text:
The Guardian for this meeting was Eliza Madrigal. The comments are by Eliza Madrigal.
Honestly, there is not much to add to this session. It was a lovely, playful session wherein Adams, Fael, and I talked about Enduring Love, Endless Compassion, and Seeing to the heart of things. What is left to say? Certainly nothing left to say for me, Eliza, who seems once again to have had way too much coffee. ;-)
Oh, well maybe that toward the end useful information about new email feature of the auto-loggger is given! Thanks Fael!
Eliza Madrigal: Hi Adams, Good Morning! :)
Adams Rubble: Good Morning Eliza :)
Eliza Madrigal: (and great shoes!)
Adams Rubble: Thanks, I like them :)
Adams Rubble: Finally a pair I really like
Adams Rubble: I wear them all the time (giggles)
Adams Rubble: they can change colors
Eliza Madrigal: Yes, we have our favorites I guess...even when we try not to.
Eliza Madrigal: Oh! That's convenient! :)
Eliza Madrigal: How are you doing this morning?
Adams Rubble: Oh, I am good, thanks
Eliza Madrigal: (Oo pink.. pretty)
Adams Rubble: How are you?
Eliza Madrigal: I'm rather thoughtful this morning actually....
Eliza Madrigal: have several things I'm turning over....
Adams Rubble listens
Eliza Madrigal: First, there is 'Compassion' as a general topic.... and the practice of seeing Kindness "everywhere"
Eliza Madrigal: Then, there is the idea of 'sin-consciousness', which as I learn and read more about certain practices, seems to come up.... and with the ethics discussion
Eliza Madrigal: And lastly, the 'approach' of Being
Eliza Madrigal: Which sounds funny as I phrase it that way
Adams Rubble: wow, that is a table full :)
Eliza Madrigal: yes, Indeed!
Eliza Madrigal: hehehe
Eliza Madrigal: Wanna pick one? Or perhaps wipe the table clean and throw out another? :)
Adams Rubble: I had the thought last night that at times it all seems very simple
Adams Rubble: I am wondering if those are the times we see best
Adams Rubble: I have been thinking much about compassion though
Adams Rubble: it seems to be the one thing that reduces our metal sufferings
Adams Rubble: more than anything else
Eliza Madrigal: Yes... it is like getting right to the Essence
Adams Rubble: I hope that is not going in too much of a different direction from what you are saying
Eliza Madrigal: Not at all!
Adams Rubble: Of couse I am influenced by my former Christian background and Paul
Adams Rubble: It seems that Love is the one place where my exdperience in PaB and Christianity come together
Eliza Madrigal: "Love never fails"
Adams Rubble: Love helps us see better
Adams Rubble: and yes about not failing :)
Eliza Madrigal: Yes.... everything will pass away....
Adams Rubble: only we fail (giggles)
Eliza Madrigal: Am hearing that Biblical phrasing that Paul uses to describe Love
Adams Rubble: yes :)
Adams Rubble: It is great!
Eliza Madrigal: "Though I speak with the tongues of men and angels and have not love it profits me nothing"
Adams Rubble: I still suffer
Eliza Madrigal: yes. Hmm
Eliza Madrigal: We struggle to 'attain' things... whether those things are knowledge or the illusion of safety....
Eliza Madrigal: and that is suffering
Adams Rubble nods
Adams Rubble: and notices Eliza has slipped in a little Buddhism :)
Adams Rubble: hehe
Eliza Madrigal: heheh... a little Buddhism has slipped into Eliza
Adams Rubble: :)
Adams Rubble admits "me too"
Eliza Madrigal: I think I've always read the Bible in a Buddhist way..
Eliza Madrigal: It is perhaps what made church difficult for me in my earlist 'spiritual discovery'years
Eliza Madrigal: *earliest
Eliza Madrigal: The other phrase that comes to mind... is "Love is stronger than death"
Adams Rubble: Hmmm
Adams Rubble: Yes
Adams Rubble: the legacy of love
Eliza Madrigal: That was a particularly nice place to pause wasn't it? :)
Eliza Madrigal: One angle I've been considering also, is that Love's Capacity is so great....
Adams Rubble listens
Eliza Madrigal: Perhaps we have people in our lives who are challenging...or we find ourselve challenging with....
Adams Rubble sees where Eliza is going
Eliza Madrigal: and if we surrender to Love (and I suppose this context Being), then we can 'actually' act with love
Adams Rubble: I am finding in my life that Love is filtering slowly but yes, in a way it is surrendering to Love
Eliza Madrigal: And it has nothing to do with 'anything that has happened'
Adams Rubble: falling into Love
Eliza Madrigal: Yes! Scary somehow. Marvelously Frightening.
Adams Rubble: as opposed to falling in love. hehe
Eliza Madrigal: !! :)
Eliza Madrigal: Conscious
Eliza Madrigal: Mindful
Eliza Madrigal: Awake
Adams Rubble: :)
Adams Rubble admits that I am not mindful most of the time
Adams Rubble: hopefully am conscious
Adams Rubble: but love CAN go bridge those times I think
Adams Rubble: "go" not supposed to be there
Eliza Madrigal: Yes.... I hear what you mean I think...
Eliza Madrigal: that we can't 'rely' on our'selves' to be consistent or mindful...we make more and more room perhaps
Adams Rubble: It seems to filter in slowly but once there it is fairly robust
Eliza Madrigal: It ripens :)
Adams Rubble: love that is
Adams Rubble: and stays
Eliza Madrigal: Love Endures
Eliza Madrigal: :)
Adams Rubble: :)
Adams Rubble: That's what I mean. hehe
Eliza Madrigal: yes :)
Adams Rubble: they come together
Adams Rubble: PaB and Christianity
Eliza Madrigal: Essentially.... the practice we intend to engage in here I think....
Eliza Madrigal: *is* love
Adams Rubble: Hello fael :)
Eliza Madrigal: We intend to accept and listen and learn and expand our capacities to Love
Eliza Madrigal: Hi Fael! :)
Eliza Madrigal: Oooo Pretty Faun
Fael Illyar: Hi Eliza, Adams :)
Fael Illyar smiles.
Eliza Madrigal: Fael, Adams and I are talking about Love this morning... Compassion :) Thoughts?
Fael Illyar: Just saying Love doesn't really bring up much :)
Fael Illyar: or, to be exacy, brings too much :)
Adams Rubble: ?
Eliza Madrigal: Yes! An Ocean.
Eliza Madrigal: And how does one talk about the ocean.... salt, water, movement... Or Bliss, Submergence, Emergence...
Eliza Madrigal: *does
Fael Illyar: Well, this came up... I prefer to love over not loving :)
Fael Illyar: to deny love is to deny yourself.
Eliza Madrigal: Hmm, to trap yourself in suffering
Eliza Madrigal: And others with you perhaps
Fael Illyar: yes, that might be one way to put it.
Fael Illyar: because, love doesn't have a target. It just is. If you try to deny it, you stop it from flowing through yourself.
Eliza Madrigal: A personal note... I realized recently that I was managing someone... or thought that I kept distance for one reason, when really... it was a punishment. It was very humbling to see that.
Eliza Madrigal: Allowing Love means seeing things in a different way often.
Adams Rubble: yes, it is connected to seeing from as different perspective
Fael Illyar: Yes... Allowing Love is also allowing yourself to see the truth.
Adams Rubble: they compliment one another
Eliza Madrigal nods... then freedom :)
Adams Rubble: seeing and compassion
Fael Illyar: I realized yesterday what has caused my tendency to leave many things to the last possible moment.
Eliza Madrigal: We arrive back and Biblical phrasing "You shall know/see the truth..."
Fael Illyar: (and with things without real deadlines, postponing them endlessly)
Eliza Madrigal: Yes Fael? That seems a big discovery.
Adams Rubble: someone once said deadlines are our best friend otherwise we would never get anything done
Eliza Madrigal: yesterday I heard "No one has a cleaner house than a writer on a deadline." hehe
Fael Illyar: The root at the bottom of it all was a fear of my father giving me a spanking if I did not force myself to do some things.
Adams Rubble: hehe
Eliza Madrigal: Ahhh
Adams Rubble: the hehe was meant for Eliza's statement
Adams Rubble: Yes, our parents can remain powerful forces in our lives
Eliza Madrigal: This created a resistence and rebellion Fael?
Fael Illyar: So, I was attempting to force myself to do certain things I thought should or must be done
Fael Illyar: and yes, this caused a rebellion that was the element that was on the surface and in my actions.
Eliza Madrigal: yes... you weren't allowed to have your own rhythm or way, in a sense.
Adams Rubble: great insight for you fael :)
Fael Illyar: rebellion against the "must" and "shoulds" I was having in me.
Fael Illyar: I only ever followed them when they became undeniable real practical necessities.
Adams Rubble: It is wonderful when we can do things because we enjoy them rather than they have to be done
Fael Illyar: Yes :)
Eliza Madrigal: Certainly
Fael Illyar: I cleaned my kitchen yesterday... feeling happy while doing it :)
Adams Rubble: :)
Eliza Madrigal: : )
Fael Illyar: it's quite shiny now :)
Fael Illyar: used to be a bit chaotic :)
Eliza Madrigal: A quote in my 1st life profile "We can do no great things, only small things with great love"
Eliza Madrigal: Mother Tereaa
Adams Rubble: :)
Eliza Madrigal: Teresa, rather
Fael Illyar: the rebellion could be put in other words as "trying to force myself to not force"
Eliza Madrigal: I think at some point, that rhythm and way of seeing things that we seem to be born with...
Eliza Madrigal: is going to emerge
Fael Illyar: It was quite a surprise to me to learn yesterday that I was actually attempting to force myself still :)
Adams Rubble: Old habits are hard to break
Eliza Madrigal nods
Adams Rubble: but they slowly fade away
Eliza Madrigal: When you can see through them clearly
Fael Illyar: yes, it ebbs and flows :)
Adams Rubble nods
Fael Illyar: I first managed to do something about this last summer when I got past an attachment to not having attachments :P
Eliza Madrigal: hehehhe....
Eliza Madrigal: is that like a fear of commitment?
Fael Illyar: but it seems to have reshaped itself and returned.
Fael Illyar: yes, fear of commitment is likely correct... because that'd cause me to force myself.
Adams Rubble: if we persist, each time around, the view is a little clearer :)
Eliza Madrigal: Ah. Yes!
Fael Illyar: Yes, feels like I actually understand the root now :)
Fael Illyar: still left to clean up the leaves and branches.
Adams Rubble: :)
Fael Illyar: I was especially surprised about the fear of my father giving me a spanking being at the bottom, considering the last one was around 20 years ago :)
Adams Rubble: it is like cleaning up real leaves and branches, it never stays clean :)
Eliza Madrigal: It is often many years before one is able to begin to see those things Fael
Adams Rubble: Fael, we can be cleaning up after our parents when we are in our 60s :)
Eliza Madrigal: heheheh
Fael Illyar: Yes, I've seen that :)
Eliza Madrigal: See above reference to person I was punishing :)
Fael Illyar: Yes :)
Adams Rubble nods
Adams Rubble: I am afraid I must run. I have some of those deadlines (giggles)
Eliza Madrigal: Bye Adams, thanks for stopping by!! :))
Adams Rubble: bye Eliza and fael...nice talking to you :)
Fael Illyar: See you later Adams :)
Fael Illyar: It's difficult to avoid the punishing ways.
Eliza Madrigal: Yes, and sometimes we are so short sighted about who we punish....
Eliza Madrigal: I punished the one who stayed... not the one who left. :)
Fael Illyar: Yes, sometimes it's even ourselves.
Eliza Madrigal: Indeed!!!
Fael Illyar: no-one suffers alone.
Eliza Madrigal: We are all connected in a very powerful way....
Fael Illyar: Yes
Eliza Madrigal: have been dwelling on this... I know it and yet I see it everwhere now... We are absolutely dependent
Eliza Madrigal: on others... on kindnesses...
Fael Illyar: Yes, especially because we so rarely give ourselves kindness.
Eliza Madrigal: this is true. It is part of our conditioning
Eliza Madrigal: When you talk about your childhood fears, that's what I hear...
Eliza Madrigal: we fall 'in line'
Fael Illyar: conditioning?
Fael Illyar: yes
Eliza Madrigal: yes. If you interrupt a pattern and lead it in a different direction all the time...
Eliza Madrigal: as with a child getting a spanking...
Eliza Madrigal: the tendency is 'set'
Eliza Madrigal: But that need to follow through with discovery and learning is incomplete
Fael Illyar: Yes and often we see what is wrong and try to fix it but overreact and go to the other extreme, like my case.
Fael Illyar: hmmh... I'm suddenly reminded of something my grandmother said often.
Eliza Madrigal: yes?
Fael Illyar: "A child must not be forced."
Eliza Madrigal: In earlier generations, it was considered loving to spank children... the 'real' world is harsh and one had to 'get ready'
Fael Illyar: one sided sentence that though...
Eliza Madrigal: Of course it is more complex.
Fael Illyar: yes, I remember a saying in Finnish to that effect.
Fael Illyar: to try to translate it would be "the one who saves the switch, hates his/her child"
Eliza Madrigal: I way overdid that (not forcing) with my oldest d. We would spend 3 hrs in the grocery store talking about everything and if there were circumstances that didn't enable that time, she would react strongly to that.
Eliza Madrigal: Fael, yes exactly!!
Fael Illyar: phone
Eliza Madrigal: :) No worries
Fael Illyar: ok, done
Fael Illyar: ah, yes, the other side of the coin is that if you go too far in that direction, you're mishandling yourself.
Eliza Madrigal: yes, it took many years to find a balance. I was a young mom and had many determinations to do everything 'differently'... start from scratch! Mostly, I'm happy about that ... must admit. :)
Eliza Madrigal: (Btw, Faun is great.. when you walked up I thought she was a separate avatar swimming in the sky approaching us :)
Fael Illyar: :)
Fael Illyar: Just something I got while fishing :P
Eliza Madrigal: Am thinking... great things can come from rebellion
Eliza Madrigal: Do you think so?
Fael Illyar: Yes, as long as it's not overblown :)
Fael Illyar: one way to characterize my no-forcing approach would be to shoot a fly with a nuke thinking it's something worse than a fly.
Eliza Madrigal: YES!
Eliza Madrigal: I think often that way of doing things... the saw to the butter sort of way... is a result of our disconnection from
Fael Illyar: well false no-forcing approach to be strict but :)
Eliza Madrigal: our true-er selves...
Eliza Madrigal: we are oveten severed from our own instincts
Eliza Madrigal: *often
Fael Illyar: Yes
Eliza Madrigal: Have you heard of the His Dark Materials books?
Eliza Madrigal: those came to mind using the word severed... and seeing Faun swimming around you :)
Eliza Madrigal: In those books, daemons are creatures connected to people... they are a vital part of them needed to be creative and rebellious...questioning and daring :)
Fael Illyar: :)
Fael Illyar: Yes, I guess it's accurate to say that daemons are creatures created by our imagination.
Eliza Madrigal: :) yes
Fael Illyar: people often do their best to transmit their own daemons to others.
Eliza Madrigal: well this is true too.... to force connection
Eliza Madrigal: rather than allow it to be or not be
Fael Illyar has catches herself doing that too.
Eliza Madrigal: we 'project'.. yup. Difficult not to
Fael Illyar: we 'project' them on others trying to shield ourselves.
Fael Illyar: when it's really our own daemon we need shielding from
Eliza Madrigal: perhaps... we need to 'make peace' with our own and everyone else's too... see through them
Fael Illyar: the daemon's certainly resist that peace hard.
Fael Illyar: in a way, it's actually the daemon trying to transmit itself to others by controlling us.
Eliza Madrigal: I'm thinking of a girl who was so harsh on the outside... for years and years... It was to cover deep pain. .. deep fear. Truly loving people I think... cut right through that. A skill to cultivate.
Fael Illyar: it does it by fooling us that we're defending ourselves.
Eliza Madrigal: Ah, yes!!!
Fael Illyar: Fear is what allows these daemons to control us.
Eliza Madrigal nods
Fael Illyar: and in a way, their food.
Eliza Madrigal: Ah... and wrapping back to the topic "Perfect love casts out fear"
Fael Illyar: Yes, Love is the best way to win fear.
Eliza Madrigal: hehhe... hope no one is offended with the Bibical references
Fael Illyar: Well, if they are, let them :) It's their daemon's being offended :)
Eliza Madrigal: I had thrown my Christianity in a closet for a long time, and Buddha gave it back. Hehhehe
Eliza Madrigal: Now it is just 'one thing' among many.... no closed doors
Fael Illyar: Yes, mine was in the closet too... still is to some extent but I've been taking it back out at times.
Eliza Madrigal: That seems to be the idea behind detachment
Fael Illyar: that? taking it out of the closet only sometimes?
Eliza Madrigal: Well... that everything is up for grabs....
Fael Illyar: sort of :)
Eliza Madrigal: nothing is overly important or unimportant
Eliza Madrigal: with regard to what we've learned or labeled ourselves
Eliza Madrigal: or others have labeled us :)
Eliza Madrigal: Not the clearest thought.. but something that seems to be about love and unity and connectedness
Eliza Madrigal: and acceptance
Eliza Madrigal: we put aspects of ourselves in the closet along with those labels we reject sometimes
Fael Illyar: labels have only the meaning you give to them.
Eliza Madrigal: yes indeed
Eliza Madrigal: So what are your plans now that the kitchen is clean Fael? :)
Fael Illyar: Well, my apartment overall still needs some work :)
Fael Illyar: now comes the problem of deciding what to do again. My list of things to do that I can do just grew a notch
Eliza Madrigal: ah, the 'that I can do' seems pertinent there?
Fael Illyar: Yes, the things I don't have a daemon blocking me from doing :)
Eliza Madrigal: :) give the daemon a job too ... hehe
Fael Illyar: they do have jobs ... the ones I've catched :)
Eliza Madrigal: heheh
Fael Illyar: although, I'm not sure if calling them daemons is suitable anymore.
Eliza Madrigal: yes, we may have exhasted that :)
Eliza Madrigal: exhausted*
Eliza Madrigal: hehe
Fael Illyar: the ones that are still controlling me instead of working for me are the ones preventing me from doing things :)
Eliza Madrigal: Sometimes we simply don't have the skills yet...
Eliza Madrigal: Sometimes all we can do is breathe and allow everything to unfold....
Eliza Madrigal: Most of the time actually.
Fael Illyar: Is that not the most important skill?
Eliza Madrigal: It feels great to do what we can do at the same time though!
Fael Illyar: interesting observation here... now that there is more sunlight here... it seems to be easier to deal with my daemons than during the winter when there was very little
Eliza Madrigal: That IS an interesting observation!
Eliza Madrigal: Light helps us see through
Fael Illyar: Yes, seems it does :)
Eliza Madrigal: :) Don't know why but I have Joni Mitchell playing in my head....
Eliza Madrigal: "its cloud illusions I recall... I really don't know love at all..."
Fael Illyar: I see...
The song is a little long to post in this rather long session. Here's a link though : http://www.lyricsfreak.com/j/joni+mitchell/both+sides+now_20075289.html
The New Auto-Logging System:
Fael Illyar: Did you notice the new session logs always in email functionality?
Eliza Madrigal: Do you mean that I've always gotten them?
Eliza Madrigal: I've never had a problem, but sometimes I stay online when posting the logs and writing during the day.... like to sit in a nice place... so usually don't have to rely on email.
Fael Illyar: Ah, no, before today, it sent them with object IMs that you get to your email only if you're offline when they're sent (and your offline IMs get emailed to you)
Eliza Madrigal: Ah... so now it will always send to email??
Fael Illyar: if you were online, they show up in local chat as a green line
Fael Illyar: and no email
Eliza Madrigal: yes, that is usually what I follow.
Fael Illyar: now you can register an email address and it will always send a message there too
Fael Illyar: is this your meeting?
Eliza Madrigal: That message I saw at the beginning.... it said to "say" something...somewhere...
Eliza Madrigal: Where is that? :)
Eliza Madrigal: (forgive my lack of tech savvy)
Fael Illyar: right here :)
Fael Illyar: on channel 17
Eliza Madrigal: yes, this is my slot now. :)
Fael Illyar: so if you say /17 firstname.lastname@example.com it will in the future try to send the log links to firstname.lastname@example.com
Fael Illyar: it will also send a test email right after you register an address
Fael Illyar: if you don't receive it, something doesn't work :)
Eliza Madrigal: Ahhhh! Thanks. I hope it doesn't have to be the same email that is registered for SL
Eliza Madrigal: Oh, how interesting....
Fael Illyar: no, it can be any email
Eliza Madrigal: it didn't show the email publicly
Eliza Madrigal: when I wrote it in chat
Fael Illyar: yes, channel 17 is not visible to people :)
Fael Illyar: just to scripts
Fael Illyar: that have decided to listen on it.
Eliza Madrigal: :)
Fael Illyar: only channel 0 is heard by other people
Fael Illyar: also, any green messages you saw with your email address, were messages only you saw :)
Eliza Madrigal: AH... layering
Eliza Madrigal: Thank you
Fael Illyar: also, for now since it's a testing period for the new script, it'll send you it both as email and as before.
Eliza Madrigal: great. I should have no worries. Shall I stop the log now and we will see?
Fael Illyar: if you wish :)
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Viewing Details:


