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2013 fantasy football quick hitters
Fowler's Take
Aside from the obvious discussion surrounding the fantasy football value of second-year starting quarterbacks Andrew Luck, RG3, Russell Wilson, and Colin Kaepernick, I'll be keeping a close eye on the following storylines now through the end of August:
Steven Jackson's role with the Falcons
Jackson's 2012 FOXSports.com average draft position was the fourth round and approximately 31st pick overall. I could easily see his ADP jump as high as the second round in some leagues.
Sure, the Rams clocked plenty of miles on the 29-year-old running back, but that's not to say he's washed up. Jackson has finished with over 1,000 rushing for eight consecutive years despite only playing in 12 games in both the 2007 and 2008 seasons.
Considering the Falcons' pass-first offense, Jackson probably won't rack up 300 carries in 2013, but if Atlanta uses him the same way they used Michael Turner in 2012, S-Jax should receive plenty of touches inside the opponent's 10-yard line. Turner averaged 10-plus touchdowns over the last four seasons. Jackson's season-high during that stretch was six rushing scores (2010).
Only six running backs rushed for over 1,000 yards with double-digit touchdowns last season. Jackson may be on the verge of reinvention down in Hotlanta.
Ryan Tannehill and Mike Wallace
Despite his quarterback numbers not matching his rookie brethren, I believe there is cautious optimism surrounding the fantasy future of Mr. Tannehill. That is to say some owners want to believe he possesses enough potential to take a flier on him.
Thus, the offseason addition of Mike Wallace becomes the focal point of fantasy analysis and cynicism over the next five months. Tannehill averaged 216 passing yards per game with 12 touchdowns and 13 interceptions thrown. It's worth noting that Reggie Bush averaged just over 18 receiving yards per game with two touchdowns last season. So, you may want to factor those numbers into Tannehill's 2012 stats.
Although Davone Bess and Brian Hartline were targeted over 230 times, racked up over 1,800 receiving yards with 35 receptions of 20 yards-or-more, the duo only scored two touchdowns in 2012. Two! Tight end Anthony Fasano, now with the Chiefs, led the team with five receiving touchdowns.
Wallace's fantasy value may have regressed last season, but his value is so directly linked to the health of Ben Roethlisberger, Big Ben's absence definitely impacted Wallace's end production. Over the last four seasons, dating back to his rookie year in 2009, Wallace has hauled in 67 passes of 20-or-more yards with 32 touchdowns. Tannehill has the deep threat to go along with his possession receivers and a reliable tight end in Dustin Keller.
Dwayne Bowe and Alex Smith
In Todd Haley's first season as head coach of the Kansas City Chiefs, Matt Cassel passed for over 3,100 yards with 27 touchdowns, 15 of which fell into the hands of Dwayne Bowe. The once exhilarating wide receiver was placed on mute during the 2012 season with just over 800 receiving yards and only three scores.
However, I'm intrigued as to Bowe's fantasy football value in 2013 with the arrival of Andy Reid, a noted lover of the pass game, and game manager/quarterback, Alex Smith, behind center.
Smith finished with a 2-to-1 touchdown to interception ratio his final two seasons with the 49ers. I think if Bowe plays a full 16-game slate in 2013, he could reemerge as one of the top fantasy receivers in the game. Note the word "could."
Daryl Richardson vs. Isaiah Pead position battle
From 1999-2012, only two names led the Rams' offense in rushing: Marshall Faulk and Steven Jackson. With Jackson now in Atlanta, the door swings open for two NFL sophomores in Richardson and Pead.
Not much separates one from the other as duo are both 23 years old, stand 5-foot-10-inches and Pead only outweighs Richardson by two pounds (197-195). Richardson did receive 98 carries compared to Pead's 10, but it's hard to give an edge to either at this point. I believe a RBBC may be coming to St. Louis this fall. Don't be shocked if the Rams select a running back in the 2013 Draft to add to the confusion..
New York Jets options on offense
By the end of training camp, I want to know if any fantasy football owner would dare take a flier on a Jet.
>>>MUST READ: 2013 Fantasy Football Pre-Draft Rankings
Beall's Take
The Return of the Running Back
Once the focal point of fantasy football, the running back has gone the way of Myspace the past few seasons, as the changing landscape of the NFL redefined the roles and utilization of the position. With offensive strategies more centered on pass-happy attacks and heavy-duty rushers replaced with halfback committees, uncovering a rotisserie workhorse was quite the endeavor. Note: the 2006 campaign saw 23 rushers cross the 1,000-yard plateau, with 10 backs racking up 300 or more carries. In 2012, those figures dropped to 16 in the 1,000-yard club and five hitting 300 attempts on their barometer. Moreover, factor in the job's conduciveness to injury, and drafting a running back in the early rounds transformed from a foregone conclusion to a risky proposition.
Yet could 2013 see a rejuvenation of power to the running back? Make no mistake, the league remains an orchestra based on the aerial stylings of maestros like Aaron Rodgers, Tom Brady, Drew Brees and Peyton Manning, and rushers are just as susceptible to ailment as ever. Nevertheless, judging by this year's collection of fantasy backs, the aptitude has never been higher and the depth has never been deeper.
Adrian Peterson, Doug Martin, Arian Foster and Jamaal Charles could all make compelling cases as a No. 1 overall pick. Sound entities like LeSean McCoy, Maurice Jones-Drew and Alfred Morris might be available in the mid-to-late second round. Chris Johnson, who has rushed for over 3,600 yards and hauled in nearly 900 receiving yards the past three years, will find himself outside the top-15 rankings at the position in some experts' standings. Even an effective asset like BenJarvus Green-Ellis, who posted over 1,094 yards and six scores last season, can be obtained at a juncture where owners are normally scrambling for back-ups or part-timers.
A storyline that will be beaten into the ground this season will be young guns like Robert Griffin III, Andrew Luck, Russell Wilson and Colin Kaepernick trying to dethrone venerable arms like Manning, Brady and Brees, understandable given the importance and excitement of the league's field generals. But in the ensuing hoopla, don't let the return of the ground game escape your grasp.
>>>MUST READ: 2013 Fantasy Football Pre-Draft Rankings
FOX Sports Go


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Australia's tax system is too big and too important not to be subject to continual discussion and scrutiny. This is why the recently completed strategic review of the Treasury is a welcome addition to this debate.
There are too often "unknowns" in tax law changes and how they affect business.
The first type of unknown is the timeline for implementing announced tax changes; and the second is the inherent uncertainty and volatility that can be caused by sudden and rapid change after government announcements.
So, as former US defence secretary Donald Rumsfeld might say, these are two of the known unknowns when it comes to tax law changes; and this is before we consider the possibility of the world of tax producing some previously unknown unknowns.
So what does all of this mean?
It is relatively straightforward – when tax changes are announced, as a community we have the not unrealistic assumption that the government will proceed down a path leading to their enactment into law. The exact nature of that path can vary but after initial policy development, it is often one of: announcement, perhaps the release of a discussion paper, consultation, exposure draft legislation, further consultation, bill finalised and introduced into Parliament, and finally it is debated in Parliament until it is passed by both houses and given the royal assent, turning it into law.
Simple, right?
So, does this happen?
In the majority of cases, yes.
In fact, the Treasury is to be congratulated for regularly issuing its "Forward Work Program for Consultation on Tax Measures".
What this does is give details of the matters open for consultation and those about to be consulted upon.
However, what is not at all clear is what the implementation time-line looks like after these consultation periods. This is where the perennial black boxes emerge and add to the unknowns. These include the government's legislative priorities and the availability of legislative drafting resources.
These aspects of the process serve to increase the uncertainty for those trying to deal with the impacts of the proposed changes.
Even worse is when there is apparently no movement at all following an announcement. An example is legislation regarding "earn-outs" that is designed to remove uncertainty as to their tax treatment.
In the minister's press release when announcing the measure in 2010, the change was said to begin from the date of royal assent, but with transitional relief to be provided in some cases back to 2007.
Industry engaged in consultations with Treasury soon after the announcement in 2010, but the ensuing silence suggests that little is now happening.
On the other hand, there is an example that at this early stage looks to be a model of an open, public legislative timeline. The recently announced discussion paper on trust tax reform changes includes both a "project objective" and a "consultation objective". It then goes on to outline "communication principles" that Treasury will adopt in dealing with interested parties.
This is followed by detailed next steps, outlining indicative time frames for consultation and release of further papers ultimately leading to legislation being introduced late this year, for application in July next year.
So everyone knows where they stand in terms of when each step in the process is expected and what the goal date is for introducing the legislation. This minimises the effect of tax law changes on taxpayers and tax practitioners.
For business, tax law changes can bring with them issues including extra compliance costs to sort out the effect of the changes and a greater need for caution in the context of pending transactions.
So a really good, open consultation timeline for legislative change gives taxpayers that extra level of certainty when it comes to dealing with that change.
Now to the "unknown" of sudden and rapid change after government announcements. A clear and detailed timeline may, in some cases, be provided, but taxpayers can only realistically deal with so much change in a short amount of time.
Changes can be accommodated and in some cases financially budgeted for if there is enough time to do so.
Of course there is nothing more rapid than the announcement of a change that applies to a period before the announcement itself – retrospective changes.
There has been an extremely concerning trend in recent months of the government announcing retrospective changes to the tax law. Changes to reverse consolidation tax laws were preceded by amendments to the petroleum resource rent tax backdated to 1990; and an overhaul of transfer pricing laws, with effect from 2004.
Taxpayers enter into transactions on the basis of the law as it is, not the law as it is rewritten after transactions have occurred.
Retrospective changes in tax law are likely to interfere with bargains struck between taxpayers who have made every effort to comply with the prevailing law at the time of their agreement.
During this period of significant market volatility, the last thing that businesses need is another hit to their profit-and-loss accounts from such significant changes to the taxation regime.
The pace of change must be foremost in the minds of the Treasury and the government when devising and implementing tax law changes.
The Australian Financial Review

