MYCN mediates cysteine addiction and sensitizes neuroblastoma to ferroptosis

Aberrant expression of MYC transcription factor family members predicts poor clinical outcome in many human cancers. Oncogenic MYC profoundly alters metabolism and mediates an antioxidant response to maintain redox balance. Here we show that MYCN induces massive lipid peroxidation on depletion of cysteine, the rate-limiting amino acid for glutathione (GSH) biosynthesis, and sensitizes cells to ferroptosis, an oxidative, non-apoptotic and iron-dependent type of cell death. The high cysteine demand of MYCN-amplified childhood neuroblastoma is met by uptake and transsulfuration. When uptake is limited, cysteine usage for protein synthesis is maintained at the expense of GSH triggering ferroptosis and potentially contributing to spontaneous tumor regression in low-risk neuroblastomas. Pharmacological inhibition of both cystine uptake and transsulfuration combined with GPX4 inactivation resulted in tumor remission in an orthotopic MYCN-amplified neuroblastoma model. These findings provide a proof of concept of combining multiple ferroptosis targets as a promising therapeutic strategy for aggressive MYCN-amplified tumors

Pre-symptomatic transcriptome changes during cold storage of chilling sensitive and resistant peach cultivars to elucidate chilling injury mechanisms

Background: Cold storage induces chilling injury (CI) disorders in peach fruit (woolliness/mealiness, flesh browning and reddening/bleeding) manifested when ripened at shelf life. To gain insight into the mechanisms underlying CI, we analyzed the transcriptome of 'Oded' (high tolerant) and 'Hermoza' (relatively tolerant to woolliness, but sensitive to browning and bleeding) peach cultivars at pre-symptomatic stages. The expression profiles were compared and validated with two previously analyzed pools (high and low sensitive to woolliness) from the Pop-DG population. The four fruit types cover a wide range of sensitivity to CI. The four fruit types were also investigated with the ROSMETER that provides information on the specificity of the transcriptomic response to oxidative stress. Results: We identified quantitative differences in a subset of core cold responsive genes that correlated with sensitivity or tolerance to CI at harvest and during cold storage, and also subsets of genes correlating specifically with high sensitivity to woolliness and browning. Functional analysis indicated that elevated levels, at harvest and during cold storage, of genes related to antioxidant systems and the biosynthesis of metabolites with antioxidant activity correlates with tolerance. Consistent with these results, ROSMETER analysis revealed oxidative stress in 'Hermoza' and the progeny pools, but not in the cold resistant 'Oded'. By contrast, cold storage induced, in sensitivity to woolliness dependant manner, a gene expression program involving the biosynthesis of secondary cell wall and pectins. Furthermore, our results indicated that while ethylene is related to CI tolerance, differential auxin subcellular accumulation and signaling may play a role in determining chilling sensitivity/tolerance. In addition, sugar partitioning and demand during cold storage may also play a role in the tolerance/sensitive mechanism. The analysis also indicates that vesicle trafficking, membrane dynamics and cytoskeleton organization could have a role in the tolerance/sensitive mechanism. In the case of browning, our results suggest that elevated acetaldehyde related genes together with the core cold responses may increase sensitivity to browning in shelf life. Conclusions: Our data suggest that in sensitive fruit a cold response program is activated and regulated by auxin distribution and ethylene and these hormones have a role in sensitivity to CI even before fruit are cold stored.This research was funded by US-Israel Binational Agriculture Research and Development Fund (BARD) Grant no. US-4027-07. We thank the European Science Foundation for Short Term Scientific Mission grants to A. Dagar (COST Action 924, reference codes COST-STSM-924-04254 and Quality Fruit COST FA1106 for networking.Pons Puig, C.; Dagar, A.; Martí Ibáñez, MC.; Singh, V.; Crisosto, CH.; Friedman, H.; Lurie, S.... (2015). Pre-symptomatic transcriptome changes during cold storage of chilling sensitive and resistant peach cultivars to elucidate chilling injury mechanisms. BMC Genomics. 