Identification of <i>Stim1</i> as a Candidate Gene for Exaggerated Sympathetic Response to Stress in the Stroke-Prone Spontaneously Hypertensive Rat

<div><p>The stroke-prone spontaneously hypertensive rat (SHRSP) is known to have exaggerated sympathetic nerve activity to various types of stress, which might contribute to the pathogenesis of severe hypertension and stroke observed in this strain. Previously, by using a congenic strain (called SPwch1.72) constructed between SHRSP and the normotensive Wistar-Kyoto rat (WKY), we showed that a 1.8-Mbp fragment on chromosome 1 (Chr1) of SHRSP harbored the responsible gene(s) for the exaggerated sympathetic response to stress. To further narrow down the candidate region, in this study, another congenic strain (SPwch1.71) harboring a smaller fragment on Chr1 including two functional candidate genes, <i>Phox2a</i> and <i>Ship2,</i> was generated. Sympathetic response to cold and restraint stress was compared among SHRSP, SPwch1.71, SPwch1.72 and WKY by three different methods (urinary norepinephrine excretion, blood pressure measurement by the telemetry system and the power spectral analysis on heart rate variability). The results indicated that the response in SPwch1.71 did not significantly differ from that in SHRSP, excluding <i>Phox2a</i> and <i>Ship2</i> from the candidate genes. As the stress response in SPwch1.72 was significantly less than that in SHRSP, it was concluded that the 1.2-Mbp congenic region covered by SPwch1.72 (and not by SPwch1.71) was responsible for the sympathetic stress response. The sequence analysis of 12 potential candidate genes in this region in WKY/Izm and SHRSP/Izm identified a nonsense mutation in the stromal interaction molecule 1 (<i>Stim1</i>) gene of SHRSP/Izm which was shared among 4 substrains of SHRSP. A western blot analysis confirmed a truncated form of STIM1 in SHRSP/Izm. In addition, the analysis revealed that the protein level of STIM1 in the brainstem of SHRSP/Izm was significantly lower when compared with WKY/Izm. Our results suggested that <i>Stim1</i> is a strong candidate gene responsible for the exaggerated sympathetic response to stress in SHRSP.</p></div

Expression analysis of candidate genes in the brainstem of WKY and SHRSP.

<p>Five rats of the each strain were used. Ventrolateral part of the brainstem including RVLM was dissected and RNA was extracted. Relative levels of gene expression were evaluated by quantitative RT-PCR analysis. The expression levels of each gene were normalized with β-actin mRNA. Each column shows the expression level of WKY and SHRSP under the room temperature (RT) or under the cold stress (Cold) as indicated in the panel for <i>Nup98</i>. SP: SHRSP, *<i>P</i><0.05 vs. WKY under the cold stress by Student’s t-test.</p

Western blot analysis of STIM1.

<p>A) Western blotting was performed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095091#s2" target="_blank">Materials and Methods</a>. The size of STIM1 in SHRSP was obviously smaller than that in WKY. A representative data is shown. B) Semi-quantitative evaluation of the STIM1 protein level was performed using ImageJ software. The relative amount of STIM1 was standardized with the level of β-actin. Room temp: room temperature, *<i>P</i><0.05 vs. WKY by Student’s t-test.</p

Genetic map of the congenic region and candidate genes located in the region.

<p>Closed columns indicate the regions transferred from the WKY genome. Vertical lines on the both ends of the columns show intervals including recombination breakpoints. Supplementary <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095091#pone.0095091.s004" target="_blank">Table S3</a> is available for further information about the candidate genes. Genomic position of each simple sequence repeat marker and SNPs in the <i>Trpc2</i>, <i>Art5</i> and <i>Olr111</i> are defined based on RGSC Genome Assembly v3.4.</p

Nonsynonymous substitutions identified in candidate genes located in a 1.8-Mbp region on chromosome 1.

<p>*The genomic position of SNP is defined based on the RGSC Genome Assembly v3.4.</p

Allelic difference of the two sequence variations identified in <i>Stim1</i> in various rat strains.

<p>Genomic position of each SNP is defined based on RGSC Genome Assembly v3.4.</p

Cross sections of muscle fibers of (A) one FSHD patient and (B) one healthy control (20×magnification, H&E-stained).

<p>Myofiber diameter size difference indicates hypertrophy in the <i>Vastus lateralis</i> muscle of the patient.</p

Cyclin A1 expression levels in FSHD and other myopathies (microarrays).

<p><i>CCNA1</i> Human Exon 1.0 ST Array signal levels in FSHD (n = 4), healthy controls (n = 7), CAV3 (n = 4), DYSF (n = 4) and FHL1 (n = 3).</p

Distribution patterns of myofiber diameters derived from FSHD patients (n = 5) and age-matched healthy controls (n = 5).

<p>Diameters (µm) from 1000 myofibers each were measured in H&E-stained cross sections under a light microscope by using the Image J software. Cumulative data are presented (FSHD: mean 71 µm, SD 23 µm; Control: mean 59 µm, SD 17 µm), <i>p</i><0.001, Wilcoxon test.</p

The issue of child soldiers in international law with regard to the African regional regulation

The main purpose of this thesis was to give a general overview of the problem of child soldiers and their protection among international law conventions and among regional law agreements with the focus on the African continent. The use of child soldiers in armed conflict is qualified as one of the worst forms of child labour.The majority of child soldiers are active in Africa and, to a lesser extent, in the Middle East and Asia. Besides introduction and conclusion, the study consists of five chapters. The first chapter describes the protection of child soldiers on the international level. Therefore it is focused on individual conventions, especially on Geneva Conventions, Additional Protocols to the Geneva Conventions, the Convention on the Rights of the Child, the Optional Protocol to the Convention on the Rights of the Child on the involvement of children in armed conflict, the Optional Protocol to the Convention on the Rights of the Child on a Communications Procedure, but as well on the International Labour Convention No. 182 or the Rome Statute. The soft law represents Paris principles. Discussed is particularly the question of the age limit for child participation in armed conflict. The second chapter is concerned with the international control mechanisms, resulting from the above-mentioned..