subset: commoncrawl


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Alaska Fish & Wildlife News
October 2013
Aerial Photo Census Team Bags Five Big Herds
Counting Caribou in Alaska
Counting caribou via aerial photography depends on a heap of good planning and equipment plus a good bit of weather luck.
In the period of time following calving, caribou can form large aggregations while they seek relief from insects. Insect harassment intensifies under warm, calm weather conditions and caribou tend to group in places where cooler temperatures or more wind may exist such as along the coast or at higher elevations. When these large aggregations form, it allows a photocensus crew to take photos while an entire herd may be contained in relatively few densely packed groups. If a caribou herd is sufficiently grouped, a photocensus plane flies over each group and takes pictures ensuring that the entire group is photographed, including shooting so that there is overlap among photographs.
It takes two specially equipped state aircraft and a fleet of contract aircraft and pilots, numerous biologists and an analyst-programmer, lots of preparation and testing, $20,000 in film and developing and hundreds of hours counting caribou dots on photographs. The respective Area Biologists vigilantly monitor their herds from mid-June through mid-July watching for the aggregations that may last a matter of hours to a few days. A change in wind speed or direction or a cumulus rain cloud can scatter the caribou. The two photo-plane crews remain on standby throughout the period, sometimes staging separately in strategic areas - such as the remote Eagle Creek airstrip on the Kukpowruk River on the western arctic slope - to be ready to depart when and if the manager says it is a go.
This past summer was a demanding, but successful photocensus season. Favorable weather conditions occurred early in the season, but a lack of insect harassment (perhaps due to late spring) led to poor aggregations for the herds that in the past had aggregated early. Things started coming together on the 3rd and 4th of July on the North Slope with the Central Arctic herd. The Palmer based photo-plane crew team got the call the night before and launched in the wee hours to rendezvous with the others 600 miles away on the North Slope. Fog, mist, and multiple cloud layers in Broad Pass prevented two attempts to cross the Alaska Range.
Coordinating from Fairbanks, Biologist Jason Caikoski initiated Plan B. He contacted the second photo-plane crew that was staging at Eagle Creek in anticipation of surveying the Western Arctic Herd. The second crew traversed 350 miles across the North Slope to successfully photograph the Central herd that evening. That survey started a 12-day, five-photocensus run including an international shoot of the Porcupine herd using two ADF&G photo planes on the same day.
It is a testament to their preparation and dedication that these crews – at a moment's notice – can be enroute to conduct these surveys anywhere in the state and sometimes western Canada. This year's effort for the Porcupine herd found one crew in Palmer one afternoon and over the Richardson Mountains in Canada the next.
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Hannah gets herself in trouble with her father, when she is forced to tell him her boyfriend from Chicago has moved to New York, and they've been seeing eachother. The boyfriend mysteriously disappears and after his initial hesitation, Jack assigns the team to the case. Further investigation uncovers his horrible relationship with his father, as well as his involvement in an accident resulting in one of his best friends being in a coma. Trying to find some money to give to the boy's parents, he gets in trouble with car thieves and drug dealers.
Hannah is in the typical teenage angst situation, believing her father wants to break her up from her boyfriend, and reacting in an aggressive manner. Jack soon realizes his authoritative behaviour isn't getting him anywhere, and changes attitude. Samantha helps out, earning Hannah's trust and making her feel safer towards her father. While Jack starts out being completely disapproving, he ends up understanding his daughter is in love, and even giving her boyfriend the benefit of a doubt. "I'm kinda hoping you're the guy my daughter thinks you are", he tells him in the interrogation room, after he is found trying to escape the country. It turns out he didn't kill his friend after all, and that he was carried away by the third passenger to hide everything from the police. Hannah meets him again, and everything is okay with them.
On a more personal level however, Brian makes a move for Samantha and she seems very much in doubt as to whether she will choose him or Jack. Jack's obligations with his daughters (he plans to take a trip to Chicago to see his other daughter as well), and providing a safe environment for Finn will obviously play a big part in her decision. Will she choose Jack or will she take the safe bet, and decide to raise Finn in a more quiet environment, with his real father? We'll see in the next episode... Danny and Elena on the other hand are going to land safely, as their marriage is a done deal.


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ESPN Apps
Max: Red Bull needs a competitive car to keep me
Max Verstappen says a long-term future at Red Bull hinges on whether the team can provide him with a competitive car before his contract expires in 2019.
Ferrari team orders: From 'may the best man win' to number one and two
Sebastian Vettel has the undoubted backing of Ferrari in his quest for the 2017 title. But Ferrari's reputation for always having a number one and number two driver is unfounded, writes Maurice Hamilton.
F1 launches eSports world championship
Formula One has launched an eSports series which will culminate at the season-ending Abu Dhabi Grand Prix in November.
Pritchett, DeJoria winners at NHRA Nationals
Leah Pritchett won the Lucas Oil NHRA Nationals on Sunday at Brainerd International Raceway for her fourth Top Fuel victory of the season, and Alexis DeJoria made it a female double with a win in Funny Car.
Power holds off Newgarden to win at Pocono
Will Power gave his championship run a serious boost on Sunday, as he held off Team Penske teammate Josef Newgarden to win the IndyCar race at Pocono Raceway for the second straight year.
Hunter-Reay cleared to race following wreck
Following a violent accident during qualifying that sent him to the hospital, Ryan Hunter-Reay has been cleared to drive in Sunday's IndyCar race at Pocono Raceway.
Formula 1
Grosjean: 'Grown up' Haas has upped its game
Romain Grosjean has praised Haas for its performance during the first half of the 2017 Formula One season having matched its points tally from the entirety of 2016.
Formula 1
Horner: Red Bull can aim to outscore Ferrari
Red Bull team principal Christian Horner believes outscoring Ferrari in the remainder of the 2017 Formula One season is a "realistic and aggressive target" for his team.
Whincup survives Supercars shunting
Jamie Whincup has taken his Red Bull Holden to a podium finish despite impeded vision for a third of Saturday's Supercars race at Sydney Motorsport Park.
McLaughlin, SVG hit by Supercars ruling
Scott McLaughlin and Shane Van Gisbergen have lost championship points in the wake of a Supercars ruling from Sydney Motorsport Park.
Pritchett's Top Fuel record holds up in qualifying
Leah Pritchett's Friday night run of 3.640 seconds at 330.63 mph, the quickest pass in NHRA history, held up in qualifying Saturday at Brainerd International Speedway.
Hunter-Reay released from hospital after crash
Ryan Hunter-Reay limped to an ambulance and was taken to the hospital after his car slammed the protective soft wall and then hit the inside wall during IndyCar qualifying at Pocono. Hunter-Reay has been treated and released.
Formula 1
Force India nearing renewed Perez deal
Sergio Perez is close to agreeing a new deal to remain with Force India for 2018, according to the team's chief operating officer Otmar Szafnauer.
Formula 1
Sainz learning from F1 'mentor' Alonso
Carlos Sainz says his wheel-to-wheel on-track battles with Fernando Alonso have enabled him to learn and improve his race-craft in Formula One.
Formula 1
McLaren: Misunderstood Alonso not hard to manage
McLaren boss Eric Boullier has refuted claims that Fernando Alonso is a difficult driver to manage, insisting the two-time Formula One world champion is merely focussed on being successful.
Pritchett has quickest run in NHRA history
Leah Pritchett (Top Fuel) and Robert Hight (Funny Car) had the quickest runs in NHRA history Friday night at Brainerd in Lucas Oil NHRA Nationals qualifying.
Top ten dream F1 venues in the United States
With owners Liberty Media looking at the possibility of a second U.S. Grand Prix to the Formula One calendar, ESPN looks at some of the best -- and most optimistic -- options available.
Formula 1
Hard to predict F1's 'weird' midfield fight, says Haas
Haas boss Guenther Steiner says everyone in Formula One's tight midfield should be considered a threat in the second half of the season due to the unpredictable nature of the fight so far.
Formula 1
Why Marko thinks Vettel will win the 2017 title
Helmut Marko thinks former prodigy Sebastian Vettel's mental strength will be the key factor in the remainder of the 2017 Formula One championship.
Formula 1
Alonso to consider 'all possibilities' before leaving F1
Fernando Alonso says he will explore all opportunities to win again within Formula One before looking outside the series for a drive in 2018.
Formula 1
F1 assessing options for second US GP
New York, Miami, Los Angeles and Las Vegas all have the potential to host a second U.S. Grand Prix, according to Formula One chairman Chase Carey.
Formula 1
Horner: F1 will end up with V6 twin turbos
If Christian Horner were in charge of Formula One he'd bring back high-revving V12 engines, but the Red Bull team boss doubts the sport's decision makers will agree with his vision for F1's future.
Bourdais gets medical clearance, 'let's go racing'
IndyCar driver Sebastien Bourdais, who has not raced since a scary crash at Indy in May, said he's gotten medical clearance to return. "All clear," he said. "Let's go racing!"
Formula 1
Vettel: Good to see other teams copying Ferrari
Sebastian Vettel is a strong believer in imitation being the sincerest form of flattery after seeing a number of Ferrari design elements pop up on rival cars this year.
Editor's Picks
ESPNF1.com: Steve McQueen and the making of 'Le Mans'
Maurice Hamilton looks at the remarkable tale behind Steve McQueen's 'Le Mans' film, as revealed in a recently-released documentary.
espnW: Over the wall and into history
After training for a year, Brehanna Daniels got her shot changing tires in the Trucks and Xfinity races, becoming the first African-American woman to work the pits in a NASCAR Series event.


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Assessment |
Biopsychology |
Comparative |
Cognitive |
Developmental |
Language |
Individual differences |
Personality |
Philosophy |
Social |
Methods | Statistics | Clinical | Educational | Industrial | Professional items | World psychology |
Social psychology: Altruism · Attribution · Attitudes · Conformity · Discrimination · Groups · Interpersonal relations · Obedience · Prejudice · Norms · Perception · Index · Outline
Conscientization refers to a type of learning which is focused on perceiving and exposing social and political contradictions. Conscientization also includes taking action against oppressive elements in one's life as part of that learning.
The English term "conscientization" is a translation of the Portuguese term conscientização, which is also translated as "conscienceness raising" and "critical consciousness". The term was coined by Brazilian educator, activist, and theorist Paulo Freire in his 1970 work Pedagogy of the Oppressed. Freire was teaching the poor and illiterate members of Brazilian society to read at a time when literacy was a requirement for suffrage and dictators ruled many South American countries.
Conscientization proceeds through the identification of "generative themes", which Freire identifies as "iconic representations that have a powerful emotional impact in the daily lives of learners." In this way, individual consciousness helps end the "culture of silence" in which the socially dispossessed internalize the negative images of themselves created and propagated by the oppressor in situations of extreme poverty. Liberating learners from this mimicry of the powerful, and the fratricidal violence that results therefrom is a major goal of conscientization. Conscientization is a fundamental aspect of Freire's concept of popular education.
Arlene Goldbard, an author on the subject of community cultural development finds the concept of conscientization to be a foundation of community cultural development. From the glossary of Goldbard's 2006 book New Creative Community.: "Conscientization is an ongoing process by which a learner moves toward critical consciousness. This process is the heart of liberatory education. It differs from "consciousness raising" in that the latter may involve transmission of preselected knowledge. Conscientization means breaking through prevailing mythologies to reach new levels of awareness—in particular, awareness of oppression, being an "object" of others' will rather than a self-determining "subject." The process of conscientization involves identifying contradictions in experience through dialogue and becoming part of the process of changing the world."[1]
- ↑ Creative Communication New Village Press.
2. Aspiazu, Gary G. (1998). "Improving the academic performance of Hispanic youth: A community education model." Bilingual Research Journal, 22:2, 3, & 4, Spring, Summer, & Fall, 1998. Available at: http://brj.asu.edu/v22234/abstract.html
3. Freire, Paulo. Pedagogy of the Oppressed. New York: Continuum, 2007.
See alsoEdit
- Critical consciousness
- Critical pedagogy
- Popular education
- Teaching for social justice
- Adult literacy
- Adult education
- Praxis
- Praxis intervention
|This page uses Creative Commons Licensed content from Wikipedia (view authors).