16:1-35. https://doi.org/10.1186/s12864-015-1395-6S13516Crisosto C, Mitchell F, Ju Z. Susceptibility to chilling injury of peach, nectarine, and plum cultivars grown in California. Hort Sci. 1999;34:1116–8.Lurie S, Crisosto C. Chilling injury in peach and nectarine. Postharvest Biol Technol. 2005;37:195–208.Crisosto C, Mitchell F, Johnson S. Factors in fresh market stone fruit quality. Postharvest News Inform. 1995;6(2):17–21.Dawson DM, Melton LD, Watkins CB. Cell wall changes in nectarines (Prunus persica): solubilization and depolymerization of pectic and neutral polymers during ripening and in mealy fruit. Plant Physiol. 1992;100(3):1203–10.Zhou H, Sonego L, Khalchitski A, Ben Arie R, Lers A, Lurie A. Cell wall enzymes and cell wall changes in ‘Flavortop’ nectarines: mRNA abundance, enzyme activity, and changes in pectic and neutral polymers during ripening and in woolly fruit. J Am Soc Hort Sci. 2000;125:630–7.Jarvis MC, Briggs SPH, Knox JP. Intercellular adhesion and cell separation in plants. Plant Cell Environ. 2003;26(7):977–89.Zhou H, Ben-Arie R, Lurie S. Pectin esterase, polygalacturonase and gel formation in peach pectin fractions. Phytochemistry. 2000;55(3):191–5.Brummell DA, Dal Cin V, Lurie S, Crisosto CH, Labavitch JM. Cell wall metabolism during the development of chilling injury in cold-stored peach fruit: association of mealiness with arrested disassembly of cell wall pectins. J Exp Bot. 2004;55(405):2041–52.Kader AA, Chordas A. Evaluating the browning potential of peaches. Calif Agric. 1984;38:14–5.Cevallos-Casals BA, Byrne D, Okie WR, Cisneros-Zevallos L. Selecting new peach and plum genotypes rich in phenolic compounds and enhanced functional properties. Food Chem. 2006;96(2):273–80.Rojas G, Méndez MA, Muñoz C, Lemus G, Hinrichsen P. Identification of a minimal microsatellite marker panel for the fingerprinting of peach and nectarine cultivars. Electron J Biotechnol. 2008;11:4–5.Scorza R, Sherman WB, Lightner GW. Inbreeding and co-ancestry of low chill short fruit development period freestone peaches and nectarines produced by the University of Florida breeding program. Fruit Varieties J. 1988;43:79–85.Brooks R, Olmo HP. Register of New Fruit and Nut Varieties, 2nd edition edn: Univ of California Press; 1972.Okie WR, Service USAR. Handbook of peach and nectarine varieties: performance in the southeastern United States and index of names: U.S. Dept. of Agriculture, Agricultural Research Service; 1998Martínez-García P, Peace C, Parfitt D, Ogundiwin E, Fresnedo-Ramírez J, Dandekar A, et al. Influence of year and genetic factors on chilling injury susceptibility in peach (Prunus persica (L.) Batsch). Euphytica. 2012;185(2):267–80.Ogundiwin E, Martí C, Forment J, Pons C, Granell A, Gradziel T, et al. Development of ChillPeach genomic tools and identification of cold-responsive genes in peach fruit. Plant Mol Biol. 2008;68(4–5):379–97.Pons C, Martí C, Forment J, Crisosto CH, Dandekar AM, Granell A. A Bulk Segregant Gene Expression Analysis of a Peach Population Reveals Components of the Underlying Mechanism of the Fruit Cold Response. PLoS ONE. 2014;9(3):e90706.Rosenwasser S, Fluhr R, Joshi JR, Leviatan N, Sela N, Hetzroni A, et al. ROSMETER: A Bioinformatic Tool for the Identification of Transcriptomic Imprints Related to Reactive Oxygen Species Type and Origin Provides New Insights into Stress Responses. Plant Physiol. 2013;163(2):1071–83.Kader AA, Mitchell FG. Maturity and quality. In: James H. LaRue RSJ, vol. Publication No. 3331, editor. Peaches, Plums, and Nectarines: Growing and Handling for Fresh Market. Oakland, Calif: Cooperative Extension, University of California, Division of Agriculture and Natural Resources; 1989. p. 191–6.Dagar A, Friedman H, Lurie S. Thaumatin-like proteins and their possible role in protection against chilling injury in peach fruit. Postharvest Biol Technol. 2010;57(2):77–85.Lill RE, Van Der Mespel GJ. A method for measuring the juice content of mealy nectarines. Sci Hortic. 