Cinacalcet suppresses calcification of the aorta and heart in uremic rats

High serum parathyroid hormone levels are associated with vascular calcification. Cinacalcet is a calcimimetic agent that inhibits parathyroid hormone secretion and is used to treat patients with secondary hyperparathyroidism. Here we measured the effects of oral cinacalcet on calcification of the aorta and heart in rats with a remnant kidney (5/6 nephrectomy) model of uremia that were fed a high-phosphate diet containing lactose to accelerate the process of aortic calcification. Alizarin red staining showed that the smooth muscle in the aortic arch of rats with a remnant kidney was calcified. The tissue levels of calcium and phosphorus in the aorta and hearts of these rats were significantly increased compared to sham-operated rats. Expression of the osteoblastic lineage genes osteocalcin, osteopontin and runt-related gene 2 were also increased in the aorta of these rats. Cinacalcet suppressed these calcification-related changes by reducing serum parathyroid hormone, calcium, phosphorus, and the calcium-phosphorus product. Parathyroidectomy also suppressed calcification in this model. We suggest that cinacalcet inhibits calcification of the aorta and heart in uremic patients with secondary hyperparathyroidism by decreasing serum parathyroid hormone levels

小核移行トリガー蛋白質の探索と後天的トリソミックレスキューへの応用

application/pdf小核移行トリガー蛋白質の特定は、二次元電気泳動法にて小核特異的蛋白質の分離に難航したため、CRISPR/Cas9を用いて標的染色体を特異的に切断、小核へ移行後、後天的トリソミックレスキューを誘導する方略に変更した。13箇所の切断で最もdisomy細胞が多く出現し、後天的トリソミックレスキューが誘導されたと考えられる。今後、更に再現性を確認する必要があるが、二本鎖切断された標的染色体は、DNA修復しきれず小核へ移行・分解される頻度が増し、結果として後天的トリソミックレスキューが誘導出来たと推察される。現在これらの誘導disomy細胞に対し、STR解析やMLPS法などの詳細な解析を実施中である。Identification of small nuclear migration trigger protein, since it was difficult to separate the small nuclear-specific protein by two-dimensional electrophoresis, specifically cleavage the target chromosome using CRISPR/Cas9, after the transition to a small nucleus, it was changed to a strategy to induce acquired trisomic rescue. Most disomy cells appeared in 13 cleavage, it is believed that acquired trisomic rescue was induced. In the future, it is necessary to further confirm the reproducibility, double-stranded cut target chromosome, the frequency of migration and decomposition to the small nucleus can not be dna repair is increased, it is presumed that acquired trisomic rescue was able to induce as a result. Currently, for these induced disomy cells, detailed analysis such as STR analysis and MLPA method is being carried out.2018年度～2019年度科学研究費補助金(挑戦的萌芽研究)研究成果報告書18K1951

ゲノム編集技術を利用したアレル特異的染色体切断によるトリソミックレスキュー誘導

application/pdfダウン症候群の人由来iPS細胞から、ゲノム編集技術を用いて任意の21番染色体1本を消去し、21番染色体の組み合わせの異なる誘導型disomy 21細胞を複数株樹立した。これら誘導型disomy 21細胞における、消去された21番染色体の特定がSTR解析によりなされ、結果、3本の21番染色体のうちの2本から構成される、3通りの組み合わせの細胞株に分類された。これらの細胞株の配列情報の比較から、最終的に3本の21番染色体を染色体全長にわたり区別するフェイジングに成功した。以上の解析情報から、21番染色体をアレル特異的に複数部位で切断するCRISPR/Cas9システムの構築に成功した。We have successfully established three induced disomy 21 iPS cell lines by genome editing technology from the original trisomy 21 iPS cells derived from a single individual with Down's syndrome. These induced three types of cells have different combinations of 21 chromosomes. The karyotypes of the induced disomy cells have been confirmed by means of chromosome spreading G-banding, short tandem repeat analysis, multiplex ligation-dependent probe amplification, and fluorescent in situ hybridization. Furthermore, we also classified them based upon the origin of deleted chromosome 21 by STR analysis. Following chromosome phasing by comparison of sequencing data with the 3 cell lines, we have subsequently succeeded in constructing a CRISPR/Cas 9 system for allele specific cleavage in multiple sites.2016年度～2017年度科学研究費補助金(挑戦的萌芽研究)研究成果報告書16K1524

