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Villa Grevigiana
Villa with 4 bathrooms and private pool in Ponte Agli Stolli, Italy
Booking cost: £0.00
Pay onlybooking deposit
From £1,300 per week
Villa to rent in Ponte Agli Stolli, Italy
Villa overview - owner's description
Traditional country villa located in the hills outside of Florence in a pleasant position between the Valdarno and the most famous towns in the Chianti Classico area, such as as Greve, Castellina and
Traditional country villa located in the hills outside of Florence in a pleasant position between the Valdarno and the most famous towns in the Chianti Classico area, such as as Greve, Castellina and Radda in Chianti, all within a half hour drive away. The villa is an ideal solution for those who want to spend a holiday in Tuscany to discover the artistic heritage of this region. The property is surrounded on three sides by a well-kept garden that also offers a pleasant amount of shaded on hot summer days. A few meters from the villa is a swimming pool for the exclusive use of guests (10x5, 1.40 m deep) (01/04-30/09) with a sunbathing area and views of the vineyards and the countryside stretching to the hills of Pratomagno. Near the swimming pool a pleasant paved area and an equipped loggia (barbecue) make the space comfortable for relaxing moments.
A chef, maid services and cooking courses are available and to to be requested when booking and to be paid for on location. The villa is equipped with a baby-bed and Wi-Fi Internet access is included in the price. Parking in the property. Central heating. PLEASE NOTE: Check-in: between 4 p.m. and 8 p.m. For late arrivals a 40 Euros fee will be applied.
ACCOMMODATION: (250 m2) Gr. fl.: the main entrance is accessed through a porch. Entrance characterized by ancient Tuscan elements, large living room (satellite TV, DVD player), dining room with fireplace, large sunny kitchen (oven, freezer, dishwasher, microwave, American coffee machine, iron and ironing board), 1 bathroom with shower, small utility room (washing machine).
1st fl.: 1 double room with double sofa bed, 1 double bedroom with bathroom with shower and bath, 1 twin bedded room with an additional 3rd bed (suitable for a child ).
2nd fl.: 1 double bedroom decorated by hand, with a bathroom with shower and a large wardrobe.
LOCATION: 1 km grocery shop, 7 km Figline Valdarno, 13 km Greve in Chinati, 20 km Radda in Chianti, 31 km Castellina in Chianti, 43 km Florence, 50 km Siena, 60 km Arezzo, 65 km San Gimignano.Please note that all extra costs must be paid in Euro on the spot
This villa does not have any reviews but the agent has 4 reviews for their other properties.
This villa is:
Outside this villa in Ponte Agli Stolli, ItalyThis villa is in the countryside. It has a pool. Car hire is necessary as the renter will need a car to get about for shopping, eating out and the beach; however parking is available if required.
Prices and availability
Select your travel dates on the calendar
Heating per m3 (cubic meter) upon consumption
per m3 (cubic meter) upon consumption, paid locally
Gas per m3 (cubic meter) upon consumption
per m3 (cubic meter) upon consumption, paid locally
Domestic help (must be requested at booking time)
Owner defined extra, paid locally
Cook (must be requested at booking time)
Owner defined extra, paid locally
4 bathrooms
Swimming pool
Private pool
Satellite / cable
Central heating, Clothes drier, Iron / board, Towels / linen, Washing machine, Car hire essential
Cooker, Crockery, Cutlery, Dishwasher, Fridge, Freezer, Glassware, Kettle, Microwave, Oven, Pots / pans, Toaster
Suitable for
Infants, Pets
Agency From Colle Di Val D'elsa, Italy Member since Mar 2013
Past bookings: 13 bookings
Response rate: 85%
Response time: within 16 hours
Last logged in: a month ago


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'Achieving 17 Goals for the Future We Want'
Since 1992, the United Nations International Day of Persons with Disabilities (IDPD) has been celebrated annually on 3 December around the world.
The theme for this year's International Day is "Achieving 17 Goals for the Future We Want". This theme notes the recent adoption of the 17 Sustainable Development Goals (SDGs) and the role of these goals in building a more inclusive and equitable world for persons with disabilities.
This year's objectives include assessing the current status of the Convention on the Rights of Persons with Disabilities (CRPD) and SDGs and laying the foundation for a future of greater inclusion for persons with disabilities.
Observance of the 2016 IDPD coincides with the 10-year anniversary of the adoption of the CRPD – one of the most quickly and widely ratified international treaties put forth by the United Nations to date.
Information and Resources:
General information, documents and resources are available at http://www.un.org/en/events/disabilitiesday/
Posters, media resources and more are available athttps://www.un.org/development/desa/disabilities/
WFOT actively supports the inclusion of Persons with Disabilities. The WFOT Resource Centre offers the following related resources:
World Report on Disability 2011 – launched
Statement on Occupational Therapy April 2010 Position Statements:
Activities of Daily Living (2012)
Client-centredness in Occupational Therapy (2010)
Community Based Rehabilitation (2004)
Consumer Interface with Occupational Therapy (2010)
Diversity and Culture (2010)
Environmental Sustainability Sustainable Practice within Occupational Therapy (2012)
Human Displacement revised (2014)
Human Rights (2006)
Occupational Therapy in Disaster, Preparedness and Response (2014)
Telehealth (2014)
Universal Design (2012)
Vocational Rehabilitation (2012)
Occupational Therapy in End of Life Care (2016)
Occupational Therapy Services in School-Based Practice for Children and Youth (2016)Other Resources:
• More UN and WHO information.

subset: commoncrawl


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TOP O' THE MORNING, 1949. Original Half Sheet (22 x 28" / 56 x 71 cm) rolled movie poster starring Bing Crosby, Ann Blyth, Barry Fitzgerald and directed by David Miller. The famous Blarney Stone of Ireland has gone missing and Bing is sent in to sing and settle the insurance claim.
Poster is in fine, rolled condition – a few pinholes.
Condition C8. For Sale $100
To see this poster and others visit our gallery the Limited Edition in old town Katy, Texas. Providing poster sales, Linen Backing, Restoration and Framing services to the Houston, San Antonio, Austin and Dallas regions since 1994.
The Limited Edition Art and Antiques was established in 1994 in Historic Downtown Katy, Texas and features an eclectic variety of antiques, ephemera and original folk art created by the proprietor Elizabeth Proctor.
We specialize in the sales, linen backing, restoration and framing of vintage posters, We purchase collections and offer consignment services for your collections.
Follow us on Facebook at
"About Limited Edition"
Gallery Phone: 281-391-1993
EMail: [email protected]
Location: 5717 Second Street Katy, TX. 77493
Find Us – Google Map


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Haiti: Jesuit declaration exposes humanitarian crisis
A group of Jesuits working in Haiti has published a declaration condemning the humanitarian crisis related to food shortages in the country. The declaration, issued on 12 April, urges Haitians to continue raising their voices and crying out against the prevailing injustices that rattle the country. Rising food prices is only one problem, one that means that many go hungry. Poverty, insecurity, mismanagement and the inability of successive governments to address society's problems are among the other reasons for the widespread violence, misery and desperation in Haitian society. Add to this the "irresponsibility of the international community [...] which has not kept its promises to Haiti and cynically looks on while Haitian society slips into hell". The declaration calls those who carry positions of responsibility in Haitian society to do their part to remedy the situation, each according to their sphere of influence and abilities. The international community needs to "respect their commitments to Haiti, especially their many promises of cooperation and effective help for the country to emerge from this quagmire." Haitians are reminded that their "strength lies in organised and sustained non-violence. Violence is never effective." In response to the declaration, the Conference of Jesuit Provincials of Latin America and the Caribbean (CPAL) has issued a statement of its own that supports and endorses the declaration and expresses its sadness at the ongoing turmoil. It also expresses its commitment to spreading the word about the declaration. Click on this link or copy it into your web browser to read the declaration: www.sjweb.info/documents/sjs/docs/Haiti_ENG.pdf


text: The new REGULATION (EU) 2015/760 OF THE EUROPEAN PARLIAMENT AND OF THE COUNCIL of 29 April 2015 on European long-term investment funds has been published in the official gazette of the EU (see http://eur-lex.europa.eu/legal-content/EN/TXT/HTML/?uri=OJ:JOL_2015_123_R_0010&from=DE). The focus of this Regulation is to boost European long-term investments in the real economy. European long-term investment funds (ELTIFs) can provide finance to various infrastructure projects, unlisted companies, or listed small and medium-sized enterprises (SMEs) that issue equity or debt instruments for which there is no readily identifiable buyer. By providing finance to such projects, ELTIFs contribute to the financing of the EU's real economy and the implementation of its policies.


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because despite his flaws he gave pleasure to millions of people or were your first thoughts "How can I cash in on this" ?
sort by best latest
When i heard that new i thought i didn´t hear well but then i realised that Michael Jackson really died. Well, i were not shocked but i was surprised.
To be completely honest, I didn't know the man personally so I wasn't saddened... but shocked ...DEFINITELY! This is something that I never expected to happen. Can we really just brush aside the shocking things this man is said to have done just because he has crossed over to the other side?
I agree with Grunge Hedgehog (and feel free to read my Hub on Jackson's passing, RIP Michael Jackson...We'll Miss You).
Let's focus on the man, the legend, the music, the videos on YouTube (which generate millions upon millions of views).
Let's put aside the scandal...the allegations...and what did or did not happen and just think about why he was so loved and what our favorite song was back when he was in his prime.
Remember how everybody at school was Moonwalking? Let's concentrate on that.


