Cytotoxic effects of two Iranian scorpions odontobuthusdoriae and bothutus saulcyi on five human cultured cell lines and fractions of toxic venom

Scorpion venom toxicity is of major concern due to its influence on human activities and public health. We investigated the in-vitro process of cell death caused by two Iranian scorpions Odontobuthus doriae and Bothutus salceyi venom on human cell lines. The aim of this study was to provide further information about triggering cell death and suggestion of methods for the elimination of unwanted cells such as tumor cells. The cytotoxicity and apoptosis induced by effect of scorpion venoms on five established eukaryotic cell lines are analyzed on different human cell lines. All cultured cell lines were incubated with varying doses of scorpion venom for 24 h at 37°C. Control culture was treated with an equal amount of SFM. The percentage of cell survival was measured using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium [MTT] colorimetric assay. Our data demonstrated that Bothutus saulcyi, does not show cytotoxic effect on any of the used cell lines. Odontobuthus doriae, however, has resulted a dose dependent cytotoxic effect with maximum at 1 ug/mL on 1321N1 glioma like cell line. Then the cytotoxic venom of O. doriae was fractionated using Sephadex G50 gel chromatography. The toxic fractions on mouse used to Cytotoxicity assay on 1321 N1 cell line and data demonstrated that, the fraction F3 showed a dose dependent Cytotoxicity assay. Further studies to explode the mode of action of these venoms are recommended and purification of the toxic fraction should be done

Cloning, expression and purification of Pwo polymerase from Pyrococcus woesei

Pyrococcus woesei is a hyperthermophilic archaea and produces a heat stable polymerase [Pwo polymerase] that has proofreading activity. In this study, this microorganism was cultured, its DNA was extracted and the pwo gene polymerase was cloned, expressed and purified. The DNA sequence of the cloned gene was verified by sequencing. The pwo polymerase gene consists of 2,328 bps [775 amino acids with about 90 kD molecular weight]. Cloning was done by GATEWAY Cloning System and for purification of recombinant protein; His6x-Tag was added to the C-terminus of the recombinant protein. We could purify Pwo polymerase enzyme by Ni-NTA resin. PCR assay showed that Pwo polymerase activity is comparable to a commercial Pfu polymerase activity