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RICHMOND, VA - The Richmond Flying Squirrels (14-15) posted a season-high 15 runs to shell the Trenton Thunder (16-13), 15-8, in front of 6,762 at The Diamond on Saturday night. The win was the first for the Squirrels against the Thunder this season after dropping the first five meetings of the year between the two clubs.
Trenton jumped out front in the first inning against Richmond lefty Chris Gloor. With one out, Jose Pirela singled into left field. Tyler Austin then singled into center field. Pirela advanced to third base when Ryan Lollis bobbled the ball in center. He scored for a 1-0 lead when J.R. Murphy lofted a sacrifice fly deep into right field.
Richmond answered in bizarre fashion in the bottom of the first inning against Trenton left-hander Francisco Rondon. Javier Herrera led off and reached on an error on first baseman Casey Stevenson. Herrera moved to second base when Joe Panik grounded out. Mark Minicozzi then struck out on a pitch in the dirt. Herrera broke for third and Murphy, the Thunder catcher, threw to third unsuccessfully as Minicozzi reached at first base. Andrew Susac followed and pulled a double just fair up the third-base line and Herrera came in to tie the ballgame, 1-1. An out later, Brett Krill sent a single bouncing into center field. Minicozzi and Susac both scored for a 3-1 Richmond advantage. All three runs in the inning charged to Rondon were unearned.
The Thunder got an unearned run back to close within a run in the third. Pirela reached on an inning-opening error on Panik. Pirela moved to second on a bounce out and scored for the second time in the ballgame when Murphy singled.
The Squirrels immediately responded once again, plating two more against Rondon in the bottom of the third. An error opened the door again, as Susac started the frame by reaching on an error on shortstop Reegie Corona. A two-out walk to Krill allowed Jarrett Parker to bat. He delivered a two-run double to deep left field to extend the Squirrels' lead to 5-2.
Richmond briefly lost the lead in the top of the fourth inning. With Ramon Flores aboard and one out, Pirela tripled off the wall in center field. Austin followed with a two-run home run to left field that tied the ballgame, 5-5.
For the third time, the Squirrels answered back in a hurry. And the third time was the most emphatic. Richmond grabbed ahold of the ballgame with five runs to finally knock Rondon from the ballgame. Travious Relaford started the inning and took a walk from Rondon. Lollis singled into right to put two aboard and Herrera worked an 11-pitch walk to load the bases. Panik flew out, but with Minicozzi batting, Relaford scored on a wild pitch. Minicozzi then stung a two-run double into the right-field corner. An out later, Ricky Oropesa punctuated the uprising with a massive two-run home run to right field. Oropesa's second home run of the season gave Richmond a 10-5 lead.
Oropesa's blast also chased Rondon (2-2), as he departed to the loss over 3 2/3 innings. He was tagged for 10 runs - five earned - on six hits. He walked three and struck out three, suffering his second loss of the year.
Gloor (1-1) worked five innings for his first victory of the season. He surrendered five runs - four earned - on eight hits. He walked one and struck out a season-high seven batters.
Richmond lefty Tom Vessella balked in a run in the top of the sixth inning, leading to the ejection of Squirrels manager Dave Machemer. His departure ignited the Squirrels, as they once against immediately answered at the plate. Herrera doubled to start the inning and Panik followed with a single. After Panik swiped second base, Minicozzi laced his second two-run double of the night. The shot through the alley in right-center put the Squirrels in front, 15-6.
Parker capped the scoring for the Squirrels when he tripled to score two more Richmond runs in the bottom of the eighth inning. The final five runs scored by the Squirrels were charged to Trenton lefty Shaeffer Hall. The Thunder picked up two runs in the top of the ninth inning to alter the final margin. Vessella and right-hander Jose Valdez combined to finish the ballgame in relief for Richmond.
Minicozzi and Parker each finished with four RBIs in the ballgame, while three Squirrels (Herrera, Minicozzi, Susac) each scored three runs. In addition to the setting a season-high for runs, the Squirrels also established a season-high for hits (14).
The finale of the three-game series with the Trenton Thunder (New York Yankees) is scheduled for 2:05 p.m. at The Diamond on Sunday afternoon. Richmond RHP Justin Fitzgerald (2-0, 1.38) will make the start against Trenton RHP Caleb Cotham (1-1, 4.30). Pregame coverage begins on Sports Radio 910, SportsRadio910.com and IHeart Radio at 1:35 p.m.
Today on MiLB.com
Top Stories on MiLB.com
- With Tebow on roster, Fireflies glow brighter
- Nationals' Fedde outduels Mets' Gsellman
- Hoffman, Bauers heading back to Minors
- Astros Prospect Primer: Martes at the top
- Tigers Prospect Primer: Stewart slugs on
- Phillies' Pivetta talks WBC on podcast
- Royals Prospect Primer: Arms in focus
- Roundup: Sisco makes difference for Orioles


text: Nuclear Slide Rules: The Old Fashioned Way To Calculate ArmageddonSean Fallon12/16/08 1:30pmFiled to: RetromodoNuclear slide rulesNukesArmageddonCalculators18EditPromoteShare to KinjaToggle Conversation toolsGo to permalink In an age when nukes existed but pocket calculators did not, the potential damage of a nuclear strike could be quantified using cardboard slide rules like this one. Advertisement Using data gleaned from the book The Effects of Nuclear Weapons, these circular "computers" could calculate weapons effects like yield and range when the big one dropped. The instrument pictured here is circa 1960, and is one of several pictured in a gallery on the Oak Ridge Associated Universities website. [ORAU via Dinosaurs and Robots via Boing Boing]Reply18 repliesLeave a reply

subset: commoncrawl


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Take a look for yourself and post up your thoughts.
Drinking beer I enjoy for good time and make relaxation for everyone. Disney World is my land favorite time for me. Horror movies? Yes. That's me for a nutshell.
One Thursday a month, Sophie lays out the rules for a horror film drinking game! Browse our past entires and be on the look out for new ones.
Tag along as our spooky patriots give you a tour of the greatest horror settings from around the U-S-of-A
Isn't it neat how everyone's tastes are different? Check out which spooky flicks our crew thought were the best, and worst, in 2016.
Whether you're a dusty Baby Boomer or a filthy Millenial, you'll no doubt appreciate Andrew's look back into the best horror TV shows since the 1950's


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2006 Baseline/The Hackett Group ROI Awards
By Kim S. Nash | Posted 2006-09-05 Email PrintThe winning projects in the Baseline/The Hackett Group ROI Leadership Awards pulled in relatively quick returns with hosted, Web-based systems.
What's the measure of success? Call us old-fashioned, but we think an information-technology system should deliver more value to an organization than it costs to put in and maintain. That's the classic definition of return on investment, and it was the key metric used to select the winners of the 2006 Baseline/The Hackett Group ROI Leadership Awards.All entrants submitted detailed, three-year cost and return information on a significant information systems project. In addition, this year we required that the results be verified by the company's finance department. The entries were then analyzed and validated by The Hackett Group, a business and technology research and consulting firm based in Atlanta.
The winner: The U.S. unit of camera maker Nikon, which saw major
benefits with a customer relationship management project that yielded
a plump 3,203% return. We also selected a solid runner-up, Kennametal. The industrial tools manufacturer used an e-procurement
system to encourage employees to buy from preferred suppliers, saving
an estimated $2 million over three years. Kennametal's three-year ROI: 161%.
Download Baseline's tool to calculate the ROI of your own I.T. projects
Both companies, notes Erik Dorr, senior business adviser at The Hackett Group, "utilized Web-based technologies, delivered benefits relatively quickly and involved process transformation rather than just automation."
Because the projects were geared around hosted applications, Nikon and Kennametal had relatively low startup costs and, as a consequence, saw fairly swift ROI.
"When you have an on-demand solution, your monthly fees are far less than if you bring a full ERP [enterprise resource planning] system in behind your firewall," says Jim Cebula, Kennametal's director of global purchasing and travel, who oversaw the e-procurement project. "Our infrastructure support cost
is Internet access. We have no I.T. depreciation at all."
Of course, not every software-as-a-service project will be a winner. But if a project isn't working out the way a company expectedif, say, you're heading into negative-ROI territoryit's less painful to pull the plug on a hosted application.
Read about the 2006 winning projects:
Read about previous years' winners:
How We Picked the Winners
All entries were independently judged for accuracy, validity and completeness by The Hackett Group, a strategic advisory firm that provides best-practices research, benchmarking and business-transformation services. The company, a subsidiary of Answerthink, specializes in analyzing performance of sales, general and administrative and supply chain activities, and has published 3,500 benchmark studies over 14 years.
How Outdated Technology Impedes Busin...
Only a minority of technology and business leaders believe that the...Watch Now


