured by CD169-expressing HeLa cells or mature DCs, and are sequestered within non-lysosomal tetraspanin-positive compartments. This distribution is reminiscent of CD169-dependent HIV-1 sequestration in mature DCs. Our results highlight GM3-CD169 binding as a gp120-independent signal for sequestration and preservation of HIV-1 infectivity. They also indicate that plasmonic AVNs offer improved features over liposome-based systems and represent a versatile tool for probing specific virus-cell interactions.

  1. Assuring the safety of genetically modified (GM) foods: the importance of an holistic, integrative approach.

    PubMed

    Cockburn, Andrew

    2002-09-11

    Genes change continuously by natural mutation and recombination enabling man to select and breed crops having the most desirable traits such as yield or flavour. Genetic modification (GM) is a recent development which allows specific genes to be identified, isolated, copied and inserted into other plants with a high level of specificity. The food safety considerations for GM crops are basically the same as those arising from conventionally bred crops, very few of which have been subject to any testing yet are generally regarded as being safe to eat. In contrast a rigorous safety testing paradigm has been developed for GM crops, which utilises a systematic, stepwise and holistic approach. The resultant science based process, focuses on a classical evaluation of the toxic potential of the introduced novel trait and the wholesomeness of the transformed crop. In addition, detailed consideration is given to the history and safe use of the parent crop as well as that of the gene donor. The overall safety evaluation is conducted under the concept known as substantial equivalence which is enshrined in all international crop biotechnology guidelines. This provides the framework for a comparative approach to identify the similarities and differences between the GM product and its comparator which has a known history of safe use. By building a detailed profile on each step in the transformation process, from parent to new crop, and by thoroughly evaluating the significance from a safety perspective, of any differences that may be detected, a very comprehensive matrix of information is constructed which enables the conclusion as to whether the GM crop, derived food or feed is as safe as its traditional counterpart. Using this approach in the evaluation of more than 50 GM crops which have been approved worldwide, the conclusion has been that foods and feeds derived from genetically modified crops are as safe and nutritious as those derived from traditional crops. The lack of

  2. Are GM Crops for Yield and Resilience Possible?

    PubMed

    Paul, Matthew J; Nuccio, Michael L; Basu, Shib Sankar

    2018-01-01

    Crop yield improvements need to accelerate to avoid future food insecurity. Outside Europe, genetically modified (GM) crops for herbicide- and insect-resistance have been transformative in agriculture; other traits have also come to market. However, GM of yield potential and stress resilience has yet to impact on food security. Genes have been identified for yield such as grain number, size, leaf growth, resource allocation, and signaling for drought tolerance, but there is only one commercialized drought-tolerant GM variety. For GM and genome editing to impact on yield and resilience there is a need to understand yield-determining processes in a cell and developmental context combined with evaluation in the grower environment. We highlight a sugar signaling mechanism as a paradigm for this approach. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Neuroradiological findings in GM2 gangliosidosis variant B1.

    PubMed

    Bano, Shahina; Prasad, Akhila; Yadav, Sachchida Nand; Chaudhary, Vikas; Garga, Umesh Chandra

    2011-07-01

    GM2 gangliosidosis variant B1 is a very rare lysosomal disorder. As per our knowledge, to date, only one article depicting the magnetic resonance imaging (MRI) findings of GM2 gangliosidosis variant B1 is available in the literature. We are the first to describe the neuroradiological findings in an Indian patient diagnosed with GM2 gangliosidosis variant B1.

  4. A profile of sphingolipids and related compounds tentatively identified in yak milk.

    PubMed

    Qu, S; Barrett-Wilt, G; Fonseca, L M; Rankin, S A

    2016-07-01

    This work characterized a fraction of constituents in yak milk within the realm of approximately 1,000 to 3,000 Da using matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry. Eleven samples of yak milk powder from the Sichuan province of China were received by the Department of Food Science, University of Wisconsin-Madison, and stored at room temperature until analysis. Sample preparation involved delipidation and deproteinization of yak milk samples and cold ethanol precipitation. Subsequently, MALDI time-of-flight mass spectrometry was performed in positive ion, reflector mode (AB Sciex TOF/TOF 4800 MALDI; AB Sciex, Foster City, CA). The instrument was first calibrated with the manufacturer's 6-peptide mixture, and each spectrum was internally calibrated using the accurate mass of ACTH Fragment 18-39 standard peptide (protonated mass at m/z 2464.199) present in each sample. Laser power was adjusted for the calibration standards and for each sample so that the signal obtained for the most-abundant ion in each spectrum could be maximized, or kept below ~2×10(4) to preserve spectral quality. Structure and name based on mass were matched using the Metlin metabolite database (https://metlin.scripps.edu/index.php). Results of the current work for yak milk powder showed a large variety of sphingolipid structures with clusters around 1,200, 1,600, and 2,000 Da. The profiling matched several glycosphingolipids, such as gangliosides GA1, GD1a, GD1b, GD3, GM1, GM2, GM3, and GT2 and several other unique moieties, including deaminated neuraminic acid (KDN) oligosaccharides, and fucose containing gangliosides. Matrix preparation and MALDI time-of-flight parameters were important factors established in this work to allow high resolution profiling of complex sphingolipids in yak powder milk. Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  5. ZO-1 expression is suppressed by GM-CSF via miR-96/ERG in brain microvascular endothelial cells.

    PubMed

    Zhang, Hu; Zhang, Shuhong; Zhang, Jilin; Liu, Dongxin; Wei, Jiayi; Fang, Wengang; Zhao, Weidong; Chen, Yuhua; Shang, Deshu

    2018-05-01

    The level of granulocyte-macrophage colony-stimulating factor (GM-CSF) increases in some disorders such as vascular dementia, Alzheimer's disease, and multiple sclerosis. We previously reported that in Alzheimer's disease patients, a high level of GM-CSF in the brain parenchyma downregulated expression of ZO-1, a blood-brain barrier tight junction protein, and facilitated the infiltration of peripheral monocytes across the blood-brain barrier. However, the molecular mechanism underlying regulation of ZO-1 expression by GM-CSF is unclear. Herein, we found that the erythroblast transformation-specific (ETS) transcription factor ERG cooperated with the proto-oncogene protein c-MYC in regulation of ZO-1 transcription in brain microvascular endothelial cells (BMECs). The ERG expression was suppressed by miR-96 which was increased by GM-CSF through the phosphoinositide-3 kinase (PI3K)/Akt pathway. Inhibition of miR-96 prevented ZO-1 down-regulation induced by GM-CSF both in vitro and in vivo. Our results revealed the mechanism of ZO-1 expression reduced by GM-CSF, and provided a potential target, miR-96, which could block ZO-1 down-regulation caused by GM-CSF in BMECs.

  6. Delivery of GM-CSF to Protect against Influenza Pneumonia

    PubMed Central

    Subramaniam, Renuka; Hillberry, Zachary; Chen, Han; Feng, Yan; Fletcher, Kalyn; Neuenschwander, Pierre; Shams, Homayoun

    2015-01-01

    Background Since adaptive immunity is thought to be central to immunity against influenza A virus (IAV) pneumonias, preventive strategies have focused primarily on vaccines. However, vaccine efficacy has been variable, in part because of antigenic shift and drift in circulating influenza viruses. Recent studies have highlighted the importance of innate immunity in protecting against influenza. Methods Granulocyte-macrophage colony stimulating factor (GM-CSF) contributes to maturation of mononuclear phagocytes, enhancing their capacity for phagocytosis and cytokine production. Results Overexpression of granulocyte macrophage-colony stimulating factor (GM-CSF) in the lung of transgenic mice provides remarkable protection against IAV, which depends on alveolar macrophages (AM). In this study, we report that pulmonary delivery of GM-CSF to wild type young and aged mice abrogated mortality from IAV. Conclusion We also demonstrate that protection is species specific and human GM-CSF do not protect the mice nor stimulates mouse immunity. We also show that IAV-induced lung injury is the culprit for side-effects of GM-CSF in treating mice after IAV infection, and introduce a novel strategy to deliver the GM-CSF to and retain it in the alveolar space even after IAV infection. PMID:25923215

  7. Media attention to GM food cases: An innovation perspective.

    PubMed

    Flipse, Steven M; Osseweijer, Patricia

    2013-02-01

    Media attention to genetically modified (GM) foods has been described as negative, especially in Europe. At the turn of the century appreciation of GM foods was at an all-time low in Europe. Food manufacturers are still careful in the use, development and communication of GM based food products, and their caution influences innovation processes. In this study we explore the link between media attention and innovation practice. Media attention to three specific high-profile GM food cases is described and linked to innovation practice. We elucidate the order of events in these cases and show that publics could only to a limited extent have formed an opinion on GM based food products based on scientifically valid data through written English media. Innovators in food biotechnology may benefit from this knowledge for future product development and marketing, and we suggest that innovation may benefit from early stakeholder involvement and communication activities.

  8. Accurate GM atrophy quantification in MS using lesion-filling with co-registered 2D lesion masks☆

    PubMed Central

    Popescu, V.; Ran, N.C.G.; Barkhof, F.; Chard, D.T.; Wheeler-Kingshott, C.A.; Vrenken, H.

    2014-01-01

    Background In multiple sclerosis (MS), brain atrophy quantification is affected by white matter lesions. LEAP and FSL-lesion_filling, replace lesion voxels with white matter intensities; however, they require precise lesion identification on 3DT1-images. Aim To determine whether 2DT2 lesion masks co-registered to 3DT1 images, yield grey and white matter volumes comparable to precise lesion masks. Methods 2DT2 lesion masks were linearly co-registered to 20 3DT1-images of MS patients, with nearest-neighbor (NNI), and tri-linear interpolation. As gold-standard, lesion masks were manually outlined on 3DT1-images. LEAP and FSL-lesion_filling were applied with each lesion mask. Grey (GM) and white matter (WM) volumes were quantified with FSL-FAST, and deep gray matter (DGM) volumes using FSL-FIRST. Volumes were compared between lesion mask types using paired Wilcoxon tests. Results Lesion-filling with gold-standard lesion masks compared to native images reduced GM overestimation by 1.93 mL (p < .001) for LEAP, and 1.21 mL (p = .002) for FSL-lesion_filling. Similar effects were achieved with NNI lesion masks from 2DT2. Global WM underestimation was not significantly influenced. GM and WM volumes from NNI, did not differ significantly from gold-standard. GM segmentation differed between lesion masks in the lesion area, and also elsewhere. Using the gold-standard, FSL-FAST quantified as GM on average 0.4% of the lesion area with LEAP and 24.5% with FSL-lesion_filling. Lesion-filling did not influence DGM volumes from FSL-FIRST. Discussion These results demonstrate that for global GM volumetry, precise lesion masks on 3DT1 images can be replaced by co-registered 2DT2 lesion masks. This makes lesion-filling a feasible method for GM atrophy measurements in MS. PMID:24567908

  9. Smart cushion innovations (ACI) 100gm crash attenuator.

    DOT National Transportation Integrated Search

    2015-03-01

    Determine the effectiveness of the SCI100GM in a mainline application. The : SCI100GM is a fully redirective, speed-dependent, non-gating, bi-directional crash : attenuator with a reverse-tapered design to eliminate side panel stress during a : colla...

  10. Overexpression of the Transcription Factors GmSHN1 and GmSHN9 Differentially Regulates Wax and Cutin Biosynthesis, Alters Cuticle Properties, and Changes Leaf Phenotypes in Arabidopsis.

    PubMed

    Xu, Yangyang; Wu, Hanying; Zhao, Mingming; Wu, Wang; Xu, Yinong; Gu, Dan

    2016-04-21

    SHINE (SHN/WIN) clade proteins, transcription factors of the plant-specific APETALA 2/ethylene-responsive element binding factor (AP2/ERF) family, have been proven to be involved in wax and cutin biosynthesis. Glycine max is an important economic crop, but its molecular mechanism of wax biosynthesis is rarely characterized. In this study, 10 homologs of Arabidopsis SHN genes were identified from soybean. These homologs were different in gene structures and organ expression patterns. Constitutive expression of each of the soybean SHN genes in Arabidopsis led to different leaf phenotypes, as well as different levels of glossiness on leaf surfaces. Overexpression of GmSHN1 and GmSHN9 in Arabidopsis exhibited 7.8-fold and 9.9-fold up-regulation of leaf cuticle wax productions, respectively. C31 and C29 alkanes contributed most to the increased wax contents. Total cutin contents of leaves were increased 11.4-fold in GmSHN1 overexpressors and 5.7-fold in GmSHN9 overexpressors, mainly through increasing C16:0 di-OH and dioic acids. GmSHN1 and GmSHN9 also altered leaf cuticle membrane ultrastructure and increased water loss rate in transgenic Arabidopsis plants. Transcript levels of many wax and cutin biosynthesis and leaf development related genes were altered in GmSHN1 and GmSHN9 overexpressors. Overall, these results suggest that GmSHN1 and GmSHN9 may differentially regulate the leaf development process as well as wax and cutin biosynthesis.

  11. Targeting the GM-CSF receptor for the treatment of CNS autoimmunity

    PubMed Central

    Ifergan, Igal; Davidson, Todd S.; Kebir, Hania; Xu, Dan; Palacios-Macapagal, Daphne; Cann, Jennifer; Rodgers, Jane M.; Hunter, Zoe N.; Pittet, Camille L.; Beddow, Sara; Jones, Clare A.; Prat, Alexandre; Sleeman, Matthew A.; Miller, Stephen D.

    2017-01-01

    In multiple sclerosis (MS), there is a growing interest in inhibiting the pro-inflammatory effects of granulocyte-macrophage colony-stimulating factor (GM-CSF). We sought to evaluate the therapeutic potential and underlying mechanisms of GM-CSF receptor alpha (Rα) blockade in animal models of MS. We show that GM-CSF signaling inhibition at peak of chronic experimental autoimmune encephalomyelitis (EAE) results in amelioration of disease progression. Similarly, GM-CSF Rα blockade in relapsing-remitting (RR)-EAE model prevented disease relapses and inhibited T cell responses specific for both the inducing and spread myelin peptides, while reducing activation of mDCs and inflammatory monocytes. In situ immunostaining of lesions from human secondary progressive MS (SPMS), but not primary progressive MS patients shows extensive recruitment of GM-CSF Rα+ myeloid cells. Collectively, this study reveals a pivotal role of GM-CSF in disease relapses and the benefit of GM-CSF Rα blockade as a potential novel therapeutic approach for treatment of RRMS and SPMS. PMID:28641926

  12. Lipidomic Profiling Links the Fanconi Anemia Pathway to Glycosphingolipid Metabolism in Head and Neck Cancer Cells.

    PubMed

    Zhao, Xueheng; Brusadelli, Marion G; Sauter, Sharon; Butsch Kovacic, Melinda; Zhang, Wujuan; Romick-Rosendale, Lindsey E; Lambert, Paul F; Setchell, Kenneth D R; Wells, Susanne I

    2018-06-01

    Purpose: Mutations in Fanconi anemia (FA) genes are common in sporadic squamous cell carcinoma of the head and neck (HNSCC), and we have previously demonstrated that FA pathway depletion in HNSCC cell lines stimulates invasion. The goal of our studies was to use a systems approach in order to define FA pathway-dependent lipid metabolism and to extract lipid-based signatures and effectors of invasion in FA-deficient cells. Experimental Design: We subjected FA-isogenic HNSCC keratinocyte cell lines to untargeted and targeted lipidomics analyses to discover novel biomarkers and candidate therapeutic targets in FA-deficient cells. Cellular invasion assays were carried out in the presence and absence of N-butyldeoxynojirimycin (NB-DNJ), a biosynthetic inhibitor of the newly identified class of gangliosides, to investigate the requirement of ganglioside upregulation in FA-deficient HNSCC cells. Results: The most notable element of the lipid profiling results was a consistent elevation of glycosphingolipids, and particularly the accumulation of gangliosides. Conversely, repression of this same class of lipids was observed upon genetic correction of FA patient-derived HNSCC cells. Functional studies demonstrate that ganglioside upregulation is required for HNSCC cell invasion driven by FA pathway loss. The motility of nontransformed keratinocytes in response to FA loss displayed a similar dependence, thus supporting early and late roles for the FA pathway in controlling keratinocyte invasion through lipid regulation. Conclusions: Elevation of glycosphingolipids including the ganglioside GM3 in response to FA loss stimulates invasive characteristics of immortalized and transformed keratinocytes. An inhibitor of glycosphingolipid biosynthesis NB-DNJ attenuates invasive characteristics of FA-deficient HNSCC cells. Clin Cancer Res; 24(11); 2700-9. ©2018 AACR . ©2018 American Association for Cancer Research.

  13. Functional interaction analysis of GM1-related carbohydrates and Vibrio cholerae toxins using carbohydrate microarray.

