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Properties For Sale in Glais, Swansea, Swansea (County of)
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Elio Petri: A Quiet Place in the Country
Ostensibly a ghost story, A Quiet Place in the Country is, in fact, an atmospheric rumination upon the nature of art, pornography, insanity and – heck – life itself.Read More
Ostensibly a ghost story, A Quiet Place in the Country is, in fact, an atmospheric rumination upon the nature of art, pornography, insanity and – heck – life itself.Read More
War Games (2011) fits in neatly with a handful of other Italian films which followed the survivalist horror template and were made at around the same time (Shadow, In the Market).
Craig Fairbrass plays a security consultant in the pay of one of those super-slick pharma companies who you just know are accidentally going to bring about the apocalypse, and so it proves
Although Neil Marshall has taken a step back from directing films in recent years – a great shame, as he is amongst our most talented filmmakers – he does occasionally dip his toe back into the industry, for instance by producing his wife Axelle Carolyn's debut release Soulmate in 2013
I'm all for any attempt to revive that venerable cinematic form that is the Italian peplum, but by any stretch of the imagination Day of the Siege ain't the film that's going to do it.
A lot of people reviewing Mindscape have mentioned the similarities to Christopher Nolan's Inception. For those with memories that span further back than five years ago, though, the production to which it is perhaps most indebted is – as is indicated by the title – the 1984 film Dreamscape
For 1969's Investigation of a Citizen above Suspicion, Elio Petri turned his eye to the then nascent urban crime thriller, a genre that reflected – and some would say preyed upon – the political and economic uncertainties rife in Metropolitan Italy at the time.

subset: commoncrawl


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Dell is an American multinational computer technology corporation named after its founder Michael Dell. They are the third largest personal computer vendor in the world, behind Lenovo and HP. Dell offers services such as developing, selling, repairing and providing related products to its vast consumer base. Most companies use Dell computers in their office due to the fast and reliable services they provide, especially when you have a system issue. Dell specializes in computer hardware, computer software, IT consulting and IT related services. Alienware and Perot Systems were taken over by Dell to give a boost to its already vast portfolio of products and services. Alienware computers are systems that are specially designed for gamers who prefer high-end graphics and a comfortable layout to game in. Innovative cooling systems inside the computer cabinet ensure peak performance even when playing new games at the highest resolution and graphics setting. Dell product lineup includes laptops, ultrabooks, desktops, all-in-one computers, electronic accessories, printers, workstations, tablets, servers, storage devices, networking devices and smartphones. Dell's success lies in the fact that they provide excellent customer service and take very little time to resolve issues with their systems.
Dell Inspiron laptops, XPS laptops and Alienware laptops are designed for everyday use due to their reliability and performance. Buy Dell computers online from Flipkart after browsing through the extensive catalog to find one that suits your needs and budget. Shop online from the convenience of your home and pay using your credit or debit card through Flipkart's secure payment processing system. In case you want to pay only when you receive the product, you can avail the cash on delivery option. Use the 30 day replacement or refund policy in case there is an issue with the product.


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86 of 90 people found the following review helpful
OK For general Ideas,
This review is from: Building a Web Site For Dummies (For Dummies (Computer/Tech)) (Paperback)
If you are looking to code a webpage or site, buy this book along with HTML 4 for the WWW by E. Castro. Castro's book is easy to follow and deals with multi-browser webpage construction. This Dummies book does give you a good feel about the practical things to consider when constructing a website. Not just about creating a page with links and pictures, but about practical navigation construction and how to build websites for a particular audience (ie. websites for your family versus an e-commerce site). Some useful info here, but they should not jump into HTML without creating a good feel for it in this book--try Castro's or HTML for Dummies--becuase this book could have served one better if it stuck with the key ingredients for a decent site, then had a small primer for HTML or recommended good HTML books. For other website design books, try Web Usability by J. Neilsen. This is an ok book that deal with the problems associated with website construction--Web Usability has color pictures and is not dependent on any one design software package.
Review Details


text: Hello! My name is Ambicrow. Although my contributions are low to smogon, i have been around with the smogon community for quite some time. I had lurked around here for around 6 months before actually creating an account, and I only created this account at first to create a RMT, and a bad one at that. After a while in the BW1 metagame, I went to the hermit caves and theorymonned for BW2, since the Japanese release was approaching. After finally coming out of my cave, I had discovered all of these new threats, like Tornadus-T and Thick Fat Mamoswine with SuperPower. I realized these threats are only as good as their respective weathers allowed them to be, so I made my own weather team, so these threats wouldn't be as useful. I was debating whether to make a team around a lilligant sweep in the sun or a nasty plot jinx in hail. As you can probably tell by the picture above and the title, I opted for the sooner, rather than the later. Without further ado, here is the team that Burned Water. Team Preview Team Building Process Hide (Move your mouse to the hide area to reveal the content) Show Hide Hide Hide I decided to base he team around a Quiver Dance Lilligant. The best way to abuse lilligant is usually under the sun, so i added a Ninetales to abuse Lilligant's chlorophyll ability. I then decided to add Genesect and Dugtrio to make the infamous Heatran trapping core. I find this core to work very well on this team, since after genesects u-turn, dugtrio 2hko's all choiced politoeds, something that rain has trouble with. Although this core can't really take down bulky Politoed, Lilligant easily sets up 1-2 quiver dances on defensive politoed, so it's isn't a threat. After that, I realized my team couldn't really take any fire moves, and had a terrible weakness to Mamoswine, a threat that I'd gladly like to avoid. So, I added offensive Traptran on a balloon. With a timid nature, this outruns all Adamant Mamoswines, and doesn't give 2 shits for its Stabs while on a balloon. My final Pokemon was Golurk. Surprisingly, it did exactly what I needed in the last slot. As of this point in the team building, I was at a road block. Before I picked Golurk, I needed a physically offensive Stealth Rocker. At first I thought Terrakion, but then my team would be very weak to scarf Terrakion, in which none of my team would want to come in on that monster. So I chose Golurk, who has a very respectable base 124 Attack, amazing typing, especially under the sun, can set up SR, and can spin block. I've made some changes! All changes are outlined below in the "In Depth" section in red. In Depth (Changes in Red) Phosphorus (Ninetales) (F) @ Shed Shell Trait: Drought EVs: 100 HP/ 200 Def/ 108 Sp. Def/ 100 Spd Calm Nature - Fire Blast - Will-O-Wisp - Roar - Pain Split Appropriately named, my Ninetales lights the match under the stomachs of its teamates. As many of you may know, the tips of matches are made of Phosphorus. I consider my team a match. Ninetales is the tip of the match, slowly sending the flame downward, until it reaches the bottom, where then it it burns the match unto itself. Once the flame goes out, there isn't much left for that match to do. So I make the tip of my match long lasting, so it doesn't spread down. Now that the analogy is done, I'll move on to the thinking behind the moves. I started out right away knowing I'd use Fire Blast. It has a very strong base power, and double Stab under the sun. The extra power was definitely needed, since my set is more supportive. Will-o-Wisp is to burn incoming weather inducers, and also incoming physical threats, such as Terrakion, who switches in often to the more Specially oriented dog. Roar is to keep switches going, and rack up SR damage, which Ninetales does quite effectively. It also makes sure Volcarona doesn't set up on me, which can be hazardous while under the sun. Finally, Pain Split is great, as it whittles down Blissey/Chansey's HP, while putting yours back up to full. Also, this plays so many mindgames with your opponent while playing the weather war. When you send your low hp Ninetales out against a low hp Forretress(for example, there are more defensive threats, like Skarmory), you just use Pain Split, now you're on equal grounds for the weather war, as the hp's are equal(in a sense). I have changed the EVs for my Ninetales. With this EV spread it tanks a +2 Mach Punch from Adamant Techniloom, and outspeeds Mamoswine. Also, it has a low Hp number, so pain split will be more advantageous, because the defense per hit point value will be higher(that's odd to say). Also, it still retains a lot of special bulk, with its natural base 100 Sp. Def, and 108 EVs. Lead (Genesect) @ Choice