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Mineral makeup has become popular for many reasons: It's eco-friendly, looks good, and feels light on the face. And because TV infomercials and the celebrities who use and recommend it, mineral makeup has gotten a lot of media attention.
"Mineral makeup has been around for 30 years or more, but has recently regained popularity in the cosmetics industry," says Scott Gerrish, MD, of Gerrish and Associates, PC, a nonsurgical skin care specialist with offices in Virginia and Maryland. "Mineral makeup was originally used by plastic surgeons and dermatologists on patients after cosmetic procedures to cover the redness and soreness."
Is mineral makeup right for you? Read on to find out.
The Magic Behind Mineral Makeup
Mineral makeup is made from pure, crushed minerals and will not cake on the skin: It allows the skin to breathe and gives you a lighter, more natural look than traditional makeup. "Mineral makeup comes in powdered, pressed, and liquid forms and has beneficial properties for your skin," says Helga Surratt, President of About Faces Day Spa & Salon, in Towson, Md.
It is ideal for all skin types, all skin tones, and women of all ages. Mineral makeup looks great, feels great, and helps to bring out your natural glow. "But take care to read the labels and make sure you're getting pure mineral makeup," Surratt says.
Why Mineral Makeup May Be Better
- Mineral makeup won't clog pores or irritate. As Pam Messy of Mary Kay Cosmetics in Owings Mills, Md., says, "Regular makeup contains artificial chemicals or preservatives, whereas true mineral makeup does not. Mineral makeup is hypoallergenic and usually safe to use on any skin." It's also free of oil, talc, perfume, dyes, alcohol, and other potentially irritating and comedogenic, or pore-clogging, ingredients, Surratt says.
- Mineral makeup ingredients can soothe skin. What it does contain are natural anti-inflammatory ingredients, such as zinc and titanium oxides, which help calm the skin when it's irritated. These ingredients also offer protection from UVA, UVB, and infrared sun rays. "Read the label, as you want at least an SPF of 15," Surratt says.
- Mineral makeup acts as a great concealer. Need to hide imperfections? Mineral makeup is lightweight and conceals, corrects, and covers pigmentations and lines on your face while still allowing your skin to breathe. "It offers skin-enhancing benefits. It smoothes the appearance of fine lines and wrinkles, conceals blotchiness, and improves the appearance of skin with acne or rosacea," Gerrish says. Mineral makeup doesn't need many touch-ups because it has water-resistant qualities that provide long-lasting coverage, another plus.
"Pure mineral makeup is so harmless you can almost sleep in it," says Messy, "though I always recommend removing all makeup before going to bed, and applying a good moisturizer."
While no form of makeup is perfect, mineral makeup products can help you avoid harsh preservatives and chemicals while hiding those fine lines and little flaws. You may never go back to regular makeup because of the way mineral makeup looks and feels on your face.
Learn more in the Everyday Health Skin and Beauty Center.


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Ben Hecht , was a Broadway playwright and prolific Hollywood screenwriter, even though he professed disdain for the motion picture industry. He was nominated six times for the Academy Award, winning twice, in 1929 and in 1936.
More on Wikipedia…
(29 quotes found)
"Hollywood held this double lure for me, tremendous sums of money for work that required no more effort than a game of pinochle."
Ben Hecht
"In moderating, not satisfying desires, lies peace."
"In the court of the movie Owner, none criticized, none doubted. And none dared speak of art. In the Owner's mind art was a synonym for bankruptcy. The movie Owners are the only troupe in the history of entertainment that has never been seduced by the adventure of the entertainment world."
"In Hollywood, a starlet is the name for any woman under thirty who is not actively employed in a brothel."
"I lost my own son (Steven) at 18, five years ago, in a car accident, so I can really sympathize with what the families have gone through."
"We are helping to make a market of low-income people, but making sure that the market is one these people can participate in, that the costs are affordable. And once they're online, there is something to do online, a destination where they can go and improve their lives."
"We are going to make sure that there is high-speed broadband available to everyone, and affordable to people historically kept out of it. It means they have not just access, but can also understand how to buy low-cost, affordable computers."
"Once you're online and you see its power, you never go back. What we want to make sure of is that the nation has 21st century infrastructure for information for low-income people."
"When you overpay small people you frighten them. They know that their merits or activities entitle them to no such sums as they are receiving. As a result their boss soars out of economic into magic significance. He becomes a source of blessings rather than wages. Criticism is sacrilege, doubt is heresy."


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This month, Cinema Asian America on Xfinity On Demand presents Duc Nguyen's Emmy Award-winning documentary, Bolinao 52, a powerful account of the experiences of Vietnamese boat people refugees, and of one boat in particular, whose tragic circumstances magnified the challenges facing those displaced by the Vietnam War.
Richard Threlkeld, Newsman at CBS and ABC, Dies in Car Accident
Richard Threlkeld, veteran TV newsman who worked at ABC and CBS, died Friday following a car crash in Long Island, N.Y. He witnessed and reported on countless big stories in a long, distinguished career in network news.
Ken Burns' Next Project for PBS: Vietnam War
NEW YORK – Having already done the Civil War and World War II, Ken Burns is working on a documentary about the Vietnam War. PBS said the 10- to 12-hour film by Burns and longtime partner Lynn Novick will be broadcast in 2016. Burns said his film will tell the human stories of Americans and […]


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Reform the filibuster. Do it now, before the new Congress is sworn in.
And then get on to something equally important
Also at the top of the list
Operationalizing ACA. Will McCrory collaborate to improve health for NC or will he turn his back on those most in need? I'm betting the latter.


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(Adds details, background)
BEIRUT, July 11 (Reuters) - At least two rockets were fired on Friday from southern Lebanon into Israel, Lebanon's news agency and security sources said, adding that it was unclear who had fired them.
Southern Lebanon is a stronghold of Hezbollah, a Shi'ite Muslim group that battled Israel seven years ago and is engaged in Syria's civil war in support of President Bashar al-Assad. Palestinian factions are also in the same area.
The news agency NNA, citing its correspondent in Tyre, said the rockets were fired towards "occupied territories" at 6.30 a.m. (0430 GMT) from the Hasbaya area in Lebanon.
Israel responded with military shells, security sources said.
In December, rockets launched from south Lebanon struck northern Israel, provoking a response across a border that had been largely quiet since a war in 2006. (Reporting by Sylvia Westall; Editing by Paul Tait; Editing by Clarence Fernandez)


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See Things Differently: Introducing Our First Guest Instagrammer
In December 2011, we launched the Chronicle Books Instagram feed. The first post was a blurry, low-lit snapshot of our catalog archive wall here in the office. (Oh, how the iPhone camera has improved since the 3G!) Since then, our photo stream has taken followers behind the scenes at Chronicle HQ, documented life in San Francisco, offered sneak peeks of books fresh off the press, and even traveled across the country to conferences, shops and trade shows.
With each Instagram that I post, my aim is to capture that special way of looking at the world that we cultivate here at Chronicle. Our company motto is "See Things Differently," and each day, in every corner of the company, we strive to find the unexpected possibilities, creative potential, and distinctiveness in everything we do. It's this perspective, this way of seeing, that enables us to consistently create beautiful books, stationery, and gifts.
Now, 62 weeks and 418 photos later, I'm handing over the reins of our Instagram feed, temporarily. One week each month, we'll be inviting a guest instagrammer to take over the Chronicle feed. These guest igers will be fellow employees, authors, illustrators, artists, and others who share that uniquely Chronicle way of looking at the world. Each month, I'll introduce them here on the blog, and you'll always be able to tell a guest post on our Instagram feed by the #SeeThingsDifferently tag followed by their personal handle.
So without further ado, let me introduce you to Benjamin Kasman, our first guest instagrammer. Ben's an avid iger with a great eye, a connoisseur of fine food and drink, and an instrumental lead in our production department. He travels the globe sourcing forest-friendly paper and sniffing out ink to ensure that every book we publish is gorgeous. Through his lens, I hope you'll get a new perspective on Chronicle Books, and see things a little differently.
Name: Benjamin Kasman
Instagram Handle: @millionairehobo
Role at Chronicle Books: Production Lead, Business Development
Your favorite photo that you've Instagrammed: http://instagram.com/p/M17kI8E1ZA/
Your favorite filter: Rise or Mayfair
Your favorite word: dilly dally
Your favorite Chronicle Book: Hoshino's Alaska
Your idea of happiness: omakase at an amazing sushi restaurant
Where you would like to live: I live in my favorite place, San Francisco. Second choice: Bora Bora
Finish this sentence: I can't stop taking photos of… the Pacific Ocean
Thank you, Ben! Follow @ChronicleBooks on Instagram or online at instagram.com/ChronicleBooks to see his photos all week long.
Guinevere de la Mare
Senior Community Manager
Guinevere de la Mare
Latest posts by Guinevere de la Mare (see all)
- When Grumpy Cat Gives You Advice, Duck - July 9, 2014
- Put an Egg On It - July 8, 2014
- Friday Reads: Always Emily - June 27, 2014
How This Flower Bouquet Pop-Up Book Was DesignedMarch 14th, 2017
An Interview with Nicole LaRue, Designer of the Women's March LogoMarch 8th, 2017
Stationery for People Who Say F*ck a LotFebruary 23rd, 2017
Grab a Slice of These Pizza Party NotecardsFebruary 20th, 2017
The Chronicle Books Design Fellowship: Poster Design + ProcessJanuary 24th, 2017


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GFive K777 Specifications
Size109 x 48 x 12 mm
Weight70 g
Screen240 x 320 px
Networks850 / 900 / 1800 / 1900
MP3 PlayerYes
Memory CardmicroSD
Java SupportYes
Radio SupportYes
Date AddedSeptember 24th, 2010
Disclaimer: We cannot guarantee that all phone specifications are correct. All GFive K777 content is created and uploaded by the users. We cannot gurantee that all of the content and Zedge services will work 100% on your GFive K777 handset as it depends on operator and network conditions, including but not limited to, your WAP/GPRS services and data plans.