    PubMed

    Kim, Chang Sup; Seo, Jeong Hyun; Cha, Hyung Joon

    2012-08-07

    The development of analytical tools is important for understanding the infection mechanisms of pathogenic bacteria or viruses. In the present work, a functional carbohydrate microarray combined with a fluorescence immunoassay was developed to analyze the interactions of Vibrio cholerae toxin (ctx) proteins and GM1-related carbohydrates. Ctx proteins were loaded onto the surface-immobilized GM1 pentasaccharide and six related carbohydrates, and their binding affinities were detected immunologically. The analysis of the ctx-carbohydrate interactions revealed that the intrinsic selectivity of ctx was GM1 pentasaccharide ≫ GM2 tetrasaccharide > asialo GM1 tetrasaccharide ≥ GM3trisaccharide, indicating that a two-finger grip formation and the terminal monosaccharides play important roles in the ctx-GM1 interaction. In addition, whole cholera toxin (ctxAB(5)) had a stricter substrate specificity and a stronger binding affinity than only the cholera toxin B subunit (ctxB). On the basis of the quantitative analysis, the carbohydrate microarray showed the sensitivity of detection of the ctxAB(5)-GM1 interaction with a limit-of-detection (LOD) of 2 ng mL(-1) (23 pM), which is comparable to other reported high sensitivity assay tools. In addition, the carbohydrate microarray successfully detected the actual toxin directly secreted from V. cholerae, without showing cross-reactivity to other bacteria. Collectively, these results demonstrate that the functional carbohydrate microarray is suitable for analyzing toxin protein-carbohydrate interactions and can be applied as a biosensor for toxin detection.

  14. Modeling gene flow distribution within conventional fields and development of a simplified sampling method to quantify adventitious GM contents in maize

    PubMed Central

    Melé, Enric; Nadal, Anna; Messeguer, Joaquima; Melé-Messeguer, Marina; Palaudelmàs, Montserrat; Peñas, Gisela; Piferrer, Xavier; Capellades, Gemma; Serra, Joan; Pla, Maria

    2015-01-01

    Genetically modified (GM) crops have been commercially grown for two decades. GM maize is one of 3 species with the highest acreage and specific events. Many countries established a mandatory labeling of products containing GM material, with thresholds for adventitious presence, to support consumers’ freedom of choice. In consequence, coexistence systems need to be introduced to facilitate commercial culture of GM and non-GM crops in the same agricultural area. On modeling adventitious GM cross-pollination distribution within maize fields, we deduced a simple equation to estimate overall GM contents (%GM) of conventional fields, irrespective of its shape and size, and with no previous information on possible GM pollen donor fields. A sampling strategy was designed and experimentally validated in 19 agricultural fields. With 9 samples, %GM quantification requires just one analytical GM determination while identification of the pollen source needs 9 additional analyses. A decision support tool is provided. PMID:26596213

  15. Use of an oncolytic virus secreting GM-CSF as combined oncolytic and immunotherapy for treatment of colorectal and hepatic adenocarcinomas.

    PubMed

    Malhotra, Sandeep; Kim, Teresa; Zager, Jonathan; Bennett, Joseph; Ebright, Michael; D'Angelica, Michael; Fong, Yuman

    2007-04-01

    Oncolytic cancer therapy using herpes simplex viruses (HSV) that have direct tumoricidal effects and cancer immunotherapy using the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) have each been effective in preclinical testing. NV1034 is a multimutated oncolytic HSV carrying the gene for murine GM-CSF that attempts to combine these 2 anticancer strategies. The purpose of this study was to compare NV1034 to NV1023, the parent HSV mutants lacking GM-CSF, to determine if such combined oncolytic and immunotherapy using a single vector has advantages over oncolytic therapy alone. Expression GM-CSF in vitro did not alter the infectivity, cytotoxicity, or replication of NV1034 compared to the noncytokine-secreting control. Tumors infected with NV1034 produced GM-CSF in picogram quantities. In vivo efficacy of the viruses against murine colorectal carcinoma CT26 and murine hepatoma Hepa l-6 was then tested in subcutaneous tumors in syngeneic Balb/c and C57 L/J mice, respectively. In these immune-competent models, NV1034 and NV1023 each demonstrated potent antitumor activity. Treatment with NV1034 had significantly better antitumor effect compared to treatment with NV1023. Furthermore, there was no difference in the antitumor efficacy of these viruses in mice depleted of CD4+ and CD8+ T lymphocytes. Viral vectors combining oncolytic and immunotherapy are promising agents in treatment of colorectal carcinoma and hepatoma.

  16. Overexpression of the Transcription Factors GmSHN1 and GmSHN9 Differentially Regulates Wax and Cutin Biosynthesis, Alters Cuticle Properties, and Changes Leaf Phenotypes in Arabidopsis

    PubMed Central

    Xu, Yangyang; Wu, Hanying; Zhao, Mingming; Wu, Wang; Xu, Yinong; Gu, Dan

    2016-01-01

    SHINE (SHN/WIN) clade proteins, transcription factors of the plant-specific APETALA 2/ethylene-responsive element binding factor (AP2/ERF) family, have been proven to be involved in wax and cutin biosynthesis. Glycine max is an important economic crop, but its molecular mechanism of wax biosynthesis is rarely characterized. In this study, 10 homologs of Arabidopsis SHN genes were identified from soybean. These homologs were different in gene structures and organ expression patterns. Constitutive expression of each of the soybean SHN genes in Arabidopsis led to different leaf phenotypes, as well as different levels of glossiness on leaf surfaces. Overexpression of GmSHN1 and GmSHN9 in Arabidopsis exhibited 7.8-fold and 9.9-fold up-regulation of leaf cuticle wax productions, respectively. C31 and C29 alkanes contributed most to the increased wax contents. Total cutin contents of leaves were increased 11.4-fold in GmSHN1 overexpressors and 5.7-fold in GmSHN9 overexpressors, mainly through increasing C16:0 di-OH and dioic acids. GmSHN1 and GmSHN9 also altered leaf cuticle membrane ultrastructure and increased water loss rate in transgenic Arabidopsis plants. Transcript levels of many wax and cutin biosynthesis and leaf development related genes were altered in GmSHN1 and GmSHN9 overexpressors. Overall, these results suggest that GmSHN1 and GmSHN9 may differentially regulate the leaf development process as well as wax and cutin biosynthesis. PMID:27110768

  17. CDP-choline liposomes provide significant reduction in infarction over free CDP-choline in stroke

    PubMed Central

    Adibhatla, Rao Muralikrishna; Hatcher, J.F.; Tureyen, K.

    2007-01-01

    Cytidine-5′-diphosphocholine (CDP-choline, Citicoline, Somazina) is in clinical use (intravenous administration) for stroke treatment in Europe and Japan, while USA phase III stroke clinical trials (oral administration) were disappointing. Others showed that CDP-choline liposomes significantly increased brain uptake over the free drug in cerebral ischemia models. Liposomes were formulated as DPPC, DPPS, cholesterol, GM1 ganglioside; 7/4/7/1.57 molar ratio or 35.8/20.4/35.8/8.0 mol%. GM1 ganglioside confers long-circulating properties to the liposomes by suppressing phagocytosis. CDP-choline liposomes deliver the agent intact to the brain, circumventing the rate-limiting, cytidine triphosphate:phosphocholine cytidylyltransferase in phosphatidylcholine synthesis. Our data show that CDP-choline liposomes significantly ( P < 0.01) decreased cerebral infarction (by 62%) compared to the equivalent dose of free CDP-choline (by 26%) after 1 h focal cerebral ischemia and 24 h reperfusion in spontaneously hypertensive rats. Beneficial effects of CDP-choline liposomes in stroke may derive from a synergistic effect between the phospholipid components of the liposomes and the encapsulated CDP-choline. PMID:16153613

  18. INF-gamma rearranges membrane topography of MHC-I and ICAM-1 in colon carcinoma cells.

    PubMed

    Bacsó, Zsolt; Bene, László; Damjanovich, László; Damjanovich, Sándor

    2002-01-18

    Flow-cytometric fluorescence energy transfer (FCET) measurements between fluorescently labeled cell surface MHC-I and ICAM-1 molecules indicated similar receptor patterns in the plasma membrane of interferon-gamma (INF-gamma)-treated colon carcinoma cells as those observed earlier at the surface of lymphoid cells. INF-gamma activation significantly increased the density of MHC-I and ICAM-1 proteins in the membrane. This increase in receptor density was accompanied by decreased proximity level of the homo-associated MHC-I receptors. Hetero-association of MHC-I and ICAM-1 molecules was increased by INF-gamma treatment. INF-gamma changed neither hetero- nor homo-association of transferrin receptors. By staining the sphingomyelin/cholesterol-enriched lipid microdomains with fluorescently labeled cholera toxin B subunit, we found an increase in the amount of lipid-raft associated G(M1)-gangliosides due to INF-gamma treatment. Confocal microscopic results and FCET measurements show that MHC-I and ICAM-1 are components of G(M1)-ganglioside containing lipid-rafts and also support an increase in the size of these lipid-rafts upon INF-gamma treatment.

  19. The GmFAD7 gene family from soybean: identification of novel genes and tissue-specific conformations of the FAD7 enzyme involved in desaturase activity.

    PubMed

    Andreu, Vanesa; Lagunas, Beatriz; Collados, Raquel; Picorel, Rafael; Alfonso, Miguel

    2010-07-01

    The FAD7 gene encodes a omega3 fatty acid desaturase which catalyses the production of trienoic fatty acids (TAs) in plant chloroplasts. A novel GmFAD7 gene (named GmFAD7-2) has been identified in soybean, with high homology to the previously annotated GmFAD7 gene. Genomic sequencing analysis together with searches at the soybean genome database further confirmed that both GmFAD7 genes were located in two different loci within the soybean genome, suggesting that the soybean omega3 plastidial desaturase FAD7 is encoded by two different paralogous genes. Both GmFAD7-1 and GmFAD7-2 genes were expressed in all soybean tissues examined, displaying their highest mRNA accumulation in leaves. This expression profile contrasted with GmFAD3A and GmFAD3B mRNA accumulation, which was very low in this tissue. These results suggested a concerted control of plastidial and reticular omega3 desaturase gene expression in soybean mature leaves. Analysis of GmFAD7 protein distribution in different soybean tissues showed that, in mature leaves, two bands were detected, coincident with the higher expression level of both GmFAD7 genes and the highest 18:3 fatty acid accumulation. By contrast, in seeds, where FAD7 activity is low, specific GmFAD7 protein conformations were observed. These GmFAD7 protein conformations were affected in vitro by changes in the redox conditions of thiol groups and iron availability. These results suggest the existence of tissue-specific post-translational regulatory mechanisms affecting the distribution and conformation of the FAD7 enzymes related with the control of its activity.

  20. Targeting the GM-CSF receptor for the treatment of CNS autoimmunity.

    PubMed

    Ifergan, Igal; Davidson, Todd S; Kebir, Hania; Xu, Dan; Palacios-Macapagal, Daphne; Cann, Jennifer; Rodgers, Jane M; Hunter, Zoe N; Pittet, Camille L; Beddow, Sara; Jones, Clare A; Prat, Alexandre; Sleeman, Matthew A; Miller, Stephen D

    2017-11-01

    In multiple sclerosis (MS), there is a growing interest in inhibiting the pro-inflammatory effects of granulocyte-macrophage colony-stimulating factor (GM-CSF). We sought to evaluate the therapeutic potential and underlying mechanisms of GM-CSF receptor alpha (Rα) blockade in animal models of MS. We show that GM-CSF signaling inhibition at peak of chronic experimental autoimmune encephalomyelitis (EAE) results in amelioration of disease progression. Similarly, GM-CSF Rα blockade in relapsing-remitting (RR)-EAE model prevented disease relapses and inhibited T cell responses specific for both the inducing and spread myelin peptides, while reducing activation of mDCs and inflammatory monocytes. In situ immunostaining of lesions from human secondary progressive MS (SPMS), but not primary progressive MS patients shows extensive recruitment of GM-CSF Rα + myeloid cells. Collectively, this study reveals a pivotal role of GM-CSF in disease relapses and the benefit of GM-CSF Rα blockade as a potential novel therapeutic approach for treatment of RRMS and SPMS. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. GM-CSF modulates autoantibody production and skin blistering in experimental epidermolysis bullosa acquisita.

    PubMed

    Samavedam, Unni Krishna S R L; Iwata, Hiroaki; Müller, Susen; Schulze, Franziska S; Recke, Andreas; Schmidt, Enno; Zillikens, Detlef; Ludwig, Ralf J

    2014-01-15

    GM-CSF activates hematopoietic cells and recruits neutrophils and macrophages to sites of inflammation. Inhibition of GM-CSF attenuates disease activity in models of chronic inflammatory disease. Effects of GM-CSF blockade were linked to modulation of the effector phase, whereas effects on early pathogenic events, for example, Ab production, have not been identified. To evaluate yet uncharacterized effects of GM-CSF on early pathogenic events in chronic inflammation, we employed immunization-induced epidermolysis bullosa acquisita (EBA), an autoimmune bullous disease caused by autoantibodies to type VII collagen. Compared to wild-type mice, upon immunization, GM-CSF(-/-) mice produced lower serum autoantibody titers, which were associated with reduced neutrophil numbers in draining lymph nodes. The same effect was observed in neutrophil-depleted wild-type mice. Neutrophil depletion in GM-CSF(-/-) mice led to a stronger inhibition, indicating that GM-CSF and neutrophils have additive functions. To characterize the contribution of GM-CSF specifically in the effector phase of EBA, disease was induced by transfer of anti-type VII collagen IgG into mice. We observed an increased GM-CSF expression, and GM-CSF blockade reduced skin blistering. Additionally, GM-CSF enhanced reactive oxygen species release and neutrophil migration in vitro. In immunization-induced murine EBA, treatment with anti-GM-CSF had a beneficial effect on established disease. We demonstrate that GM-CSF modulates both autoantibody production and skin blistering in a prototypical organ-specific autoimmune disease.

  2. Biochemical characterization of GM1 micelles-Amphotericin B interaction.

    PubMed

    Leonhard, Victoria; Alasino, Roxana V; Bianco, Ismael D; Garro, Ariel G; Heredia, Valeria; Beltramo, Dante M

    2015-01-01

    In this work a thorough characterization of the GM1 micelle-Amphotericin B (AmB) interaction was performed. The micelle formation as well as the drug loading occurs spontaneously, although influenced by the physicochemical conditions, pH and temperature. The chromatographic profile of GM1-AmB complexes at different molar ratios shows the existence of two populations. The differential absorbance of GM1, monomeric and aggregate AmB, allowed us to discriminate the presence of all of them in both fractions. Thus, we noted that at higher proportion of AmB in the complex, increases the larger population which is composed mainly of aggregated AmB. The physical behavior of these micelles shows that both GM1- AmB complexes were stable in solution for at least 30 days. However upon freeze-thawing or lyophilization-solubilization cycles, only the smallest population, enriched in monomeric AmB, showed a complete solubilization. In vitro, GM1-AmB micelles were significantly less toxic on cultured cells than other commercial micellar formulations as Fungizone, but had a similar behavior to liposomal formulations as Ambisome. Regarding the antifungal activity of the new formulation, it was very similar to that of other formulations. The characterization of these GM1-AmB complexes is discussed as a potential new formulation able to improve the antifungal therapeutic efficiency of AmB.

  3. Characterization of a cis-Golgi matrix protein, GM130

    PubMed Central

    1995-01-01

    Antisera raised to a detergent- and salt-resistant matrix fraction from rat liver Golgi stacks were used to screen an expression library from rat liver cDNA. A full-length clone was obtained encoding a protein of 130 kD (termed GM130), the COOH-terminal domain of which was highly homologous to a Golgi human auto-antigen, golgin-95 (Fritzler et al., 1993). Biochemical data showed that GM130 is a peripheral cytoplasmic protein that is tightly bound to Golgi membranes and part of a larger oligomeric complex. Predictions from the protein sequence suggest that GM130 is an extended rod-like protein with coiled-coil domains. Immunofluorescence microscopy showed partial overlap with medial- and trans-Golgi markers but almost complete overlap with the cis-Golgi network (CGN) marker, syntaxin5. Immunoelectron microscopy confirmed this location showing that most of the GM130 was located in the CGN and in one or two cisternae on the cis-side of the Golgi stack. GM130 was not re-distributed to the ER in the presence of brefeldin A but maintained its overlap with syntaxin5 and a partial overlap with the ER- Golgi intermediate compartment marker, p53. Together these results suggest that GM130 is part of a cis-Golgi matrix and has a role in maintaining cis-Golgi structure. PMID:8557739

  4. Improved purification process for cholera toxin and its application to the quantification of residual toxin in cholera vaccines.

    PubMed

    Jang, Hyun; Kim, Hyo Seung; Kim, Jeong Ah; Seo, Jin Ho; Carbis, Rodney

    2009-01-01

    A simplified method for the purification of cholera toxin was developed. The 569B strain of Vibrio cholerae, a recognized hyper-producer of cholera toxin, was propagated in a bioreactor under conditions that promote the production of the toxin. The toxin was separated from the bacterial cells using 0.2-microm crossflow microfiltration, the clarified toxin was passed through the membrane into the permeate, and the bacterial cells were retained in the retentate. The 0.2-microm permeate was then concentrated 3-fold and diafiltered against 10 mM phosphate buffer, pH 7.6, using 30-kDa crossflow ultrafiltration. The concentrated toxin was loaded onto a cation exchange column, the toxin was bound to the column, and most of the impurities were passed unimpeded through the column. The toxin was eluted with a salt gradient of phosphate buffer, pH7.0, containing 1.0M NaCl. The peak containing the toxin was assayed for cholera toxin and protein and the purity was determined to be 92%. The toxin peak had a low endotoxin level of 3.1 EU/microg of toxin. The purified toxin was used to prepare antiserum against whole toxin, which was used in a G(M1) ganglioside-binding ELISA to determine residual levels of toxin in an oral inactivated whole-cell cholera vaccine. The G(M1) ganglioside-binding ELISA was shown to be very sensitive and capable of detecting as little as 1 ng/ml of cholera toxin.

