On-site disinfection techniques are beneficial during a pandemic when there is a marked shortage of personal protective equipment (PPE), as experienced during the coronavirus disease 2019 outbreak. Ozone gas has been considered an alternative on-site disinfectant during a pandemic because it has antimicrobial activities, can be produced from air by electricity without the need for storage, and can be easily deactivated after use. However, ozone gas might become distributed at the lower layer because it has a larger molecular weight than air. This study aimed to reveal the applicability of ozone gas for the on-site disinfection of PPE. The lockers meant for changing dresses were used as ozone gas exposure boxes, and the distribution of ozone was assayed. Considering that the determined ozone levels were not consistent in the types of ozone analysers, we studied the chemical and biological activities of ozone, which were evenly detected in the locker. The gown in the locker was also uniformly exposed to ozone. Results showed that ozone gas could be used for the on-site disinfection of PPE in a closed box, such as a locker. This finding is valuable during a pandemic when PPE is in short supply.

背景:アンプルのような容器からの医薬品の汚染は、医療従事者の曝露や吸収をひき起こし、これにより職業病を引き起こす可能性がある。アンプルからの医薬品の飛散は想定されてきたが、アンプルからの飛散量を定量的に測定できる方法に関する報告はなかった。方法:私達は、入れ子にしたクリーンベンチを用いて、アンプルから放出されるエアロゾル量を測定した。この入れ子にしたクリーンベンチの外側のクリーンベンチは層流によって清浄な空気を供給し、内側のクリーンベンチでは層流が止められているために無風の状態を維持する構造となっている。結果:外側のクリーンベンチの層流を維持しながら、内側のクリーンベンチ内のエアロゾルを測定したところ、他の条件と比較しては無視できるエアロゾル量であった。アンプル開栓時には、小さいサイズのエアロゾルが大きいものより多く測定された。アンプルを開栓した位置から垂直および水平方向に40cm離れた位置においてもエアロゾルは確認された。このことはエアロゾルが両方向に飛散することを示唆している。ほとんどのエアロゾルははじめの15秒間に計測された。薬剤師としての経験年数と飛散するエアロゾル量との間に相関は認められなかった。結論:確立した方法によってアンプル開栓時に放出されるエアロゾルを定量することが可能になり、薬剤師や他の医療従事者の技術の向上や曝露防止に寄与できるものと考えられる。(著者抄録)

厚生労働省より発出された「高齢者の医薬品適正使用の指針」における処方見直しのプロセスで挙げられているポリファーマシーの関連因子は,薬剤数,服薬アドヒアランス不良,複数の医療機関への受診などがある.急性期病院では入院期間が短いことも多く,短期間で処方の適正化が必要であり,留意すべき因子抽出のために急性期病院に入院中に減薬となる患者に関連する因子を解析した.減薬群76人,非減薬群136人の解析結果では,後期高齢者,5剤以上の内服などに有意な関連は見られず,「同効薬の重複」(p=0.012),「用法の複雑性」(p=0.016),「服薬アドヒアランス不良」(p=0.005)に有意な関連が見られた.本研究において得られた関連性の高い因子を持つ患者は減薬の可能性を考慮し,介入することで,急性期病院における薬剤師業務の効率化に寄与することが考えられる.(著者抄録)

背景:シクロホスファミドはナイトロジェンマスタードから開発され、アルキル化抗がん剤として使用されている。シクロホスファミドはガス化することが疑われており、職業曝露を防ぐために注射薬調製時には閉鎖式混合調製器具の使用が推奨されている。しかし、これまで空気中のシクロホスファミド量に関する報告は相反していた。方法:シクロホスファミドの最大気化量を測定するため、外部から天井の移動をコントロールできる小箱が入った気化箱を各温度に置いた。シクロホスファミドガスの分布は、ファンを中で回していない気化箱を使って調べた。結果:シクロホスファミドの最大気化量は23℃で0.99ng/mLであり、この値は医療従事者が被ばくしうる上限値と考えられる。シクロホスファミドの分布に関しては、気化室の下層と中層では高さ依存的であったが、上層では検出限界以下であった。結論:私たちの研究は各温度下でのシクロホスファミドの最大気化量および分布を示したものである。これらの結果はシクロホスファミドの職業曝露を防ぐために寄与するであろう。(著者抄録)

薬剤師が様々な業務の中で患者の急変する場面に遭遇する可能性は高く、救急時の知識、対応についての習得は重要であるため急変時対応講習会を開催し、講習前後の知識、理解度について勤務施設により比較検討した。講習前の知識は病院勤務薬剤師の知識が有意に高いことが示唆されたが、講習後、有意差なく高評価が得られたことにより、勤務施設にかかわらず知識とスキルが修得できる講習会であることが明らかとなった。(著者抄録)

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy with a high incidence of distant metastasis and recurrence. Cancer stem cells (CSCs), which are pluripotent, self-renewable, and capable of forming tumors, contribute to PDAC initiation and metastasis and are responsible for resistance to chemotherapy and radiation. Three types of experimental methods are commonly used to identify CSCs: CSC-specific marker detection, a sphere-formation assay that reveals cell proliferation under non-adherent conditions, and detection of side-population (SP) cells that possess high intracellular-to-extracellular pump functions. Several CSC-specific markers have been reported in PDACs, including CD133, CD24, CD44, CXCR4, EpCAM, ABCG2, c-Met, ALDH-1, and nestin. There remains controversy regarding which markers are specific to PDAC CSCs and which are expressed alone or in combination in CSCs. Examining characteristics of isolated CSCs and discovering CSC-specific treatment options are important to improve the prognosis of PDAC cases. This review summarizes CSC-detection methods for PDAC, including CSC-marker detection, the sphere-formation assay, and detection of SP cells.

薬学部4年次生798名を対象とし、「アメリカ心臓協会(AHA)ファミリー&フレンズコース」での一次救命処置(BLS)実習による教育の有用性と問題点を検討した。AHA認定インストラクターによる実習前後の評価では、救急対応システム通報(前:88.9%、後:99.6%)、人工呼吸(前:35.7%、後:78.0%)、自動体外式除細動器(AED)要請(前:75.7%、後:99.7%)など、すべての実施評価項目に関して、実習後の実施率が有意に向上した(p<0.05)。さらに、通報・AED要請終了までの時間(前:13.5±8.7秒、後:9.2±28.5秒)、胸骨圧迫の中断時間(前:10.6±5.8秒、後:9.0±4.9秒)、AED到着後からショックまでの平均時間(前:76.5±16.5秒、後:54.7±8.1秒)についても実習後に有意に短くなった(p<0.05)。実習実施により手技の向上が示されたが、人工呼吸の評価項目は実施率が低い結果となった。人工呼吸の実技練習は胸骨圧迫の実技練習より回数が少ないことや、人工呼吸の実技に際し、インストラクターによる個々の実技修正が大人数でのトレーニングでは十分に実施できないことが原因であると考えられ、今後、実習内容や実施規模による人工呼吸の手技習得の検討が示唆された。(著者抄録)

