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https://w.atwiki.jp/duke15/pages/15.html
Apache projects now use Subversion for version control. It looks there is no CVS interface, at least from the web pages like http //jakarta.apache.org/site/cvsindex.html. To build Tomcat from source, we need many related projects in binary and source. To obtain a minor Apache/Jakarta project, it looks we need to create a Subversion. At least, the developer of Subversion considers it is a successor of CVS. It is a native program, thus we need to obtain source and compile it. Subversion itself depends on some apache projects, like APR... http //apr.apache.org/ http //apache.acnova.com/apr/apr-1.2.2.tar.gz APR compilation, installation was not so difficult. download http //subversion.tigris.org/ If APR is available, configure and make looks to work, but it shows message saying it s better to configure APR with Berkley DB. http //www.sleepycat.com/index.html OK. Downloaded latest release db-4.4.20. $ cd build_unix $ ../dist/configure $ make # make install Tried to rebuild apr-1.2.2. $ ./configure $ make # make install Then, try to make subversion again. However, I got the same error. You don t seem to have Berkeley DB version 4.0.14 or newer installed and linked to APR-UTIL. We have created Makefiles which will build without the Berkeley DB back-end; your repositories will use FSFS as the default back-end. You can find the latest version of Berkeley DB here http //www.sleepycat.com/download/index.shtml Oh, it s talking about apr-util!! Configureing apr-util-1.2.2 $ ./configure checking build system type... hppa2.0w-hp-hpux11.11 checking host system type... hppa2.0w-hp-hpux11.11 checking target system type... hppa2.0w-hp-hpux11.11 checking for a BSD-compatible install... /opt/imake/bin/install -c checking for working mkdir -p... yes APR-util Version 1.2.2 checking for chosen layout... apr-util Applying apr-util hints file rules for hppa2.0w-hp-hpux11.11 setting apu_crypt_threadsafe to "1" checking for APR... no configure error APR could not be located. Please use the --with-apr option. OK. Need to use --with-apr option $ ./configure --with-apr checking for APR... configure error --with-apr requires a directory or file to be provided The option needs a parameter. $ ./configure --with-apr=/usr/local/apr $ make chmod 644 /usr/local/apr/lib/libaprutil-1.a Libraries have been installed in /usr/local/apr/lib If you ever happen to want to link against installed libraries in a given directory, LIBDIR, you must either use libtool, and specify the full pathname of the library, or use the `-LLIBDIR flag during linking and do at least one of the following - add LIBDIR to the `SHLIB_PATH environment variable during execution - use the `-Wl,+b -Wl,LIBDIR linker flag See any operating system documentation about shared libraries for more information, such as the ld(1) and ld.so(8) manual pages. /opt/imake/bin/install -c -m 644 aprutil.exp /usr/local/apr/lib /opt/imake/bin/install -c -m 755 apu-config.out /usr/local/apr/bin/apu-1-config Oh, I missed a configure option... --with-berkeley-db=PATH Find the Berkeley DB header and library in \`PATH/include and \`PATH/lib . If PATH is of the form \`HEADER LIB , then search for header files in HEADER, and the library in LIB. If you omit the \`=PATH part completely, the configure script will search for Berkeley DB in a number of standard places. Trying configure again. $ ./configure --with-apr=/usr/local/apr --with-berkeley-db=/usr/local/BerkeleyDB.4.4 $ make Finally... # make install ... chmod 555 /usr/local/apr/lib/libaprutil-1.sl.2.2 /opt/imake/bin/install -c -m 755 .libs/libaprutil-1.lai /usr/local/apr/lib/libaprutil-1.la /opt/imake/bin/install -c -m 755 .libs/libaprutil-1.a /usr/local/apr/lib/libaprutil-1.a ranlib /usr/local/apr/lib/libaprutil-1.a chmod 644 /usr/local/apr/lib/libaprutil-1.a Libraries have been installed in /usr/local/apr/lib If you ever happen to want to link against installed libraries in a given directory, LIBDIR, you must either use libtool, and specify the full pathname of the library, or use the `-LLIBDIR flag during linking and do at least one of the following - add LIBDIR to the `SHLIB_PATH environment variable during execution - use the `-Wl,+b -Wl,LIBDIR linker flag See any operating system documentation about shared libraries for more information, such as the ld(1) and ld.so(8) manual pages. /opt/imake/bin/install -c -m 644 aprutil.exp /usr/local/apr/lib /opt/imake/bin/install -c -m 755 apu-config.out /usr/local/apr/bin/apu-1-config
https://w.atwiki.jp/93727/pages/51.html
