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Reviews & Features
Chris Roberts Recognises the Star Citizens of TOG
| 26 October 2013
The rising interest in Star Citizen in TOG has gained the attention and thanks form Chris Roberts and Sandi Gardiner
Warthunder heard throughout TOG
| 16 July 2013
The sounds of Warthunder is being heard throughout TOG
Dota 2 Release
| 12 July 2013
DOTA2 Released.
Planetside 2 Release 20-Nov-2012 PST
| 18 November 2012
The long awaited and critically acclaimed PlanetSide 2 will be released on 20-Nov-2012 PST
Guild Wars 2 Launch Date: 28th August
| 28 June 2012
Guild Wars 2 will officially launch on Tuesday, August 28th
Source Wars End in an Epic Battle!
| 17 June 2012
Ever watch the movie Rocky? Who hasn't, right? We had a battle for the ages last Sunday night to determine this year's Source Wars champion. It was a good one. Just like the movie!
Review: Ducky DK 9008 Mechanical Keyboard (Cherry Red switches)
| 15 April 2012 |
The Ducky DK 9008 is a mechanical keyboard designed with the Cherry (Mx) Red switches for lower actuation forces and smoother/softer keystrokes.
The MechWarrior division wants you
| 6 April 2012
Mechwarrior pilots new and old call to arms
TOG Source Wars IV
| 28 March 2012
Tom and Jerry Meets The Terrorists Meets Inglorious Basterds... Must be Source Wars IV
TERA Online
| 19 February 2012
New MMO to be released May 1st 2012
Newsfeed Selector
» GameSpot Headlines
New Dead Rising 4 Video Features a Slurpee Tornado
27 July 2016 |Capcom has released a new behind-the-scenes development diary for Dead Rising 4, looking at what makes its protagonist unique and showcasing some of the crazy weapons in the game.Among these is a military-grade exosuit, which you ...
Pokemon Go's Watch Accessory Delayed
27 July 2016 |Pokemon Go Plus, the watch-like accessory that allows players to catch monsters without looking at their phone will now be available in September, instead of July.The announcement was made by Nintendo on Twitter, but a more ...
Professor Layton's Daughter Stars in The Newest Game in The Series
27 July 2016 |Professor Layton developer Level-5 has announced a new entry in the series called Lady Layton: The Conspiracy of King Millionaire Ariadne. The game is scheduled to launch in Spring 2017 in Japan for 3DS and iOS.The game was ...
Former Burnout Devs Working on New Driving Game
27 July 2016 |Alex Ward, the director of the Burnout series, has confirmed work has begun on a new driving game. He announced the project with a short and sweet message on Twitter, but no further details on the game were provided.Work has begun ...
Nintendo Reports Big Losses as Wii U Sales Stall
27 July 2016 |Nintendo has detailed its financial results for the three-month period spanning April 1 to June 30 2016, reporting a big decline in sales compared to the same period in 2015.Net sales were cited as $587 million, which represents a ...
New PS4-Exclusive Yakuza 6 Trailer Features Famous Japanese Actors
27 July 2016 |Sega has released a dramatic new Yakuza 6 trailer featuring a host of established Japanese actors. The story-focused trailer was shared on YouTube, and can be viewed in the embed below.<iframe ...
Pokemon Go Estimated To Have Been Downloaded 75 Million Times
27 July 2016 |By now, you probably know that Pokemon Go is wildly popular. It was downloaded more times in its first week than any other app in history, in fact. Now, an analytics firm has estimated that the game has crossed another milestone, ...
All the Cosplay We Saw at Comic-Con 2016
27 July 2016 |Besides its bustling crowds of people and hype-filled panels, one of the major highlights of every Comic-Con is its cosplay. This year brought on hundreds of cosplayers dressed up as characters from some of the most popular and ...
New Overwatch Patch Buffs Ana and Adjusts McCree Again
27 July 2016 |A new update for the PC version of Overwatch is now available, and its main components deal with additions and changes from the last patch.Ana, the game's newest character, receives a small buff in this patch. Specifically, her ...
Awesome New Comics This Week: Batgirl, Black Panther, Captain America, and More
27 July 2016 |Every Wednesday, hundreds of new comic books are released, and figuring out what books are worth reading can be a daunting task. Luckily, we're here to help you out with that. Here are five upcoming issues, in no particular order, ...
» IGN Headlines
» 1UP Headlines
Review: Bending Genres in Luigi's Mansion: Dark Moon
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Review: Bending Genres in Luigi's Mansion: Dark Moon
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Review: Shambling Mirror of Fate Signals Time to Jam a Stake in Castlevania's Heart
8 March 2013 | <!--Begin feature--> <!-- for syndication --> Castlevania: Lords of Shadow: Mirror of Fate brings the series back to Nintendo's portable systems, ...
Review: Shambling Mirror of Fate Signals Time to Jam a Stake in Castlevania's Heart
8 March 2013 | <!--Begin feature--> <!-- for syndication --> Castlevania: Lords of Shadow: Mirror of Fate brings the series back to Nintendo's portable systems, ...
An Unflattering Boll Cut
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An Unflattering Boll Cut
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The Trials and Tribulations of Gears of War: Judgment
6 March 2013 | <!--Begin feature--> <!-- for syndication --> <body> As the next generation of home consoles slowly emerge into the limelight and enter the ...
The Trials and Tribulations of Gears of War: Judgment
6 March 2013 | <!--Begin feature--> <!-- for syndication --> <body> As the next generation of home consoles slowly emerge into the limelight and enter the ...
The Cinematic Nature of Parasite Eve
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The Cinematic Nature of Parasite Eve
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» GameDaily Headlines
» Gamasutra Headlines
Nintendo stays to course despite flagging sales and profits
27 July 2016 |Nintendo's financials for the quarter ended June 30, 2016, are out, and sales and profits have both taken a hit. ...
Blog: Testing for game development
27 July 2016 |"It doesn't matter what language, engine, API or platform you use. It doesn't matter how many games you've made or plan on making or even the size of your games. This article is for you" ...
Blog: Pros and cons of Pokemon Go's monetization system
27 July 2016 |It's incredibly popular, but can Pokemon Go cash in on its fame? Radiant World's monetization director Ramin Shokrizade finds out. ...
Writing romances in (non-romance) games
27 July 2016 |Ex-BioWare video game writer Alex Free (Star Wars: The Old Republic, Shadow Realms) explores the unique challenges writers face in creating meaningful romances for video games. ...
Apple iPads make a brief surge in 2016 third-quarter report
27 July 2016 |Apple's numbers remain steady as iPad revenues move upward for the first time in a while thanks to the launch of the iPad Pro. ...
Get a job: Deep Silver Volition seeks a senior Technical Artist
27 July 2016 |Saints Row developer Deep Silver Volition is looking to bring on an experienced technical artist to take a senior position at its headquarters in Champaign, Illinois. ...
Video: Tips from Paradox on mixing history with game design
27 July 2016 |At GDC 2016, Paradox Development Studio's Chris King shares solutions his studio has for bringing history into your game, while at the same time ensuring the game itself is fun and compelling to play. ...
Rad adds newer, faster game data compressors to its Oodle offerings
27 July 2016 |Fun names alert: RAD Game Tools has updated its Oodle library of data compression tools for game developers with two new compressors, Selkie and Mermaid, that are faster than Oodle's extant Kraken. ...
Political snarls drive prominent game educators out of Wisconsin
27 July 2016 |Frustrated with an inhospitable political climate, University of Wisconsin-Madison game researchers/educators Constance Steinkuehler and Kurt Squire are planning to decamp for California. ...
Blog: Game UX Summit sessions roundup
27 July 2016 |Epic Games director of user experience Celia Hodent brings us a detailed roundup of the Game UX Summit. ...
» GamesIndustry.Biz Headlines
Qualcomm settles class-action gender bias suit for $19.5 million
27 July 2016 |Chip-maker will now address imbalance within its ranks, implementing reforms to improve pay and opportunities for women
Superdata predicts Battleborn will go F2P
27 July 2016 |Shooter has been "struggling in the presence of Overwatch"
Nintendo posts biggest Q1 loss in five years
27 July 2016 |Last year's profit turns to $232 million loss in the face of collapsing Wii U sales and the absence of new revenue streams
Apple reports quarterly revenue of $42.4 billion
27 July 2016 |"Reflect stronger customer demand and business performance than we anticipated"
Microsoft plans to sabotage Steam - Sweeney
27 July 2016 |Epic co-founder says Windows 10 updates will progressively break Valve's storefront until Windows Store seems like a more stable alternative
Nintendo NX is a portable console powered by a mobile processor - report
27 July 2016 |Handheld component has detachable controllers, can be hooked up to a TV, with cartridges used for games
Tom Kalinske gets back in the game
27 July 2016 |After 20 years away from the industry, Sega's Genesis-era CEO takes chairman role at Marvel Heroes developer Gazillion
Adam Boyes joins Iron Galaxy as CEO
27 July 2016 |Ex-Sony dev relations head signs on as Killer Instinct Season 2 and 3 developer shakes up leadership team
Is Pokémon Go success going to come to a Weezing halt?
