text:
Dr. Byron Willis received his B.S.
in engineering from Columbia
University and his medical degree
from the State University of
New York at Buffalo. He
completed his neurosurgery
residency training at the Cleveland
Clinic with special training in
complex spinal disorders. He also
received additional training in
the treatment of cerebrovascular
disorders and intracranial tumors during a one year
fellowship at Derriford Hospital, England.
Dr. Willis is the president and founder of ANSS. He is
board certified by the American Board of Neurological
Surgery. He serves as the Chairman of the Department of Surgery and the Chairman of the Division of Neurosciences at Banner Good Samaritan Medical Center.
Dr. Willis's interests include complex spine, tumors,
cerebrovascular disorders and neurotrauma.
Notice of Patient Information Practices
1331 N 7th St, Ste 275 | Phoenix, AZ 85006 | USA | Phone: (602) 254-3151
copyright © (2008), Arizona Neurosurgery & Spine Specialists, P.C.


text:
"I feel it's important to spend time listening to my patients. Communication is one of the most important aspects of medicine."
Dr. Dolan enjoys reading, spending time with his family and the outdoors. He also has enjoyed teaching medical students and participating in arthritis research and volunteer work. He is a founding fellow of the American College of Rheumatology and a Co-founder of the American Belicit's Foundation.
This physician is a member of MemorialCare Physician Society which drives clinical outcomes and performance for MemorialCare Health System.
- Rheumatology
- Internal Medicine
Medical EducationInstitution:
Creighton University School of MedicineDate Completed:
May 31 1973

subset: commoncrawl


text:
I am not going into raptures about WordPress simply because so many have done it before. This post is practical, down to earth advice to those bloggers who are not yet into WordPress and why they ought to be.
WordPress is simple to install and setup
The first hurdle which a blogger must cross is setting it all up. Those of you who are using traditional website for a blog are acutely aware


text:
Skip to Main Content
Your Community
Your Government
City Services
How Do I...
City Services
Parks & Recreation
Parks & Recreation
Sherman Parks and Recreation has recently relocated. The administrative offices are now located at the Glennie O. Ham Center on 1002 N Music.
This is the location that you will come to reserve a park pavilion or ball field or to register for a recreation activity. This building and the adjacent Activity Center will house the majority of Parks and Recreation activity programming. Please refer to the
for directions.
Mission Statement
The mission of the City of Sherman Parks and Recreation Department is to enhance the quality of life in Sherman by providing the finest parks, facilities, programs and services in the area.
Notify Me
Activity Guide
Recreational Activities
Hawn Spray Ground
Pecan Grove Disc Golf
Parks & Facilities
Park Maintenance
Sponsorship Opportunities
The Splash (Swimming Pool)
Parks and Recreation Board


text:
Tweets from @TBHomeTeam/writers
MORE from our HomeTeam writers.
Wed. October 14, 2009 | Keith Niebuhr
Thursday night, Countryside faces Tarpon Springs in a football game that will showcase two of the state's most productive running backs -- the Spongers' Adrian "Bubba" Golden (824 rushing yards, 8 TDs) and the Cougars' Alton Taylor (884 rushing yards, 19 TDs). To give you an idea of what Countryside must contend with, check out this clip of Golden that was sent to me this morning from the Tarpon Springs coaching staff:


text:
Ford Model T for Sale
Selected Filters:Type of Ad: Vehicles for Sale Make: Ford Model: Model T Remove All
1925 Ford Model T Runabout
St. Louis, MO
1925 Ford Model T Runabout Roadster Gorgeous professional nut and bolt restoration! Runabout model with soft top All steel body Black exterior and interior 177 CID inline four-cylinder engine... More
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1926 Ford Model T
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The legendary Ford Model T. The humble beginnings of the modern production car as we know it today. While the Model T wasn't the... More
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1927 Ford Model T Sedan
Staunton, IL
Tudor Sedan, older resto, good body, nice redone int, runs/drives good... More
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1926 Ford Model T Touring Convertible
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1926 Ford Model T Touring 4dr Touring Convertible, nice recent resto, good body and paint, nice redone int, 4cyl, runs/drives good... More
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1925 Ford Model T
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1926 Ford Model T Limousine
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The car offered here is a Ford Model T from 1926.The vehicle is in a good condition, and a worthwhile investment.Several additional items are... More
1920 Ford Model T Huckster
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For sale is a 1920 Ford Model T Huckster. This thing starts up first try, and runs and drives beautifully. It is perfect to... More
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More information on this 1923 Ford Model T Roadster to come. If you have any questions feel free to call 615-213-1800 or visit our... More
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1912 Ford Model T Speedster
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1912 Ford Model T Speedster Wood Spoke Wheels Fenders... More
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This is considered in working and running condition. Car is located and pick up will be in Carthage North Carolina. This vehicle is part of... More
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Fiberglass Alloway-style front clip with hood scoop- 351 CI Ford engine- 4-barrel carburetor- Headers and side exhaust- Aluminum radiator with fan- Automatic transmission- Independent... More
1918 Ford Model T Open Touring
Portsmouth, RI
1918 Ford Model T touring runabout 4 passenger open convertible. 177 cubic inch 4 cylinder20 horse power. 2 speed Planetary Gear. Frame off full... More


text:
Beyond Meat, a new company based in Maryland, has come up with an alternative to chicken meat that it claims is a dead ringer for the real thing. And unlike other meat alternatives on the market, this one aims to be cheap, as well as tasty.
The inspiration for Beyond Meat (formerly known as Savage River Farms) started, oddly enough, in "chicken country." Founder Ethan Brown grew up spending weekends on his family's farm in Grantsville, Md., near the Pennsylvania border.
A Farmer Bets Better Fake Chicken Meat Will Be As Good As The Real Thing : The Salt : NPR
Current Status: Blessed (1)
Seeded on Thu May 17, 2012 6:41 AM
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text:
Makes the EU Say FU to the Bush Administration
14, 2001
by Richard Prasad
The Bush Administration has warned us since 9/11 to be vigilant
against terrorist attacks, including the anthrax attacks following
the 9/11 tragedy. But has the Bush Administration been vigilant
in preventing the proliferation of germ warfare agents throughout
the world? The shocking and disturbing answer is no.
The Bush Administration on December 7th forced a germ warfare
conference to break up. The meeting was established to try
to add enforcement provisions to the 1972 Biological Weapons
Convention. The current meeting was held in Geneva to discuss
the progress of a group who have been trying for six years
to negotiate legally enforceable compliance with the Biological
Weapons Convention.
The Biological Weapons Convention was ratified by the U.S.
and 143 other nations almost thirty years ago. Article One
of the Biological Weapons Convention states that each member
who is party to this convention will undertake to NEVER develop
produce stockpile or otherwise retain microbial or any other
biological agents or toxins. It further bans the development
of delivery systems for these toxins. Article 2 of the BWC
requires the destruction of these banned items, or their conversion
into peaceful uses within 9 months.
The Biological Weapons Convention sounds like a good framework
to build upon to try to ban these horrible weapons of mass
destruction, so why would the Bush administration prematurely
end a meeting trying to enhance enforcement of the Biological
Weapons Convention, at a time where banning such weapons has
gained increased urgency because of these anthrax attacks?
Simple, say Bush administration representatives, because the
Biological Weapons Convention is impossible to enforce. Bush
Administration officials have stated that nothing in the protocol
would prevent rogue nations from acquiring biological weapons
if the desired.
That answer has left members of the European Union shocked
and angry to say the least. In a statement the E.U. says they
are deeply committed to multilateral arms negotioations. This
action by Bush representatives only reinforces the European
view that the Bush Administration is unilateralist in its
world view, willing to go it alone on issues rather than join
with other nations.
We all know the litany of treaties that the Bush Administration
has either succeeded in killing or has backed away from. The
Kyoto Protocol, which requires strict environmental standards,
which Bush and his oil burning pals aren't willing to adhere
to. And let us not forget the Anti Ballistic Missile Treaty,
which stands in the way of Bush wanting to build SDI. But
all this unilateralism occurred before 9/11.
Many thought that since 9/11 that President Bush would understand
the importance of coalition building, since he experienced
the value of coalition building during our current war with
Afghanistan. Could he not take the leadership role that the
US has taken in the war, and take the lead role in enforcing
a ban on germ warfare as well? The argument that this convention
cannot be enforced seems weak when the US can enforce no fly
zones in Iraq and hunt down terrorists in Afghanistan. We
can do whatever we aim to do in this country, if we as a nation
have the political will to do so. President Bush does not
have the political will to do so. Why? I do not know.
Further, we are ready to declare war on Iraq for not letting
UN inspection teams in to inspect possible biological weapons
sites. It is reasonable to think we should go to war with
Iraq if they don't let us inspect those sites, but how can
we go to war with Iraq on this issue, when we are not stringently
supporting the Biological Weapons Convention, which bans the
very weapons Iraq is said to have. The hypocrisy of the Bush
Administration here is intolerable.
Finally, the lack of press coverage on this Biological Weapons
Conference is astounding. None of the major tv news organizations
covered it that I know. CNBC, and it's clone CNN never covered
it, and Fox, well, they don't cover anything. This is a huge
story with huge international ramifications, and all I could
find was a couple of newspaper articles, and that is it. I
know the press, in general, has taken to saluting the flag
and telling us what a great job the President is doing in
prosecuting the war. That is all well and good, but how about
putting the pom poms away, and informing the public about
matters of grave concern to everyone.
I am asking all the readers of Democratic Underground to
call or e-mail your local House Member or Senator and ask
them why the US walked out on a meeting to enforce the biological
weapons treaty. We the people can make enough noise to make
a difference. Maybe we can make newsworthy what the mainstream
media chooses to ignore.


text: I was going to order on Amazon but it said it was not going to ship until after Christmas. I looked around the web and came upon myotcstore.com and it said I would receive it about 3 days after shipping. I ordered it on the 12th of December and received an email update that it was processed and ready to ship on the 14th. Then on the 21st I received an email that due to an inventory problem my order shipment is being delayed. Really, 7 days later I'm being told there is a problem after I was told it was ready to ship? I received it on the 26th. I should have known better, if Amazon can't get it out by Christmas no one can. Too bad as I thought I found a new site to shop.


