text:
Dear friends,
I have a terrifically simple idea that is making me stupidly excited. I'm now four years gone from my thesis writing; a frenzied semester of coffee shops, writer meet-ups, and frantic scribbling. I'm happy to be where I am in my life now, but I really miss having a community of writers around me to inspire, challenge, edit, talk books, and drink coffee/wine with. I miss the coffee house culture, the long evenings spent with laptops touching back to back and postal service playing in the background for inspiration (I'm thinking of you, Sarah N. and Travis V!).
I'm lucky to have several friends with whom I have writerly exchanges over email, sometimes with longer gaps in between stories sent. They don't live in the Twin Cities however, and I regret not being able to interact with them face to face. Their support is sometimes the only force propelling me forward and causing me to challenge myself.
My idea is to form a Twin Cities micro-community of writer-friends who do exactly that: support and challenge each other. It's really powerful to physically sit next to a like-minded friend who is doing exactly what you are doing; yanking words, sentences, and images from their brains into typed or written word. Feeling that energy and focus can often make one more productive than if they were writing alone.
A beatboxing artist told me yesterday that the key to mastering any art (or at least improving at it) is repetition. He was returning from the restroom and I noticed that he was beatboxing under his breath as he walked back to the classroom. He lives his art right up to the very mundane moments of life. It's become part of his stride, his breath, his daily patterns. Right, I've heard that, and I know it, but it still stuck with me. Of course. Of course it's hard to just sit down and write randomly in the middle of the week and expect good results and easy flow. It's something that requires practice, repetition, consistency.
I propose the start of Writerday. It's a day of the week, like any other. It's a day started or ended with a few hours of solid focus, creativity, and of course, writing. Hopefully around a coffee table, grassy knoll, or bar with other like-minded friends. I'm thinking that the best day for Writerday would be Saturday. Perhaps starting in the late morning and lasting for several hours, or however long it takes to get some writing out. I want to make it consistent and around the same time every week so that I gain some muscle memory whenever I sit down with a computer or notepad on that day. Writerday does not have to be an all-day affair. Saturday will still also be heard and respected, and indulged in. I just want to start that day off every week with a specific mindset and focus and community that supports and challenges me to simply write more. It would also be a great way to get out on a Saturday and see more of the cities, take walks, eat delicious food, and see each other.
Any interest or feedback? Is Saturday a good day for this?
Love and Lunch,
Tuesday, July 23, 2013
Friday, July 5, 2013
Dear America
Dear America,
6 months back as your resident, as a pedestrian to your streets, an observer to your rituals, a face in your crowd, I have a lot of questions. How is it that your history has been rewritten and immortalized in colorful school textbooks when its omissions scream silently from the sidelines? How is it that a country like you can still have sidelines?
We prize ourselves on liberty, justice, freedom, in a country where we imprison and alienate many. Our prisons are overflowing. Our schools are failing. Our children are divided. We have accomplished little on this day besides setting some things on fire. We are on fire. Dear America, killer of nations, imprisoner, slave-holder, rapist—your many 'accomplishments' belie the true nature of things. A country of convenience, we have covered up a litany of crimes against humanity. We have conveniently written entire communities out of our history. It's not convenient to remember these things. We raise our white privileged children well. They learn about Benjamin Franklin and George Washington, the first of many white men who take front and center in American history. If you want to learn about African Americans, there is an alternative class you can take at university, but until then, you won't see people of color as makers of history, as people who breathed life into communities, who struggled and saved and loved and cried tears into the rivers of the Midwest and the deltas of the south. As people who lived. As the Native American people who were clawed, murdered, and torn out of their native land. America, we are liars. History isn't a book, or a page you can turn or rip out. History is us, and is written into our daily interactions, into our income, into our relationships, and into our quality of life.
I'm so goddamn ashamed of you, America. Ashamed of me. Our history is not a complicated one. It's main theme; the othering and subjugation of others. It's the only story our America has. Since the raising of the smoky and bloody star spangled banner to the fireworks and beer of yesterday, it's our only story. If we want to tell it the way we have, with only white Europeans starring front and center, then it will always be a story of murder and oppression. Until we change the uniquely narrow view that we frame our world around, America will be a place purely framed off of acts of genocide and slavery.
On the other hand, if we start America's story thousands of years ago, we would have different sort of history altogether. A tapestry of stories about a land inhabited and worshipped by thousands of families until.
Until. And that's when our America comes in.
If we aren't careful, Americans, we will always be the bad news to come after 'until.' We will always provide the rupture, the tear, the trauma in others lives. There's nothing remotely impressive or noteworthy about July 4th. For most people, it was just another day to watch family members die at the hands of white Europeans, and another day to lose their former freedom and life.
These borders are carved in loss and violence. What freedom we have is not shared by all.
Instead of seeing every fourth of July as a day to wear patriotic colors and wave flags, lets try to make it a day of discussion and reflection. It's an easy day for complacence, but the only way things will ever get better for communities of color is if we shake off that complacence and stop teaching out of the same history books we all learned from. We need a massive paradigm shift in the way we choose to remember our nation, and the way we choose to frame ourselves in the story. We should all grow up feeling connected to America's past, whether it's our past as slavers and imperialists, or our past as native people. It is what happened and it is what we as communities wrought upon others. We all have convenient stories of ancestors immigrating to America a hundred years ago, long after the civil war, long after slavery, long after the trail of tears…we all have safe dates. Those dates don't matter. We aren't exempt. We are all implicit in the very fact that racism, oppression, and slavery still exist to this day. We can't say things are better or that there is freedom until it is that way for all people in America. We can't change the past. We can however change the way we tell our story as Americans, as a start, in hopes that if we tell the true story it can help us move towards rewriting the part we are in now.
Monday, July 1, 2013
We talk of cultures around the world, enviously, as if we have lost ours somewhere far in the past. Not remembering, that to live is to have a culture. We have our personal cultures and our family cultures and our national cultures. I'm working on my own now. I'm an intricate archeological dig of my past, my ancestors, my interests, my friends, my peculiarities, my love, my hope, my present. Every day I work to uncover more of myself until someday I will unfold completely into a heavenly shape with a divine ratio of surface area to volume. Love in, love out. Love in, love out.
I want to wrap myself, like a fat vine, around the experiences and living things around me. I want to sit on a shady bench and observe what goes by for hours. I'm hungry for friendship these days. Like a satisfying growl in my stomach, I feel content with my friends. I long for them, but I enjoy them when they are around, too. I'm crazy about what they do and how they smile. I'm here to exchange love with others and act like a complete doofus along the way. I'm also here to be introspective, silent, watchful, at peace. I'm here to make decisions that are neither good nor bad but strange and of my making, and see how the universe reacts. I'm here to admire, dance, smile, and seek. I'm here to dissolve boundaries and categories and expectations and color outside of the lines. I have never understood the importance of adhering to these things. Everyone gets around in their own way and sees different scenery along the way. I want to hear about the stories that world has, the stories a single human holds within them, the sound of summer at night. We have such rich cultures within us.
I once formed a small but essential part of a dinosaur's tail. My atoms were there, and I facilitated the slow swinging, the sacred sense of balance that a tail provides. A long-neck dinosaur, with its thick grace and large eyes. I was there. Everyone else was, too. We were different but still us. We've passed through so many forms of ourselves, so many different rearrangements of our molecules. We have been here since there was a here. We've been humble plants, elegant animals, sad humans. We've been dirt and air-bound and eggplant. We have such memories that it's a blessing we can't access all of them. We are part of each other and inseparable from each other. What a relief to remember on days of stagnant selfish ipad isolation. We are each other and we are ourselves. I don't have a good sense of balance now---I stumble clumsily throughout my life---but it's a relief to realize that I once provided an essential element of balance in a single dinosaur's life as it lumbered its large feet over dusty ground.
You are infinite.
Also, HAPPY PRIDE WEEKEND!
Also, HAPPY PRIDE WEEKEND!
Subscribe to: Posts (Atom)


text:
Tim's Pumpkin Patch
Just try and stay out of my way!
I'll get you,my pretty & your little do
- The Wicked Witch of the West
More interpretations at Skywatch Friday.
what a sight - love itReplyDelete
Fantastic sight, the sky is perfect, too!ReplyDelete
What a great background sky for this wicked witch! I love your photo!ReplyDelete
Ha, a perfect skywatch shot for Halloween!! Well done.ReplyDelete
My apt is bubbling all over at Senior Halloween for to-morrow.
Have a peek and make a comment. The witches are out.
Superb capture Doug. Have a great weekend.ReplyDelete
Love it! You have created the perfect atmosphere for this shot! Happy Halloween!ReplyDelete

subset: commoncrawl


text:
As Orange County's 28 school districts prepare to begin fall classes, a statistical blip has many administrators smiling.
The blip forecasts a slight increase in the number of Orange County students this year. If correct, it would be the second year in a row--and only the second time since 1975--that the county's student enrollment has grown.
"We won't know positively until our October census, but it appears Orange County will have at least 335,000 (elementary and high school) students enrolling this fall," said Audrey Capasso, apportionment technician for the Orange County Department of Education.
Classes About to Begin
"The county had 386,086 students in 1975, but since then the total has dropped every year until last year," Capasso said. The 1984 tally was 334,803.


text:
The ISSF Rifle and Pistol World Cup in Fort Benning was closed by the 50m Rifle Prone Men final, which was won by France's Valerian Sauveplane at the very last shot.
Valerian Sauveplane won the all-new ISSF 50m Rifle Prone Men final, securing the highest step of the podium at the last shot.
How does the new final work? The qualification scores are not carried forward into the final. And, during the final, after the eighth shot - and then after every two shots - an athlete is eliminated, until the medals are decided.
The 32-year old two-time Olympian started off the final in eighth place, climbing then the scoreboard up to the top, while his opponents were dropping out of the match.
On the last two shots, Sauveplane faced Germany's Daniel Brodmeier beating him at the last shot (a 10.3) to win the Gold medal with a final score of 206.5 points.
Brodmeier - who had won the 50m Rifle 3 Positions Men event earlier this week and who set a new 50m Rifle Prone World Record of 628.8 points during the qualifications - finished in second, collecting the Silver medal with 206.3 points.
The Bronze went to Russia's Fedro Vlasov, 29, who gained his very first medal in this event with a final score of 185.6 points. Vlasov had already finished upon Fort Benning's podium on the 9th of May, when he had pocketed the 10m Air Rifle Men Bronze medal.
"I am feeling very good. Things went well, so I am really satisfied by the outcome of this match!" Sauveplane said after the competition.
"Starting off the match in eighth position, I was quite relaxed. But then, when I realized that I was actually climbing up the scoreboard, I became more and more nervous..." The champion from France explained.
"The last shot has been really difficult, because there were a few decimals between me and the German finalist. I shot a 10.3, and at the beginning I thought this was not going to be enough. But fortunately for me, that was good enough to win the match!"
Following the three medal winners, the 2012 Olympic Bronze medallist Rajmond Debevec of Slovenia, 50, placed in fourth, closing the final with 164.2 points. Behind him, in fifth place, Brazil's first-time finalist Cassio Cesar Rippel, 35, scored 143.4 points.
And today's want't the day of Henri Junghaenel, the 25-year old German shooter who had won both the 50m Rifle 3 Positions and the 50m Rifle Prone competitions at the previous ISSF World Cup Stage, in Changwon, last April. Today, after qualifying for the medal match in second with 626.9 points, he scored 122.7 points in the final, and was eliminated after the fifth series (after the 12th shot).
The home shooter Michal McPhail and the second-time finallist Stian Bogar of Norway finished in seventh and eighth place, with 102.6 and 80.9 points, respectively.
Posted By Thom Erik Syrdahl
Souce ISSF Website http://www.issf-sports.org/


