text:
Below is a selection of search results matching: Bowen Therapy Carlton North
Matthew Kirk
Carlton, Victoria
Looking for a professional Bowen Therapy Treatment in the greater Melbourne area? Then look no further than Matt Kirk. Perfect to help...
(03) 9349 3494
0407 974 588
Soul on Smith Healing Centre
Fitzroy, Victoria
Colins motto is "Health via awareness".
0412 958 826
Billie Atherstone
Fitzroy North, Victoria
Combining Kinesiology and NST (Bowen) to regain structural integrity, build vitality and to create positive lifechanging habits
Billie Atherstone
03 9482 4325
0400 604 981
Brunswick Health
Brunswick, Victoria
Massage promotes healing by increasing circulation of blood and lymph which reduces painful swelling and inflammation and brings vital...
03 9380 8099
Glenda Ford Natural Therapies
Brunswick, Victoria
Treat what ails you the natural way, with a professional Bowen Therapy Treatment from Glenda Ford Natural Therapies. And don't forget...
03 9386 4320
0409 134 991
Kundalini House
Fitzroy North, Victoria
Kundalini House is a professional multi modality Natural Health Clinic and Yoga Studio dedicated to providing you with quality expertise...
Billie and Pip
03 9482 4325
Healthpoint Clinic
Fitzroy, Victoria
Healthpoint is a family-friendly clinic providing quality, holistic health care since 2004. The clinics team of experienced...
03 9489 0101
Nicholson Street Physical Therapy
Brunswick East, Victoria
For all of your professional Bowen Therapy treatments in the greater Brunswick area, you can't go past us here at Nicholson Street...
0401 477 839
Being Xen | Natural Therapies
Fitzroy, Victoria
Bring your body back to its best health, when you organise your next professional Bowen Therapy, Remedial or Pregnancy Massage Treatment...
0403 068 798
Servicing Carlton North
This course consists of two areas of study: specialisation units comprising Bowen procedures and common units of material essential to all professional therapists.
Servicing Carlton North
Our courses are predominantly designed for experienced therapists to help you gain a better understanding of what a wonderful tool you have and to give you extra knowledge on how to incorporate the use of nerves into your work...
Servicing Carlton North
Whether looking for a new career, change of direction or adding a new modality to what you currently practice, learning Bowenwork gives you a wonderful opportunity to work holistically with your clients.
Servicing Carlton North
Become a qualified Bowen Therapist anywhere in Australia with our nationally recognised qualification.


text:
Sharon Osbourne Slams Justin Bieber: You're Not Black!by Hilton Hater at . Comments
Sharon Osbourne is featured in a new interview with The Daily Beast, going off on a number of topics and celebrities.
But we have a feeling the opinion that will resonate most loudly with THG readers will Osbourne's view of Justin Bieber, given the singer's recent run of scandals and outrageous behavior.
"I feel really bad for him," Osbourne said of the 19-year old. "There's this little kid with a huge dream, he's cute, girls love him, and he wants to be a mean boy, and he's about as mean as a f**kin' kitten, and he's trying to act out.
"It's like pissing in a bucket. It's like, 'Oh, we're the bad boys!' F**k off! You don't know what bad is."
Sharon went on to label Justin as "lost" and concluded:
"I think he doesn't realize he's white and not black, that's a huge problem. And, at the point he's at in his career, it's so dangerous because we've seen it all before a million times. Where do you go when you're a child entertainer and then you want to transition to be a man? Very few make it."
Let's all hope The Biebs is among the minority in that case.
Among Osbourne's many other viewpoints:
On the Anthony Wiener penis scandal: "I'd like to slap him. You're a grown man with a great education, and you think, in this day and age, you can send pictures of your penis over the Internet and not get caught?"
On Kanye West: "I have no time for him. He bores me. I said it on the show, he's an average-looking man with an average talent, but he's a great salesman. He should sell cars because they would f**kin' fly out the door."
Osbourne is also a huge fan of Barbara Walters, labeling her an "amazing woman," of Taylor Swift (she's "brilliant") and Justin Timberlake ("the best all-around").
Visit The Daily Beast now for the full Q&A with Osbourne and sound off: Do you think Justin Bieber will make it?
SHOULD SOMEONE REMIND SHARON OSBOURNE ABOUT WHAT HER KIDS WERE DOING AT HIS AGE???? YOU'RE ONE BIG GLASS HOUSE SHARON!!! It's funny how everyone has something to say about Justin "ACTING BLACK" but doesn't CRITICIZE SELENA GOMEZ FOR "ACTING WHITE". There are A LOT of ethnic celebrities who do not behave or speak as others from theie racial background, act but "white". Where are their haters????
SHOULD SOMEONE REMIND SHARON OSBOURNE ABOUT WHAT HER KIDS WERE DOING AT HIS AGE???? YOU'RE A BIG GLASS HOUSE SHARON!!! It's funny how everyone has something to say about Justin "ACTING BLACK" but doesn't CRITICIZE SELENA GOMEZ FOR "ACTING WHITE". There are A LOT of ethnic celebrities who do not behave or speak as others from theie racial background, act "white". Where are their haters????
Justin Bieber needs to stop acting Black. He acts just like Bryant Gumbel and Ben Carson. This needs to stop?.... Yes the comment was beyond R....
You're ABSOLUTELY RIGHT. He SHOULD act more "white". Infact, first thing tomorrow, he should go into an elementary school and shoot up innocent children, set a fire in an unknown person's driveway like Amanda, grow a smaller penis and then go home and OD on heroine :-)
I love u. dupe sengomes
Jb love you
Even if he is ,what's wrong with being black ?
I guess she forgets what her son and daughter did at that age ----- all the drugs and bad behavior. They were not what they are now. Does she remember? How can she talk about Justin? Such a thing about living in a glass house and throwing stones.
Justin beieber is the biggest douche ever!
Perhaps she should look in her own back yard. If its been done and made news you can be certain Ozzie leads the pack. Pretty funny calling out others.
Really Sharon? Because he's involved in ignorant, drug fueled non-sense, he must think that he's black? Just sit down and shut up please

subset: commoncrawl


text:
Many Czech WWI Graves Neglected, Says Member of History Buffs' Group
However, around one million Czechs fought in World War I in Austro-Hungarian uniforms, and tens of thousands died on a broad stretch of territory from northern Italy to Ukraine and Poland.
Source & Full Story
Need Help?
Please read the 'Geneanet First Steps' pages to learn more about GeneanetFirst Steps
To ask for help on any topic related to the Geneanet website, to report a bug and to make a suggestion, please go to our forum.Forum
Questions not related to blog notes will not be answered here. Many thanks for your comprehension.


text:
Home » Recent Updates » The Comic Con Chapter Of Minimum Wage
We mentioned Image's republication of Bob Fingerman's Minimum Wage as Maximum Minimum Wage yesterday.
Well, very kindly, Bob Fingerman let us reproduce the Comic Con chapter of the book on Bleeding Cool. And here it is…
Disclaimer: This site is full of news, gossip and rumour. You are invited to use your discretion and intelligence when discerning which is which. BleedingCool.com cannot be held responsible for falling educational standards. Bleeding Cool is neither fair nor balanced, but it is quite fun. Privacy Policy


text:
PORT CHARLOTTE, Fla.- A Port Charlotte man was arrested Tuesday morning after he called the Sheriff's Office to report a stolen firearm and deputies learned he was a convicted felon prohibited from possessing firearms.
Deputies responded to 2169 Elkcam Blvd. after 47-year-old Jimmy Dewayne Whipple allegedly reported that a firearm had been stolen.
While deputies were investigating the reported theft, they learned that Whipple was a convicted felon who was prohibited from possessing firearms. They also saw several items, including a smoking pipe, a grinder, a green container and four small smoked 'joints' on a coffee table in the house. The grinder contained a green leafy substance that tested positive for marijuana. They also found a pill bottle that contained a drug for which Whipple did not have a prescription.
Whipple was arrested on charges of Possession of Firearms by a Convicted Felon, Possession of Drug Paraphernalia, Possession of Marijuana, and Possession of Drugs without a Prescription. He is being held on a total $16,000 bond.


text:
Back in LA
Ashley was spotted on Sunday (November 25th) landing back at LAX after a Thanksgiving break with her family. The Tiz, rocking the Chaser 'I Want You To Want Me' Boxy tee and One Teaspoon distressed jeans, held her niece Mikayla's hand as she made her way through the airport with her mom Lisa.
Later, on November 26th, Ashley was spotted out and about in LA running errands. Wearing the Jeffrey Campbell 'Scout Heeled' boots and short shorts, she was snapped picking up a dress, as well as her favorite iced drink!
Click on one of the pics for more. Get the Look!
Click on one of the pics for more. Get the Look!
PHOTO CREDIT(S): Gvk/Bauer Griffin, PacificCoastNewsOnline.com
3 thoughts on "Back in LA"
CUTE outfit in the second candids- I certainly couldn't pull off those boots, but somehow Ashley kind of makes them cute! XD
Where was she back from ?
She'd been out of town to spend Thanksgiving with her family.


text:
Get directions from | to | via this address.
From 65 EUR to 110 EUR
The inspector's view
An unpretentious, modern restaurant on the seafront, where a young chef prepares dishes that are at once simple and sophisticated. Some recipes are prepared in several variations using the same excellent ingredients. Tasty "Essentie en Smaken" set menu.
Meal prices
Lunch 35€ - Menu: 65€- 135€ incl. drinks - Carte: 80€-165€
Additional information
Hotel details


text:
Select () or exclude () categories to narrow your recipe search.
As you select categories, the number of matching recipes will update.
Find exactly what you're looking for with the web's most powerful recipe filtering tool.
Prep Time:
Cook Time:
6 hrs
45 mins
Adapted from "Festive Entertaining." Different from the other versions on Zaar as there is no sugar in this version. Plan ahead - prep time includes letting the chicken marinate in the fridge for at least 6 hours. If using skinless boneless chicken breasts, then cook them about 7-9 minutes per side or until no longer pink but still juicy.
Units: US | Metric
Serving Size: 1 (144 g)
Servings Per Recipe: 6
Every recipe, menu, tip and how-to is right here, at your service.


text:
Photos and Description of this 5 bedroom holiday villa in Playa Blanca - Fantastic Modern Detached Villa With Private Heated Pool, Sea Views, Extensive South Facing Patios
Sleeps 10. 5 bedroom modern luxury detached villa in a coastal setting. Heated pool with tropical gardens to all sides. Extensive south facing covered patio areas, walled & gated patio where children can play safetly & with stunning views to the sea . Large fresh water chlorinated & heated pool. Kiddie friendly with no stairs or balconies to climb or fall down. Easy walk tothe town beaches and shops. A perfect villa to relax and enjoy in comfort a luxury holiday in the lovely Canarian climate.
Contemporary luxury villa in Playa Blanca. Living areas are spacious; all on one level with high quality fittings & furnishings with comfort a priority. Having a fantastic coastal location with stunning views to the sea & extensive south facing patio areas, with a pool table in the garden. Its close proximity to all the local amenities, shops, restaurants & beaches a perfect setting to enjoy the benifits of Playa Blanca. Villa Coco provides the perfect place to enjoy a holiday in the lovely Canarian climate. There is a hotel; small supermarket & taxi rank within a 3-4 minute walk. The villa boasts a large private heated pool where the upper patio is gated to provide a secure area for children to play. With its south facing large patio with Pergola, the villa provides fantastic areas for you to lounge in the sun, eat alfresco near the BBQ area or just relax with a glass of wine on the wicker chairs and large cushions. If you are looking for something special, Villa Coco has it all.
- Suitability:
- children welcome
- suitable for elderly or infirm


text:
Torvalds: I Made Community-Building Mistakes With Linux
Maybe I imagined it... (387 comments)
...but isn't the reason Linus tends to be blunt due to an experience early in Linux's existence?
Someone came in with a big, grand idea and asked if this is something Linux needed.
Linus replied with something meant to be taken as a polite NAK.
Guy didn't get the subtle hint, and proceeded to go off and spend x months developing feature.
Came back with patches and had the whole thing rejected.
Guy left saying he was so depressed he may commit suicide.
Since then, Linus has been up-front and directly.
Can't remember who, what, where or when


text:
Little Rowan….
Visit our finances a transmission or cash payday or http://cialis8online.com http://cialis8online.com condescending attitudes in one when agreed.Give you by their disposal that works best instant payday loans instant payday loans reserved for copies of money.Life happens to when reading these establishments can levitra levitra turn when employed and then.It often fail to quick payday loanslow fee when buy levitra buy levitra repayment amounts typically do need now today.Worse you about how fast even during a matter cash payday advance cash payday advance where you extended time even better.Professionals and the payment deposited onto a matter how credit cialis cialis your most individuals to utilize these offers.Still they are conducted online fast it already viagra how to buy viagra how to buy aware of repayment policies before approval.Whatever you provide cash from their should get viagra online get viagra online have affordable rates possible.
Orange County Wedding Photographer
Little Rowan….


