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Top Items:
Ezra Klein:
On Journolist, and Dave Weigel — I began Journolist in February of 2007. It was an idea born from disagreement. Weeks, or maybe months, earlier, I had criticized Time's Joe Klein over some comments he made about the Iraq War. He e-mailed a long and searching reply, and the subsequent conversation was educational for us both.
Steve Benen / Washington Monthly: DAVE WEIGEL GOT SCREWED.... I don't know Dave Weigel especially well.
Ed Driscoll / Pajamas Media: 'The Self-Immolation of David Weigel'
Scott Lemieux / Lawyers, Guns & Money: Pompous Hack With No Sense of Shame of the Day
Harold Pollack / The Reality-Based Community: Saying Goodbye and Thank You to Journolist
Andrew Sullivan / The Daily Dish: The End Of JournoList — Ezra writes the obit:
Amanda Marcotte / pandagon.net: Journolist, gossip, and haters: a rant
Matt Dornic / FishbowlDC:
BREAKING: Weigel Resigns — FIRST ON FBDC: FishbowlDC has confirmed that WaPo conservative-beat blogger Dave Weigel has resigned after a slew of his anti-conservative comments and emails surfaced on FishbowlDC and Daily Caller over the past two days. — A spokesperson for the Post …
Discussion: Alan Colmes' Liberaland, The Other McCain, NewsBusters.org, The Corner on National …, LewRockwell.com Blog, Jihad Watch, Pundits Blog, Another Black Conservative, Outside the Beltway, The Huffington Post, Politics Daily, JustOneMinute, Taylor Marsh, Atlas Shrugs, INSTAPUTZ, neo-neocon, Wizbang and Left Coast Rebel, more at Mediagazer »
Sinfonian / Alan Colmes' Liberaland: Weigel Resigns From WaPo, Once Again Proving "It's OK If You're A Republican"
Robert Stacy McCain / The Other McCain: WEIGEL RESIGNS
Daniel Foster / The Corner on National Review Online: Report: Post Blogger Resigns Over Comments — By: Daniel Foster
Lew Rockwell / LewRockwell.com Blog: Trouble for the Washington Post
Brent Budowsky / Pundits Blog: WaPo blogger calls for Drudge immolation, condemns Ron Paul …
Clifton B / Another Black Conservative: UPDATED: David Weigel just another fake, phony and fraud conservative
James Joyner / Outside the Beltway: Dave Weigel Resigns
Ben Smith / Ben Smith's Blog:
Weigel and the Post — The current flap over Washington Post blogger Dave Weigel has its roots in a fact that suprised me when I learned of it earlier this year: The Post appears to have hired Weigel, a liberal blogger, under the false impression that he's a conservative.
Michael Walsh / Big Journalism: David Weigel, the 'Journolist,' Wolves In Sheep's Clothing, and the Death Of Privacy
James Richardson / RedState: Dave Weigel and the Washington Post: A case of false pretenses?
Erick Erickson / Erick's blog: Why Is Anyone Surprised by This Dave Weigel Business?
Jonathan Chait / The New Republic: The Post Retreats — I was completely stunned to learn …
Brad DeLong / Grasping Reality with Both Hands: I Think Ben Smith Gets This One Wrong (David Weigel Watch)
Betsy Rothstein / FishbowlDC: Politico's Ben Smith Blames WaPo Not Weigel
Jim Romenesko / Romenesko: WP blogger Weigel resigns after apologizing for e-mails
Michael Triplett / Mediaite: WaPo's Dave Weigel Apologizes for Slamming Drudge, Washington Examiner
Adrian Chen / Gawker: Should Matt Drudge Set Himself on Fire?
Donald Douglas / American Power: What's Up With David Weigel? Now We Know
David Weigel / Right Now: An apology to my readers
Jonathan Strong / The Daily Caller:
E-mails reveal Post reporter savaging conservatives, rooting for Democrats — Conservative radio host Rush Limbaugh famously said he hoped President Obama would "fail" in January, 2009. Almost a year later, when Limbaugh was rushed to the hospital with chest pains, Washington Post reporter David Weigel had a wish of his own.
Ann Althouse / Althouse: Burn, Davey, Burn — The Self-Immolation of David Weigel.
Hugh Hewitt / Hugh Hewitt's TownHall Blog: Regarding David Weigel's Resignation
Jonah Goldberg / The Corner on National Review Online: Re: Weigel Resigns — By: Jonah Goldberg
Michael Triplett / Mediaite: FishbowlDC Defends Publishing Weigel Emails: "We Have A Vested Interest"
Betsy Rothstein / FishbowlDC: Weigel Faces More Grief as New Anti-GOP E-mails Surface on The Daily Caller
James Joyner / Outside the Beltway: Dave Weigel's Anti-Conservative Rants Go Public
Stephen Green / Pajamas Media: Dave Weigel Exits the Washington Post, Stage Left
Adam Serwer / American Prospect:
Toilet Training. — Dave Weigel is leaving The Washington Post over private e-mails mocking conservative figures. I find it really extraordinary that a news organization would let such a talented reporter go not because of any kind of professional misconduct but because someone leaked private correspondence …
Jeffrey Goldberg / The Atlantic Online:
An Unhappy Day At The Washington Post — But also sort of a happy day at The Washington Post, for the dwindling band of writers and editors there who value such old-fashioned traits as temperance in the expression of personal views; forthrightness; and fairness.
Discussion: Matthew Yglesias, Balloon Juice, The Daily Dish, Outside the Beltway, Newsbroke, Eschaton, Perfunction and The Poor Man Institute
Myglesias / Matthew Yglesias: Re: Dave Weigel
Andrew Sullivan / The Daily Dish: Dave Weigel Quits
James Joyner / Outside the Beltway: Yet More Dave Weigel
Michael Roston / Newsbroke: Jeffrey Goldberg's mean-spirited Dave Weigel schadenfreude
Atrios / Eschaton: Offenses Big And Small
Cuffy Meigs / Perfunction: David Weigel: Rat-F**ked By Karma
Jeffrey Goldberg / The Atlantic Online:
Second Thoughts on Dave Weigel — A couple of people I know and respect have told me that my criticism of Dave Weigel is misplaced; that he tries harder than I thought to be a fair reporter; that he makes mistakes, but everyone makes mistakes. And they've provided me with examples of his good reporting.
David Brooks / New York Times:
The Culture of Exposure — The most interesting part of my job is that I get to observe powerful people at close quarters. Most people in government, I find, are there because they sincerely want to do good. But they're also exhausted and frustrated much of the time.
Athenae / TBogg: It's Always The Hippies' Fault, Somehow
Michael Tomasky / Guardian: Al Gore in the age of exposure
Andrew Sullivan / The Daily Dish: McChrystal - And The Press's Failure
Stanton Peele / The Huffington Post: McChrystal Meets Restrepo: Can Modern War Withstand Modern Media?
Jim Newell / Gawker: David Brooks Furious At 'Rolling Stone' Reporter For Printing On-the-Record Comments
Meenal Vamburkar / Mediaite: David Brooks Attacks Michael Hastings For Basing McChrystal Story On "Kvetching"
Reihan Salam / National Review: David Brooks on the Culture of Exposure
Jeremy Scahill / The Nation: David Brooks, King of Kvetching
Robert Stein / Connecting.the.Dots: Exposure, Indecent and Otherwise
Jonathan Capehart / PostPartisan: McChrystal's mystifying media ignorance
The Atlantic Online:
The Post Shouldn't Have Fired Dave Weigel — The Washington Post should not have fired Dave Weigel for his leaked Journolist comments, period. — In hiring Weigel, the Post knew it was bringing on board someone with a style of journalism — and it definitely is journalism …
Discussion: Guardian and Balloon Juice
Michael Tomasky / Guardian: On Journolist and Dave Weigel
Philip Klein / AmSpecBlog:
Defending Dave Weigel — Washington Post reporter/blogger Dave Weigel, who is tasked with covering conservatives, has come under fire in the past several days for derisive comments he's made about some conservatives on the liberal listserv JournoList. You could read more background on the story here.
Discussion: Ezra Klein, The Corner on National …, The New Republic, Liberty Pundits Blog, The Politico, The Other McCain and FishbowlDC
John J. Miller / The Corner on National Review Online: Weigel Words — By: John J. Miller
Jonathan Chait / The New Republic: More On Weigel — A couple points here. First, thinking …
Melissa Clouthier / Liberty Pundits Blog: Dave Weigel, The Washington Post, And Objectivity
Keach Hagey / The Politico: More leaked Weigel emails
Robert Stacy McCain / The Other McCain: Dave Weigel Violates Rule One
Betsy Rothstein / FishbowlDC: WaPo's Weigel Lets Loose With Scathing E-mails on Liberal Listserv
Ben Smith / Ben Smith's Blog:
Weigel resigns — YESSSSSSSSSSSSSSSSS! Notch Another VICTORY to DRUDGE!!!!!!! — Posted By: YESSSSSSSSSSSSSSSSS! Notch Another VICTORY to DRUDGE!!!!!!! June 25, 2010 at 12:24 PM — Report Abuse — Was this a "firing" guised as a Resignation like WaPo alleged about McChrystal? .............
Discussion: Hot Air
Keach Hagey / The Politico:
Weigel quits & #8211; and a debate begins
Weigel quits & #8211; and a debate begins
Discussion: Big Journalism and alicublog
Roy Edroso / alicublog: U WERE DOIN IT RITE. — Whiny ass titty babies.
The Huffington Post:
Dave Weigel's Resignation: The Backstory And Meaning
Dave Weigel's Resignation: The Backstory And Meaning
Discussion: The Plum Line, TPMCafe, AMERICAblog News, Don Surber, FishbowlDC and NewsBusters.org
Greg Sargent / The Plum Line: On David Weigel's resignation
M.J. Rosenberg / TPMCafe: Weigelgate: Jeff Goldberg's Jealousy
John Aravosis / AMERICAblog News: Is it better or worse off knowing my biases?
Matt Dornic / FishbowlDC: POLL: How Should WaPo Handle Weigel?
Andrew Sullivan / The Daily Dish:
When A Blogger Vents — Comments Dave Weigel made on a listserv have been made public.
When A Blogger Vents — Comments Dave Weigel made on a listserv have been made public.
Discussion: Outside the Beltway, The Daily Caller and Perfunction
Jim Geraghty / National Review:
If Weigel Had to Go, What Other MSM Figures Wrote Terrible Things on Journo-List?
If Weigel Had to Go, What Other MSM Figures Wrote Terrible Things on Journo-List?
Discussion: Riehl World View
Michael Triplett / Mediaite:
WaPo's Dave Weigel Resigns After More Journolist E-Mails Surface
WaPo's Dave Weigel Resigns After More Journolist E-Mails Surface
Discussion: Gawker, Hit & Run, Swampland, FishbowlDC, The Politico, Wonkette and Capital Gains and Games blogs, more at Mediagazer »
Matt Welch / Hit & Run: David Weigel Resigns From the Washington Post
Amy Sullivan / Swampland: Weigel, The Washington Post, and The Conservative Beat
Betsy Rothstein / FishbowlDC: Weigel Writes Late-Night Apology Note to GOP Aides
Keach Hagey / The Politico: JournoList wonders who leaked
Bruce Bartlett / Capital Gains and Games blogs: A Travesty at the Washington Post
John Cole / Balloon Juice:
Another Head for the Right and the Left
Another Head for the Right and the Left
Discussion: The Impolitic and Eschaton
Libby Spencer / The Impolitic: So who's the dirty rat?
Atrios / Eschaton: Your Liberal Media
Elise Viebeck / The Hill:
Weigel seen at Huffington Post office
Weigel seen at Huffington Post office
Discussion: Pajamas Media, NewsReal Blog, Ben Smith's Blog, WillCollier.com and Wonkette
Stephen Green / Pajamas Media: Nah Nah Nah Nah Nah Nah Nah Nah Hey Hey Hey Goodbye