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'The Giver' Movie Trailer [VIDEO]
Most high school students are required to read Lois Lowry's 'The Giver.' The difference between this novel and many of the others on required reading lists is that students are usually interested in this one, and finally it's becoming a movie.
The novel focuses on a 12-year-old Jonas who lives in a utopian society where no one has knowledge or makes choices and where everyone lives according to "Sameness." When Jonas is assigned his job, he is given the position of "Receiver of Memory" which means that he is responsible for all of the knowledge of life prior to the transition to "Sameness."
Upon learning all of the things about how society used to be, Jonas is faced with the decision to either stay and live miserably knowing everything about society that his world is missing out on or to go out and leave everything he's ever known in search of more knowledge and life.
The movie is set to debut August 15, 2014 – including a star-studded cast of Meryl Streep, Jeff Bridges, Alexander Skarsgard, Taylor Swift and more.
Check out the trailer here:
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WASHINGTON—Legal experts say Roger Clemens' battle against perjury charges probably will be the subject of a new trial.
To avoid that, the experts say the baseball star would have to convince the judge that prosecutors intentionally provoked him into asking for a mistrial by showing jurors inadmissible evidence.
But attorneys and scholars who reviewed transcripts of the case provided by The Associated Press say there's no indication prosecutors wanted to start over.
U.S. District Judge Reggie Walton is considering whether another trial would subject Clemens to double jeopardy. A mistrial was declared July 14.
Walton has ordered both sides to submit their arguments in writing and has set a Sept. 2 hearing on the issue. Experts say a second trial seems likely under rules established by the Supreme Court.


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Contractor Groups Plan Golf Outing, Software Training
April 24, 2006
The South Florida Airconditioning Contractors Association (SFACA) is sponsoring a golf tournament on Tuesday, May 2, 2006 at the Bonaventure Country Club in Weston, Fla. Registrations are needed by Thursday, April 27, as there are no walk-ons.
Also, the Mechanical Contractors Association of South Florida (MCASF) has contracted with Wrightsoft to bring a two-day software training session to the association's Miami training center on May 2-3, 2006. Registration should be made as soon as possible as space availability will be finalized on Wednesday, April 26.
For more information on the SFACA golf tournament, visit www.sfaca.org or call 561-750-8558. For more information on the MCASF training session, visit www.mcasf.org or call 305-651-7570.
Publication date: 04/24/2006
Did you enjoy this article? Click here to subscribe to The NEWS Magazine


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Have a Tax Question? Ask a Tax Expert
Hello crapser,
Since your son is over the age of 24, the only way you can claim him as a dependent is if all of the following rules can be met:
1. He does not have income of his own of more than $3,650 for the year.
2. You provide more than half of his support
3. He is not married filing a joint return with his spouse
4. He is not filing his own return and claiming himself as a dependent
If all of the above rules can be met, then you can claim your son as a dependent on your return.
If this was helpful please press the Accept button. Positive feedback is also appreciated.
Thank you crapser


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An ABC employee who tried to earn virtual currency by installing Bitcoin "mining" software on one of the broadcaster's most popular sites has kept his job.
The employee, who had "high-level IT access privileges", was disciplined after placing the unauthorised code on the ABC Grandstand Sports website.
Visitors looking for sports scores or watching videos of a popular match "may have been exposed to the Bitcoin software", the ABC said. The software uses idle computer processing power from computers on a peer-to-peer network, to generate Bitcoins, which can be exchanged for cash.
"The placement of the code was detected by internal ABC checks within 30 minutes and was removed immediately," the ABC wrote recently in response to questions put on notice by Senator Eric Abetz during Senate Estimates in February.
Bitcoins can be spent at retailers that display the Bitcoin "101symbol" at the cash register or virtual checkout. For example, the online games store, JJGames.com, has a page explaining how visitors can use their Bitcoins to buy video games.
While there was no impact on the ABC's internal and external online distribution infrastructure, it was not possible for the ABC to "ascertain whether any audience members were affected by the Bitcoin software".
Security firms warned last year of potential networks of zombie computers - botnets - running Bitcoin mining software and it spreading like malware to other people's computers.
The ABC had not received any such complaints from visitors.
In addition to being disciplined, the employee's access to all productions systems has been restricted.
The employee, who has not been named, is now being closely supervised, the ABC said in its response to the Senate Estimates questions.
Senator Abetz will pursue the matter when the ABC appears before Senate Estimates on Wednesday.
It is understood he will ask for details on how many visitors accessed the infected site, which pages were affected by the code, and what code was used.
A spokesman for Senator Abetz said it appeared that there had been a serious breach of security on a publicly funded website.
"It's not just ABC computers that were potentially affected - it was computers owned and operated by members of the general public that may have been, for lack of a better word, infected by this code," the spokesman said.
The spokesman said that, despite initial concerns that the processing power of ABC servers was being used to mine for Bitcoins, "it now appears they were trying to use the computers of visitors to the ABC Grandstand website to mine these".
"Presumably that would have been through a Javascript that users would have downloaded inadvertently to their machines and, while they were on that website, the apparent intent of whoever put this code there would have been that people would be generating Bitcoins for their private use."
Senator Abetz's spokesman said he was also concerned at the lack of public disclosure over the breach, and that the opposition would be seeking more information from the ABC through the Senate Estimates process.


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THE shadow minister for public health visited Wirral for a HIV test today as part of a campaign to highlight the importance of being tested.
Luciana Berger, Labour MP for Liverpool Wavertree, was at sexual health charity Terrence Higgins Trust's centre in Birkenhead this morning.
While there, she met the charity's chief executive, Dr Rosemary Gillespie, as well as staff and volunteers, before taking the finger prick test.
Current figures from the trust show that across Merseyside and Cheshire, just under 2,000 people have the disease. Around a quarter of those are unaware of it.
Ms Berger, who admitted she has taken a HIV test, which tested negative in the past, told the Globe: "It's really important that people know how easy and accessible it is to come and have an HIV test. It literally takes an hour in total to have the test done.
"There's no stigma attached. The staff here are so friendly and will provide the support that people need.
"You don't have to wait weeks for get your result, as you may have had to in the past. It's done within about an hour of taking the test. If it's a positive result, there is support available and, if you need it, will get the treatment.
"In and around this region, last year, 70 per cent of people were diagnosed late. The later you're diagnosed, the later you will get the treatment you need. That's why it's so important people are diagnosed quickly."
The centre, in Bridge Street, also provides information, advice and support on all aspects of sexual health and HIV.
Dr Rosemary Gillespie, chief executive of Terrence Higgins Trust, added: "We're delighted that Luciana agreed to come and take a test.
"it shows real leadership and she was able to demonstrates how quick and easy it is to take the test.
"In Cheshire and Merseyside, there are just under 2,000 people living with HIV; almost a quarter of those don't know that they're infected.
"It's really important that those people are aware of their status, so that we can get them in for treatment and support, because that's the very best way that people can stay healthy and stay fit."
For more information on the services that Terrence Higgins Trust offers from its Birkenhead centre, call 666 9890 or email firstname.lastname@example.com.
The centre is open on Wednesday from 5-8pm, but appointments can be made for outside those hours.


