text:
Community shield67' Arsenal still leading 1-0.
Arsenal 1-0 Chelsea
Oxlade Chamberlain scored
Makan Udang 1-0 Totem Hamburger?Yes. Full time MU 1-0. Good start for MU :clap:
Leicester 1-0Leicester 2-0, score by vardy and mahrez
Defending Champion Chelsea vs Swansea is up next :clap:
Full time:
Arsenal 0-2 Westham :thumbsup: :thumbsup: :thumbsup:
Newcastle 2-2 Southampton
Aiseh.... :SArsenal never lose to Westham many many many years already
Last match of the week tonight
WBA VS MC :clap: :clap:
10 minutes: Wba 0-0 Mc
Yaya Toure puts MC in front :thumbsup:MC 2-0, Yaya Toure scores twice
MC 2-0, Yaya Toure scores twiceHalf time MC 2-0
Second half pick Manchester City again. Should be able to extend their lead. :clap:57 minutes 2-0
Second half pick Manchester City again. Should be able to extend their lead. :clap:Full time 3-0. Yeah I won 3000
Tonight also got BPL watch
Aston Villa vs Manchester United :clap:
Southampton vs Everton (7:45pm)
Ronald Koeman has revealed that the frustration he felt at having to watch Southampton's 2-2 draw with Newcastle from afar led to him shouting at the television.
The Southampton manager was forbidden from travelling as he recovered from surgery after rupturing his Achilles so could only watch his team's opening Barclays Premier League fixture from his home in Holland.
He remains reliant on crutches and expects to soon progress from a cast to a boot but said that his greatest relief will be to return to the bench when Saints play Everton at St Mary's on Saturday.
''It was strange, because normally I'm part of the game, and watching television is not the best you can do,'' Koeman said. ''Even worse, if the team is not playing good, it makes it more difficult.
''In the first half [I shouted at the television], because we didn't play well, we did a lot of mistakes in our ball positions.
''If you stay at home you don't have control. I had contact with Scott [Waters, the performance analyst]. It was two times in the first half.
''I didn't really like to have to contact the bench because we know what we want and how we like to play, but it's more difficult, I like to be part of that, you can shout, you can do something, you can do substitutes if you're not happy but staying at home is different.
''We didn't have a long time to prepare for the Newcastle game after the Thursday game [a 2-0 win in the Europa League away to] Vitesse [Arnhem], to recover and prepare the team. For them it was strange, because it was the first time without the manager, and also strange for the technical staff.
''You think it's more easier [to be at the game] because you think you're more part of the team than you are watching television.''
New £5million signing Oriol Romeu is expected to make his Southampton debut after arriving from Chelsea.
Koeman will be without two other summer recruits, Steven Caulker (sinus infection) and Jordie Clasie (*), however, as well as Ryan Bertrand (knee) and long-term absentee Fraser Forster.
Everton midfielder Gareth Barry believes Ross Barkley has benefited from last season's difficulties.
The 21-year-old's struggles to recapture his scintillating form of manager Roberto Martinez's first campaign were almost symptomatic of the entire team's troubles which saw them finish a disappointing 11th.
Barkley managed just two goals, taking 11 matches to get off the mark, but has already opened his account with a fine strike in an encouraging performance in the opening 2-2 draw at home to Watford.
''Ross burst onto the scene and set his standards so high but inevitably it is impossible to keep those going continuously,'' Barry told Press Association Sport.
''I am sure last season he would have wanted to score more goals and have more assists but you can't continuously keep that upward curve going.
''But it will have been a good year for him in terms of his future and he started well with a fantastic goal.''
While Everton undoubtedly need more goals from midfield, and the arrival of Tom Cleverley and Gerard Deulofeu should assist in that, their fortunes are likely to be determined by the form of striker Romelu Lukaku.
The Belgium international scored 20 times last season but eight of those were in the Europa League and without the distraction of European football this season he will be expected to have much more of an impact in the Premier League.
''Every top team needs a goalscorer scoring 20 goals and in Rom I think we have a player who can do that year in, year out,'' added Barry.
''It is important he stays fit and gets his confidence going and he can achieve great things too.''
Everton defender Leighton Baines has been ruled out for three months after ankle surgery so 19-year-old Brendan Galloway is expected to deputise at left-back again.
Forward Kevin Mirallas has recovered from the ankle injury which forced him off against Watford last weekend.
Gerard Deulofeu, Aiden McGeady and Muhamed Besic all returned to training this week after missing last weekend's season-opener with * injuries.
25" Southampton 0-1 Everton45" Southampton 0-2 Everton
Everyone was betting their monies on Arsenal because of their active transfer market and good pre seasons results. :giggle:Arsenal only got one signing this season, which is Cech from Chelsea
Lets give a big applause of appreciation to Mr Orient for his hard work and effort to report on the football newsHaha that's fine. It's my interest :handshake:
:clap: :clap: :clap: :clap: :clap: :clap: :clap: :clap: :clap:
Haha that's fine. It's my interest :handshake:
My other interest is to see Arsenal wins :DTomorrow we will see. Arsenal will visit Crystal Palace
Tomorrow we will see. Arsenal will visit Crystal Palace
Its like Maserati racing Proton Kancil, no sweat. :DDont judge so fast, they lost at home to Westham last week. And Westham lost at home to Leicester just now. Afterall ball is round in shape
LINEUPSArsenal 1-0. Giroud scores
Crystal Palace: McCarthy; Ward, Dann, Delaney, Souaré; McArthur, Cabaye; Zaha, Puncheon, Bolasie; Wickham.
Subs: Hennessey, Mariappa, Jedinak, Mutch, Lee, Bamford, Murray.
Arsenal: Cech; Bellerin, Mertesacker, Koscielny, Monreal; Coquelin, Ramsey; Cazorla, Ozil, Alexis; Giroud
Subs: Ospina, Debuchy, Gibbs, Gabriel, Arteta, Oxlade-Chamberlain, Walcott.
Arsenal 1-0. Giroud scores28' Crystal 1-1 Arsenal
28' Crystal 1-1 Arsenal
22' MC 0-0 Chelsea50' MC 1-0
70' Liverpool 1-0 BournemouthFT Liverpool won 1-0. Goal from Benteke :clap:
I don't care if Arsenal does not win anymore.This week they are against in form Liverpool :shake:
This week they are against in form Liverpool :shake:
Monday night blockbuster
Arsenal vs Liverpool :thumbsup:
Arsenal vs LiverpoolGoalless draw :shake:
Mesut Ozil has called on Arsenal to underline their Barclays Premier League title credentials with victory over Liverpool at the Emirates Stadium on Monday night.
The Gunners recovered from their opening-day home defeat by West Ham to record a battling 2-1 win at Crystal Palace last weekend.
Liverpool, though, head to north London on the back of successive victories from their first two matches.
The Reds were defeated 4-1 at the Emirates Stadium in April, with Arsenal going on to claim third place before lifting the FA Cup again at Wembley.
Ozil, who netted a fine 25-yard free-kick in that encounter, believes Monday night provides another opportunity for the Gunners to show just what they are capable of.
"I remember that we delivered a really convincing display. In the first half we decided the game because we had a high level of concentration and wanted to utilise our potential. We want to do that again this time," Ozil told Arsenal Player.
"We are at home and we know exactly how we can beat Liverpool; that is what we want to do on Monday.
"We were highly concentrated. The manager gave us tasks on the pitch and we executed them perfectly. That's what we will have to do on Monday again.
"We have to believe in ourselves as we have the potential to beat every team and we will show that on Monday."
Ozil delivered a virtuoso display at Selhurst Park last Sunday, completing a 98 per cent pass accuracy which included setting up Olivier Giroud for the opening goal.
It has been a solid transition for the German playmaker, who became the club-record signing when he joined from Real Madrid in September 2013.
Gunners boss Arsene Wenger has been impressed by the progression of the World Cup winner.
"Mesut is now completely committed to the club. Maybe in the first year he didn't know a lot about it. He came from Real Madrid and he didn't know how long and whether it would work for him, whether he would enjoy it or not," Wenger said.
"I believe now he is completely on board, committed and focused on the results of the club."
Arsenal have no fresh injury worries, although they remain without Jack Wilshere (leg), Danny Welbeck (knee) and Tomas Rosicky (both knee).
Liverpool can land a psychological blow with victory at the Emirates, according to manager Brendan Rodgers.
The Reds travel to the capital for an early meeting with another side who have top-four aspirations this season aiming to continue their bright start after back-to-back 1-0 victories over Stoke and Bournemouth.
Rodgers admits the result is unlikely to have too much impact on the final standings at this stage, yet appreciates an away win over Wenger's men can greatly enhance the confidence among his group.
"You're playing one of the rivals that you're hoping to be competing with at the top end of the table; these games are important," Rodgers acknowledged.
"They don't give the full picture in terms of how it will finish up - you can draw or lose these games and still go on and achieve what you want to achieve. Of course it's for the confidence and psychologically they are important.
"No matter when these games come in the season they are always good for the psychological effect.
"We've got a tough game away from home but we'll arrive into the game with confidence. We know they're an outstanding group of players and some quality individual players but there's a real focus in this group for this season, a real toughness, mentally.
"We know that we've got that ability to scrap and fight to get a result as well as play good football. If we can go there and perform like we think we can and keep to our game-plan we're confident we can get a result."
Liverpool will assess the fitness of captain Jordan Henderson after he was withdrawn early in the second half of the Bournemouth game with a foot injury.
The return of midfielder Joe Allen (*) remains uncertain while striker Daniel Sturridge continues to recover from hip surgery and defender Jon Flanagan (knee) is out until December
Newcastle United vs. Arsenal
G Tim Krul
D Daryl Janmaat
D F. Coloccini
D Chancel Mbemba
D Massadio Haidara
M Florian Thauvin
M Jack Colback
M Vurnon Anita
M Moussa Sissoko
M G. Wijnaldum
F A. Mitrovic
G Petr Cech
D Nacho Monreal
D Gabriel
D Laurent Koscielny
D Hector Bellerin
M A. Oxlade-Chamberlain
M Francis Coquelin
M Theo Walcott
M Aaron Ramsey
M Santi Cazorla
F Alexis Sanchez
Newcastle United vs. Arsenal25 minutes Newcastle 0-0 Arsenal
G Tim Krul
D Daryl Janmaat
D F. Coloccini
D Chancel Mbemba
D Massadio Haidara
M Florian Thauvin
M Jack Colback
M Vurnon Anita
M Moussa Sissoko
M G. Wijnaldum
F A. Mitrovic
G Petr Cech
D Nacho Monreal
D Gabriel
D Laurent Koscielny
D Hector Bellerin
M A. Oxlade-Chamberlain
M Francis Coquelin
M Theo Walcott
M Aaron Ramsey
M Santi Cazorla
F Alexis Sanchez
25 minutes Newcastle 0-0 Arsenal40 minutes still 0-0. Hang on Newcastle :clap:
Goal! Aston Villa 0, Sunderland 1. Yann M'Vila (Sunderland) from a free kick with a right footed shot to the top right corner.
Goal! Liverpool 0, West Ham United 1. Manuel Lanzini (West Ham United) right footed shot from the centre of the box to the bottom right corner. Assisted by Aaron Cresswell with a cross.
Goal! Manchester City 1, Watford 0. Raheem Sterling (Manchester City) right footed shot from very close range to the bottom right corner. Assisted by Bacary Sagna with a cross.
Goal! Chelsea 0, Crystal Palace 1. Bakary Sako (Crystal Palace) right footed shot from the centre of the box to the high centre of the goal.
Tottenham Hotspur vs. Everton
G Hugo Lloris
D Jan Vertonghen
D Danny Rose
D Eric Dier
D Kyle Walker
D Toby Alderweireld
M Nacer Chadli
M Nabil Bentaleb
M Mousa Dembele
M Ryan Mason
F Harry Kane
G Tim Howard
D Phil Jagielka
D Bryan Oviedo
D John Stones
D Seamus Coleman
M Gareth Barry
M Ross Barkley
M Tom Cleverley
M James McCarthy
F Romelu Lukaku
F Arouna Kone
First Half ends, Tottenham Hotspur 0, Everton 0.70' Tottenham 0-0 Everton
Southampton vs. Norwich City
G M. Stekelenburg
D Maya Yoshida
D Cedric
D Jose Fonte
D Matt Targett
M James Ward-Prowse
M Sadio Mane
M Oriol Romeu
M Dusan Tadic
M Steven Davis
F Graziano Pelle
G John Ruddy
D Steven Whittaker
D Sebastien Bassong
D Russell Martin
M Wesley Hoolahan
M Nathan Redmond
M Robert Brady
M Alexander Tettey
M Graham Dorrans
M Jonathan Howson
F Cameron Jerome
Swansea City vs. Manchester United
G Lukasz Fabianski
D F. Fernandez
D Neil Taylor
D Ashley Williams
D Kyle Naughton
M Gylfi Sigurdsson
M Jack Cork
M Jonjo Shelvey
M Wayne Routledge
F Bafetimbi Gomis
F Andre Ayew
G Sergio Romero
D Matteo Darmian
D Chris Smalling
D Luke Shaw
M B. Schweinsteiger
M Juan Mata
M M. Schneiderlin
M Ander Herrera
M Daley Blind
F Memphis Depay
F Wayne Rooney
Second Half ends, Swansea City 2, Manchester United 1.
49'Go Man U
Goal! Manchester United 1, Liverpool 0. Daley Blind (Manchester United) left footed shot from the centre of the box to the top left corner. Assisted by Juan Mata following a set piece situation.
Go Man UWithout Rooney and Depay just played half time, they still manage to beat Liverpool EASILY :clap:
Sunderland vs.Tottenham Hotspur
G Costel Pantilimon
D John O'Shea
D Younes Kaboul
D Billy Jones
D P. van Aanholt
M Jordi Gomez
M Yann M'Vila
M Ola Toivonen
F/M Jeremain Lens
F Jermain Defoe
F Fabio Borini
G Hugo Lloris
D Jan Vertonghen
D Kyle Walker
D Eric Dier
D Ben Davies
D Toby Alderweireld
M Nacer Chadli
M Dele Alli
M Ryan Mason
F Harry Kane
F Heung-Min Son
Just had a look at the Premier League table, surprise to see Chelsea at 17th position !!!Nobody will believe that before the season kick off.
Tottenham Hotspur vs Crystal Palace
G Hugo Lloris
D Jan Vertonghen
D Kyle Walker
D Eric Dier
D Ben Davies
D Toby Alderweireld
M Nacer Chadli
M Dele Alli
M Erik Lamela
F Heung-Min Son
F Harry Kane
G Alex McCarthy
D Pape Souare
D Brede Hangeland
D Martin Kelly
D Damien Delaney
M Bakary Sako
M Yohan Cabaye
M James McArthur
M Jason Puncheon
M Yannick Bolasie
F/M Wilfried Zaha
Crystal Palace vs West Bromwich AlbionHt 0-0
GWayne Hennessey
DScott Dann
DMartin Kelly
DPape Souare
DBrede Hangeland
MYannick Bolasie
MYohan Cabaye
MJames McArthur
MJason Puncheon
F/MWilfried Zaha
FDwight Gayle
GBoaz Myhill
DGareth McAuley
DJonny Evans
DCraig Dawson
M/DChris Brunt
MClaudio Yacob
MDarren Fletcher
MJames Morrison
FSaido Berahino
FSalomon Rondon
FJames McClean
Everton 1-1 Liverpool
Arsenal 3-0 MU
Swansea 2-2 Tottenham
Arsenal 3-0 MUArsenal is not going to be champion anyhow :clap:
Arsenal 3-0 MU
Arsenal 3-0 MU
Arsenal 3-0 MU
Arsenal 3-0 MU
Arsenal 3-0 MU
Arsenal 3-0 MU
Arsenal 3-0 MU
Arsenal 3-0 MU
:clap: :clap: :clap: :clap: :clap:
Tottenham Hotspur vs. Liverpool
GHugo Lloris
DJan Vertonghen
DDanny Rose
DKyle Walker
DToby Alderweireld
MNacer Chadli
MChristian Eriksen
MErik Lamela
MDele Alli
MMousa Dembele
FHarry Kane
GSimon Mignolet
DNathaniel Clyne
DMamadou Sakho
DMartin Skrtel
DAlberto Moreno
MEmre Can
MPhilippe Coutinho
MJames Milner
MAdam Lallana
FDivock Origi
Watford vs ArsenalHalftime 0-0
DCraig Cathcart
DSebastian Prodl
DNathan Ake
DAllan Nyom
M/DIkechi Anya
MAlmen Abdi
MEtienne Capoue
MBen Watson
FTroy Deeney
FOdion Ighalo
GPetr Cech
DPer Mertesacker
DNacho Monreal
DLaurent Koscielny
DHector Bellerin
MFrancis Coquelin
MMesut Ozil
MSanti Cazorla
MTheo Walcott
MAaron Ramsey
FAlexis Sanchez
Newcastle vs Norwich
Steve McClaren is hoping Jekyll and not Hyde turns up on Sunday as Newcastle attempt to end their wait for a first Barclays Premier League victory of the season.
McClaren, who has seen his side flit between the impressive and the abysmal through the opening weeks of his reign, is desperate to claim a league win at the ninth attempt against Norwich to dissipate the growing unrest surrounding the club.
But he needs the kind of performance which secured a 0-0 draw at Manchester United or which resulted in a 2-0 lead over Chelsea rather than the one which saw them trounced 6-1 at Manchester City last time out if the rot is to be stopped.
McClaren said: "We need that belief, and that comes from performances, playing well, results. We have kind of had that - not the results. The players just need to focus on doing their job, making sure they fight for 95 minutes and give the crowd what they want.
"They want to see fight, they want to see attitude, they want to see good football. Give them that and generally - some things are out of your control, the result is - but generally if you get that, you win games."
To date, Newcastle have collected a paltry three points and went into the weekend rooted to the foot of the table and in danger of being cast adrift.
Norwich arrive on Tyneside six points better off having made a decent fist of life back in the top flight.
McClaren said: "They've been very good, very good. It's not surprised me at all that the three [promoted] teams have started quite well.
"They have got the buoyancy and the confidence of promotion, the nucleus of the squad stays, they add very well and they can be a handful at the start, which they are proving.
"I saw them [Norwich] against West Ham - they could have won that game, should have won that game, and Leicester, they shouldn't have lost that game, so this is not going to be easy."
McClaren has received a fitness boost ahead of Sunday's Barclays Premier League clash with Norwich despite losing goalkeeper Tim Krul for the rest of the season.
Defenders Daryl Janmaat and Massadio Haidara (both knee) and Paul Dummett (*), as well as midfielder Jack Colback (*), have returned to training and are in contention as the Magpies attempt to secure their first league win of the season at the ninth attempt.
However, keepers Krul (cruciate ligament) and Karl Darlow (ankle), defenders Steven Taylor and Kevin Mbabu (both *), midfielders Rolando Aarons (calf) and Gabriel Obertan (*) and striker Emmanuel Riviere (knee) remain on the sidelines.
Norwich expect to have playmaker Wes Hoolahan available after shrugging off a heel problem while on international duty with the Republic of Ireland.
Striker Gary Hooper has stepped up his rehabilitation from an ankle injury, but is unlikely to feature this weekend.
Midfielder Youssouf Mulumbu has also returned to training this week following recovery from a fractured metatarsal, but will not be rushed into action.
Winger Matt Jarvis hopes Norwich can finally get a fortnight of frustration out of their system with victory on Sunday.
The Canaries were beaten 2-1 at home by Leicester before the international break, which was a first defeat in a promising run of four matches.
Jarvis, on loan from West Ham, admits not having another game to quickly try to put things right has made it a difficult spell for those left at the Norfolk club's training base.
"As a group it wasn't nice to lose before the international break as you have that feeling for two weeks," Jarvis said.
"We have had time to reflect on Leicester and want to put things right now."
Jarvis added: "We want to keep our away record going and have shown in games how well we can play.
"Everyone can see how good the team is, but you have to be consistent too.
"In every match this season we are looking to make a strong start to ensure the other team doesn't pick up momentum, and Sunday is no different."
AFC Bournemouth vs Newcastle United
GAdam Federici
DCharlie Daniels
DSylvain Distin
DAdam Smith
DSimon Francis
MMatt Ritchie
MAndrew Surman
MHarry Arter
MDan Gosling
MJunior Stanislas
FJoshua King
GRobert Elliot
DChancel Mbemba
DPaul Dummett
DDaryl Janmaat
DF. Coloccini
MCheick Tiote
MVurnon Anita
MMoussa Sissoko
MG. Wijnaldum
FAyoze Perez
FA. Mitrovic
Aston Villa vs Manchester City
GBrad Guzan
DMicah Richards
DAlan Hutton
DCiaran Clark
DJordan Amavi
M/DCarlos Sanchez
MCarles Gil
MIdrissa Gueye
MJordan Veretout
F/MJordan Ayew
FScott Sinclair
GJoe Hart
DBacary Sagna
DVincent Kompany
DA. Kolarov
DNicolas Otamendi
MKevin De Bruyne
MRaheem Sterling
MYaya Toure
FWilfried Bony
Watford vs Manchester United11' Watford 0-1 MU (Depay)
DMiguel Britos
DCraig Cathcart
DAllan Nyom
M/DIkechi Anya
MAlmen Abdi
MBen Watson
MJ. Manuel Jurado
MEtienne Capoue
FTroy Deeney
FOdion Ighalo
GDavid de Gea
DPhil Jones
DChris Smalling
MAnder Herrera
MM. Schneiderlin
MJesse Lingard
MJuan Mata
MAshley Young
MB. Schweinsteiger
MDaley Blind
FMemphis Depay
Watford vs Manchester UnitedHT
DMiguel Britos
DCraig Cathcart
DAllan Nyom
M/DIkechi Anya
MAlmen Abdi
MBen Watson
MJ. Manuel Jurado
MEtienne Capoue
FTroy Deeney
FOdion Ighalo
GDavid de Gea
DPhil Jones
DChris Smalling
MAnder Herrera
MM. Schneiderlin
MJesse Lingard
MJuan Mata
MAshley Young
MB. Schweinsteiger
MDaley Blind
FMemphis Depay
HT87' Troy Deeney (pen.) 1 - 1
Watford 0-1 MU
87' Troy Deeney (pen.) 1 - 1FT Watford 1-2 Manchester United
Manchester City vs Liverpool
GJoe Hart
DBacary Sagna
DMartin Demichelis
DA. Kolarov
DEliaquim Mangala
MKevin De Bruyne
MRaheem Sterling
MYaya Toure
MJesus Navas
FSergio Aguero
GSimon Mignolet
DNathaniel Clyne
DDejan Lovren
DAlberto Moreno
DMartin Skrtel
MPhilippe Coutinho
MRoberto Firmino
MJames Milner
MEmre Can
MAdam Lallana
Tottenham Hotspur vs ChelseaFT 0-0
GHugo Lloris
DJan Vertonghen
DDanny Rose
DKyle Walker
DToby Alderweireld
M/DEric Dier
MMousa Dembele
MRyan Mason
MChristian Eriksen
FHeung-Min Son
FHarry Kane
GAsmir Begovic
DB. Ivanovic
DKurt Zouma
DGary Cahill
DCesar Azpilicueta
MEden Hazard
MNemanja Matic
MCesc Fabregas
West Ham United vs West Bromwich AlbionHT 1-0
DCarl Jenkinson
DAngelo Ogbonna
DWinston Reid
DAaron Cresswell
MPedro Obiang
MManuel Lanzini
MCheikhou Kouyate
MVictor Moses
FDiafra Sakho
FMauro Zarate
GBoaz Myhill
DCraig Dawson
DGareth McAuley
DJonny Evans
DJonas Olsson
MS. Sessegnon
MDarren Fletcher
MJames Morrison
MClaudio Yacob
FSalomon Rondon
FJames McClean
Top 5 after 14 rounds
1. Manchester City 29
2. Leicester 29
3. Manchester United 28
4. Arsenal 27
5. Tottenham 25
history showed no cold horse like Leicester can win. they will break apart towards the endEvery season there will be some black horse in the beginning. Soon they will move down from the table. But if Leicester manage to finish at the top four, its already a miracle to them
Stoke City vs Manchester City7' Marko Arnautovic 1 - 0
GJack Butland
DP. Wollscheid
DRyan Shawcross
DGeoff Cameron
DErik Pieters
DGlen Johnson
MIbrahim Afellay
MXherdan Shaqiri
MGlenn Whelan
F/MMarko Arnautovic
GJoe Hart
DBacary Sagna
DMartin Demichelis
DA. Kolarov
DNicolas Otamendi
MKevin De Bruyne
MRaheem Sterling
MDavid Silva
FWilfried Bony
7' Marko Arnautovic 1 - 015' Marko Arnautovic 2 - 0
Stoke City 2-0 Manchester City
Arsenal 3-1 Sunderland
Manchester United 0-0 West Ham United
Southampton 1-1 Aston Villa
Swansea City 0-3 Leicester City
Watford 2-0 Norwich City
West Bromwich Albion 1-1 Tottenham Hotspur
Chelsea 0-1 AFC Bournemouth
I thought Vardy, Leicester City is very good for English standard, anytime better than the useless RooneyYes, I do think that he is better than Rooney to represents England in next year UEFA cup
Vardy is very fast and sharp, he has the skills of Ronaldo and Messi, good future
Yes, I do think that he is better than Rooney to represents England in next year UEFA cup
Leicester City vs Chelsea
I wish that Leicester will be the winner
vardy vardy ady ady :thumbsup:
I wish that Leicester will be the winnerLeicester 2-1 Chelsea :thumbsup:
Leicester 2-1 Chelsea :thumbsup::thumbsup:


