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Categories: Babe
Jessica Enjoys Dark-Hued And Milky MP4 - Pornography pioneer Jessica Jaymes
With super model looks and the bedside manner of a super bi-atch, pornography vet Jessica Jaymes does it again! After watching her glob dead fabulous, nearly mesmerizing looks. She turns it on in yet another jaw ripping off solo spectacle. Truly, is there anytime this nymph doesn't get those penises rock rigid and vaginas raw? So see Jessica in all her glory wiggle those flawless breasts and play with that fat clitorous as she screams with orgasmic excitement!586 Views
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Categories: Babe


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Published/Aired:September 2013
September 04, 2013
A live video feed from a bbq 24 hours a day. Every now and then the grill gets filled with steaks and meat products. The rules are simple - be the first to grab a steak from the grill - and it's yours! The exact product you tagged yourself on gets packed and sent to your house in 45 minutes.
Ambient advertisment created by MRM, Romania for Lidl, within the category: Retail Services.
Advertising Agency: MRM Worldwide Romania
Chief Creative Officer: Nir Refuah
VP: Ionela Buta
Art Director: Vlad Macarie
Copywriter: Mihaela Coman
Chief Creative Officer: Nir Refuah
VP: Ionela Buta
Art Director: Vlad Macarie
Copywriter: Mihaela Coman
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subset: commoncrawl


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Much is being made about a possible 2014 match between incumbent Maryland senator Jim Mathias (D-38) and Del. Mike McDermott (R-38B). While McDermott would have to overcome Mathias' huge money advantage (Senate President Mike Miller will THROW hundreds of thousands of dollars Mathias' way), a Mathias vs. McDermott race will show the unfathomable divide between "Annapolis Values" vs. "Eastern Shore Values".
Listen to McDermott on the recently passed budget bill:
While I am a fan of Public Radio Delmarva (actually WSDL), I don't support Maryland tax dollars going to support it. While I actually believe that SU needs a new library, I can't imagine where $135 million is going. What about all of that money in the SU foundation? Why can't it go to help build and equip a new library?
If you attended the recent town hall on Gov. O'Malley's gun bill you would have witnessed a stark contrast in approaches. McDermott has FOUGHT this bill from the start. Mathias just voted against it (I would suggest for political reasons). You would have seen and heard the passion McDermott brings to this issue. While Mathias wasn't in attendance, he sent an envoy to read a letter. The categorical difference was apparent to all – McDermott was fighting to protect one of our most sacred liberties while Mathias was tinkering around the edges to make a loathsome bill slightly less loathsome.
As a political junkie, I think a Mathias – McDermott race in 2014 will be exciting. As a citizen of Maryland, I believe that a McDermott candidacy and victory will send a message to those here and in Annapolis who believe that giving up our God-given liberty in pursuit of a few cups from the public trough is a worthwhile pursuit.
DelMarVa Dealings appears each Monday and discusses local politics and its impact on the Delmarva peninsula.


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Grammy-winning rapper T.I. appeared on Atlanta's Streetz 94.5 Monday Morning with fellow rapper Young Joc and he playfully shut down any talk of a divorce from his wife Tiny.
"That ain't your business, man," Tip said in an interview. "I know we on air. I'ma tell you on-air, it ain't your business. There you go."
Tiny reportedly filed divorce docs in December, but since then T.I.'s put on a full-court press to get her to reconsider. Things were looking good just last week when they had a nice family dinner with their 10-month-old.


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INQ, the London-based company that produced the INQ1 Facebook mobile phone last year, is now proposing to release another social networking handset but, this time, devoted the messaging phenomenom that is Twitter.
The outfit stated that since Twitter is such as popular service, it seemed reasonable to produced a phone that featured the web-based client, according to the Reuters website. It is also attempting to stay one step ahead of the competition, such as LG, which is releasing it's own social networking handsets.
Apparently, the new phone design will utilise Internet connections to send the Tweets, the 140-character messages, as opposed to the text messages that Twitter itself uses.
"This can really help open up and drive Twitter use on mobile when usage becomes part of your data package like on the PC," said Frank Meehan, chief executive of INQ.
The new handset is not expected to feature many high-end features as its proposed price of around £85 will indicate.
Click to read the Reuters story


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The global media have never had a conclave without a papal burial and the solemn majesty of bidding farewell to one pontiff before the election of another.
I am packing my bags and arrive in Rome next week, but already it's clear what is happening.
The media are running hard with reports on the pasts and backgrounds of the papabile, assessing the leading candidates, their chances and their faults. This will be the first time that the internecine and complex process of selecting The Vicar of Christ will run headlong into the fast, furious coverage in mainstream media, much less the often snarky writers of the blogosphere and new news organization in the digital realm.
Already, Cardinal Peter Turkson of Ghana has taken hits in the New York Times for his statements on gays and other reports on an inflammatory anti-Islamic film he is reported to have shown.
Contrast the coverage of Turkson with the initial analysis of Cardinal Leonardo Sandri who has the great advantage of being an Argentine whose parents emigrated from Italy. A perfect combination: Third World and Italian fused into one man. He also has long experience in the Roman Curia. The Curia — unlike the College of Cardinals — is a heavily Italian institution and the Italian cardinals will coalesce behind one candidate by the time the conclave doors close.
The question for now is whether it will be Cardinal Angelo Scola, the erudite, multilingual Cardinal Archbishop of Milan, the largest archdiocese in the world with 5 million congregants? Or Gianfranco Ravasi, president of the Pontifical Council on Culture, who quotes Kierkegaard and the Psalms in equal measure and whose encyclicals, were he to be elected pope, might even rise to scrutiny by the secular high priests in the New York Review of Books?
We have no odds yet from Las Vegas, but the media coverage of the coming of the cardinals is the campaign created before our eyes.
I rest my case for now, with an obligatory bow to John Allen for well-textured profiles in his series on cardinals in National Catholic Reporter.
As Yogi Berra so aptly said, it's not over till it's over.


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SBI PO Recrutiment 2016, SBI PO Online Form-Exam Date, Pattern & Syllabus
State Bank Of India (SBI) Recruitment 2016
State Bank of India (SBI) is a Global, Public Sector and Financial Sector Company. With 200 years history, it has more assets, customer, employee, deposits, profits and branches. All people believe in this bank. SBI is ranked 60th in the list of 1000 banks in the world.
The State Bank of India is the India's oldest banks. It's continuously including many polices like- Advisor Services, Structured Products, Mobile banking, Net Banking, General Insurance, Private Quality, Custodial Services and Point of Sale Merchant. Each one of this leadership has a huge growth of company.
SBI PO Latest vacancy Details
The State Bank of India announced 2428 vacancy for the Probationary Officer (P.O) for all the candidates, who has completed their Graduation Degree and has in the final year of Graduation also. So all the job hunters this is good news for you. You can apply within 4th May 2016 to 24th May 2016. As per your Recruitment we will give you all details:
Name of Organization: State Bank of India (SBI)
Name of Post: Probationary Officer
Eligibility: Graduation degree in any reputed University/Board
Job Location: All over India
No. of Seats: 2428
Salary /Pay Scale: 16900/- (salary will be varies from city to another)
Grade Pay: candidates will get D.A, H.R.A and C.C.A with salary.
Age Limitation: 21 years to 30 years old on 1st January 2016
Relaxation in Age: As the government Rule categories wise Relaxation.
- SC/ST-5 years
- OBC-3 years
- PWB-15 years
Application Fees: All general candidates will pay Rs600/- and SC/ST /PWD candidates will pay Rs 100/-.
Method to Apply: All the candidates, who want to apply SBI P.O 2016 vacancy, are submitting their forms before 24th May 2016 on online mode. Forms are not accepted without application fees.
Candidates must follow the given steps for apply SBI P.O post:
- Firstly Visit the Official Websites sbi.co.on.
- Click On the 'Career with us 'link.
- Carefully read the Advertisement details for SBI P.O and click on apply online.
- Candidates will fill all the required details before uploading photo and Sign.
- Click on the Submit Button After filling required details.
- Note your Registration no and Password for further login.
- You take a print out of fee challan after submission of application form.
Selection Procedure:
The candidates will be selected on the basis of written exam and interview. In the written exam Preliminary and Main Exam is included where as in interview Personal test and group discussion is included.
- Preliminary Examination
- Main Examination
- Interview
- Personal Test
- Group Discussion
SBI PO Important Dates
- Starting Date: 4th May 2016
- Closing Date: 24th May 2016
- Examination Date of SBI PO 2016:2, 3, 9, 10 July 2016
SBI PO 2016 Syllabus
- Analogy
- Coding & Decoding
- Blood Relations
- Sitting Arrangements
- Series Completion
- Syllogisms
- Decision Making
- Statement Reasoning
- Classification
- Series Completion
General English:
- Vocabulary
- Synonyms
- Antonyms
- Idioms
- Phrases
- Passage Completion
- Error Detection
- Passage Correction
- Sentence Correction
- Comprehension
- Vocabulary
- Grammar Correction
- Identification of error in sentence Construction
- Jumbled Sentence
Quantitative Aptitude-
- Number System
- Simplifications
- Roots and Averages
- Surds and indices
- Percentages
- Profits and Loss
- Ratio and Proportion
- Partnership
- Time and works
- Pipe and Cisterns
- Time and Distances
- Problems on trains
- Boats and Streams
- Allegation & Mixtures
- Simple and Compound Interest
- Volume and Surface Area
- Mensuration
Interpretation and Analysis:
- Line Charts
- Pie Charts
- Bar charts
- Tabular Data
- Probability
- Statistics
SBI PO Exam Patterns
The SBI P.O Exam will be conducted in three phase, phase is given below:-
- Preliminary Exam
- Main Exam
SBI PO Phase -I (Preliminary Exam) Pattern
Name of Subjects No. of Questions Marks
Quantitative Aptitude 35 35
Reasoning Ability 35 35
English Language 30 30
SBI PO Phase -II (Main Exam) Pattern
Name of Subjects No. of Questions Marks
General Awareness, Marketing 50 50
& Computer
Reasoning Ability 50 50
English Language 50 50
Data Analysis & Interpretation 50 50
SBI PO Phase – III (Interview) Pattern
This is the final and last round of selection in SBI P.O. In this round Personal Test and Group Discussion Round will be conducted. After qualifying this round, you will become State Bank of India Probationary Officer.
Latest Govt Job Updates
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Product Spotlight
Featured Research
Quick Stats
You are here
Whole Foods Stock Hits Seven-Year Low
Downgraded one day, upgraded the next, this grocer's stock has had a wild ride on Wall Street.
Share prices for Whole Foods Market's stock hit a seven-year low on Wednesday, following a Wall Street analyst's downgrade of the stock to "under-perform."
While Wall Street has been a rough ride for many stocks, the natural/organic grocer's stock has fallen 15 percent since last Friday's close.
An analyst with Jefferies & Co. painted a negative picture for the grocer's stock due to the economy. "We anticipate a steep falloff in spending by Whole Foods' consumers who are under mounting pressure from declining asset prices and falling disposable income," Scott Mushkin wrote. The analyst lowered his earnings estimates, citing a "near lack of visibility" on consumer spending.
On Thursday, USB upgraded Whole Foods' stock from "Sell" to "Neutral." However, the grocer's stock still closed at almost the same price as it did on Wednesday.