text:
9.0 / 1
|Mario Fart
|One day wake up,mario brothers found their tools gone.In order to find these tools back,they learn fart skill from the king of fart legendary.This skill is make use of fart to push them fly up sky,so they begin a long trip.Be careful all kinds of traps、monster、Use your skills and various items get all the tools back.
|P1-AD to move,W to fart
P2-←→ to move,↑ to fart
Tip:Fart rhythmically allow you fly higher or stay in the air.
|Action Adventure
|fart mario

subset: commoncrawl


text:
Christi's still not feeling that great and much prefers the idea of reading and checking out the latest gossip on Facebook to exploring more local Inca ruins. Indeed, she warns me that I'll be Inca'd out before we even reach Machu Picchu. Perhaps, but who can resist a site called Sexy Woman (officially, Saqsaywaman), which is located on a steep hill to the north of Cusco. This fortress was built during Inca times to protect Cusco from attack. In addition, to being an interesting archeological site, this area offers tremendous views over Cusco and the giant graffiti-carved hills to the south of town.
Sexy Woman's most impressive features are, again, the terraces, although these were used purely for military purposes. Each of the three massive stone tiers rise to a height of almost 20 feet. The stones used in the construction of these terraces are among the largest of any pre-Columbian era building (weighing anything from 100 – 200 tons). The craftsmanship is also astonishing: the terraces were built without mortar and the stones are so closely spaced that a single sheet of paper won't fit between them. Indeed, these Inca structures are so sturdy they've largely survived the devastating earthquakes that frequently menace Cusco – unlike many Spanish buildings of the same period.
Now I may be mistaken here, but at a site as provocatively named as Sexy Woman, wouldn't you think some enterprising (and suitably clad) Peruvian gal would be willing to pose for photos – for a fee, of course. All I could find was an alpaca (a cousin of the llama) although I suppose it might be female. Difficult to tell with all that wool.
Blog post by Roderick Phillips, author of Weary Heart – a gut-wrenching tale of love and test tubes.