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- Read the latest issue of the ProbesOnline E-Newsletter.
In This Issue
Fluorescence-Based Tools for Visualizing Autophagy — Premo™ Autophagy Sensors
|New Primary Anti-Mouse Qdot® Antibody Conjugate for Flow Cytometry — Mouse CD19 Qdot® 655 Conjugate
New Recombinant Proteins for Stem Cell Research — Recombinant Mouse LIF
See all of this month's New Products for Cell & Tissue Analysis
- Buzzworthy — Loss of Autophagy in Erythroid Cells Leads to Defective Removal of Mitochondria and Severe Anemia In Vivo
- The View — Imaging Autophagy
- Proven Performers — FIX & PERM® Cell Permeabilization Reagents
- On the Web — Alexa Fluor® Dyes, Autophagy
Check out the latest issue of BioProbes
what they are
LC3B plays a critical role in autophagy, and its localization can be used as a general marker for this vital process. Premo™ Autophagy Sensors enable the temporal and spatial visualization of LC3B.
what they offer
- Real-time analysis—observe autophagy in live cells
Ease of use—one-step protocol
Flexibility—compatible with live- or fixed-cell analyses
how they work
Premo™ Autophagy Sensors combine the selectivity of an LC3B–fluorescent protein (LC3B-FP) chimera with the transduction efficiency of BacMam technology, facilitating unambiguous visualization of this protein with an easy one-step protocol. Simply add the BacMam LC3B-FP reagent to cells, incubate overnight for protein expression, then image and analyze. The kits include a LC3B-FP mutant, which serves to indicate whether punctate staining may be due to FP aggregation, and chloroquine, which causes accumulation of autophagosomes.
- Learn More about Autophagy
Detecting autophagy with the Premo™ Autophagy Sensor in live cells.
Read More +
U2OS cells were cotransduced with the Premo™ Autopahagy Sensor LC3-RFP and Cellular Lights™ MAP4-GFP. The following day, cells were treated with 50 μM chloroquine. Twenty-four hours later, cells were incubated with Hoechst 33342 before imaging.
|Cat. No.
|Premo™ Autophagy Sensor LC3B-GFP
|1 kit
|Premo™ Autophagy Sensor LC3B-RFP
|1 kit
Rat anti–mouse CD19 antibody conjugated to Qdot® 655 nanocrystals, validated for flow cytometry.
what it offers
- More information with less sample, in less time
- Minimal single-laser compensation
- Compatibility with existing organic dyes
Qdot® conjugated primary antibodies are designed for use on flow cytometers with a violet (405 nm) laser, such as the Attune™ Acoustic Focusing Cytometer. They increase the number of available dyes and provide greater flexibility when designing multicolor panels. Qdot® nanocrystals can be used in combination with existing organic dyes to increase the number of detectable parameters. They also provide the advantages of increased brightness and photostability.
- See all of our Qdot® Antibody Conjugates
|Rat anti–Mouse CD19 Qdot® 655 Conjugate.
Read More +
Log fluorescence intensity profiles of mouse splenocytes gated on lymphocytes and analyzed on a BD LSR™ II flow cytometer using 405 nm excitation and the specified emission filters. The black line represents cells stained with Mouse CD19 Qdot® 655 Conjugate, and the gray line represents unstained cells.
|Cat. No.
|Rat Anti–Mouse CD19 Qdot® 655 Conjugate
|100 µL/100 tests (min)
what it is
The leukemia inhibitory factor (LIF) protein is most notable for its ability to prevent the differentiation of mouse embryonic stem cells (mESCs), enabling them to maintain pluripotency. LIF is also involved in a wide variety of processes, including inflammation, bone metabolism, development, and hematopoiesis.
what it offers
- High biological activity—more results with less protein
- High purity—no interference from other proteins or contaminants
- Proven compatibility—GIBCO® growth factors are bioassayed with GIBCO® media
how it works
Recombinant mouse LIF is available in a lyophilized format in 10 µg and 100 µg pack sizes. The biological activity of mouse LIF was measured via dose-dependent reduction of MTS within stimulated mouse M1 cells. In addition, inhibition of differentiation of mouse embryonic stem cells was observed using a concentration of 10 ng/mL of recombinant mouse LIF. The optimal concentration for each specific application should be determined by a dose response assay.
Dose-dependent reduction of MTS in LIF-treated mouse M1 cells.
Read More +
Mouse M1 cells were stimulated with GIBCO® Recombinant Mouse LIF and a competitor's recombinant mouse LIF. Biological activity was measured via dose-dependent reduction of MTS. The ED 50 of the GIBCO® recombinant mouse LIF was determined to be 2.99 pg/mL, while the ED 50 of the competitor's product was 10.86 pg/mL, which demonstrates higher bioactivity of GIBCO® recombinant mouse LIF.
The Click-iT® EdU assay is a superior alternative to traditional methods for detecting and quantitating newly synthesized DNA. Historically, active DNA synthesis has been measured by following the incorporation of the radioactive nucleoside [3H]-thymidine into DNA. This method was later replaced by immunodetection of the nucleoside analog bromodeoxyuridine (BrdU). The Click-iT® EdU assay also uses a nucleoside analog, but in this case it is detected not with an antibody but a click reaction—a copper-catalyzed covalent reaction between an azide and an alkyne. In the Click-iT® EdU assay, the EdU nucleoside contains the alkyne, and the fluorescent detection reagent contains the azide.
The small size of the detection reagent compared to an anti-BrdU antibody eliminates the need to denature the DNA for access to the incorporated nucleoside. Eliminating the harsh denaturation step—a step that often destroys antigen recognition sites and sample morphology—provides a method that is easier and more reliable to perform, even on delicate stem cells.
- Learn More about Click-iT® EdU Cell Proliferation Assays
- Learn More about other products for Stem Cell Research
|Labeling stem cell proliferation with Click-iT® EdU. Human mesenchymal stem cells were incubated with the nucleoside analog 5-ethynyl-2´-deoxyuridine (EdU). Following fixation and permeabilization, EdU was detected with a click reaction using the Click-iT® EdU Alexa Fluor® 594 Imaging Kit. Nuclei were counterstained with blue-fluorescent Hoechst 33342 before imaging with a DeltaVision® Core microscope using fluorescence and differential interference contrast (DIC).
Loss of autophagy in erythroid cells leads to defective removal of mitochondria and severe anemia in vivo.
Mortensen M et al. (2010) Proc Natl Acad Sci U S A 107:832–837.
Autophagy is critical for red blood cell differentiation
Removal of organelles is necessary for the differentiation of mammalian red blood cells from precursor cells. Mitochondria are known to be removed through the autophagy pathway. In this process, the cytoplasmic components targeted for removal are engulfed in a double-membraned vesicle (autophagosome), which then fuses with lysosomes for degradation of its contents. Atg7 encodes the E1-like enzyme required in both of the conjugation systems required for autophagosome formation.
Atg7 is a key player in erythrocyte development
To test the role of Atg7 in erythroid cells, Mortensen et al. produced a hematological Atg7 knockout mouse (organism-wide knockouts of autophagy genes are lethal). They observed that CFSE-stained erythrocytes, harvested from Atg7 knockout mice and injected into wild type mice, disappeared from the peripheral blood supply much more quickly than wild type erythrocytes injected at the same time. Also, these same peripheral blood erythrocytes were stained to a much higher degree with annexin V, indicating an increase in cell death. Taken together, these data suggest that an erythroid-intrinsic defect is responsible for the increased cell death observed.
Atg7-deficient erythrocytes accumulate damaged mitochondria
Over the course of development, erythrocytes from Atg7 knockout mice accumulated mitochondrion-derived superoxide (detected with MitoSOX™ Red Superoxide Indicator), and the mitochondria within the cells showed increasing depolarization (as judged by tetramethylrhodamine, methyl ester, perchlorate (TMRM)), which indicated that erythroid cells from Atg7 knockout mice were accumulating damaged mitochondria. The authors further showed that damaged mitochondria accumulated in the erythroid lineage but not in the myeloid lineage of the same animals. These data suggest that the removal of defective mitochondria is an essential step during erythropoiesis, and that Atg7 is a key molecular player in this process.
View the bibliography reference
- Learn More about Products for Mitochondria
|Cat. No.
|CellTrace™ CFSE Cell Proliferation Kit
|1 kit
|MitoSOX™ Red Mitochondrial Superoxide Indicator
|10 x 50 µg
|Tetramethylrhodamine, methyl ester, perchlorate (TMRM)
|25 mg
Imaging autophagy with the Premo™ Autophagy Sensor, Organelle Lights™ reagents, and MitoTracker® dye. A549 cells were transduced with the Premo™ Autophagy Sensor LC3B-GFP (green) and Organelle Lights™ Golgi-RFP (red), and then treated with 50 µM chloroquine. Twenty-four hours later, cells were loaded with 50 nM MitoTracker™ Deep Red Dye (cyan) for 5 min at 37°C and washed in dye-free DPBS. Nuclei were labeled with Hoechst 33342 (blue). Cells were imaged on a DeltaVision® Core microscope with DAPI/FITC/TRITC/Cy5 filter sets.
FIX & PERM® Cell Permeabilization Reagents: Great Data Require Great Starting Material
To accurately analyze intracellular markers by flow cytometry, cells must be fixed and permeabilized to enable detection antibodies to reach intracellular proteins. Preparation of cells for these protocols requires a delicate balance of adequately permeabilizing the cells to allow antibody access while maintaining the overall morphology of the cells. The extensively referenced FIX & PERM® Cell Permeabilization Reagents are the recognized gold standard for fixing and permeabilizing cells prior to analysis by flow cytometry.
Benefits of the reagents include:
- Superior access to intracellular structures
- No alteration of morphological scatter
- Manufactured under GMP standards for reliable performance
- Fast, easy protocols with consistent results
- Well-referenced standard for intracellular staining
The FIX & PERM® Cell Permeabilization Reagents allow mild fixation and permeabilization of cells, leaving their morphological scatter characteristics intact. This flexibility enables accurate identification of previously undetectable intracellular markers such as cytoplasmic or nuclear enzymes, phosphoproteins, oncoproteins, cytokines, and immunoglobulins.
Numerous references confirm many technical advantages of these reagents:
|Allow simultaneous addition of permeabilization medium and fluorochrome-labeled antibodies
|Eliminate the need for additional lysing solutions
|Compatible with analysis of most cellular antigens
|Reduce background staining
|Perfect for use with Invitrogen™ LIVE/DEAD® Fixable Dead Cell Stains for dead-cell discrimination
FIX & PERM® reagents are suitable for flow cytometry analysis of normal and malignant leukocyte populations derived from various human biological samples (e.g., blood, bone marrow, etc.).
- Learn More about FIX & PERM® Cell Permeabilization Reagents
|Cat. No.
|FIX & PERM® Fixation and Permeabilization Reagents
|4 x 5 mL, 200 tests
|Alexa Fluor® Dyes
It's now easier than ever to find information about Alexa Fluor® dyes—the leading and most trusted fluorescent dyes available today. With our newly redesigned web pages, you can quickly navigate to product information, performance data, and comparisons to other dyes.
|New Autophagy Web Resource
Autophagy—literally, "self-eating"—is an essential cellular process that involves degradation of cellular components or entire cells. In normal cells, it helps regulate such fundamental processes as cell growth and homeostasis, but autophagy has also been implicated in pathological processes. Our new web resource makes it easy to find tools for autophagy research—including the first commercially available reagent for autophagy research in live cells, as well as a validated antibody kit.
Molecular Probes® The Handbook
©2010 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners. Feel free to distribute ProbesOnline to friends and colleagues, but please keep this copyright statement intact.


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Non-profit CEO salaries are up
Despite all the talk that charities are in trouble because of the weakening economy and layoffs at non-profits, it does not yet appear that the people running those organizations are hurting. According to a study released Aug. 1 by Charitynavigator.org, the CEOs of non-profits actually saw an increase in salaries last year. Overall, the average… More ▸


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Digital Communications Manager
- Recruiter
- Ellwood Atfield
- Location
- North London / Hertfordshire
- Salary
- £40,000 - £50,000 plus bonus and benefits
- Posted
- 19 Nov 2013
- Closes
- 19 Dec 2013
- Ref
- Function
- Manager (In-House)
- Discipline
- Digital & Online
- Hours
- Full Time
- Contract Type
- Permanent
The Company:
Digital Communications is one of the fasted growing teams in the corporate HQ. It is seen as a core part of their international communications strategy. They are recruiting for a Digital Communications Manager to join their team.
The Role:
As the Digital Communications Manager you will be responsible for:
- Working with external agencies to deliver high quality websites and other digital projects as well as managing the projects from start to finish and ensure they are delivered on time and in budget
- Identifying the gaps in our use of digital and providing advice and guidance on how best to take advantage of these opportunities
- Working closely with the Corporate Affairs teams across the Group to improve how we influence key external stakeholders through digital and social channels
- Support the running of various corporate-level social media accounts (Twitter, YouTube and LinkedIn)
- Help implement the social and digital content platform across with the internal communications department.
To qualify as the Digital Communications Manager you must have experience of the following:
- Working effectively with colleagues / clients across Corporate Affairs and partner functions / multiple stakeholder groups to shape a joined-up approach to digital communications, engagement and brand.
- Proven track record of creating, delivering, tracking and evaluating effective digital communications plans.
- Highly motivated and creative individual able to use your initiative, to meet tight deadlines and deliver projects in budget.
- Experience at working in large, complex and fast-moving matrix organisations or having a client list that fits this profile.
- Passion about digital and corporate communications
To Apply:
To apply for the position of Digital Communications Manager please send your CV to firstname.lastname@example.com please quote the reference TE/GSK in your application.