  5. Global value of GM rice: a review of expected agronomic and consumer benefits.

    PubMed

    Demont, Matty; Stein, Alexander J

    2013-06-25

    Unlike the other major crops, no genetically modified (GM) varieties of rice have been commercialized at a large scale. Within the next 2-3 years new transgenic rice varieties could be ready for regulatory approval and subsequent commercialization, though. Given the importance of rice as staple crop for many of the world's poorest people, this will have implications for the alleviation of poverty, hunger and malnutrition. Thus, policy-makers need to be aware of the potential benefits of GM rice. We provide an overview of the literature and discuss the evidence on expected agronomic and consumer benefits of genetically engineered rice. We find that while GM rice with improved agronomic traits could deliver benefits similar to already commercialized biotechnology crops, expected benefits of consumer traits could be higher by an order of magnitude. By aggregating the expected annual benefits, we estimate the global value of GM rice to be US$64 billion per year. This is only an indicative value, as more GM varieties will become available in future. Nevertheless, such a figure can help guide policy-makers when deciding on the approval or funding of biotechnology crops and it may also raise awareness among consumers about what is at stake for their societies. Copyright © 2013 Elsevier B.V. All rights reserved.

  6. High-gain dipole antenna using polydimethylsiloxane-glass microsphere (PDMS-GM) substrate for 5G applications

    NASA Astrophysics Data System (ADS)

    Muhamad, Wan Asilah Wan; Ngah, Razali; Jamlos, Mohd Faizal; Soh, Ping Jack; Ali, Mohd Tarmizi

    2017-01-01

    A new dipole antenna designed using polydimethylsiloxane-glass microsphere (PDMS-GM) substrate is presented. The PDMS-GM substrate offered a lower permittivity of 1.85 compared to pure PDMS of 2.7. This resulted in a wide operating frequency range from 19 GHz up to more than 45 GHz, indicating a bandwidth of more than 28 GHz. The proposed PDMS-GM antenna featured a gain of up to 13.3 dB compared to pure PDMS which only produced 13 GHz of bandwidth and 5.5 dB gain. Instead of wide bandwidth and high gain, the proposed antenna is capable of becoming water resistant by covering its radiator and SMA connector. Such capabilities of the new PDMS-GM antenna indicated suitability for the fifth-generation (5G) wireless communication systems.

  7. Cerebellar Alterations and Gait Defects as Therapeutic Outcome Measures for Enzyme Replacement Therapy in α-Mannosidosis

    PubMed Central

    Damme, Markus; Stroobants, Stijn; Walkley, Steven U.; Lüllmann-Rauch, Renate; D`Hooge, Rudi; Fogh, Jens; Saftig, Paul; Lübke, Torben; Blanz, Judith

    2011-01-01

    α-Mannosidosis is a rare lysosomal storage disease with accumulation of undegraded mannosyl-linked oligosaccharides in cells throughout the body, most notably in the CNS. This leads to a broad spectrum of neurological manifestations, including progressive intellectual impairment, disturbed motor functions and cerebellar atrophy. To develop therapeutic outcome measures for enzyme replacement therapy (ERT) that could be used for human patients, a gene knockout model of α-mannosidosis in mice was analyzed for CNS pathology and motor deficits. In the cerebellar molecular layer, α-mannosidosis mice display clusters of activated Bergman glia, infiltration of phagocytic macrophages and accumulation of free cholesterol and gangliosides (GM1), notably in regions lacking Purkinje cells. α-mannosidosis brain lysates also displayed increased expression of Lamp1 and hyperglycosylation of the cholesterol binding protein NPC2. Detailed assessment of motor function revealed age-dependent gait defects in the mice that resemble the disturbed motor function in human patients. Short-term ERT partially reversed the observed cerebellar pathology with fewer activated macrophages and astrocytes but unchanged levels of hyperglycosylated NPC2, gangliosides and cholesterol. The present study demonstrates cerebellar alterations in α-mannosidosis mice that relate to the motor deficits and pathological changes seen in human patients and can be used as therapeutic outcome measures. PMID:21157375

  8. Functional paralysis of GM-CSF-derived bone marrow cells productively infected with ectromelia virus.

    PubMed

    Szulc-Dąbrowska, Lidia; Struzik, Justyna; Ostrowska, Agnieszka; Guzera, Maciej; Toka, Felix N; Bossowska-Nowicka, Magdalena; Gieryńska, Małgorzata M; Winnicka, Anna; Nowak, Zuzanna; Niemiałtowski, Marek G

    2017-01-01

    Ectromelia virus (ECTV) is an orthopoxvirus responsible for mousepox, a lethal disease of certain strains of mice that is similar to smallpox in humans, caused by variola virus (VARV). ECTV, similar to VARV, exhibits a narrow host range and has co-evolved with its natural host. Consequently, ECTV employs sophisticated and host-specific strategies to control the immune cells that are important for induction of antiviral immune response. In the present study we investigated the influence of ECTV infection on immune functions of murine GM-CSF-derived bone marrow cells (GM-BM), comprised of conventional dendritic cells (cDCs) and macrophages. Our results showed for the first time that ECTV is able to replicate productively in GM-BM and severely impaired their innate and adaptive immune functions. Infected GM-BM exhibited dramatic changes in morphology and increased apoptosis during the late stages of infection. Moreover, GM-BM cells were unable to uptake and process antigen, reach full maturity and mount a proinflammatory response. Inhibition of cytokine/chemokine response may result from the alteration of nuclear translocation of NF-κB, IRF3 and IRF7 transcription factors and down-regulation of many genes involved in TLR, RLR, NLR and type I IFN signaling pathways. Consequently, GM-BM show inability to stimulate proliferation of purified allogeneic CD4+ T cells in a primary mixed leukocyte reaction (MLR). Taken together, our data clearly indicate that ECTV induces immunosuppressive mechanisms in GM-BM leading to their functional paralysis, thus compromising their ability to initiate downstream T-cell activation events.

  9. Neutralization and clearance of GM-CSF by autoantibodies in pulmonary alveolar proteinosis

    PubMed Central

    Piccoli, Luca; Campo, Ilaria; Fregni, Chiara Silacci; Rodriguez, Blanca Maria Fernandez; Minola, Andrea; Sallusto, Federica; Luisetti, Maurizio; Corti, Davide; Lanzavecchia, Antonio

    2015-01-01

    Pulmonary alveolar proteinosis (PAP) is a severe autoimmune disease caused by autoantibodies that neutralize GM-CSF resulting in impaired function of alveolar macrophages. In this study, we characterize 21 GM-CSF autoantibodies from PAP patients and find that somatic mutations critically determine their specificity for the self-antigen. Individual antibodies only partially neutralize GM-CSF activity using an in vitro bioassay, depending on the experimental conditions, while, when injected in mice together with human GM-CSF, they lead to the accumulation of a large pool of circulating GM-CSF that remains partially bioavailable. In contrast, a combination of three non-cross-competing antibodies completely neutralizes GM-CSF activity in vitro by sequestering the cytokine in high-molecular-weight complexes, and in vivo promotes the rapid degradation of GM-CSF-containing immune complexes in an Fc-dependent manner. Taken together, these findings provide a plausible explanation for the severe phenotype of PAP patients and for the safety of treatments based on single anti-GM-CSF monoclonal antibodies. PMID:26077231

  10. The Structure of the GM-CSF Receptor Complex Reveals a Distinct Mode of Cytokine Receptor Activation

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Hansen, Guido; Hercus, Timothy R.; McClure, Barbara J.

    Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pleiotropic cytokine that controls the production and function of blood cells, is deregulated in clinical conditions such as rheumatoid arthritis and leukemia, yet offers therapeutic value for other diseases. Its receptors are heterodimers consisting of a ligand-specific {alpha} subunit and a {beta}c subunit that is shared with the interleukin (IL)-3 and IL-5 receptors. How signaling is initiated remains an enigma. We report here the crystal structure of the human GM-CSF/GM-CSF receptor ternary complex and its assembly into an unexpected dodecamer or higher-order complex. Importantly, mutagenesis of the GM-CSF receptor at the dodecamer interface andmore » functional studies reveal that dodecamer formation is required for receptor activation and signaling. This unusual form of receptor assembly likely applies also to IL-3 and IL-5 receptors, providing a structural basis for understanding their mechanism of activation and for the development of therapeutics.« less

  11. Utilization of satellite-satellite tracking data for determination of the geocentric gravitational constant (GM)

    NASA Technical Reports Server (NTRS)

    Martin, C. F.; Oh, I. H.

    1979-01-01

    Range rate tracking of GEOS 3 through the ATS 6 satellite was used, along with ground tracking of GEOS 3, to estimate the geocentric gravitational constant (GM). Using multiple half day arcs, a GM of 398600.52 + or - 0.12 cu km/sq sec was estimated using the GEM 10 gravity model, based on speed of light of 299792.458 km/sec. Tracking station coordinates were simultaneously adjusted, leaving geopotential model error as the dominant error source. Baselines between the adjusted NASA laser sites show better than 15 cm agreement with multiple short arc GEOS 3 solutions.

  12. Recombinant rabies virus expressing dog GM-CSF is an efficacious oral rabies vaccine for dogs.

    PubMed

    Zhou, Ming; Wang, Lei; Zhou, Songqin; Wang, Zhao; Ruan, Juncheng; Tang, Lijun; Jia, Ziming; Cui, Min; Zhao, Ling; Fu, Zhen F

    2015-11-17

    Developing efficacious oral rabies vaccines is an important step to increase immunization coverage for stray dogs, which are not accessible for parenteral vaccination. Our previous studies have demonstrated that recombinant rabies virus (RABV) expressing cytokines/chemokines induces robust protective immune responses after oral immunization in mice by recruiting and activating dendritic cells (DCs) and B cells. To develop an effective oral rabies vaccine for dogs, a recombinant attenuated RABV expressing dog GM-CSF, designated as LBNSE-dGM-CSF was constructed and used for oral vaccination in a dog model. Significantly more DCs or B cells were activated in the peripheral blood of dogs vaccinated orally with LBNSE-dGM-CSF than those vaccinated with the parent virus LBNSE, particularly at 3 days post immunization (dpi). As a result, significantly higher levels of virus neutralizing antibodies (VNAs) were detected in dogs immunized with LBNSE-dGM-CSF than with the parent virus. All the immunized dogs were protected against a lethal challenge with 4500 MICLD50 of wild-type RABV SXTYD01. LBNSE-dGM-CSF was found to replicate mainly in the tonsils after oral vaccination as detected by nested RT-PCR and immunohistochemistry. Taken together, our results indicate that LBNSE-dGM-CSF could be a promising oral rabies vaccine candidate for dogs.

  13. Risk, regulation and biotechnology: the case of GM crops.

    PubMed

    Smyth, Stuart J; Phillips, Peter W B

    2014-07-03

    The global regulation of products of biotechnology is increasingly divided. Regulatory decisions for genetically modified (GM) crops in North America are predictable and efficient, with numerous countries in Latin and South America, Australia and Asia following this lead. While it might have been possible to argue that Europe's regulations were at one time based on real concerns about minimizing risks and ensuring health and safety, it is increasingly apparent that the entire European Union (EU) regulatory system for GM crops and foods is now driven by political agendas. Countries within the EU are at odds with each other as some have commercial production of GM crops, while others refuse to even develop regulations that could provide for the commercial release of GM crops. This divide in regulatory decision-making is affecting international grain trade, creating challenges for feeding an increasing global population.

  14. Nitrogen dioxide induced changes in level of free fatty acids, triglyceride, esterified fatty acid, ganglioside and lipase activity in the guinea pig brain

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Farahani, H.; Hasan, M.

    1992-02-01

    The biochemical response to controlled inhalation of nitrogen dioxide (NO2) was studied in 18 male guinea pigs. Animals were exposed to 2.5, 5.0, and 10 ppm NO2 for 2h daily for 35 consecutive days, and the results compared with six control animals exposed to filtered air for 2h daily for same period. Five biochemical parameters, including triglyceride, free fatty acids, esterified fatty acid, ganglioside and lipase activity were measured immediately after the last day of exposure. At 2.5 ppm NO2 inhalation no significant changes occurred in any region of the central nervous system (CNS). While as the dose concentration wasmore » increased to 5 and 10 ppm nitrogen dioxide, significant dose-related alteration were observed in the levels of triglyceride, free fatty acid, esterified fatty acid, ganglioside and lipase activity in the different regions of the guinea pig CNS.« less

  15. Functional analysis of the GmESR1 gene associated with soybean regeneration

    PubMed Central

    Chen, Qingshan; Liu, Ming; Xin, Dawei; Qi, Zhaoming; Li, Sinan; Ma, Yanlong; Wang, Lingshuang; Jin, Yangmei; Li, Wenbin; Wu, Xiaoxia; Su, An-yu

    2017-01-01

    Plant regeneration can occur via in vitro tissue culture through somatic embryogenesis or de novo shoot organogenesis. Transformation of soybean (Glycine max) is difficult, hence optimization of the transformation system for soybean regeneration is required. This study investigated ENHANCER OF SHOOT REGENERATION 1 (GmESR1), a soybean transcription factor that targets regeneration-associated genes. Sequence analysis showed that GmESR1 contained a conserved 57 amino acid APETALA 2 (AP2)/ETHYLENE RESPONSE FACTOR (ERF) DNA-binding domain. The relative expression level of GmESR1 was highest in young embryos, flowers and stems in the soybean cultivar ‘Dongnong 50’. To examine the function of GmESR1, transgenic Arabidopsis (Arabidopsis thaliana) and soybean plants overexpressing GmESR1 were generated. In Arabidopsis, overexpression of GmESR1 resulted in accelerated seed germination, and seedling shoot and root elongation. In soybean overexpression of GmESR1 also led to faster seed germination, and shoot and root elongation. GmESR1 specifically bound to the GCC-box. The results provide a foundation for the establishment of an efficient and stable transformation system for soybean. PMID:28403182

  16. Glycolipids as indicators of tumorigenesis.

    PubMed

    Morré, D J; Kloppel, T M; Merritt, W D; Keenan, T W

    1978-01-01

    Hyperplastic liver nodules and hepatocellular carcinomas were induced in rats by oral administration of the carcinogen N-2-fluorenylacetamide. Neoplastic tissue was compared with control, fetal, neonatal, and precancerous liver tissues. The development of the tumors was slow, such that temporal changes in the biochemical and morphologic development of carcinogenesis could be identified. Ganglioside sialic acid levels were elevated in all but the most poorly differentiated tumors. Experiments to monitor individual enzymes suggested that the alterations in glycolipid composition were a direct effect of alterations in biosynthetic activities. The pattern during tumorigenesis was the inverse of that during normal development. Also, ganglioside patterns showed a progressive simplification from hyperplastic nodules to well-differentiated hepatomas and through two grades of poorly differentiated hepatomas. An increase in the activity of the branchpoint enzyme of ganglioside biosynthesis preceded both a decrease in the branchpoint enzyme of the disialoganglioside pathway and a marked increase in the galactosyltranferase of GM1 formation. The results indicate that ganglioside deletions are the end result of a cascade of events in the tumorigenic transformation. The onset of ganglioside deletions but not of the cascade per se may correlate with the onset of malignancy. Glycolipid levels are elevated early in certain surrounding tissues especially in the blood. In rats bearing transplantable hepatomas, serum levels of lipid-bound sialic acid were elevated 2.5-fold. Similar results were obtained with sera of mice bearing transplantable mammary carcinomas and of cancer patients. These findings provide new emphasis for gangliosides in both cancer detection and as regulatory signals for growth and multiplication of cells.

  17. Exploitation of molecular profiling techniques for GM food safety assessment.

    PubMed

    Kuiper, Harry A; Kok, Esther J; Engel, Karl-Heinz

    2003-04-01

    Several strategies have been developed to identify unintended alterations in the composition of genetically modified (GM) food crops that may occur as a result of the genetic modification process. These include comparative chemical analysis of single compounds in GM food crops and their conventional non-GM counterparts, and profiling methods such as DNA/RNA microarray technologies, proteomics and metabolite profiling. The potential of profiling methods is obvious, but further exploration of specificity, sensitivity and validation is needed. Moreover, the successful application of profiling techniques to the safety evaluation of GM foods will require linked databases to be built that contain information on variations in profiles associated with differences in developmental stages and environmental conditions.

  18. Constantly evolving safety assessment protocols for GM foods.

    PubMed

    Sesikeran, B; Vasanthi, Siruguri

    2008-01-01

    he introduction of GM foods has led to the evolution of a food safety assessment paradigm that establishes safety of the GM food relative to its conventional counterpart. The GM foods currently approved and marketed in several countries have undergone extensive safety testing under a structured safety assessment framework evolved by international organizations like FAO, WHO, Codex and OECD. The major elements of safety assessment include molecular characterization of inserted genes and stability of the trait, toxicity and allergenicity potential of the expressed substances, compositional analysis, potential for gene transfer to gut microflora and unintentional effects of the genetic modification. As more number and type of food crops are being brought under the genetic modification regime, the adequacy of existing safety assessment protocols for establishing safety of these foods has been questioned. Such crops comprise GM crops with higher agronomic vigour, nutritional or health benefit/ by modification of plant metabolic pathways and those expressing bioactive substances and pharmaceuticals. The safety assessment challenges of these foods are the potential of the methods to detect unintentional effects with higher sensitivity and rigor. Development of databases on food compositions, toxicants and allergens is currently seen as an important aid to development of safety protocols. With the changing global trends in genetic modification technology future challenge would be to develop GM crops with minimum amount of inserted foreign DNA so as to reduce the burden of complex safety assessments while ensuring safety and utility of the technology.