1人1体のマネキンを使用した「アメリカ心臓協会(AHA)ファミリー&フレンズコース」を実施し,心肺蘇生(CPR)手技測定システムにより胸骨圧迫手技を評価した。胸骨圧迫について実習後有意に手技が向上した(平均深さ 前;43.2±10.8mm,後:50.5±7.2mm,p<0.05)。実習前後ともに,「適切な手の位置」および「完全な圧迫解除」の実施率は,女性において有意に実施率が高かった(p<0.05)。「適切な深さ」での実施率,胸骨圧迫の平均深さおよび平均テンポについては実習前後ともに男性の実施率が高く,深さ,テンポについても女性より有意に深く,早いことが認められた(p<0.05)。胸骨圧迫の平均テンポと平均深さを3群に分類した割合を検討した結果,実習前の女性についてのみ有意差が認められ(p<0.05),胸骨圧迫のテンポが早い群が,深さが深い傾向となった。実習実施により手技の向上が示されたが,胸骨圧迫において平均深さが50mm未満である女性が57.1%認められたことから,体力的な要因も考慮した圧迫手技の習得可能な実習内容の検討が必要と考えられた。今後,ガイドラインの変更点を強調した実習内容での手技の習得の検討をするとともに,手技の評価検証方法や,手技の維持について再トレーニングの時期の検証をする必要があると考える。(著者抄録)

薬学部4年次生798名を対象とし、「アメリカ心臓協会(AHA)ファミリー&フレンズコース」での一次救命処置(BLS)実習による教育の有用性と問題点を検討した。AHA認定インストラクターによる実習前後の評価では、救急対応システム通報(前:88.9%、後:99.6%)、人工呼吸(前:35.7%、後:78.0%)、自動体外式除細動器(AED)要請(前:75.7%、後:99.7%)など、すべての実施評価項目に関して、実習後の実施率が有意に向上した(p<0.05)。さらに、通報・AED要請終了までの時間(前:13.5±8.7秒、後:9.2±28.5秒)、胸骨圧迫の中断時間(前:10.6±5.8秒、後:9.0±4.9秒)、AED到着後からショックまでの平均時間(前:76.5±16.5秒、後:54.7±8.1秒)についても実習後に有意に短くなった(p<0.05)。実習実施により手技の向上が示されたが、人工呼吸の評価項目は実施率が低い結果となった。人工呼吸の実技練習は胸骨圧迫の実技練習より回数が少ないことや、人工呼吸の実技に際し、インストラクターによる個々の実技修正が大人数でのトレーニングでは十分に実施できないことが原因であると考えられ、今後、実習内容や実施規模による人工呼吸の手技習得の検討が示唆された。(著者抄録)

1人1体のマネキンを使用した「アメリカ心臓協会(AHA)ファミリー&フレンズコース」を実施し,心肺蘇生(CPR)手技測定システムにより胸骨圧迫手技を評価した。胸骨圧迫について実習後有意に手技が向上した(平均深さ 前;43.2±10.8mm,後:50.5±7.2mm,p<0.05)。実習前後ともに,「適切な手の位置」および「完全な圧迫解除」の実施率は,女性において有意に実施率が高かった(p<0.05)。「適切な深さ」での実施率,胸骨圧迫の平均深さおよび平均テンポについては実習前後ともに男性の実施率が高く,深さ,テンポについても女性より有意に深く,早いことが認められた(p<0.05)。胸骨圧迫の平均テンポと平均深さを3群に分類した割合を検討した結果,実習前の女性についてのみ有意差が認められ(p<0.05),胸骨圧迫のテンポが早い群が,深さが深い傾向となった。実習実施により手技の向上が示されたが,胸骨圧迫において平均深さが50mm未満である女性が57.1%認められたことから,体力的な要因も考慮した圧迫手技の習得可能な実習内容の検討が必要と考えられた。今後,ガイドラインの変更点を強調した実習内容での手技の習得の検討をするとともに,手技の評価検証方法や,手技の維持について再トレーニングの時期の検証をする必要があると考える。(著者抄録)

発熱性好中球減少症(FN)の治療において、ガイドラインで提唱されているcefepime(CFPM)の治療効果を先発医薬品と後発医薬品で比較した。高速液体クロマトグラフィー(high performance liquid chromatography:HPLC)による製剤的同等性試験では、先発医薬品と後発医薬品に違いは認められなかった。有効率は先発医薬品使用群67.8%(著効32%、有効27%、やや有効8%)、後発医薬品使用群60%(著効20%、有効25%、やや有効16%)(p=0.222)、有害事象発現率は先発医薬品使用群8.0%、後発医薬品使用群7.5%(p=0.923)で2群間に統計的に有意差は認められなかったが、先発医薬品使用群は著効割合が後発医薬品より高く、有効性において優れている傾向が示された。その要因として、2群間の原疾患背景の偏りから後発医薬品使用群が治療学的に不利であった可能性が示唆されたことから、先発医薬品が必ずしも優れているとはいえなかった。(著者抄録)

わが国は2011年に東日本大震災を経験したが、災害発生時には医薬品ロジスティクスは深刻な打撃を受け、被災地の多くで医薬品不足が発生する。さらに、医薬品の需要は被災地外からは把握できないため、薬効に関係なく、多種類の医薬品が被災地に一度に送達される結果、過剰な医薬品と不要な医薬品が集積し混乱が生じることとなる。今回、われわれは災害後に生じる医薬品供給と管理などに関して、被災地での混乱を防ぐ目的で、新しい災害時支援医薬品供給管理システムをクラウド上に構築した。このシステムは、次のような長所を有している。まず、本システムによって被災地外から被災地の医薬品需要を把握することができ、これによって適切な種類と量の医薬品を送達できるようになる。第2点目として、本システムは医薬品のバーコードリーダーとしてスマートフォンを利用しており、新たな機器の調達や使用方法の習得などの必要が軽減される。最後に、被災地において容易に医薬品リストを作成することができるようになる。医薬品リストの重要性は、医師や薬剤師以外の医療従事者にも認識されているが、これまでその作成には過大な時間と手間がかかっていた現状があり、本システムは被災地の医薬品ロジスティクスを制御し、薬剤師が被災地でその他の活動をするための時間を生み出す一助となるものである。(著者抄録)

BACKGROUND: Exposure to anticancer drugs is hazardous and may lead to chromosomal abnormalities and spontaneous abortion in healthcare workers. Guidelines recommend surface decontamination and cleaning in order to minimize the occupational exposure to anticancer drugs, although no single process has been found to deactivate all currently available hazardous drugs. Ozone gas is oxidative and a decontaminant for bacteria; its characteristic as a gas has advantages in that it does not need to be wiped off or neutralized after use. METHODS: The nucleoside anticancer drugs, cytarabine and fluorouracil, were exposed to ozone gas on plates under controlled humidity. The levels of exposed ozone were evaluated using the concentration-time (CT) value, which is the mathematical product of ozone concentration and exposure time. The effects of exposure to ozone on levels of the anticancer drugs were determined by high-performance liquid chromatography (HPLC). RESULTS: The levels of cytarabine decreased with increasing CT value and were not detected beyond 40,000 CT. The decomposition levels of the anticancer drug by ozone were CT-dependent irrespective of the maximum concentration of ozone. Higher humidity in the range from 70 to 90 % accelerated the decomposition of cytarabine and fluorouracil, and neither of the drugs were detected at 90 % humidity after exposure to ozone gas. CONCLUSIONS: Ozone gas decomposed these nucleoside anticancer drugs. This is the first report of the applicability of ozone gas as a decontaminator for anticancer drugs.