text:
The Rev. John Lo Schiavo, who spent 14 years as president of the University of San Francisco, notably making headlines when he took the rare step of suspending the men's basketball program after repeated violations of National Collegiate Athletic Association rules, died on Friday in Los Gatos, Calif. He was 90.
The death was confirmed in an email by a spokeswoman for the university, Anne-Marie Devine Tasto, who said Father Lo Schiavo had dementia.
Father Lo Schiavo, a San Francisco native, was affiliated with the university, a Jesuit school, almost continuously for more than 60 years. He started as a philosophy instructor in 1950 and was later dean of students, vice president for student affairs, chairman of the board of trustees and rector of the university Jesuit community. A popular campus figure who was known as Father Lo, he was president from 1977 to 1991.
Nationally, Father Lo Schiavo is best known for his decision in 1982 to forgo the revenue, publicity and acclaim of the university's successful men's basketball program and instead stand up for institutional rectitude. His suspension of the program indefinitely was considered the first time a university had shut down a major sports program, without external pressure to do so, because of N.C.A.A. rules violations, some of which preceded his tenure.
Once a national power, the University of San Francisco Dons won 60 consecutive games from 1954 to 1956 and two national championships. Players included Bill Russell and K. C. Jones, who starred together in the National Basketball Association with the great Boston Celtics teams of the late 1950s and 1960s.
In the 1970s, led by players including Bill Cartwright, who went on to play for the Knicks and the Chicago Bulls, the Dons returned to prominence, making several appearances in the N.C.A.A. tournament. However, for a series of violations, including boosters' payments to athletes, questionable recruiting practices and tutors' taking tests for players, the basketball program was twice put on probation by the N.C.A.A., in the 1979-80 and 1980-81 seasons. One coach resigned; another was fired.
Then, in June 1982, Quintin Dailey, who had averaged 25.2 points per game for the Dons the previous season, pleaded guilty to aggravated assault for an attack on a nursing student, who said she had been fearful of being raped but was not.
Dailey was sentenced to three years' probation — he went on to play 10 years in the N.B.A. — but when a court document revealed that he had acknowledged receiving $1,000 for a no-show job from a university booster, Father Lo Schiavo acted. He announced the suspension of the program in August, before the 1982-83 season began.
The basketball program, he said at the time, "was once a source of inspiration, respect and pride for this university and city."
"That tradition adds to the sadness engendered by what it has now become," he went on. "Because of it, we have now been perceived as being hypocritical or naïve or inept or duplicitous, or perhaps some combination of all those. We have even had to suffer the accusation that we attempted to obstruct justice in order to protect a basketball player and preserve him for the team.
"However unjust those perceptions are — and they are grossly unjust — everyone who cares about U.S.F. must recognize that those perceptions have developed as a product of the basketball program. We have no responsible choice but to rid the university of the burden of them."
John Joseph Lo Schiavo was born in San Francisco on Feb. 25, 1925. His parents were immigrants from Salina, a minute island north of Sicily. His father and uncle ran a restaurant and candy shop.
Young John was an all-city basketball player at St. Ignatius High School. He studied philosophy at Gonzaga University in Spokane, Wash., where he received bachelor's and master's degrees. He was ordained a priest in 1955 after he joined the faculty at San Francisco.
As president of the university, he oversaw capital campaigns that enabled it to acquire Lone Mountain College (formerly San Francisco College for Women), now a campus of the university; to expand the law school; and to build a recreation center.
His administration balanced budgets, eliminated debt and increased the endowment more than eightfold, to $38.7 million from $4.6 million. New degree programs established included hospitality management, Judaic studies, Asia Pacific studies and sport management. After stepping down as president in 1991, he served as chancellor, concentrating on fund-raising and alumni relations.
Father Lo Schiavo "has done more good, raised more money and had a greater impact than anyone ever associated with the University of San Francisco," a campus publication, USF magazine, declared in 2010. He is survived by several cousins.
In June 1983, Father Lo Schiavo announced that he would reinstate the basketball program for the 1985-86 season, limiting its recruiting to players from the Western part of the country to diminish the frenetic competition engendered by nationwide recruiting, and establishing stricter controls over booster donations and athletes' academic life.
"I am not unguarded about U.S.F.'s and this nation's intercollegiate athletics in the future," he wrote in an essay in The New York Times. "I am keenly aware that athletics — college and professional — is an intensely competitive industry in America. There are still risks for U.S.F., despite all the precautions and controls we might have.
"The real question," he added, "is, 'Can an athletic program based on the right principles survive and thrive in this stressful environment?' I think so."
Since then, the San Francisco basketball team has appeared in one N.C.A.A. tournament, in 1998, losing in the first round.

