Apoptosis of cultured granulosa-lutein cells is reduced by insulin-like growth factor I and may correlate with embryo fragmentation and pregnancy rate.

OBJECTIVE: To correlate apoptosis of cultured human granulosa-lutein cells (GL cells) with the outcome of IVF (embryo fragmentation and pregnancy rate) and to study the effect of insulin and insulin-like growth factor I (IGF-I) on apoptosis. DESIGN: In vitro assays. SETTING: University laboratory and private IVF center. PATIENT(S): Eighty-one women undergoing IVF. INTERVENTION(S): Purified human GL cells from pooled follicles were cultured for 48 hours in serum-free media with or without insulin and IGF-I. Cumulus cells and mural GL cells were evaluated separately. MAIN OUTCOME MEASURE(S): Detection of apoptosis by using caspACE FITC-VAD-FMK, a fluorescent in situ marker for activated caspases; embryo fragmentation; and pregnancy. RESULT(S): Age younger than 38 years and successful pregnancy were associated with less apoptosis (33.0% +/- 17.2% vs. 43.2% +/- 18.0% and 30.2% +/- 14.0% vs. 40.4% +/- 19.5%, respectively). There was a linear correlation between embryo fragmentation and GL cell apoptosis. Insulin-like growth factor I decreased apoptosis in a dose-dependent fashion. A statistically significant effect (17% decrease) was reached at a dose of 10 nM. Insulin (10 nM) caused a small (8%) decrease in apoptosis, but this effect did not reach statistical significance. Cumulus cells consistently had <3% apoptosis. CONCLUSION(S): [1] Apoptosis of cultured GL cells may be associated with IVF outcome and ovarian reserve and [2] IGF-I decreases apoptosis of cultured GL cells.

Introducing Wilson disease mutations into the zinc-transporting P-type ATPase of Escherichia coli. The mutation P634L in the 'hinge' motif (GDGXNDXP) perturbs the formation of the E2P state.

ZntA, a bacterial zinc-transporting P-type ATPase, is homologous to two human ATPases mutated in Menkes and Wilson diseases. To explore the roles of the bacterial ATPase residues homologous to those involved in the human diseases, we have introduced several point mutations into ZntA. The mutants P401L, D628A and P634L correspond to the Wilson disease mutations P992L, D1267A and P1273L, respectively. The mutations D628A and P634L are located in the C-terminal part of the phosphorylation domain in the so-called hinge motif conserved in all P-type ATPases. P401L resides near the N-terminal portion of the phosphorylation domain whereas the mutations H475Q and P476L affect the heavy metal ATPase-specific HP motif in the nucleotide binding domain. All mutants show reduced ATPase activity corresponding 0-37% of the wild-type activity. The mutants P401L, H475Q and P476L are poorly phosphorylated by both ATP and P(i). Their dephosphorylation rates are slow. The D628A mutant is inactive and cannot be phosphorylated at all. In contrast, the mutant P634L six residues apart in the same domain shows normal phosphorylation by ATP. However, phosphorylation by P(i) is almost absent. In the absence of added ADP the P634L mutant dephosphorylates much more slowly than the wild-type, whereas in the presence of ADP the dephosphorylation rate is faster than that of the wild-type. We conclude that the mutation P634L affects the conversion between the states E1P and E2P so that the mutant favors the E1 or E1P state.

Several polylactosamine-modifying glycosyltransferases also use internal GalNAcbeta1-4GlcNAc units of synthetic saccharides as acceptors.

The GalNAcbeta1-4GlcNAc determinant (LdN) occurs in some human and bovine glycoconjugates and also in lower vertebrates and invertebrates. It has been found in unsubstituted as well as terminally substituted forms at the distal end of conjugated glycans, but it has not been reported previously at truly internal positions of polylactosamine chains. Here, we describe enzyme-assisted conversion of LdNbeta1-OR oligosaccharides into GlcNAcbeta1-3GalNAcbeta1-4GlcNAcbeta1-OR. The extension reactions, catalyzed by human serum, were modeled after analogous beta3-GlcNAc transfer processes that generate GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1-OR. The newly synthesized GlcNAcbeta1-3GalNAc linkages were unambiguously identified by nuclear magnetic resonance data, including the appropriate long-range correlations in heteronuclear multiple bond correlation spectra. The novel GlcNAcbeta1-3'LdN determinant proved to be a functional acceptor for several mammalian glycosyltransferases, suggesting that human polylactosamines may contain internal LdN units in many distinct forms. The GlcNAcbeta1-3'LdN determinant was unusually resistant toward jackbean beta-N-acetylhexosaminidase; the slow degradation should lead to a convenient method for the search of putative internal LdN determinants in natural polylactosamine chains.