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Download aFileDialog Demo apk 1.0.7 free for Android smartphone
Posted by KudaLab in Tools | May 20, 2016 | 6 Comments
Apk file size: 79.0 KB
Demo application for "aFileDialog", an Android library which implements a simple and easy to use file chooser. More details in http://code.google.com/p/afiledialog/
Whats new
Folder filters implemented
KudaLab part of our Tools and have average installs from 500 to 1000. Last Update May 20, 2016. Google play rating is 91.1111. Current verison is 1.0.7. Actual size 79.0 KB.
Similar applications
6 Comments
Miguel Castellanos
Great!, it does what it have to do Just what I was looking for!!!
Jose F. M.
A good library Does everithing what promises and it is easy to use
Vincent J
Great This is a great library. Maybe it could benefit from a newer design
nony bright
Very luvly I will need dis...thnx a lot
Muhammad Saqib Sabir
Thanks Alot! Well documented, The Easiest one, Both dialog and Activity supported, live Demo on google play. (you rocks man), FREE / No Ads / No Limit... I'll definitely mention your reference/name in my app (whenever it will be ready to publish). Your are doing great job. Please continue.
Muhammad Saqib Sabir
Please help me to change it's theme from dark to light.


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This property has been removed by the agent. It may be sold or temporarily removed from the market
3 bedroom semi-detached house for saleChase Cottage, 21 New Road, Woolmer Green, Herts
Property Description
Key features
- 1051 sqft three bedroom semi detached house arranged over three floors
- Sought after village location
- Open plan kitchen/dining room
- Contemporary style kitchen with integrated appliances
- Bi-folding doors opening onto the rear garden
- Designer fitted bathrooms
- Rear garden laid to lawn
- Off street parking
- Great road and rail links
- Excellent range of local schools
Full description
Tenure: Freehold
Situated in the idyllic and much sought after village of Woolmer Green this three bedroom semi-detached home is arranged over three floors with the ground floor providing living room, a beautifully appointed kitchen/diner with integrated appliances and bi-folding doors opening onto the rear landscaped garden. On the first floor there are two double bedrooms one with en-suite and a family bathroom and a further double bedroom and en-suite wc on the second floor.
There are day to day amenities within a short walk and with Knebworth station in the neighbouring village, London's Kings Cross is only a 30 minute journey. The A1 provides road links to both London and surrounding areas that provides easy access to an excellent range of catchment schools.
Additional Information
For further information or to arrange an appointment to view, contact 01992 663890.
Property ref: 121_2309_4219414
More information from this agent
To view this media, please visit the on-line version of this page at www.rightmove.co.uk/new-homes-for-sale/property-44141100.html?premiumA=true
Listing History
Map & Street View
View in fullscreenView in fullscreen
Street View is unavailable in this location
Market Info
Disclaimer - Property reference 4219414. The information displayed about this property comprises a property advertisement. Rightmove.co.uk makes no warranty as to the accuracy or completeness of the advertisement or any linked or associated information, and Rightmove has no control over the content. This property advertisement does not constitute property particulars. The information is provided and maintained by Lanes New Homes, New Homes Hertfordshire. Please contact the selling agent or developer directly to obtain any information which may be available under the terms of The Energy Performance of Buildings (Certificates and Inspections) (England and Wales) Regulations 2007 or the Home Report if in relation to a residential property in Scotland.
Map data ©OpenStreetMap contributors.

subset: commoncrawl


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tag:blogger.com,1999:blog-52406430730745197012017-09-15T01:56:07.531-07:00advice and suggestionsyou can criticisms and suggestions at here. From hobbies some info include health, technology, entertainment, education get more money tooMinatushttp://firstname.lastname@example.com:blogger.com,1999:blog-5240643073074519701.post-86126313349206525692016-05-18T00:14:00.001-07:002016-05-18T00:14:48.760-07:00Kitchen Cabinets Renovation Ideas
https://youtu.be/obJQeAl1Qwc Kitchen Cabinets Renovation Ideas You who love to re renovating your kitchen with a concept and design that is different from the previous one, if you are very easily bored, you can renovate your kitchen with different types of designs that can be applied in your kitchen there. kitchen cabinets For those of you who love to cook, then you will spend your time in the kitchen longer to explore new dishes that you can practice at home. For those of you who like to cook but you very easily bored with a kitchen that was always so-so alone, you can apply some kitchen design that is easy to implement into your kitchen. kitchen renovation With some changes and also renovated room kitchen then you will get a new atmosphere in your kitchen to start creating some new dishes that you can serve to your family. kitchen ideas The following are some designs for you who want to renovate the kitchen, survivors try it at home! #kitchencabinets #renovationideas
Aji maranathahttps://firstname.lastname@example.com:blogger.com,1999:blog-5240643073074519701.post-138495097135229022016-05-18T00:12:00.001-07:002016-05-18T00:13:16.996-07:00Small Kitchen Appliances Renovation Ideas
https://youtu.be/aQcLqxSElhY Small Kitchen Appliances Renovation Ideas Own the land that is not so wide to serve as a shelter, you should take advantage of thorough and meticulous in every detail there is. You also have to pay attention to every corner in order to be used properly. If you only have enough land to be some room in your house, you have to arrange and design each room well so that does not make you bored and also make any room in your house into a look narrower and seemingly untidy. small kitchen For those of you who love designing or rearranging any room in the house, can start from the room is you enjoy. If you love to cook, of course, you will design your kitchen into a such a way that you do not easily get bored while in the kitchen. kitchen appliances If you have a small kitchen, but not too small, you can design it with some kitchen appliances that fit the needs, character, preferences, and also design your own kitchen. If you want to add some equipment for your kitchen, you can customize it with your kitchen design and layout on your kitchen so as not aesthetically more narrow. kitchen renovation The following are some ideas for designing the kitchen equipment that you can use as a reference for those of you who want to decorate your kitchen. Good luck!! kitchen ideas #smallkitchen #appliances #renovationideas
Aji maranathahttps://email@example.com:blogger.com,1999:blog-5240643073074519701.post-18429824205212534392016-05-18T00:11:00.001-07:002016-05-18T00:11:38.039-07:00Kitchen Remodel Design Ideas
https://youtu.be/4mibslwfino Kitchen Remodel Design Ideas Have their own dwelling is a thing that will make your life easier, because you do not have to think about going to stay where it is. You can design your dwelling freely with each corner of the room that you like and want to serve as anything. kitchen remodel For those of you who like to cook, you can easily determine a kitchen design that you will use. kitchen design You are happy with the cooking, but easily bored with the design of the room is just that-that can redesign your kitchen so you do not get bored if it is in the room that you enjoy. design ideas This way, you will easily get new ideas about the recipes that you can serve to your family back home. The following are some examples of kitchen designs have been redesigned for the needs and desires. Good luck!! #kitchen #remodel #designideas
Aji maranathahttps://firstname.lastname@example.com:blogger.com,1999:blog-5240643073074519701.post-22881141757037876432016-05-18T00:09:00.001-07:002016-05-18T00:10:22.495-07:00Small Kitchen Remodel Design Ideas