subset: commoncrawl


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Say, the other day I bumped into Jon Lee, the congenial manager at my favorite book house, and after he made another customer happy he asked me to remind the flock that River City Books is going to be open on Memorial Day!
But — yes, there is a but — the store will not be open as late as is usually the case for a Monday. That is, the doors will close at 5 p.m. rather than 8 p.m.
To review, RCB's Memorial Day weekend hours:
Friday, 10 a.m. to 8 p.m.
Saturday, 10 a.m. to 5 p.m.
Sunday, 11 a.m. to 5 p.m.
Monday, 10 a.m. to 5 p.m.
I hope you have a fun, safe holiday and that good books are part of a joyous weekend.


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The Chef: Matt Owens, Harvest Pizzeria
The Chef: Matt Owens, Harvest Pizzeria
The Relationship: For two years, the German Village spot's gotten weekly orders from Wayward after they close up shop at the Bexley Farmers Market.
The Influence: The head chef relies on Wayward for produce to guide the daily pizza specials. "Right now," Owens says, "we're ordering and taking what they have available. It's fun to not have a plan, to see what they have and see what we can do with it."
Past weekly specials-which can make up to 20 percent of Harvest's pizza business-have showcased Wayward snap peas (for a pizza with peas, red onion and local bacon) and arugula (white pizza spiked with anchovies and topped with fresh arugula and dressed in oil).
It's not all about the pies for Owens. "I'd actually like to work a vegetable slaw into our menu as an appetizer because their Napa cabbage is really good. I've got some kimchi fermenting right now, and I'd like to use that as well."