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The article was originally published on Soccer Iraq.
Greek Emperors
In 2004, the footballing world stood and took notice of Iraq as they participated in their fourth Olympic games. Entering the competition as real underdogs the Lions were expected to be knocked out early in the group stage due to failure of making a mark internationally. As well as not reaching a global tournament since the late eighties (World Cup 1986 & Olympic Games 1988), Iraq's Olympic track record was not the prettiest either. One medal – Bronze, weightlifting in Rome 1960. However Athens 2004 really did put Iraq on the map as an odd defying campaign saw Adnan Hamad's men inches away from an astonishing Olympic final. A defeat to Paraguay in the semis ended all hopes but convincingly cemented Usood Al-Rafiadin's place on the big stage.
Road to Rio
Fast forward to 2016 and Iraq's Olympic team became favourites to take one of three qualifying spots to the summer games in Brazil. Iraq were predicted qualification for purely one reason – being the defending champions. Having said that, it was not all plain sailing for the Lions as the side suffered some big losses. Udinese's Ali Adnan, Rizespor's Dhurgham Ismail, and Ängelholms' Rebin Asaad could not attend the tournament in Qatar due to club commitments. One man trusted to lead the line was Grasshopper Zurich striker Sherko Kareem. Kareem lit up the U-17 World Cup in 2013 which subsequently earned him a move to Switzerland from his boyhood club Al-Shorta. Although training in Doha with the squad, the striker was sent back on a plane before a game had even kicked off. Club Zurich had recalled his services. With preparation not going exactly to plan team selection wise, the side had a secret weapon. His name was Abdul Ghani-Shahad. This was the man who was responsible for gaining Naft Al-Wasat promotion to the Iraqi Premier League and winning them the title in their first season of top flight football. A simply sensational achievement. Shahad was roped in to lift the young squad and help achieve them qualification to a fifth Olympic tournament. Unbeaten in nine games prior to the championships, the U-23 manager was off to a flyer.
Next : Halilhodzic considers recalling Mike Havenaar


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Elinz Electronics sell a range of in car entertainment systems that may help keep the young ones busy, and there are a few neat gadgets for you too.
Founded in 2005, Elinz Electronics are a Melbourne based retailer specialising in the in car electronics and camera kits. They offer free shipping and have a 30-day money back guarantee in case you change your mind.
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4. Simply enter your coupon/promo code in the box and click 'Apply Coupon Code'
Elinz Review | Pros and Cons
- Free shipping.
- Wide range of products.
- Wide range of payment methods accepted.
- In-car electronics only, no home or personal items.
How can I get Elinz free shipping?
Elinz offers free shipping on all orders in Australia.
What forms of payment does Elinz accept?
- Visa
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- PayPal
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What is the Elinz returns policy?
Elinz offers returns and refunds within 30 days of purchase. They will process a refund in most cases, but will always extract a "restocking fee", which will cover their administration in processing the refund.


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We recommend upgrading to the latest Internet Explorer, Google Chrome, or Firefox.
If you are using IE 9 or later, make sure you turn off "Compatibility View".
Handpicked brands for you
Huge Congratulations Second place in the Ride Of The Year Award on #3DFINS, Oakley Big Wave Awards! Mr Zac Haynes @zac_haynes 7K later and one helluva drop,


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Results 1 to 7 of 7
August 5th, 2008, 12:24 PM #1
Get You Daily CJ Laugh Here.
- Join Date
- June 22nd, 2008
- Posts
- 59
I got just got a Dear John email from a CJ merchant saying that I was being dropped from their program ... under the label advertiser expired.
Now get this: This merchant has a 30 day EPC of EIGHTY CENTS and a 7 day EPC of 3 eggs ... 0.00.
I'm totally crushed ... NOT.
This affiliate marketing gig is getting to be funnier than South Park.
August 5th, 2008, 12:42 PM #2
Maybe that is the reason they are dropping inactive affiliates (Those who don't have sales for long time). It can really effect Merchant's EPC.
August 5th, 2008, 04:17 PM #3
Originally Posted by Nabz
- Join Date
- October 22nd, 2006
- Posts
- 1,065
But anyway, a merchant who has a 0.80 epc cannot blame the affiliates. Its totally their failure. Either they have a product that doesn't sell or they have a bunch of affiliates that drive unqualified traffic to them.
August 5th, 2008, 04:23 PM #4
- Join Date
- January 18th, 2005
- Location
- Nunya, Business
- Posts
- 23,684
"under the label advertiser expired."
If it's advertiser expired, then everybody is gettting dropped because it means they're leaving the network. Which merchant was it by the way?
August 5th, 2008, 06:18 PM #5Originally Posted by sam_park
August 5th, 2008, 06:54 PM #6Originally Posted by sam_park
August 7th, 2008, 06:14 PM #7
- Join Date
- October 22nd, 2006
- Posts
- 1,065
I got booted too from the same pgm. I didn't know I had signed up with them.
Seisma Research World-Wide Investment Leads.
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This post by Faith
Watching Agent Carter with my dad ♥
Going out to lunch with my dad and theorizing on Agent Carter
his letters are so matter-of-fact and awesome (and the whole thing is always one very long paragraph).
the POTC music for the first time years ago; I don't have the sheet music for my favorite songs
(Up Is Down, What Shall We Die For and At Wit's End), but it's ridiculously fun to play The Kraken. ♥
You Make Me Feel So Young, Frank Sinatra
Feeling Good, Micheal Buble'
unless you allow others to help you." - Edwin Jarvis, Agent Carter
I hope you all had a good week and that this coming one is a great one for each of you. xx
Why is it that all my posts are published late at night? I should go to bed. G'night.


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MOVIE SYNOPSIS [ General Audiences ] Directed by James C. Hunter, Wade Williams leads the cast of this feature film originally of USA, with original script in English, whose premiere was scheduled for 2015. You can watch The King's Disciple online on TV cable or theaters with original audio in English at different dates. This film premiered in theaters in 2015.
Rating: 5 by 10 users./
Runtime: 100 minutes
Wade Williams, Kibwe Dorsey, Kia Shaw, Tiffany Edwards and Kiara Dorsey.
100-minute film (full movie) directed by James C. Hunter.
The original audio of The King's Disciple has dialogues in Dolby Digital, it was produced in USA.
It premiered in cinemas or television in 2015.
Screenplay written by Kibwe Dorsey and Lila Michelle Dorsey.
Kibwe Dorsey.
This film was produced and / or financed by All For The Love Entertainment.
All For The Love Entertainment.
Without commercial breaks, the full movie The King's Disciple has a duration of 100 minutes; the official trailer can be watched in streaming on the Internet. You can watch this full movie free in EnglishDolby Digital in movie TV channels, renting the DVD or with VoD services (Video On Demand player, Hulu) and PPV (Pay Per View, Netflix).
See the page of this full movie, The King's Disciple in FULLTV Movies.