text: Lausanne, Switzerland, April 8, 2011 – The International Volleyball Federation (FIVB) Board of Administration confirmed on Friday that Poland would host the ninth edition of the premier beach volleyball event, the World Championships, due to be held in 2013.The FIVB Beach Volleyball World Championships were first held in Los Angeles, USA in 1997 and this year brings the eighth edition, coming up in Rome, Italy from June 13-19.Poland has an extensive history of hosting volleyball events and will stage the 2011 FIVB Volleyball World League Finals from July 6-10 in Gdansk-Sopot, as well as the 2014 Men's Volleyball World Championship."With the standing of volleyball in Poland at the moment at an extremely high level we are confident the FIVB Beach Volleyball World Championships in 2013 will be an outstanding success," FIVB Beach Volleyball World Tour council president and board member Dr Ary Graça said. "We are looking forward to carrying on the outstanding tradition of world-class beach volleyball events in Poland in 2013.""We are grateful to the FIVB for the trust and confidence they have shown in our ability to host the premier FIVB beach volleyball property," Polish Volleyball Federation president and Executive Committee co-opted member Mr Miroslaw Przedpelski said."Volleyball in Poland is at an all-time high and with three key international events in four years, volleyball fans in Poland and right around the world are going to have a fantastic experience," he added.


text: I was having 4 hot sushi which included salmon and hot chilli sauce, and another 4 crab sushi with avocado. In total, taste was really good, fulfill and rich. And customer service was really nice. This is my last night in faro, so still have some money left on eating, so I decided this restaurant, but it still a little... More


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Just received the D4 I ordered on 5/31 from best buy. They came through and two days faster than promised. Serial 202444x.
Thanks NR for the tip that let me place my order. Came with xqd card and reader.
My Adorama order for the d4 was placed on january 20. B&H shipped my d800e on 4/26, I ordered that one at 00:00:04 on 2/7.
received order placed at best buy
(2 posts) (2 voices)-
Posted 4 years ago #
Maonua, I'm glad to hear it. I'm closing this thread because there are other threads open about D800/D800e orders.
Posted 4 years ago #
Topic Closed
This topic has been closed to new replies.


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Bio (Phillip Vannini )
Phillip Vannini
Communication and Culture
Phillip Vannini is an accomplished ethnographer and author who studies subjects such as marine mobilities, off-grid energy assemblages and the social aspects of human embodiment. Vannini's research interests broadly include material culture, technology and culture, sensory studies and cultural geographies. He maintains a website to share his ethnographic work and that of his students. In 2011, Vannini published Ferry Tales, a hypermedia book that explores mobility and sense of place and time on the British Columbia coast. In November 2014, he published Off the Grid: Re-Assembling Domestic Life, the culmination of two years of research into the lives of people across Canada who live off the grid. He also collaborated on a documentary film based on his research, Life off-grid.
Vannini joined Royal Roads University in 2005 and became professor in 2011. He primarily teaches courses on research methodology, fieldwork and cultural studies. He particularly enjoys supervising theses that make use of visual media and push the boundaries of ethnographic creativity. He has authored/edited nine books and dozens of journal articles and book chapters. He is editor of the world's first hypermedia ethnography book series. Learn more about Vannini's experience on his Academia.edu profile.
Vannini started his bachelor's degree at the University of Florence and completed it at City University of Seattle. He earned his MA in communication and his PhD in Sociology from Washington State University. In 2007 and 2010, he was nominated for Royal Roads' Kelly Outstanding Teaching Award. In 2010, he was appointed Canada Research Chair in Innovative Learning and Public Ethnography by the Social Sciences and Humanities Research Council.


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Comment Re:Duality of Wozniak's Apple Versus Jobs' Apple (Score 1) 789
If we were "everyone else" we wouldn't be the ones people run to so we can explain to them which end of the USB cable is which.
Apple is successful in good part because "everyone else" is heartily sick of having to rely on people like you. It's hardly surprising they prefer computing devices they can use without needing the help of a techie.
If Apple products aren't for you, so what? Ignore Apple and just buy your stuff from other manufacturers.
When you tell me what "everyone else" wants, it doesn't exactly make me want to run out and buy it so I can be like "everyone else".
Yes, I hear that from a few people. They want to be special by not following the trend. But to not buy a product just because everyone else buys it is pretty much the same emotional behaviour buying something because everyone else has it. Fashion and anti-fashion.


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What book do you wish you were reading right now?
Where would you take it to, if you could go anywhere to read for a while?
I actually wish that I was reading just what I am reading currently... Elantris by Brandon Sanderson.
Granted, I'm reading a couple other books right now as well (Leviathan Wakes is very good!), but I can't think of anything else I'd rather be reading at the moment than what I am reading.
If I could be anywhere, I'd want to be sitting outside in a comfy chair under a shady tree with a glass of iced tea.
All I need is a chair... :D
Monday, March 12, 2012
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This showcases the earthiness of both the duck and the chard. The pumpkin butter adds the right amount of sweetness and aromatics to round it out. Serve with oven roasted root vegetables and wild rice. Paillards are simply 'thinly pounded pieces'. When pounded thin like this, it cooks in no time. It also tenderizes. I shared the recipe for pumpkin butter yesterday. I learned how it is a Southern favourite when I was in Tennessee last year. Now I am a convert.
Chard Wrapped Paillards of Duck serves 2
- 1 whole boneless duck breast
- 1/2 teaspoons salt
- 1 teaspoon ancho chili powder
- 1/2 teaspoon black pepper
- 2 large chard leaves with ribs removed
Pull skin off duck.
Remove the rib from each chard leaf and lay out with dull side up. Place the duck paillard on a piece of chard. Sprinkle generously with ancho chili powder. Roll up tightly and place in a greased ovenproof dish. Repeat with the other half of duck breast. Bake at 350F for 10-15 minutes, depending on your taste.