  19. Accumulated α-synuclein affects the progression of GM2 gangliosidoses.

    PubMed

    Suzuki, Kyoko; Yamaguchi, Akira; Yamanaka, Shoji; Kanzaki, Seiichi; Kawashima, Masato; Togo, Takashi; Katsuse, Omi; Koumitsu, Noriko; Aoki, Naoya; Iseki, Eizo; Kosaka, Kenji; Yamaguchi, Kayoko; Hashimoto, Makoto; Aoki, Ichiro; Hirayasu, Yoshio

    2016-10-01

    The accumulation of α-synuclein (ASyn) has been observed in several lysosomal storage diseases (LSDs) but it remains unclear if ASyn accumulation contributes to LSD pathology. ASyn also accumulates in the neurons of Sandhoff disease (SD) patients and SD model mice (Hexb-/- ASyn+/+ mice). SD is a lysosomal storage disorder caused by the absence of a functional β-subunit on the β-hexosaminidase A and B enzymes, which leads to the accumulation of ganglioside in the central nervous system. Here, we explored the role of accumulated ASyn in the progression of Hexb-/- mice by creating a Hexb-/- ASyn-/- double-knockout mice. Our results show that Hexb-/- ASyn-/- mice demonstrated active microglia levels and less dopaminergic neuron loss, without altering the neuronal storage of ganglioside. The autophagy and ubiquitin proteasome pathways are defective in the neurons of Hexb-/- ASyn+/+ mice. In ultrastructural physiological studies, the mitochondria structures look degenerated and dysfunctional. As a result, expression of manganese superoxide dismutase 2 are reduced, and reactive oxygen species-mediated oxidative damage in the neurons of Hexb-/- ASyn+/+ mice. Interestingly, these dysfunctions improved in Hexb-/- ASyn-/- mice. But any clinical improvement were hardly observed in Hexb-/- ASyn-/- mice. Taken together, these findings suggest that ASyn accumulation plays an important role in the pathogenesis of neuropathy in SD and other LSDs, and is therefore a target for novel therapies. Copyright © 2016 Elsevier Inc. All rights reserved.

  20. Consumer choice: Linking consumer intentions to actual purchase of GM labeled food products.

    PubMed

    Sleenhoff, Susanne; Osseweijer, Patricia

    2013-01-01

    With a mandatory labeling scheme for GM food in Europe since 2004 measuring actual consumer choice in practice has become possible. Anticipating Europeans negative attitude toward GM food, the labeling was enforced to allow consumers to make an informed choice. We studied consumers actual purchase behavior of GM food products and compared this with their attitude and behavioral intention for buying GM food. We found that despite a majority of consumers voicing a negative attitude toward GM food over 50% of our European respondents stated that they did not actively avoid the purchase of GM food and 6% actually purchased one of the few available GM labeled food products in the period between September 2006 and October 2007. Our results imply that a voiced negative attitude of consumers in responses to questionnaires about their intentions is not a reliable guide for what they actually do in supermarkets. We conclude that the assumption of a negative attitude with regard to GM food is at least in part construed.

  1. Risk, regulation and biotechnology: The case of GM crops

    PubMed Central

    Smyth, Stuart J; Phillips, Peter WB

    2014-01-01

    The global regulation of products of biotechnology is increasingly divided. Regulatory decisions for genetically modified (GM) crops in North America are predictable and efficient, with numerous countries in Latin and South America, Australia and Asia following this lead. While it might have been possible to argue that Europe's regulations were at one time based on real concerns about minimizing risks and ensuring health and safety, it is increasingly apparent that the entire European Union (EU) regulatory system for GM crops and foods is now driven by political agendas. Countries within the EU are at odds with each other as some have commercial production of GM crops, while others refuse to even develop regulations that could provide for the commercial release of GM crops. This divide in regulatory decision-making is affecting international grain trade, creating challenges for feeding an increasing global population. PMID:25437235

  2. Applying a weed risk assessment approach to GM crops.

    PubMed

    Keese, Paul K; Robold, Andrea V; Myers, Ruth C; Weisman, Sarah; Smith, Joe

    2014-12-01

    Current approaches to environmental risk assessment of genetically modified (GM) plants are modelled on chemical risk assessment methods, which have a strong focus on toxicity. There are additional types of harms posed by plants that have been extensively studied by weed scientists and incorporated into weed risk assessment methods. Weed risk assessment uses robust, validated methods that are widely applied to regulatory decision-making about potentially problematic plants. They are designed to encompass a broad variety of plant forms and traits in different environments, and can provide reliable conclusions even with limited data. The knowledge and experience that underpin weed risk assessment can be harnessed for environmental risk assessment of GM plants. A case study illustrates the application of the Australian post-border weed risk assessment approach to a representative GM plant. This approach is a valuable tool to identify potential risks from GM plants.

  3. Which AML subsets benefit from leukemic cell priming during chemotherapy? Long-term analysis of the ALFA-9802 GM-CSF study.

    PubMed

    Thomas, Xavier; Raffoux, Emmanuel; Renneville, Aline; Pautas, Cecile; de Botton, Stephane; Terre, Christine; Gardin, Claude; Hayette, Sandrine; Preudhomme, Claude; Dombret, Herve

    2010-04-01

    : Priming with granulocytic hematopoietic growth factors may modulate cell cycle kinetics of leukemic cells and render them more susceptible to phase-specific chemotherapeutic agents. In a first report, we have shown that priming with granulocyte-macrophage colony-stimulating factor (GM-CSF) may enhance complete remission (CR) rate and event-free survival (EFS) in younger adults with acute myeloid leukemia (AML). : In this randomized trial, 259 patients with AML were randomized at baseline to receive or not receive GM-CSF concurrently with all cycles of chemotherapy. The effects of GM-CSF on survival were reported herein with a long-term follow-up and studied according to distinct biological subgroups defined on cytogenetics and molecular markers. : The EFS rate was better in the GM-CSF group (43% vs 34%; P = .04). GM-CSF did not improve the outcome in patients from good risk subgroups, while patients from poor risk subgroups benefited from GM-CSF therapy. In this population, the difference in terms of EFS probability was mainly observed in patients with high initial white blood cell count and in those with FLT3-ITD or MLL rearrangement. When combining these 2 molecular abnormalities for comparison of the effect of GM-CSF priming, the difference in terms of EFS was highly significant (5-year EFS, 39% with GM-CSF vs 8% without GM-CSF; P = .007). : Sensitization of leukemic cells and their progenitors by GM-CSF appears as a plausible strategy for improving the outcome of patients with newly diagnosed AML. Patients with poor-prognosis FLT3-ITD or MLL rearrangement might be a good target population to further investigate priming strategies. Cancer 2010. (c) 2010 American Cancer Society.

  4. MafB antagonizes phenotypic alteration induced by GM-CSF in microglia

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Koshida, Ryusuke, E-mail: rkoshida-myz@umin.ac.jp; Oishi, Hisashi, E-mail: hoishi@md.tsukuba.ac.jp; Hamada, Michito

    2015-07-17

    Microglia are tissue-resident macrophages which are distributed throughout the central nervous system (CNS). Recent studies suggest that microglia are a unique myeloid population distinct from peripheral macrophages in terms of origin and gene expression signature. Granulocyte-macrophage colony-stimulating factor (GM-CSF), a pleiotropic cytokine regulating myeloid development, has been shown to stimulate proliferation and alter phenotype of microglia in vitro. However, how its signaling is modulated in microglia is poorly characterized. MafB, a bZip transcriptional factor, is highly expressed in monocyte-macrophage lineage cells including microglia, although its role in microglia is largely unknown. We investigated the crosstalk between GM-CSF signaling and MafB bymore » analyzing primary microglia. We found that Mafb-deficient microglia grew more rapidly than wild-type microglia in response to GM-CSF. Moreover, the expression of genes associated with microglial differentiation was more downregulated in Mafb-deficient microglia cultured with GM-CSF. Notably, such differences between the genotypes were not observed in the presence of M-CSF. In addition, we found that Mafb-deficient microglia cultured with GM-CSF barely extended their membrane protrusions, probably due to abnormal activation of RhoA, a key regulator of cytoskeletal remodeling. Altogether, our study reveals that MafB is a negative regulator of GM-CSF signaling in microglia. These findings could provide new insight into the modulation of cytokine signaling by transcription factors in microglia. - Highlights: • GM-CSF alters the phenotype of microglia in vitro more potently than M-CSF. • Transcription factor MafB antagonizes the effect of GM-CSF on microglia in vitro. • MafB deficiency leads to RhoA activation in microglia in response to GM-CSF. • We show for the first time the function of MafB in microglia.« less

  5. Safety assessment of GM plants: An updated review of the scientific literature.

    PubMed

    Domingo, José L

    2016-09-01

    In a wide revision of the literature conducted in 2000, I noted that the information in scientific journals on the safety of genetically modified (GM) foods in general, and GM plants in particular, was scarce. Of course, it was not sufficient to guarantee that the consumption of these products should not mean risks for the health of the consumers. Because of the scientific interest in GM organisms (GMOs), as well as the great concern that the consumption of GM foods/plants has raised in a number of countries, I conducted two subsequent revisions (2007 and 2011) on the adverse/toxic effects of GM plants. In the present review, I have updated the information on the potential adverse health effects of GM plants consumed as food and/or feed. With only a few exceptions, the reported studies in the last six years show rather similar conclusions; that is to say, the assessed GM soybeans, rice, corn/maize and wheat would be as safe as the parental species of these plants. However, in spite of the notable increase in the available information, studies on the long-term health effects of GM plants, including tests of mutagenicity, teratogenicity and carcinogenicity seem to be still clearly necessary. Copyright © 2016 Elsevier Ltd. All rights reserved.

  6. Public Acceptance of Plant Biotechnology and GM Crops.

    PubMed

    Lucht, Jan M

    2015-07-30

    A wide gap exists between the rapid acceptance of genetically modified (GM) crops for cultivation by farmers in many countries and in the global markets for food and feed, and the often-limited acceptance by consumers. This review contrasts the advances of practical applications of agricultural biotechnology with the divergent paths-also affecting the development of virus resistant transgenic crops-of political and regulatory frameworks for GM crops and food in different parts of the world. These have also shaped the different opinions of consumers. Important factors influencing consumer's attitudes are the perception of risks and benefits, knowledge and trust, and personal values. Recent political and societal developments show a hardening of the negative environment for agricultural biotechnology in Europe, a growing discussion-including calls for labeling of GM food-in the USA, and a careful development in China towards a possible authorization of GM rice that takes the societal discussions into account. New breeding techniques address some consumers' concerns with transgenic crops, but it is not clear yet how consumers' attitudes towards them will develop. Discussions about agriculture would be more productive, if they would focus less on technologies, but on common aims and underlying values.

  7. G-CSF and GM-CSF in Neutropenia

    PubMed Central

    Mehta, Hrishikesh M.; Malandra, Michael; Corey, Seth J.

    2015-01-01

    Granulocyte Colony Stimulating Factor (G-CSF) and Granulocyte/Macrophage Colony Stimulating Factor (GM-CSF) are used widely to promote the production of granulocytes or antigen presenting cells (APC). The Food and Drug Administration approved G-CSF (filgrastim) for the treatment of congenital and acquired neutropenias and for mobilization of peripheral hematopoietic progenitor cells for stem cell transplantation. A polyethylene glycol modified (PEGylated) form of G-CSF is approved for the treatment of neutropenias. Clinically significant neutropenia, rendering an individual immunocompromised, occurs when their number is less than 1500/µl. Current guidelines recommend their use when the risk of febrile neutropenia is greater than 20%. GM-CSF (sargramostim) is approved for neutropenia associated with stem cell transplantation. Because of its promotion of APC function, GM-CSF is being evaluated as an immunostimulatory adjuvant in a number of clinical trials. More than 20 million persons have benefited worldwide, and more than $5 billion sales occur annually in the United States. PMID:26254266

  8. Protoplanetary disk formation and evolution models: DM Tau and GM Aur

    NASA Astrophysics Data System (ADS)

    Hueso, R.; Guillot, T.

    2002-09-01

    We study the formation and evolution of protoplanetary disks using an axisymmetric turbulent disk model. We compare model results with observational parameters derived for the DM Tau and GM Aur systems. These are relatively old T Tauri stars with large and massive protoplanetary disks. Early disk formation is studied in the standard scenario of slowly rotating isothermal collapsing spheres and is strongly dependent on the initial angular momentum and the collapse accretion rate. The viscous evolution of the disk is integrated in time using the classical Alpha prescription of turbulence. We follow the temporal evolution of the disks until their characteristics fit the observed characteristics of DM Tau and GM Aur. We therefore obtain the set of model parameters that are able to explain the present state of these disks. We also study the disk evolution under the Beta parameterization of turbulence, recently proposed for sheared flows on protoplanetary disks. Both parameterizations allow explaining the present state of both DM Tau and GM Aur. We infer a value of Alpha between 5x10-3 to 0.02 for DM Tau and one order of magnitude smaller for GM Aur. Values of the Beta parameter are in accordance with theoretical predictions of Beta around 2x10-5 but with a larger dispersion on other model parameters, which make us favor the Alpha parameterization of turbulence. Implications for planetary system development in these systems are presented. In particular, GM Aur is a massive and slowly evolving disk where conditions are very favorable for planetesimal growth. The large value of present disk mass and the relatively small observed accretion rate of this system may also be indicative of the presence of an inner gas giant planet. Acknowledgements: This work has been supported by Programme Nationale de Planetologie. R. Hueso acknowledges a post-doctoral fellowship from Gobierno Vasco.

  9. Genome sequences of two closely related strains of Escherichia coli K-12 GM4792.

    PubMed

    Zhang, Yan-Cong; Zhang, Yan; Zhu, Bi-Ru; Zhang, Bo-Wen; Ni, Chuan; Zhang, Da-Yong; Huang, Ying; Pang, Erli; Lin, Kui

    2015-01-01

    Escherichia coli lab strains K-12 GM4792 Lac(+) and GM4792 Lac(-) carry opposite lactose markers, which are useful for distinguishing evolved lines as they produce different colored colonies. The two closely related strains are chosen as ancestors for our ongoing studies of experimental evolution. Here, we describe the genome sequences, annotation, and features of GM4792 Lac(+) and GM4792 Lac(-). GM4792 Lac(+) has a 4,622,342-bp long chromosome with 4,061 protein-coding genes and 83 RNA genes. Similarly, the genome of GM4792 Lac(-) consists of a 4,621,656-bp chromosome containing 4,043 protein-coding genes and 74 RNA genes. Genome comparison analysis reveals that the differences between GM4792 Lac(+) and GM4792 Lac(-) are minimal and limited to only the targeted lac region. Moreover, a previous study on competitive experimentation indicates the two strains are identical or nearly identical in survivability except for lactose utilization in a nitrogen-limited environment. Therefore, at both a genetic and a phenotypic level, GM4792 Lac(+) and GM4792 Lac(-), with opposite neutral markers, are ideal systems for future experimental evolution studies.

  10. GM-CSF has disparate roles during intranasal and intradermal Francisella tularensis infection.

    PubMed

    Kurtz, Sherry L; Bosio, Catharine M; De Pascalis, Roberto; Elkins, Karen L

    2016-12-01

    Our laboratory has employed in vitro and in vivo mouse models based on Francisella tularensis Live Vaccine Strain (LVS)-induced protection to elucidate immune correlates for intracellular bacteria. Among the effectors found was GM-CSF, a pleiotropic cytokine that is integral to the development and proliferation of myeloid cells, including alveolar macrophages. GM-CSF has roles in resistance to primary murine infection with several intracellular pathogens, but its role during Francisella infection is unknown. Francisella is an intracellular pathogen that infects lungs after inhalation, primarily invading alveolar macrophages. Here we show that GM-CSF has route-dependent roles during primary infection of mice with LVS. GM-CSF deficient (GM-CSF KO) mice were slightly more susceptible than wild type to intradermal infection, but had increased resistance to intranasal infection. Similarly, these mice had increased resistance to pulmonary infection with virulent F. tularensis (SchuS4). LVS-vaccinated GM-CSF KO mice had normal adaptive immune responses, as measured by T cell activities after LVS intradermal or intranasal vaccination, and survived lethal secondary LVS challenge. GM-CSF KO mice also had robust humoral responses, producing elevated levels of serum antibodies following LVS vaccination compared to wild type mice. Taken together, our data demonstrates that the absence of GM-CSF improves resistance to pulmonary, but not intradermal, infection with Francisella. Published by Elsevier Masson SAS.