text:
Random Blogging For Wednesday January 29th
Here's what you missed on today's show.
The Morning Brain Buster:
Q: According to Women's Health, 3% of women say they can't remember this. What is it?
A: Their natural hair color
Bonehead Of The Day:
A woman in Roswell, New Mexico called in a fake report of a gunman near a convenience store to help a friend avoid a traffic ticket over a taillight.
The 22-year-old woman called 911 hoping the cop who pulled over the car she was a passenger in would get dispatched to the fake crime.
She called while the officer was checking her friend's information.
The woman later admitted making the entire story up and was arrested.
Holy Snow Rollers.. Check This Out!
"Snow Rollers" have been forming in the Midwest due to the amount of snowfall and windy conditions.
The sculptures have become big hits on Twitter and Facebook.
The rollers are formed from a combination of sticky snow, fairly strong winds and cold temperatures. Most of them are too light to be picked up but some people have been able to take them inside to try to preserve their shape.
Some have gotten to be the size of basketballs.
Teen Lucky To Be Alive After Parachute Fails
A Texas teen is lucky to be alive after her parachute failed while skydiving on her 16th birthday.
She jumped right after her dad, but her primary chute didn't open.
Her dad watched his daughter spin to the ground.
She somehow survived a 3,500 fall to the earth but ended up with broken bones and internal injuries.
She broke bones in her back and was in and out of consciousness at the hospital. She's expected to make a good recovery.
Girl Scout Cookies Go Gluten Free
The Girl Scouts are now offering gluten-free cookies!
The Girl Scouts of America are rolling out their first gluten-free cookie, the Chocolate Chip Shortbread, in 20 select markets.
The cookie is reportedly "made with real chocolate chips and real butter" and that it "contains no artificial flavors, no artificial colors, no high fructose corn syrup, no palm oil and no hydrogenated oils."
If the cookies perform well in those locations, they may go national next cookie season.
There's also another new cookie in the troop this year called the Cranberry Citrus Crisp, made from whole grains, cranberry bits and citrus flavors.
Police Chief Asks Crime To Go On Hiatus During Cold Weather
A police chief in Wisconsin hopped on the department's Facebook to cancel all crime... because of the cold weather.
The post read:
Attention, Attention, due to the "Polar Vortex," "Artic Blast," " Really Cold" temperatures, Germantown has decided to cancel all criminal activity for Tuesday January 28th 2014. We are also canceling outdoor shenanigans, crazy behavior and anything else that would subject an officer to the frigid cold temperatures tomorrow.
The high yesterday in Germantown was 2 degrees with wind chills as low as -25.
Because We're Not Fat Enough.. A Burger With A Custard Soaked, Deep Fried Buns
At Umami Burger in L.A. they have a new burger you will love - the Monte Cristo Burger.
It's a beef patty, topped with busseto prosciutto and Gruyere fondue. The bun it's served on is soaked in custard, deep fried, then topped with powdered sugar.
Maple syrup is of course, served on the side.
The man behind the burger is Michael Voltaggio who you may remember from Top Chef.
~~Talk-show host/TV mogul Oprah Winfrey is 60.
~~Actor, Tom Selleck is 69 (Blue Bloods; The Rockford Files' Lance White, Magmun PI's Thomas Sullivan "Tom" Magnum, Young & the Restless' Jed (1970s), Three Men & A Baby, In & Out, The Love Letter, Friends' Richard Burke)
~~Director-actor Ed Burns is 46. (Saving Private Ryan; She's The One; The Brothers McMullen; Holiday; many more)
~~Actress Heather Graham is 44. (Boogie Nights; Austin Powers: The Spy Who Shagged Me; The Hangover)
~~Congressman Paul Ryan is 44
~~Actress, Isabel Lucas ("Transformers: Revenge of the Fallen") is 29 (Home and Away; Daybreakers)
~~Actress Ann Jillian is 64. (Mr. Mom; It's A Living)
~~Actor, Sam Trammell ("True Blood") is 45
~~Actor Sharif Atkins is 39. (White Collar; ER)
~~Actress Sara Gilbert ("Roseanne") is 39. (The Talk)
~~Actor, Justin Hartley is 37 (Smallville; Aquaman)
~~Actress, Kelly Packard is 39 (California Dreams' Tiffani Smith, Baywatch)
~~Actor, Jason James Richter is 34 (Free Willy)
~~Actress, Katharine Ross is 74 (The Graduate; Butch Cassidy and the Sundance Kid; The Stepford Wives)
~~Actor, Marc Singer is 66 (The Beastmaster; 'V' (1983)
~~Actor, Nicholas Turturro is 52 (I Now Pronounce You Chuck & Larry; NYPD Blue; Blue Bloods)
~~Actress Diane Delano ("The Ellen Show," ''Northern Exposure") is 57.
~~Actress Judy Norton Taylor ("The Waltons") is 56.
~~Actor Sam Jaeger ("Parenthood") is 37.
~~Actor Andrew Keegan ("Party of Five") is 35. (10 Things I Hate About You; 7th Heaven; more)
~~Former baseball star, Steve Sax is 54
~~Olympic diver, Greg Louganis is 54
~~Singer Adam Lambert ("American Idol") is 32.
~~Drummer Tommy Ramone (of The Ramones) is 62
~~Drummer Louie Perez (of Los Lobos) is 61
~~Singer Charlie Wilson (of The Gap Band; solo) is 61
~~Guitarist Johnny Spampinato (of NRBQ) is 55
~~Drummer David Baynton-Power (of James) is 53
~~Bassist Eddie Jackson (of Queensryche) is 53.
~~Guitarist Jonny Lang is 33.
~~Singer, Amii Stewart is 58 ("Knock On Wood")
~~Country singer Irlene Mandrell is 58.
5 Crazy Film Props You Can Buy On eBay
If you are a film memorabilia collector, or eBay hoarder, here are five items you might want to put on your "to buy" list.
These are handful of film props that are actually for sale on eBay:
1. The Velociraptor cage from the opening of "Jurassic Park", plus a raptor model. Current bid: $99,900.10.
2. The 1970 Chevy Nova from "Bullet Proof". No bids, but starting price: $39,900.
3. The saw from "Saw". Current bid: $7,000.
4. An oar from "Titanic". Current bid: $1,045
5. Arnold Schwarzenegger prop head from "Total Recall". Current bid: $560
Go Daddy Super Bowl Ad Will Feature A Woman Quiting Her Job Live
A woman will quit her job on live TV this Sunday in a Super Bowl ad for GoDaddy.
The woman's identity won't be known to the ad airs on Sunday and her boss won't know she quitting until the ad airs.
The woman is leaving to start her own business and will send in her resignation letter after the ad airs.
7 Shows That Will Probably Be Cancelled
Here are 7 TV shows you can expect to get cancelled.
1. The Michael J Fox Show
2. Sean Saves the World (announced last night)
3. Revolution
4. The Neighbors
5. Beauty and the Beast
6. The Carrie Diaries
7. Intelligence
New Trend.. Going Bra-Less
Going bra free is the new trend.
Kim Kardashian, Miranda Kerr and Gwyneth Paltrow have been going braless.
It seems that wearing bras may be the cause for back pain and sagging.
Research shows that for three months women feel discomfort when not wearing a bra and then suddenly their back pain goes away. Also, their breasts are more plush and pushed up after 3 months without a bra.
Addicted To Your Smart Phone.. There's An App For That
There's a new app that tells you if you are addicted to your smartphone.
The free Android app called Menthal monitors your smartphone usage to see if you are dependent on it. It tells you how long you spend on the phone per day and which apps you use the most.
The app will also use your results to gauge your mood. For example, if you spend a lot of time messaging and chatting with friends, this might be an indicator that you are feeling cheerful whereas fewer interactions could mean that you are feeling depressed.
Video Of The Day:
This is a pretty funny short video.. Enjoy!


text:
Layton Shumway
Layton is an assistant editor for DeseretNews.com. He also helps manage the organization's social media presence and writes sports articles. Prior to his internship and employment at the Deseret News, Layton received a Bachelor of Arts degree in journalism from Brigham Young University in 2008 and served as copy chief, editor and sports writer for the BYU Daily Universe. Layton has also written for numerous publications in the fields of sports and entertainment, including About.com, Electronic Gaming read more Monthly, SLC Dunk, and Bitmob.
Connect with Layton Shumway
Email email@example.com
twitter @theshums


