Recombinant scorpion insectotoxin AaIT kills specifically insect cells but not human cells

The nucleotide sequence deduced from the amino acid sequence of the scorpion insectotoxin AaIT was chemically synthesized and was expressed in Escherichia coli. The authenticity of this in vitro expressed peptide was confirmed by N-terminal peptide sequencing. Two groups of bioassays, artificial diet incorporation assay and contact insecticidal effect assay, were carried out separately to verify the toxicity of this recombinant toxin. At the end of a 24 h experimental period, more than 60% of the testing diamondback moth (Plutella xylostella) larvae were killed in both groups with LC50 value of 18.4 microM and 0.70 microM respectively. Cytotoxicity assay using cultured Sf9 insect cells and MCF-7 human cells demonstrated that the toxin AaIT had specific toxicity against insect cells but not human cells. Only 0.13 microM recombinant toxin was needed to kill 50% of cultured insect cells while as much as 1.3 microM toxin had absolutely no effect on human cells. Insect cells produced obvious intrusions from their plasma membrane before broken up. We infer that toxin AaIT bind to a putative sodium channel in these insect cells and open the channel persistently, which would result in Na+ influx and finally cause destruction of insect cells.

The Effect of calmodulin antagonist berbaminederivative-EBB on hepatoma in vitro and in vivo

OBJECTIVE: To evaluate the anti-hepatoma effect of Calmodulin antagonist 0 - 4-ethoxyl-butyl-Berbamine (EBB), one of the berbamine derivatives. METHODS: Monotetrazolium (MTT) method was used to analyze the effect of EBB on the proliferation and growth inhibition effect. Of a hepatoma cell line in vitro. A mouse hepatoma model was induced by injection of hepatoma cells (H22) in the abdominal cavity. The effect of EBB on survival at different concentrations as well as in combination with 5-FU were investigated in vivo. Flow cytometry analysis, dot blot hybridization, western blot, immunochemistry, enzyme-linked lectin assay (ELISA), trifluoperazine (TFP) and electron microscopic observation were used to study the effect of EBB on cell cycle process, P53 mRNA and protein levels, calmodulin content and ultrastractural changes of hepatoma cells. RESULTS: EBB exerts a very strong inhibitory effect on human hepatoma cell line 7402 and mouse hepatoma cell line H22 in vitro. The IC(50) value of EBB for the two cell lines are 3.312 microg/ml and 1.167 microg/ml, respectively. The sensitivity of H22 cells to 5-FU can be markedly enhanced: The IC(50) dosage of 5-Fu can be decreased from 0.75 microg/ml down to 0.15 microg/ml, when jointly administered with nontoxic dosages of EBB (IC(10)). In vivo, EBB can prolong the lifespan of mice with ascites H22 to more than three months. 64% of mice survived, while all animals in the control group died by the 18th day. When EBB (5 mg x kg(-1) x d(-1)) is jointly used with 5-FU (25 mg x ml(-1) x d(-1)), 73% of mice with ascites H22 survived, much higher than 27% in the 5-FU treated group. EBB can enhance the anti-hepatoma ability of 5-Fu treatment. EBB mechanism against hepatoma: P53 expression in the EBB treated group is substantially higher than that in the control group. EBB increased the translation of P53. As a calmodulin antagonist, EBB decreases amount of the CaM in hepatoma cells and blocked the hepatoma cell proliferation cycle at the G(2)M phase. Before the G(0)/G(1) phase, a diploid peak and apoptic cells in the treated groups were observed. CONCLUSIONS: The CaM antagonist, EBB, has a strong anti-hepatoma effect and enhances the effect of 5-FU, induces hepatoma cell to apoptosis, promotes the P53 protein expression and decreases the amount of CaM in the cytoplasm. All these results demonstrate that EBB is a new and potentially useful drug against hepatoma and should be researched further.