26 July 2016 |Garry Barter of PlayerXP analyses the customer feedback for Niantic's AR giant
Humble Bundle clarifies fraud policies
26 July 2016 |"Let us take care of this for you using our infrastructure" it tells devs
» GamePolitics Headlines
Good night and good luck
19 April 2016 |I'm horrible at goodbyes and ironically I'm finding it incredibly difficult to put into words how I really feel about GamePolitics shutting down. It's been a tough year for us as we struggled to balance having to make ...
Super Podcast Action Committee Episode 186 – The Finale!
18 April 2016 |In the very final episode of Super Podcast Action Committee (episode 186) hosts Andrew Eisen and E. Zachary Knight are joined by GamePolitics managing editor James Fudge for a free-form conversation about Lollipop Chainsaw's ...
Super Podcast Action Committee Episode 185 – The Finale!
18 April 2016 |In the very final episode of Super Podcast Action Committee (episode 186) hosts Andrew Eisen and E. Zachary Knight are joined by GamePolitics managing editor James Fudge for a free-form conversation about Lollipop Chainsaw's ...
GamePolitics' own E. Zachary Knight is running for Congress
18 April 2016 |Before we go dark, we'd like to take a moment to inform you that our own E. Zachary Knight, longtime contributor and co-host of Super Podcast Action Committee, is running for US Congress. "For the first time in over 30 ...
Join us tonight at 6p PST for the FINAL episode of Super Podcast Action Committee
16 April 2016 |As Andy Barclay once said to Chucky: "This is the end, friend." And then he lit him on fire but that last part isn't important. What is important is that tonight is the final episode of Super Podcast Action ...
New study examines 'violent-sexist' video games' affect on player empathy towards female victi
16 April 2016 |A new study published in the journal PLOS ONE earlier this week takes a look at how "violent-sexist" video games affect players' empathy towards female victims of violence. The study was conducted ...
Our finest hour: the Bill Kunkel Awards
15 April 2016 |Bill Kunkel is and always will be one of my heroes. Growing up in the 70's and 80's, I read a lot of his stuff in wrestling and gaming magazines. I saw his byline and imagined that someday I could do what he did. I never met ...
Take-Two sued by former Rockstar North president for $150 million in unpaid royalties
14 April 2016 |Leslie Benzies, former president of Rockstar North (Grand Theft Auto series), is suing Take-Two Interactive for $150 million in unpaid royalties. Take-Two, who has filed a counter-suit, sees things differently. "Nowhere does ...
Looking back: GamerGate
13 April 2016 |Psst. Here's a little secret: when I report on a story, I put my beliefs on hold and try to be as fair and impartial as is humanly possible. I know that's hard for some people out there to believe, but it is the truth. ...
Senator Al Franken voices Oculus Rift privacy concerns
13 April 2016 |Last week, Senator Al Franken (D-MN) penned a letter to Oculus CEO Brendan Iribe detailing a list of privacy concerns he has regarding the Oculus Rift virtual reality headset. "Oculus' creation of an immersive virtual ...
» Joystiq Headlines
Joystiq Presents: That's a wrap
4 February 2015 |Our final Joystiq Presents podcast gives members of the staff a moment to thank the Joystiq community and say a final farewell. Listen to the MP3 Listen to other Joystiq Presents episodes....
Here's a video
4 February 2015 |We've been debating publishing this video for weeks. Now, as imaginary armed men pound on the fictional doors of Joystiq's nonexistent office, we have precious little time to debate any further. So here it is, a video of Satoru ...
Off the Grid: The Witcher Adventure Game and Star Wars Imperial Assault
4 February 2015 |Like most sci-fi shows on Fox, Off the Grid is getting canceled (we're all saying goodbye, actually). In this series finale of the board and card game series I had hoped to evolve over time, we're keeping our spirits high with a ...
Joystiq Photo Album
4 February 2015 |Like all good photo albums, our look back at ten years of Joystiq is a hot mess of nostalgia, out-of-focus shots, and GIFs (you've been warned). Now, gather round and mind the edges. >>Joystiq Photo Album...
There is No End
4 February 2015 |Joystiq will not grow to be much older than ten years. That's a massive expenditure of time, if only to receive an unfinished creation in return. I, and my best friends in the world, leave Joystiq unfinished. We leave it as it was ...
Joystiq Presents: Team spirit
4 February 2015 |In this episode, Mike Suszek (@mikesuszek) chats with News Content Director Alexander Sliwinski (@Sliwinski) about Team JOY and Team Joystiq. He also discusses the "Internet dating" side of working in the industry. Listen to the ...
Joystiq Presents: Can't stop
4 February 2015 |In this episode, Mike Suszek (@mikesuszek) takes his turn in the hot seat as he tells Features Content Director Xav de Matos (@Xav) about the rant that landed him the job of his dreams. He also talks about barely surviving his ...
Joystiq Presents: Close project
4 February 2015 |Mike Suszek (@mikesuszek) talks with Joystiq's Editor in Chief Ludwig Kietzmann (@LudwigK) about the site's history as well as a newfound opportunity to grow. Listen to the MP3 Listen to other Joystiq Presents episodes....
Games of a Lifetime: Alexander's picks
4 February 2015 |After more than ten years devoted to video games and the people who make them, Joystiq is closing its doors. We won't be reporting on the best games of 2015, so join us for one last hurrah as the Joystiq family reveals their Games ...
Joystiq Streams: Once more, with feeling
4 February 2015 |Ladies and gentlemen it's time/for all the good that's in you to shine/for all the lights to lose their shade/for all the hate that's in you to fade. Ladies and gentlemen it's time/for the maracas and the ...
» Kotaku Headlines
» Next Generation Headlines
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Nintendo NX Is a Portable Console With Detachable Controllers, Says Report
27 July 2016 |An anonymous reader writes from a report via Eurogamer.net: We now have a good idea as to what the Nintendo NX will consist of thanks to a new report from Eurogamer. According to a number of sources, Nintendo's upcoming NX will be ...
Steam On Windows 10 Will Get 'Progressively Worse': Gears of War Developer
27 July 2016 |Microsoft's Universal Windows Platform, or UWP, approach isn't sitting well with many game developers. Four months after criticising UWP ecosystem for being a walled-garden, curtailing "users' freedom to install full-featured PC ...
Nintendo Shares Plummet After Investors Realize It Doesn't Actually Make Pokemon Go
26 July 2016 |Sam Byford, reporting for The Verge: Nintendo shares have skyrocketed since Pokemon Go's release and instant transformation into global cultural phenomenon, but they fell dramatically today after investors realized that Nintendo ...
Microsoft Cuts Xbox One Price To $249 - Would You Buy or Recommend One?
26 July 2016 |Tom Warren, writing for The Verge: Microsoft is cutting the price of its Xbox One console to $249. The new price marks the third price cut in less than two months, ahead of the new Xbox One S launch on August 2nd. 500GB versions of ...
Valve Threatens Counter Strike Gambling Sites
24 July 2016 |An anonymous Slashdot reader quotes a report from HNGN: Game maker Valve is threatening to shut down sites dedicated to gambling with add-ons to its popular Counter Strike game. On Thursday the company sent cease and desist letters ...
Sega Announces Two New Sonic Games That Seek To Recapture The Glory Days
23 July 2016 |An anonymous reader writes: In celebration of Sonic the Hedgehog's 25th anniversary, Sega has announced two new Sonic games at Comic-Con in San Diego. The first game is called Sonic Mania and it's a 2D platformer that features ...
Apple: Pokemon Go Sets Record For Most Downloads In Its First Week
23 July 2016 |An anonymous reader writes: Apple has confirmed to TechCrunch that Pokemon Go has attracted more downloads in the App Store during its first week than any other app in App Store history. What's even more surprisingly is that the ...
Sony Is the Only Remaining Obstacle To PS4-Xbox Cross-Play
23 July 2016 |In March, Microsoft announced native support for cross-platform play between Xbox One and Windows 10. At the time, the company also added that this support could be extended to "other console and PC networks," something which led ...
Apple To Make $3 Billion From Pokemon Go
22 July 2016 |An anonymous reader writes from a report via The Guardian: We all know what Pokemon Go is, and we all know how successful it is. The Guardian is reporting that Apple will "rake in $3 billion in revenue from Pokemon Go in the next ...
Saudi Arabia Revives 15-Year-Old Ban On 'Zionism-Promoting' Pokemon
21 July 2016 |An anonymous reader writes: Clerics in Saudi Arabia have renewed a 15-year-old ban on Pokemon, following the release of the highly popular augmented reality version of the game, Pokemon Go. According to Reuters, the General ...
Latest Blogs
Blogs Have Moved!
| 3 November 2010
Blogs can now be found in the vBulletin blogging tool, located here.
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Guild Wars 2: Division Overview
| 22 August 2012
An Introduction to Guild Wars 2 and the TOG GW2 Crew.
