Acta Biologica Hungarica
Volume/Issue:
Volume 63: Issue 1
Expression analysis of heat shock genes in the skin, spleen and blood of common carp (Cyprinus carpio) after cadmium exposure and hypothermia
Authors:
Ágnes Ferencz University of Szeged Department of Biochemistry and Molecular Biology, Faculty of Science and Informatics P.O. Box 533 H-6701 Szeged Hungary

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Renáta Juhász University of Szeged Department of Biochemistry and Molecular Biology, Faculty of Science and Informatics P.O. Box 533 H-6701 Szeged Hungary

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Monica Butnariu Banatâs University of Agricultural Sciences and Veterinary Medicine from Timisoara Department of Exact Sciences Timisoara Romania

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Aranka Deér University of Szeged Department of Biochemistry and Molecular Biology, Faculty of Science and Informatics P.O. Box 533 H-6701 Szeged Hungary

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Ilona Varga University of Szeged Department of Biochemistry and Molecular Biology, Faculty of Science and Informatics P.O. Box 533 H-6701 Szeged Hungary

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J. Nemcsók

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Pages:
15–25
Online Publication Date:
27 Mar 2012
Publication Date:
01 Mar 2012
Article Category:
Research Article
DOI:
https://doi.org/10.1556/abiol.63.2012.1.2
Keywords:
Cadmium treatment; carp; heat shock genes; RT-PCR
Restricted access

Heat shock proteins are chaperones that play a pivotal role in controling multiple regulatory pathways such as stress defense, hormone signaling, cell cycle control, cell proliferation and differentiation, and apoptosis. In this study, the expression patterns of four well-known heat shock genes (hsp70, hsc70-1, hsc70-2 and hsp90α) were characterized in the skin, spleen and blood cells of the common carp, under unstressed conditions and after Cd2+ treatment or hypothermia. The examined genes were expressed in a tissue-specific manner: hsc70-2 was expressed constitutively, and was at best only slightly inducible; hsp90α exhibited a high basic expression in all three tissues, whereas hsc70-1 did so only in the blood cells, the expression of hsp70 proved to be below the level of detection in unstressed fish. Cold shock induced the expression of hsp genes in the spleen (hsp90α) and blood cells (hsp70, hsc70-1 and hsp90α), while Cd2+ treatment has no effect on the expression pattern. The highest inducibilities were detected in the skin: for hsp70 an induction of at least 20-fold after cadmium exposure, for hsc70-1 of at least 30-fold and for hsp90α of 3-fold after hypothermia.

  • Ali, K. S., Dorgai, L., Ábrahám, M., Hermesz, E. (2003) Tissue- and stressor-specific differential expression of two hsc70 genes in carp. Biochem. Biophys. Res. Com. 307, 503–509.

    Hermesz E. , 'Tissue- and stressor-specific differential expression of two hsc70 genes in carp ' (2003 ) 307 Biochem. Biophys. Res. Com. : 503 -509 .

    • Search Google Scholar

  • Ali, K. S., Dorgai, L., Gazdag, A., Ábrahám, M., Hermesz, E. (2003) Identification and induction of hsp70 gene by heat shock and cadmium exposure in carp. Acta Biol. Hung. 54, 323–334.

    Hermesz E. , 'Identification and induction of hsp70 gene by heat shock and cadmium exposure in carp ' (2003 ) 54 Acta Biol. Hung. : 323 -334 .

    • Search Google Scholar

  • Basu, N., Todgham, A. E., Ackerman, P. A., Bibeau, M. R., Nakano, K., Schulte, P. M., Iwama, G. K. (2002) Heat shock protein genes and their functional significance in fish. Gene 295, 173–183.

    Iwama G. K. , 'Heat shock protein genes and their functional significance in fish ' (2002 ) 295 Gene : 173 -183 .

