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The new home of
0 reviews
Across to Brixham Harbour and Torbay, Cliff Cottage has a quirky triangular shape and it just oozes charm. There is no other place in Brixham quite like it. This surprisingly spacious cottage has been built to make the most of the stunning views over the harbour and coastline, and is conveniently situated as it is just 100 yards from the harbour and town centre. The property benefits from a balcony off the living area, which makes an ideal place to sit and watch the boats go by. There is also a garden with seating area, which also offers views across the harbour. This detached holiday cottage has been furnished and decorated to a good standard and makes perfect accommodation for families and couples alike. The vehicle access, for unloading luggage, is approximately 30-metres from the entrance to the property.With Agatha Christie's former home, Greenway (National Trust), being practically on the doorstep, Cliff Cottage is a great base from which to visit this wonderful piece of history. There are many ways to arrive at Greenway. One way would be to take a boat trip from Brixham Harbour and sail past some of South Devon's most beautiful coastline, where you may even see some dolphins.Visit Paignton, which has a wide range of great activities for children of all ages, from penny arcades on the pier, to seeing all the animals at the zoo. There is a central green that is home to a multiplex cinema, crazy golf and there is also the pier which has many rides and amusements to enjoy.The Paignton and Dartmouth Steam Railway departs from the centre of town, while Paignton Zoo is a mile or so from the centre. Over at Goodrington Park, there is a boating lake, go-karts and Splashdown water park. Dartmoor is on the doorstep, with its stunning scenery and heather-clad rocky tors and ponies, and is a walkers' paradise at any time of year, with Becky Falls (rocks and waterfalls) 8 miles away. With over 450 miles worth of public rights of way, there is an abundance of walking routes to suit all tastes and abilities. Dartmoor also boasts a variety of cycle routes, both on and off road as well as horse-riding, climbing, letterboxing, geo-caching or kayaking.Further afield is Plymouth, 25 miles away, where guests can spend the day to see Plymouth Hoe, the Barbican Centre and the National Marine Aquarium, which is perfect for both children and adults. The Eden Project is approximately 1½ hours drive away and is a fabulous day out. Crealy Adventure Park is 20 miles. Exeter has a magnificent cathedral and quay, whilst a boat trip can be taken up the river to two stunning pubs. There are many villages to explore and stop off for a Devon cream tea. Take a walk along the South West Coast Path which runs along the entire length of the Jurassic Coast and offers the walker stunning views, with a bird's eye view of many coastal features. Guests even receive a free parking permit, for 1 car, that allows you to park all over Torbay. Shop, pub and restaurant 100 yards.
3 steps to entrance.Ground floor:Living/dining room: With Freeview TV, DVD player with a selection of DVDs, Playstation 2 with a selection of games, Sony music system, selection of books and board games and patio doors leading to balcony.Kitchen: With gas oven, gas hob, microwave, fridge, freezer and washer/dryer.Separate toilet.First Floor:Bedroom 1: With kingsize bed.Bedroom 2: With zip and link twin beds (can be super kingsize on request).Shower room: With shower cubicle and toilet.
Gas central heating, gas, electricity, bed linen, towels and Wi-Fi included. Travel cot and highchair. Welcome pack. Enclosed decked area with garden furniture and barbecue. Parking permit for 1 car included (May-September). No smoking.
6 reviews
400 year old character cottage
This 400 year old character holiday cottage has tiny windows, low and impressive beams, several antiques, a stone staircase and sloping floors for a holiday with a difference. Cosily furnished and equipped, there are breathtaking views of the fells from the back door of the cottage. It is just a short walk from the centre of Coniston with its shops, cafes, restaurants and pubs with Coniston Water beyond. Fabulous walking, literally from the door, from a location in the heart of the Lake District. Shops 300 yards, pub 25 yards.
Ground floor: Living/dining room. Kitchen. Stone staircase to first floor: 2 bedrooms: 1 double, 1 twin. Bathroom with shower over bath and toilet. Beams throughout.
Open fire in living room included. NSH, electricity and bed linen included. Electric cooker. Microwave. Patio and furniture. Parking (1 car). No smoking. No children under 6 years.
Nestling amidst glorious Devon countryside, these stylish lodges (refs W43657, W43658 and W43659) are the perfect choice for a relaxing country retreat, yet only 1 mile away from the small market town of Holsworthy, with its local shops, pubs and cafés. These eco-friendly, detached, contemporary lodges with grass roofs are well-appointed and comfortable. They have been built to high specifications and finished to a very good standard. The spacious open plan living areas have lovely views across the fishing lakes and countryside, and the French doors lead to a decked area, where you can enjoy a lazy breakfast whilst planning your day. Each property has received a gold award from The Green Tourism Business Scheme. Guests are provided with a delightful cream tea on arrival and are free to enjoy the owners 19 acres of land, including 10 acres of nature walks and picnic spots. Wi-fi is available to all guests in the 'the hub' which is a centrally located information building. Relax and take in the sights and sounds beside the wildlife pond, or enjoy some coarse fishing on the two well-stocked fishing lakes. Children will love the large play area with a play boat, and the games room is perfect for all ages.The wide golden sands of Bude and Widemouth Bay are just 10 miles away, with excellent surfing, pretty coves and dramatic coastal walks, along with shopping and leisure facilities. Holsworthy, has a good selection of local shops and supermarkets, along with a leisure centre and weekly market. The market towns of Launceston and Okehampton are a short drive away, and the rugged coastlines of North Devon and Cornwall are just 9 miles away, where there is beautiful scenery and excellent surfing opportunities. Coastal and country walking, golf, surfing, fishing, horse riding and cycling are all available close by, and for family fun, The Big Sheep and The Milky Way Adventure Park are not to be missed. These superb lodges are a fantastic holiday choice whatever time of the year. Beach 10 miles. Shop, pub and restaurant 1 mile.
All on ground floor: Living room with French doors/dining room/kitchen with wooden floor. 2 bedrooms: 1 double with en-suite shower room and toilet, 1 twin. Bathroom with shower over bath and toilet.
Shared freezer.
All properties: Electric woodburner in living room included. Oil central heating, electricity, bed linen and towels included. Travel cot and high chair on request. Freeview TV. DVD and small library. CD. Electric cooker. Microwave. Washing machine. Dishwasher. Shared freezer. Wi-fi in information building. Enclosed garden with decked terrace, sitting-out area and furniture. Shared 19-acre natural grounds with sitting-out area. Barbecue. Parking (1 car). Cycle store. Shared games room with pool table, table football and darts. Selection of books and games. Children's play area. Fishing available on-site (licence required). Natural water supply from borehole. No smoking. NB: 2 steps in garden. Fenced lake in grounds, 10 yards.
12 reviews
Shared swimming pool
This detached holiday cottage enjoys shared use of an outdoor swimming pool and is situated on a working smallholding, amongst 2,000 acres of natural forest, with many footpaths meandering through, making an ideal base for walking and cycling. The medieval village of Backwell has a long history, appearing in the Domesday book in 1086, and is close to Bristol airport, 3 miles, and only 10 miles from the cosmopolitan city of Bristol, with its wealth of visitor attractions including Brunel's SS Great Britain, Bristol Zoo, the interactive adventure centre, the Clifton Suspension Bridge and the lively harbourside area. Also within easy reach of the Georgian city of Bath, Chew Valley, the Mendips, Cheddar Gorge, Wookey Hole Caves, Glastonbury Tor, Wells Cathedral and Clarks Village retail outlet at Street. Visit the coastal resort of Weston-super-Mare, Ashton Court Estate and deer park, Tyntesfield National Trust Country House and Noah's Ark Zoo Farm, all of which make great days out. Shop 2 miles, pub and restaurant 3 miles.
3 steps down to entrance (alternative level access). Ground floor: Living room with flagstone floor. Dining room with flagstone floor. Kitchen. Bathroom with bath, shower cubicle and toilet. First floor: 2 bedrooms: 1 double (5ft), 1 twin.
Electric central heating, electricity, bed linen and towels included. Freeview TV. DVD. Electric cooker. Microwave. Washing machine. Dishwasher. Fridge/freezer. Patio with sitting-out area and furniture. Ample parking. Shared use of outdoor swimming pool (9m x 4.5m, depth 1m to 2.5m), open May-September. Welcome pack. No smoking.
