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2017/08/25 に公開
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うきはかいねっと 農福連携「八媛かぼちゃ」第2弾 植え込みから70日目の紹介 2017/08/25
2017/08/25 に公開
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Greg,In another one of my posts entitled "", I specifically described this phenomenon. Although there hasn't been a ruling on this defense yet, I agree. An artificially-created emergency should not be a basis for issuing a move over ticket. If anyone has such a situation, please contact me and I'd be happy to pursue this theory pro bono.Matthew Weiss
Sà interesantÃsimo su artÃculo.Por cierto que me dio por pensar a raiz de su ejemplo, que los españoles tenemos especialidad en ponerle palos a las cosas: la fregona, el chupachupsm los franceses retratados en los horrores de la guerra de Goya… Nada desvarios mios.En cualquier caso uno de los supuestos que están apuntando como causa de cierre es "enlazar sin autorización"… alucinante! Yo me acuso |
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Leuke fotos en volgens mij hebben jullie heerlijk genoten. Hier oook bijna vakantie voorbij en gaat Sophie maandag weer naar de peuterklas.Liefs,Rozmeen
Exactly. She's not using cloth diapers, y'all, hence her passive-aggresive defensive claim to "privacy". After EVERYTHING else she's disclosed about that kid– including roughly a thousand pictures– it's clear she doesn't give a shit about his privacy. She just doesn't want you to ask about cloth diapering, and, moreover, she wants you to feel a little bad for asking.
Delightful interview. Thanks Brunonia! Method-characterization is definitely the way it works for me,too. It's great as long as I don't go shopping as character X then try to go out in public somewhere!Like? 0
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oh, WOW. looks like we have both had delectable double chocolate on the brain. if only I could send you part of my cheesecake in exchange for a couple of these… YUM !
Blake: I've done lots of hands-on gardening workshops in other cities and will continue to do so if the opportunities present themselves and the match is right. Too tricky to organize a venue myself though since I am coming from Canada and can't bring anything with plants, soil, seeds over the border. So in those cases I must work with a garden store, etc that can offer up the needed supplies. P.S. I am ALWAYS open to San Francisco — love that city!
c'est scandaleux ! vous voulez tuer la creation artistique en utilisant illégalement l'Internet à des fins culturelles ! Je suis sur que votre père faisait partie de ces vauriens qui copiaient illégalement des cassettes audio ! Peut être même, j'en tremble, que votre grand père écoutait la radio !!!Je suis sûr que quelque part, dans votre famille, vous cachez un de ces énergumènes qui regardent cet objet de malheur, la télévision, au lieu d'aller au cinéma.Vous tuez la création !
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– Dear H family ~ So, so very glad we were able to squeeze you in! Your little baby boy was a delight and honor to work with! He looks GREAT in hats…I see many more in his future! ♥ ♥
If he really wants to get good at it, he should know that breath control is vital and it's very important to tongue the right holes. However, I'm sure that can wait until you find out if he's really serious about the thing.
Currently reading…"The Artist's Way" by Julia Cameron"The Paris Apartment" (which I heard on your podcast and scored for a $1 at Strands NYC!)Summer reading…Anything & Everything I can get my hands on, my list is waaaay to long to type 😉
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I am looking for RICS accredited conversion courses with the aim of becoming a surveyor. Looking online I have found a PG course at Salford and one at Northumbria. I want to study online and preferably a course with a January start date. Does anyone know of any others? And which (if any) are the best?Thanks so much – your help will mean so much to me.
Every Fall, I stock up on pumpkin seeds in our lovely region of Quebec where seeds are a plenty. I really avoid the ones they usually sell in our stores from China (sorry) but they just aren't quite right.Those cookies in that captures setting look scrumptious.Have a great week Angie.Ciao for now,Claudia
Så stort og fint dere får det:) Dette tror jeg dere kommertil å blir glade for.. Også vaskerom, det er det eneste jeg savner- heldiggris:)
We were without electric for a week. No laundry got done and we lost a refrigerator full of food. You never realize how dependent you are on things until they are no more. Definitely makes you rethink priorities and come up with creative ways to get things done.
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'System'? Which system.specifically? Must we cease 'mining' asphalt lest paved roads help robbers escape too quickly? The same 'system' that mined and processed your frightfully unethical bitcoin usage also brought me my three new pair of paid-for-with-bitcoin Alpaca socks on Monday.
Thanks to Bufo for a really enjoyable puzzle, with a lovely mixture of clues ranging from the straightforward to the tricky. My favourite clues were 12a, 2d, 22d and (best of all) 26a.
I love her! I used her In and Out the Garden as my journal and look back at all the events of each year… I think I have four from the late '80's. I have never found a journal I enjoyed since hers! We are such 'twinkies'.
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I know, I'm black and liberal so I'm entitled and stupid, etc. Meanwhile you guys can't even admit to yourselves why you lost the election lol.
Ted … Please .. I do not want to sound harsh, but are you losing your mind? P is not part of our staff and to be honest, having the same people constantly on a blog does more to decrease readership than increase it …
Je résume pour ceux qui ne feront pas le détour par wikipedia : étonnamment, les poissons cartilagineux (= chondrichtyens) sont des poissons.Donc dire que les requins sont des poissons, c'est un peu comme dire que la laitue est un végétal au lieu de dire que c'est une salade… Ca n'est peut être pas faux??Et pour le reste : je confirme, cette région est une très agréable destination à cette période.
Funny, usually Al-Cheezra does not allow comments.A December 2010 poll conducted by the prestigious Emnid Institute, and reported in the German magazine "Welt der Wunder,†revealed that 89.5% of German respondents do not believe the official story of 9/11
Don't worry I understood that garlic was a metaphor. my whole point has been to try to get you to see that music is subjective. your truth is that this music is too cluttered. mine is a different truth. there's plenty of colour don't worry. peace.
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Two years ago, I decided to live green, and one thing that I enjoyed the most is the donating part. The happiness that I feel every time I see someone with a very grateful smile is just priceless.
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Mary multumesc :)Mircea ai dreptate, si e asa frumos cand le descoperi tu!Aa multumesc! sa stii ca nu a fost prima vacanta cu copilul, prima data am mers in Paris cu el cand avea 4 luni, apoi la aproape 1 an l-am dus in Maroc, vacanta aceasta a fost de fapt a-treia la 1 an si 8 luni 🙂
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Archie est désavantagé par la description omniprésente de Wolfe, mais il se rattrape très bien… en se mettant au boulot.Un autre Stout m'attend, « Trois femmes et un homme ». Il est toujours temps de découvrir !
Trees and plants are wonderful, "they grow in unison with all that is."As to Durand's theory of "concealment of pigments" as some kind of highest virtue in painting: There may be a truth in it. But I find that statement too one sided.Matisse for instance, would have been rendered impossible by it.