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The Drosophila nicotinic acetylcholine receptor subunits Dα5 and Dα7 form functional homomeric and heteromeric ion channels
- Stuart J Lansdell1,
- Toby Collins1,
- Jim Goodchild2 and
- Neil S Millar1Email author
© Lansdell et al.; licensee BioMed Central Ltd. 2012
Received: 19 January 2012
Accepted: 17 May 2012
Published: 22 June 2012
Nicotinic acetylcholine receptors (nAChRs) play an important role as excitatory neurotransmitters in vertebrate and invertebrate species. In insects, nAChRs are the site of action of commercially important insecticides and, as a consequence, there is considerable interest in examining their functional properties. However, problems have been encountered in the successful functional expression of insect nAChRs, although a number of strategies have been developed in an attempt to overcome such difficulties. Ten nAChR subunits have been identified in the model insect Drosophila melanogaster (Dα1-Dα7 and Dβ1-Dβ3) and a similar number have been identified in other insect species. The focus of the present study is the Dα5, Dα6 and Dα7 subunits, which are distinguished by their sequence similarity to one another and also by their close similarity to the vertebrate α7 nAChR subunit.
A full-length cDNA clone encoding the Drosophila nAChR Dα5 subunit has been isolated and the properties of Dα5-, Dα6- and Dα7-containing nAChRs examined in a variety of cell expression systems. We have demonstrated the functional expression, as homomeric nAChRs, of the Dα5 and Dα7 subunits in Xenopus oocytes by their co-expression with the molecular chaperone RIC-3. Also, using a similar approach, we have demonstrated the functional expression of a heteromeric 'triplet' nAChR (Dα5 + Dα6 + Dα7) with substantially higher apparent affinity for acetylcholine than is seen with other subunit combinations. In addition, specific cell-surface binding of [125I]-α-bungarotoxin was detected in both Drosophila and mammalian cell lines when Dα5 was co-expressed with Dα6 and RIC-3. In contrast, co-expression of additional subunits (including Dα7) with Dα5 and Dα6 prevented specific binding of [125I]-α-bungarotoxin in cell lines, suggesting that co-assembly with other nAChR subunits can block maturation of correctly folded nAChRs in some cellular environments.
Data are presented demonstrating the ability of the Drosophila Dα5 and Dα7 subunits to generate functional homomeric and also heteromeric nAChRs.
Nicotinic acetylcholine receptors (nAChRs) are excitatory neurotransmitter receptors that are found in both vertebrate and invertebrate species. In insects, nAChRs are expressed throughout the nervous system and are the site of action for economically important insecticides such as spinosyns and neonicotinoids [1, 2]. Detailed information is available concerning the structure of nAChRs, as a consequence of studies conducted with receptors purified from the electric organ of the marine ray Torpedo[3] and from X-ray crystallographic studies conducted with nAChR fragments [4] and also with the closely related acetylcholine binding protein [5]. Nicotinic receptors are assembled from five subunits arranged around a central cation-selective pore [6, 7]. Conventional agonists, such as acetylcholine, activate the receptor by binding at an extracellular site located at the interface between two subunits [8], although recent evidence indicates that nAChRs can also be activated by ligands binding to an allosteric transmembrane site [9].
Ten nAChR subunits (Dα1-Dα7 and Dβ1-Dβ3) have been identified in the model insect Drosophila melanogaster and a similar number of subunits have been identified in other insect species [1, 2]. Despite considerable efforts, there has been only limited success in expressing insect nAChRs in artificial expressions systems [10, 11] and, where functional expression has been achieved, ion channel currents have tended to be small or have been generated in response to relatively high agonist concentrations [12–14]. Experimental approaches that have had some success in overcoming problems associated with expression of insect nAChRs include the expression of subunit chimeras containing domains from other neurotransmitter receptors [15], co-expression of insect nAChRs with vertebrate subunits [16, 17] or a combination of these approaches [18]. Co-expression with vertebrate nAChR subunits is an approach that has been used in the characterization of nAChR subunits cloned from insect pest species such as the aphid Myzus persicae[19, 20] and the brown planthopper Nilaparvata lugens[21, 22]. However, for most insect species for which nAChRs have been cloned, there have been no reports of successful heterologous expression. This includes nAChRs cloned from the honeybee Apis mellifera[23–25], diamondback moth Plutella xylostella[26, 27], house fly Musca domestica[28–30], locust Locusta migratoria[31], mosquito Anopheles gambiae[25], red flour beetle Tribolium castaneum[25, 32], silkworm Bombyx mori[25, 33] and tobacco hornworm Manduca sexta[34].
RIC-3 is a nAChR-associated molecular chaperone that was originally characterised in the nematode Caenorhabditis elegans[35] but has also been identified in several other species, including mammals and insects [36]. It is a transmembrane protein that is able to enhance maturation (folding and assembly) of several nAChR subtypes [36]. For example, co-expression of RIC-3 with the vertebrate nAChR α7 subunit enhances levels of functional expression in Xenopus oocytes [35] and is able to facilitate the functional expression of α7 nAChRs in mammalian cell lines that are otherwise non-permissive for expression of α7 [37, 38]. In some cell types it has been found that the α7 subunit can be expressed (subunit protein can be detected) but, in the absence of RIC-3, is unable to fold into a conformation that can be detected by radioligand binding or form functional nAChRs [37, 38]. In addition, some success has been achieved in overcoming difficulties associated with expression of insect nAChRs by the co-expression with RIC-3 [39, 40].
The Dα5, Dα6 and Dα7 subunits of Drosophila show close sequence similarity to one another (53-63% amino acid identity [41]) and also have close similarity to the vertebrate nAChR α7 subunit (42-46% amino acid identity [42]). Of the three Drosophila subunits, Dα5 and Dα7 have the closest sequence similarity to one another and Dα6 has the highest sequence similarity to the vertebrate α7 [43]. In the present study, we report the molecular cloning of the Dα5 subunit, the only Drosophila nAChR subunit for which a full-length cDNA clone was not previously available in our laboratory. Heterologous expression studies with Dα5, Dα6 and Dα7 are described in three host cell types: Drosophila S2 cells, human tsA201 cells and Xenopus oocytes. Functional expression of several subunit combinations has been achieved in Xenopus oocytes and has enabled the pharmacological properties of recombinant nAChRs to be examined. Evidence is provided that demonstrates the ability of subunits to form both homomeric and heteromeric nAChRs. Of particular note is evidence that Dα5 can generate functional homomeric channels and that Dα7 can form both homomeric and heteromeric channels. We are not aware of any previous studies demonstrating the ability of Dα5 and Dα7 subunits to generate such recombinant nAChRs, either with subunits cloned from Drosophila or with analogous nAChR subunits from other insect species.
Molecular cloning of Dα5
A full-length cDNA encoding the Drosophila nAChR Dα5 subunit was isolated from a preparation of Drosophila embryo mRNA. The Dα5 cDNA encodes an open reading frame of 807 amino acids corresponding to the previously described Dα5 isoform B [41]. In agreement with previous studies [41], the Dα5 cDNA isolated in this study contains an open reading frame encoding an unusually large N-terminal domain, extending some 300 amino acids upstream of the start methionine in most nAChR subunits.
Heterologous expression of Dα5 in Drosophila and human cell lines
Radioligand binding to Drosophila nAChR subunit combinations
Subunit combination | [125I]-α-BTX Binding | [3 H]-epibatidine binding | ||
Dα5 | – | – | – | – |
Dα5/Dα1 | – | – | – | – |
Dα5/Dα2 | – | – | – | – |
Dα5/Dα3 | – | – | – | – |
Dα5/Dα4 | – | – | – | – |
Dα5/Dα6 | – | + | – | – |
Dα5/Dα7 | – | – | – | – |
Dα5/Dβ1 | – | – | – | – |
Dα5/Dβ2 | – | – | – | – |
Dα5/Dβ3 | – | – | – | – |
Dα5/Dα1/Dα2 | – | – | – | – |
Dα5/Dα1/Dβ1 | – | – | – | – |
Dα5/Dα1/Dβ2 | – | – | – | – |
Dα5/Dα1/Dβ3 | – | – | – | – |
Dα5/Dα2/Dβ1 | – | – | – | – |
Dα5/Dα2/Dβ2 | – | – | – | – |
Dα5/Dα2/Dβ3 | – | – | – | – |
Dα5/Dα3/Dβ1 | – | – | – | – |
Dα5/Dα3/Dβ2 | – | – | – | – |
Dα5/Dα3/Dβ3 | – | – | – | – |
Dα5/Dα4/Dβ1 | – | – | – | – |
Dα5/Dα4/Dβ2 | – | – | – | – |
Dα5/Dα4/Dβ3 | – | – | – | – |
Dα5/Dα6/Dα7 | – | – | – | – |
Dα5/Dβ1/Dβ2 | – | – | – | – |
Dα5/Dβ1/Dβ3 | – | – | – | – |
Dα5/Dβ2/Dβ3 | – | – | – | – |
Dα5/Dα1/Dα2/Dβ1 | – | – | – | – |
Dα5/Dα1/Dα2/Dβ2 | – | – | – | – |
Dα5/Dα1/Dα2/Dβ3 | – | – | – | – |
Dα5/Dα1/Dα3/Dβ1 | – | – | – | – |
Dα5/Dα1/Dα3/Dβ2 | – | – | – | – |
Dα5/Dα1/Dα3/Dβ3 | – | – | – | – |
Dα5/Dα1/Dα4/Dβ1 | – | – | – | – |
Dα5/Dα1/Dα4/Dβ2 | – | – | – | – |
Dα5/Dα1/Dα4/Dβ3 | – | – | – | – |
Dα5/Dα2/Dα3/Dβ1 | – | – | – | – |
Dα5/Dα2/Dα3/Dβ2 | – | – | – | – |
Dα5/Dα2/Dα3/Dβ3 | – | – | – | – |
Dα5/Dα2/Dα4/Dβ1 | – | – | – | – |
Dα5/Dα2/Dα4/Dβ2 | – | – | – | – |
Dα5/Dα2/Dα4/Dβ3 | – | – | – | – |
Dα5/Dα3/Dα4/Dβ1 | – | – | – | – |
Dα5/Dα3/Dα4/Dβ2 | – | – | – | – |
Dα5/Dα3/Dα4/Dβ3 | – | – | – | – |
Dα5/Dα6/Dα7/Dβ1 | – | – | – | – |
Dα5/Dα6/Dα7/Dβ2 | – | – | – | – |
Dα5/Dα6/Dα7/Dβ3 | – | – | – | – |
Dα5/Dβ1/Dβ2/Dβ3 | – | – | – | – |
Dα5/Dα1/Dα2/Dα3/Dβ1 | – | – | – | – |
Dα5/Dα1/Dα2/Dα3/Dβ2 | – | – | – | – |
Dα5/Dα1/Dα2/Dα3/Dβ3 | – | – | – | – |
Dα5/Dα1/Dα2/Dβ1/Dβ2 | – | – | – | – |
Dα5/Dα1/Dα3/Dβ1/Dβ2 | – | – | – | – |
Dα5/Dα1/Dα4/Dβ1/Dβ2 | – | – | – | – |
Dα5/Dα2/Dα3/Dβ1/Dβ2 | – | – | – | – |
Dα5/Dα2/Dα4/Dβ1/Dβ2 | – | – | – | – |
Dα5/Dα3/Dα4/Dβ1/Dβ2 | – | – | – | – |
Dα5/Dα1/Dα2/Dβ2/Dβ3 | – | – | – | – |
Dα5/Dα1/Dα3/Dβ2/Dβ3 | – | – | – | – |
Dα5/Dα1/Dα4/Dβ2/Dβ3 | – | – | – | – |
Dα5/Dα1/Dβ1/Dβ2/Dβ3 | – | – | – | – |
Dα5/Dα2/Dα3/Dβ1/Dβ2 | – | – | – | – |
Dα5/Dα2/Dα4/Dβ1/Dβ2 | – | – | – | – |
Dα5/Dα2/Dβ1/Dβ2/Dβ3 | – | – | – | – |
Dα5/Dα3/Dα4/Dβ1/Dβ2 | – | – | – | – |
Dα5/Dα3/Dβ1/Dβ2/Dβ3 | – | – | – | – |
Dα5/Dα1/Dα2/Dα3/Dα4/Dβ1 | – | – | – | – |
Dα5/Dα1/Dα2/Dα3/Dα4/Dβ2 | – | – | – | – |
Dα5/Dα1/Dα2/Dα3/Dα4/Dβ3 | – | – | – | – |
Dα5/Dα6/Dα7/Dβ1/Dβ2/Dβ3 | – | – | – | – |
Dα5/Dα1/Dα2/Dα3/Dα4/Dα6/Dα7/Dβ2 | – | – | – | – |
Dα5/Dα1/Dα2/Dα3/Dα4/Dα6/Dα7/Dβ1/Dβ2 | – | – | – | – |
Dα5/Dα1/Dα2/Dα3/Dα4/Dα6/Dα7/Dβ1/Dβ2/Dβ3 | – | – | – | – |
Previous studies have shown that, when co-expressed with a vertebrate β2 subunit, some Drosophila nAChR α-subunits can generate functional recombinant nAChRs and form high-affinity binding sites for nicotinic radioligands (see for example [16, 44]). However, when Dα5 was co-expressed with vertebrate β2 in Drosophila S2 cells or in human tsA201 cells, no specific radioligand binding could be detected. These findings with Dα5 are similar to those conducted previously with the closely related Drosophila Dα6 and Dα7 subunits [15]. However, in control experiments conducted in parallel, high levels of specific radioligand binding were detected after co-expression of Drosophila Dα2 and Dα3 subunits with the rat β2 (Rβ2) subunit. This is in agreement with previous studies conducted with the Dα2 + Rβ2 and Dα3 + Rβ2 subunit combinations [17, 45].
Dα5/5HT3A subunit chimera
The influence of co-expressing combinations of subunit chimeras was also examined. In comparisons to the level of 125I]-α-bungarotoxin binding detected with Dα5/5HT3A alone, higher levels of specific cell-surface binding were detected when the Dα5 chimera was co-expressed with other subunit chimeras (Dα6/5HT3A and Dα7/5HT3A; Figure 1). However, the levels of specific binding detected were not significantly higher than would have been expected from a possible additive effect of co-expressing these chimeras. Consequently, this data cannot be used as evidence to support the possibility of heteromeric co-assembly, as was the case previously for studies conducted with the Dα6 and Dα7 subunit chimeras [15].
Heterologous expression with RIC-3
Expression in Xenopus oocytes
Functional properties of recombinant nAChRs expressed in Xenopus oocytes
Subunits | EC 50(μM or nM)* | Hill slope | n ** | I max[ I mean] † (nA) |
Dα5 | 8.8 ± 2.5 μM | 1.1 ± 0.3 | 6 | 200 [141 ± 25] |
Dα6 | – | – | ‡ | – |
Dα7 | 6.7 ± 1.7 μM | 1.0 ± 0.3 | 4 | 86 [45 ± 13] |
Dα5 + Dα6 | 8.6 ± 2.4 μM | 1.0 ± 0.1 | 5 | 47 [20 ± 8] |
Dα5 + Dα7 | 1.6 ± 0.3 μM | 1.0 ± 0.1 | 3 | 53 [39 ± 8] |
Dα6 + Dα7 | – | – | ‡ | – |
Dα5 + Dα6 + Dα7 | 13.5 ± 1.7 nM* | 1.2 ± 0.3 | 6 | 150 [107 ± 12] |
The Dα5, Dα6 and Dα7 subunits differ from other Drosophila nAChR subunits in their close sequence similarity to the vertebrate α7 nAChR subunit [41, 48], a subunit that is notable for its ability to form both homomeric and heteromeric nAChRs [49–52]. In addition to being one of the best characterised homomeric nAChRs, the vertebrate α7 subunit can co-assemble into heteromeric nAChRs by co-assembly with the α8 subunit (in avian speices) [50]. Although an α8 subunit is not present in mammals, recent evidence indicates that the mammalian α7 subunit can also form functional heteromeric nAChRs by co-assembly with β2 [51, 52].
Relatively limited information is available about the physiological roles of the Dα5, Dα6 and Dα7 subunits in Drosophila, or about the role of analogous subunits in other insect species. There is, however, evidence from studies of native nAChRs in Drosophila that Dα5 forms part of a nAChR that is sensitive to α-bungarotoxin [46], Dα6 forms part of the spinosad-sensitive nAChR [53] and that Dα7 is required for the visually-mediated cholinergic escape response [54].