Scarf Trait: Download EVs: 4 Atk / 252 SAtk / 252 Spd Hasty Nature - U-turn - Flamethrower - Thunderbolt - Ice Beam Now as you read this, I'm going to make something very clear. When his nickname spells Lead, as in a pokemon lead, that's not what I mean. I mean Lead, as in the element. Although lead isn't the softest element, it's in the top ten, and more well known then the others. Also, most of the time he is my Pokemon Lead, so that's pretty nifty. When i first made this team, I was debating between LO and Scarf, and boy has scarf been the much better choice. This allows him to revenge out the sun, something Lilligant can't do, and with perfect neutral coverage. Honestly the main reason I use him over other U-turners is the fact that he attracts opposing Heatran, and other counters to my Offensive duo of Lilligant and Heatran. Flamethrower in the sun gives him pseudo-stab, which is very useful vs. steels not named Heatran, which give my Lilligant trouble. Standard Bolt-Beam so I hit everything for neutral coverage, which is very useful for an end-game clean, and also hits threats like Gyarados and Landorus for SE damage. Promethium (Dugtrio) @ Focus Sash Trait: Arena Trap EVs: 252 Atk / 252 Spd Jolly Nature IVs: 21 HP / 0 Def / 0 SDef - Earthquake - Stone Edge - Stealth Rock - Reversal As his name states, there isn't many dugtrios left in the universe, and this one is just hanging on by a thread. Just like the element Promethium, which is extinct, or next to it, Dugtrio becomes "extinct" very soon in the match, and becomes next to it even sooner. He hangs on the thread of existence with that puny sash, since he can't take any hit otherwise. Now, dugtrio only lives because I tell him too, so he lives to serve me. When I tell him to take that Banded Dragonite's outrage, he does so with pride, knowing he will be on the brink of existence afterwards. He traps so many threats that come in on Genesect to stop his rain of terror, and kills them with little to no effort. Since Dugtrio lives to let others live, that is what Dugtrio does for Genesect. The mere existence of Genesect is only there because Dugtrio is here. So now onto the more "statistical" stuff. The IVs make sure he is left at 1 hp after 2 Seismic Tosses, which is very important when disposing of Blissey/Chansey. Max Attack and Max Speed for obvious reasons. I decided to use EdgeQuake so dugtrio could do damage to more threats, and also kill weaken flying types, like Salamance, Tornadus-T, Thunderous-T, and Volcarona ( I know it isn't a flying type). Sucker Punch lets it hit the Lati twins better, but I'm thinking of switching it to Aerial Ace, since this team has been hating Techniloom lately, and I haven't found much use for Sucker Punch as of late. Reversal is there for the 1hko on Offensive Balloon Heatran, and for Tyranitar while also hitting for very good coverage, and usually replaces Stone Edge as the coverage once I reach 1 hp. So, in your rates, if you can tell me what you tink is better for dugtrio, Aerial Ace or Sucker Punch, that'd be great. I have changed Sucker Punch to Stealth Rock, since now my Golurk has been switched out for CM Keldeo. I miss Sucker Punch a little bit, but using Stealth Rock over Sucker Punch to get Keldeo in on this team is really worth it. Hydrogen (Keldeo) @ Leftovers EVs: 252 Sp. Atk/ 252 Speed/ 4 Sp. Def Nature: Timid -Hydro Pump -Secret Sword -Calm Mind -Hidden Power Fire Although a water type shouldn't see the burning of water up close, I think this lost My Little Pony can fight like the rest of the top dogs, besides its shaky past as a Water type. It takes pride in its heritage, and keeps the name Hydrogen, in honor of its great pony parents, who he kills on a regular basis. Keldeo brings me the anti-rain I really need. He resist ice and water, and has some decent defenses of 91/90/90. He still counters Terrakion(more of a check), and also sets up in the rain. Unlike the other water and ice resists, he can take down blissey/chansey, 2hkoing them at the least with Secret Sword.Hydro Pump and Secret Sword give him his Stabs,and Calm Mind lets him take advantage of those rain pokemon that can't do jack squat to him. HP Fire allows him to still be useful in the sun, giving him an alternate Stab in the sun. I haven't got to really use it yet, so it is probably replaceable. I'm thinking of HP Ghost, since that is the standard, but it doesn't give a great range of SE hits while under the sun. but in the rain or sand, it becomes a monster. I usually keep it clear skies before keldeo gets used against a non-weather team. This makes sure his Main Stab isn't halved so early in the match. Mercury (Heatran) @ Air Balloon Trait: Flash Fire EVs: 4 HP / 252 SAtk / 252 Spd Timid Nature - Magma Storm - Sunny Day - SolarBeam - Hidden Power [Ice] Mercury has one of the lowest melting points in the world. Just like Heatran, it doesn't stay solid very easily, and is easily liquid, and a very deadly one at that. Mercury looks fun to play with. It kinda calls to you to touch it, and roll it around it your hands, but at the same time, it's killing you at that very second. Heatran is the same way. It lures in opposing threats, like Politoed, and traps them to there slow demise, taking almost 3 turns to fully take effect. Surprisingly enough, although this is a trapping set, it usually becomes a late game sweeper if the balloon is still intact. With a plethora of Super Effective coverage, it easily rushes right through teams, and outspeeds bulky pokemon, like bulky Dragonite, or Adamant Dragonite, Gastrodon, and most Gliscors.With a base 130 Sp. Atk, he hits like a freakin truck with magma storm under the sun, and nothing really wants to get hit by it. This set does get walled by opposing Heatran though, but I like the HP Ice, as it lets me take care of more important threats. Usually opposing Heatran is number 1 priority, but Lilligant can handle most Heatrans after a quiver dance or 2. Olivine (Lilligant) (F) @ Life Orb Trait: Chlorophyll EVs: 72 Def/ 252 Sp. Atk/ 184 Spd Modest Nature - Quiver Dance - Giga Drain - Sleep Powder - Hidden Power [Rock] Well, this has to be the most boring analogy, but I picked Lilligant to be Olivine, because #1-It is my favorite Silicate. and #2-It can be green. Yep, I have a favorite Silicate. The reason behind this is that it was the only one I could identify in Earth Science in 10th Grade on a regular basis. I decided to pick Lilligant over the other chlorophyll sweepers because Lilligant sets up in front of Defensive Politoed, and actually likes the rain. I especially love the Politoeds with Ice Beam. They barely do 30% after a Quiver Dance, and then I can set-up another Quiver Dance. Now the EVs are pretty basic, just maximize both Speed and Sp. Atk, so it can fare well outside of sun. Giga Drain and HP Rock give great coverage together, but get walled by steel types not named Heatran and Skarmory. Although getting walled by the most defensive typing in the game usually isn't a good thing, I an usually let it slide with all the fire attacks on my team. If there really is something that lilligant can't handle, I can always sleep it, and come back to it later, just like a book. Lilligant has to be one of my favorite Sun Sweepers, as it actually takes advantage of the other weathers too. If you haven't tried anything besides cookie-cutter Venasaur, I'd recommend trying this, especially if you have trouble vs. rain. The Closing Note I hope you guys all had a great read. I tried to make it as appealing to the eye as possible. Below I'm going to give a small threat list, and please tell me if you see something that I left out. But first, the credits- Prevent- for helpin me test this team thoroughly to see weakness. Aquaa- same as above ClubbingSealCub He was the co-founder of the team. Anything I do to this team I plan on running by him first, to see if he agrees with the changes, though it ultimately goes down to what I want. Techniloom Thick Fat Mamoswine in Rain Dual Sand Users and an Abuser Exportable (Keep the Nicknames please) Code: Olivine (Lilligant) (F) @ Life Orb Trait: Chlorophyll EVs: 72 Def / 252 SAtk / 184 Spd Modest Nature - Quiver Dance - Giga Drain - Sleep Powder - Hidden Power [Rock] Lead (Genesect) @ Choice Scarf Trait: Download EVs: 4 Atk / 252 SAtk / 252 Spd Hasty Nature IVs: 30 Atk / 30 SAtk / 30 Spd - U-turn - Flamethrower - Thunderbolt - Ice Beam Promethium (Dugtrio) @ Focus Sash Trait: Arena Trap EVs: 252 Atk / 252 Spd Jolly Nature IVs: 21 HP / 0 Def / 0 SDef - Earthquake - Stone Edge - Stealth Rock - Reversal Hydrogen (Keldeo) @ Leftovers Trait: Justified EVs: 252 Spd / 252 SAtk / 4 SDef Timid Nature - Hydro Pump - Secret Sword - Calm Mind - Hidden Power [Fire] Mercury (Heatran) @ Air Balloon Trait: Flash Fire EVs: 4 HP / 252 SAtk / 252 Spd Timid Nature - Magma Storm - Sunny Day - SolarBeam - Hidden Power [Ice] Phosphorus (Ninetales) (F) @ Leftovers Trait: Drought EVs: 100 HP / 200 Def / 108 SDef / 100 Spd Bold Nature - Fire Blast - Will-O-Wisp - Roar - Pain Split


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This is a Milton Bradley Company Candy Land Game - copyright 1962. The game board and all 4 game pieces are in excellent condition - the colors are bright and there are no tears. All of the cards show normal use with a few having slight folds but they are fully functional. Originally I thought one of the cards (the gingerbread boy) was missing but I found it so the game is complete. The box shows some wear with three of the top corners and two of the bottom corners torn. They could easily be taped. The bottom corners were taped at one time. The box top has some of the printed covering torn off in the upper right corner and the lower left corner as you can see in picture #4. Overall, the game has seen some fun over the years which I always believe only adds to its charm. The box measures 16 1/2" x 8 1/2".