    • Search Google Scholar

  • Caipang, C. M., Lazado, C. C., Brinchmann, M. F., Rombout, J. H., Kiron, V. (2011) Differential expression of immune and stress genes in the skin of Atlantic cod (Gadus morhua). Comp. Biochem. Physiol. Part D Genomics. Proteomics. [Epub ahead of print]

  • Csermely, P., Schnaider, T., Soti, C., Prohaszka Z., Nardai, G. (1998) The 90-kDa molecular chaperone family: Structure, function, and clinical applications. A comprehensive review. Pharmacol. Ther. 79, 129–168.

    Nardai G. , 'The 90-kDa molecular chaperone family: Structure, function, and clinical applications. A comprehensive review ' (1998 ) 79 Pharmacol. Ther. : 129 -168 .

    • Search Google Scholar

  • Currie, S., Tufts, B. L. (1996) Synthesis of stress protein 70 (Hsp70) in rainbow trout (Oncorhynchus mykiss) red blood cells. J. Exp. Biol. 200, 607–614.

    Tufts B. L. , 'Synthesis of stress protein 70 (Hsp70) in rainbow trout (Oncorhynchus mykiss) red blood cells ' (1996 ) 200 J. Exp. Biol. : 607 -614 .

    • Search Google Scholar

  • Duffy, L. K., Scofield, E., Rodgers, T., Patton M., Bowyer, R. T. (1999) Comparative baseline levels of mercury, hsp70 and hsp60 in subsistence fish from the Yukon-Kuskokwim delta region of Alaska. Comp. Biochem. Physiol. Toxicol. Pharmacol. 124, 181–186.

    Bowyer R. T. , 'Comparative baseline levels of mercury, hsp70 and hsp60 in subsistence fish from the Yukon-Kuskokwim delta region of Alaska ' (1999 ) 124 Comp. Biochem. Physiol. Toxicol. Pharmacol. : 181 -186 .

    • Search Google Scholar

  • Ellett, F., Lieschke, G. J. (2010) Zebrafish as a model for vertebrate hematopoiesis. Curr. Opin. Pharmacol. 10, 563–570.

    Lieschke G. J. , 'Zebrafish as a model for vertebrate hematopoiesis ' (2010 ) 10 Curr. Opin. Pharmacol. : 563 -570 .

    • Search Google Scholar

  • Fänge, R., Nilsson, S. (1985) The fish spleen: structure and function. Experientia 41, 152–158.

    Nilsson S. , 'The fish spleen: structure and function ' (1985 ) 41 Experientia : 152 -158 .

    • Search Google Scholar

  • Feder, M. E., Hofmann, G. E. (1999) Heat-shock proteins, molecular chaperones, and the stress response: Evolutionary and ecological physiology. Annu. Rev. Physiol. 61, 243–282.

    Hofmann G. E. , 'Heat-shock proteins, molecular chaperones, and the stress response: Evolutionary and ecological physiology ' (1999 ) 61 Annu. Rev. Physiol. : 243 -282 .

    • Search Google Scholar

  • Fink, A. L., Goto, Y. (1998) Molecular Chaperones in the Life Cycle of Proteins: Structure, Function, and Mode of Action. Marcel Dekker, New York.

    Goto Y. , '', in Molecular Chaperones in the Life Cycle of Proteins: Structure, Function, and Mode of Action , (1998 ) -.

  • Fulladosa, E., Deane, E., Ng, A. H., Woo, N. Y., Murat, J. C., Villaescusa, I. (2006) Stress proteins induced by exposure to sublethal levels of heavy metals in sea bream (Sparus sarba) blood cells. Toxicol. In Vitro 20, 96–100.

    Villaescusa I. , 'Stress proteins induced by exposure to sublethal levels of heavy metals in sea bream (Sparus sarba) blood cells ' (2006 ) 20 Toxicol. In Vitro : 96 -100 .

    • Search Google Scholar

  • Geist, J., Werner, I., Eder, K. J., Leutenegger, C. M. (2007) Comparisons of tissue-specific transcription of stress response genes with whole animal endpoints of adverse effect in striped bass (Morone saxatilis) following treatment with copper and esfenvalerate. Aquat. Toxicol. 85, 28–39.