Ooooohhh….I'm loving that moleskin notebook! I find myself always wanting notebooks, but then never writing in them. Thanks for the tip on the Charles Schulz Museum. I had no idea there was such a place and I've added it to my list of things to do while we're traveling. Maybe we'll get a sneak peek on your blog.
I love your weight loss plan, I'm on a diet now and i workout 3 to 4 times a week. And the reason why I wanted to your diet plan because i needed a menu food diet plan, and this really helps me alot .. thank you for posting this, even though your bird is annoying lol and your funny how you say stop…thanks again..your great….
Chinese food in North America=fake. There are no such things as big crabs in Chinese food in China. You go for the meat, in China, you go for the paste.Chinese food in China=realReferences :
Pedro,Uma vez me falaram que quem tem pele oleosa tem por obrigação usar sabonete com ph ácido, porque se usar ph alcalino as coisas pioram, até hoje isso me encuca porque tem alguns sabonetes que eu curto que são ph neutro por serem direcionados a peles sensiveis. O que você acha dessa informação?
Pedro Vieira disse:«Ricardo,Com a resolução que disponho apostava que o nariz é "Regular", assim como a boca.Pedro Vieira, Braga»Ã‰ isso mesmo, Pedro, obrigado. Está completa a ficha do Don Cosme 🙂
And it's a perfect fit! A lot of the stuff I have stockpiled needs to be taken in to fit me. I keep telling myself I should sell it on etsy or alter it. But all I do is stare at the pile and think…"one day, one day".
– I actually made the business cards..I need to make some more, will post about the process later this week. Nate is working nights so I'll have some time…Let me know when you head out to the station. I would like to walk around it as well!Thanks for visiting. Did the trackback work?
ok, io ho due cani, e una si chiama Ettore e l'altra Ermione…Ma Wonder il ptoblema non è tanto dare il nome ai cani come le persone, il problema è un altro: pernsare che i cani siano come noi persone!!! Tipo non hai mai fatto un giro per i negozi per animali??? Quante c….te ci sono??? Per me, per quando gli possa voler bene, restano pur sempre dei cani!Ora csceglierò la cassa al supermecato a sexconda di cos'ha nel carello la persona che sta davanti a me 😀
Excellent advice! Thank you! Your samples for being mysterious (I mean your voice management and nonverbal behavior) are quite vivid and subtle at the same time.You are a talented teacher (if I can tell that)! It's a pity, that the majority of viewers are …
Caro Tavares Moreira, mais um alerta, mais uma queda em saco roto, mormente neste momento especÃfico. Permita-me a ousadia de pedir-lhe um escrito não já sobre os riscos mas sobre o cenário de Portugal daqui a dez ou vinte anos, altura em que todos os efeitos das loucuras do presente somadas à s do passado irão fazer-se sentir. Como imagina o Portugal dessa altura, abstraindo-nos neste exercÃcio teórico da possibilidade eventual da implosão da UE ou uma guerra em larga escala?
That's quite alright Richard I get what you were saying before ..no offense taken:)Have a set of Ambrosio deep sect' wheels to put on and the rest…..well I figure I have till the roads are clean to decide! Are you up for some spring rides with us?
Coucou Brittounette ! Que ton message me fait plaisir !! Bon courage pour la reprise ! Mes feuilletés vont te remettre d'aplomb !! ^^ ;p Je t'embrasse bien fort !! A très vite, xx
Sorry TimS, but the idea that we should somehow be understanding of and okay with innocent people being thrown in jail, having their lives ruined, and being killed because hey, 400 years ago someone else did it, is frankly repugnant. This is about the US and saying that in this country you are free to believe as you wish and to speak your opinions without governmental reprisal, and we will not be changing this state of affairs no matter how butthurt anyone else in the world gets over it.
Hi Carla – thank you for such a great holiday! I too feel revived and rested now – especially as I also have FEAST next to me to dive into and salivate again and again! I also hear that Bold and The Beautiful is featuring a wedding in Puglia on Australia TV this week….Hmmm – I might just have to tape it and watch it to continue my Puglia holiday a little longer! Grazie mille! Ciao Bella F x
> But, the formula> =SUMIF(A1:A5,â€?*â€,B1:B5)> returns 23 because no visible character is returned in cell A4.So , it sums everything EXCEPT visual blanks!I think it is time to correct the wording in your initial post: "Sum visually blank cells such as holding null strings". Do you agree?
the best thing about making your own stocks is that it's really good for your health. The Nourished Kitchen has a great explaination on the health benefits of bone marrow soup. Bone marrow soup's
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–I mostly recall the guys at work talking about business, sports, the relative attractiveness of the females in the workplace, and where to go drinking that night.— To me, this sort of discussion is purely unappealing. Yeah, I agree most of my male friends descend to this sort of discussion. I just hear them and leave them as soon as the discussion terminates :).
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today I posted Four Simple Stock Market Lessons Related to Life. After reading through the post you can enjoy these additional thoughts from The Financial Blogger
Ja, bara vi hÃ¥ller isär etiken och estetiken. I det här inlägget sätter jag etiken i första rummet. Eller som Batman säger, "It's not who you are, it's what you do."
Too old for either…but was in vocal competitions earlier in life and won a few 2nd places, thirds and once–a 1st. Was on a (small) vocal scholarship in college (the _first_ time in college back in 1979/81.)I confess to guilty pleasures like these for viewing…Hh
Hei!Saanko kysyä, että mistä olet hankkinut tuon hameen? Tarvitsisin itse juuri samanlaisen, mutta tuntuu olevan yllättävän vaikea löytää ihan tavallista mustaa lyhyttä hametta.. Siis laadukasta sellaista.Kiitos hyvästä blogista, tämä oon uusi suosikkini! Olet todella lahjakas.-Marianna
Hallo Christiene,Ja eigenlijk, toen ik gisteren deze verkreeg van Aritha, dacht ik hem aan jou door te geven. Echter, iemand was mij te snel af, wat dan ook weer bewijst dat je hem dubbel en dik hebt verdiend. Geef hem alvast een ereplaatsje op je blog.
Go you! I'm working on emerging from my comfort zone. If you knew me years ago you'd know that I'd fallen in a funk and this just isn't me.I think it's awesome about the bike!! Last year you were rather resistant to it. You are surely a much different person than I "met" 3 years ago.
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Dacia, hai la forza e il coraggio per farti valere, anche di fronte a un attacco cosi' volgare e violento di un individuo cosi' pieno di presunzione, potere e soldi, ma senza un briciolo della tua intelligenza.AG
Great post Nishant. Yeah, most of the successful bloggers do have enemies because of the common reasons you have mentioned above. BTW, I think there is a spelling mistake in the first sub header … it should be "Revealing" instead of "Reviling".