8 reviews
These attractive properties lie in the heart of the Cotswold Hills. With characteristic dry stone walling dividing sweeping sheep pastures, ancient beech woods, trout streams, charming towns and villages of honey-coloured Cotswold limestone. The Cheltenham Gold Cup, Badminton Horse Trials, (5 minutes). Famous gardens including Hidcote Manor, Kiftsgate and Westonbirt Arboretum; NT properties include Bowood House, Chedworth Roman Villa and Corinium Museum within an easy drive. Ideal for golf and horse riding, 5 miles. Shops 3 miles, pub 1 mile.
Oak beamed open-plan living/dining room with wooden floor, farmhouse-style kitchen area, French doors to garden. Sitting room with rugs on wooden floor. Double bedroom (5ft bed) with modern tiled en-suite shower room. Bathroom with over-bath Monsoon shower and toilet with stone floor. First floor: Twin bedroom (can be 6ft double) with sloping ceiling, Velux window, built-in wardrobe and elec rad.
19 reviews
Situated in open countryside within a beautiful 2-acre garden and attached to the owner's home, Woodlands offers a perfect homely holiday base from which to explore the area. It is ideally located to enjoy the Forest of Dean, Severn and Wye Valleys, Malvern Hills and the Cotswolds. Pretty market towns of Ross on Wye, Ledbury and Tewkesbury are an easy drive. Shops and pubs 2 miles.
Ground floor: Living room with French doors. Dining room. Kitchen with tiled floor. Separate toilet. Stairs or stairlift to first floor: 2 bedrooms: 1 double with en-suite bathroom with shower over walk-in spa bath, bidet and toilet, 1 twin. Bathroom with bath, shower cubicle and toilet.
Oil central heating, electricity, bed linen and towels included. Freesat TV. Video and small library. DVD and small library. Stereo/CD. Electric cooker. Microwave. Washing machine. Dishwasher. Freezer. 2-acre shared enclosed garden with patio and furniture. Parking (2 cars). No smoking. NB: 6 steps within garden and unfenced pond.
10 reviews
A stylish first floor apartment with a wraparound balcony, situated on the edge of Eastbourne. This well-equipped holiday apartment is just a short distance to the town, pier and beaches and also Sovereign Harbour Marina, one of the UK's largest marinas at the foot of the South Downs. Eastbourne is a traditional seaside resort boasting 5 miles of unspoilt beaches, palm tree-lined promenades, golf courses and the coveted Blue Flag beach award. It also boasts more sunshine hours than other holiday destinations, with its very own 'micro climate'. An indoor leisure centre with swimming pool is within walking distance. Shop, pub and restaurant ¼ mile.
Lift or stairs to first floor: All on one level: Living/dining room/kitchen with patio doors. 2 bedrooms: 1 double with en-suite shower room and toilet, 1 twin. Bathroom with shower attachment and toilet.
Gas central heating, electricity, bed linen and towels included. Two Freeview TVs. DVD. Electric cooker. Microwave. Washer/dryer. Dishwasher. Fridge/freezer. Balcony with furniture. Parking (1 car). Welcome pack. No smoking.
2 reviews
Set on the banks of the River Wissey and boasting glorious riverside views this delightful cottage, with its decking leading to the river, is ideal for fishermen or watching the waterborne world go by. Just 500 yards from the shop in the village of Stoke Ferry, it is within reach of many attractions. The fantastic Oxborough Hall NT is just 4 miles away and well worth a visit. Enjoy walking and cycling through the famous Thetford Forest, a haven for wildlife. The wonderful beach at Holkham and Wells-next-the-Sea, the queens estate at Sandringham, the Lavender Fields and Hunstanton for family fun, are all within an easy drive. The historic town of Downham Market is 7 miles and a day trip to Cambridge is not to be missed.
Ground floor: Living room with wooden floor. Dining area/kitchen. First floor: 2 bedrooms: 1 double, 1 twin with sloping ceilings. Bathroom with shower over bath and toilet.
NSH included (£25per week October-April). Electricity, bed linen and towels included. DVD. Microwave. Washing machine. Wi-fi. Decked garden and furniture. Parking. Fishing foc. Unsuitable for the infirm. No smoking. NB: Unfenced river access from decking.
5 reviews
Ground Floor: With gas central heating and comprising:Kitchen/Diner: with oven, hob, microwave, fridge, dishwasher and iron. Sitting room: with TV, DVD, radio/CD player, books, DVD's, games and picture window. Bedroom 1: with double bed and en-suite shower room with shower cubicle, WC, basin and heated towel rail.
First Floor: Bedroom 2: with twin beds, views of the castle and en-suite shower room with shower cubicle, WC, basin and heated towel rail.
Bed linen and towels provided. Wi-Fi available. Patio with table, chairs and BBQ. Ample private parking. One small well behaved pet welcome, free of charge. Can be booked with Castle View DC4837 and The Carthouse DC5414 to accommodate up to 10 people.
8 reviews
Perfect for a relaxing break, these two comfortable, spacious and charming conversions enjoy spectacular views across a beautiful deer park grazed by red and fallow deer, a wildlife haven just 6 miles from the lovely North Norfolk Coast. The park boasts a 14-acre fishing and birdwatching lake and a network of footpaths to explore - there is even an excellent restaurant within walking distance. Deer Park Cottage (ref E2754), adjoins the owners' 18th century farmhouse, whilst Deer Park Lookout (ref E3928), sits nearby, only 2 miles from Roughton village shop and Gunton's railway station, and 6 miles from Cromer's seaside attractions. Blickling and Felbrigg Halls (both NT) are nearby and the Broads are easily accessible. Shop 2 miles, restaurant within walking distance.
Ground floor: Dining room/kitchen. 2 double bedrooms, (one 5ft). Bathroom with shower attachment and toilet. First floor: Galleried living room.
Woodburner in living room included. Private garden area and bench.
Both properties: NSH, electric heaters, electricity, bed linen and towels included. DVD. Microwave. Shared grounds. Parking. Fishing by arrrangement (at cost). NB: Unfenced lake in grounds.
10 reviews
Sea Views
On the glorious North Devon coast, these pretty semi-detached chalet bungalows, Seaville and Trouville, are the perfect choice for families and couples wanting a seaside retreat and can be booked together to accommodate up to 9 guests. Each is presented and furnished to a very good standard to make your holiday a relaxing escape. Situated in Bideford Bay Holiday Park, between the lovely cliff side village of Clovelly and the popular resort of Westward Ho! and just a short distance from the breathtaking coastal path and rock pools of Bucks Mills. For a small weekly fee holidaymakers can also enjoy and make full use of the indoor and outdoor heated swimming pools, tennis courts, crazy golf, adventure playground, kid's club and the popular park entertainment (open mid Mar-Oct). There are also bars, a restaurant, takeaway and a supermarket on site. The perfect holiday choice to relax and unwind at any time of year with sea views and ideally situated to explore the breathtaking North Devon and North Cornish coast. The famous surfing beaches at Westward Ho! and Bude are nearby, and for family fun the Big Sheep and Milky Way Adventure Park are a must. The famous Eden Project is within easy reach or take the ferry from nearby Bideford to beautiful Lundy Island. Beach 3/4 mile. Shop, pub and restaurant 100 yards.
All on ground floor: Living/dining room/kitchen. 2 bedrooms: 1 double, 1 twin. Bathroom with shower over bath. Separate toilet.
Microwave. Patio and furniture. Barbecue. Parking.
Both properties: Electric central heating, electricity and bed linen included. Freeview TV. Shared laundry room with coin-operated washing machine and tumble dryer. No smoking.
2 reviews
Situated just a few steps away from the picturesque quay and bustling Staithe Street, Kingfisher is nestled in the heart of Wells-next-the-Sea. Lying in a quiet area, this holiday apartment provides an excellent place from which to relax and unwind after a fun filled day exploring. Those wishing to park up at Kingfisher and leave their car for the duration of their stay will find much to do within Wells-next-the-Sea. The much photographed quay offers boat trips to see the seals, fishing trips and the local pastime of "gillying", which is catching crabs by hand line. A small narrow gauge railway takes you down to the wide sandy beach with its pretty beach huts. Pitch and putt and seaside amusements are also to be found here and you can pass away an hour or more exploring the quaint mix of artisan and traditional seaside shops and cafés that line pedestrianised Staithe Street.Those wishing to explore the coastline can literally head east or west to find quays, marshes, beaches and pretty seaside villages. To the east lies the Norfolk Wildlife Trust's Cley Marshes, Cromer with its Edwardian seafront and famous pier and Sheringham with its bustling market and shingle beach. Head west and you will find Holkham, with its famous hall and beach, Brancaster and Hunstanton, where you will find all the fun of the fair and a wide sandy beach that literally seems to stretch on forever. History lovers can explore Blickling Hall, The Sandringham Estate, the medieval city of Norwich and the Hanseatic Old Quarter of Kings Lynn. Bird watchers will be spoilt for choice too, especially as Norfolk was recognised as "the bird watching capital of Europe". After a busy day of exploring, you can relax and unwind at Kingfisher and dream of adventures yet to come. Beach 1 mile. Shop 20 yards, pub and restaurant 75 yards.