We investigated whether the component in cataplasm transmitted into hemorrhoid ointment in the combined storage of hemorrhoid ointment and non-steroidal anti-inflammatory drugs (NSAIDs) cataplasm. The NSAIDs cataplasm was used as a commercially available methyl salicylate (MS reishippu "TAIHO", MS cataplasm) and indomethacin (Catlep^®, IMC cataplasm) cataplasm. In addition, the hemorrhoid ointment was in a polyethylene container with (Neriproct^® ointment, DFV-L ointment) or without aluminum laminate (Posterisan^® forte, HC ointment). As for the methyl salicylate, 5.68 mg / pieces in HC ointment were detected at 40 weeks of combined storage with MS cataplasm. The methyl salicylate concentration in DFV-L ointment was lower than that in HC ointment under the same conditions. On the other hand, no contamination of indomethacin in HC and DFV-L ointment was observed in the combined storage with IMC cataplasm. These results show that the methyl salicylate in cataplasm passed the polyethylene container, and provide significant information on the risk of contamination by the combined storage of cataplasm and hemorrhoid ointment.

  Radioactive cesium has strongly bound soil as time proceeded, which could not be cleaved in mild condition. We have found that serial treatment of ammonium citrate solution and ionic liquid removed radioactive cesium from soil effectively. The sequence of the treatment is crucial, since inverse serial treatment or mixture of two kinds of solution did not show such an effect, which suggested that ammonium citrate unlocked trapped cesium in soil and ionic liquid solved it. We also found that repeating serial treatment and prolonged treatment time additively removed cesium from soil.

実験用ゴム栓1は通常のバイアル用ゴム栓(対照)の脚部に厚さ2mmのシリコンゴムを貼付し、実験用ゴム栓2はブチルゴムで作製し、上下2片を嵌合させて用いた。平均漏液量は対照用ゴム栓で8.2μL、実験用ゴム栓1で1.8μLと1/5に減少した。検体数を9とした場合の平均漏液量は対照用ゴム栓で16.7μL、実験用ゴム栓2で1.0μLと1/17に減少した。次いで実験用ゴム栓2の嵌合部空間にウレタン系連泡スポンジの吸収体を入れた場合、平均漏液量は0.3μLと更に減少した。吸収体を入れた実験用ゴム栓2を装着したバイアルを用い、注射針の抜針途中でバイアルを反転正立させ、その後に抜針する方法では、全ての検体で漏液量は検出限界以下であった。今回開発した二重底構造のゴム栓は漏液防止効果を有し、無菌性や異物排除などの条件を満たし、工場出荷時に本ゴム栓が装着されれば、医療従事者は従来のゴム栓と同じ要領で操作可能と考えられた。

簡便かつ再現性よく、針の穿刺→抜針までの1単位の操作ごとの液漏れ量を測定する方法を確立し、確立した測定法を用いて、注射針の太さおよびベベルの形状と漏液との関連性を定量的に検討した。確立した抽出および測定法を用いて、注射針形状が漏液量に与える影響について調べた。18G RB針からの漏れの平均値は19.7μL、18G SB針からの漏れの平均値は14.4μLであった。22G RB針および22G SB針からの漏れ量の平均値は、それぞれ2.6μLおよび2.5μLと低値であった。18G RBと22G RB、あるいは、18G SBと22G SBとの漏液量の差は有意であった。18G RBからの漏液量は18G SBの値より高値を示したが、両者の間に有意差は認められず、22G RBからの漏液量は22G SBの値とほぼ同等の値を示した。

BACKGROUND: We had previously identified the mutant allele of apm1(+) that encodes a homolog of the mammalian µ1A subunit of the clathrin-associated adaptor protein-1 (AP-1) complex, and we demonstrated the role of Apm1 in Golgi/endosome trafficking, secretion, and vacuole fusion in fission yeast. METHODOLOGY/PRINCIPAL FINDINGS: In the present study, we isolated rho3(+), which encodes a Rho-family small GTPase, an important regulator of exocystosis, as a multicopy-suppressor of the temperature-sensitive growth of the apm1-1 mutant cells. Overexpression of Rho3 suppressed the Cl(-) sensitivity and immunosuppressant sensitivity of the apm1-1 mutant cells. Overexpression of Rho3 also suppressed the fragmentation of vacuoles, and the accumulation of v-SNARE Syb1 in Golgi/endosomes and partially suppressed the defective secretion associated with apm1-deletion cells. Notably, electron microscopic observation of the rho3-deletion cells revealed the accumulation of abnormal Golgi-like structures, vacuole fragmentation, and accumulation of secretory vesicles; these phenotypes were very similar to those of the apm1-deletion cells. Furthermore, the rho3-deletion cells and apm1-deletion cells showed very similar phenotypic characteristics, including the sensitivity to the immunosuppressant FK506, the cell wall-damaging agent micafungin, Cl(-), and valproic acid. Green fluorescent protein (GFP)-Rho3 was localized at Golgi/endosomes as well as the plasma membrane and division site. Finally, Rho3 was shown to form a complex with Apm1 as well as with other subunits of the clathrin-associated AP-1 complex in a GTP- and effector domain-dependent manner. CONCLUSIONS/SIGNIFICANCE: Taken together, our findings reveal a novel role of Rho3 in the regulation of Golgi/endosome trafficking and suggest that clathrin-associated adaptor protein-1 and Rho3 co-ordinate in intracellular transport in fission yeast. To the best of our knowledge, this study provides the first evidence of a direct link between the small GTPase Rho and the clathrin-associated adaptor protein-1 in membrane trafficking.

Mitogen-activated protein kinases (MAPKs), which are found in all eukaryotes, are signal transducing enzymes playing a central role in diverse biological processes, such as cell proliferation, sexual differentiation, and apoptosis. The MAPK signaling pathway plays a key role in the regulation of gene expression through the phosphorylation of transcription factors. Recent studies have identified several RNA-binding proteins (RBPs) as regulators of MAPK signaling because these RBPs bind to the mRNAs encoding the components of the MAPK pathway and regulate the stability of their transcripts. Moreover, RBPs also serve as targets of MAPKs because MAPK phosphorylate and regulate the ability of RBPs to bind and stabilize target mRNAs, thus controlling various cellular functions. In this review, we present evidence for the significance of the MAPK signaling in the regulation of RBPs and their target mRNAs, which provides additional information about the regulatory mechanism underlying gene expression. We further present evidence for the clinical importance of the posttranscriptional regulation of mRNA stability and its implications for drug discovery.