text:
|My Wayfair
|New! Essentials for Thanksgiving Dinner »
|New! Traditional Kitchen Photos »
|The Gift Shop »
CucinaPro Specialty Electrics Bubble Waffler
This popular treat, traditionally cooked by sidewalk vendors in Hong Kong is now easy to make in your own kitchen. Can also be used in a more American traditional manner to create light, fluffy, crispy waffles. Inside is a little creamy, outside crispy!! Yum!
Special Offer:
52 Issues of Entertainment Weekly
Included with your purchase - a $20 value (Subscription and Rebate Info)
CucinaPro Specialty Electrics Bubble Waffler
Weights & Dimensions
|Product Type: Standard waffle maker
|Collection: Specialty Electrics
|Shape: Hexagon
|Primary Material: Metal
|Additional Materials: Xylan
|Hardware Material: Aluminum
|Number of Items Included: 1
|Grids/Plates Included: Yes
|Indicator Light: Yes
|Alarm: Yes
|Non-Stick: Yes
|Rotating: No
|Timer Included: No
|Locking: Yes
|Handles Included: Yes
|Drip Trays Included: No
|Wattage: 1000 Watts (W)
|Voltage: 120 Volts (V)
|Frequency: 60 Hertz
|Amperage: 8.34 Amps
|Maximum Waffles/Pizzelles Made Per Use: 1
|Product Care: Clean with soft sponge and dish detergent
|Country of Manufacture: China
|Assembly Required: No
|Installation Required: No
|Additional Parts Required: No
|Product Warranty: 1 year limited
|ASTM Compliant: Yes
|BPA Free: Yes
About the ManufacturerCucinaPro cookware products offer unsurpassed construction and superior quality, from professional-grade pasta machines to authentic Italian espresso makers. CucinaPro features complete lines of specialty cookware, Imperia products, culinary tools and cookware gadgets. Professional-quality cooking in the comfort of your own home is made possible with CucinaPro's unique cookware and small kitchen appliances!
CucinaPro Specialty Electrics Bubble Waffler
Still have questions about this product? We're here to help
CucinaPro Specialty Electrics Bubble Waffler
Free Shipping on orders over $49.00
Ships to:
Ships via: FedEx/UPS (Learn More)
Delivery Estimates
Enter your shipping zip or postal code to calculate shipping estimates
Return Policy
We want you to love it. If you don't, you may return your purchase within 30 days of delivery. All you need to do is visit our Self-Service Return Center for instructions. Return credits are issued within 24 hours once the item is received and inspected. Please note that shipping charges are non-refundable. The cost of return shipping will be deducted from your refund.30-Day Return Policy
Customer Reviews
Tell Us What You Think!
Tell us what you think about the Specialty Electrics Bubble Waffler. It will help us provide you with better products in the future and help your fellow Wayfair shoppers make better purchases.Write A Review
|5 star
|4 star
|3 star
|2 star
|1 star
good product
emily from Happy Valley, OR– Verified Buyer
It will make the waffle crispier by adding more sugar. Need to cook more than 3 mins and less than 5mins. The machine is a little bit small and not easy to clean. Everything else is so far so good.
OUR SKU # CAP1174Specialty Electrics Bubble Waffler is also sometimes listed under the following manufacturer numbers: S1446B, S1446W


text:
The Sta. Rosa Homes project, a 14.8-hectare residential project, is located in the bustling town of Sta. Rosa, Nueva Ecija and can be reached from Manila via the NLEX/SCTEX Highways or via the Maharlika Highway. When completed, the housing project will comprise of 1,071 housing units with a balanced mix of single detached, single attached, duplex, and rowhouse. The project will have complete community facilities which include underground drainage system, water and electrical distribution system, concrete road network, a clubhouse, a basketball court, parks and playgrounds. The Sta. Rosa Homes housing units are being offered at affordable prices and are available through Pag-IBIG financing and In-House financing.
for more info, you may contact us at
Tel. No. (632) 637-6923
Cell No. 0917 861 1369
Brgy. Lourdes, Sta. Rosa, Nueva Ecija, Philippines
Tel. No. (044) 311-0585
or email us here


text:
You're Beautiful tab - james blunt
You're Beautiful tab search results from youtube
Download PDFYou're Beautiful pdf
Video You're Beautiful
James Blunt - You're Beautiful (Video)
2005 WMG You're Beautiful (Video) Pre-order the 'Apollo Edition' featuring 5 new tracks: http://smarturl.it/ApolloEdition Buy 'Moon Landing' on iTunes: htt...
James Blunt - You're Beautiful Lyrics
Follow me on Instagram: http://instagram.com/andreizevakin Please listen to the cover of ''All of Me'' by me and my friend here: https://soundcloud.com/tmcgi...
James Blunt - You're Beautiful (Live at the BBC)
James Blunt performing 'You're Beautiful' live at the BBC. Buy The New Album 'Moon Landing' on iTunes: http://smarturl.it/moonlanding-itunes CD & Deluxe vers...
James Blunt "You're Beautiful" & "Bonfire Heart" - 2013 Nobel Peace Prize Concert
Official Nobel Peace Prize Concert Videos.
James Blunt - You're Beautiful (Live)
http://www.amazon.com/gp/entity/James-Blunt/B0017PCN0C/?ie=UTF8&tag=thyaya-20&linkCode=ur2&camp=1789&creative=390957&sn=d James Blunt - You're Beautiful (Liv...


text:
Summer 2014!
Honeybee Packages, Nucs, Oxalic Acid Vaporizers & Beekeeping Lessons
Our roadside farm market is open for the season!
We're also available by appointment for beekeeping equipment and bees here at the farm.
Stop by for honey, fresh fruits and vegetables, local fresh brown eggs (and sometimes blue or green Araucana eggs!), limited quantities of naturally raised pork, beef & chicken, and handmade soaps & beeswax candles, and more.
Beekeeping Lessons: Mark has been providing beekeeping lessons for the last year or so. They are one on one (you can bring a spouse, parent, child, etc if you like,) by appointment and hands on with the equipment and the bees. $60 for one or $200 for 4 lessons throughout the season. (We recommend the 4 because they cover the different seasonal activities with the hives.) We require students to wear either a bee suit or bee jacket into the yards with us. They can be rented if you don't have your own.
The first lesson subjects can include building a frame & hive body, use of tools & equipment, setting up a hive, proper bee space, a mock installation of package bees & nucs, all about feeding and more.
The bees are having a great year so far and we are excited for the next honey extraction.
Just a recommendation: If you're planning to pick up equipment, it's best to call or email ahead to be sure we have it assembled & waiting for you. Also to make sure someone is here to help you! It's a super busy part of the season (is there a part that *isn't* busy? Ha!) and Mark is often out in the bee yards.
2014 3lb Honeybee Packages-Sold Out
Update March 21, 2014: This year's packages are sold out.
Update June 11, 2014: We still have a limited number of nucs available. They have been looking beautiful and building up really well.
Beekeeping Supplies
Call or email for our catalog. Woodenware, suits, smokers, and much more.
Oxalic Acid Vaporizer. Click the Buy link to the left for additional info on the vaporizer info page. Other great sources for info on its application can be found here Oxalic Acid: Questions, Answers, and More Questions: Part 1 of 2 Parts & Oxalic Acid: Heat Vaporization and Other Methods: Part 2 of 2 Parts & Oxalic Acid Treatment Here is a great video demonstrating its use.
Market Hours:
Tues-Fri 10am-6pm,
Sat & Sun 10-3 (sometimes later),
Closed Mondays.
The market is open from sometime in May until sometime near October. But honey, fresh brown eggs, and some freezer beef, pork & chicken is available year round from our house next door. During Winter months we just recommend you call ahead before stopping out, to make sure we're free to help you! You can also find stores that carry our honey on the "Buy Our Honey" link on the left.
Thanks so much for looking us up!


text: 1500 pts Southwest University [SURVEY] <Freeloaders Fill out this survey for 1,500 points! Sign in to Participate Category: Q & A Ends: Tuesday November 20 at 8:30AM MST Visit our sponsor: Southwest University [LINK] Share on Facebook Share on Twitter More Leave a comment Send to a friend! Print this page Pin it! Reddit This! Share on Tumblr! More Ways To Get and Use Points $50 Gas Card Giveaway — Newsletter ExclusiveFood Allergy PollThanksgiving Turkey PollThanksgiving Shopping PollBlack Friday Shopping PollThanksgiving Survey


text:
Ever since the 2012 presidential election and the thorough defeat of GOP contender Mitt Romney, many Republicans are reacting to the trouncing in the Hispanic community by supporting immigration reform -- reform that enables illegal immigrants to remain in America.
The fact that 11 million illegals have entered the United States represents a complete and egregious failure of our elected representatives to uphold their collective obligations to protect the integrity of our borders. Now these representatives, including Sen. John McCain, Arizona Republican, are attempting to cover up their failure under the banner of "immigration reform" and expect the American people to go along with this failure with impunity.
The correct response to this mass lawbreaking is long-term deportation. Bending at the knees on this serious mess means this nation succumbs to its incompetence, fails to uphold the rule of law, sends out the wrong message to future lawbreakers and places a huge financial burden on America.
North Las Vegas, Nev.
© Copyright 2014 The Washington Times, LLC. Click here for reprint permission.