9 steps to entrance.All on the first floor.Open plan living space.Living area: With 42" Freeview TV and DVD player.Dining area.Kitchen area: With electric cooker, microwave, fridge/freezer, dishwasher and washing machine.Bedroom 1: With double bed and 24" Freeview TV and DVD player.Bedroom 2: With single bed and additional pull out bed.Bathroom: With bath and toilet.
Gas central heating, electricity, bed linen and towels included. Travel cot and highchair available on request. Small courtyard with garden furniture. Private parking for 2 cars. No smoking.
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cottages.com is the new home of cottages4you (Cottages for You).
cottages.com is a trading name of Wyndham Vacation Rentals (UK) Ltd. Registered in England and Wales.
Registered Office: Spring Mill, Stoney Bank Road, Earby, Lancashire,BB94 0AA, UK
Company Registration Number 00965389. VAT Registration Number: GB 598 22 99 77


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Been in dallas for 6 years now, folks came down to visit and I thought to myself, "self, lets take the folks out for some pie". It's something we always do when we travel, try some nice pies.
wow, was I in for a disapointment. We ended up having cheesecake.
I found out in dallas, when looking for pie the first thing you have to add some qualifiers.
Not looking for:
1) pizza
2) pecan (pecan is fine but a variety of berry & fruit pies in addition to pecan)
3) merringue (see pecan statement)
Am looking for:
Variety of handmade berry/fruit pies.
Updated 1 year ago | 3
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Updated 1 day ago | 114

subset: commoncrawl


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RXi Pharmaceuticals Corp. (NASDAQ:RXII) has earned an average broker rating score of 1.00 (Strong Buy) from the two brokers that cover the stock, Zacks Investment Research reports. Two investment analysts have rated the stock with a strong buy recommendation.
Brokerages have set a 1-year consensus price target of $5.75 for the company and are anticipating that the company will post ($0.34) earnings per share for the current quarter, according to Zacks. Zacks has also given RXi Pharmaceuticals Corp. an industry rank of 108 out of 265 based on the ratings given to related companies.
Separately, Zacks Investment Research lowered shares of RXi Pharmaceuticals Corp. from a "buy" rating to a "hold" rating in a report on Friday, July 15th.
A hedge fund recently raised its stake in RXi Pharmaceuticals Corp. stock. Renaissance Technologies LLC increased its position in shares of RXi Pharmaceuticals Corp. (NASDAQ:RXII) by 22.1% during the first quarter, according to its most recent disclosure with the Securities and Exchange Commission (SEC). The institutional investor owned 950,900 shares of the biotechnology company's stock after buying an additional 172,100 shares during the period. Renaissance Technologies LLC owned 1.46% of RXi Pharmaceuticals Corp. worth $257,000 as of its most recent filing with the SEC. 11.76% of the stock is currently owned by institutional investors and hedge funds.
Shares of RXi Pharmaceuticals Corp. (NASDAQ:RXII) traded down 7.82% during mid-day trading on Tuesday, hitting $1.06. 103,480 shares of the stock were exchanged. The stock's market capitalization is $6.95 million. The stock's 50-day moving average is $1.94 and its 200-day moving average is $2.23. RXi Pharmaceuticals Corp. has a 52 week low of $0.99 and a 52 week high of $5.30.
RXi Pharmaceuticals Corp. (NASDAQ:RXII) last released its earnings results on Thursday, August 11th. The biotechnology company reported ($0.34) earnings per share (EPS) for the quarter, topping the Thomson Reuters' consensus estimate of ($0.36) by $0.02. During the same quarter in the previous year, the business earned ($0.50) earnings per share. Analysts forecast that RXi Pharmaceuticals Corp. will post ($1.40) earnings per share for the current fiscal year.
About RXi Pharmaceuticals Corp.
RXi Pharmaceuticals Corporation is a clinical-stage ribonucleic acid (RNA) interference (RNAi) company developing therapeutics in dermatology and ophthalmology that address unmet medical needs. The Company's development programs are based on its self-delivering RNAi (sd-rxRNA) platform and Samcyprone, a topical immunomodulator.
Get a free copy of the Zacks research report on RXi Pharmaceuticals Corp. (RXII)
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Explore Faith Bibleverseoftheday, Have Faith and more!
"Just because the majority believes the trinity, hell and immortality of the soul doctrine doesn't make it the truth. Many false doctrines were added in the 4th Century A.D. Many dissenters were burned at the stake or died in other horrific ways, for refusing to accept additions to God's Word. Do your research! Find the TRUTH!" - pp
The root word for Torah is Orah which means Light, so every time you see LIGHT in your Bible it means TORAH
Believe in the light while you have the light, so that you may become children of light." When he had finished speaking, Jesus left and hid himself from them. -John 12:26
I can be a good example by being.... I plan on using this to teach my prek kids about the beatitudes with simple words that they can understand.
Share the gospel. Jesus Christ is the only way. Straight and narrow is the way...few will find it. Stand firm on His truth. Grow in His word.
A mistake, is underestimating my courage, if you only looked in my eyes you could have seen my heart flames. I will only bow to truth and honor, for all else it's best not to motivate me to display what I hold within. The light is bright, strength is swift, but defeat has never been an option. Yes, I hold that within, for I fear if it is ever loosed.


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Platte Woods Precision Auto Care
ReScore Reviews™ (1)
98% Would Recommend
Latest Review 30 days ago
Featured Business
Northtowne Hyundai6750 N. Oak Trafficway
Kansas City, MO 64118
4.4 miles away
10/23/2016 - "Kaleb "
"Fast and efficient service "
Review for Platte Woods Precision Auto Care
Ian C. on 11/12/2015
Ian said this business fixed everything that needed to be fixed. They gave him quotes on things that needed to be fixed as well, but at a later time.
Vehicle: Chevrolet HHR
Category: Service
Service Date: 11/10/2015
Review Created: 11/12/2015
This review was collected via phone interview by Customer Research Inc.


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iHerb Customer Reviews
Savings of: $2.65 (29% off)
i love it
Posted by 5612782135206448467 on Jul 07, 2016
smells great. good value. nice to use
Love it
Posted by 5368140323498952901 on Aug 01, 2016
Will buy again
Posted by 5480174481400637290 on Aug 16, 2016
I spray this on my face as a treat after washing it in the summer. It is refreshing.
Love this
Posted by 4914040994383706593 on Aug 20, 2016
Use as a toner, works very well.
Love it
Posted by 5532159553750470647 on Aug 26, 2016
This is such an excellend product! The scent is so so nice and it also absorbs very fast. It's great as a toner, body mist, lotion mask and especially for soothing skin during summer time! I absolutely love it
Posted by Reviewer1405760 on Sep 17, 2016
...scent ever!!!
totally like it
Posted by 5063166492073975965 on Sep 18, 2016
There is nothing bad to say. It smells like roses, fresh and bloomy. I use it as a face cleanser (I do not use makeup normally, when I do I prefer other cleansing products). It does an amazing job as I would expect from a good rose water. It cleanses and calms the skin. After I started using rose water as a face- cleanser my skin got a lot better. The opening is perfect to put it on some cotton pads (you flip the cap up like with some shampoos).
Love this water.
Posted by 4891951635381173040 on Sep 25, 2016
It hydrates instantly. Smells like delicate petals. I use it on my skin and hair.
Posted by Reviewer2062554 on Oct 04, 2016
This has such a lovely rose scent and makes me happy when I apply it.
Lovely subtle scent!