text:
As a key intracellular metabolite, acetyl-coenzyme A (acetyl-CoA) plays a major role in various metabolic pathways that link anabolism and catabolism. In the yeast Saccharomyces cerevisiae, acetyl-CoA involving metabolism is compartmentalized, and may vary with the nutrient supply of a cell. Membranes separating intracellular compartments are impermeable to acetyl-CoA and no direct transport between the compartments occurs. Thus, without carnitine supply the glyoxylate shunt is the sole possible route for transferring acetyl-CoA from the cytosol or the peroxisomes into the mitochondria. Here, we investigate the physiological profiling of different deletion mutants of ACS1, ACS2, CIT2 and MLS1 individually or in combination under alternative carbon sources, and study how various mutations alter carbon distribution. Based on our results a detailed model of carbon distribution about cytosolic and peroxisomal acetyl-CoA metabolism in yeast is suggested. This will be useful to further develop yeast as a cell factory for the biosynthesis of acetyl-CoA-derived products.
Citation: Chen Y, Siewers V, Nielsen J (2012) Profiling of Cytosolic and Peroxisomal Acetyl-CoA Metabolism in Saccharomyces cerevisiae. PLoS ONE 7(8): e42475. https://doi.org/10.1371/journal.pone.0042475
Editor: Paul Cobine, Auburn University, United States of America
Received: May 8, 2012; Accepted: July 6, 2012; Published: August 2, 2012
Copyright: © Chen et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The authors thank Firmenich, the Knut and Alice Wallenberg Foundation and the Chalmers Foundation for funding this project. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors declare they received funding from Firmenich. There are no other declarations relating to employment, consultancy, patents, products in development or marketed products. This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials.
Acetyl-coenzyme A (acetyl-CoA) is involved in many different metabolic pathways and plays a key role in linking anabolism and catabolism. It is a precursor metabolite for de novo biosynthesis of fatty acids and sterols. It is also the end product of lipid degradation by β-oxidation, and it is used for synthesis of C4 metabolites via the glyoxylate shunt during utilization of carbon sources such as fatty acids and C2 compounds, e.g. acetate and ethanol. Furthermore, it is a crucial substrate for energy production in the tricarboxylic acid (TCA) cycle. In addition to serving as an important metabolite intermediate in central carbon metabolism, acetyl-CoA is also an important building block for the biosynthesis of many other, biotechnologically relevant metabolites, such as waxes, flavonoids, certain amino acids, carotenoids, polyhydroxyalkanoates, polyketides, butanol and isoprenoids.
In Saccharomyces cerevisiae, acetyl-CoA metabolism takes place in at least four subcellular compartments: nucleus, mitochondria, cytosol and peroxisomes. Nuclear acetyl-CoA serves as acetyl donor for histone acetylation [1], but besides this role in chromatin regulation, acetyl-CoA is mainly important for the central carbon metabolism. Depending on the supply of substrate to the cell, various mechanisms may lead to the formation and utilization of acetyl-CoA. With sugars as carbon source, direct formation of acetyl-CoA from pyruvate catalyzed by the pyruvate dehydrogenase complex (PDH) in the mitochondria serves as fuel for the TCA cycle. On the other hand, acetyl-CoA generated via direct activation of acetate in an ATP-dependent reaction by an acetyl-CoA synthetase (ACS) in the cytosol is the only source for fatty acid and sterol biosynthesis [2]. During growth on oleate, acetyl-CoA is the end product of β-oxidation of straight chain fatty acids, which in yeast exclusively takes place in the peroxisomes [3]. When cells grow on a non-fermentable substrate such as ethanol or acetate, an additional extra-mitochondrial pool of acetyl-CoA is required for the glyoxylate shunt where it is consumed by citrate synthase and malate synthase.
Since the membranes of organelles are impermeable for acetyl-CoA [4], this molecule must either be synthesized within each subcellular compartment where it is required or imported using specific transport mechanisms. Two transport systems have been identified [4]. In the first, the carnitine/acetyl-carnitine shuttle, acetyl-CoA produced in the peroxisomes or the cytosol is converted into acetylcarnitine, which is subsequently transported into the mitochondria. However, it has been shown that S. cerevisiae is not capable of de novo synthesis of carnitine and unless carnitine is supplied with the medium this transport system is non-functional [5]. The second pathway is synthesis of C4 dicarboxylic acids from acetyl-CoA via the glyoxylate shunt, followed by transport of the C4 dicarboxylic acids to the mitochondria or the cytosol where they can serve as precursor for formation of pyruvate or phosphoenolpyruvate required for either the TCA cycle or for gluconeogenesis.
A hallmark of eukaryotic cells is the organization of various specific anabolic and catabolic pathways into subcellular compartments. Cast as a central molecule in yeast metabolism, it is not surprising that the biochemical mechanism for the generation and utilization of acetyl-CoA has been studied in detail. However, the compartmental demands for acetyl-CoA vary with the nutrient supply of a cell, and it has been difficult to ascertain the cellular location and function of certain enzymes, and further complicated by multiple isoforms of some of these enzymes related to acetyl-CoA metabolism in S. cerevisiae. This holds in particular for acetyl-CoA synthetase (EC 22.214.171.124), which catalyzes the ligation of acetate and CoA. Two structural genes, ACS1 and ACS2, encoding this enzyme have been characterized in S. cerevisiae [6]. At high concentrations of glucose, ACS1 is transcriptionally repressed, whereas a severe de-repression occurs under glucose limitation or supplementation with non-fermentable carbon sources such as ethanol or acetate. Acs1p was originally thought to be located in the mitochondria [7], [8], and possibly also in the peroxisomes [9]. Green fluorescent protein (GFP) tagging however suggested a distribution between the nucleus and the cytoplasm [10]. With respect to substrate specificity Acs1p was shown to have a 30-fold higher affinity to acetate than Acs2p supporting the idea that Acs1p is primarily responsible for acetate activation during growth on C2 compounds [6]. ACS2 is constitutively expressed irrespectively of the kind of carbon source and type of metabolism (respiratory or fermentative) [2], [11]. Thus, it is essential for growth on glucose containing medium. It has also been demonstrated that ACS2 is required for histone acetylation and the biology of the endoplasmic reticulum (ER), the Golgi apparatus and the vacuole [1]. Hence, although Acs2p was predicted to be cytosolic, it may also be present in the nucleus and possibly in the ER with respect to its function in histone acetylation and global transcriptional regulation. To allow for growth on fatty acids, ethanol or acetate, the glyoxylate shunt enzymes play an essential role because of the net synthesis of C4 metabolites from acetyl-CoA units, which are either the end product of fatty acid degradation or result from activation of C2 compounds (ethanol or acetate). Of the glyoxylate shunt enzymes, citrate synthase 2 (Cit2p) and malate synthase 1 (Mls1p) catalyze the condensation reaction of acetyl-CoA with oxaloacetate or glyoxylate, respectively. It has been difficult to establish whether all glyoxylate shunt enzymes are localized in the peroxisomes or in the cytosol. Cit2p was firstly determined as extra-mitochondrial by using immunological criteria [12]–[14]. From cellular fractionation and metabolic changes of a deletion mutant, different authors suggested that Cit2p is a cytosolic enzyme [12], [15]. However, Lewin and coworkers [16] concluded that it was peroxisomal and involved in the glyoxylate shunt by sedimentation on density gradients after cultivating the yeast cells on an oleate medium. An altering distribution has also been reported for Mls1p. Immunoelectron microscopy in combination with cell fractionation showed that it was peroxisomal when cells were grown on fatty acids, while it remained in the cytosol when yeast utilized C2 compounds such as ethanol or acetate [17].
Here we map the carbon distribution of acetyl-CoA metabolism in yeast under different nutrient conditions. Among a variety of S. cerevisiae strains, the CEN.PK strain family was selected based on the physiological and genetic properties [18]. We investigate the growth phenotypes and metabolite processing in different mutants affected in acetyl-CoA metabolism. Various location and transport possibilities are discussed under different nutrient situations, and a detailed map for acetyl-CoA metabolism in yeast is proposed.
Materials and Methods
Yeast strains, plasmid construction and gene deletions
S. cerevisiae strains and plasmids used in this study are summarized in Table 1. All experiments were performed in the background of CEN.PK 113-5D (MATa SUC2 MAL2-8c ura3-52; P. Kötter, University of Frankfurt, Germany).
To remove the SKL codons from the MLS1 gene, PCR was applied to the genomic DNA of CEN.PK 113-5D using oligonucleotides as follows, MLS1-SKL-forward, GCCGCGACTAGTAAAACAATGGTTAAGGTCAGTTTGGA; MLS1-SKL-reverse, GCAGACTGAGCTCTCACAAATCAGTGGGCGTCGC (introduced restriction sites are indicated in italics, and the stop codon is indicated in bold). The amplification product obtained was digested with SpeI and SacI (Fermentas, St. Leon-Rot, Germany), and ligated to a SpeI- and SacI-digested plasmid pSP-GM1 [19], generating plasmid pIYC15. To express a SKL-truncated Mls1p, pIYC15 was introduced into SCIYC07 (mls1Δ) and SCIYC05 (acs1Δ), resulting in strains SCIYC28 and SCIYC29, respectively. Similarly, a primer pair of MLS1-SKL-forward (same as above) and MLS1-reverse, GCAGACTGAGCTCTCACAATTTGCTCAAATCAGTGG, was used to amplify the intact MLS1 gene. Then the product was cloned into vector pSP-GM1 using SpeI and SacI, resulting in plasmid pIYC16. By transformation of pIYC16 into SCIYC07 (mls1Δ) and SCIYC05 (acs1Δ), strain SCIYC35 and SCIYC36 were obtained, respectively.
Gene deletion was performed using a cloning-free PCR-based allele replacement method [20]. For deletion of ACS1, the regions upstream and downstream of the ACS1 open reading frame were individually amplified from genomic DNA of CEN.PK 113-5D by PCR, using the following oligonucleotides: ACS1-UP-forward, AGATAATGGGGCACGACCTC; ACS1-UP-reverse, GATCCCCGGGAATTGCCATGTGTAATGATGATTTCTTTCC; ACS1-DOWN-forward, GCAGGGATGCGGCCGCTGACGCTTAGAATAGCCGCCCAGT; ACS1-DOWN-reverse, TTGCGAAGGTGTTTAGGAAG. The kanMX expression module was amplified in two overlapping parts from the plasmid pUG6 [21], using the following oligonucleotides: KanMX-UP-forward, CATGGCAATTCCCGGGGATCAAGCTTCGTACGCTGCAGGTCG; KanMX-UP-reverse, CCATGAGTGACGACTGAATCCGG; KanMX-DOWN-forward, GCAAAGGTAGCGTTGCCAATG; KanMX-DOWN-reverse, GTCAGCGGCCGCATCCCTGCCGACTCACTATAGGGAGACCG.
Fragments ACS1-UP/KanMX-UP and KanMX-DOWN/ACS1-DOWN, respectively, were fused to each other via PCR. The two fusion fragments were integrated into the genome by homologous recombination yielding strain SCIYC02.
For the loop out of the KanMX expression module, procedures were followed as described previously [21]. Subsequently, correct transformants were re-streaked on synthetic complete medium containing 50 mg L−1 uracil and 750 mg L−1 5-fluoroorotic acid (5-FOA) for the loss of the plasmid pSH47 containing the URA3 marker and the reestablishment of uracil auxotrophy. Thus, strain SCIYC05 (acs1Δ) was obtained.
The same approach was used for all gene deletions in this study. All oligonucleotides used to perform gene deletions can be found in Table S1.
The deletion of each gene was verified by diagnostic PCR, which was performed by using the genomic DNA from each strain as template, and for each gene two pairs of primers were used, one outside of the integration locus and one inside the integrated fragment (see Table S2 for the primers used for strain confirmation).
Media and growth conditions
E. coli cells were grown in LB (Luria-Bertani broth) medium with ampicillin (100 mg L−1) at 37°C. Minimal media contained 0.67% yeast nitrogen base without amino acids (Difco Laboratories, Detroit, MI, USA), 2% agar, with 2% glucose, 2% glycerol, 2% ethanol or 0.1 M potassium acetate at pH 6.0 as carbon source. Uracil (20 µg ml−1) was added as required.
For selection of G418-resistant transformants, YPD plates containing 1% yeast extract, 2% Bacto-peptone and 2% glucose, were supplemented with 200 mg L−1 Geneticin (G418 sulfate, Gibco BRL, Germany). YPG medium containing 1% yeast extract, 2% Bacto-peptone and 2% galactose, was used for activation of Cre recombinase expression. Synthetic complete medium lacking uracil contained 0.67% yeast nitrogen base without amino acids (Difco Laboratories, Detroit, MI, USA), 2% drop-out powder without uracil (MP Biomedicals, Solon, OH, USA), 2% glucose and 2% agar.
Growth characterization in liquid cultures was performed in 250 ml baffled shake flasks containing 50 ml of minimal medium with 20 g L−1 glucose as carbon source, which were inoculated at OD600 0.02 from pre-cultures. Pre-culture cells were grown in YPD medium and washed twice with sterile water. Cultures were incubated at 30°C in a rotary shaker at 180 rpm. Samples were taken during the process and the growth was measured by determining the optical density at 600 nm (OD600). The filtrate was analyzed using a Summit HPLC (Dionex, Sunnyvale, CA, USA) with an Aminex HPX-87H column (Bio-Rad, Hercules, CA, USA) to measure the residual glucose and external metabolites including glycerol, ethanol, acetate, pyruvate, glyoxylate and succinate [22].
Spot assay
Strains were grown to late log-phase in liquid minimal medium at 30°C. The cells were harvested and washed twice with sterile water. After measuring the OD600 serial dilutions with final cell concentrations of 1×105, 1×104, 1×103, 1×102 cells per ml were prepared. From each dilution, 5 µl were spotted on minimal medium plates with glucose, acetate, ethanol or glycerol as carbon source. The plates were recorded photographically following 10 days incubation at 30°C.
ACS1 deletion mutant fails to grow on non-fermentable carbon sources while acs2Δ shows the opposite phenotype
Although two acetyl-CoA synthetases in S. cerevisiae, Acs1p and Acs2p, have been studied well for their function, biochemical properties and transcription regulation, there are conflicting reports on the growth phenotype of ACS1 deletion mutants [9], [11] and some of media used before were not devoid of carnitine (using complex medium) [11]. Therefore, ACS1 and ACS2 were deleted in the strain CEN.PK 113-5D to generate two mutant strains, SCIYC05 (acs1Δ) and SCIYC23 (acs2Δ), respectively. The growth phenotypes of the mutant strains were re-characterized by a spotting assay on synthetic minimal medium with different carbon sources together with the reference strain CEN.PK 113-5D (Fig. 1). SCIYC05 (acs1Δ) showed reduced growth with glucose as carbon source compared with the reference strain. However, it failed to grow on acetate, ethanol, or glycerol as sole carbon source. This might be due to the fact that: (i) acetyl-CoA produced by Acs2p cannot enter the peroxisomes, and hence cannot enter the glyoxylate shunt which will lead to formation of C4 units and from here further to glycolytic intermediates required for cell growth; or (ii) the Acs2p activity is at an insufficient rate to provide the C4 units needed for gluconeogenesis and other biosynthetic pathways to allow cells to utilize C2 compounds as sole carbon source. In contrast, the ACS2 deletion strain SCIYC23 (acs2Δ) was unable to grow on glucose media. But it grew normally on non-fermentable carbon sources (acetate, ethanol and glycerol) compared with the reference strain.
The plates were incubated at 30°C and recorded photographically 10 days after inoculation. The strains used were reference strain CEN.PK 113-5D, mutant strains SCIYC05 (acs1Δ), SCIYC23 (acs2Δ), SCIYC06 (cit2Δ) and SCIYC07 (mls1Δ).
To further examine the physiological features of the acs1Δ strain, liquid growth assays were conducted. SCIYC05 (acs1Δ) and the reference strain were cultured in shake flasks with minimal media containing 20 g L−1 glucose as carbon source. As shown in Fig. 2A and B, SCIYC05 (acs1Δ) showed an extended lag-phase, a lower growth rate, and a lower final biomass concentration during the glucose phase compared to the reference strain. The extended lag phase for an acs1 mutant during growth on glucose has been observed in previous studies as well [11]. Although ACS1 is reportedly subjected to glucose repression [6], the strongly delayed glucose consumption indicates further that Acs1p may have other function as well, e.g. Acs1p is probably involved in chromatin regulation since a nuclear localization has been reported as well [10]. After glucose depletion, SCIYC05 (acs1Δ) stopped growing, while the reference strain continued growing. HPLC analysis of metabolites revealed that SCIYC05 (acs1Δ) did not utilize ethanol and glycerol, which were produced during growth on glucose (Fig. 2B); whereas the reference strain could metabolize all non-fermentable carbon sources derived from overflow metabolism.
Cell growth of reference strain (A) and mutant strains SCIYC05 (acs1Δ) (B), SCIYC06 (cit2Δ) (C) and SCIYC07 (mls1Δ) (D), cultured in shake flask with minimal medium containing 20 g L−1 glucose as carbon source. All measurements are mean +/− standard error of three biological replicates.
Deletion of CIT2 results in differences in growth on solid and liquid culture with non-fermentable carbon sources
In S. cerevisiae, the citrate synthase isoenzymes Cit1p and Cit3p have been shown to be exclusively located in the mitochondria, whereas it has not been conclusively shown whether Cit2p is present in the cytoplasm or in the peroxisomes. Furthermore, there are conflicting reports in the literature on the growth phenotype of cit2Δ mutant strains [4], [13], [15], [23]. To investigate the effect of CIT2 disruption on cell growth and metabolism strain SCIYC06 (cit2Δ) was re-characterized on minimal medium with glucose, glycerol, ethanol or acetate as carbon source. As shown in Fig. 1, SCIYC06 (cit2Δ) did not display any severe difference in growth on glucose, but a slight growth reduction on non-fermentable carbon sources compared to the reference strain. As carnitine is not present in the media, meaning that the carnitine shuttle is not active, these results imply that CIT2 is dispensable for growth on C2 carbon sources, which is in contrast to the study by Swiegers et al. [23].