Personnel who prepare and administer chemotherapeutic agents have been reported to develop untoward effects. The use of appropriate techniques for preparing these agents is encouraged, and educational training systems that involve the use of a fluorescent or chemiluminescence reagent as placebos have been established to minimize potential exposure to these agents. However, the optimum conditions for the use and visibility of these placebos remain obscure. In this study, our results indicated that the fluorescence intensity of fluorescent reagent decreased when it was used at a concentration greater than 0.01%. Because drops created due to splashes and leaks are extremely small and easily evaporate, it is possible that the fluorescence resulting from such drops readily disappears despite using an anti-evaporation reagent. We also developed a method to evaluate the visibility of the small drop; using this method, we determined the distance at which the drop present on the pin could be seen by the observer. The distance at which the drop was clearly recognized as a pinpoint by using the fluorescence method was almost comparable to that for the chemiluminescence method. In the chemiluminescence method, the drop on the pin was faintly visible as a slightly bright area because of low background when observed at a certain distance that was much greater than that at which the drop was clearly visible; however, such an area was not observed in the fluorescence method. The results of our study will help in the selection of a training method depending on the situation.

The interaction between Rnc1, an RNA interactive protein, and a Pmp1 mRNA was investigated by affinity capillary electrophoresis (ACE). Prior to the ACE experiments, the column performances of three capillaries (an untreated fused silica capillary, a polybrene-polyacrylic acid (PB-PAA) double layer coating capillary, and a carboxylated capillary with a covalent modification) were studied with model proteins including ribonuclease B (RNase B) and bovine serum albumin (BSA). Using an untreated fused silica and a PB-PAA double layer coating capillaries, both of the protein peaks were broad and tailing. However, using a carboxylated capillary, the protein peaks were sharp and symmetric, and migration times were repeatable (RSD<0.4%). Further, the proteins in human serum also gave sharp peaks and its repeatability was kept at a high level by pre-treatment of a capillary inner wall with 1M sodium chloride solution before each run. An Rnc1 protein was analyzed by ACE with background electrolytes containing various concentrations of Pmp1 sense mRNA using a carboxylated capillary. Increase in the concentration of the mRNA was found to delay the migration time of the protein. But the migration time of the protein was kept constant with increasing Pmp1 anti-sense mRNA instead of Pmp1 sense mRNA. A straight line (r=0.987) was obtained by plotting 1/(migration time shift) versus 1/(Pmp1 sense mRNA concentration) and the association constant of Rnc1 protein with Pmp1 sense mRNA could be estimated to be 4.15x10(6)M(-1). These results suggest that the association constants of proteins with mRNAs as ligands were easily determined by the proposed method.

The highly conserved fission yeast Pmk1 MAPK pathway plays a key role in cell integrity by regulating Atf1, which belongs to the ATF/cAMP-responsive element-binding (CREB) protein family. We identified and characterized ecm33(+), which encodes a glycosyl-phosphatidylinositol (GPI)-anchored cell surface protein as a transcriptional target of Pmk1 and Atf1. We demonstrated that the gene expression of Ecm33 is regulated by two transcription factors Atf1 and a MADS-box-type transcription factor Mbx1. We identified a putative ATF/CREB-binding site and an RLM1-binding site in the ecm33(+) promoter region and monitored the transcriptional activity of Atf1 or Mbx1 in living cells using a destabilized luciferase reporter gene fused to three tandem repeats of the CRE and six tandem repeats of the Rlm1-binding sequence, respectively. These reporter genes reflect the activation of the Pmk1 pathway by various stimuli, thereby enabling the real-time monitoring of the Pmk1 cell integrity pathway. Notably, the Deltaecm33 cells displayed hyperactivation of the Pmk1 signaling together with hypersensitivity to Ca(2+) and an abnormal morphology, which were almost abolished by simultaneous deletion of the components of the Rho2/Pck2/Pmk1 pathway. Our results suggest that Ecm33 is involved in the negative feedback regulation of Pmk1 cell integrity signaling and is linked to cellular Ca(2+) signaling.

Myosin II is an essential component of the actomyosin contractile ring and plays a crucial role in cytokinesis by generating the forces necessary for contraction of the actomyosin ring. Cdc4 is an essential myosin II light chain in fission yeast and is required for cytokinesis. In various eukaryotes, the phosphorylation of myosin is well documented as a primary means of activating myosin II, but little is known about the regulatory mechanisms of Cdc4. Here, we isolated Nrd1, an RNA-binding protein with RNA-recognition motifs, as a multicopy suppressor of cdc4 mutants. Notably, we demonstrated that Nrd1 binds and stabilizes Cdc4 mRNA, thereby suppressing the cytokinesis defects of the cdc4 mutants. Importantly, Pmk1 mitogen-activated protein kinase (MAPK) directly phosphorylates Nrd1, thereby negatively regulating the binding activity of Nrd1 to Cdc4 mRNA. Consistently, the inactivation of Pmk1 MAPK signaling, as well as Nrd1 overexpression, stabilized the Cdc4 mRNA level, thereby suppressing the cytokinesis defects associated with the cdc4 mutants. In addition, we demonstrated the cell cycle-dependent regulation of Pmk1/Nrd1 signaling. Together, our results indicate that Nrd1 plays a role in the regulation of Cdc4 mRNA stability; moreover, our study is the first to demonstrate the posttranscriptional regulation of myosin expression by MAPK signaling.

The fission yeast Schizosaccharomyces pombe (S. pombe) has become a valuable model system to elucidate the mechanisms of basic cellular functions of higher eukaryotes, including cell cycle control, membrane trafficking, and signal transduction. Having the smallest genome size among eukaryotes and with its powerful genetics, this organism is also an excellent model system for drug discovery. In addition, many signaling molecules targeted by the drug or the homologues of disease-linked human genes are highly conserved. We have been studying the signal transduction pathway in fission yeast with special emphasis on calcineurin phosphatase and mitogen-activated protein kinase (MAPK) signal transduction pathways. Our molecular genetic approach, which utilizes a crosstalk between calcineurin and MAPK signaling, has identified several regulators of Pmk1 MAPK, which is a homologue of extracellular signal-regulated kinase (ERK) in mammals. As MAPK signal transduction pathways are one of the most attractive targets for cancer therapy, inhibitors that target this signaling appear to be promising drug candidates for the treatment of cancer. Here, we first give an overview of the use of yeast as a model system for drug discovery and then we introduce our molecular genetic strategy to identify regulators of MAPK signaling and the application of this approach to drug discovery. © 2008 Humana Press Inc.

