Real-time PCR for <i>Trib3</i> (VF, <i>mv</i> and <i>dmy</i> rats).

<p>Relative expression of <i>Trib3</i> in the white matter (A) and the gray matter (B) of the cervical spinal cords from the <i>dmy</i>, <i>mv</i> and VF rats by the real-time PCR. The data are presented as the mean ratio against expression in the control rats. Although expression levels of <i>Trib3</i> mRNA is significantly increased in the <i>dmy</i> rats, no significant differences are seen in the VF and <i>mv</i> rats. *, <i>p</i> <0.05, **, <i>p</i> <0.01, Tukey-Kramer test (<i>n</i> = 3 in VF and <i>dmy</i> rats. <i>n</i> = 2 in <i>mv</i> rats.).</p

Immunohistochemistry for TRIB3 in the <i>dmy</i> rat.

<p>The immunohistochemistry for TRIB3 in the lumbar spinal cords from <i>dmy</i> rats at 7-week-old. Cytoplasm of neurons and glial cells are stained with TRIB3 in the gray (A) and white matter (B) of the <i>dmy</i> rats. Bars: 20 μm.</p

Immunohistochemistry for Golgi 58K.

<p>The immunohistochemistry for Golgi 58K in the lumbar spinal cords from the <i>dmy</i> rats at 4, 6, 7 and 8 weeks of age. The immunoreactivity for the Golgi apparatus is elevated in the ventral funiculus of the <i>dmy</i> rats at 6–8 weeks of age. Bars: 20 μm.</p

Real-time PCR for <i>Trib3</i>.

<p>Expression of <i>Trib3</i> gene in the ventral funiculus in the <i>dmy</i> rats (A), gray matter (B) and dorsal funiculus (C) of the cervical spinal cords from the control (+/+ and <i>dmy</i>/+) and the <i>dmy</i> (<i>dmy</i>/<i>dmy</i>) rats. The data are presented as the mean ratio of target to reference gene. β-actin is used as an internal control. In the <i>dmy</i> rats, expression levels of <i>Trib3</i> mRNA is significantly increased in all areas at 4, 5, 6, 7 and 8 weeks of age. *, <i>p</i> <0.05, **, <i>p</i> <0.01, Tukey-Kramer test (<i>n</i> = 3 in each group).</p

Amino acid sequences of putative K⁺-binding sites of TbGMPR.

<p>The amino acid sequence of TbGMPR was aligned against Cys loops (A) and C-terminal regions (B) of mammalian IMPDHs or GMPRs. Circles and a triangle show K⁺-binding residues and a catalytic Cys residue, respectively, described previously for HsIMPDH2 and CgIMPDH2 [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004339#pntd.0004339.ref017" target="_blank">17</a>,<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004339#pntd.0004339.ref018" target="_blank">18</a>]. Asterisks above and below the TbGMPR sequence indicate the amino acid residues identical to mammalian IMPDHs and GMPRs, respectively. Dots indicate the amino acid residues with chemically similar characteristics. Hs, human; Bt, bovine; Cg, Chinese hamster. NCBI accession numbers are as follow: NP_899066 for HsIMPDH1, NP_000875 for HsIMPDH2, NP_001071309 for BtIMPDH1, NP_001029588 for BtIMPDH2, XP_007636735 for CgIMPDH1, XP_007650832 for CgIMPDH2, XP_007621414 for CgGMPR1, and EGW11548 for CgGMPR2.</p

HPLC analysis of GMPR activity of the Tb927.5.2080 gene product.

<p>A. HPLC chromatograms of the products of the reaction with the recombinant protein encoded by the Tb927.5.2080 gene. The mixtures were sampled at 0 h (top), 1 h (middle), and 5 h (bottom) after initiating the reaction. Note that the peak heights of GMP and NADPH became lower while those of IMP and NADP⁺ became higher as the reaction proceeded. B. Consumption of GMP and formation of IMP during the reaction were quantified based on the HPLC data. The amounts of GMP (open circles) and IMP (closed circles) were expressed in percent relative to their highest values.</p

Detection of Tb927.5.2080 gene product from <i>T</i>. <i>brucei</i> cells.

<p>A. A coding sequence region within Tb927.5.2080 gene was amplified by RT-PCR with <i>T</i>. <i>brucei</i> total RNA as a template. The expression of α-tubulin was also examined as a positive control. B. SDS-PAGE was performed with 5 μg of the purified recombinant protein. A single band with molecular weight of approximately 53 kDa (arrowhead) was observed after CBB staining. C. Whole cell lysate of <i>T</i>. <i>brucei</i> (20 μg protein) and recombinant protein (50 ng) were subjected to Western blot analysis with the polyclonal antibody prepared as described in Materials and Methods. A single band was detected at a position of approximately 53 kDa (arrowhead) from each sample.</p

Table_1_A missense mutation in the Hspa8 gene encoding heat shock cognate protein 70 causes neuroaxonal dystrophy in rats.pdf

Neuroaxonal dystrophy (NAD) is a neurodegenerative disease characterized by spheroid (swollen axon) formation in the nervous system. In the present study, we focused on a newly established autosomal recessive mutant strain of F344-kk/kk rats with hind limb gait abnormalities and ataxia from a young age. Histopathologically, a number of axonal spheroids were observed throughout the central nervous system, including the spinal cord (mainly in the dorsal cord), brain stem, and cerebellum in F344-kk/kk rats. Transmission electron microscopic observation of the spinal cord revealed accumulation of electron-dense bodies, degenerated abnormal mitochondria, as well as membranous or tubular structures in the axonal spheroids. Based on these neuropathological findings, F344-kk/kk rats were diagnosed with NAD. By a positional cloning approach, we identified a missense mutation (V95E) in the Hspa8 (heat shock protein family A (Hsp70) member 8) gene located on chromosome 8 of the F344-kk/kk rat genome. Furthermore, we developed the Hspa8 knock-in (KI) rats with the V95E mutation using the CRISPR-Cas system. Homozygous Hspa8-KI rats exhibited ataxia and axonal spheroids similar to those of F344-kk/kk rats. The V95E mutant HSC70 protein exhibited the significant but modest decrease in the maximum hydrolysis rate of ATPase when stimulated by co-chaperons DnaJB4 and BAG1 in vitro, which suggests the functional deficit in the V95E HSC70. Together, our findings provide the first evidence that the genetic alteration of the Hspa8 gene caused NAD in mammals.</p

Steady-state kinetics of TbGMPR.

<p>A. Plot of the initial velocity <i>versus</i> the GMP concentration in the presence of 200 μM NADPH. B. Plot of initial velocity <i>versus</i> NADPH concentration in the presence of 1 mM GMP. The data were fitted to the Michaelis-Menten equation. The values are expressed as the mean ± S.D.</p