text:
Serena V-Neck Top
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Serena V-Neck Top
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Item# 2284
Product Details
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- Healing Hands Item #2284
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text:
If your program claims reimbursement from the MDE for GED Testing Hours, you have 2 options for recording the testing hours - as block time or as a special GED testing class. For a discussion of the two methods, see Overview: GED Hours for reimbursement.
Enter testing hours as block time
- On the Students tab, search for the student name; click the desired row in the results.
- On the Students>Time screen, complete these form fields:
- Date: Select the approximate date the hours were earned - it's OK to enter official GED hrs for all subjects at once if you don't need them split out by subject for your testing center.
- Contact: select GED Testing
- Enter number of GED testing hours in the box.
Enter testing hours in a special GED Testing Hours class
GED Testing hours are different from NRS contact (instructional) time. To learn how to create a special class for GED Testing only, see Create a GED Testing Hours Class.
To enter GED Testing hours in the special class:
- On the Classes tab, search for the GED Testing Hours class; click the desired row in the results.
- Click the Classes>Attendance tab.
- From the calendar, choose the appropriate date.
- For each student, edit the Start time by adjusting the menu. Then record the total number of official testing hours. Do not record instructional time here!
- When finished, click the Save Attendance button.


text:
STAT (signal transducers and activators of transcription) proteins are transcription factors which are activated by phosphorylation on tyrosine residues upon stimulation by cytokines. Seven members of the STAT family are known, including the closely related STAT5A and STAT5B, which are activated by various cytokines. Except for prolactin-dependent β-casein production in mammary gland cells, the biological consequences of STAT5 activation in various systems are not clear. We applied PCR-driven random mutagenesis and a retrovirus-mediated expression screening system to identify constitutively active forms of STAT5. By this strategy, we have identified a constitutively active STAT5 mutant which has two amino acid substitutions; one is located upstream of the putative DNA binding domain (H299R), and the other is located in the transactivation domain (S711F). The mutant STAT5 was constitutively phosphorylated on tyrosine residues, localized in the nucleus, and was transcriptionally active. Expression of the mutant STAT5 partially dispenses with interleukin 3 (IL-3) as a growth stimulant of IL-3-dependent cell lines. Further analyses of the mutant STAT5 have demonstrated that both of the mutations are required for nuclear localization, efficient transcriptional activation, and induction of IL-3-independent growth of an IL-3-dependent cell line, Ba/F3, and have indicated that a molecular basis for the constitutive activation is the stability of the phosphorylated form of the mutant STAT5.
Stimulation of cytokine receptors leads to activation of multiple signal transduction pathways, including the Ras-Raf-MEK-mitogen-activated protein kinase (MAPK) and the JAK-STAT pathways (14, 28, 34, 42, 44). The latter signaling pathway was originally found downstream of the interferon receptors and is now recognized as a common pathway downstream of most cytokine receptors. Upon stimulation with cytokines, receptor-associated JAKs are activated and phosphorylate STAT factors on tyrosine residues. The phosphorylated STAT molecules then form homo- or heterodimers through SH2-mediated interactions and translocate into nuclei to activate transcription of various target genes. Seven members of the STAT family (STAT1 through 4, -5A, -5B, and -6) are known; STAT5A and STAT5B are closely related. With the exception of STAT4 and STAT6, which were shown to be specifically activated by only one or two cytokines, interleukin 12 (IL-12) or both IL-4 and IL-13, respectively (13, 15), most of the other STATs are activated by multiple cytokines. In particular, both STAT5A and STAT5B are activated by numerous cytokines, including prolactin, IL-2, IL-3, IL-5, IL-7, granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF, M-CSF, erythropoietin (Epo), thrombopoietin, and growth hormone (GH).
Using the receptor for the human GM-CSF as a model system, members of our group previously showed that activation of the Ras-Raf-MEK-MAPK pathway inhibits apoptosis while the region of the GM-CSF receptor, which is responsible for activation of JAK2 and STAT5 and induction of c-myc expression, plays a role in DNA synthesis (18, 41). Members of our group also demonstrated that a dominant negative STAT5 protein partially inhibited IL-3-induced cell proliferation in Ba/F3 cells, suggesting that STAT5A is involved in cell proliferation (29). A similar conclusion was reached by others who observed that IL-2 and Epo receptors defective in STAT5 activation (5, 7, 9) were also deficient in supporting a proliferative signal. In contrast, others have concluded that STAT5 is not involved in cell proliferation since mutant IL-2 (8) and Epo receptors (37) which cannot activate STAT5 are still capable of transmitting an attenuated proliferative signal. These differing interpretations of generally similar data indicate a point of controversy. Recently, mice nullizygous for STAT5A and STAT5B have been generated (24, 45). Since these mice exhibit apparently normal hematopoiesis, the contribution of STAT5 activation to proliferation or to any other biological function, such as differentiation in normal hematopoietic tissues, remains unclear.
In this paper, we report the identification and characterization of a constitutively active form of STAT5 by PCR-driven mutagenesis followed by retrovirus-mediated expression screening. The mutant STAT5 protein is constitutively phosphorylated on its tyrosine residues, is localized in the nucleus, and is transcriptionally active in the absence of growth factor stimulation. Expression of the mutant STAT5 in IL-3-dependent cells is capable of inducing cytokine-independent growth.
Cell lines.Mouse IL-3 (mIL-3)-dependent Ba/F3 cells were maintained in RPMI 1640 medium containing 10% fetal calf serum (FCS) and 1 ng of recombinant mIL-3 produced in silk worm/ml (26). An ecotropic retrovirus packaging cell line, BOSC23 (35), was maintained in Dulbecco's modified Eagle's medium containing 10% FCS and guanine phosphoribosyltransferase (GPT) selection reagents (Specialty Media). The cells were transferred into Dulbecco's modified Eagle's medium–10% FCS without GPT selection reagents 2 days before transfection.
Retrovirus vectors.A retrovirus vector, pMX, was constructed as previously described (31). Its derivative pMX-puro vector harbors a simian virus 40 early promoter-driven puromycin resistance gene between the multicloning site and the 3′ long terminal repeat of the pMX vector (16). cDNAs for mouse STAT5A and STAT5B (28) were inserted into theEcoRI and NotI sites of the pMX and the pMX-puro vectors.
Production of retroviruses carrying STAT5A sequences with random mutations and infection with these viruses.To introduce random mutations into the STAT5A sequence, a PCR was run for 35 cycles (1 min at 94°C, 2 min at 58°C, and 3 min at 72°C) under standard conditions described previously (31) except that the deoxynucleoside triphosphate concentration was 400 μM. The pMX-STAT5A plasmid was used as a template, and a 5′ vector primer, pMX5′ (1) (5′CCCGGGGGTGGACCATCCTCT3′), and a 3′ vector primer, pMX3′ (1) (5′CCCTTTTTCTGGAGACTAAAT3′), were used to amplify the full-length sequence of STAT5A. The average frequency of point mutations ranged from 1/600 to 1/1,200 under these conditions (data not shown). The PCR product was digested with restriction endonucleases EcoRI and NotI, and the excised 2.4-kbp STAT5A sequences harboring the PCR-generated mutations were ligated to the EcoRI and NotI sites of the pMX vector. The mixture of ligated plasmids was amplified inEscherichia coli DH5α, and plasmid DNA was prepared with a Maxi prep kit (Qiagen) to be used as a library of mutated STAT5A sequences in the pMX vector. High-titer retroviruses containing the randomly mutated STAT5A sequences were produced from the library DNA with a transient retrovirus packaging cell line, BOSC23 (35), as described previously (19). For infection, Ba/F3 cells (2 × 106) were incubated with 5 ml of the retroviruses harboring the mutated STAT5A sequence library in the presence of 10 μg of Polybrene/ml and 10 ng of mIL-3/ml. After 8 h, 5 ml of fresh growth medium was added to the culture, and the incubation was extended for 16 h. Twenty-four hours after infection, cells were washed, refed with growth medium, and allowed to grow for one more day before being subjected to selection in the absence of mIL-3 in 96-well plates. Under these conditions, the efficiency of infection of Ba/F3 cells was 20 to 40% as assessed by parallel infections with the test construct pMX-hIL-3Rα (31).
Recovery and reintroduction of the integrated retroviruses.In order to recover retrovirally transduced STAT5A sequences, genomic DNA was isolated from each factor- independent (FI) clone, and the integrated STAT5 sequences were amplified from 10 ng of genomic DNAs by PCR with the retroviral vector primers described above. The PCR was run for 35 cycles as described above with Taq polymerase (Perkin-Elmer Cetus) under standard conditions with 100 μM deoxynucleoside triphosphates. The recovered PCR fragments were purified from the gel with QiaexII (Qiagen), digested withEcoRI and NotI, and inserted into theEcoRI and NotI sites of the pMX vector. The STAT5A sequence subcloned into the pMX vector was analyzed withTaq DyeDeoxy Terminator (Applied Biosystems) on an Applied Biosystems model 373A sequencer. The STAT5A sequences recovered from FI Ba/F3 cells were subcloned into pMX and reintroduced into parental Ba/F3 cells via retrovirus infection to confirm the ability of the integrated STAT5A sequences to induce FI in Ba/F3 cells.
Cell proliferation assay.A cell proliferation assay was performed with cells (either bulk population or subclones) which had been stably transduced with the wild-type or the mutant STAT5 sequences and then selected by puromycin resistance, except for one experiment whose results are shown in Fig. 2. In the experiment whose results are shown in Fig. 2, we examined FI proliferation of the Ba/F3 cells in the bulk population after the transduction of STAT5 constructs via retrovirus infection (infection efficiency was 30 to 50%) without drug selection. In this particular experiment, after transducing with the wild-type and the mutant STAT5A and STAT5B sequences by using retrovirus constructs, we seeded the transduced Ba/F3 cells in the absence of mIL-3 and the growth curves were determined. Cell proliferation was quantitated either by counting the cells (see Fig. 1B and D, Fig. 2, and Fig. 6B) or by using the Alamar blue assay (see Fig.6A) as described previously (1). For the Alamar blue assay, 2 × 104 Ba/F3 cells were suspended in 100 μl of medium and were cultured in 96-well microtiter plates for 48 h at 37°C in the presence or absence of mIL-3. Ten microliters of Alamar blue solution (Alamar Biosciences Inc.) was then added to each well, and the incubation was extended for another 24 h. The optical density was measured with an enzyme-linked immunosorbent assay microplate reader (Bio-Rad) with a test wavelength of 570 nm and a reference wavelength of 600 nm.
Immunoprecipitation and Western blotting.Immunoprecipitation, gel electrophoresis, and immunoblotting were performed according to methods described previously (46), with minor modifications. Exponentially growing cells were washed free of serum and growth factors and incubated in RPMI 1640 supplemented with 0.5% bovine serum albumin (BSA) for 12 h at 37°C. After the depletion period, cells were resuspended in the same medium at a concentration of 4 × 106/ml and were stimulated with mIL-3 or FCS at 37°C for 10 min. Cells were lysed in lysis buffer (4 × 106 cells/ml) and incubated on ice for 30 min. Cell lysates were clarified by centrifugation for 15 min at 20,000 ×g before incubation with the anti-STAT5A polyclonal antibody (R & D Systems) or the anti-Flag antibody (Eastman Kodak) and protein A-Sepharose at 4°C overnight. The immunoprecipitates were washed three times with lysis buffer, subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and electrophoretically transferred onto Immobilon filters (Millipore). After blocking in solution containing 3.0% BSA, the filters were probed with anti-phosphotyrosine antibody 4G10 (Upstate Biotechnology Inc.), stripped, and reprobed with the anti-STAT5 antibody to verify the loading amount. The filter-bound antibody was detected by the enhanced chemiluminescence system (Amersham).
Luciferase reporter assay.Ba/F3 cells were transiently transfected by electroporation at 960 μF and 300 V with 10 μg of a reporter plasmid carrying a luciferase gene under the control of the β-casein promoter, 3 μg of a β-galactosidase reporter plasmid with the Rous sarcoma virus long terminal repeat promoter, and 10 μg of test DNAs at room temperature in RPMI 1640 supplemented with 10 μg of DEAE-dextran/ml. After a 12-h recovery period in the IL-3-containing medium, cells were incubated in RPMI 1640 supplemented with 0.5% BSA for 12 h, or stimulated with 10 ng of mIL-3/ml for the last 6 h before cell lysates were prepared. Cell lysates were then subjected to luciferase (Promega) and β-galactosidase (Tropix) assays. Transduction efficiency under each condition was normalized with β-galactosidase activity.
EMSA.Nuclear extracts, prepared by the Nonidet P-40 method, were mixed with radiolabeled double-stranded oligonucleotides corresponding to the prolactin-responsive element (PRE) in the bovine β-casein promoter as described previously (46). Electrophoretic mobility shift analysis (EMSA) was performed with 8 μg of nuclear extracts in 20 μl of a reaction mixture containing 12 mM HEPES (pH 7.9), 5% glycerol, 75 mM NaCl, 1 mM EDTA, 0.1% Nonidet P-40, 1 mg of BSA/ml, 1 mg of poly(dI-dC)/ml, and 10 fmol of radioactive PRE. The mixture was incubated at room temperature for 30 min, and 5 μl was placed onto a polyacrylamide gel (0.25 × Tris-borate-EDTA). For supershift experiments, 1 μg of the anti-STAT5 polyclonal antibody (R & D Systems) was added to the reaction mixture.
Construction and expression of STAT5A-EGFP fusion molecules in NIH 3T3 cells.A mutant green fluorescent protein (GFP), EGFP, was fused to the C termini of the wild-type STAT5A, STAT5A1, STAT5A6, and STAT5A1*6 with pEGFP-N1 (Clontech), and theEcoRI-NotI fragment encoding the STAT5-GFP fusion molecule was inserted into the pMX vector. The resulting constructs (pMX-STAT5A-EGFP, pMX-STAT5A1-EGFP, pMX-STAT5A6-EGFP, and pMX-STAT5A1*6-EGFP) were expressed in NIH 3T3 cells via retrovirus infection as described previously (19). Two days after infection, the NIH 3T3 cells were observed under a fluorescence microscope (IX 70; Olympus).
Stability of phosphorylated wild-type and mutant STAT5 proteins in Ba/F3 transfectants expressing Flag-tagged wild-type and mutant STAT5A.To distinguish the endogenous and the transduced STAT5 molecules, the Flag tag was added to the C termini of the wild-type and the mutant STAT5s. The sequences for the Flag-tagged STAT5s were inserted into the EcoRI and NotI sites of the pMX-neo vector with a construct by Wang et al. (47), and Ba/F3 stable transfectants expressing the Flag-tagged STAT5s were established after gene transduction via retrovirus infection followed by selection in the presence of 1 mg of G418 (Gibco-BRL)/ml. The Ba/F3 transfectants expressing the STAT51*6-Flag were cultivated in the absence of IL-3 after the establishment of transfectants because the expression of the STAT51*6-Flag decreased over time in the media containing IL-3, probably due to a growth-inhibitory effect of the STAT51*6-Flag when stimulated with IL-3 (30). The stable transfectants were deprived of mIL-3 for 12 h in the presence of 10% FCS, stimulated with 10 ng of IL-3/ml for 30 min, washed with medium, and cultured for various time periods. The cell lysates were prepared before IL-3 stimulation and 0, 0.5, 1, 2, 4, and 8 h after the stimulation. The cell lysates were subjected to immunoprecipitation and Western blotting to examine the stability of the phosphorylated STAT5 molecules.
Establishment of Ba/F3 transfectants expressing both an inducible form of Raf, ΔRaf-ER, and either the wild-type or the mutant STAT5A.We first attempted to establish Ba/F3 transfectants expressing an inducible form of Raf, ΔRaf-ER (40). This protein consists of the catalytic domain of human c-Raf-1 (ΔRaf) and the hormone-binding domain of the estrogen receptor (ER). Addition of β-estradiol or its analog ICI 182 780 (ICI) (33) to cells expressing ΔRaf-ER leads to immediate activation of the Raf-MAPK cascade. We introduced ΔRaf-ER with a retrovirus construct harboring the G418 resistance gene and established several clones in the presence of 1 mg of G418 (Gibco-BRL)/ml. We chose two clones (BR4 and BR6) in which the expression of ΔRaf-ER and the activation of MAPK by ICI were confirmed by Western blot analysis with anti-Raf antibodies (Signal Transduction Lab) and the MAPK assay with the myelin basic protein as a substrate (data not shown). We then introduced pMX-puro-STAT5A or pMX-puro-STAT5A1*6 into BR4 cells via retrovirus infection and isolated several clones as well as bulk cells expressing the transduced STAT5A sequences in the presence of 1 μg of puromycin/ml.