To reinforce the observation that CIT2 deletion still allows cell growth, SCIYC06 (cit2Δ) as well as the reference strain were tested in liquid culture with minimal medium including 20 g L−1 glucose as carbon source. SCIYC06 (cit2Δ) did not exhibit any growth difference when utilizing glucose as carbon source, whereas it failed to continue growing when glucose was depleted (Fig. 2C). As shown in Fig. 2C, metabolite analysis of the shake flask cultivation with SCIYC06 (cit2Δ) revealed that acetate was formed while ethanol was being consumed. This conversion results in net NAD(P)H production in the cells and hence allows the cells to generate ATP. The phenomenon of accumulating acetate, however, indicates that despite the presence of intact ACS1 and ACS2 the cells were not able to further catabolise acetyl-CoA resulting in acetate accumulation and no biomass formation.
MLS1 deletion affects the utilization of non-fermentable carbon sources
In yeast, two enzymes, Mls1p and Dal7p, have a 81% protein sequence identity to each other. Dal7p is thought to be involved in the degradation of allantoic acid to urea, whereas only MLS1 is highly transcribed upon growth on non-fermentable carbon sources [24]. Hence, malate synthase Mls1p is believed to be specifically involved in the glyoxylate shunt necessary for the utilization of C2 carbon sources. Although Mls1p ends with a C-terminal SKL tripeptide representing a peroxisomal targeting signal PTS1, selective distribution to the peroxisomes or the cytosol depending on the carbon source has been reported [17]. As expected for the MLS1 deletion strain, no significant changes in phenotype were observed on plate assays with glucose as carbon source (Fig. 1). In contrast, SCIYC07 (mls1Δ) was unable to grow on minimal media with glycerol, ethanol or acetate as sole carbon sources. Our results are consistent with previous work [17]. It was therefore confirmed that MLS1 is indispensable for the utilization of non-fermentable carbon sources. This also applies to the other key enzyme (Icl1p) unique to the glyoxylate shunt (data not shown).
In liquid culture, while SCIYC07 (mls1Δ) behaved similar during glucose as carbon source; the growth of SCIYC07 (mls1Δ) was remarkably slow after depletion of glucose, compared with the reference strain (Fig. 2A and 2D). However, extracellular metabolite analysis revealed that ethanol was almost completely metabolized, and after glucose consumption acetate first increased slightly until 24 hours after inoculation, and then decreased slowly. The concentration of glycerol was also shown to decrease very slowly, from 1.14 g L−1 to 0.53 g L−1 after glucose depletion. Comparing the consumption of (non-fermentable) carbon sources between SCIYC06 (cit2Δ) (Fig. 2C) and SCIYC07 (mls1Δ) (Fig. 2D), it was found that SCIYC06 (cit2Δ) metabolized ethanol to form acetate and the biomass concentration did not increase. On the other hand SCIYC07 (mls1Δ) consumed both C2 carbon sources, but still the OD600 only increased by 1.46, compared to the reference strain, for which the OD600 increased by 6.15 (Fig. 2A). This raised the question why both the accumulated ethanol and acetate were consumed, while only a small increase in biomass was observed.
Although MLS1 was deleted, isocitrate lyase was still active in these cells producing glyoxylate, which has been reported to be a toxic intermediate for mammalian cells [25] and bacteria [26]. To test the possibility of glyoxylate accumulation, extracellular glyoxylate and pyruvate were analyzed for SCIYC07 (mls1Δ) as well as reference strain. No accumulation of glyoxylate or pyruvate was detected for the reference strain. For SCIYC07 (mls1Δ), as shown in Fig. 3, when cells started to consume both C2 carbon sources, glyoxylate and pyruvate accumulated. Glyoxylate reached a concentration of 0.4 g L−1 at 40 hours after inoculation, then decreasing a little, whilst pyruvate continuously increased from the time point of glucose depletion to the end of the cultivation.
The concentrations of glyoxylate and pyruvate were measured in shake flask cultivation with minimal medium containing 20 g L−1 glucose as carbon source. All measurements are mean +/− standard error of three biological replicates.
CIT2 deletion restores growth in an acs1Δ strain
It was observed that deleting ACS1, CIT2 or MLS1, which are related to the acetyl-CoA metabolism, led to different changes in carbon source utilization. To investigate if the double deletion or triple deletions of these three genes cause different phenotypic changes in growth characteristics compared with single deletion mutants, we constructed the mutants SCIYC14 (acs1Δ cit2Δ), SCIYC15 (cit2Δ mls1Δ) and SCIYC16 (mls1Δ acs1Δ). Repeating the ACS1 deletion in SCIYC15 (cit2Δ mls1Δ) led to the triple deletion strain SCIYC21 (acs1Δ cit2Δ mls1Δ).
As shown in Fig. 4, no apparent growth was exhibited in the double deletion strains SCIYC15 (cit2Δ mls1Δ), SCIYC16 (mls1Δ acs1Δ), and the triple deletion mutant SCIYC21 (acs1Δ cit2Δ mls1Δ), supplemented with non-fermentable carbon sources. However, SCIYC14 (acs1Δ cit2Δ) was surprisingly able to grow on glycerol, ethanol or acetate as sole carbon source. Although the growth was slower compared to the reference strain, it clearly showed growth was restored in the acs1Δ and cit2Δ double deletion strain, whereas acs1Δ single deletion led to an inability to grow on such carbon sources.
Plate assay for SCIYC14 (acs1Δ cit2Δ), SCIYC15 (cit2Δ mls1Δ) and SCIYC16 (mls1Δ acs1Δ), and the triple deletion strain SCIYC21 (acs1Δ cit2Δ mls1Δ) on minimal medium with glucose, glycerol, ethanol or acetate as carbon source. The plates were recorded photographically after 10 days incubation at 30°C.
Truncated Mls1p suppresses the growth defects of the acs1Δ mutant
The inability of the acs1Δ single deletion strain to grow on C2 carbon source was overcome by further deleting another gene (CIT2) related to the acetyl-CoA metabolism. This phenotype implied that our previous hypothesis on the inefficiency of Acs2p in SCIYC05 (acs1Δ) was incorrect. This phenotypic change also indicates the possibility of flexible distribution of glyoxylate shunt enzymes. One possible assumption to explain these phenotypes would be that on non-fermentable carbon sources both Cit2p and Mls1p are localized in the peroxisomes when ACS1 is deleted, whereas Mls1p mainly stays in the cytosol in the case of the acs1Δ and cit2Δ double deletion strain. To validate this prediction, the plasmid pIYC15 encoding a truncated Mls1p (without the -SKL tripeptide), which is supposed to be located in the cytoplasm, was transformed into SCIYC07 (mls1Δ) and SCIYC05 (acs1Δ), yielding strains SCIYC28 and SCIYC29, respectively. Harboring the plasmid pIYC15 restored the growth of SCIYC07 (mls1Δ) on non-fermentable carbon sources, which confirmed that expressing a cytosolic malate synthase was efficient for maintaining a functional glyoxylate shunt (Fig. 5). As shown in Fig. 5, also SCIYC29 expressing the truncated Mls1p was able to grow on glycerol, ethanol or acetate as sole carbon source. As a control, the wild type Mls1p was also expressed in SCIYC07 (mls1Δ) and SCIYC05 (acs1Δ), resulting in strains SCIYC35 and SCIYC36, respectively. While SCIYC35 grew well on ethanol or acetate, SCIYC36 was unable to grow on C2 carbon sources (Fig. 5). Taking into account of the growth difference of SCIYC29 and SCIYC36, the results indeed support the hypothesis that both Cit2p and Mls1p are localized in the peroxisomes during growth on non-fermentable carbon sources when ACS1 is deleted.
Cells were grown on minimal medium supplemented with glucose, glycerol, ethanol or acetate as carbon source. Uracil (20 µg ml−1) was added as required (reference, SCIYC05 (acs1Δ) and SCIYC07 (mls1Δ)). The plates were recorded photographically after 10 days of incubation at 30°C.
Acetyl-CoA is a key intermediate in cellular metabolism. It is involved as a precursor metabolite; it is used for generation of Gibbs free energy, and for production of many industrially relevant compounds. In the yeast S. cerevisiae metabolism involving acetyl-CoA is compartmentalized and may vary with the nutrient supply of a cell. Where acetyl-CoA is metabolized on non-fermentable carbon sources is not fully understood, but as S. cerevisiae cannot synthesize carnitine and we did not provide this via the medium, the glyoxylate shunt is the sole possible route for transferring acetyl-CoA (in form of tricarboxylic acid cycle intermediates) into the mitochondria. Cit2p, Mls1p, Acs1p and Acs2p, are the main enzymes relevant to acetyl-CoA metabolism—either consuming or producing this molecule outside of mitochondria. Therefore, the aim of this study was to investigate the physiological effects of different mutant strains with combinations of these genes deletion (ACS1, ACS2, CIT2 and MLS1) on different carbon sources in S. cerevisiae, and try to understand intracellular transport of intermediates in various mutations with alternative carbon sources.
Irrespective of the nutrient supply to the cell the cytosolic acetyl-CoA pool is indispensable for synthesis of fatty acids and sterols, and it relies completely on acetyl-CoA synthetase activity on all carbon sources. In view of its essential role in the central carbon metabolism, at least one functional isoenzyme of acetyl-CoA synthetase must be located in the cytosol. SCIYC23 (acs2Δ) lacking ACS2 can grow on minimal medium containing a non-fermentable carbon source like ethanol or acetate, which clearly indicates that Acs1p is subjected to cytosolic distribution under these conditions. If Acs1p was exclusively localized in the peroxisomes, it could not activate acetate to form acetyl-CoA in the cytosol; and acetyl-CoA produced in the peroxisomes cannot be shuttled without the carnitine transport system. Our findings for the acs2Δ mutant are consistent with earlier reports by van den Berg and Steensma [11], i.e. that inactivation of ACS2 did not cause inability to grow on media with acetate or ethanol as sole carbon source (in complex medium with yeast extract). Since the media used in our study are devoid of carnitine it further confirms that Acs1p is distributed to the cytosol on a non-fermentable carbon source. The ability of acs2Δ mutants to grow in aerobic, glucose-limited chemostat cultures has also been reported [6], which further supports the conclusion that Acs1p is subjected to cytosolic distribution under de-repressing conditions. Even though it is reported that Cit2p has both N- and C-terminal signal for mitochondrion and peroxisomes, the potential of N-terminal signal can only be recognized by removing the C-terminal sequence [27]. Taking this into account together with that recently reported CIT2 is in peroxisomes regardless of synthetic or complex medium [28], strongly indicates that Cit2p is the peroxisomal isoenzyme of citrate synthase that is involved in the glyoxylate shunt, and it therefore seems likely that Acs1p functions in the peroxisomes in synthesis of acetyl-CoA required for use by Cit2p. Furthermore, according to the results of Kunze et al. [17], Mls1p remains in the cytosol in ethanol-grown cells and it therefore seems reasonable that there is a dual distribution of Acs1p for cells growing under non-repressing conditions. Similarly, it was also recently shown that fumarase and aconitase were dually localized in the mitochondria and the cytoplasm [29], [30]. Overall, Acs1p is suggested to show a dual distribution pattern between the cytoplasm and the peroxisomes, as shown in Fig. 6A.
(A) reference strain; Acs1p is suggested to be distributed between the cytosol and the peroxisomes, Cit2p is strongly suggested to be located in the peroxisomes, Mls1p stays in the cytosol; pathway I (in green) and II (in blue) indicate the possible shunt in the absence of CIT2 during growth on C2 compounds; (B) acs1Δ deletion strain; Cit2p and Mls1p are compartmentalized in the peroxisomes; (C) cit2Δ deletion strain; Mls1p remains in the cytosol; (D) acs1Δ and cit2Δ double deletion strain; Mls1p is targeted to the cytosol. Location of reactions marked by dashed lines was not identified in this study. Oac, oxaloacetate; Acs1p, acetyl-CoA synthetase 1; Acs2p acetyl-CoA synthetase 2; Cit2p citrate synthase 2; Mls1p malate synthase 1; Icl1p, isocitrate lyase 1; Mae1p, malic enzyme; Pck1p, phosphoenolpyruvate carboxykinase 1; Mdh2p, malate dehydrogenase 2.
The scenario in Fig. 6A does probably not apply to the acs1Δ strain. For mutant SCIYC05 (acs1Δ), the inability to grow on a non-fermentable carbon source was observed. Besides the requirement of 1.8 mmol cytosolic acetyl-CoA per gram cells for lipid synthesis in the cytosol [31], metabolism on C2 carbon sources passes through acetyl-CoA for both biosynthesis and energy metabolism. Therefore, inefficient conversion of C2 compounds (ethanol or acetate) to acetyl-CoA could be explained by low Acs2p activity, which may explain the acs1Δ strain's inability to grow on non-fermentable carbon sources. However, the double mutant strain lacking both ACS1 and CIT2 was shown to be able to grow on non-fermentable carbon sources, which contradicts this hypothesis. Furthermore, expressing the truncated but not the intact Mls1p in the cytoplasm of SCIYC05 (acs1Δ) restored the growth capacity. This observation strongly suggests that both Cit2p and Mls1p are located in the peroxisomes of cells lacking ACS1 (Fig. 6B), and low Acs2p activity therefore cannot explain the inability of growth of the acs1Δ strain.
Outside the mitochondria, acetyl-CoA synthetases serve as the main producers of acetyl-CoA, while citrate synthase 2 catalyzes one of the acetyl-CoA consuming reactions. Deletion of CIT2 breaks the integrity of the glyoxylate shunt. However, in our study the cit2Δ strain SCIYC06 (cit2Δ) was shown to be able to grow on minimal media plates containing ethanol, acetate or glycerol as sole carbon source. As shown in Fig. 6A and 5C, the disruption in the glyoxylate shunt resulting from the CIT2 deletion can be bypassed by (i) succinate being imported into the mitochondria followed by decarboxylation of malate to pyruvate via malic enzyme (as shown pathway I in Fig. 6A); or (ii) malate being converted by cytosolic malate dehydrogenase Mdh2p to form oxaloacetate, followed by synthesis of phosphoenolpyruvate (PEP) involving PEP carboxykinase (as shown pathway II in Fig. 6A). Thus, citrate or isocitrate from the mitochondria might bypass the Cit2p reaction in the glyoxylate shunt. This is supported by the fact that the mitochondrial citrate synthase Cit1p could act as a shunt for the missing Cit2p via the citrate transporter Ctp1p [32]. Although the discrepancy of growth phenotype on solid and liquid medium is unclear so far, the growth conditions are different, which might lead to the different physiology.
Being an essential enzyme, elimination of Mls1p also breaks the integrity of the glyoxylate shunt. According to the plate assay, the MLS1 deletion strain SCIYC07 (mls1Δ) was unable to grow on non-fermentable nutrient sources, indicating that Mls1p is specific and absolutely required for growth on C2 compounds as carbon source. Surprisingly, this mutant utilized ethanol and acetate derived from the overflow metabolism during growth on glucose in shake flask cultivation, while the biomass was increasing only slightly. After conversion of C2 carbon sources to acetyl-CoA, this compound can be metabolized by different pathways such as the glyoxylate shunt, or the biosynthesis of fatty acids and sterols. Analysis of the extracellular metabolite profile revealed that glyoxylate accumulated. This observation indeed showed that in absence of MLS1, the glyoxylate shunt was working partially, which is also consistent with the phenomenon that all metabolites from overflow metabolism are slowly consumed. These results suggested an alternative pathway to the activities of Mls1p: the counter-exchange of succinate and fumarate and subsequent intra-mitochondrial conversion of succinate to fumarate, together with the canalization of fumarate to malate. This is supported by the fact of that the mitochondrial succinate-fumarate carrier Sfc1p is indispensible for acetate utilization on minimal medium [28].
In contrast to the inability of SCIYC05 (acs1Δ) to grow on non-fermentable carbon sources, SCIYC14 (acs1Δ cit2Δ) was observed to be able to grow under such carbon conditions. Although the membranes of subcellular organelles are permeable to C2 compounds such as ethanol and acetate, metabolism on C2 carbon source has to pass through acetyl-CoA for biosynthesis and energy requirement. In SCIYC14 (acs1Δ cit2Δ), the conversion of a C2 carbon source to acetyl-CoA can be realized only by the sole extra-mitochondrial producer, cytosolic Acs2p. The acetyl-CoA units so produced cannot be transported into other compartments without the help of carnitine. Furthermore, the resulting acetyl-CoA units in the cytosol must be channeled into the synthesis of C4 dicarboxylic acids. Taken together with the deficiency of Cit2p activity, Mls1p which is the only candidate enzyme left can condense acetyl-CoA to form C4 metabolites, may well be localized in the cytoplasm of SCIYC14 (acs1Δ cit2Δ) (Fig. 6D).
In this study, we have investigated the physiological profiling of S. cerevisiae mutants affected in acetyl-CoA metabolism. Through analyzing the growth phenotypes and metabolite processing changes in different mutants on non-fermentable carbon sources, we have proposed a model of acetyl-CoA metabolism to speculate how various mutations alter carbon distribution. This study should be of interest to those studying acetyl-CoA production in eukaryotic cells and central carbon metabolism in fungi.
Supporting Information
Table S1.
List of primers used in this study for gene deletions.
Table S2.
List of primers used in this study for strain confirmation.
Author Contributions
Conceived and designed the experiments: YC. Performed the experiments: YC. Analyzed the data: YC VS JN. Wrote the paper: YC VS JN.
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- 31. Pronk JT, Wenzel TJ, Luttik MAH, Klaassen CCM, Scheffers WA, et al. (1994) Energetic aspects of glucose metabolism in a pyruvate-dehydrogenase-negative mutant of Saccharomyces cerevisiae. Microbiology 140: 601–610.
- 32. Kaplan RS, Mayor JA, Gremse DA, Wood DO (1995) High-level expression and characterization of the mitochondrial citrate transport protein from the yeast Saccharomyces cerevisiae. J Biol Chem 270: 4108–4114.


