Zinc is a novel intracellular second messenger

Zinc is an essential trace element required for enzymatic activity and for maintaining the conformation of many transcription factors; thus, zinc homeostasis is tightly regulated. Although zinc affects several signaling molecules and may act as a neurotransmitter, it remains unknown whether zinc acts as an intracellular second messenger capable of transducing extracellular stimuli into intracellular signaling events. In this study, we report that the cross-linking of the high affinity immunoglobin E receptor (Fcɛ receptor I [FcɛRI]) induced a release of free zinc from the perinuclear area, including the endoplasmic reticulum in mast cells, a phenomenon we call the zinc wave. The zinc wave was dependent on calcium influx and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase activation. The results suggest that the zinc wave is involved in intracellular signaling events, at least in part by modulating the duration and strength of FcɛRI-mediated signaling. Collectively, our findings indicate that zinc is a novel intracellular second messenger

血清TARC/CCL17値は薬剤性過敏症症候群(DIHS) の早期診断および病勢の指標となりうる。

BACKGROUND:Drug-induced hypersensitivity syndrome (DIHS)/drug rash with eosinophilia and systemic symptoms (DRESS) is a serious acute drug reaction with fever, cutaneous eruption, lymphadenopathy, and several visceral dysfunctions. Eosinophilia is a common hematological abnormality in DIHS/DRESS suggesting that the Th2-type immune response is involved. Thymus and activation-regulated chemokine (TARC/CCL17) is a family of CC chemokines known to play an important role in Th2-mediated immune-inflammatory processes. OBJECTIVE:We investigated the pathogenic role of TARC in patients with DIHS. METHODS:Sera were obtained from 8 patients with DIHS, 7 patients with Stevens-Johnson syndrome/Toxic epidermal necrolysis (SJS/TEN), and 14 patients with drug-induced maculopapular exanthema (MPE). Serum TARC levels were measured by ELISA. TARC levels were then compared with clinical symptoms and various hematological parameters. In addition, a biopsy was taken from the lesional skin of patients with DIHS and stained with anti-TARC Ab and anti-CD11c Ab. RESULTS:Serum TARC levels in patients with DIHS were significantly higher than those in patients with SJS/TEN and MPE during the acute phase. Serum TARC levels in DIHS patients correlated with skin eruptions, serum sIL-2R levels, eosinophil counts, and serum IL-5 levels. Immunohistochemical staining revealed that TARC was mainly expressed on CD11c+ dermal dendritic cells in patients with DIHS. CONCLUSION:Serum TARC levels may be associated with the initial presentation of DIHS as well as disease activity during the course. Thus, they could be useful as an indicator for early diagnosis and assessment of disease activity in DIHS. CD11c+ dendritic cells may be the main source of TARC in patients with DIHS.博士（医学）・甲第597号・平成25年3月15日Copyright © 2012 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved

Association of TUSC1 and DPF3 gene polymorphisms with male infertility

Purpose Recently, a genome-wide association studies of a Hutterite population in the USA revealed that five single nucleotide polymorphisms (SNPs) with a significant association with sperm quality and/or function in ethnically diverse men from Chicago were significantly correlated with family size. Of these, three SNPs (rs7867029, rs7174015, and rs12870438) were found to be significantly associated with the risk of azoospermia and/or oligozoospermia in a Japanese population. In this study, we investigated whether the rs10966811 (located in an intergenic region between the TUSC1 and IZUMO3 genes) and rs10129954 (located in the DPF3 gene) SNPs, previously related to family size, are associated with male infertility. In addition, we performed association analysis between rs12348 in TUSC1 and rs2772579 in IZUMO3 and male infertility. Methods We genotyped 145 patients with infertility (including 83 patients with azoospermia, and 62 with oligozoospermia) and 713 fertile controls by PCR-RFLP technique for polymorphism. Because rs10966811 has no restriction sites, the SNP rs12376894 with strong linkage disequilibrium was selected as an alternative to rs10966811. Results There was a statistically significant association between rs12376894 proxy SNP of rs10966811, and oligozoospermia. A statistically significant association between rs10129954 and azoospermia, and oligozoospermia were observed. When we assessed the relationship between rs12348 in TUSC1 and rs2772579 in IZUMO3 and male infertility traits, we found that rs12348 in TUSC1 was significantly associated with azoospermia and oligozoospermia, but rs2772579 in IZUMO3 was not associated with male infertility. Conclusion We found that the polymorphisms in TUSC1 and DPF3 displayed strong associations with male infertility