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Councilman Paul Cunningham wants to ensure voters are well-versed in this year's ballot measures before heading to the polls this November.
So he's inviting residents to join him and other city officials in a town hall forum on the issues Wednesday, September 25, at the Eastside City Hall located at 7575 E. Speedway.
Topics include Proposition 401, which seeks to raise the city's base spending limit by $50 million, and Proposition 402, which is the city's 10-year general plan.
The meeting will run from 5:30 p.m. to 7:00 p.m.
An RSVP is requested. If interested, contact the Ward 2 office at 791-4687 or firstname.lastname@example.com.


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Owner of Owl Wise Phil Eckett was the winner of the Simply Business 200,000 customers competition, with his irresistible story of a bird-focused business built from passion and commitment. We went to visit Phil in his Essex home to present him with his iPad prize and, accompanied by some of the beautiful owls in his care, Phil told us a bit more about what he does.
Midweek on a quiet suburban street in Essex, and I was looking into the huge, amber eyes of an Eagle Owl. She was called Isis, and she was unruffled by my presence and was a natural in front of the camera. However, she did think that the fluffy microphone looked particularly delicious. An intrigued crane of Isis' neck and a few curious swoops later, cameraman Duncan Wright decided that we would remove the piece of expensive equipment that Isis saw only as a prey-sized rodent….
Duncan and I had come to this unusual street on Canvey Island to meet Phil Eckett, and to hear a bit more about his business, [Owl Wise][2]{: target="_blank"}. As one of the largest providers of business insurance in the country, Simply Business gets a fascinating insight into UK businesses every day. When we celebrated reaching 200,000 customers in the summer and invited businesses to tell us their stories, the decision on our favourite was unanimous.
A self-proclaimed 'owl-oholic', Phil told us that his love of feathered things had begun as a child. A ten year old Phil came across a couple of orphaned barn owls, and took them in and learnt how to care for them. As his number of owls grew, so did his reputation. Soon, people from all over Essex and beyond were bringing him birds of prey that they found injured or in need of care. Looking after the birds and working on releasing them back into the right habitat was something that Phil initially juggled alongside a fulltime job and raising a family.
Watching Isis and some of the other beautiful birds swoop gracefully back and forth across the driveway and return to Phil's gloved hand, it wasn't difficult to see why these creatures had captivated him. Genesis of a business from a personal passion was a theme that ran through many of the business stories that were submitted to us. Phil eventually saw an opportunity to create a business based on his love and knowledge of the birds; Owl Wise provides demonstrations to schools, youth groups, parties and other events, with Phil's passion for conservation and energetic, dedicated explanation of bird conservation a key element. In turn, the fees for these events helps fund the expensive breeding and conservation work.
Back at the Eckett family home, Duncan and I watched Pod, the Burrowing Owl explore the living room. Cute, tiny and characterful, Pod was only a couple of months old, and was keen to see whether he could go up the staircase. Phil told us how burrowing owls dig their subterranean homes, and also about the rattlesnake-like noise that they make to frighten off predators.
I was completely won over by Owl Wise and by the dignified, intelligent charm of the beautiful birds that Phil works with. Sitting in Phil's living room, surrounded by owl memorabilia and listening to the human-like chatter of the parrot and learning about birds of prey from Phil, I felt privileged to have an insight into this business. At Simply Business, we're very proud to form even a small part of Owl Wise's story, and to get the chance to share it with you.
Ready to set up your cover?
As the UK's biggest business insurance provider, we specialise in public liability insurance and protect more trades than anybody else. Why not take a look now and build a quick, tailored quote?


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Apply Now!
Follow these steps to apply to Wartburg College:
- Complete and submit the appropriate online application.
- Send, fax or email high school and/or college transcripts, ACT or SAT scores.(International students may submit a TOEFL or IELTS instead of the ACT or SAT score.)
- Transfer students must also submit a Transfer Reference Form.
- International students must submit the Declaration of Finances Form.
- Home schooled applicants should review these special instrutions.


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London (Jun. 20)
(Jewish Telegraphic Agency)
Another appeal to the League of Nations against the Hungarian government for its continued practice of the numerus clausus, not withstanding promises of its speedy abolition, may be directed on behalf of Anglo-Jewry, it was learned yesterday at the monthly meeting of the Board of Jewish Deputies.
Lucien Wolf, secretary of the Joint Foreign Committee, in reporting on the situation in Hungary, was instructed by the Board to inquire into the expediency of another appeal to the League. This action was taken in view of the recent statement of Count Klebelshurg. Hungarian Minister of Education, that there is no prospect for an early modification of the numerus clausus and that it is even possible that the scope of the numerus clausus law may be extended.


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GENEVA – As Europe's refugee crisis continues to evolve, offers of assistance have come from some unexpected places. Interventions by governments, civil-society groups, and aid organizations have been complemented by a broad-based response from the business community. This mobilization highlights not only the role that the private sector can play in managing migration, but also the importance of extending this engagement beyond the response to the immediate crisis.
Contributions have been made by companies large and small. Shop owners have provided refugees with free food and clothing, and local transport firms have helped people move across borders. On the corporate level, FedEx, JPMorgan Chase, and Google have all made direct contributions of more than $1 million to humanitarian organizations. American Express and Daimler are matching their employees' donations, Western Union is offering ten cents per transaction made by consumers in the European Union, and Norwegian Air has raised money through inflight collections.
Meanwhile, the Bayern Munich Football Club has opened a training camp for refugees, and Siemens has launched a traineeship program in Germany for asylum-seekers.
On the web, Facebook is connecting asylum-seekers with members of their diaspora, as well as citizens who want to help, and a program called "Refugees Welcome" is helping refugees find vacant rooms in Germany, Spain, Portugal, Austria, Poland, Greece, Sweden, and the Netherlands. In France, an Amazon wish list has been established to allow people to purchase goods for refugees (portable fuel packs and warm boots are the items most in demand).