1988;36(3–4):267–71.Tusher VG, Tibshirani R, Chu G. Significance analysis of microarrays applied to the ionizing radiation response. Proc Natl Acad Sci. 2001;98(9):5116–21.Pavlidis P, Noble WS. Matrix2png: a utility for visualizing matrix data. Bioinformatics. 2003;1-9(2):295–6.Dagar A, Pons Puig C, Marti Ibanez C, Ziliotto F, Bonghi CH, Crisosto C, et al. Comparative transcript profiling of a peach and its nectarine mutant at harvest reveals differences in gene expression related to storability. Tree Genet Genomes. 2013;9(1):223–35.Doherty CJ, Van Buskirk HA, Myers SJ, Thomashow MF. Roles for Arabidopsis CAMTA transcription factors in cold-regulated gene expression and freezing tolerance. Plant Cell. 2009;21(3):972–84.Gilmour S, Zarka D, Stockinger E, Salazar M, Houghton J, Thomashow M. Low temperature regulation of the Arabidopsis CBF family of AP2 transcriptional activators as an early step in cold-induced COR gene expression. Plant J. 1998;16(4):433–42.Dong L, Zhou H-W, Sonego L, Lers A, Lurie S. Ethylene involvement in the cold storage disorder of ‘Flavortop’ nectarine. Postharvest Biol Technol. 2001;23(2):105–15.Zhou H-W, Dong L, Ben-Arie R, Lurie S. The role of ethylene in the prevention of chilling injury in nectarines. J Plant Physiol. 2001;158(1):55–61.Kilian J, Whitehead D, Horak J, Wanke D, Weinl S, Batistic O, et al. The AtGenExpress global stress expression data set: protocols, evaluation and model data analysis of UV-B light, drought and cold stress responses. Plant J. 2007;50(2):347–63.Giraldo E, Díaz A, Corral JM, García A. Applicability of 2-DE to assess differences in the protein profile between cold storage and not cold storage in nectarine fruits. J Proteome. 2012;75(18):5774–82.Obenland D, Vensel W, Hurkman Ii W. Alterations in protein expression associated with the development of mealiness in peaches. J Hortic Sci Biotechnol. 2008;83(1):85–93.Vizoso P, Meisel L, Tittarelli A, Latorre M, Saba J, Caroca R, et al. Comparative EST transcript profiling of peach fruits under different post-harvest conditions reveals candidate genes associated with peach fruit quality. BMC Genomics. 2009;10(1):423.Hannah M, Heyer A, Hincha D. A Global Survey of Gene Regulation during Cold Acclimation in Arabidopsis thaliana. PLoS Genet. 2005;1(2):e26.Walia H, Wilson C, Condamine P, Liu X, Ismail AM, Zeng L, et al. Comparative Transcriptional Profiling of Two Contrasting Rice Genotypes under Salinity Stress during the Vegetative Growth Stage. Plant Physiol. 2005;139(2):822–35.Lurie S, Zhou H-W, Lers A, Sonego L, Alexandrov S, Shomer I. Study of pectin esterase and changes in pectin methylation during normal and abnormal peach ripening. Physiol Plant. 2003;119(2):287–94.Peace C, Crisosto C, Gradziel T. Endopolygalacturonase: A candidate gene for Freestone and Melting flesh in peach. Mol Breed. 2005;16(1):21–31.Luza JG, van Gorsel R, Polito VS, Kader AA. Chilling Injury in Peaches: A Cytochemical and Ultrastructural Cell Wall Study. J Am Soc Hortic Sci. 1992;117(1):114–8.Masia A, Zanchin A, Rascio N, Ramina A. Some Biochemical and Ultrastructural Aspects of Peach Fruit Development. J Am Soc Hortic Sci. 1992;117(5):808–15.Dean GH, Zheng H, Tewari J, Huang J, Young DS, Hwang YT, et al. The Arabidopsis MUM2 Gene Encodes a β-Galactosidase Required for the Production of Seed Coat Mucilage with Correct Hydration Properties. Plant Cell Online. 2007;19(12):4007–21.Johnson CS, Kolevski B, Smyth DR. TRANSPARENT TESTA GLABRA2, a Trichome and Seed Coat Development Gene of Arabidopsis, Encodes a WRKY Transcription Factor. Plant Cell Online. 2002;14(6):1359–75.Karssen CM, der Swan DLC B-v, Breekland AE, Koornneef M. Induction of dormancy during seed development by endogenous abscisic acid: studies on abscisic acid deficient genotypes of Arabidopsis thaliana (L.) Heynh. Planta. 