text:
1 lb ground sirloin
1 lb ground chuck
1 28oz can crushed tomatoes
2-3 tbl chipotle chili powder
2-3 tbl chili powder
1-2 tbl ground cumin
1-2 tbl onion powder
6-8 cloves fresh minced garlic
1 tbl coriander
1 can refried beans
1 can pinto beans
1 can whole kernel corn
1 jalapeno chile, finely chopped
1 Anaheim chile, finely chopped
1 medium to large white onion, diced
1 medium to large red onion, diced
1/8 to 1/4 tsp cayenne pepper
1 tsp Kosher salt
4 cups water
2 cans chicken stock
Brown meat and drain fat. In an 8-quart pot (that's what I used), combine meat with the
rest of the ingredients. Bring to a boil, stirring occasionally. Reduce heat to low and
simmer for 90 minutes, stirring every 10 minutes.
Wednesday, December 8, 2010
Chili Time
Sorry for the near-abandonment of the blog. My primary computer's web browser doesn't allow me to sign in for some reason so it's a hassle to create new posts.
1 lb ground sirloin
With the weather getting colder here in Phoenix, I've had a craving for my chili. This isn't an old sacred family recipe. I cobbled this together after some trial and error during my long-ago time in Dallas. It's not even close to an authentic Texas chili recipe. I read dozens of chili recipes online and blogs and threw together the elements that sounded good. Eventually I settled on this and it usually comes out pretty tasty. I try to keep the spice level down, as I'm not a hard-core heat enthusiast. I prefer flavor over incendiary ingredients. I found grocery store generic spices work best for me usually. I tried high-quality spices and chili powders from the awesome Central Market in Plano, and man, I felt like the ill-fated at the end of Nazi in Raiders of the Lost Ark. It's nothing special and can be modified to one's tastes. I've used both beef and turkey for the meat and both work wonderfully.
Tim's Chili
1 lb ground sirloin
Posted by Yankee1969 at 4:14 PM
No comments:
Subscribe to: Post Comments (Atom)


text:
Joining an MNC or a startup depends on your goals in life, your personality and your backing. Both have advantages and disadvantages. You need to weigh yours before you choose one of the two. A wrong decision can be disastrous to your career ....and your peace of mind. Here's a check-list of ten traits that anyone opting for startups should tick-mark.
1. Seeking JD bigger than CTC
Is your fascination for job description bigger than your CTC expectations? A startup can offer you a big role, a vivid one that MNCs won't offer you with your kind of experience. You would develop expertise too on the go. The trade-off is that not every startup is well funded to offer you a CTC as great as offered by MNCs. They have to keep funds for the runway too and you being part of the startup have to accept that. That doesn't mean your in-hand will be too low to survive. But start with startup is a trade-off and you need to be ready for it. If you make yourself too expensive, the strartup won't be able to afford you.
These pointers can even be handy for startup owners while hiring. Happy hiring!!!
2. Stepping-in everywhere
Are you bored with same well-defined monotonous job? Are you looking for verity of challenging roles? Do you want to try your hands on your colleague's responsibilities too? Then you have plenty of such opportunities with startups. Yes, startups often are under-manpowered and this is not just attributed to fund crunch. The myths about startups do a pretty good job in refraining talented people from accepting offers. So, you would have plenty of opportunities to step-in for someone on leave or someone who leaves.
Do You like to work in a Startup or MNC?
Priorities change quickly. You too should be ready to change that quick. Many a times sustainability is more important than bringing an idea to life. One has to think on toes and change according to the market needs. Getting possessed with an idea or technology is not an option, specially in a startup. .
4. Challenging excellence with limited resources
Every company emphasises on innovation, which is a good thing. The question is - are you able to innovate in limited resources? Startups really have crunch of resources. On top of it, they are in true need of innovation...and that too innovation which challenges pre-set standards of excellences. Then only they can become an MNC in future. This is the opportunity where you can think & implement out-of-box ideas. Your ideas should be great but the implementation shouldn't cost a fortune.
5. Can handle empowerment
Can you handle empowerment? Most of the employees want to be empowered but can't really handle it when they be empowered. At a startup, you enjoy the state of empowerment. You get freedom to do whatever you think is the best for the company. There is less over-the-shoulder monitoring. You don't get pre-set rulebook. Neither do you have pre-defined daily routine nor is there demand of daily reports on those ppt templets. There is sometimes no answers to your emails and you are expected to take the best decisions in that scenario. You have more responsibilities than you think. You are responsible and accountable for your work as well as company's success...and failure too.
6. Being persistently patient
It takes time to build a company. Three to five years go just in getting break-even. Are you persistent enough? and patient enough? Although, being persistent looks opposite of being a change champion, but it's not. You have to move with the changing external environment, you might have to give up on some not-fruitful awesome ideas but you have to persist on building a company and taking forward its core values. In this persistence, you need a lot of patience. On top of it, you need to be persistent in being patient till your startup becomes big.
7. Lacking ego
Working at startup needs compromising ego at several levels. Are you ready to not compare you salary sheet with your batch mates? Are you happy telling anyone & everyone that you work for a small company, a startup - a company who's name they have not heard at all? Can you also let go an office clash? Do you have guts to not feel ego-hurt if your ideas are rejected? Can you handle it when you are told to execute some task lower than your capabilities. Well it is simple. You need to lack ego to be at peace with your present state, your company's present state and your present task. As important it is for your own health, as it is for the startup's health. If you can handle this then only join a startup.
8. Taking pride in every small achievements
Once you are sure that your ego is no more getting hurt, you need to take pride. Are you proud of your achievements, no matter how small? Do you feel happy telling others that you work for a promising startup? Are you proud of the fact that you are a part of building a big company from scratch? You need to let go what others think about your company and professional achievements and feel happy about what you have achieved even when you don't have a big MNC brand-name attached to your LinkedIn profile. It is essential for your work performance and INNER PEACE.
9. Having strong backbone
Yes, backbone. Just like lack of ego and presence of pride, you need a strong backbone. Are you and your support system immune enough? Everyone in society will not appreciate your efforts. You need to be strong enough. You need to not get ego-hurt and keep taking pride. It is difficult handling those who have taken oath to irritate you. For your own sake, your opinions and your work should not get influenced. I am saying it again...you need to be real strong. And so should be your support system...your family. If your family stands by you, you can win the world. They need to understand you and your goals. They shouldn't compare you with other's children/spouse but be strong for you and give you strength.
10. Planning to start a company someday
It's not necessary that everyone joining startup should have a vision of building another company. Some join just to feel proud of the fact that they played a big role in a small company or are a part of building something big for the future or just because their JD is exciting enough. But if you want to start your own startup, it's a great learning platform to work for one. A part from that, you will be able to make your own list of do's, don'ts and maybes
|About the Author
Anjali is a guest writer for startuptimes.in . She is a startup enthusiast who loves the idea of being one of the key players in the success of aspiring startups and can be found sharing knowledge and tips accumulated from startups around her. She is the Digital Marketing Executive at Rezoomex (www.rezoomex.com) - a startup project in IT recruitment that aims to change the way job search and candidate search is done in IT industry. Anjali believes that business and people should be ethical and with purpose. Join her chain of change by education - Educate Your Maid (https://www.facebook.com/educateyourmaid)
Leave a Comment and Join the Discussion


text:
In home sessions are also called Lifestyle sessions by other photographers. These types of sessions are typically very unposed and focus on the relationship between the newborn and his or her new family.
When I photograph a newborn in your home, I focus on the relationships between the newborn, parents, siblings and pets. I typically take photos all over your home, including your living room, nursery and master bedroom. Because I am using natural light, a lot of the locations in the home are dependent upon that.
Lifestyle sessions are usually very relaxed. We don't have to worry about baby being super sleepy because mom or dad is usually holding baby. The best part? You don't have to worry about packing a bag for the baby and taking them out of the home! Everything you need will be just a few steps away!
What is included in your Newborn Lifestyle Package?
Included in your lifestyle newborn in home photography session is 2-4 hours, 10 high resolution images on a disc with a print release, and shots including siblings, family, parents, and with pets. I travel to your home within the first 5-10 days of birth. Cost is $359.


text:
Wolves seem to be quite popular with these hidden animal illusions. Check this one out and let me know how many you find. Oh and this one is a little weird in that some of the wolves are immediately visible whilst others are actually quite tricky to pick out. My last count i made it 16, there could be more.
Hidden Wolves in mountain illusion | Wolves mountain illusion
Posted on Friday, June 18, 2010 by FoOliSh
Wolves seem to be quite popular with these hidden animal illusions. Check this one out and let me know how many you find. Oh and this one is a little weird in that some of the wolves are immediately visible whilst others are actually quite tricky to pick out. My last count i made it 16, there could be more.
3D Art Adds Illusion Advertisement Aeroplane Album Cover Alphabets Amazing Art Anamorphic Angry Animals Animation Aquarium Architecture Argentina Arrow Art Artist Attention Awareness Axe Towel Bag Balancing Balcony Ball Bathroom Batman Beans Beautiful Bed Sheet Bezold BiCycle Bikini Billie Joe Armstrong Binocular Birds Black Block Boat Box Brain Brightness Building Bunny Butterfly Calm Camera Car Casino Cat Celebrities Optical Illusion Celebrity Chair Charlie Chaplin Circle City Clockwise Clothes Cloud Coca Cola Color Colour Computer Confusing Sign Couple Cow Cowboy Creative Crocodile Cube Curved Dancing Death Design Devil Diamonds Dices Dick Digital Dimensional Dizzle Dog Dolphin Dot Dragon Drawing Drinking Drunked Duck Earthquake Edges Effect Einstein Mask Elephant Elisabeth Expansion Extreme Eyes Face Falling Festival Fire Fish Flag Flexible Floating Floor Flowers Flowing Flying Focus Fountain Freeze Friends Frown Fruit Funny Gadget Game Garden Ghost Girl Glass Glowing Google Earth Gorilla Graphics Great Guitar Hand Hat Head Heart Helicopter Hidden Object Horizontal Horror Horse Hot Hotel Illusion Impossible Interesting Invisible Istvan Orosz Jauria Jesus Joshua Callaghan Julian Beever Lady Language Laptop Lava Leaning Leaves Life Lift Lifting Object Illusion Light Lines Links Lion Love Magazine Magic Man Mastaches Memories Michael Jackson Mirror Monalisa Money Moniter Moon Motion Motorbike Mountain Moving Music Mysterious Island Naked Name Nature Naughty Night Numbers Old man Opposite Optical Illusion painted illusion Painting Pandas Parallel Paris People Photography Pictures Planets Possible Prongs Puzzle Queen Queen Elizabeth Radiations Random Reading Rectangle Reflection Restaurant Ring Rocket Romantic Roof Rotating Sand Scary Scary Optical Illusions School Screen Sculpture Seconds Sense Sex Sexy Sexy Lingerie Shadow Shampian Shigeo Fukuda Simultaneous Singer Size Skeleton Sketches Skull Sleeping Smoke Snail Snakes Soccer ball Soldier Soul Spider Spinning Spiral Spitting Square Star Statue Statue of Liberty Stitches Strange Street Structure Style Sun Swelling Swimming Pool Taj Mahal Taller Tattoo Designs Test Tigers Tobacco Toilet Top Tower Tracking Transparent Tree Triangle Trick Truck Unbelievable University Unusual Image Upside Down Upstairs Valentine Day Special Vampire Vehicle Videos Virgin Mary Visual Water Waterfall Watermelons Waving Weird Wheel Whirlpool Wildlife Window Wine Wolf Wolves Women Wood Words World World Map Zebra