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|Rider Strong
Ethan Edwards
|Date of birth
11 December 1979
|Place of birth
San Francisco, California, USA
|IMDb profile
Rider Strong is an actor who played the role of Ethan Edwards in the Law & Order: Criminal Intent episode "The Pilgrim".
Rider began his acting career at the young age of 9, when he starred as Gavroche in the San Francisco production of Les Misérables. After the stage production, Rider appeared in small screen roles on television. In 1992, he received his first regular television series role as Julie Andrews's son on Julie. When the series was canceled, Rider made his debut on film as Amy Irving's son in Benefit of the Doubt.
It was not until he was chosen for the role of Shawn Hunter in the ABC series Boy Meets World that he received fame and recognition. During the seven year run he was nominated twice for the Young Artist Award. However, though he enjoyed the role of Shawn Hunter, Rider was continually perceived as "the Bad Boy." In the 2006 series Child Star Confidential, Rider revealed that the fan mail he received on the set of Boy Meets World, often asked him to murder someone for them. When the series ended, Rider returned to the big screen to star as Paul in the indie horror film Cabin Fever.
Rider is currently working on Cabin Fever 2: Spring Fever, the sequel to Cabin Fever. It is slated to be released in 2008.


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Forums Lobby GENERAL I Want to Sell I Want to Sell in the EU topic #1695
Delkin adaptor SD to CF
Horus Registered since 29th Mar 2007
Sat 26-Feb-11 11:27 PM | edited Sun 27-Feb-11 01:54 PM by Horus
Hello I'm selling 1 of my 2 Delkin adaptor SD to CF.
Be aware this is a CF type II and so will not be able to introduce in a D3s or D700.
Just bought new for 22€ and not open.
I have tested the other one I keep, work perfectly well with latest SDHC cards.
Sell price will be of 15€, post fees to the charge of the buyer.
Photography is a joy, happy shooting
Nikonians Gallery
Modérateur Nikonians francophone
Nikonians French Moderator


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Pandora's Box online bingo promotion makes not so Desperate Housewives!
Opening Pandora's box is usually seen as a step into the unknown however over at Desperate Housewives Bingo during February online bingo players are being given the opportunity to win a gorgeous Pandora Bracelet
During the month of February, watch for the Valentine's Heart Pattern to play in all our rooms. Load up and max out because each time you bingo on this special pattern you'll get 1 entry in Pandora's Love Box!
Each week, February 7th, 14th, 21st, and 28th Desperate Housewives Bingo will draw two winners at random from all the weeks entries to win one of these gorgeous Pandora Bracelets.
The first name drawn will win a Pandora starter bracelet with a last name initial charm in either gold or sterling silver. The second name drawn out of the box will receive a fabulous Pandora's Valentine's bracelet
The box will be emptied each week after the drawing and we'll start filling it up again for the next weeks draw!
Here are the two lucky winners for the previous weeks draws:
7 Feb – 1st drawing jwords, 2nd drawing – crazedmomto5
14 Feb – 1st drawing ARIES63, 2nd drawing – Mojlry
If you would like to see more information about Desperate Housewives Bingo check out our MrsBingo Desperate Housewives Bingo Review


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April Fools! is the 23th episode of Season 7 of Shining Time Station episode made by SuperMalechi. Original Airdate was in April 23, 2000.
It's April Fools Day at Shining Time Station and Schemer plays new pranks on people around the station. He puts a plastic bug in Stacy's cup of water, he tricks Becky into thinking there are rats in the station, he puts a plastic spider on SpongeBob's head, he orders a big bowl of popcorn for Baby Bop, making her have a tummyache, and more.
- Mr. Conductor (Ringo Starr)
- Stacy Jones
- Barney the Purple Dinosaur
- Baby Bop the Green Dinosaur
- BJ the Yellow Dinosaur
- SpongeBob SquarePants
- Patrick Star
- Horace Schemer
- Billy Twofeathers
- Dan
- Becky
- Matt
- Kara
- JB King
- The Jukebox Band
Thomas Stories
- Percy & The Signal and James In A Mess (both told by Ringo Starr)
Jukebox Band Song
- This Old Man
- Barney has his Season 3 voice and Late 1993/1994-1997 costume.
- Baby Bop has her Season 3 voice and 1998-2002 costume.
- BJ has his Season 3 voice and 1998-2002 costume.
- SpongeBob has his 1993-1999 voice and 2000-2001 design.
- Patrick has his 1993-2000 voice and 2000-2001 design.
- The Barney costume from "Are We There Yet?" is used.
- The Barney voice from "Run, Jump, Skip and Sing" is used.
- The Baby Bop costume from "Sing & Dance with Barney" is used.
- The Baby Bop voice from "Fun & Games" is used.
- The BJ costume from "Circle of Friends" is used.
- The BJ voice from "I Can Be a Firefighter" is used, except it was mixed with BJ's 1996 voice.
- First Shining Time Station episode to use Season 2's SpongeBob and Patrick designs.
- The SpongeBob design from "Bossy Boots" is used.
- The SpongeBob voice from "Bubblestand" is used.
- The Patrick design from "Big Pink Loser" is used.
- The Patrick voice from "Tea at the Treehouse" is used.
- When SpongeBob screams after Schemer puts a plastic spider on his head, his scream is the same as Derek's scream from "A Splash Party Please!" (when Tina tells Derek there's a bug in his sack), except it was pitched down to -7 and mixed with SpongeBob's 1993-1999 voice.
- When Schemer screams as the pop sprays all over him, his scream is the same as Eric Cartman's scream from "Cartman Sucks" (when Cartman runs out of the park, holding his arm), except it was mixed with Schemer's voice.
- The Season 6 Barney & Friends Musical Arrangement from "Ready, Set, Go!" is used.
- First time Schemer thinks his arcade would be taken away.
- This episode took place in April 1, 2000.