Thanks for visiting my shop!
Thanks for visiting my shop!
1960's Vintage Candy Land Game
- Vintage item from the 1960s
- Ships from United States to select countries.
- Feedback: 84 reviews
- Favorited by: 5 people
- Listed on Sep 10, 2011
- 2214 views
- 5 favorites
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I am using Google Maps v3.
I'm currently using the autoload functionality and it's working great:
<script type="text/javascript" src='http://www.google.com/jsapi?autoload={modules:[{name:"maps",version:3,other_params:"sensor=false"}]}'></script>
Now I want to use the autoload to also include the
callback option to callback my function named
How do I modify my working code above to include the
callback option?
I've tried a few different ways and none work. I receive an "Invalid Autoload" error.
I've tried the below, as well as a ton of other variations and none work. I still cannot figure out the appropriate syntax to use the
callback method.
<script type="text/javascript" src='http://www.google.com/jsapi?autoload={modules:[{name:"maps",version:3,other_params:{"sensor=false","callback=myCallbackFunction"}]}'></script>


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If you have troubles since the update to WordPress 4.5 please read this post before opening a new thread.
Hi everyone!
Is this a custom-made-theme? Or can we download it somewhere and install it for our use?
Thank you!
Hey Dennis!
Sorry this is a custom made and highly hacked version of the old bbpress software, nothing you could (ow would like to) use, I can assure you ;D
Hey Kriesi!
Okay! I thought / hoped that' it's a quick & easy forum. :-)
Thank you!
The topic 'This Forum – where can I get the template? :-)' is closed to new replies.


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Summer SA/TO ScoopPosted on June 2, 2016
Read June's issue of the USY Social Action/Tikun Olam newsletter, the SA/TO Scoop!
USY Meets WorldPosted on June 2, 2016
SWUSY alumnus Aaron Jacobs reflects on how USY's values of social action and tikun olam effected his career later life. His experience on USY on Wheels helped inspire his first-ever credit as a writer for Disney's Girl Meets World. Watch Aaron's TV episode "Girl Meets Money," which is currently streaming on Netflix, along with the entire second season of Girl Meets World.
May SA/TO ScoopPosted on May 5, 2016
Click on the preview below for the latest updates from your International USY Social Action/ Tikun Olam Committee with their monthly newsletter, The SA/TO Scoop! IN THIS ISSUE: Meet Your Newly Elected Regional Social Action/Tikun Olam VPs Lishmoah LeShoah: Holocaust … Continue reading
Recognizing Modern Oppression at the Seder TablePosted on April 21, 2016
Every year, Jewish families from around the world gather together at their Seder tables to recount the story of the Jewish people's journey from slavery to freedom. And yet, the struggle for freedom did not cease when the Israelites fled Egypt some 3000 years ago. The LGBTQ community still faces the oppression that we sought to escape when we left Egypt. Join with other USYers as we celebrate inclusion at our seder's this year. Continue reading


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Hey gang,
I got a new job which (while amazing) has thrown my life into a sort of massive whirlwind. I'm still alive, but was feeling over whelmed. I nuked my patreon to free up some time, but in a ....moment, also nuked the tumblr, and my other dA account in an attempt to refocus.
That was probably an over reaction. hahaha.
I was close to nuking the picarto and this dA page, but I came to my senses.
I'll spool up something for that other work soon ish, along with something for limited commissions, but I'll probably let the patreon cool for awhile.
so yeah, still here ^_____^


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I have demand for them in the very very beginning of a city but then the blue bar goes empty and the rest of the time its just yellow and green. I see the saying that if they dont get shops soon then they are leaving. But the bar for demand is NEVER blue. My cities end up being mostly residential and industrial. And a very small touch of commercial. Is that normal?
Oh and other thing I havent got to work... I gifted one of my other cities 300K to get it started. Never received it. The town is well developed now and I now got it. The other town is short 300K now.
You are not allowed to request a sticky.


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Купить просто ! Добавляете товар в корзину не менее чем на 5 000 рублей, потом переходите в корзину и подтверждаете заказ !
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I had a long discussion with my broker about this one. There's simply no logical reason why puts are classified as "covered" when there's a corresponding stock position with the same exposure. It seems as if somebody who meant "calls" wrote "options" in the regulations. The concept has no regulatory meaning, but I talk about "Cash-covered puts" when I sell puts and deposit the money to buy the stock in an interest-bearing account at the same time.My broker is fond of selling deep out-of-the-money puts in parallel with a covered call position (I pay an annual fee rather than commissions). In case of disaster, the stock part of the covered call is sold at about the exercise price of the put (this makes me very nervous, but he does it with his own money as well as clients' money)


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Haiti: Jobs & Hope
Haiti: Jobs & Hope
Haiti: Jobs & Hope
Operation Blessing Builds School in Haiti
Islam Making Inroads in Haiti
Dozens of volunteers are building thousands of new houses for Haitians left homeless by January's massive earthquake.
The graffiti on a building in Belgium says it all: "Welcome to 'Belgistan." In fact, some are now calling...
A pregnant mother confronts a terrifying breast cancer diagnosis. Plus, a skydiver plummets to earth how he least expected…still...
From the most conquered city in history, Scott Ross tours the place where the past comes alive and the...
"Touchdown Tony" Nathan has a solution to reconcile a divided people. Plus, a Marine's doomed marriage is saved.


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WNGlosses (finlayson-gwc2014.zip\CICWN-1.0.zip)
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… made of polypropylene and purchased from USA/Scientific Plastics (Ocala, FL, U.S.A.); glass tubes were borosilicate … E-64 wasgenerously provided by A. F. Goldberg (Harvard Medical School,Boston, MA, U.S.A.). All other chemicals and solvents were reagent grade , obtained from commercial sources. Lobsters were purchased from commercial suppliers and held at 12- 14°C in artificial seawater.
