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The story below is originally published on Mainichi Daily News by Mainichi Shinbun (http //mdn.mainichi.jp). They admitted inventing its kinky features, or rather deliberately mistranslating them from the original gossip magazine. In fact, this is far from the general Japanese' behavior or sense of worth. このページは、毎日新聞事件の検証のための配信記事対訳ページです。直接ジャンプして来られた方は、必ずFAQをお読みください。 ※ この和訳はあくまでもボランティアの方々による一例であり、翻訳の正確さについては各自判断してください。もし誤訳(の疑い)を発見した場合には、直接ページを編集して訂正するか翻訳者連絡掲示板に報告してください。 Curtain draws on Self-Defense Force stripper who plugged for peace平和を訴え続けた自衛隊ストリッパーに死の幕が下りる 拡散状況 関連ページ Curtain draws on Self-Defense Force stripper who plugged for peace 平和を訴え続けた自衛隊ストリッパーに死の幕が下りる 0 Curtain draws on Self-Defense Force stripper who plugged for peace 2006,02,02 Shukan Asahi 2/10 By Ryann Connell 平和を訴え続けた自衛隊ストリッパーに死の幕が下りる 週刊朝日 2/10 ライアン・コネル記 1 Tomomi Sawaguchi made her name for getting into a military uniform, but she really became a star when she got out of it, judging by Shukan Asahi (2/10). 沢口友美は軍の制服を着ることで有名になったが、彼女が本当にスターになったのは、週刊朝日(2/10)から判断すれば、それを脱いだときであった。 2 But leukemia cut down Sawaguchi in the prime of her life, eventually claiming her last month aged just 44. しかし白血病が、沢口の人生の最盛期に彼女を打ちのめし、ついに先月、まだ44歳の彼女の命を奪った。 3 Sawaguchi became known across Japan for being the Self-Defense Force stripper, a title she earned following a stint in Japan's psuedo-military changing truck gears before a successful period on the stage, where this time she was getting her gear off. 沢口は、現役自衛隊ストリッパーであることで、日本中で知られるようになった。 この肩書きは、舞台上で成功した時期よりも前の、方針転換中の日本の軍隊もどきに属した期間のために彼女が獲得したものだ。 舞台では彼女は服を脱いで裸身になった。 4 Sawaguchi the SDF Stripper was born in the Hiroshima Prefecture port city of Kure, but left her hometown for Tokyo at 18. She joined the SDF shortly afterward and quickly married and had a kid, but divorced at 26. 自衛隊ストリッパー・沢口は、広島県の港湾都市呉で生まれたが、18歳のとき故郷を離れて東京に行った。 しばらくしてから彼女は自衛隊に入隊し、すばやく結婚し一児をもうけたが、26歳のときに離婚した。 5 While strolling around the seedy Tokyo entertainment district of Ikebukuro one day in the late '80s, a "talent scout" picked her up and introduced her to the world of stripping, where she quickly made a name for herself. 80年代後半のある日、東京の評判のよくない歓楽街・池袋をぶらぶら歩いているときに、「タレントスカウト」が彼女を見出し、彼女をストリップの世界へといざなった。 そこで彼女は素早く名声を得た。 6 "As far as I know, Sawaguchi was the first performer to bring 'nyotaimori,' or the serving of food on a naked woman's body, onto the stripping stage," Motoji Takasu, a publishing producer, tells Shukan Asahi. "Her pudgy little body made her a stripping star in a very short time." 「私の知る限りでは、沢口は、ストリップの舞台の上で最初に「女体盛り」――裸の女性の体の上に食べ物を置くこと――を行ったストリッパーでした」と、出版プロデューサーの高須基仁は週刊朝日に語る。 「彼女のずんぐりした小さな体のため、あっという間に彼女はストリップのスターになりました」 7 Sawaguchi had initially kept her military roots a secret, but opened up about her weapon-wielding experiences during a 1993 magazine interview, throwing her into the national spotlight. Around the same time, she started using her newly found near-fame to campaign for peace. She appeared at a number of anti-war protests and deepened ties with journalists and other peace activists. 当初沢口は自衛隊の経歴を秘密にしていたが、1993年のある雑誌インタビューにおいて、武器を取り扱った体験を公表し、国民的なスポットライトの中に自分を投げ入れた。 ほとんど同時期に、彼女は自分が新たに手に入れた名声に近いものを使って、平和運動を行いはじめた。 彼女は数々の反戦抗議に姿を現し、ジャーナリストや自分以外の平和活動家との絆を深めた。 8 "There's no room for eroticism once war starts," Shukan Asahi quotes Sawaguchi saying in what became her trademark argument. "Nudity is a symbol of peace." 「いったん戦争がはじまれば、エロティシズムを許容する余地は無くなります」と、週刊朝日は彼女のトレードマークとなった沢口の持論を引用する。「裸は平和の象徴です」 9 In February 2003, just days before the United States invaded Iraq, Sawaguchi headed off to Iraq and bared her body again, this time to act as a human shield. But she attracted considerable criticism in Japan and was forced to head back home. 2003年の2月、合衆国がイラクを侵略する本の数日前、沢口はイラクへと向い、今度は「人間の盾」の行動をするため、彼女の体をむきだしにした。 しかし彼女は日本で相当な量の批判を招いてしまい、帰国せざるを得なくなった。 10 Not long after she returned, Sawaguchi began complaining about numbness in her legs. She thought it was economy class syndrome. The pain lingered for a considerable period so the peace activist cum SDF stripper sought medical consultation and was shocked to learn in April last year that she had actually contracted leukemia. She began a blog to detail her experiences battling the disease from her hospital room. 帰国してほどなく、沢口は足の痺れを訴え始めた。彼女はそれはエコノミークラス症候群だと思った。 その痛みはかなりの期間つづいたので、この平和活動家兼自衛隊ストリッパーは、医師に診断してもらい、実は彼女は白血病にかかっていたことを去年の四月に知り、衝撃を受けた。 