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an excellent spawn rate. It's used to be busy though, so skip through some channels to find an empty one. At level 46 it's good to go to Singapore (with the plane in Kerning City) and fight Truckers.At level 52 you go to El Nath and fight Hectors and White Fangs. Take the black VIP Taxi in El Nath and you will be taken right to the correct map.After that try some monsters out in Ludibrium, and then the Haunted House in NLC. (Twisted Jesters – lvl 70 monster).
UTS, Gore won. Crime may be trying to slip Bush in..hoping we don't notice. bush was the Worst President Ever. Given the option between an AfAm man…and two wites anointed by the wingnuts…the US overwhelmingly preferred the AfAm.Still waiting for the proof. How about some evidence? Facts? Waiting. Anytime now.Mold
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Yes, I expect Agatha Christie could get away with murder in her writing career…groan…!I didn't mean 'don't risk it' as a rule, Sally. Was just summarising the majority opinion from this thread.
I'm with you… the critic sounds like he'd have prefered to see Kim Kardashian or Angelina Jolie doing an interview…. celebrities should be crass and classless shouldn't they?
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It was an absolutely gorgeous wedding. Danielle is beautiful and Matt so handsome. May God continue to bless you and have a wonderful life together. And stay as tight as the knot!!! Love Lizzy <3
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che post interessante!! non sono mai stata a un'asta, in realtà l'ho sempre immaginata una cosa triste, con tutti quegli oggetti che abbandonano il vecchio proprietario per trovarne di nuovi (se penso alla fiera della vanità ad esempio, mi mette tanta tristezza). tu invece me la fai vedere in modo del tutto nuovo, glamour anche!
Theology can just be an abstraction divorced from life but morality is something livedIn the gospel Judgment Parables , Jesus does not ask what one's theology was but how one lived and treated others , especially the most desperateReferences : Was this answer helpful?
Hi Dave,will the webinar also be available as web-video? I tried attending a google webinar once at it was (due to the webinar software) a most frustrating experience that I don't really want to repeat.Thomas
which can apparently be found in the latest issue of Japanese mag Famitsu (translated by HeartStation), indicates that there may be even more work on Nomura's plate than we thought, as he says that
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neileJanuary 29, 2013Hi, I am wondering why no lunges and why we have to lower weight. Is it not OK to just go with the "feel system"? If it feels ok, go for it or am I missing something? Thanks so much for your post.
A singapour, on trouve dans les transports des graphismes interdisant de manger boire, ou de transporter des Durians (facile à retouver sur internet).Le durian est un fruit populaire dans la région mais qui sent très fort, et il n'est même pas permis d'en transporter dans le métro.
Está tudo grosso, está tudo grosso! Ainda vem a Isabelinha dizer que a Cultura portuguesa está a ser divulgada no estrangeiro… eu só tenho conhecimento de publicidade ao sol, à praia e ao golfe e notÃcias destas que revoltam.Cumps
J'ai raté la sangria basque, je voulais en faire vace du txacoli mais il a été bu à une vitesse ! Même pas pu photographier la btl, faut dire, c'était avant notre escapade en Aubrac… Tu y étais quand ?
Rien ne sert de se crêper le chignon..Les religions sont l'opium des peuples, un guide destiné à l'origine aux populations incultes pour qu'elles s'élèvent au-dessus de la barbarie. Un païen qui ne crois en rien a beaucoup plus de chances de s'enfoncer et de faire stagner son âme, ce n'est pas ce qui est voulu par l'Autorité Supérieure, quel que soit le nom qu'on lui donne.Le but est que l'espèce humaine progresse et gravisse les échelons.
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Whether we are pre-peak of post-peak in 2010 is less relevant than the fact the oil production is asintotic since 2005, despite the surges in price. Demand grows and shrinks but similar amounts are delivered, only with a higher/lower price! Looks like supply is constraint!
hola amigos:llegué esta página buscando información sobre el j100a a pedido de mi hemanita que querÃa algunos juegos… depues de recorrer la red, he llegado a la conclusión de que sencillamente no se puede; ni juegos, ni ringstons, ni walpapper, nada; de hecho en otros foros cuentan que aún cuando tengas un cable usb para tu celular, de todos modos no funciona.quise escribir para que no sigan perdiendo su tiempo y a la vez agradecer a todos por su información.
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I agree as long as the taxes stay at the current rates, but I've afraid along with the rest of america, the government is way paste what is considered acceptable spending. So higher taxes are almost certainly in the cards for the US…A side benefit of the Roth (and one that nobody considers) is that dividends aren't taxed in a Roth IRA (or options either I believe)… It's like the ultimate loophole
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Bonjour, Le Bel Espoir devrait reprendre la mer fin avril 2013. Actuellement le chantier travaille sur les mâts, le gréement et le pont arrière.On fera son programme en janvier 2013. A bientôt. (Secrétariat)
Make sure you lisen to Brian's Untitled Radio Program (BURP) every Monday from 3pm-4pm, for the best in punk rock, garage, ska, new wave, goth, and more.
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Laundry. Dry laundry. Clothes on spare room sofa ready to sort to put away. Kids wear them for varying number of minutes/days before dirty. Laundry. Dry laundry. Clothes on spare room sofa ready to sort to put away.Kids wear them for varying number of minutes/days before dirty. Laundry…I am inspired to consider moving my family to a nudist colony…. (;-)
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Ojalá puedas cruzar el charquito y conocer parÃs, yo también quiero conocer ParÃs, creo que hay tantas cosas que admirar que se necesitan muchos viajes, algún dÃa… Espero también ver la pelÃcula la actriz es la Sherlock holmes.Saludos
( to myself) …"ohh didn't see that coming"..haha…like we talk or hang out or sip tea and talk hair. What a weird reaction! haha…"the nearsighted owl" dyed her hair bright red one night without telling us. Of course it could possibly just randomly happen one night. I'm a weirdo. :) I love it by the way! Totally a super fun addition to your already adorbs look! Have fun with it! Maybe sew yourself a shiny cape. ;)Happy Friday!!xo Jenny Holiday
Happy Thanksgiving Kim!Thanks so much for hosting WOW each week. Wonderful features. That little truck stole my heart and I am going to steal that idea.:-)
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they are here to help you is a liar, simply because they are here to help themselves. I am happy that Rick was able to point that out. Do something because you want to, don't come in the name of sacrifice in order to move humanity forward. In this life you have the choice of being a bum or being wealthy, it is up to you, no Government or church is responsible for you, just be responsible for yourself in everything, even in sex.
One day I wish to be able to pour my heart out like this. You are such a wonderful writer Kate. Sometimes we dig the whole just a wee bit too dip, don't we. Ah, motherhood…Stasha recently posted..
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Qualche idea su come mai stavolta il vettore si chiama come un dio greco della guerra? Che vogliano davvero dare battaglia agli alieni sulla Luna? Complottisti, ribellatevi!A parte gli scherzi una nuova corsa alla Luna sembra affascinante, anche perchè magari gli obiettivi potrebbero anche essere un pò più spinti delle missioni originarie. Speriamo davvero che il portafogli sia ampio, altrimenti rimarrà solo un sogno.