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I've been interested in this piece of equipment for a long time. I can't afford it right now but am wondering if any of you have had the chance to use one. If so, what are your thoughts? Besides the cost ($2,600us, I believe) what are the other drawbacks? Any other thoughts would be appreciated.
I know of a shop that has one sitting with a pile of other unused equipment so I may make an offer if I become convinced I wouldn't be wasting my money. Thanks.


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Brenda Liebman Aron
I. Introduction:
A. History of American Sign Language Storytelling:
In our generation there were no TV or captioned TV so stories were often told "orally" from older
deaf students in deaf schools: to teach, to give advice, and often older students are left to
supervise younger kids, they are to keep younger kids busy- to keep them entertained. Also
often deaf family members and friends share stories such as folklores with younger members.
B. The Importance of Telling a Story: ASL storytelling is just another form of the dramatic
arts-no! It is not just that, it is also to teach language, that is why there is the word LANGUAGE
after the word, sign. It is also to teach. It can be a moral lesson, to teach about our world, to
preserve our history and culture, or to teach about rhythm appreciation.
C. Objective/Goal: Before we discuss storytelling techniques, we must first ask ourselves:
What do we want to share with our listeners? What is our purpose of telling a tale?
1. Is it to entertain?
2. To teach a moral lesson?
3. To make them think?
4. To encourage appreciation of reading and literature?
D. Intonation = Visual Imagery: Now with visual listeners, storytelling should be done through
the visual medium. Just like voice intonations- we use our hands, arms, facial expressions and
body movements to convey what we want to share. Again that is to teach language. I call this
VISUAL IMAGERY. To visually provide language through the eyes.
II. Pre-planning Stage: We must first ask ourselves before we select a story:
A. What do we wish to impart to our audience/listeners?
B. Who are the audience/listeners? i.e.; High School students, preschools, or deaf club members
1. General age of audience: stories should be age appropriate
2. Gender of audience: i.e.; 8-10 years old boys in the dormitory
3. Check for local dialect: regional signs, signs specific to school
4. Culture of Audience: specific cultural groups or diverse cultures represented
C. What is the purpose ? i.e., holiday theme, moral value: i.e.; someone stole somebody's
money, to put them to sleep
III. Basic Techniques of American Sign Language Storytelling:
A. Signing Space: wider use of arms, hands, and signing area. Storytellers must remember to
sign according to characters in the story.
B. Setting the Scene or "Stage": relevant to the story if the story elaborates on the setting of a
story, such as the village in "A Christmas in Wales" or to provide a background- we should also
provide a visual picture prior to telling the story
C. Characterization Set-Up(s): (absent referents) Get into the role of the characters in the
story-first by describing the characters in the story and then acting out the characteristics of the
character, i.e.; snotty, timid, nervous, proud, happy etc.
D. Period of Time: Period of story, lapse of time, etc. Establish the period of time-explain if it is
set in the 1860's to provide examples and what is not available or what is available-at that time
period- i.e., using a washboard or a wringer versus a washing machine.
E. Role Shifting: Exaggerated than in normal conversation, show the difference in character
shifts by moving to a new area of floor space. Body movements are also exaggerated.
F. Changing Perspectives:
1. From narrator to a character in the story to another character. be sure to show difference in height by looking down to a smaller person/animal (i.e. Hare and Tortoise) and vice versa. Looking up at a large tree, looking sideways etc.
2. Another example of changing perspectives is when the storyteller acts out a character such as a duck and then uses classifiers to describe how a duck walks. This is a change from personification of a duck to describing the behavior of a duck. There is often a switching between these two perspectives: personification and description in a tale.
G. Inanimate Objects: acting out of inanimate objects i.e., in Beauty and the Beast: The mother
teapot and baby cup... We can act out the shape of the objects through use of classifiers,
personification, facial expression, body movements etc. The use of signing can also show
differences such as the form of speech, register, and language
H. The 4 "E's": Elaboration, Enhancement, Embroiderment and Expression
1. Elaboration: To show a visual image of each character-or to provide a
clear visual picture-i.e., a tall and large tree trunk to a small boy, steps to the Lincoln
2. Enhancement: enhance the characters way of speaking or acting, from
very timid to very bold-to show emotions i.e. fear or anger. To highlight the character's
3. Embroiderment: To weave a good tale-you need to embroider the struggle, conflict, good vs. bad, or the beauty of the tale. To digress if it would help them better understand the story. Use contrastive structure for opposites.
4. Expression: Use your expression to enhance characterization as well as character shifting, to show emotions, to show through body language
I. Timing:
1. To show relative time: Use of facial expressions and temporal aspects for long, long time ago, far into the future, etc.
2. Pacing of story: slow and leisurely i.e. strolling down the street,
suspense-someone is coming closer and closer or according to the period, character,
and region of the story i.e. Country Mouse visits City Mouse-hustle and bustle of city
life compared to pastoral countryside
J. Audience Involvement:
1. Constant awareness of audience's response by facial expression and body language for mood
2. Consultative register: ask audience questions from the story to check for understanding i.e., Who took Farmer Jones' pig?
IV. Genre:
A. Folklore: traditional "orally" transmitted beliefs, practices and tales of a people. *from
Reader's Digest Illustrated Dictionary, 1987
1. The purpose of ASL folklores is to provide a moral lesson and to share values and beliefs of our culture. Stories are best told by native users of ASL storytelling.
B. Folktale: a story or legend forming part of an oral tradition and passed on
from generation to generation. Stories can be general such as regional stories, multicultural
stories, or from our deaf history.
C. Mystery/Whodunit/Scary stories: storyteller must be careful to select stories
appropriate for age and readiness of audience
D. Romance: Light romance for younger age: i.e. Cinderella, Sleeping Beauty,
teen romance tend to be "crushes", infatuation, first love etc. Teens and older have more involved
and sometimes convoluted romances
E. Repetitive Story-Lines: excellent in teaching turn-taking, sense of rhythm, and to teach
language. Usually good with younger children.
F. Rhythmic Repetition: Rhythmic beats or handclapping and visual
onomatopeia is our way of explaining sounds and rhythm appreciation
G. Soliloquoy: Most difficult of storytelling characterization. Must have good grounding in
American Sign Language storytelling techniques.
V. Some Suggestions in Getting Started for the Storyteller:
A. Always pre-read and practice in front of a mirror
B. Important for Storyteller to become familiar in applying ASL linguistics
C. Focus first on the larger setting down to smaller details
D. Try out with friends or colleagues skilled in ASL
E. Start with wordless comic strips and storybooks: Focus on the surroundings in the story
and characters as well as characterizations.
F. Short one-two lines children's books are good stepping stone in the
development of storytelling techniques.
G. Build-up to short stories: i.e., folktales with two or three characters is excellent
H. You are ready for longer stories when you have developed confidence,
mastered the basic storytelling techniques, and have had consistently good audience response
I. Tell stories from different genres to provide you with some practice in using different
J. Fingerspelling in storytelling: Reduce the amount of fingerspelling. Fingerspell only for places,
names, and brand names, etc. Use fingerspelling for emphasis and titles too.
K. Pantomime: Do not pantomime the whole story. Pantomiming is okay if it is
related to the character or the characteristics.
L. Storytelling should be an enjoyable experience so ENJOY!
VI. Carrying on the Tradition to Our Deaf Youth:
It is very important in this age of captioning to share the art of ASL storytelling to our youngsters.
A. Schools for the deaf should take the lead in promoting by role in the classroom and teaching
storytelling skills to parents of deaf children in early intervention and preschool programs, and
likewise parents of deaf children tell stories to their deaf children.
B. Deaf ASL storytellers be matched to families with young deaf children to provide support by
modeling good storytelling techniques
C. ASL Storytellers should use opportunities such as:
1. providing ASL storytelling in local libraries, evening programs at
2. on public access TV
3. during fairs and festivals...
D. We should preserve old movies and videotapes of master storytellers to share...
E. Schools keep a collection of videotapes of excellent storytellers in the library.
1. From the community
2. Purchase from catalogs
F. We should also encourage our deaf children to spread their wings
1. By encouraging and exposing deaf children at a young age to develop good reading habits and eventually enjoy literature in different genres and styles.
2. By encouraging opportunities to develop their imaginations-tell stories in the classroom-start them young.
3. By trying their hand at telling stories themselves... children at a young age are able to re-tell a story from a favorite book their mother read to them even though they can't read yet.
4. Teaching and providing opportunities for middle-HS deaf students to read to young deaf children
5. Provide opportunities for deaf students to compete in storytelling contests not based on speech skills but on ASL techniques!