    Leutenegger C. M. , 'Comparisons of tissue-specific transcription of stress response genes with whole animal endpoints of adverse effect in striped bass (Morone saxatilis) following treatment with copper and esfenvalerate ' (2007 ) 85 Aquat. Toxicol. : 28 -39 .

    • Search Google Scholar

  • Hao, H., Naomoto, Y., Bao, X., Watanabe, N., Sakurama, K., Noma, K., Motoki, T., Tomono, Y., Fukazawa, T., Shirakawa, Y., Yamatsuji, T., Matsuoka, J., Takaoka, M. (2010) HSP90 and its inhibitors. Oncol. Rep. 23, 1483–1492.

    Takaoka M. , 'HSP90 and its inhibitors ' (2010 ) 23 Oncol. Rep. : 1483 -1492 .

  • Hartl, F. U. (1996) Molecular chaperones in cellular protein folding. Nature 381, 571–580.

    Hartl F. U. , 'Molecular chaperones in cellular protein folding ' (1996 ) 381 Nature : 571 -580 .

    • Search Google Scholar

  • Hassanein, H. M. A., Banhawy, M. A., Soliman, F. M., Abdel-Rehim, S. A., Muller, W. E. G., Schroder, H. C. (1999) Induction of Hsp70 by the herbicide oxyfluoren (goal) in the Egyptian Nile fish, Oreochromis niloticus. Arch. Environ. Contam. Toxicol. 37, 78–84.

    Schroder H. C. , 'Induction of Hsp70 by the herbicide oxyfluoren (goal) in the Egyptian Nile fish, Oreochromis niloticus ' (1999 ) 37 Arch. Environ. Contam. Toxicol. : 78 -84 .

    • Search Google Scholar

  • Hendrick, J. P., Hartl, F. U. (1993) Molecular chaperone functions of heat-shock proteins. Annu. Rev. Biochem. 62, 349–384.

    Hartl F. U. , 'Molecular chaperone functions of heat-shock proteins ' (1993 ) 62 Annu. Rev. Biochem. : 349 -384 .

    • Search Google Scholar

  • Hermesz, E., Ábrahám, M., Nemcsók, J. (2001) Identification of two hsp90 genes in carp. Comp. Biochem. Physiol. Part C. 129, 397–407.

    Nemcsók J. , 'Identification of two hsp90 genes in carp ' (2001 ) 129 Comp. Biochem. Physiol. Part C. : 397 -407 .

    • Search Google Scholar

  • Iwama, G. K., Thomas, P. T., Forsyth, R. B., Vijayan, M. M. (1998) Heat shock protein expression in fish. Review. in Fish Biol. Fish 8, 35–56.

    Vijayan M. M. , 'Heat shock protein expression in fish ' (1998 ) 8 Review. in Fish Biol. Fish : 35 -56 .

    • Search Google Scholar

  • Jonak, C., Klosner, G., Trauringer, F. (2006) Heat shock proteins in the skin. Int. J. Cosm. Sci. 28, 233–241.

    Trauringer F. , 'Heat shock proteins in the skin ' (2006 ) 28 Int. J. Cosm. Sci. : 233 -241 .

    • Search Google Scholar

  • Kiang, J. G., Tsokos, G. C. (1998) Heat shock proteins 70 kDa: Molecular biology, biochemistry, and physiology. Pharmacol. Ther. 80, 183–201.

    Tsokos G. C. , 'Heat shock proteins 70 kDa: Molecular biology, biochemistry, and physiology ' (1998 ) 80 Pharmacol. Ther. : 183 -201 .

    • Search Google Scholar

  • Krone, P. H., Sass, J. B. (1994) Hsp90α and hsp90β genes are present in the zebrafish and are differentially regulated in developing embryos. Biochem. Biophys. Res. Commun. 204, 746–752.