Identification of mutant STAT5s which induce FI growth of Ba/F3 cells.We attempted to identify constitutively active forms of STAT5A by PCR-mediated mutagenesis followed by a high-efficiency retrovirus expression screening system (19, 32). Briefly, random mutations were introduced into the STAT5A sequence by PCR, and the mutated STAT5A sequences were expressed in an mIL-3-dependent cell line (Ba/F3) with retrovirus vector pMX (31) and a transient retrovirus packaging cell line, BOSC23 (35). Ba/F3 cells infected with these viruses were then screened for IL-3-independent growth, and 13 IL-3-independent clones were isolated. The integrated STAT5A-coding sequences in FI clones were recovered by PCR with vector primers. To confirm the ability of the mutant forms of STAT5A to induce IL-3-independent proliferation of Ba/F3 cells, the PCR fragments were subcloned into pMX, reintroduced into Ba/F3 cells, and analyzed for their ability to confer FI growth. The recovered STAT5A sequences from six FI clones but not the other seven FI clones efficiently reproduced the FI phenotype in Ba/F3 cells. Clone 5 showed the best phenotype and was chosen for further experiments. Several point mutations were found in the STAT5A sequence recovered from Ba/F3 FI clone 5; three independent PCR-recovered DNA subclones designated 5-1, 5-2, and 5-3 are shown in Fig. 1A. Of these subclones, only 5-1 induced FI when reintroduced into naive Ba/F3 cells (Fig. 1B and data not shown). The other two clones (5-2 and 5-3) had several point mutations in common with subclone 5-1 (i.e., point mutations 1 and 6); however, 5-2 and 5-3 also harbored additional point mutations, including a nonsense mutation, which were probably generated during the recovery of the integrated STAT5A sequence by PCR (Fig. 1A). Common point mutation 1 was located in the STAT transactivation domain and caused an amino acid change from serine to phenylalanine (S711F). Point mutation 6, on the other hand, was located upstream of the putative DNA binding domain and resulted in an amino acid change from histidine to arginine (H299R). In the Ba/F3 stable transfectant expressing the mutant STAT5A (5-1), the mutant STAT5A was constitutively phosphorylated at tyrosine residues (Fig. 1C), but constitutive activation of JAK2 and MAPK were not observed (data not shown).
Two mutations were required for constitutive activation of STAT5A.To clarify which of the point mutations observed in subclone 5-1 was responsible for IL-3-independent growth of Ba/F3 cells, we constructed retroviruses encoding STAT5A with each single point mutation alone and assessed their ability to promote IL-3-independent proliferation of Ba/F3 cells. The sequences for these STAT5A mutants were subcloned into the pMX-puro retrovirus vector, and Ba/F3 cells transduced with these constructs (infection efficiency was 30 to 50%) were selected with 1 μg of puromycin/ml for a week. The STAT5A mutants harboring either mutation 1 (STAT5A1) or 6 (STAT5A6) alone could not support long-term growth of Ba/F3 cells (data not shown). Since the original subclone 5-1 gave rise to the FI phenotype, we reconstructed the double mutant STAT5A1*6 to examine the effect of combining the two point mutations. Interestingly, expression of the double mutant STAT5A1*6 in Ba/F3 cells induced IL-3-independent proliferation in Ba/F3 stable transfectants (BaF/5A1*6) (Fig. 1B) and was able to support long-term growth of the transfectants in the absence of IL-3 (Fig. 1D). In Fig. 1B, continuous proliferation of the BaF/5A1*6 transfectant, which is similar to that of the Ba/F3 transfectant expressing the original subclone 5-1 (BaF/5-1), is shown. Expression of STAT5A1*6 also induced FI growth of other IL-3-dependent cell lines, such as TF-1 (20) and OTT-1 (12) (data not shown). In contrast, introduction of the wild-type STAT5A never gave rise to FI growth (Fig. 2). To exclude the possibility that the growth of BaF/5A1*6 cells was mediated through an autocrine mechanism, we examined whether growth-promoting activity was present in the supernatant of BaF/5A1*6 cells. As shown in Fig. 1D, the supernatant of BaF/5A/1*6 cells did not have any growth-promoting activity for the parental Ba/F3 cells.
The same mutations in STAT5B have similar biological effects.STAT5B is 95% identical to STAT5A at the amino acid level (22, 23, 28), and although expression of STAT5B has been shown to be critical in the GH-GH receptor system (45), little is known regarding the functional difference between STAT5A and STAT5B in hematopoietic cells. Since the amino acid sequences surrounding the mutations (mutations 1 and 6) were conserved in STAT5A and STAT5B but not in other STAT proteins, we introduced the same mutations into STAT5B and examined the ability of the mutant STAT5B to induce FI. As shown in Fig. 2, expression of the mutant forms of STAT5A or STAT5B rendered Ba/F3 cells FI with the same efficiency, suggesting functional similarity between STAT5A and STAT5B. This result also confirms the efficacy of these mutations in the constitutive activation of STAT5 molecules.
Characterization of the mutant STAT5A molecules.To examine the mechanistic basis of STAT5A activation by the two mutations, we characterized the biochemical properties of the mutant STAT5As expressed in bulk populations of puromycin-selected Ba/F3 cells transduced with sequences for wild-type STAT5A, STAT5A1, STAT5A6, or STAT5A1*6. In cells depleted of mIL-3 and serum for 12 h, tyrosine phosphorylation was detected on STAT5A1 and STAT5A1*6 but not on the wild-type or STAT5A6 protein. The level of phosphorylation was, however, much weaker than that resulting from IL-3 stimulation (Fig.3A). Consistent with these results, STAT5A1 and STAT5A1*6, but not the wild-type STAT5A or STAT5A6, exhibited weak but significant DNA binding activity without IL-3 stimulation, as assessed by EMSA (Fig. 3B). Interestingly, in a transactivation assay in which Ba/F3 cells were cotransfected with various STAT5 constructs and a STAT5-responsive luciferase reporter plasmid (46), all three mutant STAT5As were able to induce luciferase activity in the absence of IL-3 stimulation (Fig. 3C). Transactivation by the mutant STAT5A1*6 was strongest, and its constitutional transcriptional activity was even stronger than that of the wild-type STAT5A stimulated by IL-3 (Fig. 3C).
Subcellular localization of the wild-type and the mutant STAT5As was also assessed by using STAT5-EGFP fusion constructs (Fig.4). While the wild-type STAT5A-EGFP as well as STAT5A1-EGFP and STAT5A6-EGFP did not show any particular localization in most NIH 3T3 cells (except for a few in which STAT5-GFP proteins localized to the nucleus) (Fig. 4A to C), the mutant STAT5A1*6-EGFP localized to the nucleus in most NIH 3T3 cells (Fig.4D).
Stability of the phosphorylated forms of the wild-type and the mutant STAT5As.To further elucidate the molecular basis for the activation of the mutant STAT5s, we established Ba/F3 transfectants expressing Flag-tagged wild-type STAT5A, STAT5A1, STAT5A6, and STAT5A1*6. These cells were deprived of mIL-3 for 12 h and stimulated with 10 ng of mIL-3/ml for 30 min, and the tyrosine phosphorylation was chased for 8 h. As shown in Fig.5, phosphorylation of STAT5A1*6 after IL-3 stimulation was much stronger and more stable than that of the wild-type STAT5A, STAT5A1, and STAT5A6, whose tyrosine phosphorylation started to decrease 2 h after IL-3 stimulation and returned to the basal level in 4 h. Significant tyrosine phosphorylation of the Flag-tagged STAT5 was observed only in the cells expressing the Flag-tagged STAT51*6 before IL-3 stimulation (Fig. 5, lower right panel, first lane). The result shown in Fig. 5 is inconsistent with the result shown in Fig. 3A, where STAT5A1*6 as well as STAT5A1 mutants showed constitutive phosphorylation. This inconsistency may be due to the addition of the Flag-tag to STAT5 in the experiment whose results are shown in Fig. 5.
Cooperation between the mutant STAT5s and the Raf-1 protein.Although both mutant STAT5A and STAT5B induced IL-3-independent growth of Ba/F3 cells, these cells still required FCS for proliferation. This observation indicates that an additional signal(s) supplied by the serum is required to support cell proliferation. Since previous results suggested that both the JAK-STAT and the Raf-MAPK pathways are necessary to stimulate cell proliferation (18, 29), we reasoned that the signal missing under the serum-free conditions may be complemented by the activation of the Raf-MAPK pathway. To test this possibility, we expressed an inducible form of Raf, ΔRaf-ER (40), together with the wild-type or mutant STAT51*6 in Ba/F3 cells.
The biological effects of coexpression of ΔRaf-ER and STAT5A were assessed both in clones (Fig. 6A) and in bulk cultures (Fig. 6B) of drug-selected Ba/F3 cells transduced with ΔRaf-ER and either the wild-type or mutant STAT5A. A Ba/F3 clone expressing only ΔRaf-ER, designated BR4, did not exhibit IL-3-independent growth in response to ICI treatment. However, BR4 subclones expressing relatively higher levels of the mutant STAT5A1*6 (M2, M3, M4, and M6) proliferated without IL-3 and serum in the presence of ICI (in a dose-dependent manner), while clones (M1 and M5) which expressed lower levels of the mutant STAT5A1*6 failed to proliferate (Fig. 6A). On the other hand, expression of the wild-type STAT5A at levels much higher than that observed for clones expressing the highest levels of mutant STAT5A (compare W1 and W2 with M2, M3, M4, and M6) did not proliferate in response to ICI (Fig. 6A). To address the possibility of clonal differences among the infected BR4 subclones, we also tested bulk cultures of puromycin-selected cells for ICI-dependent, IL-3-independent growth (Fig. 6B). A bulk population of BR4 cells transduced with the sequence for the mutant STAT5A1*6 followed by selection with puromycin proliferated in the presence of ICI. However, ICI did not stimulate the proliferation of BR4 cells and BR4 cells expressing the wild-type STAT5A. Similar results were obtained in the experiment with another ΔRaf-ER-transduced Ba/F3 clone, BR6 (data not shown). These results showed that activation of neither the Raf-MAPK pathway nor the mutant STAT5A by itself can support proliferation of Ba/F3 cells under serum-free conditions but that activation of the two together induces cytokine- and serum-independent proliferation of Ba/F3 cells.
Activation of Raf had no effect on DNA binding activity of the mutant STAT5A.To test the direct effects of the activation of the Raf-MAPK pathway on the DNA binding activity of the wild-type and the mutant STAT5A, we examined nuclear extracts prepared from BR4, W1, and M6 cells in an EMSA with the PRE as a probe (46) (Fig.7). Nuclear extract from M6 cells contained DNA binding activity, albeit at a lower level than that observed in IL-3-stimulated extracts, which was supershifted by anti-STAT5A antibody. Activation of ΔRaf-ER by ICI had no detectable effect on DNA binding activity of the double mutant, M6, suggesting that activation of the Raf-MAPK pathway is not important for DNA binding activity of the mutant STAT5. Similar results were obtained when bulk selected cells were used for the experiment (data not shown).
Using PCR-driven random mutagenesis followed by retrovirus-mediated expression screening, we identified a constitutively active form of STAT5 harboring two point mutations. In addition to being constitutively phosphorylated at tyrosine residues, the mutant STAT5A1*6 is capable of binding a target DNA sequence, stimulating transcription of a reporter plasmid, and supporting FI proliferation of IL-3-dependent cell lines (summarized in Table1). Constitutive activation of the mutant STAT5A is also associated with nuclear translocation of the molecule, as demonstrated by the experiment in which the mutant STAT5A1*6-EGFP fusion molecule was expressed in NIH 3T3 cells. The observation that a constitutively active STAT5A mutant promotes cell proliferation is consistent with the previous observation that a dominant negative form of STAT5 partially inhibits IL-3-driven cell proliferation (29). However, the property of the mutant molecule does not necessarily reflect that of the wild-type molecule. For instance, while v-abl and bcr-abl are highly mitogenic, there is no evidence to suggest that c-abl is required for proliferation (38). Thus, it is possible that the constitutively active mutation detected in STAT5A is a gain-of-function mutation. Therefore, whether STAT5 activation is physiologically involved in cell proliferation is still open to question.
Previous studies of cytokine receptor signaling have defined at least two pathways important for proliferation: the JAK-STAT and the Raf-MAPK pathways (18, 41). It was also shown that serum by itself weakly stimulated the Raf-MAPK pathway (18). On the other hand, in sharp contrast to the mutant STAT5, the constitutively active form of MPL which was able to stimulate both the JAK-STAT and the Raf-MAPK pathways could induce FI growth even in the absence of FCS (32). From these results, we predicted that the mutant STAT5A would collaborate with the Raf-MAPK pathway to support cell growth. To test this possibility, we transduced either the wild-type STAT5A or the mutant STAT5A1*6 in BR4 cells expressing the ΔRaf-ER construct. As expected, estrogen-induced Raf activation synergized with expression of the mutant STAT5A1*6, but not with the wild-type STAT5A, to induce cell proliferation in the absence of both IL-3 and serum.
Although it is generally accepted that both Myc and Ras-Raf are required for cell proliferation, the mutant STAT5A in concert with Raf was apparently able to stimulate cell proliferation without significant induction of the Myc protein. The doubling time of proliferation driven by the mutant STAT5A and Raf (24 h), however, is longer than that of IL-3-driven proliferation (12 h). One possible explanation for this difference is inadequate expression of c-myc in Ba/F3 cells expressing the mutant STAT5A and/or ΔRaf-ER. Since the expression of c-myc is tightly correlated with proliferation in many systems and is efficiently induced by IL-3, it will be interesting to test whether ectopic Myc expression can complement activation of STAT5A and Raf to yield maximal proliferation.
The same two point mutations in the corresponding region of STAT5B also induced IL-3-independent proliferation of Ba/F3 cells. This result further substantiates the significance of these mutations. In order to investigate how these two mutations constitutively activate STAT5, we constructed mutant STAT5A molecules possessing each mutation (mutation 1 or 6). Mutation 1, but not 6, was by itself able to induce constitutive phosphorylation of STAT5A and constitutive binding of STAT5A to the target DNA sequence. However, for efficient transcriptional activation, nuclear localization, and cell growth, both mutations were required (summarized in Table 1). The fact that mutation 1 (S711F) results in constitutive DNA binding activity is reminiscent of the behavior of C-terminally truncated STAT5s (2, 27, 47) that exhibited delayed dephosphorylation and sustained DNA binding. The phenotype displayed by proteins with these different alterations in the C-terminal region may thus be mechanistically related, but it is important to note that these C-terminal truncations resulted in an overall dominant negative phenotype. Alternatively, mutation 1 may somehow facilitate dimerization of STAT5 because of its close proximity to the C-terminal tyrosine (Y694 of STAT5A or Y699 of STAT5B), which becomes phosphorylated in response to cytokine stimulation and mediates STAT5 dimerization (10). These two possibilities are not mutually exclusive, because dimerization of STAT5 should facilitate DNA binding. On the other hand, it is mysterious that the N-terminal mutation 6 (H299R) is also capable of inducing constitutive transcriptional activity although this mutant lacks constitutive phosphorylation at tyrosine residues and did not show DNA binding activity without IL-3 stimulation. This discrepancy may be explained by the difference in the sensitivities of the assay systems (luciferase assay versus Western blotting and EMSA). We hypothesized that mutation 6 was involved in stabilization of the phosphorylated form of STAT5 since it was reported that the N-terminal part of STAT1 was required for its dephosphorylation (43). In addition to inhibiting dephosphorylation, mutation 6 may also interfere with other mechanisms of down-regulation, such as ubiquitin-proteasome-mediated degradation (17) or proteasome- and p53-dependent protein masking (39); these mechanisms were shown to be involved in regulation of activated STAT1 or STAT3 and STAT5, respectively. To test this hypothesis, we expressed Flag-tagged wild-type STAT5 and mutant STAT5s. However, STAT5A6 did not show any difference in the stability of phosphorylated STAT5A after IL-3 stimulation, and both mutations 1 and 6 are required for the stability of the phosphorylated STAT5. The extremely high degree of transcriptional activation by the mutant STAT51*6 after IL-3 stimulation (Fig. 3C) is probably due to the stability of the phosphorylated STAT5A1*6 (Fig. 5). In summary, the present data strongly indicate that the mechanism of constitutive phosphorylation and activation of the mutant STAT5A1*6 is the stability of the phosphorylated mutant STAT5A1*6.
Constitutive activation of STATs in patients' leukemic cells has been reported by several groups (11, 48). STAT activation has also been demonstrated in transformed cells, such as human T-cell leukemia virus type 1-infected cells (25) and cells expressing v-src (3), v-abl(6), or bcr-abl (4). While activation of STATs is usually associated with concomitant activation of JAKs or other kinases, we observed phosphorylation of mutant STAT5 without detectable activation of JAKs. Recently, a TEL-JAK2 fusion protein has been implicated in leukemogenesis in three patients with leukemia (21, 36). Lacronique et al. have also shown that the TEL-JAK2 fusion protein can induce constitutive activation of STAT5 in Ba/F3 cells (21). Therefore, it would be interesting to test whether mutation of STAT5 could also be involved in leukemogenesis. Experiments with patients' leukemic cells and transgenic mice expressing the STAT5A1*6 mutant are in progress.
We have shown in this paper the mechanism by which the mutant STAT5 is activated. To clarify whether STAT5 activation is involved in cell proliferation as well as in other biological processes, such as differentiation, it is necessary to identify the target gene(s) whose expression can promote cellular proliferation.
Tetsuya Nosaka, Kazuhide Misawa, Atsushi Miyajima, and Toshio Kitamura were partly supported by the Ministry of Education, Science and Culture of Japan. DNAX Research Institute is supported by the Schering-Plough Corporation. The Department of Hemopoietic Factors was supported in part by Chugai Pharmaceutical Company Ltd.
We thank James Ihle for the STAT5-Flag plasmid construct.
- Received 1 December 1997.
- Returned for modification 27 January 1998.
- Accepted 31 March 1998.
- Copyright © 1998 American Society for Microbiology


