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Environmental Science and Pollution Research

, Volume 21, Issue 9, pp 6044–6056 | Cite as

Assessment of Jatropha curcas L. biodiesel seed cake toxicity using the zebrafish (Danio rerio) embryo toxicity (ZFET) test

  • Arnold V. HallareEmail author
  • Paulo Lorenzo S. Ruiz
  • J. C. Earl D. Cariño
Research Article

Abstract

Consequent to the growing demand for alternative sources of energy, the seeds from Jatropha curcas remain to be the favorite for biodiesel production. However, a significant volume of the residual organic mass (seed cake) is produced during the extraction process, which raises concerns on safe waste disposal. In the present study, we assessed the toxicity of J. curcas seed cake using the zebrafish (Danio rerio) embryotoxicity test. Within 1-h post-fertilization (hpf), the fertilized eggs were exposed to five mass concentrations of J. curcas seed cake and were followed through 24, 48, and 72 hpf. Toxicity was evaluated based on lethal endpoints induced on zebrafish embryos namely egg coagulation, non-formation of somites, and non-detachment of tail. The lowest concentration tested, 1 g/L, was not able to elicit toxicity on embryos whereas 100 % mortality (based also on lethal endpoints) was recorded at the highest concentration at 2.15 g/L. The computed LC50 for the J. curcas seed cake was 1.61 g/L. No further increase in mortality was observed in the succeeding time points (48 and 72 hpf) indicating that J. curcas seed cake exerted acute toxicity on zebrafish embryos. Sublethal endpoints (yolk sac and pericardial edema) were noted at 72 hpf in zebrafish embryos exposed to higher concentrations. The observed lethal endpoints induced on zebrafish embryos were discussed in relation to the active principles, notably, phorbol esters that have remained in the seed cake even after extraction.

Keywords

Zebrafish Danio rerio Embryotoxicity Jatropha curcas Phorbol esters Seed cake ZFET test 

Notes

Acknowledgments

The authors are grateful to the National Institute of Health (NIH) of the University of the Philippines Manila for the financial grant. Special thanks also go to Dr. Elisa Co and Asst. Prof. Kimberly Benjamin, who have given invaluable inputs and saw the study through its completion.