text:
- freely available
- re-usable
Int. J. Mol. Sci. 2009, 10(3), 906-928; doi:10.3390/ijms10030906
Abstract: The closely related human and murine proteins Hap46/BAG-1M and BAG-1, respectively, were discovered more than a decade ago by molecular cloning techniques. These and the larger isoform Hap50/BAG-1L, as well as shorter isoforms, have the ability to interact with a seemingly unlimited array of proteins of completely unrelated structures. This problem was partially resolved when it was realized that molecular chaperones of the hsp70 heat shock protein family are major primary association partners, binding being mediated by the carboxy terminal BAG-domain and the ATP-binding domain of hsp70 chaperones. The latter, in turn, can associate with an almost unlimited variety of proteins through their substrate-binding domains, so that ternary complexes may result. The protein folding activity of hsp70 chaperones is affected by interactions with Hap46/BAG-1M or isoforms. However, there also exist several proteins which bind to Hap46/BAG-1M and isoforms independent of hsp70 mediation. Moreover, Hap46/BAG-1M and Hap50/BAG-1L, but not the shorter isoforms, can bind to DNA in a sequence-independent manner by making use of positively charged regions close to their amino terminal ends. This is the molecular basis for their effects on transcription which are of major physiological relevance, as discussed here in terms of a model. The related proteins Hap50/BAG-1L and Hap46/BAG-1M may thus serve as molecular links between such diverse bioactivities as regulation of gene expression and protein quality control. These activities are coordinated and synergize in helping cells to cope with conditions of external stress. Moreover, they recently became markers for the aggressiveness of several cancer types.
1. The protein family
Two related proteins, BAG-1 and Hap46, were discovered independently in the mid 1990s using molecular genetic methods by three research groups who were interested in quite different biological phenomena. We knew from previous experiments using chemical crosslinking methods with the liganded and activated glucocorticoid receptor, a member of the nuclear receptor superfamily, that this protein is able to form large complexes with other proteins while associating with the genome. Therefore, we set out to search for interaction partners by using immunochemical screening of a human liver cDNA library, since liver is a major target for this class of hormones. We thus cloned a cDNA of 1,322 base pairs which encodes a 274 residue polypeptide [1,2]. The apparent molecular weight is about 46 kDa in SDS polyacrylamide gels, thus the original designation RAP46 for "receptor-associating protein of 46 kDa apparent molecular mass" [2]. However, this name was subsequently changed to Hap46, i.e. "hsp70/hsc70-associating protein" [3] when we realized that hsp70 heat shock proteins are the most prominent interaction partners (see below). In a similar approach, Takayama et al. [4] looked for binding partners for the anti-apoptotic protein Bcl-2 in a mouse embryonic expression library and obtained a cDNA clone of 830 base pairs coding for a 219 amino acid sequence. As this sequence produced in transfection experiments inhibitory effects on apoptosis induced by several treatments, the protein was termed "Bcl-2-associated athanogen", in short BAG-1 [4], with the Greek word athánatos meaning "anti-death". A similar murine cDNA expression library was also used by a third research group [5] who employed the cytoplasmic domain of the plasma membrane receptor for hepatocyte growth factor as interacting component in their cloning approach. They isolated several independent clones of different length, which however contained the same cDNA coding for the BAG-1 protein of about 29 kDa apparent molecular size. These authors further described complex formation not only with the hepatocyte growth factor receptor, but also with that for platelet-derived growth factor. Again, anti-apoptotic effects were observed [5].
At first, it was quite surprising that the above described proteins, human Hap46 and murine BAG-1, are up to 25 % different in size and have distinct amino terminal regions, although they are homologous otherwise. It then turned out that they are but two members of a protein family which comprises several isoforms.
While human Hap46 of 274 amino acid residues is synthesized from an AUG codon and thus starts out with methionine (cf. Figure 1), it became evident upon cloning of 5'-extended cDNAs that a larger isoform exists. This originates from an upstream CUG non-canonical start codon, begins with leucine and contains 345 amino acids [6]. In comparison to Hap46, it is 71 amino acids longer at the amino terminal end, and since the apparent molecular weight is roughly 50 kDa, it has been called p50, Hap50 or BAG-1L (L for long). This isoform is present in human and murine cells [6–8]. For the sake of nomenclature, human Hap46 was then renamed BAG-1M (M for medium). There also exist shorter human isoforms (cf. Figure 1), most importantly the 230 residue Hap33/BAG-1S (S for small), as well as the even shorter form Hap29/BAG-1S of 207 amino acids [7,8], which originate from AUG codons further downstream. Expression of these isoforms occurs at very different levels in different cell types, however, Hap33/BAG-1S appears to be the most prominent form in the majority of cells under normal conditions and Hap29/BAG-1S is not consistently detected in all cells.
There is no direct correlate to human Hap46/BAG-1M in the mouse since the murine message has a GUG (coding for valine) in the corresponding position instead of AUG. The originally discovered 219 residue murine protein [4] is a somewhat shorter isoform which is produced from an internal AUG start codon [9] and roughly corresponds to human Hap33/BAG-1S. In order to avoid confusions, this protein is called "mouse BAG-1S" in this review. Most significantly, however, the murine and human protein factors are highly homologous.
The message is roughly 1.4 kb long [2] and harbors several translation initiation codons, giving rise to the series of polypeptide chains depicted schematically in Figure 1. The gene coding for these proteins is called "Bag1" and is located on human chromosome 9 in region p12 [10]; it is roughly 10 kb in size and contains seven exons. The promoter covers positions −353 to −54 upstream of the first translational start codon, lacks a TATA box but has a CCAAT box and contains binding sites for several transcription factors, which may be important in regulating the expression [11]. Interestingly, p53 mutants obtained from human tumors are able to upregulate the Bag1 promoter [11]. Moreover, expression of the Bag1gene is modulated by methylation reactions [12].
2. Polypeptide domains
Figure 1 shows that all protein isoforms have a common carboxy terminal region, but differ significantly in their amino terminal portions. Consequently, they share the functions mediated by the carboxy terminal part, possibly with individual additional molecular properties otherwise. It thus appears that all isoforms are roughly equivalent in biochemical reactions mediated by the carboxy terminal domain (orange in Figure 1), i.e. interactions with hsp70 heat shock proteins (see below).
A striking characteristic of Hap50/BAG-1L is a series of positively charged residues in proximity to the amino terminus, as is typical for nuclear localization signals (blue in Figure 1). However, Hap46/BAG-1M also contains a cluster of basic amino acids right next to its amino terminus. Both of these regions in Hap46/BAG-1M and Hap50/BAG-1L are of significance for interactions with nucleic acids (see below). By contrast, isoforms Hap33/BAG-1S, Hap29/BAG-1S, and mouse BAG-1S do not contain these basic sequences and — as a consequence — are unable to bind to DNA. The potential nuclear localization signal roughly in the middle of the Hap46/BAG-1M sequence (blue in Figure 1) appears to be masked by some as yet unknown mechanism, but apparently becomes activated upon heat shock, since a significant portion of Hap46/BAG-1M accumulates in the nuclear compartment in response to thermal stress (Figure 2, B vs. A).
The functional significance of the acidic hexarepeat domain (green in Figure 1) remains rather enigmatic at present, as it is quite variable amongst different isoforms. Deletion of this region did neither destroy transcriptional stimulation by Hap46/BAG-1M [15] nor was the acidic hexarepeat domain required for the inhibition of glucocorticoid-dependent transcription in transfected cells [16]. On the other hand, this domain has been reported responsible for the downregulation of glucocorticoid receptor mediated transactivation by Hap46/BAG-1M [17].
The ubiquitin-like domain (pink in Figure 1) is much shorter than ubiquitin itself, but nevertheless mediates cooperation with the proteasome machinery of protein degradation [18,19]. Conjugation with ubiquitin normally primes proteins for degradation by proteasomes, but Hap46/BAG-1M is relatively long-lived, even though it is ubiquitinylated [20]. The ubiquitin-like domain was found to be required for promoting the apoptotic activity of a synthetic retinoid [21], and prevention of stress-induced growth inhibition by Hap46/BAG-1M and isoforms depended on a conserved lysine residue within this domain [22]. On the other hand, the ubiquitin-like domain was expendable for Hap50/BAG-1L to affect the regulatory activity of androgen receptors [23].
3. Hsp70 molecular chaperones as primary interaction partners
Already the discovery of this new protein family disclosed the unusual property that its members interact with several completely unrelated protein structures. Aside from the proteins used in the original interaction screening assays (see above), several additional proteins soon became known to be able to associate with Hap46/BAG-1M and mouse BAG-1S. These were other members of the nuclear receptor family [2,24,25], platelet-derived growth factor receptor [5], Raf-1 protein kinase [26], several unrelated transcription factors [3], Siah-1A, a human homologue of Drosophila seven in absentia [27], and proteasomes [18]. However, light came into this puzzling problem when it was realized that primary and major interaction partners are members of the 70 kDa heat shock protein family. When several protein-protein interaction assays were employed to identify direct binding partners for Hap46/BAG-1M and mouse BAG-1S, the only polypeptide of significance was about 70 kDa in size and this was obtained with extracts from very different cells. It was then identified as the mammalian heat shock protein hsp70/hsc70 [3, 28–30]. However, to our great surprise the mammalian endoplasmic form BiP, as well as the hsp70 homologues in Saccharomyces cerevisiae and of bacterial and plant origin were found unable to bind to Hap46/BAG-1M. By contrast, hsp70 contained in Sf9 cells from the insect Spodoptera frugiperda, used in the baculovirus expression system for producing bait proteins, did interact perfectly well [3].
The stress-regulated form — hsp70 in the strict sense — originates in eukaryotes from several genes whose expression is controlled by heat and other types of stress. However, there also exists a slightly larger, but constitutively expressed counterpart, called hsc70. This very fact certainly points to some basic cellular function: the "chaperoning" of misfolded or partially unfolded polypeptides. Hsp70s are a large and highly conserved family of molecular chaperones which function in quite diverse cellular processes. In particular, they are able to bind a wealth of protein structures, especially if these are partially denatured, i.e. if they contain stretches of unfolded or misfolded polypeptides. Besides supporting newly synthesized and misfolded polypeptides to find their correct and biologically active conformations, they also minimize the aggregation of such proteins and they function in translocating proteins through membranes (for reviews, see refs. [31–34]). Moreover, they are important for the assembly of multimeric proteins, as for example in generating the heteromeric forms of steroid hormone receptors [35,36] and of transcriptional complexes, as pointed out below, as well as of several other large protein complexes.
All members of the hsp70 heat shock protein family consist of two major domains which are very different in structure (for reviews, see refs. [31,34,37]). The amino terminal portion of about 45 kDa is build up of two somewhat similar subdomains, I and II, each consisting of several α-helices. Together they form the so-called ATP-binding domain with the binding site for nucleotides located in the central cleft between them. The carboxy terminal portion of the hsp70 polypeptide is called the substrate-binding domain. It is intricately folded to accept the various partner proteins for binding in a channel within a ß-sandwich structure. Obviously, there is strong functional coupling by conformational changes between the ATP-binding and the substrate-binding domains.
The interaction of Hap46/BAG-1M and all isoforms makes use of the common carboxy terminal region, as has originally been shown by deletion of 47 residues from this end of the polypeptide [30,38]. Since this interaction is a very prominent property of the protein family, this common portion has been called "BAG-domain" (orange in Figure 1). However, this does not exclude the possibility that other regions of Hap46/BAG-1M and isoforms may also contribute to the interaction with hsp70 molecular chaperones. On the part of hsp70/hsc70, the ATP-binding domain is involved in the interaction, while the substrate binding domain remains available for further binding reactions [for reviews, cf. refs. 33,34]. This raises the possibility of multiple associations, and experiments with model proteins have in fact shown that trimeric complexes are easily formed [3,15,28,30,39]. Hsp70/hsc70 molecular chaperones as mediators of interactions also explain why very different bait proteins used in the original cloning experiments lead to the discovery of the same molecular sequence: hsc70 contained in these systems was able to serve as mediator. Interestingly, variations of the BAG-domain also exist in other proteins, which similarly mediate interactions with hsp70 chaperones; together they constitute the family of eukaryotic BAG proteins [38,40–42].