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|260 m²
|Tipo de imóvel
|Estadia Mínima
|7 noites
A Casa do Lago tem tudo que precisa para umas férias relaxantes Família
Bonitas recém-atualizados 1920 Lake House localizado diretamente em Long Pond. Acomodações, tranquila, caracterizando uma praia de areia branca exuberante. Perfeito para uma ou duas famílias pequenas. Assista ao Águias e Osprey busca americana para os peixes no crepúsculo da sua envolvente alpendre completo com uma lareira ao ar livre cheio e cadeiras de balanço de madeira. Long Pond é full-recreação, nós fornecemos dois caiaques, cadeiras de praia e toalhas de praia. Caminhadas, ciclismo, golfe, pesca, natação, canoagem, mergulho, banhos de sol e cochilando são apenas algumas das atividades que podem ser apreciadas na, casa lagoa em frente à pitoresca. Estamos a apenas 2 km dos campos de golfe Pinehills, 6 quilômetros de Praia do Cavalo Branco, 7 km de trilhas de mountain bike, Pine Hills e 8 km do centro de Plymouth, onde você pode ir observação de baleias ou pesca em alto mar ou participar de muitas das nossas atrações turísticas e 10 quilômetros de Cape Cod. E para os dias chuvosos, temos uma TV 80 'projeção com som surround em um filme de mini-teatro. The Lake House compartilha uma praia com uma pequena cabana. Crianças são bem-vindas.
We feel so fortunate to have stayed here. We are a family of five and all very particular about our vacation home. My husband loves to cook and needs a well stocked kitchen. The house offers the best of modern upgrades and conveniences while maintaing it's quaint cottage feel. There was plenty of room for us to spread out through out the house. I particularly loved having my morning coffee on the front porch. My son's loved the theater room in the basement. My daughter liked sitting by the lake taking in the mid afternoon sun reading her book. And of course, my husband loved the kitchen cooking on the viking stove and utilizing every bit of counter space (which was extensive). In the evenings we gathered around the outdoor fireplace on the porch. There is much to do in the town of Plymouth and surrounding areas. We hiked at Miles Standish State Park, Rode our bikes on the Cape Cod Rail Trail, attended a minor league baseball game in Cape Cod, checked out the town of Plymouth and of course the "rock". The afternoons we were back at the house swimming and/or fishing in the lake, playing cam jam on the lawn or just sitting and relaxing. The ages of our children are 23, 21 and 16. The older two are away at school during the year and having this one week of year to get together is very important to us. I can't say enough good things about the house and Lynn the owner who was always available by text for any questions we had. Both Lynn and Jack came to meet us our first day at the house to meet us and show us where everything was. It was such a pleasure meeting them both and we are looking forward to coming back again next year!
This was just a perfect place for our family vacation. We were blown away by the property and the gorgeous lake views with pristine lake swimming. You just don't find properties that sit on a quiet crystal clear lake with a private clean sandy beach. This is a perfect place for our vacation. We had to drag our kids away from the house to sight see and do other things. We just didn't want to leave. The house is very close to the beaches in Plymouth and the Cape but there was nothing like swimming in the lake when we returned. The kitchen is superb for anyone who loves to cook - viking range, dining area with views and all the tools you need. The house was clean and beautifully decorated. I would highly recommend this rental, it is a really unique and wonderful property. Lynn the owner was extremely helpful. We can't wait to come back!
The house and land are beautiful. The house is clean and the beds are comfortable. With that being said it is a shared property. I was informed tha it was a shared beach but when I got there I was then informed that there would be another couple staying in the beach house which shares the driveway, beach, and yard. It was very uncomfortable with the people living right there. The owner came the first three days we were there to drop something off or get trash or check the other tenants in so there was a lack of privacy. For the very high cost of the rental I would have thought it would be more private. I will not be returning with my family.
We rented the house for 7 days in July for a family reunion. The house was very nice and had all of the amenities needed to make the stay worthwhile. The house is off of the beaten path, but close enough to the main thoroughfares to make this an excellent choice, only 20 minutes to Plymouth and fresh seafood (yum). Since our mom was born and raised on the east side of Long Pond, it definitely was the place we had to be for the reunion. If you have never experienced Long Pond, do yourself a favor and stay at this house with a nice sandy beach right at the end of the lawn and wonderful scenery. We would definitely consider this place again.
Our family recently stayed at the Lake House and had a wonderful time! It is beautifully furnished with a great view of the water. Beds were very comfortable and the home theater was great! The owner has a gourmet kitchen and great attention to detail. Our family can't wait to return for another getaway!
Localizado directamente na Long Pond no meio do deserto, mas a apenas 1. 5 milhas fora da rota 3. No centro da área de Seis Lagoas, cercado por terras de conservação com marcadas trilhas para caminhada e pântanos cranberry imaculadas. Perto do Pinehills Village Center com o mercado, Long Wine Ridge e Spirits, Correios, lavanderias, Cafe Olio, e extensas trilhas para caminhadas. Dez minutos para o Cape Cod Canal (tanto o Sagamore e Bourne Pontes). Dois quilômetros de Myles Standish State Forest, com suas extensas trilhas de bicicleta pavimentadas. Dois quilômetros de campos de golfe ~ Pinehills, Waverly Oaks, Squirrel Run. Seis quilômetros do centro histórico de Plymouth. Cinco quilômetros de Plimoth. Seis quilômetros de White Horse Beach. Cinco quilômetros das famosas Pine Hills trilhas de mountain bike.
|Validade da Tarifa
|por noite
|Noite (fim de semana)
|por semana
|por mês
|Pacote ou Evento
Tarifa Básica
Dia de mudança: sábado
Estadia mínima: 7 noite(s)
|Observações: As tarifas levam em consideração a estadia de 6 hóspedes, adicione R$ 97 por noite, por pessoa adicional
|Weekly Cleaning fee
Rates are for up to 6 people - we can accommodate up to 10. There is an additional charge of $30 per day per person over 6 people.
Check-in time: 4:00 PM; Check-out time: 10:00 AM


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We have been developing 240sxlisted.com for the past few months. Our intentions are to provide a 1 stop Nissan 240sx version of craigslist. Strickly Nissan 240sx related ads.We are Nissan lovers just like you, we have been in the 240sx scene for years so we know what you are looking for in a website!
S14 Zenki1 - 20 of 27 listings
1995 Nissan 240sx S15 RB25 swappedS14 Zenki - Lenoir City (Tennessee) - August 21, 2017 11500.00 Dollar US$
I have a 1995 Nissan 240sx with 133,xxx miles. Motor is stock as well as the turbo. Intake manifold has been upgraded along with front mount intercooler and intake. Stock rb ecu, manual fuel pressure regulator and manual boost controller. Set t...
1995 240sx SE with SR20DET $6500S14 Zenki - Atkins (East Coast) - August 16, 2017 6500.00 Dollar US$
Great body, no rust, straight frame, clean title 240sxmotorset came from JGY, JGY did installKoyo Rad3inch exhaust300zx big brakesJGY flywheelCC clutchmade 215HP at 7psiwhite face gaugesto replicate this with buying the items and the shell, it would ...
Looking for a 95 or newer s14S14 Zenki - Danville (Illinois) - June 29, 2017 3000.00 Dollar US$
I'm looking for a 95 or newer S14 for sale with medium to low miles and little to no rust for under 3000
1996 240SX Base RedS14 Zenki - Lansdale (Pennsylvania) - June 24, 2017 2000.00 Dollar US$
VIN: JN1AS44D8TW051723 1996 240SX base model About 179,000 miles (odometer only reads 79,000 miles) 5 speed manual, short shifter Short intake HKS cat back exhaust No spare tire and trunk floor removed. Battery relocated to trunk. Paint wearing off o...
1996 S14 Base ModelS14 Zenki - Lansdale () - June 17, 2017 2000.00 Dollar US$
1996 240SX base modelAbout 179,000 miles (odometer only reads 79,000 miles)5 speed manual, short shifterShort intakeHKS cat back exhaustNo spare tire and trunk floor removed.Battery relocated to trunk.Paint wearing off on hood and roof.Passenger side...
1995 240sx base modelS14 Zenki - Dothan () - June 2, 2017 4000.00 Dollar US$
I have a 1995 240sx 5speed base model motor runs good and drives pretty good but could use new master or slave not sure. I bought the car back in march but it needed some work to get it running so for that reason I haven't transferred title over to m...
WTB Nissan ZenkiS14 Zenki - Chicago (Midwest) - March 23, 2017 4500.00 Dollar US$
Have cash in hand and looking for a fair s14, looking for an unmolested and fair price, located in chicago.
1995 240sx for saleS14 Zenki - ft. lauderdale (Florida) - February 13, 2017 5500.00 Dollar US$
For sale: 1995 Nissan 240sx. I CAN SEND CRAIGSLIST LINK WITH CLEAR PICTURES. sAYS PICTURES ARE TOO LARGE TO POST HERE-Clean title - RUNS STRONG with COLD AC.-Excellent Condition, NO LEAKS, a great beginner car or project car. MUST S...
1995 240SX / S14 Nissan ALL ORIGINAL by ownerS14 Zenki - Seattle (NW) - November 9, 2016 8000.00 Dollar US$
Vin: JN1AS44D7SW017481 Accident History: No Accidents Mods: Sports suspension, Multiple CD Player Changer Type: 240SX S14 Year: 1995 PRICE: 8K or best offer Color: Original triple pearl white Parts Included: Full size spare tire plus original ...
1996 240SX SE with VH45DE swap 95% doneS14 Zenki - Liberty (Southeast) - September 27, 2016 8000.00 Dollar US$
1996 S14 240SX SE, I'd estimate chassis mileage around 175k but I honestly have no clue, cluster has been changed numerous times before I even got the car 11 years ago. Chassis was originally auto and has been through 4 drivetrain swaps (KA, RB25, SR...
FOR SALE 1995 240SX ZENKI (Silvia)S14 Zenki - Fountain Valley (California) - September 5, 2016 1500.00 Dollar US$
1995 Nissan 240sx Zenki car is primmered white, door handles and trunk is shaved,with custom wrapped black white interior , car run and drives fine , needs a few minor fixes like brakes rotors , sensor , ignition , battery, and the car was last regis...
1996 240sx /seS14 Zenki - McDonough (Georgia) - August 5, 2016 1800.00 Dollar US$
S14 needs work, body is good. Must sell asap in Georgia. Do not know issues with the car. Car has very good tires, body, and interior. sunroof 5 spd. Had emissions 6 mo ago and passrf but there are issues with motor. stock KA24de
1995 with RB25DET and s15 Front and rear conversionS14 Zenki - Hanford () - April 25, 2016 11.00 Dollar US$
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1995 240sx SE rolling chassis (Americus,Ga)S14 Zenki - Americus (Georgia) - April 6, 2016 750.00 Dollar US$
I have a 1995 240sx SE rolling chassis for sale. Pickup only or you can schedule a shipping method. The car has 130k miles on it. The engine is in good condition, but had a turbo kit installed on it and I have sold the kit. The car is ready for a new...
1995 zenki 240sx w/ Kouki front 1JZ swapS14 Zenki - Augusta (Georgia) - October 10, 2015 7000.00 Dollar US$
Up for sale is a 1995 zenki 240sx w/ Kouki front 1JZ Swap. car has 190xxx miles on body. The car is in need a battery to crank up! It is been sit for couple months! Specs: -1jzgte bone stock eng w/r154 trans -Act extreme 6puck clutch -6262 precision ...
1995 Nissan 240sx 5spd SR20DET TomeiS14 Zenki - Missoula (Montana) - August 7, 2015 9400.00 Dollar US$
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Wtb 240sxS14 Zenki - (Florida) - June 30, 2015 Check with seller
Looking for a Zenki s14 or a s13 with manual transmission only. The more stock the better, must have power steering and ac <-I know it it's a long shot to find one like that. I don't mind if it has body damage or not so good paint (as long as it i...
Nissan silvia 240sx for sale ConnecticutS14 Zenki - Wallingford (Connecticut) - June 27, 2015 11000.00 Dollar US$
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Nissan s14 240sx for sale in AlabamaS14 Zenki - Mobile (Alabama) - June 27, 2015 2500.00 Dollar US$
Bone stock 240sx 5spd with an exception of a Injen intake but do have stock intake. 260000 mileage, medium size dents on both fenders, driver side window off track due to some bummy ass thief who stole radio and amp, one fog light don't work, does ha...
Original, Clean, Rust-Free, SES14 Zenki - Knightdale (Southeast) - June 22, 2015 7.00 Dollar US$
1995 Nissan 240SX SE edition. Original paint. Everything is stock/OEM. No one has chopped this car up or attempted to mod it out. No collisions. All panels are original. NO RUST. Automatic transmission. Original 2.4 liter engine. Pristine interior. N...


