Distribution of allele frequencies at TTN g.231054C > T, RPL27A g.3109537C > T and AKIRIN2 c.*188G > A between Japanese Black and four other cattle breeds with differing historical selection for marbling

<p>Abstract</p> <p>Background</p> <p>Marbling defined by the amount and distribution of intramuscular fat, so-called <it>Shimofuri</it>, is an economically important trait of beef cattle in Japan. Our previous study detected 3 single nucleotide polymorphisms (SNPs), <it>g.231054C > T</it>, <it>g.3109537C > T </it>and <it>c.*188G > A</it>, respectively, in the 5' flanking region of the <it>titin </it>(<it>TTN</it>), the 5' flanking region of the <it>ribosomal protein L27a </it>(<it>RPL27A</it>) and the 3' untranslated region of the <it>akirin 2 </it>genes (<it>AKIRIN2</it>), which have been considered as positional functional candidates for the genes responsible for marbling, and showed association of these SNPs with marbling in Japanese Black beef cattle. In the present study, we investigated the allele frequency distribution of the 3 SNPs among the 5 cattle breeds, Japanese Black, Japanese Brown, Japanese Shorthorn, Holstein and Brown Swiss breeds.</p> <p>Findings</p> <p>We genotyped the <it>TTN g.231054C > T</it>, <it>RPL27A g.3109537C > T </it>and <it>AKIRIN2 c.*188G > A </it>SNPs by polymerase chain reaction-restriction fragment length polymorphism method, using 101 sires and 1,705 paternal half sib progeny steers from 8 sires for Japanese Black, 86 sires and 27 paternal half sib progeny steers from 3 sires for Japanese Brown, 79 sires and 264 paternal half sib progeny steers from 14 sires for Japanese Shorthorn, 119 unrelated cows for Holstein, and 118 unrelated cows for Brown Swiss breeds. As compared to the frequencies of the <it>g.231054C > T T</it>, <it>g.3109537C > T T </it>and <it>c.*188G > A A </it>alleles, associated with high marbling, in Japanese Black breed that has been subjected to a strong selection for high marbling, those in the breeds, Japanese Shorthorn, Holstein and Brown Swiss breeds, that have not been selected for high marbling were null or lower. The Japanese Brown breed selected slightly for high marbling showed lower frequency than Japanese Black breed in the <it>g.3109537C > T T </it>allele, whereas no differences were detected between the 2 breeds in the frequencies of the <it>g.231054C > T T </it>and <it>c.*188G > A A </it>alleles.</p> <p>Conclusions</p> <p>Based on this finding, we hypothesized that the pressure of the strong selection for high marbling in Japanese Black breed has increased the frequencies of the <it>T</it>, <it>T </it>and <it>A </it>alleles at the <it>TTN g.231054C > T</it>, <it>RPL27A g.3109537C > T </it>and <it>AKIRIN2 c.*188G > A </it>SNPs, respectively. This study, together with the previous association studies, suggested that the 3 SNPs may be useful for effective marker-assisted selection to increase the levels of marbling.</p

Superoxide Flux in Endothelial Cells via the Chloride Channel-3 Mediates Intracellular Signaling

Reactive oxygen species (ROS) have been implicated in both cell signaling and pathology. A major source of ROS in endothelial cells is NADPH oxidase, which generates superoxide (O2.−) on the extracellular side of the plasma membrane but can result in intracellular signaling. To study possible transmembrane flux of O2.−, pulmonary microvascular endothelial cells were preloaded with the O2.−-sensitive fluorophore hydroethidine (HE). Application of an extracellular bolus of O2.− resulted in rapid and concentration-dependent transient HE oxidation that was followed by a progressive and nonreversible increase in nuclear HE fluorescence. These fluorescence changes were inhibited by superoxide dismutase (SOD), the anion channel blocker DIDS, and selective silencing of the chloride channel-3 (ClC-3) by treatment with siRNA. Extracellular O2.− triggered Ca2+ release in turn triggered mitochondrial membrane potential alterations that were followed by mitochondrial O2.− production and cellular apoptosis. These “signaling” effects of O2.− were prevented by DIDS treatment, by depletion of intracellular Ca2+ stores with thapsigargin and by chelation of intracellular Ca2+. This study demonstrates that O2.− flux across the endothelial cell plasma membrane occurs through ClC-3 channels and induces intracellular Ca2+ release, which activates mitochondrial O2.− generation