Get to know your guildmate interview #4: Serah De Vere
| 29 December 2012
Ral Partha interviews TOG member and Guild Wars 2 fan "Serah De Vere", in the latest installment of "Get to know your guildmates with Ral Partha"!
Get to know your guildmate interview #3: Pumpkin
| 19 December 2012
Ral Partha interviews TOG member and Guild Wars 2 fan Pumpkin, in the latest installment of "Get to know your guildmates with Ral Partha"!
| 10 September 2012
Weekly WvW Events Wednesday night and Saturday your time zone
GW2 Member Interview #2
| 10 September 2012
Icehawke - who is he and what does he do?
TF2 Unveils a new Co-Op Mode - Mann vs Machine
| 26 August 2012
The game that keeps on giving unveils new co-op mode. Red and Blue join forces to fight off a marauding mob of malevolent machines. TOG TF2 has 2 MvsM servers up and running for your machinations. See the ...
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| 24 August 2012
Get to know GW2's 501st sign up: Klobberella
2 Sep: GW2 Crafting Carnival
| 24 August 2012
2 Sep: Meet and Greet, Chat and Craft!
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| 23 August 2012
Specs gives a humorous but serious run down on how to stay healthy in the first weekend of Guild Wars 2.
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Change is coming to Cantha


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subset: commoncrawl


text:
We have issues with utf8-string comparisons in MySQL 5, regarding case and accents :
from what I gathered, what MySQL implements collations by considering that "groups of characters should be considered equal".
For example, in the
utf8_unicode_ci collation, all the letters "EÉÈÊeéèê" are in the same box (together with other variants of "e").
So if you have a table containing ["video", "vidéo", "vidÉo", "vidÊo", "vidêo", "vidÈo", "vidèo", "vidEo"] (in a varchar column declared with ut8_general_ci collation) :
- when asking MySQL to sort the rows according to this column, the sorting is random (MySQL does not enforce a sorting rule between "é" and "É" for example),
- when asking MySQL to add a Unique Key on this column, it raises an error because it considers all the values are equal.
What setting can we fiddle with to fix these two points ?
PS : on a related note, I do not see any case sensitive collation for the utf8 charset. Did I miss something ?
[edit] I think my initial question still holds some interest, and I will leave it as is (and maybe one day get a positive answer).
It turned out, however, that our problems with string comparisons regarding accents was not linked to the collation of our text columns. It was linked to a configuration problem with the
character_set_client parameter when talking with MySQL - which defaulted to
Here is the article that explained it all to us, and allowed us to fix the problem :
Getting out of MySQL character set hell
It is lengthy, but trust me, you need this length to explain both the problem and the fix.


text:
The latest version of the Syntax highlighter (when using v3.x) breaks a ton of my source code.
Here is the plugin running in v3.x mode (broke):
Here is the plugin running in v2.x mode (prefect, works FANTASTIC):
Here is the source page, currently running v2.x:
Can we get this fixed? This is crazy that it just ignored the formatting of my code! Note to others, you may want to flip back to v2.x until these early issues are resolved.


text:
Via Sakis Koukouvis
Rescooped by
Alistair Parker
from Science News
onto Art meets Technology
3-D Printing is going to have a dramatic impact on our lives and I find the possibilities very exciting!
I feel like this is something out of a science fiction. But it is evident to expect the greatest technology advancements to be in the medical sector. This is an example of integrating medical technology with CAD (Computer Aided Design) styled software.


text:
Your products, customized just the way you want them!
Looking for
Team Uniforms,
Embroidered Clothing?
Groups & Teams apparel on our sister site
Transform your creativity into beautiful products, sell them on CafePress and make money. It's easy and fun.
Tyrannosaurus Rex hates push-ups, lifting, rowing, jump rope, swimming, bowling, basketball and everything that sucks with short tiny arms. Funny parody prehistoric dinosaurs dinosaur humor.


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|Aug 20 - 21
| 2016 Yu-Gi-Oh! TRADING CARD GAME World Championship
|Orange County Convention Center
West Concourse
Orlando, FL
|FREE Public events all weekend! More info to come.
|Aug 27 - 28
|Yu-Gi-Oh! Championship Series Toronto, Canada
|Metro Toronto Convention Centre,
Toronto, Ontario
|Check back later
|Sep 17 - 18
|Yu-Gi-Oh! Championship Series Mexico City, Mexico
|Centro Banamex,
Mexico City, Mexico
|Sep 17 - 18
|Ultimate Duelist Series 2016 Summer Invitational
|Embassy Suites by Hilton Chicago-Downtown Magnificent Mile
Chicago, IL
|Check back later
|Oct 15 - 16
|Yu-Gi-Oh! Championship Series Minneapolis, MN
|Minneapolis Convention Center
Minneapolis, MN
|Check back later
|Nov 12 - 13
|Yu-Gi-Oh! Championship Series Rio de Janeiro, Brazil
|Centro de Convenções SulAmérica,
Rio de Janeiro, Brazil
|Check back later


text:
Tapioca Pudding
|Vanilla bean soy beverage powder
|240 Milliliter (1 Cup)
|720 Milliliter (3 Cups)
|180 Milliliter (3/4 Cup)
|Granulated sweetener
|120 Milliliter (1/2 Cup)
|Maple syrup
|45 Milliliter (3 Tablespoon)
Blend soy beverage powder and water in double boiler.
Mix with wire whisk.
Add tapioca and cook the mixture in the double boiler until thickened.
Add Devansweet and maple syrup.
Mix well.
Transfer the pudding to a covered container and refrigerate until chilled.
The pudding will thicken slightly as it chills.
Rate It
Nutrition Rank
Nutrition Facts
Serving size: Complete recipe
Calories 1685 Calories from Fat 56
% Daily Value*
Total Fat 6 g9.7%
Saturated Fat 0.03 g0.13%
Trans Fat 0 g
Cholesterol 0 mg
Sodium 1482.8 mg61.8%
Total Carbohydrates 319 g106.4%
Dietary Fiber 1.6 g6.5%
Sugars 125.1 g
Protein 80 g160.7%
Vitamin A Vitamin C
Calcium 6.6% Iron 18.8%
*Based on a 2000 Calorie diet


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Molecular cloning and analysis of the gene encoding the thermostable penicillin G acylase from Alcaligenes faecalis.
Verhaert, R M
Riemens, A M
van der Laan, J M
van Duin, J
Quax, W J
Applied and Environmental Microbiology
Alcaligenes faecalis penicillin G acylase is more stable than the Escherichia coli enzyme. The activity of the A. faecalis enzyme was not affected by incubation at 50 degrees C for 20 min, whereas more than 50% of the E. coli enzyme was irreversibly inactivated by the same treatment. To study the molecular basis of this higher stability, the A. faecalis enzyme was isolated and its gene was cloned and sequenced. The gene encodes a polypeptide that is characteristic of periplasmic penicillin G acylase (signal peptide-alpha subunit-spacer-beta subunit). Purification, N-terminal amino acid analysis, and molecular mass determination of the penicillin G acylase showed that the alpha and beta subunits have molecular masses of 23.0 and 62.7 kDa, respectively. The length of the spacer is 37 amino acids. Amino acid sequence alignment demonstrated significant homology with the penicillin G acylase from E. coli A unique feature of the A. faecalis enzyme is the presence of two cysteines that form a disulfide bridge. The stability of the A. faecalis penicillin G acylase, but not that of the E. coli enzyme, which has no cysteines, was decreased by a reductant. Thus, the improved thermostability is attributed to the presence of the disulfide bridge.
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Lark, Cynthia
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Journal of Bacteriology
Lark, Cynthia (Saint Louis University, St. Louis, Mo.) and Robert Schichtel. Comparison of spheroplast induction in Alcaligenes faecalis by three different agents. J. Bacteriol. 84:1241–1244. 1962.—Alcaligenes faecalis strain LB was exposed to different concentrations of cycloserine, d-methionine, or penicillin. The time course of spheroplast induction by these agents was measured as a function of their concentration. The results were consistent with models in which cycloserine reversibly inhibited an intracellular enzyme, and d-methionine was built into a defective cell-wall unit. Penicillin simulated cycloserine. This was taken as further evidence that penicillin inhibits an enzyme associated with cell-wall synthesis.
Related Articles
Crystallization and X-ray structure analysis of a thermostable penicillin G acylase from Alcaligenes faecalis
Varshney, Nishant Kumar
Suresh Kumar, R.
Ignatova, Zoya
Prabhune, Asmita
Pundle, Archana
Dodson, Eleanor
Suresh, C. G.
Acta Crystallographica Section F: Structural Biology and Crystallization Communications
2012;68(Pt 3):273-277.
A thermostable penicillin G acylase from A. faecalis has been crystallized in two space groups: C2221 and P41212. X-ray diffraction data were collected to 3.3 and 3.5 Å resolution, respectively.
