Monochloramine inhibits ultraviolet B-induced p53 activation and DNA repair response in human fibroblasts

AbstractMonochloramine (NH2Cl) is one of the inflammation-derived oxidants, and has various effects on cell cycle, apoptosis and signal transduction. We studied the effects of NH2Cl on DNA repair response induced by ultraviolet B (UVB) irradiation in normal human diploid fibroblasts, TIG-1. TIG-1 irradiated with 20 mJ/cm2 UVB showed marked increase in thymine dimer, which decreased by about 50% after 24 h. This decrease in thymine dimer was significantly attenuated (P<0.05) by the pretreatment of NH2Cl (200 μM), which indicated DNA repair inhibition. UVB induced p53 phosphorylation at Ser15, Ser20 and Ser37, and p53 accumulation, and NH2Cl also inhibited these changes. Consequently, UVB-induced increase in the downstream effectors of p53, namely p21Cip1 and Gadd45a, were almost completely inhibited by NH2Cl. Immunoprecipitation study indicated that the association of p53 and MDM2, an E3 ubiquitin ligase for p53, did not change substantially by NH2Cl and/or UVB. The phosphorylation of p53 (Ser15 and Ser37) by UVB is catalyzed by ATR (ataxia telangiectasia mutated and Rad3 related kinase), which works as DNA damage sensor, and ATR also phosphorylates checkpoint kinase 1(Chk1) at Ser345. NH2Cl also inhibited the phosphorylation of Chk1 (Ser345). As UVB-induced DNA damage is repaired by nucleotide excision repair (NER) in human cells, these findings indicated that NH2Cl inhibited NER through the inhibition of p53 phosphorylation and accumulation, and NH2Cl probably impaired DNA damage recognition and/or ATR activation. NH2Cl may facilitate carcinogenesis through the inhibition of NER that repairs DNA damages from various carcinogens

Oxidative modification of IκB by monochloramine inhibits tumor necrosis factor α-induced NF-κB activation

AbstractWe have previously reported that monochloramine (NH2Cl), a neutrophil-derived oxidant, inhibited tumor necrosis factor α (TNFα)-induced expression of cell adhesion molecules and nuclear factor-κB (NF-κB) activation (Free Radical Research 36 (2002) 845–852). Here, we studied the mechanism how NH2Cl inhibited TNFα-induced NF-κB activation, and compared the effects with taurine chloramine (Tau–NHCl). Pretreatment of Jurkat cells with NH2Cl at 70 μM resulted in suppression of TNFα-induced IκB phosphorylation and degradation, and inhibited NF-κB activation. In addition, a slow-moving IκB band appeared on SDS-PAGE. By contrast, Tau–NHCl for up to 200 μM had no effects. Interestingly, NH2Cl did not inhibit IκB kinase activation by TNFα. Protein phosphatase activity did not show apparent change. When recombinant IκB was oxidized by NH2Cl in vitro and phosphorylated by TNFα-stimulated Jurkat cell lysate, its phosphorylation occurred less effectively than non-oxidized IκB. In addition, when NF-κB–IκB complex was immunoprecipitated from NH2Cl-treated cells and phosphorylated in vitro by recombinant active IκB kinase, native IκB but not oxidized IκB was phosphorylated. Amino acid analysis of the in vitro oxidized IκB showed methionine oxidation to methionine sulfoxide. Although Tau–NHCl alone had little effects on TNFα-induced NF-κB activation, simultaneous presence of Tau–NHCl and ammonium ion significantly inhibited the NF-κB activation, probably through the conversion of Tau–NHCl to NH2Cl. These results indicated that NH2Cl inhibited TNFα-induced NF-κB activation through the oxidation of IκB, and that NH2Cl is physiologically more relevant than Tau–NHCl in modifying NF-κB-mediated cellular responses

The relation between subjective symptom and circulation during orthostatic stress using a tilt table

