A variety of approaches have been studied to overcome the problems encountered with using antibiotics, which are ineffective in treating Helicobacter pylori infections. In our study, chitosan/poly-gamma-glutamic acid nanoparticles incorporated into pH-sensitive hydrogels were developed as an efficient carrier for amoxicillin delivery. Our results indicate that hydrogels are pH-sensitive, leading to protecting nanoparticles from being destructed by gastric acid. The results of drug releasing in vitro study clearly indicate that the amount of amoxicillin released from nanoparticles incorporated in hydrogels at pH 1.2 was relatively low (14%), compared to that from only nanoparticles (50%). Confocal laser scanning microscopy revealed that nanoparticles could infiltrate cell-cell junctions and interact with H. pylori infection sites in the intercellular spaces. Additionally, the incorporation of amoxicillin-loaded nanoparticles in a hydrogel protected the drug from the actions of the gastric juice and facilitated amoxicillin interaction specifically with intercellular spaces, the site of H. pylori infection.
The structural gene encoding the extracellular lipase of Aeromonas hydrophila MCC-2 was cloned and found to be expressed in Escherichia coli using its own promoter. When the cloned gene (lip) was expressed in E. coli minicells, an 80 kDa protein was identified. Subcellular fractionation of E. coli carrying the lip gene indicated that the Lip protein was mainly associated with the membrane fraction. Nucleotide sequence analysis revealed that the gene is 2253 bp long, coding for a 79.9 kDa protein with an estimated pl of 1036. The deduced protein contains two putative signal peptide cleavage sites; one is a typical signal peptidase cleavage site and the other bears a strong resemblance to known lipoprotein leader sequences. Radioactivity from [3H]palmitate was incorporated into the Lip protein when expressed in E. coli.The deduced protein contains a sequence of VHFLGHSLGA which is very well conserved among lipases. It shows 67% and 65% overall identity to the amino acid sequences of lipase from A. hydrophila strains H3 and JMP636, respectively, but shows little homology to those of other lipases. The Lip protein was purified to homogeneity from both A. hydrophila and recombinant E. coli. In hydrolysis of p-nitrophenyl esters and triacylglycerols, using purified enzyme, the optimum chain lengths for the acyl moiety on the substrate were C,, to C,, for ester hydrolysis and C, to C,,, for triacylglycerol hydrolysis.
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