Skip to main content
SpringerLink
Log in

Localization of granulatimide alkaloids in the tissues of the ascidian Didemnum granulatum

  • Research Article
  • Published:
Marine Biology Aims and scope Submit manuscript

Abstract

Ascidians are a notable source of nitrogen-bearing secondary metabolites with a wide range of biological activities. Although many biologically active compounds have been isolated from ascidians, it is often unclear whether the animal or associated microbial symbionts such as bacteria or fungi are the true biosynthetic source of the metabolites. We have addressed the question of the biosynthetic source of the alkaloids granulatimide and isogranulatimides by localizing these compounds within the ascidian. In this work, we demonstrate that granulatimide is stored in Didemnum granulatum tunic bladder cells. Analysis by confocal fluorescence microscopy at the granulatimide emission range indicated the presence of fluorescent cells as highly vacuolated cells found dispersed in the ascidian tunic. Since this is the most exposed ascidian tissue, it is possible that these alkaloids may have a protective role, either as sunscreens and/or as feeding deterrents.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

References

  • Amador ML, Jimeno J, Paz-Ares L, Cortes-Funes H, Hidalgo M (2003) Progress in the development and acquisition of anticancer agents from marine sources. Ann Oncol 14:1607–1615

    Article  CAS  Google Scholar 

  • Behmer OA, Tolosa EMC, Freitas Neto AG (1976) Manual de técnicas de histologia normal e patológica. EDART/EDUSP, Sao Paulo

  • Berlinck RGS, Britton R, Piers E, Lim L, Roberge M, Rocha RM, Andersen RJ (1998) Granulatimide and isogranulatimide, aromatic alkaloids with G2 checkpoint inhibition activity isolated from the Brazilian ascidian Didemnum granulatum: structure elucidation and synthesis. J Org Chem 63:9850–9856

    Article  CAS  Google Scholar 

  • Bibby TS, Nield J, Chen M, Larkum AWD, Barber J (2003) Structure of a photosystem II supercomplex isolated from Prochloron didemni retaining its chlorophyll a/b light-harvesting system. Proc Natl Acad Sci USA 100:9050–9054

    Article  CAS  Google Scholar 

  • Blunt JW, Copp BR, Munro MHG, Northcote PT, Princep MR (2005) Marine natural products. Nat Prod Rep 22:15–61

    Article  CAS  Google Scholar 

  • Britton R, de Oliveira JHHL, Andersen RJ, Berlinck RGS (2001) Granulatimide and 6-bromogranulatimide, minor alkaloids of the Brazilian ascidian Didemnum granulatum. J Nat Prod 64:254–255

    Article  CAS  Google Scholar 

  • Bugni TS, Ireland CM (2004) Marine-derived fungi: a chemically and biologically diverse group of microorganisms. Nat Prod Rep 21:143–163

    Article  CAS  Google Scholar 

  • Dunham P, Weissmann G (1986) Aggregation of marine sponge cells induced by Ca pulses, Ca ionophores, and phorbol esters proceeds in the absence of external Ca. Biochem Biophys Res Commun 134:1319–1326

    Article  CAS  Google Scholar 

  • Faulkner DJ (2002) Marine natural products. Nat Prod Rep 19:1–48

    CAS  PubMed  Google Scholar 

  • Faulkner DJ, Newman DJ, Cragg GM (2004) Investigations of the marine flora and fauna of the Islands of Palau. Nat Prod Rep 21:50–76

    Article  CAS  Google Scholar 

  • Groepler W, Schuett C (2003) Bacterial community in the tunic matrix of a colonial ascidian Diplosoma migrans. Helgoland Mar Res 57:139–143

    Article  Google Scholar 

  • Hildebrand M, Waggoner LE, Lim GE, Sharp KH, Ridley CP, Haygood MG (2004) Approaches to identify, clone, and express symbiont bioactive metabolite genes. Nat Prod Rep 21:122–142

    Article  CAS  Google Scholar 

  • Hirose E (1992) Tunic cells in Leptoclinides echinatus (Didemnidae, Ascidiacea): an application of scanning electron microscopy for paraffin embedding specimens. Hiyoshi Rev Natur Sci 11:5–8

