Stimulation of CK2-dependent Grp94 phosphorylation by the nuclear localization signal peptide.

The nuclear localization signal sequence (NLS) of SV40 Large T antigen is essential and sufficient for the nuclear translocation of the protein. Phosphorylation often modulates the intracellular distribution of signaling proteins. In this study, we investigated effects of the NLS-peptide of Large T antigen on protein phosphorylation. When crude cell lysates were incubated with [γ-(32)P]ATP, phosphorylation of several endogenous substrates with molecular masses of 100, 80, 50, and 45 kDa by an endogenous kinase was stimulated by the addition of the wild type NLS-peptide (CPKKKRKVEDP). The mutated NLS-peptide (CPKTKRKVEDP) and the reversed NLS-peptide (PDEVKRKKKPC) are weak in the nuclear localization activity, and they only weakly stimulated phosphorylation of these substrates. The mobility of the 100 kDa phosphoprotein was indistinguishable with that of an endoplasmic reticulum (ER)-resident molecular chaperone glucose-regulated protein 94 (Grp94) belonging to the Hsp90 family, and purified Grp94 was phosphorylated by a kinase in cell lysates in an NLS-dependent fashion. The 100 kDa protein was identified as Grp94 by immunoprecipitation and reconstitution experiments. Purification of the NLS-dependent Grp94 kinase by sequential biochemical column chromatography steps resulted in isolation of two polypeptides with molecular masses of 42 and 27 kDa, which were identified as α and β subunit of protein kinase CK2, respectively, by western blotting analysis and biochemical characterization. Moreover, effect of an excess amount of GTP and V8 peptide mapping showed that the NLS-dependent Grp94 kinase in the cell lysate is identical with CK2. Surprisingly purified CK2 did phosphorylate Grp94 even without the NLS-peptide, suggesting that an additional suppressive factor is required for NLS-dependent phosphorylation of Grp94 by CK2. We suggest a possible general role for CK2-catalyzed phosphorylation in the regulation of NLS-dependent protein nuclear translocation

Physical Relation of Source I to IRc2 in the Orion KL Region

We present mid-infrared narrow-band images of the Orion BN/KL region, and N-band low-resolution spectra of IRc2 and the nearby radio source "I." The distributions of the silicate absorption strength and the color temperature have been revealed with a sub-arcsecond resolution. The detailed structure of the 7.8 micron/12.4 micron color temperature distribution was resolved in the vicinity of IRc2. A mid-infrared counterpart to source I has been detected as a large color temperature peak. The color temperature distribution shows an increasing gradient from IRc2 toward source I, and no dominant temperature peak is seen at IRc2. The spectral energy distribution of IRc2 could be fitted by a two-temperature component model, and the "warmer component" of the infrared emission from IRc2 could be reproduced by scattering of radiation from source I. IRc2 itself is not self-luminous, but is illuminated and heated by an embedded luminous young stellar object located at source I.Comment: 20 pages, 11 figures. Minor corrections had been done in the ver.2. Accepted for publication in PAS

In vitro import of peroxisome-targeting signal type 2 (PTS2) receptor Pex7p into peroxisomes