https://youtu.be/mR6ZyOW5X5Q Small Kitchen Remodel Design Ideas For those of you who have a dwelling with land that is not so wide, you can work around this by designing each room into a smaller but still comfortable to live in. small kitchen Design the meticulously and carefully so that you get a room that suits your needs. If you just rely on preferences alone, then you will only find any room that is not appropriate. kitchen remodel If you love to cook, then design the room of your kitchen better so when you're using it, you feel comfortable and not too bored to be lingering there. kitchen design If you want to make your kitchen a little more comfortable when you are cooking, then you should adjust any equipment that is there with the size of the room that is not too tight and you can also freely use it without feeling bored. kitchen ideas The following are some examples of ideas for designing or rearranging your kitchen space in the house. Good luck!! #smallkitchen #remodel #designideas
Aji maranathahttps://email@example.com:blogger.com,1999:blog-5240643073074519701.post-80333416947214009322016-05-13T00:02:00.001-07:002016-05-13T00:03:06.564-07:00Funny Cats Video Watching for A Thing

https://youtu.be/sB-BYxFNohM Funny Cats Video Watching for A Thing Have a pet at home will make you happy, because with your pet will make your pet happy with this, are you tired when I got home would be lost when your pet look cute and adorable. Cute and adorable animals that may be kept by most people is the cat, because it is funny and lazy, makes everyone happy to see it and maintain it. funny cat video If you are a fan of cats and have it at home, you'll be happy to take care of and also make your cat feel pampered by their owners. You can also document the activity cute your cat at home. cat video watching The following are some of the cute cat videos that you can make as a spectacle at home. #funnycat #watching
Aji maranathahttps://email@example.com:blogger.com,1999:blog-5240643073074519701.post-90037751378851372852016-05-12T23:59:00.001-07:002016-05-13T00:00:19.242-07:00Funny Cats Video Laying in The Bed Couch And Mum
https://youtu.be/OeN1HXJNNyQ Funny Cats Video Laying in The Bed Couch And Mum Watch videos of animals, especially cats are very cute with her behavior is very adorable and it is spoiled. funny cats video Animals synonymous with laziness makes us happy to see the video and acting silly, if you like cats, all activities will invite the attention of your cat when he saw it. laying down Cats lazing will make you happy with the acting weird but still funny. The following is being lazy cat video that you can watch and you make a video collection of cute cat in your home. #funnycat #layingdown
Aji maranathahttps://email@example.com:blogger.com,1999:blog-5240643073074519701.post-87312923439308951902016-05-12T23:58:00.001-07:002016-05-12T23:58:52.795-07:00Funny Cats Trying To Eat Something
https://youtu.be/AqpY1ruFcGA Funny Cats Trying To Eat Something Watch videos cat was eating, very funny if you see it well. You will be pleased when watching videos cat was eating. funny cat video If you have a cat at home or you're maintain it, you'll easily find your cat when hungry and you gave him food. Or maybe you can also teased by providing goods that easily attracts attention. cat video eating You will feel comforted by his behavior funny when you're eating, you can easily find your cat while it is seducing you when you're hungry. The following are some of the compilation of videos made for you who love the cat was eating. cat trying to eat something #funnycat #eating
Aji maranathahttps://email@example.com:blogger.com,1999:blog-5240643073074519701.post-60589982067524499792016-05-12T23:56:00.001-07:002016-05-12T23:57:18.519-07:00Funny Cats Are Playing Around
https://youtu.be/x1xjH5i42z4 Funny Cats Are Playing Around Watch funny videos of cats, for those who just love cats would be very nice to do it at home while relaxing from all the activities already carried out a full day. funny cat videos If you like cats who are doing activities, you can maintain one or several cats that you can later immortalized activities. Cats can also be your best friend in the house. cat video If you like cats, but you can not keep it at home, you can watch some videos of cats on the internet. You can also download some cat videos to serve as your collection at home. If you like it, you can also collect some objects associated with cats that can make you happy. Following some cat videos that are playing that you can use as a reference in the home. cat are playing around #funnycat #playingaround
Aji maranathahttps://firstname.lastname@example.com:blogger.com,1999:blog-5240643073074519701.post-84298735343571614462016-04-29T22:48:00.001-07:002016-04-29T22:49:22.658-07:00Hydroponic System In Greenhouse
https://youtu.be/ZNYLdwrGWeU
Hydroponics System In Greenhouse By means of hydroponic gardening requires a lot of knowledge to avoid all bad possibilities that could happen. Usually, for those beginners, they will grow plants hydroponically indoors in their homes. They also usually will grow hydroponic plant them on the front porch or in the yard. For those who are already proficient and also have a lot of knowledge about growing plants hydroponic system, they will be taking to build greenhouses devoted to developing their hydroponic plants. Most of them also use their expertise to open businesses become suppliers of hydroponic crops or vegetables. Those who do this will require a lot of land, so that many of them will use the greenhouse as farming land for them. hydroponics system in greenhouse If you want to start by growing plants with hydroponics system, you can start from your home, but if you are going to open a business like most people, you can start with your greenhouse. Good luck! #hydroponics #system #greenhouse
Aji maranathahttps://firstname.lastname@example.com:blogger.com,1999:blog-5240643073074519701.post-35798332827418449932016-04-29T22:46:00.001-07:002016-04-29T22:47:22.942-07:00Garden Edging Ideas For Your House
https://youtu.be/O05SPtLsj4k
Garden Edging Ideas For Your House If you have a garden and you love to plant various types of unusual plants, you can add the edges of your garden. It serves so that your plants do not interfere with other plants and you will easily distinguish every existing plants. garden edging ideas You can also provide a useful edges so that your plants do not get out of the path of your estate. If you have some items that are not used or are able to serve the banks of the goods for your garden, you can apply easily in your garden.
#gardenedging #ideas
Aji maranathahttps://firstname.lastname@example.com:blogger.com,1999:blog-5240643073074519701.post-4291369701162282082016-04-29T22:44:00.001-07:002016-04-29T22:45:15.926-07:00Hydroponics Vegetable Plants System
https://youtu.be/821NbBya2_o
Hydroponics Vegetable Plants System planting a vegetable crop with a hydroponic system requires a lot of expertise, because you are growing vegetables with this system, you will get many benefits if it is successful, but you will also get a lot of harm if you do not succeed. hydroponics vegetable you who are familiar with the life of gardening and is very fond of vegetables, growing vegetables will be happy. you will grow vegetables by hydroponics system, but before you plant it, you have to know about all the rules so you do not have a lot of losses. If you are already proficient in gardening, especially with a hydroponics system, then you will easily apply it in your hydroponic garden. You will also get many benefits of gardening with this water media. hydroponics plants system if you do not understand it, do not hesitate to ask people who are experts in their field, you can also find some of the resources from the internet or from books. you can apply in your hydroponic system vegetable garden. Good luck!
#hydroponics #vegetable #plant #system
Aji maranathahttps://firstname.lastname@example.com:blogger.com,1999:blog-5240643073074519701.post-30873579748910193092016-04-29T22:41:00.001-07:002016-04-29T22:42:46.772-07:00Garden Edging Stone Ideas
https://youtu.be/XQaic1uk8R8
Garden Edging Stone Ideas Impose limits on the garden you can use a variety of media types. As usual most people, they will provide a barrier on their gardens with stone. garden edging If you think you have a lot of inventory stone in the house, you can set it so as to form a barrier that can limit your garden. garden edging stone If you like cleanliness and having some funds to renovate your garden, you can also make your garden with rock ledges and a mixture of several ingredients that can make the ledges not easily destroyed. If you do not have a lot of expenses, you can also set it in such a way. This enables you if you want to rearrange your garden, you do not need a lot of money to renovate it again.