1983;157(2):158–65.Bui M, Lim N, Sijacic P, Liu Z. LEUNIG_HOMOLOG and LEUNIG Regulate Seed Mucilage Extrusion in ArabidopsisF. J Integr Plant Biol. 2011;53(5):399–408.Hussey S, Mizrachi E, Spokevicius A, Bossinger G, Berger D, Myburg A. SND2, a NAC transcription factor gene, regulates genes involved in secondary cell wall development in Arabidopsis fibres and increases fibre cell area in Eucalyptus. BMC Plant Biol. 2011;11(1):173.Jin H, Cominelli E, Bailey P, Parr A, Mehrtens F, Jones J, et al. Transcriptional repression by AtMYB4 controls production of UV‐protecting sunscreens in Arabidopsis. EMBO J. 2000;19(22):6150–61.Romera-Branchat M, Ripoll JJ, Yanofsky MF, Pelaz S. The WOX13 homeobox gene promotes replum formation in the Arabidopsis thaliana fruit. Plant J. 2013;73(1):37–49.Itkin M, Seybold H, Breitel D, Rogachev I, Meir S, Aharoni A. TOMATO AGAMOUS-LIKE 1 is a component of the fruit ripening regulatory network. Plant J. 2009;60(6):1081–95.Bemer M, Karlova R, Ballester AR, Tikunov YM, Bovy AG, Wolters-Arts M, et al. The Tomato FRUITFULL Homologs TDR4/FUL1 and MBP7/FUL2 Regulate Ethylene-Independent Aspects of Fruit Ripening. Plant Cell Online. 2012;24(11):4437–51.Jaakola L, Poole M, Jones MO, Kämäräinen-Karppinen T, Koskimäki JJ, Hohtola A, et al. A SQUAMOSA MADS Box Gene Involved in the Regulation of Anthocyanin Accumulation in Bilberry Fruits. Plant Physiol. 2010;153(4):1619–29.Ogundiwin EA, Peace CP, Nicolet CM, Rashbrook VK, Gradziel TM, Bliss FA, et al. Leucoanthocyanidin dioxygenase gene (PpLDOX): a potential functional marker for cold storage browning in peach. Tree Genetics Genomes. 2008;4(3):543–54.Baxter IR, Young JC, Armstrong G, Foster N, Bogenschutz N, Cordova T, et al. A plasma membrane H + −ATPase is required for the formation of proanthocyanidins in the seed coat endothelium of Arabidopsis thaliana. Proc Natl Acad Sci U S A. 2005;102(7):2649–54.Cheng GW, Crisosto CH. Browning Potential, Phenolic Composition, and Polyphenoloxidase Activity of Buffer Extracts of Peach and Nectarine Skin Tissue. J Am Soc Hortic Sci. 1995;120(5):835–8.Wang Y-S, Tian S-P, Xu Y. Effects of high oxygen concentration on pro- and anti-oxidant enzymes in peach fruits during postharvest periods. Food Chem. 2005;91(1):99–104.Sevillano L, Sanchez-Ballesta MT, Romojaro F, Flores FB. Physiological, hormonal and molecular mechanisms regulating chilling injury in horticultural species. Postharvest technologies applied to reduce its impact. J Sci Food Agric. 2009;89(4):555–73.Provart NJ, Gil P, Chen W, Han B, Chang HS, Wang X, et al. Gene expression phenotypes of Arabidopsis associated with sensitivity to low temperatures. Plant Physiol. 2003;132(2):893–906.Prasad T, Anderson M, Stewart C. Acclimation, Hydrogen Peroxide, and Abscisic Acid Protect Mitochondria against Irreversible Chilling Injury in Maize Seedlings. Plant Physiol. 1994;105(2):619–27.Mhamdi A, Queval G, Chaouch S, Vanderauwera S, Van Breusegem F, Noctor G. Catalase function in plants: a focus on Arabidopsis mutants as stress-mimic models. J Exp Bot. 2010;61(15):4197–220.Ulmasov T, Murfett J, Hagen G, Guilfoyle TJ. Aux/IAA proteins repress expression of reporter genes containing natural and highly active synthetic auxin response elements. Plant Cell Online. 1997;9(11):1963–71.Schepetilnikov M, Dimitrova M, Mancera E, Martínez AG, Keller M, Ryabova LA. TOR and S6K1 promote translation reinitiation of uORF containing mRNAs via phosphorylation of eIF3h. EMBO J. 2013;32(8):1087–102.Xiong Y, Sheen J. The Role of Target of Rapamycin Signaling Networks in Plant Growth and Metabolism. Plant Physiol. 2014;164(2):499–512.Murray JAH, Jones A, Godin C, Traas J. Systems Analysis of Shoot Apical Meristem Growth and Development: Integrating Hormonal and Mechanical Signaling. Plant Cell Online. 2012;24(10):3907–19.Leiber R-M, John F, Verhertbruggen Y, Diet A, Knox JP, Ringli C. The TOR Pathway Modulates the Structure of Cell Walls in Arabidopsis. Plant Cell Online. 2010;22(6):1898–908.Garcia-Hernandez M, Davies E, Baskin TI, Staswick PE. Association of Plant p40 Protein with Ribosomes Is Enhanced When Polyribosomes Form during Periods of Active Tissue Growth. Plant Physiol. 1996;111(2):559–68.Cunningham JT, Rodgers JT, Arlow DH, Vazquez F, Mootha VK, Puigserver P. mTOR controls mitochondrial oxidative function through a YY1-PGC-1[agr] transcriptional complex. Nature. 2007;450(7170):736–40.Baur AH, Yang SF. Methionine metabolism in apple tissue in relation to ethylene biosynthesis. Phytochemistry. 1972;11(11):3207–14.Peiser GD, Wang T-T, Hoffman NE, Yang SF, Liu H-w, Walsh CT. Formation of cyanide from carbon 1 of 1-aminocyclopropane-1-carboxylic acid during its conversion to ethylene. Proc Natl Acad Sci. 1984;81(10):3059–63.Begheldo M, Manganaris GA, Bonghi C, Tonutti P. Different postharvest conditions modulate ripening and ethylene biosynthetic and signal transduction pathways in Stony Hard peaches. Postharvest Biol Technol. 