Formation of covalently bound protein adducts with lithocholic acid (LCA) might explain LCA's known carcinogenic properties and hepatotoxicity. We performed studies aimed at isolating and identifying hepatic proteins tagged with LCA, presumably via the epsilon-amino group of lysine residues. Antibodies recognizing the 3alpha-hydroxy-5beta-steroid moiety of LCA were generated by immunizing rabbits with immunogens in which the carboxyl group of LCA was coupled to BSA via a 6-aminohexanoic acid and/or succinic acid spacer. The resulting antibodies reacted with N-alpha-(t-butoxycarbonyl)-l-lysine-epsilon-LCA, the amidated and nonamidated forms of LCA, as well as synthetically prepared LCA adducts with ovalbumin and lysozyme. Proteins tagged with LCA in the liver of bile duct-ligated rats were isolated by immunoprecipitation using these antibodies. Proteins were isolated by two-dimensional electrophoresis, and their structure was identified using matrix-assisted laser desorption ionization time-of-flight mass spectrometry and computer-assisted programs. Proteins labeled with LCA were Rab-3, Rab-12, Rab-16, and M-Ras. Rab proteins are Ras-like small GTP binding proteins that regulate vesicle trafficking pathways. The covalent binding of the Rab proteins with LCA may influence vesicular transport or binding of vesicles to their cognate membrane and may contribute to LCA-induced liver toxicity.

Reactive metabolic-modified proteins have been proposed to play an important role in the mechanism(s) of the hepatotoxicity and colon cancer of lithocholic acid (LCA). To identify cellular proteins chemically modified with LCA, we have generated a monoclonal antibody that recognizes the 3alpha-hydroxy-5beta-steroid moiety of LCA. The spleen cells from a BALB/c mouse, which was immunized with an immunogen in which the side chain of LCA was coupled to bovine serum albumin (BSA) via a succinic acid spacer, was fused with SP2/0 myeloma cells to generate antibody-secreting hybridoma clones. The resulting monoclonal antibody (gamma2b, kappa) was specific to LCA-N(alpha)-BOC-lysine as well as the amidated and nonamidated forms of LCA. The immunoblot enabled the detection of LCA residues anchored on BSA and lysozyme. The antibody will be useful for monitoring the generation, localization, and capture of proteins tagged with LCA, which may be the cause of LCA-induced toxicity.

In fission yeast, knockout of the calcineurin gene resulted in hypersensitivity to Cl(-), and the overexpression of pmp1(+) encoding a dual-specificity phosphatase for Pmk1 mitogen-activated protein kinase (MAPK) or the knockout of the components of the Pmk1 pathway complemented the Cl(-) hypersensitivity of calcineurin deletion. Here, we showed that the overexpression of ptc1(+) and ptc3(+), both encoding type 2C protein phosphatase (PP2C), previously known to inactivate the Wis1-Spc1-Atf1 stress-activated MAPK signaling pathway, suppressed the Cl(-) hypersensitivity of calcineurin deletion. We also demonstrated that the mRNA levels of these two PP2Cs and pyp2(+), another negative regulator of Spc1, are dependent on Pmk1. Notably, the deletion of Atf1, but not that of Spc1, displayed hypersensitivity to the cell wall-damaging agents and also suppressed the Cl(-) hypersensitivity of calcineurin deletion, both of which are characteristic phenotypes shared by the mutation of the components of the Pmk1 MAPK pathway. Moreover, micafungin treatment induced Pmk1 hyperactivation that resulted in Atf1 hyperphosphorylation. Together, our results suggest that PP2C is involved in a negative feedback loop of the Pmk1 signaling, and results also demonstrate that Atf1 is a key component of the cell integrity signaling downstream of Pmk1 MAPK.

Ubiquitin-specific protease 14, also known as the 60 kDa subunit of tRNA-guanine transglycosylase (USP14/TGT60 kD), belongs to the ubiquitin-specific processing protease (UBP) family. USP14/TGT60 kD expression in leukemic and colorectal cancer cell lines, and the suppression of such an expression after the induction of cell differentiation have been reported. In the present study, we attempted to clarify whether USP14/TGT60 kD overexpression affects the clinicopathological features of colorectal cancer. Immunohistochemically, USP14/TGT60 kD was absent or weakly localized in the cytoplasm of normal colorectal epithelial cells. In 18 of 99 (18.2%) colorectal cancer patients, USP14/TGT60 kD was strongly detected in the cytoplasm of cancer cells. USP14/TGT60 kD expression correlated with pathological stage (P=0.03), and lymph node (P=0.03) and liver (P=0.03) metastases. Furthermore, the percentage of patients strongly positive for USP14/TGT60 kD expression increased with pathological stage. The overall survival rate was worse in patients with a high USP14/TGT60 kD expression level than in those with a low USP14/TGT60 kD expression level. Our results suggest that USP14/TGT60 kD also controls the fate of proteins that regulate tumor invasion and metastasis.

tRNA-guanine transglycosylase (TGT) is an enzyme which synthesizes a modified nucleoside, queuosine, by exchanging the base moiety of guanosine for queuine in tRNA. We have reported that the expression level of the 60-kDa subunit of TGT (TGT60kD) is elevated in leukemic cells, however, there is no other report on the expression of TGT60kD in cancer cells. The expression levels of the TGT60kD protein are elevated in four of the five colon cancer cell lines and 83% of colon cancer tissues compared with normal tissues. The expression levels of the TGT60kD protein decreased in two colon cancer cell lines, after cell differentiation was induced. A marked positive staining of cancer cells in colon tissues was observed, and the subcellular staining pattern was mainly cytosolic. These data suggest that the role of TGT60kD in colon carcinogenesis.

Formation of covalently bound protein adducts with 2-arylpropionic acids (2-APAs) has been proposed as a possible explanation for hypersensitivity and toxic responses to chiral carboxylic acid drugs. To identify the cellular proteins chemically modified with optically active (S)-ibuprofen, we generate polyclonal antibodies by immunizing rabbits with immunogen coupled to bovine serum albumin (BSA) via the spacer of 4-aminobutyric acid. The resulting antibodies largely cross-reacted with N-alpha-(t-butoxycarbonyl)--(S)-ibuprofenyl lysine as well as with the conjuguated (S)-ibuprofen with glycine and taurine and unconjugated (S)-ibuprofen, enabling enantioselective detection of (S)-ibuprofen residues anchored on ovalbumin molecules, introduced by the reaction of the ibuprofen p-nitrophenyl ester. Furthermore, immunoblotting with an antibody allows the enantioselective detection of (S)-ibuprofen-introduced glutathione-S-transferase (GST). These results indicate that the developed method will be useful for monitoring the generation and localization of protein covalently bound with (S)-ibuprofen, which may be the cause of ibuprofen-induced toxicity.