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text: Dengue virus infection induces autophagy: an in vivo studyYing-Ray Lee, Hsuan-Yun Hu, +5 authors Hsiao-Sheng LiuJournal of Biomedical Science2013We and others have reported that autophagy is induced by dengue viruses (DVs) in various cell lines, and that it plays a supportive role in DV replication. This study intended to clarify whether DV infection could induce autophagy in vivo. Furthermore, the effect of DV induced autophagy on viral replication and DV-related pathogenesis was investigated. The… (More)


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Congenital distal renal tubular acidosis (RTA) from mutations of the B1 subunit of V-ATPase is considered an autosomal recessive disease. We analyzed a distal RTA kindred with a truncation mutation of B1 (p.Phe468fsX487) previously shown to have failure of assembly into the V1 domain of V-ATPase. All heterozygous carriers in this kindred have normal plasma HCO3− concentrations and thus evaded the diagnosis of RTA. However, inappropriately high urine pH, hypocitraturia, and hypercalciuria were present either individually or in combination in the heterozygotes at baseline. Two of the heterozygotes studied also had inappropriate urinary acidification with acute ammonium chloride loading and an impaired urine-blood Pco2 gradient during bicarbonaturia, indicating the presence of a H+ gradient and flux defects. In normal human renal papillae, wild-type B1 is located primarily on the plasma membrane, but papilla from one of the heterozygote who had kidney stones but not nephrocalcinosis showed B1 in both the plasma membrane as well as diffuse intracellular staining. Titration of increasing amounts of the mutant B1 subunit did not exhibit negative dominance over the expression, cellular distribution, or H+ pump activity of wild-type B1 in mammalian human embryonic kidney-293 cells and in V-ATPase-deficient Saccharomyces cerevisiae. This is the first demonstration of renal acidification defects and nephrolithiasis in heterozygous carriers of a mutant B1 subunit that cannot be attributable to negative dominance. We propose that heterozygosity may lead to mild real acidification defects due to haploinsufficiency. B1 heterozygosity should be considered in patients with calcium nephrolithiasis and urinary abnormalities such as alkalinuria or hypocitraturia.
- V-ATPase
- haploinsufficiency
- kidney stones
- distal renal tubular acidosis
renal tubular acidoses (RTAs) are tubulopathies where nonvolatile acid accumulates because of decreased tubular secretion of acid by the proximal or distal nephron, which can result from congenital or acquired causes (19, 31). Distal H+ excretion requires carbonic anhydrase to generate an acid-base pair (H+ and HCO3−) to furnish substrate for acid-base transporters. Luminal translocation of H+ from the cell into the urine is mediated by V-ATPase, and concurrent basolateral exit of HCO3− into the plasma occurs via anion exchanger 1 (AE1) (68). It is not mere coincidence that congenital forms of distal RTA (dRTA) in humans result from inactivating mutations of carbonic anhydrase II enzyme (23, 53, 55), the B1 or a4 subunits of V-ATPase (60, 67), or the AE1 Cl−-base exchanger (6, 7, 11).
These candidate genes were identified using classical genetics on kindreds derived from probands initially identified by clinicians. In general, phenotypic information is limited to clinical diagnosis of RTA based on hypobicarbonatemia without elevation of plasma anion gap or creatinine and concomitant unduly alkaline urine pH. The disorder may be brought to clinical attention by complications such as growth retardation, nephrocalcinosis, and nephrolithiasis (31). Most clinical data are limited to plasma HCO3− concentration and spot urinary pH with no further phenotypic characterizations.
Congenital dRTA usually follow autosomal recessive inheritance with the exception of some mutations of AE1, which are autosomal dominant. The basis for the recessive nature is usually attributed to adequacy of the normal functioning transporters provided by the single wild-type allele, or haplosufficiency. In the case of AE1-related autosomal dominant dRTA (6, 11, 25, 29, 57, 69), in vitro data have suggested that mutant AE1 may prevent wild-type AE1 from reaching the membrane via heterodimerization or by mistargeting of the mutant to the apical membrane, thereby nullifying apical H+ secretion (13, 49, 50, 64). This dominant model by mutant AE1 remains to be proven in vivo. Other than these AE1 mutations, the accepted paradigm is that one needs two mutant alleles to produce dRTA. However, the foundation of this paradigm is based on the absence of overt clinical diagnoses in heterozygotes.
The clinical diagnosis of dRTA depends on the stringency of the definition and how one sets the threshold for defining renal acid underexcretion. Since metabolic acidosis is not a dichotomous trait, a forme fruste of dRTA can conceivably eclipse detection by conventional clinical tests, and indepth metabolic studies are not usually performed by practitioners. Wrong and coworkers (16, 71) recognized that defective distal renal acidification can be associated with a normal plasma HCO3− concentration and set the precedence for using acute acid loading to uncover renal acidification defects. Interestingly, a considerable fraction of the literature on this type of incomplete dRTA actually stems from patients with kidney stones. While most patients with calcium oxalate stones can maximally acidify their urine, up to 30% of patients with calcium phosphate stones have reduced ability to lower their urine pH (2, 22, 45, 46). Impaired maximal acidification is also more common in recurrent stone formers and those with bilateral disease (2, 12, 15, 30, 43, 44, 54, 61, 63). No one investigated the basis for these subtle acidification anomalies.
Distal apical H+ secretion is mediated by V-ATPase (5, 27), which belongs to a family of ATP-energized H+-translocating ion pumps (18). V-ATPases acidify intracellular organelles, which is essential for receptor-mediated endocytosis, lysosomal hydrolysis, intracellular protein targeting, processing of hormones, and uptake and storage of neurotransmitters. In specialized cells, plasma membrane-situated V-ATPases are instrumental in the regulation of bone resorption, synaptic transmission, endolymph pH regulation, and renal acid excretion. V-ATPases are complex proteins composed of at least 13 different subunits organized into 2 distinct functional domains: a peripheral catalytic sector 650-kDa V1 domain (A3B3C1D1E3F1G3H1) and an intramembranous 260-kDa proteolipid V0 domain [a1d1c″1(c′,c)6] (24). In mammals, B1 is restricted to epithelia of the inner ear, epididymis, and distal renal tubule. Mutations in the B1 subunit gene ATP6V1B1 are responsible for autosomal recessive dRTA with deafness (19, 28, 56, 60). The mechanisms by which some B1 subunit mutations lead to V-ATPase dysfunction were unknown until recently, when the majority of mutant B1 subunits were shown to fail to incorporate into the V1 subunit (20, 73).
We posed the simple but fundamental question of whether heterozygous carriers of mutant B1 have any detectable renal acidification abnormalities. In addition, if they do, is there evidence for negative dominance? We took a family known to harbor an inactivating truncation mutation of B1 (21) and studied the heterozygous members for abnormalities in renal acidification, prompted by the fact that one of the heterozygotes developed severe kidney stones. Despite the fact that none of the heterozygotes carry the clinical diagnosis of RTA, we obtained evidence that the heterozygotes have abnormal baseline steady-state urinary chemistry, and two heterozygotes showed abnormal urinary acidification upon provocation. Because of the previous postulation of negative dominance of the mutant B1 subunit over the wild-type subunit (73), we used in vitro biochemical, mammalian cell expression, and yeast complementation approaches to examine for this possibility. We conclude that the affected heterozygotes in our kindred are in a state of haploinsufficiency where the pump from the normal allele can sustain normal clinical chemistry but the burden of heterozygosity in this family is incomplete dRTA. Individuals with incomplete dRTA or calcareous nephrolithiasis with distinct chemical abnormalities should be considered as potential heterozygous carriers of mutant B1 alleles.
Patient experiments.
Subjects were recruited from University of Texas Southwestern Medical Center clinics with approval by the Institutional Review Board, and all participants gave informed consent. Normal subjects comprised staff and students on campus. A systemic multichannel analysis was used for serum analytes (Beckman CX9ALX, Beckman, Fullerton, CA). Serum pH and Pco2 were measured using a Radiometer ABL 5 (Radiometer America, Westlake, OH). Urine creatinine was measured via the picric acid method, sulfate was assessed by ion chromatography, ammonium was assessed by the glutamate dehydrogenase method, and citrate was assessed enzymatically (Boehringer-Mannheim Biochemicals, Indianapolis, IN).
The ammonium chloride load test was conducted as previously described (33). Fasted subjects had urine collected hourly from 0700 to 1200 hours under mineral oil. At 0800 hours, 50-meq ammonium chloride gelatin capsules were administered orally in two to four divided doses over 30 min. Water (250 ml) was given at 0700, 0800, 0900, 1000, and 1100 hours. Arterialized venous blood was obtained for chemistry and pH and blood gases at 0700, 1000, and 1200 hours. Urine was collected for 4–6 h depending on the response to the acid load. To perform urine minus blood Pco2, which yields a surrogate for H+ flux in the presence of bicarbonaturia (17, 62), two forearm intravenous catheters were inserted: one for NaHCO3 infusion and the other for blood sampling. Sodium bicarbonate (250 meq) in 750 ml was infused, and urine was collected every 30 min until the urine pH was >7.0, and the urine was then collected every 15 min for three times and analyzed for Pco2, pH, total volume, and creatinine. Venous blood was drawn before the infusion and at the time of each of the last three urine collections for Pco2 and pH.
For immunohistochemistry of the human kidney, intraoperative medullary biopsy samples (approved by the Institution Review Board of the University of Texas Southwestern Medical Center) were immersion fixed in 4% paraformaldehyde (4°C for 4 h). After being washied with PBS (4°C overnight), tissues were frozen in OCT, and 4-μm sections were cut and washed with PBS (for 15 min) followed by 0.1% Triton X-100 incubation (10 min). Sections were incubated with a blocking solution (PBS, 3% BSA, and 10% goat serum for 60 min) and then rabbit polyclonal anti-B1 antibody (1:100; 4°C overnight) (47). After being washed with PBS, sections were incubated with Alexa fluor 488-conjugated goat anti-rabbit IgG (Invitrogen, Carlsbad, CA) and then with 1:80 rhodamine phalloidin (Invitrogen). After additional PBS washes, sections were mounted and then visualized with a Zeiss LSM510 microscope.