#gardenedging #gardenstone
Aji maranathahttps://firstname.lastname@example.com:blogger.com,1999:blog-5240643073074519701.post-52897887338326306522016-04-26T00:21:00.001-07:002016-04-26T00:21:06.909-07:00Video Smart Cats

Video Smart Cats, not only a human being smart, animals also smart in every way. especially cats, the cat is an animal that is awesome, the animals are very cute and adorable. If employers want to train and teach a cat to do things that are funny, the cat will definitely doing happy. and over time you will become clever cat. cat like this is that you must opt in cat show. https://www.youtube.com/watch?v=OT2yJhaFFRk
Aji maranathahttps://email@example.com Vegas, NV, USA36.1699412 -115.1398295999999835.7602007 -115.78527659999999 36.579681699999995 -114.49438259999998tag:blogger.com,1999:blog-5240643073074519701.post-36805615612700977752016-03-22T02:58:00.001-07:002016-03-22T02:58:31.441-07:00fashion design todayfashion design today more hits you know , know not many clothes design her beautiful brilliant and always attract the attention of the devotees https://youtu.be/CrzqdeqGQ84Aji maranathahttps://email@example.com:blogger.com,1999:blog-5240643073074519701.post-12248835113444623202016-01-27T01:55:00.001-08:002016-01-27T01:55:55.614-08:00ideas garden border edgingedging garden is that part of a home where you can interact with nature, spend time outdoors and even gain a new hobby in the form of gardening. https://youtu.be/tzazVoypoBoAji maranathahttps://email@example.com:blogger.com,1999:blog-5240643073074519701.post-87295368032912213032015-05-26T05:07:00.001-07:002015-05-26T05:07:19.853-07:00Scooter: FRIENDLY'S LUX - INTROFRIENDLY'S LUX - INTRO one of motorbike parts details. Here motorcycles and motor bike information, happy to read FRIENDLY'S LUX - INTRO information.
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Own Your Success (Paperback Pod)
By Ben Newman, B Newman, Michael Ed. Newman
What if you could make each and every day victorious by focusing on daily activities rather than obsessing over results that you can't control? Based on author Ben Newman's popular program, "Own YOUR Success" gives you the power to make each day a triumph. The most successful people find great success when they focus on having a passion for the process. The key: make today victorious regardless of the obstacles that come your way. Figure out what fires YOU up without exception and ignite that passion so that you can routinely create your prizefighter day. Told through the eyes of an executive at the top of his game, yet the bottom of his existenceOffers original, practical, and proven exercises to transform challenges into maximum performanceIncludes real-world advice and proven strategies to help individuals in the business world achieve even greater success
"Own YOUR Success" will lead you to uncover your true potential and create a life that belongs to YOU.
1 $22.95 9781119089810 No volume discount available.
About the Paperback
|John Wiley & Sons
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Photo essay: Once-grand Gratiot Avenue in Detroit is deteriorating
More than half of the buildings on Gratiot Ave. in Detroit are abandoned.
More than half of the buildings on Gratiot Ave. in Detroit are abandoned.
As a towering claw dug into one of four remaining high-rises, Prince forlornly posed as cameras chronicled the beginning of the end for the nation's first publicly funded housing project for black people.
The 2,500-seat theater once hosted the Kinks, the Who, Jefferson Airplane, Cream, Yes, Fleetwood Mac, Steppenwolf, King Crimson, MC5 and the Stooges.
The tagger was spotted late this afternoon atop the high-rise that once hosted The Beatles, the Rolling Stones, actress Mae West and playboy Horace Dodge Jr.
The map is so precise and detailed that you can zoom in on each parcel and collect information such as owner, year built, assessment value, tax status and size.
"Out of an album fell this passport," the man said. "It literally fell into my hands."
Parks will later move to Detroit and work as an assistant to Congressman John Conyers, Jr.
The holiday was initially proposed by Rep. John Conyers (D., MI) and Sen. Edward Brooke (D., Mass) in 1979.
Once lined with opulent homes, Alfred Street in Brush Park is largely vacant, an early victim of residential flight.
The east-side home was the site of a famous confrontation between a black family and a violent white mob.


text:
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|This Just In
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|casual dresses
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A flirty one-shoulder dress with a '70s-inspired ruffle. Made of soft silk, this one easily crosses the day-to-night divide.
Free shipping and free returns exclusively for Madewell Insiders
(hi there, risk-free try-ons). *For complete details, see our Shipping Policy and Return Policy.
Silk One-Shoulder Dress
Silk One-Shoulder Dress is rated out of 5 by 11.
Rated 5 out of 5 by JLRN from A-mazing! Girls, if you haven't bought this Madewell dress yet, BUY IT! Seriously, it's amazing. I'm 5'3", 125lbs, small on top, curvy on bottom and a size 2 was perfect for me, which is what I usually buy at Madewell. I did try on the 4, but I found the top was looser than I like for my 32C chest. The fabric feels so soft! The pockets were fine. If the dress is hanging loose on you, then you don't notice the pockets. If the dress is more fitting on you, then the pockets will bulge out a bit as they did on me when I tried on the size 2.
Date published: 2016-10-25
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Date published: 2016-09-06
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Date published: 2016-09-05
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Date published: 2016-10-29


text:
This 8-day leisurely cruise visits the celebrated sights of Upper Egypt and some
of the finest monuments in ancient antiquity. Included are visits to the Valley of
the Kings, the Colossi of Memnon and the temples of Karnak and Luxor. Stay on
board the MS Mayfair, one of the finest cruise vessels operating on the Nile with
74 cabins featuring panoramic windows, some with balconies, as well as a pool,
sun deck and spa facilities.
View more climate information & imperial units
Fly from London to Luxor with Egyptair. Transfer to the MS Mayfair for a 7-night
cruise. (N)
Morning visit to the Valley of the Kings, the Temple of Queen Hatshepsut and the
Colossi of Memnon. Sail to Edfu via Esna lock. (B, L, D)
Morning visit to the Temple of Horus. Afternoon cruise to to Aswan. (B, L, D)
Morning visits to the Aswan High Dam, the Unfinished Obelisk and the Temple of
Philae. In the afternoon, take a felucca trip on the Nile. (B, L, D)
Full day at leisure. (B, L, D)
Morning visit to the temples of Sobek and Haroeris. Cruise to Luxor. (B, L, D)
Morning visit to the temples of Luxor and Karnak. Afternoon at leisure to visit
the house of Howard Carter or the Valley of the Queens. (B, L, D)
Morning transfer and fly to London with Egyptair. (B)
As per itinerary – B: Breakfast, L: Lunch, D: Dinner, N: No meals.
This tour is led in each destination en route by a local tour guide.
This tour is based in 5-star cruise accommodation.
When on board the Nile cruise, transfers and excursions
are operated as a group with other passengers who may or may not be Cox &
Kings clients.
This tour departs every Monday from 2 January 2017 to 11
December 2017.
For a price quote based on alternative accommodation
standards or flight options, or for a cruise-only price, please call 020 7808 6784
or email email@example.com
The MS Mayfair is a Superior Plus cruiser with superb facilities and excellent levels of service. There are 74 cabins on the ship including Executive and Presidential suites. Higher category cabins offer more spacious accommodation and a balcony. All cabins offer either twin or double beds and large panoramic windows. Leisure facilities include an outdoor swimming pool, two jacuzzis, cabanas and sunbeds for relaxation on the sun deck, and a spa featuring a sauna, steam room and massage suites. There is also a gift shop selling souvenirs and a library, which also has board games. The Nile Restaurant offers a fine selection of international and traditional dishes. A buffet breakfast is served daily while lunch and dinner are served à la carte. The Bel Air lounge is the perfect venue to enjoy a glass of wine or cocktail before or after dinner. There is also a pool bar for afternoon...
Read more
Regional air connections are available from within the UK. Please contact your travel consultant for current prices from your nearest city.
We can also arrange flights from overseas, simply give us a call to find out more.