As has been discussed elsewhere [10, 11], difficulties have been encountered in the efficient functional expression of insect nAChRs. Here we report the cloning of a full-length cDNA of the Drosophila Dα5 subunit corresponding to a previously described isoform B [41]. Other isoforms of Dα5 described previously (isoforms A and C) [41] are a consequence of alternative splicing and have fewer exons than isoform B. Isoform A lacks exon 7, which codes for part of the second transmembrane domain, whilst isoform C lacks exon 5, which codes for the region containing the extracellular Cys-loop. The cloning of the Dα5 subunit was first reported in 2002 [41] but no expression studies were described at that time. More recently, it has been reported that Dα5 does not generate functional homomeric nAChRs when expressed in Xenopus oocytes, even when co-expressed with RIC-3 [40]. Functional expression was, however, reported in the same study when Dα5 was co-expressed with Dα6 and RIC-3 [40]. In the present study, we have detected functional responses when Dα5 is co-expressed with Dα6 but, in contrast to the previous study [40], we have also obtained evidence for the functional expression of homomeric Dα5 nAChRs. Similarly, we have demonstrated that Dα7 can form both homomeric and heteromeric nAChRs. As far as we are aware, there have been no previous reports of the successful functional expression of Dα7, as either a homomeric or a heteromeric nAChR. Given the difficulties encountered in obtaining reproducible functional expression of insect recombinant nAChRs, it is not surprising that there may be some apparent differences in subunit combinations found to generate functional receptors in this and previous studies, particularly since the focus of the most detailed previous study was the identification of a spinosyn-sensitive nAChRs [40].
Our studies conducted in cell lines provided evidence that the pairwise combination Dα5 + Dα6 generates a high affinity radioligand binding site, a finding that agrees with previous studies demonstrating functional expression of Dα5 + Dα6 nAChRs in oocytes [40]. Interestingly, we have found no evidence of specific binding when Dα7 was co-expressed with Dα5 and Dα6 in the same cell lines. This lack of specific binding would seem to suggest that, in the two cell lines examined, co-assembly of Dα7 with the Dα5 and Dα6 subunit interferes with the formation of correctly assembled complexes. We observed a somewhat similar situation in oocytes, where expression of Dα7 alone generates functional nAChRs but it fails to do so when co-expressed with Dα6. This may reflect a tendency for Dα6 and Dα7 to assemble into non-functional complexes. The one situation where this tendency is not dominant is when all three subunits (Dα5 + Dα6 + Dα7) are co-expressed with RIC-3 in oocytes, where they are able to form a functional 'triplet' nAChR with high apparent affinity for acetylcholine.
The present findings suggest that the environment provided by the host cell exerts a substantial effect on the assembly of these nAChR subtypes, a phenomena that has been reported previously for other nAChRs [47, 55, 56]. Previous studies by another research group [40] support the conclusion that co-assembly of Dα5 + Dα6 nAChRs is somewhat inefficient. Not only was functional expression of the Dα5 + Dα6 subunit combination found to be inconsistent in the previous study, but it also appeared to be dependent on the ratio of cRNAs injected [40]. Perhaps this inconsistent functional expression reflects a tendency for some subunit combinations to assemble into non-functional complexes and that this may be more prevalent in certain subunit stoichiometries. It is possible that, in the native cellular environment, factors determining efficiency of subunit assembly and maturation may differ, perhaps as a consequence of a different array of endogenous chaperone proteins. This conclusion is supported by previous studies that have indicated that influence of RIC-3 on maturation of nAChRs is influenced by the host cell [39] and may help to explain the differences that we have observed in the ability of some subunit combinations to assemble into nAChRs in different expression systems.
The data obtained from expression studies in Drosophila and human cell lines is broadly similar. However, successful expression in human cells required incubation at a temperature lower than they would normally be maintained at (25°C, rather than 37°C) [note: Drosophila S2 cells are routinely maintained at 25°C]. Previous studies have demonstrated that the folding and assembly of the nAChRs from insects [17] and from some other non-insect species, such as the cold-water ray Torpedo[57], can be influenced by temperature. This temperature dependence appears to be a consequence of inefficient protein folding and/or subunit assembly at higher temperatures. Previously, due to difficulties in expression of Dα6 and Dα7 nAChR subunits, we examined the ability of subunit chimeras to assemble into complexes capable of binding 125I]-α-bungarotoxin [15]. From such studies, it was possible to conclude that the Dα6 and Dα7 subunits were capable of heterometic co-assembly. In the present study the data from subunit chimeras is less clear cut. Although higher levels of 125I]-α-bungarotoxin were seen consistently when the Dα5 chimera was co-expressed with either the Dα6 and Dα7 chimeras, it was not clear in all cases whether this was greater than an additive effect. Nevertheless these findings are consistent with the conclusion that Dα5 is able to co-assemble into heteromeric complexes. For all subunit combinations examined, responses to acetylcholine were completely blocked by α-bungarotoxin, a finding that is consistent with previous studies conducted with native nAChRs purified from Drosophila which demonstrated that Dα5 is part of an α-bungarotoxin binding nAChR [46].
As mentioned above, a previous study has reported the functional expression of heteromeric Dα5 + Dα6 nAChRs (co-expressed with RIC-3) in Xenopus oocytes and also the inability of either Dα5 or Dα6 to form functional homomeric nAChRs [40]. Significantly, the authors of this earlier study describe substantial difficulties in achieving reliable functional expression. In the present study, despite demonstrating the functional expression of several combinations of the Dα5, Dα6 and Dα7 subunits, we have also encountered a much lower success rate than is typically achieved with other nAChRs. In both transfected cell lines and in Xenopus oocytes, we occasionally failed to detect evidence of radioligand binding or functional expression, despite success with other nAChRs that were expressed as positive controls (for example the mammalian α7 nAChR). The difficulties that we and others have encountered may be associated with a tendency for these subunits to co-assemble into non-functional complexes. It is possible that this may reflect a requirement for additional chaperone proteins. Indeed, a study conducted with a C. elegans nAChR has demonstrated a requirement for three different chaperone proteins for efficient functional heterologous expression [58].
In summary, whereas it has been reported previously that Dα5 and Dα6 can form a functional heteromeric nAChR (albeit inefficiently) when expressed in Xenopus oocytes [40], this is the first evidence that either Dα5 or Dα7 can form functional homomeric nAChRs. It is also the first demonstration that Dα7 can form a functional heteromeric nAChR. Of particular interest is the evidence that the three subunits examined in this study can co-assemble to form a functional triplet (Dα5 + Dα6 + Dα7) nAChR with a high apparent affinity for acetylcholine.
Plasmids and cRNA synthesis
Subcloning of Drosophila nAChR subunit cDNAs Dα1(ALS), Dα2(SAD), Dα3, Dα4, Dα6, Dα7, Dβ1(ARD), Dβ2(SBD) and Dβ3 [alternative subunit nomenclature in parenthesis] into expression vectors pRmHa3 and pRK5 has been described previously [15, 17, 59, 60]. Construction and subcloning of Dα6/5HT3A and Dα7/5HT3A subunit chimeras has also been described previously [15]. For expression studies in Xenopus oocytes, subunit cDNAs were subcloned into pGEMHE [61] downstream of the SP6 promoter. Plasmid constructs (pGEMHE) containing nAChR subunit cDNAs were linearized with NheI and purified with QIAQuik PCR purification kit (Qiagen). In vitro synthesis of cRNA was performed using mMessage mMachine SP6 transcription kit (Ambion). C. elegans RIC-3 (CeRIC-3) cDNA [35] was provided by Millet Treinin (Hebrew University, Israel). The D. melanogaster RIC-3 (DmRIC-3) cDNA used in this study corresponds to the previously described splice variant DmRIC-37a,9[39].
Molecular cloning of Dα5
Oligonucleotide primers were synthesized which correspond to the predicted 5′ and 3′ untranslated regions of transcript CG32975 identified by the GadFly Drosophila genome annotation project (flybase.org). A first-strand cDNA synthesis kit (G.E. Healthcare) was used to isolate cDNA from Drosophila melanogaster embryo Poly A + RNA (Clontech). A 2425 bp fragment was amplified using KOD hot start polymerase (Novagen) and was subcloned into plasmid pCRII (Invitrogen). The cDNA construct was sequenced and found to correspond to the previously identified isoform B [41] [note: isoforms A and C show alternative splicing and have fewer exons than isoform B]. The cDNA fragment was subcloned into the EcoRI site of pRmHa3 and pGEMHE to create pRmHa3-Dα5 and pGEMHE-Dα5.
Construction of Dα5/5HT3A chimera
To construct a Dα5/5HT3A chimera, a similar approach was used to that described previously for the Drosophila Dα6 and Dα7 subunits [15] and for mammalian nAChR subunits [47, 62]. A BclI site was introduced into pRmHa3-Dα5 by means of the QuikChange site-directed mutagenesis method (Stratagene) at a position equivalent to V201 in the previously described mammalian α7/5HT3A chimera [62]. The C-terminal region of Dα5 was removed by digestion with BclI and SmaI and the corresponding region of the mouse 5HT3A subunit [63] ligated to create the construct pRmHa3-Dα5/5HT3A. The chimeric cDNA was subcloned into plasmid pRK5 by excising the construct from pRmHa3 with restriction enzymes EcoRI and XbaI to create pRK5-Dα5/5HT3A.
Heterologous expression in cultured cell lines
Schneider's Drosophila S2 cells [64] were obtained from Dr Thomas Bunch, University of Arizona, and grown in Shields and Sang M3 medium (Sigma) containing 12.5% heat inactivated foetal calf serum (First Link), 100U/ml penicillin and 100 μg/ml streptomycin (Invitrogen) at 25°C. Exponentially growing S2 cells were transfected by a modified calcium phosphate method as described previously [65]. Cells were transiently transfected with plasmid pRmHa3 were induced by the addition of CuSO4 (0.6 mM) for 24 h. Human kidney tsA201 cells [66] were obtained from Dr William Green, University of Chicago, and cultured in Dulbecco's modified Eagle's medium (Invitrogen) containing 10% foetal calf serum (First Link) and 100U/ml penicillin and 100 μg/ml streptomycin (Invitrogen). Cells were maintained in a humidified incubator containing 5% CO2 at 37°C. Cells were transfected using Effectene (Qiagen) according to the manufacturer's instructions and incubated overnight at 37°C. To facilitate efficient folding and assembly of Drosophila nAChR subunits, cells were incubated at 25°C for a further 24 hours, before being assayed for radioligand binding.
Radioligand binding
3 H]-epibatidine (56.31 Ci/mmol) and 125I]-α-bungarotoxin (2200 Ci/mmol) were purchased from Perkin Elmer. 3 H]-methyllycaconitine (100 Ci/mmol) was purchased from American Radiolabeled Chemicals. Radioligand binding to transiently transfected S2 or tsA201 cells (both whole cell or membrane preparations) using tritiated ligands has been described previously [45]. Samples were assayed by filtration onto Whatman GF/B filters pre-soaked in 0.5% polyethylenimine (PEI) followed by rapid washing using a Brandel cell harvester. Samples assayed using the ligand 125I]-α-bungarotoxin were incubated in buffer containing 0.5% BSA and harvested onto Whatman GF/A filters pre-soaked in 0.5% PEI, as described previously [15]. Preliminary experiments were carried out to ensure that incubation times were long enough to enable radioligand binding to reach equilibrium. Amounts of total cellular protein were determined by a Bio-Rad DC protein assay using bovine serum albumin standards.
Oocyte electrophysiology
Adult female Xenopus laevis frogs were obtained from the European Xenopus Resource Centre (University of Portsmouth). Oocytes were isolated and defolliculated as described previously [67] following procedures that have been approved by both UCL's Biological Services Management Group and the UK Home Office (under licences PIL70/23585 and PPL70/06819). For heterologous expression, cRNA (6–12 ng) was injected into the oocyte cytoplasm in a volume of 32.2 nl, using a Nanoject II microinjector (Drummond Scientific). Experiments were performed, typically, 2–4 days after injection of oocytes. Two electrode voltage-clamp recordings (with the oocyte membrane potential held at -60 mV) were performed essentially as described previously [67], using a Warner Instruments OC-725 C amplifier (Harvard Apparatus), PowerLab 8SP and Chart 5 software (AD Instruments). Agonists were applied to oocytes using a BPS-8 solenoid valve solution exchange system (ALA Scientific), controlled by Chart software. Data were analyzed using GraphPad Prism software. For multiple comparisons, statistical significance was determined by ANOVA with Tukey's post-hoc test. Additional pair-wise comparisons were performed by Student's t-test.
- nAChR:
nicotinic acetylcholine receptor.
This work was supported by the Biotechnology and Biological Sciences Research Council (BBSRC) via an Industrial Partnership Award grant (BB/G009392/1) that was co-funded by Syngenta. TC was supported by a BBSRC doctoral training account PhD studentship.
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This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