A Novel Role of the L-Type Calcium Channel α1D Subunit as a Gatekeeper for Intracellular Zinc Signaling: Zinc Wave

Recent studies have shown that zinc ion (Zn) can behave as an intracellular signaling molecule. We previously demonstrated that mast cells stimulated through the high-affinity IgE receptor (FcεRI) rapidly release intracellular Zn from the endoplasmic reticulum (ER), and we named this phenomenon the “Zn wave”. However, the molecules responsible for releasing Zn and the roles of the Zn wave were elusive. Here we identified the pore-forming α1 subunit of the Cav1.3 (α1D) L-type calcium channel (LTCC) as the gatekeeper for the Zn wave. LTCC antagonists inhibited the Zn wave, and an agonist was sufficient to induce it. Notably, α1D was mainly localized to the ER rather than the plasma membrane in mast cells, and the Zn wave was impaired by α1D knockdown. We further found that the LTCC-mediated Zn wave positively controlled cytokine gene induction by enhancing the DNA-binding activity of NF- κB. Consistent with this finding, LTCC antagonists inhibited the cytokine-mediated delayed-type allergic reaction in mice without affecting the immediate-type allergic reaction. These findings indicated that the LTCC α1D subunit located on the ER membrane has a novel function as a gatekeeper for the Zn wave, which is involved in regulating NF-κB signaling and the delayed-type allergic reaction

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

PreS領域の変異による肝細胞内のHBs抗原蓄積のメカニズム

これまでに,HBs抗原の肝細胞への蓄積は肝障害の重症度と関連していることが,トランスジェニックマウスを用いた実験モデルで証明されている.またenvelope蛋白の分泌の制御の一部は,large S,middle S,small Sなどによって担われていることが知られている.従って,preS領域の塩基変異によるenvelope蛋白の産生の変化は,envelope蛋白の分泌と細胞内の蓄積に影響を与えることが予想される.我々は,B型慢性肝炎患者における肝細胞内のHBs抗原の蓄積,preS領域の塩基配列の関連について検討した.慢性B型肝炎患者15例を対象とし血清よりDNAを抽出後,PreS領域をPCR増幅した.肝細胞中のHBs抗原強陽性は9例で変異はSP1結合部周辺に強くみられ,それらの症例は,肝細胞内にエンベロープ蛋白の蓄積を多く認めた.SP1結合部周囲の変異は,HBs抗原プロモーターと他のエンベロープ蛋白との活性化に影響を与える可能性が示唆された.It has been shown that accumulation of hepatitis B surface antigen (HBsAg) in hepatocytes is associated with liver cell damage. Release and retention of envelope protein is controlled by the major surface antigen promoter and the balance of large, middle and small envelope proteins. To investigate the mechanism of accumulation of envelope protein in vivo, we analyzed the relationship between the sequence of the preS region and retention of envelope protein histologically in patients with chronic hepatitis B. We studied 15 patients with histologically diagnosed chronic hepatitis B. Accumulation of envelope protein was studied immunohistochemically using polyclonal anti-HBs antibody. Sequence analysis was performed using direct sequencing methods following polymerase chain reaction (PCR) amplification. Strongly positive HBsAg staining was observed in 9 patients, and the remaining patients were negative for HBsAg. Sequence analysis revealed that the mutations were clustered in and around the Sp1 binding site within the major surface promoter region in the patients who exhibited accumulation of envelope protein. Mutations in and around the Sp1 binding site may affect activity of the major surface antigen promoter and the balance between the various envelope proteins, resulting in accumulation of envelope protein in hepatocytes