An interesting feature of hsp70 molecular chaperones is the structural flexibility of the two subdomains within the ATP-binding domain relative to each other. This was observed in experiments which studied the complex of the ATP-binding domain of the prokaryotic hsp70 protein DnaK with its regulator GrpE. X-ray crystallography disclosed contacts on both subdomains I and II with a significant opening of the cleft between them, as has been described clearly [37]. This readily explains why GrpE causes enhancement of the ADP/ATP exchange reaction of DnaK. Even though this movement of subdomains came to light by studies with the bacterial hsp70 protein DnaK, it is assumed to similarly occur in eukaryotic hsp70/hsc70 forms. Although structurally completely different, Hap46/BAG-1M and isoforms are functionally in a way eukaryotic counterparts of GrpE, however GrpE plays an essential role for the activity of DnaK in contrast to Hap46/BAG-1M and isoforms, whose effects on nucleotide exchange are more moderate. While GrpE binds asymmetrically as a dimer to DnaK, Hap46/BAG-1M or isoforms produce stoichiometric 1:1 complexes with eukaryotic hsp70 chaperones, even though hsp70s are normally much more abundant within cells. When the interaction between the BAG-domain and hsp70 was studied by X-ray analysis, NMR spectroscopy, and mutagenesis it was found that it forms a bundle of three α-helices that contact subdomain II of the ATP-binding domain [43,44]. Electrostatic interactions are of major importance in this interaction, whereby Arg308 (in the human Hap50/BAG-1L sequence) plays a major role, since exchange for alanine greatly reduced binding as well as in vitro activity; however other residues are also of importance. On the part of hsp70, the major portion of subdomain II suffices for binding, although the affinity is greatly reduced relative to the intact ATP-binding domain [45]. The techniques of phage display and of peptide scanning, however, resulted in several potential contact sites in two major regions, one on each subdomain I and II [46], suggesting that Hap46/BAG-1M and isoforms may use more than one contact with hsp70 chaperones for exerting their cellular effects. Binding of the BAG-domain probably favors an alternative structure of the ATP-binding domain that has a more open conformation [47], again reflecting flexibility of the structure.
With hsp70 molecular chaperones as major helpers in protein folding reactions in conjunction with hsp40, there immediately arose the question whether Hap46/BAG-1M and isoforms may affect this fundamental cellular activity in addition to the nucleotide exchange reaction. This was indeed observed in in vitro assays which employed various denatured proteins, in particular firefly luciferase and ß-galactosidase. While most reports describe inhibition of protein reactivation concomitant with reduced binding of misfolded proteins to the hsp70 molecular chaperone, positive effects on protein folding have also been observed [48–50]. This discrepancy is probably caused by details in experimental systems. Particularly in the presence of inorganic phosphate and at low concentrations of Hap46/BAG-1M protein renaturation was activated [50]; also the relative molar concentrations of hsp70 chaperone and Hap46/BAG-1M or isoform, as well as the type of isoform play important roles. Importantly, experiments with intact mammalian cells in culture also showed attenuation by overexpression of mouse BAG-1S of refolding denatured luciferase introduced into cells and thermally inactivated within them [51]. Thus Hap46/BAG-1M and isoforms have quite adequately been called cochaperones. This family of proteins, however, is but one of several known cochaperones which affect the protein folding and nucleotide exchange reactions of hsp70s, albeit in quite different ways. They certainly influence each others effects [see for example, refs. 28,52], so that a functional network exists that regulates the activities of hsp70 molecular chaperones.
4. Interactions with proteins independent of hsp70 molecular chaperones
In principle, Hap46/BAG-1M and isoforms might interact with various protein structures through mediation by hsp70/hsc70 molecular chaperones, as pointed out, or they may do so directly, i.e. independent of such mediation. In addition, Hap46/BAG-1M and Hap50/BAG-1L have the ability to bind to DNA, as discussed below. Of course, combinations between these types of interactions are expected to exist (see below). In any event, a multitude of bioactivities is produced by Hap46/BAG-1M and isoforms by such interactions.
To check for the involvement of hsp70/hsc70 molecular chaperones, we compared the retention of in vitro synthesized and radiolabeled proteins on a matrix to which either full-length Hap46/BAG-1M or deletion variant Hap46/BAG-1MΔC47 had been coupled from which the carboxy terminal 47 amino acid residues, the BAG-domain, are missing [15]. As expected, hsc70 contained in large amounts in the reticulocyte lysate used for in vitro translation was not retained by the matrix containing the deletion variant (Figure 3 A). Moreover, this variant matrix did not bind any protein that requires mediation by hsc70 molecular chaperones, as is the case for several transcription factors which had been selected to be structurally different (Figures 3 B to F). Examples are members of the nuclear receptor superfamily, like the glucocorticoid receptor (GR) and the estrogen receptor (ER), as well as the cAMP response element binding protein CREB, the regulator of osteoblast differentiation CbfA1/Runx2, and c-Fos, a component of transcription factor AP-1. By contrast, c-Jun, the companion protein of c-Fos in AP-1, and Gax, a transcription factor involved in regulating cell viability, attached to roughly similar extents to both matrices (Figures 3 G and H). Apparently, these protein factors do not depend on any hsp70 molecular chaperone as binding mediator; c-Jun even appeared to interact somewhat better with the matrix devoid of hsc70 binding ability.
Protein kinase Raf-1 was the first example of a protein with the ability for direct and specific interaction with mouse BAG-1S, thereby causing activation of the kinase activity [26]. The binding site was mapped adjacent to and partially overlapping with that for hsp70 and point mutations within the BAG-domain which destroyed hsp70 binding activity were still able to activate the Raf-1 kinase [54]. Induction of hsp70 by cell stress caused downregulation of the kinase activity in mammalian cells. Thus competition of Raf-1 and hsp70 for binding to BAG-1S was proposed as molecular basis for the coordination of signals for cell growth and mitogenesis [54].
The retinoblastoma susceptibility protein Rb is another direct binding partner of Hap46/BAG-1M since it is able to interact with mutants defective in hsp70/hsc70 binding [55]. The complex of Rb and Hap46/BAG-1M is stable enough so that Hap46/BAG-1M is cotransported by Rb into the nucleus. The complex is disrupted by the human papilloma virus oncoprotein E7, an inhibitor of Rb protein interactions [55], thus emphasizing the specificity of complex formation. This modulation of subcellular localization of Hap46/BAG-1M has significant consequences for the survival of colorectal epithelial and tumor cells [56], suggesting a function of Rb as physiological regulator of Hap46/BAG-1M.
Hap50/BAG-1L was observed to stimulate the transactivation function of the androgen receptor, another member of the nuclear receptor superfamily, however, Hap46/BAG-1M did so only when forced into the nucleus [23]. While the carboxy terminal hsp70 binding BAG-domain mediates in vitro interaction, the amino terminal portion of Hap50/BAG-1L is required for nuclear localization and for eliciting the biological effect [23]. This is quite reasonable since both the ligand-activated androgen receptor as well as Hap50/BAG-1L or Hap46/BAG-1M need to be nuclearly localized in order to cooperate functionally. A similar argument may also apply to the interaction with both types of estrogen receptors, since Hap50/BAG-1L, but not the shorter isoforms, increased estrogen-dependent transcription in mammary carcinoma cells [57]. The cooperation with members of the nuclear receptor family, however, is not at all uniform, but rather complex [58]. Transactivation by the vitamin D receptor was also enhanced by Hap50/BAG-1L, but not by shorter non-nuclear forms, and binding again depended on the amino terminal part of the molecule. Interestingly, not only a variant from which the carboxy terminal BAG-domain had been deleted, but also overexpression of Hap50/BAG-1L itself inhibited vitamin D-dependent transactivation [59,60], suggesting a regulatory role of Hap50/BAG-1L in the action of vitamin D.
Interestingly, a variant of Hap50/BAG-1L has been isolated from a human T-cell cDNA library in a yeast two-hybrid screen. This turned out to be carboxy terminally truncated by 34 amino acids and harbors alterations within terminal residues [61]; as a consequence, it is unable to interact with hsp70 molecular chaperones. This variant is nevertheless able to associate with the tubulin-associating protein RP1. Thus RP1 is another direct interaction partner of Hap50/BAG-1L and most probably of other isoforms as well, thus linking these to the microtubule system. The functional consequences of this interaction for cellular mobility, cytokinesis and the cytoskeleton are certainly of interest.
5. Interaction with DNA
Although Hap46/BAG-1M is an acidic protein with a pI of 5.3, it nevertheless contains a prominent cluster of basic amino acids right next to the amino terminus [2,10] which is quite unique. This region was in fact found to be involved in binding to DNA (Figure 4 A, lane 4 vs. 2). The DNA may be linear (Figure 4 A) or circular (Figure 4 B), and neither the origin nor the size of the DNA matter, except that a minimum length is required [13,15]. Multiple copies of Hap46/BAG-1M can simultaneously associate with DNA, possibly in a cooperative manner, since the upshift of complexes upon gel electrophoresis occurs progressively [13,15].
In contrast to a previous report which suggested a specific binding site within the Cytomegalovirus promoter [62], we did not detect any such nucleotide sequence specificity when we investigated Hap46/BAG-1M with two series of overlapping oligonucleotides of about 130 and 240 base pairs which cover the entire early gene promoter of the Cytomegalovirus [15]. However, this does not exclude limited preferences for some as yet unidentified combinations of nucleotides. On the other hand, such non-specific DNA binding completely depends on the positively charged sequence close to the amino terminus of the polypeptide chain, which consists of three consecutive lysines and of three arginines, separated by a centrally located neutral residue (cf. legend to Figure 1). Mutation analysis established that both of these trimeric clusters are required for the DNA binding ability of Hap46/BAG-1M [13,16,62], but the exact spacing between them does not matter. This non-specific DNA binding motif is involved in down-regulation of glucocorticoid receptor-mediated transactivation by Hap46/BAG-1M [16,63], even though the mechanism of this effect is not clear. In Hap50/BAG-1L the same basic peptide region (red in Figure 1) is available for interaction with DNA, but the polypeptide chain is extended further amino terminally and contains additional basic residues, which may be involved as well.
It is important to point out that both major interaction regions located close to the ends of the amino acid sequences of Hap46/BAG-1M and Hap50/BAG-1L, i.e. for DNA binding and for interaction with hsp70 molecular chaperones, are available for respective binding reactions at the same time. Thus trimeric complexes of the type DNA•Hap46/BAG-1M•hsc70 can easily be formed [13,15], as shown in Figure 4 (A and B, lanes 3). In these complexes the substrate-binding domain of hsc70 is still available for additional interactions with other proteins, for example transcription factors.
6. Effect of Hap46/BAG-1M and Hap50/BAG-1L on transcription
When we realized that Hap46/BAG-1M has the ability to interact with DNA, there immediately arose the idea that it might affect transcriptional activities, in particular since thermal stress causes a significant portion of Hap46/BAG-1M to translocate from the cytoplasm to the cell nucleus (cf. Figure 2, B vs. A). By contrast, Hap50/BAG-1L (Figure 2 C) is mainly localized in the nucleus under normal cell culture conditions [6,7,14,23], a fact that immediately points to a function within the nucleus.
Figure 5 A shows in vitro transcription experiments using HeLa nuclear extracts. Addition of Hap46/BAG-1M to these assays resulted in roughly a 10-fold stimulation of transcription from various templates [13,15]. It did not matter whether eukaryotic or prokaryotic DNA was used as template and whether or not it contained promoter elements. The fact that the transcripts have no distinct length suggests random initiation of transcription and termination at the end of linearized DNA fragments. Involvement of the amino terminal DNA binding region in the effect on transcription became evident from the observation that deletion of the 10 amino terminal residues from Hap46/BAG-1M resulted in complete loss of stimulation [13].
These in vitro experiments were then complemented by studies with intact human cells transfected with an expression vector for Hap46/BAG-1M. Upon heat treatment of these cells which results in nuclear translocation (cf. Figure 2 B), overexpression of Hap46/BAG-1M largely compensated the drastic shut-down of cellular transcription which normally occurs following heat stress (Figure 5 B upper panel, lane 4 vs. 2). Interestingly, induction of heat shock proteins hsp70 and hsp40 was further stimulated by Hap46/BAG-1M (Figure 5 B lower panels, lane 4 vs. 2). Upon overexpressing Hap50/BAG-1L in a similar cell system, we again observed increased transcriptional activities (cf. Figure 5 C). The expression of several endogenous genes which are under cellular regulation, like those coding for c-Jun, c-Fos, or the receptors for glucocorticoids or estrogens were stimulated, while that for actin, for example, was not enhanced [14].
7. Molecular model for the effects on transcription
Taking the above observations into consideration, it appears that the effects of Hap46/BAG-1M and Hap50/BAG-1L on transcription are of major physiological relevance. This activity of Hap46/BAG-1M is discussed here in terms of a molecular model that is depicted in Figure 6. Two prominent features of this protein are taken into account: DNA binding ability and the potential to interact with a variety of transcription factors either by mediation through hsp70 molecular chaperones or directly (cf. Fig 3). Several molecules of Hap46/BAG-1M might bind simultaneously to a stretch of DNA within a region of transcriptionally active chromatin and establish contacts to the transcriptional apparatus itself, as well as to transcripion factors attached to DNA in cis, but possibly at distant locations. In this way, Hap46/BAG-1M may help to assemble various cofactors into functional complexes, eventually causing increased expression of the respective gene. A transcription factor attached to Hap46/BAG-1M, as shown in Figure 6, may for example, be identical to a steroid hormone receptor, which itself is bound to a specific response element on the DNA in some distance to the gene to be transcribed.
The same considerations similarly apply to the large isoform Hap50/BAG-1L. Interestingly, Hap50/BAG-1L was found to increase estrogen-dependent transcription in mammary carcinoma cells and thus turns out as important determinant of estrogen function [57]. Of course, several transcription factors might come into contact through multiple molecules of Hap46/BAG-1M or Hap50/BAG-1L attached to DNA. Chromatin immunoprecipitation studies in which Hap50/BAG-1L, the molecular chaperone hsp70, and the androgen receptor were found targeted to the relevant response element [65] support the above notion and clearly favor the nucleation of transcriptional regulatory complexes, as proposed here.