    Sass J. B. , 'Hsp90α and hsp90β genes are present in the zebrafish and are differentially regulated in developing embryos ' (1994 ) 204 Biochem. Biophys. Res. Commun. : 746 -752 .

    • Search Google Scholar

  • Lertkiatmongkol, P., Pethuan, S., Jirakanjanakit, N., Rongnoparut, P. (2010) Transcription analysis of differentially expressed genes in insecticide-resistant Aedes aegypti mosquitoes after deltamethrin exposure. J. Vector. Ecol. 35, 197–203.

    Rongnoparut P. , 'Transcription analysis of differentially expressed genes in insecticide-resistant Aedes aegypti mosquitoes after deltamethrin exposure ' (2010 ) 35 J. Vector. Ecol. : 197 -203 .

    • Search Google Scholar

  • Lund, S. G., Phillips, M. C., Moyes, C. D., Tufts, B. L. (2000) The effects of cell ageing on protein synthesis in rainbow trout (Oncorhynchus mykiss) red blood cells. J. Exp. Biol. 203, 2219–2228.

    Tufts B. L. , 'The effects of cell ageing on protein synthesis in rainbow trout (Oncorhynchus mykiss) red blood cells ' (2000 ) 203 J. Exp. Biol. : 2219 -2228 .

    • Search Google Scholar

  • Manchado, M., Salas-Leiton, E., Infante, C., Ponce, M., Asensio, E., Crespo, A., Zuasti, E., Cañavate, J. P. (2008) Molecular characterization, gene expression and transcriptional regulation of cytosolic HSP90 genes in the flatfish Senegalese sole (Solea senegalensis Kaup). Gene 416, 77–84.

    Cañavate J. P. , 'Molecular characterization, gene expression and transcriptional regulation of cytosolic HSP90 genes in the flatfish Senegalese sole (Solea senegalensis Kaup) ' (2008 ) 416 Gene : 77 -84 .

    • Search Google Scholar

  • Mladineo, I., Block, B. A. (2010) Expression of cytokines IL-1beta and TNF-alpha in tissues and cysts surrounding Didymocystis wedli (Digenea, Didymozoidae) in the Pacific bluefin tuna (Thunnus orientalis). Fish Shellfish Immunol. 29, 487–493.

    Block B. A. , 'Expression of cytokines IL-1beta and TNF-alpha in tissues and cysts surrounding Didymocystis wedli (Digenea, Didymozoidae) in the Pacific bluefin tuna (Thunnus orientalis) ' (2010 ) 29 Fish Shellfish Immunol. : 487 -493 .

    • Search Google Scholar

  • Morimoto, R. I., Santoro, M. G. (1998) Stress-inducible responses and heat shock proteins: New pharmacological targets for cytoprotection. Nat. Biotechnol. 16, 833–838.

    Santoro M. G. , 'Stress-inducible responses and heat shock proteins: New pharmacological targets for cytoprotection ' (1998 ) 16 Nat. Biotechnol. : 833 -838 .

    • Search Google Scholar

  • Morimoto, R. I., Tissieres, A., Georgopoulos, C. (1990) Stress Proteins in Biology and Medicine. Cold Spring Harbor Press, New York.

    Georgopoulos C. , '', in Stress Proteins in Biology and Medicine , (1990 ) -.

  • Mukhopadhyay, I., Nazir, A., Saxena, D. K., Chowdhuri, D. K. (2002) Toxicity of cypermethrin: hsp70 as a biomarker of response in transgenic Drosophila. Biomarkers 7, 501–510.

    Chowdhuri D. K. , 'Toxicity of cypermethrin: hsp70 as a biomarker of response in transgenic Drosophila ' (2002 ) 7 Biomarkers : 501 -510 .

    • Search Google Scholar

  • Oruc, E. U., Usta, D. (2007) Evaluation of oxidative stress responses and neurotoxicity potential of diazinon in different tissues of Cyprinus carpio. Environ. Toxicol. Pharmacol. 23, 48–55.