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John, that image is VERY DISTURBING in that this dude looks just like CHRISTIANS depict JESUS to look like, though we all know Jesus was really a dark skinned man (disclaimer.... funny ha)
Are you sure this guy has all his marbles in one bag?
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Back to Nature Women's Yoga Retreat in Oregon, July 2020 Session 1
- Tipi Village Retreat
- Marcola Road, Marcola, OR, USA
- Jul 17 - 19, 2020 (3 days)
Event Highlights
- All Meals (2 B, 1 L, 2 D)
- Water, coffee, Metolius Tea Apothecary loose leaf tea and Yogi hot tea, Metolius Tea Apothecary cold brewed tea, and Pacific Northwest wines
- 4 x 90 minute yoga sessions co-taught by Autumn and Toni
- Guided meditation and yoga nidra (deep relaxation/yogic sleep)
- Foundations of Asana Workshop with Toni: Alignment & modifications for the individual student (90 minutes)
- Roll & Release Workshop with Autumn: Reduce tension and tightness in the physical body (60-90 minutes)
- Two nights accommodation at the Tipi Village Retreat
- Gift bag of local goodies.
ScheduleDaily Schedule
6:30-8:00 coffee and tea available
8:00-9:30 morning practice
9:30 breakfast
10:30 bag lunches will be available for you to take on an adventure
11:00 optional workshop
3:30 optional workshop
5:00 evening practice
7:30 dinner
*schedule is subject to change*
About This Event
Join Ambuja Yoga for our Seventh Annual Women's "Glamping" Back to Nature Yoga Retreat in Oregon in July 2020. This women's yoga retreat has been so successful we are offering three sessions: July 17-19, 2020, July 19-21, 2020, and September 11-13, 2020. We would love to see you in July or September!
Located just outside of Eugene, Oregon, at the Tipi Village Retreat in Marcola, this all-inclusive three day women's retreat is designed to help you unwind, deepen your yoga practice, and enjoy Oregon's beautiful outdoors.
Book Early! This retreat will sell out just as quickly as last year and group size is capped at 22.
The Tipi Village Retreat, nestled within the Pacific Northwest woods, is the perfect place for our "glamping" retreat as we celebrate the dog days of summer with creekside outdoor yoga, a beautiful local day-hike, and gourmet al fresco meals. At night you will find comfort within one of the Tipi Village's beautiful tipis or cozy indoor rooms. Our women's yoga retreat offers you the opportunity to deepen your yoga practice, recharge your soul, and find greater gratitude; an opportunity to shine your light and share your love on and off the mat.
A woman in harmony with her spirit is like a river flowing. She goes where she will without pretense and arrives at her destination prepared to be herself and only herself.
~Maya Angelou
Meet Your Instructors
Autumn Adams, E-RYT 200, RYT 500, CPNYT, YACEP
Autumn founded Ambuja Yoga in 2014 as a way to not only share her love of yoga, travel & adventure, but as a way to cultivate intention, courage and grace in her students. She hopes her retreat attendees will leave her retreats with a feeling of purpose and with the ability to invite their intention into all aspects of their lives. She encourages her students to be conscious creators… to have the courage to take risks (both big and small), the courage to listen deeply to their body, to their gut, to their heart and the grace to "roll with the punches" on this messy path we call life.
Autumn's retreats have been written up in online publications like (contact info blocked), (contact info blocked), Asia Spa, Direct Holiday, and Bend Nest. Her retreats provide the perfect balance of fun, nature and soul work.
Toni Larson, E-RYT 200, RYT 500, YACEP
Beauty moves Toni and touches her heart. Toni's yoga practice keeps her doing the things she loves most. It has given her a recipe for life. She is a better mother, partner and human being because of it. Toni's teaching provides a relaxed atmosphere. She approaches yoga with a very real and down to earth style and recognizes that each person's body, range of motion, strength and energy are different. Toni is able to create and hold space so students can approach their practice with wisdom, courage and most importantly unconditional love. Toni has been teaching yoga for over 6 years and is the owner of Barefoot Bliss Yoga.
All Meals (2 B, 1 L, 2 D)Water, coffee, Metolius Tea Apothecary loose leaf tea and Yogi hot tea, Metolius Tea Apothecary cold brewed tea, and Pacific Northwest wines4 x 90 minute yoga sessions co-taught by Autumn and ToniGuided meditation and yoga nidra (deep relaxation/yogic sleep)Foundations of Asana Workshop with Toni: Alignment & modifications for the individual student (90 minutes)Roll & Release Workshop with Autumn: Reduce tension and tightness in the physical body (60-90 minutes)Two nights accommodation at the Tipi Village RetreatGift bag of local goodies.
Back to Nature Women's Yoga Retreat Includes:
Back to Nature Women's Yoga Retreat Excludes:Transportation to/from the Tipi Village RetreatHard Alcohol and BeerWifiJunk Food
Women's Yoga Retreat Pricing Per Person:
Superior AccommodationSitting Bull Tipi with King Bed Double Occupancy: $569 per personChief Joseph Tipi with Queen Bed Double Occupancy: $569 per personSacagawea Tipi with King Bed Double Occupancy: $569 per person
Standard Accommodation
Tipi accommodation at the Tipi Village Retreat in Marcola, Oregon" data-large-file="(contact info blocked)2014/03/Tipi-Village-Retreat-e(contact info blocked).jpg?fit=768%2C576" src="(contact info blocked)177054/original/(contact info blocked).jpg" data-lazy-src="(contact info blocked)300%2C225" alt="Tipi Village Retreat, Marcola, Oregon" width="300" height="225" class="fr-fic fr-dii" data-id="177054">
Sun rays through the trees.Hiawatha Tipi with Queen Bed Double Occupancy: $532 per personSession 1 seeking roommate.Calapooya Tipi with Queen Bed Double Occupancy: $532 per person
Budget Accommodation: Crazy Horse Large Shared TipiDouble Bed- Shared: $478 per personFoam Sleeping Mat on Raised Platform: $478 per person**** For those who choose the foam sleeping mat option you will need to bring your own sleeping bag. A towel and pillow will be provided.**
Accommodation Descriptions:Double Occupancy: This option is perfect for two friends or sisters; with a shared full, queen or king bedShared Tipi: Perfect for those on a tighter budget that still want a weekend getaway or a group of friends. Sleeps 8.Solo Yoginis: I recommend the shared tipi. It is a wonderful way to connect with your fellow yoginis. Due to limited space we are not offering single occupancy rooms.Groups: Additional foam sleeping mats with platforms are available for groups that would like triple occupancy (Weekend rate for third or fourth person: $478).
Thank you for your guidance, presence and sharing your gifts and purpose with me. I am honored. I've learned the importance of taking time for myself — for retreats like this, yoga classes, meditation. Ensure I do things that bring me joy. I've grown with presence and self awareness. My favorite part of this weekend was the whole weekend, the spiritual connection, the connection with others, and embracing the unknown. LOVE and BE LOVED! XOXO Sarah B., Back to Nature Retreat 2017
What's Included
- Accommodation, meals, coffee, tea, wine, yoga, meditation, swag bag and more.
What's Not IncludedTransportation to the Tipi Village
Venue HighlightsGlamping, amazing food, and close to nature! We practice yoga creekside and among towering trees.
AccommodationsAt this "glamping" retreat you will be staying in one of the Tipi Village's many tipis. Tipis have either a queen or king size bed with plenty of warm, cozy bedding. Shared bathrooms are spacious and appealing.
- Towels
- Hot Tub
- Coffee/Tea
Menu Types
- Vegan
- Vegetarian
- Pescetarian
- Meat
- Gluten Free
- Dairy Free
- Nut Free
Menu/CuisineHealthy, delicious meals that may include paella, salads, a mixed grill, or curry.
Lunch is a light affair that can be easily taken away on an adventure.
DirectionsThe nearest airport is in Eugene (approx 40 minutes away).
Portland (2-3 hours drive)
Bend (2-3 hours drive)
Cancellation Policy
100% deposit refund for cancellation 60+ days before event.
50% deposit refund for cancellation 30-59 days before event.
0% deposit refund for cancellation 0-29 days before event.
The remaining balance (total price minus deposit) is due upon arrival.
Get full details...
Reviews To review this event, visit the page for the venue or retreat leaders (above).
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Thank you!
We're glad you've joined our retreat community. See you around :)Return to my event ×