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dinner is waiting for you
dinner is waiting for you
100% In-Home Culinary Experience. Chef Inspired. Customized. Convenience.
• 1 x 2.5 # side of salmon, skin on
• ½ T. caraway seed
• ½ T. coriander seed
• 1 tsp. black peppercorns
• 1.5 oz. salt
• 1 oz. sugar
• 4 oz. fresh dill
1. In a saute pan, toast the caraway and coriander seeds for 2-3 minutes. Allow to cool and transfer to a spice grinder with the black pepper and grind until smooth. Mix together with the salt and sugar. Set aside.
2. Cut the belly meat off of the salmon so you are left with 1 even strip of salmon side. Fill a large bowl with salted water and soak the salmon side for 15 minutes. Remove from the water, pat very dry and season, skin side with an equal amount of the seasoning mixture on 1 side. Lay out a long sheet of plastic wrap and place half of the dill down and place the seasoned side of the salmon on top of the dill evenly. Season the skin with the remaining seasoning and top evenly with the remaining dill. Wrap the salmon side up tightly and place on a sheet try, skin side down with a heavy baking pan on top. Refrigerate for 24 hours.
3. After 24 hours, flip the salmon over so that the skin side is up, place the heavy baking pan on top and put back in the refrigerator for 24 more hours.
4. Remove the wrap and remove the dill and remove as much seasoning as possible. Pat both sides with a paper towel.
5. Slice very thin with a very sharp knife diagonally.


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Often estimating a project can feel overly complex and difficult, but leadership coach Susanne Madsen shows you how to make project estimation simple and easy.
Here's a shot of the whiteboard for your reference!
In Review: How to Estimate Projects, Simply
The first thing Susanne acknowledged is that project estimates are often wrong. But she didn't leave it there. She offered these four reason why they can be miscalculated.
- Being too optimistic
- Not being clear about what we're estimating
- Not factoring in everything that needs doing
- Don't distinguish between effort and duration
To avoid these pitfalls she offered using Program Evaluation and Review Technique (PERT), which uses three points to get better estimations. Basically it takes the most optimistic, most likely and most pessimistic estimates and finds the sweet spot.
By factoring in a full range of possibilities, you give yourself a more accurate peek into the future of your project.
Pro-Tip: You may also want to have your team trained to estimate. By sharing the responsibilities of the job you can get more perspectives and a closer approximation to how long it will take and how much it will cost.
Thanks for watching!
Hi, I'm Susanne Madsen [SP]. Welcome to this White Board session on estimation. We will take something that can at times be quite complex and make it simple and easy for you to use.
The thing about estimation is that we often get it wrong. There are a number of reasons for that and I've written them up on the board behind me. The first one is that we tend to be overoptimistic. I think this is human behavior. If I ask you how long it takes you to decorate your spare bedroom you might say, "Oh, one day, easily." But once you get there you realize that it takes you a lot longer.
Other reasons are that we are not good enough at looking at all the detail of what needs to get done, and we don't factor in all the aspects. If I ask a builder how long it takes him to build a house for me he might say three months, but we haven't discussed the fact that I also expect a landscaped garden, a beautiful driveway, a specially-designed roof, and three bathrooms. So we have to talk about the detail of the work before we can reasonably provide an estimate for it.
The last reason I've written up behind me on the board for why we get estimates wrong is that we confuse effort and duration. They are two very different types of units. If I ask my builder how long it takes him to build my house and he maintains three months, then great. I think I'll get my house in three months' time.
But what I don't know is that my builder is also going to work on my neighbor's house, so he can only be with me every second day. So instead of getting my house in three months' time, I'm actually getting it in six months. This happens on projects all the time. You forget that your developers are also working on other projects. So do distinguish between effort and duration.
Now let's turn our attention to the PERT estimation technique. It's a three-point technique that can help you to estimate more realistically. PERT stands for Program Evaluation and Review Technique. What we're doing here is that we're taking the most optimistic estimate, plus four times the most likely, plus the most pessimistic estimate, divided by six. It's a weighted average.
Let's look at an example. Imagine that you're asking your developer how long it takes to create a new web page for you. He gives you a most optimistic estimate of two days and the most pessimistic estimate of seven days, but the most likely, he says, is three. If you use the PERT estimation technique, you'll see that that gives you an outcome of three and a half days. If this is an effort estimate, meaning that he is working fully for you, your next task is to find out by when you can get those three and a half days by.
Thank you for watching. Please visit us again at projectmanager.com.


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Wages, Profits, and Macroeconomic Adjustment: A Comparative Study
NBER Reprint No. 74
This paper is not currently available on-line.
Machine-readable bibliographic record - MARC, RIS, BibTeX
Published: Sachs, Jeffrey D. "Wages, Profits, and Macroeconomic Adjustment: A Comparative Study." Brookings Papers on Economic Inquiry, edited by Arthur M. Okunand George L. Perry, Vol. 2, pp. 269-332. Washington, D.C.: The Brookings Institution, 1979.
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text: JOIN THE TEAM! We're currently hiring a Managing Director of the NLG Foundation. This is a full-time, salaried position with benefits based in the NLG National Office in New York City. Candidates should have a strong background in fundraising and development. Click here for the full announcement and to apply.


text: This introduction to the ideas and methods of linear functional analysis shows how familiar and useful concepts from finite-dimensional linear algebra can be extended or generalized to infinite-dimensional spaces. Aimed at advanced undergraduates in mathematics and physics, the book assumes a standard background of linear algebra, real analysis (including the theory of metric spaces), and Lebesgue integration, although an introductory chapter summarizes the requisite material. Highlights of the second edition include - a new chapter on the Hahn-Banach theorem and its applications to the theory of duality. This chapter also introduces the basic properties of projection operators on Banach spaces, and weak convergence of sequences in Banach spaces; - topics that have applications to both linear and nonlinear functional analysis; - extended coverage of the uniform boundedness theorem; - plenty of exercises, with solutions provided at the back of the book.


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Hi all, I have a bride with celiac's disease (no gluten). I normally use the Henry & Henry sleeve fillings. The box says they contain modified food starch, which sounds like they are not gluten free. Anyone know how to make a filling with no gluten? It will be for a small tier of the cake, the rest of it will be a regular cake.


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Toronto, Ontario – Left Field Brewery have released a new one-off beer today – Bang-Bang Dry Hopped Sour. Named for a close play that happens so fast you might miss it – or have to take a second look.
This refreshing sour pops with aromas of freshly sliced lemon and papaya, with flavours of bright fruit punch, sour candy, tangerine and subtle hints of pineapple. Dry hopped with Citra, Summer and Simcoe, a fruity, tropical hop punch and crisp, tart finish make Bang-Bang a perfect summer refresher worthy of a replay.
Available in their Tap Room and Bottle Shop today.


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Home decor in India is extremely underrated. Functional, premium home decor, even more so. Very few people in the country realise that there's a certain sense of beauty associated with it – after all, the job of home decor is to add (and increase!) the aesthetic value of your room, and your home.
Trouble is, while there are scores of decor stores in the country, mostly catering to pieces of art that can be stored or hung in a place that falls in your peripheral vision, the people in India have never seen the humble ceiling fan as a potential decor item.
A perennial problem for the aesthetically conscious, the drab old ceiling fan poses quite a challenge. It's certainly necessary unless one wishes to be broiled alive in the (dis)comfort of their unventilated living space.
But having a fan in an exquisitely hideous shade of brown bang in the centre of the ceiling leaves little room for more elegant embellishment.
How often have you wished you could have something nicer to look at than three dust-coated blades that turn into a dust-coated blur every time you look at your ceiling?
Maybe wished for something truly impressive when your snooty distant relatives come to visit?
Well, all of that is about to change.
After months of research, planning, implementation, engineering, and execution, Fanzart has created its showstopper, and a true masterpiece – the Fandelier!
And yes, as the name itself suggests, the Fandelier is a portmanteau; a combination of two things we all need and want on our ceiling – a functional fan, and a stunning, gorgeous chandelier.
But the real question on everyone's mind, we're sure, is this: how does it work? Well, let's break it down!
Let's start with its design. Unique, one of a kind, and oh-so-stunning, the design for the Fandelier has been inspired by the gorgeous city of Venice. Its beauty has been embedded into the look and feel of the fan, and thus, adds more than a touch of resplendence to your space. Furthermore, given its radiance, the Fandelier makes for the perfect 'centrepiece' of sorts. The luxury fan has also been equipped with as many as 70 LED lights and 120 crystals – all of which as to its striking beauty. This comes in handy in more ways than one, particularly when not in use, because it doubles up as a home decor product!
When it comes to the fan aspect of the creation, especially its technical features, the Fandelier knocks it out of the park there as well. Called the 'Venetian', it has an incredible 14 foldable acrylic blades around its gorgeous LED mounted crystal dome – which means, when not in use, they collapse, fold, and don't use up any space! This ceiling fan with lights also comes equipped with a strong and reliable DC motor, which means you won't have to worry about replacing the core machine for a while after purchasing it. The fan also come with a remote control kit –– which ensures that you can control the fan, and its speed, right from the comforts of your couch.
All of this sounds like a dream, doesn't it? Well, that's not all the fan offers, and we're not done yet!
The Fandelier is also bi-directional –– and if you're confused what that means, let us explain it to you further. The direction in which your fan rotates plays a crucial role in determining the airflow. So if the fan rotates anti-clockwise, the breeze will come directly at you, whereas in the case of a clockwise rotation, the air will vortex upwards, and circulate in the room without giving you the cool breeze. The former comes in handy in the hot, humid, sweltering summers, whereas the latter comes in handy come the nippy winters! And thus, we can establish that the Fandelier has been tailor-made for every season –– and that's not where it ends either.
Despite the fact it boasts the splendid bi-directional technology, contains an incredible DC motor, and is embellished and adorned with 70 LED lights and 120 crystals, the Fandelier consumes only 55 watts of power – which is extraordinary levels of efficiency, that is unmatchable in the Indian market today.
The Founder Speaks
The founder of Fanzart, Mr. Anil Lala, says, "With mesmerizing craftsmanship, inspired by the iconic city of Venice, regarded by connoisseurs as one of the greatest hubs of art, Venetian has as many as 14 foldable acrylic blades around a stunning LED mounted crystal dome and remote control kit for comfort and convenience."
Explaining further, he adds, "With its unique construction, the Venetian is priced at Rs. 69,990, and is equipped with 70 LED lights and 120 crystals, that consumes only 55 watts of power. When not in use, the Venetian ceiling fan looks like an astonishing ceiling light fixture, with 14 foldable blades which can also be collapsible blades around a stunning mounted crystal dome. Its exclusive motor technology ensures that the fan will remain quiet, while in operation.
"Our range of Fandelier Fans, are perfect for the upcoming festive season. Not only do Fandelier Fans illuminate your room in style but also brings the breezy flair to your celebrations!"
The Features
- At 82 cms, the fan is perfect for a large bedroom, drawing room, living room, dining area, and even hotels and bars.
- Remote Control Kit
- 14 blades, each of which is retractable and collapsible when not in us
- 52 inches long
- A 20 mm, powerful DC motor
- 38 watts LED
- Multiple colours
And well, there you have it. With a ton of top-notch features, flawless, stunning designs, and aimed to please the trained eye, you can't miss out on installing one. Or more than one, really –– we're more than happy to help you there!
For what makes four walls a house are the functional appliances, true. But what makes a house a home is when the appliances are no longer just bland and forgettable, but beautiful pieces of art that make you want to come back every time you leave.
You can learn more about Fanzart and its beautiful Fandelier on our website here: https://www.fanzartfans/