Posted by 4686926107687940315 on Oct 07, 2016
I have dry sensitive skin, I can't use moisturizers or anything else on my face as it causes me to break out. I've been using this rose water for over a month now, it's light and has a subtle scent of roses. My skin absorbs it quickly which is great! No break outs and my skin feels a lot softer. The bottle looks like it would last 2 month if used twice a day. I'm going to have to buy these in bulk next time :)


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Coexpression of Mutant p53 and p193 Renders Embryonic Stem Cell–Derived Cardiomyocytes Responsive to the Growth-Promoting Activities of Adenoviral E1A
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Abstract—Expression of adenoviral E1A in cardiomyocytes results in the activation of DNA synthesis followed by apoptosis. In contrast, expression of simian virus 40 large T antigen induces sustained cardiomyocyte proliferation. Previous studies have shown that T antigen binds to 2 proapoptotic proteins in cardiomyocytes, namely the p53 tumor suppressor and p193 (a new member of the BH3-only proapoptosis subfamily). Structure-function analyses identified a p193 C-terminal truncation mutant that encodes prosurvival activity. This mutant was used to test the role of p193 in E1A-induced cardiomyocyte apoptosis. E1A induced apoptosis in cardiomyocytes derived from differentiating embryonic stem cells. Expression of the prosurvival p193 mutant alone or a mutant p53 alone did not block E1A-induced apoptosis. In contrast, combinatorial expression of mutant p193 and mutant p53 blocked E1A-induced apoptosis, resulting in a proliferative response indistinguishable from that seen with T antigen. These results confirm the hypothesis that there are 2 proapoptotic pathways, encoded by p53 and p193, respectively, which restrict cardiomyocyte cell cycle activity in differentiating embryonic stem cell cultures. Furthermore, these results explain in molecular terms the phenotypic differences of E1A versus T-antigen gene transfer in cardiomyocytes.
The capacity of adult mammalian cardiomyocytes to initiate DNA synthesis and to subsequently divide appears to be quite limited.1 2 Consequently, the ability of the heart to undergo regenerative growth in disease states is also limited, a fact underscored by the high morbidity and mortality rates for individuals with severe cardiovascular disease. It is generally believed that increasing the number of functional cardiomyocytes in a diseased heart would have a positive impact on contractile activity provided that the new cells participate in a functional syncytium. This belief has fostered the development of a number of novel approaches to increase cardiomyocyte number.3 4 One approach has focused on the identification of key cardiomyocyte cell cycle–regulatory proteins, with the hope that exploitation of these proteins (or their respective regulatory pathways) could promote regenerative myocardial growth.3 Toward that end, a number of gene transfer approaches have been used to target expression of the simian virus 40 (SV40) large T antigen (T-Ag) or the adenoviral E1A oncoproteins to cardiomyocytes in an effort to induce proliferation. Targeted expression of T-Ag to atrial or ventricular myocardium in transgenic mice can induce a proliferative response,5 6 7 which in most instances results in the formation of tumors comprising differentiated, proliferating cardiomyocytes. In contrast, the ability of E1A to induce cardiomyocyte proliferation appears to be more restricted. Although E1A expression in cultured fetal or neonatal cardiomyocytes can reactivate DNA synthesis, this is followed rapidly by apoptosis.8 9 10 11 12
It is well established that DNA tumor virus oncoproteins subjugate cell cycle activity by binding to and altering the activity of endogenous cell cycle regulators.13 In light of this paradigm, identification of the cardiomyocyte T-Ag and E1A binding partners could provide insight into cell cycle regulation in the myocardium. In the case of T-Ag, 3 prominent cardiomyocyte binding proteins have been identified. They are the p53 tumor suppressor,14 15 p107 (a member of the retinoblastoma protein gene family),14 16 and p193 (a novel member of the BH3-only family of proapoptosis proteins).14 17 The same set of T-Ag binding proteins was detected in multiple independently derived tumor cell lines, as well as in primary cardiomyocyte cultures from the transgenic tumors. In the case of E1A, retinoblastoma protein family members (pRB, p107, and p130) and p300 (a transcriptional coactivator with intrinsic histone acetylase activity) were identified as cardiomyocyte binding proteins.10 Thus, T-Ag possesses several antiapoptotic activities that are lacking in E1A (namely the ability to bind to 2 proapoptotic proteins, p53 and p193). Given the cell death observed in response to E1A expression, we would anticipate that expression of E1A in conjunction with blockade of both the p53 and the p193 pathways would induce cardiomyocyte proliferation, rather than apoptosis, as was reported previously where one or both of these proapoptotic pathways were intact.8 9 10
In this report, a preliminary structure-function study was initiated to further characterize the proapoptotic activity encoded by p193. Surprisingly, a mutant molecule encoding p193 amino acid residues 1 to 1152 possessed growth-promoting activity in an NIH-3T3 assay similar to that observed with a p193 antisense expression construct. Additional analyses revealed that expression of this mutant rendered NIH-3T3 cells resistant to methyl methanesulfonate (MMS)–induced apoptosis. The availability of a prosurvival p193 variant and a mutant p53 (which is able to block p53-induced apoptosis) enabled us to directly test the hypothesis that DNA tumor viral oncoprotein activation of the cardiomyocyte cell cycle requires blockade of both the p193 and the p53 pathways. Expression of E1A in embryonic stem (ES) cell–derived cardiomyocytes led to widespread apoptosis. E1A induced similar levels of apoptosis in cardiomyocytes expressing mutant p53 or mutant p193. In contrast, coexpression of mutant p53 and mutant p193 blocked E1A-induced apoptosis, resulting in a proliferative response indistinguishable from that seen with SV40 T-Ag gene transfer. These results provide an explanation in molecular terms for the different phenotypes observed in cardiomyocytes expressing T-Ag versus E1A.
Materials and Methods
Recombinant Clones
cDNAs encoding full-length17 and C-terminal–deleted p193 were subcloned into the pRC/cytomegalovirus (CMV) expression vector (Invitrogen). The mouse α-cardiac myosin heavy chain (MHC) promoter18 was used to generate expression constructs for the ES cell–derived cardiomyocyte colony growth assay. For generation of a mutant p53 expression cassette, a p53 genomic clone from CB7 cells was used.19 20 For generation of an E1A expression cassette, a wild-type E1A genomic clone was used.
NIH-3T3 Colony Growth Assay
NIH-3T3 cells (American Type Culture Collection) were transfected using a calcium phosphate approach,21 selected in G418 (300 μg/mL) for 15 days, and stained with gentian violet. Stable cell lines were obtained by prolonged G418 selection. Transfection efficiencies were calculated by monitoring expression of a cotransfected CMV-βGAL reporter gene at 48 hours after transfection.
Assessment of Apoptosis
NIH-3T3 cells were grown at a concentration of 3×106 cells per dish and treated with MMS. In other experiments, cells from microdissected beating clusters from the ES cell–derived cardiomyocyte colony growth assay were analyzed. Cells were processed using a DNA ladder kit (Roche). For terminal deoxynucleotidyltransferase–mediated dUTP nick-end labeling (TUNEL), dispersed cell preparations were plated onto fibronectin-coated chamber slides and processed using an in situ cell death detection kit (Roche).
ES Cell Culture Conditions and Cardiomyocyte Colony Growth Assay
Undifferentiated R1 ES cells were transfected via electroporation with a construct carrying both an α-MHC–aminoglycoside phosphotransferase (MHC-neor) and a phosphoglycerate kinase (pGK)-hygromycin–resistant transgene in a common pBM20 vector backbone (Boehringer-Mannheim).22 In some studies, the cells were cotransfected with multiple expression vectors. Transfected cells were selected by incubation in growth medium containing hygromycin B (200 μg/mL) for 7 days and dissociated, and 4×106 cells were plated per 100-mm bacterial dish and cultured in growth medium lacking leukemia inhibitory factor (LIF). After 4 days of culture, the resulting embryoid bodies were plated onto cell culture dishes. When spontaneous contractile activity was noticed, growth medium supplemented with G418 (200 μg/mL) was added to eliminate the noncardiomyocytes. Transfection efficiencies were calculated by monitoring expression of a cotransfected CMV-βGAL reporter gene at 48 hours after transfection. To visualize cardiomyogenic induction, the plates were stained with periodic acid–Schiff (PAS) reagent.