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text:
The FoodSaver® Vacuum Sealing System Kit
Certain Restrictions Apply* Details
Certain Restrictions Apply* Details
For full shipping terms and restrictions, please visit our FAQ. Free shipping offer is for orders shipped within the 48 continental United States automatically applied at checkout . Limited-time offer subject to change without notice. Applicable sales taxes apply. Excludes replacement parts and accessories.
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The FoodSaver® Vacuum Sealing System Kit
$99.99- See final price in cart
The FoodSaver® Manual Kit features the FoodSaver® V2244 Vacuum Sealing System to keep food fresh up to 5x longer and saves up to $2700 per year based on buying in bulk, on sale and preventing waste. Form and function combine with a sleek, compact design and easy-to-use manual operation to preserve a variety of foods. Includes starter kit of bags and rolls.
Included In Offer:
2 - 11 inch Vacuum-Seal Rolls
20 - Quart Vacuum-Seal Bags
1- Accessory HoseSee More...
Additional Information
This FoodSaver® Vacuum Sealing kit keeps food fresh up to 5x longer and saves up to $2700 per year based on buying in bulk, on sale and preventing waste. Form and function combine with a sleek, compact design and easy-to-use manual operation to preserve a variety of foods.
- Compact size with manual operation
- CrushFree™ Instant Seal helps protect delicate foods during sealing process
- Patented, removable, dishwasher-safe, drip tray
- Accessory port and hose vacuum seals canisters and marinates food in minutes
- 5-year limited warranty
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The FoodSaver® Vacuum Sealing System Kit is rated out of 5 by 63.
Rated 5 out of 5 by Rickthe3 from 15 years I bought my foodsaver about fifteen years ago, and it is still working very well.
Date published: 2016-05-30
Rated 1 out of 5 by Cis 57 from It did not work. I purchased this Food Saver and from day one, it did not work. It was returned for a refund.
Date published: 2016-05-15
Rated 5 out of 5 by Jgarr from It's a money saver I bought this a few months ago and have used it more than I expected. I have been getting fresh asparagus out of the garden and vacuum sealing them for later this season and it works great
Date published: 2016-04-30
Rated 5 out of 5 by Jim1492 from I Bought This As A Gift I have been using the FoodSaver for over a year and I am so impressed that I gave this as a gift to my son. He roasts and grinds his own coffee beans and this will be excellent with the 3 set canisters to preserve freshness.
Date published: 2016-02-06
Rated 5 out of 5 by Golfhandds from Great value I bought this for my mother and she really loves it. She has used a foodsaver for years but it finally died. This is simple to use and effective. Highly recommended.
Date published: 2016-01-29
Rated 5 out of 5 by Jack777 from Simple and easy to use. The use of this product is very easy and the process is so simple. Lovin it!
Date published: 2016-01-21
Rated 5 out of 5 by VeryOldMan from Greatest thing in the kitchen since the spoon! Many years ago, I received a vacuum bagger from the kids as a gift. I thought at the time, "What a waste of counter space." I figured that I would just use it until I had to actually buy bags, then store it away in the basement next to all of the other "One month wonder" appliances like the pasta extruder, dehydrator, air popper, ice shaver, hot dog roller, yogurt maker, etc., etc.. Then I started to use it. A little at first, then a lot. The more I used it, the more I learned of its capabilities, many of which go way beyond simply saving leftovers or buying in bulk for $$ savings. One day, over a decade later, after having sealed a Zillion bags, it finally broke. After a sad funeral into the dumpster, I set out to find its identical replacement. No such luck. Ebay, garage sales, secondhand stores, nothing was coming up. The Department store from where it was originally purchased no longer carried that exact model even though they had several to choose from. Due to my very limited counter space, I needed the same size and footprint that the now kaput one had. On to the Internet to find a suitable contender. About a half hour later, with a list of possibilities, I weeded it down to The V2244, based on the size, price and included options. There were only two down down marks in my review. I wanted a white one, they only had black in that model, and the cord is too short for my application. My old one's cord was plenty long enough. As for performance, it works even better than my old one that required two hands to operate. This one is hands free. Couldn't be happier! (Unless it was white with a longer cord.) Other than my coffee maker, it is the most used appliance on the counter top. Yup, even more than the microwave. I have even been known to bag soup. (Oh yes you can!) What else do I do with with this little marvel? Glad you asked. Camping with the great grand kids, we always pack a spare pair of socks, underwear, and T shirt for each kid in a bag. Food for camping is assured to stay dry. Wet foods like steaks, chicken, hot dogs, chili, etc. are frozen and now act as ice packs to keep everything else cold. When traveling and space is of a premium, bag it! Cut the roll a little longer than you need and you can even re-seal/reuse it. We have sealed a down comforter in a 6 long foot bag. You will never believe how little room it needs once all of the air is removed. (Don't forget to add an extra foot of bag so that you can re-seal it next season.) I collect antique model airplane engines. After they are cleaned and oiled I put them in a vac bag for long term storage and protection from moisture. I also store old photographs in vac bags. Being old people we have tons of unused old people drugs. Who knows, might need them some day. Vac bag'm, store in a cool dark closet and they will stay effective well past the expiration date. Did I mention re-sealing chip bags, cereal bags, almost anything that you want to protect from the elements and preserve for long periods should be vac bagged. It also makes a great wedding or house warming gift! Would I recommend this thing? Of course! Click on picture to enlarge.
Date published: 2016-01-20
Rated 3 out of 5 by Paula55 from Product just okay This product is a replacement since I can no longer find the wrap for my older unit. The Food Saver does the trick, but not as easily as my previous machine. I wish the Food Saver would store wrap in the unit, instead of me cutting sizes. I will look at other models, but I really am not that thrilled with this model.
Date published: 2016-01-19


text:
|Publication number
|Publication type
|Application number
|Publication date
|Jun 29, 1993
|Filing date
|Jun 1, 1989
|Priority date
|Jun 1, 1989
|Fee status
|Also published as
|Publication number
|Victor Ling, Elias Georges
|Original Assignee
|The Ontario Cancer Institute
|Export Citation
|BiBTeX, EndNote, RefMan
|Non-Patent Citations (6), Referenced by (4), Classifications (9), Legal Events (4)
|External Links: USPTO, USPTO Assignment, Espacenet
The invention is an immunoassay method for determining the specificity of binding of a monoclonal antibody to an antigen, and thereby enhancing the sensitivity of the assay. The invention makes use of epitope characterization to provide peptides encoding epitopes. Comparative binding studies of antibody to antigenic substances with or without the addition of the epitopic peptide enables the determination of the specificity of antibody binding.
Immunoassays are routinely used in a wide variety of diagnostic and analytical applications. With the relatively recent availability of monoclonal antibodies, the power of immunoassays to focus on a particular antigen target is greatly increased. However, when antibody binding is observed in one of these assays, it is possible that such binding may be nonspecific, that is, does not result from epitope specific antibody-antigen binding; and therefore, gives a false positive result. Accordingly, it is desirable to be able to augment the power of a monoclonal antibody immunoassay with the determination that the observed binding is epitope specific. Such a determination also serves to enhance the sensitivity of the assay.
The invention addresses this need and is applicable to virtually any immunoassay method as, for example, ELISA, RIA or immunohistochemical staining. The present method enhances the power of immunoassays employing monoclonal antibodies for which epitope encoding peptides are known by enabling the user of the assay to ensure the specificity of binding being observed
Accordingly, the present invention provides an immunoassay method for determining the specificity of binding of a monoclonal antibody to an antigen, comprising the steps of:
a) contacting a first portion of a test substance with the monoclonal antibody, incubating the resulting mixture to promote binding of the antibody to available antigen associated with the test substance, and testing for binding of antibody to the test substance;
b) if binding of antibody to the first portion of test substance is demonstrated, contacting a second portion of the test substance with the monoclonal antibody which has been pre-incubated with at least an excess amount of a peptide encoding the antibody epitope for the antigen sufficient to ensure occupation of substantially all antibody binding sites for the epitope, incubating the resulting mixture to promote binding of the antibody to any available antigen associated with the test substance, and testing for binding of antibody to the test substance; and
c) comparing the results for the binding of the antibody to the first and second portions of the test substance to determine the specificity of antibody binding.