    Google Scholar 

  • Hirose E (2001) Acid containers and cellular networks in the ascidian tunic with special remarks on ascidian phylogeny. Zool Sci 18:723–731

    Article  Google Scholar 

  • Hirose E, Aoki M, Chiba K (1996) Fine structure of tunic cells and distribution of bacteria in the tunic of luminescent ascidian Clavelina miniata (Ascidiacea. Urochordata). Zool Sci 13:519–523

    Article  Google Scholar 

  • Hirose E, Ishii T, Saito Y, Taneda Y (1994) Seven types of tunic cells in the colonial ascidian Aplidium yamazii (Polyclinidae, Aplousobranchia): morphology, classification and possible functions. Zool Sci 11:737–743

    Google Scholar 

  • Hirose E, Ohtsuka K, Ishikura M, Maruyama T (2004) Ultraviolet absorption in ascidian tunic and ascidian-Prochloron symbiosis. J Mar Biol Assoc UK 84:789–794

    Article  CAS  Google Scholar 

  • Hirose E, Saito Y, Watanabe H (1991) Tunic cell morphology and classification in Botryllid ascidians. Zool Sci 8:951–958

    Google Scholar 

  • Hirose E, Yamashiro H, Mori Y (2001) Properties of tunic acid in the ascidian Phallusia nigra (Ascidiidae, Phlebobranchia). Zool Sci 18:309–314

    Article  Google Scholar 

  • Horton PA, Longley RE, McConnell OJ, Ballas LM (1994) Staurosporine aglycone (K252-C) and arcyriaflavin-A from the marine ascidian, Eudistoma sp. Experientia 50:843–845

    Article  CAS  Google Scholar 

  • Jeedigunta S, Krenisky JM, Kerr RG (2000) Diketopiperazines as advanced intermediates in the biosynthesis of ecteinascidins. Tetrahedron 56:3303–3307

    Article  CAS  Google Scholar 

  • Jiang X, Zhao B, Britton R, Lim LY, Leong D, Sanghera JS, Zhou B-BS, Piers E, Andersen RJ, Roberge M (2004) Inhibition of Chk1 by the DNA damage checkpoint inhibitor isogranulatimide. Mol Cancer Ther 3:1221–1227

    CAS  PubMed  Google Scholar 

  • Jimeno J, Lopez-Martin JA, Ruiz-Casado A, Izquierdo MA, Scheuer PJ, Rinehart KL (2004) Progress in the clinical development of new marine-derived anticancer compounds. Anti Cancer Drug 15:321–329

    Article  CAS  Google Scholar 

  • Kerr RG, Miranda NF (1995) Biosynthetic studies of ecteinascidins in the tunicate Ecteinascidia turbinata. J Nat Prod 58:1618–1621

    Article  CAS  Google Scholar 

  • Kühl M, Larkum AWD (2002) The microenvironment and photosynthetic performance of Prochloron sp. in symbiosis with didemnid ascidians. In: Seckbach J (eds) Cellular origin and life in extreme habitats: symbiosis, mechanisms and model systems. Kluwer Academic, Dordrecht, pp 273–290

    Google Scholar 

  • Lindquist N, Fenical W (1991) New tambjamine class alkaloids from the marine ascidian Atapazoa sp. and its nudibranch predators—origin of the tambjamines in Atapazoa. Experientia 47:504–506

    Article  CAS  Google Scholar 

  • Long PF, Dunlap WC, Battershill CN, Jaspars M (2005) Shotgun cloning and heterologous expression of the patellamide gene cluster as a strategy to achieving sustained metabolite production. ChemBioChem 6:1760–1765

    Article  CAS  Google Scholar 

  • Maruyama T, Hirose E, Ishikura M (2003) Ultraviolet-light-absorbing tunic cells in didemnid ascidians hosting a symbiotic photo-oxygenic prokaryote, Prochloron. Biol Bull 204:109–113

    Article  Google Scholar 

  • Newman DJ, Cragg GM (2004a) Advanced preclinical and clinical trials of natural products and related compounds from marine sources. Curr Med Chem 11:1693–1713