Wish I lived in the Bay area! Waah!Have fun, Nathan, and congratulations again on your (multiple) book deal and publication with Penguin.I loved the entire series this week. Thanks again. Wishing you the very best!
I still think this theory is plausible, given Obama's and the State Department's record of selling out American interests. And as to Morsi/Egypt 'making things happen' in Libya: well, if not for the accidental appearance of the SEALS, all would have gone as planned. Libya is right next door to Egypt, and has oil which Egypt would really really want, and I expect Obama has offered to aid Egypt in placing Libya into its sphere of influence. After the Election, of course.
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sep14Vanessa Es lamentable esta negociación o casi negociación. Por mi parte estoy totalmente desilucionada pero no especÃficamente del sindicato sino de mis compañeros ya que nunca se peleo lo que merecemos con convicción siempre estuvo el comentario desalentador y mala onda que pena por ellos. Por mi parte si hubiese existido la unión sindical hubiéramos conseguido esto y mucho mas. Hubo huelga o fue un recreo…… Este acontecimiento quedara en el recuerdo ( mmmm burla) de muchos. Que poco sindicales fuimos cero peso.
/ I am really impressed not only with the "call to arms" referenced in this article but also in the storyline itself. I think this is a quality piece that shows that word of mouth still works – it looks a little differently these days but when one concerned parent talks to another – Great things can happen. I don't know the book that was involved but trust that my good friends from Social Media know what they are doing. Three cheers to all involved!
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Burkina faso is one of the poorest countries in the world. please take that into consiceration mr & mrs architect if you discuss and plan somthing like this.i'm deeply agitated by anger and concern if people are planing something like this. AND MAKE A HUGE FENCE AROUND IT.ARE YOU CRAZY?
JamesYou're like a man obsessed!The National Conversation was a wee bit more open than Calman. In any case, your post is about Calman, but you can't resist shoehorning the SNP in for a swift kick on the way past?Did Alex Salmond steal your seat in the Holyrood restaurant or something?The basic thing is that Calman is merely the SVR writ large. A meaningless sop and a trap. Cleverer people than me have analysed it and deduced a trap in Calman that, if the tax powers were used, you would be worse off than before
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I'm not sure we can be friends.Mike Huckaby, Groove Podcast 13I first listened to this during hour 13 of a 15-hour overnight drive and for the first 20 minutes of the mix, it was the worst decision of the trip. Huckaby
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I can only imagine how hard it must have been to say goodby to your sister. My family mostly live only a couple hours away and sometimes that's hard I hope this next week brings you some smiles!
Já už sem nechci psát, protože jsem si pÅ™eÄetla v pravidlech, že pod nickem se nesmÃ, ale nedá mi to:)Teoreticky: je v pořádku, když je esenciálnà náležitostà smlouvy úplata a strany smluvnÄ› uÄinà úplatu závislou na nejisté události budoucÃ? Pokud tedy pÅ™idajà podmÃnku, na jejÃmž splnÄ›nà závisÃ, zda splatnost vůbec kdy nastane? Podle mne to v pořádku nenÃ. Cena může být splatná v dlouhé lhůtÄ›, ale musà být splatná v konkrétnÃm okamžiku, který nastane. Takto chápu podstatu problému já.Opravdu se omlouvám a už mizÃm:)
Va be'… poi si meravigliano che la gente non compra i giornali, quando si esce dagli argomenti principe dei giornali (calcio, paraculismo politico e "sbatti il mostro in prima pagina") si precipita in un baratro senza fondo piastrellato di incompetenza, ignoranza e malafede.Che tristezza.
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This is no conspiracy. I have never believed in any conspiracy. Everything that we know is either a huge coincidence or that Obama is just the luckiest man on earth. I know that ORYR readers are very smart people. Can anyone come up with a math equation that states the evidence we have vs the government media complex?
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Orly is the embodiment of this verse:1 Corinthians 1:2727But God hath chosen the foolish things of the world to confound the wise; and God hath chosen the weak things of the world to confound the things which are mighty;
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If you are heading to Florida for vacation, chances are the first thing you are going to do is check into your hotel room and head to the beach. Who can blame you? Warm golden sand, rhythmic waves, the sunlight cascading down on your bright-colored ray bans,there are plenty of reasons to fall in love with the beach. We will get to the tanning and sandcastle building soon. First we have to make sure we are properly protected from the sun. Just like wearing a helmet while riding a bike or having an escape plan when fighting breaks out during Monopoly, going to the beach without the right sunscreen could leave you bright red. When you head to the store to grab a bottle you may find there are different brands and strengths. How do you know which of these is right for you?
First you have to know what the SPF (Sun Protection Factor) rating is. SPF rating tells how effective the sunscreen is at blocking the sun's UVB rays. UVB rays are what cause sunburns, and potentially skin cancer in large amounts. The basic SPF rating is 15 SPF. For the average person 15 SPF will do just fine. If you find that you are more susceptible to sunburn, or have a skin condition that causes you to be sensitive to light, it could be beneficial to move to at least 30 SPF.
You may also want to see rather or not a sunscreen protects against UVA rays. These are also known to cause skin cancer if you spend prolonged time in them without protection. Many bottles will not openly say rather or not they will protect against UVA rays, so it is better to search for sunscreen that has any of these active ingredients.
- Avobenzone
- Oxybenzone
- Titanium dioxide
- Sulisobenzone
- Zinc Oxide
- Ecamsule
Any sunscreen with at least one of these ingredients will help protect against UVA rays. This way you get the whole package while you lay on your back soaking in the rays.
One last thing to look for are added perks, such as being sweat or water-resistant. If you plan on spending time in the water, or working up a sweat playing volleyball, make sure your sunscreen can handle it. You don't want to spend half your vacation afraid to move because of a bad sunburn. It's also not too wonderful when it comes to vacation photos. Luckily the Ocean Walk Daytona vacation spot is just down the road from plenty of stores that sell sun screen. It may take a few more minutes before you can dive fully into your ocean side activities, but a few minutes is better than a few days resembling a man-sized lobster.

subset: commoncrawl


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" Stop worrying about the beta, and just get the product out. If you are not embarrased by your first product, then you're doing it wrong. It should be ugly, it should be clunky, it should be what you think it should be... but less. And once you get it in the hands of the customer, the customer is going to tell you what they like and what they don't like. And that's what we realized - we were trying to be perfectionists. You want your product to be perfect, but you make these assumptions that it's going to be valuable. And the best way to do that is get it in the hands of the customer who will tell you if they find value in it."
– Josh Carter
Josh Carter is the Co-Founder & CEO of Brightwork, a microservices platform that enables developers to build faster on a reliable and scalable solution. Since their founding they've raised over $300K in funding and have gone through Techstars in Chicago. Josh started out in the Navy, where he served for about 3 years. Since his time in the Navy he's held multiple engineering roles in the Telecom industry and eventually a Senior Support Engineer at the startup, Twilio, a communication startup that went public earlier this year. Josh founded his own digital marketing agency - Plunk - and is also a former founding board member of Operation Code.
