Four recombinant Sco-CHH mutants exhibit hyperglycemic activities significantly different from those elicited by wild-type Sco-CHH.

<p>Eyestalk-ablated animals (<i>S</i>. <i>olivacea</i>) received a 50-μl injection of (A) I2A rSco-CHH (▼: 10 pmol/animal), (B) F3A rSco-CHH (▼: 10 pmol/animal), (C) D12A rSco-CHH (▼: 10 pmol/animal), (D) D60A rSco-CHH (▼: 10 pmol/animal), (A-D) wild-type rSco-CHH (○: 10 pmol/animal) or saline (●). Hemolymph was withdrawn at designated time points and processed for determination of glucose levels. Data are given as mean ± S.E.M. For the sake of clarity, one-sided SEM bars are given. Sample size (n) is 8 for each time point. *,** indicate significant differences from corresponding zero time controls at 5% and 1% levels, respectively. Parenthesized * indicates significant differences between the wild-type and mutants at the same time-point at 5%. No significant change over time was observed in saline-treated animals.</p

The <i>p</i>-values derived from statistical analyses in early stage of diseases when SARA scores were below 10 unit points.

<p>Comparison of MRS ratios in the cerebellum between subgroups of SCAs.</p

The <i>p</i>-values derived from statistical analyses in early stage of diseases when SARA scores were below 10 unit points.

<p>Comparison of MRS ratios in the cerebellum between patients with subtypes of SCA and MSA-C.</p

Identification and characterization of wild-type and alanine-substituted rSco-CHH peptides.

<p>^a Data of % α-helix were calculated from the CD spectral data of peptides not yet amidated.</p><p>^b Percentage of acetonitrile at which each peptide was eluted.</p><p>^c Monoisotopic values are given.</p><p>Identification and characterization of wild-type and alanine-substituted rSco-CHH peptides.</p

Demographic features of the subjects.

#<p>Kruskal-Wallis test, <i>p</i><0.05.</p>§<p>Patients with SCA2 or SCA3 had a longer disease duration than those with SCA17, <i>p</i><0.05.</p>§§<p>Patients with SCA3 had a longer disease duration than those with MSA-C.</p>*<p>Patients with SCA2 or SCA3 were younger than those with SCA6.</p>**<p>Patients with SCA were younger than those with MSA-C.</p

Representative MR spectra.

<p>(A) The MR spectroscopy in the cerebellar hemispheres (upper row) and vermis (lower row) in healthy controls and patients with SCA6, SCA3, SCA17, SCA1, SCA2 or MSA-C. (B) Group comparisons of NAA/Cr, Cho/Cr and NAA/Cho between the patients and controls.</p

Circular dichroism spectra of wild-type and alanine-substituted rSco-CHH-Gly.

<p>CD spectral data of each recombinant peptide, dissolved in 10 mM PBS (150 mM NaCl, pH 6.8), were collected from 260 nm to 200 nm at 25°C using an AVIV 202 spectropolarimeter.</p

The MR spectroscopy features of the study participants.

*<p>Mann-Whitney test, <i>p</i><0.05, compared with the healthy controls.</p

Position and residue of CHH, ITP, MIH peptides which point-mutated peptides have been tested for importance in biological activity.^a

<p>^a Data are summarized from the present study (Sco-CHH), [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134983#pone.0134983.ref034" target="_blank">34</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134983#pone.0134983.ref035" target="_blank">35</a>] (Scg-ITP), and [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134983#pone.0134983.ref033" target="_blank">33</a>] (Maj-MIH). Note that for ITP and MIH, only mutants and residues relevant for discussion are presented.</p><p>^b Non-parenthesized number indicates the position of the residue for CHH and ITP sequences, and parenthesized number that for MIH sequence. Underlined number indicates position of the residues that is suggested to be functionally discriminating Group I and Group II peptides, with the exceptions that residue at position 2 being suggested to be functionally discriminating CHH and ITP and that residues at positions 55/56 among CHH, ITP, and Group II peptides. Motif names in which the residues are located according to Lacombe et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134983#pone.0134983.ref030" target="_blank">30</a>] are given above residue numbers.</p><p>^c Underlined mutant indicates significant change in activity, with all mutants exhibited no activity or activity lower than wild-type, except I2A, which exhibited higher activity; single letter indicates the residue at that position.</p><p>^d,e amidated and non-amidated mutants, respectively.</p><p>Position and residue of CHH, ITP, MIH peptides which point-mutated peptides have been tested for importance in biological activity.<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134983#t002fn001" target="_blank">^a</a></p

Two recombinant Sco-CHH mutants completely lose hyperglycemic activity.

<p>Eyestalk-ablated animals (<i>S</i>. <i>olivacea</i>) received a 50-μl injection of (A) R13A rSco-CHH (▼: 10 pmol/animal), (B) I69A rSco-CHH (▼: 10 pmol/animal), (A, B) wild-type rSco-CHH (○: 10 pmol/animal) or saline (●). Hemolymph was withdrawn at designated time points and processed for determination of glucose levels. Data are given as mean ± S.E.M. For the sake of clarity, one-sided SEM bars are given. Sample size (n) is 8 for each time point. *,** indicate significant differences from corresponding zero time controls at 5% and 1% levels, respectively. Parenthesized *,** indicate significant differences between the wild-type and mutants at the same time-point at 5% and 1% levels, respectively. No significant change over time was observed in saline-treated animals.</p