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Penname: | anitamarie | Member Since: | 2006.05.11 |
Track This Author: | |
Stories Posted: | 2 |
Reviews Posted: | 30 |
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Like everone on this site, I just like to escape once in awhile. I'm *personal info removed by staff* from Quebec who's working hard to improve her writting skills. |
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: In the Midst of War by anitamarie
Novella Thanks to Belle_moi for the beautiful banner Discovering a pregnancy in the midst of war can be difficult, particularly when the father is presumed dead. A closer look at the friendship between Sirius and Lily at a very hopeless moment in the war. Rating upped mainly for language and violence. Final chapter is up! Genres: Humor, Action/Adventure, Angst Era: Marauders Characters: Dumbledore, Hagrid, Harry, James, Lily, Lupin, Pettigrew, Sirius Pairings: James/Lily, Sirius/Lily, Other Pairing Advisory: Strong Language, Strong Violence, Scenes of a Mild Sexual Nature, Sensitive Topic/Issue/Theme, Published: 2006.05.11 Latest Chapter: 2006.08.19 Updated: 2006.08.19 Status: COMPLETED (14998 words) |
: Release by anitamarie
One-shot Sometimes love is not enough. A Lily/Sirius fic - sharing a moment they both needed. Written in character and in a way so there is no betrayal to James. (First Fanfic - Please Review). Special thanks to anora tonks for the beautiful banner. Genres: Angst Era: Marauders Characters: Lily, Sirius, Snape Pairings: Sirius/Lily Advisory: Strong Language, Strong Violence, Scenes of a Sexual Nature, Substance Use or Abuse Published: 2006.05.11 Latest Chapter: 2006.07.29 Updated: 2006.07.29 Status: COMPLETED (2035 words) |