If you choose to take the tour without flights arranged through Cox & Kings, please contact one of our travel consultants prior to booking the flights to ensure that you are aware of the tour timings and all the implications of not booking through us. We strongly advise you not to book flights independently of Cox & Kings if significant charges would apply in case of cancellation.
Flight details may be subject to change prior to departure.
This tour can be booked / reserved online by clicking on one of the coloured links below.
CNO2017: 7 Nights
Luxor is a modern city, full of the monuments of both ancient Egyptian and other, more...


text:
|Publication number
|Publication type
|Application number
|PCT number
|Publication date
|Feb 3, 2009
|Filing date
|May 31, 2000
|Priority date
|Jun 3, 1999
|Fee status
|Also published as
|Publication number
|Donald Gullberg
|Original Assignee
|Cartela R&D Ab
|Export Citation
|BiBTeX, EndNote, RefMan
|Patent Citations (17), Non-Patent Citations (13), Referenced by (2), Classifications (35), Legal Events (6)
|External Links: USPTO, USPTO Assignment, Espacenet
The present invention relates to a recombinant or isolated integrin heterodimer comprising a subunit α11 and a subunit β, the subunit α11 thereof, homologues and fragments of said integrin and of said subunit α11, processes of producing the same, polynucleotides and oligonucleotides encoding the same, vectors and cells comprising the same, binding entities binding specifically to binding sites of the same, and the use of the same.
Integrins are heterodimers composed of non-covalently associated α- and β-chains which connect cells to the extracellular matrix or to other cells (1). In addition to acting as mechanical links between the cytoskeleton and extracellular ligands, integrins are signal transducing receptors which influence processes such as cell proliferation, cell migration and cell differentiation (2-4). Integrins can be grouped into subfamilies based on shared β-chains, shared ligand binding properties, or shared structural features of the α-chains. Currently 17 α-chains and 8 β-chains have been identified (5). Of the subfamilies with shared β-chains, the Pβ1 subfamily has the most members. To date, 11 integrin α-chains associated with the β1-chain have been identified and characterized, α1-α10 and αv (5).
Several integrins bind the sequence RGD in their respective ligands (1). Of those integrins identified so far, α4-, α5-, α8-, αIIb- and αv-chains form heterodimers that mediate RGD-dependent interactions. The ligands containing RGD are generally found in the interstitial type of extracellular matrix. Major non-RGD dependent ligands include various collagen and laminin isoforms. Although both collagens and laminins contain the RGD sequence in their primary sequences, these RGD sequences are cryptic (6-9) and normally not accessible to cells in the native proteins, but they may be exposed during growth and reorganization events of the extracellular matrix.
Another subdivision of integrins can be made based on structural similarities of the α-chains. A number of integrins contain an extracellular I-domain (10, 11) which is homologous to collagen binding A-domains present in von Willebrand factor (12). The I-domain constitutes an inserted domain of approximately 200 amino acids which is present in 8 known integrins (α1, α2, α10, αL, αM, αX, αD and αE) (5, 10). Structural analysis of integrin I-domains crystallized in the presence of Mg2+ have revealed the presence of a characteristic "MIDAS" (metal ion dependent adhesion site) motif, shown to be critical for ligand binding (13). Integrin α-chains containing the I-domain are not cleaved into heavy and light chains, although the rat α1 chain possesses a proteolytic cleavage site near the membrane spanning region (14, 15). For I-domain integrins the principal ligand binding sites are found within the I-domain (10). Known ligands for I-domains found within the β1 integrin subfamily include laminins and collagens (α1β1 and α2β1 integrins) (16-19), and Echovirus (α2β1 integrin) (20).
Structure comparisons have suggested that integrins fold into a so-called 7-bladed β-propeller structure which forms one globular domain with the ligand binding region on the upper surface (21). The I-domain is inserted between blade 2 and 3 in this propeller and divalent cation binding sites are located on the lower surface in blades 4-7 (22, 23). Studies of β2 integrins have revealed that proper folding of the β2-chain is dependent on the presence of the αL-chain but that the I-domain folds independently of other structural elements in the α- and β-chains (24). In integrin α-chains, a less conserved stalk region separates the predicted β-propeller from the short transmembrane region. This stalk region is possibly involved in transducing conformational changes between the extracellular and intracellular regions, as well as mediating protein-protein interactions. Although integrins take part in cell signalling events, the cytoplasmic tail is short and lacks enzymatic activity. The sequence GFFKR is conserved in a majority of integrin α-subunits cytoplasmic tails and has been shown to be important for calreticulin binding (25).
Cellular interactions with the extracellular matrix during muscle formation and in muscular dystrophy have received increased interest during the past years. In the early 1960's a mutant was described in Drosophila which was characterized by the detachment of muscles from their attachment points at the time of the first embryonic muscle contraction, causing the embryos to assume a spheroid shape (26). The mapping of the molecular defect in the lethal myospheroid mutant in 1988 to an integrin β-chain (27), was the first evidence for a role of integrins in maintaining muscle integrity. More recently, refined analysis of Drosophila mutants have indicated distinct roles for integrins in muscle endpoint attachments and sarcomere structure (28). The Drosophila integrins are all cleaved α-chains and share many features with vertebrate integrins such as the ability to cluster into focal contacts (29).
The finding that inactivation of the α7 integrin gene in mouse (30), as well as mutations in the human ITGA7 gene (31), both cause muscular dystrophy affecting mainly muscle attachment points, indicates a striking conservation of integrin function during evolution. Of the 11 members of the β1 subfamily, α7 exists as a major integrin α-chain (32, 33) associated with the β1D integrin chain in the adult skeletal muscle sarcolemma (34). Intriguingly, mutations in the basement membrane protein laminin α2-chain (35-37) cause a more severe disease than that observed for the laminin receptor integrin α7β1 (30). This indicates that other receptors for laminins exist in muscle.
A novel integrin has recently been identified on cultured human fetal muscle cells (38). The present invention is related to, inter alia, the cloning and characterization of this novel I-domain containing, β1-associated integrin chain, which is expressed in muscle tissues.
The full-length cDNA for this integrin subunit, α11, has now been isolated. The open reading frame of the cDNA encodes a precursor of 1188 amino acids. The predicted mature protein of 1166 amino acids contains 7 conserved FG-GAP repeats, an I-domain with a MIDAS motif, a short transmembrane region and a unique cytoplasmic domain of 24 amino acids containing the sequence GFFRS. α11, like other I-domain integrins, lacks a dibasic cleavage site for generation of a heavy and a light chain, and contains three potentional divalent cation binding sites in repeats 5-7. The presence of 22 inserted amino acids in the extracellular stalk portion (amino acids 804-826) distinguishes the α11 integrin sequence from other integrin α-chains. Amino acid sequence comparisons reveal the highest identity of 42% with α10 integrin chain. Immunoprecipitation with antibodies to α11 integrin captures a 145 kD protein, distinctly larger than the 140 kD α2 integrin chain when analyzed by SDS-PAGE under non-reducing conditions. Fluorescense in situ hybridization maps the integrin α11 gene to chromosome 15q23, in the vicinity of an identified locus for Bardet-Biedl syndrome. Based on Northern blotting integrin α11 mRNA levels are high in adult human uterus and in heart, and intermediate in skeletal muscle and some other tissues tested. During in vitro myogenic differentiation, α11 mRNA and protein are up-regulated. Studies of ligand binding properties show that α11β1 binds collagen type I Sepharose and cultured muscle cells localize α11β1 into focal contacts on collagen type I.
The present invention relates in its different aspects to the following:
A recombinant or isolated integrin subunit α11 comprising essentially the amino acid sequence shown in SEQ ID No. 1, or homologues or fragments thereof.