The enzyme penicillin G acylase (EC 18.104.22.168) catalyzes amide-bond cleavage in benzylpenicillin (penicillin G) to yield 6-aminopenicillanic acid, an intermediate chemical used in the production of semisynthetic penicillins. A thermostable penicillin G acylase from Alcaligenes faecalis (AfPGA) has been crystallized using the hanging-drop vapour-diffusion method in two different space groups: C2221, with unit-cell parameters a = 72.9, b = 86.0, c = 260.2 Å, and P41212, with unit-cell parameters a = b = 85.6, c = 298.8 Å. Data were collected at 293 K and the structure was determined using the molecular-replacement method. Like other penicillin acylases, AfPGA belongs to the N-terminal nucleophilic hydrolase superfamily, has undergone post-translational processing and has a serine as the N-terminal residue of the β-chain. A disulfide bridge has been identified in the structure that was not found in the other two known penicillin G acylase structures. The presence of the disulfide bridge is perceived to be one factor that confers higher stability to this enzyme.
thermostability; disulfide bridges; Ntn hydrolases; orthorhombic form; tetragonal form; calcium binding
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Purification and properties of inducible penicillin beta-lactamase isolated from Alcaligenes faecalis.
Fujii, T
Sato, K
Inoue, M
Mitsuhashi, S
Antimicrobial Agents and Chemotherapy
An inducible penicillin beta-lactamase was purified from a strain of Alcaligenes faecalis resistant to beta-lactam antibiotics. The purified enzyme preparation gave a single protein band on polyacrylamide gel electrophoresis, and its molecular weight was 29,000 based on sodium dodecyl sulfate-acrylamide gel electrophoresis. Its isoelectric point was 5.9. The enzyme more rapidly hydrolyzed penicillins, such as penicillin G, ampicillin, carbenicillin, piperacillin, and cloxacillin, than it hydrolyzed cephalosporins. For the hydrolysis of penicillin G, the optimal pH was 5.5, and the optimal temperature was 35 degrees C. The enzyme activity was inhibited by iodine, Cu2+, Hg2+, and EDTA but was not inhibited by clavulanic acid and sulbactam.
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Production of Cephalosporin C by Paecilomyces persicinus P-10
Pisano, M. A.
Vellozzi, E. M.
Antimicrobial Agents and Chemotherapy
After the growth of Paecilomyces persicinus P-10 in a glucose-peptone medium, filtrates were collected and analyzed for antibiotic antivity. Activities against Salmonella gallinarum ATCC 3030 and Alcaligenes faecalis ATCC 8750 (penicillin N-resistant strain) were obtained. Part of the former activity was readily inactivated by penicillinase. The fraction active against A. faecalis was isolated by passage through Amberlite XAD-2 and Amberlite IRA-68. The powder eventually obtained was subjected to paper chromatography followed by bioautography, and the activity obtained corresponded to that of a sample of cephalosporin C. Thin-layer chromatography was also employed to verify the presence of cephalosporin C in the P-10 powder. The active solids were further purified by means of paper chromatography in a solvent system consisting of n-butanol-acetic acid-water (60:15:25, vol/vol). The material obtained from this procedure yielded an infrared absorption spectrum identical to that of cephalosporin C. Similarly, the ultraviolet absorption of the purified preparation coincided with that of cephalosporin C. Exposure of the purified solids to cephalosporinase resulted in rapid inactivation of the antibiotic. In addition to penicillin N and cephalosporin C, filtrates of P. persicinus P-10 also contained deacetylcephalosporin C, deacetoxycephalosporin C, and cephalosporin P.
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Unusual Effects of Penicillin G and Chloramphenicol on the Growth of Moraxella osloensis
DeLeys, Robert J.
Juni, Elliot
Antimicrobial Agents and Chemotherapy
Growth of exponential-phase liquid cultures of Moraxella osloensis was inhibited by 0.5 U of penicillin G per ml. For this organism, low concentrations of penicillin acted primarily in a bacteriostatic rather than in a bactericidal manner. At higher concentrations of penicillin some killing did take place, but the rate of killing was rather slow and appeared to be independent of penicillin concentration. Microscopic observation of cells from penicillin-treated cultures showed little or no cellular swelling or lysis. The total cell count did not decrease significantly during 6 h of incubation in 5,000 U of penicillin per ml. The rates of respiration, nucleic acid synthesis, and protein synthesis were not affected by the presence of penicillin. Attempts to counteract the bactericidal action of high concentrations of penicillin with growth inhibitory concentrations of chloramphenicol were unsuccessful, since chloramphenicol itself was more bactericidal than penicillin for M. osloensis.
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Cloning, Overexpression, and Characterization of a Novel Thermostable Penicillin G Acylase from Achromobacter xylosoxidans: Probing the Molecular Basis for Its High Thermostability
Cai, Gang
Zhu, Songcheng
Yang, Sheng
Zhao, Guoping
Jiang, Weihong
Applied and Environmental Microbiology
The gene encoding a novel penicillin G acylase (PGA), designated pgaW, was cloned from Achromobacter xylosoxidans and overexpressed in Escherichia coli. The pgaW gene contains an open reading frame of 2,586 nucleotides. The deduced protein sequence encoded by pgaW has about 50% amino acid identity to several well-characterized PGAs, including those of Providencia rettgeri, Kluyvera cryocrescens, and Escherichia coli. Biochemical studies showed that the optimal temperature for this novel PGA (PGA650) activity is greater than 60°C and its half-life of inactivation at 55°C is four times longer than that of another previously reported thermostable PGA from Alcaligenes faecalis (R. M. D. Verhaert, A. M. Riemens, J. V. R. Laan, J. V. Duin, and W. J. Quax, Appl. Environ. Microbiol. 63:3412-3418, 1997). To our knowledge, this is the most thermostable PGA ever characterized. To explore the molecular basis of the higher thermostability of PGA650, homology structural modeling and amino acid composition analyses were performed. The results suggested that the increased number of buried ion pair networks, lower N and Q contents, excessive arginine residues, and remarkably high content of proline residues in the structure of PGA650 could contribute to its high thermostability. The unique characteristic of higher thermostability of this novel PGA provides some advantages for its potential application in industry.
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Isolation and characterization of a penicillinase from Pseudomonas cepacia 249.
Prince, A
Wood, M S
Cacalano, G S
Chin, N X
Antimicrobial Agents and Chemotherapy
Pseudomonas cepacia has an inducible beta-lactamase which is responsible for its novel ability to catabolize beta-lactam compounds. The gene encoding this enzyme, penA, was cloned from a genomic library of P. cepacia 249 on the broad-host-range cosmid pLAFR. This separated the penA gene from the gene encoding a second beta-lactamase in P. cepacia 249. Expression of penA was inducible in an Escherichia coli host strain by low levels of penicillin. The 33,500-molecular-weight enzyme had penicillinase activity not inhibited by clavulanic acid or sulbactam and was highly active against piperacillin and azlocillin. In comparison with other inducible beta-lactamases produced by gram-negative organisms, the penA enzyme had many properties which were similar to those of the penicillinase produced by Alcaligenes faecalis. It was unlike the ampC-type cephalosporinase produced by Pseudomonas aeruginosa.
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Identification of dimethyl disulfide-forming bacteria isolated from activated sludge.
Tomita, B
Inoue, H
Chaya, K
Nakamura, A
Hamamura, N
Ueno, K
Watanabe, K
Ose, Y
Applied and Environmental Microbiology
Twenty-four strains with high dimethyl disulfide (DMDS)-forming ability were isolated from activated sludge and identified to the genus level. These bacteria were classified into four groups (A, B, C, and D) by the API ZYM System (API System S.A., Montalieu, France). Group A (three strains) was identified as genus Lactobacillus by the API 20B System, by the method of Cowan and Steel, and by production of lactic acid as confirmed by gas-liquid chromatography. Group B (eight strains) was identified as genus Corynebacterium by API 20B and the Cowan and Steel method. Group C (one strain) was suggested to belong to genus Corynebacterium by the API 20B System. Group D (12 strains) was identified as genus Pseudomonas or Alcaligenes by the API 20B System, as genus Alcaligenes by the Cowan and Steel method, and as Achromobacter group Vd by the API 20NE System. However, on the basis of guanine-plus-cytosine contents in DNA and form of flagella, these strains were identified as genus Pseudomonas. Formation of DMDS from DL-methionine and S-methyl-L-cysteine was tested. DMDS-forming bacteria isolated from activated sludge formed DMDS from both precursors. In genus Pseudomonas, P. aeruginosa could not form DMDS from either precursor, but P. acidovorans, P. alcaligenes, P. pseudoalcaligenes, and P. testosteroni formed DMDS. In genus Alcaligenes, A. denitrificans subsp. xylosoxydans, A. denitrificans subsp. denitrificans, A. faecalis, and A. odorans formed DMDS from both precursors. Achromobacter group Vd formed DMDS from S-methyl-L-cysteine, but could not from DL-methionine.
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Contribution of permeability and sensitivity to inhibition of DNA synthesis in determining susceptibilities of Escherichia coli, Pseudomonas aeruginosa, and Alcaligenes faecalis to ciprofloxacin.