The top reason to listen to today's show is:


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Abrigall Masella is a great hostel fully adapted for your leisure travel, winter sports and adventure activities in one of the best surroundings of the Cerdanya.Abrigall Masella has the same services that you can find in a hotel. Our hostel includes a cafe, bar, disco, TV room, reading / games room, disabled access and a parking for buses. Among our facilities we highlight the restaurant "Xup Xup", a large and complete buffet with maximum capacity for 250 people.Organize your event at the Abrigall Masella and choose the two room that will best suit your needs.
Adaptable rooms for different types of events.
At Abrigall Masella we offer parking for large groups who are eager to enjoy the Masella by bus.
It has the best quality in cooked dishes of the area thanks to its local products.
Enjoy the tranquility and relaxation of a nice coffee in the cafeteria of our Abrigall Masella.
Registered Name: SAGATU ASOCIADOS COMERCIAL HOTELERA, S.L. CIF: B-63751853 Address: C/ Paris, nº 120, 1ª, C.P. 08036, Barcelona E-mail: firstname.lastname@example.com
The processing of data is authenticated according to the GRDP (EU) 2016/679 as required for the execution of the contractual relationship, to formalise the reservation of SERCOTEL services and/or products.
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In the case that SERCOTEL is authorised to send promotional commercial communications via email, the user's consent will be the legitimising factor.
The purpose of collecting and managing your personal data through the registration form is to allow the management of a commercial, financial and administrative relationship with the clients for everything related to the services and accommodation at our hotels.
The purpose of collection or spontaneous mailing of your Curriculum Vitae is to study the various candidates, so that our Human Resources Department can proceed to the selection of the candidates with the ideal profile to be incorporated into the company.
Additionally, the user can authorise SERCOTEL to manage their data for promotions and for marketing, including emailing of commercial communications relating to the mix of products and services that we offer, as well as those related to the hotel sector (travel, gastronomy, leisure, etc.)
Data will be retained during the lifetime of the administrative and/or commercial relationship between the interested party and SERCOTEL. Furthermore, the timescale provided by legislation specifies the minimum period that this data should be kept available.
If the user wishes to exercise their right to suppress or oppose use of the data, their data will be blocked and then destroyed in a way that respects their confidentiality.
Recipients of the data are third parties who are responsible for its management and who provide their services to SERCOTEL, with whom a corresponding agreement has been drawn which guarantees their compliance to RGPD and the appropriate security measures.< p>
Your data can be provided to the rest of the companies within the SERCOTEL GROUP for the correct development or provision of reserved services.
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- This event has passed.
Trunk or Treat 2019
October 27, 2019 @ 10:00 am - 2:00 pmFree – $15
Join us at the Automobile Driving Museum for our annual "Trunk or Treat" Halloween Party!
FREE for kids 10 and under – this is a SAFE Family Fun event!
Hearse cars, games, treats, and tricks all day at the museum!
Food provided by Taco Taxi!
Plus, presentations from the Grandeza Mexicana Folk Ballet Company
Oriental Trading Company is a proud sponsor of Trunk or Treat 2019! Shop their Halloween section for all your spooky needs!
GoGo squeeZ will be here giving away FREE SAMPLES of delicious applesauce and yogurt!
Emceed by the one and only Ken Vela!


text:
Links for Learning
These links are to assist the students in furthering their learning. Seeley Union School District is not responsible for the content of external websites.
Renaissance Learning Program
Our school uses this program to assist their success in language arts and literacy.
AR Book Finder
Does your student want to read a certain book, but they do not know if it is AR? Click on this link to find out if it is an AR book, what level it is, and how many points it is worth!
Common Sense Media: Age-Based Media Reviews for Families
Not sure what is age appropriate for your child? Common Sense Media is a non-profit organization that "provides education and advocacy to families to promote safe technology and media for children."
Epic! is the Leading Digital Library for Kids 12 & Under. Instantly access 35,000 of the best books, learning videos, quizzes & more!
Educational games for grades PreK through 6 that will keep kids engaged and having fun. Topics include math, reading, typing, just-for-fun logic games and more!


text:
Prostate cancer is one of the malignant tumors which exhibit resistance to anticancer drugs, at least in part due to enhanced antiapoptotic mechanisms. Therefore, the understanding of such mechanisms should improve the design of chemotherapy against prostate cancer. Galectin-3 (Gal-3), a multifunctional oncogenic protein involved in the regulation of tumor proliferation, angiogenesis, and apoptosis has shown antiapoptotic effects in certain cell types. Here, we show that the expression of exogenous Gal-3 in human prostate cancer LNCaP cells, which do not express Gal-3 constitutively, inhibits anticancer drug–induced apoptosis by stabilizing the mitochondria. Thus, Gal-3-negative cells showed 66.31% apoptosis after treatment with 50 μmol/L cis-diammine-dichloroplatinum for 48 hours, whereas two clones of Gal-3-expressing cells show only 2.92% and 1.42% apoptotic cells. Similarly, Gal-3-negative cells showed 43.8% apoptosis after treatment with 300 μmol/L etoposide for 48 hours, whereas only 15.38% and 14.51% of Gal-3-expressing LNCaP cells were apoptotic. The expression of Gal-3 stimulated the phosphorylation of Ser112 of Bcl-2-associated death (Bad) protein and down-regulated Bad expression after treatment with cis-diammine-dichloroplatinum. Gal-3 also inhibited mitochondrial depolarization and damage after translocation from the nuclei to the cytoplasm, resulting in inhibition of cytochrome c release and caspase-3 activation. These findings indicate that Gal-3 inhibits anticancer drug–induced apoptosis through regulation of Bad protein and suppression of the mitochondrial apoptosis pathway. Therefore, targeting Gal-3 could improve the efficacy of anticancer drug chemotherapy in prostate cancer. (Cancer Res 2006; 66(6): 3114-9)
- Prostate cancer
- Galectin-3
- Apoptosis
- Drug resistance
Prostate cancer is the most common cancer in men, with ∼230,110 new cases and 29,900 deaths annually in the U.S. in 2004 ( 1). About 10% to 20% of men with prostate cancer present with metastatic disease. Initially, primary androgen ablation therapy leads to a reduction in serum levels of prostate-specific antigen in patients with metastasis of prostate cancer, but in almost every patient, the disease eventually becomes hormone-refractory and apoptosis-resistant ( 2). The prognosis for these patients is poor because conventional chemotherapeutic drugs have little effect on hormone-refractory prostate cancer and do not provide a marked survival advantage ( 3). Therefore, it is necessary to clarify the mechanism of chemotherapeutic drug resistance of prostate cancer and develop a novel strategy for the treatment of prostate cancer.