Mammalian cell expression experiments.
PCR-generated cDNA fragments were inserted in frame into COOH-terminal single hemagglutinin (HA)-tagged PMH (Roche) or NH2-terminal FLAG-tagged p3xFLAG-CMV-10 (Sigma-Aldrich, St. Louis, MO) vectors for mammalian expression. Synonomous mutations in B1 were created by a QuikChange Multi Site-Directed Mutagenesis Kit according to the manufacturer's instructions (Agilent Technologies). All constructs were verified by sequencing. Human embryonic kidney (HEK)-293 cells [American Type Culture Collection (ATCC)] were cultured (37°C, 5% CO2) in high-glucose (450 mg/dl) DMEM (GIBCO-BRL) supplemented with 10% (vol/vol) FBS, penicillin (100 units/ml), and streptomycin (100 μg/ml). Transfections were performed with Lipofectamine 2000 (Invitrogen), and cells were used 48 h later. The design of the small interfering (si)RNAs is shown in Fig. 3A, and siRNAs were custom synthesized by Invitrogen and added to cells at a final concentration of 200 pM.
Immunoblots and immunoprecipitation were performed as previously described for these cells (20). The primary antibodies used were as follows: anti-B1 antibody, rabbit polyclonal (Xie Lab) and mouse monoclonal (SC-55544, Santa Cruz Biotechnology); anti-FLAG antibody, rabbit polyclonal antibody (F7425, Sigma) and mouse monoclonal (F3165, Sigma); and anti-HA antibody, rabbit polyclonal (SAB4300603, Sigma) and mouse monoclonal (H3663, Sigma). Secondary horseradish peroxidase-conjugated sheep anti-mouse and donkey anti-rabbit IgG antibodies were used at 1:20,000 to 1:10,000 dilutions, and signals were detected using enhanced chemiluminescence (Amersham, Piscataway, NJ).
For immunoprecipitation, cells were lyzed in buffer [containing 150 mM NaCl, 50 mM Tris·HCl (pH 8), 5 mM EDTA, 1 mM EGTA, 1% Triton X-100, 100 μg/ml PMSF, 2 μg/ml leupeptin, 2 μg/ml aprotinin, and 2 μg/ml pepstatin A], and lysates were cleared by centrifugation (14,000 g for 30 min). Precipitating antibodies (1:200 dilution) followed by prewashed protein G-agarose (50% slurry) were added (4 h at 4°C). After being washed with lysis buffer (3 times), proteins were eluted from the beads with 2.5× loading buffer [2.5 mM Tris·Cl (pH 6.8), 2.5% SDS, 2.5% β-mercaptoethanol, and 25% glycerol], heated (95°C for 2 min), resolved by SDS-PAGE, transferred to polyvinylidene difluoride membranes, and probed with the stated primary antibodies.
For immunocytochemistry, HEK-293 cells were fixed 48 h after transfection with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked by 3% BSA and 10% goat serum for 1 h. Specimens were incubated with anti-FLAG mouse monoclonal antibody. Fluorescence images were acquired through a Zeiss ×100 objective lens using a Zeiss LSM-510 laser scanning confocal microscope.
Microsomal vesicles were isolated from HEK-293 cells (two 10-cm dishes) as previously described (32). The cell pellet was resuspended in ice-cold hypotonic lysis buffer [10 mM HEPES (pH 7.2), 1 mM EDTA, 5 μM diisopropyl fluorophosphate, 10 μg/ml pepstatin A, 10 μg/ml aprotinin, and 10 mg/ml benzamidine] and then homogenized by 10 strokes in a tight fitting Dounce followed by 15 strokes after the addition of an equal volume of sucrose buffer (500 mM sucrose and 10 mM HEPES, pH 7.2). The lysate was centrifuged (600 g for 15 min), the secondary supernatant was centrifuged again (100,000 g for 45 min), and the pellet was resuspended in buffer (250 mM sucrose and 10 mM HEPES, pH 7.2) at 2–6 mg/ml protein. Vesicular H+ pumping was assessed by measurements of acridine orange absorbance quenching in a SLM-Amino DW2-C dual wavelength spectrophotometer (change in absorbance at 492–540 nm) (14, 72). Vesicles (300 μg) were diluted into 1.6 ml assay buffer [200 mM NaCl, 30 mM Na-tricine (pH 7.5), 3 mM MgCl2, 0.5 mM EDTA, and 6 μM acridine orange]. Reactions were initiated by the addition of ATP (final concentration: 1.3 mM) and valinomycin (final concentration: 1 μM) and were terminated by the addition of 1.6 μM of H+ ionophore 1799.
Yeast transformation and growth selection.
COOH-terminally HA-tagged expression constructs in p426TEF or p425TEF vectors (35) were transformed into Saccharomyces cerevisiae strain YBR127C (genotype MATa/MATalpha his3Δ1/his3Δ1 leu2Δ0/leu2Δ0 lys2Δ0/+ met15Δ0/+ ura3Δ0/ura3Δ0 ΔVMA2/ΔVMA2, ATCC) and grown at 30°C in YEPD medium (10 g/l yeast extract, 20 g/l peptone, 20 g/l d-glucose, and 100 mg/l adenine hemisulfate) or synthetic minimal uracil and leucine (SD -UL) dropout medium (-uracil and leucine DO supplement, 6.7 g/l nitrogen base, 20 g/l d-glucose, 100 mg/l adenine hemisulfate, and 50 mM MES, pH 5.5). All yeast media contained 200 μg/ml geneticin. Transformation was performed as previously described by Gietz et al. (52). Single colonies were isolated on pH 5.5 SD -UL plates, and the growth of transformants was assessed on YEPD plates buffered to pH 5.5 with 50 mM MES or pH 7.5 with 50 mM MOPS.
For immunoblot analysis, 10 optical density units of yeast were washed once with 1 mM EDTA-H2O and lysed in 2 M NaOH (10 min, 4°C). Proteins were isolated by trichloroacetic acid/acetone precipitation, resuspended in SDS sample buffer [25 mM Tris·Cl (pH 6.8), 9 M urea, 1 mM EDTA, 1% (wt/vol) SDS, 0.7 M β-mercaptoethanol, and 10% (vol/vol) glycerol], separated by SDS-PAGE, and analyzed by immunoblot analysis as described above.
Pedigree, clinical phenotype, and 24-h urine chemistry.
Figure 1 shows the pedigree previously partially described (3, 21). The family members have advanced in age since the original publication (3), but the clinical diagnoses have not changed, with six family members carrying the diagnosis of dRTA. One heterozygote carrier presented with urolithiasis with a heavy stone load, which required surgical intervention (asterisk in Fig. 1). Interestingly, she also had unduly alkaline urine and unexplained hypocitraturia, which cannot be explained by unusual acid intake (urinary SO4 excretion: 31 meq/day) or alkali intake (urinary K+ excretion: 67 meq/day, estimated net gastrointestinal alkali absorption: 27 meq/day) (65). The combination of high urine pH and hypocitraturia suggest defective urinary acidification. This prompted us to screen the rest of the family. Stone risk profiles from 24-h or spot urines are shown in Fig. 1. Although none of the heterozygotes carriers were previously recognized by their physicians as having dRTA, all of them showed some degree of urinary abnormalities, such as hypocitraturia and hypercalciuria, that cannot be attributable to secondary causes. Some of them also have urine pHs that are slightly alkaline, but since these were obtained on outpatient random diets, one cannot definitely conclude on abnormality. We could not identify asymptomatic stones in the rest of the family members because the subjects declined imaging experiments.
H+ gradient and H+ flux measurements in heterozygotes.
Some but not all family members consented to participate in more detailed metabolic experiments. To test whether the collecting duct can generate and sustain a high H+ gradient, we loaded the subjects with ammonium chloride to induce maximal H+ pumping. This maneuver has been shown to maximally lower urine pH in normal individuals (16, 51, 71). The two heterozygotes lowered their urine pH and increased ammonium excretion, but their urine pHs were not as low as the normal sibling (Fig. 2, A and B). The urinary ammonium excretion was numerically lower than but probably not that different from the wild type. The small numbers did not permit meaningful statistical analysis. The response in the homozygous patient was basically flat.
Acidic urine implies adequate pump and epithelial integrity to generate and maintain pH gradients. Since secretion of a small amount of H+ into the urine across a tight epithelium can theoretically maintain a high H+ gradient, this does not guarantee adequate flux of H+ to excrete the acid load. To examine that, we use the urine minus blood Pco2 test to estimate H+ flux (62). Under high luminal HCO3− concentration, the generation of luminal CO2 can only result from titration of luminal HCO3− from high rates of apical H+ secretion. Of the heterozygous subjects tested, none of them mounted a normal Pco2 gradient (Fig. 2C).
The combination of abnormal steady-state baseline 24-h urine parameters and abnormal H+ gradient and H+ flux upon challenge beget the conclusion that these heterozygous individuals do not have normal urinary acidification and actually fit the criteria for incomplete dRTA.
Calcareous kidney stone and B1 expression in situ in a heterozygote.
This investigation was initiated by one family member who presented with calcareous stones of significant load that eventually required surgical intervention. Somewhat unexpected was that the stone composition was not the calcium phosphate typically expected of dRTA patients. Instead, the subject had 80% calcium oxalate and 20% calcium phosphate stones. There was no evidence of nephrocalcinosis by a preoperative noncontrast computerized tomography scan (not shown), intraoperative inspection, and tissue histology. An intraoperative biopsy of the papillary tip in this patient revealed an abnormal cellular pattern of the B1 subunit. While the distribution of B1 in an unrelated normal individual showed mostly apical staining, the affected heterozygote showed a mixture of both apical and rather prominent diffuse intracellular staining (Fig. 2D). Our anti-B1 antibody does not recognize truncated B1; thus the signal likely represents wild-type B1 in V1 domains that did not assemble into V0V1. The rest of the family members had no documented history of stones, but they did not consent to imaging experiments in the absence of a clinical diagnosis.
Examination for negative dominance in mammalian cells.
The metabolic phenotypes indicated that the only truly normal individual in this family was the homozygous wild-type male member. In this kindred, most heterozygotes seemed to exhibit some abnormal (albeit mild) renal acidification. This can be due to either a dominant negative effect of mutant truncated B1 or haploinsufficiency of wild-type B1.
We previously characterized the p.Phe468fsX487 truncation mutation as one that prevents proper assembly of B1 into V1, and the defect is not due to the loss of the PDZ binding motif at the COOH terminus (21). HEK-293 cells express both B1 and B2 subunits (RT-PCR; data not shown), which do not furnish a clean system to study transfected proteins. We knocked down the endogenous B subunits with siRNAs and engineered synonomous nucleotide mutations in transfected human B1 to protect it from the siRNA (Fig. 3A). siRNA reduced endogenous V-ATPase activity to ∼25–30% of normal without affecting the expression of transfected FLAG-tagged B1 (Fig. 3B). This knockdown is necessary to examine the function of heterologously expressed B1 as the endogenous B activity is strong and precludes functional readout of the transfected B1 (Fig. 4A).