Bedard, J
Chamberland, S
Wong, S
Schollaardt, T
Bryan, L E
Antimicrobial Agents and Chemotherapy
To examine the correlation between bacterial cell susceptibility to ciprofloxacin and the magnitude of uptake and cell target sensitivity, the relative contribution of ciprofloxacin accumulation in intact cells and its ability to inhibit DNA synthesis were investigated among strains of Escherichia coli, Pseudomonas aeruginosa, and Alcaligenes faecalis. Uptake studies of [14C]ciprofloxacin demonstrated diffusion kinetics for P. aeruginosa and E. coli. Ciprofloxacin was more readily removed from E. coli J53 and A. faecalis ATCC 19018 by washing than from P. aeruginosa PAO503. These results indicate that the process of cell accumulation is different for P. aeruginosa in that the drug is firmly bound at an extracellular site. Whatever the washing conditions, A. faecalis accumulated less drug than either of the other two bacteria. Magnesium chloride (10 mM) caused a substantial decrease of ciprofloxacin accumulated and an increase in the MIC, depending upon the nature of the medium. The addition of carbonyl cyanide m-chlorophenylhydrazone caused a variable increase in drug accumulated, depending on the medium and the bacterial strain. The concentration of ciprofloxacin required to obtain 50% inhibition (ID50) of DNA synthesis for P. aeruginosa PAO503 and A. faecalis ATCC 19018 did not correlate with their corresponding MICs but did for E. coli J53. Treatment with EDTA decreased the ID50 of ciprofloxacin for P. aeruginosa PAO503 and its gyrA derivative by 5- and 2-fold, respectively, and decreased the ID50 for E. coli JB5R, a strain with a known decrease in OmpF, by 1.4-fold but did not decrease the ID50 for the normally susceptible E. coli J53. The ID(50) for P. aeruginosa obtained after EDTA treatment or in ether-permeabilized cells was higher than that obtained for the other two strains. The protonophore carbonyl cyanide m-chlorophenylhydrazone prevented killing by low ciprofloxacin concentrtaions, but sodium azide did not. The latter compound did not enhance killing in association with inhibition of a previously described energy-dependent efflux of ciprofloxacin susceptibility being the susceptibility to inhibition of DNA synthesis in E. coli, poor premeability associated with the small pore size of A. faecalis, and a combination of low permeability and reduced susceptibility of DNA synthesis to inhibition for P. aeruginosa.
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Paradoxical response of Enterococcus faecalis to the bactericidal activity of penicillin is associated with reduced activity of one autolysin.
Fontana, R
Boaretti, M
Grossato, A
Tonin, E A
Lleò, M M
Satta, G
Antimicrobial Agents and Chemotherapy
Ten clinical isolates of Enterococcus faecalis were examined for susceptibility to the bactericidal activity of penicillin. Four of these had MBCs of penicillin equal to 2 to 4 x the MIC, and six exhibited a paradoxical response to penicillin, i.e., the bactericidal activity of the antibiotic had a concentration optimum at 2 to 4 x the MIC and decreased significantly at concentrations above this. We found that the paradoxical response to penicillin was an intrinsic and stable property of a strain, but that its phenotypic expression was not homogeneous; only a fraction of the cell population that died at low concentrations was able to survive at high penicillin concentrations. The size of this fraction increased with increasing antibiotic concentration and reached a maximum in the late-log phase of growth. All 10 strains produced a lytic enzyme that was active on Micrococcus luteus heat-killed cells, whereas only some strains lysed E. faecalis heat-killed cells. Strains producing large amounts of the latter enzyme did not show the paradoxical response to penicillin, whereas mutants of these strains that lacked this enzymatic activity paradoxically responded to the antibiotic activity. In addition, from strains that showed paradoxical response to penicillin and produced only the enzyme that was active on M. luteus, it was possible to isolate mutants that were also capable of lysing E. faecalis cells and that were killed with similar efficiency by all concentrations above the MBC. On the basis of these findings, the paradoxical response to penicillin is explained as a property of certain strains of E. faecalis; this property is genetically characterized by alterations in synthesis or activity of one autolysin but phenotypically expressed only by a few cells that are in a particular physiological condition when exposed to high concentrations of antibiotics.
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Microbial Purification of Postfermentation Medium after 1,3-PD Production from Raw Glycerol
Szymanowska-Powałowska, Daria
Piątkowska, Joanna
Leja, Katarzyna
BioMed Research International
1,3-Propanediol (1,3-PD) is an important chemical product which can be used to produce polyesters, polyether, and polyurethanes. In the process of conversion of glycerol to 1,3-PD by Clostridium large number of byproducts (butyric, acetic and lactic acid) are generated in the fermentation medium. The aim of this work was to isolate bacteria strains capable of the utilization of these byproducts. Screening of 30 bacterial strains was performed using organic acids as carbon source. Selected isolates were taxonomically characterized and identified as Alcaligenes faecalis and Bacillus licheniformis. The most active strains, Alcaligenes faecalis JP1 and Bacillus licheniformis JP19, were able to utilize organic acids almost totally. Finally, it was find out that by the use of coculture (C. butyricum DSP1 and A. faecalis JP1) increased volumetric productivity of 1,3-PD production (1.07 g/L/h) and the yield equal to 0.53 g/g were obtained in bioreactor fermentation. Moreover, the only by-product present was butyric acid in a concentration below 1 g/L.
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Identification of Amino Acid Residues Responsible for the Enantioselectivity and Amide Formation Capacity of the Arylacetonitrilase from Pseudomonas fluorescens EBC191 ▿ †
Kiziak, Christoph
Stolz, Andreas
Applied and Environmental Microbiology
The nitrilase from Pseudomonas fluorescens EBC191 converted (R,S)-mandelonitrile with a low enantioselectivity to (R)-mandelic acid and (S)-mandeloamide in a ratio of about 4:1. In contrast, the same substrate was hydrolyzed by the homologous nitrilase from Alcaligenes faecalis ATCC 8750 almost exclusively to (R)-mandelic acid. A chimeric enzyme between both nitrilases was constructed, which represented in total 16 amino acid exchanges in the central part of the nitrilase from P. fluorescens EBC191. The chimeric enzyme clearly resembled the nitrilase from A. faecalis ATCC 8750 in its turnover characteristics for (R,S)-mandelonitrile and (R,S)-2-phenylpropionitrile (2-PPN) and demonstrated an even higher enantioselectivity for the formation of (R)-mandelic acid than the nitrilase from A. faecalis. An alanine residue (Ala165) in direct proximity to the catalytically active cysteine residue was replaced in the nitrilase from P. fluorescens by a tryptophan residue (as found in the nitrilase from A. faecalis ATCC 8750 and most other bacterial nitrilases) and several other amino acid residues. Those enzyme variants that possessed a larger substituent in position 165 (tryptophan, phenylalanine, tyrosine, or histidine) converted racemic mandelonitrile and 2-PPN to increased amounts of the R enantiomers of the corresponding acids. The enzyme variant Ala165His showed a significantly increased relative activity for mandelonitrile (compared to 2-PPN), and the opposite was found for the enzyme variants carrying aromatic residues in the relevant position. The mutant forms carrying an aromatic substituent in position 165 generally formed significantly reduced amounts of mandeloamide from mandelonitrile. The important effect of the corresponding amino acid residue on the reaction specificity and enantiospecificity of arylacetonitrilases was confirmed by the construction of a Trp164Ala variant of the nitrilase from A. faecalis ATCC 8750. This point mutation converted the highly R-specific nitrilase into an enzyme that converted (R,S)-mandelonitrile preferentially to (S)-mandeloamide.
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Chemical Characterization, Crossfeeding and Uptake Studies on Hydroxamate Siderophore of Alcaligenes faecalis
Sayyed, R. Z.
Chincholkar, S. B.
Meyer, J. M.
Kale, S. P.
Indian Journal of Microbiology
We report the production of two types of siderophores namely catecholate and hydroxamate in modified succinic acid medium (SM) from Alcaligenes faecalis. Two fractions of siderophores were purified on amberlite XAD, major fraction was hydroxamate type having a λmax at 224 nm and minor fraction appeared as catecholate with a λmax of 264 nm. The recovery yield obtained from major and minor fractions was 297 and 50 mg ml−1 respectively. The IEF pattern of XAD-4 purified siderophore suggested the pI value of 6.5. Cross feeding studies revealed that A. faecalis accepts heterologous as well as self (hydroxamate) siderophore in both free and iron complexed forms however; the rate of siderophore uptake was more in case of siderophores complexed to iron. Siderophore iron uptake studies indicated the differences between hydroxamate siderophore of A. faecalis and Alc E, a siderophore of Alcaligenes eutrophus.
Siderophores; IEF; Crossfeeding; Iron uptake
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Cellular fatty acids of Alcaligenes and Pseudomonas species isolated from clinical specimens.
