22:T537,Casticin, a polymethoxyflavone, present in natural plants, has been shown to have biological activities including anti-cancer activities. Herein, we investigated the anti-oral cancer activity of casticin on SCC-4 cells in vitro. Viable cells, cell cycle distribution, apoptotic cell death, reactive oxygen species (ROS) production, and Ca²⁺ production, levels of ΔΨ_m and caspase activity were measured by flow cytometric assay. Cell apoptosis associated protein expressions were examined by Western blotting and confocal laser microscopy. Results indicated that casticin induced cell morphological changes, DNA condensation and damage, decreased the total viable cells, induced G₂ /M phase arrest in SCC-4 cells. Casticin promoted ROS and Ca²⁺ productions, decreases the levels of ΔΨ_m , promoted caspase-3, -8, and -9 activities in SCC-4 cells. Western blotting assay demonstrated that casticin affect protein level associated with G2/M phase arrest and apoptosis. Confocal laser microscopy also confirmed that casticin increased the translocation of AIF and cytochrome c in SCC-4 cells. In conclusion, casticin decreased cell number through G₂ /M phase arrest and the induction of cell apoptosis through caspase- and mitochondria-dependent pathways in SCC-4 cells.23:T44b,DNase I of tilapia (Oreochromis mossambicus) was purified to homogeneity. Tilapia DNase I is most active at pH 8.5 with Mg2+ as activator. The Ca2+/Mg2+ pair has a synergistic effect on activation. The enzyme is readily inactivated by heating above 55 degrees C, but is not inactivated by trypsin or 2-mercaptoethanol under alkaline conditions, with or without CaCl2. Its isoelectric point is 6.0. The 258-amino-acid sequence of tilapia DNase I was derived from overlapping sequences of tryptic, chymotryptic and CNBr peptides. The purified enzyme has two variants differing by a single Lys-->Arg mutation at position 125. The polypeptide chain has one disulfide bridge and one carbohydrate side chain. By mass spectrometry, the purified enzyme shows many molecular mass forms differing by Lys/Arg substitution and sugar-chain length. The major form has a molecular mass of 30,914 Da. A 1061-bp nucleotide sequence for the cDNA of tilapia DNase I, obtained by gene cloning and DNA sequencing, contains an ORF coding for a putative 26-residue transmembrane peptide and the mature DNase I polypeptide.24:T52f,It is known, from the zymogram method of nuclease activity assay, that the crude extracts of Cunninghamella echinulata var. echinulata contained at least three distinct extracellular nucleases. Among them, the major form was 30 kDa in molecular mass and termed nuclease C1. In this report, nuclease C1 was purified to apparent homogeneity by chromatography on Cibacron blue-3GA, phenyl-Sepharose 4B and HiTrap Heparin. Nuclease C1 acquired enzymatic activity in the presence of Mn2+ or Mg2+ and was inhibited by EDTA. The activity was maximal at pH 7-8.5. The primary structure of nuclease C1 was completely determined using enzymatic digestion and gene cloning. The N-terminal 49 residues of nuclease C1 were first elucidated from a tryptic digest. Two degenerate upstream primers were subsequently designed to amplify the cDNA encoding nuclease C1. The resulting protein sequence of nuclease C1 was shown to be composed of 252 residues. It was intriguing to find that the protein sequence of nuclease C1 showed significant similarities with the sequences of the mitochondrial nucleases of Saccharomyces cerevisiae (44% identity) and Schizosaccharomyces pombe (42% identity). Residue His87 of nuclease C1 was postulated to be located at the active site from sequence similarity with secreted nuclease from Serratia marcescens.

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