text:
Chairperson of International Immigration Organization Eman Oraiqat has requested the concerned Kuwaiti officials to give a grace period to the expatriates who failed to update the data of their passports, prior to imposing fines on them, in line with the ideology followed by HH the Amir of Kuwait Sheikh Sabah Al-Ahmad Al- Jaber Al-Sabah for which he won the UN 'Global Humanitarian Leader', reports Al-Rai daily.
During a TV interview, she stressed the need to respect the laws of the country because they are part of the country's sovereignty, indicating that she is following up the case with the concerned officials. She said she is certain that the Kuwaiti officials will consider the ideology of HH the Amir Sheikh Sabah Al-Ahmad Al- Jaber Al-Sabah who was first known as the 'Leader of Diplomacy' before he was granted the title 'Global Humanitarian Leader' by the United Nations.
"We hope the expatriates who are forced to pay fines will be given a grace period to rectify their situation similar to the amnesty granted by HH the Amir to the violators of residency law in 2008 and 2012, which is in line with Kuwait's status of being the 'International Humanitarian Center", she affirmed.
Regarding the statements issued by some Kuwaiti officials to reduce the number of expatriates in the country, she said, "We support any country that is keen about correcting its internal balance in order to protect its security and social infrastructure.
Therefore, we are cooperating with the concerned ministries to ensure implementation of a fair mechanism for reducing the number of expatriate personnel in Kuwait". She added that human trafficking is considered as the third profitable illegal trade after drugs and weapons due to which the process of controlling the illegal companies that bring marginal personnel to Kuwait will be the first step on the right path.
"It is hard to fail, but it is worse never to have tried to succeed."
"Envy comes from wanting something that isn't yours. But grief comes from losing something you've already had."


text:
by Susan Hutton
Poetry Foundation Media Services
It's a warm, windy day in the Mexican War Streets district of Pittsburgh, and I am looking for the house where the exiled Chinese poet Huang Xiang has been settled for nearly two years. The street is so narrow that it might easily be mistaken for an alley. Finally I find what I've heard described: a house with Huang Xiang's poetry painted on its brown clapboard exterior in vast, spidery characters. Some of the characters are almost six feet tall.
Huang Xiang is a Chinese poet who is sponsored by the Pittsburgh branch of the North American Network of Cities of Asylum (NANCA), an organization that seeks to aid and defend writers who are persecuted in their home countries. Founded in 2003 by Russell Banks, Wole Soyinka, and Salman Rushdie, NANCA was born out of the International Parliament of Writers. Huang Xiang began his residency in Pittsburgh in 2004 and writes from the house he refers to as "Poet's House, Dream Nest."
The ancient Chinese poets wrote on walls and in caves, and carved their words in stone. "I want to preserve and expand this Chinese tradition," he says, referring to the poems painted on his house, "where the poem is on the street, on the stones, in the gardens. I want to beautify every corner."
Born in 1941 in the Chinese province of Hunan, Huang Xiang is a compact, well-kept man who meets me at the door in a white pressed shirt. Nothing about his appearance betrays the 12 years he spent in Chinese prisons and labor camps. He was first arrested in 1959 for leaving one province without official permission and seeking employment in another. For this he was sentenced to four years in laogai, a reform camp similar to the Russian gulag. In 1965 he was arrested for engaging in counterrevolutionary activities—primarily writing, reading, and discussing issues related to human rights—and was sentenced to three years of hard labor in laogai and forbidden to read or write. By the time he was 25, he'd served more than seven years in laogai. His writings were banned in China for 40 years.
Though he avoided prison for the next decade, he was officially forbidden to write. He continued to do so anyway, secretly; his rooms were regularly searched, and any discovered writing was confiscated. Out of necessity, he made it a habit to commit his poems to memory, sometimes reciting them privately for a small circle of friends.
In 1978 Huang Xiang traveled 1,500 miles to Beijing to post his poems in huge character posters on what became known as the Democracy Wall. His act sparked the Democracy Wall Movement, in which dissidents posted news and ideas on a wall in the Xidan district of Beijing. Over a six-month period, Huang Xiang returned to Beijing on three separate occasions to post more poems, to advocate for democracy and human rights, and to criticize Mao Zedong and the Cultural Revolution. Arrests and imprisonments followed for decades until 1996, when Huang Xiang discovered he'd again been named the leader of a counterrevolutionary clique because of his literary pursuits. Fearing another arrest, he and his wife, Zhang Ling, fled China and were granted asylum in the United States in February 1997.
Huang Xiang is widely regarded as China's Walt Whitman. His poetry ranges widely in subject matter, touching on politics, philosophy, love, the beauty of the rural provinces, spiritual life, and his beloved literary ancestors—Wang Wei, Li Bai, and Du Fu in particular. There is a quality of feeling in his poems that is consistently, essentially human. Take, for example, the closing lines of his poem "Dry Bones":
After millions of years,
Millions of years in the layered earth
A future anthropologist
Or archeologist
When digging up my dead bones
Will, please, under this same burning sun
Raise up these remains of water and air, and
Seek out the Man.
He wrote this poem when he was 27 years old. It's a striking thing by itself, but it is even more striking when one considers the context in which it was written. Millions died as the People's Republic gained power, and their stories were lost forever. Even as a young man, Huang Xiang was determined to keep his story alive even as his persecuted body became mere "remains."
It is moving to see his house, covered in characters as it is, as an echo of the act of the young man who traveled to Beijing in order to post his poems, holding a bucket of flour paste in front of a growing crowd of sympathizers who linked arms to protect him. Like the poems on the Democracy Wall, the house poems are public objects of art.
"When I posted my poems on the Democracy Wall, a huge crowd gathered. It was very risky, and the consequences were dear. Here, on the house, it was not risky. It was safe. The first time it was an act of rebellion; this time it is an expression of art. And if the two are combined, it reflects my pursuit of spiritual, artistic freedom."
This is his ultimate aim, he says, leaning forward intently. "I want to write my poems on the sky," he says, "so everyone can read them."
Later, reviewing my notes, I recognized the line. It comes from the poem for which he is best known: "Song of the Torches." The translation I have reads: "It seemed to me that there was nobody else in the church, nor / In the city, nor in the whole world. The sky was my paper, / And I was holding an immense brush to write on it."
Susan Hutton's first book of poems is On the Vanishing of Large Creatures. She lives in Ann Arbor.
© 2007 by Susan Hutton. All rights reserved.
EDITOR'S NOTE: Huang Xiang has recently moved to New York City.
Distributed by the Poetry Foundation at www.poetryfoundation.org.


text:
From the University of South Carolina School of Medicine Genetic Counselibg Program:
Help future young, high-risk individuals like me and you by participating in a research study! We would love to hear your story! We are looking for women or men under the age of 25 who have undergone genetic testing and know that they carry a BRCA1 or BRCA2 gene mutation to take our online survey! For more details and information or to participate in our research please click here:http://www.surveymonkey.com/s/8HQNVDD


text:
Today's FREE MP3 is doubly on-time. One, it fits with Hispanic Heritage Month; alto saxophonist Miguel Zenón was born in San Juan, Puerto Rico.
Two, the cat was just named a MacArthur Fellow. That means he'll receive $500,000, no strings attached... a so-called "genius grant."
(The MacArthur Foundation always manages to reward one badass musician amongst the scientists, writers and visual artists it blesses likewise. Last year it was bluesman Corey Harris. In 2006, jazz violinist Regina Carter.)
If you follow this link to Miguel Zenón's website and scroll to the bottom, you can download a full 54-minute set that Zenón's quartet recorded live in Paris (for radio broadcast) on May 16, 2008.
Click here to hear a portion on my Vox blog. This tune is titled "Ulysses in Slow Motion."
The other compositions on the MP3 are "Santo" and "Third Dimension." All three are from Zenón's latest album, "Awake," which was produced by Branford Marsalis.
If you like what you hear, check out the 5-minute promo video at Amazon.com. It includes a testimonial from Marsalis.
Monday, September 29, 2008
A free Miguel Zenón download
Posted by Undercover Black Man at 11:16 AM
Labels: Free MP3s
3 comments:
- DeAngelo Starnes said...
This guy is a great talent on the saxophone. Check the Liberation Music Orchestra's Not in Our Name and he plays some baad shit. Thanks for this Dave.
- September 29, 2008 at 12:08 PM
- Undercover Black Man said...
^ Cool. I assumed you might be familiar with him, DeAng. I bought "No in Our Name" on your recommendation, but haven't had a chance to spin it yet.
- September 29, 2008 at 2:08 PM
- DeAngelo Starnes said...
Spin that thing, brotha. You will love the medley they do on America the Beautiful/Lift Ev'ry Voice/Skies Over America. Some beautiful dissonance that I know a musicologist like yourself will dig.
Is it me or did Barack beat McCain down like Larry Holmes did Muhammed Ali? The commentators seemed to think it was even. I didn't see it, and I'm not being biased about it. McCain put me to sleep.
- September 29, 2008 at 5:59 PM
Subscribe to: Post Comments (Atom)


text:
Countries Publishing Free Photo Guides
Monday, August 30, 2010
New Photoguide Site - Bali
Hey it's been a while since we have launched a new site, but I'm happy to announce that we have an administrator for the Bali site. Graham Grocott has taken up the reigns and knows a few things about the places to photograph in this beautiful paradise.
So if you have a minute, pop over to the Bali site and check out his first photoguide.
Welcome Graham
Posted by Brent Pearson at 5:08 PM
1 comment:
- Sydney Photographer said...
good one..
- May 10, 2011 at 3:57 AM
Subscribe to: Post Comments (Atom)


text:
Connor loves Thomas the Train so we asked Heidi to create a train wall mural with Connor's name incorporated into it. She sent us an initial draft and, after a few e-mails back and forth, she came up with the final product which we are very happy with. Most importantly, Connor loves his new train and talks about it every time he goes into his room.
Heidi's business is very unique in that she will take any idea you come up with and create an original design just for you. Then, you can always provide additional suggestions and tweak the design (within reason of course). Our suggestions were adding the train cars to the engine and a bit of steam puffing from the engine to add some balance. She was willing to try anything and provided a few different options in our back and forth communications. I have posted a few photos to show the final product. I love that the letters are nice and large so Connor can practice saying and spelling his name.
One of the most important things about vinyl wall art is the actual application. I am certainly no expert but Jeremy is since his mom owns a graphics shop and he has worked with his fair share of vinyl in the past. We actually had some trouble putting the mural up the first time around because the masking layer wasn't quite sticky enough. After letting Heidi know, she promptly sent some new masking and it worked just fine. The customer service throughout the process of designing and shipping the wall art has been fantastic. Along with the wall art, she also sent detailed instructions and a small squeegee to help with the application. The last time we bought wall art, we had to use a credit card so I appreciate the extra tool!
As I said, I am no expert at wall art or vinyl but Jeremy mentioned that he was very impressed with the details and variety of colours. You don't often see wall art that allows for so many colours or such fine detail. It's really quite exceptional and particularly impressive that you are able to choose exactly what you'd like.
I have posted a couple more examples to show some of the other pieces that she has done. Each custom wall art is roughly 30" in length and 18-22" tall. Wall art is available in any size/colour and is completely customizable to you.
The Giveaway:
One lucky blog reader will receive a customized piece of wall art that is roughly 30" in length and 18-22" tall. You will have your choice of design and Heidi would be happy to create something unique just for you.
As usual, I will use www.random.org to select a winner and all entries must be made in separate comments.
How to Enter:
1. Tell us what type of design you would choose for your child's wall art.
2. "Like" Willmek Sign Works on Facebook. Feel free to leave a compliment while you're there!
3. Follow A Sophisticated Mommy via Google Friend Connect.
4. "Like" A Sophisticated Mommy on Facebook.
5. Share this giveaway. (e.g. Facebook, Twitter, Blog, Forum)
This giveaway is open to Canada only. Contest will be open until Friday, June 17th, 2011 at Midnight SK Time.
|Here's Connor checking out his train for the first time.