起立負荷時における気分不良の有無と体循環,脳循環との関係について検討することを目的とし,電動チルト台を用いて起立負荷を行った.対象者は20代の自律神経疾患を有さない健常女性12名とし,起立負荷によって気分不良を示さなかった群を正常群とし,示した群を気分不良群とした.電動tilt tableを0°→ 30°→ 45°→ 60°→ 0°と変化させ,各段階を約3分ずつ保持した.その際,平均動脈血圧(MBP),心拍出量,心拍数,1回拍出量,総末梢血管抵抗,腓腹筋内側頭部のTotal Hb,中大脳動脈の平均血流速度(FV)と末梢血管抵抗(PI)を測定し,気分不良尺度を10段階評価でもって記録した.その結果,正常群は起立負荷に伴いFVは低下を示したが,MBP,PIに著変はなく,気分不良群はMBPの上昇に対してPIは減少し,FVはほぼ変化はみられなかった.一般的にめまいなどの気分不良症状は脳血流量の減少により生じるとされていたが,今回の結果では気分不良には脳血流量の増加による脳細動脈へのストレスなどが考えられた.This study aimed at considering the relation between subjective symptoms and the circulation of healthy women during orthostatic stress using a tilt table. From 12 healthy women in there twenties who don't have autonomic nervous disorders, two groups were formed: 1) a normal group which didn't feel ill during orthostatic stress, and 2) a FI group which feel ill during orthostatic stress. An electric tilt table was changed from 0°→30°→45°→60°→0°, and each stage was held for about 3 minutes. Mean artery blood pressure (MBP), cardiac output, heart rate, stroke volume, total peripheral resistance and total hemoglobin at the part of interior gastrocnemius (Total Hb), flow velocity (FV) and peripheral resistance (PI) of the middle cerebral artery (MCA) were measured. The scale of poor feeling was also recorded by10 stage evaluations. Consequently, although the normal group showed an FV fall with orthostatic stress,there were no significant changes in MBP and PI. In the FI group, PI decreased but FV didn't show muchchange with the rise of MBP. According to this result, the stress to the arteriola caused not by a fall but anincrease in the cerebral blood flows etc. seems thus to have been the source of the feeling

Detection of Strong Millimeter Emission from the Circumstellar Dust Disk Around V1094 Sco: Cold and Massive Disk around a T Tauri Star in a Quiescent Accretion Phase?

We present the discovery of a cold massive dust disk around the T Tauri star V1094 Sco in the Lupus molecular cloud from the 1.1 millimeter continuum observations with AzTEC on ASTE. A compact ($r\lesssim$320 AU) continuum emission coincides with the stellar position having a flux density of 272 mJy which is largest among T Tauri stars in Lupus. We also present the detection of molecular gas associated with the star in the five-point observations in $^{12}$CO J=3--2 and $^{13}$CO J=3--2. Since our $^{12}$CO and $^{13}$CO observations did not show any signature of a large-scale outflow or a massive envelope, the compact dust emission is likely to come from a disk around the star. The observed SED of V1094 Sco shows no distinct turnover from near infrared to millimeter wavelengths, which can be well described by a flattened disk for the dust component, and no clear dip feature around 10 \micron suggestive of absence of an inner hole in the disk. We fit a simple power-law disk model to the observed SED. The estimated disk mass ranges from 0.03 to $\gtrsim$0.12 M_\sun, which is one or two orders of magnitude larger than the median disk mass of T Tauri stars in Taurus.Comment: 18 pages, 4 figures, accepted for publication in Ap

A Novel Gene, fudoh, in the SCCmec Region Suppresses the Colony Spreading Ability and Virulence of Staphylococcus aureus

Staphylococcus aureus colonies can spread on soft agar plates. We compared colony spreading of clinically isolated methicillin-sensitive S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA). All MSSA strains showed colony spreading, but most MRSA strains (73%) carrying SCCmec type-II showed little colony spreading. Deletion of the entire SCCmec type-II region from these MRSA strains restored colony spreading. Introduction of a novel gene, fudoh, carried by SCCmec type-II into Newman strain suppressed colony spreading. MRSA strains with high spreading ability (27%) had no fudoh or a point-mutated fudoh that did not suppress colony spreading. The fudoh-transformed Newman strain had decreased exotoxin production and attenuated virulence in mice. Most community-acquired MRSA strains carried SCCmec type-IV, which does not include fudoh, and showed high colony spreading ability. These findings suggest that fudoh in the SCCmec type-II region suppresses colony spreading and exotoxin production, and is involved in S. aureus pathogenesis

Transcription and Translation Products of the Cytolysin Gene psm-mec on the Mobile Genetic Element SCCmec Regulate Staphylococcus aureus Virulence