The invention will be described in relation to a particular antibody/antigen system, but it should be understood that the following description is not intended to limit the scope of the general method of the invention which has broad application.
FIG. 1 provides a schematic illustration of 205 overlapping hexapeptides derived from the 211 amino acid residue polypeptide for P-glycoprotein as encoded by the clone pCHP1. Also shown are three graphs representing the optical density (absorbance) for the various hexapeptides in the presence of three different monoclonal antibodies to P-glycoprotein (C219, C494 and C32) developed by ELISA.
FIG. 2 shows schematically the epitope location of the three monoclonal antibodies C219, C494 and C32 on P-glycoprotein (hamster pgp2). Also shown are twelve putative transmembrane domains numbers 1-6 and 1'-6'. The two proposed A and B ATP-binding domains are indicated by the shadowed squares. The sites and the amino acid sequences (epitopes) recognized by the three monoclonal antibodies are indicated by arrows. The peptide number corresponds to the hexapeptides of FIG. 1. The absorbance signals from the ELISA are indicated as a relative value with 5 pluses representing the highest absorbance with a particular antibody.
FIG. 3 shows a comparison of the potential C219, C494, and C32 binding sequences among P-glycoprotein gene family in hamster, human, and mouse. The P-glycoprotein tandem repeats (as illustrated in FIG. 2) are referred to as C- and N-terminal domains. The potential antibody binding sequences from the different gene members in hamster, human, and mouse have been classified based on the hamster numbering system. The notation in the closed brackets correspond to the designation given to each gene member. Bold letter amino acids for each epitope indicate the position of the critical residues for antibody binding.
FIG. 4 shows a comparison of antibody binding to peptide analogues of their binding sequences. The different peptide analogues for C219, C494, and C32 have been tested for their binding to their respective antibodies. The values of the substrate absorbance at 405-630 nm have been converted to plus signs as in FIG. 2. The species origin and the gene member of the P-glycoprotein gene family as found in the literature are indicated in the closed brackets. The bold letter amino acids represent some of the different amino acids between the analogue and the antibody binding sequence.
FIGS. 5a-f show epitope specific staining of P-glycoprotein isoforms in relation to normal hamster tissues. FIG. 5a shows ascending colon stained with C494 mAb, and FIG. 5b shows the same system in the presence of the peptide KPNTLEGNVKC. FIG. 5c shows adrenal gland stained with C32 mAb, and FIG. 5d shows the same system in the presence of the peptide GDNSRVVSQDEIERAAC. FIG. 5e shows skeletal muscle from the chest wall stained with C219 mAb, and FIG. 5f shows the same system in the presence of the peptide VVQEALDKAREGRTC.
The development of multidrug resistant tumour cells during malignant progression is thought to be a major factor contributing to non-response in chemotherapeutic treatment of cancer. The increased expression of the membrane P-glycoprotein (relative molecular mass, Mr, 170,000) is the most consistent change observed in multidrug resistant cells in vitro, and it has been demonstrated by gene transfer studies to be causative of multidrug resistance (MDR). P-glycoprotein (Pgp) is made up of a tandem repeat containing twelve potential transmembrane domains and two putative cytoplasmic ATP binding sites (see FIG. 2). The role of P-glycoprotein as an energy dependent efflux pump was proposed from its primary sequence and structural similarity to many membrane-associated transport proteins, most notably the bacterial transport protein, hemolysin B (Gerlach, J. H. et al., Nature 324, 485-489 (1986), Gros, P. et al., Cell 47, 371-380 (1986)). The presence of P-glycoprotein has been demonstrated in a variety of human malignant tumuors. However, whether or not P-glycoprotein predicts non-response to chemotherapy remains a critical issue and requires more correlative studies (Ling, V. J. Nat. Cancer Inst. 81, 84-85 (1989)). P-glycoprotein is also found in certain normal tissues, including large intestine, adrenal glands, kidney, liver and brain. The expression of Pgp in different tissues had led to the speculation that it may play a role in normal detoxification and transport of lipophilic molecules.
Recent data indicate that P-glycoprotein is encoded by a family of three genes in rodents, and two in human (Ng, W. F. et al. Mol. Cell. Biol. 9, 1224-1232 (1989)). A comparison of the amino acid sequences among the different gene family members, or isoforms, indicate a similar overall structure (Endicott, J. A. et al. Mol. Cell Biol. 7, 4075-4081 (1987)). However, transfection studies using full-length cDNAs have suggested that only some of the Pgp isoforms confer a MDR phenotype on otherwise drug sensitive cells. The class I and II isoforms have been directly implicated in drug resistance, while the function of the class III isoform is not known (Ng supra).
Immunohistochemical staining of Pgp expression has the advantages of single cell localization and detection of polarized distribution. Until now, however, the monoclonal antibodies (mAbs) used could not address the question of differential gene expression. In relation to the present invention, the epitopes of three P-glycoprotein-specific monoclonal antibodies have been mapped to a resolution of a single amino acid. Monoclonal antibody C494 is gene specific, binding to a sequence present only in the class I isoform of hamster and human. The monoclonal antibody C32 recognizes a sequence that is conserved in hamster class I and II isoforms In contrast, the monoclonal antibody C219 recognizes a highly conserved amino acid sequence found in all Pgp isoforms characterized to date. In accordance with the invention, the epitope specific staining of these antibodies in immunohistochemical studies was confirmed by competition with specific peptides Using such reagents to examine Pgp expression in normal hamster tissues, it was observed that colonic epithelial cells express predominantly the class I isoform in a polarized manner, adrenal cortical cells express predominantly the class II isoform, while only a small percentage of skeletal muscle fibers express P-glycoprotein, the class III isoform. The occurrence of this isoform in muscle is unexpected, and suggests the presence of a specialized subset of muscle fibers which have not been identified previously.
The epitope sequences of the three monoclonal antibodies C219, C32, and C494, were determined using a synthetic strategy devised by Geysen and his coworkers (Proc. Natl. Acad. Sci. USA 81, 3998-4002 (1984)). Overlapping hexapeptides covering the entire 211 amino acid fragment from the C-terminal cytoplasmic domain (shown previously to contain all three mAbs binding sites (Riordan, J. R. et al. Nature 316, 817-819 (1985))) Were synthesized on polypropylene pins (see Materials and Methods). Each successive pin contained the last five residues of the preceding one and the following amino acid in the sequence (FIG. 1). A library of about 250 peptides was screened by ELISA with each monoclonal antibody to determine its binding sequence C219 mAb reacted with two peptides (198 and 199), and only these peptides yielded a strong signal out of the peptides tested. The monoclonal antibody C494 reacted with four successive peptides giving a stronger signal than C219 mAb. In contrast, C32 mAb binding to synthetic hexapeptides occurred over two peaks. Although the binding of the antibody C32 to peptides 119, 120 and 125-129 resulted in weaker signals than that seen for the other two antibodies, it was nevertheless reproducible, and significantly above the background.