The transfected wild-type B1 conferred robust V-ATPase activity, whereas mutant B1 was inactive (Fig. 4A). Cotransfection of mutant B1 appeared to reduce the H+ pump activity of wild-type B1 (Fig. 4A). At face value, this appears to support a dominant negative effect of the mutant over wild-type B1. However, one needs to exercise caution in this finding. The reduction of wild-type activity can be explained by the fact that cotransfection of mutant B1 decreases the expression of wild-type B1 (Fig. 4, B–D). When normalized for the wild-type B1 antigen, the V-ATPase activity of cotransfected cells was actually normal.
To further confirm this point, we kept the amount of wild-type B1 constant and gradually increased the truncated mutant and searched for the presence of interference. When transfected mutant B1 expression was increased, the transfected wild-type B1 expression appeared to be decreased (Fig. 4C). However, a similar effect was seen in the converse experiment, where increasing wild-type B1 expression decreased mutant B1 expression (Fig. 4C). When both B1 subunits are driven to extremely high levels by strong promoters in these transient expression experiments, the two subunits may be competing for the same limited translational machinery. We fathom that this is in fact the more likely explanation than a dominant negative effect of the mutant on translation of wild-type B1.
To further test this hypothesis, we examined whether transfection of exogenous mutant B1 affects the expression of native B1 and its ability to assemble. As shown in Fig. 5, exogenous mutant B1 did not affect the level of native B1 expression, assembly, or function. This speaks strongly against a dominant negative effect of mutant B1 on native wild-type B1.
Examination for evidence of negative dominance in yeast.
To thoroughly refute the dominant negative hypothesis, we used a second system to test for dominance. Yeast provides an ideal system for studying transporters in general because of the ease in obtaining definitive genetic null cells and the ability to use growth as a functional screen; thus, this host has been used extensively for studying V-ATPase subunits (26, 48). Therefore, an independent test of negative dominance by the mutant B1 subunit is H+ pumping function in yeast, which is critical to confer the ability to grow in pH 7.5. As previously shown, human B1 complements the yeast homolog well in a growth assay (21). In null yeast, the presence of mutant B1 did not affect the expression level of B1 or the ability of wild-type B1 to support growth in pH 7.5 (Fig. 6A). This provides additional evidence showing that the truncated mutant protein exerts no significant effects on wild-type B1. We further examined whether there was any evidence of negative dominance in 12 other known human mutations to complete an exhaustive search using the yeast model. None of these mutants affected the ability of wild-type B1 to complement pump function in null yeast, and none of these mutations affected the level of B1 expression (Fig. 6B).
Inactivating mutations of the V1 subunit B1 and the V0 subunit a4 are associated with congenital autosomal recessive dRTA with sensorineural hearing loss (28, 56, 58, 60, 67). The mechanism of dysregulated acidification from B1 mutations was unclear until recently, when disrupted V-ATPase assembly in vitro was described as one mechanism for seven known ATP6V1B1 missense mutations (20, 73). The finding of abnormal acidification in the heterozygous carriers in this kindred was unexpected but unequivocal. The question is why these subjects should be abnormal given that they have one normal copy of B1 and, as far as we know, B1 expression is biallelic.
The current stoichiometric model of V1 (A3B3C1D1E3F1G3H1) confers a 0.875 (1–0.53) probability of incorporating at least one abnormal B1 subunit. A heterogeneous wild-type/mutant B1 can conceivably affect ATP hydrolysis by A3B3 and impair pump function. However, we cannot find evidence showing that the truncated B1 is capable at all of incorporation into V1. However, mutant B1 can theoretically affect the synthetic pathway of wild-type B1 if both alleles are translated together. Based on the observation that overexpressed mutant B1 subunits can lower Na+-independent cell pH recovery after acid load in a rat IMCD cell line, Yang and coworkers (73) proposed a dominant negative mechanism of transfected mutant B1 subunits over native wild-type B1 subunits. However, in the background of native B1, the effect of the transfected protein is not easy to assay and certainly difficult to interpret. We reduced endogenous B1 to levels where we could see the activity of the transfected protein, and the initial results hinted at the possibility of negative dominance (Fig. 4A). However, any reduction in wild-type B1 activity was in fact seen only in the situation where both transfected proteins were driven to very high levels. Mutant B1 did not exert any dominant effect over wild-type B1 expression, assembly, or function (Figs. 5 and 6). To complete our search, we studied 12 naturally occurring human B1 mutations in null yeast and failed to find any dominant negative action for any of them. Although this was a single sample, the heterozygous carrier exhibited an abnormal pattern of staining of B1 in the renal medulla, which we interpret as unassembled mutant B1. The higher than expected intracellular staining likely represents the failure of mutant B1 to incorporate into V1, as we have observed in cell culture (20). At the moment, the data in concert support the de facto conclusion that the clinical burden of heterozygosity in this family is likely due to haploinsufficiency.
We are cognizant of the fact that not all families with dRTA are linked to either ATP6V1B1 or ATP6V0A4 (56), suggesting possible additional loci and mechanisms for congenital dRTA. We cannot rule out the possibility that our clinical and metabolic findings can be related to other putative loci that modify urinary acidification. Nephrolithiasis or nephrocalcinosis are common in subjects with dRTA (4, 8, 9, 66) due to the combination of alkaline urine and hypocitraturia and, to some extent, hypercalciuria (8, 37, 39, 40), although abnormalities in stone inhibitors, such as glycosaminoglycans and nephrocalcin, have also been proposed (36). These urinary abnormalities promote all calcium stones but, in particular, calcium phosphate stones, which is compatible with human population data that show increased prevalence of renal acidification defect in calcium phosphate stone formers (22, 45, 46).
Although there is universal agreement that calcium stones are common in dRTA, the consensus is that dRTA is a rare cause of kidney stones. However, decades of patient data have provided unequivocal evidence for impaired renal acidification in the general stone-forming population without frank Mendelian dRTA. In 7 studies (12, 38, 41, 42, 59, 61, 70) amounting to >1,000 stone formers, abnormalities in urinary acidification have been found in an average of 17% (range: 6–26%) of all-comers. Whereas the first stone suffer may have a <10% chance of abnormal renal acidification (43), in recurrent stone formers, the frequency has been estimated from 7% to 100% in 7 studies totaling >200 patients (1, 2, 10, 34, 44, 54, 63). Abnormalities in acidification are also more common in bilateral stones, ranging in prevalence from 36% to 64% in a total of 228 patients (15, 44).
The clinical data suggest prevalent defective renal acidification even in the general stone-forming population and is staggeringly high in specific subgroups. In a small number of patients with the triple feature of bilateral recurrent calcium phosphate stones, Konnak and coworkers (30) found that all of them had defective renal acidification. Although these individuals may have acquired incomplete dRTA, they can also be heterozygous carriers of candidate genes. However, no genotypic data were presented in any of these studies mentioned above. In fact, based on the supposition, albeit unverified, that heterozygous carriers of the dRTA candidate genes are completely normal individuals, genetic causes for subtle defects in acidification in stone formers were likely never examined. If heterozygous careers of mutant candidate genes of dRTA harbor subclinical defects that elude routine clinical chemistry and confer a propensity for disease rather causing disease, then one will not succeed in recognizing inheritance patterns at all. There is a need for database of sequence variance in candidate genes for dRTA in the general stone-forming population.
The authors are supported by National Institutes of Health (NIH) Grants R01-DK-081523, R01-AI-041612, R01-HL-72034, the O'Brien Kidney Research Center (NIH Grant P30-DK-079328), the Simmons Family Foundation, and the Charles Pak and Donald Seldin Endowment for Metabolic Research. D. G. Fuster was supported by the Swiss National Science Foundation (31003A_135503 and NCCR Kidney.CH) and by a Medical Research Position Award of the Foundation Prof. Dr. Max Cloëtta.
No conflicts of interest, financial or otherwise, are declared by the author(s).
Author contributions: J.Z., D.G.F., M.A.C., X.-S.X., and O.W.M. conception and design of research; J.Z., D.G.F., M.A.C., H.Q., C.G., X.-S.X., and O.W.M. performed experiments; J.Z., D.G.F., M.A.C., H.Q., C.G., X.-S.X., and O.W.M. analyzed data; J.Z., D.G.F., M.A.C., H.Q., X.-S.X., and O.W.M. interpreted results of experiments; J.Z., D.G.F., M.A.C., H.Q., X.-S.X., and O.W.M. prepared figures; J.Z., D.G.F., M.A.C., H.Q., X.-S.X., and O.W.M. drafted manuscript; J.Z., D.G.F., M.A.C., H.Q., X.-S.X., and O.W.M. edited and revised manuscript; J.Z., D.G.F., M.A.C., H.Q., X.-S.X., and O.W.M. approved final version of manuscript.
The authors are grateful to Dr. Margaret Pearle (Department of Urology) for obtaining the kidney biopsy and the Mineral Metabolism Clinical Laboratory staff at the Pak Center at University of Texas Southwestern Medical Center for processing of the patient samples.
- Copyright © 2014 the American Physiological Society
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Inexact title. See the list below. We don't have an article named Main/FeliciaSorceressOfKatara, exactly. We do have:
If you meant one of those, just click and go. If you want to start a Main/FeliciaSorceressOfKatara page, just click the edit button above. Be careful, though, the only things that go in the Main namespace are tropes and should be created through the YKTTW system. Don't put in redirects for shows, books, etc.. Use the right namespace for those.