Dees, S B
Moss, C W
Journal of Clinical Microbiology
The cellular fatty acid composition of 25 clinical isolates of Alcaligenes and Pseudomonas was determined by gas-liquid chromatography (GLC). The GLC fatty acid profiles of three species of Pseudomonas were markedly different from those of Alcaligenes. The most significant differences were the presence and relative amounts of hydroxy, branched-chain, and cyclopropane fatty acids. One of the major fatty acids in A. faecalis was a 17-carbon cyclopropane (17 delta) acid, whereas a 15-carbon branched-chain acid (13-methyl tetradecanoate) characterized isolates of P. putrefaciens. The determination of these fatty acids by GLC provides a rapid and specific means of distinguishing clinical isolates of Pseudomonas and Alcaligenes.
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Metabolism of fensulfothion by a soil bacterium, Pseudomonas alcaligenes C1.
Sheela, S
Pai, S B
Applied and Environmental Microbiology
Fensulfothion (O,O-diethyl O-[4-(methylsulfinyl)phenyl]phosphorothioate), an organophosphorus pesticide used to control the golden nematode Heterodera rostochiensis, is used as a source of carbon by microorganisms isolated from soils treated with the pesticide. Two of the microbial isolates, Pseudomonas alcaligenes C1 and Alcaligenes sp. strain NC3, used more than 80% of the pesticide in 120 h in culture when supplemented as a source of carbon. P. alcaligenes C1, which showed maximal growth on fensulfothion, degraded the compound to p-methylsulfinyl phenol and diethyl phosphorothioic acid. The phenolic metabolite could be identified by conventional spectral analysis, whereas the spectral patterns of the phosphorus-containing metabolite suggested that the compound was complexed with some cellular molecules. However, utilization of the phosphoric acid ester and ethanol by P. alcaligenes C1 suggested that the microbe attacks fensulfothion by an initial hydrolysis of the compound and subsequent utilization of the phosphoric acid ester. The pathway of degradation of fensulfothion by P. alcaligenes is of great value in the detoxification of the pesticide residues and also in the environmentally stable phosphoric acid esters.
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Booth, James S.
Appleman, Milo D.
Journal of Bacteriology
Booth, James S. (University of Southern California, Los Angeles) and Milo D. Appleman. Degradation of ergothioneine by cell-free extracts of Alcaligenes faecalis. II. Production of glutamic acid. J. Bacteriol. 85:654–657. 1963.—On the basis of oxidation and paper chromatographic procedures, glutamic acid was identified as the end product of ergothioneine degradation by cell-free extracts of Alcaligenes faecalis. Hydrogen sulfide and ammonia yields were determined. Several differences between the metabolism of whole cells and cell-free extracts were noted. Cleavage of the imidazole ring by cell-free extracts appeared to be hydrolytic rather than oxidative.
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Size of diffusion pore of Alcaligenes faecalis.
Ishii, J
Nakae, T
Antimicrobial Agents and Chemotherapy
The diffusion pore of the outer membrane of Alcaligenes faecalis was shown to be substantially smaller than the Escherichia coli porin pore. In experiments with intact cells, pentoses and hexoses penetrated into the NaCl-expanded periplasm, whereas saccharides of Mr greater than 342 did not. Cells treated with 0.5 M saccharides of Mr greater than 342 weighed 33 to 38% less than cells treated with isotonic solution, suggesting that these saccharides do not permeate through the outer membrane. The diffusion rates of various solutes through the liposome membranes reconstituted from the Mr-43,000 outer membrane protein showed the following characteristics. (i) The relative diffusion rates of pentoses, hexoses, and methylhexoses appeared to be about 1.0, 0.6, and negligibly small, respectively. (ii) The diffusion rate of glucose appeared to be about 1/10th that with the E. coli B porin. (iii) The diffusion rate of gluconic acid was five to seven times higher than that of glucose. (iv) The diffusion rates of beta-lactam antibiotics appeared to be 40 to less than 10% of those with the E. coli B porin.
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Microbial Fermentation of Rice Straw: Nutritive Composition and In Vitro Digestibility of the Fermentation Products
Han, Youn W.
Applied Microbiology
Rice straw was fermented with Cellulomonas sp. and Alcaligenes faecalis. Microbial cells and undigested residue, as well as chemically treated (NaOH or NH4OH) and untreated straws, were analyzed for nutrient composition and in vitro digestibility. In a typical fermentation, 75% of the rice straw substrate was digested, and 18.6% of the total substrate weight that disappeared was recovered as microbial protein. The microbial cell fraction was 37% protein and 5% crude fiber; the residue was 12% protein and 45% crude fiber. The microbial protein amino acid profile was similar to alfalfa, except for less cysteine. The microbial cells had more thiamine and less niacin than Torula yeast. In vitro digestibility of the microbial protein was 41.2 to 55%; that of cellulose was 52%.
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Unbalanced growth and macromolecular synthesis in Streptococcus mutans FA-1.
Mattingly, S J
Dipersio, J R
Higgins, M L
Shockman, G D
Infection and Immunity
The continued synthesis of deoxyribonucleic acid, protein, cell wall peptidoglycan and intracellular iodophilic polysaccharide (IPS) by Streptococcus mutans strain FA-1 after several treatments intended to inhibit protein synthesis was studied. Exponential-phase cultures were: (i) simultaneously deprived of two required amino acids (cystine and leucine) that are not present in the cell wall peptidoglycan of this species; (ii) depreived of required amino acids (lysine or glutamate plus glutamine ) that are present in both peptidoglycan and protein; or (iii) treated with tetracycline. Each of these three types of treatment was accompanied by a different pattern of unbalanced growth. The patterns of unbalanced growth that accompanied treatments (i) or (ii) differed substantially from the patterns observed previously for Streptococcus faecalis ATCC 9790, a noncariogenic organism that does not contain IPS. In contrast to S. faecalis 9790, S. mutans FA-1 failed to accumulate peptidoglycan and thicken its wall when deprived of non-wall amino acids. Instead, S. mutans FA-1 continued to accumulate IPS to levels substantially higher than those found in exponential-phase cells. Again, in contrast to S. faecalis, S. mutans FA-1 failed to autolyze upon deprivation of essential precursors of wall peptidoglycan. Under conditions of lysine of glutamate/glutamine deprivation, S. mutans FA-1 continued to accumulate IPS to very high levels. Treatment with tetracycline did result in peptidoglycan accumulation and wall thickening in a manner very similar to that observed previously for inhibition of protein synthesis in S. faecalis. Realtively little IPS synthesis continued after tetracycline treatment. Accumulation of IPS appeared to occur when both ribonucleic acid and peptidoglycan synthesis were severely inhibited. The observations are discussed in terms of the survival of cariogenic organisms in the oral environment.
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Cloning, nucleotide sequence, and expression in Escherichia coli of the gene for poly(3-hydroxybutyrate) depolymerase from Alcaligenes faecalis.
Saito, T
Suzuki, K
Yamamoto, J
Fukui, T
Miwa, K
Tomita, K
Nakanishi, S
Odani, S
Suzuki, J
Ishikawa, K
Journal of Bacteriology
The extracellular poly(3-hydroxybutyrate) depolymerase gene from Alcaligenes faecalis T1 was cloned into Escherichia coli DH1 by using the plasmid pUC8. An A. faecalis T1 genomic library was prepared in E. coli from a partial Sau3AI digest and screened with antibody against the depolymerase. Of the 29 antibody-positive clones, 1 (pDP14), containing about 4 kilobase pairs of A. faecalis T1 DNA, caused expression of a high level of depolymerase activity in E. coli. The enzyme purified from E. coli was not significantly different from the depolymerase of A. faecalis in molecular weight, immunological properties, peptide map, specific activity, or substrate specificity. Most of the expressed enzyme was found to be localized in the periplasmic space of E. coli, although about 10% of the total activity was found in the culture medium. Results of a deletion experiment with pDP14 showed that a large SalI fragment of about 2 kilobase pairs was responsible for expression of the enzyme in E. coli. The nucleotide sequence of the large SalI fragment has been determined. Comparison of the deduced amino terminus with that obtained from sequence analysis of the purified protein indicated that poly(3-hydroxybutyrate) depolymerase exists as a 488-amino-acid precursor with a signal peptide of 27 amino acids.
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Replacement of Lysine by Hydroxylysine and Its Effects on Cell Lysis in Streptococcus faecalis
Shockman, Gerald D.
Thompson, J. Stuart
Conover, Margaret J.
Journal of Bacteriology
Shockman, Gerald D. (Temple University, Philadelphia, Pa.), J. Stuart Thompson, and Margaret J. Conover. Replacement of lysine by hydroxylysine and its effects on cell lysis in Streptococcus faecalis. J. Bacteriol. 90:575–588. 1965.—Hydroxylysine was not significantly incorporated by Streptococcus faecalis ATCC 9790 or 8043 until exponential growth ceased as a result of lysine exhaustion. Uptake was then rapid and virtually complete within 1 hr. Lysine absence, rather than physiological age, seemed to be the governing factor. Hydroxylysine uptake rapidly reached a peak in the acid-soluble fraction, suggesting a precursor role for substances in this fraction. Substitution of hydroxylysine for lysine was much more efficient in mucopeptide synthesis than in protein synthesis. In wall medium, less than 1% of the incorporated hydroxylysine was found in the protein fraction. Addition of lysine to both growth and wall media inhibited both further hydroxylysine uptake and transfer of hydroxylysine from acid-soluble to mucopeptide or protein fractions. Hydroxylysine resulted in decreased penicillin susceptibility only after it was postexponentially incorporated. This effect was physiologically similar to that seen after threonine deprivation or chloramphenicol treatment. Hydroxylysine incorporation increased resistance to autolysis, but failed to decrease lysozyme susceptibility when measured after heat inactivation of autolysis. Electron microscopy of negatively stained cells showed increased thickness of cell walls containing hydroxylysine. Thus, most of the effects of replacement of lysine by hydroxylysine resemble those seen after deprivation of a nonwall amino acid (e.g., threonine or valine) or after chloramphenicol treatment. Each of these conditions results in inhibition of protein synthesis while permitting cell-wall synthesis to continue, resulting in autolysis-resistant, thick-walled cells.