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Norwood Group design, manufacture and install demountable and relocatable steel & glass partitions, cleanrooms, pods, steel doors, walk-on-ceilings, wall lining/ cladding and integrated services.
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Air Science cover a broad range of environments with its air treatment products and services. Cleanrooms, Allergen, Heathpro, air filters and various other air cleaning technologies are available for babies, hospitals, workplaces, educational establishments, pets at home, people with allergies, etc.
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I really hope this column will bless you. You can read the most recent column below and previous columns at the archive.
Essentialism reigned for 2,000 years.
Beginning with Aristotle, philosophers had an almost standard belief that all things and beings had an essence that defined their purpose and existence. For instance, a knife's essence is the blade, and its purpose is to cut. They argued over what essence was, but never about whether things or people have an essence.
I love Legos. As a kid, if I wasn't playing with my slingshot or toy guns outside, I was playing with my Legos inside. I love how open Legos are. I could create any other toy I wanted from them. A bucket full of Legos was the only toy I ever needed because Legos let me create, and I could do anything I wanted with the things I built. Interestingly, I could never create another me.
Even as far as artificial intelligence has advanced in recent years, we, as a species, have yet to create anything that can rival the creativity, intuition and learning power of humans. It seems to be a rule of being that the created is always inferior to the creator.
I have a 4-year-old daughter who has an amazing imagination.
One day I sent her to timeout. My children sit in timeout in the corner between the door to our garage and the coat closet in our house. Her few minutes were up, so I went to go get her when I heard her voice whispering. I paused to listen.
"OK, are you ready to marry your husband? You're going to have a very happy life with the closet door knob. OK, here's your flowers."
She had made pretend friends out of the doorknobs to the two doors, and was presenting a leaf she found on the floor as a bridal bouquet. I didn't even know how to respond to her indomitable imagination. I just asked her if I could officiate the wedding. My closet and garage door knobs have been happily married for several months now.
"Alexander and the Terrible, Horrible, No Good, Very Bad Day" by Judith Viorst was one of my favorite books as a child. Alexander woke up on the wrong side of the bed with gum in his hair, and every page of the book chronicles how everything that can go wrong in the daily life of a young boy did go wrong for Alexander. I got picked on a lot as a kid and I had more than a few bad days myself. There was always something cathartic about someone else's bad day.
God loved Nebuchadnezzar. God loved him so much, he gave Nebuchadnezzar dreams about the future.
The first time we actually read Nebuchadnezzar's words in the book of Daniel, he laments a mysterious dream that he had in Daniel 2. It was of a large statue made of different materials that was destroyed by a rock and replaced by a mountain. Though Nebuchadnezzar had not told him what the dream was about, Daniel revealed that God himself had given Nebuchadnezzar this dream. God used the dream to show Nebuchadnezzar the kingdoms of history that would follow his and to show him how the Kingdom of God — represented by the mountain — would supplant them all. Nebuchadnezzar praised Daniel's God because only a true and powerful God could reveal another man's dream along with its meaning.
There's a new girl in my youth group. The other day at church, she told me she had "the Force." Being the Star Wars nerd that I am, I understood this to mean she was claiming to be able to move things without touching them and do mind tricks.
And she was right.
In the state of Texas, it is illegal to shoot a buffalo from the second story of a hotel. Neither are you allowed to sell you own eye. In our state capitol, you may not carry wire cutters in your pocket.
I discovered each of these when I searched for Texas' strangest laws. You can find list upon list of laws for almost every jurisdiction in America that we might call "strange" because governments around the country passed laws that seem to exceed, or at least no longer perform, the proper function of government in comic ways.
On Christmas Eve in 1814, British and American delegates met in Ghent, Belgium, to sign a treaty. The Treaty of Ghent formally ceased hostilities between England and the United States of America, putting an end to the War of 1812. But no one told our soon-to-be seventh President Andrew Jackson nor the British forces sent to capture New Orleans until several weeks later.
On Jan. 8, 1815, the Battle of New Orleans broke out with British soldiers outnumbering Americans by more than 2 to 1. The American victory, however, was decisive. The number of British killed, wounded or captured was 2,034 — about a fifth of the total force. By contrast, only 62 Americans were killed, wounded or missing when the engagement ended on Jan. 18.
Humans are experts at destruction. In our history on this planet, we have demonstrated an amazing ability to break and kill. When you consider the things we actually manage to build and create, we seem to excel most at building things that destroy.
Most of the inventions we now enjoy in the modern era were the product of war efforts. Planes, computers, the Internet, orbital flight, nuclear power, GPS, wristwatches, stainless steel and even the microwave oven all have their origins in war in the 20th century alone. Our most successful efforts at creativity were stumbled upon as we intentionally sought out ways to kill each other better.
My first career was in information technology services. I did a lot of different things from desktop support to systems administration to even a little bit of IT sales. I am so thankful for those years. I learned so much about serving people during those years. I learned how to prioritize people over the machines. I even learned how to calm frustrated people down. It's actually quite easy.
"I am not your enemy. I just want to help you."
I have a friend who lived most of her life in Japan. After she married an American, her husband put considerable effort into understanding the culture that formed the way his wife thinks and sees the world. After all, as a Christian, he took the biblical injunction to dwell with his wife "with understanding" (1 Peter 3:7).
He once explained to me what he thought was the core difference between Eastern and Western thought. In Western thinking, the question is, "Is it right or wrong?" But in the Japan, he said, the core question is, "Is this honorable or shameful?"
Ironically, in my unmitigated Western mode of thinking, I immediately began to think, "Is it right or wrong to think that way?" In fact, I need to admit that I don't love anything more than being right. Conversely, I don't hate anything more than being wrong. For most of my life, this has informed my arguments. I would argue absolutely anything simply to win the argument — to be perceived as right whether I was or not, and to prove someone else was wrong whether they were or not.
I love to argue. It might be a personality flaw. There's something I have always loved about jousting with words.
And that's exactly what arguing used to be to me: Jousting with words. Poking at someone with my words in order to knock them down for no good reason beyond sport, entertainment or the release of aggression. I suspect many people think of arguing the same way. "Arguing" has become a negative word and activity. I would like to rescue the word.
I was alone on an island, hungry and miles of paddling away from any help. I had unwisely decided to spend a week of solo beach camping by living off the hook, and I hadn't caught a single fish in days.
I was hungry in a way I had never experienced hunger before.
I went with some friends to see the latest Christian movie in the theaters the other night. We sat in an air-conditioned building in large, comfortable, reclining chairs and watched a high-definition projected display while our brothers and sisters in other parts of the world sat in jail cells and torture chambers, or at least on a dirt floor hoping police don't discover their church gathering.
It strikes me as odd that in a country where movies appealing to "Christians" are cranked out several times a year, churches still struggle to grow. We've all heard the statistics about how Christianity is on the decline in the West. We are seeing the same historical pattern which led Europe into a post-Christian era play out here in America, even with a church on every corner and "Christian" movies in every theater.
A little more than a year ago (Jan. 1, 2015), one of the largest churches in the United States disbanded.
Mars Hill Church, boasting more than 12,000 in attendance across 15 campuses in five states at its peak, dissolved as an organization. Allegations of abusive leadership and plagiarism against Mark Driscoll, the founder and lead pastor of the church, surfaced and the ensuing controversy led the celebrity pastor to resign. Leaving a massive leadership vacuum in his wake, the church liquidated its assets, dismissed its staff and helped satellite sites become their own independent congregations.


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Fetish Girls
Categories: Erotic, Fetish
Marisela - Fabulous Smoking Honey Gives A Bj - [Low]
Smoking is an inherently softcore activity, rife with symbolism. But when you mix up the symbolic action with the actual action, you get the ultimate smoking fetish wish script. Marisela and her beau enjoy making smoking pornography tapes for each other, and Marisela has had a lot of practice providing her beau head while smoking cigarettes and they both truly get off on the practice. Marisela is clothed in a promiscuous cheerleader apparel, and she makes some muddy chat at her bf while she lights her cigarette and blows the smoke in his face. She gropes the smoke with her lips, puckering them around the filter like its the head of his prick while she fellates the smoke into her lungs. She eventually quits frolicking with him and pulls out his trouser snake and she gives him an awesome fellatio. She likes gulping his stiffy, and she stops often to take a haul on her cigarette and exhale directly on the head of his rock-hard immense schlong. The couples glamour smoking porno flick resumes until Marisela has her beau on the verge of climax and she milks his penis until he shoots a exploding geyser throughout her face and she proceeds to fellate on her cigarette with her jizm packed jaws.374 Views
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Categories: Erotic, Fetish


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Roasted Tomato and Pasta Soup
READY IN: 1hr
Recipe by Lorac
Roasting intensifies the flavor of the vegetables and adds a wonderful smoky taste. The roasting can be done a day in advance and the soup can be frozen with out the pasta.
Top Review by Melissa Bell
This is an amazing soup. I broiled my veggies on low, because high charred them right away. I also added fresh basil and parsley. Really delicious! Thanks for the recipe.
Ingredients Nutrition
- 453.59 g ripe Italian plum tomato, halved lengthwise
- 1 large red pepper, quartered lengthwise and seeded
- 1 large red onion, quartered lengthwise
- 3 clove garlic, unpeeled
- 29.58 ml olive oil
- 1301.24 ml vegetable stock
- 4-5 fresh sage leaves, chopped
- 0.25 ml sugar
- 236.59 ml tubetti
- salt and pepper
- balsamic vinegar
Directions See How It's Made
- Preheat oven to 375°F.
- Spread vegetables in a roasting pan and drizzle with oil.
- Roast for 30 to 40 minutes or until vegetable are soft and lightly charred, strring and turning them after 15 minutes.
- Process the vegetables with 1 cup of stock until pureed.
- Pour into a sieve placed over a large saucepan and press the puree through into the pan.
- Add stock, sage, sugar, salt and pepper and bring to a boil.
- Add pasta and cook according to package directions for al dente.
- Ladle into serving bowls and drizzle with a little balsamic vinegar.


text:
Five Regions of the Future: Preparing Your Business for Tomorrow's Technology Revolution
Futurist Joel Barker was the first to apply the scientific concept of paradigm shifts to the world of business, which helped make his 1992 book, Future Edge (later republished as Paradigms), a national bestseller. He has spent more than twenty- five years studying how companies adapt (or fail to) to new breakthroughs.
Now Barker and fellow futurist Scott Erickson offer a bold new way of looking at today s rapidly evolving technologies: as five distinct "ecosystems" that each operates with a distinct set of values, advantages, and disadvantages: Super Tech: Bigger, better, more! (e.g., fusion power) Limits Tech: Use what you ve got (e.g., aerogel insulation) Local Tech: Think small, think home (e.g., electric wind turbines) Nature Tech: Be one with nature (e.g., organic plastics) Human Tech: What lies within us (e.g., stem cells)
From pet robots to hypersonic planes, from wave power to waterless toilets, Barker and Erickson give readers a totally new way to understand and take advantage of the future of technology. Five Regions of the Future is an essential book for anyone baffled by today s technological onslaught.
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text:
"Old Guard Avant-garde:
Friday, January 02 2009 @ 07:35 PM MST
Contributed by: Admin
Views: 61,229
"Old Guard Avant-garde:
Getting Real About What is - and what isn't the real thing.'"
Joseph Bean. Frontiers, October 4, 1996.
The fuss about the Old Guard of leather and the new forms leathersex has taken in recent years is not going to go away any time soon, I'm sure. Nor do I think there is much I could say that would settle many of the disagreements about what the Old Guard is or isn't and how it relates to
the younger community and those who are newer to S&M. I took a run at explaining my point of view in "Leathersex: A Guide...," and undertook a few related questions in "Leathersex Q&A," but I doubt I've made any inroads there. So, rather than attempt a complete explanation of anything and rather than attempt to settle disputes, I'd like to just put a few thoughts in a familiar order, and leave others to determine whether I've given them anything useful.
Here then, in my hand, from the home office buried in my past and located
somewhere in Southern California are two Top 10 Lists, offered with apologies only to David Letterman.
Top 10 Reasons a Man Might Claim to be a Member of Leather's Old Guard
10) He imagines that wrinkles, rigidity, discomfort with modern music and the absence of a tan are all excused in Old Guard leathermen.
9) He doesn't have the gumption to say, "I'd like to beat you 'til we both come," and he believes that giving his sex-style a fancy name like Old Guard is a higher road (but, of course, hopes the effect will be much the same no matter what he says.)
8) He doesn't want to have to negotiate a safe scene and, as a bottom, imagines that Old Guard means "without limits," so no negotiation will be expected or required.
7) He doesn't want his authority as a Top questioned, and imagines that the moniker Old Guard will deflect any questioning of authority (not to mention any doubts about his skill, etc.,.)
6.) He confuses Old Guard with old hand, and believes that others will not require that he demonstrate caring, understanding or trustworthiness if he can pass of his experienced skill as Old Guard (by which he means something more than mere skill.)
5.) He confuses Old Guard with serious and like to think of himself as a serious leatherman, often actually meaning that he is looking for severe or extreme scenes, even to the point of getting into unnecessarily risky situations.
4.) He's no longer young or no longer sees himself as attractive and believes that his claim to be Old Guard makes him attractive in some other way.
3.) He has heard one or more highly-respected leathermen accept reference to themselves as Old Guard and wants to appear to be like them.
2.) He figures he can get away with anything if you believe he's Old Guard. You'll blame any confusion, discomfort, apparently unsafe situation or even complete failure of the scene on your own lack of Old Guard training.
1.) He wants to seem to have been in the leathersex life as long as his bought used leather jacket might have been even if he just discovered S&M last weekend.
Top 10 Signs You've Actually Met an Old Guard Leatherman
10.) He'll readily admit his age, even if he's old, but is unlikely to volunteer a self-appraisal that involves the words "Old Guard."
9.) He would rather be himself than be your fantasy man, no matter how much he wants you.
8.) He has spent all of the time and effort necessary (money too, if any) to be fully trained, fully equipped and fully prepared for a scene before he undertakes to do it.
7.) He is more likely to speak of the men in his circle in terms of the debt he owes them for his position in leather society, for his skills and for his toys than to speak of his exalted (or not) position, his developed (or wished for) skills and his great (or small) toy collection.
6.) He assumes nothing until conditions are such that he knows no assumptions are required for him to step into his earned and maintained position among his peers, those who aspire to be his peers, and those he
accepts as his superiors.
5.) He is a gentleman in the simplest and truest sense of the word so that courtesy is a habit, not a weakness; grace is a strength, not an effeminacy; power is a fact, not a claim; dominance and submission are acts of will, not role-plays; and, while self-assertion is a given, self-aggrandizement is never intended.
4.) He is careful to develop a functioning dynamic based on trust/trustworthiness, submission/ dominance and the famous power exchange before threatening anyone with the storming of his limits or the
opportunity to enter an ecstatic state.
3.) Top or bottom, he falls in love with every play partner, if only for an hour or two, and is unashamed to share that love openly even when speaking of it might undermine the nature of the interplay.
2.) His body, his knowledge, conversational skills and courtesy are
nothing compared with what it is that actually compels your interest in
1.) He values his reputation above his orgasm and his integrity above
his reputation.
If I seem to idealize Old Guard leathermen it is only because - if they are understood to be the military-style, semi-secret society of my novitiate in leather, circa 1964 - then that is truly my view of who they were and are, as well as what I learned to aspire to.
Maybe no one ever achieved perfection in all of this, but we dreamed (and still do) that a brotherhood based on shared values and earned honor was worthwhile because it gave us a clear sense of ourselves and provided a social context that we found lacking elsewhere.
We could just as well have been Elks or Oddfellows but for the fact that an important part of what we shared was sexual.
We certainly could have been accused of separatism, but that was based as
much on our being rejected as on our wish to close anyone out. In fact, the older and more respected leathermen in my coming-out circle seemed eager to have me succeed in every way. They wanted me to achieve full membership in their structured fraternity, and did everything reasonable to help me. The driving idea was that I should achieve it, not expect it to be given me.
Things are different today, but I am not sure I could say it better, only differently. Sorry for the interruption. Let the war resume.
Joseph Bean
Copyright 1996