The results tabulated in FIG. 2 represent the relative intensity of the ELISA signal obtained for the binding of the three monoclonal antibodies to the generated synthetic peptides. The movement of the six amino acid window defines the boundaries of the continuous epitopes, and identifies some of the amino acids that are critical for binding. For example, the antibody C219 bound strongly to the amino acid sequence (in single letter code) VQEALD with val506 and asp511 representing two critical amino acids required for antigen recognition. C494 mAb bound to four hexapeptides covering the amino acid sequence KPNTLEGNV with thr323 and glu325 as the critical residues. Differences in the number of hexapeptides involved in antibody binding using this assay are likely related to the size of the epitope recognized by a given antibody, and the number of residues (distance) between the critical amino acids in a given epitope. The C32 mAb binding domain covers a stretch of thirteen amino acids (GDNSRVVSQDEIER), with four critical amino acids associated with its binding, asp427 and val431 from the first domain, and glu436 and glu438 from the second. The weak signals seen for C32 mAb in FIG. 1 was due to the small size of the synthetic hexapeptides used to probe for its epitope. A comparable ELISA signal to that seen with C494 mAb was obtained with C32 mAb when the complete sequence of thirteen amino acids (GDNSRVVSQDEIER) was synthesized on a pin (results not shown). A similar extension of the peptide sequences for C219 and C494 epitopes, however, did not result in an increased signal.
The amino acid sequences of the antigenic peptides from FIG. 1 were located on the full structure of P-glycoprotein as shown in FIG. 2. The antibody C219 binds to an amino acid sequence six residues away from the consensus sequence of the B site of the proposed ATP binding domain. A homologous amino acid sequence for C219 mAb is also found in the N-terminal half of Pgp. The epitope recognized by C494 mAb is on the other side of the ATP binding domain to that of C219. C32 monoclonal antibody binds to a region positioned between the A and B sites of the postulated ATP binding domain. However, the N-terminal half of Pgp does not contain a homologous sequence for the C494 or C32 epitope.
FIG. 3 shows a comparison of the antibodies binding regions among the different members of the Pgp gene family characterized to date from hamster, human, and mouse. The C219 binding site is conserved in both the C- and N-terminal halves of all Pgp isoforms. The finding that C219 epitope sequences are conserved in all mammalian P-glycoproteins points to the usefulness of this antibody as an immunological probe to quantitate the levels of P-glycoprotein in cell lines and tumour tissues. Moreover, since the epitope for C219 is found in both halves of the P-glycoprotein molecule, in different gene members, and in different species, it must have been conserved through evolutionary history. It is interesting that the bacterial transport protein hemolysin B which is structurally very similar to P-glycoprotein, especially in the ATP binding domain, does not contain this particular epitope. Thus, this epitope sequence in P-glycoprotein may represent a functional difference between these highly homologous proteins.
The C494 epitope is found only in the human (mdr1) and hamster (pgp1) class I gene product. The monoclonal antibody C494 does not recognize the other Pgp isoforms since their analogous sequences contain a substitution of a critical amino acid (thr323). This finding demonstrates the specificity of the antibody C494, and argues for its use as a class I gene specific probe. In vitro transfection studies have demonstrated that human class I gene (mdr1) can confer MDR in a variety of cells. Thus, positive staining with the C494 antibody may be diagnostic of cells expressing the MDR phenotype in human tissues.
A comparison of sequences analogous to the C32 mAb binding site from the different members of the Pgp gene family reveals that the hamster class I and II molecules have the requisite amino acid sequence for this monoclonal antibody. This sequence differs from those of the other P-glycoproteins characterized to date at a critical amino acid (glu438 to val438). Therefore, this antibody is expected to bind very strongly to hamster class I and II isoforms, but only weakly to hamster class III isoform and the Pgp isoforms of human and mouse.
FIG. 4 lists the relative signals from the ELISA tests performed using analogous peptide sequences from the different Pgp isoforms. The binding of C219 mAb to the peptide sequence VQAALD in the N-terminal half was stronger than that seen for the epitope sequence VQEALD in the C-terminal half, due to a single amino acid substitution (ala substituted for glu508). The substitution of val for glu508 in the N-terminal half of human class I Pgp isoform (VQVALD) reduced its binding to the antibody (FIG. 4). The ability to immunoprecipitate a protein fragment containing the N-terminal ATP binding domain from an in vitro expression system with C219 antibody (Endicott, Georges, and Ling unpublished observation) further corroborate the results seen using the synthetic peptides.
The specificity of the monoclonal antibody C219 was investigated further using a series of synthetic hexapeptide analogues starting with the C219 epitope sequence and replacing in turn each residue by one of the other 19 genetically coded amino acids (results will be detailed elsewhere). For example, it appears that the third position in the C219 epitope sequence (VQEALD) can tolerate a number of substitutions such as val, ala, or asp without a great loss in binding. However replacement with other amino acids such as his, gly, lys, or pro resulted in minimal or no binding to the antibody. A search through the Genbank database for amino acid sequences similar to that of C219 epitope sequence (VQEALD) revealed an amino acid sequence VQHELD in myosin heavy chains (rat cardiac muscle), and the sequence VQEALE in DNA polymerase (Bacteriophage T4). These sequences are not recognized by the antibody C219 since such amino acid substitutions did not produce peptides that bound to the antibody. Myosin heavy chain protein used in the molecular weight standards (Amersham) mix did not stain with C219 mAb by Western blot analysis.
The results from the ELISA for the binding of peptide analogues of the C494 epitope indicated that a substitution of a single critical amino acid (thr323) to trp in class II hamster (pgp2), or lys in class III hamster (pgp3) and human (mdr3) results in complete loss of antibody recognition. These results confirm that the monoclonal antibody C494 is a specific immunological probe for the expression of human (mdr1) and hamster (pgp1) P-glycoprotein class I isoform (FIG. 4). Analogues for only part of the C32 mAb binding domain (SQDEIER) were synthesized since the remainder of the same antibody binding domain is conserved among the different members of Pgp gene family (FIG. 3). The values tabulated in FIG. 4 demonstrate the effect of a single critical amino acid substitution on the binding of C32 mAb to this peptide (e.g. val substituted for glu438 fail to bind the antibody). Therefore, the monoclonal antibody C32 should bind most strongly to hamster class I and II products, and much more weakly to the other Pgp isoforms. In agreement with the above conclusion, previous results have demonstrated that hamster P-glycoprotein binds more strongly to C32 mAb than that of mouse and human using a Western blot technique.
An immunohistochemical study of normal hamster tissues was undertaken, in which serial sections of each tissue were incubated with each of the three mAbs, either in the absence or in the presence of the peptide containing the respective epitope sequence. Specific staining by monoclonal antibody against Pgp was defined as staining that could be completely abolished by competition with a 100-fold molar excess of the peptide containing the antibody epitope. This method allows for the positive identification of epitope specific staining, and is in contrast to conventional immunohistochemical staining whereby an "irrelevant" antibody is used as a negative control.
Uncompetable staining with all three antibodies was frequently observed. Notably, this included strong membrane staining of epithelial cells of seminal vesicles, distinct staining of cells of colonic crypts, and moderate intracytoplasmic staining of hepatocytes. Such staining is likely due to non-specific interactions with the antibody through regions other than those containing the paratope domain. Thus, the use of peptides in a competitive binding assay can clearly result in enhanced specificity and sensitivity of the immunohistochemical procedure.
The epitope mapping studies predict that three distinct patterns of reactivity with the mAb panel would result from the expression of each of the three hamster Pgp isoforms (Table I). The staining of intestinal tissue sections with C494 mAb alone (FIG. 5a), and in the presence of a peptide containing the C494 mAb epitope (FIG. 5b) revealed a strong signal for the luminal surface of colonic epithelial cells which is abolished when the free peptide was added. A similar staining is observed with C219 and C32 mAbs (results not shown). This staining pattern is consistent with the predominant expression of Pgp1 isoform (see Table I). The immunohistochemical staining shows that the class I isoform has a polarized distribution in the colonic epithelial cell membrane. This pattern of pgp isoform expression are consistent with a membrane transport function, possibly for the active extrusion of toxic molecules.