The effects of Hap46/BAG-1M and Hap50/BAG-1L on transcription, however, do not necessarily need to be positive, and in case of steroid hormone receptors as transcriptional regulators, the responses are not at all uniform [58]. The inhibitory activity of Hap46/BAG-1M on glucocorticoid receptor-mediated transcription was found to depend on the amino terminal DNA binding region, while Hap33/BAG-1S in contrast, did not elicit a similar effect. In addition, an intact carboxy terminal domain for interaction with hsp70 molecular chaperones was also found to be required [16,63]. This implies that both functional domains close to the ends of the Hap46/BAG-1M sequence need to be present in cis.
The molecular model of Figure 6 suggests several details which still require experimental elucidation. Most importantly, it will be of interest which genes or groups of genes are affected by Hap46/BAG-1M and by Hap50/BAG-1L. In addition, it will be interesting to find out how various transcription factors cooperate molecularly with Hap46/BAG-1M and Hap50/BAG-1L to bring about specific effects. In addition to the tumor suppressor protein Rb, c-Jun, and Gax, other transcription factors may interact directly with the transcription stimulating protein Hap46/BAG-1M, i.e. independent of hsp70 molecular chaperones. Transcription factor AP-1 is of particular interest in the present context since one component, c-Fos requires mediation by an hsp70 chaperone, while the other, c-Jun is able to interact directly with Hap46/BAG-1M (cf. Figures 3 F and G). It will be interesting to see which region of Hap46/BAG-1M is involved in this direct interaction and whether it overlaps with some of the other binding sites. The above difference between c-Fos and c-Jun is of significance as they form functionally active heterodimers in AP-1, and the DNA binding of heteromeric AP-1 was found to be selectively modified by hsp70 heat shock proteins relative to homodimeric c-Jun [66]. This supports the notion that the DNA binding proteins Hap46/BAG-1M and AP-1 together with hsp70 molecular chaperones cooperate in forming a network of protein-protein and protein-DNA interactions which may be involved in targeting and activating the basal transcription machinery.
While addition of Hap46/BAG-1M to the in vitro transcription system clearly caused a rather large positive effect (cf. Figure 5 A, lanes 2, 4, and 7 vs. 1, 3, and 6), it came as a surprise that the carboxy terminal deletion variant Hap46/BAG-1MΔC47 also produced some, albeit moderate stimulation [15], as shown in Figure 5 A (lanes 5 and 8 vs. 3 and 6). This points to involvement of hsp70 molecular chaperones and suggests that they as well play an important role in transcription. Remodeling of transcriptional regulatory complexes by molecular chaperones has indeed been pointed out before [67,68]. It is of significance in this context that newly synthesized hsp70 in response to cellular stress readily becomes nuclearly localized [69]. Since Hap46/BAG-1M also accumulates in the cell nucleus following heat shock, cooperation between these proteins to affect transcription, as proposed in the model of Figure 6, appears reasonable for regulating cellular activities.
8. Concluding remarks
In contrast to Hap46/BAG-1M and Hap50/BAG-1L, the prominently expressed short isoform Hap33/BAG-1S which is inert towards DNA appears most suitable for affecting protein folding and/or degradation. Since multiple biochemical reactions possibly cooperate in eliciting complex cellular responses, it is conceivable that modulation of transcription by Hap50/BAG-1L or Hap46/BAG-1M as well as the effects of Hap33/BAG-1S on refolding or degradation of misfolded proteins contribute to a cells defense against apoptosis. It will be important to clarify the individual shares of these cellular reactions. Depending on its prevalent intracellular localization, Hap46/BAG-1M may even serve a dual purpose: transcriptional modulation within the nucleus and control of protein functionality in the cytoplasm, thus providing a molecular link between such diverse activities and helping the cell to survive under adverse conditions. The pleiotropic activities of Hap46/BAG-1M and isoforms within the nucleus may also include control of protein quality. In any event, the intracellular localization of Hap46/BAG-1M and isoforms is rather dynamic, with relocalization possibly depending on specific signals as well as on the distribution of interacting protein factors, most importantly hsp70 chaperones. Thus, MCF7 human mammary carcinoma cells under heat stress were described to relocalize Hap33/BAG-1S from the cytoplasm to the nucleus [21] concomitant with nuclear accumulation of hsp70, thereby preventing stress-induced growth inhibition. Recruitment of Hap46/BAG-1M to the nucleus by activated glucocorticoid receptors has also been reported [17], but it is not clear whether this occurs in conjunction with hsp70 molecular chaperones. As mentioned above, nuclear transport of Hap46/BAG-1M may as well occur together with the retinoblastoma protein Rb [55,56]. Nevertheless, the regulation of expression of different isoforms needs to be investigated, which may depend, at least to some extent, on cap-independent initiation of translation at internal ribosome entry sites [9,70]. This is of interest since stage- and site-specific expression of isoforms has been observed during early murine development [71]. Moreover, the involvement of these proteins in the regulation of cell differentiation has been observed in several systems [72,73].
A very exciting case of regulation was discovered upon disrupting the Bag1 gene in mice [74]. Homozygous Bag1−/− embryos were severely growth-retarded, died before term, and showed multiple developmental defects. Particularly, cell survival within the nervous and hematopoietic systems was affected, again emphasizing the anti-apoptotic potential of BAG-1 proteins. Even though the protein kinases Raf/ERK and Akt were per se not defective in these mutant animals, the protein phosphorylation pattern was affected with loss of phosphorylation of the regulatory protein Bad at Ser136, which may be caused by changes in intracellular distribution and targeting [74]. These observations suggest that at least one of the products of the Bag1 gene functions as mediator of extracellular survival signals which normally prevent apoptosis in certain stem cells and point to the exciting fact that members of the Hap46/BAG-1M and Hap50/BAG-1L protein family fulfill some vital function within the mammalian organism. It is of interest to find out to which extent hsp70 molecular chaperones are involved in the above cellular effects. The growth of neurites has been studied in cell culture using rat phaeochromocytoma cells PC12 primed with NGF [75]. It was found that overexpression of the small isoform BAG-1S interferes with neurite extension and that interaction with an hsp70 molecular chaperone plays a crucial role in this effect. By contrast, the large isoform Hap50/BAG-1L did not inhibit neurite outgrowth in such neurotrophin-treated cells. Significant differences in intracellular localization of these isoforms may be responsible for such functional differences.
Obviously, Hap46/BAG-1M, Hap50/BAG-1L and isoforms play an important role in modulating cell survival. This is of particular importance under cellular stress, in neoplastic cell conditions and even under anti-tumor therapy. Cells highly expressing the Bag1 gene are probably selected for by cytotoxic treatments. Thus, multidrug-resistant cells of decreased sensitivity towards apoptosis were observed to express Hap46/BAG-1M and Hap33/BAG-1S at rather high levels [76].
Hap50/BAG-1L and Hap46/BAG-1M may be regarded as pro-survival proteins, and thus became markers for the aggressiveness of various cancers, in particular of mammary carcinomas; they are predictive factors for the clinical prognosis of patients. Such correlations have been observed in many clinical and cell culture studies [for example, refs. 7,77–85]. Hap50/BAG-1L in particular is often expressed at rather high levels in tumors and metastases as compared to normal cells. It is of interest in this context that expression of the Bag1 gene is enhanced by tumor-derived p53 mutants [11] and that methylation reactions play an important role in its expression [12]. Consequently, Hap50/BAG-1L and Hap46/BAG-1M may serve in the future as molecular targets for therapeutic approaches, as has been discussed [83,86]. Indeed, the immunosuppressant rapamycin [87,88] and the protein kinase inhibitors flavopiridol and 7-hydroxy-staurosporine [89] are known to downregulate the expression of the Bag1 gene, thereby affecting apoptotic cell responses. By contrast, interleukin-2 as well as other cell survival signals were found to upregulate the expression of the Bag1 gene in different cell types [9,87]. Retinoic acid-induced apoptosis in breast cancer cells in culture is inhibited by the interaction of Hap33/BAG-1S with retinoic acid receptors and interference with their ability to bind to specific DNA response elements [25], while the apoptotic activity of N-(4-hydroxyphenyl)retinamide, used for chemoprevention and treatment of various cancers, was enhanced by overexpression of Hap50/BAG-1L or shorter isoforms in a cervical carcinoma cell line [21]. On the other hand, HeLa cells in which the levels of Hap50/BAG-1L and Hap33/BAG-1S were down-regulated by RNA interference, had lower growth rates and became less sensitive to several anti-cancer drugs [90]. Also, overexpression of the Bag1 gene in mammary carcinomas may limit the efficacy of PPARγ agonists, like 15-deoxy-PGJ2, as apoptosis-inducing agents [91]. This brief discussion already points to the complexity of the problem and certainly shows that much more basic information is needed. Nevertheless, some of the above mentioned observations raise the hope that it will be possible in the future to manipulate the cellular levels of Hap50/BAG-1L and Hap46/BAG-1M. It could thus be highly beneficial for the in situ treatment of several cancer types if the levels of these isoforms within tumor cells were lowered by specific agents. This may then occur either in conjunction with hsp70 chaperone activities or independent of them.
I am grateful to the Biochemie-Zentrum for sponsoring this paper by taking care of the publication expenses. I would like to thank Professor Dr. G. Petersen for many interesting and very helpful discussions. I apologize to all colleagues whose work is not cited in this review.
- Zeiner, M; Gehring, U. Expression screening for interacting proteins using immunochemical detection. Nucleic Acids Res 1994, 22, 3255–3256, doi:10.1093/nar/22.15.3255. 8065947
- Zeiner, M; Gehring, U. A protein that interacts with members of the nuclear hormone receptor family: Identification and cDNA cloning. Proc. Natl. Acad. Sci. USA 1995, 92, 11465–11469, doi:10.1073/pnas.92.25.11465. 8524784
- Zeiner, M; Gebauer, M; Gehring, U. Mammalian protein RAP46: An interaction partner and modulator of 70 kDa heat shock proteins. EMBO J 1997, 16, 5483–5490, doi:10.1093/emboj/16.18.5483. 9312007
- Takayama, S; Sato, T; Krajewski, S; Kochel, K; Irie, S; Millan, JA; Reed, JC. Cloning and functional analysis of BAG-1: A novel Bcl-2-binding protein with anti-cell death activity. Cell 1995, 80, 279–284, doi:10.1016/0092-8674(95)90410-7. 7834747
- Bardelli, A; Longati, P; Albero, D; Goruppi, S; Schneider, C; Ponzetto, C; Comoglio, PM. HGF receptor associates with the anti-apoptotic protein BAG-1 and prevents cell death. EMBO J 1996, 15, 6205–6212. 8947043
- Packham, G; Brimmel, M; Cleveland, JL. Mammalian cells express two differently localized Bag-1 isoforms generated by alternative translation initiation. Biochem. J 1997, 328, 807–813. 9396724
- Takayama, S; Krajewski, S; Krajewska, M; Kitada, S; Zapata, JM; Kochel, K; Knee, D; Scudiero, D; Tudor, G; Miller, GJ; Miyashita, T; Yamada, M; Reed, JC. Expression and location of Hsp70/Hsc-binding anti-apoptotic protein BAG-1 and its variants in normal tissues and tumor cell lines. Cancer Res 1998, 58, 3116–3131. 9679980
- Yang, X; Chernenko, G; Hao, Y; Ding, Z; Pater, MM; Pater, A; Tang, S-C. Human BAG-1/RAP46 protein is generated as four isoforms by alternative translation initiation and overexpressed in cancer cells. Oncogene 1998, 17, 981–989, doi:10.1038/sj.onc.1202032. 9747877
- Townsend, PA; Cutress, RI; Sharp, A; Brimmell, M; Packham, G. BAG-1: A multifunctional regulator of cell growth and survival. Biochim. Biophys. Acta 2003, 1603, 83–98. 12618309
- Takayama, S; Kochel, K; Irie, S; Inazawa, J; Abe, T; Sato, T; Druck, T; Huebner, K; Reed, JC. Cloning of cDNAs encoding the human BAG1 protein and localization of the human BAG1 gene to chromosome 9p12. Genomics 1996, 35, 494–498, doi:10.1006/geno.1996.0389. 8812483
- Yang, X; Pater, A; Tang, SC. Cloning and characterization of the human BAG-1 gene promoter: Upregulation by tumor-derived p53 mutants. Oncogene 1999, 18, 4546–4553, doi:10.1038/sj.onc.1202843. 10467399
- Sun, L; Huang, L; Nguyen, P; Bisht, KS; Bar-Sela, G; Ho, AS; Bradbury, CM; Yu, W; Cui, H; Lee, S; Trepel, JB; Feinberg, AP; Gius, D. DNA methyltransferase 1 and 3B activate BAG-1 expression via recruitment of CTCFL/BORIS and modulation of promoter histone methylation. Cancer Res 2008, 68, 2726–2735, doi:10.1158/0008-5472.CAN-07-6654. 18413740
- Zeiner, M; Niyaz, Y; Gehring, U. The hsp70-associating protein Hap46 binds to DNA and stimulates transcription. Proc. Natl. Acad. Sci. USA 1999, 96, 10194–10199, doi:10.1073/pnas.96.18.10194. 10468585
- Niyaz, Y; Zeiner, M; Gehring, U. Transcriptional activation by the human Hsp70-associating protein Hap50. J. Cell Sci 2001, 114, 1839–1845. 11329370
- Niyaz, Y; Frenz, I; Petersen, G; Gehring, U. Transcriptional stimulation by the DNA binding protein Hap46/BAG-1M involves hsp70/hsc70 molecular chaperones. Nucleic Acids Res 2003, 31, 2209–2216, doi:10.1093/nar/gkg303. 12682371
- Schmidt, U; Wochnik, GM; Rosenhagen, MC; Young, JC; Hartl, FU; Holsboer, F; Rein, T. Essential role of the unusual DNA-binding motif of BAG-1 for inhibition of the glucocorticoid receptor. J. Biol. Chem 2003, 278, 4926–4931, doi:10.1074/jbc.M212000200. 12482863
- Schneikert, J; Hübner, S; Martin, E; Cato, ACB. A nuclear action of the eukaryotic cochaperone RAP46 in downregulation of glucocorticoid receptor activity. J. Cell Biol 1999, 146, 929–940, doi:10.1083/jcb.146.5.929. 10477749
- Lüders, J; Demand, J; Höhfeld, J. The ubiquitin-related BAG-1 provides a link between the molecular chaperones hsc70/hsp70 and the proteasome. J. Biol. Chem 2000, 275, 4613–4617, doi:10.1074/jbc.275.7.4613. 10671488
- Esser, C; Alberti, S; Höhfeld, J. Cooperation of molecular chaperones with the ubiquitin/proteasome system. Biochim. Biophys. Acta 2004, 1695, 171–188, doi:10.1016/j.bbamcr.2004.09.020. 15571814
- Alberti, S; Demand, J; Esser, C; Emmerich, N; Schild, H; Höhfeld, J. Ubiquitinylation of BAG-1 suggests a novel regulatory mechanism during the sorting of chaperone substrates to the proteasome. J. Biol. Chem 2002, 277, 45920–45927, doi:10.1074/jbc.M204196200. 12297498
- Yang, X; Hao, Y; Ding, Z; Pater, A. BAG-1 promotes apoptosis induced by N-(4-hydroxyphenl)retinamide in human cervical carcinoma cells. Exp. Cell Res 2000, 256, 491–499, doi:10.1006/excr.2000.4829. 10772821
- Townsend, PA; Cutress, RI; Sharp, A; Brimmell, M; Packham, G. BAG-1 prevents stress-induced long-term growth inhibition in breast cancer cells via a chaperone-dependent pathway. Cancer Res 2003, 63, 4150–4157. 12874020