Lipid Peroxidation Generates Body Odor Component trans-2-Nonenal Covalently Bound to Protein in Vivo*

trans-2-Nonenal is an unsaturated aldehyde with an unpleasant greasy and grassy odor endogenously generated during the peroxidation of polyunsaturated fatty acids. 2-Nonenal covalently modified human serum albumin through a reaction in which the aldehyde preferentially reacted with the lysine residues. Modified proteins were immunogenic, and a specific monoclonal antibody (mAb) 27Q4 that cross-reacted with the protein covalently modified with 2-nonenal was raised from mouse. To verify the presence of the protein-bound 2-nonenal in vivo, the mAb 27Q4 against the 2-nonenal-modified keyhole limpet hemocyanin was raised. It was found that a novel 2-nonenal-lysine adduct, cis- and trans-Nϵ-3-[(hept-1-enyl)-4-hexylpyridinium]lysine (HHP-lysine), constitutes an epitope of the antibody. The immunoreactive materials with mAb 27Q4 were detected in the kidney of rats exposed to ferric nitrilotriacetate, an iron chelate that induces free radical-mediated oxidative tissue damage. Using high performance liquid chromatography with on-line electrospray ionization tandem mass spectrometry, we also established a highly sensitive method for detection of the cis- and trans-HHP-lysine and confirmed that the 2-nonenal-lysine adducts were indeed formed during the lipid peroxidation-mediated modification of protein in vitro and in vivo. Furthermore, we examined the involvement of the scavenger receptor lectin-like oxidized low density lipoprotein receptor-1 in the recognition of 2-nonenal-modified proteins and established that the receptor recognized the HHP-lysine adducts as a ligand

Final 3-year Results of the Dasatinib Discontinuation Trial in Patients With Chronic Myeloid Leukemia Who Received Dasatinib as a Second-line Treatment

Introduction: We previously reported an interim analysis of the DADI (dasatinib discontinuation) trial. The results showed that 48% of patients with chronic myeloid leukemia in the chronic phase who maintained a deep molecular response (DMR) for ≥ 1 year could discontinue second- or subsequent-line dasatinib treatment safely at a median follow-up of 20 months. However, the results from longer follow-up periods would be much more useful from a clinical perspective. Patients and Methods: The DADI trial was a prospective, multicenter trial conducted in Japan. After confirming a stable DMR for ≥ 1 year, dasatinib treatment subsequent to imatinib or nilotinib was discontinued. After discontinuation, the loss of DMR (even of 1 point) was defined as stringent molecular relapse, thereby triggering therapy resumption. The predictive factors of treatment-free remission (TFR) were analyzed. Results: The median follow-up period was 44.0 months (interquartile range, 40.5-48.0 months). The estimated overall TFR rate at 36 months was 44.4% (95% confidence interval, 32.0%-56.2%). Only 2 patients developed a molecular relapse after the 1-year cutoff point. The presence of imatinib resistance was a significant risk factor for molecular relapse. Moreover, high natural killer cell and low γδ+ T-cell and CD4+ regulatory T-cell (CD25+CD127low) counts before discontinuation correlated significantly with successful therapy discontinuation. Conclusion: These findings suggest that discontinuation of second- or subsequent-line dasatinib after a sustained DMR of ≥ 1 year is feasible, especially for patients with no history of imatinib resistance. In addition, the natural killer cell count was associated with the TFR