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East Hampton Santa ParadeSaturday, December 6, 2008 |
East Hampton - The East Hampton Santa Parade wound festively down Main Street Saturday morning, making its way on to Newtown Lane in East Hampton Village to the delight of youngsters and the young at heart, as temperatures were mild and hundreds turned out to welcome the holiday season. Representatives from local schools, churches and civic groups drove floats, trucks, tractors and classic cars along the main thoroughfares, some tossing candies to the multitude of children and families lining the broad streets. |


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The bullying of academics follows a pattern of horrendous, Orwellian elimination rituals, often hidden from the public. Despite the anti-bullying policies (often token), bullying is rife across campuses, and the victims (targets) often pay a heavy price. "Nothing strengthens authority as much as silence." Leonardo da Vinci - "All that is necessary for evil to succeed is that good men [or good women] do nothing." Edmund Burke
July 07, 2007
3 comments:
- Anonymous said...
University of Glamorgan, according to UCU wales, has the worst record for dealing with employee complaints
- Anonymous said...
racism is evident at the highest levels.
In an investigation into a case of racism, the investigating committee completely failed to report the distress and illness of the complainant but made sure to refer to the distress of the racist, which was brought on by the investigation.
The complainant decided to leave this university well before the end of his contract. - 5:43 AM
- Anonymous said...
this university should have never been granted the license to teach...no ethics no boundaries...only an animalistic type of management


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Cereal isn't one of those foods that I typically eat–or at least not since high school (back then I used to practically live on cereal); but recently I've been on a kick with this particular mix. I often enjoy it on lazy weekends, because it's a cinch to pull together. To make it even easier, you can double, or triple, or quadruple! the amounts and store it in a sealed airtight container for whenever a hankering for an easy meal strikes.
Super Simple Breakfast Cereal
makes 2 to 3 servings
Simply combine all ingredients and top with almond milk when serving.
To easily toast pecans, bake pecans in a preheated oven at 375 °F on an ungreased baking sheet for about 7 minutes, or until fragrant.
[…] 3. Super Simple Breakfast Cereal […]
That does look a lot easier than making granola! Thanks for reminding me about the existence of kasha– great idea for a nutty-tasting, gluten-free addition to a breakfast cereal.
This would make a great hot cereal, too! As soon as I return from traveling, I am making this :)
Looks great! I've never had kasha.
So the kasha is raw? It never looks very edible to me!


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Alice Waters to write three new books
She is the culinary genius behind Chez Panisse in Berkeley. Along with friend and chef Jeremiah Tower, she pioneered California Cuisine and has had a heavy influence in promoting sustainable, locally grown food sources for restaurants. Her Chez Panisse Foundation introduced the Edible Schoolyard, a program that provides funding and materials needed for a one-acre garden on the grounds of Martin Luther King Junior Middle School in Berkeley, and teaches students of all ages about nutrition and the value of fresh fruits and vegetables.
And now Alice Waters will be authoring three new books to add to her collection of tomes about food and cooking.
In 2011, Clarkson Potter, the publisher responsible for the eleven books Waters has already penned, will release Classic Chez Panisse, a fortieth anniversary celebration of the restaurant. Waters has also agreed to write a retrospective memoir, as well as a sequel to 2007's The Art of Simple Food.
Before long, we just might have the California edition of Julie and Julia, replacing a Queens apartment kitchen and beef bourgnigon with a communal garden and striped bass with lemon sauce.
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Oude Liederen (Old Hymns) is a collection of twelve hymns composed by Joachim Oudaen, Jan Bredenburg Jr., and Reinier Rooleeuw, which was introduced with a new rhymed version of the Psalms in 1684 in the Lam and Toren Mennonite congregation at Amsterdam and used there until 1797. There were a number of reprints: the following unchanged editions are known: Amsterdam 1685 (with notes), Haarlem 1713, Haarlem 1713 (with notes), Amsterdam 1721, Amsterdam 1727, Haarlem 1734, Haarlem 1756, Haarlem 1756 (increased by seven hymns; this edition is entitled Opwekkende Gezangen), Rotterdam 1775 (entitled Liederen en Gezangen), Amsterdam 1793 (two different editions). A greatly revised edition was published at Amsterdam in 1791. (Copies of most of these editions are found in [[Amsterdam Mennonite Library (Bibliotheek en Archief van de Vereenigde Doopsgezinde Gemeente te Amsterdam)|Amsterdam Mennonite Library]].)
Author(s) | Nanne van der Zijpp |
Date Published | 1959 |
Cite This Article
MLA style
Zijpp, Nanne van der. "Oude Liederen." Global Anabaptist Mennonite Encyclopedia Online. 1959. Web. 25 Mar 2017. http://gameo.org/index.php?title=Oude_Liederen&oldid=109185.
APA style
Zijpp, Nanne van der. (1959). Oude Liederen. Global Anabaptist Mennonite Encyclopedia Online. Retrieved 25 March 2017, from http://gameo.org/index.php?title=Oude_Liederen&oldid=109185.
Adapted by permission of Herald Press, Harrisonburg, Virginia, and Kitchener, Ontario, from Mennonite Encyclopedia, Vol. 4, p. 97. All rights reserved.
©1996-2017 by the Global Anabaptist Mennonite Encyclopedia Online. All rights reserved.


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Without Fair Trade prices and credit programs, sugarcane farmers earn on average between $750-1200 per year and many have to sell their belongings in order to finance next year's crop.
Sugar can sweeten anyone's day! Fair Trade sugar increases wages and community development funds for struggling farmers. Sugar cane farmers are some of the most impoverished in the world as a result of fluctuating prices and difficulty accessing the U.S. market. By connecting directly to buyers, Fair Trade farmers are able to improve the lives of their families and the quality of their products. Add Fair Trade sweetness to your day, and bring a smile to the faces of farmers.
Looking for business information on the Fair Trade Certified Sugar program?


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Tuesday, September 16, 2008
6 comments:
- Julia said...
Maryellen, I had just read this blog this morning and my heart broke for what they must be going through, I've been thinking and praying for them all morning. I so hope they can bring their little girl home!
- September 16, 2008 at 2:27 PM
- pugmama said...
oh my God......
- September 16, 2008 at 7:43 PM
- Rebecca said...
Her blog is private... what is going on? I heard about a family who was caring for a sick child in China, is this that family?
- September 17, 2008 at 9:21 AM
- Kristin said...
I have been following this blog and then all of a sudden this morning it is private. :( Will you keep us updated please?
- September 17, 2008 at 10:53 AM
- A Beautiful Mess said...
The blog is no longer private. Continue to pray for this child and her family!
- September 17, 2008 at 3:46 PM
- Rebecca said...
Just visited. Oh my word... I am praying. Who could forget the faces of this sweet family?? Praying that they can bring her home SOON.
- September 18, 2008 at 4:07 PM
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ABUJA, Nigeria (AP) — A large explosion struck the United Nations' main office in Nigeria's capital Friday, flattening one wing of the building and killing several people. A U.N. official in Geneva called it a bomb attack.
"I saw scattered bodies," said Michael Ofilaje, a UNICEF worker at the building. "Many people are dead."
He said it felt like "the blast came from the basement and shook the building."
The building houses about 400 employees of the U.N. in Nigeria, including the majority of its offices. A local U.N. spokesman declined to comment.
Alessandra Vellucci, a spokeswoman for the U.N. office in Geneva, said the global body's offices in Abuja had been bombed.
She told The Associated Press that there was no word yet on casualties.
The building, located in the same neighborhood as the U.S. embassy and other diplomatic posts in Abuja, had a huge hole punched in it.
Local police spokesman Jimoh Moshood confirmed the blast, but said police were still investigating the cause.
No one immediately claimed responsibility for the blast, but oil-rich Nigeria faces terrorism threats on multiple fronts. Last year, a militant group from the country's crude-producing Niger Delta blew up car bombs in the capital during Nigeria's 50th independence anniversary ceremony, killing at least 12.
Nigeria, a nation of 150 million, is split between a largely Christian south and Muslim north. In recent months, the country has faced an increasing threat from a radical Muslim sect called Boko Haram, which wants to implement a strict version of Shariah law in the nation. The sect has carried out assassinations and bombings, including the June car bombing of the national headquarters of Nigeria's federal police that killed at least two people.
Earlier this month, the commander for U.S. military operations in Africa said Boko Haram may be trying to link with two al-Qaida-linked groups in other African countries to mount joint attacks in Nigeria.
Gen. Carter Ham told AP on Aug. 17 during a visit to Nigeria that "multiple sources" indicate Boko Haram made contacts with al-Qaida in the Islamic Maghreb, which operates in northwest Africa, and with al-Shabab in Somalia.
"I think it would be the most dangerous thing to happen not only to the Africans, but to us as well," Carter said.
Associated Press writer Jon Gambrell in Lagos, Nigeria and Frank Jordans in Geneva contributed to this report.