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Give us a call! 303-922-4304 www.AnnVanderslice.com Hours: 8-5 M-F
1 Denver Federal Center Building 45, Entrance E-9, Room 1050 Lakewood, CO 80225
National and Personal Independence W hen I was growing up, Fourth of July was a day when the entire community got together. As I mentioned THEIMPORTANCEOFPROACTIVEPLANNING
last month, I was raised in a small town, and on Independence Day we gathered at the football field for fireworks. These celebrations are etched in my memory, and they bring me back to my childhood. These days, I can't help but relate Independence Day to the importance of preserving your own independence during retirement, whether that be financial security or the freedom to spend your days how you want. The best advice I can give people to achieve this goal is to develop a plan. This may seem like a given, but most people haven't really done proactive planning since they've been in school. A lot of us think about retirement in terms of absence, in particular the absence of work, but I urge you to contemplate it in a positive way.
What will my days consist of? How much money do I need? Have I planned for unexpected events like illness and long- term care? Only after you envision your retirement lifestyle can you properly tailor a savings plan to achieve your goals. We all want a comfortable, enjoyable, and secure retirement, but that doesn't happen by accident. You'll notice one common thread to these questions: They assume you're in control. Sure, you can't predict illness, but you can ensure you have insurance in case it happens. Too often, we dwell on things we can't control. Recently, I've received many calls about proposed budget changes. I'm not saying these considerations don't affect planning, but they shouldn't be your primary concern.
www.AnnVanderslice.com | 1 – Ann Vanderslice As a federal employee, you've weathered more change than you probably realize. The next change always seems the scariest, but things are always scarier on the horizon than they are when they arrive. Change, whether we like or not, is a fact of life. Discussing this reminds me of one of my favorite quotes from author John C. Maxwell: "Change is inevitable. Growth is optional." With that in mind, let's dedicate this summer to focusing on the aspects of life we can control and setting our sights on growth. Our forefathers fought tirelessly to gain independence from Britain and protect our freedoms. The work to protect your own independence might not be quite as historic, but it does make a significant difference.
Everyone should ask a few specific questions as they approach retirement.
Published by The Newsletter Pro . www.NewsletterPro.com
Made with FlippingBook - Online magazine maker