text:
This lock retro-fits to timber front doors to secure your home without the need for keys. Instead, you memorise a four- to 12-digit PIN code. No more losing keys, no need to carry keys when you go for a run, and if you want to let a friend into the house simply tell them the code. You can even set up a temporary code for house guests or cat sitters and then delete it once they leave. From £99.99. Visit yale.co.uk.
A camera system that makes it easy to watch over several homes at once. If a VueZone camera detects motion, it sends a short video clip to your smartphone, tablet or PC. Or you can log in to view a live video feed at any time and see what your teenage kids are up to. The starter kit costs £247.17 and includes two battery-powered cameras plus a gadget that connects them to your broadband. Subscriptions start for free and range up to £69.99 a year for the Elite service, which lets you keep track of up to 15 cameras across three locations. Visit netgear.co.uk.
One of the best burglar deterrents is to make your home look occupied when you are out. QMotion shades, from £674, are as easy to install as a normal blind because their technology is hidden inside the roller. Even the batteries, which have a five-year life, are hidden. And there are no cords, so the blinds are safer for young children. Adjust by remote control — from £63 — or use QSync, at £359, for smartphone or tablet control of up to 450 blinds at once... which may be a little over the top. You can also set times and heights for each blind, so they open and close daily, even if you are away. Visit qmotionshades.co.uk.
This smoke and carbon monoxide alarm from Nest is both beautiful and clever. If you have several alarms in the house, they can be wirelessly linked to go off at once. They also link to a smartphone app, so if Nest Protect notices a tiny amount of smoke — because you've burnt the toast, for example — it sends a "heads up" message to your phone so you can take action before the alarm proper activates. Should the alarm go off, simply wave to silence it. The alarm has a human voice as well as a loud horn, because children can easily sleep through beeping alarms. Priced £109 from nest.com/uk.
Keep your entire home at your fingertips while you are away with LIGHTWAVERF's Modular range of intelligent dimmers, radiator valves, sockets and sensors. Use your smartphone to control heating, lighting and power. Working late at the office? Use the app to close the curtains and turn on the lights but leave the heating off until half an hour before you get home. Meanwhile, motion sensors can trigger a siren, or launch a security camera that then sends live video to your phone or tablet. Starter kits from £179.99. Visit lightwaverf.com.
A HomeMonitor security camera, £149.99, needs no disruptive wiring — it simply plugs into the mains and uses wifi. It records constantly and can see in the dark. Videos are stored free online for seven days, or view live video via any smart device. You get free alerts when the camera detects motion, letting you log on to check whether it's an intruder and not the cat. Visit homemonitor.me.
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text:
Satomi Kawakita Large Pom Pom Studs
Satomi Kawakita Large Pom Pom Studs
The oxidized silver pom pom studs by Satomi Kawakita are at once organic, delicate and timeless. For casual wear or dressy these are subtle and unique and ideal for those on the go that want a unique earring that can be worn and then forgotten. • 11mm sterling silver hand crafted stud earring • Comes with box
What's in a name? For this New York-based boutique, a whole lot of love. Elizabeth Jeffer started the business with her beloved grandmother Rosalind Tiger's artistic spirit and style in mind. The online shop's curated edit of travel-inspired wares is the stuff dreams are made of. With a keen focus on long-lasting craftsmanship and production, this shop is filled to the brim with amazing, hard-to-find labels. Elizabeth developed her fine eye for design and style early in life and honed it further at UC Berkeley as an art history major. After working as a sales representative for years she decided to make the leap and launch her own boutique in September of 2011. Combining her extensive sales experience with her love for fashion and design, you can count on Roztayger to seek out under-the-radar labels and rad new designers to love.
Do you have questions about this product? We're here to help!


text:
Two Pinkies One Gummy by Doggie999Watch
©2012-2017 Doggie999
Gummy: Why me? XD
Gummy: ........help me.....they're multiplying....
Some cute Pinkies =3
Gummy is having a wtf moment.
Dat title. o.o
nice picture. i like it.
Is it me or does Gummy seem totally freaked lol
This is so cute.
The other one is really Berriswaner.
Soooooo kayoooooooot!
Love this!
Love this!
This is sweet!
Yea, making bubbles in the tub is fun, aint it Pinkie?
This is incredibly cute. Great work!


text:
|Reviews for Tactile
| soratori42 chapter 1 . 11/29/2013
this was absolutely adorable
I really can't say that any better than any of the other reviewers have though
| Kasey chapter 1 . 7/20/2013
This is so amazing it's almost painful
| Istealurfrenchfries chapter 1 . 12/2/2012
Aww! Why couldn't they have gotten together in the show! I love this.
| RoseKatnissWeasley7 chapter 1 . 11/17/2012
this is so cute!
| ZekksGoddess chapter 1 . 11/3/2012
These little moments are my favorites, so finding this little beauty delighted me. Wonderful work. :)
| A Pirate By Any Other Name chapter 1 . 10/10/2012
This is sweetness and goodness and David Tennant all over. And true. Nine wasn't much of a touchy-feeling kind of guy. But Ten...Ten was. And now I must cry because I have to talk about them in the past tense...
| sindea.d chapter 1 . 10/8/2012
Loved your story :)
| ReprobaVir chapter 1 . 10/8/2012
As always, this was spectacular.
The imagery used was incredibly fresh, and the whole piece flowed brilliantly. I loved perusing this poetic analysis of the Doctor and Rose's relationship, and the comparison between it and the relationship between her and the last generation of the Doctor was stunning.
Please keep writing, full length works, little pieces like this, all of them are wondrous.