Assessment of Gene Expression
For Western blots, protein samples were displayed on polyacrylamide gels; transferred to nitrocellulose; and reacted with anti-p53 PC35 (Oncogene), anti-E1A M73 (Santa Cruz Biotechnology), anti–T-Ag PAB-416 (Oncogene), or anti–sarcomeric myosin MF20 (Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, Iowa) as described.17 23 24 For Northern blots, total RNA was purified and processed as described.17 21 25
An expanded Materials and Methods section can be found in the online data supplement available at http://www.circresaha.org.
Identification of a p193 Mutant Encoding Prosurvival Activity
In a preliminary effort to establish structure-function relationships, a series of expression vectors encoding p193 molecules with nested C-terminal truncations was generated and tested in an NIH colony growth assay. Transfection with a control expression vector lacking a cDNA insert indicates the cumulative sum of cell growth and death under the conditions used. In agreement with previous results,17 transfection with an expression vector encoding full-length p193 failed to give rise to colonies because of its proapoptotic activity, and transfection with an expression vector encoding a full-length p193 in the antisense orientation resulted in enhanced growth (as evidenced by a marked increase in both colony size and number) compared with the control construct (Figure 1A⇓). Surprisingly, transfection with an expression vector encoding p193 amino acid residues 1 through 1343 (1343stp) resulted in slightly enhanced growth in a subset of the resultant colonies. This effect was even more pronounced with an expression vector encoding p193 amino acid residues 1 through 1152 (1152stp); transfection with this construct resulted in the formation of a large number of colonies with, on average, markedly increased size as compared with the other constructs tested. Progressively greater C-terminal truncations (912stp, 309stp, and 243stp) resulted in a loss of the growth-promoting phenotype seen with 1152stp. The level of growth enhancement obtained with 1152stp was similar to that obtained with the p193 antisense construct (Figure 1⇓).17 Importantly, similar transfection efficiencies were obtained with all of the p193 constructs tested, as determined by cotransfection with a reporter gene encoding β-galactosidase activity (Figure 1⇓ legend).
Because p193 encodes a BH3-only proapoptosis activity, the marked growth enhancement observed with the 1152stp construct was rather surprising. Stable cell lines carrying either the control or the 1152stp expression vectors were generated to determine whether 1152stp encoded prosurvival activity. The cells were incubated with increasing concentrations of MMS, an agent that promotes DNA damage and that has previously been shown to induce apoptosis via both p53-dependent and p53-independent pathways.26 27 28 29 30 31 DNA prepared from the control cells incubated in MMS at high concentration exhibited pronounced internucleosomal cleavage (Figure 2⇓), indicative of a strong apoptotic response. In contrast, DNA prepared from the 1152stp cells incubated with MMS showed no evidence of internucleosomal cleavage (Figure 2⇓), suggesting that apoptosis was either delayed or blocked in cells expressing 1152stp. These data indicate that C-terminal truncation of p193 at amino acid residue 1152 bestows a prosurvival activity on the molecule.
Coexpression of Mutant p53, p193, and E1A Markedly Increases the Yield of ES Cell–Derived Cardiomyocytes
To directly determine the importance of the p53 and p193 proapoptotic pathways in cardiomyocyte cell cycle regulation, expression constructs encoding a mutant p53 (designated CB7; see Reference 1919 ), the prosurvival p193 mutant (1152stp), and E1A under the regulation of the mouse α-cardiac MHC promoter were generated and used in an ES cell–derived cardiomyocyte colony growth assay. The cardiomyocyte colony growth assay was based on the previous observation that essentially pure cardiomyocyte cultures suitable for long-term studies can easily be generated from differentiating ES cells that carry an MHC-neor/pGK-hygror transgene.22 Undifferentiated ES cells were transfected with an MHC-neor/pGK-hygror transgene alone or in combination with the MHC-CB7, MHC-1152stp, and/or MHC-E1A transgenes. The presence of pGK-hygror sequences allowed for the selection of colonies of transfected, undifferentiated ES cells. The resulting colonies were pooled and used to generate embryoid bodies for in vitro differentiation. The presence of MHC-neor sequences allowed for enrichment of cardiomyocytes after in vitro differentiation; once cardiomyogenic induction was observed (as evidenced by the presence of spontaneous contractile activity), G418 was added to the cultures to eliminate nonmyocytes. After 60 days of culture, the dishes were stained with PAS reagent to visualize the cardiomyocytes (PAS stains glycogen-containing cells intense violet).
The results are shown in Figure 3⇓. The control dish depicts cardiomyocytes obtained by transfection with the MHC-neor/pGK-hygror transgene alone and is indicative of the baseline rate of ES cell–derived cardiomyocyte growth under the conditions used. Expression of CB7 or 1152stp did not have a marked impact on the yield of cardiomyocytes. In contrast, expression of E1A dramatically decreased cardiomyocyte yield, consistent with the proapoptotic activity of this molecule in cardiomyocytes as described by others. Coexpression of E1A and CB7 or E1A and 1152stp did not result in enhanced cardiomyocyte yield as compared with expression of CB7 or 1152stp alone, although the yield was increased as compared with cultures transfected with E1A only. In contrast, coexpression of E1A, CB7, and 1152stp markedly increased the yield of cardiomyocytes, with levels similar to those obtained from transfection with T-Ag.
Coexpression of CB7 and 1152stp Inhibits E1A-Induced Apoptosis in ES Cell–Derived Cardiomyocytes
The results presented above are consistent with the hypothesis that coexpression of CB7 and 1152stp blocks cardiomyocyte apoptosis in response to E1A expression. Several analyses were performed to further test this hypothesis. DNA was prepared from parallel cultures of ES cell–derived cardiomyocytes after 15 days of differentiation. Gel electrophoresis revealed the presence of marked internucleosomal DNA fragmentation from cultures expressing E1A alone, E1A and CB7, or E1A and 1152stp (Figure 4⇓). In contrast, no evidence for internucleosomal DNA fragmentation was observed in cultures coexpressing E1A, CB7, and 1152stp, consistent with the absence of apoptosis in these cells. DNAs prepared from the other cultures in this series were included as additional controls (Figure 4⇓).
TUNEL analysis was also used to monitor apoptosis. Dispersed cell preparations were generated after 15 days of differentiation, and the cardiomyocytes were replated at low density on chamber slides. After an additional 24 hours of culture, the samples were processed for TUNEL analysis. TUNEL-positive cardiomyocytes were readily detected in cultures expressing E1A alone, but not in cultures coexpressing E1A, CB7, and 1152stp (Figure 5A⇓). The frequency of TUNEL positivity was scored in all of the cultures (Figure 5B⇓). Frequencies of TUNEL positivity ranging from 40% to 50% were observed in cultures expressing E1A alone, E1A and CB7, or E1A and 1152stp, whereas the frequency observed in cultures cotransfected with E1A, CB7, and 1152stp was comparable with the control cultures. These data collectively indicate that coexpression of CB7 and 1152stp blocks E1A-induced apoptosis in ES cell–derived cardiomyocytes.
ES Cell–Derived Cardiomyocytes Expressing E1A and CB7 or E1A and 1152stp Are Not Viable
Transgene expression was monitored using protein and RNA prepared from cultures after 60 days of differentiation (Figure 6⇓). Seventy-five micrograms of protein or 10 μg of RNA from each sample was analyzed. High levels of CB7 protein and 1152stp mRNA were observed in all cultures transfected with the MHC-CB7 and MHC-1152stp constructs, respectively. In contrast, E1A protein was detected only in the cultures cotransfected with MHC-E1A, MHC-CB7, and MHC-1152stp. As expected, T-Ag protein was only detected in cultures transfected with the MHC–T-Ag construct. The absence of E1A protein in colonies arising from ES cells transfected with the MHC-E1A alone, or from ES cells cotransfected with MHC-E1A and MHC-CB7 or MHC-E1A and MHC-1152stp, is consistent with the high levels of DNA degradation and cardiomyocyte TUNEL positivity observed in these cultures. Importantly, these expression analyses confirm that cardiomyocytes expressing E1A are only viable if both CB7 and 1152stp are expressed.
