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F. Mangia - Molecular regulation of cell proliferation and apoptosis in early embryo blastomeres
by cDNA coding sequence (Fiorenza et al., Gene 2001,
278:125-30). In contrast, western blot analysis displayed
the presence of 4 electrophoretic bands having
apparent MW of 42, 55, 67 and 72 kDa, respectively,
the relative abundance of which varied during cerebellum
development and in vitro CGN culture. In
detail, the 72 kDa band relatively increased in amount
during postnatal cerebellum development, suggesting
a functional role in the fully mature cerebellum,
whereas, among other THG1-pit bands, the 42 kDa
one corresponded to the MW expected by the encoded
amino acid sequence (including a number of putative
phosphorylation and/or O-glycosylation sites)
and thus was likely to represent the precursor of
higher MW bands. This possibility was investigated
by digesting CGN protein extracts with alkaline
phosphatase and/or a mixture of glycosidases, leading
to the conclusion that the 42 kDa protein gave origin
to the 67 kDa by O-glycosylation and that this band
was then converted to the 72 kDa by phosphorylation.
Because these findings suggested that THG1-pit functions
were finely regulated, we investigated the effects
of: i) TGF-β1 on CGN in vitro differentiation; and ii)
external K + concentration (25 mM K + ext , depolarizing
and maintaining cell viability; 5 mM K + ext ,
hyperpolarizing and committing CGN to apoptosis;
D'Mello et al., Proc Natl Acad Sci USA 1993, 90:10989-
93) on Thg-1pit transcritpion and intracellular THG-
1pit localization. CGN incubation in the presence of 5
60
mM K + ext and TGF- β1 transiently elicited both an
early (peaking at 30 min) and a late (peaking at 3 hr)
transcriptional waves, rapidly resulting in a significant
increase in THG-1pit content. We have recently
investigated the function(s) of different THG-1pit
forms also by biochemically fractionating CGN cytoplasmic,
chromatin and nuclear matrix fractions,
showing the specific presence of the 72 kDa band in
the chromatin (indipendent of K + ext ) and the 67 kDa
in the nuclear matrix (transiently increasing under
apoptotic condition). The functional meaning of 72
kDa and 67 kDa subnuclear localizations are currently
under investigation.
Selected Pubblications
Puglisi R, Bevilacqua A, Carlomagno G, Lenzi A,
Gandini L, Stefanini M, Mangia F, Boitani C. Mice
overexpressing the mitochondrial phospholipid
hydroperoxide glutathione peroxidase in male germ
cells show abnormal spermatogenesis and reduced
fertility. Endocrinology 2007, 148:4302-9.
Fiorenza MT, Torcia S, Canterini S, Bevilacqua A,
Narducci MG, Ragone G, Croce CM, Russo G,
Mangia F. TCL1 promotes blastomere proliferation
through nuclear transfer, but not direct phosphorylation,
of AKT/PKB in early mouse embryos. Cell
Death Differ. 2008, 15:420-2.
Fig. 1 - Nuclear translocation of THG-1pit landmarks GN committment
to apoptosis. DIV7 CGNs cultured on coverslips were
maintained for 1 hr in the presence of 25 mM or 5 mM K+ext supplemented
with TGF- 1 and were eventually processed for
immunofluorescence detection of THG-1pit and the early apoptosis
marker AIF. Nuclei were stained with Hoechst 33258. Note THG-
1pit and AIF colocalization.