text:
Localization of the Carnation Italian ringspot virus replication protein p36 to the mitochondrial outer membrane is mediated by an internal targeting signal and the TOM complex
Here we report on the molecular signal responsible for sorting p36 to the mitochondrial outer membrane. Using a combination of gain-of-function assays with portions of p36 fused to reporter proteins and domain-swapping assays with p36 and another closely-related viral RNA-binding protein, p33, that sorts specifically to the peroxisomal boundary membrane, we show that the mitochondrial targeting information in p36 resides within its two transmembrane domains (TMDs) and intervening hydrophilic loop sequence. Comprehensive mutational analysis of these regions in p36 revealed that the primary targeting determinants are the moderate hydrophobicity of both TMDs and the positively-charged face of an amphipathic helix within the intervening loop sequence. We show also using bimolecular fluorescence complementation (BiFC) that p36 interacts with certain components of the translocase complex in the mitochondrial outer membrane (TOM), but not with the sorting and assembly machinery (SAM).
Our results provide insight to how viruses, such as CIRV, exploit specific host-cell protein sorting pathways to facilitate their replication. The characterization of the targeting and insertion of p36 into the mitochondrial outer membrane also sheds light on the mechanisms involved in sorting of host-cell membrane proteins to mitochondria, a process that has been largely unexplored in plants.
The hallmark of positive-strand RNA viruses is their ability to recruit distinct host-cell organelle membranes in order to create unique compartments at which viral RNA replication takes place
While the cytolopathological features of MVB biogenesis have been relatively well studied, many fundamental questions remain about the molecular mechanisms underlying the interaction of viral replication factors with host-cell membranes. For instance, the significance of the diversity of intracellular membranes used by different viruses is unknown. Likewise, the events involved in the specific intracellular targeting and membrane integration/assembly of the viral replication proteins, as well as the host-cell factors that facilitate these processes and/or mediate membrane remodelling are, in most cases, poorly studied and unclear.
CIRV is a member of the
In CIRV-infected cells, MVBs are derived from mitochondria
Here, we describe the results of a comprehensive study of the molecular signals involved in the mitochondrial targeting of p36. We show using a combination of p36-reporter fusion proteins and p36-p33 hybrid proteins, wherein specific regions of p36 were replaced with those that constitute the peroxisomal targeting information in p33 (and vice versa), that the mitochondrial sorting of p36 is mediated by an internal targeting signal consisting of its two moderately hydrophobic TMDs and a positively-charged face of an amphipathic helix located within the intervening loop sequence. Notably, this targeting signal in p36 resembles the targeting determinants in several authentic mitochondrial outer membrane proteins from evolutionarily diverse organisms. We show also that p36 interacts with certain components of the
Results and discussion
p36 expressed transiently in tobacco BY-2 cells is localized to mitochondrial outer membranes
To begin to decipher the intracellular targeting information within p36, we took advantage of tobacco (
Figure 1
Localization of p36 to the outer mitochondrial membrane in BY-2 cells
Localization of p36 to the outer mitochondrial membrane in BY-2 cells. Tobacco BY-2 cells were either non-transformed or transformed transiently (via biolistic bombardment) with either myc-p36 or p36-myc and then processed for immunofluorescence CLSM. Hatched boxes represent the portion of the cells shown at higher magnification in the panels to the right. Arrowheads indicate obvious examples of the torus fluorescent structures containing myc-p36 or p36-myc either delineating the spherical structures containing endogenous mitochondrial matrix-localized E1β or colocalizing with the torus structures containing the endogenous mitochondrial outer membrane protein porin. The bottom row of images show representative immunofluorescence staining patterns attributable to endogenous porin and E1β in non-transformed cells; arrowheads in the high magnified images indicate obvious examples of porin-containing torus structures (similar to those containing myc-p36 or p36-myc [compare with cells above]) enclosing the E1β-containing spheres. Bar = 10 μm.
While it remains to be determined whether the aggregated mitochondria in p36-transformed cells were modified also in terms of their ultrastructure, this possibility is likely since expression of p36 (e.g., p36-GFP) in various other cell types (e.g., leaf mesophyll protoplasts and yeast cells) led to similar aggregations of mitochondria, as well as a proliferation of their outer membranes
The localization of myc-p36 and p36-myc to mitochondria indicates also that appending the myc epitope to the N or C terminus of p36 did not disrupt the protein's normal targeting behaviour, since non-tagged (wild-type) p36 colocalized also with endogenous porin in the outer membranes of aggregated mitochondria (refer to Additional file
Additional file 1
Localization of p36 expressed alone or together with the p95 replication protein in BY-2 cells and electron microscopic analysis of
Click here for file
The localization of epitope-tagged and wild-type p36 to mitochondrial outer membranes, and their subsequent effects on mitochondrial morphology and distribution, were similar also to when p36 was co-expressed with its allied replication protein, p95, either together alone (Rep) or together in the context of full-length CIRV, i.e., a CIRV cDNA positioned within a plant expression plasmid (Additional file
p36 is orientated in mitochondrial outer membranes in an Nout-Cout topology
The topological orientation of p36 in mitochondrial outer membranes was assessed using a differential detergent permeabilization assay
As shown in Figure
Figure 2
Topological orientation of p36 in differentially permeabilized BY-2 cells
Topological orientation of p36 in differentially permeabilized BY-2 cells. BY-2 cells were either non-transformed or transformed transiently (via biolistic bombardment) with myc-p36 or p36-myc, fixed, and then permeabilized with either triton X-100 (which permeabilizes both the plasma membrane and organellar membranes) or digitonin (which permeabilizes only the plasma membrane). Permeabilized cells were then processed for (immuno)epifluorescence microscopy using antibodies raised against (as indicated by the labelling at the top left of each micrograph) either cytosolic α-tubulin, mitochondrial matrix-localized E1β, the myc epitope and/or the p36 C-terminal peptide sequence (amino acids 218–237) located downstream of the protein's second (of two) predicted TMDs. Bar = 10 μm.
Overall, these data are in agreement with the previously proposed model for the membrane topology of p36 based on the hydropathy profile of the protein's primary amino acid sequence
An internal domain of p36 composed of two TMDs and an intervening loop sequence functions as a mitochondrial targeting signal
While previous studies using chimeras of CIRV p36 and CymRSV p33, expressed either in the context of the full-length virus or alone, revealed that the N terminal half of each protein is responsible for its sorting to mitochondria and peroxisomes, respectively
Figure 3
Localization of p36-CAT fusion proteins in BY-2 cells
Localization of p36-CAT fusion proteins in BY-2 cells. (A) Schematic illustrations of CAT and various p36-CAT fusion proteins and their corresponding subcellular localizations in transformed (via biolistic bombardment) BY-2 cells. The numbers in the name of each fusion construct denote the specific amino acid residues from p36 that were fused to the N terminus of CAT. Portions of the p36 ORF are colored blue or black, the latter denoting the two putative TMDs in p36 and the numbers shown above p36-CAT include the position of each TMD's left and right amino acid border. Grey boxes denote CAT. Mito, mitochondria; Cyt, cytosol. (B) Representative (immuno)epifluorescence micrographs illustrating the localizations of the various constructs shown in (A). Each micrograph is labelled at the top left with the name of the transiently-expressed fusion protein or the endogenous mitochondrial marker protein, E1β. Hatched boxes represent the portion of the cells shown at higher magnification in the panels to the right. Arrowheads indicate obvious colocalizations. Bar = 10 μm.
Figure 4
Localization of p36-p33 hybrid proteins in BY-2 cells
Localization of p36-p33 hybrid proteins in BY-2 cells. (A) Alignment of the deduced amino acid sequences of CIRV p36 and TBSV p33. Sequences were obtained from GenBank and aligned using the ClustalW
As shown in Figure
Similar to p36-CAT, p36 1-190-CAT, containing residues 1–190 of p36 including the protein's N-terminal hydrophilic domain, TMDs 1 and 2, and the intervening loop sequence appended to CAT, localized to (aggregated) mitochondria (Figure
Other p36-CAT fusion proteins comprised of smaller portions of the p36 N-terminal half also did not target to mitochondria. That is, both p36 90-164-CAT and p36 120-164-CAT, consisting of TMD1 and the intervening loop sequence and the intervening loop sequence alone, respectively, localized to the cytosol (Figure
Additional file 2
Localization of p36-CAT and topological orientation of p36 90-190-CAT in BY-2 cells. (A) Tobacco BY-2 cells were transformed transiently (via biolistic bombardment) with p36 90-190-CAT (consisting of the p36 amino acid residues 90–190 fused to the N-terminus of CAT) and then processed for immunofluorescence CLSM using primary antibodies raised against CAT. Hatched boxes represent the portion of the cells shown at higher magnification in the panels to the right. The merged image shows that the torus fluorescent structures containing p36 90-190-CAT delineate the spherical structures attributable to mitochondrial matrix-localized E1β. Arrowheads indicate obvious examples of a toroidal enclosure of a sphere. Bar = 10 μm. (B) BY-2 cells were transformed transiently (via biolistic bombardment) with p36 90-190-CAT, fixed, and then permeabilized with either triton X-100 (which permeabilizes both the plasma membrane and organellar membranes) or digitonin (which permeabilizes only the plasma membrane). Permeabilized cells were then processed for (immuno)epifluorescence microscopy using antibodies raised against (as indicated by the labelling at the top left of each micrograph) either cytosolic α-tubulin, mitochondrial matrix E1β, or CAT (in p36 90-190-CAT). Note that, similar to endogenous cytosolic α-tubulin, p36 90-190-CAT, but not endogenous mitochondrial matrix E1β, were immunodetected in both triton X-100- and digititon-permeabilized cells, indicating that the C-terminal-appended CAT moiety was exposed to the cytosol. Although the relative position of the N terminus of p36 90-190-CAT was not directly tested in these differential permeabilization experiments, this fusion protein, similar to full-length p36 (refer to Figure
Click here for file
Characterization of the mitochondrial targeting information in p36 was also carried out using domain-swapping assays with TBSV p33, an ortholog of CymRSV that has been recently well characterized in terms of its peroxisomal membrane targeting signal
As shown in Figures
Replacement of the N-terminal half of p33 (residues 1–156) with the corresponding sequences from p36 (residues 1–190), including the N-terminal hydrophilic domain and both TMDs plus the intervening loop sequence, resulted in the hybrid protein (p36 1–190 p33-myc) being sorted to aggregated mitochondria, but not to peroxisomes (Figure
Overall, the data presented in Figures
The moderate hydrophobicity of TMD1 and TMD2 is essential for targeting p36 to mitochondria
The results presented above indicate that the sorting of p36 to mitochondria is, at least in part, dependent upon its TMDs. Thus, to test whether specific amino acid sequences or more global properties, e.g., overall hydrophobicity and/or length, within one or both of the TMDs contribute important mitochondrial targeting information, we generated several different mutant versions of p36-myc containing altered TMD sequences (Figure
Figure 5
Localization of p36 and Cb5 proteins with modified TMDs in BY-2 cells
Localization of p36 and Cb5 proteins with modified TMDs in BY-2 cells. (A) Schematic illustrations of various p36-myc and myc-Cb5 proteins with modified TMDs and their corresponding subcellular localizations in transformed (via biolistic bombardment) BY-2 cells. The name of each mutant construct denotes p36-myc or myc-Cb5 and the specific alterations to its TMD(s). Hydrophilic portions of the p36 ORF are colorized blue, with TMD1 and TMD2 in p36 colored black and white, respectively. The single TMD in Cb5 is colorized red. Grey boxes denote the position of the myc epitope. Cyt, cytosol; ER, endoplasmic reticulum; Mito, mitochondria; ?, unknown subcellular compartment(s). (B) Representative (immuno)epifluorescence micrographs illustrating the localizations of the various constructs shown in (A). Each micrograph is labelled at the top left with the name of either the transiently-expressed protein, the endogenous mitochondrial marker protein, E1β, the endogenous peroxisomal marker protein, catalase, or ConA (fluor-conjugated Concanavalin A) serving as stain for the ER
As shown in Figure
An examination of the TMD amino acid sequences within p36 (underlined, Figure
As shown also in Figure
Together, the data presented in Figure