References

  1. ADFG. (2013). Alaska Department of Fish & Game (ADFG) Education Resources. Retrieved from Coagulated Yolk Disease: http://www.adfg.alaska.gov/static/species/disease/pdfs/fishdiseases/coagulated_yolk_disease.pdf. Accessed 4 Mar 2013.
  2. Adolf W, Opferkuch H, Hecker E (1984) Irritant phorbol derivatives from four Jatropha species. Phytochemistry 23:129–132CrossRefGoogle Scholar
  3. Ahmad A, Mat Yasin N, Derek CL (2011) Microalgae as a sustainable energy source for biodiesel production: a review. Renew Sust Energ Rev 15:584–593CrossRefGoogle Scholar
  4. Ali S, van Mil H, Richardson M. (2011). Large-scale assessment of the zebrafish embryo as a possible predictive model in toxicity testing. PloS ONE, 6(6):e21076. doi: 10.1371/journal 6(6): e21076
  5. Ali S, Champagne D, Richardson M (2012) Behavioral profiling of zebrafish embryos exposed to a panel of 60 water-soluble compounds. Behav Brain Res 228:272–283CrossRefGoogle Scholar
  6. Atabani A, Silitonga A, Badruddin I, Mahlia T, Masjuki H, Mekhilef S (2012) A comprehensive review on biodiesel as an alternative energy resource and its characteristics. Renew Sust Energ Rev 16:2070–2093CrossRefGoogle Scholar
  7. Atadashi I, Aroua M, Aziz A (2010) High quality biodiesel and its diesel engine application: a review. Renew Sust Energ Rev 14:1999–2008CrossRefGoogle Scholar
  8. Berry J, Gantar M, Gibbs P, Schmale M (2007) The zebrafish (Danio rerio) embryo as a model system for identification and characterization of developmental toxins from marine and freshwater microalgae. Comput Biochem Phys A 145:61–72Google Scholar
  9. Bertolini T, Giorgione J, Harvey D, Newton A (2003) Protein kinase C translocation by modified phorbol esters with functionalized lipophilic regions. J Org Chem 68:5028–5036CrossRefGoogle Scholar
  10. Bluhm K, Heger S, Seiler T, Hallare AV, Schäffer A, Hollert H (2012) Toxicological and ecotoxicological potencies of biofuels used for the transport sector—a literature review. Energy Environ Sci 5:7381–7392CrossRefGoogle Scholar
  11. Brose N, Rosenmund C (2002) Move over protein kinase C, you’ve got company: alternative cellular effectors of diacylglycerol and phorbol esters. J Cell Sci 115:4399–4411CrossRefGoogle Scholar
  12. Bui A, Xiao R, Perveen Z, Kleinow K, Penn A (2012) Zebrafish embryos sequester and retain petrochemical combustion products: developmental and transcriptome consequences. Aquat Toxicol 108:23–32CrossRefGoogle Scholar
  13. Bustros A, Baylin S, Berger C, Roos B, Leong S, Nelkin B (1985) Phorbol esters increase calcitonin gene transcription and decrease c-mycm RNA levels in cultured human medullary thyroid carcinoma. J Biol Chem 260:98–104Google Scholar
  14. Caloca M, Fernandez S, Lewin N, Ching D, Modalii R, Blumberg P, Kazanietz M (1997) b2-Chimaerin Is a high affinity receptor for the phorbol ester tumor promoters. J Biol Chem 272:26488–26496CrossRefGoogle Scholar
  15. Carlsson G, Patring J, Ullerås E, Oskarsson A (2011) Developmental toxicity of albendazole and its three main metabolites in zebrafish embryos. Reprod Toxicol 32:129–137CrossRefGoogle Scholar
  16. Chen Y, Huang Y, Wen C, Wang Y, Chen W, Chen L, Tsay H (2008) Movement disorder and neuromuscular change in zebrafish embryos after exposure to caffeine. Neurotoxicol Teratol 30:440–447CrossRefGoogle Scholar
  17. Cheng J, Miller A, Webb S (2004) Organization and function of microfilaments during late epiboly in zebrafish embryos. Dev Dyn 23:313–323CrossRefGoogle Scholar
  18. Colon-González F, Kazanietz MG (2006) C1 domains exposed: from diacylglycerol binding to protein–protein interactions. Biochim Biophys Acta 1761:827–837CrossRefGoogle Scholar
  19. Colon-Gonzalez F, Leskow FC, Kazanietz MG (2008) Identification of an autoinhibitory mechanism that restricts C1 domain-mediated activation of the Rac-GAP alpha2-chimaerin. J Biol Chem 283:35247–35257CrossRefGoogle Scholar
