Ty1 integrase overexpression leads to integration of non-Ty1 DNA fragments into the genome of Saccharomyces cerevisiae

The integrase of the Saccharomyces cerevisiae retrotransposon Ty1 integrates Ty1 cDNA into genomic DNA likely via a transesterification reaction. Little is known about the mechanisms ensuring that integrase does not integrate non-Ty DNA fragments. In an effort to elucidate the conditions under which Ty1 integrase accepts non-Ty DNA as substrate, PCR fragments encompassing a selectable marker gene were transformed into yeast strains overexpressing Ty1 integrase. These fragments do not exhibit similarity to Ty1 cDNA except for the presence of the conserved terminal dinucleotide 5′-TG-CA-3′. The frequency of fragment insertion events increased upon integrase overexpression. Characterization of insertion events by genomic sequencing revealed that most insertion events exhibited clear hallmarks of integrase-mediated reactions, such as 5 bp target site duplication and target site preferences. Alteration of the terminal dinucleotide abolished the suitability of the PCR fragments to serve as substrates. We hypothesize that substrate specificity under normal conditions is mainly due to compartmentalization of integrase and Ty cDNA, which meet in virus-like particles. In contrast, recombinant integrase, which is not confined to virus-like particles, is able to accept non-Ty DNA, provided that it terminates in the proper dinucleotide sequence

Formulation and Evaluation of Levofloxacin Hemihydrate Loaded Mucoadhesive Alginate Beads for the Treatment of Helicobacter Pylori Infection

H. pylori colonize the gastric mucosa leading to gastritis, gastric ulcer, and gastric carcinoma. To increase the efficacy of eradicating the infection, a localized delivery system of anti‐H. pylori agents in the stomach is required. Mucoadhesive beads of levofloxacin were prepared to increase the local concentration of the antibiotic in the stomach to eradicate H. pylori infection. The main goal of this study was to optimize mucoadhesiveness and controlled release property. The optimized formulation for levofloxacin mucoadhesive beads was obtained with Sodium alginate, Carbopol 934P and calcium chloride. Based on the mucus turnover rate and dissolution time, formulations LM 5 (formulations consisting of 2% w/v Sodium alginate, 1% w/v carbopol 974P and 3% w/v Calcium chloride) were selected as best formulations Invitro studies clearly indicates that the prepared formulations possess good bioadhesive properties. These properties enable the microspheres to adhere to the gastric mucosal surface and stay in stomach for prolonged periods, which eventually resulted in better eradication of H. pylori than the conventional dosage forms. This study proves the possibility of delivering levofloxacin for the treatment of H. pylori infection