  11. Verification of Global Assimilation of Ionospheric Measurements Gauss Markov (GAIM-GM) Model Forecast Accuracy

    DTIC Science & Technology

    2011-09-01

    m b e r o f O cc u rr e n ce s 50 ( a ) Kp 0-3 (b) Kp 4-9 Figure 25. Scatter plot of...dependent physics based model that uses the Ionospheric Forecast Model ( IFM ) as a background model upon which perturbations are imposed via a Kalman filter...vertical output resolution as the IFM . GAIM-GM can also be run in a regional mode with a finer resolution (Scherliess et al., 2006). GAIM-GM is

  12. Endocytosis and recycling of AMPA receptors lacking GluR2/3.

    PubMed

    Biou, Virginie; Bhattacharyya, Samarjit; Malenka, Robert C

    2008-01-22

    Excitatory synapses in the mammalian brain contain two types of ligand-gated ion channels: AMPA receptors (AMPARs) and NMDA receptors (NMDARs). AMPARs are responsible for generating excitatory synaptic responses, whereas NMDAR activation triggers long-lasting changes in these responses by modulating the trafficking of AMPARs toward and away from synapses. AMPARs are tetramers composed of four subunits (GluR1-GluR4), which current models suggest govern distinct AMPAR trafficking behavior during synaptic plasticity. Here, we address the roles of GluR2 and GluR3 in controlling the recycling- and activity-dependent endocytosis of AMPARs by using cultured hippocampal neurons prepared from knockout (KO) mice lacking these subunits. We find that synapses and dendritic spines form normally in cells lacking GluR2/3 and that upon NMDAR activation, GluR2/3-lacking AMPARs are endocytosed in a manner indistinguishable from GluR2-containing AMPARs in wild-type (WT) neurons. AMPARs lacking GluR2/3 also recycle to the plasma membrane identically to WT AMPARs. However, because of their permeability to calcium, GluR2-lacking but not WT AMPARs exhibited robust internalization throughout the dendritic tree in response to AMPA application. Dendritic endocytosis of AMPARs also was observed in GABAergic neurons, which express a high proportion of GluR2-lacking AMPARs. These results demonstrate that GluR2 and GluR3 are not required for activity-dependent endocytosis of AMPARs and suggest that the most important property of GluR2 in the context of AMPAR trafficking may be its influence on calcium permeability.

  13. GM-CSF primes cardiac inflammation in a mouse model of Kawasaki disease

    PubMed Central

    McKenzie, Brent S.

    2016-01-01

    Kawasaki disease (KD) is the leading cause of pediatric heart disease in developed countries. KD patients develop cardiac inflammation, characterized by an early infiltrate of neutrophils and monocytes that precipitates coronary arteritis. Although the early inflammatory processes are linked to cardiac pathology, the factors that regulate cardiac inflammation and immune cell recruitment to the heart remain obscure. In this study, using a mouse model of KD (induced by a cell wall Candida albicans water-soluble fraction [CAWS]), we identify an essential role for granulocyte/macrophage colony-stimulating factor (GM-CSF) in orchestrating these events. GM-CSF is rapidly produced by cardiac fibroblasts after CAWS challenge, precipitating cardiac inflammation. Mechanistically, GM-CSF acts upon the local macrophage compartment, driving the expression of inflammatory cytokines and chemokines, whereas therapeutically, GM-CSF blockade markedly reduces cardiac disease. Our findings describe a novel role for GM-CSF as an essential initiating cytokine in cardiac inflammation and implicate GM-CSF as a potential target for therapeutic intervention in KD. PMID:27595596

  14. Apprehending ganglioside diversity: a comprehensive methodological approach[S

    PubMed Central

    Masson, Elodie A. Y.; Sibille, Estelle; Martine, Lucy; Chaux-Picquet, Fanny; Bretillon, Lionel; Berdeaux, Olivier

    2015-01-01

    Gangliosides (GGs) make a wide family of glycosphingolipids ubiquitously expressed in mammalian tissues and particularly abundant in the brain and nervous system. They exhibit a huge diversity due to structural variations in both their oligosaccharidic chain and ceramide moiety, which represent a real analytical challenge. Since their discovery in the 1940s, methods have persistently improved until the emergence of LC/MS, which offers a high level of specificity and sensitivity and is suitable with high-throughput profiling studies. We describe here a comprehensive approach relying on various techniques and aiming at fully characterizing GGs in biological samples. First, total GG content was determined by a biochemical assay. Second, GG class composition was assessed by high-performance thin-layer chromatography followed by colorimetric revelation. Then, ceramide types of GG classes were identified, and their relative quantification was performed thanks to the development of a powerful and reliable LC/MS method. Finally, ceramides were structurally characterized, and minor and less common GG classes were identified using high-resolution MS. These methods were applied to the rat retina to provide an exhaustive description of its GG composition, giving the base for a better understanding of the precise roles of GGs in this tissue. PMID:26142958

  15. Comparison of the glycosphingolipids of human-induced pluripotent stem cells and human embryonic stem cells.

    PubMed

    Säljö, Karin; Barone, Angela; Vizlin-Hodzic, Dzeneta; Johansson, Bengt R; Breimer, Michael E; Funa, Keiko; Teneberg, Susann

    2017-04-01

    High expectations are held for human-induced pluripotent stem cells (hiPSC) since they are established from autologous tissues thus overcoming the risk of allogeneic immune rejection when used in regenerative medicine. However, little is known regarding the cell-surface carbohydrate antigen profile of hiPSC compared with human embryonic stem cells (hESC). Here, glycosphingolipids were isolated from an adipocyte-derived hiPSC line, and hiPSC and hESC glycosphingolipids were compared by concurrent characterization by binding assays with carbohydrate-recognizing ligands and mass spectrometry. A high similarity between the nonacid glycosphingolipids of hiPSC and hESC was found. The nonacid glycosphingolipids P1 pentaosylceramide, x2 pentaosylceramide and H type 1 heptaosylceramide, not previously described in human pluripotent stem cells (hPSC), were characterized in both hiPSC and hESC. The composition of acid glycosphingolipids differed, with increased levels of GM3 ganglioside, and reduced levels of GD1a/GD1b in hiPSC when compared with hESC. In addition, the hESC glycosphingolipids sulf-globopentaosylceramide and sialyl-globotetraosylceramide were lacking in hiPSC. Neural stem cells differentiating from hiPSC had a reduced expression of sialyl-lactotetra, whereas expression of the GD1a ganglioside was significantly increased. Thus, while sialyl-lactotetra is a marker of undifferentiated hPSC, GD1a is a novel marker of neural differentiation. © The Author 2017. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  16. The distinct properties of natural and GM cry insecticidal proteins.

    PubMed

    Latham, Jonathan R; Love, Madeleine; Hilbeck, Angelika

    2017-04-01

    The Cry toxins are a family of crystal-forming proteins produced by the bacterium Bacillus thuringiensis. Their mode of action is thought to be to create pores that disrupt the gut epithelial membranes of juvenile insects. These pores allow pathogen entry into the hemocoel, thereby killing the insect. Genes encoding a spectrum of Cry toxins, including Cry mutants, Cry chimaeras and other Cry derivatives, are used commercially to enhance insect resistance in genetically modified (GM) crops. In most countries of the world, such GM crops are regulated and must be assessed for human and environmental safety. However, such risk assessments often do not test the GM crop or its tissues directly. Instead, assessments rely primarily on historical information from naturally occurring Cry proteins and on data collected on Cry proteins (called 'surrogates') purified from laboratory strains of bacteria engineered to express Cry protein. However, neither surrogates nor naturally occurring Cry proteins are identical to the proteins to which humans or other nontarget organisms are exposed by the production and consumption of GM plants. To-date there has been no systematic survey of these differences. This review fills this knowledge gap with respect to the most commonly grown GM Cry-containing crops approved for international use. Having described the specific differences between natural, surrogate and GM Cry proteins this review assesses these differences for their potential to undermine the reliability of risk assessments. Lastly, we make specific recommendations for improving risk assessments.

  17. Public Acceptance of Plant Biotechnology and GM Crops

    PubMed Central

    Lucht, Jan M.

    2015-01-01

    A wide gap exists between the rapid acceptance of genetically modified (GM) crops for cultivation by farmers in many countries and in the global markets for food and feed, and the often-limited acceptance by consumers. This review contrasts the advances of practical applications of agricultural biotechnology with the divergent paths—also affecting the development of virus resistant transgenic crops—of political and regulatory frameworks for GM crops and food in different parts of the world. These have also shaped the different opinions of consumers. Important factors influencing consumer’s attitudes are the perception of risks and benefits, knowledge and trust, and personal values. Recent political and societal developments show a hardening of the negative environment for agricultural biotechnology in Europe, a growing discussion—including calls for labeling of GM food—in the USA, and a careful development in China towards a possible authorization of GM rice that takes the societal discussions into account. New breeding techniques address some consumers’ concerns with transgenic crops, but it is not clear yet how consumers’ attitudes towards them will develop. Discussions about agriculture would be more productive, if they would focus less on technologies, but on common aims and underlying values. PMID:26264020

  18. Association of anti-gangliosides antibodies and anti-CMV antibodies in Guillain-Barré syndrome.

    PubMed

    Wang, Lijuan; Shao, Chunqing; Yang, Chunjiao; Kang, Xixiong; Zhang, Guojun

    2017-05-01

    Numerous types of infection were closely related to GBS, mainly including Campylobacter jejuni , Cytomegalovirus, which may lead to the production of anti-gangliosides antibodies (AGA) . Currently, although there are increased studies on the AGA and a few studies of anti-CMV antibodies in GBS, the association between them remains poorly documented. Therefore, our research aims to analyze the correlation of anti-CMV antibodies and AGA in GBS. A total of 29 patients with GBS were enrolled in this study. The CMV antibodies were tested by the electrochemiluminescence immunoassay "ECLIA" (Roche Diagnostics GmbH). The serum gangliosides were determined by The EUROLINE test kit. Of the 29 patients with GBS, 9 (31%) were AGA-seropositive, in which 22 were CMV-IgG positive in CSF at the same time, but all 29 samples were CMV-IgM negative in both serum and CSF. In the AGA-positive group, the rate of both serum and CSF positive was 87.5% (7/8), higher than 50% (7/14) of the negative group, although no statistical significance was found. In addition, we found that there was a trend of higher ratio of men, a younger age onset, less frequent preceding infection, a higher level of CSF proteins, and less frequent cranial nerve deficits, although the data did not reach a statistical significance. In spite of no statistical significance association was found between serum AGA and CMV-IgG in serum and CSF. However, we found that there was a trend of high positive rate of both serum and CSF-CMV-IgG in AGA-positive than the negative group. So we should further expand the sample size to analyze the association between AGA and CMV or other neurotropic virus antibodies in various diseases, to observe whether they could be serological marker of these diseases (especially GBS) or the underlying pathogenesis.

  19. Characterization of GM events by insert knowledge adapted re-sequencing approaches

    PubMed Central

    Yang, Litao; Wang, Congmao; Holst-Jensen, Arne; Morisset, Dany; Lin, Yongjun; Zhang, Dabing

    2013-01-01

    Detection methods and data from molecular characterization of genetically modified (GM) events are needed by stakeholders of public risk assessors and regulators. Generally, the molecular characteristics of GM events are incomprehensively revealed by current approaches and biased towards detecting transformation vector derived sequences. GM events are classified based on available knowledge of the sequences of vectors and inserts (insert knowledge). Herein we present three insert knowledge-adapted approaches for characterization GM events (TT51-1 and T1c-19 rice as examples) based on paired-end re-sequencing with the advantages of comprehensiveness, accuracy, and automation. The comprehensive molecular characteristics of two rice events were revealed with additional unintended insertions comparing with the results from PCR and Southern blotting. Comprehensive transgene characterization of TT51-1 and T1c-19 is shown to be independent of a priori knowledge of the insert and vector sequences employing the developed approaches. This provides an opportunity to identify and characterize also unknown GM events. PMID:24088728

  20. Characterization of GM events by insert knowledge adapted re-sequencing approaches.

    PubMed

    Yang, Litao; Wang, Congmao; Holst-Jensen, Arne; Morisset, Dany; Lin, Yongjun; Zhang, Dabing

    2013-10-03

    Detection methods and data from molecular characterization of genetically modified (GM) events are needed by stakeholders of public risk assessors and regulators. Generally, the molecular characteristics of GM events are incomprehensively revealed by current approaches and biased towards detecting transformation vector derived sequences. GM events are classified based on available knowledge of the sequences of vectors and inserts (insert knowledge). Herein we present three insert knowledge-adapted approaches for characterization GM events (TT51-1 and T1c-19 rice as examples) based on paired-end re-sequencing with the advantages of comprehensiveness, accuracy, and automation. The comprehensive molecular characteristics of two rice events were revealed with additional unintended insertions comparing with the results from PCR and Southern blotting. Comprehensive transgene characterization of TT51-1 and T1c-19 is shown to be independent of a priori knowledge of the insert and vector sequences employing the developed approaches. This provides an opportunity to identify and characterize also unknown GM events.

  1. Functional Analysis of GmCPDs and Investigation of Their Roles in Flowering

    PubMed Central

    Wang, Miao; Xu, Xin; Zhang, Xinxin; Sun, Shi; Wu, Cunxiang; Hou, Wensheng; Wang, Qingyu; Han, Tianfu

    2015-01-01

    The onset of floral development is a pivotal switch in the life of soybean. Brassinosteroids (BRs), a group of steroidal phytohormones with essential roles in plant growth and development, are associated with flowering induction. Genes involved in BR biosynthesis have been studied to a great extent in Arabidopsis, but the study of these genes has been limited in soybean. In this study, four CPD homologs (GmCPDs) catalyzing BR synthesis were isolated from soybean. Transcripts were mainly confined to cotyledons and leaves and were down-regulated in response to exogenous BR. Bioinformatic analysis showed strong sequence and structure similarity between GmCPDs and AtCPD as well as CPDs of other species. Overexpression of GmCPDs in an Arabidopsis BR-deficient mutant rescued the phenotype by restoring the biosynthesis pathway, revealing the functional roles of each GmCPDs in. Except for the rescue of root development, leaf expansion and plant type architecture, GmCPDs in expression also complemented the late flowering phenotype of Arabidopsis mutants deficient in CPD. Further evidence in soybean plants is that the expression levels of GmCPDs in are under photoperiod control in Zigongdongdou, a photoperiod-sensitive variety, and show a sudden peak upon floral meristem initiation. Together with increased GmCPDs in expression in the leaves and cotyledons of photoperiod-insensitive early-maturity soybean, it is clear that GmCPDs in contribute to flowering development and are essential in the early stages of flowering regulation. PMID:25734273

  2. GM1 antibodies in post-polio syndrome and previous paralytic polio.

    PubMed

    Farbu, E; Rekand, T; Tysnes, O-B; Aarli, J A; Gilhus, N E; Vedeler, C A

    2003-06-01

    We studied the relationship between post-polio syndrome (PPS) and GM1 antibodies, since such antibodies have been associated with PPS and motor neuron disorders. Sera from 144 patients with previous poliomyelitis (105 paralytic, 22 nonparalytic and 17 PPS), 60 with previous Guillain-Barré syndrome, 44 with amyotrophic lateral sclerosis (ALS) and 22 healthy blood donors were analyzed with ELISA for GM1 IgM, IgG and IgA antibodies. GM1 antibodies were present in 14% of the PPS patients, but the prevalence did not differ significantly from that of the other groups. Our study does not support the hypothesis that GM1 antibodies are involved in the pathogenesis of PPS.

  3. NMR-Metabolic Methodology in the Study of GM Foods

    PubMed Central

    Sobolev, Anatoly P.; Capitani, Donatella; Giannino, Donato; Nicolodi, Chiara; Testone, Giulio; Santoro, Flavio; Frugis, Giovanna; Iannelli, Maria A.; Mattoo, Autar K.; Brosio, Elvino; Gianferri, Raffaella; D’Amico, Irene; Mannina, Luisa

    2010-01-01

    The 1H-NMR methodology used in the study of genetically modified (GM) foods is discussed. Transgenic lettuce (Lactuca sativa cv "Luxor") over-expressing the ArabidopsisKNAT1 gene is presented as a case study. Twenty-two water-soluble metabolites (amino acids, organic acids, sugars) present in leaves of conventional and GM lettuce were monitored by NMR and quantified at two developmental stages. The NMR spectra did not reveal any difference in metabolite composition between the GM lettuce and the wild type counterpart. Statistical analyses of metabolite variables highlighted metabolism variation as a function of leaf development as well as the transgene. A main effect of the transgene was in altering sugar metabolism. PMID:22253988

  4. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is released by female mouse bladder urothelial cells and expressed by the urothelium as an early response to lipopolysaccharides (LPS).