AbstractPex7p, the peroxisome-targeting signal type 2 (PTS2) receptor, transports PTS2 proteins to peroxisomes from the cytosol. We here established a cell-free Pex7p translocation system. In assays using post-nuclear supernatant fractions each from wild-type CHO-K1 and pex7 ZPG207 cells, 35S-labeled Pex7p was imported into peroxisomes. 35S-Pex7p import was also evident using rat liver peroxisomes. 35S-Pex7p was not imported into peroxisomal remnants from a pex5 ZPG231 defective in PTS2 import and pex2 Z65. When the import of 35S-Pex5pL was inhibited with an excess amount of recombinant Pex5pS, 35S-Pex7p import was concomitantly abrogated, suggesting that Pex5pL was a transporter for Pex7p, unlike a yeast cochaperone, Pex18p. 35S-Pex7p as well as 35S-Pex5p was imported in an ATP-independent manner, whilst the import of PTS1 and PTS2 cargo-proteins was ATP-dependent. Thereby, ATP-independent import of Pex7p implicated that Pex5p export requiring ATP hydrolysis is not a limiting step for its cargo recruitment to peroxisomes. PTS1 protein import was indeed insensitive to N-ethylmaleimide, whereas Pex5p export was N-ethylmaleimide-sensitive. Taken together, the cargo-protein translocation through peroxisomal membrane more likely involves another ATP-requiring step in addition to the Pex5p export. Moreover, upon concurrent import into peroxisomes, 35S-Pex5pL and 35S-Pex7p were detected at mutually distinct ratios in the immunoprecipitates each of the import machinery peroxins including Pex14p, Pex13p, and Pex2p, hence suggesting that Pex7p as well as Pex5p translocated from the initial docking complex to RING complex on peroxisomes

A C₃-symmetric macrocycle-based, hydrogen-bonded, multiporous hexagonal network as a motif of porous molecular crystals

A C₃-symmetric π-conjugated macrocycle combined with an appropriate hydrogen bonding module (phenylene triangle) allowed the construction of crystalline supramolecular frameworks with a cavity volume of up to 58 %. The frameworks were obtained through non-interpenetrated stacking of a hexagonal sheet possessing three kinds of pores with different sizes and shapes. The activated porous material absorbed CO₂ up to 96 cm³ g-¹ at 195 K under 1 atm.This is the accepted version of the following article: Hisaki I., Nakagawa S., Tohnai N., et al. A C₃-symmetric macrocycle-based, hydrogen-bonded, multiporous hexagonal network as a motif of porous molecular crystals. Angewandte Chemie - International Edition 54, 3008 (2015), which has been published in final form at https://doi.org/10.1002/anie.201411438.[Link to final article]. This article may be used for non-commercialpurposes in accordance with the Wiley Self-ArchivingPolicy [https://authorservices.wiley.com/author-resources/Journal-Authors/licensing/self-archiving.html

Physicians' attitudes about artificial feeding in older patients with severe cognitive impairment in Japan: a qualitative study

<p>Abstract</p> <p>Background</p> <p>The question of whether to withhold artificial nutrition and hydration (ANH) from severely cognitively impaired older adults has remained nearly unexplored in Japan, where provision of ANH is considered standard care. The objective of this study was to identify and analyze factors related to the decision to provide ANH through percutaneous endoscopic gastrostomy (PEG) in older Japanese adults with severe cognitive impairment.</p> <p>Methods</p> <p>Retrospective, in-depth interviews with thirty physicians experienced in the care of older, bed-ridden, non-communicative patients with severe cognitive impairment. Interview content included questions about factors influencing the decision to provide or withhold ANH, concerns and dilemmas concerning ANH and the choice of PEG feeding as an ANH method. The process of data collection and analysis followed the Grounded Theory approach.</p> <p>Results</p> <p>Data analysis identified five factors that influence Japanese physicians' decision to provide ANH through PEG tubes: (1) the national health insurance system that allows elderly patients to become long-term hospital in-patients; (2) legal barriers with regard to limiting treatment, including the risk of prosecution; (3) emotional barriers, especially abhorrence of death by 'starvation'; (4) cultural values that promote family-oriented end-of-life decision making; and (5) reimbursement-related factors involved in the choice of PEG. However, a small number of physicians did offer patients' families the option of withholding ANH. These physicians shared certain characteristics, such as a different perception of ANH and repeated communication with families concerning end-of-life care. These qualities were found to reduce some of the effects of the factors that favor provision of ANH.</p> <p>Conclusion</p> <p>The framework of Japan's medical-legal system unintentionally provides many physicians an incentive to routinely offer ANH for this patient group through PEG tubes. It seems apparent that end-of-life education should be provided to medical providers in Japan to change the automatic assumption that ANH must be provided.</p