    Usta D. , 'Evaluation of oxidative stress responses and neurotoxicity potential of diazinon in different tissues of Cyprinus carpio ' (2007 ) 23 Environ. Toxicol. Pharmacol. : 48 -55 .

    • Search Google Scholar

  • Padmini, E. (2010) Physiological adaptations of stressed fish to polluted environments: role of heat shock proteins. Rev. Environ. Contam. Toxicol. 206, 1–27.

    Padmini E. , 'Physiological adaptations of stressed fish to polluted environments: role of heat shock proteins ' (2010 ) 206 Rev. Environ. Contam. Toxicol. : 1 -27 .

    • Search Google Scholar

  • Park, J. H., Lee, J. J., Yoon, S., Lee, J. S., Choe, S. Y., Choe, J., Park, E. H., Kim, C. G. (2001) Genomic cloning of the Hsc71 gene in the hermaphroditic teleost Rivulus marmoratus and analysis of its expression in skeletal muscle: Identification of a novel muscle-preferred regulatory element. Nucleic Acids Res. 29, 3041–3050.

    Kim C. G. , 'Genomic cloning of the Hsc71 gene in the hermaphroditic teleost Rivulus marmoratus and analysis of its expression in skeletal muscle: Identification of a novel muscle-preferred regulatory element ' (2001 ) 29 Nucleic Acids Res. : 3041 -3050 .

    • Search Google Scholar

  • Pearl, L. H., Prodromou, C. (2000) Structure and in vivo function of hsp90. Curr. Opin. Struct. Biol. 10, 46–51.

    Prodromou C. , 'Structure and in vivo function of hsp90 ' (2000 ) 10 Curr. Opin. Struct. Biol. : 46 -51 .

    • Search Google Scholar

  • Rakers, S., Gebert, M., Uppalapati, S., Meyer, W., Maderson, P., Sell, A. F., Kruse, C., Paus, R. (2010) ’Fish matters’: the relevance of fish skin biology to investigative dermatology. Exp. Dermatol. 19, 313–324.

    Paus R. , '’Fish matters’: the relevance of fish skin biology to investigative dermatology ' (2010 ) 19 Exp. Dermatol. : 313 -324 .

    • Search Google Scholar

  • Roberts, R. J., Agius, C., Saliba, C., Bossier, P., Sung, Y. Y. (2010) Heat shock proteins (chaperones) in fish and shellfish and their potential role in relation to fish health: a review. J. Fish. Dis. 33, 789–801.

    Sung Y. Y. , 'Heat shock proteins (chaperones) in fish and shellfish and their potential role in relation to fish health: a review ' (2010 ) 33 J. Fish. Dis. : 789 -801 .

    • Search Google Scholar

  • Sanders, B. M., Nguyen, J., Martin, L. S., Howe, S. R., Coventry, S. (1995) Induction and subcellular localization of two major stress proteins in response to copper in the fathead minnow Pimephales promelas. Comp. Biochem. Physiol. C Pharmacol. Toxicol. 112, 335–343.

    Coventry S. , 'Induction and subcellular localization of two major stress proteins in response to copper in the fathead minnow Pimephales promelas ' (1995 ) 112 Comp. Biochem. Physiol. C Pharmacol. Toxicol. : 335 -343 .

    • Search Google Scholar

  • Santacruz, H., Vriz, S., Angelier, N. (1997) Molecular characterization of a heat shock cognate cDNA of zebrafish, hsc70, and developmental expression of the corresponding transcripts. Dev. Genet. 21, 223–233.

    Angelier N. , 'Molecular characterization of a heat shock cognate cDNA of zebrafish, hsc70, and developmental expression of the corresponding transcripts ' (1997 ) 21 Dev. Genet. : 223 -233 .

    • Search Google Scholar

  • Vijayan, M. M., Pereira, C., Kruzynski, G., Iwama, G. K. (1998) Sublethal concentrations of contaminant induce the expression of hepatic heat-shock protein 70 in 2 salmonids. Aquat. Toxicol. 40, 101–108.