  20. Cowden J, Padnos B, Hunter B, MacPhail R, Jensen K, Padilla S (2012) Developmental exposure to valproate and ethanol alters locomotor activity and retino-tectal projection area in zebrafish embryos. Reprod Teratol 33:165–173Google Scholar
  21. da Cruz A, Leite M, Rodrigues L, Nascimento I (2012) Estimation of biodiesel cytotoxicity by using acid phosphatase as a biomarker of lysosomal integrity. Bull Environ Contam Toxicol 89:219–224CrossRefGoogle Scholar
  22. Davis R, McKernan L, Rhodes J, Kulkosky J (2011) In vivo effects of antiviral protein kinase C modulators on zebrafish development and survival. Int Schol Res Netw 1–7Google Scholar
  23. Devappa R, Makkar H, Becker K (2010) Jatropha toxicity—a review. J Toxicol Environ Health B 13:476–507CrossRefGoogle Scholar
  24. Devappa R, Rajesh S, Kumar V, Makkar H, Becker K (2012) Activities of Jatropha curcas phorbol esters in various bioassays. Ecotoxicol Environ Safe 78:57–62CrossRefGoogle Scholar
  25. El Rafei S, El Diwani G, Hawash S (2011) Ozone for phorbol esters removal from Egyptian Jatropha oil seed cake. Adv Appl Sci Res 4:221–232Google Scholar
  26. Embry M, Belanger S, Braunbeck T, Galay-Burgos M, Halder M, Hinton D, Léonard M, Lillicrap A, Norberg-King T, Whale G (2010) The fish embryo toxicity test as an animal alternative method in hazard and risk assessment and scientific research. Aquat Toxicol 97:79–87CrossRefGoogle Scholar
  27. Francis G, Edinger R, Becker K (2005) A concept for simultaneous wasteland reclamation, fuel production, and socio-economic development in degraded areas in India; need, potential, and perspectives of Jatropha plantations. Nat Resour Forum 29:19–24Google Scholar
  28. Fraysse B, Mons R, Garric J (2006) Development of a zebrafish 4-day embryo-larval bioassay to assess toxicity of chemicals. Ecotoxicol Environ Safe 63:253–267CrossRefGoogle Scholar
  29. Gandhi V, Cherian K, Mulky M (1995) Toxicological studies on Ratanjyot Oil. Food Chem Toxicol 33:39–42CrossRefGoogle Scholar
  30. Gilbert S (2010) Developmental Biology, 9th edn. Sinauer Associates Inc., SunderlandGoogle Scholar
  31. Goel G, Makkar H, Francis G, Becker K (2007) Phorbol esters: structure, biological activity, and toxicity in animals. Int J Toxicol 26:279–288CrossRefGoogle Scholar
  32. Gubitz G, Mittelbach M, Trabi M (1999) Exploitation of the tropical oil seed plant Jatropha curcas L. Bioresour Technol 67:73–82CrossRefGoogle Scholar
  33. Hallare A, Nagel K, Kohler H-R, Triebskorn R (2006) Comparative embryotoxicity and proteotoxicity of three carrier solvents to zebrafish (Danio rerio) embryos. Ecotoxicol Environ Safe 63:378–388CrossRefGoogle Scholar
  34. He W (2011) Biochemical and genetic analyses of Jatropha curcas L, seed composition. University of York, Department of Biology, YorkGoogle Scholar
  35. Heger S, Bluhm K, Agler MT, Maletz S, Schäffer A, Seiler T-B, Angenent LT, Hollert H (2012) Biotests for hazard assessment of biofuel fermentation. Energy Environ Sci 5:9778–9788CrossRefGoogle Scholar
  36. Henn K, Braunbeck T (2011) Dechorionation as a tool to improve the fish embryo toxicity test (FET) with the zebrafish (Danio rerio). Comp Biochem Physiol 153:91–98Google Scholar
  37. Hill J, Nelson E, Tilman D, Polasky S, Tiffany D (2006) Environmental, economic, and energetic costs and benefits of biodiesel and ethanol biofuels. Proc Natl Acad Sci U S A 103:11203–11210Google Scholar
  38. Hollert H, Keiter S, König NK, Rudolf M, Ulrich T, Braunbeck T (2003) A new sediment contact assay to assess particle-bound pollutants using zebrafish (Danio rerio) embryos. J Soils Sediments 3:197–207CrossRefGoogle Scholar
  39. Hsu T, Tsai H, Huang K, Luan M, Hsieh C (2010) Sublethal levels of cadmium down-regulate the gene expression of DNA mismatch recognition protein MutS homolog 6 (MSH6) in zebrafish (Danio rerio) embryos. Chemosphere 81:748–754CrossRefGoogle Scholar
  40. Kaiser D, Sieratowicz A, Zielke H, Oetken M, Hollert H, Oehlmann J (2012) Ecotoxicological effect characterisation of widely used organic UV filters. Environ Pollut 163:84–90CrossRefGoogle Scholar