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Glutamine (Gln) is the most potent of the amino acids (AAs) that regulate liver anabolism, and its effect is similar to that of insulin in peripheral tissues. However, the influence of AAs on regulation of metabolic enzyme-encoding genes is not known at the molecular level in liver. We now report that Gln and some essential AAs activate the human GAPDH gene that codes for GAPDH, a central enzyme of glycolysis and a target for insulin regulation. In HepG2 cells, Gln upregulated the GAPDH mRNA level, and this effect was additive to that of insulin. Transient transfection of GAPDH promoter/cat constructs demonstrated that a gene-specific and insulin-independent transcriptional step is involved in the Gln responsiveness of GAPDH. Transfected HepG2 cells challenged with various AAs, Gln metabolites or inhibitors of Gln metabolism showed that the Gln-induced effect is similar to that of some essential AAs and that Gln metabolism is a necessary step for GAPDH activation. Deletion mutants and site-directed mutagenesis of the GAPDH promoter indicated that the Gln responsiveness is mediated by a sequence that is distinct from insulin-responsive elements and from positively acting elements previously described in this promoter. This motif located at -126/-118 clearly differs from AA-responsive elements recently identified in other genes. Electromobility shift assay and supershifts showed that the transcription factors bound to the Gln-responsive element in the GAPDH promoter are C/EBPα and -δ. This finding is consistent with the role of C/EBP family members in controlling the hepatic expression of genes involved in nutrient metabolism.
- amino acid-responsive element
- glutamine
- insulin
- transcription factor
mammals constantly adjust their metabolism in response to various physiopathological situations such as changes in nutritional status or other stressful conditions. The associated gene regulations are controlled by complex interactions of hormonal, neuronal, and nutritional factors. Unlike nutrients such as carbohydrates, lipids, vitamins, and minerals, whose roles in controlling gene expression have been extensively studied in mammals (23), the effect of amino acids (AA) has received limited attention (22, 32, 34, 37). An AA-regulated transcription has been reported for several genes, namely collagen Iα (10), β-actin (29), asparagine synthetase (AS) (25), and the C/EBP-homologous protein (CHOP) (18). However, only AS and CHOP transcription mechanisms have been elucidated at the promoter level, and quite a limited number of AA-responsive elements (AARE) has been described (9, 17, 27, 46, 47). Contrasting with the limited knowledge of the molecular mechanisms, whereby AAs control gene expression, the signaling pathways mediating the anabolic effects of some AAs on hepatic carbohydrate, lipid, and protein metabolisms (7, 8, 11) are being identified (22, 33, 50). These anabolic effects are similar to that of insulin in peripheral tissues, and experimental evidences demonstrate that AAs can interact with the insulin-signaling pathway. A recent study (33) indicates that the anabolic effects of Gln, Leu, and insulin are mediated by different signaling pathways and the latter two likely end up with shared targets.
The liver plays a central role in adaptation of metabolic pathways associated with the changes in AA metabolism and AA plasma content that are induced by nutritional factors or stress. Among relevant AAs, Gln plays a key role in stressful conditions where its liver uptake is markedly enhanced (49). On the other hand, glycolysis is one of the metabolic pathways most extensively involved in these situations, and the glycolytic enzyme GAPDH has a central role at this stage. Up to now, regulation of GAPDH gene expression has been poorly documented. Surgical stress and hepatocellular carninoma have been shown to induce liver GAPDH mRNA expression (20, 26), whereas alcohol consumption decreased it (4). Regulation of GAPDH gene expression by nutritional and hormonal factors has also been reported. Notably, the hepatic expression of GAPDH is upregulated in fasted-refed rats (20, 38), but only stimulation by insulin has been documented (2). The insulin control of GAPDH is mediated by two independent insulin-responsive elements (IRE)-A and -B (38) that are located upstream of other regulatory elements involved in the basal activity of this gene (1). Furthermore, the GAPDH mRNA level is known to be controlled by a set of AAs (45), but a possible control of the GAPDH promoter by given AAs has not yet been documented. We have now investigated how insulin and AAs, mainly glutamine (Gln), regulate the hepatic expression of GAPDH. We demonstrate that insulin and Gln modulate the GAPDH promoter in an independent and gene-specific fashion and that Gln metabolism is required in the Gln-driven regulation. In the GAPDH promoter, an AARE that binds the C/EBPα and -δ transcription factors (TF) is responsible for this regulation by Gln.
Cultures of HepG2 hepatoma cells. The human HepG2 hepatoma cell line was plated and maintained at 37°C under a 5% CO2 and water-saturated atmosphere in an RPMI 1640 medium (Sigma) containing 2 mM l-Gln, 50 U/ml penicillin, 50 μg/ml streptomycin, and 10% (vol/vol) heat-decomplemented FCS. All induction experiments were carried out with subconfluent cells in an FCS-free medium. When indicated, a Gln-free RPMI 1640 medium was supplemented with Gln (Life Technologies) and/or insulin (Novo Nordisk), 6-diazo-5-oxo-l-norleucine (DON), glucosamine, or NH4Cl (Sigma). In induction experiments with various AAs, an MEM medium (Life Technologies) lacking a given AA was supplemented or not with this AA at the concentration that is usually found in this medium, as indicated in results.
Determination of intracellular AA content. Subconfluent HepG2 cell cultures were washed with PBS and maintained for 24 h in the presence or absence of Gln. Then they were washed with PBS and scraped in 0.1 M sulfosalicylic acid. The acid-precipitable material was discarded, and the AA content in the supernatant was measured on an amino acid autoanalyzer (Beckman).
RNA isolation and quantitation by Northern blot analysis. Total RNAs were extracted from HepG2 cells by an acidic guanidinium thiocyanate/phenol/chloroform procedure, electrophoresed (20 μg/lane), blotted, and hybridized with a [α32P]dCTP-labeled cDNA as in Ref. 21. Full-length human cDNA probes included those for GAPDH [a kind gift from Dr. J. P. Borel, Centre National de la Recherche Scientifique (CNRS), Reims, France], α1-microglobulin/bikunin precursor (AMBP) (21), albumin (a kind gift from Dr. B. Bois-Joyeux, CNRS, Meudon, France), α2-HS glycoprotein (AHSG), and α1-antichymotrypsin (A1Ach; a courtesy from P. Arnaud, Charleston, SC). A rat 18S cDNA was kindly provided by Dr. R. Vranckx (Paris, France). The autoradiographic signals obtained with a given cDNA probe were quantitated by densitometric scanning with an image analyzer (Biocom 500; Biocom, Les Ulis, France). In every experiment, we verified that the signal-to-amount ratio was linear in the range of mRNA amounts studied with each probe (not detailed). All densitometric values for a given mRNA were normalized with the 18S signals obtained on the same blot. Every value is the mean of at least three independent experiments with triplicates per experiment.
Plasmids. p47 Is a plasmid with the bacterial β-galactosidase (β-gal) gene under the control of the cytomegalovirus (CMV) immediate-early enhancer/promoter. pBLcat5 with the chloramphenicol acetyl-transferase (cat) gene driven by the Herpes simplex virus (HSV) thymidine kinase (tk) promoter (-109/+51) and the promoter-free pBLcat6 plasmid have been described elsewhere (14). The p488/cat plasmid with the human GAPDH promoter (-488/+21) fused to cat in pUC/cat, the promoter-free pUC/cat plasmid, and deletion mutants of the GAPDH promoter covering from +21 to -329 (p329/cat), -308 (p308/cat), or -269 (p269/cat) in pUC/cat were a courtesy from Dr. X. Le Liepvre (41). p489/cat With the human GAPDH promoter (-489/+21) in pBLcat6 and its deletion mutants from +21 to -181 (p181/cat), -143 (p143/cat), or -81 (p81/cat) were a courtesy from Dr. T. Aki (1). Four mutants of p181/cat designated p181M1/cat to p181M4/cat, in which eight nucleotides in the natural GAPDH sequence were replaced by the 5′-TCTCGAGG-3′ sequence (an XhoI site is underscored) were purchased from BioMethodes (Génopole Industries) and verified by sequencing. Other cat plasmids included p-4964AMBP/cat with the human AMBP enhancer/promoter (from -4964 to +57) fused to cat in pUMSV0/cat and the control, promoter-free pUMSV0/cat plasmid (43) as well as p-3147AHSG/cat with the human AHSG promoter (from -3147 to +48) fused to cat in pBL/cat6 (6).
HepG2 cell transfections with reporter plasmids and cat/ β-gal assays. The HepG2 cells were plated in 6-cm plastic petri dishes and were first cultured in the standard plating medium described above. When the cells reached 70% confluency, the standard medium was replaced with MEM, and a cat plasmid (8 μg) and p47 (1 μg) were cotransfected by CaP04 precipitation (43). After 12 h, the cells were given media containing variable amounts of insulin and/or Gln (see results), further cultured for 24 h, and finally harvested by scrapping. A cell extract was assayed for β-gal and cat activities, and the final values of cat activities were expressed as counts per minute [3H]acetyl-chloramphenicol per β-gal unit (42). The background activity provided by a matched promoterless plasmid was substracted from that obtained with the promoter/cat construct(s) under study. In every experiment, the transfection of each plasmid was done in triplicate. All final values provided in results are a mean calculated from at least three independent experiments.
EMSA. HPLC-purified oligos were purchased from Genset. A series of double-stranded oligos covered the -127/-96 area in the GAPDH promoter, the wild-type (WT), and four mutant sequences noted (M1-M4), as further detailed in results. The WT sequence was used as a probe. Another probe was made of a proven C/EBP binding site (5′-CTAGGGCTTGCGCAATCTATATTCG-3′, Geneka catalog ref. 1200009). Other double-stranded oligos harbored proven binding sites for CREB/ATF (5′-GATTCAATGACATCACGGCTGTG-3′, Geneka catalog ref. 1200007), upstream stimulatory factor (USF)-1 (5′-GGCCAGACCACGTGGTCTGTTC-3′, Geneka catalog ref. 1200059), or NF-1 (5′-GGCACCTGTTTCAATTTGGCACGGAGCCAACAG-3′, Geneka catalog ref. 12011420). An AHSG double-stranded oligo, covering the -76/-50 area of the AHSG promoter, harbored a proven binding site for C/EBP (5). Probe labeling was done by end phosphorylation of one oligo with [γ32P]ATP (5,000 Ci/mmol; Amersham). Competitors were in a 200-fold molar excess over the GAPDH probe used at 1 ng/reaction or were in increasing molar excess over the C/EBP probe as indicated. Nuclear extracts from HepG2 cells were purchased from Euromedex, and they were used at 10 μg/reaction. Electrophoresis was done in a nondenaturing 6% polyacrylamide gel (43). Rabbit polyclonal IgGs were purchased from Santa Cruz Biotechnology and included anti-CREB-1/ATF-1 (catalog ref. C-21), anti-CRE-BP1/ATF-2 (catalog ref. N-96), anti-ATF-3 (catalog ref. C-19), and anti-CREB-2 /ATF-4 (catalog ref. C-20) (28). An anti-USF-1 antiserum was kindly provided by Dr. P. Rouet (Inserm, Toulouse, France), and anti-C/EBPα, anti-C/EBPβ, anti-C/EBPδ antisera were a kind gift from S. L. McKnight (Tularik, San Francisco, CA). Supershift experiments with these IgGs or antisera or a nonimmunized rabbit serum as a control were done by pre-incubating them with the nuclear extract on ice before probe addition. Electromobility shifts and supershifts assays were repeated at least three times using independently prepared nuclear extracts.
Effects of Gln and insulin on GAPDH mRNA level. We first evaluated whether Gln, alone or in association with insulin, can regulate the level of endogenous GAPDH mRNA in the human HepG2 hepatoma cells. As a control, we also investigated this effect on other genes with an hepatic expression, namely acute-phase proteins and genes A1Achy, albumin, AMBP, and AHSG (44). In HepG2 cells cultured for 48 h (Fig. 1), the GAPDH mRNA abundance increased with an increasing insulin input and plateaued at 20 mU/ml (resulting in a 3.3-fold increase), which is consistent with a previous report (38). The A1Achy, albumin, and AMBP mRNA levels peaked at 20 mU/ml. By contrast, the AHSG mRNA level remained unchanged. Whatever the amount of insulin, the GAPDH mRNA level increased (1.5-2 fold) in cells cultured with 10 mM Gln, whereas the A1Achy, albumin, AMBP, and AHSG mRNA levels were unaffected by this AA. A Gln-induced increase in GAPDH protein enzymic activity (1.4-fold) was consistent with these results (data not shown). Accordingly, Gln upregulates the GAPDH mRNA level in a gene-specific fashion and regardless of the insulin input.
Effects of Gln and insulin on GAPDH transcription. We next studied whether the above effects take place at a transcriptional level. Given that the endogenous expressions of the genes above are low in HepG2 cells, nuclear run-on experiments turned out to be poorly informative (data not shown). Therefore, we performed experiments with the GAPDH, AMBP, or AHSG promoters fused to the bacterial cat reporter gene. These promoter/cat constructs were transiently transfected in HepG2 cells. A culture period of at least 18 h was required to observe any insulin- or Gln-associated effect (data not shown). The transfected cells were cultured for 24 h in the absence or presence of 10 mM Gln and/or 20 mU/ml insulin. This insulin input was chosen because it induces a maximum upregulation of the GAPDH mRNA level (Fig. 1) and promoter activity (data not shown). As shown in Fig. 2, the GAPDH promoter activity was significantly increased in the presence of either insulin (2.5-fold), Gln (3-fold), or both (5.5-fold). These activities were additive, which is consistent with the insulin- or Gln-induced changes in endogenous GAPDH mRNA level observed above. Furthermore, the maximum and half-maximal Gln upregulation were respectively obtained with 2 and 0.5 mM Gln (Fig. 2). Therefore, the highest increase in Gln-induced transcription of GAPDH can be observed with the Gln content that is usually included in RPMI 1640 culture medium, namely 2 mM Gln. In contrast, the transcriptional activities of the AHSG and AMBP promoters were not affected by insulin, Gln, or both (Fig. 2). Likewise, a promoter of viral origin, namely the HSV tk promoter, was not affected. These features point to a GAPDH-specific transcriptional effect of insulin and Gln in our study.
Specificity of the Gln-induced effect and associated metabolites. To determine the AA-dependent specificity of the Gln-induced effect, we compared the effects of various AAs. As shown in Fig. 3A, 0.1 mM Met upregulated the GAPDH promoter to a higher extent than Gln, whereas 0.05 mM Trp, 0.2 mM Phe, or 0.4 mM Val also upregulated this promoter, albeit to a low extent. Therefore, the Gln-induced effect is not strictly specific for this AA.
We next investigated whether the effect of Gln might be related to its transport system. Similar to Gln, Pro, His, and Asn are transported through an Na+-dependent transport system in HepG2 cells (24). Yet, these amino acids do not modulate the GAPDH promoter (Fig. 3A). Even at a 10 mM concentration, Pro as well as Ala, which is also transported by an Na+-dependent transport system (24), failed to upregulate the GAPDH promoter (data not shown). Conversely, Met and Trp, whose transport sytems are Na+ independent, increased the GAPDH promoter activity (4- and 2-fold, respectively) when used at 10 mM (data not shown). This suggests that the Gln-induced effect is not related to its Na+-dependent transport system.