text:
Here's a quick little review of a stand I came across at our farmer's market. The stand was Taco's El Vaquero (sic). The selections are limited but fresh and tasty. This day he had chicken, steak and goat meats for tacos or tortas. I had a goat and steak taco. Both were very good, loaded up with the works. I also tried a fabulous agua fresca that was a mix of apple, melons and banana. One of the best aguas frescas I've had in a long time. He just does farmer's markets in Northern Orange County (Orange, Fullerton, Garden Grove and Anaheim, I think) as well as catering.
You can see the phone numbers on his sign. Don't go to the address though - as I say, it is just catering and the markets. I think the address is his home.


text:
eIF3 binds to 40S ribosomal subunits and stimulates recruitment of Met‐tRNAiMet and mRNA to the pre‐initiation complex. Saccharomyces cerevisiae contains an ortholog of human eIF3 subunit p35, HCR1, whose interactions with yeast eIF3 are not well defined. We found that HCR1 has a dual function in translation initiation: it binds to, and stabilizes, the eIF3–eIF5– eIF1–eIF2 multifactor complex and is required for the normal level of 40S ribosomes. The RNA recognition motif (RRM) of eIF3 subunit PRT1 interacted simultaneously with HCR1 and with an internal domain of eIF3 subunit TIF32 that has sequence and functional similarity to HCR1. PRT1, HCR1 and TIF32 were also functionally linked by genetic suppressor analysis. We propose that HCR1 stabilizes or modulates interaction between TIF32 and the PRT1 RRM. Removal of the PRT1 RRM resulted in dissociation of TIF32, NIP1, HCR1 and eIF5 from eIF3 in vivo, and destroyed 40S ribosome binding by the residual PRT1–TIF34–TIF35 subcomplex. Hence, the PRT1 RRM is crucial for the integrity and ribosome‐binding activity of eIF3.
Translation initiation in eukaryotes is a complex series of reactions leading to the formation of an 80S ribosomal complex containing methionyl initiator tRNA (Met‐tRNAiMet) base paired with the initiation codon in mRNA. These reactions require the participation of numerous protein factors called initiation factors (eIFs), several of which operate at multiple steps in the process. eIF3 and eIF1A stimulate dissociation of 80S ribosomes into 40S and 60S subunits, and also promote binding of a ternary complex consisting of Met‐tRNAiMet, eIF2 and GTP to the small subunit, forming the 43S pre‐initiation complex (reviewed in Merrick and Hershey, 1996). eIF3 remains bound to the 43S complex and prevents its disruption by 60S ribosomal subunits (Chaudhuri et al., 1999). The 43S complex interacts with the m7G cap structure at the 5′ end of mRNA, forming the 48S complex, in a reaction stimulated by multiple mRNA‐binding factors [eIF4F, eIF4A, eIF4B and poly(A)‐binding protein (PAB1)] and eIF3 (Merrick and Hershey, 1996; Sachs and Varani, 2000). The 48S complex scans the mRNA until an AUG start codon is encountered, facilitated in this process by eIF1 and eIF1A (Pestova et al., 1998). Upon base‐pairing between the anticodon of Met‐tRNAiMet and the AUG start codon, eIF5 stimulates the hydrolysis of GTP bound to eIF2, followed by release of the eIF2–GDP binary complex and other initiation factors. The 60S ribosome then joins with the 48S pre‐initiation complex, dependent on eIF5B, and the entire process is completed when eIF5B is released from the 80S initiation complex (Pestova et al., 2000).
Mammalian eIF3 has the most complex structure of the initiation factors, containing 11 non‐identical subunits (Vornlocher et al., 1999). An eIF3 complex has been isolated from the yeast Saccharomyces cerevisiae by affinity purification and gel filtration that contains only five subunits, TIF32/RPG1, NIP1, PRT1, TIF34 and TIF35 (Danaie et al., 1995; Asano et al., 1998; Phan et al., 1998), which are homologs of human eIF3 subunits p170, p110, p116, p36 and p44, respectively (Asano et al., 1997). A mutation in PRT1 or depletion of NIP1 both led to defects in Met‐tRNAiMet binding to 40S ribosomes in yeast cell extracts that could be complemented with the purified yeast eIF3 complex. Thus, the yeast five‐subunit factor can associate with 40S subunits and stimulate Met‐tRNAiMet binding, two of the key activities ascribed to mammalian eIF3 (Phan et al., 1998).
We demonstrated recently that yeast eIF3, eIF1, eIF5 and the ternary complex reside in a multifactor complex (MFC) that is an important intermediate in translation initiation (Asano et al., 2000). eIF1 and eIF5 are physically associated with eIF3 via the NIP1 subunit (homolog of mammalian eIF3‐p110) (Naranda et al., 1996; Asano et al., 1998; Phan et al., 1998), whereas the Met‐tRNAiMet·eIF2·GTP–eIF3 interaction is bridged by eIF5 (Asano et al., 1999, 2000). Some of these interactions have been confirmed for the corresponding mammalian factors (Bandyopadhyay and Maitra, 1999; Fletcher et al., 1999). In yeast, eIF5 and eIF1 were shown to promote stringent selection of AUG as the start codon (Huang et al., 1997); hence, eIF3 may play a structural role in AUG recognition and GTP hydrolysis in addition to promoting initiation complex assembly.
Only one additional human eIF3 subunit (p35) has an ortholog encoded in the S.cerevisiae genome. The gene encoding this protein, HCR1, was identified as a high‐copy suppressor of the temperature‐sensitive (Ts−) phenotype of the rpg1‐1 allele of TIF32/RPG1 (Valášek et al., 1998, 1999). The hcr1Δ mutant was viable, but showed a reduction in growth rate. Combining the hcr1Δ allele with rpg1‐1 exacerbated the growth defect conferred by rpg1‐1. Moreover, the HCR1 and TIF32 gene products were co‐immunoprecipitated from yeast cell extracts. Despite the fact that purified yeast eIF3 preparations did not contain HCR1 (Phan et al., 1998), these findings suggested that HCR1 is physically associated with yeast eIF3 in vivo.
We show here that a portion of HCR1 resides in the MFC containing eIF3, eIF5, eIF1 and the ternary complex. Importantly, the hcr1Δ mutation decreased the abundance of the MFC, implicating HCR1 in pre‐initiation complex assembly. We also found that recombinant HCR1 and the internal segment of TIF32, which is 25% identical to HCR1, bound concurrently to the RNA recognition motif (RRM) of PRT1, indicating a network of physical interactions involving HCR1, TIF32 and the PRT1 RRM. A mutant PRT1 protein lacking the RRM was shown previously to interfere with translation in otherwise wild‐type cells and to reside in defective complexes incapable of stable binding to 40S ribosomes (Evans et al., 1995). In agreement with our in vitro binding data, we found that these defective complexes contained TIF34 and TIF35 but lacked TIF32, HCR1 and NIP1. We propose that HCR1 serves to stabilize or modulate the interaction between TIF32 and the PRT1 RRM, which is crucial for integrity of the MFC and its association with 40S ribosomes. Unexpectedly, deletion of HCR1 reduced the abundance of 40S ribosomes, indicating a novel function for this eIF3‐associated factor in ribosome biogenesis or stability.
HCR1 is an eIF3‐associated protein that interacts directly with PRT1 and TIF32/RPG1
To determine whether the previously described HCR1–TIF32 complex (Valášek et al., 1999) also contained the other subunits of eIF3, we prepared whole‐cell extracts (WCEs) from the hcr1Δ strain transformed with a plasmid encoding c‐Myc‐tagged HCR1 (Myc‐HCR1) or an empty vector. These extracts were immunoprecipitated with antibodies against TIF32 or c‐Myc and the immune complexes were probed with antibodies against eIF3 subunits, eIF1 and eIF5. As expected, the anti‐TIF32 antibodies immunoprecipitated a large fraction (∼60%) of TIF32, PRT1, TIF34 and TIF35 (all eIF3 subunits), ∼20% of the eIF5 and ∼10% of the eIF1 from both extracts. Additionally, ∼20% of the Myc‐HCR1 co‐immunoprecipitated with TIF32 from the HCR1‐Myc extract. The ϵ subunit of eIF2B (GCD6), which does not interact with eIF3 (Cigan et al., 1991), was not immunoprecipitated by the TIF32 antibodies from either extract (Figure 1A, lanes 3 and 5). The TIF32–HCR1 complexes detected above were judged to be part of eIF3 because the c‐Myc antibodies immunoprecipitated ∼10–20% of the other eIF3 subunits, 1–3% of eIF1 and eIF5, and no GCD6, along with ∼60% of the Myc‐HCR1 (Figure 1B, lane 3). None of these proteins was immunoprecipitated with c‐Myc antibodies from the wild‐type untagged extract (Figure 1B, lane 5). These results demonstrate that HCR1 is physically associated with the eIF3–eIF1–eIF5 complex.
The relatively small proportions of eIF3 subunits, eIF5 and eIF1 that co‐immunoprecipitated with Myc‐HCR1 compared with the larger amounts of these proteins that co‐immunoprecipitated with TIF32 suggested that HCR1 might be a substoichiometric component of eIF3. To investigate this possibility, we compared the amounts of the c‐Myc‐tagged forms of these two proteins expressed in the wild‐type strain W303 by western blot analysis of WCEs using antibodies against the c‐Myc epitope. The results suggested that Myc‐HCR1 was present at a quarter of the level of Myc‐PRT1 (data not shown). Using polyclonal antibodies against HCR1 and PRT1, we found that ratios of Myc‐HCR1 to total HCR1, and of Myc‐PRT1 to total PRT1, were very similar in these extracts. Assuming that the specific binding activity of the c‐Myc antibody was identical for the two tagged proteins, these results suggest that no more than a quarter of the total eIF3 complexes contain HCR1. We also noted that a smaller proportion of the total HCR1 was immunoprecipitated with TIF32 antibodies than was observed for other eIF3 subunits. Thus, in addition to its relatively low abundance, it appears that HCR1 is less tightly associated with eIF3 than are the five core subunits.
We next asked whether HCR1 can interact directly with an individual subunit of eIF3. A glutathione S‐transferase (GST)–HCR1 fusion protein was incubated with various 35S‐labeled eIF3 subunits synthesized in reticulocyte lysate. Large fractions of the radiolabeled PRT1 (∼40%) and TIF32 (∼20%) were bound to the immobilized GST–HCR1 (Figure 1C). Much lower amounts of [35S]HCR1 (∼1–2%) and no detectable radiolabeled NIP1, TIF34 or TIF35 bound to GST–HCR1. These data suggest that HCR1 binds directly to eIF3 subunits TIF32 and PRT1. It was reported previously that p170 is the binding partner in human eIF3 for p35, the homolog of HCR1 (Block et al., 1998). Thus, the association of HCR1/p35 with the largest subunit of eIF3 is evolutionarily conserved between yeast and humans.
HCR1 enhances formation of the MFC, the abundance of 40S ribosomes and the rate of translation initiation in vivo
It was of interest to determine whether deleting HCR1 would destabilize the MFC (Asano et al., 2000). In the first approach to addressing this issue, we immunoprecipitated the MFC from HCR1 and hcr1Δ extracts using TIF32 antibodies and quantified the amounts of the individual components (Figure 1D, bottom panel). We found that deletion of HCR1 reduced the amounts of eIF2 and eIF5 (by ∼30%) and the amount of eIF1 (by ∼75%) that co‐immunoprecipitated with eIF3 (Figure 1D, lanes 2 and 4). Considering that HCR1 is less abundant than eIF3, this represents a significant effect on the formation or stability of the MFC.
In the second approach, we separated the MFC from its individual constituents by resolving WCEs from HCR1 and hcr1Δ strains on a sucrose gradient by velocity sedimentation. At the same time, we examined the polysome profiles in these extracts to determine whether the slow growth phenotype of the hcr1Δ strain resulted from diminished translation initiation. As shown in Figure 2A, deletion of HCR1 led to only a slight reduction in the polysome/monosome ratio (2.57 in the hcr1Δ mutant versus 3.08 in the wild type). However, there was an obvious decrease in the proportion of large polysomes in the mutant, indicating a reduced rate of translation initiation. Deletion of HCR1 also led to a striking reduction in the abundance of free 40S subunits and a corresponding increase in free 60S subunits. The 60S/40S ratio for the hcr1Δ strain was 4.32 compared with 1.19 for the wild type. This last defect suggests that HCR1 is required for the biogenesis or stability of 40S ribosomes (Kressler et al., 1999) in addition to its function in eIF3.
Western blot analysis of the gradient fractions (Figure 2B) and quantification of these data (Figure 2C) revealed that eIFs 1, 2, 3 and 5 were depleted specifically in fractions 6–9 containing the MFC (Asano et al., 2000) in the hcr1Δ mutant versus the wild type. There was a corresponding increase in the proportions of eIFs in fractions 10–12 containing 43–48S initiation complexes in the hcr1Δ mutant (Figure 2B and C). Probing for the 40S subunit ribosomal protein S22 revealed the anticipated ∼2‐fold reduction in the amount of free 40S subunits in the mutant (Figure 2B). Hence, in Figure 2C, the calculated proportions of each factor in the 40–48S region were normalized for the amounts of 40S ribosomes present in the relevant fractions. These findings suggest that HCR1 promotes the formation or stability of the MFC, and that inactivation of HCR1 impedes the conversion of 43S to 48S, or 48S to 80S, initiation complexes. The results in Figure 2B (top panel) also provide additional evidence that HCR1 is a component of the MFC and is recruited to 43–48S initiation complexes along with the other constituents of the MFC.