彼女はブログを始めて、病室から闘病体験を詳しく記述した。 11 "She sounded really cheerful when I called her in the hospital.She told me jokingly how much she thought her hair was a pain and how she was always pulling it out," publishing business figure Takasu says. "For somebody who had made a living out of their beauty, the experience must have been a tough one for Sawaguchi. It was too tough for me to go and visit her." 「私が病院にいる彼女に電話をしたとき、彼女はとても快活そうな様子でした。 彼女は私に冗談っぽく、彼女は自分の髪を悩みの種と考えていて、いつも髪を引き抜いている、と私に語りました」と、出版界の大立者の高須はいう。 「自分の美しさで生計を立てている人として、この経験は沢口にとって厳しいものだったに違いありません。 厳しすぎて、私は彼女のお見舞いに行きませんでした」 12 "Sawaguchi's performances on stage were all about the fine line dividing Eros (the Greek god of love) and Thanatos (Greek mythology's personification of death)," Takasu tells Shukan Asahi. "She spent her life stripping for peace." (By Ryann Connell) 「沢口の舞台でのパフォーマンスはすべて、エロス(ギリシャの愛の神)とタナトス(ギリシャ神話における死の権化)を分ける微妙な境界線についてのものでした」と、高須は週刊朝日に語る。 「彼女は自分の人生を、平和のためにストリップして過ごしました」(ライアン・コネル記) 13 February 2, 2006 2006年2月2日 拡散状況 Psychommu Gaijin 部分転載:http //pgaijin.blogspot.com/2006/02/mainichi-daily-news-waiwai-curtain.html 英語サイト http //marc.newsvine.com/_news/2006/02/03/81556-curtain-draws-on-self-defense-force-stripper-who-plugged-for-peace 関連ページ Psychommu Gaijin WaiWaiの記事を転載した英語サイト:N 毎日新聞英語版から配信された記事2006年
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25/Nov/2011 probeの解離定数を求める。 I am gonna determine Kd value. 2 units/ml Bc-PLC 2/5 units/ml Bc-PLC 2/5*5 units/ml Bc-PLC 2/5*5*5 units/ml Bc-PLC 2/5*5*5*5 units/ml Bc-PLC 2/5*5*5*5*5 units/ml Bc-PLC 2/5*5*5*5*5*5 units/ml Bc-PLC 2/5*5*5*5*5*5*5 units/ml Bc-PLC 0 units/ml Bc-PLC At the same time, I am gonna determine real amount of DAG level by brigh and dyer method. 18/Nov/2011 SMの量におけるD609の影響をMDCK細胞で調べる。 Effect of D609 on SM level in MDCK cells 1)ctrl 2)SMase 3)3hour D609 incubation 4)6hour D609 incubation 5)20hour D609 incubation mCherry-lysenin conc is 50microg/ml. 1 30 3hour 4 30 6hour 6 00 20hour SMase 1 units/ml 10 min. After that, fixed. What I have to prepare is that HBSS and 3 % PFA Effect of DGAT inhibitor on DAG level in MDCK cells. 1microM A922500 was used for this experiment. 2pm Add A922500 start incubation 4pm Add A922500 start incubation 5pm DAG probe incubation. こんなかんじで 17/Nov/2011 Rat red blood cell experiment, At first, I did the experiment as follows 1)1U/ml Pc-PLC + 200times annexin V + 9*108 cell/ml result hemolysis within 4 min 2)0.1U/ml Pc-PLC + 40 times annexin V + 9*108 cell/ml result hemolysis within 5 min In this time, protocol was wrong. 3)real experiment 0.1U/ml Pc-PLC + 40 times annexin V + 9*108 cell/ml I get annexin V fluorescence but it is not for living RBL cells. At first, RBC was subjected to hemolysis, become ghost, and then become fluorescence. 4)0.1U/ml Bc-PLC + 40 times annexin V + 9*108 cell/ml RBC rarely give hemolysis and no fluorescence. And I tryed the experiment whether Bc-PLC makes DAG on the plasma membrane in RBC in a Bc-PLC conc. dependent manner. 1) 800 microl RBC (9*108 cell/ml) 2) Add DAG probe 24 microl here And 3)2microl 100 units/ml Bc-PLC was added to the 8 microl HBSS. 4)2microl of this solution was added to next tube, which has 8 microl HBSS. 5)this step is done one after another. this means that 5 time solution is gonna prepare. Next in each tube, 75 microl RBC solution was added and is incubated for 10 min @r.t. after that, fixed 6% (final 3%), and incubated for 15min. and spin down. こんな感じでやりましたよーん。 30/Oct/2011 D609の濃度 50microg/ml Offered as 5 mg (sc-201403) and 25 mg (sc-201403A) sizes Synonym O-Tricyclo[5.2.1.02,6]dec-9-yl dithiocarbonate potassium salt CAS Number 83373-60-8 Molecular Weight 266.5 Molecular Formula C11H15OS2K Purity 98% Physical Appearance Off-white solid Solubility Soluble in water (25 mg/ml) I used D609 with the concentration of 188microM 以下のものをやってみる。 Other experiments 1. living MDCK cells with alpha PMA and PMA 2. DAG probe calibration with Bc-PLC Bc-PLC 20 units/ml Bc-PLC 10 units/ml Bc-PLC 5units/ml Bc-PLC 0units/ml D609 3. fixed MDCK cells with SMase noSMase SMase SMase+D609 SMase+BcPLC 4. living MDCK cells with SMase + Bc-PLC 5. SM amount with SMase or nojirimycin 6. DAG amount with SMase or nojirimycin 111016 もう一度最後の実験 以下の実験を2回繰り返す。 それぞれについて10個の細胞をとる。 それでanalyzeして終わり。 Thinking about biological significance, I am gonna try this experiment as follows. 