Intracellular proteases of Yarrowia lipolytica have been scarcely studied. These enzymes may play an important role in nitrogen metabolism, posttranslational processing, nutritional stress, dimorphism, etc.; biochemical and genetic control of these enzymes can help in obtaining high-level expression of recombinant proteins in heterologous systems. In this study, we report the presence of three proteases: aminopeptidase yylAPE, carboxypeptidase yylCP and dipeptidyl aminopeptidase yylDAP, measured under several nutritional conditions. Yarrowia lipolytica produced the highest level of intracellular proteolytic enzymes, i.e. yylAPE, yylCP and yylDAP, in media with peptone during stationary growth phase. When soluble extracts were subjected to PAGE, and the three activities were revealed in gels with the corresponding substrates, only one band of activity was detected for each one. The three enzymes were affected by serine protease inhibitors. Chelating agents affected mainly APE activity. The aminopeptidase was purified by selective fractionation with ammonium sulfate and three chromatographic steps (anion exchange, hydrophobic interaction and gel filtration chromatography). The enzyme had a molecular mass of 97 kDa; optimal pH and temperature were 7.0 and 37 degrees C, respectively. The aminopeptidase showed a preference for lysine in the N-position. The K(m) value was 0.86 microM and V(max) value was 990.8 micromoL min(-1) mg(-1) for Lys-pNA.

The aminopeptidase pumAPE was purified from the haploid fungus Ustilago maydis FB1 strain. The purification procedure consisted of ammonium sulfate fractionation and three chromatographic steps, which included anion-exchange, hydrophobic interaction, and gel filtration chromatography, resulting in a 23% recovery. The molecular mass of the dimeric enzyme was estimated to be 110 kDa and 58 kDa by gel filtration chromatography and SDS-PAGE, respectively. Enzymatic activity was optimal at pH 7.0 and at 35 degrees C toward Lys-pNA and the pI was determined to be 5.1. The enzyme was inhibited by EDTA-Na2, 1,10- phenanthroline, bestantin, PMSF and several divalent cations (Cu2+, Hg2+ and Zn2+). The aminopeptidase showed a preference for lysine and arginine in the N-position. The K(m) value was 54.4 microM and the Vmax value was 408 micromolmin(-1)mg(-1) for Lys-pNA.

The aminopeptidase pumAPE was purified from the haploid fungus Ustilago maydis FB1 strain. The purification procedure consisted of ammonium sulfate fractionation and three chromatographic steps, which included anion-exchange, hydrophobic interaction, and gel filtration chromatography, resulting in a 23% recovery. The molecular mass of the dimeric enzyme was estimated to be 110 kDa and 58 kDa by gel filtration chromatography and SDS-PAGE, respectively. Enzymatic activity was optimal at pH 7.0 and at 35 degrees C toward Lys-pNA and the pI was determined to be 5.1. The enzyme was inhibited by EDTA-Na2, 1,10- phenanthroline, bestantin, PMSF and several divalent cations (Cu2+, Hg2+ and Zn2+). The aminopeptidase showed a preference for lysine and arginine in the N-position. The K(m) value was 54.4 microM and the Vmax value was 408 micromolmin(-1)mg(-1) for Lys-pNA.

Lung cancer is the neoplasia with highest incidence and mortality worldwide. One of the strategies of treatment for this tumor type is based on ionizing radiation. Radiation induces cytotoxicity through some mechanisms, including gene expression changes. Recently, microRNAs (miRNAs) have been described as one of the molecules that regulate expression genes and may contributes to radioresistance in several tumor types. Global miRNAs expression profiles of radiosensitive and radioresistant lung tumor cells may elucidate the molecular mechanism related to tumoral radioresistance. In order to identify miRNAs associated with innate and acquired radioresistance, we will evaluate the miRNAs expression profiles in two lung carcinoma cell lines and in the same cell lines with acquire radioresistance by fractionated doses radiation treatment. For establish the median lethal dose radiation, H1944 and Calu-1 lung carcinoma cell lines were treated with increasing doses of X-rays (from 2 to 12 Gy). Cell survival, proliferative ability and clonogenic potential postradiation were evaluated. Moreover, we will generate a model of acquired radioresistance by treating lung tumor cells with fractionated doses of 2 Gy X-rays up to total dose of 60 Gy for to evaluate the expression of miRNAs in cells with acquired radioresistance. Results showed differential sensitivity to radiotherapy from lung tumors cells, whit 8 Gy as median lethal dose radiation. For to evaluate the expression of miRNAs during innate response to radiotherapy, total RNA was obtained from tumor cells treated with 8 Gy of X-rays. Additionally, it has been obtained lung tumor cells clones with treatment total doses of 28 Gy in a fractionated radiation scheme. These results will allow compare miRNAs expression during immediate cell response to radiation and those whose expression will modified during fractionated radiation treatment and may be associated with radioresistance acquired, which contribute to understanding the molecular mechanism to radioresistance in lung cancer.