We also investigated whether Gln metabolism is required in the process under study. Gln from the culture medium is metabolized by HepG2 cells, which, in turn, modulates the intracellular pool of free AAs. Indeed, we could observe that the total AA content of HepG2 cells increased 2.5-fold in the presence of 10 mM Gln (498 ± 76 vs. 1,482 ± 135 μmol/g protein). Whereas the contents of Gln and AAs resulting from Gln metabolism such as Glu, Ala, Asp, and Pro increased, Thr, Gly, Orn, and Asn contents were reduced and all other AA contents remained unchanged (data not shown). As shown in Fig. 3B, the inhibition of Gln metabolism by DON, an inhibitor of mitochondrial glutaminases and glutamine-dependent amidotransferases, abolished the effect of Gln on GAPDH transcription. Therefore, Gln metabolism is likely to be involved in the Gln-induced upregulation of GAPDH. Gln metabolism by glutaminase produces glutamate and NH4Cl. A possible involvement of NH4Cl was investigated. As shown in Fig. 3B, 10 mM NH4Cl largely mimicked the Gln-induced upregulation of the GAPDH promoter. Finally, a potential role for glucosamine-6-phosphate, which is produced from Gln and fructose-6-phosphate by Gln fructose-6-phosphate amidotransferase, was also sought. Glucosamine mimicked the Gln-induced effect as its input in the culture medium strongly increased the GAPDH promoter activity. These results indicate that Gln metabolism is a necessary step for the Gln-induced activation of GAPDH transcription.
Element(s) of the GAPDH promoter involved in transcriptional activation by Gln. Several functional elements including IRE-A, IRE-B, C/EBP-binding domains, and other positively acting elements have been previously described within the human GAPDH promoter, as depicted in Fig. 4. Various cat constructs with the GAPDH promoter deleted to a variable extent (p488/cat to p82/cat) were used in this study. When transfecting these constructs in HepG2 cells, deletions from -488 to -181 did not significantly affect the basal promoter activity, whereas deletions down to -143 decreased it (data not shown), which is consistent with the acknowledged presence of positively acting elements from -181 to -143 (1). The longest construct p488/cat fully retained a capacity to be upregulated by insulin (Fig. 4, right), whereas any shorter construct was barely (p329/cat) or no longer insulin inducible, which fits with a former report (2). Any deletion construct down to p143/cat was Gln inducible, whereas p82/cat had lost this response. Therefore, an AARE appears to be present in the GAPDH promoter between bp -143 and -83, which acts independently of IRE-A, IRE-B, and other upstream elements previously described.
As shown in Fig. 4 (bottom left), the -116/-105 sequence in GAPDH exhibits some similarity with the AARE at -70/-64 and the nutrient-sensing response element 1 (NSRE-1) at -68/-60 found in the AS promoter (9, 27). Also, a search with the program MatInspector combined with the TRANSFAC database software (52) revealed that the GAPDH sequence at -126/-118 exhibits a similarity with binding sites for the ATF/CREB, C/EBP, and USF families and the sequence at -110/-97 is somewhat similar to a binding site for the ATF/CREB family. To assess the possible involvement of this -127/-96 sequence in the response to Gln, a series of p181/cat mutants designated p181M1/cat to p181M4/cat were used to scan this sequence and were tested for cat activity (Fig. 5). The mutations M1 and M2 slightly decreased the basal activity of the GAPDH promoter, whereas the mutation M4 slightly increased it. Moreover, M2, M3, and M4 did not prevent the Gln-induced upregulation of the GAPDH promoter, whereas M1 suppressed it. Therefore, the -116/-105 sequence that is similar with the AARE and NSRE-1 in AS does not participate in this activation. By contrast, the -126/-118 sequence that is similar to ATF/CREB, C/EBP, and USF binding sites mediates the Gln-induced upregulation of the GAPDH promoter. This element is here below designated as the GAPDH Gln-responsive element (GlnRE).
Transcription factors involved in GAPDH activation by Gln. EMSAs were performed with a GAPDH probe covering from -127 to -96 (Fig. 6). Four bands formed with HepG2 nuclear extracts (lane 1). These bands included two specific complexes, A and B, as proven by an autologous competitor (lane 2), as well as two nonspecific complexes (*). Complex A was detected as a slow, faint band, whereas complex B appeared as a strong band of fast mobility. To identify which GAPDH sequence(s) was able to form complex A or B, mutant oligos M1-M4, whose sequences were identical to those shown in Fig. 5, were used as competitors. Complex A was abolished by M2, M3, and M4 (lanes 4-6) but not by M1 (lane 3), whereas complex B was abolished by M1 and M2 (lanes 3 and 4) but by neither M3 nor M4 (lanes 5 and 6). The locations of the mutated nucleotides in M1-M4 indicate that two sequences at -127/-120 and -110/-97 participate in the formation of complexes A and B, respectively. Because these sequences encompass the potential ATF/CREB-, C/EBP-, or USF-binding sites noted above, proven binding sites for some members of these families were used as competitors. A binding site for ATF/CREB (lane 7) or C/EBP (lane 9) abolished complex A, whereas an USF-1 binding site failed to do so (lane 8), indicating that complex A at -126/-118 is C/EBP and/or ATF/CREB related. Also, the ATF/CREB competitor abolished complex B (lane 7), indicating that complex B at -110/-97 is ATF/CREB related. A competitor with an NF-1 site used as a negative control failed to abolished either complex A or complex B.
Supershifts (Fig. 6, lanes 11-19) were performed to identify which members of the C/EBP and ATF/CREB families form complexes A and B. Anti-C/EBP-α antibodies abolished complex A (lane 12), and anti-C/EBP-δ antibodies supershifted it (lane 14), whereas anti-C/EBP-β (lane 13), -CREB-1/ATF-1 (lane 16), -CREB-2/ATF-4 (lane 17), -ATF-3 (lane 18), or -USF-1 antibodies (lane 19) did not. Therefore, complex A is made up of C/EBP-α and -δ proteins. Anti-CRE-BP1/ATF-2 antibodies supershifted complex B (lane 15), whereas anti-C/EBP-α (lane 12), -β (lane 13), -δ (lane 14), -CREB-1/ATF-1 (lane 16), -CREB-2/ATF-4 (lane 17), -ATF-3 (lane 18), or -USF antibodies (lane 19) did not. Therefore, complex B is made up of the CRE-BP1/ATF-2 protein. Of note, the supershifted complex B was relocated over the EMSA area normally occupied by complex A. Hence, it cannot be formally excluded that the anti-CRE-BP1/ATF-2 antibodies supershifted complex A as well. Finally, together, our mutagenesis experiments and EMSA data pointed to the binding site at -127/-120 and the associated complex A as the mediators of the GAPDH promoter regulation by Gln. To confirm that the binding site at -127/-120 is a bona fide C/EBP binding site, a reverse EMSA experiment with a proven C/EBP binding site used as a probe was carried out (Fig. 6, lanes 20-32). The complexes formed with this probe and an HepG2 nuclear extract included a nonspecific complex (*) as well as a major set of closed bands (noted C) whose appearance fits the pattern expected for C/EBP complexes (3). This set of bands was competed out by the autologous competitor (lanes 21-23), a heterologous, high-affinity C/EBP binding site found in the AHSG promoter (5; and data not shown) (lanes 30-32) and the WT GAPDH sequence -127/-96 (lanes 24-26) but was not competed by the GAPDH mutant M1 (lanes 27-29). This unambiguously identified the -127/-120 sequence as a C/EBP binding site. Moreover, the above competitions that were done with a variable excess of competitor pointed to a similar affinity of the C/EBP proteins for the site at -127/-120 in GAPDH and the high affinity site in AHSG, thus indicating that the former site has a strong affinity for C/EBP proteins.
Our data document the novel observation that Gln is able to upregulate GAPDH gene expression in an additive way with insulin. This event takes place, at least partly, at the transcriptional level and is consistent with an increase in in vivo transcription rate in rat hepatocytes incubated with Gln as observed in run-on experiments (data not shown). This effect is gene specific, because Gln is unable to modulate the hepatic transcription of some other genes, namely AMBP and AHSG. Studies of the AA-controlled expression of the AS and CHOP genes have shown that they specifically and differently respond to availability of various sets of AAs (25, 31). Consistent with this, the overall pattern of GAPDH gene expression in response to given AAs is different from what is observed for CHOP and AS genes. A noted exception is Met, which is a potent inducer for AS, CHOP, and GAPDH. Moreover, GAPDH expression is controlled by a set of essential AAs (i.e., Met, Phe, Trp, and Val) and by Gln, considered a "conditionally essential" AA (12). This similar effect of essential AAs and Gln underscores the Gln characteristic. Moreover, these AAs effects occur at physiological concentrations as observed in portal vein of fed rats (30). Likewise, half-maximal Gln activation of GAPDH transcription occurs at 0.5 mM, the physiological serum Gln concentration in human.
Gln exerts anabolic effects on liver metabolism by stimulating glycogen, lipid, and protein metabolisms, and most of these Gln-induced effects are mediated by cell swelling that results from a Na+-dependent transport of AAs, a subsequent accumulation of catabolic byproducts, and an associated increase in intracellular osmolarity (11, 33, 50). These Gln effects were reproduced in our experimental conditions. Indeed, in HepG2 hepatoma cell cultures, Gln is transported through an Na+-dependent transport system (24), and a Gln/glutaminase-mediated production of Glu as well as a production of Asp and Ala by Glu transamination most likely contributed to the observed accumulation of these AAs. Yet, Pro and Gly, whose intake is Na+ dependent (24), failed to regulate GAPDH transcription. Moreover, Met and Trp, which are usually transported through a Na+-independent system in mammalian cells, upregulated GAPDH transcription. Together, these features point to a cell-swelling independent mechanism in the Gln-mediated GAPDH regulation.
Gln metabolism is important in regulating GAPDH gene transcription. Indeed, 1) the Gln-induced upregulation of the GAPDH promoter was time (>18 h) and dose dependent, 2) an inhibition of Gln metabolism suppressed GAPDH activation, 3) Gln byproducts such as ammonia, which is produced by glutaminase, and glucosamine, which is produced by Gln fructose-6-phosphate amidotransferase, also activated GAPDH transcription. Glucosamine, which is likely phosphorylated into glucosamine-6-phosphate in HepG2 cells, is the precursor of the hexosamine pathway. Interestingly, this pathway leads to protein glycosylation and is known to participate in signal transduction (51).
We have mapped the GAPDH GlnRE to a GCACGTAGC motif at -126/-118. This excludes that the two upstream elements involved in the basal GAPDH expression (1) as well as the IRE-A and -B sites participate in the Gln responsiveness of this gene. Our supershift experiments have indicated that the GAPDH GlnRE binds C/EBPα and -δ. The identification of these TFs is compatible with a previous detection of a C/EBP binding site around -100 (2). All members of the C/EBP family contain a bZIP domain. The latter consists of a basic AA-rich DNA binding region and a leucine zipper dimerization motif that mediates a homo- or heterodimerization with other C/EBP proteins (40). Such a heterodimerization between C/EBPα and -δ over the GAPDH GlnRE is suggested by the complete removal of complex A by both anti-C/EBPα and -δ antibodies. Also, the C/EBP proteins can heterodimerize with other bZIP TFs such as the CREB/ATF family members (28, 40). In keeping with this, a competitor oligo with a CREB/ATF site abolished the formation of complex A in our EMSAs. Therefore, the further participation of a CREB/ATF protein in some C/EBP heterodimers bound to the GAPDH GlnRE is to be considered. Indeed, 1) the pattern of complex A over the GAPDH probe is different from that of typical complex C over a control C/EBP probe, and 2) mobility of complex A is slower than that of complex C in keeping with ATF2 size (55 kDa), which is almost twice that of the main isoforms of C/EBPα and -δ. Our finding that the Gln-driven transcription of GAPDH is mediated by C/EBP is consistent with the acknowledged role of these TFs in controlling the expression of genes that play a central role in the carbohydrate and lipid metabolisms (35, 40). C/EBP expression is regulated by numerous pathways including those for some hormones and nutrients (40). In particular, an AA limitation (Phe, Met, Leu, or Trp) increases the abundance of C/EBPα and -β mRNAs in rat hepatoma cells (37). However, neither the migration nor abundance of the (GlnRE-C/EBP) complex A seen in our EMSA changed following Gln induction (data not shown). Therefore, our data suggest that the Gln-induced upregulation of GAPDH transcription does not involve any change in C/EBP binding activity. Because GAPDH activation requires Gln metabolism and is also induced by glucosamine, the possibility that Gln controls GAPDH transcription by virtue of a change in TF glycosylation is an attractive hypothesis that deserves further investigation.
In the GAPDH promoter, the sequence starting at 8 nt downstream of the GlnRE is somewhat similar to AS or, to a lesser extent, CHOP AAREs. However, this GAPDH sequence that binds ATF-2 does not mediate the Gln responsiveness. Yet AS or CHOP AAREs are, in turn, only partially structurally and functionally related (16). Indeed, The AS AARE is made up of two responsive elements (NSRE-1 and -2) that are involved in response to AA starvation or an endoplasmic reticulum stress (notably induced by glucose starvation). C/EBPβ(h8) and ATF-4(h7) bind to NSRE-1, are the mediators of these responses, and are transcriptionally regulated by nutrients. On the other hand, ATF-2, which does not bind to NSRE-1, is necessary to obtain the full AS response to the above stress. The CHOP AARE shares homology with the AS NSRE-1, but it binds ATF-2 and responds to AA starvation only. Moreover, the AS promoter harbors three GC-rich sequences that are located about 30 nt upstream of the AARE and are required for the AA-induced transcription of this gene (36). The GAPDH promoter also contains two GC-rich sequence boxes that are located 30 nt upstream of the GlnRE (-181/-143) and allow for a high basal transcription rate (1). However, these GC-rich sequence boxes are not required for an induction of GAPDH by AAs. Collectively, our data show that the GAPDH GlnRE is structurally and functionally different from AS and CHOP AAREs.
Although extrapolation of our results to human liver must be made with caution in view of the limitations inherent to the use of a cell line as an experiment model, the hepatic regulation of GAPDH by Gln and some essential AAs might be of physiological relevance. The AA-induced GAPDH upregulation might be associated with an increased glycolytic flux yielding the substrates and energy required for some anabolic processes. This hypothesis is consistent with the observation that parenteral Gln infusion enhances glucose use in dog (13). Alternatively, an AA-induced GAPDH upregulation might also be important for the potential nonglycolytic functions of the enzyme. Indeed, over the last decade, numerous studies have identified diverse biological properties for mammalian GAPDH protein. GAPDH has a role in membrane fusion and transport; in the cytoplasm, it participates in the translational control of gene expression by phosphorylation as well as in microtubule assembly; within the nucleus, it is involved in tRNA export and DNA replication and repair. It also may play a role in apoptosis, neuronal disorders, and prostate cancer, and it appears to be involved in the toxicology of nitric oxide (46). A better knowledge of AA effects at a molecular level will likely help optimize the use of AAs as a nutritional support well beyond some current conventional treatments. In particular, it is noteworthy that the GAPDH response to Gln is insulin independent. Yet some AA-associated beneficial effects on carbohydrate and protein metabolism are emerging from clinical investigations. Indeed, an oral supplementation with essential AAs for patients with Type 2 diabetes mellitus appears to decrease postprandial plasma glucose (39). Likewise, enteral Gln stimulates gut protein anabolism as reflected by an increased mucosal fractional synthesis rate in humans (15, 19). Identification of novel target genes for AA-based adjuvant therapies remains an important challenge for the future.
We are grateful to Dr. M. C. Alexander-Bridges who made possible the use of p488/cat plasmid.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Copyright © 2003 the American Physiological Society