The galectins comprise a family of 14 members of β-galactoside-binding proteins, characterized by their affinity for β-galactosides and by a conserved sequence of the carbohydrate recognition domain that bind to the carbohydrate portion of cell surface glycoproteins or glycolipids. Galectin-3 (Gal-3), a 31-kDa chimeric gene product, is a multifunctional oncogenic protein which regulates cell growth, cell adhesion, cell proliferation, angiogenesis, and apoptosis ( 4– 12). We and others have shown that endogenous Gal-3, which contains the NWGR anti-death motif of the Bcl-2 family, inhibits epithelial cell apoptosis induced by staurosporine, chemotherapeutic agents such as cisplatin, genistein, tumor necrosis factor, and nitric oxide ( 13– 16). We also reported that nuclear export of phosphorylated Gal-3 regulates its antiapoptotic activity in response to chemotherapeutic drugs ( 17). Phosphorylated wild-type Gal-3 was exported from the nucleus to the cytoplasm and protected cancer cells from drug-induced apoptosis, whereas nonphosphorylated Ser6 mutant Gal-3 was neither exported from the nucleus nor protected cancer cells from drug-induced apoptosis ( 17). Other investigators also reported that under certain conditions, Gal-3 was found in the cytoplasm and perinuclear mitochondrial membranes ( 18, 19), where it was involved in the control of apoptosis, possibly through an interaction with the bcl-2 protein ( 20).
Expression and cellular localization of Gal-3 are important for the prognosis of a variety of cancers. Sanjuan et al. reported on the down-regulation of Gal-3 in colorectal cancer with increased cytoplasmic expression of Gal-3 at more advanced stages ( 21). Down-regulation of Gal-3 was also observed in prostate cancer, and both nuclear exclusion and cytoplasmic localization of Gal-3 are correlated with cancer progression ( 19, 22). Furthermore, Califice et al. reported that cytoplasmic Gal-3 expression in LNCaP cells induced tumor growth, invasion, angiogenesis, and decreased inducible apoptosis ( 23).
Here, we investigated the effects of introducing Gal-3 into non-expressing human prostate cancer cells (LNCaP) on their response to proapoptotic chemotherapeutic agents and explored the mechanisms involved in the apoptotic function of Gal-3. We show that the expression of Gal-3 in LNCaP cells inhibits cis-diammine-dichloroplatinum (CDDP)– and etoposide-induced apoptosis by regulation of Bcl-2-associated death (Bad) protein, mitochondrial integrity, inhibition of cytochrome c release, and caspase-3 activation.
Materials and Methods
Cell culture. The human prostate cancer cell line LNCaP was from American Type Culture Collection (Manassas, VA). LNCaP cells were maintained in RPMI 1640 containing 2 mmol/L glutamine, penicillin-streptomycin (Life Technologies, Inc., Grand Island, NY) and 10% fetal bovine serum in 5% CO2 at 37°C using standard cell culture procedure. LNCaP cells were transfected with a Gal-3 expression vector as described previously ( 24). Briefly, the plasmid pH29.BA2, which uses the human β-actin promoter to drive expression of the human Gal-3 cDNA, was provided by Dr. Douglas Cooper (University of California, San Francisco, CA). This vector was used for the transfection of subconfluent monolayers of LNCaP cells grown for 1 week prior to transfection in Opti-MEM I medium (Life Technologies) supplemented with 5% FCS. Transfection was done using Lipofectin Reagent (Life Technologies) following the manufacturer's protocol. For each dish, cotransfection was done with 9 μg of pH29.BA2 and 1 μg of pSV2neo containing the neomycin resistance gene. Selection was carried out with G418 (Life Technologies) at a concentration of 300 μg/mL, which was added 48 hours after transfection. Clones of stable transfectants have been isolated and two of them, 29-11 and 29-23, were used in the present study.
Apoptosis assay. Apoptosis was assessed by measuring propidium iodide permeability (Oncogene, San Diego, CA) using a Becton Dickinson FACScan and CellQuest software. Fragmentation of chromosomal DNA detected as the appearance of hypoploid DNA was measured as previously described ( 25). Briefly, a total of 1 × 106 parental or Gal-3-expressing LNCaP cells were treated with 50 mmol/L CDDP or 300 mmol/L etoposide for 48 hours at 37°C. After washing, cells were fixed with 80% ethanol for 30 minutes at 4°C, washed with PBS, and treated with RNase A (1 mg/mL in PBS) for 15 minutes at 37°C, followed by staining with propidium iodide (50 μg/mL) for 15 minutes at room temperature.
Caspase-3 activation assay. The active form of caspase-3 was determined by direct staining of cells with a FITC-conjugated rabbit anti-active caspase-3 monoclonal antibody (BD Biosciences, San Diego, CA) followed by FACS analysis as described previously ( 25). Briefly, cells were fixed with 1% formaldehyde for 15 minutes at 4°C, washed twice with PBS, permeabilized with 0.1% Triton X-100 in PBS for 15 minutes at 4°C and incubated with goat serum. Then, cells were stained with FITC-conjugated anti-active caspase-3 antibody (PharMingen, San Diego, CA). After washing with PBS, cells were again fixed with 1% formaldehyde and analyzed with FACScalibur. To further investigate caspase activity, we measured acetyl-Asp-Glu-Val-Asp-7-amino-4-methylcoumarin (Ac-DEVD-AMC) activity in control or Gal-3-expressing LNCaP cells treated with 100 mmol/L CDDP as described previously ( 25). Cells were lysed with cell extraction buffer [20 mmol/L HEPES (pH 7.5), 10 mmol/L KCl, 1.5 mmol/L MgCl2, 1 mmol/L EDTA, 1 mmol/L EGTA, and 1 mmol/L DTT] containing 0.03% Nonidet P-40. Lysates were centrifuged at 15,000 × g for 10 minutes, and 50 μL of the cytosolic fraction was incubated for 60 minutes at 37°C in a total volume of 200 μL of caspase buffer [10 mmol/L HEPES (pH 7.5), 50 mmol/L NaCl, and 2.5 mmol/L DTT] containing 25 μmol/L of Ac-DEVD-AMC (Bachem, King of Prussia, PA). -Amino-4-methylcoumarin fluorescence, released by caspase activity, was measured at 460 nm using 360 nm excitation wavelength using a Spectra Maxi Gemini fluorescence plate reader (Molecular Devices, Sunnyvale, CA). Caspase activity was normalized per microgram of protein determined by the bicinchoninic acid protein assay kit (Pierce Chemical Co., Rockford, IL).