    PubMed

    Li, Yan; Lu, Ming; Alvarez-Lugo, Lery; Chen, Gang; Chai, Toby C

    2017-04-01

    We studied in vitro and in vivo response of primary mouse bladder urothelial cells (mBUC) and bladder urothelium to lipopolysaccharides (LPS), focusing on granulocyte-macrophage colony-stimulating factor (GM-CSF) signaling. Female C57BL/6 mBUC were exposed for 12 hr to differing concentrations of LPS (100 ng/ml to 10 µg/ml). mBUC were also exposed to a single dose of LPS (1 µg/ml) for 3, 6, 12 hr. Neutralizing GM-CSF antibody (0.1 μg/ml) was used block GM-CSF activity in vitro. In vivo experiments were performed, whereby, LPS (1 mg/ml) was instilled intravesically and left to dwell for 30 min followed by harvest of bladder urothelium 3 to 18 hr later. ELISA measured GM-CSF. qPCR quantitated mRNA for GM-CSF, vascular endothelial growth factor-A (VEGF-A), cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), and tumor necrosis factor α (TNF-α). RT-PCR was used to detect mRNA for GM-CSF, GM-CSFRα, and β in bladder tissues. Immunohistofluorescence and Western blots for GM-CSFRα were performed on bladder tissues. LPS induced a dose-dependent release of GM-CSF by mBUC. Mouse bladder urothelium did not express GM-CSF mRNA at baseline, but expressed GM-CSF mRNA 3 hr after in vivo LPS exposure, with GM-CSF mRNA expression disappearing 18 hr later. GM-CSFRα expression was confirmed in bladder urothelium. GM-CSF neutralizing antibody significantly diminished LPS-induced increases of VEGF and COX-2 mRNA expression. Urothelium and mBUC secreted GM-CSF as an early response to LPS. GM-CSF mediated downstream expression of VEGF and COX-2. Urothelial GM-CSF may function as a signaling mediator for both inflammation and pain transduction. Neurourol. Urodynam. 36:1020-1025, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

  5. Reduced expression of granule proteins during extended survival of eosinophils in splenocyte culture with GM-CSF.

    PubMed

    Ryu, Seul Hye; Na, Hye Young; Sohn, Moah; Han, Sun Murray; Choi, Wanho; In, Hyunju; Hong, Sookyung; Jeon, Hyejin; Seo, Jun-Young; Ahn, Jongcheol; Park, Chae Gyu

    2016-05-01

    Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a multifaceted hematopoietic cytokine and the culture of mouse bone marrow with GM-CSF produces a variety of myeloid cells including granulocytes, macrophages, and dendritic cells. In the present study, we cultured mouse splenocytes with GM-CSF and examined the changes in hematopoietic cell populations over a week. Most of the splenic hematopoietic cells disappeared significantly from culture within 6days with or without the presence of GM-CSF. Among the splenic granulocyte populations, only eosinophils fully survived throughout the culture with GM-CSF for more than a week. During 10days of culture with GM-CSF, splenic eosinophils maintained their morphology as well as most of their surface molecules at high levels, including CCR3 and Siglec F. Meanwhile, the expression of mRNAs encoding major basic protein-1 (MBP-1) and eosinophil peroxidase (EPO), two major eosinophil-derived granule proteins, was diminished significantly from the cultured eosinophils. EPO assays also revealed that eosinophils in culture for more than 5days retained 30% or less EPO activity compared to those in uncultured splenocytes. In contrast, culture of splenocytes with GM-CSF did not change the capacity of eosinophils to migrate in response to eotaxin-1. Our results indicate that mouse splenic eosinophils are effectively cultured for lengthy periods while their expression of eosinophil-derived granule proteins is specifically suppressed. The relevance of these findings to eosinophilic inflammatory response is discussed. Copyright © 2016 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

  6. Measuring Positive Cooperativity Using the Direct ESI-MS Assay. Cholera Toxin B Subunit Homopentamer Binding to GM1 Pentasaccharide

    NASA Astrophysics Data System (ADS)

    Lin, Hong; Kitova, Elena N.; Klassen, John S.

    2014-01-01

    Direct electrospray ionization mass spectrometry (ESI-MS) assay was used to investigate the stepwise binding of the GM1 pentasaccharide β- D-Gal p-(1→3)-β-D-Gal pNAc-(1→4)[α-D-Neu5Ac-(2→3)]-β- D-Gal p-(1→4)-β-D-Glc p (GM1os) to the cholera toxin B subunit homopentamer (CTB5) and to establish conclusively whether GM1os binding is cooperative. Apparent association constants were measured for the stepwise addition of one to five GM1os to CTB5 at pH 6.9 and 22 °C. The intrinsic association constant, which was established from the apparent association constant for the addition of a single GM1os to CTB5, was found to be (3.2 ± 0.2) × 106 M-1. This is in reasonable agreement with the reported value of (6.4 ± 0.3) × 106 M-1, which was measured at pH 7.4 and 25 °C using isothermal titration calorimetry (ITC). Analysis of the apparent association constants provides direct and unambiguous evidence that GM1os binding exhibits small positive cooperativity. Binding was found to be sensitive to the number of ligand-bound nearest neighbor subunits, with the affinities enhanced by a factor of 1.7 and 2.9 when binding occurs next to one or two ligand-bound subunits, respectively. These findings, which provide quantitative support for the binding model proposed by Homans and coworkers [14], highlight the unique strengths of the direct ESI-MS assay for measuring cooperative ligand binding.

  7. Response to issues on GM agriculture in Africa: Are transgenic crops safe?

    PubMed

    Adenle, Ademola A

    2011-10-08

    The controversies surrounding transgenic crops, often called Genetically Modified Organisms (GMOs), call for a need to raise the level of public awareness of Genetic Modification (GM) technology in Africa. This should be accomplished by educating the public about the potential benefits and risks that may be associated with this new technology. In the last 15 years, GM crop producing countries have benefited from adoption of this new technology in the form of improved crop productivity, food security, and quality of life. The increased income to resource-poor farmers is a key benefit at the individual level especially as most countries using this technology are in the developing world, including three African countries (South Africa, Burkina Faso and Egypt). Despite clear benefits to countries and farmers who grow GMOs, many people are concerned about suspected potential risks associated with GMOs. This sparks debate as to whether GM technology should be adopted or not. Given the concerns regarding the safety of GMO products, thorough scientific investigation of safe application of GMOs is required. The objective of this paper is to respond to the issues of GM agriculture in Africa and some of the issues surrounding the adoption of GM crops between developed and developing countries. In this article, I analyse relevant papers relating to the adoption of GM technology particularly in developing countries including the few African countries that have adopted GM crops. The issues discussed span a wide range including: safety; potential benefits and risks; disputes between the United States of America (USA) and the European Union (EU) over adoption of GM crops with a focus on Africa continent. This article is concluded by summarising the issues raised and how GM technology can be adopted for agricultural development in Africa.

  8. Response to issues on GM agriculture in Africa: Are transgenic crops safe?

    PubMed Central

    2011-01-01

    The controversies surrounding transgenic crops, often called Genetically Modified Organisms (GMOs), call for a need to raise the level of public awareness of Genetic Modification (GM) technology in Africa. This should be accomplished by educating the public about the potential benefits and risks that may be associated with this new technology. In the last 15 years, GM crop producing countries have benefited from adoption of this new technology in the form of improved crop productivity, food security, and quality of life. The increased income to resource-poor farmers is a key benefit at the individual level especially as most countries using this technology are in the developing world, including three African countries (South Africa, Burkina Faso and Egypt). Despite clear benefits to countries and farmers who grow GMOs, many people are concerned about suspected potential risks associated with GMOs. This sparks debate as to whether GM technology should be adopted or not. Given the concerns regarding the safety of GMO products, thorough scientific investigation of safe application of GMOs is required. The objective of this paper is to respond to the issues of GM agriculture in Africa and some of the issues surrounding the adoption of GM crops between developed and developing countries. In this article, I analyse relevant papers relating to the adoption of GM technology particularly in developing countries including the few African countries that have adopted GM crops. The issues discussed span a wide range including: safety; potential benefits and risks; disputes between the United States of America (USA) and the European Union (EU) over adoption of GM crops with a focus on Africa continent. This article is concluded by summarising the issues raised and how GM technology can be adopted for agricultural development in Africa. PMID:21981823

  9. A novel subset of helper T cells promotes immune responses by secreting GM-CSF

    PubMed Central

    Zhang, J; Roberts, A I; Liu, C; Ren, G; Xu, G; Zhang, L; Devadas, S; Shi, Yufang

    2013-01-01

    Helper T cells are crucial for maintaining proper immune responses. Yet, they have an undefined relationship with one of the most potent immune stimulatory cytokines, granulocyte macrophage-colony-stimulating factor (GM-CSF). By depleting major cytokines during the differentiation of CD4+ T cells in vitro, we derived cells that were found to produce large amounts of GM-CSF, but little of the cytokines produced by other helper T subsets. By their secretion of GM-CSF, this novel subset of helper T cells (which we have termed ThGM cells) promoted the production of cytokines by other T-cell subtypes, including type 1 helper T cell (Th1), type 2 helper T cell (Th2), type 1 cytotoxic T cell (Tc1), type 2 cytotoxic T cell (Tc2), and naive T cells, as evidenced by the fact that antibody neutralization of GM-CSF abolished this effect. ThGM cells were found to be highly prone to activation-induced cell death (AICD). Inhibitors of TRAIL or granzymes could not block AICD in ThGM cells, whereas inhibition of FasL/Fas interaction partially rescued ThGM cells from AICD. Thus, ThGM cells are a novel subpopulation of T helper cells that produce abundant GM-CSF, exhibit exquisite susceptibility to apoptosis, and therefore play a pivotal role in the regulation of the early stages of immune responses. PMID:24076588

  10. Development of New Cooling System Using Gm/jt Cryocoolers for the SKS Magnet

    NASA Astrophysics Data System (ADS)

    Aoki, K.; Haruyama, T.; Makida, Y.; Araoka, O.; Kasami, K.; Takahashi, T.; Nagae, T.; Kakiguchi, Y.; Sekimoto, M.; Tosaka, T.; Miyazaki, H.; Kuriyama, T.; Ono, M.; Orikasa, T.; Tsuchihashi, T.; Hirata, Y.

    2008-03-01

    We plan to develop a new improved cooling system for the Superconducting Kaon Spectrometer (SKS) magnet and transfer the magnet to the K1.8 beamline of the Hadron Hall of the Japan Proton Accelerator Research Complex (J-PARC) for further use in nuclear physics experiments. To replace the present 300 W cryogenic system, we will adopt a new cooling method that uses 4 K Gifford-McMahon/Joule-Thomson (GM/JT) cryocoolers. In order to decide a practical design for the new liquid helium reservoir of the magnet, which will be equipped with GM/JT cryocoolers, cooling tests on a GM/JT cryocooler were performed from February to March 2007. We constructed a new cooling test stand with a GM/JT cryocooler and measured the cooling capacities under several thermal shield temperatures with or without a baffle, which helped prevent convection. Based on the test results, we have finally decided to adopt three GM/JT cryocoolers for the new SKS along with a baffle and an additional dedicated GM cooler to cool the thermal shield of the GM/JT ports.

  11. Anti-tumor effect of in vivo IL-2 and GM-CSF electrogene therapy in murine hepatoma model.

    PubMed

    Chi, Chau-Hwa; Wang, Yu-Shan; Lai, Yen-Shuae; Chi, Kwan-Hwa

    2003-01-01

    We evaluated the effect of in vivo electrogene therapy (EGT), a newly-developed gene transfer method using electroporation on the induction of anti-cancer immunity. The in vivo EGT was carried out by direct injection of plasmid DNAs encoding mouse interleukin-2 (IL-2) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in a subcutaneous murine hepatoma model of 1MEA.7R.1 cells. Six electric pulses were generated in situ from a square-wave electroporator fitted with a circular, six-needle electrode array. 1MEA.7R1 cells in vitro were modified to secret IL-2 (1MEA.7R.1/IL-2 cells). The 1MEA.7R.1/IL-2 cells had a similar cell doubling-time as their parent cells but showed a much slower growth rate on Balb/C mice. One, or 3 rounds of single gene EGT with IL-2 gene showed a dose-responsive effect of growth retardation. Co-administration of 3 rounds of IL-2/GM-CSF double genes EGT had a stronger growth inhibition effect than 3 rounds of IL-2 single gene EGT. Three rounds of IL-2/GM-CSF EGT rendered the tumor to a growth rate of stably transfected 1MEA.7R.1/IL-2 cells. Seven rounds of IL-2/GM-CSF EGT markedly inhibited the tumor growth. Reverse transciptase-polymerase chain reaction confirmed the expression of IL-2, GM-CSF and interferon-gamma within treated tumors. Systemic inhibitory effects can be demonstrated from tumor-re-challenged experiments on mice which received 3 rounds of double-gene EGT. The T cell proliferation assay revealed an increased T cell proliferation in double-gene EGT-treated mice. This experiment showed that partial systemic immunity can be provoked by IL-2/GM-CSF double-gene EGT. These findings suggest that our immuno-gene therapy protocol has the potential for future clinical applications.

  12. Cloning and sequence analysis of a cDNA clone coding for the mouse GM2 activator protein.

    PubMed Central

    Bellachioma, G; Stirling, J L; Orlacchio, A; Beccari, T

    1993-01-01

    A cDNA (1.1 kb) containing the complete coding sequence for the mouse GM2 activator protein was isolated from a mouse macrophage library using a cDNA for the human protein as a probe. There was a single ATG located 12 bp from the 5' end of the cDNA clone followed by an open reading frame of 579 bp. Northern blot analysis of mouse macrophage RNA showed that there was a single band with a mobility corresponding to a size of 2.3 kb. We deduce from this that the mouse mRNA, in common with the mRNA for the human GM2 activator protein, has a long 3' untranslated sequence of approx. 1.7 kb. Alignment of the mouse and human deduced amino acid sequences showed 68% identity overall and 75% identity for the sequence on the C-terminal side of the first 31 residues, which in the human GM2 activator protein contains the signal peptide. Hydropathicity plots showed great similarity between the mouse and human sequences even in regions of low sequence similarity. There is a single N-glycosylation site in the mouse GM2 activator protein sequence (Asn151-Phe-Thr) which differs in its location from the single site reported in the human GM2 activator protein sequence (Asn63-Val-Thr). Images Figure 1 PMID:7689829

  13. GM-CSF: An Immune Modulatory Cytokine that can Suppress Autoimmunity

    PubMed Central

    Bhattacharya, Palash; Thiruppathi, Muthusamy; Elshabrawy, Hatem A.; Alharshawi, Khaled; Kumar, Prabhakaran; Prabhakar, Bellur S.

    2015-01-01

    GM-CSF was originally identified as a colony stimulating factor (CSF) because of its ability to induce granulocyte and macrophage populations from precursor cells. Multiple studies have demonstrated that GM-CSF is also an immune-modulatory cytokine, capable of affecting not only the phenotype of myeloid lineage cells, but also T-cell activation through various myeloid intermediaries. This property has been implicated in the sustenance of several autoimmune diseases like arthritis and multiple sclerosis. In contrast, several studies using animal models have shown that GM-CSF is also capable of suppressing many autoimmune diseases like Crohn's disease, Type-1 diabetes, Myasthenia gravis and experimental autoimmune thyroiditis. Knockout mouse studies have suggested that the role of GM-CSF in maintaining granulocyte and macrophage populations in the physiological steady state is largely redundant. Instead, its immune-modulatory role plays a significant role in the development or resolution of autoimmune diseases. This is mediated either through the differentiation of precursor cells into specialized non-steady state granulocytes, macrophages and dendritic cells, or through the modulation of the phenotype of mature myeloid cells. Thus, outside of myelopoiesis, GM-CSF has a profound role in regulating the immune response and maintaining immunological tolerance. PMID:26113402

  14. Immunoglobulin GM and KM allotypes and prevalence of anti-LKM1 autoantibodies in patients with hepatitis C virus infection.

    PubMed

    Muratori, Paolo; Sutherland, Susan E; Muratori, Luigi; Granito, Alessandro; Guidi, Marcello; Pappas, Georges; Lenzi, Marco; Bianchi, Francesco B; Pandey, Janardan P

    2006-05-01

    GM and KM allotypes-genetic markers of immunoglobulin (Ig) gamma and kappa chains, respectively-are associated with humoral immunity to several infection- and autoimmunity-related epitopes. We hypothesized that GM and KM allotypes contribute to the generation of autoantibodies to liver/kidney microsomal antigen 1 (LKM1) in hepatitis C virus (HCV)-infected persons. To test this hypothesis, we characterized 129 persons with persistent HCV infection for several GM and KM markers and for anti-LKM1 antibodies. The heterozygous GM 1,3,17 23 5,13,21 phenotype was significantly associated with the prevalence of anti-LKM1 antibodies (odds ratio, 5.13; P=0.002), suggesting its involvement in this autoimmune phenomenon in HCV infection.

  15. Immunoglobulin GM and KM Allotypes and Prevalence of Anti-LKM1 Autoantibodies in Patients with Hepatitis C Virus Infection

    PubMed Central

    Muratori, Paolo; Sutherland, Susan E.; Muratori, Luigi; Granito, Alessandro; Guidi, Marcello; Pappas, Georges; Lenzi, Marco; Bianchi, Francesco B.; Pandey, Janardan P.