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Sickening video has emerged of yet another police officer violently slamming a handcuffed suspect face-first into the ground.
The footage shows Colorado Springs officer Tyler Walker ramming 18-year-old Alexis Acker into the concrete floor while the girl was already restrained.
The police reports, claim that Walker "escorted her to the floor," or that he "rolled her to the floor."
Indeed, the officer "escorted" Acker to the floor with such force that her front teeth were smashed in and permanent injuries were caused, including trauma to the face, head, teeth and jaw; migraine headaches, concussion, closed head injuries, memory and cognitive function problems, as well as post traumatic stress disorder.
The incident occurred in late 2013. The video has just been released as part of a $500,000 brutality lawsuit against the CSPD.
Acker had been arrested on charges of resisting arrest and assault on a police officer, with cops claiming that she was "intoxicated and verbally uncooperative with police," during an attempted arrest of her boyfriend.
The cops delivered Acker to the hospital so doctors could grant "medical clearance" for her to be taken to jail, according to the police report. It was in the hospital where the footage was captured.
The officer is seen slamming Acker to the ground after she kicked out at him when he ordered her to sit down.
While it is clear that the very slight teenage girl did not in any way harm Walker, who is 100 pounds heavier than her, he alleged that she "kicked him in the groin area" and that he felt "immediate pain".
Walker wrote in his report that "It was then evident that I had caused her injury and knocked out her front tooth while throwing her on to the hard linoleum hospital floor, while her hands [were] still handcuffed behind her back,"
Furthermore, the officer claimed that while throwing the girl to the floor, he suffered an injury to his left knee. Walker was later advised of his rights as a victim by the police department,
Walker's supervisor, Sgt. Mary Walsh, did not personally witness the incident, yet bizarrely claimed that the officer "rolled her out of the chair to the floor" — his "only means to control her." She also claimed that a hospital employee and security guard verified that "the force was necessary and justified to control Alexis."
Acker was charged with five offenses, which included two felony assaults on a police officer.
Her attorney, Cindy Hyatt, told reporters that "There's no way she kicked him that hard in the balls. He wouldn't be standing. He'd be hunched over."
"This is a very violent attack on someone who is in handcuffs, who is partially restrained and tiny, and there's just no need for it," Hyatt added. "You can't have something like this, whether it happens 100 times, 10 times or one time. It's unacceptable. It cannot be tolerated. As a patrol officer in particular, that's part of the job, dealing with that without planting someone's face in the floor."
CSPD has refused to confirm whether Walker has been disciplined, but he remains on the force.
As InfoWars has noted, there have recently been a number of disturbing cases of exactly this type of force being used by police across the country.
Indeed, there are so many cases of this type of take down occurring, often when the subject is handcuffed, some ending in paralysis or even death. It is clear that cops are being trained to deal with subjects in this manner.
Steve Watson is a London based writer and editor for Alex Jones' Infowars.com, and Prisonplanet.com. He has a Masters Degree in International Relations from the School of Politics at The University of Nottingham, and a Bachelor Of Arts Degree in Literature and Creative Writing from Nottingham Trent University.