This scenario that the mitochondrial sorting of p36 is mediated, at least in part, by the moderate hydrophobic nature of its TMDs is in agreement with results from several other studies on the sorting of authentic mitochondrial outer membrane proteins. That is, depending on the protein, a moderate hydrophobic TMD(s), combined with positive charges at its flanking soluble regions, is essential for mitochondrial outer membrane targeting [reviewed in
A notable extension of this model is that, for at least some mitochondrial outer membrane proteins, they appear to rely also on subtle sequence-specific features within their TMDs. For instance, both the yeast 5-kD and 7-kD TOM subunits (TOM5 and TOM7) possess a conserved proline residue near the center of their TMDs that is required for their efficient targeting to mitochondria
The intervening loop sequence of p36 contains an amphipathic helix with a positively-charged face that is essential for mitochondrial targeting
Since the intervening loop sequence in p36 (residues 120–165 between TMD1 and TMD2) is significantly divergent from the corresponding loop sequence in peroxisomal-targeted p33 (Figure
Figure 6
Localization of p36 intervening-loop mutants in BY-2 cells
Localization of p36 intervening-loop mutants in BY-2 cells. (A) Deduced amino acid sequence of the intervening loop sequence (residues 120–165) and portions of the immediately-adjacent TMDs (underlined) in p36. The numbers shown above individual amino acids indicate their relative positions in p36. Amino acids predicted to form an α-helix (residues 131–157) are shaded grey and residues that are proposed to constitute the positively-charged face of the α-helix are bolded. Secondary structure prediction was carried out using Jpred
Based on these observations and previous reports that the targeting of several other authentic mitochondrial outer membrane-destined proteins rely on positively-charged residues together with a flanking TMD(s)
Mitochondrial targeting of p36-myc was also abolished when glycine residues were exchanged for each of the four positively-charged residues predicted to be situated along the same face of the amphipathic α-helix within the protein's intervening loop sequence, i.e., p36-myc K134K137R144K151ΔG mislocalized to the cytosol (Figure
The results for p36-myc K93K94R98R101ΔG also indicate that not all mutations to the protein affected its mitochondrial targeting fidelity. An observation that underscores a substantive caveat of this study (and any analysis of membrane protein targeting) whereby mutations of p36 that resulted in the protein's mislocalization could have been due to aberrant protein folding, rather than a disruption of a specific targeting determinant(s). Although this possibility cannot be excluded, our combined use of loss-of-function and gain-of-function targeting assays strongly supports our definition of the
Collectively, our findings on the combined importance of positively-charged residues (Figure
p36 interacts with certain components of the mitochondrial TOM complex, but not with the mitochondrial SAM complex
Almost all mitochondrial proteins are encoded by nuclear genes, synthesized on free polyribosomes in the cytosol, and targeted post-translationally to the organelle. Thereafter, the recognition, translocation, and sorting of these proteins is usually mediated by the TOM complex and, depending on the protein's submitochondrial destination, one of several other translocase complexes located in the outer and inner mitochondrial membranes [reviewed in
While it is generally accepted that the various mitochondrial import pathways and associated machinery are conserved among evolutionarily diverse organisms including yeast, animals and plants, considerable differences exist among some of their core protein subunit compositions and functional specificities, particularly in plants
To determine the insertion pathway of p36 into the outer mitochondrial membrane we tested whether it could interact directly with a variety of protein components of the plant TOM and/or SAM complexes. Specifically, we employed bimolecular fluorescence complementation (BiFC) assays to determine whether
BY-2 cells were transiently transformed with pairs of plasmids encoding p36 fused at its C terminus to the C-terminal half of the modified yellow fluorescent protein Venus
Figure 7
BiFC analysis of interactions among p36 and components of the TOM and SAM complexes
BiFC analysis of interactions among p36 and components of the TOM and SAM complexes. (A) BY-2 cells were co-transformed (via biolistic bombardment) with either p36, the N-terminal mitochondrial matrix targeting presequence of the β subunit of the F1-ATPase (βATPase), porin, p33, or Cb5 fused at either their N or C termini to cVenus (columns) and various constituents of the TOM complex (or an N-terminal mutant version of TOM20; TOM20mut), METAXIN, or p36 fused at either their N or C termini to nVenus (rows). In addition, all cells were co-transformed with either RFP, which served as a convenient means of identifying transformed cells. At ~16 h post-bombardment, cells were formaldehyde fixed and viewed by epifluorescence microscopy. Interactions were scored based on either the presence (+) or absence (-) of a BiFC (Venus) signal relative to reconstitution controls in which individual Venus half fusion proteins were co-expressed with the corresponding "empty" vector containing nVenus or cVenus fragments alone. All BiFC signals observed co-localized with (co)expressed mitochondrial βATPase-GFP. For each pair of plasmids tested, > 25 transformed cells were scored from at least two independent transformation (biolistic bombardment) experiments. ND, not determined. (B) Predicted topologies of mitochondrial outer membrane proteins used in BiFC experiments, the results of which are presented in (A). Also shown is the localization of the soluble βATPase N-terminal mitochondrial targeting presequence in the matrix following import. The topological orientations of mitochondrial outer membrane proteins shown are based on differential permeablization results presented either in Figure
Alternatively, BY-2 cells were co-bombarded with a plasmid coding for an individual nVenus- or cVenus-tagged protein along with a plasmid encoding βATPase-GFP (consisting of the N-terminal mitochondrial matrix targeting presequence of βATPase [residues 1–60] fused to the N terminus of GFP), which served to identify transformed cells and, acting as a well-characterized mitochondrial marker protein
Additional file 3
Localization and topology of nVenus and cVenus fusion proteins used in BiFC assays. (A) Tobacco BY-2 cells were transformed transiently (via biolistic bombardment) with selected individual nVenus (and myc-tagged) or cVenus (and HA-tagged) fusion proteins as shown in Figures
Click here for file
The results presented in Figure
In a series of control experiments we also evaluated whether or not the βATPase matrix targeting presequence and/or the full-length outer mitochondrial β-barrel membrane protein, porin, interacted with the TOM and SAM complex protein subunits in a manner similar to p36. As shown in Figure
At least one unexpected finding from the BiFC experiments presented in Figure
In additional control experiments TBSV p33 (p33-cVenus) was not observed to interact with any of the TOM complex subunit proteins tested nor with p36 (Figure
Taken together, the BiFC results presented in Figure
Viruses are well known for their ability to exploit specific organelles and pathways in infected host cells in order to facilitate their replication
While the functional significance of the CIRV-mitochondria relationship and the molecular mechanisms underlying the membrane rearrangements that occur during mitochondrial MVB biogenesis in CIRV-infected cells remain to be elucidated, future studies aimed at understanding these processes, similar to results presented here for the sorting of p36, will undoubtedly provide insights to certain aspects of normal mitochondrial biogenesis in plant cells (e.g., membrane remodelling during mitochondria fission). To date, only one plant outer mitochondrial membrane protein, Cb5, has been well examined in terms of its targeting signal and insertion mechanism
Recombinant DNA procedures and reagents
Standard recombinant DNA procedures were performed as described by Sambrook et al.
Additional file 4
List of synthetic oligonucleotide primers used in the construction of plasmids.
Click here for file
Construction of plasmids
Plasmids containing p36 and/or p95
pRTL2/p36, encoding the CIRV 36 kDa replicase protein was constructed in the following manner. First, sequences encoding the p36 ORF were amplified via the polymerase chain reaction (PCR) (primers Fp135 and Rp107; Additional file
The C-terminal myc-epitope-tagged version of p36 in pRTL2 (pRTL2/p36-myc) was constructed by annealing complementary oligonucleotides (Fp177 and Rp178) that encoded the myc-epitope tag (-EQKLISEEDL-;
To construct pRTL2/Rep, sequences encoding both CIRV replicase proteins (p36 and p95) were amplified via PCR (primers Fp135 and Rp572) from pUC18/CIRV with 5'
An infectious cDNA of CIRV, whereby the sequence for the entire CIRV genome
Plasmids containing p36-CAT or p36-GFP fusion constructs
Construction of p36-CAT fusion constructs was carried out by first introducing various silent restriction sites at specific localizations within the p36 ORF. For instance, a silent
To construct pRTL2/p36-CAT, encoding the entire p36 ORF fused to the N terminus of CAT, the
Plasmids containing modified versions of p36 or p33
pRTL2/p33 1-156-p36-myc, pRTL2/p33 103-156-p36-myc and pRTL2/p33 103-131-p36-myc, consisting of p33 amino acids 1 to 156, 103 to 156, and 103 to 131, respectively, within the context of p36-myc, were constructed as follows. pRTL2/p33 1-156-p36-myc was constructed by digesting pRTL2/p33-myc (
pRTL2/p36 1-190-p33-myc, pRTL2/p36 1-120-p33-myc, and pRTL2/p36 120-190-p33-myc, consisting of p36 amino acids 1 to 190, 1 to 120, and 120 to 190, respectively, within the context of p33-myc, were constructed as follows. pRTL2/p36 1-190-p33-myc was constructed by digesting pRTL2/p36-myc (
Plasmids encoding modified versions of p36-myc in which TMD1 and TMD2 were either swapped or replaced (individually or together) with synthetic TMDs were generated as follows. pRTL2/p36-myc TMD1 ⇔ TMD2, in which TMD1 of p36 was replaced with TMD2 of p36 and vice versa, was constructed by first ligating annealed, complementary synthetic oligonucleotides (Fp654 and Rp653) coding for p36 TMD2 (amino acids 166–188) along with 5'
pRTL2/p36-myc TMD1ΔsynTMD and pRTL2/p36-myc TMD2ΔsynTMD, in which TMD1 and TMD2 of p36, respectively, were replaced with an artificial/idealized TMD consisting of multiple -LALV-amino acid repeats
pRTL2/p36-myc TMD1ΔCb5TMD was constructed by ligating annealed complementary oligonucleotides (Fp1977 and Rp1978) coding for the 18 amino-acid-long TMD of the tung mitochondrial isoform of Cb5 (isoform D)
pRTL2/p36-myc TMD1Δp33TMD was constructed by ligating annealed complementary oligonucleotides (Fp113 and Rp114) coding for the p33 TMD1 (residues 84–98) and the amino acid sequence -SYA-immediately downstream of TMD1 with 5'
pRTL2/p36-myc Δ 131–157 coding for p36-myc lacking amino acids residues 131–157 within the intervening loop sequence was generated using site-directed mutagenesis (primers Fp1966 and Rp1967), along with pRLT2/p36-myc as template DNA. pRTL2/p36-myc K93K94R98R101ΔG in which the positively-charged amino acid residues at positions 93, 94, 98 and 101 in the p36 ORF were each replaced with a glycine were constructed using site-directed mutagenesis (primers Fp412 and Rp410), along with pRTL2/p36-myc as template DNA. pRTL2/p36-myc K134K137R144K151ΔG was constructed in two steps. First, sequences encoding arginine at position 144 and lysine at position 151 in p36-myc (pRTL2/p36-myc) were mutated to glycines using site-directed mutagenesis (primers Fp2048 and Rp2049), yielding pRTL2/p36-myc R144K151ΔG. Second, sequences encoding the lysines at positions 134 and 137 in pRTL2/p36-myc R144K151ΔG were mutated to glycines using site-directed mutagenesis (primers Fp2098 and Rp2099), yielding pRTL2/p36-myc K134K137R144K151ΔG.
Plasmids used for BiFC
BiFC plasmids used in this study contained fragments derived from Venus, a variant of the yellow fluorescent protein (YFP)
To construct pSAT4/nVenus C1, sequences encoding the N-terminal 174 amino acids of Venus along with introduced 5'
Construction of BiFC plasmids containing TOM20 (or the mutant version thereof; TOM20mut), TOM22, TOM40 or METAXIN fused at their N termini to nVenus was carried out as follows. First, sequences encoding the entire TOM20, TOM22, TOM40 or METAXIN ORFs were amplified via PCR using the appropriate oligonucleotide primers (Additional file
To construct BiFC plasmids containing p36 fused at its C terminus to nVenus, sequences encoding the entire p36 ORF was amplified via PCR using the appropriate oligonucleotide primers (Additional file
pSAT4/mtOM64-nVenus, consisting of the Arabidopsis mitochondrial outer membrane protein of 64-kD (mtOM64), a protein proposed to function as mitochondrial protein receptor
Construction of BiFC plasmids containing the βATPase presequence, porin, p36, or p33 fused at their C termini to cVenus was carried out as follows. Sequences encoding either the βATPase mitochondrial targeting presequence or the entire ORF of porin, p36 or p33 were amplified via PCR using the appropriate oligonucleotide primers (Additional file
Other Plasmids
pUC18/βATPase-GFP encoding the N-terminal mitochondrial matrix targeting presequence (residues 1–60) of the β subunit of F1-ATPase from
Transient transformation of tobacco BY-2 cells and fluorescence microscopy
Tobacco (
Fixed and permeabilized cells were processed for immunofluorescence microscopy as described by Trelease et al.