アルバム曲目リストYear of the Gentleman Because of You In My Own Words Do You Artist Ne-Yo Album Because Of You Year 2007 Maybe This Decision Was A Mistake You Probably Dont Care What I Have To Say But It's Been Heavy On My Mind For Months Now Guess I'm Tryna Clear Some Mental Space I Would Love To Talk To You In Person But I Understand Why That Can't Be I'll Leave You Alone For Good I Promise If You Answer This One Question For Me I Just Wonder...Do You Ever Think Of Me...Anymore, Do You First Off Let Me Say Congratulations Heard That You Just Had A Baby Girl If She Looks Anything Like Her Mother She's The Prettiest Thing In The World Swear That I'm Not Tryna Start No Trouble Tell Your Fiance He Can Relax I'll Leave You Alone For Good I Promise There's A Question I Just Got To Ask I Just Wonder...Do You Ever Think Of Me...Anymore, Do You I Know What We Had Is Dead And Gone Too Many Times I Made You Cry And I Dont Mean To Interrupt Your Life I Just Wonder Do I Ever Cross Your Mind I Just Wonder...Do You Ever Think Of Me...Anymore, Do You I Just Wonder...Do You Ever Think Of Me...Anymore, Do You I Just Wonder...Do You Ever Think Of Me...Anymore, Do You ) アルバム Because of You 曲目リスト Year of the Gentleman 1. Closer 2. Nobody 3. Single 4. Mad 5. Miss Independent 6. Why Does She Stay 7. Fade Into The Background 8. So You Can Cry 9. Part Of The List 10. Back To What You Know 11. Lie To Me 12. Stop This World Because of You 1. Because Of You 2. Crazy (feat. JAY-Z) 3. Can We Chill 4. Do You 5. Addicted 6. Leaving Tonight (feat. Jennifer Hudson) 7. Ain t Thinking About You 8. Sex With My Ex 9.Angel 10.Make It Work 11.Say It 12.Go On Girl 13.Spotlight 14.That s What It Does In My Own Words 1.Stay 2.Let Me Get This Right 3.So Sick 4.When You re Mad 5.It Just Ain t Right 6.Mirror 7.Sign Me Up 8.I Ain t Gotta Tell You 9.Get Down Like That 10.Sexy Love 11.Let Go 12.Time 上へ
https://w.atwiki.jp/hmiku/pages/25937.html
【登録タグ NIYMORIY Y 巡音ルカ 曲】 作詞：NIYMORIY 作曲：NIYMORIY 編曲：NIYMORIY 唄：巡音ルカ 曲紹介 歌詞 （動画内歌詞より転載） (※) WHAT ABOUT BREAKING THE BRAIN? MY SOUND OF VIRUS. I REPLACED 90 PERCENT OF MY BRAIN WITH VIRUS. I'M HACKING TO YOUR GENOME. MY SOUND OF VIRUS. OPEN UP A DOOR WAY TO YOUR DEEP DIMENSION. (※) (※繰り返し) I want to go to your deepest dimension. Ok, here we go. TRANSFORM THE VIRUS CELLS REPLACE THE CELLS THEY REPLICATE I'LL REPLACE YOUR BRAIN (※繰り返し) WHAT ABOUT BREAKING THE BRAIN? MY SOUND OF VIRUS. I REPLACED 90 PERCENT OF MY BRAIN WITH VIRUS. I'M HACKING TO YOUR GENOME. MY SOUND OF VIRUS. OPEN UP A DOOR WAY TO I FOUND 10% OF YOU. (※繰り返し) コメント かっこいい曲だよな -- 名無しさん (2013-11-09 16 09 08) ボカロ嫌いを黙らせれそう笑 -- 名無しさん (2013-11-12 21 05 13) 久しぶりに聞いた。当時マイリスしてた自分にGJと言ってやりたい。 -- 名無しさん (2015-08-02 23 03 15) 名前 コメント
https://w.atwiki.jp/mtgflavortext/pages/5044.html
「眼は可能なことしか見ようとせぬ。鍛えた精神は不可能をも探求できる。」 ――知識の神、ケフネト "Eyes see only what is possible. A trained mind can explore the impossible." ――Kefnet, god of knowledge アモンケット 【M TG Wiki】 名前
https://w.atwiki.jp/letsgotoharvard/pages/14.html
Transfer Program The Committee on Transfer Admissions welcomes inquiries from all qualified students interested in transferring to Harvard College. Over the years we have found that transfer students contribute a great deal to college life here as well as gain much from their experience. We hope the information that follows will enable you to give careful consideration to Harvard as you make plans to continue your education. This year the Committee will admit a small number of transfer students who present a clearly defined academic need for transfer, supported by both a proven record of achievement at the college level and strong faculty recommendations. Candidates for transfer admission will be considered for Fall 2011 enrollment. Our admissions procedures are designed to give you maximum freedom and flexibility to present yourself in your own words. We hope you will respond in whatever ways you feel will best demonstrate your interests and accomplishments. Here are some recommendations we hope you will consider as you complete your application. Keep copies of all materials submitted and ask your teachers to do the same. Materials can be lost in the mail. Supplementary materials or portfolios may be submitted, but you should do so only if you have an unusual talent. Such materials are neither required nor expected as the required components of the application provide ample basis on which to make our decisions. Because we cannot return materials, applicants should send only duplicates. The answers to many questions about admissions requirements and deadlines are included in this website. Each admission decision is made without any regard for a candidate s financial need — a policy we call "need-blind admission." Indeed the Admissions Committee may respond favorably to evidence that a candidate has overcome significant obstacles, financial or otherwise. Once an applicant is admitted, we create an individual financial aid package that will enable that student s family to meet the cost of attendance. Providing financial access to Harvard for every admitted student is one of our highest priorities. We hope you will take every opportunity to explore whether Harvard might be a good match for your academic, extracurricular and personal interests. Please let us know if we can be of assistance to you during the admissions process. Best wishes for a happy and productive year.