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A Wisconsin couple said they may be evicted from their apartment for displaying a gay pride flag.
"This is a place we live. It's our home," Kevin Kollman said.
He and Merle Malterer have called the Country Oaks Apartments home for six years, taking pride in the property and themselves.
"This is who I am, this is who we are," Kollman added.
They've always decorated outside, but they said it's recently become a problem.
"Personally, I feel like we're being kind of singled out here because it's what it is," Kollman said.
On Wednesday, they got a note saying they breached their lease and have five days to take down the gay pride American flag.
"It's a symbol of America, it's a symbol of our pride in America being a gay couple here in Oak Creek," Kollmann said.
Local media reached out to the property owner who said the couple was asked to take down a Green Bay Packers flag back in January, and that this is not an issue of discrimination, but an issue with the property's appearance.
He added that nobody is allowed to have flags anywhere, and it's against the lease agreement.
"I've never been told to take them down," said Ken Miller, another apartment tenant.
When local media looked around the property, they found several flags, including outside of Miller's place.
"I've been here for three years, and I've had these flags up for at least two years and I've never had any issues with the flags," Miller said.
Kollmann and Malterer plan not to keep the flag flying.
The property owner said he doesn't want to evict the couple and he'd be willing to sit down with them.
The couple said they're looking into taking legal action.
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Divorcing Your Mortgage
In our day and age, divorce and separation is an inevitable occurrence that affects us all. We have all known someone directly, or have been that someone, who has experienced a divorce. It can be a very stressful and messy time, but it doesn't have to be overwhelming. Knowing your options when it comes to dealing with your home, and it's existing mortgage, will alleviate large concerns.
When you go through a divorce, your assets and finances get distributed according to your separation agreement or court order. Usually, people's largest asset is their home and any other properties owned within the marriage or partnership. If you find yourself holding a mortgage on the property(ies) you own during a divorce or separation, rest assured you have options.
An assumable mortgage is one that can be transferred without changing the terms of the original mortgage. One partner can assume the current mortgage if the mortgage loan is assumable and both agree and sign an assumption agreement. It is best to check with your mortgage broker to find out further details and processes for assuming the mortgage. If the mortgage is assumable, this can be a cheaper alternative to refinancing.
Canada Mortgage and Housing Corporation (CMHC) recently changed the guidelines on refinancing; now, homeowners are only allowed to refinance up to 80% of their home's value. However, what most people are not aware of is that in circumstances of separation or divorce, CMHC will allow you to refinance up to 95% of your home's value. In this case it allows one partner to buy the home back from the other partner with only 5% down. The partner keeping the home can, therefore, pays the share of equity owed to their partner and refinances the home in their name only.
If one person keeps possession of the home while both partner's names are on the mortgage and any payments are missed it can damage both individuals' credit scores.
Most commonly, when people go through a separation or divorce they decide to sell the partnership home. The equity in the home will then be distributed between both partners as set out in a separation agreement or by the courts. When you are considering selling the home, consider any penalties that may arise from eliminating your contract early and redoing a new one on your own. Also, it is important to remember that child and spousal support payments that are paid, count as an expense; likewise, child and spousal support payments that are received, count as income. This may affect your ability to get approved for another mortgage on your own.
Going through a separation or divorce can be very costly. It's often a good time to draw on the equity in your property, consolidate other debts, and be realistic about what you can afford in a new mortgage. Contact AC Lending for expert advice on how to manage your mortgage through a separation or divorce.


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Handy Interior Design Tips That Will Enhances Your Home
Are you looking to brighten up a dull room and searching for interior design tips? One great way to help you liven up a room is by painting and giving it a whole new look. The following article will give some helpful information on ways you can go about adding color through paint to help turn a dull room into one that pops.
Set a budget for your interior-design project before you start any work. Costs for home-improvement projects can quickly spiral out of control if you don't set a spending limit. While you can be a little flexible, you shouldn't spend more than you can afford. Having a budget can help you be more creative as well.
Be sure that every room has excellent lighting. Quality lighting will help you show off your home, and can even help a room feel bigger. If there's a room in your home that has dark corners even with the lights turned on, invest in a stylish new lamp. It'll make the whole room look better.
When you are getting ready to paint a room, paint just a portion of a wall first, and wait a few days before you decide. You can avoid the hassle and added cost of re-painting a poor color choice by seeing how you feel about it after a couple of days.
Take your sofa cushion shopping with you. It might feel silly, but taking along part of your sofa can save you a lot of time any money. Bring it with you when shopping for curtains or other decorating materials. This way, you will be able to ensure that everything matches.
It can be difficult to decorate a basement because you cannot possibly imagine what you could do in such a dark and gloomy place. If you use some brighter colors and fabrics, you can turn your dark, damp, depressing basement into a place where you will want to spend time with your family.
It's very common for a home to have at least one small room that needs to feel larger than it really is. Light colored walls and ceilings create the illusion of a larger space. Dark colors have the tendency to shrink an already little space.
As you plan your interior design layout, keep it realistic. While you want to add some personality to your home, you don't want to do anything drastic that you will regret later. Selling your home will become more difficult if a buyer would have to remodel something just to move in. If there is something that you just must do to your home that might not be considered normal, just remember that you may have to remove it later on.
Now that you read the article from above, it is very simple to turn an ordinary room into a room that dazzles by adding some color through painting. Stick to the tips presented in this article to help give you some solutions to turning that boring room into a room that nice and relaxing.


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Mas Finance's aims to be one of the most efficient distributor of financial services. Efficiency in general describes the extent to which time, effort (labor) or cost (money) is well used for the intended task or purpose. It means optimum use of available resources & to generate the best possible productivity by them.
We firmly believe that even the biggest company can collapse if they are not efficiency-conscious and even the smallest company can be a superpower if they pay attention to increasing their efficiency.
Banking & Finance
ASP.Net, Bootstrap, jQuery
Ahmedabad, India


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- This event has passed.
Pride Picnic
Wednesday, June 29, 2016 @ 1:00 pm - 5:00 pm
The fifth annual Pride Picnic is here!
On June 29, We would like to invite you to Pride Picnic 2016! This free event features food, music, games, prizes, and more. Make sure to swing by to enjoy some good food, good music and good people! U of T Pride t-shirts will also be available for pickup. We will have paint ready for you to decorate your t-shirts at the picnic!
Date: June 29, 2015
Time: 1 – 5 pm
Venue: Woodsworth College Courtyard (Bronfman) at 119 St.George Street (Bloor and St.George Intersection)
This event is wheelchair accessible. Accessible washrooms are located on the first floor and gender-neutral washrooms are located on the second floor of Woodsworth. There will be signage for both.
Food will be served at this event, including vegan and gluten-free, halal and vegetarian options.
If you have any questions, accessibility requests, or want to volunteer with us, please contact firstname.lastname@example.com.
Association of Part-time Undergraduate Students (APUS) University of Toronto Mississauga Students' Union (UTMSU) Woodsworth College Student Association (WCSA)
UT Graduate Students' Union (GSU),
Scarborough Campus Students' Union (SCSU)
Students' for Barrier Free Access (SBA)
Woodsworth Inclusive (WINC)
University of Toronto Students' Union (UTSU)
Move U
Equity Movement