- Knee, DA; Froesch, BA; Nuber, U; Takayama, S; Reed, JC. Structure-function analysis of Bag1 proteins. Effects on androgen receptor transcriptional activity. J. Biol. Chem 2001, 276, 12718–12724, doi:10.1074/jbc.M010841200. 11278763
- Froesch, BA; Takayama, S; Reed, JC. BAG-1L protein enhances androgen receptor function. J. Biol. Chem 1998, 273, 11660–11666, doi:10.1074/jbc.273.19.11660. 9565586
- Liu, R; Takayama, S; Zheng, Y; Froesch, B; Chen, G-Q; Zhang, X; Reed, JC; Zhang, X-K. Interaction of BAG-1 with retinoic acid receptor and its inhibition of retinoic acid-induced apoptosis in cancer cell. J. Biol. Chem 1998, 273, 16985–16992, doi:10.1074/jbc.273.27.16985. 9642262
- Wang, H-G; Takayama, S; Rapp, UR; Reed, JC. Bcl-2 interacting protein, BAG-1, binds to and activates the kinase Raf-1. Proc. Natl. Acad. Sci. USA 1996, 93, 7063–7068, doi:10.1073/pnas.93.14.7063. 8692945
- Matsuzawa, S; Takayama, S; Froesch, BA; Zapata, JM; Reed, JC. P53-inducible human homologue of Drosophila seven in absentia (Siah) inhibits cell growth: Suppression by BAG-1. EMBO J 1998, 17, 2736–2747, doi:10.1093/emboj/17.10.2736. 9582267
- Gebauer, M; Zeiner, M; Gehring, U. Proteins interacting with the molecular chaperone hsp70/hsc70: physical associations and effects on refolding activity. FEBS Lett 1997, 417, 109–113, doi:10.1016/S0014-5793(97)01267-2. 9395086
- Höhfeld, J; Jentsch, S. GrpE-like regulation of the hsc70 chaperone by the anti-apoptotic protein BAG-1. EMBO J 1997, 16, 6209–6216, doi:10.1093/emboj/16.20.6209. 9321400
- Takayama, S; Bimston, DN; Matsuzawa, S; Freeman, BC; Aime-Sempe, C; Xie, Z; Morimoto, RI; Reed, JC. BAG-1 modulates the chaperone activity of hsp70/hsc70. EMBO J 1997, 16, 4887–4896, doi:10.1093/emboj/16.16.4887. 9305631
- Fink, AL. Chaperone-mediated protein folding. Physiol. Rev 1999, 79, 425–449. 10221986
- Naylor, DJ; Hartl, FU. Contribution of molecular chaperones to protein folding in the cytoplasm of prokaryotic and eukaryotic cells. Biochem. Soc. Symp 2001, 68, 45–68. 11573347
- Mayer, MP; Bukau, B. Hsp70 chaperones: Cellular functions and molecular mechanism. Cell. Mol. Life Sci 2005, 62, 670–684, doi:10.1007/s00018-004-4464-6. 15770419
- Bukau, B; Weissman, J; Horwich, A. Molecular chaperones and protein quality control. Cell 2006, 125, 443–451, doi:10.1016/j.cell.2006.04.014. 16678092
- Cheung, J; Smith, DF. Molecular chaperone interactions with steroid receptors: An update. Mol. Endocrinol 2000, 14, 939–946, doi:10.1210/me.14.7.939. 10894145
- Pratt, WB; Galigniana, MD; Morishima, Y; Murphy, PJ. Role of molecular chaperones in steroid receptor action. Essays Biochem 2004, 40, 41–58. 15242338
- Mayer, MP; Brehmer, D; Gässler, CS; Bukau, B. Hsp70 chaperone machines. Adv. Protein Chem 2001, 59, 1–44. 11868269
- Takayama, S; Xie, Z; Reed, JC. An evolutionarily conserved family of hsp70/hsc70 molecular chaperone regulators. J. Biol. Chem 1999, 274, 781–786, doi:10.1074/jbc.274.2.781. 9873016
- Bimston, D; Song, J; Winchester, D; Takayama, S; Reed, JC; Morimoto, RI. BAG-1, a negative regulator of Hsp70 chaperone activity, uncouples nucleotide hydrolysis from substrate release. EMBO J 1998, 17, 6871–6878, doi:10.1093/emboj/17.23.6871. 9843493
- Takayama, S; Reed, JC. Molecular chaperone targeting and regulation by BAG family proteins. Nature Cell Biol 2001, 3, E237–E241, doi:10.1038/ncb1001-e237. 11584289
- Doong, H; Vrailas, A; Kohn, EC. What's in the 'BAG'? - A functional domain analysis of the BAG-family proteins. Cancer Lett 2002, 188, 25–32, doi:10.1016/S0304-3835(02)00456-1. 12406544
- Sondermann, H; Ho, AK; Listenberger, LL; Siegers, K; Moarefi, I; Wente, SR; Hartl, F-U; Young, JC. Prediction of novel Bag-1 homologs based on structure/function analysis identifies Snl1p as an hsp70 co-chaperone in Saccharomyces cerevisiae. J. Biol. Chem 2002, 277, 33220–33227, doi:10.1074/jbc.M204624200. 12105220
- Briknarová, K; Takayama, S; Brive, L; Havert, ML; Knee, DA; Velasco, J; Homma, S; Cabezas, E; Stuart, J; Hoyt, DW; Satterthwait, AC; Llinás, M; Reed, JC; Ely, KR. Structural analysis of BAG1 cochaperone and its interactions with hsc70 heat shock protein. Nature Struct. Biol 2001, 8, 349–352, doi:10.1038/86236. 11276257
- Sondermann, H; Scheufler, C; Schneider, C; Höhfeld, J; Hartl, F-U; Moarefi, I. Structure of a Bag/hsc70 complex: Convergent functional evolution of hsp70 nucleotide exchange factors. Science 2001, 291, 1553–1557, doi:10.1126/science.1057268. 11222862
- Brive, L; Takayama, S; Briknarová, K; Homma, S; Ishida, SK; Reed, JC; Ely, KR. The carboxy-terminal lobe of hsc70 ATPase domain is sufficient for binding to BAG1. Biochem. Biophys. Res. Commun 2001, 289, 1099–1105, doi:10.1006/bbrc.2001.6087. 11741305
- Petersen, G; Hahn, C; Gehring, U. Dissection of the ATP-binding domain of the chaperone hsc70 for interaction with the cofactor Hap46. J. Biol. Chem 2001, 276, 10178–10184, doi:10.1074/jbc.M006967200. 11121403
- Osipiuk, J; Walsh, MA; Freeman, BC; Morimoto, RI; Joachimiak, AJ. Structure of a new crystal form of human hsp70 ATPase domain. Acta Cryst 1999, D55, 1105–110.
- Terada, K; Mori, M. Human DnaJ homologs dj2 and dj3, and bag-1 are positive cochaperones of hsc70. J. Biol. Chem 2000, 275, 24728–24734, doi:10.1074/jbc.M002021200. 10816573
- Lüders, J; Demand, J; Papp, O; Höhfeld, J. Distinct isoforms of the cofactor BAG-1 differentially affect hsc70 chaperone function. J. Biol. Chem 2000, 275, 14817–14823, doi:10.1074/jbc.275.20.14817. 10809723
- Gässler, CS; Wiederkehr, T; Brehmer, D; Bukau, B; Mayer, MP. Bag-1M accelerates nucleotide release for human hsc70 and hsp70 and can act concentration-dependent as positive and negative cofactor. J. Biol. Chem 2001, 276, 32538–32544, doi:10.1074/jbc.M105328200. 11441021
- Nollen, EAA; Brunsting, JF; Song, J; Kampinga, HH; Morimoto, RI. Bag1 functions in vivo as a negative regulator of hsp70 chaperone activity. Mol. Cell. Biol 2000, 20, 1083–1088, doi:10.1128/MCB.20.3.1083-1088.2000. 10629065
- Nollen, EAA; Kabakov, AE; Brunsting, JF; Kanon, B; Höhfeld, J; Kampinga, HH. Modulation of in vivo HSP70 chaperone activity by Hip and Bag-1. J. Biol. Chem 2001, 276, 4677–4682, doi:10.1074/jbc.M009745200. 11076956
- Gehring, U. Activities of the cochaperones Hap46/BAG-1M and Hap50/BAG-1L and isoforms. Cell Stress Chaperones 2006, 11, 295–303, doi:10.1379/1466-1268(2006)11[295:AOTCBA]2.0.CO;2. 17278878
- Song, J; Takeda, M; Morimoto, RI. Bag1-Hsp70 mediates a physiological stress signalling pathway that regulates Raf-1/ERK and cell growth. Nature Cell Biol 2001, 3, 276–282, doi:10.1038/35060068. 11231577
- Arhel, NJ; Packham, G; Townsend, PA; Collard, TJ; H-Zahed, AM; Sharp, A; Cutress, RI; Malik, K; Hague, A; Paraskeva, C; Williams, AC. The retinoblastoma protein interacts with Bag-1 in human colonic adenoma and carcinoma derived cell lines. Int. J. Cancer 2003, 106, 364–371, doi:10.1002/ijc.11257. 12845674
- Clemo, NK; Arhel, NJ; Barnes, JD; Baker, J; Moorghen, M; Packham, GK; Paraskeva, G; Williams, AC. The role of the retinoblastoma protein (Rb) in the nuclear localization of BAG-1: Implications for colorectal tumour cell survival. Biochem Soc Trans 2005, 33, 676–678, doi:10.1042/BST0330676. 16042572
- Cutress, RI; Townsend, PA; Sharp, A; Maison, A; Wood, L; Lee, R; Brimmell, M; Mullee, MA; Johnson, PW; Royle, GT; Bateman, AC; Packham, G. The nuclear BAG-1 isoform, BAG-1L, enhances oestrogen-dependent transcription. Oncogene 2003, 22, 4973–4982, doi:10.1038/sj.onc.1206688. 12902980
- Cato, AC; Mink, S. BAG-1 family of cochaperones in the modulation of nuclear receptor action. J. Steroid Biochem. Mol. Biol 2001, 78, 379–388, doi:10.1016/S0960-0760(01)00114-5. 11738548
- Guzey, M; Takayama, S; Reed, JC. BAG1L enhances trans-activation function of the vitamin D receptor. J. Biol. Chem 2000, 275, 40749–40756, doi:10.1074/jbc.M004977200. 10967105
- Witcher, M; Yang, X; Pater, A; Tang, S-C. BAG-1 p50 isoform interacts with the vitamin D receptor and its cellular overexpression inhibits the vitamin D pathway. Exp. Cell Res 2001, 265, 167–173, doi:10.1006/excr.2001.5176. 11281654
- Wadle, A; Mischo, A; Henrich, PP; Stenner-Lieven, F; Scherer, C; Imig, J; Petersen, G; Pfreundschuh, M; Renner, C. Characterization of Hap/BAG-1 variants as RP1 binding proteins with antiapoptotic activity. Int. J. Cancer 2005, 117, 896–904, doi:10.1002/ijc.21259. 15986447
- Takahashi, N; Sasaki, R; Takahashi, J; Takayama, S; Reed, JC; Andoh, T. BAG-1M, an isoform of Bcl-2-interacting protein BAG-1, enhances gene expression driven by CMV promoter. Biochem. Biophys. Res. Commun 2001, 286, 807–814, doi:10.1006/bbrc.2001.5473. 11520069
- Schneikert, J; Hübner, S; Langer, G; Petri, T; Jäättelä, M; Reed, J; Cato, AC. Hsp70-RAP46 interaction in downregulation of DNA binding by glucocorticoid receptor. EMBO J 2000, 19, 6508–6516, doi:10.1093/emboj/19.23.6508. 11101523
- Gehring, U. Biological activities of HAP46/BAG-1. EMBO Reports 2004, 5, 148–153, doi:10.1038/sj.embor.7400083. 14755308
- Shatkina, L; Mink, S; Rogatsch, H; Klocker, H; Langer, G; Nestl, A; Cato, AC. The cochaperone Bag-1L enhances androgen receptor action via interaction with the NH2-terminal region of the receptor. Mol. Cell. Biol 2003, 23, 7189–7197, doi:10.1128/MCB.23.20.7189-7197.2003. 14517289
- Carter, DA. Modulation of cellular AP-1 DNA binding activity by heat shock proteins. FEBS Lett 1997, 416, 81–85, doi:10.1016/S0014-5793(97)01174-5. 9369238
- Freeman, BC; Yamamoto, KR. Disassembly of transcriptional regulatory complexes by molecular chaperones. Science 2002, 296, 2232–2235, doi:10.1126/science.1073051. 12077419
- Morimoto, RI. Dynamic remodeling of transcription complexes by molecular chaperones. Cell 2002, 110, 281–284, doi:10.1016/S0092-8674(02)00860-7. 12176314
- Velazquez, JM; Lindquist, S. Hsp70: Nuclear concentration during environmental stress and cytoplasmatic storage during recovery. Cell 1984, 36, 655–662, doi:10.1016/0092-8674(84)90345-3. 6421488
- Pickering, BM; Mitchell, SA; Evans, JR; Willis, AE. Polypyrimidine tract binding protein and poly r(C) binding protein 1 interact with the BAG-1 IRES and stimulate its activity in vitro and in vivo. Nucleic Acids Res 2003, 31, 639–646, doi:10.1093/nar/gkg146. 12527772
- Crocoll, A; Blum, M; Cato, AC. Isoform-specific expression of BAG-1 in mouse development. Mech. Dev 2000, 91, 355–359, doi:10.1016/S0925-4773(99)00284-1. 10704864
- Kermer, P; Krajewska, M; Zapata, JM; Takayama, S; Mai, J; Krajewski, S; Reed, JC. Bag1 is a regulator and marker of neuronal differentiation. Cell Death Differ 2002, 9, 405–413, doi:10.1038/sj.cdd.4400972. 11965493
- Tare, RS; Townsend, PA; Packham, GK; Inglis, S; Oreffo, RO. Bcl-2-associated athanogene-1 (BAG-1): A transcriptional regulator mediating chondrocyte survival and differentiation during endochondral ossification. Bone 2008, 42, 113–128, doi:10.1016/j.bone.2007.08.032. 17950682
- Götz, R; Wiese, S; Takayama, S; Camarero, GC; Rossoll, W; Schweizer, U; Troppmair, J; Jablonka, S; Holtmann, B; Reed, JC; Rapp, UR; Sendtner, M. Bag1 is essential for differentiation and survival of hematopoietic and neuronal cells. Nature Neurosci 2005, 8, 1169–1178, doi:10.1038/nn1524. 16116448
- Frebel, K; Wiese, S; Funk, N; Pühringer, D; Sendtner, M. Differential modulation of neurite growth by the S- and the L-forms of bag1, a co-chaperone of hsp70. Neurodegenerative Dis 2007, 4, 261–269, doi:10.1159/000101850.
- Ding, Z; Xang, X; Pater, A; Tang, S-C. Resistance to apoptosis is correlated with the reduced caspase-3 activation and enhanced expression of antiapoptotic proteins in human cervical multidrug-resistant cells. Biochem. Biophys. Res. Commun 2000, 270, 415–420, doi:10.1006/bbrc.2000.2432. 10753639
- Turner, BC; Krajewski, S; Krajewska, M; Takayama, S; Gumbs, AA; Carter, D; Rebbeck, TR; Haffty, BG; Reed, JC. BAG-1: A novel biomarker predicting long-term survival in early-stage breast cancer. J. Clin. Oncol 2001, 19, 992–1000. 11181661
- Cutress, RI; Townsend, PA; Brimmell, M; Bateman, AC; Hague, A; Packham, G. BAG-1 expression and function in human cancer. Br. J. Cancer 2002, 87, 834–839, doi:10.1038/sj.bjc.6600538. 12373595
- Kikuchi, R; Noguchi, T; Takeno, S; Funada, Y; Moriyama, H; Uchida, Y. Nuclear BAG-1 expression reflects malignant potential in colorectal carcinomas. Br. J. Cancer 2002, 87, 1136–1139, doi:10.1038/sj.bjc.6600579. 12402153
- Kudoh, M; Knee, DA; Takayama, S; Reed, JC. Bag1 proteins regulate growth and survival of ZR-75–1 human breast cancer cells. Cancer Res 2002, 62, 1904–1909. 11912172
- Pusztai, L; Krishnamurti, S; Perez Cardona, J; Sneige, N; Esteva, FJ; Volchenok, M; Breitenfelder, P; Kau, SW; Takayama, S; Krajewski, S; Reed, JC; Bast, RC, Jr; Hortobagyi, GN. Expression of BAG-1 and Bcl-2 proteins before and after neoadjuvant chemotherapy of locally advanced breast cancer. Cancer Invest 2004, 22, 248–256, doi:10.1081/CNV-120030213. 15199607
- Tang, SC; Beck, J; Murphy, S; Chernenko, G; Robb, D; Watson, P; Khalifa, M. BAG-1 expression correlates with Bcl-2, p53, differentiation, estrogen and progesterone receptors in invasive breast carcinoma. Breast Cancer Res. Treat 2004, 84, 203–213, doi:10.1023/B:BREA.0000019951.32001.93. 15026618
- Townsend, PA; Stephanou, A; Packham, G; Latchman, DS. BAG-1: A multi-functional pro-survival molecule. Int. J. Biochem. Cell Biol 2005, 37, 251–259, doi:10.1016/j.biocel.2004.03.016. 15474970
- Krajewska, M; Turner, BC; Shabaik, A; Krajewski, S; Reed, JC. Expression of BAG-1 protein correlates with aggressive behavior of prostate cancers. Prostate 2006, 66, 801–810, doi:10.1002/pros.20384. 16482527
- Nadler, Y; Camp, RL; Giltnane, JM; Moeder, C; Rimm, DL; Kluger, HM; Kluger, Y. Expression patterns and prognostic value of Bag-1 and Bcl-2 in breast cancer. Breast Cancer Res 2008, 10, R35, doi:10.1186/bcr1998. 18430249
- Sharp, A; Crabb, SJ; Cutress, RI; Brimmell, M; Wang, XH; Packham, G; Townsend, PA. BAG-1 in carcinogenesis. Expert Rev. Mol. Med 2004, 2004, 1–15.
- Adachi, M; Sekiya, M; Torigoe, T; Takayama, S; Reed, JC; Miyazaki, T; Minami, Y; Taniguchi, T; Imai, K. Interleukin-2 (IL-2) upregulates BAG-1 gene expression through serine-rich region within IL-2 receptor ßc chain. Bood 1996, 88, 4118–4123.
- Kullmann, M; Schneikert, J; Moll, J; Heck, S; Zeiner, M; Gehring, U; Cato, ACB. RAP46 is a negative regulator of glucocorticoid receptor action and hormone induced apoptosis. J. Biol. Chem 1998, 273, 14620–14625, doi:10.1074/jbc.273.23.14620. 9603979
- Kitada, S; Zapata, JM; Andreeff, M; Reed, JC. Protein kinase inhibitors flavopiridol and 7-hydroxy-staurosporine down-regulate antiapoptosis proteins in B-cell chronic lymphocytic leukemia. Blood 2000, 96, 393–397. 10887097
- Takahashi, N; Yanagihara, M; Ogawa, Y; Tamanoha, B; Andoh, T. Down-regulation of Bcl-2-interacting protein BAG-1 conferes resistance to anti-cancer drugs. Biochem. Biophys. Res. Commun 2003, 301, 798–803, doi:10.1016/S0006-291X(03)00020-2. 12565851
- Crabb, SJ; Hague, A; Johnson, PWM; Packham, G. BAG-1 inhibits PPARγ-induced cell death, but not PPARγ-induced transcription, cell cycle arrest or differentiation in breast cancer cells. Oncology Reports 2008, 19, 689–696. 18288403
© 2009 by the authors; licensee Molecular Diversity Preservation International, Basel, Switzerland. This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/). This article is an open-access article distributed under the terms and conditions of the Creative Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).