屠場の低温室内におけるToxoplasma gondiiの生存期間に関する研究

Examination was made of the survival period of Toxoplasma gondii in the low temperature rooms of a slaughterhouse. Materials for the examination were the mouse bodies themselves and the organs, such as the liver and brain, of mice infected with the RH or Beverley strain. These materials were stored in both of the refrigerating and the cold-storage rooms, then taken out of the rooms one after another at a short interval and examined on the existence of live Toxoplasma in them with the intraperitoneal inoculation into healthy mice. In the first experiment, it was revealed that the survival of the proliferative form of Toxoplasma in an infected mouse body was 8 days in the refrigerating room and 4 days in the cold-storage room, but a putrefactive sign was manifesting slowly in the mice stored more than 13 days in the refrigerating room and 6 days in the other. In the following experiment, the livers excised from RH-infected mice and the brains from Beverley-infected ones were stored only in the refrigerating room. It was recognized as the result that cysts were capable of survival for as long as 67 days and proliferative forms could survive for 11 days in the room. A control experiment was attempted on the resistance of T. gondii to -14℃ in a freezer and it was shown that both forms of this protozoa in the infected mouse organs could remain alive more than an hour but did not for 3 hours in a freezer of -14℃. Temperatures in both rooms were continuously measured by auto-recording thermometers. In the refrigerating room, it was 0.47℃ in average and the cold-storage room always had 3 to 4℃ higher temperature than the refrigerating room.Toxoplasma gondiiの低温に対する抵抗性についての報告は少なくない。しかし、これらの研究では、実験室内の冷蔵庫またはフリーザーなどの精確に調節された温度条件下に実施されたものである。屠畜肉やその内臓中に潜在する本原虫が、もっとも重要な感染源と見なされている現在では、屠場の低温室中で畜肉中の本原虫が、どれ程の期間生存しつづけるかが疫学上重要な意味を持つことはいうまでもない。事実、屠場の冷却室あるいは冷蔵室の温度は、頻回の扉の開閉や大量の温かい大動物肉塊の搬入などのため、相当な変動を受けることが予測され、実験室の冷蔵庫内温度条件とはかなり異なるものであると考えられた。　本研究は、以上の趣旨に沿って長崎市営屠場内の低温室を利用し、T. gondiiの増殖型および？子の生存期間を検討した。被検材料は、大動物肉塊や内臓を用いることができなかったので、RHおよびBeverley株感染マウスおよびその臓器を使用した。　第1実験では、PH株感染マウス自体を冷却室および冷蔵室に保存した。以後、毎日または隔日に保存マウス体を取り出し、その肝および脾乳剤を健常マウス腹膣内接種し、そのマウスからの原虫検出を試みた。接種ご30日以内に死亡したマウスは即時に、それ以上生存したものは屠殺して、その腹膣液および脳中の原虫の有無を顕微鏡下に検討した。その成績では、マウス体内のRH株増殖型は冷却室中で8日間、冷蔵室中で4日間生存することを認めた。しかし、保存マウスの腹膣内臓器の腐敗が冷却室では13日め以降から、また冷蔵室では6日めから認められたことから、その成績にはマウスの腐敗が影響を及ぼしていることが想像された。実験中の室温は、冷却室では平均0.9℃、最高2.5℃、最低-1.1℃、冷蔵室では平均4.2℃、最高低巾は5.1～3.2℃であった。　第2実験では、RH株感染マウス肝とBeverley株感染マウス脳を冷却室中に保存し、一定期間毎に取り出し、乳剤として健常マウスに接種した。本実験によりマウス肝中のRH株増殖型は11日間、マウスの脳中のBeverley株囊子は実に67日間冷却室で生存することが判明した。実験期間中の冷却室温度は平均0.47℃、最高低巾は3.2～-3.5℃におよんだ。対照実験として、-14℃のフリーザーの中で第2実験と同一材料を用いて検査したが、その結果、囊子、増殖型とも、1時間保存材料中に生存することを認めたが、3時間材料からは証明できなかった。　以上の実験で、屠場冷却室中で囊子は67日間、増殖型は11日間生存し、かつ、感染力を保有していることが判明したが、大動物肉塊や臓器中では、より長時間生存することが想定された