text:
Why no outcry from Perkasa over instant bumis?
YOURSAY 'The bumiputeras' share of the economic cake has become smaller with these type of foreigners who have become instant bumiputeras.'
Pakistani with blue IC 'was threatened' when voting
Multi Racial: I wonder why those so-called patriotic NGOs and political parties are silence on this issue of citizenship being fraudulently given to foreigners.
Why none of them call for former PM Dr Mahathir Mohamad, the Project IC mastermind, and those executing his Project IC directives to be charged immediately? What they did is treason.
The Malay rulers should not keep quiet on this issue, too. If the authorities do not act, the rulers should give royal orders for action to be taken.
Gotcha: The bumiputeras' share of the economic cake has become smaller with these type of foreigners who have become instant bumiputeras. Umno-linked NGOs, what have you got to say about this?
Homesick: The important question is: What is to be done about the problems and how to stop it from ever happening again?
My prediction is nothing, as long as Umno-BN is in charge of Putrajaya. So, to the real Malays of Malaysia, please wake up and kick out Umno Baru Malays now before you become the minority.
If nothing is done now, in 20 or 30 years' time, a new breed of foreign bumiputeras will become the majority.
And all the Umno Baru Malays would be long gone with all their stolen loot and living a life of luxury in London, Paris, Cayman Island, etc, and the real Malaysians will be second-class citizens in their own country.
Heavywater: How many witness does royal commission of inquiry (RCI) really need before addressing the problems.
It doesn't need 147 witnesses, just a handful would be enough for the commission to reflect what went wrong in Sabah.
Anonymous #07247310: There are around 50,000 Pakistanis doing business in and around Johor Bahru area and they are buying houses 15 percent cheaper than me.
No raising Kugan case, deputy speaker orders MPs
Cala: The death of A Kugan under the watchful eye of the Subang Jaya police was only part of the larger social phenomenon that points to the lack of rule of law and unequal protection under the law.
By right, if the Umno-led BN regime has been sincere and steadfast to the functions of a democratic government, following from justice VT Singham's reprimand of the police for their role in causing Kugan's death, an admission of guilt and responsibility would be PM Najib Razak's best way to display his 1Malaysia concept.
To challenge the verdict of judge Singham is to defend the guilty party, which is in effect ignored "the rule of law", and thereby giving rise to the suspicion of the practice of "unequal protection under the law".
At this rate, one will see further deterioration of Malaysia's worldwide governance indicators prepared by the World Bank annually.
The West progresses so much over the East because of its culture of freedom of speech. The West believes that any issue can be debated freely and in the end, the reality (the truth) shall prevail.
Malaysia clearly is a Third World state as evidence from the Dewan Rakyat speaker's short-sightedness when he disallowed the parliamentary debate on Kugan's death. He wins in the short run, but Malaysia loses in the long run.
Old Timer: Kugan 's case and the other deaths in jail is an important national issue and should be debated in the Parliament, so that deaths in custody will no longer occur.
How can they bar this issue from being discussed in Parliament? Is this Parliament of BN or the rakyat?
Fighting for Justice: Why then do we have MPs for? They have been chosen to voice the rakyat's grievances.
If the deputy speaker can dictate what the MPs can talk or cannot talk in Parliament, then why hold elections and pretend to practice democracy.
The opposition cannot talk about haze, cannot talk about Kugan, cannot talk about indelible ink, cannot talk about Independent Police Complaints and Misconduct Commission (IPCMC)...
Anonymous #33877536: Whenever the government is caught with their pants down on issues unfavourable to them, then it cannot be debated in Parliament.
This is because they cannot counter the opposition with facts and figures. It's as simple as that.
Wira: Mr Deputy Speaker, how can you say this is still sub judice as the Kugan trial is already over? Moreover, it is not known if the government will appeal.
You are clearly putting the cart before the horse in disallowing discussion on the case in Parliament.
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text:
Open enrollment interest in Pierce County mixed
Open enrollment applications recorded by area schools last month showed mixed interest.
Last week, the Ellsworth and Plum City districts reported applicant activity this February was above that from February of 2010, while activity in the Elmwood and Spring Valley districts was below their reports from a year ago.
However, only in Ellsworth and Plum City were the number of students who indicated they're considering transferring out this fall higher than the previous year. Ellsworth alone had a fewer number of students signaling they may transfer in than it did at this time in '10.
Read more in the print version of the Herald March 9.


text:
Renewed interest in antifols for the treatment of childhood cancers has resulted from identification of novel antifols with broad spectrums of anti-cancer activity and from re-evaluation of the original clinical antifol, aminopterin. In this pre-clinical study we evaluated the in vitro activity of both traditional antifols (methotrexate, aminopterin) and novel antifols (pemetrexed, talotrexin) in childhood acute leukemias and lymphomas.
[ hide abstract ]