Cloning and in vitro expression of TPK3, a Toxoplasma gondii homologue of shaggy/glycogen synthase kinase-3 kinases

PURPOSE: An established method of cryostorage that might preserve the vascular and endothelial responses of human femoral arteries (HFAs) to be transplanted as allografts was studied. METHODS: HFAs were harvested from multiorgan donors and stored at 4 degrees C in Belzer solution before cryostorage. One hundred eleven HFA rings were isolated and randomly assigned to 1 control group of unfrozen HFAs and 2 groups of HFAs cryopreserved for 7 and 30 days, respectively. Cryopreservation was performed in Elohes solution containing dimethyl sulfoxide (1.8 mmol/L), and the rate of cooling was 1.6 degrees C/min, until -141 degrees C was reached. The contractile and relaxant responses of unfrozen and frozen/thawed arteries were assessed in organ bath by measurement of isometric force generated by the HFAs. RESULTS: After thawing, the maximal contractile responses to all the contracting agonists tested (KCl, U46619 a thromboxane A2-mimetic], norepinephrine, serotonin, and endothelin-1) were in the range of 7% to 34% of the responses in unfrozen HFAs. The endothelium-independent relaxant responses to forskolin and verapamil were weakly altered, whereas the endothelium-independent relaxant responses to sodium nitroprusside were markedly reduced. Cryostorage of HFAs also resulted in a loss of the endothelium-dependent relaxant response to acetylcholine. The vascular and endothelial responses were similarly altered in the HFAs cryopreserved for 7 and 30 days. CONCLUSION: The cryopreservation method used provided a limited preservation of HFAs contractility, a good preservation of the endothelium-independent relaxant responses, but no apparent preservation of the endothelium-dependent relaxation. It is possible that further refinements of the cryopreservation protocol, such as a slower rate of cooling and a more controlled stepwise addition of dimethyl sulfoxide, might allow better post-thaw functional recovery of HFAs.