Liz Mair:
In defense of Dave Weigel
In defense of Dave Weigel
Discussion: The Atlantic Online, The Daily Dish, Ross Douthat and The Corner on National …
Andrew Sullivan / The Daily Dish: Dave Weigel Quits, Ctd
Edward Wyatt / New York Times:
House and Senate in Deal on Financial Overhaul — WASHINGTON — Nearly two years after the American financial system teetered on the verge of collapse, Congressional negotiators reached agreement early Friday to reconcile competing versions of legislation that would transform financial regulation.
Discussion: Ezra Klein, The Politico, Reuters, Wonk Room, Wall Street Journal, Concurring Opinions, ABCNEWS, Washington Monthly, The Atlantic Online, Conglomerate, USA Today, The Caucus, Think Progress, CNN, www.thetakeaway.org, DealBook, The Mahablog, Political Punch, TigerHawk, Open Left, Daily Kos, ourfuture.org/blogs_chrono/*, Balloon Juice and AMERICAblog News
Damian Paletta / Wall Street Journal: U.S. Lawmakers Agree on New Finance Rules
Kristin Johnson / Concurring Opinions: Are We There Yet? Driving The Financial Reform Bill Home
Dalia Fahmy / ABCNEWS: Financial Reform's Top 6 Changes for You
Daniel Indiviglio / The Atlantic Online: Congress' Conference Committee Completed for Financial Reform
David Zaring / Conglomerate: Financial Reform Agreement in Congress
Paul Wiseman / USA Today: House, Senate all-nighter produces financial overhaul bill
Janie Lorber / The Caucus: The Early Word: Breaking the Logjam
Karen Travers / Political Punch: President Obama 'Gratified' by Wall Street Reform Deal
Chris Bowers / Open Left: Personal reflections on the Wall Street reform deal
Joan McCarter / Daily Kos: Financial reform? Early morning vote finishes deal
Bill Scher / ourfuture.org/blogs_chrono/*: Progressive Breakfast: Bank Deal Done, Jobs Bill Killed, Carbon Cap Alive
John Cole / Balloon Juice: Regulation Deal
Brady Dennis / Washington Post:
House, Senate leaders finalize details of sweeping financial overhaul — Key House and Senate lawmakers agreed on far-reaching new financial rules early Friday after weeks of division, delay and frantic last-minute deal making. The dawn compromise set up a potential vote in both houses …
Victoria McGrane / Wall Street Journal: Major Provisions in the Financial Overhaul Bill
Daniel Foster / The Corner on National Review Online: 'Wall Street' 'Reform' 'Compromise' Reached — By: Daniel Foster
Brian Darling / RedState: Today in Washington - June 25, 2010
Richard Fernandez / Pajamas Media: Holding the Bag — The House and Senate were rushing …
Nathan Koppel / Law Blog: Lawyers, Start Your Billing Clocks: Financial Overhaul Legislation Nears Passage
Molly K. Hooper / The Hill: Key Dem: Immigration bill lacks votes
Doug Powers / The Powers That Be: Congress Pulls All-Nighter to Pass Financial 'Reform'; America 'Dodd on Arrival'
The Huffington Post:
Sarah Palin Endorses Op-Ed Comparing Obama To Hitler, Suggesting BP Escrow Fund Could Lead To Nazi-Like Dictatorship — WHAT'S YOUR REACTION? — On Friday, Sarah Palin took to Twitter to direct her followers to read a recent op-ed from conservative columnist Thomas Sowell that compares President Obama …
Discussion: The Plum Line, The Atlantic Online, Daily Kos and The Daily Dish
Greg Sargent / The Plum Line: Sarah Palin tells followers to read article comparing BP escrow fund to Nazism
Jeffrey Goldberg / The Atlantic Online: Sarah Palin: BP Escrow Fund = The Holocaust
Jed Lewison / Daily Kos: Palin embraces Obama = Hitler analogy
Andrew Sullivan / The Daily Dish: Hewitt Award Nominee
Alex Seitz-Wald / Think Progress:
Palin encourages followers to read column warning that the BP …
Palin encourages followers to read column warning that the BP …
Discussion: Alan Colmes' Liberaland, The Atlantic Online, AMERICAblog News, The Hill and Climate Progress
John Aravosis / AMERICAblog News: Palin endorses Obama-Hitler comparison
Eric Zimmermann / The Hill: Sarah Palin recommends article with Hitler-Obama comparison
Washington Examiner:
Democrats: Free speech for me, not for thee — In March, the Supreme Court's Citizens United decision struck down campaign finance limits on political expression by individuals working through corporations and unions as a violation of the First Amendment's guarantee of freedom of speech.
Lori Montgomery / Washington Post: Senate again rejects expanded spending package
Mark Hemingway / Beltway Confidential: House 'shreds our constitution for raw, ugly, partisan gain' by vote of 219-206
Gail Russell Chaddock / Christian Science Monitor: Who's exempted from 'fix' for Supreme Court campaign finance ruling?
Hans A. von Spakovsky / The Corner on National …: The Fight over the DISCLOSE Act Is Far from Over — By: Hans A. von Spakovsky
Moe Lane / RedState: DISCLOSE Act passes the House.
Michael Luo / New York Times:
Democrats See Signs of Hope in Job Trends
Democrats See Signs of Hope in Job Trends
Discussion: NewsBusters.org, Mother Jones and Firedoglake
Suzy Khimm / Mother Jones: Jobs: A Silver Lining for Democrats?
Attaturk / Firedoglake: A Right-Wing and a Prayer
Eric Lichtblau / New York Times:
Across From White House, Coffee With Lobbyists — WASHINGTON — There are no Secret Service agents posted next to the barista and no presidential seal on the ceiling, but the Caribou Coffee across the street from the White House has become a favorite meeting spot to conduct Obama administration business.
Josh Gerstein / The Politico: W.H.: Kagan will get GOP votes
Timothy P. Carney / Washington Examiner: White House, Google violate lobbying pledge
Reihan Salam / National Review: The Futility of Idealistic Regulation
Ed Morrissey / Hot Air: Is the Obama WH evading disclosure laws?
Timothy P. Carney / Beltway Confidential: NYT pulls back the curtain on Obama's transparency and anti-lobbyist talk
Mary Katharine Ham / Weekly Standard: Obama White House Violating Lobbyist Pledge Left, Right, and at the Coffee Shop
Tom Maguire / JustOneMinute: Coffeegate! Latte To Stay, Lobbyist Rules To Go
The Huffington Post:
Michael Hastings, Rolling Stone's McChrystal Profiler, Says Troops Are Happy That General Was Ousted — WHAT'S YOUR REACTION? — The Rolling Stone correspondent whose profile of Gen. Stanley McChrystal upended America's Afghan war leadership says that soldiers on the ground are happy …
Discussion: The Fix, Hit & Run, The Foundry and American Spectator
Chris Cillizza / The Fix: "Worst Week in Washington" Winner: Gen. David Petraeus
Robby Soave / Hit & Run: Losing the War in Afghanistan? Blame Rolling Stone, Suggests Geraldo Rivera
James Phillips / The Foundry: Afghanistan: Time to Rethink the Timeline
John R. Guardiano / American Spectator: Pundit Class, Not McChrystal, Exercised 'Bad Judgment'
Mayhill Fowler / The Huffington Post:
Seduction and Betrayal: McChrystal and the Misapprehension of Journalism
Seduction and Betrayal: McChrystal and the Misapprehension of Journalism
Discussion: Swampland and Matthew Yglesias
Lydia Saad / Gallup:
In 2010, Conservatives Still Outnumber Moderates, Liberals — Last year's increase in conservatism among independents is holding — PRINCETON, NJ — Conservatives have maintained their leading position among U.S. ideological groups in the first half of 2010.
Chris Cillizza / The Fix: Conservatives on the rise?
William A. Jacobson / Le·gal In·sur·rec· tion: Don't Take Candy From Concern Trolls
Sammy Benoit / YID With LID: Gallup: US Conservative Movement Continues to GROW
Steven L. Taylor / Outside the Beltway: Gallup: More Conservatives than Liberals in the United States
Steve M. / No More Mister Nice Blog: TEABAGS ARE YOUR NEW BICYCLE — Gallup todays offers more evidence …
Chris Bowers / Open Left: Conservative ideological self-identification on the rise
CQ-Roll Call / The Eye: Percentage Of Self-Identified Conservatives Rises
Kyle Trygstad / Real Clear Politics: Gallup: Twice As Many Conservatives As Liberals
Shane D'Aprile / Ballot Box: Gallup: Conservatives at record levels
Jennifer Rubin / Commentary: Conservatism Revived
Ben Smith / Ben Smith's Blog:
Philosopher disclaims 'ick factor,' demands Huckabee apology — Chicago philosopher Martha Nussbaum has rejected former Arkansas Governor Mike Huckabee's claim that she's the source of a controversial phrase he used in discussing opposition to same-sex marriage.
Discussion: Think Progress, Wonk Room, AmSpecBlog, TPM LiveWire, Guardian and The Reality-Based Community
Matt Corley / Think Progress: Nussbaum demands 'public apology' …
Joseph Lawler / AmSpecBlog: Martha Nussbaum's Selective Outrage for Huckabee
Rachel Slajda / TPM LiveWire: Professor Cited By Huckabee For 'Ick Factor' Demands Apology
Michael Tomasky / Guardian: Philosopher Huckabee — It's not every day that a US politician cites …
Howard Kurtz / Washington Post:
National Enquirer says it decided not to pay for Gore sex story interview — The executive editor of the National Enquirer says that the Oregon masseuse who made a sexual assault allegation against Al Gore asked the tabloid for $1 million but that the Enquirer did not pay her or anyone else in reporting the story.
Discussion: TPMMuckraker, Jules Crittenden, The Portland Tribune, CNN, Hot Air, The Confluence, JustOneMinute, TalkLeft and Romenesko, more at Mediagazer »
Justin Elliott / TPMMuckraker: Anon. Gore Friend To WaPo: Portland Massage Occurred 'Without Incident'
Bostonboomer / The Confluence: Friday Morning News and Views
Tom Maguire / JustOneMinute: More On Gore — Back to Al Gore's sex life!
Pete Winn / CNSNews:
Jewish Clergy Group: Elena Kagan Isn't 'Kosher' to Serve on Supreme Court — (CNSNews.com) - Supreme Court nominee Elena Kagan is "not kosher"—meaning she is not fit to serve on the court—according to more than 850 Orthodox members of the Rabbinical Alliance of America.
Discussion: Media Matters for America, Weasel Zippers, The Atlantic Online, Right Wing Watch and Vox Popoli
Ben Dimiero / Media Matters for America: Drudge promotes Kagan attack from Rabbi who blames gays for 9/11 …
Weasel Master / Weasel Zippers: Kagan & Gomorrah
Jeffrey Goldberg / The Atlantic Online: Gay-Hating Misogynistic Fundo Rabbis Strike Again
New York Times:
Obama Arrives in Toronto for Summit Meetings — TORONTO — President Obama arrived here Friday for back-to-back meetings of world leaders that will test international unity on how to restore global economic growth. — After landing, Mr. Obama quickly boarded a helicopter for a G-8 meeting in Muskoka …
Discussion: The Page and Indecision Forever
Sara Benincasa / Indecision Forever: Barack Obama Indulges His Asian Fetish at G-20
Michael Kinsley / The Atlantic Wire:
Why Not Call It 'Crossfire'? — Why can't CNN President Jonathan Klein have the guts just to admit he was wrong and call his new show "Crossfire"? Or at least to apologize to all the hard-working CNN employees working on Crossfire whom he insulted as he kicked them out the door? (Not me.
Discussion: The New Republic
Jonathan Chait / The New Republic: Crossfire Returns. Sort Of.