Lumican is a member of the small-leucine-rich proteoglycan (SLRP) family and is overexpressed during wound healing of the cornea, in ischemic and reperfused heart, and in several cancer tissues. Lumican is considered to regulate the collagen fibril diameter and interfibrillar spacing. However, the effect of lumican on cell growth has not been adequately examined. In the present study, we attempted to clarify whether lumican contributes to human embryonic kidney (HEK) 293 cell growth, using the morpholino antisense oligonucleotide (m-anti oligo) against lumican mRNA. M-anti oligo is a novel oligonucleotide and exhibits a higher antisense activity, higher water solubility, and greater resistance to nucleases in target cells than phosphorothioate types of oligonucleotide. After delivery of m-anti oligo against lumican mRNA, the fluorescein 5-isothiocyanate (FITC) conjugated oligonucleotides were observed in the cytoplasm and nucleus of HEK 293 cells at 24 h by confocal laser microscopy. M-anti oligo for lumican mRNA strongly inhibited the synthesis of lumican protein in the HEK 293 cells, and the HEK cell growth rate was higher than those in the control groups. These findings may indicate that lumican protein has an inhibitory effect on HEK 293 cell growth in vitro.

Queuosine is a modified nucleoside located at the first position of the tRNA anticodon, which is synthesized by tRNA-guanine transglycosylase (TGT), Although the levels of queuosine in cancer cells have been reported to be lower than those in normal cells, the expression levels of TGT remain to be determined, We determined the expression levels of a subunit of TGT (TGT60KD). Contrary of our expectations, the results revealed higher levels of expression of TGT60KD than that in normal cells, and the level of queuosine in the tRNA fraction corresponded with that of TGT60KD expression. These results suggest the possibilities that the expression levels of TGT60KD regulate TGT activity and the levels of queuosine, and that TGT60KD plays significant roles in carcinogenesis. To our knowledge, this is a first report of increased expression levels of TGT60KD in human cancer cells.

A simple and sensitive semi-micro high-performance liquid chromatography (WPLC) was established for determining the serum levels of clycyrrhizin (GL) in humans. Butyl p-hydroxybenzoate was used as the internal standard and serum was deproteinized by methanol. The samples were separated on a Capcell Pak C18 UG120 column (150 x 1.5 mm i.d.: particle size, 5 mu m). The detection limit of GL in serum was 100 ng/ml, which enables determination of serum levels of GL after administration of a therapeutic dose. The time-course study suggested that the elimination rate of GL differed between subjects for the same administered dose, although the sample was too small to allow a meaningful comment, In clinical practice, GL is used for its antiviral and anti-inflammatory effects. Excessive administration of GL can induce pseudoaldosteronism; however the optimal GL concentration in serum remains to be determined. The determination method reported here is expected to aid in the safe and efficient use of the drug in clinical practice.

We demonstrate that glycyrrhizin (GL) enhanced Fas-mediated apoptotic body formation and DNA fragmentation in T cell lines although GL alone did not induce apoptosis. The enhancement effect of Fas-mediated apoptosis by GL was dose-dependent above 0.3 mu M. Time course study revealed that simultaneous co-treatment of GL and anti-Fas antibody was crucial for the enhancement of apoptosis and pretreatment with GL was not effective. Anti-Fas antibody elicited caspase-3-like activity. However caspase-3-like activity with co-treatment of GL and anti-Fas antibody was the same level as the antibody alone. Glycyrrhetic acid, the aglycon of GL, did not enhance Fas-mediated apoptosis. The amphipathic property of GL might enable it to interact with the plasma membrane and lead to the enhancement of apoptosis.

A monoclonal antibody specific for a modified nucleoside, 5-methyt-2'-deoxycytidine (m5dCyd), was prepared using 5-methylcytidine (m5Cyd)-keyhole limpet haemocyanin (KLH) conjugate, and was characterized, Termed FMC9, the antibody reacts with m5dCyd and slightly with m5Cyd and 5-methylcytosine (m5Cyt) but not with other nucleosides tested in this investigation, FMC-9 was used in an enzyme-linked immunosorbent assay (ELISA) system for the quantitation of m5dCyd levels, Sensitivity was in the picomole range. Methylation levels in peripheral blood cells of healthy donors were determined by inhibition ELISA, The percentage of m5dCyd in peripheral blood cells of 10 healthy donors was 5.08 +/- 0.50%, These results suggest that the inhibition ELISA using FMC9 is useful to monitor m5dCyd levels in the peripheral blood cells.

We found that ibuprofen (IBU) had a potential for releasing serum albumin-bound glycyrrhetic acid(GA). Based on this observation, IBU was used to pretreat samples for the determination of serum GA levels by an inhibition ELISA. This method, termed IBU method was evaluated by the recovery of GA from human serum albumin (HSA) or normal human serum (NHS) that contained the exogeneously added GA (37-1000 ng/ml). The mean recovery of GA from HSA and NHS samples treated with IBU were 104.7 and 105.2%, respectively, whereas those without IBU pretreatment were 2.8 and 10.7%, respectively. Comparison of IBU method and chloroform extraction method revealed that the GA content of serum samples pretreated by each method were almost the same. These results suggest that IBU method is useful as a serum processing procedure for the determination of serum GA levels by an inhibition ELISA.

It is known that in cancer patients elevated levels of modified nucleosides originated from RNA are excreted in the urine. Modified nucleosides in the serum are thought to be more useful than those in the urine as tumor markers because they are not influenced by other factors. However the determination of these nucleosides is difficult because of their low amounts. To examine the efficacy of the modified nucleosides in the serum as tumor markers, ascites and solid tumor mouse models were prepared, and the amounts of 1-methyladenosine and pseudouridine in the serum were determined. Along with the growth of ascites tumor, the amounts of 1-methyladenosine and pseudouridine in the serum increased. The modified nucleosides in the serum in a solid tumor model also increased. This is the first report on the variation in the amount of 1-methyladenosine in the serum of tumor models, and the results suggest the usefulness of measuring the amounts of 1-methyladenosine and pseudouridine in the serum as tumor markers.

To evaluate the clinical usefulness of serum 1-methyladenosine, several modifications have been made in our previously established inhibition ELISA system. Horseradish peroxidase (HRP) labeled anti-mouse IgG and 3, 3', 5, 5'-tetramethylbenzidine (TMBZ) mere used as a secondary antibody and a substrate, respectively. The second blocking was done just before the addition of the secondary antibody. The standard curve of the modified ELISA system showed good linearity between 1 and 1,000 ng/ml, and the detection limit was 50 pg/well. Using the ultrafiltrated-serum samples, serum 1-methyladenosine levels in healthy individuals and cancer patients mere determined. The mean level of 1-methyladenosine in 31 healthy individuals was 28.3 +/- 7.9 ng/ml, and cut off value was set at 44.1 ng/ml (Mean+ 2SD). In cancer patients, elevated levels of serum 1-methyladenosine above the cut off value were detected in 4 out of 25 cases tested, though 11 cases had elevated urinary 1-methyladenosine levels above the cut off value (3.23 nmol/mu mol creatinine). Since 1-methyladenosine has no interaction with serum proteins and its molecular weight is quite low, it might be rapidly excreted into the urine.