1. noSMase 2. SMase 3. SMase+D609 4. SMase+BcPLC 16/June/2011 Now I started to write the papers. But I have several experiments I have to do and I want to do. 1. test if the DAG which exist already on the membrane is abolished by incubated with D609. 2. I am gonna take beautiful data of cholesterol, DMS and NB-DNJ. SMS2がPC-PLCであるか否か?とういうことに関係するのですが、 D609処理した細胞で、ライセニンで染まるかどうかという実験を過去にラボでやった人はいらっしゃいますか? あと、SMS1およびSMS2が欠損したMEF-ZS2とSMS1もしくは、SMS2が発現させた細胞では、ライセニンで染めるとどのように違いがでますか? こちらについては、やっているのではないかと思い聴いて見ました。 And then I want to get the PC-PLC antibody. 20May2011 I am gonna do the experiment. I don t miss to write but in the previous experiment. UV was irradiated to MDCK cells for 1 min. 30 min, 1hour 2hout later, the cells were fixed and observed. 30min there is 30% cells in which DAG probe was translocated. But not so obvious. So I am gonna do 10 min incubation after UVB irradiation. Because some papers says that 10 min is more DAG. So, 1. UVB 10min 2. UVB D609 10 min 3. No UVB And then the previous experiment that when MDCK was fixed, DAG probe localized at the plasma membrane incubated with 12.5 Bc-PLC 10 min. So, I need to play around the concentration of Bc-PLC. 4. Bc-PLC 0 microunit/ml 5. Bc-PLC 0.1 microunit/ml 6. Bc-PLC 0.5 microunit/ml 7. Bc-PLC 1 microunit/ml 8. Bc-PLC 5 microunit/ml 9. Bc-PLC 12.5 microunit/ml And then, U73122 doesn t abolish ATP-induced DAG on the outer leaflet of plasma membrane. So, I suspect PAP-induced DAG. I am gonna use the propranolol. 10. ctrl 11. D609 12. D609+ATP 13. D609+ATP+propranolol At the same time, I am gonna use the MDCK cells expressing mGluR5 and bath application of glutamate. 14. ctrl 15. glutamate 06May2011 I gonna do the experiment about DAG flip in fixed cells. A. EYFP-C1AB and DsRed B. EYFP-C1AB and DsRed with 10mM MbCD + 12.5unit/ml BcPLC C. EYFP-C1AB and DsRed with 20microM Fumonisin B1 + 12.5unit/ml BcPLC D. EYFP-C1AB and DsRed with 50nM ISP1 + 12.5unit/ml BcPLC E. EYFP-C1AB and DsRed with 6 microM DMS + 12.5unit/ml BcPLC F. EYFP-C1AB and DsRed with 100 microM NB-DNJ + 12.5unit/ml BcPLC G. EYFP-C1AB and DsRed with Bc-PLC as ctrl H. EYFP-C1AB and DsRed with PMA 28Apr2011 How to irradiate UVB (290-320nm) 1. reduce the volume of medium, DMEM 100 microl. Go to program 2. Set 10 mJoules/cm2 for 1min 3. replace the medium with appropriate media. That s it. 27Apr2011 Now Francoise kindly decide the condition of UVB irradiation. So I am doing the following experiments under the condition. I will observe DG probe and PKCdelta antibody accumulation. 1. Ctrl(negative) Just MDCK cells. 2. Ctrl(positive) PMA 3. UBV and leave 30 min 4. UBV and leave 1 hour 5. UBV and leave 3 hour 7. UBV and leave 30 min with D609 (D609 nothing after UVB) 8. UBV and leave 1 hour with D609 (D609 nothing after UVB) 9. UBV and leave 3 hour with D609 (D609 nothing after UVB) 10. UBV and leave 3 hour with D609 (D609 still incubation after UVB) 11. no D609 12. D609+100microM ATP 13. D609+100microM ATP+1microM U73122 22Apr2011 Thinking about the experiment I have to do. 1. I can get PKCgamma antibody within next week. So I gonna do the following experiment. A. EYFP-C1AB, PKCgamma antibody, DsRed. B. EYFP-C1AB, PKCgamma antibody, DsRed with SMase. C. EYFP-C1AB, PKCgamma antibody, DsRed with SMase and D609. 2. check whether DAG flop is inhbited in the presence of U73122. Actually when I did this expeirment previously using 100 microM U73122, unexpectedly more bright fluorescence comes up. Maybe some artifact. So I gonna reduce the concentration of U73122. A. 100 microM ATP B. 100 microM ATP, 10 microM U73122 C. 10 microM ATP D. 10 microM ATP, 10 microM U73122 3. I am gonna observe the effect of MbCD, FumonisinB1, ISP1, DMS and NB-DNJ in fixed cells. A. EYFP-C1AB and DsRed B. EYFP-C1AB and DsRed with 10mM MbCD + 12.5unit/ml BcPLC C. EYFP-C1AB and DsRed with 10microM(?)Fumonisin B1 + 12.5unit/ml BcPLC D. EYFP-C1AB and DsRed with 50nM ISP1 + 12.5unit/ml BcPLC E. EYFP-C1AB and DsRed with 6 microM DMS + 12.5unit/ml BcPLC F. EYFP-C1AB and DsRed with 100 microM NB-DNJ + 12.5unit/ml BcPLC 07Apr2011 Thinking about discussion with Francoise. 1. I am giving Francoise MDCK cells. 2. I gonna show the paper I am preparing now. 3. I gonna give her D609, rottelin Reiko has. 4. I gonna give her the paper relationship with D609 and apoptosis, with rottelin and apoptosis, and MDCK and apoptosis, SMase and apoptosis. 