Immunohistochemistry of Adult OC1 Knockout Retinas.

<p>Changes in mature retinal cell populations resulting from the loss of OC1 were examined using immunohistochemistry. Most retinal populations were unchanged between wildtype and OC1-KO littermates, including rod photoreceptors (A,A’), amacrine cells (B,B’,C,C’), bipolar cells (D,D'), and Muller glia (E,E’). However, anti-Calb28k-staining shows a decrease in horizontal cells in OC1-KO retinas as compared to a wildtype littermate (F,F'). Arrowheads indicate horizontal cell nuclei. Scale bars represent 100 µm.</p

Unbiased search for changes in the OC2-deficient retina.

<p>(A) Quantitative PCR (qPCR) analyses determining the relative amounts of Lhx1 in OC1-KO, and OC2-KO retinas at E16.5 or adult compared to WT littermate retinas. (B) qPCR determining the relative amounts of various genes identified in models of retinal damage or disease expressed in adult OC2 retinas. Each marker known to be involved in retinal stress is significantly upregulated in OC2 compared to their WT littermates, including GFAP, Edn2, C4b, and Cebpd. (C) WT and OC2-KO retinas were stained with an anti-GFAP antibody. Scale bars represent 100 µm. (D) qPCR determining the relative amounts of Opn4 in E14.5 OC1-KO retinas compared to WT littermate retinas. * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.005. Scale bars represent 100 µm. All quantitative results are plotted on logarithmic scale.</p

Examination of early postnatal stages in OC2-KO retinas.

<p>To determine whether this decrease in horizontal cells was confined to the fully mature retina, or was present earlier in development, immunohistochemistry was performed on the early postnatal retina. Age-matched animals were processed for either retinal sections (P5) or flatmounts (P10) and stained with an anti-Rhodopsin antibody (A,A’), an anti-Chx10 antibody (B,B’), or an anti-Calbindin 28k antibody (C,C’,D,D’). Scale bars represent 100 µm.</p

Expression of Onecut1 and Onecut2 in the developing murine retina.

<p>The expression of Onecut transcription factors was analyzed throughout retinal development (A). A heatmap indicating the genes (rows) expressed in isolated single retinal progenitor cells (columns) at various stages of development, from embryonic day (E)12.5 to E16.5. Increased expression of a gene in a given cell is indicated in shades of red, while the absence of expression is indicated with a black square. Expression patterns of Onecut1 (B) and Onecut2 (C) mRNAs were determined via in situ hybridization at various stages of retinal development and in the adult retina. Arrowheads indicate expression in the ganglion cell layer. Adult scale bars represent 100 µm; all others 200 µm. (D,E) Dissociated cell in situ hybridization was performed at E14.5 using a probe targeting either Onecut1 or Onecut2 and Math5. Arrowheads indicate overlapping Math5 and Onecut family member expression. (F) Quantification of dissociated retinal cells expressing Math5, Onecut1, and Onecut2.</p

Immunohistochemistry of Adult OC2 Knockout Retinas.

<p>Changes in mature retinal cell populations resulting from the loss of OC2 were examined using immunohistochemistry. Rod photoreceptors (A,A'), amacrine cells (B,B',C,C'), bipolar cells (D,D'), and Muller glia (E,E') were unchanged, whereas the horizontal cell population was greatly decreased (F,F'; arrowheads indicate horizontal cell bodies). Scale bars represent 100 µm. (G) The results of the immunohistochemical analyses were quantified for identically-sized fields of cryosectioned retinal tissue. Although staining for Brn3b, Chx10, Pax6, and the Calb28k that marks amacrine cell bodies were unchanged, horizontal cell bodies marked by Calb28k were significantly decreased in OC2-KO retinas (n = 3, p<0.005).</p

Common Hydrogen Bond Interactions in Diverse Phosphoryl Transfer Active Sites

<div><p>Phosphoryl transfer reactions figure prominently in energy metabolism, signaling, transport and motility. Prior detailed studies of selected systems have highlighted mechanistic features that distinguish different phosphoryl transfer enzymes. Here, a top-down approach is developed for comparing statistically the active site configurations between populations of diverse structures in the Protein Data Bank, and it reveals patterns of hydrogen bonding that transcend enzyme families. Through analysis of large samples of structures, insights are drawn at a level of detail exceeding the experimental precision of an individual structure. In phosphagen kinases, for example, hydrogen bonds with the O_3β of the nucleotide substrate are revealed as analogous to those in unrelated G proteins. In G proteins and other enzymes, interactions with O_3β have been understood in terms of electrostatic favoring of the transition state. Ground state quantum mechanical calculations on model compounds show that the active site interactions highlighted in our database analysis can affect substrate phosphate charge and bond length, in ways that are consistent with prior experimental observations, by modulating hyperconjugative orbital interactions that weaken the scissile bond. Testing experimentally the inference about the importance of O_3β interactions in phosphagen kinases, mutation of arginine kinase Arg₂₈₀ decreases k_cat, as predicted, with little impact upon K_M.</p></div

Effects of a hydrogen bond at O_3β on orbital and interaction energies in Structure1.

<p>Shortening the hydrogen bond between N-methylacetamide and methyl triphosphate: A) decreases the orbital energy of the σ*(O_3β—P_γ) anti-bonding orbital; while (B) leaving unchanged both the n(O_γ) donor orbital energies and (C) F_i,j, a measure of the overlap between the n(O_γ) lone pair orbitals and σ*(O_3β—P_γ). D denotes hydrogen bond donor. σ* denotes σ*(O_3β—P_γ).</p

Mean number of enzyme-ligand interactions.

<p>Interactions are shown with nonbridging β- (A), bridging β- (B) and bridging α-oxygens (C) in the O_3β―P_γ cleaving-, P_α―O_3α cleaving- and non-catalytic-NTP-binding sites. The resolution cutoff of structures used is 2.7 Å, and water is not included. Bars show standard errors.</p

Saturation of NTP oxygen lone pairs by enzyme or solvent interactions in O_3β—P_γ-cleaving active sites.

<p>Bars denote standard errors.</p

Enzyme structures can be categorized according to the fate of the bound nucleoside triphosphate (NTP).

<p>A) Phosphoryl transfer in which the O_3β―P_γ bond is cleaved. B) Reactions in which the P_α―O_3α bond is cleaved and C) Structures where the bound NTP does not undergo a chemical reaction. Red lettering indicates the atoms in the scissile bond and red arrows depict the transfer of electrons in going from reactants to products.</p