  41. Karaj S, Muller J (2011) Optimizing mechanical oil extraction of Jatropha curcas L. seeds with respect to press capacity, oil recovery and energy efficiency. Ind Crop Prod 34:1010–1016CrossRefGoogle Scholar
  42. Kimmel C, Ballard W, Kimmel S, Ullmann B, Schilling T (1995) Stages of embryonic development of the zebrafish. Dev Dyn 203:253–310CrossRefGoogle Scholar
  43. Kouame S (2011) Comparative characterization of Jatropha, soybean and commercial biodiesel. J Fuel Chem Technol 39:258–264CrossRefGoogle Scholar
  44. Krone P, Blechinger S, Evans T, Ryan J, Noonan E, Hightower L (2005) Use of fish liver PLHC-1 cells and zebrafish embryos in cytotoxicity assays. Methods 35:176–187CrossRefGoogle Scholar
  45. Kumar S, Chaube A, Jain S (2012a) Sustainability issues for promotion of Jatropha biodiesel in Indian scenario: a review. Renew Sust Energ Rev 16:1089–1098CrossRefGoogle Scholar
  46. Kumar S, Chaube A, Jain S (2012b) Critical review of jatropha biodiesel promotion policies in India. Energ Policy 41:775–781CrossRefGoogle Scholar
  47. Lahnsteiner F (2008) The effect of internal and external cryoprotectants on zebrafish (Danio rerio) embryos. Theriogenology 69:384–396CrossRefGoogle Scholar
  48. Lahnsteiner F, Urbanyi B, Horvath A, Weismann T (2001) Bio-markers for egg quality determination in cyprinid fishes. Aquaculture 195:331–352CrossRefGoogle Scholar
  49. Leme D, Grummt T, Heinze R, Sehr A, Renz S, Reinel S, Oliveira D, Ferraz E, de Marchi MR, Machado M, Zocolo G, Morales M (2012) An overview of biodiesel soil pollution: data based on cytotoxicity and genotoxicity assessments. J Hazard Mater 199–200:343–349CrossRefGoogle Scholar
  50. Leskow F, Holloway B, Wang H, Mullins M, Kazanietz M (2006) The zebrafish homologue of mammalian chimerin Rac-GAPs is implicated in epiboly progression during development. Proc Natl Acad Sci U S A 103:5373–5378CrossRefGoogle Scholar
  51. Li D, Lu C, Wang J, Hu W, Cao Z, Sun D, Xia H, Ma X (2009) Developmental mechanisms of arsenite toxicity in zebrafish (Danio rerio) embryos. Aquat Toxicol 91:229–237CrossRefGoogle Scholar
  52. Li C, Devappa R, Liu J, Lu J, Makkar H, Becker K (2010) Toxicity of Jatropha curcas phorbol esters in mice. Food Chem Toxicol 48:620–625CrossRefGoogle Scholar
  53. Li X, Ma Y, Li D, Gao X, Li P, Bai N, Luo M, Tan X, Lu C, Ma X (2012) Arsenic impairs embryo development via down-regulating Dvr1 expression in zebrafish. Toxicol Lett 212:161–168CrossRefGoogle Scholar
  54. Livingston B, Wilt F (1992) Phorbol esters alter cell fate during development of sea urchin embryos. J Cell Biol 119:1641–1648CrossRefGoogle Scholar
  55. Makkar H, Becker J. (1997). Potential of J. curcas seed meal as a protein supplement to livestock feed; constraints to its utilisation and possible strategies to overcome constraints. In: Jatropha 1997 Symposium. Managua, Nicaragua. Feb 23–27, 1997Google Scholar
  56. Makkar H, Becker K (2009) Jatropha curcas, a promising crop for the generation of biodiesel and value-added co-products. Eur J Lipid Sci Technol 111:773–787CrossRefGoogle Scholar
  57. Makkar H, Francis G, Becker K (2008) Protein concentrate from Jatropha curcas screw-pressed seed cake and toxic and antinutritional factors in protein concentrate. J Sci Food Agric 88:1542–1548CrossRefGoogle Scholar
  58. Makkar H, Maes J, De Greyt W, Becker K (2009) Removal and degradation of phorbol esters during pre-treatment and transesterification of Jatropha curcas oil. J Am Oil Chem Soc 86:173–181CrossRefGoogle Scholar
  59. Marguerie A, Buckley C, Fleming A (2006) Validation of an embryotoxicology screen using zebrafish. Toxicol Lett 164:15–37CrossRefGoogle Scholar
  60. McKernan L, Momjian D, Kulkosky J (2012) Protein kinase C: one pathway towards the eradication of latent HIV-1 reservoirs. Adv Virol 1–8Google Scholar
  61. Menezes R, Rao N, Karanth S, Kamath A, Manipady S, Pillay V (2006) Jatropha curcas poisoning. Indian J Pediatr 73:63–64CrossRefGoogle Scholar