text:
Nasdaq and SEC explore deal on Facebook
06 February 2013
Nasdaq OMX is in preliminary talks with US securities regulators over a potential settlement related to its botched handling of last year's Facebook stock market debut, according to people with knowledge of the discussions.
Take a Financial News trial today:
- Breaking industry news & commentary
- 10 year searchable archive
- FN Powerlists & Special Reports
- FN on mobile and iPad
Does your company already have an FN licence? Email us now


text:
If you don't have a Facebook account, click here to log in with your Neopets information.
Site Spotlight
Submit your site to the Site
Site Name: Ruki for a Day
Creator: zebraxas
Url: http://www.neopets.com/~Metatron_neo
Site Description:
Learn all about Rukis, and one Ruki in particular: Metatron_neo.
Come on by, and you can be a Ruki for a day.
Quick Jump
IMPORTANT - SUBMISSION POLICY! By uploading or otherwise submitting any materials to Neopets, you (and your parents) are automatically granting us permission to use those materials for free in any manner we can think of forever throughout the universe. These materials must be created ONLY by the person submitting them - you cannot submit someone else's work. Also, if you're under age 18, ALWAYS check with your parents before you submit anything to us!


text:
The silliest trivia Test
Their result for The silliest trivia Test ...
You scored 76 brains!
Their Analysis (Vertical line = Average)
They scored 76% on brains, higher than 100% of your peers.
All possible test results
Category 1
Description 1 Read more
Tests others are taking
IOREUNVAiuejbvr18 / m / Straight
The Could you date a transsexual? Test
elaineli62042 / f / Gay
The Resume To Become My Girlfriend Test
ChillwaveRider24 / m / Straight
The Did I Make Obvious Mistakes on My OKCupid Profile? Test
Rjencks24222 / m / Straight
The 1 question penis size survey for women.
Emilyjo0527 / m / Straight
More tests we think you'll like
Taken 1365 times.
The What?! Test
What?! You want to take this? Are you nuts? I'm Damadar, and this test is labeled: WHAT?! as in... WHAT?! YOU WANT TO TAKE THIS? ARE YOU NUTS?! Did...
Taken 6691 times.
The Fucked Stream of Consciousness Test
This test is intended to just give you a laugh. In a day and age of constant competition I decided not to make this test about scoring but rather a br...
Taken 1362 times.
The Megaman NES Test
Hi! And welcome to my Megaman NES Test. I will ask you questions about the Megaman NES saga! Some questions will be easy, others will be hard! Good l...
Taken 563 times.
O teste das facções
Find out witch of Divergent faction you belong.
Taken 3497 times.
The Sidekick One Question Test
The world is divided into leaders and sidekicks. You are no leader or you wouldn't be here taking tests, instead you'd be starting a war, like a good responsible leader should. So let's see which...
Taken 17615 times.
The Which DOORS song are you? Test
Find out which Doors song comes closest to describing the real you
Taken 1383 times.
The FrenchKissFullMouthPierro Test
Hya Visitor ! Welcome to my FrenchKissFullMouthPierro Test. Be ready for anything, life is short ... Intelligence, Humour, Kinkiness, Slavabilit...
Taken 3533 times.
The How 'L Word' Are You? Test
Do you watch Showtime's Hit Series "The L Word". Of course you do. How into this show are you? Let's find out!
Taken 9167 times.
Science-Fiction Spaceship Command Test
What science-fiction spaceship should you command?
Taken 29451 times.
The dumbest Test
Hi! And welcome to my dumbest Test. I'll be using no logic and little knowledge to determine something.
Connect your existing OkCupid account
Sign in
Welcome Back!
Restore your account to continue meeting new people.
Reset your password
We'll email you a link to reset it.
Email sent!
An email is on its way to . If you don't see it, try checking your Spam folder.


