御所本『三十六人集』（宮内庁書陵部蔵）の筆蹟と筆者

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Diazotization of kynurenine by acidified nitrite secreted from indoleamine 2,3-dioxygenase-expressing myeloid dendritic cells

Indoleamine 2,3-dioxygenase (IDO)-initiated tryptophan metabolism along the kynurenine (Kyn) pathway regulates T-cell responses in some dendritic cells (DC) such as plasmacytoid DC. A Kyn assay using HPLC showed that samples were frequently deproteinized with trichloroacetic acid (TCA). In the present study, bone marrow derived myeloid DC (BMDC) were differentiated from mouse bone marrow cells with GM-CSF. CpG oligodeoxynucleotides (CpG) induced the expression of IDO protein with NO production in BMDC cultured for 24 hr. The concentrations of Kyn in the culture supernatants were not increased by stimulation with CpG but rather decreased by based on the Kyn assay after deproteinization with TCA. The level of Kyn exogenously added into the cell-free culture supernatant of BMDC stimulated with CpG was severely decreased by deproteinization with TCA but not methanol, and the decrease was prevented when BMDC was stimulated with CpG in the presence of a NOS inhibitor. Under acidic conditions, Kyn reacted with nitrite produced by BMDC, and generated a new compound that was not detected by Ehrlich reagent reacting with the aromatic amino residue of Kyn. An analysis by mass spectrometry showed the new compound to be a diazotization form of Kyn. In conclusion, the deproteinization of samples by acidic treatment should be avoided for the Kyn assay when NO is produced.journal articl

Upper Bound on the Decay τ→μγ from the Belle Detector

journal articl

XRN2-deficient cells display-increased amounts of R loops.

(A-C) Basal levels of R loops were monitored and quantitated by immunofluorescence with S9.6 antibody in shXRN2 and shScr fibroblasts and MCF-7 cells transfected with a siRNA control (sicont) or targeting XRN2 (siXRN2). (D) Levels of nuclear R-loop formation in control and XRN2 KD cells as indicated examined by dot-blot analysis using the S9.6 antibody. (**p<0.01).</p

NRDE-2, the human homolog of fission yeast Nrl1, prevents DNA damage accumulation in human cells

<p>The RNA helicase Mtr4 is a versatile protein that is a crucial component of several distinct RNA surveillance complexes. Here we describe a novel complex that contains Mtr4, but has a role distinct from any of those previously described. We found that Mtr4 association with the human homolog of fission yeast Nrl1, NRDE-2, defines a novel function for Mtr4 in the DNA damage response pathway. We provide biochemical evidence that Mtr4 and NRDE-2 are part of the same complex and show that both proteins play a role in the DNA damage response by maintaining low DNA double-strand break levels. Importantly, the DNA damage response function of the Mtr4/NRDE-2 complex does not depend on the formation of R loops. We show however that NRDE-2 and Mtr4 can affect R-loop signals at a subset of distinct genes, possibly regulating their expression. Our work not only expands the wide range of Mtr4 functions, but also elucidates an important role of the less characterized human NRDE-2 protein.</p

XRN2-deficient cells are hypersensitive to various chemotherapeutic agents.

<p>(<b>A-D</b>) shScr and shXRN2 fibroblasts and MDA-MB-231 cells transfected with a siRNA control or targeting XRN2 were either mock-treated or exposed to: (<b>A, B</b>) ionizing radiation (IR); (<b>C, D</b>) ultraviolet light (UV). (<b>E-F</b>) shScr and shXRN2 fibroblasts were either mock-treated or exposed to: (<b>E</b>) H₂O₂ or (<b>F</b>) Aphidicolin (APH). Cells were then monitored for survival using colony forming assays. Colonies of >50 normal-appearing cells were quantified for mock- versus agent-exposed cells. (**p<0.01).</p

DDR regulators accumulate at the 3’ end of genes after XRN2 loss.

<p>(<b>A-E</b>) Accumulation of the DNA damage regulators, ATM, BRCA1, γ-H2AX, 53BP1, and CtIP, was monitored at the 3’ pause sites of the (<b>A</b>) <i>ENSA</i>, (<b>B</b>) <i>β-actin</i>, (<b>C</b>) <i>Akirin</i>,<i>1</i> (<b>D</b>) <i>Gemin7</i> genes and (<b>E</b>) an intronic region of <i>Gemin7</i> by chromatin immunoprecipitation. n = 3, S.E. is indicated. (<b>F</b>) Model for XRN2 functions in DNA repair pathway choice. In normal conditions, XRN2 binds to the NHEJ factor 53BP1 promoting DSB repair via the NHEJ pathway. In the absence of XRN2, NHEJ is inhibited downstream of 53BP1, allowing DSB repair via the HR pathway.</p

Loss of XRN2 leads to increased DSB formation and genomic instability.

<p>(<b>A</b>) Steady state levels of XRN2 protein in shScr (+) compared to shXRN2 (-) fibroblast cells were monitored by western blotting and immunofluorescence. (<b>B, C</b>) Basal levels of 53BP1, pATM^ser1981, γ-H2AX, and BRCA1 foci/nuclei were quantitated in shScr, shXRN2, and MCF-7 cells treated with control and XRN2 specific siRNA. (<b>D, E</b>) Genomic aberrations were quantified in shScr, shXRN2, and shk-h cells using derived metaphase spreads. (**p<0.01).</p

R-loop formation and transcription contribute to delayed DSB repair kinetics in XRN2-deficient cells.

<p><b>(A)</b> Levels of R loops were monitored in mock- or IR (1 Gy)-exposed shXRN2 compared to shScr fibroblast cells by immunofluorescence. <b>(B-C)</b> Regression of 53BP1 foci/nucleus was monitored by IF in IR (1 Gy)-treated shXRN2 and shScr cells that were exposed to α-amanitin (α-aman) or transfected with GFP-RNaseH. (*p<0.05).</p

Systematic Profiling of Poly(A)+ Transcripts Modulated by Core 3’ End Processing and Splicing Factors Reveals Regulatory Rules of Alternative Cleavage and Polyadenylation

<div><p>Alternative cleavage and polyadenylation (APA) results in mRNA isoforms containing different 3’ untranslated regions (3’UTRs) and/or coding sequences. How core cleavage/polyadenylation (C/P) factors regulate APA is not well understood. Using siRNA knockdown coupled with deep sequencing, we found that several C/P factors can play significant roles in 3’UTR-APA. Whereas Pcf11 and Fip1 enhance usage of proximal poly(A) sites (pAs), CFI-25/68, PABPN1 and PABPC1 promote usage of distal pAs. Strong cis element biases were found for pAs regulated by CFI-25/68 or Fip1, and the distance between pAs plays an important role in APA regulation. In addition, intronic pAs are substantially regulated by splicing factors, with U1 mostly inhibiting C/P events in introns near the 5’ end of gene and U2 suppressing those in introns with features for efficient splicing. Furthermore, PABPN1 inhibits expression of transcripts with pAs near the transcription start site (TSS), a property possibly related to its role in RNA degradation. Finally, we found that groups of APA events regulated by C/P factors are also modulated in cell differentiation and development with distinct trends. Together, our results support an APA code where an APA event in a given cellular context is regulated by a number of parameters, including relative location to the TSS, splicing context, distance between competing pAs, surrounding cis elements and concentrations of core C/P factors.</p></div