The invention also encompasses integrin homologues of said integrin, isolated from other species, such as bovine integrin heterodimer comprising a subunit α11 in association with a subunit β, preferably β1, as well as homologues isolated from other types of human cells or from cells originating from other species.
The term "homologues" in the context of the present invention is meant to imply proteins of a common evolutionary origin, having identical or similar functions, specifically requiring evidence based on gene structure and not merely a similarity of protein structure.
The invention also encompasses a process of producing a recombinant integrin subunit α11 comprising essentially the amino acid sequence shown in SEQ ID No. 1, or homologues or fragments thereof, which process comprises the steps of
a) isolating a polynucleotide comprising a nucleotide sequence coding for an integrin subunit α11, or homologues or fragments thereof,
b) constructing an expression vector comprising the isolated polynucleotide,
c) transforming a host cell with said expression vector,
d) culturing said transformed host cell in a culture medium under conditions suitable for expression of integrin subunit α11, or homologues or fragments thereof, in said transformed host cell, and, optionally,
e) isolating the integrin subunit α11, or homologues or fragments thereof, from said transformed host cell or said culture medium. The transformation can be performed in vitro, in situ or in vivo.
In further aspects, the invention encomppases:
a) isolating one polynucleotide comprising a nucleotide sequence coding for a subunit α11 of an integrin heterodimer and, optionally, another polynucleotide comprising a nucleotide sequence coding for a subunit β of an integrin heterodimer, or polynucleotides or oligonucleotides coding for homologues or fragments thereof having similar biological activity,
b) constructing an expression vector comprising said isolated polynucleotide coding for said subunit α11 optionally in combination with an expression vector comprising said isolated nucleotide coding for said subunit β,
c) transforming a host cell with said expression vector or vectors, which transformation may be performed in vitro, in situ or in vivo,
d) culturing said transformed host cell in a culture medium under conditions suitable for expression of an integrin heterodimer comprising a subunit α11 and a sub-unit β, or homologues or fragments thereof, in said transformed host cell, and, optionally,
e) isolating the integrin heterodimer comprising a subunit α11 and a subunit β, or homologues or fragments thereof, or the α11 subunit thereof from said transformed host cell or said culture medium.
i) a first vector, said first vector comprising a polynucleotide or oligonucleotide coding for a subunit α11 of an integrin heterodimer, or for homologues or parts thereof, which polynucleotide or oligonucleotide comprises essentially the nucleotide sequence shown in SEQ ID No. 1 or parts thereof, and
ii) a second vector, said second vector comprising a polynucleotide or oligonucleotide coding for a subunit β of an integrin heterodimer, or for homologues or fragments thereof.
In embodiments of this process, said fragment is a peptide chosen from the above defined group.
In one embodiment of said process, the cells are chosen from the group comprising fibroblasts, muscle cells, chondrocytes, osteoblasts, mesenchymally derived cells and stem cells.
Especially, said process is used during pathological conditions involving said subunit α11. Said pathological conditions comprise in one embodiment damage of muscles, muscle dystrophy, fibrosis or wound healing. In another embodiment, said pathological conditions comprise damage of cartilage and/or bone, or cartilage and/or bone diseases. In a still further embodiment, said pathological conditions comprise trauma, rheumatoid arthritis, osteoarthritis or osteoporosis.
In a further embodiment, said process is a process for detecting the formation of cartilage during embryonic development, or for detecting physiological or therapeutic reparation of cartilage and/or muscle, or for selection and analysis, or for sorting, isolating or purification of chondrocytes and/or muscle cells, or for detecting regeneration of cartilage or chondrocytes during transplantation of cartilage or chondrocytes, respectively, or of muscle or muscle cells during transplantation of muscle or muscle cells, respectively, or for studies of differentiation of condrocytes or muscle cells.
Said process may be and in vitro, an in situ or an in vivo process.
In embodiments of this process, said fragment is as defined above.
In one embodiment, said process is a process for detecting the presence of an integrin subunit α11 comprising the amino acid sequence shown in SEQ ID No. 1, or of an integrin heterodimer comprising said subunit α11 and a subunit β, or of homologues or fragments thereof.
Furthermore, embodiments of this process encompass similar embodiments as defined above in connection with the process of using the integrin subunit α11 as a marker or target molecule.
In embodiments of this process, said fragment is as defined above.
Furthermore, embodiments of this process encompass similar embodiments as defined above in connection with the process of using the integrin subunit α11 as a marker or target molecule.
A method of gene therapy, whereby a vector comprising a polynucleotide or oligonucleotide coding for a subunit α11 of an integrin heterodimer, or for homologues or fragments thereof, which polynucleotide or oligonucleotide comprises essentially the nucleotide sequence shown in SEQ ID NO: 1 or parts thereof, and optionally a second vector comprising a polynucleotide or oligonucleotide coding for a subunit β of said integrin heterodimer, is administered to a subject suffering from pathological conditions involving said subunit α11.
One embodiment of this method comprises hybridising a polynucleotide or oligonucleotide to the DNA or RNA encoding the integrin subunit α11 or homologue or fragment thereof, which polynucleotide or oligonucleotide fails to hybridise to a polynucleotide or oligonucleotide encoding an integrin subunit α10.
Cell Cultures
The human fetal myoblast/myotube cultures were derived from clone G6 originating from a thigh muscle of a 73-day old aborted fetus ((39); referred to as G6 hereafter). Cultures of G6 and 2.5 years postnatal human satellite cells XXVI, a gift from Dr. Helen Blau (Stanford University, CA), were grown as reported earlier (39). Human rhabdomyosarcoma cell lines RD (ATCC No. CCL-136) and A204 (ATCC No. CRL-7900) were grown in DMEM (Swedish Agricultural University, Uppsala) supplemented with 10% fetal calf serum.
RNA Isolation and cDNA Synthesis
Total RNA from G6 and XXVI myoblasts, the same cells differentiated for 3 or 7 days, and RD and A204 cell lines, was isolated using the RNeasy Midi kit (Qiagen) according to the manufacturer's instructions. Poly-A RNA was extracted from total RNA of G6 and XXVI cells using Dynabeads mRNA DIRECT kit (DYNAL A.S., Norway).
PCR Based Cloning and Generation of Human α11 Probes
First strand cDNA was generated from 1 μg of G6 mRNA using a reverse transcription PCR-kit (Perkin-Elmer). Advantage cDNA Polymerase Mix (Clontech) was used in PCR amplifications using two different pairs of primers: (1) 5′ ACG GGA GAC GTG TAC AAG TG 3′ (forward), 5′-AAA GTG CTG AAC CTC CAC CC-3′ (reverse) and (2) 5′-CAC CAT CCA CCA GGC TAT GC-3′ (forward), 5′-TTA GCG TTC CGT TAT AAA CA-3′ (reverse). The PCR conditions were: 94� C., 4 min. ("hot start"); 94� C., 30 s; 55� C., 30 s; and 72� C., 1 min., for 25 cycles. Two products, named PCR1 and PCR2, were obtained (
Screening of cDNA Libraries
A λZAP custom made G6 cDNA library (Stratagene, USA) was screened with PCR2 (see above) as a probe. The screening procedure (carried out as described in (40)) resulted in two clones representing the 5′ non-coding region and the beginning of the coding part of integrin α11 (
Northern Hybridization
A filter containing 6 μg of the poly-A RNA from G6 and XXVI cells and 10 μg of the total RNA from RD and A204 cell lines, and a Human Multiple Tissue Northern Blot containing poly-A RNA from adult human tissues (Clontech), were hybridized at 68� C. in ExpressHyb solution (Clontech) with probes labeled as described above. The probes used were PCR1, PCR2, cDNA clone 1.3 (
cDNA Sequencing and Sequence Analysis
All PCR fragments and cDNA clones were sequenced on both strands either manually (29) or using ABI 310 Genetic Analyzer automatic sequencer. Sequences were analyzed with the aid of MacVector™ 6.0, DNA Star, Faktura™NEW 1.2.0, and Sequence Navigator 1.0.1 software programs. A distance tree of all I-domain containing integrin a subunits was assembled using SEAVIEW and PHYLO-WIN softwares (41). Percent similarity between every two members in the I-domain integrin subfamily was calculated by a formula I=(1−D)�100, where "I" is identity and "D" is distance.
