Surface Grafting of Poly(L-glutamates). 2. Helix Orientation

In this paper the average helix orientation of surface-grafted poly(γ-benzyl L-glutamate) (PBLG), poly(γ-methyl L-glutamate) (PMLG), and poly(γ-methyl L-glutamate)-co-(γ-n-stearyl L-glutamate) (PMLGSLG 70/30) was investigated by means of FT-IR transmission spectroscopy. The theoretical relation between the average tilt angle (θ) and the absorption peak areas of three different backbone amide bands could be calculated because their transition dipole moment directions with respect to the helix axis were known. From the normalized absorptions, the average tilt angles of grafted helices of PBLG, PMLG, and PMLGSLG 70/30 were determined. The somewhat larger average angle of PMLG helices of 35 ± 5° with respect to the substrate compared to the value of 32 ± 5° of PBLG was due to the higher grafting density of PMLG. Because of the smaller helix diameter as a result of the smaller size of the methyl side group, more PMLG helices grew on the same surface area. Sterical hindrance and unfavorable polar interactions between unidirectional aligned helices forced the PMLG helices in a more upright arrangement. The even more perpendicular orientation of PMLGSLG 70/30 (48 ± 6°) could be the result of incorporation of mainly γ-methyl L-glutamate N-carboxyanhydride (MLG-NCA) monomers during the initiation step. Incorporation of the much larger γ-n-stearyl L-glutamate N-carboxyanhydride (SLG-NCA) monomers afterward lead to enlarged angles with respect to the substrate. Due to swelling, a pronounced change in helix orientation of grafted PMLGSLG 70/30 in n-hexadecane was observed, resulting in an almost perpendicular helix orientation.

Transcription Factor FoxO1 Is Essential for Enamel Biomineralization

The Transforming growth factor β (Tgf-β) pathway, by signaling via the activation of Smad transcription factors, induces the expression of many diverse downstream target genes thereby regulating a vast array of cellular events essential for proper development and homeostasis. In order for a specific cell type to properly interpret the Tgf-β signal and elicit a specific cellular response, cell-specific transcriptional co-factors often cooperate with the Smads to activate a discrete set of genes in the appropriate temporal and spatial manner. Here, via a conditional knockout approach, we show that mice mutant for Forkhead Box O transcription factor FoxO1 exhibit an enamel hypomaturation defect which phenocopies that of the Smad3 mutant mice. Furthermore, we determined that both the FoxO1 and Smad3 mutant teeth exhibit changes in the expression of similar cohort of genes encoding enamel matrix proteins required for proper enamel development. These data raise the possibility that FoxO1 and Smad3 act in concert to regulate a common repertoire of genes necessary for complete enamel maturation. This study is the first to define an essential role for the FoxO family of transcription factors in tooth development and provides a new molecular entry point which will allow researchers to delineate novel genetic pathways regulating the process of biomineralization which may also have significance for studies of human tooth diseases such as amelogenesis imperfecta

<i>FoxO1</i> mutant teeth deposit an enamel matrix and form enamel rods.

<p>Scanning electron microscopy of fractured 15 month old <i>FoxO1</i> mutant incisors revealed that, in regions where enamel had not chipped away, the mutant teeth initially had an enamel thickness that was comparable to their littermate controls (compare A and B). Higher magnification revealed that the mutant enamel also exhibited the typical decussating pattern of enamel rods (compare C and D).</p

<i>FoxO1</i> mutant enamel is softer than controls.

<p>The Vickers microhardness test revealed that the adult <i>FoxO1</i> mutant teeth are significantly softer than their littermate controls.</p

<i>Smad3</i> mutants suffer from a white, chalky tooth phenotype reminiscent of the <i>FoxO1</i> mutants.

<p><i>Smad3</i> mutants suffer from a white, chalky tooth phenotype reminiscent of the <i>FoxO1</i> mutants.</p

Loss of <i>FoxO1</i> and <i>Smad3</i> results in the down-regulation of a common set of genes.

<p>Quantitative rtPCR was performed on mRNA isolated from adult <i>FoxO1</i> mutant (A) and <i>Smad3</i> mutant (B) incisors and compared to control littermates. Both mutants exhibited a similar trend in the down-regulation of genes known to be necessary for proper enamel development and maturation (*p<0.05, **p<0.01, ***p<0.001).</p

Mapping of putative FoxO/Smad genomic binding sites.

<p><i>In silico</i> analysis of putative FoxO, Smad, and CEBPβ binding elements, conserved between mice and humans and residing within several genes down-regulated in the <i>FoxO1</i> and <i>Smad</i> mutants (see text for further details). The colored numbers indicate additional SBEs or CBEs that, due to scaling, could not be represented on the gene tracks.</p

Within the tooth, Rx-Cre activity is confined to the ameloblast layer.

<p><i>Rx-Cre^+/tg</i> mice were crossed to the <i>ROSA26^+/lacZ</i> Cre reporter line and Cre activity was assessed via X-gal staining. Rx-Cre exhibited broad activity throughout the anterior head in such tissues as the retina, tongue, skin, hair follicles, and teeth (A). Closer inspection of the incisors (B and C) and the molars (D and E) revealed that Cre activity was confined to the ameloblast layer (Am) and completely absent from the odontoblast layer (Od). The Cre activity in the molars was more mosaic than the incisors (arrows in E).</p