TABLE I______________________________________Epitope Distribution of Pgp IsoformsP-glycoprotein isoforms EpitopesClasses C219 mAb C32 mAb C494 mAb______________________________________I (Hamster pgp1) + + +II (Hamster pgp2) + + -III (Hamster pgp3) + - -I (Human mdr1) + - +III (Human mdr3) + - -II (Mouse mdr1) + - -III (Mouse mdr2) + - -______________________________________
FIGS. 5c and 5d show the staining of adrenal tissue sections with C32 mAb in the absence, and presence of its epitope, respectively. The plasma membrane of adrenal cortical cells is stained with C32 and C219 mAbs only, indicating the predominant expression of the class II Pgp isoform (see Table I). Previous reports of P-glycoprotein expression in the adrenal gland have raised the possibility that Pgp may be involved in the transport of corticosteroids. In this study the detection of Pgp in the steroid producing zona fasciculata and zona reticularis, agree with the above hypothesis. However, cortical cells in the zona glomerulosa, also known to produce steroids, do not express detectable levels of P-glycoprotein and this suggests that Pgp is not a general mechanism for secreting steroids, but that it may be a marker of differentiation of specialized cortical cells. Since the immunohistochemical technique described here is semiquantitative, it was not possible to determine if tissues exhibiting the class I pattern of staining also expressed much lower levels of the other two isoforms. Similarly, tissues exhibiting the class II staining pattern may also express the class III isoform at low levels.
Serial sections of hamster skeletal muscles incubated with C219 mAb (FIG. 5e) stained approximately 5% of muscle fibers. The staining signal of these fibers was completely abolished in the presence of the peptide containing C219 mAb epitope (FIG. 5f). Staining of skeletal muscle with C32 or C494 did not reveal epitope specific staining. This staining pattern is specific to a class III isoform expression alone (Table I). The expression of the class III isoform was previously observed in Northern blot analysis (mdr2 positive) of mouse muscle tissue, and Western blot analysis (C219 positive and C494 negative) of human muscle tissue. However, it could not be determined in those studies that the expression of the class III isoform was localized to a subset of muscle fibers as shown here. Skeletal muscle from a variety of locations (chest wall, paraspinal, diaphragm, thigh, and anterior abdominal wall) show two distinct types of fibers, with the majority of fibers lacking detectable amounts of Pgp while less than 10% of fibers express high levels of the class III isoform. The distribution of this isoform within muscle fibers follows a fairly regular, coarse, transverse pattern, in addition to patchy areas of plasma membrane staining. Ultrastructural studies are ultimately required to localize Pgp to subcellular structures in skeletal muscle fibers, but it is likely a membrane component, for example, a component of T-tubules. The finding that the class III isoform is expressed only in a subset of muscle fibers is unexpected. It could be speculated that muscle fibers expressing the class III Pgp isoform perform specialized function not previously recognized. For example, it may be involved in the energy-dependent transport of metabolites across the complex membrane system of some skeletal muscle fibers.
Competition experiments with hexapeptides precisely mapped the epitopes of the three different monoclonal antibodies against P-glycoprotein. The monoclonal antibodies were shown to bind to "continuous" epitopes consisting of critical amino acids located within a few residues from each other. The fact that all these epitopes are of this type may be due to the original screening (SDS-denatured proteins in a dot blot assay) for these antibodies. However, all these mAbs can bind to the native form of the P-glycoprotein. It is likely that this mapping technique will be applicable for defining epitopes of other mAbs generated in this manner.
The expression of P-glycoprotein in normal tissues and cell lines has been analyzed using various biochemical techniques. Immunohistochemical staining of tissues has the advantages of detecting polarized expression, as well as, the simultaneous visualization of P-glycoprotein in tissue sections containing both normal and tumour cells. However, as in any immunohistochemical detection assay, a high level of staining specificity is sometimes compromised in an effort to increase sensitivity. In this study a high level of staining specificity and sensitivity has been achieved through the combined use of monoclonal antibodies with epitope-specific peptides in a competitive immunohistochemical assay. For example, as pointed out earlier, non-competable staining was frequently observed in many tissues, and this was assumed to be due to nonspecific interaction. The staining of a subset of muscle fibers with C219 mAb in muscle tissue was unexpected and this was initially assumed to be due to crossreactivity of the mAb with myosin (Thiebaut, F. et al. J. Histo. & Cyto, 37, 159-164 (1989)). However, in the present study epitope specific peptides were used in a competitive immunohistochemical analysis which demonstrated that this staining is most likely due to the presence of the class III Pgp isoform in a subset of muscle fibers. Previous immunohistochemical studies of P-glycoprotein expression in normal tissues and tumour samples have provided insight into the cellular localization of this membrane glycoprotein, however it has not been possible to distinguish what classes of Pgp isoforms are expressed at the protein level. The results of the epitope mapping studies described here suggest that the three classes of mAbs, represented by C219, C32, and C494, may be used in combination to determine the pattern of expression of the Pgp isoforms in normal and tumour tissues. This may have applications in studies for investigating the correlation of P-glycoprotein expression with patient response to cancer chemotherapy.
Prederivatized plastic pins and polypropylene trays were obtained from Cambridge Research Biochemicals (Valley Stream, N.Y.). Active esters of Fmoc amino acids were supplied by MilliGen (Millipore, Bedford, Mass.). Glass-distilled dimethylformamide (DMF) was purchased from Anachemia (Montreal) and kept on 4 A molecular sieves at 4� C. for 2 to 3 weeks. The amine content was monitored by the dinitrofluorobenzene assay. Only DMF giving an absorbance value at 381 nm below 0.10 as compared to a reagent blank was used in the synthesis. Reagent grade dichloromethane (DCM) and methanol (MeOH) were purchased from BDH Chemicals (Montreal) and used without further purification. Other suppliers are listed as follows: piperidine from Fisher Scientific (Toronto, Ontario), PAM resins from Applied BioSystems (Foster City, Calif.), Boc amino acids from IAF Biochemicals (Montreal, Quebec), dicyclohexylcarbodiimide (DCC) from Aldrich Chemical Co. (Milwaukee, Wisc.), sequencing-grade trifluoroacetic acid (TFA) and diisopropylethylamine (DIEA) from Chemical Dynamics Corp. (South Plainfield, N.J.).
Overlapping hexapeptides were synthesized on polyethylene pins as described earlier (Geysen, H. M. et al. supra). The peptides were assembled on the pins in the C-to-N terminus direction using 9-fluorenylmethyloxycarbonyl (Fmoc) protected amino acids. Briefly, the plastic pins were arranged on polypropylene block supports in a pattern suitable for soaking the tip of each pin into individual well of 96-well polypropylene plates. The pins were prederivatized with a non-detachable 15-atom long spacer ending with a Fmoc-b-alanine group. All steps in the synthesis were performed at room temperature. The pin blocks were initially soaked for 30 minutes in a 20% (v/v) piperidine/DMF bath to remove the Fmoc group generating a free terminal amino group. The pins were then cycled through the following steps: DMF washes (2 times; 2 minutes), methanol washes (3 times, 2 minutes), the pins were air-dried for 15 minutes, soaked in DMF (5 minutes) and blotted gently with tissue paper. The preformed active esters (oxobenzotriazine esters for serine and threonine; pentafluorophenyl esters for all other amino acids) of Fmoc amino acids (30 mM) were then dissolved in DMF containing 1-hydroxybenzotriazole (30 mM). The solutions were then dispensed immediately in the appropriate wells of 96-well polypropylene plates. The coupling step was initiated by placing the tip of the pins in their respective wells. The pin blocks and the polypropylene plates were carefully placed in sealed plastic trays and the coupling reactions were left to proceed overnight. The Fmoc deprotection and subsequent steps were then repeated until all hexapeptides were completed. The final Fmoc group on the completed peptides was removed as described above and the resulting free amino group was acetylated (DMF: acetic anhydride: diisopropylethylamine 50:5:1 (v/v/v); 90 minutes). The side-chain protecting groups of all peptides were simultaneously cleaved by soaking the pins in trifluoroacetic acid:phenol:ethanedithiol 95:2.5:2.5 (v/v/v) for 4 hours. The pins were successively washed with dichloromethane (twice for 2 minutes), neutralized with 5% DIEA/DCM (twice for 5 minutes), followed by single dichloromethane wash (5 minutes), air dried (15 minutes), wetted in water for 2 minutes, and soaked in methanol overnight. Remaining traces of solvent were evaporated under vacuum and the blocks were stored in plastic containers at room temperature in the presence of dessicant. Amino acid analysis was performed on ten pins. The peptide substitution per pin ranged in value from 2 to 4 nanomoles.