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I just read an excellent New York Times Magazine profile of Damon Lindelof, and I have a few thoughts about it.
1. The profile describes Lindelof's tumultuous experience with the fans of Lost, the show he co-created and ran for all of its much-scrutinized run. For years, Lindelof endured outraged cries from devoted fans of the show who felt that the series finale failed to wrap up the mysteries the show had allegedly set out to solve. The feedback turned so sour that Lindelof deleted his Twitter account with a flourish on October 14th, 2013, explaining later that the resurgence of negativity that followed the polarizing Breaking Bad series finale left him feeling psychologically battered.
I never watched Lost to solve the mysteries. I was certainly interested in finding answers, and I enjoyed delving into Jeff Jensen's exhaustive analyses for Entertainment Weekly, but when it came time to watch the finale, I was far more invested in where the characters would find themselves at the end of the episode, and how the journeys we'd watched unfold for six years would conclude. I realize I'm in the minority, and that the frustrating banality of the Smoke Monster, the Whispers and the Magic Cork left people frustrated to the point of dismissing the entire show. I think that's an unfair response, given that the show always cared about people as much as it did smoke and mirrors. I was glad I'd spent six years with Jack, Kate, Sawyer, Locke, Hurley, Ben, Juliet and the dozens of other characters who popped in and out. It's not wrong to want more from a show famous for its mysteries, but it's also not right to condemn the creator for having other ideas about how he wanted his show to end. There's no use crucifying Lindelof for "wasting our time." That we were invested in the first place means that he was doing his job right for quite a while, and that counts for something.
2. The Leftovers has massive potential, and I'm really curious to see how it adheres to and departs from the Lindelof conventions Brodesser-Akner describes in her piece. I'm intrigued by the idea that the pilot establishes the mystery and future episodes explore the ramifications of that mystery, providing a distraction from the pressing desire to answer the questions. Again, though, Lost at its best pulled off the same trick. The first season of Lost had exquisitely strange moments of mysterious wonder, but it also had extraordinary explorations of character and theme in episodes like "Walkabout" and "Solidarity." The show at its worst got bogged down in circumvention after circumvention, at the expense of logical character development and legitimate suspense. I sincerely hope that The Leftovers, unburdened by the same fervent audience of 15 million viewers (network TV circa 2004, people!) that analyzed every frame of Lost, will have time to tell stories about people, so that the mysteries are in support of something larger and more meaningful.
3. Brodesser-Akner spends much of the piece linking Lindelof's family background with the themes and characters in his shows, as with his explicit similarity to Jack Shepard, the Man of Science on Lost. This section got me thinking about an increasingly prevalent television equivalent of the auteur theory in film. This theory essentially credits the successes and failures of a movie to its director, who conjures his singular vision onscreen with the help of the cast and crew. On television, the writer is held up as the analogous authority figure, asserting his vision over the rest of the creative team and influencing the TV landscape with his characters and words.
Based on Lindelof's conflicted feelings about being a celebrity at the center of a controversial television show, perhaps television criticism should strive more energetically to spread the credit to the directors who execute the vision from page to screen, the cinematographers who capture the look and feel of the show, the editors who shape a show's rhythms, the composers who use music to define a show's personality, and all of the other creative people whose input and technical expertise contributes to our appreciation of a show's merits. It's not that these people never get credit, but they probably don't get enough, and a systematic critical shift might persuade some viewers to criticize shows in a way that won't do psychological damage to the people behind the shows we love, like and even hate. If the ultimate purpose of television criticism is to think more deeply about the things we do to enrich our lives when we're not working, perhaps we ought to pay more attention to the people who view television as a job, not a pastime.


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तन्मनो मयि युञ्जाना अचिरान्मामवाप्स्यथ ॥ ३२ ॥
hy aṅga-saṅgo nṛṇām iha
tan mano mayi yuñjānā
acirān mām avāpsyatha
na — not; prītaye — for satisfaction; anurāgāya — for loving attraction; hi — certainly; aṅga-saṅgaḥ — physical association; nṛṇām — for people; iha — in this world; tat — therefore; manaḥ — your minds; mayi — upon Me; yuñjānāḥ — fixing; acirāt — very quickly; mām — Me; avāpsyatha — you will achieve.
For you to remain in My bodily association would certainly not please people in this world, nor would it be the best way for you to increase your love for Me. Rather, you should fix your minds on Me, and very soon you will achieve Me.
The Lord pointed out that people in general would not appreciate a loving affair between Lord Kṛṣṇa, who superficially was appearing as a cowherd boy, and the wives from the brāhmaṇa community. Also, the brāhmaṇa ladies' own devotion and love would increase most efficiently in separation. In other words, it would be best all around if they continued to fix their minds on Lord Kṛṣṇa and thus went on with the process they had been practicing throughout their lives. The Lord and His bona fide representative, the spiritual master, expertly engage the Lord's devotees in different types of service so that all of them can quickly return to His lotus feet.


text: For Arburianpelarota (wall | block log | uploads | logs | Chat ban log) Search for contributions IP address or username: Namespace: all (Main) Talk User User talk B-Daman Wiki B-Daman Wiki talk File File talk MediaWiki MediaWiki talk Template Template talk Help Help talk Category Category talk Forum Forum talk User blog User blog comment Blog Blog talk Module Module talk Message Wall Thread Message Wall Greeting Board Board Thread Topic Exclude namespace Associated namespace Deleted only Only show edits that are latest revisionsFrom year (and earlier): From month (and earlier): all January February March April May June July August September October November December 05:00, April 12, 2014 (diff | hist) . . (+149) . . B-Daman Wiki Retrieved from "https://bdaman.fandom.com/wiki/Special:Contributions/Arburianpelarota" Community content is available under CC-BY-SA unless otherwise noted.


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It's that time of the year again. We're busy buying presents for our family and friends. Malls are filled with shoppers, and parking lots are filled with cars. Even in the midst of holiday chaos, there's something everyone can do for their school from the comfort of their home. There is no need to compete for a parking space or wrestle through a busy mall; and most of all, it's free! It only takes 3 minutes of your time.
Lysol is doing a Spread Holiday Cheer (NO GERMS) Sweepstakes until 12/29/2017. Enter for a chance to win 10,000 Bonus Box Tops. There will be 10 winning schools, so the chance for your school to win just got 10 times higher! Click on the image to enter the sweepstakes now!
Also, for those who want to keep your home or office clean. Lysol is still doing a bonus offer, where you get 100 Bonus BoxTops when you buy 5 participating Lysol products. Tis the season to stock up on Lysol!
Happy Box Tops clipping!


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Information on the Placement Tests
The prerequisite for taking all English courses at the University of Potsdam, without exception, is the placement test.
For law students in the 3rd semester and higher who have not yet taken the test: Friday, Oct 04, 11:00-12:00).
For first-semester law students, the test date will be announced during the introductory week of the FSR Jura
Placement Tests for Students of Law
For first-semester students, the test is held both during the introductory events at Zessko's language departments in October, as well as on another date in November. For students in later semesters, the test is offered at least once each semester, during the registration week before courses begin.
In the field of law, the following course assignments are made on the basis of the placement test results:
Placement test results
Course description
Information and registration
50-59 points
UNIcert II/1 EAP
60-69 points
UNIcert II/2 EAP
70-100 points
UNIcert III/1
80-100 points
UNIcert III/1
Only after Part 1
UNIcert III/2
* EAP: English for Academic Purposes
For further information about course occupancy, please see the "Frequently Asked Questions."