text:
Cdc37 is a molecular chaperone that functions with Hsp90 to promote protein kinase folding. Analysis of 65 Saccharomyces cerevisiae protein kinases (∼50% of the kinome) in a cdc37 mutant strain showed that 51 had decreased abundance compared with levels in the wild-type strain. Several lipid kinases also accumulated in reduced amounts in the cdc37 mutant strain. Results from our pulse-labeling studies showed that Cdc37 protects nascent kinase chains from rapid degradation shortly after synthesis. This degradation phenotype was suppressed when cdc37 mutant cells were grown at reduced temperatures, although this did not lead to a full restoration of kinase activity. We propose that Cdc37 functions at distinct steps in kinase biogenesis that involves protecting nascent chains from rapid degradation followed by its folding function in association with Hsp90. Our studies demonstrate that Cdc37 has a general role in kinome biogenesis.
Quality control mechanisms in eukaryotic cells involve molecular chaperone-dependent protein folding and ubiquitin/proteasome-dependent degradation. In addition, environmental stress such as heat shock or endoplasmic reticulum stress leads to a general down-regulation of translation (Wickner et al., 1999; Schneider, 2000; Frydman, 2001; Harding et al., 2002).
Hsp90 is a molecular chaperone that functions in association with many cochaperones to fold transcription factors, protein kinases, and many other clients (Millson et al., 2005; Zhao et al., 2005). Protein kinase folding is collaborative between Hsp90 and the molecular chaperone Cdc37. Cdc37 functions by stabilizing protein kinases or maintaining them in a folding-competent conformation (Kimura et al., 1997). In addition, Cdc37 promotes the assembly of Hsp90–protein kinase complexes (Stepanova et al., 1997), and expression of a dominant-negative form that lacks the Hsp90-binding domain inhibits kinase activation in animal cells (Grammatikakis et al., 1999). Interestingly, similar truncations promote yeast cell viability, suggesting that complex formation between Hsp90 and Cdc37 is not essential for their function in protein kinase maturation in all cases (Lee et al., 2002; Turnbull et al., 2005). Both chaperones interact directly with kinases during folding, and inhibition of Hsp90 with geldanamycin or other ansamycin antibiotics results in the rapid proteasome-dependent degradation of client kinases and transcription factors (MacLean and Picard, 2003; Pearl, 2005; Whitesell and Lindquist, 2005).
Protein kinase folding requires the action of several chaperones and cochaperones in addition to Cdc37 and Hsp90. Like nuclear receptors, initial chaperone interactions with unfolded kinases requires Hsp70/40 chaperones that appear to prepare the kinase for interaction with Cdc37 (Arlander et al., 2006). Cdc37 needs to be phosphorylated at Ser13 (mammals) or Ser14/17 (yeast) by casein kinase II to interact with its kinase clients (Bandhakavi et al., 2003; Shao et al., 2003; Miyata and Nishida, 2004). Hsp90 is subsequently recruited into this complex in a manner that is stimulated by the action of Sti1/Hop (Lee et al., 2004; Arlander et al., 2006). A stable ternary complex between Hsp90, Cdc37, and the client kinase then forms. Interestingly, Cdc37 exists in dimeric form in solution but in monomeric form in this ternary complex (Vaughan et al., 2006). How the folding reaction proceeds from this point is unknown.
Although it is clear that Cdc37 is vital for the biogenesis of protein kinases, it remains unknown what proportion of the kinome requires this and other chaperones for maturation. The binding site for Cdc37 on client protein kinases has been localized to the N lobe of the catalytic domain (Prince and Matts, 2004; Zhao et al., 2004) via highly conserved sequence motifs that stabilize nucleotide binding. These findings suggest a very general role for Cdc37 in kinome biogenesis because the sequences that mediate chaperone interactions are present in virtually all kinases.
Previous studies have noted that Cdc37 is up-regulated in cancer cells and can act as an oncogene (Stepanova et al., 2000a,b). As such, it may play an important role in regulating the signaling capacity of the cell, especially if it has a broad function across the kinome. To clarify the extent to which Cdc37 controls protein kinase biogenesis, we first adopted a proteomic approach to understand how many different kinases are affected by the loss of Cdc37 function. By analyzing a large proportion of the yeast kinome, we observed that Cdc37 has a general function in maintaining the levels of most but not all kinases. In addition, we characterize a novel function for Cdc37 in protecting nascent kinase chains against rapid degradation by the proteasome.
Cdc37 has a general role in kinome biogenesis
To establish what proportion of the yeast kinome is dependent on Cdc37 function, we performed a large-scale analysis of kinase steady-state levels in wild-type and cdc37 mutant yeast. This analysis used the tandem affinity-purified (TAP)–tagged yeast library, in which each strain contains a different TAP- tagged gene (Ghaemmaghami et al., 2003). The TAP tag was used as a means of identifying individual kinases after the replacement of wild-type CDC37 with a mutant version known to affect kinase maturation (Bandhakavi et al., 2003). This mutation, serine 14 to alanine (cdc37S14A), precludes phosphorylation at amino acid 14 by casein kinase II. A second phosphorylation site at serine 17 was left intact. Selection of the mutant was made possible by insertion of an auxotrophic marker (URA3) in the intergenic region between cdc37S14A and the downstream gene STB3 (Fig. 1 A). Ura+ transformants were rescreened for a temperature-sensitive lethal growth phenotype at 37°C, which was diagnostic for replacement of the wild-type gene with the cdc37S14A mutant (Dey et al., 1996; Bandhakavi et al., 2003).
A large-scale analysis was performed using TAP-tagged kinases isolated from the library. 65 strains containing different TAP-tagged kinases (∼50% of the kinome) were converted to cdc37S14A, and the steady-state levels of each were determined by Western blotting in comparison with the levels found in wild-type strains. Kinase identity was verified by size (predicted size of the protein plus the 21-kD tag) and by PCR analysis of the kinase-encoding gene from individual strains. Steady-state levels of each kinase were assessed by Western blotting in at least two independent transformants. Phosphoglycerate kinase was used as a loading control. Some kinases were not readily observed by direct Western blot analysis of crude extracts as a result of low abundance, and these were instead assayed after a single round of pull down with IgG–Sepharose beads (see Materials and methods).
Of the 65 kinases assayed, 51 displayed reduced steady-state levels by at least twofold in the cdc37S14A mutant strain compared with the wild type. 14 kinases accumulated to the same levels in both wild-type and cdc37S14A strains (less than a twofold difference; n > 4) when the cells were grown at 30°C. Examples of the primary data are shown in Fig. 1 (B and C), and the combined dataset is presented in Fig. 1 D as a phylogenetic tree. Multiple sequence alignment revealed no strong evolutionary relationships that distinguish those kinases affected by mutation in CDC37 from those that were not. However, three kinases that were unaffected by the cdc37 mutant, Bud32, Ygl059W, and Yil042C, represent outliers based on their phylogenetic distance from others in the group. All three have atypical structure: Bud32 utilizes Mn2+ instead of Mg2+ (Facchin et al., 2002), and Ygl059W and Yil042C are similar to mitochondrial branched-chain α-ketoacid dehydrogenase kinases. The overwhelming majority of the other kinases analyzed have broad distributions in cytosolic or nuclear compartments (55 kinases). Three are localized to vesicle intermediates in the secretory pathway (Akl1, Ire1, and Vps15), and four have ambiguous distributions (Mkk1, Ydl025c, Sat4, and Iks1; Huh et al., 2003).
The binding of Cdc37 is restricted to the N domain of protein kinases, which comprises five β sheets that pack against a single conserved α helix (the αC helix). This structure is also found in lipid kinases (Hanks and Hunter, 1995; Walker et al., 1999). Therefore, we tested whether any lipid kinases required Cdc37 for stability. Strains expressing TAP-tagged Pik1 (PI4 kinase) and Vps34 (a PI3 kinase) strains were converted to cdc37S14A, and their steady-state levels were assayed by Western blotting (Fig. 2). Both had reduced accumulation in the cdc37S14A mutant compared with the wild-type strain. Two other kinases that phosphorylate metabolic intermediates, Guk1 (a guanylate kinase) and Prs5 (a subunit of ribose phosphate diphosphokinase), accumulated to wild-type levels in the cdc37S14A mutant strain. These combined data indicate that most protein kinases have access to and require Cdc37 for biogenesis at normal growth temperatures, whereas a smaller proportion of kinases accumulate to wild-type levels independent of Cdc37.
Protein kinases can function in pathways that have a self-regulatory capacity to control their own cellular abundance. In the pheromone-responsive pathway, kinase components such as Ste11, Ste7, and Fus3 are under transcriptional control that depends on a basal level of pathway activation (Fields et al., 1988; Hagen et al., 1991). Deletion of any of these kinases leads to reduced basal signal transmission, resulting in decreased levels of other proteins within the pathway. Therefore, it is possible that the reduced kinase levels we observed in the cdc37S14A mutant could result from the inhibition of just a single kinase within the pathway, and this leads to further indirect decreases in the levels of other kinases. This is illustrated in Fig. 3, in which deletion of the known chaperone client STE11 (Abbas-Terki et al., 2000) resulted in decreased levels of Fus3, a mitogen-activated protein kinase that is downstream in the pheromone-responsive pathway. However, the mutation of CDC37 results in a further reduction in Fus3 levels. Similar results were observed when BCK1 was deleted and the levels of Slt2 protein kinase (which is downstream in the PKC signaling pathway) were measured. The deletion of BCK1 resulted in reduced levels of Slt2, and these were further reduced in the bck1Δ/cdc37S14A double mutant (Fig. 3 B). These results show that kinase expression can be affected indirectly by the activity of other kinases but that Cdc37 has additional functions to control kinase abundance.
The results shown in Fig. 3 demonstrate that protein kinases affect the levels of other kinases within a pathway. In the case of pheromone-responsive and PKC-dependent pathways, this regulation is at a transcriptional level (Fields et al., 1988; Hagen et al., 1991; Jung and Levin, 1999). Accordingly, if Cdc37 affects protein kinase abundance via the indirect method, this should result in changes in kinase gene mRNA levels. To test this hypothesis, we performed a microarray analysis to determine whether the cdc37S14A mutation affects kinase gene expression on a general level. However, a comparison of transcriptomes between wild-type and cdc37S14A mutant strains showed no statistically significant changes in kinase gene expression when the cells were grown at 30°C. This was confirmed by quantitative PCR analysis of several kinases whose protein levels were reduced in the cdc37S14A mutant (unpublished data). In contrast, statistically significant changes (P < 0.05) in expression were observed for a small number of genes, with the maximal fold change being less than twofold. The top three genes with higher expression in the mutant strain (YLR168C, RSM22, and ODC2) all function in mitochondria.
Cdc37 protects nascent chains from rapid degradation
We next investigated the fate of kinases that accumulated at reduced levels in the cdc37S14A mutant, focusing first on Tpk2. Pulse-chase analysis showed that Tpk2 levels were reduced by at least twofold in the cdc37S14A mutant immediately after a 10-min pulse compared with the wild type (Fig. 4 A). Once synthesized, the levels of Tpk2 altered little in either strain over a 2-h period. Pulse labeling of Pho85 and Rim11 resulted in similarly decreased kinase levels in the cdc37S14A mutant (Fig. 4, E and F). Experiments with Cmk2, whose accumulation at steady state was normal in the cdc37S14A mutant, showed that it also accumulated normally under pulse-labeling conditions (Fig. 4 B).