To quantify the differential effect of transgene expression on cardiomyocyte yield, MHC expression was monitored. One percent of the total protein from each culture dish was subjected to Western blot analysis using an anti-MHC antibody (MF20; see Figure 6⇑). Consistent with the qualitative observations from PAS-stained cultures, the cardiomyocyte yield was markedly enhanced in cultures coexpressing E1A, CB7, and 1152stp, with total yields similar to that seen with cultures expressing T-Ag. Importantly, all of the ES cell–derived cultures contained differentiated, spontaneously contracting cardiomyocytes. The relatively low MHC signal in the control and singly transfected cultures is due to the low protein input on the Western blot; because of the marked enhancement of cardiomyocyte yield in the cotransfected and T-Ag cultures, low protein input was necessary to capture signals in the linear range of the film. Finally, the ES cell–derived cardiomyocyte colony growth assay was highly reproducible, and a similar effect of transgene expression on cardiomyocyte growth has been obtained in 5 independent experiments.
The results presented here indicate that E1A-induced cell cycle activation in ES cell–derived cardiomyocytes provokes an apoptotic response similar to that observed after adenovirus-mediated E1A gene transfer in fetal or neonatal cardiomyocyte cultures. Furthermore, coexpression of mutant p53 and mutant p193 blocked E1A-induced apoptosis in an ES cell–derived cardiomyocyte colony growth assay, resulting in a proliferative response indistinguishable from that seen with SV40 T-Ag gene transfer. These results provide an explanation in molecular terms for some of the phenotypic differences observed in response to T-Ag versus E1A expression in cardiomyocytes. Studies with transgenic mice have indicated that targeted expression of T-Ag promotes sustained proliferation of differentiated cardiomyocytes.5 6 7 Biochemical analyses of cardiomyocytes derived from the transgenic animals identified 2 proapoptotic T-Ag binding proteins, p53 and p193,14 17 leading to the hypothesis that there are 2 prominent proapoptotic activities, encoded by p53 and p193, respectively, that function to block cardiomyocyte proliferation induced by targeted expression of DNA tumor virus oncoproteins.17
In contrast, targeted expression of E1A using either the atrial natriuretic factor (ANF) or α-cardiac actin promoters failed to produce viable transgenic mice (K.B.S.P. and L.J.F., unpublished results, 1991). This observation is consistent with the profound cardiomyocyte apoptosis observed in response to adenoviral delivery of E1A to fetal cardiomyocytes8 9 10 11 12 or in response to E1A expression in ES cell–derived cardiomyocytes as described in this report. The observation that the combinatorial activities encoded by mutant p53 and mutant p193 block E1A-induced apoptosis in ES cell–derived cardiomyocytes and thereby renders the cells proliferative supports the hypothesis that these 2 proapoptotic pathways must be compromised for successful cell cycle progression to occur in response to DNA tumor virus oncoprotein expression. This scenario is further supported by the previous observations that E1A expression induced apoptosis in fetal or neonatal cardiomyocytes in which one or both of these proapoptotic pathways were intact.8 9 10 However, because the molecular activities of 1152stp are not fully characterized, we cannot at present rule out the possibility that expression of mutant p193 has a direct cell cycle effect in ES cell–derived cardiomyocytes, in addition to the clearly documented antiapoptotic effects. Indeed, although an overt cell cycle effect was not evident in the singly transfected ES cell–derived cardiomyocyte cultures (see Figure 3⇑), expression of 1152stp appeared to have a cell cycle effect in NIH-3T3 cells (see the colony growth assay in Figure 1⇑). It is also noteworthy that our studies used a genomic E1A clone, which can encode the 9S, 12S, and 13S splice variants. Most early cardiomyocyte E1A studies used cDNAs encoding the 12S splice variant only. Western analyses clearly indicate that the products of both the 12S and 13S transcripts are expressed in our study (Figure 6⇑). Finally, preliminary studies revealed that expression of Bcl-2 and E2F-1 are not markedly increased in response to coexpression of E1A, CB7, and/or 1152stp in the ES cell–derived cardiomyocytes (see the online data supplement available at http://www.circresaha.org), which suggests that the altered survival and proliferation observed in this model do not simply result from altered expression of these molecules.
Members of the Bcl family of apoptosis regulators share homology to Bcl-2, the prototypical family member, at one or more motifs known as Bcl-2 Homology domains (BH1, BH2, BH3, and/or BH4). Family members with the greatest homology to Bcl-2 tend to promote cell survival, whereas those more distantly related tend to promote apoptosis.32 The proapoptosis activity of the BH3-only proteins resides largely in their ability to heterodimerize with, and thereby alter the activity of, prosurvival Bcl-2 family members. Deletion of the BH3 domain blocks the ability of BH-3–only proteins to form heterodimers and in most instances concomitantly abolishes their apoptotic activity.33 34 It was therefore not surprising that deletion of the C-terminal BH3 domain abolished the proapoptotic activity of p193.17 In contrast, the observation that 1152stp encodes prosurvival activity was quite surprising.
There are several instances wherein modification of a prosurvival Bcl family member produced a proapoptotic variant. For example, alternative splicing of Bcl-XL produces a proapoptotic variant, Bcl-xS, containing BH3 and BH4 domains.35 Similarly, alternative splicing of Mcl-1 and Bok-L (prosurvival proteins that contain BH1, BH2, and BH3 domains) disrupts the BH1 and BH2 domains in these molecules, resulting in the formation of proapoptotic BH3-only splice variants.36 37 38 In contrast, we are unaware of previous examples in which modification of a proapoptotic BH3-only protein resulted in a prosurvival phenotype. At present the molecular basis for the prosurvival and growth-enhancing phenotypes encoded by 1152stp is not clear. It is unlikely that these activities result from simple titration of the normal p193 binding partners, as marked and consistent growth enhancement was only observed with the 1152stp construct. Rather, disequilibrium resulting from the titration of only a subset of the proteins that bind to full-length p193 might underlie the activities encoded by 1152stp. Unfortunately, the primary amino acid sequence and subcellular localization of p193 do not suggest an obvious mechanistic explanation for the activity encoded by 1152stp.
Expression of p193-antisense constructs in an NIH-3T3 colony growth assay resulted in decreased levels of the endogenous p193 transcript with a concomitant increase in cell growth17 (see also Figure 1⇑). The phenotype of cells expressing 1152stp closely mimicked that of cells with antisense-mediated p193 loss of function, raising the possibility that 1152stp may directly antagonize the proapoptotic activity of endogenous p193. This view is supported by the observation that 1152stp expression bestows a potent prosurvival phenotype in 2 independent model systems (resistance to MMS-induced apoptosis in NIH-3T3 cells and, in combination with coexpression of mutant p53, resistance to E1A-induced apoptosis in ES cell–derived cardiomyocytes). The enhanced proliferation and prosurvival phenotypes resulting from 1152stp expression are both consistent with the notion that this molecule encodes dominant negative p193 activity. Despite these circumstantial arguments, additional experimentation is required before 1153stp can be designated as a dominant negative p193.
The ES cell–derived cardiomyocyte colony growth assay relied on cotransfection of an MHC-neor/pGK-hygror transgene in combination with the genes of interest. On differentiation, imposition of G418 selection resulted in cultures that were markedly enriched for cardiomyocytes. This approach is quite analogous to and enjoys the benefits of standard fibroblast colony growth assays and also provides a relatively rapid system with which to examine the effects of a given gene on cardiomyocyte proliferation and/or survival. Moreover, the cultures are not subject to temporal limitations resulting from proliferation of noncardiomyocytes. However, because multiple genes were introduced via cotransfection, the approach as described does suffer from the possibility of partial gene transfer. For example, it is likely that the presence of CB7-positive/E1A-negative and 1152stp-positive/E1A-negative cardiomyocytes (Figures 3⇑ and 6⇑) is attributable to the differentiation of progenitor cells that were not transfected with the E1A expression construct. In support of this, polymerase chain reaction analysis of DNA prepared from these cultures indicated that the E1A transgene was absent (see the online data supplement). Similarly, the slight increase in TUNEL positivity in cultures cotransfected with E1A, CB7, and 1152stp as compared with control cultures is also likely attributable to partial gene transfer (Figure 5⇑). Importantly, coexpression of E1A, CB7, and 1152stp had a similar effect on cardiomyocyte proliferation when using either ANF-promoted transgenes or a combination of ANF- and MHC-promoted transgenes (data not shown), indicating that promoter squelching is not a problem with this assay.