Considering that the hcr1Δ mutation led to reduced amounts of 40S subunits, we asked whether the decreased amount of the MFC or the reduced rate of converting 43S/48S to 80S complexes seen in the mutant could be a secondary consequence of diminished 40S subunits. Inconsistent with this possibility, we found that deletion of the 40S ribosomal protein gene RP51A did not alter the distribution of eIFs between the MFC and 43–48S initiation complexes (data not shown). Importantly, rp51aΔ leads to an even greater reduction in the pool of 40S subunits and growth rate than does hcr1Δ (Foiani et al., 1991).
High‐copy HCR1 is an allele‐specific suppressor of prt1 mutations
To obtain evidence that HCR1 interacts with PRT1 in vivo, we asked whether high‐copy HCR1 could suppress the Ts− phenotypes of various prt1 mutations. As shown in Figure 3B, high‐copy HCR1 strongly enhanced growth at 36°C in the prt1‐63 and prt1‐3 mutants, and weakly stimulated growth of the prt1‐1 strain. The Ts− phenotypes of the prt1‐2 and prt1‐26 mutants were not suppressed by high‐copy HCR1, even though the latter mutant has a less severe phenotype than does the prt1‐63 mutant, which was strongly suppressed by high‐copy HCR1 (Figure 3B). The allele‐specific suppression of different prt1 mutations by high‐copy HCR1 is consistent with a direct interaction between PRT1 and HCR1 in vivo.
The RRM of PRT1 is necessary and sufficient for interaction with TIF32 and HCR1 in vitro
We showed previously that PRT1 could interact with all core eIF3 subunits except NIP1 (Asano et al., 1998). Having found that HCR1 also interacts with PRT1, we set out to locate precisely the binding domains in PRT1 for HCR1 and other eIF3 subunits. A battery of N‐ or C‐terminal truncations of [35S]PRT1 (depicted in Figure 3A) was tested for in vitro binding to GST fusions containing full‐length HCR1 or other eIF3 subunits. Only the first 136 residues of PRT1 (in fragment ΔA‐PRT1), encompassing the RRM, were sufficient for binding to GST–TIF32 and GST–HCR1 (Figure 3C, lanes 3 and 6). Consistently, deletion of 42 or 100 residues from the C‐terminus of PRT1 (ΔM‐PRT1 and ΔS‐PRT1) did not reduce its interaction with GST–HCR1. It was shown previously that these deletions disrupted interactions of PRT1 with GST–TIF34 and GST–TIF35, but not with GST–TIF32 (Asano et al., 1998). Removal of the first 27 residues from the PRT1 segment (yielding ΔB‐PRT1) abolished its interaction with GST–TIF32 without affecting its binding to GST–HCR1. Binding to GST–TIF32 in the absence of the first 27 residues of PRT1 was restored, however, by including an additional 125 residues C‐terminal to the RRM (yielding ΔC‐PRT1). Deletion of the N‐terminal 110 residues including most of the RRM (yielding fragment Δ1‐PRT1), or residues 1–260 (Δ2‐PRT1), abolished binding of PRT1 to GST–HCR1 and GST–TIF32 (Figure 3C). Thus, the RRM in PRT1 is necessary and sufficient for binding HCR1, whereas TIF32 binding requires additional PRT1 residues immediately flanking the RRM.
Previously, we showed that the C‐terminal 83 residues of PRT1 were sufficient for interaction with TIF34 and TIF35 in yeast two‐hybrid assays, and we confirmed this finding for TIF34 by showing that [35S]TIF34 synthesized in vitro bound to the GST–PRT1[641–724] fusion containing the C‐terminal 83 residues of PRT1 (Asano et al., 1998). Additionally, deletion of residues 625–724 or 683–724 from [35S]PRT1 (ΔM‐PRT1 and ΔS‐PRT1) abolished its interaction with GST–TIF34 and GST–TIF35 (Asano et al., 1998). In the same study, however, [35S]TIF35 failed to interact with GST– PRT1[641–724], suggesting that the binding domain for TIF35 extends further towards the N‐terminus of PRT1 than that for TIF34. However, we found here that GST–TIF35 bound strongly to [35S]PRT1 segments containing only the C‐terminal 123 or 83 residues of the protein (Figure 3C, Δ6‐PRT1 and Δ7‐PRT1). Presumably, fusion of the C‐terminal fragment of PRT1[641–724] to GST masked its TIF35‐binding site without affecting that of TIF34. We conclude that HCR1 and TIF32 bind to the N‐terminal segment of PRT1 harboring the RRM, whereas TIF34 and TIF35 interact with the extreme C‐terminus of PRT1 (Figure 3A).
An internal segment of TIF32 similar in sequence to HCR1 is sufficient for interaction with the RRM of PRT1
Prompted by our finding that HCR1 and TIF32 both interacted with the RRM in PRT1, we aligned the HCR1 and TIF32 sequences and discovered that residues 490–790 in TIF32 are 25% identical to the entire sequence of HCR1 (Table I). Accordingly, we tested whether the HCR1‐like domain (HLD) in TIF32 binds to the PRT1 RRM. A 35S‐labeled fragment comprising the HLD of TIF32 (Δ1‐TIF32, Figure 4A) was sufficient for strong binding to GST fusions containing either full‐length PRT1 or the N‐terminal 136 residues of PRT1 (GST–PRT1 RRM[1–136]) (Figure 4B). Simultaneous removal of 61 and 40 residues from the N‐ and C‐terminus, respectively, of this internal TIF32 fragment (yielding Δ2‐TIF32) reduced its binding to GST–PRT1 RRM[1–136]. TIF32 fragments Δ1 and Δ2 showed no binding to GST–NIP1 or GST–HCR1 (Figure 4B), indicating the specificity of their interaction with the PRT1 RRM.
An internal deletion in TIF32 removing most of the HLD (ΔSal‐TIF32) significantly reduced, but did not abolish, binding to the GST–PRT1 fusions. Thus, the HLD of TIF32 is sufficient, but not absolutely required, for TIF32 binding to the RRM domain in PRT1. Moreover, the C‐terminal domain of TIF32 (Δ4‐TIF32) bound to GST–PRT1 RRM[1–136] but not to GST–NIP1 or GST–HCR1, identifying a second region in TIF32 sufficient for binding to the PRT1 RRM. Deletion of both the C‐terminal domain and the HLD (Δ3‐TIF32 and Δ5‐TIF32) abolished interaction of TIF32 with GST–PRT1 and GST–PRT1 RRM (Figure 4B). These findings suggest that TIF32 can bind to the RRM domain of PRT1 through its HLD or C‐terminus. The fact that full‐length GST–PRT1 did not bind to the C‐terminal segment of TIF32, whereas GST–PRT1 RRM did (Figure 4B), may indicate that intramolecular interactions in PRT1 compete for interaction between the PRT1 RRM and C‐terminal domain of TIF32.
The TIF32 fragments containing the N‐terminal half of the protein (ΔSal‐, Δ3‐ and Δ5‐TIF32) bound to GST–NIP1 and GST–HCR1, whereas the C‐terminal fragments (Δ1‐, Δ2‐, Δ4‐ and Δ1+4‐TIF32) did not (Figure 4B). These data suggest that NIP1 and HCR1 interact primarily with the N‐terminal half of TIF32 (Figure 4A). However, considering that none of the N‐terminal fragments of TIF32 bound as strongly as full‐length TIF32 to GST–HCR1, the C‐terminus of TIF32 may make additional contacts with HCR1, or it may stabilize the conformation of the N‐terminus of TIF32 needed for strong interaction.
We showed above that PRT1 RRM[1–136] binds both to the HLD in TIF32 and to HCR1 itself, and wished to determine whether these interactions could occur simultaneously. Having found that the HLD of TIF32 (in [35S]Δ1‐TIF32) did not bind to GST–HCR1 (Figure 4B), we asked whether the PRT1 RRM could bridge an interaction between these two polypeptides. The PRT1 RRM[1–136] fragment used in these experiments was produced by factor Xa digestion of the GST–PRT1 RRM[1–136] fusion and purified from the GST moiety. As shown in Figure 4C (lane 5), the purified PRT1 RRM fragment bound to GST–HCR1 and bridged an interaction between GST–HCR1 and [35S]Δ1‐TIF32. As expected, the PRT1 RRM fragment did not mediate binding of [35S]Δ1‐TIF32 to GST alone (lane 3). Thus, the PRT1 RRM can interact simultaneously with HCR1 and the HLD of TIF32.
Genetic evidence for the functional significance of sequence similarity between TIF32 and HCR1
The Ts− phenotype of the rpg1‐1 allele of TIF32 is conferred by a single amino acid substitution of arginine to isoleucine at position 731 (Valášek et al., 1998) in the HLD of TIF32. This arginine residue is conserved in HCR1 at position 215 (Table I) and corresponds to the C‐terminal residue in a K‐x5‐ER‐x2‐R motif that is completely conserved among S.cerevisiae HCR1 and all known TIF32 orthologs (data not shown). A similar motif (K‐x3‐EK‐x2‐K) occurs in human eIF3‐p35. To explore the physiological significance of the sequence similarity between HCR1 and TIF32, we asked whether substitution of Arg215 in HCR1 with isoleucine (hcr1‐R215I) would alter the ability of HCR1 to function in vivo as a dosage suppressor of rpg1‐1 and prt1 mutations. In parallel, we substituted the second conserved arginine present in the K‐x5‐ER‐x2‐R motif located three residues upstream of Arg215 (producing hcr1‐R212I), and separately introduced a frameshift mutation that replaced the last 57 residues of HCR1 (including the K‐x5‐ER‐x2‐R motif) with 42 novel amino acids encoded in a different reading frame (hcr1‐3).
The hcr1‐3 frameshift mutation destroyed the ability of high‐copy HCR1 to suppress rpg1‐1, prt1‐63, prt1‐3 and prt1‐1, whereas the R212I substitution had no effect on suppression of these mutations by high‐copy HCR1. Interestingly, the R215I substitution eliminated suppression of rpg1‐1, enhanced suppression of prt1‐63, and had no effect on suppression of prt1‐3 and prt1‐1 by high‐copy HCR1 (Figure 4D and data not shown). Furthermore, HCR1‐R215I on a low‐copy‐number plasmid fully complemented the slow growth phenotype of hcr1Δ strain YLVH13, whereas low‐copy hcr1‐3 did not (data not shown). This last finding indicates that HCR1‐R215I is not a null allele. We conclude that the Ile215 substitution alters the function of HCR1 in a manner that influences its ability to compensate for defects in PRT1 and TIF32 when overexpressed. The fact that mutating the equivalent amino acids in HCR1 and the HLD of TIF32 altered the functions of both proteins in vivo supports the physiological relevance of sequence similarity between HCR1 and TIF32. The different phenotypes observed when high‐copy HCR1‐R215I was combined with rpg1‐1 or various prt1 alleles provides additional evidence for functional interactions between HCR1 and the TIF32 and PRT1 subunits of eIF3.
Interaction of TIF32 with the PRT1 RRM is required for eIF3 integrity and binding of eIF3 to the 40S ribosome
It was shown previously that deletion of the N‐terminal 100 amino acids of PRT1 was lethal in vivo and that expressing the mutant protein (encoded by prt1‐Δ100) in otherwise wild‐type cells inhibited translation initiation through formation of PRT1‐containing complexes incapable of binding to 40S ribosomes (Evans et al., 1995). Having found that the PRT1 RRM is critical for its interaction with TIF32 and HCR1 in vitro (Figure 3B), we predicted that expression of prt1‐Δ100 from a low‐copy plasmid (low‐copy prt1‐Δ100) in PRT1 cells would lead to the formation of incomplete eIF3 complexes containing TIF34 and TIF35 bound to the C‐terminal domain of prt1‐Δ100 but lacking TIF32, HCR1 and NIP1. As a consequence, the pool of TIF34 and TIF35 available for association with wild‐type PRT1 would be decreased, leading to reduced amounts of intact eIF3.
We reasoned that if this interpretation is correct, it should be possible to restore high levels of intact eIF3 in cells containing prt1‐Δ100 by overexpressing TIF34 and TIF35. Indeed, TIF34 and TIF35 present together on a high‐copy plasmid fully suppressed the dominant Slg− phenotype of low‐copy prt1‐Δ100 in a PRT1 strain, whereas high‐copy‐number TIF32, NIP1 or HCR1 alone did not (Figure 5B). As high‐copy TIF34 alone partially suppressed the Slg− of prt1‐Δ100, whereas high‐copy TIF35 did not (Figure 5B and not shown), it seems that TIF34 is the limiting factor in PRT1 cells containing the prt1‐Δ100 product.
To demonstrate directly that the prt1‐Δ100 product is defective for interaction with TIF32 but retains the ability to bind TIF34 and TIF35, we inserted polyhistidine tags at the C‐termini of prt1‐Δ100 and wild‐type PRT1, and introduced the tagged alleles on high‐copy plasmids into a wild‐type strain. The tagged proteins were purified on nickel–agarose resin and analyzed by western blotting. As expected, His‐tagged wild‐type PRT1 (PRT1‐His) co‐purified with all four eIF3 subunits, HCR1 and eIF5 (Figure 5C, lanes 2 and 3), whereas none of these proteins was recovered from a control strain containing only untagged PRT1 (Figure 5C, lanes 8 and 9). Importantly, TIF34 and TIF35 co‐purified with prt1‐Δ100‐His to the same extent observed for PRT1‐His, whereas no detectable TIF32, NIP1, HCR1 or eIF5 co‐purified with the tagged mutant protein (Figure 5C, lanes 5 and 6). Thus, the RRM domain in PRT1 is essential for association of HCR1 and TIF32 (and consequently of NIP1 and eIF5) with eIF3 in vivo, but dispensable for the formation of a PRT1–TIF34–TIF35 subcomplex.
We wished to confirm that the stable subcomplexes containing TIF34, TIF35 and prt1‐Δ100 were unable to bind to 40S ribosomes in vivo. We prepared extracts from PRT1 strains containing low‐copy prt1‐Δ100‐His or an empty vector, resolved them on sucrose density gradients and probed for eIF3 subunits by western blotting. In both strains, a significant fraction of the core eIF3 subunits, HCR1 and the γ subunit of eIF2 (GCD11) were in fractions 10–12 containing the 43–48S initiation complexes (Figure 6A and B). In the wild‐type extract, most of the remainder of these proteins was in fractions 6–9, corresponding to the MFC (Figure 6B). In contrast, nearly all of the prt1‐Δ100‐His in the mutant extract was at the top of the gradient in fractions 2–4 (Figure 6A, anti‐His) along with a higher proportion of the total TIF34 and TIF35 than was seen in the corresponding fractions for the wild‐type extract (Figure 6B). Interestingly, there was a decreased proportion of eIF2 in fractions 6–9 containing the MFC and an increased proportion of eIF2 at the top of the gradient (fractions 1–4) in the mutant extract (Figure 6). Together, these data are consistent with the idea that prt1‐Δ100‐His sequesters a fraction of TIF34 and TIF35 in defective subcomplexes that cannot form the MFC or bind to 40S ribosomes. The fact that no TIF32, HCR1 or full‐length PRT1 was present at the top of the gradient in the mutant extract (Figure 6A) could indicate that the pools of these proteins expected to occur free of TIF34 and TIF35 when prt1‐Δ100‐His is being expressed are either degraded or form a subcomplex that binds to 40S subunits.