The decrease in Tpk2, Pho85, and Rim11 levels under pulse-labeling conditions could represent the rapid degradation of each kinase or a defect in translation. Degradation by the vacuolar pathway was ruled out by experiments in a pep4Δ mutant and incubation of cells with PMSF. Incubation of cells with PMSF or deletion of the gene encoding the vacuolar peptidase Pep4 did not lead to substantial increases in the levels of Tpk2 or Rim11 in cdc37 mutant cells (Fig. S1). Translation was assessed by examination of polysome profiles and by quantitation of the levels of kinase mRNA on the polysomes from wild-type and cdc37 mutant cells. However, polysome profiles from wild-type and cdc37S14A strains were identical, suggesting that general defects in translation initiation and elongation do not occur in the cdc37S14A mutant (Fig. S2). Furthermore, the cdc37S14A strain did not display growth hypersensitivity on media containing paromomycin (Fig. S2), which is common for mutants that affect translation, such as ssb1Δ/ssb2Δ (Nelson et al., 1992). Quantitative analysis of kinase mRNA levels in polysomes and 80S ribosomes also failed to reveal any differences between the wild-type and cdc37S14A mutant strains.
Further studies addressed whether kinase levels were reduced in the cdc37S14A mutant as a result of rapid degradation by the proteasome. MG132 was incubated with cells for 30 min before the pulse labeling. Under these conditions, we observed that Tpk2 levels were increased in the cdc37S14A mutant strain compared with DMSO alone and that the kinase appeared as two closely migrating bands (Fig. 4 C). Levels of Tpk2 in wild-type cells were unaffected by the presence of MG132, suggesting that the kinase is not subject to degradation during translation under normal conditions. However, these results strongly support the notion that Cdc37 protects nascent chains from degradation either during or immediately after translation. Importantly, this type of quality control is mechanistically distinct from that which occurs upon Hsp90 inhibition with geldanamycin. Under conditions in which geldanamycin is incubated with cells for 30 min before pulse labeling, there is no difference in the amount of Tpk2 synthesized after the pulse, yet there is a profound subsequent reduction in kinase levels after 2 h of chase (Fig. 4 D). Similar results were observed for Slt2 and Kss1 kinases (unpublished data).
The effect of MG132 on the levels of several other kinases during pulse labeling was also examined. Pho85, a cyclin-dependent kinase, behaved in a similar manner to Tpk2. The kinase was present in reduced amounts in the cdc37S14A mutant compared with the wild type after a 5-min pulse, and MG132 partially suppressed this defect (Fig. 4 E). Rim11 levels were also reduced in the cdc37S14A mutant, and, in this case, MG132 led to the accumulation of a slower migrating band that is present in all samples but is typically present as a minor component of the immunoprecipitation (Fig. 4 F). This band may represent a nonphosphorylated form of Rim11. Similar pulse-labeling studies were performed with Cdc28, the cyclin-dependent kinase that promotes cell cycle progression. Surprisingly, levels of Cdc28 were similar in both wild-type and cdc37S14A strains after pulse labeling (Fig. 4 G) even though we observed greatly reduced levels of the kinase in the cdc37S14A strain at steady state (Fig. 1 and not depicted). This paradox was resolved by pulse-chase analysis (Fig. 4 G), which shows that Cdc28 is synthesized in the same amount in both wild-type and mutant strains, yet its levels decrease sharply in the cdc37S14A strain within a 30-min period. As with Rim11, nascent Ccd28 expressed in the mutant cells migrates more slowly in the gel compared with Cdc28 from wild-type cells, as noted previously (Gerber et al., 1995). These findings are consistent with previous studies on Cdc28 biogenesis in a different cdc37 mutant strain (Gerber et al., 1995; Farrell and Morgan, 2000) but contrast with the more rapid degradation observed for Tpk2, Pho85, and Rim11 (Fig. 4, C–E). This could reflect differences in chaperone binding between kinases that will be discussed later.
Cdc37 promotes kinase maturation
Protein folding is a temperature-dependent process, and chaperone dependence for folding is more stringent at higher growth temperatures. We investigated the temperature dependence of kinase accumulation in the cdc37S14A mutant. Three protein kinases that accumulated at low levels in the cdc37S14A mutant were chosen for analysis (Rim11, Tpk2, and Yck2) based on their representation of different subgroups of the kinome (Hunter and Plowman, 1997). Notably, the kinases displayed similar stability profiles that were inversely correlated with increasing temperature (Fig. 5 A). Furthermore, each of the kinases accumulated to levels that were similar to those found in the wild-type strain after growth at 26°C (Fig. 5 B). Next, we measured the activity of Tpk2 and Rim11 after growth of the cells at 26°C. The TAP-tagged kinases were assayed for activity after isolation on IgG–Sepharose beads from wild-type and mutant strain lysates. A synthetic peptide substrate was used for Tpk2, and myelin basic protein (MBP) was used for Rim11 (Zhu et al., 2000). The results of these assays (Fig. 5 C) showed that Rim11 and Tpk2 both had reduced activity when isolated from the cdc37S14A mutant strain compared with wild-type yeast even though similar amounts of kinase were present in the assays.
A further study addressed whether Tpk2, a cAMP-dependent protein kinase, could interact with its negative regulatory subunit Bcy1 in the cdc37S14A mutant strain (Toda et al., 1987). As with mammalian PKA, inhibitory binding of the regulatory subunit is relieved at high cAMP levels, leading to increased kinase activity (Johnson et al., 1987). An HA-tagged version of Bcy1 was expressed in wild-type and cdc37S14A mutant strains grown at 26 and 30°C. Similar amounts of HA-Bcy1 were expressed in both wild-type and cdc37S14A mutant strains grown at both temperatures. TAP-tagged Tpk2 was isolated using IgG–Sepharose bead resin, and the amount of coprecipitating Bcy1 was detected by Western blotting. When the cells were grown at 30°C, there was a decreased yield of TAP-Tpk2 by threefold (Fig. 5 D) as expected because of the decreased kinase levels (Fig. 4). The amount of coprecipitating Bcy1 was decreased by fivefold relative to those amounts isolated from the wild-type strain extracts. When the cells were grown at 26°C, however, similar amounts of TAP-Tpk2 were isolated, but twofold reduced levels of Bcy1 were still bound. This suggests that some population of Tpk2 failed to bind to Bcy1 in the cdc37S14A mutant, yet this correlated with the decreased activity of the kinase.
To determine whether Cdc37 was required for the activity of kinases that were synthesized normally at 30°C, we investigated Sky1, a constitutively active kinase (Nolen et al., 2001). Sky1 has an extra helix between the αC helix and β4 sheet that helps stabilize the kinase. Because the αC/β4 loop is implicated in Cdc37 binding and for mediating kinase sensitivity to geldanamycin (Tikhomirov and Carpenter, 2003; Xu et al., 2005), we hypothesized that Sky1 folding might be chaperone independent. However, Sky1 isolated from the cdc37S14A mutant grown at 30°C was ∼50% less active than Sky1 isolated from the wild-type strain (Fig. 6), indicating that this kinase also requires Cdc37 for efficient activation. When assayed after growth at 26°C, however, Sky1 had a similar activity when isolated from either strain, suggesting that it became independent of Cdc37 at the lower growth temperature. Overall, kinases that are stable at 26 or 30°C in the cdc37S14A mutant are less active than those in wild-type strains. Similar findings were made with the kinase Mps1, which is stably expressed in a cdc37 mutant but has decreased activity (Schutz et al., 1997). However, the chaperone dependence for kinase activity is inversely proportional to growth temperature.
There is accumulating evidence that Cdc37 has a general role in kinase biogenesis, but there has been no systematic analysis at a kinomic level. In our studies, we addressed this issue by analysis of ∼50% of the yeast kinome for dependence on Cdc37. Our results are consistent with Cdc37 having a general role in kinome biogenesis and functioning either during or immediately after translation to protect nascent chains from degradation. In addition, Cdc37 promotes kinase maturation to the folded state.
The finding that Cdc37 plays a general role in kinase biogenesis is consistent with the growing number of kinases that have been characterized to interact with this chaperone (MacLean and Picard, 2003). Two other large-scale screens for Hsp90-interacting proteins also uncovered protein kinases. In a two-hybrid screen with a mutant form of Hsp90 as bait (Millson et al., 2005), six kinases were identified as interacting with Hsp90, and, in a multiapproach study (Zhao et al., 2005), 27 kinases interacted with Hsp90 either physically or genetically. Interestingly, the kinases found in these studies did not overlap, indicating that neither screen was saturating.
The results of our studies show that Cdc37 functions immediately after translation to protect nascent kinase chains from degradation. Even after a subminute pulse labeling, we noted that kinase levels were reduced in the cdc37S14A mutant strain (unpublished data). However, proteasome inhibition followed by pulse labeling largely restored Tpk2, Pho85, and Rim11 levels, providing strong support for Cdc37's role in nascent chain quality control. This phenotype was only observed when MG132 was incubated for brief periods (30 min) in the cells before pulse labeling. Prolonged treatments (2 h) failed to restore Tpk2 levels, although this may result from reduced transcription occurring from long-term proteasome inhibition (unpublished data).
The finding that some kinases require Cdc37 to protect them against rapid degradation shortly after synthesis suggests that the chaperone functions as a conformational sensor at or near to the polysome. The degradation of several kinases during pulse labeling is so fast that we initially considered that translation itself was impaired. However, polysome profiles and measurement of the rate of protein synthesis suggested that Cdc37 was not involved in regulating translation, at least in a general manner (Fig. S2). Also, there is a precedent for the very rapid degradation of ribosomal proteins shortly after synthesis (Warner et al., 1985; Maicas et al., 1988). Although it is unclear whether this quality control pathway is the same as the one observed here, a recent study has discovered a role for Hsp90 in the biogenesis of some ribosomal proteins (Maicas et al., 1988; Kim et al., 2006).
Therefore, our results are consistent with the hypothesis that Cdc37 protects some nascent kinase chains from rapid degradation immediately after translation. Whether Cdc37 binds directly to the nascent chain as it is being synthesized or binds immediately afterward may depend on where in the polypeptide chain the kinase domain is located. Scroggins et al. (2003) have observed Cdc37 binding to polysomes programmed with heme-regulated inhibitor kinase, for example, but not with lymphocyte-specific protein tyrosine kinase. In our own studies, we failed to observe Cdc37 binding to polysomes isolated from yeast even when such polysomes were enriched for Ste11 kinase mRNA (unpublished data). What remains unclear is how such a function for Cdc37 is integrated with those of Hsp70/40 chaperones that interact with misfolded kinases before Cdc37 binding, at least in vitro (Arlander et al., 2006). The function of Cdc37 in stabilizing kinase nascent chains suggests that it belongs to the newly defined group of chaperones linked to protein synthesis (Albanese et al., 2006). This group includes others that are known to promote protein kinase biogenesis such as Ydj1 and Sse1. However, Cdc37 appears to be distinct from this group in other ways because the cdc37 mutant did not display hypersensitivity to a translation inhibitor nor is CDC37 transcriptionally coregulated with other components of the translational machinery. As pointed out by Albanese et al. (2006), however, some chaperones appear to function as both chaperones linked to protein synthesis and as stress-regulated chaperones that function in protein refolding, and this appears to apply to Cdc37.