25/Mar/2011 Thinking about biological significance, I am gonna try this experiment as follows. 1. noSMase 2. noSMase+U73122 3. noSMase+D609 4. SMase 5. SMase+U73122 6. SMase+D609 7. SMase+BcPLC 8. SMase+BcPLC+U73122 Express EYFP-C1AB, DsRed and PLCepsilon-HA Express EYFP-C1AB, DsRed and PLCepsilon-HA(dominega) Express EYFP-C1AB, DsRed and PLCalpha-HA Express EYFP-C1AB, DsRed and PLCalpha-HA(dominega) 24/Mar/2011 And then, I found another contraversy. In IPS1 experiment, flip doesn t occure even though without SM. I need to do same experiment with SMase. 22/Mar/2011 I am gonna do the following experiments from the data I took on 15/Mar/2011. At first, 10 microM U73122 experiment doesn t work. So I am gonna do increase its concentration or decreases in ATP concentration. This experiment is very critical to say that the EYFP-C1AB fluorescence is caused by DAG drived from PI-PLC. 1. 100 microM ATP 2. 100 microM ATP, 100 microM U73122 3. 100 microM ATP, 100 microM U73343 4. 10 microM ATP 5. 10 microM ATP, 100 microM U73122 6. 10 microM ATP, 100 microM U73343 Another point I have to mention is that 24 hour incubation with U73122 did not give the difference in outer DAG signal compared to ctrl. This means that PI-PLC doesn t contribute to outer DAG. But, I can see PI-PLC dependent outer DAG sigal. So I want to make clear the signal from PI-PLC caused by ATP addition is transient or sustained. I guess this signal is transient. I am gonna do 10min, 30min, 1 hour, 2 hour. In some case I am gonna remove the ATP in the middle of reaction. 15/Mar/2011 I am gonna take reproduciblity. 1. MDCK Cameleon with ATP no D609 2. MDCK Cameleon with ATP with D609 3. MDCK control no D609 4. MDCK D609 only 5. MDCK D609 ATP 6. MDCK D609 ATP U73122 7. MDCK D609 ATP U73343 8. MDCK D609 ATP R59949 9. MDCK D609 ATP SMase 10. MDCK D609 PMA 11. MDCK D609 DiC8 12. MDCK C1AB inner no D609 13. MDCK C1AB inner no D609 ATP 14. MDCK C1AB inner D609 15. MDCK C1AB inner D609 ATP 16. MDCK mGluR no D609 17. MDCK mGluR D609 18. MDCK mGluR D609 Result Patially success. I got good result about that in the presence of D609 and ATP the DAG signal was detected, but no signal in the presence of D609 only. Of course, no D609 gives very bright fluorescence. But disappointed point is that U73112 did not inhibit the fluorescence which is caused by addition of ATP. Interpretation of it is due to week effect of U73122. I need to increase in concentration of U73122 or decrease of concentration of ATP. And then, the points I need to condider is that in the presence of U73122, which is 24 hours incubation, DAG signal did not decreases. But transient increase caused by inner DAG caused by ATP through PI-PLC is detected. How do I come to term with these. 09/Mar/2011 The idea to observe DAG flop. 1. no D609 2. ctrl 3. ATP 10 min 4. ATP U73122 10 min 5. ATP R59949 10 min 6. ATP SMase 10 min 7. PMA 10 min 8. DiC8 10 min 9. Inner C1AB ATP 10.Inner C1AB ATP 1. About 9 and 10, C1AB is transfected one day before. 2. 2-10 is incubated with D609 for 6 hours. 3. 1067microl(97times11)HBSS is prepared 4. 33microl C1AB is put, means total 1100microl 5. 100microl pick up. Number1 6. 4microl D609 is put, total 1004 microl 7. Divide in half 8. first half, 5 microl ATP is added. 9. 100 microl pick up. Number2. 10. From remaining 400microl, each 100 microl is taken, add 1 microl PMA(10microM) and 1 microl DiC8 (100microM).Number 7 and Number 8. 11. About second half, 4microl ATP is added. 12. 100microl is picked up, Number3. 13. 1 microl U723122(15microM), R59949(10microM) or 2microl SMase(1unit) is added to each 100 microl, Number 4,Number5 and Number 6. Result In some part it is works well. But I missed to enter D609 in the cell which was supposed to be in the presence of U73122. So I need to do again about U73122. And My concern is C1AB probe expressed into the cells localized to the membrane in the presence of only D609. I need to chech this is really real or not. And I would be better to confirm that P2Y2R stay on the surface on the plasma membrane, by doing measure increase in calcium concentration. c 04/Mar/2011 I prepared WR19L cells, lymphocyte. I am gonna do the following experiments. I am gonna add 10 microM PMA, 100 microM DiC8, 12.5 units/ml Bc-PLC, 10 microM C6-Cer mixed with EYFP-C1AB probe. Experimental procedure WR19Lcells were spind down and collected. Cells were in total 815 microl(163microl*5 in each)HBSS solution. Add 25microl 3.8mg/ml EYFP-C1AB. 2microl 1mM PMA, 1mM alphaPMA, 100mM DiC8, 8.5microl of 12.5unit/ml Bc-PLC are put in the 1.5 ml tubes in advace. 