    2006-01-01

    GM and KM allotypes—genetic markers of immunoglobulin (Ig) γ and κ chains, respectively—are associated with humoral immunity to several infection- and autoimmunity-related epitopes. We hypothesized that GM and KM allotypes contribute to the generation of autoantibodies to liver/kidney microsomal antigen 1 (LKM1) in hepatitis C virus (HCV)-infected persons. To test this hypothesis, we characterized 129 persons with persistent HCV infection for several GM and KM markers and for anti-LKM1 antibodies. The heterozygous GM 1,3,17 23 5,13,21 phenotype was significantly associated with the prevalence of anti-LKM1 antibodies (odds ratio, 5.13; P = 0.002), suggesting its involvement in this autoimmune phenomenon in HCV infection. PMID:16641304

  16. Methylmercury causes neuronal cell death through the suppression of the TrkA pathway: In vitro and in vivo effects of TrkA pathway activators

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Fujimura, Masatake, E-mail: fujimura@nimd.go.jp; Usuki, Fusako

    Methylmercury (MeHg) is an environmental toxin which induces cell death specific for the nervous systems. Here we show that MeHg causes neuronal cell death through the suppression of the tropomyosin receptor kinase A (TrkA) pathway, and that compounds activating the TrkA pathway prevent MeHg-induced nerve damage in vitro and in vivo. We first investigated the mechanism of MeHg-induced neurotoxicity in differentiating neurons using PC12 cells. Exposure to 100 nM MeHg for 1 day induced apoptosis in differentiating PC12 cells. Further, MeHg-induced apoptosis was preceded by inhibition of neurite extension, as determined by ELISA analyses of the neurite-specific protein neurofilament tripletmore » H protein (NF-H). To determine the mechanism of MeHg-induced apoptosis, we evaluated the effects of MeHg on the TrkA pathway, which is known to regulate neuronal differentiation and viability. Western blot analysis demonstrated that, like the TrkA phosphorylation inhibitor K252a, MeHg inhibited phosphorylation of TrkA and its downstream effectors. Furthermore, GM1 ganglioside and its analog MCC-257, which enhance TrkA phosphorylation, overcame the effect of MeHg in neurons, supporting the involvement of the TrkA pathway in MeHg-induced nerve damage. Finally, we demonstrated that MCC-257 rescued the clinical sign and pathological changes in MeHg-exposed rats. These findings indicate that MeHg-induced apoptosis in neuron is triggered by inhibition of the TrkA pathway, and that GM1 ganglioside and MCC-257 effectively prevent MeHg-induced nerve damage. - Highlights: • Exposure to 100 nM MeHg for 1 day induced apoptosis in differentiating PC12 cells. • Inhibition of neurite extension was involved in MeHg-induced apoptosis. • Like the TrkA phosphorylation inhibitor, MeHg inhibited phosphorylation of TrkA. • GM1 ganglioside and its analog effectively prevented MeHg-induced nerve damage.« less

  17. Standardization of the CFU-GM assay: Advantages of plating a fixed number of CD34+ cells in collagen gels.

    PubMed

    Dobo, Irène; Pineau, Danielle; Robillard, Nelly; Geneviève, Frank; Piard, Nicole; Zandecki, Marc; Hermouet, Sylvie

    2003-10-01

    We investigated whether plating a stable amount of CD34(+) cells improves the CFU-GM assay. Data of CFU-GM assays performed with leukaphereses products in two transplant centers using a commercial collagen-based medium and unified CFU-GM scoring criteria were pooled and analyzed according to the numbers of CD34(+) cells plated. A first series of 113 CFU-GM assays was performed with a fixed number of mononuclear cells (i.e., a variable number of CD34(+) cells). In these cultures the CFU-GM/CD34 ratio varied according to the number of CD34(+) cells plated: median CFUGM/CD34 ratios were 1/6.2 to 1/6.6 for grafts containing <2% CD34(+) cells, vs. 1/10.2 for grafts containing > or =2% CD34(+) cells. The median CFU-GM/CD34 ratio also varied depending on pathology: 1/9.3 for multiple myeloma (MM), 1/6.8 for Hodgkin's disease (HD), 1/6.5 for non-Hodgkin lymphoma (NHL), and 1/4.5 for solid tumors (ST). A second series of 95 CFU-GM assays was performed with a fixed number of CD34(+) cells (220/ml). The range of median CFU-GM/CD34 ratios was narrowed to 1/7.0 to 1/5.2, and coefficients of variation for CFU-GM counts decreased by half to 38.1% (NHL), 36.1% (MM), 49.9% (HD), and 22.4% (ST). In addition, CFU-GM scoring was facilitated as the percentages of cultures with >50 CFU/GM/ml decreased from 6.7% to 43.8% when a variable number of CD34(+) cells was plated, to 4.5% to 16.7% when 220 CD34(+) cells/ml were plated. Hence, plating a fixed number of CD34(+) cells in collagen gels improves the CFU-GM assay by eliminating cell number-related variability and reducing pathology-related variability in colony growth.

  18. MDSCs are involved in the protumorigenic potentials of GM-CSF in colitis-associated cancer.

    PubMed

    Ma, Ning; Liu, Qilin; Hou, Lin; Wang, Yalin; Liu, Ziling

    2017-06-01

    Chronic inflammation is thought to be a major driving force for the development of colitis-associated colorectal cancer (CAC). As one member of proinflammatory cytokine family, granulocyte macrophage colony-stimulating factor (GM-CSF) has been identified to play a key role in CAC pathogenesis recently. The underlying mechanisms, however, remain largely unknown. In this study, we found that myeloid-derived suppressor cells (MDSCs) accumulated increasingly in the lesions during the progression from colitis to cancer, which was critical for CAC formation. Importantly, this MDSC accumulation was controlled by GM-CSF. MDSC number decreased significantly in GM-CSF-deficient mice suffering from CAC induction, and transfusion of MDSCs from wild-type CAC-bearing mice into GM-CSF-deficient counterparts led to recurrence of CAC. Furthermore, the supernatants of CAC lesions or GM-CSF alone was sufficient to differentiate hematopoietic precursors into MDSCs. Addition of neutralizing anti-GM-CSF antibody impaired the MDSC-differentiating effects of the supernatants of CAC lesions. Overall, these findings shed new insights into the mechanisms of GM-CSF underlying CAC development, by inducing/recruiting CAC-promoting MDSCs. Blocking GM-CSF activity or MDSC function may represent new therapeutic strategies for CAC in clinic.

  19. Chimeric HIV-1 Envelope Glycoproteins with Potent Intrinsic Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) Activity*

    PubMed Central

    Boot, Maikel; Cobos Jiménez, Viviana; Kootstra, Neeltje A.; Sanders, Rogier W.

    2013-01-01

    HIV-1 acquisition can be prevented by broadly neutralizing antibodies (BrNAbs) that target the envelope glycoprotein complex (Env). An ideal vaccine should therefore be able to induce BrNAbs that can provide immunity over a prolonged period of time, but the low intrinsic immunogenicity of HIV-1 Env makes the elicitation of such BrNAbs challenging. Co-stimulatory molecules can increase the immunogenicity of Env and we have engineered a soluble chimeric Env trimer with an embedded granulocyte-macrophage colony-stimulating factor (GM-CSF) domain. This chimeric molecule induced enhanced B and helper T cell responses in mice compared to Env without GM-CSF. We studied whether we could optimize the activity of the embedded GM-CSF as well as the antigenic structure of the Env component of the chimeric molecule. We assessed the effect of truncating GM-CSF, removing glycosylation-sites in GM-CSF, and adjusting the linker length between GM-CSF and Env. One of our designed EnvGM-CSF chimeras improved GM-CSF-dependent cell proliferation by 6-fold, reaching the same activity as soluble recombinant GM-CSF. In addition, we incorporated GM-CSF into a cleavable Env trimer and found that insertion of GM-CSF did not compromise Env cleavage, while Env cleavage did not compromise GM-CSF activity. Importantly, these optimized EnvGM-CSF proteins were able to differentiate human monocytes into cells with a macrophage-like phenotype. Chimeric EnvGM-CSF should be useful for improving humoral immunity against HIV-1 and these studies should inform the design of other chimeric proteins. PMID:23565193

  20. Cell to cell contact through ICAM-1-LFA-1 and TNF-alpha synergistically contributes to GM-CSF and subsequent cytokine synthesis in DBA/2 mice induced by 1,3-beta-D-Glucan SCG.

    PubMed

    Harada, Toshie; Kawaminami, Hiromi; Miura, Noriko N; Adachi, Yoshiyuki; Nakajima, Mitsuhiro; Yadomae, Toshiro; Ohno, Naohito

    2006-04-01

    SCG is a major 6-branched 1,3-beta-D-glucan in Sparassis crispa Fr. showing antitumor activity. We recently found that the splenocytes from naive DBA/1 and DBA/2 mice are potently induced by SCG to produce interferon- gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin-12p70 (IL-12p70), and that GM-CSF plays a key biologic role among these cytokines. In this study, we investigated the contribution of cell-cell contact and soluble factors to cytokine induction by SCG in DBA/2 mice. Cell-cell contact involving intercellular adhesion molecule-1 (ICAM-1) and lymphocyte function-associated antigen-1 (LFA-1) was an essential step for the induction of GM-CSF and IFN-gamma by SCG but not for the induction of TNF-alpha or IL-12p70 by SCG. SCG directly induced adherent splenocytes to produce TNF-alpha and IL-12p70. GM-CSF was required for the induction of TNF-alpha by SCG, and in turn, TNF-alpha enhanced the release of GM-CSF and thereby augmented the induction of IL-12p70 and IFN-gamma by SCG. Neutralization of IL-12 significantly inhibited the induction of IFN-gamma by SCG. We concluded that induction of GM-CSF production by SCG was mediated through ICAM-1 and LFA-1 interaction, GM-CSF subsequently contributed to further cytokine induction by SCG, and reciprocal actions of the cytokines were essential for enhancement of the overall response to SCG in DBA/2 mice.

  1. 5 CFR 531.245 - Computing locality rates and special rates for GM employees.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 5 Administrative Personnel 1 2010-01-01 2010-01-01 false Computing locality rates and special... Gm Employees § 531.245 Computing locality rates and special rates for GM employees. Locality rates and special rates are computed for GM employees in the same manner as locality rates and special rates...

  2. Overexpression of a GmGBP1 ortholog of soybean enhances the responses to flowering, stem elongation and heat tolerance in transgenic tobaccos.

    PubMed

    Zhao, Lin; Wang, Zhixin; Lu, Qingyao; Wang, Pengpeng; Li, Yongguang; Lv, Qingxue; Song, Xianping; Li, Dongmei; Gu, Yuejiao; Liu, Lixue; Li, Wenbin

    2013-06-01

    Soybean is a typical short-day crop, and its photoperiodic and gibberellin (GA) responses for the control of flowering are critical to seed yield. The GmGBP1 mRNA abundance in leaves was dramatically increased in short-days (SDs) compared to that in long-days in which it was consistently low at all time points from 0 to 6 days (days after transfer to SDs). GmGBP1 was highly expressed in leaves and exhibited a circadian rhythm in SDs. Ectopic overexpression of GmGBP1 in tobaccos caused photoperiod-insensitive early flowering by increasing NtCO mRNA levels. GmGBP1 mRNA abundance was also increased by GAs. Transgenic GmGBP1 overexpressing (-ox) tobacco plants exhibited increased GA signaling-related phenotypes including flowering and plant height promotion. Furthermore, the hypocotyl elongation, early-flowering and longer internode phenotypes were largely accelerated by GA3 application in the GmGBP1-ox tobacco seedlings. Being consistent, overexpression of GmGBP1 resulted in significantly enhanced GA signaling (evidenced suppressed expression of NtGA20ox) both with and without GA treatments. GmGBP1 was a positive regulator of both photoperiod and GA-mediated flowering responses. In addition, GmGBP1-ox tobaccos were hypersensitive to ABA, salt and osmotic stresses during seed germination. Heat-inducible GmGBP1 also enhanced thermotolerance in transgenic GmGBP1-ox tobaccos during seed germination and growth. GmGBP1 protein was localized in the nucleus. Analyses of a series of 5'-deletions of the GmGBP1 promoter suggested that several cis-acting elements, including P-BOX, TCA-motif and three HSE elements necessary to induce gene expression by GA, salicic acid and heat stress, were specifically localized in the GmGBP1 promoter region.

  3. Continuity of Monolayer-Bilayer Junctions for Localization of Lipid Raft Microdomains in Model Membranes

    PubMed Central

    Ryu, Yong-Sang; Wittenberg, Nathan J.; Suh, Jeng-Hun; Lee, Sang-Wook; Sohn, Youngjoo; Oh, Sang-Hyun; Parikh, Atul N.; Lee, Sin-Doo

    2016-01-01

    We show that the selective localization of cholesterol-rich domains and associated ganglioside receptors prefer to occur in the monolayer across continuous monolayer-bilayer junctions (MBJs) in supported lipid membranes. For the MBJs, glass substrates were patterned with poly(dimethylsiloxane) (PDMS) oligomers by thermally-assisted contact printing, leaving behind 3 nm-thick PDMS patterns. The hydrophobicity of the transferred PDMS patterns was precisely tuned by the stamping temperature. Lipid monolayers were formed on the PDMS patterned surface while lipid bilayers were on the bare glass surface. Due to the continuity of the lipid membranes over the MBJs, essentially free diffusion of lipids was allowed between the monolayer on the PDMS surface and the upper leaflet of the bilayer on the glass substrate. The preferential localization of sphingomyelin, ganglioside GM1 and cholesterol in the monolayer region enabled to develop raft microdomains through coarsening of nanorafts. Our methodology provides a simple and effective scheme of non-disruptive manipulation of the chemical landscape associated with lipid phase separations, which leads to more sophisticated applications in biosensors and as cell culture substrates. PMID:27230411

  4. The Soybean GmNARK Affects ABA and Salt Responses in Transgenic Arabidopsis thaliana

    PubMed Central

    Cheng, Chunhong; Li, Changman; Wang, Diandong; Zhai, Lifeng; Cai, Zhaoming

    2018-01-01

    GmNARK (Glycine max nodule autoregulation receptor kinase) is the homolog of Arabidopsis thaliana CLAVATA1 (CLV1) and one of the most important regulators in the process of AON (Autoregulation of Nodulation), a process that restricts excessive nodule numbers in soybean. However, except for the function in AON, little is known about this gene. Here, we report that GmNARK plays important roles in process of plant response to abiotic stresses. Bioinformatic analysis and subcellular localization experiment results showed that GmNARK was a putative receptor like kinase and located at membrane. The promoter of GmNARK contains manifold cis regulatory elements that are responsive to hormone and stresses. Gene transcript expression pattern analysis in soybean revealed GmNARK was induced by ABA and NaCl treatment in both shoot and root. Overexpression of GmNARK in Arabidopsis resulted in higher sensitivity to ABA and salt treatment during seed germination and greening stages. We also checked the expression levels of some ABA response genes in the transgenic lines; the results showed that the transcript level of all the ABA response genes were much higher than that of wild type under ABA treatment. Our results revealed a novel role of GmNARK in response to abiotic stresses during plant growth and development. PMID:29720993

  5. Integration of Ganglioside GT1b Receptor into DPPE and DPPC Phospholipid Monolayers: An X-Ray Reflectivity and Grazing-Incidence Diffraction Study

    PubMed Central

    Miller, C. E.; Busath, D. D.; Strongin, B.; Majewski, J.

    2008-01-01

    Using synchrotron grazing-incidence x-ray diffraction (GIXD) and reflectivity, the in-plane and out-of-plane structures of mixed-ganglioside GT1b-phospholipid monolayers were investigated at the air-liquid interface and compared with monolayers of the pure components. The receptor GT1b is involved in the binding of lectins and toxins, including botulinum neurotoxin, to cell membranes. Monolayers composed of 20 mol % ganglioside GT1b, the phospholipid dipalmitoyl phosphatidylethanolamine (DPPE), and the phospholipid dipalmitoyl phosphatidylcholine (DPPC) were studied in the gel phase at 23°C and at surface pressures of 20 and 40 mN/m, and at pH 7.4 and 5. Under these conditions, the two components did not phase-separate, and no evidence of domain formation was observed. The x-ray scattering measurements revealed that GT1b was intercalated within the host DPPE/DPPC monolayers, and slightly expanded DPPE but condensed the DPPC matrix. The oligosaccharide headgroups extended normally from the monolayer surfaces into the subphase. This study demonstrated that these monolayers can serve as platforms for investigating toxin membrane binding and penetration. PMID:18599631

  6. Generalized shrunken type-GM estimator and its application

    NASA Astrophysics Data System (ADS)

    Ma, C. Z.; Du, Y. L.

    2014-03-01

    The parameter estimation problem in linear model is considered when multicollinearity and outliers exist simultaneously. A class of new robust biased estimator, Generalized Shrunken Type-GM Estimation, with their calculated methods are established by combination of GM estimator and biased estimator include Ridge estimate, Principal components estimate and Liu estimate and so on. A numerical example shows that the most attractive advantage of these new estimators is that they can not only overcome the multicollinearity of coefficient matrix and outliers but also have the ability to control the influence of leverage points.

  7. Zinc oxide nanoparticles provide anti-cholera activity by disrupting the interaction of cholera toxin with the human GM1 receptor.

    PubMed

    Sarwar, Shamila; Ali, Asif; Pal, Mahadeb; Chakrabarti, Pinak

    2017-11-03

    Vibrio cholerae causes cholera and is the leading cause of diarrhea in developing countries, highlighting the need for the development of new treatment strategies to combat this disease agent. While exploring the possibility of using zinc oxide (ZnO) nanoparticles (NPs) in cholera treatment, we previously found that ZnO NPs reduce fluid accumulation in mouse ileum induced by the cholera toxin (CT) protein. To uncover the mechanism of action of ZnO NPs on CT activity, here we used classical (O395) and El Tor (C6706) V. cholerae biotypes in growth and biochemical assays. We found that a ZnO NP concentration of 10 μg/ml did not affect the growth rates of these two strains, nor did we observe that ZnO NPs reduce the expression levels of CT mRNA and protein. It was observed that ZnO NPs form a complex with CT, appear to disrupt the CT secondary structure, and block its interaction with the GM1 ganglioside receptor in the outer leaflet of the plasma membrane in intestinal (HT-29) cells and thereby reduce CT uptake into the cells. In the range of 2.5-10 μg/ml, ZnO NPs exhibited no cytotoxicity on kidney (HEK293) and HT-29 cells. We conclude that ZnO NPs prevent the first step in the translocation of cholera toxin into intestinal epithelial cells without exerting measurable toxic effects on HEK293 and HT-29 cells. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Purification and biochemical characterization of a highly thermostable bacteriocin isolated from Brevibacillus brevis strain GM100.