Epifluorescent images of BY-2 cells were acquired using a Zeiss Axioskop 2 MOT epifluorescence microscope (Carl Zeiss, Toronto, Canada) with a Zeiss 63× Plan Apochromat oil-immersion objective. Image captures were performed using a Retiga 1300 charge-coupled device camera (Qimaging, Burnaby, Canada) and Northern Eclipse 5.0 (Empix Imaging, Mississauga, Canada) or OpenLab 5.0 (Improvision Inc., Lexington, MA, USA) software. CLSM images of BY-2 cells were acquired using a Leica DM RBE microscope with a Leica 63× Plan Apochromat oil-immersion objective, a Leica TCS SP2 scanning head, and the Leica TCS NT software package (Version 2.61) (Leica, Heidelberg, Germany). Fluorophore emissions were collected sequentially in double- and triple-labeling experiments; single-labeling experiments showed no detectable crossover at the settings used for data collection. Confocal images were acquired as a z-series of representative cells and single optical sections were saved as 512 × 512-pixel digital images. All fluorescence images of cells shown in individual figures are representative of > 50 independent (transient) transformations from at least two independent transformation experiments. Figure compositions were generated using Adobe Photoshop CS (Adobe Systems, San Jose, CA).
Biolistic bombardment of cells for BiFC experiments was carried out essentially as described above with the exception that tungsten particles were coated with 200 ng each of plasmids encoding the appropriate combinations of proteins. Tungsten particles were coated also with 200 ng of pRTL2/RFP serving as an internal reference marker to help identify cells that were transformed and, thus, could be examined for the BiFC signal. Alternatively, particles were coated also with 200 ng of pUC18/βATPase-GFP or pRTL2/RFP-MFP serving as a mitochondrial or peroxisomal marker protein, respectively, and for assessing (mitochondrial or peroxisomal) localization of co-expressed Venus half fusion proteins.
Approximately 16 h after bombardment, cells were formaldehyde fixed and either examined (via fluorescence microscopy) for BiFC (and RFP) or processed for immunofluorescence microscopy as described above. The 16 h time point and the amounts of plasmid DNA employed in BiFC assays were chosen based on preliminary optimization experiments aimed at eliminating the possibility of any non-specific (false positive) reconstitutions that may occur due to protein (over)expression [reviewed in
Topological orientations of Venus half fusion proteins were determined using digitonin (differential) permeabilization as described above, with the exception that biolistic bombardments were performed with gold particles (Sigma-Aldrich Ltd.) coated with 5 μg of plasmid DNA, and cells were formaldehyde fixed ~4 h after bombardment.
Rub inoculation of C. quinoa and electron microscopy
CIRV cDNA rub-inoculated
Accession numbers
National Center for Biotechnology Information (NCBI) and/or Arabidopsis Genome Initiative (AGI) database accession numbers for proteins described in this study are as follows: βATPase (P17614), Cb5D (AAT84461), CIRV p36 (CAA59477), CIRV p95 (NP_612580), METAXIN (NP_565446, At2g19080), MFP (AAL35606), mtOM64 (NP_196504; At5g09420), porin (Q9SRH5; At3g01280), TBSV p33 (NP_062898), TOM20 (NP_189344; At3g27080), TOM22 (NP_563699; At1g04070), TOM40 (NP_188634; At3g2000).
βATPase: β subunit of the F1-ATPase; BiFC: bimolecular fluorescence complementation; BY-2: Bright Yellow-2; CAT: chloramphenicol acetyltransferase; Cb5: cytochrome
Authors' contributions
YTH, AWM and SKG conducted the experiments. YTH, AWM and RTM designed and planned the experiments, YTH and RTM were involved in writing the paper, and all authors read, provided critical comments, and approved the final manuscript.
The authors thank Jan Mierynk (University of Missouri-Columbia) for anti-E1β antibodies, Tom Elthon (University of Nebraska-Lincoln) for anti-porin antibodies, Stanton Gelvin (Purdue University) for pSATN BiFC plasmids, Scott Bingham (Arizona State University) for assistance with sequencing, and John Greenwood (University of Guelph) for guidance with electron microscopy. We also thank Kimberley Gibson and Graham Smith for cell culture maintenance, Ian Smith for assistance with designing Figure
Viral RNA replication in association with cellular membranes
Wrapping things up about virus RNA replication
Virus factories: association of cell organelles for viral replication and morphogenesis
The plant viruses: polyhedral virions with monopartite RNA genomes
The role of peroxisomes in viral replication
The molecular biology of
Advances in the molecular biology of tombusviruses: gene expression, genome replication, and recombination
Molecular cloning and complete nucleotide sequence of carnation Italian ringspot tombusvirus genomic and defective interfering RNAs
Cytochemical and autoradiographic observations on tomato bushy stunt virus-induced multivesicular bodies
Origin and function of tomato bushy stunt virus-induced inclusion bodies. II. Quantitative electron microscope autoradiography and immunogold cytochemistry
Immunodetection of the 33K/92K polymerase proteins in cymbidium ringspot virus-infected and in transgenic plant tissue extracts
The tomato bushy stunt virus replicase proteins are coordinately expressed and membrane associated
Ultrastructure and origin of cytoplasmic multivesicular bodies induced by carnation Italian ringspot virus
The 5'-terminal region of a tombusvirus genome determines the origin of multivesicular bodies
Membrane targeting sequences in tombusvirus infections
Expression of a plant virus non-structural protein in
The open reading frame 1-encoded ('36K') protein of carnation Italian ringspot virus localizes to mitochondria
Mitochondrial targeting and membrane anchoring of a viral replicase in plant and yeast cells
Development and application of an
BY-2 cells: culture and transformation for live-cell imaging
Transient expression of fluorescent fusion proteins in protoplasts of suspension cultured cells
Epitope tagging: general method for tracking recombinant proteins
Characterization of a monoclonal antibody directed against the E1α subunit of plant pyruvate dehydrogenase
Effects of turnip crinkle virus infection on the structure and function of mitochondria and expression of stress proteins in turnips
Localization of the tomato bushy stunt virus replication protein p33 reveals a peroxisome-to-endoplasmic reticulum sorting pathway
Oilseed isocitrate lyases lacking their essential type 1 peroxisomal targeting signal are piggybacked to glyoxysomes
Novel targeting signals mediate the sorting of different isoforms of the tail-anchored membrane protein cytochrome
A simple method for displaying the hydropathic character of a protein
An artificial anchor domain: hydrophobicity suffices to stop transfer
Expression of the
The role of the p33:p33/p92 interaction domain in RNA replication and intracellular localization of p33 and p92 proteins of cucumber necrosis tombusvirus
Mitochondrial biogenesis: protein import into and across the outer membrane
Biogenesis of mitochondrial outer membrane proteins
Signal-anchor domains of proteins of the outer membrane of mitochondria: structural and functional characteristics
Characterization of the signal that directs Tom20 to the mitochondrial outer membrane
Bipartite signals mediate subcellular targeting of tail-anchored membrane proteins in
An artificial mitochondrial tail signal/anchor sequence confirms a requirement for moderate hydrophobicity for targeting
A conserved proline residue is present in the transmembrane-spanning domain of Tom7 and other tail-anchored protein subunits of the TOM translocase
Targeting and assembly of mitochondrial tail-anchored protein Tom5 to the TOM complex depend on a signal distinct from that of tail-anchored proteins dispersed in the membrane
Import and insertion of proteins into the mitochondrial outer membrane
Connection of mitochondrial outer and inner membranes by Fzo1 is critical for organellar fusion
Membrane topology and mitochondrial targeting of mitofusins, ubiquitous mammalian homologs of the transmembrane GTPase Fzo
A dynamic machinery for import of mitochondrial precursor proteins
Translocation of proteins into mitochondria
Multiple pathways for sorting mitochondrial precursor proteins
A novel insertion pathway of mitochondrial outer membrane proteins with multiple transmembrane segments
Alternative function for the mitochondrial SAM complex in biogenesis of α-helical TOM proteins
Biogenesis of beta-barrel membrane proteins of mitochondria
Cytosolic factor- and TOM-independent import of C-tail-anchored mitochondrial outer membrane proteins
Integration of tail-anchored proteins into the mitochondrial outer membrane does not require any known import components
Protein import into mitochondria: origins and functions today
Functional definition of outer membrane proteins involved in preprotein import into mitochondria
Translocation and insertion of precursor proteins into isolated outer membranes of mitochondria
MOM22 is a receptor for mitochondrial targeting sequences and cooperates with MOM19
Mitochondrial protein import. Bypass of proteinaceous surface receptors can occur with low specificity and efficiency
Purification and characterization of the preprotein translocase of the outer mitochondrial membrane from Arabidopsis. Identification of multiple forms of TOM20
Mitochondrial protein import: convergent solutions for receptor structure
Convergent evolution of receptors for protein import into mitochondria
Tom22', an 8-kDa trans-site receptor in plants and protozoans, is a conserved feature of the TOM complex that appeared early in the evolution of eukaryotes
A plant outer mitochondrial membrane protein with high amino acid sequence identity to a chloroplast protein import receptor
A transcriptomic and proteomic characterisation of the Arabidopsis mitochondrial protein import apparatus and its response to mitochondrial dysfunction
A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications
Subcellular localization of interacting proteins by bimolecular fluorescence complementation in planta
Identification of new fluorescent protein fragments for bimolecular fluorescence complementation analysis under physiological conditions
Bimolecular fluorescence complementation: visualization of molecular interactions in living cells
Truncated presequences of mitochondrial F1-ATPase beta-subunit from
Identification and characterization of a plastid-localized Arabidopsis glyoxylate reductase isoform: comparison with a cytosolic isoform and implications for cellular redox homeostasis and aldehyde detoxification
Surface plasmon resonance analysis of interactions between replicase proteins of tomato bushy stunt virus
The p36 and p95 replicase proteins of carnation Italian ringspot virus cooperate in stabilizing defective interfering RNA
The C-terminal TPR domain of Tom70 defines a family of mitochondrial protein import receptors found only in animals and fungi
Flock house virus RNA polymerase is a transmembrane protein with amino-terminal sequences sufficient for mitochondrial localization and membrane insertion
Membrane association of greasy grouper nervous necrosis virus protein A and characterization of its mitochondrial localization targeting signal
Targeting of Hepatitis C virus core protein to mitochondria through a novel C-terminal localization motif
Molecular basis for mitochondrial localization of viral particles during Beet Necrosis Yellow Vein virus infection
RNA-dependent RNA polymerase from Atlantic Halibut Nodavirus contains two signals for localization to the mitochondria
Dual subcellular distribution of cytochrome
The C-terminus of cytochrome
Intracellular sorting of the tail-anchored protein cytochrome
Prediction of protein submitochondria locations by hybridizing pseudo-amino acid composition with various physicochemical features of segmented sequence
Analysis and prediction of mitochondrial targeting signals
Arabidopsis mitochondrial proteomics
Experimental analysis of the Arabidopsis mitochondrial proteome highlights signaling and regulatory components, provides assessment of targeting prediction programs, and indicates plant-specific mitochondrial proteins
Mapping the Arabidopsis organelle proteome
Molecular cloning: a laboratory manual
Nuclear transport of plant potyviral proteins
Rapid delivery of foreign genes into plants by direct rub-innoculation with intact plasmid DNA of a tomato bushy stunt virus gene vector
Simultaneous visualization of multiple protein interactions in living cells using multicolor fluorescence complementation analysis
Engineered GFP as a vital reporter in plants
Tung tree DGAT1 and DGAT2 have nonredundant functions in triacylglycerol biosynthesis and are localized to different subdomains of the endoplasmic reticulum
The peroxisomal multifunctional protein interacts with cortical microtubules in plant cells
C-terminal polypeptides are necessary and sufficient for in-vivo targeting of transiently-expressed proteins to peroxisomes in suspension-cultured cells
Purification and biosynthesis of cottonseed (
Developmental expression of the mitochondrial pyruvate dehydrogenease complex in pea (
The visible touch: in planta visualization of protein-protein interactions by fluorophore-based methods
Molecular and cellular approaches for the detection of protein-protein interactions: latest techniques and current limitations
A low-viscosity epoxy resin embedding medium for electron microscopy
Simplified lead citrate stain for use in electron microscopy
Lectin-binding sites as markers of Golgi subcompartments: Proximal to distal maturation of oligosaccharides
Identification of a rice RNA- and MT-binding protein as the multifunctional protein (MFP), a peroxisomal enzyme involved in the β-oxidation of fatty acids