HCR1 associates with eIF3 and stabilizes the multifactor complex
HCR1 is a non‐essential yeast protein similar in sequence to human eIF3 subunit p35. Previously, it was shown that HCR1 interacted genetically and physically with TIF32 (Valášek et al., 1999), but it was unclear whether this interaction occurred in the context of eIF3. Here we showed that a proportion of the entire eIF3 complex, and also fractions of eIF5 and eIF1, were co‐immunoprecipitated with HCR1 (Figure 1). In addition, we provided evidence that HCR1 resides in the MFC (Figure 2), which is an important translation initiation intermediate in vivo (Asano et al., 2000). HCR1 was detected in 43S or 48S initiation complexes, but not in 80S ribosomes, consistent with a direct role in the initiation phase of translation. In vitro binding assays with recombinant proteins indicated that HCR1 binds directly to eIF3 subunits TIF32 and PRT1. However, it appears that HCR1 is less abundant and less tightly bound to the eIF3 complex than are the five core subunits we identified previously (Phan et al., 1998).
Deletion of HCR1 led to a slow growth phenotype and produced abnormalities in the polysome profile, indicating a reduced rate of translation initiation. Coincidentally, we observed a sharp decrease in the association of eIF1 with eIF3, and a moderate decrease in the amount of eIF5 and eIF2 associated with eIF3 in the hcr1Δ mutant (Figure 1D). The latter phenotype suggests that the abundance of the MFC is diminished in hcr1Δ cells, which we confirmed by comparing the amounts of MFC resolved on sucrose gradients between mutant and wild‐type extracts (Figure 2B). Considering that both eIF1 and eIF5 bind to eIF3‐NIP1, and that eIF5 bridges the interaction between eIF2 and eIF3 in the MFC (Asano et al., 2000), these defects suggest that HCR1 promotes the known interactions between NIP1 and eIFs 1 and 5. Although HCR1 did not interact with NIP1 in our binding experiments, we recently obtained evidence that NIP1 enhances the binding of HCR1 to PRT1 and TIF32 (L.Phan, L.Schoenfeld, L.Valášek and A.G.Hinnebusch, unpublished observations).
We detected an accumulation of the eIFs comprising the MFC in 43–48S complexes in hcr1Δ cells (Figure 2B and C), suggesting that recruitment of these factors to 40S ribosomes is not rate limiting in this mutant. Rather, it appears that conversion of 43S to 48S, or 48S to 80S, initiation complexes occurs inefficiently in the absence of HCR1. Recently, we observed a defect in the conversion of 48S to 80S complexes resulting from a mutation in eIF5 (tif5‐7A) that also destabilized the MFC. It appears that AUG recognition or GTP hydrolysis by eIF2 is impaired when the integrity of the MFC complex is disrupted by the tif5‐7A mutation (K.Asano, L.Phan, L.Valášek and A.G.Hinnebusch, unpublished observations). Similarly, the impaired interaction of eIFs 1 and 5 with eIF3 produced by hcr1Δ may have a deleterious effect on the scanning process. This would be consistent with the known roles of eIF1 and eIF5 in selection of the start codon by scanning ribosomes (Huang et al., 1997; Pestova et al., 1998).
Unexpectedly, deletion of HCR1 decreased the steady‐state level of 40S subunits and produced a concomitant increase in free 60S subunits. This reduction in 40S subunits probably contributes to the diminished polysome size and slow growth phenotype of the hcr1Δ strain. It is unlikely to account for the decreased amounts of the MFC or the accumulation of 43S or 48S complexes observed in hcr1Δ cells, as these defects were not observed in the rp51aΔ mutant with an even greater depletion of 40S subunits. Hence, we conclude that HCR1 has a dual function in translation initiation: enhancing the activity of eIF3 and its associated factors, and promoting the biosynthesis or stability of 40S subunits. An attractive hypothesis is that both functions depend on the ability of HCR1 to bind to a specific site on the 40S ribosome. In this way, HCR1 could interact with factors involved in 40S biogenesis and also promote the correct orientation of the MFC on the ribosome in 43–48S initiation complexes. Its dual interactions with 40S ribosomes and eIF3 might help explain why HCR1 is less tightly associated with eIF3 than are the five core subunits. Interestingly, eIF6 may be another protein with a dual function in ribosome biogenesis and translation initiation. It was isolated originally as an anti‐association factor that binds to 60S ribosomes (Merrick and Hershey, 1996) but was implicated more recently in promoting the stability or biogenesis of 60S subunits (Si and Maitra, 1999).
A network of interactions linking HCR1, TIF32 and the RRM domain at the N‐terminus of PRT1
We noted that HCR1 is related in sequence to a functionally important segment of TIF32, and showed that HCR1 and the HLD in TIF32 both interact with the N‐terminal segment of PRT1 containing an RRM motif. As these interactions can occur simultaneously in vitro, we propose that HCR1 stabilizes or modulates the interaction between TIF32 and the PRT1 RRM (Figure 7). HCR1 additionally interacted with TIF32 in vitro. It is unlikely that the interactions of TIF32 and HCR1 with the PRT1 RRM domain were mediated by RNA, because RNase A treatment did not reduce these interactions in GST pull‐down experiments of the type shown in Figure 3 (data not shown). The interaction between TIF32 and the RRM domain of PRT1 is conserved in the human homologs of TIF32 and PRT1 (Methot et al., 1997). PRT1 also contains binding domains for TIF34 and TIF35, and thus seems to function at least partly as a scaffold in the eIF3 complex. Only the extreme C‐terminus of PRT1 (residues 641–724) was required for its interactions with TIF34 and TIF35 in vitro (Figure 7).
In keeping with our in vitro binding data, the prt1‐Δ100 product lacking the RRM domain formed a stable subcomplex in vivo containing TIF34 and TIF35 but lacking HCR1, TIF32, NIP1 and eIF5 (Figure 5C). These results provide in vivo evidence that the PRT1 RRM is required for incorporation of TIF32 into eIF3. Since NIP1 seems to interact only with TIF32 (Asano et al., 1998), and contains the binding domain for eIF5 (Phan et al., 1998) (Figure 7), deleting the RRM from PRT1 should lead simultaneously to loss of TIF32, NIP1 and eIF5 from the complex, as we observed.
Role of the PRT1 RRM domain in 40S subunit binding
We showed that expression of prt1‐Δ100 sequesters a fraction of TIF34 and TIF35 in defective subcomplexes lacking TIF32 and NIP1, which fail to bind eIF5 and HCR1 (Figure 5C) and cannot interact tightly with 40S ribosomes. Consistently, expression of these prt1‐Δ100–TIF34–TIF35 subcomplexes reduced formation of the multifactor complex containing eIF2 (Figure 6A). Our conclusion that the decreased level of intact eIF3 accounts for the growth defect conferred by prt1‐Δ100 is supported by the fact that this phenotype was suppressed by overexpressing TIF34 and TIF35 (Figure 5B). Presumably, providing excess amounts of these two subunits restored the concentration of intact eIF3 containing full‐length PRT1 to nearly wild‐type levels.
The sequestration of TIF34 and TIF35 in defective eIF3 subcomplexes incapable of forming the MFC or binding to 40S ribosomes might be expected to decrease the formation of functional 43–48S complexes. Ostensibly at odds with this expectation, we did not observe a reduced proportion of eIF3 subunits or other eIFs in the 43–48S region of the gradient in the mutant extract containing prt1‐Δ100 (Figure 6). However, the predicted decrease in the rate of 43–48S complex formation in the mutant could be masked if binding of the MFC complex to 40S subunits continued after cell lysis while conversion of 48S to 80S initiation complexes occurred slowly in the extracts. In this event, there would be similar amounts of 48S complexes in mutant and wild‐type extracts plus an excess of free MFCs in the wild‐type extract, as we observed (Figure 6). This explanation is consistent with the recent finding that in vitro binding of the ternary complex to the 40S ribosome is ∼3‐fold faster than conversion of 48S to 80S complexes (Lorsch and Herschlag, 1999). This phenomenon may also have influenced the results we obtained for the hcr1Δ extract in Figure 2B, but there we observed an excess of 43–48S complexes in the mutant, suggesting a defect at a subsequent step of the pathway.
It is possible that the ribosome‐binding defect of the eIF3 subcomplex containing prt1‐Δ100, TIF34 and TIF35 results from loss of interaction between the PRT1 RRM and 18S rRNA. However, the human PRT1 homolog failed to bind 18S rRNA in vitro (Methot et al., 1997; Block et al., 1998) and yeast PRT1 also showed no RNA‐binding activity in vitro (Naranda et al., 1994). It is known that RRM2 of PAB1 is required for protein–protein interaction with eIF4G (Kessler and Sachs, 1998). Thus, an alternative hypothesis would be that the PRT1 RRM is the binding domain for TIF32, and TIF32 or NIP1 interacts directly with the 40S ribosome. Consistent with this last possibility, hTIF32 (p170) interacted with both 18S rRNA and β‐globin mRNA (Block et al., 1998). Another interesting possibility, for which there is precedent (Scherly et al., 1990; Kessler et al., 1997), would be that binding of TIF32 to the PRT1 RRM enhances interaction of the RRM with 18S rRNA. TIF35 also contains an RRM that interacted with both 18S rRNA and β‐globin mRNA in vitro (Hanachi et al., 1999). Our results indicate that the TIF35 RRM was insufficient to mediate strong ribosome association of the prt1‐Δ100–TIF34–TIF35 subcomplex. Consistently, deletion of the TIF35 RRM conferred a Slg− phenotype, but was not lethal in yeast (Hanachi et al., 1999). It will be important to determine which subunits of eIF3 bind directly to the 40S ribosome to mediate recruitment of the MFC to the pre‐initiation complex.
Materials and methods
Yeast strains
Details of the yeast strains used in this study are given in Table II.
Plasmid constructions and site‐directed mutagenesis of HCR1
Table Table III contains brief descriptions of all plasmids employed in this study. Details of their construction are available as supplementary data at The EMBO Journal Online.
WCE preparation and immunoprecipitation
Yeast cells of strains W303 and YLVH13 alone or YLVH13 (hcr1Δ) transformed with either YCpLVHM‐T or YCplac22 were grown in 100 ml of YPD or SC medium lacking tryptophan, respectively, to an A260 of ∼1.0. Yeast WCEs were prepared and immunoprecipitations were conducted as described previously (Asano et al., 1999) except that a different breaking buffer was used (Valášek et al., 1998) for immunoprecipitations using c‐Myc antibodies. Our modifications of this protocol are available as supplementary data at The EMBO Journal online.
Polysome profile analysis
Preparation of WCEs and subsequent polysome analysis were conducted essentially as described previously (Foiani et al., 1991). Our modifications of this protocol are available as supplementary data at The EMBO Journal online.
GST pull‐down experiments
Preparation of recombinant proteins, in vitro translation and in vitro binding experiments were carried out as described previously (Asano et al., 1998).
Ni2+ affinity purification of eIF3 complexes containing His‐tagged PRT1
Preparation of WCEs and subsequent Ni2+ affinity purification were conducted essentially as described previously (Phan et al., 1998). Our modifications of this protocol are available as supplementary data at The EMBO Journal online.
Preparation of antibodies against HCR1
The GST–HCR1 fusion protein encoded by pGEX‐HCR1 was expressed in Escherichia coli and purified from the WCE by incubation with glutathione–Sepharose 4B beads (Pharmacia). The isolated protein was resolved by SDS–PAGE (4–20% gels), excised from the gel and washed with 1× phosphate‐buffered saline (PBS). Rabbits were injected with the purified protein and serum containing polyclonal antibodies against HCR1 was obtained commercially from Covance Research Products (Denver, PA).
Antibodies against TIF32 (Valášek et al., 1998), PRT1, NIP1, TIF34, eIF1, eIF5, GCD6, GCD11 (Phan et al., 1998) and TIF35 (L.Phan, L.W.Schoenfeld, L.Valášek, K.Nielsen and A.G.Hinnebusch, submitted) used in this study were described previously. Antibodies against S22 were kindly provided by Jan van't Riet.
Supplementary data
Supplementary data for this paper are available at The EMBO Journal Online.
Supplementary Information
Supplementary data [emboj7593594-sup-0001.htm]
We are indebted to Chris Barnes, Harald Nierlich, Jiri Hašek, Gerard Griffioen, Vladimir Reiser and Jan van't Riet for sending us yeast strains, plasmids and other materials used in this study. We are grateful to Thomas Dever, Beatriz Castilho and Katsura Asano for critical reading of the manuscript, and to the members of the Hinnebusch and Dever laboratories for helpful discussions and suggestions during the course of this work.
- Copyright © 2001 European Molecular Biology Organization