168microl solution containing EYFP-C1AB is added in each tube and incubate 10 min. 30micro 20% PFA is added and fix for 10 min. After that, spine down cells and remove supernatant. Add 50mM NH4Cl for 5min. Spine down and suck up supernatant. Add 100microl HBSS. Result I could do it! I can see brighter fluorescence in PMA than that in control.About DiC8, It seems that there is no difference compared to control.About Bc-PLC, I could see very bright fluorescence in the cells which is aggregated, indicating that DAG is important for membrane fusion. 04/Mar/2011 I prepared WR19L cells, lymphocyte. I am gonna do the following experiments. I am gonna add 10 microM PMA, 100 microM DiC8, 12.5 units/ml Bc-PLC, 10 microM C6-Cer mixed with EYFP-C1AB probe. Experimental procedure WR19Lcells were spind down and collected. Cells were in total 815 microl(163microl*5 in each)HBSS solution. Add 25microl 3.8mg/ml EYFP-C1AB. 2microl 1mM PMA, 1mM alphaPMA, 100mM DiC8, 8.5microl of 12.5unit/ml Bc-PLC are put in the 1.5 ml tubes in advace. 168microl solution containing EYFP-C1AB is added in each tube and incubate 10 min. 30micro 20% PFA is added and fix for 10 min. After that, spine down cells and remove supernatant. Add 50mM NH4Cl for 5min. Spine down and suck up supernatant. Add 100microl HBSS. 18/Feb/2011 I am gonna follow Malhotra s procedure. I prepared the liposomes as follows. 1. POPC only=60 2. POPC/PS=60/20 3. POPC/GM1=60/20 These liposome includes 2mol% Rhodamine-PE 1. POPC(0.2micromol) avanti 13.7mM 14.6microl 2. PS(0.04microl)avanti 100721 2.3mM 0.45 microl 3. GM1(0.04micromol) sigma G7641 1mM 1.128 microl 4. Phodamin-PE(4nmol) Avanti 0.15 mM 28 microl 1. I diluted these liposomes in 666microl, 888microl, 888microl in each. This means that 300 microM liposomes. 2. I am gonna pick up 100 microl in each. 3. EYFPC1AB domain is 1 mg/ml I am gonna add 1microl into 100 microl. This means that 10 microg/ml. 4. About DiC8, I have 1M DiC8 now (DMSO). I am gonna dilute this solution 200 times(PBS(-)), this means 5mM. Add 1microl into 100 microl. I want to prepare as follow. A. POPC B. POPC+probe C. POPC+probe+DiC8 D. POPC/PS E. POPC/PS+probe F. POPC/PS+probe+DiC8 G. POPC/GM1 H. POPC/GM1+probe I. POPC/GM1+probe+DiC8 5. FRET mesurement.30 min incubation. excitation 488 nm emission 500-650nm 6. Flotation assay (I can only use 4 samples for ultracentrifugation) B. POPC+probe C. POPC+probe+DiC8 H. POPC/GM1+probe I. POPC/GM1+probe+DiC8 7. Normalize with fluorescence of liposomes. 8. Apply sample solution to SDS-Page. 9. Stain with GFP antiboly. 06/Feb/2011 I couldn t make liposomes previously in 03/Feb/2011. However, I leave these lipid films for 1 week in the room condition. This might cause some bad effect on these membranes. So, I gonna make liposome again with same condition with littele modification. Because previous study use fluorescence PE was used 2mol%. I am gonna increase the volume of Rhodamine-PE from 0.5 to 2 mol%. So, 1. POPC only 2. POPC/DAG=60/6 3. POPC/DAG/GM1=60/6/20 4. POPC/DAG/PS=60/6/20 5. POPC/PS=60/20 6. POPC/GM1=60/20 Phodamine-PE is inluded all 2 mol%. 1. POPC(0.2micromol) avanti 13.7mM 14.6microl 2. PODG(0.033micromol) avanti 800815C 3.4 mM 9.8 microl 3. PS(0.04microl)avanti 100721 2.3mM 0.45 microl 4. GM1(0.04micromol) sigma G7641 1mM 1.128 microl 5. Phodamin-PE(4nmol) Avanti 0.15 mM 28 microl So then, I am gonna prepare following. 1. Take 102 microl POPC and 196 microl PE. 2. Mix and take 42.6 microl each in 6 tubes. 3. 9.8microl DAG is added 2,3,4 tubes. 4. 1.1microl GM1 is added 3,6 tubes. 5. 0.5microl PS is added 4,5 tubes. result I try sonication extensivly. But didn t make liposomes containing DAG even though biophysical journal paper shows that they could make liposomes. 03/Feb/2011 Lipid filmes prepared in 20/Jan/2011 was voltexed. But 5,6 didn t make liposomes. 