Cytochrome c release. A total of 2 × 108 cells were harvested 24 hours following treatment with 50 mmol/L CDDP, washed twice with ice-cold PBS, resuspended in ice-cold cell extraction buffer [20 mmol/L HEPES-KOH (pH 7.5), 10 mmol/L KCl, 1.5 mmol/L MgCl2, 1 mmol/L sodium EDTA, 1 mmol/L sodium EGTA, and 4 mmol/L DTT] containing 250 mmol/L sucrose and protease inhibitor cocktail (Sigma, St. Louis, MO), and incubated for 1 hour at 4°C as described previously ( 25). The lysates were then passed through a 27 1/2-gauge syringe 10 times and centrifuged at 15,000 × g for 30 minutes at 4°C. The resulting supernatant was analyzed by immunoblot analysis using anti–cytochrome c antibody (Zymed Laboratories, Inc., San Francisco, CA).
Immunofluorescence. Immunofluorescent staining of cells was done as described previously ( 17). Briefly, the cells were fixed with 4% paraformaldehyde for 5 minutes, permeabilized with 0.5% Triton X-100 for 5 minutes and blocked with 1% bovine serum albumin in PBS for 30 minutes. After the blocking, rabbit polyclonal anti-Gal-3 antibody was added at a 1:50 dilution and incubated for 1 hour. Secondary antibody (FITC goat anti-rabbit IgG; Zymed) was added at 1:200 dilutions and incubated for 1 hour. The stained cells were analyzed by confocal immunofluorescence microscopy using a Zeiss laser scanning microscope 310 (Zeiss, Chester, VA).
Mitochondrial staining. Cells on glass coverslips were incubated with complete medium containing 1.0 μg/mL JC-1 (Molecular Probes, Inc., Eugene, OR) for 30 minutes at 37°C. After washing with PBS at 37°C, cells were placed on a special device, which changes medium from PBS to 100 mmol/L CDDP in PBS. The stained cells were analyzed using confocal immunofluorescence microscopy using a Zeiss laser scanning microscope 310 (Zeiss). The cells were scanned by dual excitation of 488 nm (green) and 568 nm (red) laser lines.
Western blot analysis of cytoplasmic and nuclear protein. Cytoplasmic and nuclear proteins were extracted as described previously ( 17). Cells were grown to a subconfluent state on 150 mm diameter plates and then exposed to anticancer drugs, CDDP (Sigma) for 48 hours. The cells were then harvested, washed with PBS and resuspended in hypotonic buffer (20 mmol/L HEPES, 10 mmol/L KCl, 1 mmol/L MgCl2, 0.5 mmol/L DTT, 0.1% Triton X-100, 20% glycerol, 5 μg/mL leupeptin, 10 μg/mL aprotinin, and 500 μmol/L phenylmethylsulfonyl fluoride; Sigma). The suspensions were centrifuged at 3,000 rpm for 5 minutes. The supernatant was used as the cytoplasmic fraction. The pellet was resuspended in extraction buffer (20 mmol/L HEPES, 10 mmol/L KCl, 1 mmol/L MgCl2, 0.5 mmol/L DTT, 0.1% Triton X-100, 20% glycerol, 420 mmol/L NaCl, 5 μg/mL leupeptin, 10 μg/mL aprotinin, and 500 μmol/L phenylmethylsulfonyl fluoride). This suspension was centrifuged at 15,000 × g for 30 minutes. The supernatant contained the nuclear fraction. Protein concentrations were measured using protein assay reagent (Bio-Rad, Hercules, CA). Ten micrograms of cytoplasmic protein and 20 μg of nuclear protein were separated by 12.5% SDS-PAGE by the method of Laemmli and transferred to a polyvinylidene difluoride membrane. This membrane was subjected to immunoblot analysis using rat anti-Gal-3 antibody TIB166 (American Type Culture Collection), anti-Bcl-2 antibody, anti-active Bad antibody (Promega, Madison, WI), anti-phospho-Bad (Ser112) antibody (Santa Cruz Biotechnology, Santa Cruz, CA), anti-cytochrome c antibody (Zymed), anti-β-actin antibody (Sigma), horseradish peroxidase (HRP) anti-rat antibody, HRP anti-mouse antibody, and HRP anti-rabbit antibody (Zymed). Western blot analyses were done using the enhanced chemiluminescence detection system (Amersham Biosciences, Piscataway, NJ) as described previously ( 16, 17, 25). For densitometric analysis of Western blots, Scion Image (Scion, Frederick, MD) was used.
Gal-3 suppresses anticancer drug–induced apoptosis. Two clones of Gal-3-expressing LNCaP cells were isolated. Western blot analysis of Gal-3 transfectants in LNCaP confirmed that the control cells do not express protein, whereas the transfectants express abundant levels of the protein ( Fig. 1 ). To determine whether the expression of Gal-5 in LNCaP cells results in antiapoptotic effects, control and Gal-3-expressing LNCaP cells were treated with 50 μmol/L CDDP or 300 μmol/L etoposide, and cell death was analyzed by flow cytometry after staining with propidium iodide. Following treatment with 50 μmol/L CDDP for 48 hours, control Gal-3-null LNCaP cells underwent 66.31% apoptotic cell death, whereas in the Gal-3-expressing cells, only 2.92% and 1.42% of the cells were apoptotic ( Fig. 2A ). Similarly, exposure of these cells to 300 μmol/L of etoposide for 48 hours resulted in apoptosis of 43.8% of control LNCaP cells, whereas only 15.38% and 14.51% of Gal-3-expressing LNCaP cells were apoptotic ( Fig. 2B). These results indicate that Gal-3 could inhibit apoptosis induced by both CDDP and etoposide.
Gal-3 inhibits cytochrome c release and subsequent caspase-3 activation. Mitochondrial events critical for apoptosis include the disruption of electron transport, loss of mitochondrial transmembrane potential, and the release of cytochrome c ( 25, 26), resulting in caspase-9 and caspase-3 activation. To examine whether intracellular Gal-3 inhibits the loss of mitochondrial integrity, we analyzed cytochrome c release from the mitochondria. The results show that intracellular Gal-3 inhibited cytochrome c release from the mitochondria ( Fig. 3A ). Densitometric tracing analysis shows that the level of cytochrome c treated with CDDP in Gal-3-expressing LNCaP decreased by 3.3-fold compared with control cells ( Fig. 3B).
Caspase-3 is a critical downstream protease in the apoptotic cascade ( 27, 28), which is involved in cell death in response to numerous apoptotic stimuli including Fas ligand or tumor necrosis factor-α ligation with its receptor ( 26, 29). To investigate whether Gal-3-inhibited apoptosis induced by CDDP is the result of inhibition of the caspase pathway, we evaluated the levels of active caspase-3 after treatment with 50 μmol/L CDDP for 48 hours. The results show that Gal-3 inhibited active caspase-3 expression ( Fig. 4A ). We further measured DEVDase activity in control and Gal-3-expressing LNCaP cells treated with 100 μmol/L CDDP 6 hours after the induction of apoptosis. DEVDase activity in control LNCaP cells treated with CDDP increased by >13-fold above basal levels, whereas its activity in Gal-3-expressing LNCaP cells increased by only 2.5- or 3.0-fold above basal levels ( Fig. 4B). These results showed that Gal-3 inhibited cytochrome c release induced by CDDP resulting in inhibition of the caspase pathway. These results suggest that intracellular Gal-3 may directly affect mitochondria integrity leading to the inhibition of the downstream effector caspase-3.
