Influence of the 15-HETE on cytosolic [ Ca2+] i of the rabbit pulmonary artery smooth muscle cells

Extracellular Ca2+ influx was blocked by L-type Ca2+ channel blocker nifedipine to observe the effects of 15-hydroxyeicosatetraenoic acid on the constriction of rabbit pulmonary artery rings and on the changes of Ca2+ level in the rabbit pulmonary artery smooth muscle cells, and further to investigate the mechanism of the calcium mobilization induced by the 15-HETE under hypoxic conditions. The effect of extracellular Ca2+ on tension of the rabbit PA rings was also studied. Nifedipine (10 µ mol/L) had no effect on 1 µ mol/L 15-hydroxyeicosatetraenoic acid induced vasoconstriction under normoxic and hypoxic conditions. Intracellular Ca2+ increased markedly in the 15-HETE group (cells were exposed to 1 µ mol/L 15-HETE for 8 min during culture) compared to the control group (P < 0.05). The study demonstrated that the 15-HETE could induce the elevation of Ca2+ in the pulmonary artery smooth muscle cells and the elevated calcium came from the release of the intracellular calcium.

Cloning, expression and characterization of aerolysin from Aeromonas hydrophila in Escherichia coli.

Aerolysin is a toxin (protein in nature) secreted by the strains of Aeromonas spp. and plays an important role in the virulence of Aeromonas strains. It has also found several applications such as for detection of glycosylphosphatidylinositol (GPI)-anchored proteins etc. A. hydrophila is a ubiquitous Gram-negative bacterium which causes frequent harm to the aquaculture. To obtain a significant amount of recombinant aerolysin in the active form, in this study, we expressed the aerolysin in E. coli under the control of T7 RNase promoter. The coding region (AerA-W) of the aerA gene of A. hydrophila XS91-4-1, excluding partial coding region of the signal peptide was cloned into the vector pET32a and then transformed into E. coli b121. After optimizing the expression conditions, the recombinant protein AerA-W was expressed in a soluble form and purified using His.Bind resin affinity chromatography. Recombinant aerolysin showed hemolytic activity in the agar diffusive hemolysis test. Western blot analysis demonstrated good antigenicity of the recombinant protein.