Contribution of hypothermia to cerebroprotective effects of TAK-937 after transient MCAO in rats.

<p>Brain temperature (<b>A</b>) and infarct volume (<b>B</b>). Data are indicated as the means ± SEM. Significant differences from the corresponding vehicle-treated group are indicated by ^***<i>P</i><0.001 and ^*<i>P</i><0.05 (Dunnett’s test), and from the corresponding TAK-937-treated group is indicated by ^##<i>P</i><0.01 (Student’s <i>t</i> test). Numbers of rats used are shown in parentheses.</p

Effect of TAK-937 on rectal and brain temperature in rats with transient MCAO.

<p>Effect of TAK-937 on rectal and brain temperature in rats with transient MCAO.</p

Reversal of cerebroprotection of TAK-937 by AM251, a CB₁ antagonist, after transient MCAO in rats.

<p>Rectal temperature (<b>A</b>) and infarct volume (<b>B</b>). Data are indicated as the means ± SEM. Significant differences from the corresponding vehicle-treated group are indicated by^***<i>P</i><0.001 (Dunnett’s test), and from the corresponding TAK-937-treated group is indicated by ^###<i>P</i><0.001 (Student’s <i>t</i> test). Numbers of rats used are shown in parentheses.</p

Phylogenetic relationship of birds used in this study.

<p>The phylogenetic tree is based on documented literature <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059291#pone.0059291-Dimcheff1" target="_blank">[27]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059291#pone.0059291-Griffin1" target="_blank">[28]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059291#pone.0059291-Kimball1" target="_blank">[29]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059291#pone.0059291-Shibusawa1" target="_blank">[30]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059291#pone.0059291-Cracraft1" target="_blank">[32]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059291#pone.0059291-Hackett1" target="_blank">[34]</a>, but the relationships within Phasianidae (quail, bamboo partridge, chicken, peafowl, and turkey) were simplified due to the lack of consensus among proposed phylogeny. The presence (+) and absence (−) of Galα1-4Gal or Galβ1-4Gal on glycoproteins are based on detections in various tissues from species indicated with an <i>asterisk</i> (*) or on detections on egg while/yolk glycoproteins from all species except bamboo partridge. mya, million years ago.</p

Antibody/lectin-staining of protein extracts from various tissues of quail (A) and bamboo partridge (B).

<p>Proteins (20 µg/lane) blotted onto PVDF membranes were visualized with CBB-staining. Pigeon IgG (for CBB and anti-P₁ mAb stainings) and α-galactosidase-treated pigeon IgG (for anti-(Galβ1-4Gal) mAb and ECA stainings) were used as controls.</p

List of birds whose eggs and/or tissues were used.

a<p>Based on the DNA-DNA hybridization method by Sibley, <i>et al</i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059291#pone.0059291-Sibley1" target="_blank">[9]</a>.</p>b<p>Red Junglefowl is believed to be the direct ancestor of the domestic chicken.</p>c<p>Mallard is believed to be the ancestor of domestic ducks.</p

Identification of avian egg yolk IgG^a.

a<p>Molecular masses of whole isolated avian IgGs, also called IgYs, were approximately 170 kDa, and are larger than those of mammals (approximately 150 kDa).</p>b<p>Emu IgG showed two bands of around 65 kDa in SDS-PAGE at reducing conditions, with CBB-staining (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059291#pone-0059291-g002" target="_blank">Figure 2B</a>). The higher band shifted to the lower band by treatment with glycoamidase F (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059291#pone-0059291-g003" target="_blank">Figure 3B</a>), suggesting that the different molecular sizes are mainly due to differences in glycosylation.</p

Analysis of the tissue distributions of α4GalT(Gal), β4GalT(Gal), and β4GalT(GlcNAc) from quail and bamboo partridge.

<p>Microsomal fractions or tissue extracts were prepared from various tissues of quail (A) or bamboo partridge (B). The values represent the means ±S.D. of duplicate samples. Scales for β4GalT(Gal) (<i>open bar</i>) activities were indicated on the left <i>y</i>-axes, and those for β4GalT(GlcNAc) (<i>hatched bar</i>) activities on the right <i>y</i>-axes of each graph. No α4GalT(Gal) activities were detected in any tissues from quail or bamboo partridge. β4GalT(Gal) activities were detected only in tissues from quail.</p

Digestion of quail ovomucoid and avian egg yolk IgG with exogalactosidases or glycoamidase F (GAF).

<p><i>A</i>, Quail egg white proteins and quail ovomucoid were untreated (−) or treated with β4-galactosidase (β). <i>B</i>, The isolated egg yolk IgGs from duck, emu, guineafowl, ostrich, peafowl, and turkey were untreated (−) or treated with β4-galactosidase (β) or GAF (<i>N</i>). In <i>B</i>, pigeon egg yolk IgG was treated with α-galactosidase (α), α-galactosidase and then β4-galactosidase (αβ), or GAF (<i>N</i>). Each sample was separated by SDS-PAGE, transferred onto PVDF membranes, and stained with CBB or anti-(Galβ1-4Gal) mAb.</p

MALDI-MS profiles of permethylated total IgG <i>N</i>-glycans from turkey, guineafowl, and peafowl.

<p>The avian IgG <i>N</i>-glycans before (A) and after (B) β4-galactosidase digestion were permethylated and analyzed with MALDI-MS. The most probable structures corresponding to the afforded major [M+Na]⁺ molecular ion signals are annotated along with their monoisotopic values. Assignments are based on a combination of inferred glycosyl compositions, susceptibility to β-galactosidase digestion (B) and MS/MS data obtained on the more abundant components (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059291#pone-0059291-g005" target="_blank">Figure 5</a>). The MS/MS data positively identify <i>N</i>-glycans with a bisecting GlcNAc as the major complex type structures but do not rule out the presence of alternative non-bisected structures. Unassigned signals that did not give interpretable glycan-like MS/MS spectra are marked with an asterisk (*). Monosaccharide symbols used conform to the recommendations by the Consortium for Functional Glycomics.</p