text:
Some say weekends are the best, which i do agree. Some say drinking is the best, which also might be true, but asians have one big disadvantage...they cant hold their liquor. One sip of it and they have the "asian glow", which is when an asian turns bright red. Can't hide it wortha shit. so gayyy
And i had dinner with some friends and my friend A told this attrctive girl to eat dinner with us. We all talked for a bit except my friend B. HE was dead silent for the whole dinner until we were ready to go. The one comment he said to the very attractive girl was, " WOW! your gonna finish that whole apple by yourself????"...smh
Were college students...they should have "how to talk to girls 101"


text:
To the Times of Ti:
An article that appeared in the Sunday, May 6, 2012 Press Republican was titled: Essex County Supervisors fear sitting on a Powder Keg."
Perhaps this is a message for them to get off their "chairs" and start addressing some of the many problems of Essex County.
First - A study was made concerning Horace Nye which addressed costs, etc., several years ago. Why not review the study to see if the findings could be used to make operational costs more attainable? The results might possibly indicate a procedure to enable Essex County to continue with local management rather than selling to an outside entity. Local people who manage the home understand the cares and needs of North Country residents. With outside ownership the familiarity that the patients now have would be lost. Review the study and save one of our most treasured facilities if possible.
Second - If Essex County had five legislators instead of 18 supervisors, we would have to purchase only five chairs, pay five salaries versus 18 and disperse expense accounts and benefits for five legislators rather than 18 supervisors. This study has also been done and has proven to be more effective in other counties.
Get out of those chairs and address two pressing issues rather than sitting back in new comfortable chairs doing little to address Essex County and its problems. There are more pressing problems than outdated chairs.
Walter P. Huchro