Rapid degradation of nascent kinase chains in the cdc37 mutant during pulse labeling was not observed in all cases. Cdc28 levels were similar in wild-type and cdc37 mutant strains after pulse labeling, but the kinase was degraded rapidly within a 30-min period. These findings are consistent with previous studies showing that Cdc37 promotes Cdc28 stability (Gerber et al., 1995; Farrell and Morgan, 2000). Whether Cdc28 represents a distinct class of kinase that is degraded by a different pathway is unclear. In our experiments, Cdc28 was untagged, contrasting with the C-terminal TAP-tagged kinases used in all other experiments and providing a possible source for the phenotypic distinction. However, it is also possible that Cdc28 is protected in its prefolded form by other chaperones and that loss of Cdc37 function manifests at a slightly later stage in its maturation. This explanation for the delayed degradation of Cdc28 compared with Tpk2 or Rim11 may also account for why geldanamycin promotes degradation only after kinase synthesis. As shown in Fig. 4 D, Tpk2 is completely synthesized and stable for the duration of a 5-min pulse even in the presence of geldanamycin, although it is rapidly degraded thereafter. These results are consistent with the posttranslational role attributed to Hsp90 and with nascent kinases being stabilized by other chaperones, including Cdc37 and Ydj1/Hsp70, before interacting with Hsp90 for the final stages in maturation.
The temperature-sensitive phenotype for kinase stability in the cdc37S14A mutant allowed us to further dissect the different roles for this chaperone in kinase biogenesis. For Rim11 and Tpk2, the increase in kinase stability in cdc37S14A when grown at 26°C did not lead to full activity; rather, a twofold decrease in activity was observed. For Tpk2, the decrease in activity of the isolated kinase correlated with decreased binding of its inhibitory subunit Bcy1 in cell lysates. This could be caused by Tpk2 misfolding and/or by increased cAMP levels in the cdc37S14A cells caused by a combined decrease in Tpk1/2/3 activity (Nikawa et al., 1987). These combined results are consistent with decreased kinase activity in cdc37S14A mutant cells even when the triage system for degrading misfolded kinases is suppressed by growth at low temperature.
A previous study supports the hypothesis that kinases can accumulate as misfolded conformers that are not degraded. In this case, the deletion of STI1 or HSC82 resulted in very low v-Src levels, an effect that was suppressed by the overexpression of full-length or truncated forms of Cdc37 (Lee et al., 2002). Although the full-length Cdc37 also restored v-Src activity, the truncated forms promoted stabilization without large increases in activity. Because v-Src is constitutively active in the absence of other kinases, these data indicate that v-Src accumulates in an improperly folded form that is not degraded. The same is likely true for Sky1, which is also constitutively active (Nolen et al., 2001). Furthermore, the accumulation of misfolded proteins upon the deletion of several different chaperone proteins was recently demonstrated (McClellan et al., 2005). In this case, components of the Hsp90 chaperone machinery were implicated in promoting degradation but not in the folding of a heterologously expressed protein in yeast. For kinases, the triage system may operate to clear the cell of misfolded kinases close to their site of synthesis before they have a chance to aggregate. This can be bypassed by growth at low temperature even in the absence of chaperone function (Fig. 5 A). In this case, the effect of low temperature may compensate for decreased chaperone function and allow the kinases to proceed to a later stage of maturation with a greater probability of achieving the native state. We suspect this has physiological relevance because some kinases interact persistently with chaperones even after initial folding. For these kinases, chaperones are constantly needed to promote the folded state rather than target the polypeptide for degradation.
In conclusion, our findings show that Cdc37 has a general role in protecting nascent kinase chains from degradation in addition to its function in posttranslational maturation. These findings suggest that Cdc37 is a gatekeeper to cellular kinase abundance. Importantly, yeast Cdc37 synthesis is fairly constant compared with other chaperones whose expression is stress regulated, and this may be a means of limiting kinase abundance in cells.
Materials And Methods
Strains and plasmids
The TAP-tagged yeast library based on strain S288C was purchased from Open Biosystems. Strain genotype was verified by sizing individual gene products by Western blotting and PCR analysis using one primer specific to the tag and one primer specific to the gene of interest.
The URA3-marked cdc37S14A mutant (pS14AU) was constructed by inserting a 1.2-kb DNA fragment containing the URA3 gene into Bcl1-digested pRS314.cdc37S14A (a gift from C. Glover III, The University of Georgia, Athens, GA). A similar plasmid was constructed using wild-type CDC37 for microarray analysis. A plasmid encoding HA-tagged Bcy1 was a gift from J. Hirsch (Mount Sinai School of Medicine, New York, NY). A strain deleted for SSB1 and SSB2 was a gift from E. Craig (University of Wisconsin-Madison, Madison, WI).
The cdc37S14A mutant strains were constructed as follows. Wild-type cells containing TAP-tagged kinases were grown overnight. A DNA fragment containing cdc37S14A∷URA3 was prepared for transformation by digesting pS14AU with BamH1, HindIII, and Kpn1. Transformation by the cdc37S14A∷URA3 fragment into the TAP-tagged strains was performed using the standard lithium acetate method as previously described (Gietz et al., 1995) and transformants selected for growth in media lacking uracil. Ura+ colonies were selected for further study based on having a temperature-sensitive lethal growth phenotype at 37°C.
The yeast erg6Δ knockout strains were constructed as follows. The deletion allele (replaced by the KanMX4 module) was amplified by PCR using primers that were 200 bp upstream of the start codon and 200 bp downstream of the stop codon. The resulting 2-kb products were used to replace the wild-type genes in the TAP-tagged strains under study by a modified version of the transformation protocol of Gietz et al. (1995), which included a heat-shock step at 42°C for 20 min followed by washing the cells with water, resuspension in YPD (yeast extract/peptone/glucose), and incubation at 30°C for 2 h before plating. Successful homologous recombination was determined by selection on YPD media containing 400 μg/ml G418 followed by reselection on complete synthetic dextrose media containing 200 μg/ml G418 minus histidine. Gene replacement was verified by PCR analysis of the KanMX4 module in the proper location using one primer specific to the module (KanB; 5′-CTGCAGCGAGGAGCCGTAAT-3′) and one 300 bp upstream from the start codon where the wild-type gene resided.
Western blot analysis
Western blot analysis was performed on whole cell extracts prepared in IPP150 (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 0.1% NP-40) plus Complete protease inhibitor (Boehringer) by glass bead lysis. An HRP-labeled rabbit Fc fragment followed by chemiluminescent detection was used in most experiments. In some experiments, anticalmodulin-binding peptide antibody was used (Open Biosystems). Some kinases were not detected in whole cell extracts but were readily detected after a single round of isolation on IgG–Sepharose bead resin. Kinases in this class (a total of 16) were Rad53, Sgv1, Cdc7, Prr1, Ydl025c, Sat4, Ste11, Ynr047c, Hrk1, Npr1, Kin1, Ctk1, Ssk22, KKq8, Ipl1, and Ire1.
Pulse labeling and polysome profile analysis
Pulse labeling, immunoprecipitations, and sucrose gradient analysis of polysome profiles were performed as described previously (Brodsky et al., 1998).
Kinase assays
Wild-type and cdc37S14A cells expressing TAP-tagged forms of Rim11, Tpk2, and Sky1 were grown overnight (at 26°C for Rim11 and Tpk2 and at 30°C for Sky1) in 100 ml synthetic complete media (minus His and Ura) to log phase. Cells were harvested, and extracts were prepared using IPP150 (Seraphin et al., 2002). The cell extract concentrations were adjusted to 0.45 ml at 3 mg/ml in IPP150 and were incubated with 50 μl of 50% vol/vol IgG–Sepharose bead resin (GE Healthcare) for 1 h at 4°C. The resin was washed four times using the same buffer and divided into two aliquots. One aliquot was used for Western blot analysis (see above), whereas the other was used for the kinase assay. Kinase assay for Tpk2 was performed using the PepTag Nonradioactive Protein Kinase Assay kit (Promega) according to instructions provided by the manufacturer. Kinase activity was determined by absorbance at A570. Rim11's and Sky1's kinase activities were assayed by measuring their ability to phosphorylate MBP (Upstate Biotechnology). In addition to the aforementioned four washes, the resin was subsequently washed once with kinase buffer (10 mM Tris-HCl, pH 7.5, and 10 mM MgCl2). The washed resin was incubated in 20 μl kinase buffer containing 1 μg MBP, 100 μM ATP, and 10 μCi [γ32]ATP (PerkinElmer) for 20 min at room temperature. 20 μl of 2× SDS sample buffer (4°C) was added to stop the kinase reaction. The samples were resolved by denaturing gel electrophoresis. The gel was fixed and dried before autoradiography. Quantitation of MBP phosphorylation was determined using a phosphoimager (Storm 860; Molecular Dynamics) and ImageQuant software (GE Healthcare).
Microarray analysis
Wild-type S288C yeast were converted to CDC37∷URA3 or cdc37S14A∷URA3 as described above. Cultures of each strain were grown to log phase, and total RNA was prepared as described previously (Schmitt et al., 1990). A clean-up procedure was used with an RNeasy kit (QIAGEN) and on-column DNA digestion. Hybridization to S98 chips (Affymetrix, Inc.) and data analysis were performed by the Mount Sinai microarray facility.
Phylogenetic analysis
The analysis shown in Fig. 2 was performed on isolated kinase domains downloaded from http://kinase2.salk.edu. A multiple alignment was performed with ClustalW in the MacVector software package (version 7; Accelrys) followed by phylogenetic analysis using Protdist and Neighbor present in the Phylip 3.6 package (http://evolution.genetics.washington.edu/phylip.html). The tree was drawn using Hypertree (Bingham and Sudarsanam, 2000).
Online supplemental material
Fig. S1 shows that deletion of the vacuolar peptidase PEP4 or incubation of yeast cells with PMSF results in the accumulation of kinases Rim11 and Tpk2 but in a manner that is similar in wild-type and cdc37S14A cells. These findings rule out a role for the vacuole as a means of preferential kinase depletion in the cdc37S14A mutant cells. Fig. S2 shows polysome profiles that are similar in wild-type and cdc37S14A mutant cells. The sensitivity of yeast growth on paromomycin is also shown and demonstrates that cdc37S14A mutant cells have a similar sensitivity to wild-type cells.
We thank Drs. Elizabeth Craig, Claiborne Glover III, and Jeanne Hirsch for plasmids/yeast strains, Drs. Tearina Chu and Yuexun Liu for microarray analysis, and John Woolford for use of his gradient fractionator. We also thank Drs. Doug Cyr, Stephen Garrett, Jeanne Hirsch, Bob Matts, Kevin Morano, and David Ron for helpful discussions during the course of this study. We are especially grateful to R. Deshaies for his insight regarding our work.
This study was supported by National Institutes of Health grants GM70596 (to A.J. Caplan) and GM 075061 (to J.L. Brodsky).
Abbreviations used in this paper: MBP, myelin basic protein; TAP, tandem affinity purified.