    Article  CAS  Google Scholar 

  • Newman DJ, Cragg GM (2004b) Marine natural products and related compounds in clinical and advanced preclinical trials. J Nat Prod 67:1216–1238

    Article  CAS  Google Scholar 

  • Omura S, Sasaki Y, Iwai Y, Takeshima H (1995) Staurosporine, a potentially important gift from a microorganism. J Antibiot 48:535–548

    Article  CAS  Google Scholar 

  • Piers E, Britton R, Andersen RJ (2000) Improved synthesis of isogranulatimide, a G2 checkpoint inhibitor. Syntheses of didemnimide C, isodidemnimide A, neodidemnimide A, 17-methylgranulatimide, and isogranulatimides A-C. J Org Chem 65:530–535

    Article  CAS  Google Scholar 

  • Roberge M, Berlinck RGS, Xu L, Anderson H, Lim LY, Curman D, Stringer CM, Friend SH, Davies P, Vincent I, Haggarty SJ, Kelly MT, Britton R, Piers E, Andersen RJ (1998) High throughtput assay for G2 checkpoint inhibitors and identification of the structurally novel compound isogranulatimide. Cancer Res 58:5701–5706

    CAS  PubMed  Google Scholar 

  • Rottmayr EM, Steffan B, Wanner GG (2001) Pigmentation and tunic cells in Cystodytes dellechiajei (Urochordata, Ascidiacea). Zoomorphology 120:159–170

    Article  Google Scholar 

  • Sakai R, Jares-Erijman EA, Elipe MVS, Rinehart KL (1996) Ecteinascidins: putative biosynthetic precursors and absolute stereochemistry. J Am Chem Soc 118:9017–9023

    Article  CAS  Google Scholar 

  • Saleh MB, Kerr RG (2004) Oxidation of tyrosine diketopiperazine to DOPA diketopiperazine with tyrosine hydroxylase. J Nat Prod 67:1390–1391

    Article  CAS  Google Scholar 

  • Salomon CE, Faulkner DJ (2002) Localization studies of bioactive cyclic peptides in the ascidian Lissoclinum patella. J Nat Prod 65:689–692

    Article  CAS  Google Scholar 

  • Salomon CE, Magarvey NA, Sherman DH (2004) Merging the potential of microbial genetics with biological and chemical diversity: an even brighter future for marine natural product drug discovery. Nat Prod Rep 21:105–121

    Article  CAS  Google Scholar 

  • Schmidt EW, Suddek S, Haygood MG (2004) Genetic evidence supports secondary metabolic diversity in Prochloron spp., the cyanobacterial symbiont of a tropical ascidian. J Nat Prod 67:1341–1345

    Article  CAS  Google Scholar 

  • Schmidt EW, Nelson JT, Rasko DA, Sudek S, Eisen JA, Haygood MG, Ravel J (2005) Patellamide A and C biosynthesis by a microcin-like pathway in Prochloron didemni, the cyanobacterial symbiont of Lissoclinum patella. Proc Natl Acad Sci 102:7315–7320

    Article  CAS  Google Scholar 

  • Schreiber U, Gademann R, Ralph PJ, Larkum AWD (1997) Assessment of photosynthetic performance of Prochloron in Lissoclinum patella in hospite by chlorophyll fluorescence measurements. Plant Cell Physiol 38:945–951

    Article  CAS  Google Scholar 

  • Schupp P, Eder C, Proksch P, Wray V, Schneider B, Herderich M, Paul V (1999) Staurosporine derivatives from the ascidian Eudistoma toealensis and its predatory flatworm Pseudoceros sp. J Nat Prod 62:959–962

    Article  CAS  Google Scholar 

  • Schupp P, Proksch P, Wray V (2002) Further new staurosporine derivatives from the ascidian Eudistoma toealensis and its predatory flatworm Pseudoceros sp. J Nat Prod 65:295–298

    Article  CAS  Google Scholar 

  • Shen GQ, Baker BJ (1994) Biosynthetic studies of the eudistomins in the tunicate Eudistoma olivaceum. Tetrahedron Lett 35:1141–1144