2008;48(1):8–8.Shi Y, Tian S, Hou L, Huang X, Zhang X, Guo H, et al. Ethylene signaling negatively regulates freezing tolerance by repressing expression of CBF and type-A ARR genes in Arabidopsis. Plant Cell. 2012;24(6):2578–95.Thain SC, Vandenbussche F, Laarhoven LJJ, Dowson-Day MJ, Wang Z-Y, Tobin EM, et al. Circadian Rhythms of Ethylene Emission in Arabidopsis. Plant Physiol. 2004;136(3):3751–61.Wang KLC, Yoshida H, Lurin C, Ecker JR. Regulation of ethylene gas biosynthesis by the Arabidopsis ETO1 protein. Nature. 2004;428(6986):945–50.Zheng Z, Guo Y, Novák O, Dai X, Zhao Y, Ljung K, et al. Coordination of auxin and ethylene biosynthesis by the aminotransferase VAS1. Nat Chem Biol. 2013;9(4):244–6.Poschet G, Hannich B, Raab S, Jungkunz I, Klemens PAW, Krueger S, et al. A Novel Arabidopsis Vacuolar Glucose Exporter is involved in cellular Sugar Homeostasis and affects Composition of Seed Storage Compounds. Plant Physiol. 2011;157(4):1664–76.Wang K, Shao X, Gong Y, Zhu Y, Wang H, Zhang X, et al. The metabolism of soluble carbohydrates related to chilling injury in peach fruit exposed to cold stress. Postharvest Biol Technol. 2013;86:53–61.Liu Y-H, Offler CE, Ruan Y-L. Regulation of fruit and seed response to heat and drought by sugars as nutrients and signals. Frontiers Plant Sci. 2013;4:282.Coello P, Hey SJ, Halford NG. The sucrose non-fermenting-1-related (SnRK) family of protein kinases: potential for manipulation to improve stress tolerance and increase yield. J Exp Bot. 2011;62(3):883–93.Baena-González E, Sheen J. Convergent energy and stress signaling. Trends Plant Sci. 2008;13(9):474–82.Baena-González E. Energy Signaling in the Regulation of Gene Expression during Stress. Mol Plant. 2010;3(2):300–13.Robaglia C, Thomas M, Meyer C. Sensing nutrient and energy status by SnRK1 and TOR kinases. Curr Opin Plant Biol. 2012;15(3):301–7.Uemura M, Joseph RA, Steponkus PL. Cold Acclimation of Arabidopsis thaliana (Effect on Plasma Membrane Lipid Composition and Freeze-Induced Lesions). Plant Physiol. 1995;109(1):15–30.Zhang C, Tian S. Crucial contribution of membrane lipids’ unsaturation to acquisition of chilling-tolerance in peach fruit stored at 0°c. Food Chem. 2009;115(2):405–11.Abdrakhamanova A, Wang QY, Khokhlova L, Nick P. Is Microtubule Disassembly a Trigger for Cold Acclimation? Plant Cell Physiol. 2003;44(7):676–86.Baluška F, Hlavacka A, Šamaj J, Palme K, Robinson DG, Matoh T, et al. F-Actin-Dependent Endocytosis of Cell Wall Pectins in Meristematic Root Cells. Insights from Brefeldin A-Induced Compartments. Plant Physiology. 2002;130(1):422–31.Baluška F, Liners F, Hlavačka A, Schlicht M, Van Cutsem P, McCurdy DW, et al. Cell wall pectins and xyloglucans are internalized into dividing root cells and accumulate within cell plates during cytokinesis. Protoplasma. 2005;225(3–4):141–55.Gonzalez-Aguero M, Pavez L, Ibanez F, Pacheco I, Campos-Vargas R, Meisel L, et al. Identification of woolliness response genes in peach fruit after post-harvest treatments. J Exp Bot. 2008;59(8):1973–86.Bashline L, Lei L, Li S, Gu Y. Cell Wall, Cytoskeleton, and Cell Expansion in Higher Plants. Mol Plant. 2014;4:586–600.Dhonukshe P, Grigoriev I, Fischer R, Tominaga M, Robinson DG, Hašek J, et al. Auxin transport inhibitors impair vesicle motility and actin cytoskeleton dynamics in diverse eukaryotes. Proc Natl Acad Sci. 2008;105(11):4489–94.Fischer U, Men S, Grebe M. Lipid function in plant cell polarity. Curr Opin Plant Biol. 2004;7(6):670–6.Schrick K, Fujioka S, Takatsuto S, Stierhof Y-D, Stransky H, Yoshida S, et al. A link between sterol biosynthesis, the cell wall, and cellulose in Arabidopsis. Plant J. 2004;38(2):227–43.Cheng GW, Crisosto CH. Iron—Polyphenol Complex Formation and Skin Discoloration in Peaches and Nectarines. J Am Soc Hortic Sci. 1997;122(1):95–9.Bouché N, Fait A, Zik M, Fromm H. The root-specific glutamate decarboxylase (GAD1) is essential for sustaining GABA levels in Arabidopsis. Plant Mol Biol. 2004;55(3):315–25.Pedreschi R, Franck C, Lammertyn J, Erban A, Kopka J, Hertog M, et al. Metabolic profiling of ‘Conference’ pears under low oxygen stress. Postharvest Biol Technol. 2009;51(2):123–30

