Using a yeast two-hybrid assay system, it was demonstrated that the four-helix bundle of the Rhodobacter sphaeroides PrrB histidine kinase both serves as the interaction site for the regulatory domain of its cognate response regulator PrrA and is the primary determinant of the interaction specificity. The α-helix 1 and its flanking turn region within the dimerization domain (DD) of the PrrB histidine kinase appear to play an important role in conferring the recognition specificity for the PrrA response regulator on the DD. The catalytic ATP-binding domain of the histidine kinase, which functions as the catalytic unit for the phosphotransfer reaction from ATP to the conserved histidine residue in the DD, also appears to contribute to the enhancement of the recognition specificity conferred by the DD. It was also revealed that replacement of Asp-63 and Lys-113 of the PrrA response regulator by alanine abolished protein–protein interactions between PrrA and its cognate histidine kinase PrrB, whereas mutations of Asp-19, Asp-20 and Thr-87 to alanine did not affect protein–protein interactions, indicating that among the active site residues of PrrA, Asp-63 and Lys-113 are important not only in the function of PrrA but also for protein–protein interactions between PrrA and PrrB.

ObjectiveRevascularization is critical for successful ovarian tissue transplantation. Vascular endothelial growth factor (VEGF) and angiopoietin-2 (angpt-2) are the principal mediators of neovascularization. This study was designed to assess VEGF and angpt-2 levels in cryopreserved ovarian tissue after heterotopic autotransplantation.MethodsOvarian tissues harvested from ICR mice at 5 to 6 weeks of age were stratified as follows: no cryopreservation (controls, group I); vitrification in VFS-40 (vitrification, group II); and gradual freezing in dimethyl sulfoxide (slow-freezing, group III). Frozen specimens were thawed at room temperature, assaying VEGF and angpt-2 levels 1 week after cryopreservation and 2 weeks after autotransplantation.ResultsVEGF and angpt-2 protein levels were significantly lower in cryopreserved ovaries of groups II and III than in controls (group I, P<0.05), whereas groups II and III did not differ significantly in this regard. After autotransplantation of cryopreserved ovarian tissue, VEGF and angpt-2 protein levels did not differ significantly by technique but tended to be lower than corresponding levels in controls.ConclusionExpression of angiogenic factors in ovarian tissue is thought to vary by method of cryopreservation. Our findings indicate that levels of angiogenic factors expressed in cryopreserved ovarian tissue after autotransplantation do not differ appreciably from control levels, regardless of cryopreservation technique.

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