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I am Creative, Nostalgic, and Humorous
Well, I should say I'm also looking for something more committed and not just a hook-up. Sure, "getting some" would be nice, but there needs to be more than that, like having some similar interests to converse about.
Though on an online forum recently I was told, "You have a convoluted, and somewhat bizarre thought process." All things considered, I won't argue with that. In my "about me" section all it says is, "More jaded and cynical than the average room full of people." I think that was a line from a Rollins spoken word performance I saw years ago.
I have a major fetish for womens' clothing and crossdressing and like to get dressed up as often as possible, and sometimes even go out dressed if it is appropriate, like to shows and occasionally to LaCage. I have a vast collection of clothes and almost 50 pairs of shoes. That said I absolutely LOVE high heels, the higher the better, and luckily I have what seems to be the most common shoes size of 8.5/9. Though I have certain styles I prefer, and others I can't stand. I started dressing while in high school and still do so today; though my taste in outfits is better now. I just find most girly clothes to be so cute, and dressing is fun. I wear a size 0 juniors, and have a girls size 10 skirt I fit in. Sometimes I do think about just giving it up, but now it's so much a part of who I am I find it difficult to do so. Just being honest.
Added that last part to get it up to 1000 words. Rambling done...
Also volunteer at Brew City Bruisers Roller Derby bouts.
Actually, my legs when I'm in drag.
Movies: The Die Hard Series (4 is my fave and I don't care what anyone says), The Kevin Smith Movies, Anchorman, Goodfellas, Hobo with a Shotgun, I'm Gonna Git You Sucka, Infra-Man, Kick Ass, Leon: The Professional, Otaku No Video, Robocop, Romy and Michelle's High School Reunion, Sucker Punch, True Romance, Very Bad Things, Wasabi, just about anything with Ellen Page.
Music: 80's One Hit Wonders, Alice in Videoland, Crystal Method, Electric Light Orchestra, Juno Reactor, Leftfield, Scooter,.
Food: Pretty bland, actually. Lots of turkey, chicken, or steak. And soup. Not much for trying new things.