    Article  Google Scholar 

  • Shirae M, Saito Y (2000) A comparison of hemocytes and their phenoloxidase activity among botryllid ascidians. Zool Sci 17:881–891

    Article  CAS  Google Scholar 

  • Sings HL, Rinehart KL (1996) Compounds produced from potential tunicate-blue-green algal symbiosis: a review. J Ind Microbiol Biotechnol 17:385–396

    Article  CAS  Google Scholar 

  • Steffan B, Brix K, Pütz W (1993) Biosynthesis of shermilamine B. Tetrahedron 49:6223–6228

    Article  CAS  Google Scholar 

  • Tincu JA, Craig AG, Taylor SW (2000) Plicatamide: a lead to the biosynthetic origins of the tunichromes? Biochem Biophys Res Commun 270:421–424

    Article  CAS  Google Scholar 

  • Tincu JA, Menzel LP, Azimov R, Sands J, Hong T, Waring AJ, Taylor SW, Lehrer RI (2003) Plicatamide, an antimicrobial octapeptide from Styela plicata hemocytes. J Biol Chem 278:13546–13553

    Article  CAS  Google Scholar 

  • Vera MD, Joullié MM (2002) Natural products as probes of cell biology: 20 years of didemnin research. Med Res Rev 22:102–145

    Article  CAS  Google Scholar 

  • Vervoort HC, Gross SER, Fenical W, Lee AY, Clardy J (1997) Didemnimides A-D: novel predator-deterrent alkaloids from the Caribbean mangrove ascidian Didemnum conchyliatum. J Org Chem 62:1486–1490

    Article  CAS  Google Scholar 

  • Vervoort HC, Pawlik JR, Fenical W (1998) Chemical defense of the caribbean ascidian Didemnum conchyliatum. Mar Ecol Prog Ser 164:221–228

    Article  CAS  Google Scholar 

Download references

Acknowledgments

The authors are particularly grateful to Dr. Christine Salomon (University of Minnesota) for a careful reading of the manuscript and many suggestions to improve it. The authors thank the staff of CEBIMar—USP for the provision of laboratory facilities and technical support during field trips; the staff of the Laboratório de Fisiologia Celular do Plasmodium (IB-USP) for the assistance with confocal microscopy; and Gustavo M. Diaz (Universidade Estadual de Campinas, Brazil) for valuable discussions. Financial support was provided by FAPESP as grants to M.R.C. (99/11443-1) and R.G.S.B. (01/06202-7), and as a scholarship to S.P.L. The authors also thank the Universidade de São Paulo within the PROCONTES program to M.H.R.S. and R.G.S.B. All experiments complied with current Brazilian laws. The authors dedicate this work to the late Professor D. John Faulkner (Scripps Institution of Oceanography), in memoriam.

Author information

Authors and Affiliations

  1. Instituto de Química de São Carlos, Universidade de São Paulo, Caixa Postal 780, 13560-970, São Carlos, SP, Brazil

    Mirna H. R. Seleghim, Simone P. de Lira & Roberto G. S. Berlinck

  2. Faculdade de Informática e Administração Paulista, 01538-001, São Paulo, SP, Brazil

    Patrícia T. Campana

  3. Departamento de Fisiologia, Instituto de Biociências, Universidade de São Paulo, Cidade Universitária, Rua do Matão, Travessa 14, n. 321, 05508-900, São Paulo, SP, Brazil

    Marcio R. Custódio

Authors
  1. Mirna H. R. Seleghim
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Simone P. de Lira
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Patrícia T. Campana
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Roberto G. S. Berlinck
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Marcio R. Custódio
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Marcio R. Custódio.

Additional information

Communicated by M. Kühl, Helsingør

Rights and permissions

Reprints and permissions

About this article

Cite this article

Seleghim, M.H.R., de Lira, S.P., Campana, P.T. et al. Localization of granulatimide alkaloids in the tissues of the ascidian Didemnum granulatum . Mar Biol 150, 967–975 (2007). https://doi.org/10.1007/s00227-006-0410-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00227-006-0410-5

Keywords

Search

Navigation