Ageenko NV et al. (2014), Pigment cell differentiation in sea urchin blas...

ECB-ART-43481

Mar Drugs 2014 Jun 27;127:3874-91. doi: 10.3390/md12073874.

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Pigment cell differentiation in sea urchin blastula-derived primary cell cultures.

Ageenko NV , Kiselev KV , Dmitrenok PS , Odintsova NA .

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The quinone pigments of sea urchins, specifically echinochrome and spinochromes, are known for their effective antioxidant, antibacterial, antifungal, and antitumor activities. We developed in vitro technology for inducing pigment differentiation in cell culture. The intensification of the pigment differentiation was accompanied by a simultaneous decrease in cell proliferation. The number of pigment cells was two-fold higher in the cells cultivated in the coelomic fluids of injured sea urchins than in those intact. The possible roles of the specific components of the coelomic fluids in the pigment differentiation process and the quantitative measurement of the production of naphthoquinone pigments during cultivation were examined by MALDI and electrospray ionization mass spectrometry. Echinochrome A and spinochrome E were produced by the cultivated cells of the sand dollar Scaphechinus mirabilis in all tested media, while only spinochromes were found in the cultivated cells of another sea urchin, Strongylocentrotus intermedius. The expression of genes associated with the induction of pigment differentiation was increased in cells cultivated in the presence of shikimic acid, a precursor of naphthoquinone pigments. Our results should contribute to the development of new techniques in marine biotechnology, including the generation of cell cultures producing complex bioactive compounds with therapeutic potential.

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Genes referenced: LOC100887844 LOC115919910 tubgcp2

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References [+] :

Ageenko, Expression of Pigment Cell-Specific Genes in the Ontogenesis of the Sea Urchin Strongylocentrotus intermedius. 2011, Pubmed, Echinobase

	Figure 1. The structures of shikimic acid, a precursor of naphthoquinone pigments, and echinochrome and spinochromes in accordance with a previous report [7].
	Figure 2. Embryonic pigment cells in a blastula-derived cell culture of the sea urchin Scaphechinus mirabilis cultivated for 3 days. The cells were cultivated in seawater (A); the coelomic fluid of intact sea urchins (B); or the coelomic fluid of injured sea urchins (C). All culture media were supplemented with 2% fetal bovine serum. Insets in B and C: higher magnifications. Note the change in morphology of the pigment cells depending on the medium tested. Scale bar, 10 μm.
	Figure 3. Embryonic pigment cells in a blastula-derived cell culture of the sea urchin Strongylocentrotus intermedius cultivated for 5 (A,B); 17 (C,D) or 41 days (E,F). A, C, E—cells cultivated in the coelomic fluid of intact sea urchins; B, D, F—cells cultivated in the coelomic fluid of injured sea urchins. The coelomic fluid was supplemented with 2% fetal bovine serum. Note the change in morphology of the pigment cells during cultivation. Scale bar, 10 μm.
	Figure 4. Cellular dynamics of pigment cells cultivated in coelomic fluids of intact (blue line) and injured (red line) sea urchins for 2.5 months. At least 500 cells were counted in each tested culture medium.

	Figure 6. Immunofluorescence detection of dividing (phospho-H3-histone-positive) cells in a blastula-derived culture. The preparations were imaged via confocal microscopy. The cells were cultivated on fibronectin-coated coverslips in coelomic fluid obtained from injured sea urchins (A) and in seawater (B) for 12 h and then labeled with Abs against phospho-H3-histone Abs for the detection of dividing cells (red) and tubulin (green) for the detection of microtubules. The nuclei were stained with DAPI (blue). Arrows show ph-H3-histone-positive cells. Scale bar, 10 µm.
	Figure 7. The mass spectra of coelomic fluids obtained from intact (A) and injured (B) sea urchins by MALDI TOF MS using a mass spectrometer (Ultraflex-III TOF/TOF, Bruker Daltonics, Germany). Note a new peak in the coelomic fluid of the injured sea urchins, corresponding to a protein with MW near 4500 Da (marked by an arrow), as well as the shift of the basic components of the intact coelomic fluid to approximately 40–120 Da. The coelomic fluids from three independent experiments were analyzed.
	Figure 8. ESI MS profiles of naphthoquinone pigments from sea urchin cell cultures: A—the pigment extracts from the cells of the sea urchin S. mirabilis after 3-day cultivation in seawater; B—the pigment extracts from the cells of the sea urchin S. intermedius after 6-day cultivation in seawater.