2,3,4 did but rim of glass tube have fluorescence lipids, I am afraid of phase separation of DAG on the glass tube. Only 1 made liposomes well. 20/Jan/2011 I did the experiment of 09/Jan/2011 But this experiment didn t work well. Even positive control didn t work well, which is same result of ctrl. Toshi-sensei said that the distance between fluorescent PE and EYFP-C1AB doesn t become close enough. So I am gonna give up this experiment. I am gonna do following expeiment. I will prepare following liposomes. 1. POPC only 2. POPC/DAG=60/6 3. POPC/DAG/GM1=60/6/20 4. POPC/DAG/PS=60/6/20 5. POPC/PS=60/20 6. POPC/GM1=60/20 Phodamine-PE is inluded all 0.5 mol%. 1. POPC(0.2micromol) avanti 13.7mM 14.6microl 2. PODG(0.033micromol) avanti 800815C 3.4 mM 9.8 microl 3. PS(0.04microl)avanti 100721 2.3mM 0.45 microl 4. GM1(0.04micromol) sigma G7641 1mM 1.128 microl 5. Phodamin-PE(1nmol) Avanti 0.15 mM 7 microl So then, I am gonna prepare following. 1. Take 102 microl POPC and 49 microl PE. 2. Mix and take 21.6 microl each in 6 tubes. 3. 9.8microl DAG is added 2,3,4 tubes. 4. 1.1microl GM1 is added 3,6 tubes. 5. 0.5microl PS is added 4,5 tubes. 09/Jan/2011 I read a paper in which they made liposomes. I will modify a little bit. And I am gonna detect FRET signal. ex 488 nm, em 560 nm. 1.POPC/BODIPYPE(530/560)=60/0.6 2.POPC only (as ctrl) 3.POPC/PS/BODIPYPE(530/560)=60/6/0.6 (positive ctrl) 4.POPC/GM1/BODIPYPE(530/560)=60/6/0.6 5.POPC/GM1=60/6 (as ctrl) Each is divided into two samples. One is for ctrl. The other is for Bc-PLC addition. 1. POPC(0.2micromol) avanti 13.7mM 14.6microl 2. BODIPYPE(530/560)(0.004micromol) Molecular probe 1 mM 2microl 3. PS(0.02microl)avanti 100721 2.3mM 0.245 microl 4. GM1(0.02micromol) sigma G7641 1mM 0.564 microl 03/Jan/2011 I did the condition of 02/Dec. But this lipid mixture did not make liposomes more than previous condition. So Next thinking is that remove cholesterol. That menas that POPC/SM/DAG=60 20 10 and so on. And I did the experiment in which I see the effect of C1AB on cell growth. MDCK and HEK cells. I cultured HEK cells as following condition. cell concentration is 1.62*106 cells/ml. 10microl solution was added to 500microl medium. After that 10microl 3.8mg/ml C1AB was added. Ctrl is only 10microl 200mM imidazole buffer solution. About HEK cells, 3.0*105 cells/ml was prepared. 30microl this solution was added to 500microl DMEM solution. After that, 25microl 3.8mg/ml C1AB solution was added. 1. POPC(0.2micromol) avanti 13.7mM 14.6microl 2. brainSM(0.066micromol) avanti 860062C 31.4mM 2.1 microl 3. chol(0.2micromol) unknown 10.2mM 19.6microl 4. PODG(0.033, 0.066micromol) avanti 800815C 3.4 mM 9.8, 19.6 microl So, I made such liposomes. 1. POPC=60 2. POPC/DAG=60/5 3. POPC/DAG=60/10 4. POPC/SM=60/20 5. POPC/SM/DAG=60/20/10 6. POPC/SM/DAG=60/20/20 02/Dec/2010 It turns out that these lipid mixture I prepared in 30/Dec did not form liposomes. So, I asked Toshi-sensei how to make liposomes well. うえだくん、 コレステロールを増やせばうまくいくかも知れません。 POPC/SM/chol/DAG=60 20 60 10とか。 試してみてもらえますか? 小林 So I made liposome as follows. 1. POPC(0.2micromol) avanti 13.7mM 14.6microl 2. brainSM(0.066micromol) avanti 860062C 31.4mM 2.1 microl 3. chol(0.2micromol) unknown 10.2mM 19.6microl 4. PODG(0.033, 0.066micromol) avanti 800815C 3.4 mM 9.8, 19.6 microl So, I made such liposomes. 1. POPC/SM/chol=60/20/60 2. POPC/SM/chol/DAG=60/20/60/10 3. POPC/SM/chol/DAG=60/20/60/20 30/Dec/2010 I prepared liposomes newly. SM was included because SM is important for distribution of DAG on the membrane. And then, I mimicked the outer leaflet of the plasma membrane. So, I used as follows 1. POPC(0.6micromol) avanti 13.7mM 43.8microl 2. brainSM(0.2micromol) avanti 860062C 31.4mM 6.3 microl 3. chol(0.2micromol) unknown 10.2mM 19.6microl 4. PODG(0.1, 0.2, 0.3,0.4 micromol) avanti 800815C 3.4 mM 29.4, 58.8,88.2,117microl So, I made such liposomes. 1. POPC/SM/chol=60/20/20 2. POPC/SM/chol/DAG=60/20/20/10 3. POPC/SM/chol/DAG=60/20/20/20 4. POPC/SM/chol/DAG=60/20/20/30 5. POPC/SM/chol/DAG=60/20/20/40 28/Dec/2010 EYFP-C1AB 1.77 mg/ml now (M.W=38,000) I took 3 microl this means 1.77 mg*3microl/1000microl=5.31 microg. So 5.31/38000=0.0001397micromol=0.1397nmol. 1 cell have 1 fmol DAG. glass base dish maybe has 10000 cells/1 well. So 1 fmol*10000=10pmol DAG. but outer DAG is less assume 1%. So 100fmol DAG. liposome 19mM DOPC 52.6microl this means 19 mmol/1000000microl*52.6microl=1micromol. 