  62. Menna P, Skilton G, Leskow F (2003) Inhibition of aggressiveness of metastatic mouse mammary carcinoma cells by the β2-chimaerin GAP domain. Cancer Res 63:2284–2291Google Scholar
  63. Mondala A, Liang K, Toghiani H, Hernandez R, French T (2009) Biodiesel production by in situ transesterification of municipal primary and secondary sludges. Bioresour Technol 100:1203–1210CrossRefGoogle Scholar
  64. Morin B, Filatreau J, Vicquelin L, Barjhoux I, Guinel S, Leray-Forget J, Cachot J (2011) Detection of DNA damage in yolk-sac larvae of the Japanese Medaka, Oryzias latipes, by the comet assay. Anal Bioanal Chem 399:2235–2242CrossRefGoogle Scholar
  65. Mosior M, Newton A (1995) Mechanism of interaction of protein kinase C with phorbol esters: reversibility and nature of membrane association. J Biol Chem 270:25526–25533CrossRefGoogle Scholar
  66. OECD (2006) OECD guideline for the testing of chemicals. Organization for Economic Cooperation and Development, ParisGoogle Scholar
  67. OECD (2011) Validation report (phase 1) for the zebrafish embryo toxicity test part 2. Organization for Economic Cooperation and Development, ParisGoogle Scholar
  68. OECD (2012) Validation report (phase 2) for the zebrafish embryo toxicity test. Organization for Economic Cooperation and Development, ParisGoogle Scholar
  69. Ohtani M, Nakano Y, Usami T, Demura T (2012) Comparative metabolome analysis of seed kernels in phorbol ester-containing and phorbol ester-free accessions of Jatropha curcas L. Plant Biotechnol 29:171–174CrossRefGoogle Scholar
  70. Oliveira R, Domingues I, Grisolia C, Soares A (2009) Effects of triclosan on zebrafish early-life stages and adults. Environ Sci Pollut Res 16:679–688CrossRefGoogle Scholar
  71. Osman A, Wuertz S, Mekkawy I, Exner H, Kirschbaum F (2007) Lead induced malformations in embryos of the African catfish Clarias gariepinus (Burchell, 1822). Environ Toxicol 375–389Google Scholar
  72. Pandey V, Singh K, Singh J, Kumar A, Singh B, Singh R (2012) Jatropha curcas: A potential biofuel plant for sustainable environmental development. Renew Sust Energ Rev 16:2970–2983Google Scholar
  73. Phasukarratchai N, Tontayakom V, Tongcumpou C (2012) Reduction of phorbol esters in Jatropha curcas L. pressed meal by surfactant solutions extraction. Biomass Bioenerg 44:48–56CrossRefGoogle Scholar
  74. Punsuvon V, Nokkaew R, Karnasuta S (2012) Determination of toxic phorbol esters in biofertilizer produced with Jatropha curcas seed cake. Sci Asia 28:223–225CrossRefGoogle Scholar
  75. Pyati U, Look T, Hammerschmidt M (2007) Zebrafish as a powerful vertebrate model system for in vivo studies of cell death. Semin Cancer Biol 17:154–165CrossRefGoogle Scholar
  76. Rakshit K, Darukeshwara J, Raj K, Narasimhamurthy K, Saibaba P, Bhagya S (2008) Toxicity studies of detoxified Jatropha meal (Jatropha curcas) in rats. Food Chem Toxicol 46:3621–3625CrossRefGoogle Scholar
  77. Ratnadass A, Wink M (2012) The phorbol ester fraction from jatropha curcas seed oil: potential and limits for crop protection against insect pests. Int J Mol Sci 13:16157–16171CrossRefGoogle Scholar
  78. Reimers M, Flockton A, Tanguay R (2004) Ethanol- and acetaldehyde-mediated developmental toxicity in zebrafish. Neurotoxicol Teratol 26:769–781CrossRefGoogle Scholar
  79. Reimers M, La Du J, Periera C, Giovanini J, Tanguay R (2006) Ethanol-dependent toxicity in zebrafish is partially attenuated by antioxidants. Neurotoxicol Teratol 28:497–508CrossRefGoogle Scholar
  80. Rifes P, Carvalho L, Lopes C, Andrade R, Palmeirim I, Thorsteinsdóttir S (2007) Redefining the role of ectoderm in somitogenesis: a player in the formation of the fibronectin matrix of presomitic mesoderm. Development 134:3155–3165CrossRefGoogle Scholar
  81. Sahoo P, Das L (2009) Process optimization for biodiesel production from Jatropha, Karanja and Polanga oils. Fuel 88:1588–1594CrossRefGoogle Scholar
  82. Segner H (2009) Zebrafish (Danio rerio) as a model organism for investigating endocrine disruption. Comp Biochem Physiol 149:187–195Google Scholar