The observation that E1A induces proliferation only in ES cell–derived cardiomyocytes that also express mutant p53 and mutant p193 supports the notion that there are 2 proapoptotic pathways that can negatively regulate cell cycle activity in ES cell–derived cardiomyocytes. This finding may have important implications for therapeutic myocardial regeneration. For example, if no additional cell cycle checkpoints are activated on terminal differentiation, the results presented here would predict that abrogation of p53 and p193 activity should render adult cardiomyocytes responsive to cell cycle activation. In light of this, it is of interest to note that several groups have shown that E1A and E2F-1 have very similar activities when expressed in fetal cardiomyocytes in vitro.8 9 10 11 12 39 Furthermore, E2F-1 expression in adult cardiomyocytes results in initiation of DNA synthesis followed by apoptosis.40 It will thus be of great interest to determine whether coexpression of E2F-1 (or E1A), CB7, and 1152stp is sufficient to promote proliferation in adult cardiomyocytes. The p53 and p193 may also impact on pathophysiologic apoptosis in the heart. Forced expression of p53 is sufficient to induce cardiomyocyte apoptosis, and although p53 has been implicated in some forms of myocardial apoptosis, its role as a causative agent in this process remains controversial. Combinatorial blockade of p53 and p193 may have a greater protective effect during pathophysiologic apoptosis, similar to that seen after E1A gene transfer.
This work was supported by grants from the National Heart, Lung, and Blood Institute. We thank Dr Betty Moran (Fels Institute for Cancer Research and Molecular Biology, Philadelphia, Pa) for sequences encoding E1A, Alan Bernstein (Canadian Institutes of Health Research, Ottawa, Ontario, Canada) for the CB7 genomic clone, and Andres Nagy (Samuel Luenfeld Research Institute, Toronto, Ontario, Canada) for R1 ES cells.
Original received February 2, 2001; revision received April 2, 2001; accepted April 2, 2001.
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- Coexpression of Mutant p53 and p193 Renders Embryonic Stem Cell–Derived Cardiomyocytes Responsive to the Growth-Promoting Activities of Adenoviral E1AKishore B. S. Pasumarthi, Shih-Chong Tsai and Loren J. FieldCirculation Research. 2001;88:1004-1011, originally published May 25, 2001http://dx.doi.org/10.1161/hh1001.090878
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- Coexpression of Mutant p53 and p193 Renders Embryonic Stem Cell–Derived Cardiomyocytes Responsive to the Growth-Promoting Activities of Adenoviral E1AKishore B. S. Pasumarthi, Shih-Chong Tsai and Loren J. FieldCirculation Research. 2001;88:1004-1011, originally published May 25, 2001http://dx.doi.org/10.1161/hh1001.090878
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- Expression of Mutant p193 and p53 Permits Cardiomyocyte Cell Cycle Reentry After Myocardial Infarction in Transgenic Mice
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Item G25631
Seven Second War German Badges and Insignia
Tombac, aluminum, tin. Eagle is marked with the RZM insignia and maker marked "M1/149" (Lentwerk Brüder Schneider AG., Wien) on the prongs, Army cap badge is marked "GES. GESCH.", ranging from fine to extremely fine.
Item G23551
A Badge of the Croatian/German Einsatzstaffel (ES) ES Units
Badge of the Einsatzstaffel (ES) der Deutschen Mannschaft, in die stamped bronze, with characteristic Croatian style pin on reverse, 32.5mmx43.3mm, unmarked, missing catch, otherwise in in very fine condition, and not a ground dug example; very rare.
Footnote: Established in July of 1941, this force comprised of ethnic German minority was officially part of the Croatian Ustasha Army, although led by local German Officer cadre. After fall of Jugoslavia, in July 1941, Einsatzstaffel (ES) der Deutschen Mannschaft was established as part of Deutsche Mannschaft (main organization of ethnic Germans in Croatia). Most of the soldiers of Einsatzstaffel (ES) were from north-western part of Croatia (and northern part of Serbia), namely Slavonia and Srijem. First commander of this unit was local German Jakob Lichtenberger, appointed by the leader of ethnic Germans in Croatia Volksgruppenfhrer Branimir Altgayer. Initially this unit was equipped with locally made (in Osijek) uniforms similar to uniforms of Croatian Home Guard, however at the beginning of 1942 Waffen SS style uniforms were introduced. Many members of these units served on the Russian Front in 1941-42; however in 1943 this entire unit was incorporated into 7.SS-Freiwilligen-Gebrigs-Division "Prinz Eugen".
Item G21327
A RAD Reichsarbeitsdienst Badge
A two-piece constructed RAD "Reichs" Arbeitsdiest badge; In Bakalite, 40 mm pin; unmarked; measuring 44 mm x 38 mm; pin previously repaired with hot glue, but otherwise better than fine condition.
Item G24340
You have no obligation to purchase the product once you know the price. You can simply remove the item from your cart.
You have no obligation to purchase the product once you know the price. You can simply remove the item from your cart.


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A tumor or tumour is the name for a neoplasm or a solid lesion formed by an abnormal growth of cells (termed neoplastic) which looks like a swelling. Tumor is not synonymous with cancer. A tumor can...
Stridor is a high pitched wheezing sound resulting from turbulent air flow in the upper airway. It is primarily inspiratory. It can be indicative of serious airway obstruction from severe conditions...
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Contract Research Organization news
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Pope claims GMOs could have 'ruinous impact' on environment
Pope Francis has warned against the use of genetically modified foods in a message to mark World Food Day. …[T]he Pope said that a growing number of people seem to "believe they are omnipoten...
Former pope Benedict makes rare appearance with successor Francis
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At the Vatican, Biden Seeks Common Cause with Pope on Cancer
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FAO and WFP Leaders Urged by Pope Francis to Continue Efforts to Combat Hunger
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Pope Francis to attend cardiologists' meet in Rome
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Mediaplanet, Alyssa Milano, Rosie Pope and more come together for New and Expecting Parents Campaign
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The statin wars expose 2 factions in medicine
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If the pope can retire, why can't Japan's elderly emperor?
By Linda Sieg TOKYO (Reuters) - Pope Benedict XVI did it. Dutch Queen Beatrix did it. Public broadcaster NHK reported last month that Emperor Akihito, 82, wanted to abdicate "in a few years"...
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Law of corresponding states for open collaborations.
We study the relation between number of contributors and product size in Wikipedia and GitHub. In contrast to traditional production, this is strongly probabilistic, but is characterized by two quanti...
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There's a really good Scottish fiddler named Ian Hardie that I've been listening to and I've put a couple of samples below. The cool thing is that he publishes most of his scores on his web site. So, if you want to try to play them, he makes it easy for you.
You can see them here: http://www.ian-hardie.co.uk/scores.php
A positive attitude may not solve all your problems, but it will annoy enough people to make it worth the effort. ~Herm Albright
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text: Insane cyclists in Budapest risking their lives between tramsAttila Nagy2/05/14 1:07pmFiled to: crazybudapesthungarybicyclecyclists101EditPromoteDismissUndismissHideShare to KinjaToggle Conversation toolsGo to permalink Cyclists in Budapest, Hungary, can be as insane as in the rest of the world. Or even a bit more, judging from this video in which two guys—one from New York, truth be told—risk their lives going through the thin trenches crated by running trams.SPLOID is a new blog about awesome stuff. Join us on FacebookGear from Kinja DealsSpray and Scrub Your Disgusting Screens with AM Lab ProductsFlexi Vario: A Better, Compact, Modular Pet LeashThis Essential Cree LED Flashlight Is Just $3, If You HurryReply10 repliesLeave a reply


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read moreFebruary 15 2016:Paul Newman, Barrister and Mediator of 3PB writes an informative article on when claims consultants become lawyers (or do they). (UK) Ltd Terms and Conditions and the Privacy Policy Digital edition Subscribe goodtoknow is part of the Time Inc. These days just disagreeing with somebody online can be construed as "hate speech" by the sjw cyber grievance mongers.
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It was a very successful application for Birte Höcker. The researcher from the Max Planck Institute for Developmental Biology has now received an ERC Consolidator Grant and prize money of nearly 2 Million Euros from the European Research Council (ERC). She and her team can now follow up on new ideas about protein design, their scientific discipline.
Höcker, a biochemist, studies the evolution of proteins and uses her knowledge to construct new ones. She could already show that the diversity of proteins formed from smaller fragments, which led her to a new scientific approach: Use these parts and recombine them according to a construction kit. Fittingly, her ERC project application is called "Protein Lego".