https://w.atwiki.jp/h_session/pages/1009.html
■ゲームシステム：迷宮キングダム ■ＧＭ：やりたい人。主には俺。 ■開催頻度：ＧＭがやりたい気分になってＰＬが集まったら。 あるいはＰＬがやりたい気分になってＧＭが承諾したら。つまるところ不定期。 ■突発セッションの有無：有 ■制限人数：上限はなし。多くなったら別の国を作る。 ■新規参加：可 ■キャンペーン方針：国家運営したいのでだらだらと。 ■エロ方針：セルフサービス。 ■禁止事項：マナー違反全般。 ■他キャンペーンからの持込：不可 ■他キャンペーンへの持込：不可 ■備考：サプリメントとかバリバリ使いたいＧＧＭなので持ってると嬉しいにゃー、とか。 あと人間以外のランドメイカーは初期では認めていないのであしからず。 ■チャンネル：#Make_the_Kingdom! および #Make_the_Kingdom!裏 ■国家データ 【国名】 スキゾパラレル子供帝国 【国教】 【ランドメイカー】 “いい大人が泣いて許しを乞う”ロータスエンブレイス女帝陛下（ラヂヲヘッド） “あとは野となれ山となれ”のミッドナイト （GRIFIS） 『涙なしには語れない』カテーテル（神谷涼） 『ロリと貧乳と眼鏡っ娘の守護者』マシリト（ゆき） “湯上りは親でも惚れる”ノワール（no.marcy） “万死に一生を得る”パープル（みはいる） “栗の花薫る”シュペル＝マリオン（ドルフ☆レーゲン） 【逸材】 名前 ジョブ 効果 「クスコの娘」ダイス 衛視 (常駐)「治安レベル」が１上がる。 【国力】（総人口／５０＋ランドメイカー・逸材・施設の修正） 生活：３ 文化：３ 治安：２ 軍事：２ 【人口】 ＬＭ ：７ 逸材 ：１ 民 ：４２ 総人口 ：５０ 【予算】 【ＭＡＰ】 既知の土地 【コメント投稿】 名前 コメント
https://w.atwiki.jp/ohden/pages/187.html
ライブラリの指定 『-lhoge』とすると『libhoge.a』を指定したことになる。 更新日： 2011年02月08日 (火) 20時13分36秒 http //www.unixuser.org/~euske/doc/makefile/ -- (s1n) 2010-03-09 19 44 28 http //www.c.csce.kyushu-u.ac.jp/~seiichirou/wiki/index.php?Makefile%A4%CE%BD%F1%A4%AD%CA%FD -- (s1n) 2010-03-09 19 45 42 make -pn 2 /dev/null make内部で定義済の生成ルールが見れる -- (s1n) 2010-03-26 09 52 21 名前 コメント すべてのコメントを見る
https://w.atwiki.jp/decision2/pages/8.html
動画(youtube) @wikiのwikiモードでは #video(動画のURL) と入力することで、動画を貼り付けることが出来ます。 詳しくはこちらをご覧ください。 ＝＞http //atwiki.jp/guide/17_209_ja.html また動画のURLはYoutubeのURLをご利用ください。 ＝＞http //www.youtube.com/ たとえば、#video(http //youtube.com/watch?v=kTV1CcS53JQ)と入力すると以下のように表示されます。
https://w.atwiki.jp/carstereo/pages/138.html
Hey, Gimme some of that there guitar stuff.. (gimme some more, Gimme some more..) I remembered to hold her close, I remembered to stick around. I remember when we made love, I remembered her favorite sounds. I remembered her favorite jeans, I wear them all the time, I remembered to make good love, but I forgot to make her mine. I remember when she was hurt, I remembered to soohe her soul. I remember when she would cry, I would never, ever let go. I remember the special touch, I remember the favorite lies, I remembered to make good love, but I forgot to make her mine. OH! Too late she s gone, gone, gone..... My baby s gone.. ..She hurt me so.. We were the chosen ones, I let it slip around. I was the last to know when the rain came down. She had a way with me, but I never gave her the sign. I remember everything, I forgot to make her mine! Too late she s gone, gone, gone..... She s gone, oh. My baby took me to school! ..The rain came down.. See, I remembered EVERYTHING. I remembered to make good love, but I... ..Ahh you know what I forgot. Too late she s gone, gone, gone.....
https://w.atwiki.jp/happyhappyhappyhappy/pages/39.html
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.