  83. Selderslaghs I, Rompay A, Coen W, Witters H (2009) Development of a screening assay to identify teratogenic and embryotoxic chemicals using the zebrafish embryo. Reprod Toxicol 28:308–320CrossRefGoogle Scholar
  84. Shuit S, Lee K, Kamaruddin A, Yusup S (2010) Reactive extraction and in situ esterification of Jatropha curcas L. seeds for the production of biodiesel. Fuel 89:527–530CrossRefGoogle Scholar
  85. Siang C. (2009). Jatropha curcas L.: development of a new oil crop for biofuel. http://www.eaber.org/sites/default/files/documents/IEEJ_Siang_2009.pdf Accessed 26 Oct 2012
  86. Silitonga A, Atabani A, Mahlia T, Masjuki H, Badruddin I, Mekhilef S (2011) A review on prospect of Jatropha curcas for biodiesel in Indonesia. Renew Sust Energ Rev 15:3733–3756CrossRefGoogle Scholar
  87. Singh R, Vyas D, Srivastava N, Narra M (2008) SPRERI experience on holistic approach to utilize all parts of Jatropha curcas fruit for energy. Renew Energy 33:1868–1873CrossRefGoogle Scholar
  88. Skjaerven K, Finn R, Kryvi H, Fyhn H (2003) Yolk resorption in developing plaice (Pleronectes platessa). University of Bergen, Department of Biology. Institute of Marine Research, BergenGoogle Scholar
  89. Smith B, Sturm R, Carchman R (1983) Calcium modulation of phorbol ester-induced alterations in murine macrophage morphology. Cancer Res 43:3385–3391Google Scholar
  90. Sosa M, Lewin N, Choi S, Blumberg P, Kazanietz M (2009) Biochemical characterization of hyperactive 2-chimaerin mutants revealed an enhanced exposure of C1 and Rac-GAP domains. Biochemistry 34:8171–8178CrossRefGoogle Scholar
  91. Strahle U, Jesuthasan S (1993) Ultraradiation irradiation impairs epiboly in zebrafish embryos: evidence for a microtubule-dependent mechanism of epiboly. Development 119:909–919Google Scholar
  92. Strair R, Schaar D, Goodell L (2002) Administration of a phorbol ester to patients with hematological malignancies: preliminary results from a phase i clinical trial of 12-O-tetradecanoylphorbol-13-acetate. Clin Cancer Res 8:2512–2518Google Scholar
  93. Subramanian K, Singal S, Saxena M, Singhal S (2005) Utilization of liquid biofuels in automotive diesel engines: an Indian perspective. Biomass Bioenergy 29:65–72CrossRefGoogle Scholar
  94. Travis C, Bishop W, Clarke D (2003) The genomic revolution: what does it mean for human and ecological risk assessment? Ecotoxicology 12:489–495CrossRefGoogle Scholar
  95. Voelker D, Vess C, Tillmann M, Nagel R, Otto G, Geisler R (2007) Differential gene expression as a toxicant-sensitive endpoint in zebrafish embryos and larvae. Aquat Toxicol 81:355–364CrossRefGoogle Scholar
  96. Wakandigara A, Nhamo LRM, Kugara J (2013) Chemistry of phorbol ester toxicity in Jatropha curcas seed—a review. Int J Biochem Res Rev 3(3):146–161Google Scholar
  97. Weigt S, Huebler N, Strecker R, Braunbeck T, Broschard T (2011) Zebrafish (Danio rerio) embryos as a model for testing proteratogens. Toxicology 281:25–36CrossRefGoogle Scholar
  98. Yang C, Kazanietz M (2007) Chimaerins: GAPs that bridge diacylglycerol signalling and the small G-protein Rac. Biochem J 403:1–12Google Scholar
  99. Yang C, Liu Y, Leskow F, Weaver V, Kazanietz M (2005) Rac-GAP-dependent inhibition of breast cancer cell proliferation by a2-chimerin. J Biol Chem 280:24363–24370CrossRefGoogle Scholar
  100. Yasuda S, Kai M, Imai S, Kanoh H, Sakane F (2007) Diacylglycerol kinase c interacts with and activates b2-chimaerin, a Rac-specific GAP, in response to epidermal growth factor. FEBS Lett 581:551–557CrossRefGoogle Scholar
  101. Yuan S, Miller D, Barnett G, Hahn J, Williams B (1995) Identification and characterization of human ß2-chimaerin: association with malignant transformation in astrocytoma. Cancer Res 55:3456–3461Google Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Arnold V. Hallare
    • 1
    Email author
  • Paulo Lorenzo S. Ruiz
    • 1
  • J. C. Earl D. Cariño
    • 1
  1. 1.Department of Biology, CASUniversity of the PhilippinesManilaPhilippines

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