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1. 1. The enzyme fructose-1,6-bisphosphatase was purified from the mantle of the sea mussel Mytilus galloprovincialis Lmk. The purified enzyme showed a single band in SDS-polyacrylamide gel electrophoresis. The mol. wt and subunit mol. wt of the enzyme were 105,000 and 27,000, respectively. 2. 2. Divalent cations are essential for the enzyme activity. In the absence of chelating agents, FBPase 1 exhibits hyperbolic kinetics with respect to Mn 2+ , Zn 2+ and Mg 2+ . The K m for Mg 2+ is lower than the physiological concentration of cation in the tissue, whereas its K m for Mn 2+ and Zn 2+ is greater than the respective in vivo concentrations. 3. 3. The joint action of Mg 2+ and Zn 2+ increases the affinity of the enzyme for the substrate Fru-1,6-P 2 , though V max is reduced. 4. 4. Na + strongly inhibits the enzyme even at very low concentrations. K + has no effect whatsoever.

Purpose Persistent cloaca is a rare, severe congenital anomaly. The Eph family receptor tyrosine kinases and their ligands, the ephrin family, mediate diverse embryonic cell-cell recognition events. We previously reported ephrin-B2lacZ/lacZ mice manifest male high imperforate anus and female persistent cloaca (Dravis et al. Dev Biol 2004; 271(2): 272-290). We studied and demonstrated via 3D-reconstruction whether altered ephrin-B2 signaling in female ephrin-B2 lacZ/lacZ mutant mice affect apoptosis, thereby possibly contributing to persistent cloaca malformation. Material and methods Female embryonic days 11.5, 13.5 and 16.5 (E11.5, E13.5 and E16.5) wild type (WT), ephrin-B2lacZ/+ (Z/+) and ephrin-B2 lacZ/lacZ (Z/Z) mice sections were dual labeled for ephrin-B2 and TUNEL immunohistochemistry. Anti-galactosidase antibody detected the ephrin-B2-gal fusion protein in Z/+ and Z/Z mice. WinSurf software was used for 3D reconstructions of the cloacal region. Results In WT E11.5 females, the cloacal membrane (CM) is intact. The urorectal septum (URS) is partially separating the cloaca into urogenital sinus and hindgut; no vagina has developed. In contrast, Z/Z females had poor cloacal development. Z/+ embryos showed widened URS. Apoptosis in WT females occurred prominently in CM, dorsal cloaca, and hindgut. In contrast, apoptosis in Z/Z females was limited to the endoderm of the poorly formed cloaca. These changes progressed throughout E13.5 and finally at E16.5, Z/Z females exhibited persistent cloaca. Conclusions The 3D reconstruction of the cloacal region provides a better understanding of the mechanism by which altered ephrin-B2 signaling yields persistent cloaca via diminished apoptosis in Z/Z mice.