    Iwama G. K. , 'Sublethal concentrations of contaminant induce the expression of hepatic heat-shock protein 70 in 2 salmonids ' (1998 ) 40 Aquat. Toxicol. : 101 -108 .

    • Search Google Scholar

  • Welch, W. J. (1993) How cells respond to stress. Sci. Am. 269, 56–64.

    Welch W. J. , 'How cells respond to stress ' (1993 ) 269 Sci. Am. : 56 -64 .

  • Williams, J. H., Farag, A. M., Stansbury, M. A., Young, P. A., Bergman, H. L., Petersen, N. S. (1996) Accumulation of hsp70 in juvenile and adult rainbow trout gill exposed to metal-contaminated water and/or diet. Environ. Toxicol. Chem. 15, 1324–1328.

    Petersen N. S. , 'Accumulation of hsp70 in juvenile and adult rainbow trout gill exposed to metal-contaminated water and/or diet ' (1996 ) 15 Environ. Toxicol. Chem. : 1324 -1328 .

    • Search Google Scholar

  • Young, J. C. (2010) Mechanisms of the Hsp70 chaperone system. Biochem. Cell. Biol. 88, 291–300.

    Young J. C. , 'Mechanisms of the Hsp70 chaperone system ' (2010 ) 88 Biochem. Cell. Biol. : 291 -300 .

    • Search Google Scholar

  • Young, J. C., Moarefi, I., Hartl, F. U. (2001) Hsp90: A specialized but essential protein-folding tool. J. Cell Biol. 154, 267–273.

    Hartl F. U. , 'Hsp90: A specialized but essential protein-folding tool ' (2001 ) 154 J. Cell Biol. : 267 -273 .

    • Search Google Scholar

  • Zafarullah, M., Wisniewski, J., Shworak, N. W., Schieman, S., Misra, S., Gedamu, L. (1992) Molecular cloning and characterization of a constitutively expressed heat-shock-cognate hsc71 gene from rainbow trout. Eur. J. Biochem. 204, 893–900.

    Gedamu L. , 'Molecular cloning and characterization of a constitutively expressed heat-shock-cognate hsc71 gene from rainbow trout ' (1992 ) 204 Eur. J. Biochem. : 893 -900 .

    • Search Google Scholar
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Cover Acta Biologica Hungarica
Acta Biologica Hungarica
Print ISSN:
0236-5383
Online ISSN:
1588-256X

Senior editors

Editor(s)-in-Chief: Elekes, Károly

Editorial Board

    1. Csányi, Vilmos (Göd)
    1. Dudits, Dénes (Szeged)
    1. Falus, András (Budapest)
    1. Fischer, Ernő (Pécs)
    1. Gábriel, Róbert (Pécs)
    1. Gulya, Károly (Szeged)
    1. Gulyás, Balázs (Stockholm)
    1. Hajós, Ferenc (Budapest)
    1. Hámori, József (Budapest)
    1. Heszky, László (Gödöllő)
    1. Hideg, Éva (Szeged)
    1. E. Ito (Sanuki)
    1. Janda, Tibor (Martonvásár)
    1. Kavanaugh, Michael P. (Missoula)
    1. Kása, Péter (Szeged)
    1. Klein, Éva (Stockholm)
    1. Kovács, János (Budapest)
    1. Brigitte Mauch-Mani (Neuchâtel)
    1. Nässel, Dick R. (Stockholm)
    1. Nemcsók, János (Szeged)
    1. Péczely, Péter (Gödöllő)
    1. Roberts, D. F. (Newcastle-upon-Tyne)
    1. Sakharov, Dimitri A. (Moscow)
    1. Singh, Meharvan (Fort Worth)
    1. Sipiczky, Mátyás (Debrecen)
    1. Szeberényi, József (Pécs)
    1. Székely, György (Debrecen)
    1. Tari, Irma (Szeged)
    1. Vágvölgyi, Csaba (Szeged),
    1. L. Zaborszky (Newark)

 

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Acta Biologica Hungarica
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