. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. Time (s) Figure 2. Responses of ctenophores to physical stimulation. Lumi- nous ctenophores produced bright flashes when disturbed (A-C), even if only briefly touched (arrowhead). In contrast, Pleurohrachia pileits showed no light emission even during continuous stirring (D, E). The y-axis shows counts per 20-ms bin. produce light, we attempted to regenerate extracts with synthesized coelenterazine (provided by O. Shimomura), the luciferin found in luminous ctenophores and cnidar- ians (Ward and Cormier, 1975; Shimomura, 198
. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. Time (s) Figure 2. Responses of ctenophores to physical stimulation. Lumi- nous ctenophores produced bright flashes when disturbed (A-C), even if only briefly touched (arrowhead). In contrast, Pleurohrachia pileits showed no light emission even during continuous stirring (D, E). The y-axis shows counts per 20-ms bin. produce light, we attempted to regenerate extracts with synthesized coelenterazine (provided by O. Shimomura), the luciferin found in luminous ctenophores and cnidar- ians (Ward and Cormier, 1975; Shimomura, 1985). Specimens were homogenized in 100 mM Tris, 50 mA/ EDTA, 500 mM NaCl, pH , filtered through a What- man GF/C glass-fiber filter to remove debris, and centri- fuged for 30 min at 35,000 X g. Photoprotein present in one ml of supernatant was triggered by the addition of 50 mM CaCl2 until no further light was produced (typi- cally 250 ^1 was sufficient, although no light was emitted by Pleurobrachia preparations). This was followed by 250 n\ of 200 mM EDTA to chelate the added Ca2+, and the solution was saturated with ammonium sulfate to precipitate the reacted protein. For the regeneration, one ml of the saturated solution was centrifuged at 15,000 RPM in an Eppendorf minicentrifuge for 15 min. The pellet of precipitate was resuspended in 200 ^1 of 10 mA/Tris, 5 mM EDTA, 500 mA/ NaCl, and 5 mM 0- mercaptoethanol (techniques based on Campbell and Herring, 1990). Each treatment was incubated for 6 h at 4°C with 2 n\ methanol either containing coelenterazine or with no luciferin for the negative controls. The light produced upon final addition of CaCl2 indicated the extent of regeneration. This experiment was conducted using the hydromedusa Haliscera conica as a positive control. We replicated this experiment once using Haliscera, the hydroid Obelia sp., and an undescribed luminous ctenophore; and again using the ctenophores Beroe cuciimis, I 'elamen parallelum, and Haeckelia
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