A polyclonal antiserum (α11 cyt) was produced against the peptide CRREPGLDPTPKVLE from the integrin α11 cytoplasmic domain. Peptide synthesis and conjugation to Keyhole limpet hemocyanin, immunization of rabbits and affinity purification was performed at Innovagen AB (Lund, Sweden). The monoclonal antibody Mab 13 against integrin β1 was obtained from S. K. Akiyama (NIEHS, NIH). Monoclonal antibodies to integrin α1 (clone FB12, sold as MAB 1973) and integrin α2 (clone BHA2.1 sold as Mab 1998) were both obtained from Chemicon, Temecula, Calif. The monoclonal antibody to vinculin (clone hVIN-1) was from Sigma (Saint Louis, Mo., USA). Secondary fluorescent antibodies (CY3™-coupled goat-anti rabbit IgG and FITCcoupled goat anti-mouse IgG of multiple labeling grade) were from Jackson ImmunoResearch Laboratories, Inc. (West Grove, Pa., USA).
Immunoprecipitation and SDS-PAGE
G6 and XXVI cells were labeled with [35S] cysteine/methionine and subjected to immunoprecipitation and SDS-PAGE as reported previously (38). The two-step procedure used to dissociate integrin heterodimers was carried out as follows. After incubation of samples with β1 antibody and capture with GammaBind G Sepharose (Amersham Pharmacia Biotech, Uppsala, Sweden), 100 μl of 1% SDS was added to the washed beads which were then boiled for 5 minutes. 10 mM Tris-HCl, pH 7.4, 0.15 M NaCl and 1% Triton X-100 was added to a final volume of 1 ml and the lysate was incubated with GammaBind G Sepharose for 1 hour. The incubation with GammaBind G was performed in order to ensure that no reactive β1 antibodies remained. After removal of GammaBind G Sepharose, α11 integrin antibody was added for additional 2 hours, followed by capture with protein A Sepharose (Amersham Pharmacia Biotech) and boiling in SDS-PAGE sample buffer.
Chromosomal Localization
Chromosomal localization of the human integrin α11 was performed by using a combination of FISH (Fluorescent In Situ Hybridization) technique and DAPI (4′,6-diamidino-2-phenylindole) banding essentially as described earlier (42). As a hybridization probe, the 1.4 kb RT-PCR product PCR3 was used.
Surface Iodination and Affinity Chromatography
Cultured XXVI cells were surface iodinated as described (38). Labeled cells were solubilized in 1 ml of solubilization buffer (10 mM Tris-HCl pH 7.4, 15 mM NaCl, 1% Triton X-100, 1 mM MgCl2, 1 mM CaCl2, 1 mM MnCl2), centrifuged at 14000 g for 20 min., and soluble membrane proteins were applied to a collagen type I Sepharose (bovine collagen type I from Vitrogen (Collagen Corp., Palo Alto) coupled to CNBr-activated Sepharose CL-4B at 3 mg/ml gel as described (14)), equilibrated in solubilization buffer. Following a one hour incubation the column was washed extensively with buffer A (10 mM Tris-HCl pH 7.4, 50 mM NaCl, 1 mM MnCl2, 0.1% Triton X-100) and by 10 column volumes of buffer A without NaCl. Bound proteins were eluted with 20 mM EDTA, 10 mM Tris-HCl pH 7.4, 0.1% Triton X-100. Peak fractions were pooled and concentrated by immunoprecipitation with β1 integrin and α11 integrin antibodies as described under Immunoprecipitation and SDS-PAGE. Eluted frations and captured proteins were analyzed on 7.5% SDS-PAGE gels followed by autoradiography.
Indirect Immunofluorescence
Cells cultured on coverslips were washed in serumfree medium and fixed for 8 min. in acetone at −20� C. Non-specific binding sites were blocked by incubating with 10% goat serum diluted in phosphate buffered saline. In the double immunofluorescence staining protocol, primary antibodies (anti-α11 cyt (rabbit antibody) and anti-vinculin (mouse antibody)) were simultaneously incubated with fixed cells for 1.5 hours at +37� C. Specifically bound antibodies were detected using anti-rabbit Cy3 IgG and anti-mouse FITC IgG. Stained cells were mounted in Vectashield™ mounting medium (Vector Laboratories, Inc., Burlingame, Calif., USA) and visualized and photographed under a Zeiss light microscipe equipped with optics for observing fluorescence.
cDNA Cloning of a Novel Integrin α-Chain
In order to determine the nature of the integrin chain that we had previously characterized on human fetal muscle cells and named αmt (38), a number of approaches were used. Applying PCR with mRNA from fetal muscle cells as template together with degenerate primers to conserved regions of integrin α subunits (43) we amplified cDNA for α1, α4, α5, α6 and αv integrin chains (data not shown), but failed to amplify the novel integrin. However, while searching through the literature we came across two integrin sequences obtained in a subtractive hybridization protocol comparing human primary myoblasts with the rhabdomyosarcoma cell line RD (44). After having confirmed that these sequences could be amplified by PCR from human fetal G6 myoblast cDNA, PCR was performed assuming that these sequences were derived from the same transcript. In this manner a 1.4 kb cDNA fragment with integrin-like sequence was obtained. Screening of a human fetal myoblast cDNA library and 5′ RACE yielded additional 5′ sequence. We determined the mRNA expression pattern in a number of human tissues (see below) and observed a high mRNA expression in the uterus. Screening of a uterus cDNA library resulted in the identification of the complete open reading frame. A schematic illustration of the cloning strategy is shown in
cDNA Sequence and Predicted Amino Acid Sequence of α11 Integrin Chain
By sequence analysis of cDNA clones and 5′ RACE products we obtained a continuous sequence of 3983 nucleotides (nt) composed of 90 nt 5′ non-coding sequence, 3564 nt open reading frame, and 326 nt 3′ non-coding sequence. Translation of the sequence predicts an integrin α-chain like precursor of 1188 amino acids including a 22 amino acid long signal peptide (
The transmembrane region (amino acids 1142-1164) is 23 amino acids long and is followed by a cytoplasmic tail of 24 amino acids. The cytoplasmic tail contains the sequence (SEQ ID No. 4) GFFRS instead of the conserved GFFKR sequence (SEQ ID No. 5), found in all other α8-α10. It will be interesting to determine the importance of this sequence in defining the cytoplasmic domain as well as its possible ability to bind calretriculin and other intra-cellular components.
Comparison of Integrin α11 Chain with Other Integrin a
Alignment of the predicted α11 integrin amino acid sequence with other integrin sequences shows the highest overall identity with α10 (42% identity), α1 (37% identity), and α2 (35% identity), followed by the remaining I-domain containing integrin subunits. Of the non I-domain containing integrins, α4 and α9 are the most similar to α11. A distance tree shows that α10 and α11 form a separate branch from the most closely related α1 and α2 integrin chains (
Analysis of sequences identified during screening for genes upregulated during tadpole regression revealed a partial sequence, which at the time was reported to show the highest similarity to integrin α1 (41% identity) (50). This sequence, when translated (amino acids 1-116), shows 71% identity to human α11 and thus most likely represents the Xenopus orthologue of α11 rather than that of the α1. These data suggest that α11 is well conserved during evolution.