text:
WikiLeaks releases 573.000 pager intercepts from 9/11 2001.
8:51:31 AM email@example.com|Please call Pentagon Weather|UNCLASSIFIED
Please call Pentagon Weather.......reference 1030 Meeting.....703-695-0406
ANDREW J. TERZAKIS, Lt
8:53:44 AM "NYPD Ops Div" <|1 PCT WORLD TRADE CENTER|--- 1 PCT - WORLD TRADE CENTER - POSSIBLE EXPLOSION WORLD TRADE CENTER BUILDING. LEVEL 3 MOBILIZATION TO CHURCH AND VESSY. 10:05:57 AM Please don't leave the building. One of the towers just collapsed! PLease, please be careful. Repeat,
From 3AM on Wednesday November 25, 2009, until 3AM the following day (US east coast time), WikiLeaks will release over half a million US national text pager intercepts.
The intercepts cover a 24 hour period surrounding the September 11, 2001 attacks in New York and Washington.
To foster a deeper understanding, the messages will be released to the global community "live". That is, the first message, corresponding to 3AM September 11, 2001, five hours before the first attack, will be released at 3AM November 25, 2009 and the last, corresponding to 3AM September 12, 2001 at 3AM November 26, 2009.
To follow the release, please visit http://911.wikileaks.org


text:
Sigh! I have to accept that I am very bad at maintaining a regularly scheduled blog post.
But I like blogging and I miss blogging. And I'll try to blog on a more frequent basis. So be prepared coz I will be posting about the mundane happenings of my life again. (^_^)


text: [For recent brief comment on the tone and mood of opening day at Sochi, click here.] I had not planned a eulogy for Pete Seeger. He was never an idol of mine, although I admired both the man and his music. Cynical Bob Dylan was more my speed. But Seeger made an impact. He wrote his masterpiece on war and renewal—"Where have all the flowers gone?"—early in the Atomic Age. The Cold War turned it into an existential question: what kind of renewal might emerge from a radioactive wasteland? It was not Pete Seeger who answered that question. It was two Russians and an American, whose cool judgment under immense pressure saved our species from self-extinction. Their names were Khrushchev, Arkhipov, and Kennedy. I like to think that Seeger's song helped motivate at least one of them to keep his twitching finger off the button and let us live. Seeger wrote other songs that inspired the American labor movement. For several decades, that movement produced the most equitable and widely prosperous society the world has ever known. In the last two decades, along with what is left of British industry, it has helped lift nearly a billion people out of extreme poverty. (If you want to know what difference a strong labor movement makes, just look at Bangladesh and its tragic garment workers today.) The plutocrats and pols who accommodated the US movement were smart enough to consider workers' welfare a means to their own prosperity. And they, including Henry Ford, were right. Today their successors struggle just to see the problem of inequality. They are too preoccupied, as they travel the world in their private jets, trading their derivatives and regaling the press with Marie-Antionette-like bon mots about doing "God's work." Seeger's lyrics also inspired generations of African-Americans and their sympathizers. Not only did they help reduce racism here and abroad. They ultimately helped give us an African-American leader of the free world, who incidentally does not treat Britain as his poodle. No, I didn't idolize Seeger. But when I read in the British newsweekly The Economist that he had been a "Bolshie with a banjo," I saw red, and not in Seeger. Only an arrogant pipspeak of a British twit, in complete ignorance of American culture, and with pretensions toward nobility that he could never attain, could have written that caption. Real British nobility—of which precious few remain today—kept the people's feelings and their welfare at least marginally in view. They also had some manners and knew Latin, including "de mortuis, nihil nisi bonum." The bilious obit continued with slander after slander, which the safely anonymous author no doubt considered "cute." It ended by dinging Seeger for renouncing Communism late, and so for failing to "Turn, Turn Turn." All in all, the obit was brutally and obnoxiously dismissive. Just a few years ago, The Economist had been a paragon of journalism. It did its best to mimic the dismal science from which it drew its name. It put data first, calculations second, and ideology a distant third. That's why I subscribed to it, beginning about two decades ago (with a long hiatus). Now, The Economist's order of precedence appears to have been reversed. Ideology comes first, calculation second, and facts a distant third. In this respect it appears to be reliving the process by which Rupert Murdoch annihilated the Wall Street Journal as a serious newspaper. It all started, in my view, with a nauseatingly triumphalist issue, in June-July 2012, lauding London as the best city in Europe for its banking and real-estate bubbles. When I read that, I wondered why The Economist didn't also tout Russian spooks' plain-view murder (albeit delayed) of Litvinenko by polonium in 2006. If you're going to sell your soul and your open society to the highest bidder, why not go all the way? The snarky Seeger obit was not enough, by itself, to get me to cancel my subscription. Not yet, anyway. But the writing appears to be on the rest-room wall. And not just for British news media. While on fellowship in Cambridge four decades ago, I rode the Tube every chance I got. I loved it for its convenience, speed, reliability, cleanliness, and quiet. Now all those things, apparently, are gone. The last time I rode the Tube, several years ago, huge crowds were waiting at our station. The trains on the line we were to use were over half an hour late. We waited outside, in the rain, as clueless clerks tried to line the crowd up so exiting passengers could leave a dangerously overcrowded platform. We finally made it to our destination, about an hour late. When we returned home in the late evening, the trains were full of drunk, rowdy and obnoxious youth. As I exited our train with my Asian-origin wife, walking next to a drunk and grinning spitting image of a skinhead, I steeled myself to deliver a swift kick to his crotch. Fortunately, the youth limited his lack of civility to drunkenness. We exited the Tube without incident, though not without the type of unease that I had never felt on any subway in New York or Moscow. Maybe if the Brits had had a Pete Seeger of their own, their twits' knees might jerk a little less violently for conservative pablum. They might show a little respect for, if not insight into, foreign cultures divided by a common language, let alone the perpetual struggle of working people for a fair shake. And with a Pete Seeger to give their aimless louts a bit of hope for the future, the Tube might regain its impressive order from the seventies, if not run on time. Footnote 1: If you like slow, lugubrious singing, you can watch and listen to a young Seeger singing the song here. Like many great song writers, Seeger was not the best performer of his own tunes. If you want a more effective performance, try the one by Bob Dylan or by Peter, Paul and Mary, for which you'll have to pay. Footnote 2: The very same issue with the nasty Seeger obit had a slanted story on just the previous page. Summarizing recent academic research on US economic inequality and social mobility from Harvard and Berkeley, its subhead read "America is no less socially mobile than it was a generation ago." But the penultimate paragraph reported enormous geographic disparities in social mobility within the US. And its anonymous writer failed to point out (or perhaps even to notice) that the data reported ended more than a generation ago, years before the more recent inflection point at which US inequality took off. Murdoch, who has been notorious for rewriting his minions' headlines, could not have done a better job of slanting the story and distorting its conclusion if he had rewritten it himself. And this bit of pro-plutocrat propaganda is no doubt the source of what has become conventional Yankee wisdom: that US social mobility has not changed, at least up to 1983! [see the graph on printed page 23] Epitaph (to Journalism, not Seeger!): Rewrite the headline, and you control the lead. Control the lead, and you control the story. Control the story and control the thought. Control the thought, and you control the mind. This is Murdoch's Orwellian formula. This is what gave us Yanks Fox, destroyed the Wall Street Journal, and has turned our Yankee politics into a farce that precocious three-year-olds would laugh at. I canceled my Journal subscription after reading too many stories whose real meaning became clear only after two or three readings. I sincerely hope that doesn't happen to The Economist, or to our Mother Country. Despite its economic shortcomings, Britain has always seemed to meet or exceed us Yanks in taste and intelligence. But just read the story on page 23 of The Economist's February 1, 2014 issue. Then read the subhead and scrutinize the graph. If an Orwellian shiver doesn't crawl down your spine as one did mine, then you're a better (or more sanguine) man than I am, Gunga Din. You can't take frequent error for mere sloppiness, not when it consistently points in a rightward direction. permalink


text:
Home Tags Painpig
Tag: painpig
Pain Pigs – A Masochist's View
A talk with Master Walter's boi about pain and his need of being tortured. MasterMarc: Hello Boi. Some weeks ago I've talked with your master about...
sadOsam contains homosexual content. Just enter the site if you haven't any problems with homosexuality and Porn. This web-site contains material of an ADULT nature. By entering the site you are confirming you are of the legal age in your state/country to view such material and that you will not be offended by it's contents. All persons pictured within over 18. All content herein Copyright WANKDAY 2017. If you do not agree or are under legal age to view this site, please leave.


text:
Memo Pad: Sharp Words... Shutter Fly... Close, But No Cigar...
Published: Friday, January 18, 2008
SHARP WORDS: Don't mess with Anna Wintour. A lesson learned by many in fashion, but obviously not by Sen. Hillary Clinton or her advisers. Clinton was to appear in Vogue as the presidential race reached high gear, but backed out late last fall before a photo shoot was scheduled for fear of appearing too alluring. New York Post columnist Liz Smith reported Nov. 1 that "the astute [Vogue contributing editor] Julia Reed hung ten waiting to write about her and the giant fotog Annie Leibovitz had her cameras at the ready for nothing." A Vogue spokesman confirmed: "We were told by Ms. Clinton's camp that they were concerned if Clinton appeared in Vogue that she would appear too feminine." (Clearly, though, the presidential candidate didn't worry about that when she cried in New Hampshire.)But Wintour didn't take Clinton's dis lightly. In her February editor's letter, Wintour takes Clinton to task for being behind the times.
"Imagine my amazement, then, when I learned that Hillary Clinton, our only female president hopeful, had decided to steer clear of our pages at this point in her campaign for fear of looking too feminine. The notion that a contemporary woman must look mannish in order to be taken seriously as a seeker of power is frankly dismaying." Wintour continues: "This is America, not Saudi Arabia. It's also 2008: Margaret Thatcher may have looked terrific in a blue power suit, but that was 20 years ago. I do think Americans have moved on from the power-suit mentality, which served as a bridge for a generation of women to reach boardrooms filled with men. Political campaigns that do not recognize this are making a serious misjudgment." Calls and e-mails to the Clinton camp went unreturned as of press time.Vogue has featured Sen. Clinton with her family and by herself in sizeable features six times since 1992, and she was the first first lady to appear on the magazine's cover, in December 1998. Despite Wintour's lashing, bridges have not been permanently burned: Vogue's spokesman said the magazine and the Clintons are "working on something together for Vogue in the near future."
A presidential cover in January 2009, perhaps?
— Stephanie D. Smith of WWD


text:
"I have heard there are troubles of more than one kind. Some come from ahead and some come from behind. But I've bought a big bat. I'm all ready you see. Now my troubles are going to have trouble with me!"
Thursday, June 23, 2011
so i haven't blogged for a while but honestly life has been too busy to say a word and all of you know that if i have the option to be on line i will accomplish nothing that day so i try to abstain as much as possible :)
anyway loooong story short i have soo much to catch everyone up on and don't have the time
i've missed telling you about easter, prom, graduation, my scholarships, kelsey's scholarships, kelsey's trip to state, our AC dying along with the washing machine, softball, baseball, and ella-sophie's birthday
we've had alot going on and i will post more later, i'm trying to get back to blogging more regularly so be patient. in the mean time here's a few shots of this past month.
love and blessings to everyone,
2 comments:
- Indigo Children said...
love the pics. hope you guys have air conditioning now. can't wait to see you guys.
- June 24, 2011 at 5:42 AM
- High in Demand said...
Love you!
I'm excited about the prospect of more blog post from you! I have to get my McCurdy fix somehow! : )
- July 15, 2011 at 6:52 PM
Subscribe to: Post Comments (Atom)