The combination of loss of glyoxalase1 and obesity results in hyperglycemia.

The increased formation of methylglyoxal (MG) under hyperglycemia is associated with the development of microvascular complications in patients with diabetes mellitus; however, the effects of elevated MG levels in vivo are poorly understood. In zebrafish, a transient knockdown of glyoxalase 1, the main MG detoxifying system, led to the elevation of endogenous MG levels and blood vessel alterations. To evaluate effects of a permanent knockout of glyoxalase 1 in vivo, glo1-/- zebrafish mutants were generated using CRISPR/Cas9. In addition, a diet-induced-obesity zebrafish model was used to analyze glo1-/- zebrafish under high nutrient intake. Glo1-/- zebrafish survived until adulthood without growth deficit and showed increased tissue MG concentrations. Impaired glucose tolerance developed in adult glo1-/- zebrafish and was indicated by increased postprandial blood glucose levels and postprandial S6 kinase activation. Challenged by an overfeeding period, fasting blood glucose levels in glo1-/- zebrafish were increased which translated into retinal blood vessel alterations. Thus, the data have identified a defective MG detoxification as a metabolic prerequisite and glyoxalase 1 alterations as a genetic susceptibility to the development of type 2 diabetes mellitus under high nutrition intake

Molecular basis for defective glycosylation and Pseudomonas pathogenesis in cystic fibrosis lung