    PubMed

    Ghadbane, Mouloud; Harzallah, Daoud; Laribi, Atef Ibn; Jaouadi, Bassem; Belhadj, Hani

    2013-01-01

    A bacteriocin-producing (11,000 AU mL(-1)) strain was isolated from the rhizosphere of healthy Algerian plants Ononis angustissima Lam., and identified as Brevibacillus brevis strain GM100. The bacteriocin, called Bac-GM100, was purified to homogeneity from the culture supernatant, and, based on MALDI-TOF/MS analysis, was a monomer protein with a molecular mass of 4375.66 Da. The 21 N-terminal residues of Bac-GM100 displayed 65% homology with thurincin H from Bacillus thuringiensis. Bac-GM100 was extremely heat-stable (20 min at 120 °C), and was stable within a pH range of 3-10. It proved sensitive to various proteases, which demonstrated its protein nature. It was also found to display a bactericidal mode of action against gram-negative (Salmonella enteric ATCC 43972, Pseudomonas aeruginosa ATCC 49189, and Agrobacterium tumefaciens C58) and gram-positive (Enterococcus faecalis ENSAIA 631 and Staphylococcus aureus ATCC 6538) bacteria, and a fungistatic mode of action against the pathogenic fungus Candida tropicalis R2 CIP 203.

  9. Epistatic interactions between Fc (GM) and FcγR genes and the host control of human immunodeficiency virus replication.

    PubMed

    Deepe, Raymond N; Kistner-Griffin, Emily; Martin, Jeffrey N; Deeks, Steven G; Pandey, Janardan P

    2012-03-01

    Host genetic factors are thought to contribute to the interindividual differences in the control of human immunodeficiency virus (HIV) replication. The aim of the present investigation was to determine whether genes encoding GM and KM allotypes-genetic markers of immunoglobulin γ and κ chains, respectively-and those encoding Fcγ receptor (FcγR) IIa and IIIa are associated with the host control of HIV replication. A case-control design was employed among HIV-infected subjects, with a group that spontaneously controlled HIV replication ("controllers") as cases (n = 73) and those who did not control replication as controls (n = 100). Genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism, direct DNA sequencing, and TaqMan genotyping assays. In Caucasian Americans, certain combinations of FcγR and GM genotypes were differentially distributed between controllers and noncontrollers. Among the noncarriers of the FcγRIIa arginine allele, GM21 noncarriers had over 7-fold greater odds of being controllers than the carriers of this allele (odds ratio [OR] = 7.47). These GM determinants also interacted with FcγRIIIa alleles. Among the carriers of the FcγRIIIa valine allele, GM21 noncarriers had over 3-fold greater odds of being controllers than the carriers of this allele (OR = 3.26). These results demonstrate epistatic interactions of genes on chromosomes 14 (GM) and 1 (FcγR) in influencing the control of HIV replication. Copyright © 2012 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.

  10. T Cell Production of GM-CSF Protects the Host during Experimental Tuberculosis.

    PubMed

    Robinson, Richard T

    2017-12-12

    Although classically associated with myelopoiesis, granulocyte-macrophage colony-stimulating factor (GM-CSF) is increasingly recognized as being important for tuberculosis (TB) resistance. GM-CSF is expressed by nonhematopoietic and hematopoietic lineages following infection with Mycobacterium tuberculosis and is necessary to restrict M. tuberculosis growth in experimental models. Until the recent study by Rothchild et al. (mBio 8:e01514-17, 2017, https://doi.org/10.1128/mBio.01514-17), it was unknown whether GM-CSF-producing T cells contribute to TB resistance. Rothchild et al. identify which conventional and nonconventional T cell subsets produce GM-CSF during experimental TB, establish their protective nature using a variety of approaches, and provide a mechanistic basis for their ability to restrict M. tuberculosis growth. This commentary discusses the significance of these findings to basic and applied TB research. As translated to human disease, these findings suggest vaccine-mediated expansion of GM-CSF-producing T cells could be an effective prophylactic or therapeutic TB strategy. Copyright © 2017 Robinson.

  11. GM2-Gangliosidosis, AB Variant: Clinical, Ophthalmological, MRI, and Molecular Findings.

    PubMed

    Renaud, Deborah; Brodsky, Michael

    2016-01-01

    GM2-gangliosidosis, AB variant is a very rare form of GM2 gangliosidosis due to a deficiency of GM2 activator protein, associated with autosomal recessive mutations in GM2A. Less than ten patients, confirmed by molecular analysis, have been described in the literature.A 12-month-old Hmong girl presented to the neurometabolic clinic for evaluation of global developmental delay, hypotonia, and cherry red spots. The parents were not known to be consanguineous. Her examination was remarkable for hypotonia with hyperreflexia and excessive startling. The head circumference was normal. An extensive neurometabolic evaluation was negative.Developmental regression began at 14 months of age. Retinal examination at 16 months of age disclosed 4+ cherry red/black spots with "heaped up" ring of whitish infiltrate surrounding both foveae but no evidence of optic atrophy or peripheral retinal abnormalities. Repeat magnetic resonance imaging (MRI) scan at 17 months of age revealed delayed but interval myelination associated with abnormal signal intensity of the bilateral thalami presenting as T2 hyperintensity of the posterior thalami in the region of the pulvinar nuclei and T2 hypointensity in the anterior thalami. Sequencing of the GM2A gene revealed a homozygous c.160 G>T mutation, predicted to result in a premature protein termination p. Glu54*.

  12. Stimulatory versus suppressive effects of GM-CSF on tumor progression in multiple cancer types

    PubMed Central

    Hong, In-Sun

    2016-01-01

    Granulocyte-macrophage colony-stimulating factor (GM-CSF, also called CSF-2) is best known for its critical role in immune modulation and hematopoiesis. A large body of experimental evidence indicates that GM-CSF, which is frequently upregulated in multiple types of human cancers, effectively marks cancer cells with a ‘danger flag' for the immune system. In this context, most studies have focused on its function as an immunomodulator, namely its ability to stimulate dendritic cell (DC) maturation and monocyte/macrophage activity. However, recent studies have suggested that GM-CSF also promotes immune-independent tumor progression by supporting tumor microenvironments and stimulating tumor growth and metastasis. Although some studies have suggested that GM-CSF has inhibitory effects on tumor growth and metastasis, an even greater number of studies show that GM-CSF exerts stimulatory effects on tumor progression. In this review, we summarize a number of findings to provide the currently available information regarding the anticancer immune response of GM-CSG. We then discuss the potential roles of GM-CSF in the progression of multiple types of cancer to provide insights into some of the complexities of its clinical applications. PMID:27364892

  13. Soybean Salt Tolerance 1 (GmST1) Reduces ROS Production, Enhances ABA Sensitivity, and Abiotic Stress Tolerance in Arabidopsis thaliana.

    PubMed

    Ren, Shuxin; Lyle, Chimera; Jiang, Guo-Liang; Penumala, Abhishek

    2016-01-01

    Abiotic stresses, including high soil salinity, significantly reduce crop production worldwide. Salt tolerance in plants is a complex trait and is regulated by multiple mechanisms. Understanding the mechanisms and dissecting the components on their regulatory pathways will provide new insights, leading to novel strategies for the improvement of salt tolerance in agricultural and economic crops of importance. Here we report that soybean salt tolerance 1, named GmST1, exhibited strong tolerance to salt stress in the Arabidopsis transgenic lines. The GmST1-overexpressed Arabidopsis also increased sensitivity to ABA and decreased production of reactive oxygen species under salt stress. In addition, GmST1 significantly improved drought tolerance in Arabidopsis transgenic lines. GmST1 belongs to a 3-prime part of Glyma.03g171600 gene in the current version of soybean genome sequence annotation. However, comparative reverse transcription-polymerase chain reaction analysis around Glyma.03g171600 genomic region confirmed that GmST1 might serve as an intact gene in soybean leaf tissues. Unlike Glyma.03g171600 which was not expressed in leaves, GmST1 was strongly induced by salt treatment in the leaf tissues. By promoter analysis, a TATA box was detected to be positioned close to GmST1 start codon and a putative ABRE and a DRE cis-acting elements were identified at about 1 kb upstream of GmST1 gene. The data also indicated that GmST1-transgenic lines survived under drought stress and showed a significantly lower water loss than non-transgenic lines. In summary, our results suggest that overexpression of GmST1 significantly improves Arabidopsis tolerance to both salt and drought stresses and the gene may be a potential candidate for genetic engineering of salt- and drought-tolerant crops.

  14. Uptake of raft components into amyloid β-peptide aggregates and membrane damage.

    PubMed

    Sasahara, Kenji; Morigaki, Kenichi; Mori, Yasuko

    2015-07-15

    Amyloid aggregation and deposition of amyloid β-peptide (Aβ) are pathologic characteristics of Alzheimer's disease (AD). Recent reports have shown that the association of Aβ with membranes containing ganglioside GM1 (GM1) plays a pivotal role in amyloid deposition and the pathogenesis of AD. However, the molecular interactions responsible for membrane damage associated with Aβ deposition are not fully understood. In this study, we microscopically observed amyloid aggregation of Aβ in the presence of lipid vesicles and on a substrate-supported planar membrane containing raft components and GM1. The experimental system enabled us to observe lipid-associated aggregation of Aβ, uptake of the raft components into Aβ aggregates, and relevant membrane damage. The results indicate that uptake of raft components from the membrane into Aβ deposits induces macroscopic heterogeneity of the membrane structure. Copyright © 2015 Elsevier Inc. All rights reserved.

  15. Alteration of Electrostatic Surface Potential Enhances Affinity and Tumor Killing Properties of Anti-ganglioside GD2 Monoclonal Antibody hu3F8.

    PubMed

    Zhao, Qi; Ahmed, Mahiuddin; Guo, Hong-fen; Cheung, Irene Y; Cheung, Nai-Kong V

    2015-05-22

    Ganglioside GD2 is highly expressed on neuroectodermal tumors and an attractive therapeutic target for antibodies that have already shown some clinical efficacy. To further improve the current antibodies, which have modest affinity, we sought to improve affinity by using a combined method of random mutagenesis and in silico assisted design to affinity-mature the anti-GD2 monoclonal antibody hu3F8. Using yeast display, mutants in the Fv with enhanced binding over the parental clone were FACS-sorted and cloned. In silico modeling identified the minimal key interacting residues involved in the important charged interactions with the sialic acid groups of GD2. Two mutations, D32H (L-CDR1) and E1K (L-FR1) altered the electrostatic surface potential of the antigen binding site, allowing for an increase in positive charge to enhance the interaction with the negatively charged GD2-pentasaccharide headgroup. Purified scFv and IgG mutant forms were then tested for antigen specificity by ELISA, for tissue specificity by immunohistochemistry, for affinity by BIACORE, for antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-mediated cytotoxicity in vitro, and for anti-tumor efficacy in xenografted humanized mice. The nearly 7-fold improvement in affinity of hu3F8 with a single D32H (L-CDR1) mutation translated into a ∼12-fold improvement in NK92MI-transfected CD16-mediated ADCC, a 6-fold improvement in CD32-mediated ADCC, and a 2.5-fold improvement in complement-mediated cytotoxicity while maintaining restricted normal tissue cross-reactivity and achieving substantial improvement in tumor ablation in vivo. Despite increasing GD2 affinity, the double mutation D32H (L-CDR1) and E1K (L-FR1) did not further improve anti-tumor efficacy. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. Impact of GM crops on biodiversity.

    PubMed

    Carpenter, Janet E

    2011-01-01

    The potential impact of GM crops on biodiversity has been a topic of interest both in general as well as specifically in the context of the Convention on Biological Diversity. Agricultural biodiversity has been defined at levels from genes to ecosystems that are involved or impacted by agricultural production (www.cbd.int/agro/whatis.shtml). After fifteen years of commercial cultivation, a substantial body of literature now exists addressing the potential impacts of GM crops on the environment. This review takes a biodiversity lens to this literature, considering the impacts at three levels: the crop, farm and landscape scales. Within that framework, this review covers potential impacts of the introduction of genetically engineered crops on: crop diversity, biodiversity of wild relatives, non-target soil organisms, weeds, land use, non-target above-ground organisms, and area-wide pest suppression. The emphasis of the review is peer-reviewed literature that presents direct measures of impacts on biodiversity. In addition, possible impacts of changes in management practises such as tillage and pesticide use are also discussed to complement the literature on direct measures. The focus of the review is on technologies that have been commercialized somewhere in the world, while results may emanate from non-adopting countries and regions. Overall, the review finds that currently commercialized GM crops have reduced the impacts of agriculture on biodiversity, through enhanced adoption of conservation tillage practices, reduction of insecticide use and use of more environmentally benign herbicides and increasing yields to alleviate pressure to convert additional land into agricultural use.

  17. New measurement of G_E/GM for the proton

    NASA Astrophysics Data System (ADS)

    Segel, Ralph

    2003-10-01

    Recent polarization transfer measurements of the ratio of the proton electric to magnetic form factor, G E /G_M, find μ_pG E /GM = 1 - 0.13Q ^2 while a long series of L-T separations are fit by μ_pG_E/GM ≈ 1. Jefferson Lab experiment E01-001 used a new technique for making L-T separations that greatly reduces the dominant systematic uncertainties present in previous determinations. Protons from ep scattering were measured over a wide range in ɛ at Q^2 = 2.64, 3.20 and 4.10 GeV^2 and, simultaneously, protons scattered at Q^2 = 0.5 GeV^2 were measured over a small range in ɛ. The Q^2 = 0.5 GeV^2 measurements provided an internal monitor and only kinematic factors and ratios of simultaneously measured cross sections enter into the determinations of G_E/G_M. Measuring the proton cross sections has the advantage that for the same Q^2, count rates change very little with ɛ and also proton momentum is the same at all ɛ thus eliminating the effect of any momentum-dependent inefficiencies. Neither of these is true for L-T separations performed by measuring electron cross sections. Furthermore, the radiative corrections for the proton cross sections are a factor of about 2.5 smaller. All previous L-T separations measured electron cross sections and none had the advantage of an internal monitor. Therefore, the results of E01-001 stringently test whether systematic uncertainties in previous L-T separations may have been sufficient to explain the discrepancy with the recent polarization transfer results.

  18. Influence of one-year neurologic outcome of treatment on newborns with moderate and severe hypoxic-ischemic encephalopathy by rhuEP0 combined with ganglioside (GM1).

    PubMed

    Zhu, X-Y; Ye, M-Y; Zhang, A-M; Wang, W-D; Zeng, F; Li, J-L; Fang, F

    2015-10-01

    To observe the one-year neurologic prognostic outcome of newborns with moderate and severe hypoxic-ischemic encephalopathy (HIE) who received recombinant human erythropoietin (rhuEPO) combined with exogenous monosialotetrahexosylganglioside (GM1) treatment to provide new guidelines for clinical treatment. Seventy-six newborns with moderate and severe HIE were selected from February 2011 to February 2014 in our hospital. This study received the informed consent of our hospital's Ethics Committee and the newborns' guardians. The newborns were divided to an observation group (n = 34 cases) and a control group (n = 42 cases). All newborns underwent hypothermia and conventional treatment for their conditions. The control group received GMl treatment and observation group received rhuEPO combined with GMl treatment. The curative differences and neural behavior from these two groups were compared. The excellent, efficient proportion and total effective rate of the newborns from the observation group were higher than the control group. The death rate, cerebral palsy and the invalid ratio of the newborns from the observation group were lower than that of the control group. Awareness, muscle tension, primitive reflex and increased intracranial pressure recovery time of the newborns in the observation group were less than those of the control group. The Neonatal Behavior Neurological Assessment (NBNA) score of both groups after the treatment of 7, 14 and 28 days were significantly higher and increased with time (p < 0.05). The MDI, PDI and DQ score of newborns from the two groups all increased after treatment of 3, 6 and 12 months than those of before, which increased with time (p < 0.05). The rhuEPO + GMl treatment in newborns with HIE improves short-term clinical effects and long-term neurological symptoms.

  19. 5 CFR 531.247 - Maximum payable rate rule for GM employees.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... rate is a special rate, the highest previous rate (after any geographic conversion) must be compared... 5 Administrative Personnel 1 2010-01-01 2010-01-01 false Maximum payable rate rule for GM... REGULATIONS PAY UNDER THE GENERAL SCHEDULE Determining Rate of Basic Pay Special Rules for Gm Employees § 531...

  20. Assessment of the safety of foods derived from genetically modified (GM) crops.

    PubMed

    König, A; Cockburn, A; Crevel, R W R; Debruyne, E; Grafstroem, R; Hammerling, U; Kimber, I; Knudsen, I; Kuiper, H A; Peijnenburg, A A C M; Penninks, A H; Poulsen, M; Schauzu, M; Wal, J M

    2004-07-01

    This paper provides guidance on how to assess the safety of foods derived from genetically modified crops (GM crops); it summarises conclusions and recommendations of Working Group 1 of the ENTRANSFOOD project. The paper provides an approach for adapting the test strategy to the characteristics of the modified crop and the introduced trait, and assessing potential unintended effects from the genetic modification. The proposed approach to safety assessment starts with the comparison of the new GM crop with a traditional counterpart that is generally accepted as safe based on a history of human food use (the concept of substantial equivalence). This case-focused approach ensures that foods derived from GM crops that have passed this extensive test-regime are as safe and nutritious as currently consumed plant-derived foods. The approach is suitable for current and future GM crops with more complex modifications. First, the paper reviews test methods developed for the risk assessment of chemicals, including food additives and pesticides, discussing which of these methods are suitable for the assessment of recombinant proteins and whole foods. Second, the paper presents a systematic approach to combine test methods for the safety assessment of foods derived from a specific GM crop. Third, the paper provides an overview on developments in this area that may prove of use in the safety assessment of GM crops, and recommendations for research priorities. It is concluded that the combination of existing test methods provides a sound test-regime to assess the safety of GM crops. Advances in our understanding of molecular biology, biochemistry, and nutrition may in future allow further improvement of test methods that will over time render the safety assessment of foods even more effective and informative. Copryright 2004 Elsevier Ltd.