Gal-3 translocation. Gal-3 can shuttle between the nucleus and cytoplasm and can be translocated to the perinuclear membrane following a variety of apoptotic stimuli such as CDDP, serum withdrawal, and staurosporine ( 17, 18, 30). To determine the effects of CDDP on the localization of the transfected Gal-3 in LNCaP cells, we preformed immunofluorescent staining following exposure to CDDP. Before CDDP treatment, Gal-3 was randomly localized in both the nuclear and cytoplasmic compartments ( Fig. 5A ). However, after 48 hours of exposure to 50 μmol/L of CDDP at 37°C, it was localized exclusively in the cytoplasm ( Fig. 5B). The observed change in Gal-3 localization was confirmed by Western blot analysis, which showed that the level of nuclear Gal-3 in LNCaP transfectants decreased after treatment with CDDP ( Fig. 5C), whereas the increase of cytoplasmic Gal-3 expression was not obvious, as described previously ( Fig. 5D). Western blot analysis of whole cell lysates revealed that exposure to CDDP didn't change the total levels of Gal-3 (data not shown), indicating that down-regulation in nuclear Gal-3 expression was not due to the degradation of Gal-3, but was due to its nuclear export. To conclude, CDDP exposure induced Gal-3 translocation from the nucleus to the cytoplasm in LNCaP cells.
Gal-3 regulates mitochondrial integrity. We measured and evaluated mitochondrial membrane potential by confocal microscopy after staining living cells with the cationic dye, which exhibits potential-dependent accumulation in mitochondria. Mitochondrial depolarization occurring at the early stages of apoptosis is indicated in JC-1-stained cells by a decrease in the red/green fluorescence intensity ratio. Before exposure to CDDP, the mitochondria in both control and Gal-3-expressing LNCaP cells seemed to be intact. Mitochondrial integrity was evident from the formation of red fluorescence in JC-1 stained cells ( Fig. 6A ). After exposure to CDDP for 15 minutes, fluorescence of mitochondria in Gal-3-deficient LNCaP cells began to shift from red to green, and by 60 minutes, the mitochondria appeared green in most cells ( Fig. 6B and C), indicating depolarization of the mitochondrial membrane. In contrast, mitochondrial membrane potential in Gal-3-expressing LNCaP cells was maintained partially after exposure to CDDP for 60 minutes because many mitochondria seemed to emit red and yellow fluorescence ( Fig. 6D-F, 0, 15, and 60 minutes, respectively). These data suggest that intracellular Gal-3 inhibited mitochondrial depolarization and damage induced by anticancer drug.
Regulation of Bad protein by Gal-3. Bad protein is a member of the Bcl-2 protein family that plays an important role in apoptosis. The balance between proapoptotic (e.g., Bad and Bax) and antiapoptotic (e.g., Bcl-2 and Bcl-XL) members of the Bcl-2 family is critical to control mitochondria-induced apoptosis ( 31). In our study, Bad protein was expressed in both Gal-3-deficient LNCaP cells and Gal-3-expressing LNCaP cells. The level of Bad was higher in the Gal-3-expressing cells ( Fig. 7 , compare lanes 3 and 1). Interestingly, treatment with CDDP had an opposite effect on the level of Bad in these cells; it decreased in Gal-3-expressing cells and increased in Gal-3-deficient cells. The level of phospho-Bad increased in Gal-3-expressing cells, whereas phospho-Bad expression was dramatically suppressed in Gal-3-deficient cells ( Fig. 7). Bcl-2, Bax, and phospho-MEK expression could not be evaluated because their levels in the LNCaP cells were very low (data not shown).
There is no effective treatment for patients with metastasized hormone refractory prostate cancer because this cancer is resistant to anticancer drugs, probably due to enhanced resistance to apoptosis. We hypothesized that Gal-3, which exhibits antiapoptotic effects in a variety of cells and renders resistance to various proapoptotic agents, may play a role in the drug resistance of prostate cancer. To test this hypothesis, we exploited our finding that LNCaP prostate cancer cells do not express Gal-3 constitutively, as opposed to other prostate cancer cell lines (e.g., DU145 and PC3; ref. 32). By transfecting the LNCaP cells with a Gal-3 expression vector, we were able to compare and contrast the response to proapoptotic agents of isogenic cells without and with Gal-3 protein and thereby assess the potential role and mechanism by which Gal-3 could affect the response to chemotherapeutic agents.
We report here that Gal-3 inhibited apoptosis induced by chemotherapeutic agents such as cisplatin and etoposide in prostate cancer cells, similarly to what we had observed previously in breast cancer cells ( 13). However, in the present study, we were able to obtain some clues about the mechanism of this effect, as we found that after CDDP treatment, Gal-3 was translocated from the nucleus to the cytoplasm and decreased the level of Bad expression, increased phosphorylation of Bad, and attenuated the depolarization of the mitochondrial membrane. Bad expression and its phosphorylation regulates Bcl-2 expression and the balance between proapoptotic and antiapoptotic proteins is critical to regulate mitochondria-induced apoptosis ( 30). Based on these data, we propose the following model for the mechanism by which Gal-3 can protect cells against proapoptotic anticancer drugs ( Fig. 8 ). Proapoptotic agents like CDDP induce DNA damage leading to apoptosis by decreasing phosphorylation of Bad and increasing Bad expression causing mitochondrial membrane depolarization, resulting in the release of cytochrome c and activation of caspase-3 (via activation of caspase-9). However, when cells express Gal-3, the treatment with CDDP induces translocation of Gal-3 from the nucleus to the cytoplasm. The increase in cytoplasmic Gal-3 then induced phosphorylation of Bad and suppresses Bad expression as well as its function (possibly acting like Bcl-2). Consequently, the mitochondrial membrane maintains its potential, the resulting cytochrome c is not released from the mitochondria, caspase-3 is not activated, and the cells are protected from undergoing apoptosis.
Our data provides the theoretical foundation for a new therapeutic target for improving chemotherapy of prostate cancer. Down-regulation of intracellular Gal-3 by antisense short hairpin RNA approaches may be a useful strategy to enhance the response of prostate cancer to proapoptotic anticancer drugs.
Grant support: CA46120 from the NIH/National Cancer Institute (A. Raz).
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
We thank Vivian Powell for manuscript preparation and editing, Victor Hogan for editing, and Akiko Yoshida for technical assistance.
- Received October 17, 2005.
- Revision received January 4, 2006.
- Accepted January 18, 2006.
- ©2006 American Association for Cancer Research.