A monoclonal antibody against 5-methylcytidine was prepared and characterized. This antibody, termed AMC, was reactive with compounds that had 5-methylcytosine structure (i.e. 5-methyl-2′-deoxycytidine, 5-methylcytidine and 5-methylcytosine). AMC had the highest reactivity to 5-methyl-2′-deoxycytidine among reactive compounds and had no or very slight cross-reactivity to cytidine-related compounds and any other compounds. Analysis of immunoreactive materials in urine revealed that 5-methyl-2′-deoxycytidine rather than 5-methylcytidine was, contrary to our expectation, the major component. Then the inhibition ELISA system using AMC was established and urinary levels of 5-methyl-2′-deoxycytidine in healthy individuals and cancer patients were determined. The mean excretion levels of healthy individuals was 0.90 ± 0.43 nmol/μmol creatinine and the cut-off value was set at the mean + 2 S.D. of healthy individuals (1.76 nmol/μmol creatinine). Among various types of cancer tested, elevated levels of 5-methyl-2′-deoxycytidine were detected in leukemia patients. From these results, urinary 5-methyl-2′-deoxycytidine might be applicable as a biologic marker for leukemia. © 1995.

[N-11C-methyl]-cocaine ([11C]cocaine), synthesized by N-methylation of norcocaine with [11C]CH3I, was used to assist in imaging the variety of local distribution by positron emission tomography (PET). The radiochemical yield and the radiochemical purity after purification of [11C]cocaine by high performance liquid chromatography (HPLC) at a sp. act. of 814GBq/mmol were 47-58% and > 99%, respectively. The time required for synthesis including the purification was 25-30 min from the end of [11C]CH3I trapping. The physical distribution of [11C]cocaine in organ was also investigated in mice at various time after i.v. injection. The main accumulation of radioactivity occurred in the lung, kidney and brain within l min after the injection. In the brain, no differences in the organ were observed except the radioactivity level in each section increased for the first 5 min, since then radioactivity decreased dramatically. Furthermore, in the behavioral sensitization model of cocaine, the peak of [11C]cocaine uptake in each brain area was shown to be 5-15 min. © 1994.

By use of monoclonal antibodies (MoAbs) termed APU-6 and AMA-2, we determined the usefulness of urinary modified nucleosides, pseudouridine and 1-methyladenosine, as markers for malignancy. In patients with leukemia and other forms of cancer, these nucleosides elevated significantly and reflected the disease status of patients. The immunohistochemical analysis showed that cancer cells were specifically stained with the MoAbs. Chemical identification of the cellular components reactive with the MoAbs revealed that APU-6-associated antigens were mainly rRNA and AMA-2-associated antigens were mainly tRNA. These results suggest that APU-6 and AMA-2 would be useful tools for clinical and biological studies of cancer.

Urinary levels of pseudouridine and l-methyladenosine in patients with leukemia and lymphoma were measured by the inhibition ELISA using monoclonal antibodies to determine the correlation of nucleosides excretion with disease activity. Significantly elevated levels of these nucleosides were detected in patients with all types of disease tested. Seventy-seven percent ( 46 60) and 62% ( 37 62) of patients had elevated levels of pseudouridine and l-methyladenosine above normal mean + 2S. D., respectively, and combination assay of these nucleosides gave higher positive rate (87% 52 60) than either single assay. The changes of urinary pseudouridine and l-methyladenosine reflected the disease status of patients in remission or in relapse and the effect of chemotherapy. These results suggest that urinary pseudouridine and l-methyladenosine might be clinically useful as complementary markers to the monitoring of the disease status of patients with leukemia and lymphoma by hematological examination. © 1992.

4年制の病院実務実習における実務実習モデル・コアカリキュラムの到達目標の実施状況について学生に調査を行い、平成19年度と20年度の結果を比較した。その結果、現在の4週間病院実習においてもモデル・コアカリキュラムの学習項目が導入、実施され、改善されていることがわかった。

実務実習モデル・コアカリキュラムに示されている到達目標の実施状況について、保険薬局および学生に対して調査を行った。現在の短期実習においてもモデル・コアカリキュラムの学習項目が導入・実施されていることがわかった。さらに、学習項目の実施について、指導薬剤師と学生の認識の違いはなかった。

6年制教育における長期実務実習および事前実習をより充実させる目的で、学生に対して実務実習に関する意識調査を行った。実習内容については充実していたとの感想が80％以上を占めた。さらに深く学びたいと思った内容としては服薬指導が高く、次いで在宅医療(薬局実習)、チーム医療(病院実習)であった。指導については、約90％の学生がわかりやすかったと高い評価をしていた。

We have researched the needs and the supply of the drugs in disaster. 1) The prediction of the needs for the drug: We researched the needs in the pharmacies of Shingu, Wakayama, since the medical region of Shingu is isolated from others because of its location. The drugs were classified by active ingredients and analyzed the decrease of them on the assumption that the supply of the drugs is stopped by disaster. It is revealed that the circulatory and antipsychotic drugs, which are often prescribed for long duration, become insufficient shortly after the disaster occurs. 2) The supply of the drugs: We have constructed the system, in which data can be registered through barcode and are managed as the crowd services. In addition to the management of the stock of drugs, the system can easily make a drug list in an aid station, show a map of aid stations and search and request for a drug in other aid stations regarding the distance.

本研究では,高等生物と極めて近い細胞周期システムを有する分裂酵母モデル生物を用いた<分子遺伝学的アプローチ>と<リン酸化プロテオーム解析>を軸として、細胞増殖と分化のスイッチ機構を分子レベルで明らかにすることを試みた。 特に、細胞増殖と細胞質分裂に重要な働きをするMAPキナーゼであるPmk1の細胞周期における働きに焦点をあてた解析を行った。Pmk1 MAPキナーゼの標的基質として、従来減数分裂において重要な働きをしていると報告されていたRNA結合タンパク質であるNrdlを同定した。また、Pmk1は外界からの刺激に応じてリン酸化、活性化されることは報告されていたが、今回Pmk1 MAPキナーゼの活性化が細胞周期依存的に変動することも発見した。このような細胞周期依存的なMAPキナーゼの活性や標的遺伝子群のリン酸化あるいは発現状態を定量的にアッセイできるレポーターシステムも構築した。Pmk1 MAPKはミオシン重鎖や軽鎖の変異体と遺伝学的な関係を示すことも明らかとなった。すなわち、ミオシン軽鎖の変異体や重鎖変異体の示す温度感受性がPmk 1MAPKノックアウト細胞では回復した。これらの結果から、Pmk1 MAPKはミオシンとの機能的な関係を通して、細胞質分裂を制御する可能性が示唆された。 また、新たに細胞質分裂に異常を示す変異体の取得を行い、高等生物のPSTPIPとホモロジーの高いタンパク質を同定した。このタンパク質はPmk1 MAPKと機能的な関係があることも明らかにした。具体的にはPSTPIPホモログを過剰発現するとMAPキナーゼが活性化することから、PSTPIPホモログはMAPキナーゼの上流で細胞質分裂シグナルを伝達すると考えられる。