Peptides coupled to solid support polypropylene pins were incubated in phosphate buffered saline, pH 7.4 (PBS) for 30 min. at room temperature. Pins were then soaked for 1 hour in a blocking buffer (1% w/v ovalbumin, 1% w/v bovine serum albumin (BSA), 0.1% v/v Tween 20 in PBS) to reduce non-specific adsorption of antibodies. Alternatively, the wells of a 96-well ELISA plates were coated with solutions of the free peptides dissolved in PBS. The plates were incubated overnight at room temperature and washed once with PBS. All unreacted sites were blocked with 3% BSA solution. The pins were incubated overnight at 4� C. in wells containing 100 μl aliquots of primary antibody solutions (0.5-2.0 μg/ml dissolved in coating buffer). After one hour incubation with peroxidase conjugated goat anti-mouse antibody, pins were washed four times with PBS solution containing 0.05% (v/v) Tween 20. The binding of antibody to peptides was detected by incubating the pins for 30 minutes with a freshly prepared solution of azino-di-3 -ethylbenzthiazdinsulphonate (ABTS) in 1M citric acid pH 4.0. Measurements of color development were made at 405-630 nm using the microplate reader (EL30 Bio-Tek instrument). Antibodies bound to the pins were stripped off the solid-supports by sonicating the pins for 30 minutes at 65� C. in 0.1M sodium phosphate solution containing 1% sodium dodecyl sulfate (SDS) and 0.1% (v/v) 2-mercaptoethanol. The pins were rinsed in warm distilled water (50�-60� C.) and finally washed in a bath of boiling methanol.
Normal adult Chinese hamsters were sacrificed in a carbon dioxide chamber. Organs and tissues were removed at 4� C. within 1 hour of sacrifice, and frozen in isopentane chilled with dry ice. Frozen sections (8 μm) were cut and fixed in cold acetone (10 minutes at 4� C.), then stained for Pgp using an avidin-biotin-peroxidase complex technique. The primary antibody (C219, C32, or C494) was used at 10 μg/ml in 1% BSA/PBS. One hour prior to incubation of tissue sections with the primary antibody, the antibody solution was pre-incubated with 100-fold molar excess of either the peptide encoding the antibody epitope or an irrelevant peptide. This sufficiency of excess peptide was based on a competitive binding assay in which Pgp immobilized on nitrocellulose filter was allowed to react with each mAb in the presence of increasing concentrations of either the peptide encoding the mAb epitope or an irrelevant peptide. Competitive binding of the primary antibody to the peptide and to Pgp present in tissues proceeded for 1 hour at room temperature in a humidified chamber. The sections were washed with PBS and incubated sequentially with biotinylated horse-antimouse antibody and with avidin-biotin-peroxidase complex (Vector Laboratories, Burlingame, Calif.) according to manufacturer's instructions. The binding of the antibody to tissues was detected by 5 minutes' incubation with 3,3'-diaminobenzidine tetrahydrochloride (1 mg/ml; Sigma) and hydrogen peroxide (0.003%). Tissues were counterstained with hematoxylin, dehydrated and mounted in Permount.
|Endicott et al. Mol. Cell Biol. 7, 4075 4081 (1987).
|Endicott et al. Mol. Cell Biol. 7, 4075-4081 (1987).
|Geysen et al. PNAS 81:3998 4002 (1984).
|Geysen et al. PNAS 81:3998-4002 (1984).
|Riordan et al. Nature 316, 817 819 (1985).
|Riordan et al. Nature 316, 817-819 (1985).
|Citing Patent
|Filing date
|Publication date
|May 20, 1997
|Aug 6, 2002
|Chiron Corporation
|Hepatitis C virus multiple copy epitope fusion antigens
|Jun 14, 2000
|Aug 20, 2002
|Syde A. Taheri
|Autogenous cell patch cardio myoplasty and advanced muscle graft implantation system
|Jun 17, 2002
|Jun 6, 2006
|Chiron Corporation
|Multiple epitope fusion protein
|May 18, 2007
|Jan 3, 2008
|Xiaomin Fan
|Methods for the identification and the isolation of epitope specific antibodies
|U.S. Classification
|435/7.93, 435/7.1, 435/5
|International Classification
|Cooperative Classification
|European Classification
|Jan 25, 1993
Owner name: ONTARIO CANCER INSTITUTE, THE, CANADA
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST.;ASSIGNORS:LING, VICTOR;GEORGES, ELIAS;REEL/FRAME:006404/0652
Effective date: 19890801
|Nov 25, 1996
|Fee payment
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|Dec 22, 2000
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|Dec 17, 2004
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By A Customer on Dec 15 2000
Perfect for the living room voyeur. This is what a lot of guys imagine when visiting the gym and seeing an attractive woman working out. They're lifting weights. They're doing squats. They're doing sit-ups. And what's that... they're naked?! The video features three reasonably fit women who take you thru a series of instructional exercise clips. It opens with the three of them in aerobics outfits doing warm-ups. Then it goes into the individual segments, featuring a single girl doing a particular exercise. They start with some pectoral exercises. The blonde is lying on her back on the bench with small weights in her hands. In a surprising and pleasing shift from the opening segment, she's suddenly wearing no clothes! The female narrator talks calmly thru the exercise, while the camera shows her demonstration, which happily includes close-ups of her bare breasts. This isn't porn per se; nobody struts in front of the camera or spreads their legs. But there are plenty of full-frontal nude shots, and the woman aren't shy about trying to hide anything. To finish things off, the closing segment features all three doing standing stretches and cool-down exercises in the nude. I know the video was supposed to be instructional, but it only left me wanting instructions on how to get the sequel!
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By A Customer on July 30 2000
I liked the other three video productions in this series but this one was cheap compared to them. I like the fact the other videos had an integrity and obvious benefit to veiwers and the fact that the models were nude added to the sexiness of the videos. In this one the women introduce themselves and pose while doing some of the exercises. To me , that cheapens the production and takes away the value of the video. It's more like a porno. If they would maintain the standards of Nude Yoga, Nude Tai Chi, and Nude Stretching videos, I would have been much more impressed.
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By Jeffrey Strain on June 2 2002
I jump rope to this tape with soundturned down. I listen to music asthe tape plays and am totally distracted from the monotony of exercise and the pain in my calves.All three girls are beautiful andthe photography avoids the sleazinessof Nude Tai Chi and Nude Stretching,which have all the class of any issueof Hustler. Nude Personal Training, Nude Yoga andNude Cigar Smoking are the best of theseries.
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By A Customer on Oct. 5 1999
Unlike the other "Nude" exercise tapes that chew up a third of their running length promoting other tapes in the series, you get a full half-hour's worth of three gorgeous models...naked from the ankles up..working out with hand weights and doing light calisthenics. The production quality is top-notch, body display is good, and the girls actually have pubic hair - imagine that!
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Excellent exercise video. The women in this video not only have the nice bodies, they're also the real life aerobic instructors. Very professional. Full nudity.Published on Feb. 15 2002
While the production quality is good, the females act like zombies following some forced script. There is no sense of their being present or spontaneous, so for me this fails as... Read morePublished on May 30 2000
I like this video because is very resursefull and gives you same ideeas how you can do it,and wherePublished on Feb. 22 2000 by george777
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I have a free ping service - it keeps saying that the site is up and then down.
When I visit the site and ask friends to, and also check via "is it down for me" the site is up?
Why would the ping service report false positive?
Any ideas? Is it perhaps .htaccess script? Ideas?