Cytotoxic cyplasin of the sea hare, Aaplysia punctata, cDNA cloning, and expression of bioactive recombinants in insect cells.

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.

Xenon prevents cellular damage in differentiated PC-12 cells exposed to hypoxia.

BACKGROUND: The neuroprotective effect of xenon has been demonstrated for glutamatergic neurons. In the present study it is investigated if dopaminergic neurons, i.e. nerve-growth-factor differentiated PC-12 cells, are protected as well against hypoxia-induced cell damage in the presence of xenon. RESULTS: Pheochromocytoma cells differentiated by addition of nerve growth factor were placed in a N2-saturated atmosphere, a treatment that induced release of dopamine, reaching a maximum after 30 min. By determining extracellular lactate dehydrogenase concentration as marker for concomitant cellular damage, a substantial increase of enzymatic activity was found for N2-treated cells. Replacement of N2 by xenon in such a hypoxic atmosphere resulted in complete protection against cellular damage and prevention of hypoxia-induced dopamine release. Intracellular buffering of Ca2+ using the Ca-chelator 1, 2-bis(2-Aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis(acetoxymethyl) ester (BAPTA) reduced the neuroprotective effect of xenon indicating the essential participation of intracellular Ca2+-ions in the process of xenon-induced neuroprotection. CONCLUSIONS: The results presented demonstrate the outstanding property of xenon to protect neuron-like cells in a hypoxic situation.

Prevention of neurotoxicity in hypoxic cortical neurons by the noble gas xenon.

Hypoxia-induced neuronal damage and glutamate release were investigated in a N(2)- or in xenon-atmosphere for embryonic rat cortical neurons; cellular damage and glutamate over-release were observed in N(2)-treated cells whereas xenon protected the cells from the hypoxic insult. The protective effect of xenon was strongly reduced by pre-incubating neurons with the calcium-chelator BAPTA-AM indicating a role for calcium in this process. The results demonstrate (a) the neuroprotective properties of xenon, suggest (b) a relationship between the prevention of neurotransmitter release in a hypoxic situation and neuroprotection and present (c) evidence that such neuroprotection may be based on yet other xenon-dependent mechanisms.

[Inhibition and induction of protein syntheses by actinomycin in the growing oocytes ofMusca domestica]. / Hemmung und Induktion von Proteinsynthesen durch Actinomycin in den wachsenden Oocyten vonMusca domestica.

The protein synthesis of growing oocytes ofMusca domestica L. is investigated by radioautographic and electrophoretic methods on an intermediate developmental stage (stage 3), and after the end of growth (stage 6) in the normal development and after treatment with Actinomycin. The very high RNA synthesis of the nurse cells and of the follicle epithelium of growing oocytes is completely blocked by injection of 16 Μg/g body weight Actinomycin. The protein synthesis in the euplasm of a growing oocyte is relatively high, the stage 6 shows a high protein synthesis in spite of the absence of RNA synthesis. The proteins of the ovary are separated electrophoretically on Cellogel-strips. The ratio of the newly-synthetized proteins is determined after incorporation (30 min) of14C-labelled amino acids. The protein synthesis is stimulated after 4-6 hours treatment with Actinomycin. After a longer influence of the antibiotic, synthesis of the egg-nurse cell unit decreases to below the control value. The ripe oocyte does not react autoradiographically to Actinomycin. Only some fractions of the newly-synthetized euplasmatic proteins are sensitive to treatment with Actinomycin. After intermediate incubation times with Actinomycin new proteins appear in the ovary and their synthesis is demonstrable in part even after treatment for 48 hours. There is no newly-synthetized RNA in the ripe oocyte, which, nevertheless, reacts with Actinomycin to inhibit some and to stimulate some new protein synthesis. In this case the action of the antibiotic is unknown.

[Synthesis of haemolymph proteine and the uptake of the yolk fraction in the oocyte during Actinomycin-treatment. (Studies onMusca domestica)]. / Synthese der Hämolymphproteine und die Aufnahme der Dotterfraktion in die Oocyte unter Actinomycin-Einflu\ (Untersuchungen anMusca domestica).

By means of radioautographic and electrophoretic techniques yolk protein uptake in the growing oocytes ofMusca domestica was investigated. After 30 min yolk protein becomes visible in the radioautograms. By stainability and labeling the yolk fraction could be demonstrated in the pherogram of ovary and haemolymph in an intermediate developmental stage (stage 3). After the end of vitellogenesis it does not appear in the haemolymph. The yolk protein uptake is inhibited by Actinomycin, but the synthesis goes on nearly as normal. This inhibition can be interpretated as a new accessory effect of Actinomycin.