高等生物に極めて近い細胞内情報伝達経路を有する分裂酵母モデル生物を用いて、細胞内シグナル伝達による細胞内輸送の制御機構を解析した。特にカルシウム依存性に活性化されるタンパク質脱リン酸化酵素であるカルシニューリンを介するシグナル経路を中心に解析を行った。カルシニューリンの特異的阻害薬であるFK506に対して感受性を示す変異体を取得した。これらの変異体は<細胞増殖に対してカルシニューリン活性が必須である>ことからこれらの変異体の原因遺伝子を同定することで、カルシニューリン経路と密接に関連する遺伝子が同定できると考えられる。我々は現在までにこれらの変異体の原因遺伝子として、低分子量Gタンパク質であるRab11のホモログであるYpt3,クラスリンアダプター複合体のμ1サブユニットであるApm1などを同定してきた。さらに、Apmはゴルジ・エンドゾームにおいて小胞形成に重要な働きをするとともに、分泌の過程においても関与することで細胞質分裂や細胞壁合成を制御すること、これらの働きにカルシニューリン活性が必須であることを示した。さらに、Apm1はゴルジ・エンドゾームのみならずmedial regionに局在するとともに、核とSpindle pole bodyにも局在することを明らかにした。 また、低分子量Gタンパク質Ypt6のホモログRyh1を同定し、Ryh1とYpt3という二つの低分子量Gタンパク質がゴルジから細胞膜へいたる分泌の過程で協同的に機能することを証明した。新たに免疫抑制薬感受性変異体原因遺伝子としてRab GDIも同定した。Gdi1変異蛋白質は細胞質におけるタンパク量が激減していることを見出すとともに、gdi1変異体の示す細胞内輸送の異常をPhosphatidyl inositol transfer proteinであるSpo20が回復できるということを明らかにした。

高等生物に極めて近い細胞内情報伝達経路を有する分裂酵母モデル生物を用いて、カルシウムシグナル伝達経路による細胞周期制御機構を解析した。特にカルシウム依存性に活性化されるカルシニューリン、およびProtein Kinase C, MAPキナーゼなどを中心に解析を行った。カルシニューリンの特異的阻害薬であるFK506に対して感受性を示す変異体を取得した。これらの変異体は<細胞増殖に対してカルシニューリン活性が必須である>ことからこれらの変異体の原因遺伝子を同定することで、カルシニューリン経路と密接に関連する遺伝子が同定できると考えられる。現在までにこれらの変異体の原因遺伝子として、細胞内輸送に関与する低分子量Gタンパク質Rab11のホモログ,Ypt6のホモログ、クラスリンアダプター複合体のμ1サブユニットApm1、などを同定してきた。 さらに、これらのタンパク質は細胞内輸送におけるゴルジ・エンドゾームから細胞膜へ至る分泌の過程を制御することによってカルシニューリンと共同的に細胞質分裂に関与することを報告した。 また、Protein Kinase C経路において機能する分子を同定した結果、細胞質分裂に関与するセプチンを同定した。セプチンノックアウト細胞は細胞質分裂の異常を示し、細胞の成長端やmedial regionに局在するが、これらの局在がProtein kinase Cをノックアウトすることで異常になること、さらに、Protein Kinase Cとセプチンが複合体を形成することも見出した。またセプチンのPKCコンセンサス配列に変異を入れるとセプチンの機能低下が起こることを証明した。これらの結果から、セプチンはProtein kinase C経路において細胞質分裂をコントロールしている可能性が示唆された。

甘草の成分であるグリチルリチン(GL)は、抗ウィルス作用や抗アレルギー作用を有することが知られている。一方、Fasを介するアポトーシスのシグナル伝達系は、自己反応性リンパ球の除去等に関与することが報告されている。これまでに研究代表者らは、GLが単独ではアポトーシスを誘導することなく、Fasを介したアポトーシスを増強することを見出しており、今回その奏功機構について検討した。 GLのリジン結合体より、GLのFITC蛍光標識体(GL-Lys-F1)を調製した。GL-Lys-FlもGLと同様に、Fas介在アポトーシスを増強した。ヒト白血病細胞株Jurkat及びMolt-4FをGL-Lys-Flとインキュベートし、フローサイトメトリーにより解析した結果、GL-Lys-Flは細胞と弱く結合するものの、界面活性剤を含まない緩衝液による洗浄で容易に解離することが明らかになった。GLは細胞膜と弱く結合することにより、アポトーシス増強をすることが示唆された。 次に、GLの構造体類似体によるアポトーシス増強作用を検討した。GLのアグリコンであるグリチルレチン酸は増強活性を示さなかったが、GLのモノグルクロナイドはGLより弱い増強活性を示した。しかし、グルクロン酸にはこのような活性は認められなかった。GLによるアポトーシス増強には、アグリコン部分とグルクロン酸の両方が必要であることが明らかになった。 つづいて、GLによるアポトーシス関連タンパク質の量的変化を検討した。GLはFas、Fas ligand、FADDなどのタンパク質の発現量に影響を与えなかった。これらのことから、GLによるアポトーシス増強には、タンパク質の量的変動以外の要因が大きく関与していることが考えられた。

tRNA-グアニントランスグリコシラーゼ(TGT)は、修飾核酸であるキューオシンの合成酵素である。TGTはヘテロダイマーとして存在していると考えられており、TGTの60kDaのサブユニット(TGT60kDa)がクローニングされている。また、最近TGT60kDaは脱ユビキチン化活性を有することが報告された。一方、我々はTGT60kDaが白血病、大腸がん、乳がんなどにおいて高度に発現していることを初めて明らかにした。しかし、TGTのがん治療への応用性についての報告は極めて乏しい。そこで、TGT60kDaのがんの個性診断への応用性を検討した。 TGT60kDaタンパクを高発現させた細胞株のマイトマイシンに対する感受性は、対照の低発現株と比較して低下していた。検討した他の制がん剤ではこのような効果は認められなかったことから、マイトマイシンに特異的な細胞傷害機構にTGT60kDaが関与していることが考えられる。また、TGT60kDaタンパク高発現細胞株と低発現細胞株のmRNAブロファイルを比較した結果、高発現株ではCDK2、acyl-Coenzyme A oxidaseなどのmRNA量の増加とubiquitin-conjugating enzymeであるHBUCE1などのmRNA量の減少が認められた。TGT60kDaが高発現することにより、細胞分裂の刺激とユビキチン化タンパクの減少がともに誘導されている可能性が考えられた。さらに、TGT60kDaの血清中腫瘍マーカーとしての応用性を検討した結果、血清中TGT60kDa量は担がん日数の経過によって変化しなかった。TGT60kDaは血清マーカーとしてより、組織診断などのがん細胞の個性診断に応用すべきことが明らかになった。