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Novel techniques were used to determine when in the cell cycle of proliferating NIH 3T3 cells cellular Ras and cyclin D1 are required. For comparison, in quiescent cells, all four of the inhibitors of cell cycle progression tested (anti-Ras, anti-cyclin D1, serum removal, and cycloheximide) became ineffective at essentially the same point in G1 phase, approximately 4 h prior to the beginning of DNA synthesis. To extend these studies to cycling cells, a time-lapse approach was used to determine the approximate cell cycle position of individual cells in an asynchronous culture at the time of inhibitor treatment and then to determine the effects of the inhibitor upon recipient cells. With this approach, anti-Ras antibody efficiently inhibited entry into S phase only when introduced into cells prior to the preceding mitosis, several hours before the beginning of S phase. Anti-cyclin D1, on the other hand, was an efficient inhibitor when introduced up until just before the initiation of DNA synthesis. Cycloheximide treatment, like anti-cyclin D1 microinjection, was inhibitory throughout G1 phase (which lasts a total of 4 to 5 h in these cells). Finally, serum removal blocked entry into S phase only during the first hour following mitosis. Kinetic analysis and a novel dual-labeling technique were used to confirm the differences in cell cycle requirements for Ras, cyclin D1, and cycloheximide. These studies demonstrate a fundamental difference in mitogenic signal transduction between quiescent and cycling NIH 3T3 cells and reveal a sequence of signaling events required for cell cycle progression in proliferating NIH 3T3 cells.
The cell cycle requirements for peptide growth factors have been demonstrated in cells rendered quiescent by growth factor deprivation (24). At a certain point following stimulation to reenter the cell cycle, the continued presence of a variety of growth factors became unnecessary for continuation through G1 phase and the initiation of DNA synthesis (25). This point of commitment to cell cycle progression, termed the restriction point, is generally observed several hours before the initiation of S phase. The restriction point is thus defined as the last point at which positive proliferative signals are required for completion of the cell cycle. Interestingly, in quiescent cells the restriction point is identical to the G1 arrest point induced by other conditions, including protein synthesis inhibitors and amino acid deprivation (29).
While results obtained with quiescent cells are important, there is reason to believe the situation might be different for cells continuously progressing through the cell cycle. For example, passage through G1 phase in quiescent and proliferating cells exhibits several important molecular differences, including expression of the cyclin-inhibitory protein p27kip1(3), Cdc6 (42), and Fos and related proteins (14), and formation of a complex between E2F and the retinoblastoma protein (Rb)-related p130 (19, 34). In addition, the time required between the restriction point and initiation of S phase (near 4 h in the case of the cells used in this study) is nearly as long as the entire G1 phase when these cells are actively cycling. In cells with a short G1phase, a prolonged period between completion of all required signaling events and the initiation of S phase would not appear warranted or perhaps even allowed. Indeed, since the overall length of the cell cycle appears to depend in large part on the length of the G1 phase, it is likely that this cell cycle period must be compressed as much as possible for rapid proliferation, potentially requiring the transfer of activities present in the G1phase of quiescent cells to other cell cycle periods when they continuously cycle (26).
Foremost among the positive proliferative signaling pathways in fibroblast-like cells is the one involving peptide growth factors and cellular Ras (30). Upon binding to their receptors, peptide growth factors initiate a cascade of signaling events which leads to the activation of cellular Ras, which in turn functions as a molecular switch to transmit mitogenic signals to a cascade of cytoplasmic kinases (35), ultimately resulting in the activation of mitogen-activated protein kinases (MAPKs) (20). MAPKs stimulate transcription factors which in many cells turn on the expression of cyclin D1 (1). Cyclin D1 binds to the cyclin-dependent kinases 4 and 6 to form an Rb kinase (31). Upon phosphorylation, Rb loses its repressive activity for the E2F-DP transcription factor complex, which then activates transcription of many genes required for transition from G1 to S phase and for DNA replication (22). Therefore, cyclin D1 is essential for G1/S transition in Rb-positive cells (17, 23). It is believed that this molecular sequence forms a cascade vital to the positive stimulation of cell cycle progression.
Because Ras and its downstream targets play a central role in regulating mitogenic signaling within the cell, it is conceivable that Ras plays a role in progression past the restriction point. This has been verified by using microinjection of an efficiently neutralizing anti-Ras antibody. While Ras activity is required multiple times during the stimulation of quiescent cells to proliferate, the final of these requirements appears to be at or near the restriction point (5, 21, 38). In addition, cyclin D1, the cyclin-inhibitory protein p27kip1 (32), and phosphorylation of Rb (41) have each been proposed to be critical for passage through the restriction point.
Although it is apparent that growth factors and Ras activity are required for proliferation in actively cycling cells (21, 27), the timing of these requirements remains to be determined. Such analyses are complicated on one hand by the fact that cell cycle synchronization induces alterations in cell cycle characteristics and on the other hand by the difficulty in performing cell cycle analyses without synchronization. Time-lapse analyses have been used to determine in asynchronous cultures a point in G1 phase at which growth factors become unnecessary for continued proliferation (15, 44). These studies avoided the necessity of synchronization and perturbation of the cell cycle resulting therefrom. We have extended these studies by using time-lapse analyses combined with quantitative fluorescence cytochemistry and microinjection to analyze the requirements for signaling molecules in continuously proliferating cells. The results reveal a requirement for positive signaling molecules fundamentally different from that previously reported for quiescent cells.
Materials.Cy3-conjugated anti-rat immunoglobulin G (IgG), Cy2-conjugated anti-rat IgG with minimal cross-reactivity against mouse IgG, Cy3-conjugated anti-mouse IgG, and Cy3-conjugated anti-mouse IgG with minimal cross-reactivity against rat IgG were purchased from Jackson ImmunoResearch (West Grove, Pa.). Anti-ACTIVE MAPK polyclonal antibody was obtained from Promega (Madison, Wis.). Mouse monoclonal anti-mouse cyclin D1 antibody 72-13G (Santa Cruz Biotechnology, Santa Cruz, Calif.) was used for all staining applications. Nonimmune rat IgG was obtained from Sigma (St. Louis, Mo.). [methyl-3H]thymidine (80 Ci/mmol) was purchased from Amersham (Arlington Heights, Ill.). Autoradiography emulsion-type NTB2 was obtained from Eastman Kodak Company (New York, N.Y.). DAPI (4′,6-diamidino-2-phenylindole, dilactate) was a product of Molecular Probes (Eugene, Oreg.).
Antibody preparation.Hybridomas expressing neutralizing antibodies against either Ras (Y13-259, rat IgG) or cyclin D1 (DCS-6, mouse IgG; a generous gift from Michele Pagano) were grown in serum-free medium (Hybridoma-SFM; Gibco BRL, Gaithersburg, Md.). Each secreted antibody was purified accordingly, using ammonium sulfate precipitation followed by DEAE chromatography (13). The purified antibodies were dialyzed against 25 mM Tris-HCl (pH 7.5) and concentrated to 10 mg/ml in a Centricon 30 (Amicon, Bedford, Mass.). These antibodies were used exclusively for microinjection.
Cell culture and microinjection.NIH 3T3 cells were maintained and made quiescent as described elsewhere (11). To determine the requirement for protein synthesis, cells were treated with 2 μM cycloheximide, which suppressed overall protein synthesis by 95 to 98% (33). The antibodies indicated were injected into cells grown on a coverslip as described previously (37).
Time-lapse video photography.Digital images were obtained with a charge-coupled device (CCD) camera (Sony; Tokyo, Japan) attached to a frame capture board controlled by the NIH Image program. Individual frames of 640 by 480 pixels were captured every 5 to 15 min. Up to 310 individual frames were captured in a single stack, which was then replayed by the NIH Image program at various speeds in the forward or backward direction to create a movie for analysis. Images of cells were obtained with phase-contrast optics and a 4× objective to allow analysis of the largest possible area of the coverslip. The area of the coverslip to be analyzed was marked with two contiguous circles of varying size by using a diamond object marker (Leitz). This allowed realignment of the area of analysis and identification of individual cells following immunostaining, DAPI staining, and/or autoradiography. When a second time-lapse analysis followed microinjection, care was taken to ensure that the same area of the coverslip and same alignment were viewed in each movie. Antibodies to be studied were microinjected into all the cells, and only the cells, within the designated circular area. In this way it was possible to follow the analysis from the beginning and determine which cells had arisen from originally injected cells. The injection was further confirmed by immunofluorescence staining for injected antibody.
To avoid possible complications with this constant illumination, several experiments were duplicated with a similar approach except that the light was shuttered. In this case, the cells were illuminated only during the time of exposure, less than 1 s every 10 min. These images were collected with a highly sensitive, cooled CCD camera (Princeton Instrument) and controlled by the Metamorph software package (Universal Imaging). Because of the sensitivity of the cooled CCD camera, the cells were illuminated with extremely faint light. In these cases, there is little possibility that the illumination of cells would have any effects on the results obtained. In no case were the results obtained with the two methods of illumination different.
Immunofluorescence staining and detection of DNA synthesis.For immunostaining, the cells were fixed with methanol for 5 min at −20°C. The cells were rehydrated with phosphate-buffered saline (PBS) and blocked with PBS containing 3% (vol/vol) normal goat serum (Gibco BRL) for 1 h at room temperature. The antibody solution was made in blocking solution. After incubation with primary antibody for 1 h at room temperature or overnight at 4°C, the monolayer was rinsed with PBS for 5 min thrice. The cells were incubated with fluorochrome-conjugated specific secondary antibody (1:1,000 dilution). After three washes with PBS, cells were stained with DAPI (10 mg/ml) and mounted with 50% glycerol in PBS. To detect DNA synthesis, 5-bromo-2′-deoxyuridine (BrdU) labeling was performed with a 5-Bromo-2′-deoxy-uridine Labeling and Detection Kit II (Boehringer Mannheim, Indianapolis, Ind.) according to the manufacturer's instructions except that positive cells were visualized by Cy3-conjugated anti-mouse IgG antibody. S phase was also detected by labeling with [3H]thymidine followed by autoradiography as described before (11). Measurement of immunofluorescence or DAPI signal was performed before autoradiography.
Measurement of fluorescent signal of each stained cell.A Leica model DM 900 fluorescent microscope was used to quantitate fluorescence intensity. The filter cubes A, L4, and N2.1 were used to detect signals from DAPI, Cy2, and Cy3, respectively. With those cubes, no significant crossover signal was detected. Digital images were captured with a cooled (−25°C) CCD camera (Princeton Instrument) controlled with Metamorph software. The exposure time was adjusted so that the brightest signal in the specimen gave less than 90% of the maximum linear range for the camera (a gray scale of 0 to 4,096; 12-bit gray scale). Integration of the fluorescence signal from each nucleus was obtained by processing the image by using Metamorph analysis features.
Determination of the restriction point in quiescent cells.For the purposes of comparison with cycling cells, it was first necessary to carefully determine the point at which various inhibitory treatments lose their ability to block cell cycle progression following serum addition to quiescent cells. Quiescence was induced by culture for 48 h in 0.5% serum. One set of these plates was stimulated with serum and injected with anti-Ras antibody Y13-259 at various times thereafter. Cells were treated with tritiated thymidine immediately following injection until 24 h after the original addition of serum. Thus, labeling of these cells with thymidine would indicate that the injected antibody had failed to block cell cycle progression to S phase. Thymidine labeling would indicate that the injection had taken place after the critical point in G1 phase at which cellular Ras is last required prior to commitment to enter DNA synthesis (Fig. 1E). In the second set of plates, tritiated thymidine was added together with serum, and the cells were fixed various times thereafter. In these plates, thymidine labeling would indicate that the cells had progressed into S phase by the time of fixation (Fig. 1E). From the injected set of plates, the point of Ras requirement following serum addition was determined; from the second set of plates, the timing of entry into S phase was determined.
Anti-Ras efficiently blocked thymidine labeling when injected within 5 h of serum addition and inhibited by 50% entry into S phase when injected near 8 h following serum addition. In these cultures, half of the cells entered S phase near 12 h following addition of serum (Fig. 1A). Thus, the last point at which cellular Ras was required was approximately 4 h prior to the initiation of S phase. For comparison, in the same experiment, a third set of plates was treated as described above except that anti-cyclin D1 antibody (from M. Pagano) was injected instead of anti-Ras antibody. In these cultures, the pattern of inhibition of entry into S phase was indistinguishable from that observed with anti-Ras injection, indicating that within the limits of this determination, the last requirement for cyclin D1 prior to entry into S phase was identical to the last point at which cellular Ras was required (Fig. 1A). Similar analyses were performed to determine the last point at which protein synthesis or serum was required by treatment with 2 μM cycloheximide (Fig. 1B) or by serum removal (Fig. 1C). The timing of inhibition with these two treatments was indistinguishable from the timing of inhibition by injected anti-Ras or anti-cyclin D1 antibody.
Finally, the inhibition points identified above were compared to the timing of appearance of cyclin D1 in the nuclei of quiescent cells following treatment with serum. The level of cyclin D1 in individual cells was determined by indirect immunofluorescence using anti-cyclin D1 at various times following serum addition. The amounts of cyclin D1 were then determined by quantitating the fluorescence levels with a sensitive CCD camera and image analysis using the Metamorph software program. Nuclei were identified by DAPI staining, and only fluorescence over the nuclei was measured. Numbers reported represent the averages of approximately 300 cells. The levels of cyclin D1 in these cultures began to increase at 5.5 h following serum addition and reached a peak at 11 h, after which the cyclin D1 returned to modest levels (Fig. 1D). Half-maximal cyclin D1 levels were observed slightly after the time of half-maximal inhibition by anti-Ras and anti-D1 (Fig. 1A and D). It is not possible to determine the minimal level of cyclin D1 required to promote cell cycle progression, and therefore it is not possible to strictly relate the data on D1 levels to inhibition by anti-D1 antibody, but the timing of cyclin D1 expression and the requirement for cyclin D1 as determined by antibody injections were similar. To confirm the analyses described above, the total amount of cellular cyclin D1 protein was determined at various times following serum addition to quiescent NIH 3T3 cultures by Western analysis. The total amounts of cellular cyclin D1 as determined by Western analysis were similar to the results obtained as described above with antibody staining (data not shown). The close correlation between total cellular cyclin D1 protein and nuclear staining confirms the quantitative nature of the fluorescence staining measurements.
Cell cycle characteristics of cycling NIH 3T3 cells.Any previously identified means of synchronizing continuously proliferating cells would result in alterations of their cell cycle characteristics. For example, mitotic shake-off of NIH 3T3 cells resulted in a G1 phase of near 10 h (unpublished data), while the G1 phase determined by time-lapse analysis was about 5 h (see below). We therefore performed all studies without synchronization. Instead, time-lapse analyses of an asynchronous culture was used to identify the approximate cell cycle position of individual cells. The time-lapse analysis made it possible to determine how long prior to the termination of the experiment an individual cell had passed through mitosis. This value, termed the age of the cell, was used as an indication of the approximate cell cycle position (Fig.2) (44). To relate age with cell cycle position, a culture was followed in time lapse for more than 20 h, with the final 30 min in the presence of BrdU. The cells were then fixed and stained with a fluorescent antibody against BrdU. The age of each cell was determined individually by following that cell in the time-lapse movie beginning at the time of fixation and running the movie backwards until the cell could be observed to pass through mitosis (Fig. 2). Labeling with BrdU would indicate that the cell had been in S phase during the last 30 min. This approach allowed an analysis of cells in all cell cycle phases at the same time.
To determine the age at the time of entry into S phase, we grouped together all cells within a given age period and determined the proportion of these which remained unlabeled with BrdU (Fig.3A). Cells began to enter S phase within 4 h, approximately half were labeled at 5 h, and most cells had entered S phase within 7 h following mitosis. At 17 h following mitosis (the average generation time of the culture), the proportion of BrdU-labeled cells had declined to near zero (Fig. 3A). This indicates that cells from 4 to 7 h of age were in G1 phase, while G2 phase was first observed in cells 11 to 12 h of age. Cells weakly labeled with BrdU would have been in transitions to or from S phase at the time of labeling. Their appearance was taken as an indication of early and late S phase, and they were observed with peaks at 5 and 10 h of age, which indicates the general boundaries of S phase (Fig. 3B). While the cycling characteristics of individual cells vary (with cell cycle times of 13 to 23 h), the overall characteristics of the entire culture were quite consistent between analyses. Therefore, from 100 to 250 cells were analyzed in each experiment to compensate for random variations between individual cells.
Ras and cyclin D1 requirements in cycling cells.To determine the cell cycle point at which Ras is required in proliferating NIH 3T3 cells, a culture was followed in two sequential time-lapse movies of the same area of the coverslip. Anti-Ras antibody was microinjected between the two movies into cells in the area of analysis (localized within a circle scribed on the back of the coverslip). The age of cells at the time of injection was determined from the first movie, while the fate of injected cells was determined from the second (Fig.4D). For comparison, the behavior of neighboring uninjected cells was also determined. Surprisingly, almost all of the injected cells passed through mitosis following anti-Ras injection, regardless of the cell cycle position at the time of injection. To more carefully analyze the data, the length of the cell cycle in which the injection took place was calculated by determining the time of the mitoses before and after injection for each cell (Fig.4D). The combined data from five separate, highly similar experiments clearly indicate that while some injected as well as uninjected cells were delayed in passage through mitosis following treatment, most injected cells passed through the cell cycle without delay compared to uninjected cells, regardless of the cell cycle position at the time of injection (Fig. 4A).