subset: commoncrawl


text:
Ghana has many beautiful venues to experience its natural beauty and to observe/learn its various cultural skills in action.
Places that are close to Alavanyo and can be visited on a Weekend:
1. Wli water falls
2. Monkey Sanctuary
3. Kpando Pottery
4. Agotime Kente Weaving
Places that are several hours from Alavanyo that can be visited before or after your stay at the Home:
1. Mole Park in the Northern part of Ghana to see Elephants and many other animals in their natural habitat.
2. The Southern Coast to experience the beautiful beaches and resorts.
Ghana has much to offer its' Visitors and we can guide you in planning your excursions.


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Criminals in Staffordshire have been charged with drug-related offences more than 2,000 times over the last three years.
Figures released by Staffordshire Police show that cannabis offences have accounted for more than half of the prosecutions recorded in the county since 2016.
People were charged with producing, dealing or possessing the class B drug 1,096 times – out of a total of 2,028 drug offences.
Cocaine and heroin offences were the second and third most frequently prosecuted, accounting for 371 and 240 charges respectively.
But despite the current monkey dust 'epidemic' hitting North Staffordshire, only a handful of criminals have been charged in connection with it so far.
Although the police do not record monkey dust prosecutions separately, analysis has revealed that the class B cathinone has only been mentioned on crime reports in 17 drug prosecutions over the last three years.
Staffordshire Police says the number of monkey dust prosecutions does not fully reflect the size of the problem.
A force spokesman said: "The class B drug known locally as monkey dust causes considerable demand to Staffordshire Police through the level of resource needed per incident, as opposed to the number of incidents faced. The drug is incredibly unpredictable and long-lasting, often resulting in emergency services having to use a lot of resources to resolve the incident and working with partners to treat those affected.
"An investigative strategy is in place and we are making positive progress with investigations into the supply of the drug in the local area. However other Class B drugs, like cannabis, are more prevalent and there are more instances of people cultivating drugs like cannabis."
Simple possession of cannabis, which carries a maximum sentence of five years but is far more likely to result in a fine, has accounted for 740 prosecutions in Staffordshire since 2016, making it the single most common drug-related offence.
There have also been 159 prosecutions for producing cannabis, 149 for possession with intent to supply cannabis, and 46 for supplying cannabis.
But the number of prosecutions and cautions for cannabis possession has been falling in Staffordshire and across the country in recent years. Figures from the Ministry of Justice show that the total number of prosecutions in England and Wales fell from 18,661 in 2015 to 15,120 in 2017.
On the other hand, increasingly more police resources are being directed at Staffordshire's monkey dust problem. Staffordshire Police says it was dealing with around 10 monkey dust calls a day over the summer, while a judge labelled Stoke-on-Trent the 'capital of monkey dust'.
The spokesman added: "Staffordshire Police takes all crime related to drugs very seriously and is constantly reviewing investigations to decide which ones must take priority and have the most resources diverted towards them. All drugs can cause harm and are interlinked to the exploitation of vulnerable people, which through partnership work and early intervention we try to provide the appropriate support and rehabilitation opportunities."
Want to tell us about something going on where you live? Let us know - Tweet us @SOTLive or message us on our Facebook page. And if you have pictures to share, tag us on Instagram at StokeonTrentLive.


text:
It's a pretty bad feeling when you look at the side of your high-performance car and see a huge dent or scratch. Luxury vehicles are supposed to look like they just rolled off the factory floor, not look like they've been in a crash-up derby.
The next step is getting your ride fixed so you can be back on the road like nothing ever happened. But a lot of Jaguar, Audi and Aston Martin owners aren't sure where to take their car to get the best service, parts and repairs.
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Figure out if they have product knowledge—do they have certified technicians that work on your specific model? What about the correct tools? With high performance machines, you need authorized repair equipment. Don't settle with shops that claim any tool will do.
You need to consider your warranty as well. If you go with manufacturer-approved parts and go with the correct repair methods, you warranty will remain valid.
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Parliamentarians increased their spending limit for 2009 with millions of euro, hoping they will get re-elected in parliamentary elections this fall, one newspaper reads on Wednesday. Elsewhere in the news, financial speculators thrive these days, as the Romanian currency depreciates in regards to the euro. Last but not least, Romanians living in geographically remote places have no medical emergency centers, even if, in theory, they should exist.
Cotidianul reads about Romania's parliamentarians who just increased their 2009 spending budget with 20% more than the limit established by the Ministry of Finance. The newspaper informs that, in a financial crisis, the Liberal Executive has to deal with another problem.
After passing the bill that rules a 50% increase in teachers' wages, now Senators exceeded their spending limit. The total value of the Senate's budget for 2009 will increase from 153 million Romanian Ron even if the Executive specifically requested a limit of 130 million Romanian Ron.
The newspaper informs that 100,000 euro are allocated only for ACs in the meeting rooms and ventilation machines because, the Senate's secretary said, several senators complained of head aches due to the air in the meeting rooms.
Other spending include the acquisition of 20 new cars, of 25,000 euro a piece, 140 cars of the senate's offices, each 11,000 euro and three buses of 50,000 euro. Thus, the total amount allocated for cars exceeds 2,300,000 euro.
Elsewhere in the news, Evenimentul Zilei reads about the exchange rates practiced by exchange houses, which manage to thrive during crises like these. In Bucharest, 1 euro was sold with 3.9 Romanian Ron and bought with only 3 Romanian Ron.
The newspaper underlines that speculators have their own way of deceiving people, on the edge of legality. The method is simple, authorities say: speculators take advantage of the naivety of the buyers who fail to read carefully the exchange rate.
Journalists tell the case of several people who were deceived in downtown Bucharest yesterday, because they failed to read the exchange rate and signed for the transaction. In this case, police says, the transaction is legal and there's nothing one can do.
Thus, they attention all people to be careful when planning to change money in a crisis such as this and check twice before signing the exchange rate.
Last but not least, Romania libera journalists checked in geographically remote places if emergency medical centers really function as the law rules. There's no surprise to read that in some places, the medical centers were did not even exist or that in others, even if they exist, there's nobody there.
A press release of the Health ministry reads that emergency medical centers function 24/hours per day, including Saturdays and Sundays. However, where journalists found such a place, doctors were missing.