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Hi there! Is this your first time?
Did you know if you Register you have access to free search tools including the ability to save listings and property searches? Did you know that you can bypass the search altogether and have listings sent directly to your email address? Check out our how-to page for more info.
- Price$43,000
- Acres0.33
33 Cessna Court, Greenville
Vacant commercial parcel in Haywood Road area. 1/3 acre parcel is in the Byrdland Park commercial park and is also adjacent to 3 acres (on the market) along Byrdland Drive as well as the Redemption World Outreach parking lot.
Essential Information
- Price$43,000
- Acres0.33
- TypeLots/Land
- Sub-TypeCommercial Site
- StatusActive
- AmenitiesNone
- FeaturesAdditional Acreage
School Information
- ElementarySara Collins
- MiddleBeck
- HighJ. L. Mann
Community Information
- Address33 Cessna Court
- SubdivisionNone
- CityGreenville
- CountyGreenville
- StateSC
- Zip Code29607
- Lot DescriptionHeavily Wooded, Level
Listing Details
- Listing AgentJohnie Devore
- Listing OfficeRealty One Group Freedom
Listings provided courtesy of the Greater Greenville Association of REALTORS®, Copyright 2020.
IDX information is provided exclusively for consumers' personal, non-commercial use and may not be used for any purpose other than to identify prospective properties consumers may be interested in purchasing.
Listing information last updated on July 13th, 2020 at 10:01pm EDT.


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The International Chamber of Shipping's (ICS') newly-published Flag State Performance Table features positive indicators across the 12 largest flag states, with not more than one negative indicator per flag.
The results showed that, recorded over the course of the 15-year initiative, ongoing improvement continues unabated.
However, the table does indicate that Panama, the world's largest ship registry with double the tonnage of its nearest competitor, Liberia, has received a red indicator in respect of the USCG Target List (safety). This shows that Panama is on the USCG's list of targeted flag organisations, with an above-average detention ratio and more than one detention in the course of three years.
Helio Vicente, senior adviser (Policy) at ICS, assured Safety at Sea that the result would not by itself be a negative indictment of the Panama registry or its almost 7,100 registered vessels. "In the event that a flag state, in this case Panama, is missing a single positive indicator in itself this should not be seen as a measurement of overall performance, noting that in some cases there might be legitimate reasons for a flag state to be missing a few positive indicators," he said.
"While ICS allows the data to speak for itself, if a large number of positive indicators are shown as being absent, this could suggest that performance is unsatisfactory and therefore shipping companies might wish to have further discussions with the flag state concerned to effect any improvements that might be necessary."
The table also showed various major flag states with no red indicators at all, which included Panama's nearest competitor Liberia, the Marshall Islands, Hong Kong, Greece and the Bahamas.


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The Supreme Court (SC) has issued a stay order on the corruption case against suspended Director General of the Inland Revenue Department, Chudamani Sharma, filed at the Special Court.
A divisional bench of Justice Cholendra Shumsher JBR and Prakash Raut ordered the Special Court to put the case on hold and send it to full justice, according to SC Spokesperson Mahendranath Upadhyay.
As some legal complications have arisen in the case relating to Chudamani Sharma, the divisional bench Justice duo ordered to send the case to SC's full bench, said Spokesperson Upadhyay.
Sharma was arrested by the anti-graft body CIAA on June 2 for his alleged involvement in helping telecommunications service provider NCell exempt Rs 21 billion capital gains tax.


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Tensions between YouTube's management team and the platform's creators are symptomatic of the platform's greater identity crisis. Can the company create more commercial opportunities without losing itself in the process?
YouTube holds a unique place in the social media and content creation landscape. It's by far the largest open content platform on the internet, with 1.8 billion logged-in users and a #1 ranking among teens over other social media platforms. At the same time, the company is trying to court larger advertising partners, which requires asserting a higher level of control over the platform and its users.
YouTube is caught between a rock and a hard place; to keep the advertisers it has and continually attract more, the platform has to evolve. But this evolution may compromise the very principles on which it was founded. How can YouTube retain what makes it so popular with its viewer base — namely, high-quality content from its creators — while increasing its commercial appeal?
A Need for Control
It's safe to say that YouTube is in the midst of an identity crisis. The company was founded as an open platform for creators to express themselves without being beholden to anyone. This freedom, in part, is why viewers flock to the platform. But the very thing that makes YouTube successful — unfiltered commentary within a close-knit community — makes it a minefield for advertisers trying to protect their brands' reputations.
In the past year alone, YouTube has been plagued by PR nightmares, ranging from ads showing up on ISIS recruitment videos to reports of disturbing content targeted at children. YouTube has even faced controversies surrounding some of its most popular creators, including Logan Paul and PewDiePie, both of whom attract millions of views every month.
Many prominent advertisers have dropped the platform from their media mixes altogether, while others have significantly scaled back their contracts. To win these partners back, YouTube has been forced to implement stricter policies around user-generated content — arguably at the expense of the creators themselves.
But What About the Creators?
YouTube has rolled out changes that increase the subscriber and viewership threshold a channel must achieve to be monetized (i.e. eligible for ads), and videos are now subject to stricter review and automated flagging. In theory, this is a good thing — why wouldn't creators want a safer, friendlier YouTube?
The problem comes with implementation. On a platform as large as YouTube, it's nearly impossible to create an algorithm that accurately flags problematic content. Smaller creators are being shut out from monetization altogether by long review times, while established YouTube celebrities contend with inexplicable demonetization of their content. News shows, LGBTQ+ content, and educational videos about women's health have all been flagged and demonetized, often with little explanation or recourse for the creators.
To make things worse, YouTube is also experimenting with personalized algorithmic feeds (the way that Facebook and Instagram currently organize posts) to replace the chronological home page. Creators worry that subscribers won't see new videos as a result of this change, making it even harder to maintain a steady stream of revenue.
Most creators will admit that nobody is guaranteed ad revenue. But because YouTube's success is almost entirely attributable to the time, energy, and creativity of its users, creators expect to be consulted when the company makes major changes to monetization or payout structures.
By all accounts, that expectation has not been met. Major creators including Philip DeFranco and Casey Neistat have been outspoken about the lack of communication from YouTube management. Many are seeking out alternative revenue streams, including sites like Patreon and Twitch. Some are leaving YouTube altogether, and creator burnout is at an all-time high.
Creators Left in the Dust
Sure, the changes to monetization are frustrating. But creators have a bigger fear: that YouTube will turn away from them altogether.
For YouTube, which hasn't been shy about its ambitions to become more like a television network, courting advertisers has become a priority. At this year's Brandcast, the company's yearly conference for advertisers, YouTube announced several new initiatives: the option to run ads on YouTube TV, the introduction of human reviewers for the Google Preferred advertising network, and an expansion of its music offerings.
Notably absent from the Brandcast announcement was a call to invest in the platform's most popular creators, including people like Neistat and DeFranco. While some of the planned developments feature creators on- and off-camera, several do not, leading creators to wonder whether YouTube will deliver on its promise to increase transparency and opportunities for engagement.
There's also the question of how users will respond to the changes. Can YouTube convince its users to pay for a TV streaming or music service? Those offerings are certainly more advertiser-friendly, but they aren't necessarily the reason that users visit YouTube in the first place. If updates to the platform favor advertisers too heavily, YouTube risks losing the two things that catapulted it to greatness: strong user communities and original, organic content.
How Do We Move Forward?
It's clear that YouTube has reached a tipping point. Will it be able to grow and offer advertisers new opportunities while maintaining the "DIY" organic feel of its platform?
A few takeaways stand out:
- Brand partnerships with individual creators still work. Though they can be time-consuming, they're brand-safe, allow creators to parlay the brand to their audiences in a meaningful way, and generate great returns.
- Creators need to be front-and-center as YouTube makes changes. Otherwise, they'll leave the platform — and take their audiences with them. Reversing the slow death of creator networks may open lines of communication between YouTube management and individual users.
- YouTube must continue to refine its algorithms and invest in human reviewers to perfect its flagging system, weeding out problematic content without damaging legitimate channels.
- For brands that cannot accept risk, the new corporate partnerships on YouTube TV and the nascent streaming music service might be the best options. These channels allow cautious brands to advertise on YouTube without compromising the integrity of their platform.
What YouTube is trying to do is really hard, and they're not going to get it right the first time. But hopefully — with trial and error and lots of input from creators — the company can reach an equilibrium that keeps brands interested and its users happy.


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How Marvin helps you beat procrastination
Procrastination harms you in many ways. But Marvin is here to help. Find out how Marvin can help you beat procrastination.
Raise awareness
You can't fix what you can't see
You usually know when you are not getting as much done as you want. But without knowing the cause of your low productivity you can't do much about it.
In Marvin, you work from a daily plan which will quickly uncover if procrastination is a problem for you.
If there is a large gap in what you intend to get done and what you actually get done (that cannot be explained by other reasons), the culprit is procrastination.
Marvin helped me realize that I actually have a procrastination problem. I always thought planning is the problem… like I don't know what to do. But it turns out when I do make a great plan, I barely complete any of the tasks on it and only log about 1h of actual work time. Yikes!
- Nils P.
Uncover procrastination patterns
Knowledge is power
Marvin has many tools to help you uncover patterns in your procrastination.
These insights help you to be strategic about fixing your procrastination.
For example: The "Procrastination Count" feature adds an exclamation mark to a scheduled task for each day that you put off the task.
After a few weeks you will start to notice which kinds of tasks you tend to put off.
I realized that every task I procrastinate is longer than 30min (I use time estimates). So I started breaking the tasks down more and limiting each task to 30min with time boxing. I now finish my daily lists almost every day! I guess I really like short tasks.
- Jennifer H.
Make an intention
Increasing the chance you will do something
Many studies have shown that you are significantly more likely to do something if you make a specific intention on how and when you will do it.
Making an intention for action is at the very core of the Marvin productivity concept.
Working from a daily plan means you make an intention to do each task on a specific day and perhaps even a specific time.
I learned how to make tasty tasks and day planning made me really mindful of what I want to get done each day. It's so much easier to make progress on my goals when the steps are literally laid out right in front of me.
- Kyle O.
Reduce overwhelm
Fighting the big procrastination trigger
Procrastination is our attempt to avoid things that cause negative emotions.
When our todo list overwhelms us, our instinct is to avoid it.
While overwhelm isn't the only procratination trigger, it is a very common one. Marvin reduces overwhelm by:
- Storing your master list out of plain view
- Making it easy to break down large projects into small steps
- Allowing you to break down your day into manageable chunks
- Letting you see one task at a time by using the super focus mode or task jar
I can't believe how much of a difference the super focus mode has made in my life. I just breeze though my tasks now, whereas before I would often stare at my todo list and just feel paralyzed by overwhelm.
- Mia S.
Clever features
Shifting the procrastination equation in your favor
We are always coming up with new features that help to shift the procrastination equation in your favor by:
- Decreasing aversion to a task
- Making progress feel more satisfying
- Adding additional motivation for task completion
- Increasing the negative consequences for inaction
Speaking of procrastination... you might be interested in our Procrastination Bootcamp.


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Events for 30 June 2019
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Summer Reading Challenge (ages 0-18)
Ages 0-18 You don't need to be in Paris this summer to keep track of your reading and collect prizes while you're at it! […]Find out more »
23 h 00 min
Teen Advisory Group: Deadline to Apply (ages 12-18)
Open to Library members ages 12-18 Provide valuable feedback and earn community service hours! Provide feedback and earn community service hours by joining […]Find out more »