These data were summarized by calculating the proportion of cells with lengthened cell cycle times (greater than 30 h) versus the age at the time of injection. The level of inhibition was generally less than 20% even for cells soon after mitosis (Fig. 4C). Surprisingly, these data indicate that there was no requirement for cellular Ras during the first G1 phase of the cell cycle following injection in asynchronous NIH 3T3 cells. This conclusion was apparent from the combined data and from each of the five individual experiments. The effectiveness of the injected antibody was confirmed by the fact that while most all injected cells passed through one mitosis following injection, very few cells passed through a second mitosis (Fig. 4C). Thus, anti-Ras was inhibitory for the progression of cycling cells, but only if it was introduced into the cell prior to mitosis, in the preceding cell cycle.
The cell cycle requirement for cyclin D1 was next determined by microinjecting a neutralizing, highly specific monoclonal antibody. The results from three highly similar (Fig. 4A) experiments were combined to indicate that the inhibitory characteristics of anti-cyclin D1 were quite different from those of anti-Ras. Cells injected with anti-D1 within the first few hours following mitosis were essentially all delayed in passage through the current cell cycle compared to uninjected cells. By 8 h of age, however, the proportion of cells which were inhibited in cell cycle passage by anti-D1 injection was low (Fig. 4B). Summary of these data (Fig. 4C) shows that the inhibitory behavior of injected anti-D1 was strikingly similar to the profile of entry into S phase (Fig. 3A). Inhibition by anti-cyclin D1 antibody was high in the first 3 h following mitosis, when the cells were all in G1 phase. From 4 to 7 h after mitosis, the proportion of cells in S phase increased, while inhibition by anti-cyclin D1 decreased. Therefore, cyclin D1 was required during G1 phase of cycling NIH 3T3 cells and was apparently required up until near the beginning of S phase. Clearly, cell cycle progression in cycling cells is much different with respect to molecular requirements from that in quiescent cells.
Based upon previous studies (7, 36), it would appear extremely unlikely that the delay in cell cycle inhibition by anti-Ras compared to anti-D1 resulted from a delayed action of anti-Ras antibody following introduction into the cell. To directly test this possibility, however, we determined how rapidly anti-Ras antibody was able to block ERK phosphorylation (one of the most rapid effects of Ras activity). Quiescent NIH 3T3 cells were injected with anti-Ras antibody and stimulated with serum at various time thereafter. At 15 min following serum addition, the cells were fixed and stained with an antibody which recognized only phosphorylated ERK proteins. The anti-Ras antibody blocked serum-induced phosphorylation in all plates analyzed, including those cells which were stimulated with serum immediately following antibody injection (Fig.5). As a control, nonspecific immunoglobulin injected at the same time had no inhibitory effects on ERK phosphorylation (Fig. 5). Thus, the above results must not have been due to delayed activity of injected anti-Ras antibody. Moreover, the effects of anti-Ras differed morphologically from the effects of anti-cyclin D1. Even within the first cell cycle period, the anti-Ras antibody caused the cells to flatten and stop migrating. This fact is curious when it is considered that these morphological changes did not lead to altered cell cycle progression, while anti-cyclin D1, which did not cause altered morphology or interfere with cellular migration, dramatically inhibited cell cycle progression when introduced early in the cell cycle (data not shown).
Inhibition of cycling cells with cycloheximide and serum removal.To determine the restriction point in cycling cells for cycloheximide and serum removal, experiments similar to those described above were performed. The results of two separate experiments with each reagent were summarized and compared to the results obtained as reported above with anti-cyclin D1 (Fig. 4B). The inhibition characteristics of anti-D1 (Fig. 6A), cycloheximide (Fig. 6B), and entry into S phase (Fig. 3A) were indistinguishable within the level of accuracy possible in these types of experiment. Cells within 3 h of mitosis were essentially all inhibited in cell cycle progression by anti-cyclin D1 or cycloheximide, while both inhibitors lost activity by the time cells in the culture had entered S phase (Fig. 6B). On the other hand, the inhibitory effect of serum removal was intermediate between the effects of anti-Ras and anti-cyclin D1 inhibitions. In this case, cells were consistently and efficiently inhibited only within the first hour following mitosis, after which cell cycle inhibitions became minimal (Fig. 6C). In all cases, few cells passed through a second mitosis following any of these treatments, indicating that each was an efficient inhibitor of cell cycle progression in cycling cells. These results not only emphasize the differences in cell cycle points of inhibition in cycling compared to quiescent cells but also reveal differences even between separate inhibitors. It is apparent that a complex signaling sequence operates to regulate cell cycle progression in cycling cells.
Direct comparison of the timing of Ras, cyclin D1, and cycloheximide requirements.The above results reveal for the first time differences in the cell cycle time at which Ras and cyclin D1 are required in continuously proliferating cells. To confirm this result, and to more accurately determine the differences between these times, anti-Ras and anti-cyclin D1 were each injected into several plates of asynchronous NIH 3T3 cells. After incubation for various lengths of time, individual plates were then pulsed for 1 h with tritiated thymidine. The cells were then fixed and stained for injected immunoglobulin (to identify injected cells), followed by autoradiography to detect cells in S phase. Little inhibition of thymidine labeling was observed soon after either of these injections, since neither antibody would be expected to interfere with an ongoing S phase, and most labeling soon after injection would be by cells already in S phase when injection took place. On the other hand, with increasing incubation time between injection and thymidine labeling, cells synthesizing DNA at the time of injection would complete S phase, such that new cells would be required to initiate DNA synthesis if the proportion of labeled cells were to be maintained. Inhibition of thymidine labeling in this experiment would be seen most rapidly following injection of an antibody able to immediately inhibit entry into S phase (Fig. 7B). In individual experiments, anti-cyclin D1 reduced the labeling of injected cells between 2 and 4 h sooner than did injected anti-Ras. These results indicate that anti-Ras had to be in the cells from 2 to 4 h longer than anti-cyclin D1 in order to block entry into S phase (Fig. 7). In other words, anti-Ras inhibited cell cycle progression 2 to 4 h prior to the point at which anti-cyclin D1 was inhibitory.
In the above experiment, the time of anti-Ras and anti-cyclin D1 inhibition were compared to each other, without direct reference to the cell cycle. For example, the same result would be obtained if the restriction point for Ras and cyclin D1 were separated by 2 to 4 h in G2 phase. Therefore, we designed a separate experimental strategy to directly determine the time of inhibitor action relative to the beginning of S phase. A double-labeling procedure was used to identify cells which initiated a new cycle of DNA synthesis as follows. Cells were pulsed for 1 h with BrdU, washed, and immediately pulsed with tritiated thymidine for the next 5 h. A cell labeled with thymidine but not with BrdU must have initiated a new cycle of DNA synthesis during the 5 h of thymidine labeling following removal of BrdU. To relate the point of Ras and protein synthesis requirements to the beginning of S phase, cells were injected with anti-Ras or treated with cycloheximide (2 μM) and at various times thereafter were subjected to the double-labeling procedure (Fig.8B). With this protocol, approximately 15% of untreated cells scored positive for entry into S phase at all times tested. (The slight underestimate from the expected number was apparently due to the manipulations required.) Cycloheximide treatment, as predicted based on the above data, blocked entry into S phase almost immediately. Even cells treated with cycloheximide immediately after removal of BrdU were reduced to approximately half of the level of untreated cells; cells were efficiently inhibited from entering S phase if treated with cycloheximide just prior to the 1-h BrdU treatment. On the other hand, anti-Ras required longer to inhibit entry into S phase. Cells injected immediately following BrdU treatment exhibited little inhibition, while the number of cells able to initiate S phase 4 h after anti-Ras injection was reduced to approximately half of the level of untreated cells (Fig. 8).
These data confirm previous results and indicate that while cycloheximide blocks entry into S phase up until just prior to the beginning of DNA synthesis, cellular Ras activity becomes unnecessary to the cell several hours prior to the beginning of S phase. Taken together these results confirm the difference between cycling and quiescent cells and between the timing at which different inhibitory treatments block the progression of cycling cells.
When serum is added to quiescent cells, it is continuously required until the cells progress to a point in G1 phase after which the cells become committed to entry into S phase even in the absence of serum (25). A number of other inhibitory treatments also lose their ability to block entry into S phase at this same point, several hours prior to the beginning of DNA synthesis (5, 11, 12, 23, 26). Consistent with this pattern, we found that injection of anti-Ras and anti-cyclin D1 antibodies together with serum removal and cycloheximide treatment all blocked entry into S phase following release from quiescence at essentially the same point, approximately 4 h prior to the beginning of S phase in these NIH 3T3 cells. It was the purpose of this study to extend this type of analysis to cells continuously passing through the cell cycle without introducing the perturbations required for inducing cell synchrony. For this purpose, the approximate cell cycle position of individual cells in an asynchronous culture was determined by following them in time-lapse prior to treatment or injection. This allowed determination of the age of each cell and therefore its approximate cell cycle position without synchronization. In addition, time-lapse analysis after injection allowed a determination of the fate of injected cells.
Surprisingly, we found that the injected anti-Ras antibody did not delay passage of injected cells through the next scheduled mitosis, regardless of the cell cycle position at the time of injection. Efficient inhibition was eventually observed, but only after each injected cell had passed through one mitosis. Thus, in order to block entry into S phase, the anti-Ras antibody had to be present within the cell prior to the preceding mitosis. By contrast, anti-cyclin D1 antibody efficiently inhibited the number and timing of the next mitoses when introduced during G1 phase of asynchronous cells. Our concern that these differences might result from a delay in the ability of the injected anti-Ras to neutralize cellular Ras activity following microinjection was unfounded, based on work performed here as well as previous studies (7, 36). Another possibility is that Y13-259 could not rapidly neutralize Ras because a Ras effector molecule, Raf-1, had already bound to Ras preventing antibody binding. This, however, is not likely because of the following lines of evidences. (i) The Raf-Ras complex has an off rate of about 1 min in an equilibrium system (8). If this were the case in vivo, injected anti-Ras antibody would quickly replace Raf already bound to Ras. (ii) Binding of Raf to Ras does not change the intrinsic GTPase activity of Ras (50% conversion of GTP to GDP in about 30 min) (10) or slightly enhances it (40). Because Raf would be released upon hydrolysis of the GTP bound to Ras, the half-life of the Ras-Raf complex in Y13-259 injected cells could not be longer than 30 min. Once the Ras-Raf complex dissociates, anti-Ras would be able to bind and inactivate the Ras protein (9). (iii) The anti-Ras antibody Y13-259 used here can inhibit the proliferation of oncogenic Ras-transformed cells with kinetics similar to those of nontransformed cells (35). Moreover, injection of anti-Ras antibody into cells transformed by oncogenic Ras weas found to stop migration within 15 min (unpublished data), despite the fact that oncogenic Ras has very slow intrinsic GTPase activity, and would be expected to be continuously bound to Raf protein prior to injection of anti-Ras. Migration has previously been shown to be dependent on Ras activity and to be stimulated by oncogenic Ras (7). Therefore, even though Raf was in a complex with Ras at the time of anti-Ras injection, the antibody was able to replace Raf and neutralize Ras activity in a time frame much shorter than the delay in antibody neutralization reported here. Binding of Ras to Raf could not, therefore, account for the delay in cell cycle inhibition following anti-Ras injection compared to anti-cyclin D1 injection. Thus, cellular Ras and cyclin D1 were apparently required at different times in cycling cells, while cycloheximide was similar to anti-cyclin D1 and serum removal was intermediate between anti-Ras and anti-cyclin D1.
This conclusion was confirmed with two separate experimental strategies. In the first, a kinetic labeling procedure was used to determine the time required for each antibody to block thymidine labeling in an asynchronous culture. In the second, a novel BrdU-tritiated thymidine labeling sequence was utilized to identify cells which initiate S phase within a given time period. In both cases, the requirement for cellular Ras activity was found to precede that for cyclin D1 or cycloheximide. For example, it required almost 4 h for anti-Ras to block entry into S phase by 50%, while cycloheximide (which functions at a time similar to that of anti-cyclin D1) inhibited entry into S phase almost immediately. Therefore, these data from three separate techniques together indicate that in cycling NIH 3T3 cells, cellular Ras activity and cyclin D1 are required at distinct cell cycle points.
The difference in timing of inhibition between cycling and quiescent cells most probably results from the fact that stimulation of quiescent cells with serum not only involves a stimulation of cell cycle progression but also requires a change in growth phase. This fact might also account for the long period between (i) the point when all protein synthesis required for the initiation of S phase is complete and (ii) the actual initiation of DNA synthesis. Such a delay would apparently not be tolerated in cycling cells with a short G1 phase. Indeed, a short delay between the cyclin D1 or the protein synthesis requirement and the beginning of S phase might be essential to reduce the overall length of the cell cycle in rapidly proliferating cells.
The time at which serum is required in cycling NIH 3T3 cells is slightly different from than for Ras. Serum was required during early G1 phase. Similar results have been obtained with a time-lapse approach similar to the one used in this study (43, 44). In that work, the G1 phase was divided into two parts by the requirement for serum. The fact that anti-Ras and serum removal have profiles of inhibition in cycling cells which show subtle but important differences indicates that Ras must not be the only important target of serum stimulation. Thus, another signaling pathway induced by serum, which is distinct from the Ras pathway, is apparently required after Ras activity becomes unnecessary for continued cell cycle progression. This alternative pathway might control the stability of cyclin D1. Serum withdrawal is reported to activate the calpain-dependent degradation of cyclin D1 protein (2). In addition, the phosphatidylinositol 3-kinase-Akt-glycogen synthase kinase-3β pathway has been shown to involve posttranslational control of cyclin D1 protein stability (4). Phosphatidylinositol 3-kinase is activated by Ras through direct interaction with its catalytic subunit, p110 (28). However, it can also be activated independently of Ras through interaction of its regulatory subunit with the growth factor receptor (39). Thus, it is possible that the continued presence of activated growth factor receptors can stabilize cyclin D1 proteins, or promote their continued synthesis, even in the absence of Ras activity.
Cyclin D1, however, is not the only target of growth factor action. Cell cycle progression requires a number of other signaling activities, including the suppression of cyclin-inhibitory proteins, the activation and coordination of multiple MAPK pathways, the activation of transcription factors, the regulation of other small G proteins such as those involved in controlling the cytoskeleton, and controls over translation and apoptosis. It will be essential to consider a variety of other signaling molecules before we are able to fully understand these results. Moreover, the cyclin D family contains three members, at least two of which are likely to be expressed in fibroblastic cells. The anti-cyclin D1 antibody used here has been shown in previous studies to be highly specific for the D1 isoform in neutralizing activity (18). The potential role of other cyclin D molecules will also have to be determined by future experimentation.
These data, therefore, provide novel insight into the control of cell cycle progression in actively cycling cells. Cellular Ras activity is required prior to mitosis to allow a cell to make a commitment to progress through the entire subsequent cell cycle, while cyclin D1 activity is required for the G1/S-phase transition. These data suggest the following model for control of cell cycle progression in continuously cycling cells. Continued cell cycle progression requires the presence of growth factors and other appropriate proliferative conditions. These conditions are communicated to the cell, at least in part, through the activation of cellular Ras. In a cell with a relatively short G1 phase, such as the NIH 3T3 cells studied here, Ras must become activated during G2phase to allow the cell to proceed into the next cell cycle after passage through mitosis. Since Ras activity becomes largely unnecessary by the time a cell has passed through mitosis, it must stimulate the production of another molecule(s) whose continued production is independent of continued Ras activity and which is required until the cell begins DNA synthesis, after which completion of the cell cycle is normally ensured. The data suggest the possibility that cyclin D1 is this downstream target of Ras. It is reported to be stimulated by Ras activity (1, 6, 16, 27), and these studies demonstrate that it is required through G1 phase. This possibility is supported by data which indicate that cyclin D1 is induced during G2 phase of the NIH 3T3 cell cycle in a Ras-dependent manner. This expression of cyclin D1 then continues independently of Ras for over 6 h and is required for initiation of S phase (unpublished data).
We thank Michele Pagano for the generous gift of a neutralizing anti-cyclin D1 monoclonal antibody. We also thank Guan Chen for helpful suggestions throughout this work and Alan Wolfman and Seiji Kondo for helpful suggestions in preparation of the manuscript.
This work was supported by NIH grant GM52271.
- Received 9 February 1999.
- Returned for modification 19 March 1999.
- Accepted 5 April 1999.
- Copyright © 1999 American Society for Microbiology