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Mirpur, Mar 21: Pakistan all-rounder Shahid Afridi is on the verge of becoming only the second cricketer in history to achieve a rare milestone.
When Afridi takes to the field against India today in ICC World Twenty20 at Shere-e-Bangla National Stadium, he will be aiming to join South Africa all-rounder Jacques Kallis with a unique record.
The 34-year-old Afridi is on 499 international wickets. If he gets one more scalp today, he will be the only second player after Kallis to achieve a rare double of 500 wickets and 10,000 plus runs in international cricket.
Afridi has so far taken 48 Test, 378 ODI and 73 T20I wickets (all put together 499). Since making his debut in international cricket in 1998, Afridi has been one of the best all-rounders in the shorter format for Pakistan.
He has featured in 27 Tests, 378 ODIs and 70 T20Is. He has so far scored over 10,000 international runs (1,716 in Tests, 7,619 in ODIs and 1,044 in T20Is - total 10,379).
The 38-year-old Kallis, who recently retired from Test cricket, has taken 577 international wickets (292 Test, 273 ODI, 12 T20I) besides scoring over 25,000 international runs (13,289 Test, 11,574 ODI, 666 T20I - total 25,529).
Afridi is one of the key players in today's blockbuster India-Pakistan World T20 clash. On current form, he should reach the milestone in Mirpur tonight.


text:
From Our 2010 Archives
Health Tip: Managing Shoulder Arthritis
Latest Arthritis News
(HealthDay News) -- Arthritis can cripple joints in the shoulder, causing significant pain and difficulty moving the affected arm.
The American Academy of Orthopaedic Surgeons suggests these ways to manage pain caused by arthritis in the shoulder:
-- Diana Kohnle
Copyright © 2010 HealthDay. All rights reserved.


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WICF FRI 1030PM Studio Theater
Women in Comedy Festival
Friday, March 23, 2012 at 10:30 PM (EDT)
Friday March 23, 2012 1030pm
ImprovBoston Studio Theater featuring:
Mary Dolan Comedy Hour featuring:
Andrea Alton, Brianne Berkson, Rob Crean, Lillian DeVane, Aaron Haber, Connie Howe, Carmen Morales, Jessimae Peluso, Jessica Sutich, Sasheer Zamata
For details please visit: womenincomedyfestival.com
The Women in Comedy Festival celebrates Boston comedy and the women who create it, support it, and perform it. Bringing together women and men from all walks of comedy: improv, sketch, film, stand up, storytelling and humor writing, the festival gives Boston comedy audiences a chance to see both local and nationally known comedians.
Buy tickets for shows presented at ImprovBoston.
In order to purchase these tickets in installments, you'll need an Eventbrite account. Log in or sign up for a free account to continue.


text:
Florida: The Final Stretch; Update: Romney Wins
And I mean "the final stretch" as far as the Sunshine State's primary goes — not in the "God's waiting room" sense.
The latest polls put Romney back on top by about a dozen over Gingrich. Will Mitt take it, or will Newt have the eye of the tiger? Oops, I mean, will he have what it takes to win (I knew Survivor would eventually find a way to get back in the news).
Gingrich had been up in Florida for a short time by nearly double digits, but that disappeared so quickly the lead was either never actually there or Romney's not really as far ahead as they're now saying he is. It probably won't be a runaway for Romney, though he seems to have gotten out to a lead among the early voters. And let's not forget about Santorum. There's always a chance for a surprise due to some last-minute disenchantment with the top two.
Meghan McCain writes that Newt should like totally quit the race after the Florida primary. I'm serious you guys!
Make your own predictions and place your bets! I'll update tonight as the results come in (and I may be blogging at Michelle's place as well… not sure yet).
Final question: How will Romney perform in the "cracker counties"?
Update: The Florida primary has been called for Romney. I'm writing a little about it at Michelle's site.
9 Responses to "Florida: The Final Stretch; Update: Romney Wins"
maladroit on
January 31st, 2012 7:04 pm
Disenchantment with the top two? The past week has been a torturous blow-by-blow, televised competition to see who can sink lower. Even during the AFC/NFC Playoffs we were slapped across the face with Newt doot and Mitt schitt.
I was so PO'd by this morning that I looked at my ballot and put my hanging chad on Rick Santorum. And I can tell you; I wasn't the only one! There was a table set up in the parking lot with free coffee, donuts and danish (love this country!) and there were others who voted for Rick or Dr. Paul because of the horrible sliming of the Sunshine State by Mittens and Newty.
They had better change their approach to TV advertising — quick.
Truesoldier on
January 31st, 2012 7:20 pm
Agreed. We all know they both have dirty laundry (most politicians do), but please of please start telling us why we should vote FOR you and not AGAINST the other guy.
SignPainterGuy on
January 31st, 2012 8:34 pm
Good point ! As I`ve said many times, there are only "FOR" boxes, there are no "AGAINST" boxes on a ballot ! (except in referendums, of course)
SignPainterGuy on
January 31st, 2012 8:37 pm
Negatividity turns me off as quickly as most anything, short of lying !
If your opponent has negatives, sure, point them out, but make a bigger deal out of your own positives !
Granny55 on
January 31st, 2012 9:30 pm
I so agree with all of your sentiments and it is sending me in Santorums direction because of it.
Erin on
February 2nd, 2012 5:08 am
That's typically how it works…for those who lack control, they try and control every one else…dysfunctional and emotionally stunted.
Lubbock Self Storage
maladroit on
February 2nd, 2012 9:44 pm
Think of all the wasted $$ that could have been spent attacking the REAL PROBLEM; the Kenyan boy-king in DC.
rain on
December 6th, 2012 4:37 am
well that all what politics is all about..what else is new.
PassbeeMedia on
March 15th, 2014 5:50 am
My partner and i and so go along with all of your current comments and it's transmitting everyone throughout Santorums direction because of it.
Leave a Reply
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Ergotron WorkFit-PD. Colour of product: Black, Grey. Lift: 50 cm. Width: 800 mm, Depth: 600 mm, Height: 820 mm
The WorkFit Personal Desk (PD) enables an effortless sit-and-stand work style—move from a sitting to a standing position whenever you want. Customize with accessories to fit individual user needs and workflow.Features 19.5-inch (50 cm) height adjustment—patented CF technology allows instantaneous, tool-free, non-motorized re-positioning while you work! An integrated brake secures table in place and easily releases to change heights.