The CFTR gene encodes a transmembrane conductance regulator, which is dysfunctional in patients with cystic fibrosis (CF). The mechanism by which defective CFTR (CF transmembrane conductance regulator) leads to undersialylation of plasma membrane glycoconjugates, which in turn promote lung pathology and colonization with Pseudomonas aeruginosa causing lethal bacterial infections in CF, is not known. Here we show by ratiometric imaging with lumenally exposed pH-sensitive green fluorescent protein that dysfunctional CFTR leads to hyperacidification of the trans-Golgi network (TGN) in CF lung epithelial cells. The hyperacidification of TGN, glycosylation defect of plasma membrane glycoconjugates, and increased P. aeruginosa adherence were corrected by incubating CF respiratory epithelial cells with weak bases. Studies with pharmacological agents indicated a role for sodium conductance, modulated by CFTR regulatory function, in determining the pH of TGN. These studies demonstrate the molecular basis for defective glycosylation of lung epithelial cells and bacterial pathogenesis in CF, and suggest a cure by normalizing the pH of intracellular compartments

<em>CNDP1</em> knockout in zebrafish alters the amino acid metabolism, restrains weight gain, but does not protect from diabetic complications.

The gene CNDP1 was associated with the development of diabetic nephropathy. Its enzyme carnosinase 1 (CN1) primarily hydrolyzes the histidine-containing dipeptide carnosine but other organ and metabolic functions are mainly unknown. In our study we generated CNDP1 knockout zebrafish, which showed strongly decreased CN1 activity and increased intracellular carnosine levels. Vasculature and kidneys of CNDP1 −/− zebrafish were not affected, except for a transient glomerular alteration. Amino acid profiling showed a decrease of certain amino acids in CNDP1 −/− zebrafish, suggesting a specific function for CN1 in the amino acid metabolisms. Indeed, we identified a CN1 activity for Ala–His and Ser–His. Under diabetic conditions increased carnosine levels in CNDP1 −/− embryos could not protect from respective organ alterations. Although, weight gain through overfeeding was restrained by CNDP1 loss. Together, zebrafish exhibits CN1 functions, while CNDP1 knockout alters the amino acid metabolism, attenuates weight gain but cannot protect organs from diabetic complications

Langzeitverhalten von Fensterfalz-Dichtungsmaterialien Schlussbericht

TIB: RN 5973 (2007) / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekSIGLEDEGerman

Endosomal hyperacidification in cystic fibrosis is due to defective nitric oxide–cylic GMP signalling cascade

Endosomal hyperacidification in cystic fibrosis (CF) respiratory epithelial cells is secondary to a loss of sodium transport control owing to a defective form of the CF transmembrane conductance regulator CFTR. Here, we show that endosomal hyperacidification can be corrected by activating the signalling cascade controlling sodium channels through cyclic GMP. Nitric oxide (NO) donors corrected the endosomal hyperacidification in CF cells. Stimulation of CF cells with guanylate cyclase agonists corrected the pH in endosomes. Exposure of CF cells to an inhibitor of cGMP-specific phosphodiesterase PDE5, Sildenafil, normalized the endosomal pH. Treatment with Sildenafil reduced secretion by CF cells of the proinflammatory chemokine interleukin 8 following stimulation with Pseudomonas aeruginosa products. Thus, the endosomal hyperacidification and excessive proinflammatory response in CF are in part due to deficiencies in NO- and cGMP-regulated processes and can be pharmacologically reversed using PDE5 inhibitors