PreS領域の変異による肝細胞内のHBs抗原蓄積のメカニズム

これまでに,HBs抗原の肝細胞への蓄積は肝障害の重症度と関連していることが,トランスジェニックマウスを用いた実験モデルで証明されている.またenvelope蛋白の分泌の制御の一部は,large S,middle S,small Sなどによって担われていることが知られている.従って,preS領域の塩基変異によるenvelope蛋白の産生の変化は,envelope蛋白の分泌と細胞内の蓄積に影響を与えることが予想される.我々は,B型慢性肝炎患者における肝細胞内のHBs抗原の蓄積,preS領域の塩基配列の関連について検討した.慢性B型肝炎患者15例を対象とし血清よりDNAを抽出後,PreS領域をPCR増幅した.肝細胞中のHBs抗原強陽性は9例で変異はSP1結合部周辺に強くみられ,それらの症例は,肝細胞内にエンベロープ蛋白の蓄積を多く認めた.SP1結合部周囲の変異は,HBs抗原プロモーターと他のエンベロープ蛋白との活性化に影響を与える可能性が示唆された.It has been shown that accumulation of hepatitis B surface antigen (HBsAg) in hepatocytes is associated with liver cell damage. Release and retention of envelope protein is controlled by the major surface antigen promoter and the balance of large, middle and small envelope proteins. To investigate the mechanism of accumulation of envelope protein in vivo, we analyzed the relationship between the sequence of the preS region and retention of envelope protein histologically in patients with chronic hepatitis B. We studied 15 patients with histologically diagnosed chronic hepatitis B. Accumulation of envelope protein was studied immunohistochemically using polyclonal anti-HBs antibody. Sequence analysis was performed using direct sequencing methods following polymerase chain reaction (PCR) amplification. Strongly positive HBsAg staining was observed in 9 patients, and the remaining patients were negative for HBsAg. Sequence analysis revealed that the mutations were clustered in and around the Sp1 binding site within the major surface promoter region in the patients who exhibited accumulation of envelope protein. Mutations in and around the Sp1 binding site may affect activity of the major surface antigen promoter and the balance between the various envelope proteins, resulting in accumulation of envelope protein in hepatocytes

B型肝炎ウイルスのコアプロモーター活性と急性B型肝炎の重症度との関連

[目的] 劇症肝炎を含む,急性B型肝炎のcore promoterの塩基配列を解析し,さらに,肝炎の重症度と変異株のpromoter活性の相関につき検討を行った.[対象と方法] 劇症肝炎(FH)7例,重症急性肝炎(SAH)5例,急性肝炎(AH)6例を対象とし,core promoter,precore/coreを含む領域をPCR法で増幅したのち,増幅産物をpGEM-5Zへサブクローニングし,最低5クローンの塩基配列を調べた.また,すべての症例のcore promoterを含む領域のPCR産物をCAT enhancer vector(Promega)に挿入し,リポフェクチン法で培養肝細胞(Huh7)にトランスフェクションした後,QUAN-T CAT(Amarsham)を用いてpromoter活性を測定した.[結果] core promoter領域の変異はFHで平均4.6個,SAHで3.4個,AHで0.5個と,重症化に伴い頻度が増加した.nt 1762とnt 1764のA→T,G→Aのいわゆるdouble mutation(MT62, MT64)は,FH 7例中2例,SAH 5例中3例に認めた.またFH 5例,SAH 2例にnt 1773のC→T(MT73)の変異を認めたが,FHあるいはSAHの全例に共通する変異はなかった.次にcore promoterの変異株のpromoter活性をCAT assayで調べたところ,野生株(WT)で3202 ± 122cpm,MT62,64,73 : 3625 ± 746cpm,MT62,64:3699 ± 122cpm,MT73 : 3027 ± 570cpmであり,そのほかの変異のパターンを持つ症例でも,WTと比較しpromoter活性の低下,あるいは亢進のいずれも認められなかった.[考察] core promoter領域の変異は,重症度と相関することから,肝炎の重症化との関連が示唆された.しかし,我々の用いたアッセイ系ではpromoter活性に差がみられず,重症化の要因は転写活性の変化とは別の機序によると考えられた.This study sought to identify the contribution of mutations in the core promoter region of the hepatitis B virus (HBV) to the pathogenesis of severe acute onset hepatitis B. Eighteen patients were enrolled in the study: 7 with fulminant hepatitis (FH), 5 with severe acute hepatitis (SAH), and 6 with acute self-limited hepatitis (AH). Sequences and transcriptional activity of core promoter region of HBV from the patients were studied. The mean number of base changes increased with the severity of the hepatitis: 4.6 ± 3.1 in FH, 3.4 ± 1.7 in SAH, and 0.5 ± 0.5 in AH. A double mutation, A (1762)→T and G (1764)→A, was found in 2 of FH, 3 of SAH, and none of AH. AC (1773)→T mutation was found in 5 of FH, 2 of SAH, and none of AH. There was no significant difference in chloramphenicol acetyl transferase activity between different mutated bases care promotor sequences. This finding suggests that the number of mutations in core promoter region was correlated with severity of liver disease, but development of fulminant hepatitis was not a consequence of enhanced core promoter activity