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A wave front of increased free calcium traversing the egg at fertilization is demonstrated in the sea urchin Lytechinus pictus. The use of the fluorescent calcium chelator fura-2 in combination with low-light-level TV microscopy and image processing allows the visualization of the Ca2+ wave front with high spatial and temporal resolution. Such a wave is demonstrated as increased fluorescence after an excitation of 340-nm wavelength and as the reciprocal image in form of a reduced fluorescence when excited at 380 nm. The band-like appearance of the wave resembles the Ca2+ wave described for larger eggs of other species. In a dispennic egg the high resolution of the system used allows us to recognize two waves of Ca2+ originating from the respective points of sperm entry.

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A 56-kDa protein isolated from the mucus of the European sea hare Aplysia punctata shows a prefer ential toxicity to autonomously growing transformed mammalian cells. Cell death induced by this protein differs from both apoptosis and necrosis. The cytotoxic effects are irreversible and become apparent at nanomolar concentrations in a cell type-dependent manner. In contrast, injection of micromolar concentrations into mice is tolerated without apparent negative consequences. Microsequencing of the 56-kDa protein released a peptide sequence whose corresponding nucleotide sequence was used as probe to screen A. punctata RNA-based cDNA and to select cDNA clones encoding polypeptides comprising the target peptide. Two closely related cDNA were detected. The cDNA encoding a polypeptide 558 aa in length was considered to reflect a bonafide clone encoding the cytotoxic protein. Its protein-coding section was recloned in vectors suitable for expression in Escherichia coli, in mammalian cells, and in insect cells, respectively. The E. coli-expressed polypeptide was biologically inactive. Transfected mammalian cells expressed a cytotoxic factor and died thereof as if treated with the genuine cytotoxic protein. In contrast, transfected insect cells, which proved to be much less sensitive when treated with the genuine protein, expressed the cytotoxic factor and continued to proliferate, allowing to establish stable insect cell lines expressing sufficient amounts of the cytotoxic factor for further characterization.

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Xenon is an anesthetic with very few side-effects, yet its targets at the cellular level are still unclear. It interferes with many aspects of intracellular Ca2+ homeostasis, but so far no specific event or defined regulatory complex of the Ca2+-signaling system has been identified. Specific effects of xenon were found by investigating its effects on the cell cycle in human endothelial cells: there is a relationship between two cell cycle transition points, their regulation by Ca2+, and specific blocks induced by xenon. Within the group of substances studied (xenon, isoflurane, desflurane, helium, and N2), only xenon blocks the cells almost completely at the G2-M transition after a 2-h treatment; those cells that slip through this block are then arrested at metaphase. If xenon is removed, cells that have been accumulating at the G2-M boundary move into mitosis, and cells blocked at metaphase complete their mitosis normally. No such specific block of the cell cycle was found with the other substances studied. An artificial increase of intracellular Ca2+ in the submicromolar range, using a very low dose of the Ca2+ ionophore ionomycin, or a threefold increase of the external Ca2+ concentration suffices to lift the xenon-induced metaphase block; the cells enter anaphase despite the presence of xenon and complete cell division. Thus, the specific but completely reversible inhibition by xenon of the G2-M transition and the block at metaphase suggest an interaction with a Ca2+-dependent event involved in the control of these processes. The results are consistent with the hypothesis that suppression of Ca2+ signals can be considered as a common denominator of the effects of xenon on the cell cycle and on the neuronal system during anesthesia.

Calcium ions are important in the regulation of mitotic apparatus assembly and in the control of chromosome movement. Changes in intracellular free calcium concentration, [Ca2+]i are achieved by an intracellular calcium-transport system which is highly conserved in different cell types. A membrane-bound protein of relative molecular mass (Mr) 46,000 (46K) is part of this transport system and has been implicated in the regulation of the [Ca2+]i changes associated with the course of mitosis. A monoclonal antibody against this 46K protein inhibits Ca2+-uptake into isolated Ca2+-sequestering membranes and specifically labels membranes associated with the mitotic apparatus of sea urchin embryos. Here we investigate the relationship between the intracellular calcium transport system and mitosis by injection of this monoclonal antibody into living mitotic sea urchin embryos. We find that after injection the intracellular free calcium increases up to 10(-6) M, the mitotic apparatus is rapidly destroyed and the cell is irreversibly blocked in its development.