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Biochemical and molecular characterization of the Pseudomonas lemoignei polyhydroxyalkanoate depolymerase system.
Jendrossek, D
Frisse, A
Behrends, A
Andermann, M
Kratzin, H D
Stanislawski, T
Schlegel, H G
Journal of Bacteriology
Pseudomonas lemoignei has five different polyhydroxyalkanoate (PHA) depolymerase genes (phaZ1 to phaZ5), which encode the extracellularly localized poly(3-hydroxybutyrate) (PHB) depolymerases C, B, and D, poly(3-hydroxyvalerate) (PHV) depolymerase, and PHB depolymerase A, respectively. Four of the five genes (phaZ1 to phaZ4) have been cloned, and one of them (phaZ1) was studied in detail earlier (D. Jendrossek, B. Müller, and H. G. Schlegel, Eur. J. Biochem. 218:701-710, 1993). The fifth PHA depolymerase gene (phaZ5) was identified by colony hybridization of recombinant Escherichia coli clones with a phaZ5-specific oligonucleotide. The nucleotide sequence of a 3,704-bp EcoRI fragment was determined and found to contain two large open reading frames (ORFs) which coded for a polypeptide with significant similarities to glycerol-3-phosphate dehydrogenases of various sources (313 amino acids; M(r), 32,193) and for the precursor of PHB depolymerase A (PhaZ5; 433 amino acids; M(r), 44,906). The PHV depolymerase gene (phaZ4) was subcloned, and the nucleotide sequence of a 3,109-bp BamHI fragment was determined. Two large ORFs (ORF3 and ORF4) that represent putative coding regions were identified. The deduced amino acid sequence of ORF3 (134 amino acids; M(r), 14,686) revealed significant similarities to the branched-chain amino acid aminotransferase (IlfE) of enterobacteria. ORF4 (1,712 bp) was identified as the precursor of a PHV depolymerase (567 amino acids; M(r), 59,947). Analysis of primary structures of the five PHA depolymerases of P. lemoignei and of the PHB depolymerases of Alcaligenes faecalis and Pseudomonas pickettii revealed homologies of 25 to 83% to each other and a domain structure: at their N termini, they have typical signal peptides of exoenzymes. The adjacent catalytic domains are characterized by several conserved amino acids that constitute putative catalytic triads which consist of the consensus sequence of serine-dependent hydrolases including the pentapeptide G-X-S-X-G, a conserved histidine and aspartate, and a conserved region resembling the oxyanion hole of lipases. C terminal of the catalytic domain an approximately 40-amino-acid-long threonine-rich region (22 to 27 threonine residues) is present in PhaZ1, PhaZ2, PhaZ3, and PhaZ5. Instead of the threonine-rich region PhaZ4 and the PHB depolymerases of A. faecalis and P. pickettii contain an approximately 90-amino-acid-long sequence resembling the fibronectin type III module of eucaryotic extracellular matrix proteins. The function of the fibronectin type III module in PHA depolymerases remains obscure. Two types of C-terminal sequences apparently represent substrate-binding sites; the PHB type is present in the PHB depolymerases of A. faecalis and P. pickettii and in PhaZ2, PhaZ3, and PhaZ5 and the PHV type is present in the PHV-hydrolyzing depolymerases (PhaZ4 and PhaZ1). phaZ1 was transferred to A. eutrophus H16 and JMP222. All transconjugants of both strains were able to grow with extracellular PHB as a carbon source and produced translucent halos on PHB-containing solid media. PhaZ1, PhaZ2, PhaZ4, and PhaZ5 were purified from P. lemoignei and from recombinant E. coli; the processing sites of the precursors in E. coli were the same as in P. lemoignei, and similar substrate specificities were determined for the wild-type and the recombinant proteins. All PHA depolymerases hydrolyzed PHB at high specific activities. PhaZ1 and PhaZ4 additionally cleaved PHV, and PhaZ4 hydrolyzed poly(4-hydroxybutyrate). None of the depolymerases was able to hydrolyze polyactide or PHA consisting of monomers with more than five carbon atoms. While the wild-type depolymerase proteins were glycosylated and found to contain glucose and N-acetylglucosamine, none of the recombinant proteins was glycosylated. PHB hydrolysis was dependent on divalent cations such as Ca2+ and was inhibited by the presence of EDTA.
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Excretion of Lipoteichoic Acid by Group A Streptococci
Alkan, Michael L.
Beachey, Edwin H.
Journal of Clinical Investigation
Group A streptococci were grown in the presence of [2-3H]glycerol. Concentrated suspensions of the labeled organisms were incubated with and without penicillin. [3H]Glycerol-labeled material accumulated in the supernates in increasing amounts with increasing concentrations of penicillin, ranging from 0 to 50 U/ml. The excretion of labeled material occurred in the absence of nucleic acid synthesis or bacteriolysis indicating that the phenomenon is independent of cell multiplication or decay. The accumulation of label was paralleled by an accumulation of erythrocyte-sensitizing material measured by passive hemagglutination tests for lipoteichoic acid antigen, indicating that a portion of the labeled material possessed the properties of lipoteichoic acid. Culture supernates were fractionated by column chromatography, and the materials obtained were analyzed by electrophoresis on sodium dodecyl sulfate polyacrylamide, thin-layer chromatography, and paper chromatography. The ability of the same materials to bind to human erythrocytes and epithelial cells was tested. The culture supernate contained lipoteichoic acid, deacylated lipoteichoic acid, glycerol phosphate, and free glycerol. Penicillin caused an increase in the amounts of each of the excreted materials. Streptococci that were stimulated with penicillin to lose their lipoteichoic acid (previously shown to mediate adherence of group A streptococci) lost their ability to adhere to buccal mucosal cells, suggesting that penicillin may influence bacterial ecology by mechanisms other than killing sensitive organisms.
Related Articles
Penicillin-netilmicin synergism against Streptococcus faecalis.
Korzeniowski, O M
Wennersten, C
Moellering, R C
Sande, M A
Antimicrobial Agents and Chemotherapy
The combination of penicillin plus netilmicin was synergistic in vitro against 28 strains of Streptococcus faecalis and compared favorably with penicillin in combination with gentamicin. Similarly, penicillin plus netilmicin was as effective as penicillin plus gentamicin in the therapy of 67 rabbits with enterococcal endocarditis produced with a streptomycin-susceptible (S) or a streptomycin-resistant (R) strain of S. faecalis. After 5 days of infection, control rabbits had bacterial titers of 10(10) colony-forming units (CFU)/g of vegetation. Those treated with penicillin plus netilmicin had mean titers of 10(5.2) and 10(5.1) CFU/g for S and R strains, respectively, and those treated with penicillin plus gentamicin had mean valve titers of 10(5.8) CFU/g for both strains. After 10 days of therapy, mean valve titers with penicillin plus netilmicin were 10(3.8) and 10(4.7) CFU/g, and with penicillin plus gentamicin they were 10(4.5) and 10(5.4) CFU/g for S and R strains, respectively. Thus, if netilmicin proves to be less toxic than other aminoglycoside antibiotics, it may have potential usefulness in the therapy of enterococcal endocarditis.
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I have to be more explicit now. The analysis given by stikman and few others ignores to a large extent the black responses.
I wish to challenge all players who believe that White would have a good game in the gambit.
3 days per move, 1.d4 Nf6, 2.g4 Nxg4, and I have Black.
I only analyzed my own games I've played yet. More will follow (at the moment I'm busy with the analysis of two other GWG games). At this stage I'm in a learning proces.
I would like to take you up for a challenge with the GWG. Maybe you can learn me something .
Hey BigG...thats the point...everyone of us is in a learning process dealing with this gambit (you remember our both GWG-battles...both quickly won by white ) and i think its really helpful, if a good and sceptical player liker schlechter55 joins us in analyzing the possibilties for both sides.
@Dark_Falcon: That's why I play schlechter55 at the moment with a GWG on the board .
Super article!!
The first analyzed GWG game between Dark_Falcon and me. It's an interesting game where black played 2...e5 (Oshima Variation).
The complete text of the analysis of the game (click here).
And the other game between Dark_Falcon and me ...