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Jeremy Judd, Database & Research Manager
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On Wed, 23 Jan 2008, isplist logicore net wrote:
I presume you mean Xen, rather than Zen: http://www.cl.cam.ac.uk/research/srg/netos/xen/Sorry, have not had my coffee yet :). Yes, the citrix folks seem to have bought XenSource. Wasn't that the big open source one?
It is still big and it is still OSS. I don't see either of those being affected by the Citrix acquisition.


text:
Posted on 6 April 2015
In case you're not caught up, here are some previous videos of my boyfriend Jake . It's been quite a winding road for us. :)
- The worst part is ...
... that I was sure I was never going to see you again. That they were putting me on ice, waiting for the right moment to thaw me out, and use me against you. For a while I had a, uh, bag packed. I hid it under my bed. I had it all worked out. But John would remind me of what would happen ...
... to you ...
... if I came back.
- Wait here.
- What's that?
- It's you. I mean, I wanted a funeral or memorial or something to say goodbye, but they said that your dad didn't want anything, so Pinstripe just got me that instead. Sorry.
- No, it's fine.You okay?
- Yeah, I just need some water.
- Yeah. Okay, okay. That's fine. We'll get you some water.
- Kay.
- I almost forgot. We can drink it right out of the tap.
- So, you were telling me what you were doing down there?
- Oh, yes. I open up my own black tie piano bar. But I called it "Stella's."
- Of course.
- Which you'd be happy about. You should'a seen the dames.
- Ah. Whew. I can only imagine.
- And when I wasn't running the bar, I was restoring vintage airplanes. I also did a little time as a double secret agent.
- Ah.
- For France.
- Absolutely. On the side. Then why are you here? Livin' the dream.
- No, no. There's only so much paradise one guy can take.
- Oh, yeah.
- I didn't do much, honestly. Um, about half way in, I, uh, actually did book a gig. Um, play and singing at a cantina was basically my food money, because You know, Threadgill paid my rent. I still don't understand why he did that.
- He wanted you alive.
- Any idea why? - Mhm.
For me.
- I can't imagine that guy ever doing anything for anyone else.
- Well, he's gone now.
- What do you mean he's gone?
- Last weekend, at that thing, these two guys came and
put him in handcuffs and took him away and I actually haven't heard from him since.
- I never gave up hope.
- Well, I never knew that ...
... hope was on the table.
- I'm sorry, I didn't --
- No, Jesus, You have nothing to be sorry about. I'm okay.
- What?
- I just ...
... can't believe you're here.
- Stella, let's just leave. Now, let's go anywhere. Start over. We can't have a life here, it's too dangerous. It's too close. To him.
- I know. But I can't. I'm just getting started here.
- With what? Your website? Leading sheep to the slaughter? Stella, you can't --
- Oh, wait, hey hey hey. Look, it's more complicated than that. Okay?
- Look Stella, whatever this thing is, it's not your war.
- It's everybody's war, actually.
- Just come with me.
- I can't. I don't run anymore. And I don't want you to run, either.
- Do you still love me?
- Of course I love you.
I just ...
... also ...
I let you go.
- Wow.
- No.
- No, no, no.
- Jake I didn't mean it like --
- No. I get it. You're right. This is not one of those just ... jump-right-back-into-it- and-act-like-nothing-ever-happened things.
- I wish. Do you remember our third date? You took me to the roof of the physics building.
- Must have been one of your other boyfriends, because I brought you to the English building, but go on.
- Did you? Anyway. That's when I knew. Third date. I have no idea why I'm thinking of that right now.
- So this ...
... makes you feel the way you felt on our third date.
- Oooh, yeah.
- I guess that's cool.
- That sounds WAY less elegant when you put it like that. My way was way better.
- If we're being honest ...
... I knew ...
... as soon as I saw you. I knew.
- Liar.
- No. Stella. Leaving is ...
... it's not a new idea. Not to me. I've always wanted to leave here. Take you with me I can't loose you again.
- Well, um ...
... wherever we go ...
... he will find us.
- So what then?
- Well, we do what we can. We stay here. And fight.