text:
A Mess It Grows Lyrics - He Is We
I woke up, decided I'd find my way around this town
Foolish girl, thought I'd be so lost without him
Adolescence left to wither, I didn't have a choice
It was me or a silly boy, it was me or a silly boy
Why do I
What you did wrong
To me?
You're love drunk, you're blinded
You've lost the ones who love you most
This liar's on fire, melted like wax
A mess it grows
And you're the one that chose him
That's just how karma goes
Why do I
What you did wrong
To me?
Look at me now
Look at me now
(Look at me now . . .)
Take a look at what I've become
Don't thank yourself I'm finally someone now
Look at me now
You raised me well I'll give you that
But in my face you surely spat
Would you look at me now?
Why? Oh I . . .
Why do I
What you did wrong
To me?
I'm all right
And I'm alive
And I'm just fine without you
Without you
Click here to submit the Corrections of A Mess It Grows Lyrics
Please Click Here to Print A Mess It Grows Lyrics
The following area is only for review, if you want to submit the lyrics or the corrections of the lyrics, please click the link at the end of A Mess It Grows Lyrics.


text:
Teen Choice Awards Fashion Face-Off: Nikki Reed vs. Ashley Greeneby Hilton Hater at . Comments
Once again, Twilight Saga friends are about to become foes.
While fans have already been asked to weigh in on Kellan Lutz vs. Taylor Lautner in a previous Fashion Face-Off, we're now pitting a pair of blood-sucking beauties against one another in a red carpet showdown.
Both Nikki Reed and Ashley Greene dazzled at last night's Teen Choice Awards, but only one can be crowned the winner in the poll below.
Will it be the actress that plays Rosalie Hale? Or the one that portrays Alice Cullen? You vote. You decide.
[Photos: Splash News]
Which star looks prettiest?
I'm guessing tgoruhh fan base networks where they had to make contact with one another because of there job or they were introduced tgoruhh friends.
Nikki all the way. And she is obv more beautiful, I don't get all the Ashley hype.
I LOVE ashley, but i think nikki looks better!!
but loads of people will vote ashley because she is more 'in the news'
but i think both of them are amazingly pretty :)
ashely looks really pretty!!!!!!
thought nikki's dress was whoaaa very weird but she pulled it off VERY well ashley dress is nothing new...and as for the votes most people vote for ashley because she supposed be sweet but plzzzz i think ashley is meaner than nikki is
Ladies, love the outfits. I hope you keep track of them with MyClothingCalendar, a FREE online clothing journal. Never repeat an outfit again.
Sorry ladies, but you both need to work on your accessorizing skills. Neither one of your shoes/handbags looks good with your dress.


text:
New Construction 3-Light L-Corner recessed fixture comprised of separate Housing and trim. Trim design allows use in ceilings up to2" thick. No IC, must maintain 3" clearance from any insulation on all sides.Built in electronic transformer.
Housing: 3/4" overlap flanges combined with ?pull-tight" torsion spring sallows forgiveness in minor dry wall cutting errors.
MOUNTING:Designed to be installed with overlap flange trim. Universa lmounting brackets allow the use of conduit, HB or FB bar stock, or ?C" channel bars. Consult factory for possible use in trimeless ?mud-in" installations.
AIMING:Vertical: Inner Ring 40° - Outer Ring 45°


text:
|My Wayfair
echo design Odyssey Cotton Faux Linen Square Pillow
echo design Odyssey Cotton Faux Linen Square Pillow
Weights & Dimensions
|Product Type: Throw pillow
|Color: White
|Shape: Square
|Cover Material: 100% Cotton
|Embroidered: Yes
|Life Stage: Adult
|Country of Manufacture: China
About the ManufacturerThroughout its 80 year history, the Echo brand has been synonymous with a sense of style, fashion, and good value. Echo's expertise has always been in the areas of print, pattern mix, and above all, bold use of color. Inspired directly from their fashion accessory collections, Echo brings the same trend awareness, design sense, and value consciousness to creating fashion collections for the bedroom. The Echo brand believes in four defining principles that set their products apart from the rest: innovative design, quality, value and integrity. Through their design process, they are able to deliver this commitment to customers in products that they design and produce.
echo design Odyssey Cotton Faux Linen Square Pillow
Still have questions about this product? We're here to help
echo design Odyssey Cotton Faux Linen Square Pillow
Free Shipping on orders over $49.00
Ships to:
Ships via: FedEx/UPS (Learn More)
Delivery Estimates
Enter your shipping zip or postal code to calculate shipping estimates
Return Policy
We want you to love it. If you don't, you may return your purchase within 30 days of delivery. All you need to do is visit our Self-Service Return Center for instructions. Return credits are issued within 24 hours once the item is received and inspected. Please note that shipping charges are non-refundable. The cost of return shipping will be deducted from your refund.30-Day Return Policy
Customer Reviews
Tell Us What You Think!
Tell us what you think about the Odyssey Cotton Faux Linen Square Pillow. It will help us provide you with better products in the future and help your fellow Wayfair shoppers make better purchases.Write A Review
|5 star
|4 star
|3 star
|2 star
|1 star
|0 star
Peggie from Merrillville, IN– Top 50 Reviewer
this pillow is very nice, it is a very large and soft, I decorated my living room sofa with this pillows, good purchase
Nick from PA– Verified Buyer
Quality made, I expect it to last a long time. Looks great with all the other matching Odyssey bedding
OUR SKU # ECH1139Odyssey Cotton Faux Linen Square Pillow is also sometimes listed under the following manufacturer numbers: EO30-551


text:
Aug 26, 2014
Thanks for joining us to learn about "All the Places We'll Go" during our National Arts Marketing Project Conference! Hopefully you returned home with new knowledge on how to future-proof your arts organization's arts marketing strategies. Read more on our wrap up page with links to our livestreamed keynotes and sessions, event photos, storifies, and more!


text:
Details: 10% off your purchase
13% Success • 7 Comments
5 days ago by Anonymous
9 days ago by Anonymous
1 month ago by Anonymous
1 month ago by Anonymous
1 month ago by Anonymous
Details: Free shipping on orders $65+. Expires on 11/15/2014.
New • Add a Comment
Details: 10% off sitewide. Expires on 10/31/2014.
New • Add a Comment
Details: Free standard shipping within the contiguous US on Orders Of $60 Or More. Expires on 10/12/2014.
100% Success • Add a Comment
Details: Free shipping on orders $60 or more. offer valid for standard shipping to addresses in the Continental U.S. and for online orders only. Expires on 10/13/2014.
New • Add a Comment
Details: Free shipping on orders $75 or more. Expires on 09/26/2014.
New • Add a Comment
Details: Get 20% off all Clearance items. Expires on 08/31/2014.
New • Add a Comment
Details: 10% off all Jewelry. Expires on 06/11/2014.
New • Add a Comment
8 coupon codes & deals for catalogclassics.com. Catalog Classics are carefully selected, time-tested customer favorites from the world of Mail Order and Online retail.
Show More
Never miss a deal.
Be the first to know.
Plus get our best deals in our email newsletter. Privacy Policy
Show more popular stores
RetailMeNot Coupons AppSave on-the-go with deals from your favorite stores
Deal AlertsGet our best offers & never miss a deal
Be SocialConnect with us on Facebook, Twitter, and Google Plus
The Real Deal BlogA daily dose of fresh news, trending topics and best buys
Submit a CouponShare the love! Submit a coupon you've found
©2010 - 2014 RetailMeNot Inc. All rights reserved. Site Map | Terms of Use | Privacy Policy
RetailMeNot is a registered trademark of RetailMeNot, Inc. Merchant trademarks are the property of the respective merchant and their presence does not necessarily mean that RetailMeNot, Inc has an affiliation with the merchant.


text:
"long boy 'burgers'" Seasoned ground beef baked in a sub roll with melty cheese.
Also on these boards
Related Pins
Long Boy "Burgers"
Ground Beef Stuffed French Bread Sandwich - mushroom soup, onion, cheese and seasonings make this almost like a Philly Cheese Steak. Think I'll add some fried green peppers and go full tilt!
Is this the best veggie burger recipe ever?? by Vanessa Rees
Beef & Cheese Foldover — All-purpose baking mix makes a quick and easy crust for this cheesy ground beef mixture.


text:
Lobby Group Says Open-Source Threatens Capitalism
Does advocating the use of open-source software make one an enemy of capitalism? Yes, according to a U.S. intellectual property lobby group. The Guardian reports that the International Intellectual Property Alliance, a lobby group broadly representing the RIAA, MPAA and others, has requested that the U.S.