Very interesting. For blitz it is definitely a good weapon. For longer games I am not that sure, but if the Evan's gambit is good this must be good as well.
@csalami10: I only play correspondence (long) games, and GWG works for me (even at 2000+) .
2000 - over, or below zero?
Whoever is willing to take the white pieces after 1.d4 Nf6 2.g4 (?) Nxg4 3.e4 d6 4.Be2 Nf6 5.Nc3 e5 (variant from #45 which is not mentioned) is very courageous. If White trades pawns and queens, his development advantage consists of having played Bf1-Be2 and g2-out of the board at the cost of a pawn, hardly impressive compensation. If 6.d5 White loses any central initiative he may have had. Otherwise Black trades himself on d4 and plays ...Nc6 etc. with an easy development.
I would be much more afraid of meeting the Budapest than this "gambit".
I mean, it's less awful than it seems, but there is still a good margin before being even remotely playable at 2000+ level, long games.
another senseless and respectless post from the bearded greek guy...you will never learn how to behave.
@Irontiger: Perhaps the GWG doesn't suit everybody, but some find it intriguing enough to play it . There's nothing wrong with the Budapest Gambit (I play it myself), but others can't related to that either. Most important is that you play your own style of chess. There's no right or wrong, just an enjoyable game of chess .
What about in GWG if 3. f3 and then black plays this? Drunken Knight Countergambit: 3.-e5!?4.fxg4 Dh4+ 5.Kd2 exd4 6.Rf3 Dxg4 7.Tg1 De4 8.Ke1 Rc6 9.c3
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As wild lion populations continue to decrease at alarming rates take a look at who is working hard to protect them. Nikela Volunteer Cassie finds huge challenges and some innovative solutions.
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After reading please share.
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For computers and laptops you will need Adobe reader. Download it here for free.
7 Critically Endangered Animal Species
A list of 7 critically endangered wildlife species that need special attention and how you can help. Compiled by Margrit Harris
POACHED! in Japanese
The personal account of a vet's heroic quest to save Geza the rhino. Translated from the original English into Japanese by Yuko Imaizumi.
Human Wildlife Conflict: The Red Sea Example of Unsustainable Use
While in Egypt Sylvia Lin stumbled on an unexpected human wildlife conflict issue.
Cheetah Conservation: What's Working? What isn't?
This is a follow up eReport to "Cheetah Conservation: What's Really Happening". What Sylvia discovers may surprise you.
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Since childhood Sylvia has had an idylic view of Cheetahs, so she was totally unprepared for what she discovered.
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DENVER -- Indians designated hitter Travis Hafner was advised by renowned orthopedist Dr. James Andrews to continue with a strengthening program for his ailing right shoulder.
"Dr. Andrews was very straight forward," Indians head trainer Lonnie Soloff said. "He didn't think surgery would help his condition. He is doing isometric exercises, stability exercises, lifting weights."
Hafner has been on the disabled list since May 30 with a sore shoulder. He got a second opinion Wednesday from Andrews, who agreed with the Indians' Dr. Mark Schickendantz that Hafner needs continued strengthening of the rotator cuff and shoulder girdle.
"In the absence of irritating that shoulder on a daily basis with game activities, our hope is that it will continue to get stronger," Soloff said.
Hafner will be re-evaluated in a week to 10 days. He is hitting .217 with four home runs this season, but has been bothered by the shoulder since spring training.
The 31-year-old Hafner is Cleveland's career leader at DH with 134 home runs and 464 RBIs.


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I am currently developing an application in C# where I need to write a tab separated CSV file from the data that it retrieves from a MySQL Database. The database retrieval works fine.
The problem that I am having is writing the file. Between each variable that I am writing I am using the \t which I thought put a tab into the csv, therefore when opening in excel each variable will be in its own cell.
However for some reason it is not doing this it just writes the whole line as one long string. Below is an example of the code that I am code that I have written:
while (reader.Read())
int bankID = reader.GetInt16("ban_bankID");
int userID = reader.GetInt16("ban_userID");
string bankUsername = reader.GetString("ban_username");
string accountName = reader.GetString("ban_accountName");
string accountType = reader.GetString("ban_accountType");
decimal overdraft = reader.GetDecimal("ban_overdraft");
char defaultAccount = reader.GetChar("ban_defaultAccount");
string line = bankID + "\t" + userID + "\t" + bankUsername + "\t" + accountName + "\t"
+ accountType + "\t" + overdraft + "\t" + defaultAccount + "\n";
Thanks for your help with this problem.


text:
Kona's Max Unger acclimating to life in New Orleans with the Saints
Beyond football, the Hawaii Preparatory Academy graduate told KHON2 he's trying to acclimate to life in the Big Easy.
Honolulu's Michelle Wie limps into British Open in Scotland
Michelle Wie has quite the entourage these days. The doctor at the Cleveland Clinic in Ohio who has worked on her hip. The doctor in Los Ang…
9 Hawaii natives selected to Polynesian Football H.O.F. College Player of the Year Award Watch List
The Polynesian Football Hall of Fame announced Tuesday the 2015 Watch List for the PolynesianCollege Football Player of the Year Award, and …
Clarke named preseason all MWC selection, 'Bows picked to finish 5th in West Division
University of Hawai'i senior offensive lineman Ben Clarke was selected to the Preseason All-Mountain West Team, while the Rainbow Warriors w…
Arizona Cardinals name woman as inside linebackers training camp coach
The Cardinals say she is believed to be the first woman to hold a coaching position of any kind in the NFL.
Kaiser offensive lineman Michael Eletise commits to Arizona
After receiving 42 scholarship offers, Michael Eletise announced his decision Monday night on KHON2 at 6. He is considered to be the most hi…
Maui's Shane Victorino traded from Red Sox to Angels
Maui's Shane Victorino is headed to Anaheim. The Saint Anthony graduate and 2-time All-Star Outfielder was traded Monday from the Boston Red…
Australia's Belivacqua captures 19th Molokai 2 Oahu Paddleboard World Championship
The 19th annual Molokai 2 Oahu Paddleboard World Championships took place Sunday, where Australian dominance continued.
Kaiser's Michael Eletise prepares to make collegiate commitment
Kaiser offensive lineman Michael Eletise will make his college commitment Monday night.
Waipahu's Viloria wins 4th straight with 1st round TKO
It might be time for Waipahu native Brian Viloria to get back into title contention. For the fourth straight fight, Viloria's hand was raise…
Hall of Fame won't allow Seau's daughter to give speech
CANTON, Ohio (AP) — Junior Seau's daughter will be commenting only on a video when her late father enters the Pro Football Hall of Fame on A…
Harrison thanks father for path to Team USA
Like father, like son. The saying rings especially true for the Harrisons. Following in the footsteps of Kenny's All-American career at the …
Maryknoll graduate Burnett to play college hoops at Kennesaw State
Josh Burnett says he will study broadcast journalism at the Atlanta school while accomplishing a life-long dream of playing Division I ball.
MUST SEE: 'Bows OL Clarke executes reverse backflip off diving board
University of Hawaii offensive lineman Ben Clarke "flipped out" at the team's off-season barbecue.
LeBron James signs TV, movie deal with Warner Bros.
James says connecting with his fans and "telling meaningful stories" have always been his passion.
Hawai'i football stars victorious in stars and stripes
This past weekend in Canton, Ohio, former University of Hawai'i All-American Mike Edwards, former Warrior quarterbacks coach Dan Morrison, a…
Tennessee Titans announce signing of Marcus Mariota
"Thank you to the Tennessee Titans organization as well as my agent for making a dream come true."
Hawaii girls soccer team wins Nike Crossfire Challenge tournament
Hawaii Rush Soccer Club 02 Nero defeated Pacific Blue from Washington 2-1 in Monday's final to take home the trophy.
Hawaii surfer eliminated by Mick Fanning reacts to shark attack
KHON2 spoke with Ewa Beach native Keanu Asing, who surfed that same contest and was eliminated by Fanning hours before.
'It was like me or the shark': Surfer Mick Fanning on attack day after
The confrontation took place on live TV, and video showing the dark fin approaching Fanning from behind quickly went viral.


text:
1 Review
We have enjoyed this very much, Lennie! I cut the calories and fat some by using a 6 oz. box of the sugar-free Jell-o, Splenda instead of sugar, and 8 oz. of fat-free cream cheese. It made an guilt-free treat which DH particulary liked, since he had two helping of this!;)I also made it in a 9 inch square pyrex pan, since I didn't think my ring mold was large enough.Thanks for posting this recipe!


text:
My Demon Prince Chapter 4 by Vilen vileRated 64.7%, Read 4431 times, Posted Wed 10th of July 2013
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text:
Engine Performance problem
2001 Ford F150 6 cyl Two Wheel Drive Manual 70000 miles
Upon hard or sudden acceleration the engine misses (studders). I have replaced the a/filter, fuel filter, f/pump, s/plug wires and cleaned throttle plate & mas.
It is more pronounced at low rpms at any speed. Any suggestions would be greatly appreciated!
Friday, January 23rd, 2009 AT 4:25 PM