Dr. Birte Höcker
Jörg Abendroth / Max Planck Institute for Developmental Biology
"With this project application we follow a completely new approach to protein design", says Birte Höcker. "We identify stable fragments of well-known protein structures, recombine them, and so we see new complex and functional proteins coming to life."
The scientist is fascinated by the composition of these essential proteins. "In the first place, the activity of proteins makes life possible", states Höcker. "And I am particularly interested in all these little details of their structures – how they differ from each other and why they fold the way they do."
The deeper understanding of protein structures can help to build tailor-made proteins for use in biotechnology, medicine and synthetic biology. Protein design has a broad application range, for example creating new enzymes for manufacturing fine chemicals or degrading toxic material, for new protein based drugs or even as biosensors as tools in basic research.
"Currently we focus primarily on the basics of this technology", Höcker underlines. Together with her research group she wants to lay the foundation, so that tailor-made proteins for specific applications can be constructed in the near future.
The ERC Grants are the most coveted awards in the European research landscape. Each year the European Research Council supports upcoming scientists of any nationality with Consolidator Grants. The awardees have to carry out excellent research projects and should have finished their doctoral degree at least seven years ago.
About Birte Höcker:
Dr. Birte Höcker finished her doctoral degree in biochemistry in 2003 at the University of Cologne, before she worked as a postdoc at the Duke University Medical Center in the USA. Since 2006, she is an Independent Research Group Leader at the Max Planck Institute for Developmental Biology in Tübingen, Germany.
About us:
The Max Planck Institute for Developmental Biology conducts basic research in the fields of biochemistry, genetics and evolutionary biology. It employs about 360 people and is located at the Max Planck Campus in Tübingen. The Max Planck Institute for Developmental Biology is one of 83 research institutes that the Max Planck Society for the Advancement of Science maintains in Germany.
Nadja Winter | Max-Planck-Institut für Entwicklungsbiologie
Further reports about: > Biology > Max-Planck-Institut > Protein > basic research > construction > protein structures > structures
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text:
Poll: Industry frustrated so far with 2003, but optimistic
That is the result of the quarterly poll undertaken for Automobilwoche by Wiesbaden business consultants ACON, which queried manufacturers, suppliers, dealers and financial service providers.
"At the moment the industry's upswing is still more wishful thinking than reality," said ACON board member Juergen Freitaeger summarizing the study results.
While the industry saw a glimmer of hope with the positive response of the IAA in Frankfurt, most of the people questioned think it will be spring before the increased interest in new cars translates into more sales.
Dealers, who were generally very positive about the third quarter, believe that consumer habits will improve only slightly during the coming months. Manufacturers and importers are less positive.
Financial service providers and suppliers are the most optimistic. Changes are usually perceived at an early stage for both sectors, as the number of loan applications or supply contracts increases.
Suppliers, who during the second quarter were almost depressed with their situation, seem to have new hope, bolstered by positive export prospects and some OEM announcements that they would continue to reduce vertical integration.
Freitaeger said the hoped-for economic upswing is dependant on overall conditions, including fuel prices, tax laws and unemployment.


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Pencil midi dress by TFNC. Crafted in a two tone jersey fabric with contrast contoured panelling to the sides. Featuring a cowl neckline, short cap sleeves, keyhole notch with button fastening and zip through closure to the reverse. Designed with a body-conscious fit and midi length styling.
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سكس حيوانات مع بشر It may be topical as in the case of news and some made-for-television movies or historical as in the case of such documentaries or fictional series. سكس حيوانات مع بشر Inoue and Tatsuki look around desperate to find out where the sounds are coming from. سكس صوط وصوراليله علوي Women interested in Yaoi are called Fujoshi ???, which means rotten girl or rotten woman.
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text: Home > Metro Manila MRT 3 calms public after fire ABS-CBN News Posted at Nov 05 2012 09:25 AM | Updated as of Nov 06 2012 11:04 PM Share Facebook Twitter GPlus LinkedIn Pinterest MANILA, Philippines – The Metro Rail Transit (MRT 3) management is assuring the public that its trains are still safe to ride despite the fire incident last Saturday. "Normal po ang operation natin ngayong araw na ito at walang dapat ipangamba ang ating mga mananakay dahil safe na safe po ang ating mga tren ngayong araw na ito," MRT 3 General Manager Al Vitangcol told radio dzMM. "Lahat ng natitira nating 72 cars ay ayos pong lahat at ang ating mga istasyon naman po ay secured. Wala pong dapat ipangamba ang ating mga sumasakay na pasahero," he added. Vitangcol said an investigation confirmed that an electrical wiring of the train's "traction motor" suffered a short circuit. "Nagkaroon na po kami ng preliminary investigation at napag-alaman po natin na ang sanhi noong insidente noong Sabado ay ang pagsho-short circuit ng isang traction motor," he said. "Ang supply kasi natin ng kuryente ay mataas. Imbis na ma-consume ng traction motor yung kuryenteng dumadaloy, pumasok ito doon sa distribution box. Pagpasok po sa distribution box, natunaw po yung mga high-tension wire." Aside from the investigaton done by the MRT management, the Bureau of Fire Protection also launched a separate investigation. MRT-3 suspended trips momentarily Saturday afternoon after smoke was seen coming out from one of the train's coaches. The train's northbound passengers were directed to get off the train at the Kamuning station as authorities attended to the situation. Two passengers suffered bruises due after some of the passengers panicked as they rushed out of the coaches. The injured passengers were identified as Susana Perater and Jerome Hidalgo. They were brought to the East Avenue Medical Center. Share Facebook Share on Twitter GPlus LinkedIn Pinterest Read More: Metro Rail Transit MRT-3 MRT fire /entertainment/v2/10/22/16/in-photos-star-magic-ball-2016-couples-love-teams/overseas/10/22/16/somali-pirates-free-26-sailors-including-filipinos/sports/10/22/16/ginebras-chua-on-win-binigay-ng-diyos-sa-amin-talaga/entertainment/10/22/16/after-marco-vivoree-evicted-from-pbb/entertainment/10/22/16/liza-soberano-takes-home-best-dressed-award


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steve batchelder's curator insight, August 10, 2015 2:01 PM
As creators, we need to think carefully about the longevity of the content deluge. Will people continue to "pay attention" (think about that phrase for a moment) to what we're publishing? Whatever, whenever we're publishing?
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Valuable look at how to leverage disruptive growth.
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best practice: hyper disruptive business models


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I am having problems with two of my cats. One cat insist on kicking the litter out the door everytime that she burys the waste. I swear that she is trying to dig to china. The second cat a DSH named Uncle Louie was afraid to use it until to this last week. He would always use the uncovered box down stairs. I saw Uncle Louie use the covered box today for the first time. He jumped in and proceeded to piss straight out the door onto my carpet The problem as the other cat Lita just got done a couple of minutes earlyier and kicked the litter out on the carpet. What a muddy slimey mess on my rug was the result EEEEeeeeerrrrrrrr Does anyone have any ideas how to avoid this mess from happening again?
post #1 of 5
8/16/03 at 8:35am
Thread Starter


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- For accurate prices please enter the date of your trip!
Accommodation services
Pet friendly, Safe deposit box
Hairdryer, Desk in the room, Tea/coffee-maker


text:
Chelsea stop the rot after successive Premier League defeats as they grind down Hull to earn a 2-0 win at the KCOM Stadium.
James Milner's late penalty gives Liverpool victory at Swansea, increasing the scrutiny on the hosts' manager Francesco Guidolin.
Lewis Hamilton puts in a scintillating performance to take pole position for the Malaysian Grand Prix.
Europe fight back from a 4-0 foursomes whitewash to cut the USA's lead to 5-3 after day one of the Ryder Cup.
Henrik Stenson and Justin Rose will partner rookies in Saturday's foursomes as Europe begins day two of the Ryder Cup 5-3 behind the United States.
Scottish boxer Mike Towell dies in hospital after being seriously injured in a bout on Thursday, his management says.
Europe are still down but they are on the way back up, writes BBC Sport's Tom Fordyce after watching a day of two halves at Hazeltine.
Europe win three of the afternoon fourballs matches to cut the United States' lead on the first day of the Ryder Cup.
Sam Allardyce could face an FA ban over the newspaper revelations that led to his departure as England manager.
Europe try to get on the Ryder Cup scoreboard during a good afternoon fourball session against the USA at Hazeltine.