3.2mM DODG 12.5microl this means 3.2mmol/1000000microl*12.5microl=40nmol. These two are mixed, so 4%mol DAG. suspended in 200microl HBSS. 100microl picked up, means 500nmol PC 20 nmol DAG. vesicle contains two side, so 250nmol PC 10nmol DAG I did liposome experiment, but result is mysterious. In the presence of DAG in PC liposome, fluorescence light is brighter than that in the presence of only PC liposome. I think about this. Basically, I don t know what happen, but there is some hint. 1. cells were ripped off when both liposome was added, this means that PC works as detergent because I add a lot of liposome, 500 nmPC. So I need to reduce the amount of PC liposome. 2. In this experiment, I add 4mol% DAG in PC liposome. But other experiment, for example SM-lysenin, give 20mol% DM into PC liposome. So I can add more DAG, maybe at least 10%, possbly 20%-40%. As you know, If DAG included more, this membrane doesn t make a liposome. So I need to take care about it. Next time, I am goning to prepare the liposome, リポソームですが、1mMを1ml位用意して、 蛋白の濃度にもよると思いますが、 こちらの実験では、GFP-Lysの場合、 いつも、50倍希釈で染色しているので、 (1マイクロ/50マイクロPBS) 1マイクロに、50マイクロのリポソームを混ぜて、 37度で30分してから、それで染色していました。 1mMはかなり濃い濃度なので、タンパク質はすべて 結合すると考えています。 さっき探していた論文ですが、(準備中) For the preabsorption experiment, Dronpa-θ-D4 or Dronpa-NT-Lys was incubated with MLVs at 37°C for 30 min prior to apply them to HeLa cells. としか書いてありませんでした。 これで良いですか? 牧野 Reply Forward Reply |Ueda Yoshibumi to Asami show details Dec 17 (7 days ago) リポソームを作るとき、はじめに、クロロホルムに溶けた脂質を遠沈管の中に加えて、 窒素で飛ばしてからPBSなどを加えてベシクルにするんだよね? だいたい、クロロホルムに溶けた1mM脂質(例えばDOPC)を何μl位遠沈管に入れて乾かしていますか? それは脂質のストック濃度によると思います。 10mMの脂質ストックなら100ulだし、20mMなら50です。 それで乾いてから1mlのPBSをいれて、 最終濃度1mMリポソームが 1mlできるということです。 Show quoted text - Reply Forward Reply |Ueda Yoshibumi to Asami show details Dec 17 (7 days ago) 1mMだったら脂質を1ml入れているっていうことだね。 かなり多い量を加えているんだね! なるほど、わかりました。 Reply |Asami Makino to Ueda show details Dec 17 (7 days ago) でも、結局リポソームは50マイクロLとか使わないから、 1mlも作る必要ないと思う。 200マイクロでも作れば充分なんじゃないかな。 ただ、こちらにある脂質のストックはほとんど10mM以上あるので たくさんある脂質は少量はかり取るのが大変だから、 たくさん使ってもいいと思います。 Original Message ----- From Ueda Yoshibumi To Asami Makino Show quoted text - Sent Friday, December 17, 2010 3 31 PM Subject Re MLV なるほど、了解しました! So, I prepared vesicles. I use 10microg/ml YFP-C1AB to stain the cells. YFP-C1AB M.W. 38kDa=38,000 This means 0.000263micromol/ml I add 100microl, So total amount is 0.02nmol. I want to include DAG into the vescle And then, 1 cells has 1 fmol DAG. glass based dishes is maybe 10 pmol DAG.
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TRIP MACHINE PhoeniX(踊) 曲名 アーティスト フォルダ 難易度 BPM NOTES/FA(SA) その他 TRIP MACHINE PhoeniX DE-SIRE改 SN2 踊12 (80-)160 283 / 1 STREAM VOLTAGE AIR FREEZE CHAOS 60 52 110 4 39 楽譜面(8) / 踊譜面(12) / 激譜面(16) / 鬼譜面(16) 属性 左右振り、渡り、同時踏み、ソフラン(減速)、ラス殺し 譜面 http //eba502.web.fc2.com/fumen/ddr/sn2/trip_pho_8t.html 譜面動画 https //www.youtube.com/watch?v=yrcVVOxJt20 (x3.25, NOTE ※CLAP音合成) プレイ動画 https //www.youtube.com/watch?v=KDoN_jhAuGI (x2.0, NOTE) https //www.youtube.com/watch?v=8qQmDmKxo-c (x2.75, NOTE) 解説 BPM推移:160-80-160 連続同時踏みが非常に難易度が高くスコアが出にくい。特に、八分のリズムでの連続同時踏みは遠い配置でPARANOiA HADES並。 -- 名無しさん (2011-03-07 22 25 08) 8分同時2連だけでなく8分踏みの合間に頻繁に同時が来るので慌ててバランスを崩さないように。ジャンプで体力温存できないとラストの滝も12にしては相当厳しいので一気に落とされる可能性が高い。 -- 名無しさん (2012-02-09 03 09 34) 名前 コメント コメント(私的なことや感想はこちら) 結構渡りスキルが必要。 -- 名無しさん (2009-12-10 00 09 37) 四分よりも八分の矢印の方が多いんじゃないか?これ。 -- 名無しさん (2010-03-14 01 16 22) 何だかんだでハデス楽に匹敵する難しさだと思う。アンリアル踊よりもBPMが遅い分同時が遠いし -- 名無しさん (2012-02-10 01 56 10) アンリアルとトリフェニとパラリスと伝説(いずれも踊)は足12の危険範囲内だと感じた。下手な足13よりムズいだろこれ。 -- 名無しさん (2013-11-20 20 05 43) ↑伝説の踊は弱いぞ -- 名無しさん (2014-12-14 16 25 03) ユニバーヒルズの対象になってたから足12くらいなら死にはしないだろうと思ってプレイしたら爆死したwやっぱり地雷なのね -- 名無しさん (2014-12-22 02 34 56) 道中の8分の〆が遠めの同時だったり、8分同時渡りが2連とは言え実質ハデス(楽)よりも速かったり、とどめにラス殺しの捻り折り返し8分滝はAA(激)よりも速かったりとノート数以外は足12かこれ?と思えるほどのテクニカルてんこ盛り譜面。最低でも序盤と終盤に来る裏拍同時渡り地帯が体力&ゲージ回復と思える位の地力は欲しい。13クラス。 -- 名無しさん (2020-10-28 06 47 19) 準備運動もそぞろに選んだらなす統べなく死んだ。ラス殺しどころか道中も滝に挟まる同時が厳しすぎて足が動くのを拒否した。密度以外の全てが詐称と思えるくらい強い。 -- 名無しさん (2022-09-13 13 12 55) 名前 コメント PV http //www.nicovideo.jp/watch/sm1820925 http //www.nicovideo.jp/watch/sm1820925