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Use the Beehive's Entrepreneur's Center to learn everything you need to know to make your business a success, whether you are just getting started or want to bring in new investors to expand your operations
Start Your BusinessWant to be your own boss? Check out this section. You'll find out if owning a business is right for you. Plus, you'll get all the information you need to turn your dreams into reality.Build a Business Plan | Checking Your Pulse | Is Owning a Business Right for You? |
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Grow Your BusinessUse the Beehive's Entrepreneur's Center to learn everything you need to know to start and grow a business, attract additional investments to expand your operations, and manage your day-to-day activities.Low-Cost Marketing Ideas | Using the Internet for your Business | Get more money |
Manage Your BusinessNow that your business is up and running you still might need some help. This section shows you how to manage your rapidly growing business by hiring quality staff, facing growth challenges, investing your money wisely and moreStart now with the Beehive's Career Coach. |


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Air Products is a proud partner of many non-profit organizations in the communities where we live and work. Our different type of partnerships, including volunteer involvement and financial support, are as varied as the organizations themselves. The Air Products Foundation provides philanthropic contributions that improve the lives of people in our communities. Through financial contributions, in-kind donations and volunteerism, the Air Products Foundation supports initiatives focused on enhancing opportunities in, Education, Health and Human Services, Community and Economic Development, Arts and Culture and Environment, Safety and Sustainability.
In the United States, we provide a Matching Gifts program to higher education institutions and qualifying nonprofit arts and cultural organizations and environmental and conservation organizations. Gifts to accredited colleges and universities are currently matched 1-for-1, up to $5,000 annually per eligible employee. Gifts to arts groups are currently matched 2-for-1, up to $2,000 annually per eligible employee. Gifts to environmental and conservation groups are currently matched 1-for-1, up to $1,000 annually per eligible employee.
Air Products is a socially responsible company, and that responsibility is demonstrated through our corporate contributions program. The Air Products Foundation and the company donate millions of dollars annually to nonprofit, tax-exempt organizations in need in communities around the world where the company has a significant presence or investment.
In making funding decisions, a variety of factors are considered. This year all requests should be submitted electronically. Please do not send hard copy proposals, but everything should be sent electronically to email@example.com. We can be much more efficient if all proposals are sent by email so that we can have an accurate email address for you. An email acknowledgement will also be sent once your proposal is received.
We look forward to you emailing all proposals to Corporate Relations by June 1, 2017.
A document containing the Air Products' Charitable Contribution Process for funding in 2018 will be uploaded soon. Please check back before submitting your proposal by June 1, 2017 to ensure the funding process has not changed.
Thank you for your continued work to improve the quality of life and health for all of us and our families in the greater Lehigh Valley.
If you would prefer to contact us by phone, you can call any of our Air Products locations worldwide by visiting Worldwide Locations & Phone Numbers.
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I get 14/34. Be sure the axle ratio is what you think it is.
If your speed is off by 25%, then change your tooth count ratio by 25%. Ditto for 20% or any other figure.
The percentage change per tooth changes for every gear available. For example, going from 45 to 44 changes by 2.3% while going from 36 to 35 changes by 2.8%. Same for the drive gears. None of that really matters if you just adjust your tooth counts as noted in the above paragraph. |
Be certain that not only is your Axle ratio correct, but that the speedometer itself isn't a little worn out. I went through this with a customer. I kept telling him the math was correct, and that the drive/driven gears I had chosen for him were a factory pairing as well. We even got the tire mfg to give me the true rolling diameter of the tire and checked his air pressure. To make a long story short; his speedometer was wore out.
Using the TCI calculator Speedometer Gear Calculator and Charts - TCI® Auto you need a driven gear with a theoretical 36.36 teeth, assuming your figures are indeed correct. Please understand that in order for the drive/driven system to work mechanically, there is some inaccuracy in the system. Driven gears in the middle of the range tend to be more accurate than the ones at the end of the range. In addition, the whole speedo, cable, drive/driven system seems to be most accurate in the 50-60mph range and accuracy falls off to some degree above and below that. |
It sounds like the clockspring is weak.
A speedometer can read fast and still accurately register mileage. An odometer will register 1 mile when 1000 revolutions of the input is achieved. How fast it does that is the speed in Miles per hour. If you can verify that the odometer is reading acuratley then you can determine that the speedo is or is not reading correctly. If you Put a 1000 rpm motor on the speedo drive it should register 60 MPH and clock 1 mile in 60 seconds. Getting a perfectly calibrated 1000 rpm motor can be a daunting task, but if you can find a 5,280 foot stretch of road you can verify the revolutions of the cable per mile...which should be 1000. If that is correct and your speed is not registering correctly then the spring in the speedo head is wacked. Also to get the exact tire diameter , i use a dab of grease on the tire, then roll the car untill there are 2 spots on the floor, then measure the distance between the spots ( circumference) and divide by Pi (3.14) That gets er pretty close
Fact is stranger than Fiction Last edited by LATECH; 06-26-2013 at 04:52 PM. |
The Following User Says Thank You to LATECH For This Useful Post: | ||
T Evans (08-02-2014) |
Thanks You everyone for your help,
To answer a few questions: 1. I purchased a rear end refresh with a new auburn limited slip unit and a 3.73 upgrade so that is what I expect it to be (that's what the invoice says); however, just be sure I jacked up the rear end and measured it and it is a 3.73. 2. the speedometer is the original out of the truck (1984) so I am thinking it could very well be the problem; I replaced the speedo cable a while back but never the actual unit (not that easy to source these days). I am planning on replacing the entire dash with an autometer custom mockup next winter but for now I have to make do. LATech, I do have access to a road that long so I will be experimenting shortly. 3. I got the exact tire diameter from the goodyear website (I have goodyear tires) but I did not double check their numbers as it looked pretty much what I expect it to be. 3. the TCI calculator was what I was using all along to calculate my numbers (not sure what the transmission shop used). 4. I'm ok with some inaccuracy but 20% is a little much I agree though that it seems to be pretty much bang on in the 50 mph and below range; I am just confused as to how the system can get worse the higher it gets unless there is a ratio built into the system? I would have thought it was 1 to 1. 5. Cobalt327, thanks but I would rather, at this point, try to find a combination with my 15 gear setup if at all possible. Thank You for all your input everyone! Gary |
36.4 teeth/15 teeth. Rounded off to 36, obviously. Something else is going on here if it was so far off when you have tried combinations that were close.
Thank You for all the help folks,
I experimented over the weekend by putting in a 45 tooth just to see what it would do; it 'should' have changed to becoming really far below the speed but it didn't, it did go the other way (from above to now below) and it was pretty close for some of the numbers. I then put in a 44 and that is actually pretty acurate for the low and high speeds, not so much for the standard 45 mph. In the end, the speedometer I think has gone bye bye, either through a weak spring or something else; it is a 1984 truck with original speedometer after all; and it was a farm truck so there is/was lots of dust that I cleaned up from behind the dash. I'm thinking some of it is still in there so I will look at cleaning up the speedometer. Are there kits out there to rebuild the spring? I would rather not spend the $150 that LMC truck wants for a new one. I was using my GPS which I validated against out daily driver to get mph's. My end result is that @ 35 mph it shows 30, @ 60 mph it shows 50, @ 75 it shows 75. and you can see a noticable jump when it hits 65 where it jumps right to 70 thereby causing 75 to line up when it gets there. So, question, * Should I change the speedometer cable first (it's only 2 years old) before tackling the speedometer (LMC truck has on for $150) or should both be done? Once that's done then I will be going back to the 36 that the tranny shop put in... Thanks! Last edited by ggevaert; 07-14-2013 at 02:08 PM. |
I would try lubricating the shaft that supports the indicator needle. If it is dragging, the magnet in the rotating bell cant pull it far enough to register correctly. A weak spring would allow a higher speed at low speeds, where a dragging / dry bushing would register lower.
Fact is stranger than Fiction |
wrap up
Just to wrap this up.
I pulled the gauge cluster and speedometer and it was caked with red dirt (matches the farm it came from) so I ordered and installed a new speedometer unit. While I was at it I also installed a new speedo cable and now my speed is inline with what is expected. It is still off a few teeth (needs at 39 and not 36.4 as noted) but at least now I can get it dialed in. Thank You to everyone who helped out! Cheers. Gary |
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Online now: 3 members: Kokopelli eye institute, SLinn16, timmyb; 14 anonymousDiscussion: North East Championships
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Sept. 18, 2009 MTM Men's 70 25 posts | North East Championships From a Cape May native. The Davies sports complex is multi purpose as many communitiy recreation complexes are. Some of the fields are designed for little league baseball, little league softball, adult softball, youth soccer, and youth football. So permanent fences are out on some of the fields. There are two softball fields with dugouts and two without. The complex also contains a fenced little league field, a fenced adult baseball field, a skateboard /bicycle park, a street hockey rink, a snack stand, and the community recreation center. The complex is located about two miles from town where you will find shopping centers, fast food and other restaurants and a tavern. Located about a mile away is the free Cape May County Park and Zoo the gem of NJ zoos and you are only a few miles from some of the finest beaches anywhere. The infields might not be the best but the complex and area is definitely different than the Lower Allen/ Harrisburg area. The 60 teams will be playing in Avalon NJ. Avalon NJ is one of the finest seashore towns along the entire NJ coast. If the wind is blowing off the ocean( out to left field) bombers are going to love it. It is a two field multi purpose complex with portable fences and dugouts built about 10 years ago. Resturants and taverns are nearby. SSUSA staff has a list of motels. Not all motels are open as the season is winding down but it is still busy as it is a beautiful time of the year at the Jersey Shore with many events still going on. North Wildwood has their unbelievable Irish week end 9-24-thru 9-27. Good luck to all and prey for no rain. |
Sept. 18, 2009 kbl Men's 60 544 posts | mtm, thanks for your input. do you work for the chamber of commerce?(LOL) ken |


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