The F region downstream of the mecI gene in the SCCmec element in hospital-associated methicillin-resistant Staphylococcus aureus (HA-MRSA) contains two bidirectionally overlapping open reading frames (ORFs), the fudoh ORF and the psm-mec ORF. The psm-mec ORF encodes a cytolysin, phenol-soluble modulin (PSM)-mec. Transformation of the F region into the Newman strain, which is a methicillin-sensitive S. aureus (MSSA) strain, or into the MW2 (USA400) and FRP3757 (USA300) strains, which are community-acquired MRSA (CA-MRSA) strains that lack the F region, attenuated their virulence in a mouse systemic infection model. Introducing the F region to these strains suppressed colony-spreading activity and PSMα production, and promoted biofilm formation. By producing mutations into the psm-mec ORF, we revealed that (i) both the transcription and translation products of the psm-mec ORF suppressed colony-spreading activity and promoted biofilm formation; and (ii) the transcription product of the psm-mec ORF, but not its translation product, decreased PSMα production. These findings suggest that both the psm-mec transcript, acting as a regulatory RNA, and the PSM-mec protein encoded by the gene on the mobile genetic element SCCmec regulate the virulence of Staphylococcus aureus

The whole blood transcriptional regulation landscape in 465 COVID-19 infected samples from Japan COVID-19 Task Force

「コロナ制圧タスクフォース」COVID-19患者由来の血液細胞における遺伝子発現の網羅的解析 --重症度に応じた遺伝子発現の変化には、ヒトゲノム配列の個人差が影響する--. 京都大学プレスリリース. 2022-08-23.Coronavirus disease 2019 (COVID-19) is a recently-emerged infectious disease that has caused millions of deaths, where comprehensive understanding of disease mechanisms is still unestablished. In particular, studies of gene expression dynamics and regulation landscape in COVID-19 infected individuals are limited. Here, we report on a thorough analysis of whole blood RNA-seq data from 465 genotyped samples from the Japan COVID-19 Task Force, including 359 severe and 106 non-severe COVID-19 cases. We discover 1169 putative causal expression quantitative trait loci (eQTLs) including 34 possible colocalizations with biobank fine-mapping results of hematopoietic traits in a Japanese population, 1549 putative causal splice QTLs (sQTLs; e.g. two independent sQTLs at TOR1AIP1), as well as biologically interpretable trans-eQTL examples (e.g., REST and STING1), all fine-mapped at single variant resolution. We perform differential gene expression analysis to elucidate 198 genes with increased expression in severe COVID-19 cases and enriched for innate immune-related functions. Finally, we evaluate the limited but non-zero effect of COVID-19 phenotype on eQTL discovery, and highlight the presence of COVID-19 severity-interaction eQTLs (ieQTLs; e.g., CLEC4C and MYBL2). Our study provides a comprehensive catalog of whole blood regulatory variants in Japanese, as well as a reference for transcriptional landscapes in response to COVID-19 infection

DOCK2 is involved in the host genetics and biology of severe COVID-19

「コロナ制圧タスクフォース」COVID-19疾患感受性遺伝子DOCK2の重症化機序を解明 --アジア最大のバイオレポジトリーでCOVID-19の治療標的を発見--. 京都大学プレスリリース. 2022-08-10.Identifying the host genetic factors underlying severe COVID-19 is an emerging challenge. Here we conducted a genome-wide association study (GWAS) involving 2, 393 cases of COVID-19 in a cohort of Japanese individuals collected during the initial waves of the pandemic, with 3, 289 unaffected controls. We identified a variant on chromosome 5 at 5q35 (rs60200309-A), close to the dedicator of cytokinesis 2 gene (DOCK2), which was associated with severe COVID-19 in patients less than 65 years of age. This risk allele was prevalent in East Asian individuals but rare in Europeans, highlighting the value of genome-wide association studies in non-European populations. RNA-sequencing analysis of 473 bulk peripheral blood samples identified decreased expression of DOCK2 associated with the risk allele in these younger patients. DOCK2 expression was suppressed in patients with severe cases of COVID-19. Single-cell RNA-sequencing analysis (n = 61 individuals) identified cell-type-specific downregulation of DOCK2 and a COVID-19-specific decreasing effect of the risk allele on DOCK2 expression in non-classical monocytes. Immunohistochemistry of lung specimens from patients with severe COVID-19 pneumonia showed suppressed DOCK2 expression. Moreover, inhibition of DOCK2 function with CPYPP increased the severity of pneumonia in a Syrian hamster model of SARS-CoV-2 infection, characterized by weight loss, lung oedema, enhanced viral loads, impaired macrophage recruitment and dysregulated type I interferon responses. We conclude that DOCK2 has an important role in the host immune response to SARS-CoV-2 infection and the development of severe COVID-19, and could be further explored as a potential biomarker and/or therapeutic target