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このワードは、検索してはいけなかった言葉に登録されています。 登録タグ PC被害 偽警告 危険度5 検索してはいけなかった言葉 詐欺 非常識 【警告】詐欺サイトの為、検索する際は注意 yowwinnerprize.comというサイトがTOPにヒットする。 そのサイトはSafariで入ると詐欺サイトだという赤い警告画面が映るが、Chromeでは映らず、そのままアクセスしてしまう。 警告を無視してサイトに入ると、ビジターアンケートや偽のウイルス警告に飛ばされる。 gredでチェックするとフィッシング詐欺だという判定が出たため、絶対に個人情報を入力したりしないこと。 分類:PC被害、非常識 危険度:5 コメント サイトの手口は「ドメイン売り出し中に見せかけてクレジットカード番号を入力させる」、「ビジターアンケート(偽ウイルス警告)にリダイレクトする」の2通りあるっぽい -- rule 17 (2020-04-18 11 06 56) これは結構目にしたことがあります。同人サイトとかによく出るらしいので皆さん注意 -- 唐紅 (2020-04-18 14 13 19) すげぇ危険なワードが追加されたな -- なナス (2020-04-18 15 36 52) douse -- 名無しさん (2020-04-19 13 58 35) どうせいつもの偽警告やろwwwと高をくくっていたら全画面偽警告であと変な文字化けしたようなファイルをインストールしてこようとしたので注意です -- 名無しさん (2020-04-19 13 59 52) ちなみに、インストールされそうになるファイルのファイル名に使われている文字はZalgo文字といって、文字そのもののデータが大きいためクラッシュさせやすくなるから使われているのだと推測できる。FireFoxでこのサイトを開いた場合には、ブラクラが発動するため注意。Xで閉じようとすると更にウィンドウが開くyouareanidiot方式である。 -- FireFoxでアクセスは控えて (2020-05-21 16 30 08) なるほどZalgo文字。そのようなものがあるのですね。ありがとうございます。 -- 名無しさん (2020-05-24 23 40 01) よくアダルトサイトの高難易度の広告の×押せなかったら必ずこれに飛ぶからくそイライラする。これを交わすプロいずれか現れそう。 -- お前らの父 (2020-07-26 20 56 49) 何であんなiPhone 11 Pro推すの?? -- tt (2020-09-26 22 36 24) ビジターアンケートがこのwikiだと悪質すぎて良いサイトもこいつのせいで台無しになってるんじゃないかっていうレベル -- 海草ライト君 (2020-09-27 01 51 27) マカフィーの公式サイトにリダイレクトしたww -- 名無しさん (2021-04-28 00 54 03) 特別レポート:中居正広の最新の投資は、専門家に畏怖の念を抱き、大手銀行を恐怖に陥れた←こんなのが出たwww しかもマカフィー検知しなかったwww -- ムーシュ (2021-06-18 20 17 22) 何回も見たことあるわ・・ -- ヌガー (2021-08-22 12 01 09) ZALGOっていう文字化けみたいなみたいなファイルウイルス検査にかけたけど無害だったからお土産として持ってるww -- Believe115 (2022-01-19 09 14 02) 学校のパソコンでやったらフィッシングサイトがブロックされました -- 名無しさん (2022-02-11 17 33 36) ヒットしません -- 名無しさん (2022-02-16 12 48 52) ロボットでない場合は、[許可]をクリックします -- ▢I'm not a robot (2022-09-12 21 22 16) 学校のpcではcisco umblallaでブロックされた、、() pornhub見れるくせによぉ -- SuperNanasiManEX (2023-02-20 12 33 54) 似たようなサイトないんか -- 名無しさん (2023-08-16 05 19 42) 名前 コメント
https://w.atwiki.jp/zitaku_server/pages/9.html
2013/5/30 Tripwireとは? Tripwireはホスト型IDSである。IDS(Intrusion Detection System)とは侵入を検知するシステムである。Tripwireはホストのファイルの改竄を監視してくれる。 TripwireをEPELからインストール # yum --enablerepo=epel -y install tripwire Tripwireの設定 # tripwire-setup-keyfiles Enter the site keyfile passphrase # 任意のパスフレーズ Verify the site keyfile passphrase # 再入力 Enter the local keyfile passphrase # 任意のパスフレーズを設定 Verify the local keyfile passphrase # 再入力 Please enter your site passphrase # パスフレーズで応答 Please enter your site passphrase # パスフレーズで応答 # cd /etc/tripwire # vi twcfg.txt # 9行目の値をtrueにする。 LOOSEDIRECTORYCHECKING = true # 12行目:報告レベル4にする。 REPORTLEVEL = 4 暗号署名設定ファイル作成 # cd /etc/tripwire # twadmin -m F -c tw.cfg -S site.key twcfg.txt Please enter your site passphrase #パスフレーズ入力 Wrote configuration file /etc/tripwire/tw.cfg # ポリシーファイル最適化スクリプト作成 # cd /etc/tripwire # vi twpolmake.pl #!/usr/bin/perl # Tripwire Policy File customize tool # ---------------------------------------------------------------- # Copyright (C) 2003 Hiroaki Izumi # This program is free software; you can redistribute it and/or # modify it under the terms of the GNU General Public License # as published by the Free Software Foundation; either version 2 # of the License, or (at your option) any later version. # This program is distributed in the hope that it will be useful, # but WITHOUT ANY WARRANTY; without even the implied warranty of # MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the # GNU General Public License for more details. # You should have received a copy of the GNU General Public License # along with this program; if not, write to the Free Software # Foundation, Inc., 59 Temple Place - Suite 330, Boston, MA 02111-1307, USA. # ---------------------------------------------------------------- # Usage # perl twpolmake.pl {Pol file} # ---------------------------------------------------------------- # $POLFILE=$ARGV[0]; open(POL,"$POLFILE") or die "open error $POLFILE" ; my($myhost,$thost) ; my($sharp,$tpath,$cond) ; my($INRULE) = 0 ; while ( POL ) { chomp; if (($thost) = /^HOSTNAME\s*=\s*(.*)\s*;/) { $myhost = `hostname` ; chomp($myhost) ; if ($thost ne $myhost) { $_="HOSTNAME=\"$myhost\";" ; } } elsif ( /^{/ ) { $INRULE=1 ; } elsif ( /^}/ ) { $INRULE=0 ; } elsif ($INRULE == 1 and ($sharp,$tpath,$cond) = /^(\s*\#?\s*)(\/\S+)\b(\s+- \s+.+)$/) { $ret = ($sharp =~ s/\#//g) ; if ($tpath eq /sbin/e2fsadm ) { $cond =~ s/;\s+(tune2fs.*)$/; \#$1/ ; } if (! -s $tpath) { $_ = "$sharp#$tpath$cond" if ($ret == 0) ; } else { $_ = "$sharp$tpath$cond" ; } } print "$_\n" ; } close(POL) ; # perl twpolmake.pl twpol.txt twpol.txt.new # twadmin -m P -c tw.cfg -p tw.pol -S site.key twpol.txt.new Please enter your site passphrase # パスフレーズ入力 Wrote policy file /etc/tripwire/tw.pol # tripwire -m i -s -c tw.cfg Please enter your local passphrase # パスフレーズ入力 実行してみる # cd /etc/tripwire # tripwire -m c -s -c tw.cfg ※時間がかかります