マウス接種法および螢光抗体法による屠殺豚からのToxoplasma gondiiの検出について

Ninety five pigs suspicious of Toxoplasma-infection were selected from 18,867 ones killed at Isahaya City Slaughterhouse and used for the isolation of T. gondii with mouse inoculations of their hilar or hepatic lymph nodes and also for the microscopic detection of the parasite in the lymph nodes with direct fluorescent antibody technic. In the mouse inoculation method, Toxoplasma hemagglutination test was carried out with sera of mice killed 6 weeks after the inoculation of the lymph nodes into the mice. Further, T. gondii strains newly isolated were subinoculated into mice and hamsters to investigate their virulence. The isolation rate of T. gondii was 8/95 or 8.4%, while 33 of 95 (34.7%) were positive in hemagglutination test. Fluorescent antibody technic indicated a positive response in 19 of 95 pig lymph nodes (20.0%). Eight T. gondii strains were isolated and demonstrated a high virulence for mice and hamsters. In this paper, the methods used and the above mentioned results are stated in detail and discussed.長崎県諫早市立屠場に1966年12月より1967年3月までに搬入された豚18,867頭より,屠場獣医師の協力により選抜されたトキソプラズマ症の疑いある病変豚(肺水腫,肝壊死斑,腸充血など)95頭の肝または肺門リンパ腺の螢光抗体法(直接法)により原虫検出,マウス接種法による原虫分離,および接種マウスのHA抗体価を測定し,それらの成績を比較検討した.また分離された株の毒性についてもRH株と比較し検討した.1.マウス接種法によるトキソプラズマ原虫分離は95例中8例(原虫分離率8.4%)で,いずれも栄養型が検出された.分離8株中5株は継代初期においては,シスト型も検出された.2.マウス接種法によるHA抗体価陽性(256倍以上陽性)は95例中33(陽性率34.7%)であった.原虫分離8株はいずれもHA陽性であった.3.豚の肝または肺リンパ腺の割面スタンプ標本の,螢光抗体法(直接法)によるトキソプラズマ原虫の検出率は95例中19例(検出率20.0%)であった.この19例中4例からマウス接種法により原虫が分離できた.4.分離株のマウスおよびハムスターに対する毒性はRH株と同じ程度かやや弱毒であった

Prevalence and safety of robotic surgery for gastrointestinal malignant tumors in Japan

[Aim] The National Health Insurance system has reimbursed robotic gastrointestinal surgery since April 2018 in Japan. Additionally, strict facility and surgeon standards were established by the government and the academic society. This study aimed to evaluate the prevalence and safety of robotic surgery using a Japanese nationwide web-based database. [Methods] Patients who underwent the following robotic surgeries for malignant tumors in 2018 were included: esophagectomy (RE), total gastrectomy (RTG), distal gastrectomy (RDG), proximal gastrectomy (RPG), low anterior resection (RLAR), and rectal resections other than RLAR (RRR). The number of cases and surgical mortality rates each month were calculated to evaluate the prevalence and safety of robotic procedures. [Results] A total of 3281 patients underwent robotic gastrointestinal surgery. The monthly number of robotic surgeries nearly doubled in April 2018 when they were initially reimbursed by the National Health Insurance system. Operative mortality rates were 0.9%, 0.4%, 0.2%, and 2.8% for RE (n = 330), RTG (n = 239), RDG (n = 1167), and RPG (n = 109), respectively. No mortality was observed in RLAR (n = 1062) or RRR (n = 374). [Conclusion] Robotic surgery for gastrointestinal malignant tumors was safely introduced into daily clinical practice along with rigorous surgeon and facility standards in Japan