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Have Relationship Questions? Ask a Counselor for Answers ASAP
can you chat with me ?
I am available right now to chat if you would like. If you are not available you can ask me any questions you would like and I would be happy to answer them for you.
hey how are you
I am available now if you are online
are you online?
ughh im always missing you ((( and you seemed to help a lot of people
ok im going to wright you my situation and see what think ab it:
ok here is my uneasy situation ,im a lesbian i had a girlfriend for 6 months she is 19 im 26 ,young girls love me a lot i always had yonger girlfriends i was a dj a bartender im sexy and feminime russian girl ,but back to the point ...My gf is working for political party of democracy and she was invited on the conference to Puerto Vallarta mexico and she could take one friend with her ,so she took me ,along with her went her friend Daniel ,he took his friend also ,her name was Alejandra ,she turned out to be a lesbian very beautiful and smart and i liked her a lot ...first time i saw kind of met her when she came down in the evening to eat with us ,we were all in the same Sheraton hotel and Daniel had his guitar and i played and she really showed interest in me ,she was smiling and this eye contact that can tel you some things too and she played guitar too ...but my girlfriend was sitting next to me so i couldnt do much ...Later on daniel(i have to say he wasnt aware that im a lesbian,neither he knew that my gf was cause we are very fenime )and his friend that i liked they came down next day ,but my gf told me i shouldnt flirt with people so i was very closed this time with this girl ...but i was feeling she liked me...later on Daniel he expressed interest in me ,he spent a llot of time w me we were playing volleyball talking a lot and stuff like that ...we all came on one bus and when we were coming back me and my gf were late ,so we couldnt sit toogether and she also got kind of mad with me because she was jelaous about this girl and the only sit i had was right next to daniel and her ..so i set there daniel and her were talking to me cracking jokes she was asking me things and listening pretty close and laughing and smiling and that good stuff ...later sit that w me was empty and daniel set in it and started talking a lot of stuff ,we were listening to the music and talking like friends ,st this time his friend falled asleep and i talled him "Daniel i really like your friend i cant hold it down " he was like "wau really ,(by the way daniel has a gf too)" then he asked do you like her more or me ? wasnt in jelaous way more like a friendly playing way ,i sad "your friend but please do not tell my gf " he said do not worry and i will tell Alejandra ,she likes european girls he said and im russian ) ...my gf was very pissed that he was sitting and taling to me but she was in the other part of autobus ,at some point of timeDaniel tool headphones of Alejandra and sayd pretend like you took it without intention and then you will have a reason to see her agai and ill tell her that you like her i was like "ok",when autobus arrived it was a late night ...so me and my gf and Daniel took one taxi toogether ,he kissed us goodbuy ,my gf pretended like noth happened but she was pissed ...so next day im wrighting daniel in FB "hey how are you whats up have you talked to alejandra ?" and he was like "oh yes and she ws very interested in you and she gave her phone number so you can text or call her " and i was like wau cool...so i texted her i sayd hey how are you remeber me? and answer was "oh yes i do off caurse whats up " she sounded super excited but she was entering her class so she said "can i text you later ? cause im going inthe class " i sayd off caurse ) and she texted me around 8 pm and sayd "send me some russian music to download"i was like sure i will and the i asked her what she likes to do in her free time ? she didnt answer for a while and then she said she was playing tennis w her best friend and it was night so i didnt bother any more ...Next day i wright Daniel on FB and say "hey whats up "(oh i forgot to mention my gf found out me and him became friends on FB and she was really mad and they work toogether )...Daniel wrighting back"yes " nothing else i say "how are you today ?"the answer from him was "?"and then i was like -"do u want coffee im inviting " no answer -are you ok? no answer are you mad for something no answer
so i texted his friend Alejandra "hey how are you ?" no answer do you want come out and have beer this weekend ? no answer i sent her a friend request in FB no answer ....after that my gf took headphoes that i took and gave to daniel and sayd "if you want a russian gf look in the internet and tell your friend he same "...and daniel showed his phone and sayd "look i dont need her she is texting me "....ok after that passed one day ...and i dont know what to do IM SOOOO LIKING THIS GIRL ANDI KNOW SHE LIKED ME TOO ...i saw it in her eyes in her smile in everyth ...and she was the one who passed me her number ..and she did to her friend she ws very interested in me ...and she sounded happy when i texted her ...so what should i do ,what can i text her what you advice me to talk to her she is a lawyer very 24 y.o. very pretty ,smart w ambitions girl ,me too lol...but what to do ?
how to get her to answer i gave it 4 days break after she didnt answer now im thinking to send her this letter can you read it and tell me if you think its a good one or ahouls i take someth out or should i add something :
Dear Alejandra,
I thoroughly enjoyed your company recently at the Sheraton in Puerto Vallarta. I was very taken with your wit and intelligence. Your beauty and warm personality have totally captivated me. And the eyes you have are a hell of a mystery) i really did want spend more time with you ..but i couldnt ...
You seem to be really Sharp ,Classy,private and at the same time open person who actually cares for the feeling of the other people ..I have a girl friend and she is a wonderful person and we ve been in a relationship for 4 months(you had a pleasure to meet her ) , and its not my intention to hurt her ,neither do i have intention to play with you ...she does know that i really liked you and she is not happy about it ,but neither we are happy in a relationship at the moment because of the age difference i guess and some other things ,so im not a player,im not a dishonest person ...i really enjoy relationship with a person...not the one time things...im just not happy in my current relationship ..and since we are not trees we have this unik opportunity to change things around us ... and i tend to use it ..I would rather said all that to you in person ...but again i couldnt and it wouldnt be fair to the person im with cause i had to tell that that to her first before telling that to you ...
I dont know what Majo told to Daniel ,i dont i know ,what he told to you about all the situation ...so if i have to defend myself ...i cant ..cause i simply dont know what went in between ... but neither is my intention to do so ...i just want be honest and forward with you and doesnt matter what other people say ..."its all about us" queen said)
What i want do tho is to take other people off this equision for a minute and to have at least one chance to talk to you in person ,as we never had this chance and i had a feeling, that we had some spark in the eyes when we saw each other ...i might be wrong and misread the signals ..you can let me know right away and it will be the end of it.. just tell me .
But if im right and we have a beautiful connection,why not to give it a chance ? its a rare occurrence why to let it go and wonder all life what could ve have happened ?I am usually more prudent than this, but forgive me for throwing all the caution to the wind. I must ask you if you would consider getting together with me sometime very soon, for dinner and drinks, or just coffee, if that is your preference.
I know that I am being forward and hope that you don't find me foolish,i m only expressing the way i feel toward You and i would love to get the same from you , and nothing more i can do ,so im leaving everything else up to you to deside ...,
but please consider my request. This could be a remarkable turning point in both of our lives, or could just be a short and pleasant meeting for one time.
I would be grateful and delighted if you accept my invitation.
In friendship,
am i being to straight foerward or no what do you think ?
are you online
i see ou helped a lot of people ...that s why i really wanted your opinion on my case i appreciate it in advance
When you wrote to Alejandra you told he that you had a girlfriend and that she is wonderful. You were very honest with telling her how you felt. But you also said that you were not happy in your current relationship. I feel that Alejandra would be worried and scared to get involved with someone that has a current girlfriend. She would be concerned that if she invested all her time getting to know you and fall in love that you might not leave your girlfriend.
I am sure she wi wondering why you are still in a current relationship with your girlfriend if you are not happy. I want you to think of your current relationship because some times when something is missing you tend to look outside the relationship.
I want you to think about what is missing and if it could be fixed. Alejandra could be someone that you image as the perfect woman, but you would need to spend more time with her in order to figure out your true feelings.
It is important that you look at your current relationship and ask yourself this important question. Can you picture your life without your current girlfriend? If you woke up tomorrow and she was no longer your girlfriend how would you feel. I feel that Alejandra knows that you would have to go through a break up in order to move on with her and I feel she is scared that you might still have feelings for your current girlfriend.
I want you to really think about what you are looking for in a relationship? What type of person are you looking for that you can spend the rest of your life with, it is important to know exactly what you want.
I am sure you look for certain qualities in a person and Alejandra has qualities that sparked your interest, you sound like you had an instant attraction too her.
But I want you to make sure that this is what you fully want, because it would mean breaking p with your current girlfriend. Some times what happens is you realized you love the person you were with all along. This is why you really have to think about what you want. If you want to give this a chance with Alejandra or if you want to stay to work things out with your girlfriend. You are very honest and that is key in a successful relationship. Admitting you had feelings for someone else was very honest.
I feel she is not answering you because she doesn't want to be that person that break two people up. She doesn't want to be the cause. If you were single I don't think there would have been any problem.
You talk very positive about your current girlfriend and Alejandra could be wondering why you are not staying with her and trying to move on. Maybe you should explain why you are not happy in the relationship, why you found her to be someone that you could see a life with.
I am sure Alejandra has a lot of questions why you want to leave your current situation.
I am here to help you and answer all your questions. I want to guide you in making the best choices for your life. If you could please rate me again, I am here to help.


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By Jim Corbett, USA TODAY
Linebacker Karlos Dansby and the Miami Dolphins never got the memo, the one about Baltimore Ravens running back Ray Rice being voted the third-most underrated player (tied with Houston Texans receiver Andre Johnson) in a Sports Illustrated poll of players.
Rest assured that Atlanta Falcons coach Mike Smith will make sure his guys shadow the Ravens dynamo when the teams meet in Thursday night's date between Ravens vs. Falcons (8 p.m. ET on NFL Network).
The multiple-threat tailback gashed the Dolphins for 180 yards from scrimmage, including 97 yards on seven receptions to key Sunday's 26-10 Ravens runaway.
THE HUDDLE: Ray Lewis says Thursday games unfair to players
Asked why the Dolphins didn't shadow Rice with a linebacker, Dansby said dismissively, "For what? Why would we shadow him? He didn't do anything.
"They checked it down to him. But he was the last resort. Why would we need to shadow him? They just kept drives alive like that."
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With quarterback Joe Flacco patiently re-setting his feet and checking down after going through his progressions against Miami's coverage that's designed to take away receivers Anquan Boldin and Derrick Mason, Rice skewered Miami.
Flacco completed 20 of 27 passes for 266 yards on a day when a whipping wind caused a potential Chad Henne touchdown pass to sail beyond wide-open tight end Anthony Fasano.
Flacco, Rice and the 6-2 Ravens against the 6-2 Falcons makes for a compelling NFL Network debut of their Thursday night schedule of games.
"It wouldn't surprise me if our opening Thursday night game could be a precursor to the Super Bowl," NFL Network analyst Joe Theismann said. "Because in the NFC, I think the Giants are playing very good football. But certainly the Atlanta Falcons are in control. They have a lot of things going for them.
"Their defense will continue to do the job. They run the football very well.
"Every time somebody makes the comment about Ray Lewis not being able to do the things that he could do, he does something even more spectacular. And now with (safety) Ed Reed back, it gives them that little bit of swagger, that little bit of uncertainty every team has to have."
The showdown matches fellow 2008 draft classmate, Flacco and Matt Ryan— "Joe Cool" vs. "Matty Ice."
Flacco is 29-16 as a starter. Ryan is 26-12, including 17-1 inside the Georgia Dome.
"Both those quarterbacks are going to be stars in this league," NFL Network analyst Kurt Warner said.
"They've developed extremely quickly."
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