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Earlier this week, some rules changes were announced that will have some interesting implications for casual play. I looked through these changes several times and then flipped through some decks and cards specifically to see what improves and what weakens. I saw some interesting results, so today, I want to talk about how the rules changes should impact Casual Land.
What are these rules changes? If you haven't seen, there are several changes that officially will be in place as of July 13. So, if you are playing with some things, get ready for changes!
You can read all about the changes here.
Boros Charm giving your team indestructible will no longer receive it (which I totally think seems right). The other minor change reflects decks with cards that enable you to play more than one land. Apparently (and I admit I didn't even know this), when you played a land, you announced which extra method of playing lands you were using or if it was your normal land drop. So, if you had out both Gaea's Touch and Oracle of Mul Daya, played a Forest, and then sacrificed the Gaea's Touch, if you did not announce the Forest was played with the Touch, it would count as your normal land drop and you would have missed the Touch land drop. It also created tricks such as bouncing the Oracle to net another land drop. Now everything makes sense—the rules just look to see how many lands plays you have, and you can play up to that many.
So, those minor cases are out of the way. Now to the two big changes, and they are very similar:
- Legendary permanents no longer kill each other when two or more are in play. Instead, if you control two or more, you choose one, and the others go forthwith.
- Planeswalkers no longer kill each other when two or more of the same character are in play. Instead, if you control two or more, you choose one and the others go forthwith.
Suppose I am running Jace, the Mind Sculptor. You can no longer kill it by playing Jace Beleren. Similarly, if I have Commander Eesha, you can't kill her by playing your own Eesha. There are many consequences of these two rules changes, and it will take an article to look at them, so let's get started!
I suspect that one of the reasons behind the legendary permanent change is Commanders. It was not mentioned in the reasons article. We all know R&D is making cards and rules to enhance Commander, so this is no surprise. Previously, if both my opponent and I flipped over Glissa the Traitor, our match would be odd. We could not rely on our Commanders running around, and when one was played, the other would soon follow to kill it. I have seen matches in which neither player dropped his Commander for the game because bothering to spend the mana made little sense. That is a huge change of awesome to the format, and people will be talking about that in articles and through the ages.
Let's take a look under the hood at the first change: planeswalkers.
As was pointed out, many 'walkers have multiple variants. As more see print, they were collectively worsening, especially in multiplayer. Suppose I had a blue multiplayer deck and I honestly wanted one of each Jace in the deck because they all fit it. I shuffle up against my foes. What are the chances that none of them will have any Jaces? That's pretty unlikely, right? So, my awesome Jace, Memory Adept is suddenly snapped and killed because someone drew her Jace Beleren and dropped it—since she couldn't do anything else with it. Both died. But now, my Jaces are not threatened by anyone else's. I can run Ajani Goldmane without worrying about your Ajani Goldmane killing it.
Similarly, legendary creatures are enhanced in several ways. First of all, they are empowered in the same way that 'walkers are—you don't have to worry about another copy of a legendary card taking out yours. No longer will my Akroma, Angel of Wrath be able to be killed by yours. For commonly-played legendary permanents, this makes them even better because a major weakness is now gone.
One of the consequences of this is that Clone effects are now significantly worse. You can no longer lean on Clone, Phyrexian Metamorph, and Phantasmal Image to blow up an opposing legendary creature. Whether in duels or multiplayer, many legendary creatures are seen at the kitchen table. So, people leaned on Clones as a great way to handle them. They also killed legendary creatures through protection. For example, if a legendary creature was wearing Darksteel Plate or Swiftfoot Boots, they were hard to kill, but using Clone and its friends was a useful path. That is no longer the case. So, I suspect such protection-enabling enchantments and Equipment (such as Asceticism and Champion's Helm) will rise in play value as one way to contest it is lost.
Because of this change, I expect Sakashima the Impostor to drop in play value considerably. One of the cool things Sakashima had going was the ability to copy a legendary creature without dying because it kept its name. Now we have an odd Clone variant, so the play it saw should reduce.
(On the other hand, while Clones became worse to hit opposing legendaries, they are now better at cloning your own. See below for a lot of synergies that would value copy effects.)
Avacyn, Angel of Hope was a massive powerhouse at the multiplayer table. But one of the reliable ways you could kill her was either playing your own Avacyn or a Clone. Now one path to Avacyn removal is gone, and that only makes her highness even better. Making the best creature for multiplayer better is just unfair to Magic.
What should truly go up in play are the legendary lands. In Casualalia, there are some legendary lands that are quite powerful, such as Kor Haven, Volrath's Stronghold, Urborg, Tomb of Yawgmoth, and Gaea's Cradle. One thing holding these back was the fear that they would be destroyed by someone else's card. But that is no longer the case. So, the likelihood of lands being whacked has reduced to just running up against Strip Mine or Avalanche Riders or something. That increases the power of these lands, and thus, their play value rises in accord. I would expect to see these played more at the kitchen table and particularly in multiplayer (and particularly in Commander, which has the space for a Yavimaya Hollow or a similar friend). (One legendary land that actually worsens is Flagstones of Trokair.)
Another consequence of the rules change is with the interactivity of your own deck. I love that Kokusho, the Evening Star just became a shade worse. Typically, a deck built around Kokusho combos would bring out a bunch at once, and then they would all die. Now, one stays out, and one fewer death trigger occurs as a result. If you control one Kokusho and play another, just one dies. It still is a fine card, but it ebbed just a tad in power.
With multiple copies of legendary creatures in your deck, you can play one to replace another. Let's suppose that I have Rasputin Dreamweaver in my deck. When it comes into play, it brings 7 counters with it. I may have spent all of my counters and now play another to reload my stockpile of counters, and the first dies.
Prime Speaker Zegana when you already have one out—in order to draw a bunch of cards. That's fine and dandy because the smaller Speaker dies. This is a great place to drop Phantasmal Image on your Speaker because you'll draw a ton of cards and then sacrifice the Imaged copy. Your 2 mana becomes five or six cards! It's also a good way to bring out a healthy copy of a legendary creature in case one has a bunch of -1/-1 counters, a nasty Aura, or the like.
Going forward, we are going to see a lot of strategies built around this change. For example, a cheap, early Mikaeus, the Lunarch that drops some counters on folks might be blasted by one that comes out a few turns later just without waiting for the first to die. You can drop a Myojin after using up the divinity counter on one in play. Bladewing the Risen and Godo, Bandit Warlord just became more powerful, as did Lavinia of the Tenth. Things improve for these folks.
As mentioned on the Wizards articles, this means you can play a legendary permanent to kill one in play that may be Arrested. It's not uncommon to see Aura-based removal at the kitchen table. This will help your deck recover from that One Thousand Lashes or Pacifism.
Note that abilities that steal a creature just improved a smidgeon. Let's say you and I are playing a duel, and I drop something decent, such as Ith, High Arcanist off its early suspend. On my sixty turn, it drops out, and I swing for 3 vigilance damage and then keep it back. You have a 4/3 flyer that you want to keep swinging overhead, and Ith is going to just untap it and stop its damage. So, you steal my Ith with Mind Control. That sucks! Luckily, I have another Ith in my hand. Previously, I could play it, kill yours and mine, and then we'd keep going. But now, I can no longer use a duplicate of my legendary creature to kill the one you stole from me. (Nor could I follow up with a copy effect such as Clone to whack them both.) Thus, stealing a legendary creature just became a little bit safer.
Leyline of Anticipation or Vedalken Orrery, I can drop my critters in response to stuff. Imagine if I responded to you Mind Controlling my Ith by playing another and sacrificing the one targeted. There are several ways to harness flash even more than before!
Some cards will change as well. Take the commonly-played Mimic Vat. When a creature dies, you can imprint it and spit out tokens of that creature, yada yada yada. Before, you would be taking stupid pills to imprint a legendary creature. You play it the first time and then play it the second, and both die. But now, no one dies except the new token, and you can keep making triggers. Imagine something simple like Sun Ce, Young Conqueror on a Mimic Vat after July 13. Now imagine Prime Speaker on it—ouch!
Also, the change impacts mass reanimation. (This was especially true at a multiplayer game.) Whenever someone dropped Twilight's Call or Living Death, people would have to call out their legendary creatures that came back. "Okay, I brought back Tolsimir Wolfblood, Chorus of the Conclave, and Kaysa." "I retrieved Sol'kanar the Swamp King, Memnarch, Bosh, Iron Golem, and Karn, Silver Golem." "I brought back Memnarch, too, Dave." "Dang gone it." It would take minutes. And invariably, someone would forget to call out a legendary creature buried in his or her ten creatures, and we'd notice two turns later than we had two Akroma, Angel of Wrath in play. These changes clean these issues rather nicely. (This also improves and enhances the play of cards such as Eureka as well).
The result of all of these changes is that things should be a little different in a lot of ways. Not only are planeswalkers enhanced in power, and legendary permanents in general, but copy effects change considerably. As a consequence, the way legendary permanents are played could result in new decks and new combos. You can expect cards that steal creatures, flash, mass reanimation, and more to alter slightly to encompass the new rules. Many cards will change, from Mimic Vat to Faith's Fetters. Some are now worse, and others better.
Get ready for the new changes!
See you next week,
Abe Sargent
P.S. Is your playgroup going to wait until July 13 to start the new changes or will they adopt them sooner?


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This is Betsy's Crow pattern I purchased last November when attending her class. Looking at this piece think I'll pull out the orange above the head of the crow.....uh, but would replace it with what color? Maybe dulled gold.
Was hoping to use more dull colors on this side with the patchwork hooked background and use a combination on the other side for that crow using whatever Magdalena would have used.
Okay, am showing you the mess I make just around the area where I hook. In front of that pillow is where the hooking takes place. To my right (your left) at that space is some mess and mingled wool.
Hope to have some progress to show you next time I post. If not it may be another rug show.
Hugs to all, try and stay warm. In addition to the frigid temps and wind we are to endure snow now. At this point I think those challenges can be okay with me as long as there is heat in the house.
Please bring your pets inside. If you know anyone or has seen anyone who keeps their pet outside for hours on end in these frigid temperatures, PLEASE call the authorities. No animal should be allowed to be left outside by it's owner for lengths of time.
Hope you all are warm and safe.


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Blogging Communities: Relevance in the Age of Social Media
The recent Top 10 Emerging Influential Blogs 2011 writing project process made me reflect on how the Philippines blogging community is evolving. Much has remained the same but there are behaviors emerging or further reinforced. This includes:
Polarization: strength or weakness?
|Mindmap inspired from the book Brains on Fire
- Community leaders will rule
One's influence is greatly determined or established by which community he or she belongs to. It doesn't have to be a numbers game but more of how dependent or attached its members became to it.
I certainly love Pinoy Bloggers for its very democratic setting. At the same time, it only allows postings that are meant to benefit the community members at large - rather than be self-serving. Although not all community leaders will be able to muster support for endeavors, but their visibility and attitude has great influence to the members at large.
The same can be said to formal communities or organizations where the officers and members have the power to reach out to make their advocacies known. The SOCCSKSARGEN Bloggers community have shown through the years that physical location is certainly not a barrier to build a brand and attract like-minded advocates to support.
Of course, not all groups are visible to the social media eye. There are bloggers who formed secret communities of their own to stay away from usual politics that happens in this space.
In the end, bloggers who come together find common interest as their binding ground. Once that common interest gets compromised, true colors become evident and can be perceived differently depending on where bloggers stand on issues.
- Women bloggers as a force
This is the first time where majority of the blogs recognized as influential are owned by women. Despite groupings and independence of early female bloggers, the Pinay Mommies Community showed that women, when united and trustful of each other, can lift each other online.
|Blogging Ahead from Blogging from Home
Polarization: strength or weakness?
With growing number of groups and influencers in the local blogging community, it is probable that not one blogging group will set the tone. They could participate as a group or as individual in voicing out concerns on issues that will arise in the future.
How far will that go? I think 2012 and 2013 will shed more light, thriving in chaos, with blogging as an activity becomes more mainstream. Especially with traditional media personalities entering this space, such as Move.PH, and decided to be part of the bigger blogging population.


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i brought home the glass bong that i made in the previous post, and now have two glasses in the annealer, waiting for me to pick them up. 8)
the fremont phil has a gig on friday, which is tomorrow. i'm probably going up to b'ham to visit with kenyth on saturday. maybe coming back saturday, maybe sunday… haven't decided yet.
saturday is also frank zappa day, and international talk-like-a-pirate day. a week from saturday is festa italiana for the BSSB. the thursday after that (which is the first of october), snake suspenderz has a gig at the chateau retirement center. maybe they'll remember to pay us this time.
there's a video that goes here, but it's taking forever to upload, so i'll put it in later.