text:
A Texas mayor and her mayor pro tem have resigned from a small Texas town after being caught using city funds to pay for their expensive tickets to go see Michelle Obama at a book signing event.
Forest Hill, Texas, Mayor Lyndia Thomas and Mayor Pro Tem Beckie Hayes both submitted their resignations after they were told the city council intended to begin an investigation into the untoward use of city funds.
The two reportedly used city money to pay for their tickets to see Obama during her book tour event in Dallas last year, according to ABC News.
Trending: 77-Year-Old Veteran Beaten By Liberals Over MAGA Hat
According to city officials, the two paid for the tickets out of their own pockets but then turned in vouchers for the $1,090.00 price tag and received reimbursement.
The expenditure was approved by the city manager, but Hayes claims she later repaid the ticket price to the city.
Mayor Thomas claimed she resigned to avoid having the city fire her.
"I will not leave my fate in the hands of other individuals," Hayes told ABC affiliate WFAA. "I am a woman of integrity, and the allegations, they have no substance. They are false."
Thomas had asked for a three-month period to "prepare" for her defense, but the city council decided to move ahead with a hearing anyway, thus prompting Thomas' resignation.
According to the Star-Telegram:
Thomas said she received a letter on Jan. 8 listing the allegations against her, and argued that she had less than six weeks to prepare. "I am asking the council to pull the charges against myself and the mayor pro tem because we did not do anything illegally. If you don't pull the charges, I ask that you you give us a chance to prepare."
The council announced ahead of time that its meeting was set to consider dismissing the mayor over the abuse of city funds.
The council also pointed to other questionable expenses:
Meanwhile, Joubert, Jones and Boardingham said other questionable expenses include the purchase of school supplies, an event sponsored by the Links Foundation and meals at Pappadeaux Seafood Kitchen and Texas Roadhouse. They said the events and meals were not related to their duties as elected officials.
The resigned officials claimed they were subjected to a political witch hunt.
They also noted that neither take a salary from the city and are entitled to have expenses reimbursed.
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