Chromosomal Localization of the Integrin α11 Gene
A fluorescent cDNA probe was used for in situ hybridization on metaphase chromosome spreads. The analysis shows that the integrin α11 gene (ITGA11) is located on chromosome 15q23 (
Expression Pattern of α11 mRNA in Adult Tissues
Northern blot analysis of mRNA from various adult human tissues shows the highest level of expression of α11 in adult human uterus. A strong signal is also noted in heart, while intermediate levels of α11 mRNA are present in skeletal muscle and intermediate to low levels in other adult tissues tested (
Biochemical Characterization of α11 Protein
Following the cloning of the full-length α11 integrin cDNA it was essential to determine if the predicted amino acid sequence was identical to the novel uncleaved β1 integrin-associated α-chain that we had previously noted to be upregulated during in vitro differentiation of human myoblasts (38). To answer this question we raised antibodies to the cytoplasmic tail of the integrin α11 chain. Immunoprecipitation from the human satellite cells showed that the antibodies precipitated a 145 kDa α11 band associated with a 115 kDa β1 band (
To show that α11 is associated with the β1 subunit a two-step immunoprecipitation procedure was performed. Integrins were first precipitated with a monoclonal antiβ1 integrin antibody and GammaBind G captured integrins were then dissociated by boiling in 1% SDS. In the second step, SDS was diluted tenfold and antibodies to α11 were added. As shown in
Induction of α11 mRNA and Protein During Myogenic Differentiation In Vitro
It has previously been determined that αmt is the major integrin α-chain that is up-regulated during myogenic differentiation on human fetal myoblasts in vitro (38). To compare α11 levels in myoblasts and myotubes, immuno-precipitates were analyzed from myoblast cultures in pro-liferation medium, and from parallel cultures allowed to differentiate and form myotubes in differentiation medium for 7 days. Immunoprecipitation with both β1 and α11 antibodies showed that α11, like αmt, is strongly up-regulated at the protein level in differentiation cultures of human fetal muscle cells and satellite cells (
Analysis of mRNA from the two rhabdomyosarcoma cell lines RD and A204 (
Ligand Binding Specificity of α11β1 Integrin
So far identified I-domain containing integrins of the β1 integrin subfamily all bind collagens (5, 15, 59). For α1 and α2 this binding capacity has been shown to reside within the I-domain (17, 18). To determine if α11β1 also binds collagen we performed collagen type I Sepharose chromatography of membrane proteins from surfaceiodinated XXVI satellite cells. Direct analysis of the EDTA eluate revealed weak bands corresponding to the positions of α1, α2, α11 and β1 in parallel immunoprecipitations (
α11 Integrin Protein Distribution in Human Embryo
Morphologically normal human embryos (aged from 4 to 8 post-ovulatory weeks) were obtained from legal abortions induced by Mifepristone (RU486) at Hopital Broussais in Paris. All procedures were approved by the Ethical Committee of Saint-Vincent de Paul Hospital in Paris.
Each sample was first examined macroscopically during dissection under a stereo-microscope. The development stage of the embryos was determined using established criteria. Tissues were collected shortly after delivery and frozen within the first 24 h post mortem on dry ice and stored at −80� C. until used. Seven micron-thick cryostat sections were mounted on slides previously coated with a 2% 3-aminopropyl-triethoxysilane solution in acetone. The cryosection was left unfixed prior to blocking of non-specific binding sites with 10% goat serum diluted in phosphate buffered saline. For immunofluorscence, the section was incubated with α11 antibodies 1.5 h at +37� C. Specifically bound antibodies were detected using goat anti-rabbit Cy3 IgG (Jackson Immunoresearch). The stained tissue section was mounted in Vectashield™ mounting medium (Vector Laboratories Inc.) and visualized and photographed under a Zeiss ligth microscope equipped with optics for observing fluorescence.
The results obtained are shown in
A. Clones 1.1-1.3 and 2.1-2.3 are from the first and the second round of screening, respectively. Fragment 0.0 represents a 5′ RACE product as well as a clone obtained from screening of the G6 library. PCR fragments 1-3 and a SacI fragment of a clone 1.3, λ290, are marked with thick lines. Names and positions of all the clones on a scheme are shown in tabulated form in B.
B. Names of the PCR-amplified fragments and cDNA clones shown in A are in the left column, and their positions in the full length cDNA of integrin α11 in the right column.
The putative signal peptide is underlined in bold, I-domain is boxed, potential N-linked glycosylation sites are marked with asterisks, cysteines are underlined, potential divalent cation binding motifs are double underlined and the transmembrane domain is underlined with dashes. A 22 amino acid insert is boxed in bold.
A tree was assembled based using ClustalW multiple alignment-based SEAVIEW and PHYLOWIN softwares. A scale at the bottom shows percent identity.
A. Left panel shows the FISH signals on human chromosome 15; right panel shows the same mitotic figure stained with 4′,6-diamino-2-phenylindole to identify human chromosome 15.
B. Diagram of FISH mapping result for the probe PCR3 based on a detailed analyses of 10 different images. Each dot represents the double FISH signals detected on human chromosome 15.
Integrin α11 mRNA and integrin α1 mRNA were analyzed on a membrane with RNA from various adult human tissues where mRNA loading was normalized with respect to β-actin. Probes used for hybridizations are marked on the left. Size of molecular weight standard is marked to the right. Note that the β-actin probe reacts with 2 kb β/γ actin transcripts and the muscle specific 1.8 kb α-actin message.
A. α11 associates with β1 integrin chain. Human XXVI and G6 muscle cells were metabolically labeled with [35S] cysteine/methionine and integrins were immunoprecipitated with the indicated antibodies (β1, α2 and α11). Evidence for the association of integrin α11 with the β1 subunit obtained by treating proteins precipitated with anti-β1 antibodies with SDS followed by a second precipitation with α11 antibodies (ant-α11+SDS). Precipitated proteins were resolved on 7.5% SDS-PAGE gels in the absence of reducing agents, followed by fluorography.
B. Induction of Integrin α11 Upon Myogenic Differentiation In Vitro.
G6 muscle cells were metabolically labeled with [35S] cysteine/methionine when growing in proliferation medium (mb-proliferating myoblasts) and after 7 days in differentiation medium) (mt-myotubes). Integrins were precipitated with antibodies to β1 and α11 and the precipitates were resolved on 7.5% SDS-PAGE gels both under non-reducing (UNREDUCED) and reducing (REDUCED) conditions. Lanes 1, 3, 5 and 7 are immunoprecipitations with the antibody to integrin β1, and lanes 2, 4, 6 and 8 with the antibody to integrin α11.
C. Upregulation of Integrin α11 mRNA in Differentiated Myogenic Cells.
mRNA was extracted from G6 and XXVI cells growing under proliferating (p) or differentiating (d) conditions for 3 days (d3) or 7 days (d7). Total RNA was isolated from RD and A204 cells. Following separation of RNA on agarose gel and transfer to the membrane, the filter was hybridized with probes to α11 integrin (α11) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Size of bands in RNA standard (in kb) are marked to the right.