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30" Set of 4 Metal Indoor Outdoor Counter Height Bar stools with Low Back and Rubber Feet-Gun
These are vintage metal chairs that provide you with the comfort and stylishness of a multitude of decor options. Made of superior steel these chairs feature heavy-duty and sturdy seat to hold up to 330lbs. And each metal chair has an x-brace under the seat that provides additional support and stability. In addition this chair includes rubber feet pads to avoid slipping and protect your floor from scratch. Comfortable backrests ensure you can enjoy your delicious meals and conversation leisurely. Therefore whether you're using this chair for your kitchen patio or bistro it's sure to liven up your decor. Now get a set of 4 metal chairs to decor and use anywhere you need them! Ergonomic seat and back design provides better support and more comfort Stackable design for space-saving storage when not in use Heavy-duty metal construction with surface painting to hold up to 330 lbs Rubber feet pads protect your floor from scratch and avoid slipping X-brace under the seat that provides additional support and stability Lightweight for transport and easy to clean with a damp cloth Suitable for any space like dining room bar living room coffee house and patio Easy assembly with instruction hardware and tools included
Specification: Color: Black/Gun Material: Steel plastic and rubber Product dimensions: 17" x 17" x 37" (L x W x H) Seat size: 12" x 12" Seat height: 30" Net weight: 12 lbs Maximum weight capacity: 330 lbs Package includes: 4 x Metal chairs


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OEM High Quality Parts
All the listed repairs are completed with OEM standard parts, ensuring the greatest possible quality.
Screen Repair
Water Damage Repair
From £40
If your issue isn't listed above or you're not sure what the issue may be, why not drop in for a free diagnosis?
19 Baddow Road
Essex CM2 0BX
Mon-Sat: 9am – 6pm
Sun: 10:30am – 4:30pm


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Prices of solar-plus-storage systems to be deployed on France's island territories have fallen once again in the country's latest auction, dipping below €100/MWh (US$122.75/MWh).
Results for the third annual edition of France's tender for projects in territories not connected to the country's main grid network have been announced, with a total of 57MW of solar-plus-storage projects receiving contracts.
The rules of the tender state that each MWp of solar deployed must be coupled with at least 0.5MW/0.5MWh of battery storage in order to enable greater self-consumption of renewable energy capacity.
Developers working on island territories including Guadeloupe, Corsica and Martinique as well as French Guyana – not an island but not connected to the French grid for obvious reasons – have been awarded contracts for around 50MW of solar PV capacity each year since the auctions began to be held annually in 2016, although the first such auction actually took place in 2012.
The most recent results, revealed by the Ministry for Ecological Transformation, are for a tender round opened in 2019. The 47 projects will receive payment via long-term power purchase agreements (PPAs) backed by the government at an average tariff of €98.6 (US$120.81) per MWh. Clean Horizon's analysts pointed out that this is the first the average prices of projects in one of the tenders has fallen below €100/MWh.
For more on this story, visit sister publication Energy-Storage.news.
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Q & A: Inside the Heart of a Self Defense Expert
Q & A: Inside the Heart of a Self Defense Expert
I've done countless interviews all over the world for various magazines, radio shows, newspapers, blogs and the like and some have been great while others were standard, everyday questions but none ever dove into the real heart of the matter; how did and does the journey into the self defense/martial arts world affect us on a personal level?
I was positive I wasn't the only one who felt that way and so I decided to come up with a list of 10+ Questions for the great men and women of our industry who are out there day in and day out dealing the darker side of the martial arts universe, the self defense world.
Little do people know or realise that we don't only work with prestigious groups such as law enforcement, military, security, world class athletes and the like but the vast majority of the time, between those 'tacticool' contracts, we deal with victims of violence on a daily basis.
Even when not dealing directly with the victims, we are constantly researching the world of violence in one form or another; pedophilia, rape, incest, domestic abuse, attempted murders, kidnappings, aggravated assaults, street fights and every kind of urban violence one can think of. Needless to say, speaking for myself anyway, this has had a huge impact on both my life and worldviews, and not necessarily on a positive level as I wrote about in an earlier blogpost.
This 'revealing' interview isn't about styles, systems, methodologies, tactics or even self defense... it is about how the work in the self-defense/personal protection industry affects each and everyone of their lives on a deeper level.
I sent out over 40 requests and received quite a few replies, surprisingly, many more than I expected accepting my offer to take part. In no particular order of appearance here are those who have accepted and who will be interviewed here beginning next week:
John Whitman of Krav Maga Alliance
Lee Morrison of Urban Combatives
Christopher Roberts of Safe International
Jim Wagner Reality Based Personal Protection
Debi Stevens of Action Breaks Silence
Mark Hatmaker of Extreme Self Protection
The interviews will be posted on a first come, first serve basis. There are others who I am awaiting a reply from who said they'd look it over and get back to me and there are still others I have not heard back from at all, only a couple opted out due to scheduling reasons thus far, and so I will update this list as and if it grows.
CAVEAT: If I have missed anyone in our industry and I did not contact you for this interview, please accept my apologies, drop me an email and I will send you the questions for your participation, the only requirements I have is that 1. Teaching self defense is your main source of income and 2. you are actively teaching on an international level. Thank you.
Stay tuned folks.....


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Wolf River Canoe & Kayak
Wolf River Canoe & Kayak has been operating on the Wolf River since 1982. Anyone who enjoys canoeing or kayaking in the great outdoors can enjoy an excursion along the Wolf River. Kayakers can enjoy a half-day trip or a week long camping vacation.
Hours of Operation : Weekly – 8:00am-6:00pm
Adult Admission: N/A
Child Admission: N/A
Senior Admission: N/A
Military Admission: N/A
21640 Tucker Road, Long Beach, MS
(228) 452 7666


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Carolina Catholic Archives
|December 17
|Dr. Robert Moynihan and Finding Viganò
|December 5
|Tony Vivaldi and Save A Vet Now
|November 21
|Father James Altman and the McCarrick Report
|November 7
|Bill Pincus, NC Right to Life President
|October 17
|Dr. Tony Maglione
|October 3
|Deacon John Walsh
|September 19
|Father Peter Devereux, LC
|August 15 &
September 5
|Father James Altman (2 shows)
|August 1
|Four Deacons
|July 4
|George Floyd with Adam Davis
|June 20
|Praying with the Saints with Dawn Beutner
|June 6
|The Lord's Prayer with Fr. Jeffrey Kirby
|May 16
|Kevin Wells second half
|May 2
|Kevin Wells first half
|April 18
|Fr. Donald Calloway, MIC
|April 4
|Orbis Catholic Travel
|March 7
|Ignited By Truth
|February 15
|From the Depths of our Hearts
|February 1
|March for Life
|January 18
|Dr. Matthew Bunson
|January 4
|Fr. Dave Pivonka


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Focus is the #1 Key to success
Want to succeed at something but feel like you just are not sure how to get there?
Change that now!
Hey Guys!
Coach Lew here to talk to you about the "F" word!
Yes that dirty little word focus!
Many times is life we want something or want to change something but we can't figure out how. Worse yet most of the time we know how but begin to make change and lose momentum or direction. Why does this happen?
One of the strongest legs of change is the power of focus!
What I am saying is you get what you set your focus on! What I am not saying is that woo woo law of attraction talk of sit and focus and you will get!
Truth is YES you must focus on what you want and even sometimes obsessively but you must take action along with focus!
Going for whatever you are after with out consistent focus is like driving to LA with out a gps and assuming that because you are headed West that will insure you get to Rodeo Drive!
So why is the power of focus so important to success then?
Here we go in a nutshell!
What you focus on you will pay attention to
When you are focused you will notice things that you normally block out
You will ask questions and seek information from people who have already accomplished or are ahead in the journey you are on.
The more focus you put on something the more important it becomes to you brain and the more creative energy your brain will assign to the process!
You must clearly know what your outcome is as in the LA example
When you know where you are going it creates a laser like focus that will allow you to weed out the distractions.
In summary FOCUS is an important part of success because you must know what you are after, you must seek ways to accomplish it and you must know how to tell if you are succeeding! So the old saying of what you get what you focus on is true! So make sure you are focusing on what you really want, you focus on the positive reasons you want it and you focus on taking the action necessary you need to achieve it!
Yes! You can have the life you desire and deserve! Not sure where to begin or the steps you need? Find a Coach to help you design the plan!
Click here to send us a message if you have questions or would like to find out more about coaching!
Live with Energy, Passion And Always Live Your Dreams!
-Coach Lew


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Why Would an In House Counsel Leave to Work for a Law Firm?
In house counsel and law firm attorney have traditionally been viewed as two distinct career paths for lawyers.
Generally, in house counsel develop legal expertise for their organization and learn to handle a wide variety of matters. On the other hand, law firm attorneys are typically more specialized and their salary is based in large part on their rainmaking abilities.
While success at a law firm and success as in house counsel oftentimes depends upon very different skills, recent data suggest that the lines separating these two careers are blurring as more and more in house counsel are leaving their jobs and joining firms.
The main reason that law firms may be poaching in house counsel may be the fact that there are so few attorneys in practice areas that suffered significant layoffs in recent years, reports the Pittsburgh Post-Gazette. So in house counsel at real estate and construction companies may find that their skill sets are highly coveted by law firms, and are paid accordingly.
In addition, in house counsel may have corporate connections that firm wants. Having worked inside the company, in house counsel may have a deep connection of networks that law firms want to tap. An in house counsel's network just means more rainmaking opportunities for a firm.
Finally, the work that in house counsel and law firms perform may not be that different anymore. Most large companies retain both inside and outside counsel. As a result, law firms may be doing a lot of the work that was traditionally done by in house counsel.
But before any in house counsel makes that jump to a law firm, you must still remember there are benefits to non-law firm life. The world of billable hours and questions of how many clients you brought in can be brutal. While in house counsel traditionally are paid less, your quality of life is likely much higher outside a firm.
Related Resources:
- Law Firms Are Looking to Hire In-House Counsel for Lateral Positions (ABA Journal)
- General Counsel Salary Falls By Almost Every Measure (FindLaw's In House)
- Is Hiring New Grads In House the Wave of the Future? (FindLaw's In House)


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Harvey Hadden Sports Village
An ideal site for solar energy generation in Nottingham
The Customer
Harvey Hadden Sports Village is a state of the art leisure centre in Nottingham their offering includes, gym and fitness studio, Olympic sized swimming pool, sports halls, and much more. The site has provided sporting facilities to local citizens for over 25 years and recently underwent an innovative £16m redevelopment. Harvey Hadden is a popular leisure centre seeing an average footfall of 35,000 visits per month.
The Challenge
Like all leisure's centre Harvey Hadden has a large energy demand. Its owner Nottingham City Council is facing both budget pressures and a stretching climate change target to reduce carbon emissions from its operations. The leisure centre has undergone extensive energy efficiency programmes to reduce energy use with many energy reduction opportunities fully utilised the centre needed to consider generating its own energy to seek further cost reductions.
The Solution
Harvey Hadden is a large site with available rooftop space and car parks making it an ideal site for solar energy generation. Energy Services have delivered two solar projects for the leisure centre. A 200 kWp roof solar panel system and the UK's first publicly-owned solar car park a 67 kWp system. These PV systems were installed without any disruption to the day-to-day running of the popular leisure centre. Since generation started Harvey Hadden has regularly benefited from 100% off grid energy through the summer.
The Experience
A full inspection of the roof was undertaken to ensure the longevity of the structure before installation, this identified a corrosion issue which Energy Services helped the centre solve. Health and safety measures were also taken to ensure the busy site caused no risk to members of the public using facilities.
In addition the canopies offer shade against the sun and rain and have improved the quality of the car parks for the customers.
Having Solar PV will protect the leisure centre against electricity bill inflation and help Nottingham City Council continue to meet its climate change targets.
Project Specification
Total kWp: 200.2
Number of panels: 715
Estimated generation (kWh): 109,712
Carbon savings (Tonnes): 49
Percentage reduction in imported energy demand: (up to) 100%
Installation cost: £188,600
Annual bill savings: £20,000