XXVI cells were surface iodinated and integrins were analyzed by immunoprecipitation and collagen I Sepharose affinity chromatography. Immunoprecipitation reveals the presence of β1 integrins (lane 1), α1β1 (lane 2), α11β1 (lane 3) and α2β1 (lane 4) at the surface of XXVI cells. EDTA eluted proteins bound to collagen I Sepharose contain weak band in the position of α1, α11, α2 and β1 integrin chains (lane 5). Immunoprecipitations with β1 integrin antibodies (lane 6) and α11 integrin antibodies (lane 7) confirm the presence of α11 and β1 in the EDTA eluate.
Panel 2: α11β1 Localizes to Focal Contacts on Collagen.
Indirect immunofluorescent visualization of vinculin (A, B) and α11 integrin chain (C, D) in human XXVI satellite cells seeded on collagen type I (A and C) and fibronectin (B and D). Note the localization of integrin α11 chain to focal contacts of cells allowed to attach to collagen and its complete absence on cells seeded on fibronectin. Vinculin is found in focal contacts on both substrates. A and C show the same cell double stained for both antigens. Scale bar is 20 μm.
Composite of immunohistochemical staining of sagital section of human embryo at 8 weeks of gestation. Note high levels of α11 protein around vetrebrae (arrows), in intervertebral disc (asterisks), around ribs (thin arrows) and around forming cartilage in the forelimb (arrowhead).
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|Citing Patent
|Filing date
|Publication date
|Nov 3, 2008
|Feb 12, 2013
|Human Genome Sciences, Inc.
|12 human secreted proteins
|Nov 3, 2008
|Dec 17, 2009
|Human Genome Sciences, Inc.
|12 Human Secreted Proteins
|U.S. Classification
|435/7.1, 530/324, 530/395, 530/350
|International Classification
|Cooperative Classification
|European Classification
|Apr 15, 2002
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text:
Game Thoughts From Ruston – From the Bird's Nest
The loss to Tech wasn't a surprise. But the Cajuns got exposed in some areas.
The loss to Tech wasn't a surprise. But the Cajuns got exposed in some areas.
After a decade, long time rivals finally meet
Two fierce formal rivals finally renew series
Some good inter conference action in week one
Louisiana Tech has hired ex-South Florida head football coach Skip Holtz to fill the vacant position in Ruston. Holtz replaces...
Louisiana Tech football coach Sonny Dykes' name was mentioned as a potential candidate for several jobs that came open or would come open...
Last year the University of Houston was very close from going unranked in the preseason, to a BCS bowl. The Cougars dream ended when Southern Miss crushed them in the C-USA title game. Of all the unranked teams, who might break through into the BCS this year?


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Socket Bit, 3/8 in. Dr, 3/16 in. Hex
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text: Bugs item #963587, was opened at 2004-05-31 13:34 Message generated for change (Tracker Item Submitted) made by Item Submitter You can respond by visiting: https://sourceforge.net/tracker/?func=detail&atid=109655&aid=963587&group_id=9655 Category: xine Group: v0.9.9 Status: Open Resolution: None Priority: 5 Submitted By: Gavin Charles Flower (nivag) Assigned to: Nobody/Anonymous (nobody) Summary: loss of colour (color for the Americans) Initial Comment: Initially Xine plays .avi and vcd's in colour. However, without changing any settings, it will suddenly displays only in grayscale. This usuially happens on the 3rd or fourth time it is used in the same logged in session. Logging out and in clears the problem. It has happened with both Fedora Core 1 & 2. I'm using the latest CVS snapshot as of Sunday 20-May-2004. ---------------------------------------------------------------------- You can respond by visiting: https://sourceforge.net/tracker/?func=detail&atid=109655&aid=963587&group_id=9655


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Ok, so I just moved to Morningside Heights from Brooklyn, and am learning that there are not a huge number of good restaurants in the area. I also just went from being employed to living on a students budget. That being said, I am desperate for decent Thai and Indian that is at least somewhere near to my new residence (Morningside Heights or UWS). I love Angon, but I'm a first year law student who rarely has the free time needed to get down there.
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In a pinch I rented a tuxedos at Teds. And then discovered that this place is awesome. It's really simply, Teds is a family owned men's clothing store with...more
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Find great deals on eBay for Rock Crusher in Mining Equipment Shop with confidence , Skip to main content , All of the parts of the crusher are replaceable, except for the crusher body and This crusher uses a 5 hp electric , Impact mill rock crusher grinding milling gold mining equipment machine jaw ball $4,80000Know More
What is the working principle of ball mill? - Quora
Ball Mill Working principle: When the ball mill works, motor and speed reducer transmit torque , New Endurable wearing parts to increase service life of vulnerable parts 7Alternative PLC automatic control , Mineral and Mining Engineering: What is a ball mill, and main parts of ball mill? Mining Industry: What are good.Know More
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This includes used ball, grinding and sag mills, used crushing equipment, ship , managers, and contacts within most major mining equipment manufacturersKnow More
Manufacturing process < All about Cement < Cement : Lafarge
The sand and iron ore are transport from outside the plant (from different suppliers) , Step 2: Crushing, stacking, and reclaiming of raw materials , The clinker is taken from the clinker storage to cement ball mill hoppers for cement grinding Part of the hot air extracted from the cooler is utilized as a secondary and tertiary air.Know More
Ball mill parts - JYS ADVANCED WEAR RESISTANT , - Crusher Parts
Ball mill plants are widely used in the metal scrap crushing, mining crushing , The steel ball is an important basic components in the ball mill machineKnow More
Mill machining - Bacchus Marsh Grammar Wiki
29 May 2014 , 1 Definition; 2 History; 3 Grist Mill; 4 Hammer Mill; 5 Ball Mill; 6 Disc , It covers a wide variety of different operations and machines, , processes in industry and machine shops today for machining parts to precise sizes and shap , The hammermill can be used as a primary, secondary, or tertiary crusherKnow More
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Ball Mills Shenyang Huayue Brilliant Machinery & Equipment Co,Ltd have been , Big Rock Consulting has partnered with a major bearing wholesale supply , Standard & Short Head Cone Crushers HYB has purchased the drawings , Mills, Feeding Equipment, Vibrating Screen, and all related spare partsKnow More
Hammer mill by SaraswathiB - Pharmainfo
29 May 2014 , A hammer mill is a crusher that can grind, pulverize, and crush a wide range of , The hammer mill consist of three basic parts as follows:Know More
EMAX - High Energy Ball Mill - Retsch
The Emax is an entirely new type of ball mill for high energy milling , the Emax unites the advantages of different mill types: high-frequency impact (mixer mill),.Know More
Ball mill maintenance - Prime Machine, Inc
Industrial Crusher maintenance and rebuilds Kiln Maintenance Gearboxes and Drives , Ball mill maintenance- our shop and field crews make a unique combination to , fabrication, and engineering capabilities to rebuild your mill components , Our babbitt facility is capable of rebuilding the largest of your bearingsKnow More
Grinding Mills: Ball Mill & Rod Mill Design & Parts - Mineral ,
10 Jul 2016 , Common types of grinding mills include Ball Mills and Rod Mills , only on the largest particles, thereby expending most of their crushing force.Know More
DESIGN AND FABRICATION OF MINI BALL MILL (Part 1) (PDF ,
30 May 2016 , The main component of ball mill is a jar with the diameter and length , For the grinding balls to crush the raw material, stainless steel balls areKnow More
Size reduction equipments - Part II, Fabrizio Sarghini « Equipment ,
Food materials size reduction: Crushing Rolls , forces: however, in choke feeding conditions, also attrition forces can play a major role in size reduction , can be processed by using ball mills, hammer mills, roller mills, and attrition millsKnow More
Planetary Mills – fritschde
Planetary Mills for fine grinding of various materials, mixing, homogenising of , through the high-energy impact of grinding balls in rotating grinding bowlsKnow More