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Amazon Forest More Resilient to Climate Change Than Feared
The Amazon rainforest is less vulnerable to die off because of global warming than widely believed because the greenhouse gas carbon dioxide also acts as an airborne fertilizer, a study showed on Wednesday.
The boost in growth due to the CO2 this century is likely to exceed any damaging effects of rising temperatures, it said.
"I am no longer so worried about a catastrophic die-back due to CO2-induced climate change," Professor Peter Cox of the University of Exeter in England told Reuters of the study he led in the journal Nature. "In that sense it's good news."
(READ the story from Reuters)


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Speech by Sir Mark Walport on the future of climate science advice in government.
Climate science review - Sir Mark Walport
View the slides from the presentation.
2 October 2013
Government Office for Science
Professor Sir Mark Walport
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Team Building
In most companies, when you get results, you get rewards. And if you can't lead teams to success, you'll end up stuck in a job with no exit. Use this strategic blending of common-sense strategies and implementable team building exercises to build and bolster your winning team…
You want to improve teamwork. So you reward group performance, praise any signs of collaboration and prod loners to become joiners. That's a good start, but why stop there?
Make sure your team is working more like the Manhattan Project and less like Enron… Use these articles, exercises and strategies to get your team building training up and running!
Consider making a new employee's first day at work a culture-only affair.
Real magic happens for organizations whose interests branch out beyond their physical walls and into the community around them.
The tendency to credit team leaders for a group's success—and blame them for a group's failure—is often misguided.
As you work to build a team, keep these points in mind.
Garry Ridge's goal for the last 20 years has been to transform WD-40 into a global, diversified brand.
One powerful way to extract your team members' best ideas, suggestions and efforts is to conduct a problem-solving meeting based on a definite agenda and guidelines.
Steve Kerr has built the Golden State Warriors into a formidable team of selfless contributors.
It's better to use the limited time you have as a team to examine important issues.
How to get your employees to believe in you, the organization and its mission.
On a mid-October night in an alternate universe, the Cleveland Indians actually defeated the Chicago Cubs in game seven of the World Series to take baseball's 2016 crown, and in doing so, denied millions of diehard Cubs fans from realizing their century-old dream of winning a league title. What could manager Joe Maddon possibly have said to his team to make such a fictional defeat less crushing? Maybe he could have tried something that, with a little modification, might work for any leader trying to motivate a staff in their worst moment.


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"Life is not the way it's supposed to be. It's the way it is. The way you cope with it is what makes the difference."
I hope you will spend whatever time you can with your gran in her last days. Not eating or drinking is often the first indication that the body is shutting down and death is imminent. Her urine output will likely decrease and become concentrated as her kidneys shut down. You can wet her mouth with ice chips or offer her water through a straw. You can gently apply lotion to her face and limbs. That will feel good to her. I did this with my favorite aunt in her final days last Dec. She was 82, but we were very close and it was hard to watch her die. However, she knew I was there and appreciated it.
My thoughts are with you at this difficult time.
saw gran yesterday. she has this toothless smile which is just priceless. i have taken in some old photos and put them upon the wall where she can see them.
doctors say her kidneys are still functioning ok, but she does not want to eat. she has told the nurses that she wants to die. they think she is dying. i have told her i loved her and if she decides not to eat then i respect her wishes. doctor says she might not last longer than a few weeks, but it is in gods hands. i am just trying to spend as much time with her as possible. she has barely eaten in 3 weeks. if it is her time i hope she goes soon. she does not want tv, radio she just sits in her room staring at the wall sleeping alot.
my mum has been out a week, she is weak, tired all the time and very frustrated at not being ableto use herhand properly cause of the stroke. she had been sick yesterday and her nose was bleeding. as far as i know she has not been able to get out for a drink. her partner is watching her like a hawk, he even has the neighbours watchin that she does not go out for a drink.
Allie, I am glad you are going to the funeral parlor today to discuss your gran's funeral in advance.
As to your mum, the alcohol could be making her that sick. If her body just can't take any more of it, it will try to get rid of it by vomiting. This may be a blessing in disguise if the drinking makes her sick to her stomach...though I have to say it never stopped my mother, who would drink, go to the bathroom and be sick, then trot back to her room and chug the bottle again. This would go on all night.
Hang in there!
My wee gran passed away on Saturday. she went over 6 weeks with no eating, refusing medication and taking only sips at atime. i am gutted that she is away, but i know she is at peace it has been a long wait. i was with her at the time, which i am gladcause she was scared.
my mum is still in hospital due to get out last week, until she started vomiting, they were very worriedabout her as her neurilogical signs were very low, they say she might have blood posioning. is this what happens when the liver is in a bad way, i do not know what her future is to be.
i have decided tostill go away on holiday its on Thursday, the funeral will be after i come back, hopefully my mum willbe strong enough.
thanks for the support


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SmartEtailing's POS Sync can push your inventory to customers shopping on the Giant consumer site and we provide this service for free. With a compatible POS system, you can now offer your in-stock bikes through Giant WebLink; they handle the transaction and then send that customer into the store for product pickup. How do you participate?
You can opt-in to this program with one of the following methods:
You have a SmartEtailing website with POS Sync enabled.
If you are an authorized Giant retailer using SmartEtailing POS Sync, you're able to fully participate in this program by changing one setting in the backend of your site that will share your inventory with Giant. We will do this for you if you give us a call or fill out the form on this page.
You have a compatible POS System and sign up for a Free Buy Local Now account. Give us a call or fill out this form and we'll get you set up.
If you are already signed up for Giant's Where to Buy program there is one additional setting to configure, if you fill out this form or call us we will do it for you.
Giant uses the inventory data from your POS System to refer shoppers on Giant's website to your local store and sell product that you have in stock. This is a very important and FREE tool for Giant retailers. Sign-up today to get connected!


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Stock Graphic >> Illustration - Office Workspace 153883 by karnoff @ CreativeMarket
File Size: 5.26 MB; Layered: Yes; Vector: Yes.
Flat modern design vector illustration concept of creative office workspace, workplace. Business work flow items and elements, office things, equipment, objects. Perfect office desk.
All illustrations are easy to use and highly customizable, to fit your needs.
Features: ? 4 modern long shadow flat design illustrations for SEO services, Marketing, Business and Web design, Web development, Cloud Computing ? Available in: EPS, AI and high resolution 5000X7000px JPG format. ? Easy to edit & customize ? 100% resizable
Keywords: flat, desk, design, camera, vector, icon, coffee, business, concept, designer, marketing, technology, development, computer, management, devices, tools, workplace, workspace, notebook.


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My Nonprofit Reviews
159 profile views
1 reviews
Review for Keiki Cupboard, Haiku, HI, USA
Every child should feel worthy, and one cannot when they don't have the hygeine, clothes or school supplies to spend a proper day at school. Keiki cupboard fills greatly needed support for the child to succeed.
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I've personally experienced the results of this organization in...
Seeing school children with smiles on their face and a look of confidence.
If I had to make changes to this organization, I would...
Help it grow!
Role: Volunteer & I've been on non profits on the mainland that support well being.


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Crusher Run In Albany Ny
Recent News,Mellott Company,Mobile Crushing Service
designed the ® HP5 cone crusher with safety, simplicity and ..... When you run a rock crusher repair shop like we do, you see a lot of consequences ... showcase on February 25th 2013 in Albany, NY with Callanan Industries.Read More
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Granite Crushing Plant. After first crushing, the material will be fed into cone crusher by belt conveyor for secondary crushing;Read More
Full Public Notice,New York District,U.S. Army
Feb 25, 2015 ... City of Albany, Albany County, New York. A detailed .... Plaza, 99 Washington Avenue, Albany, New York 12231, Telephone (518) 474-6000. Comments ... City of Albany, Albany County NY .... GRADATION, CRUSHER RUN).Read More
Home [www.colonieblock.com]
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Velocipede,Wikipedia, the free encyclopedia
... the bearings to help them run smoothly. Like the High Wheel bicycles that became popular later in the 19th century, boneshakers were front-wheel drive, but in ...Read More
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HV-Rob,Hudson Valley Ruins and related topics by. Welcome to the (Post)-Halloween Blog Part 5, the final installment I have planned for this years series.Read More
Stone dust vs sand for laying pavers? Help!,GardenWeb
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Contact Info. Address: No.416 Jianye Road, South Jinqiao Area, Pudong, Shanghai, China Tel: 0086-21-58386256 Fax:0086-21-58385887 [email protected]Read More
Crusher Run Prices Albany Ny
Granite Crushing Plant. After first crushing, the material will be fed into cone crusher by belt conveyor for secondary crushing;Read More
Warren W. Fane
Warren W. Fane, Inc. sells directly to both residential ... we also have various approved New York State materials. Look to us for all ... Quarry Stone Gravel Stone Item 4 Septic Gravel Road Salt & Sand/Salt Mix Screened Topsoil Crusher Run Concrete Sand Mason ...Read More
Crusher Run in Albany, New York with Reviews & Ratings,YP.com
Find 6 listings related to Crusher Run in Albany on YP.com. See reviews, photos, directions, phone numbers and more for the best Crushed Stone in Albany, NY.Read More
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-There have been fiber optic lines run in certain areas throughout the town already so mapping will need ... April 1, 2014-March 31, 2015 from Town & County Bridge and Rail, Albany, NY, and. THEREFORE, be it ... Crusher Run. Troy Sand & ...Read More
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Covey Run Winery · CR Cellars ..... The Chook · The Crusher ... 1440 Central Ave :: Albany, NY 12205 :: 877-77-WINE-1 (877-779-4631) :: (518) 694-8503Read More
New York State Assembly,Peter D. Lopez
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Find 37 listings related to Crush And Run Gravel in Albany on YP.com. See reviews, photos, directions, phone numbers and more for the best Crushed Stone in Albany, NY.Read More
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This report is a bonus feature from the Nov. 12 issue of Nation's Restaurant News, typically available to subscribers only. Subscribe today.
For fine dining, the Great Recession underscored the fact that necessity is the mother of invention — or reinvention, as the case may be.
Confronted with pervasive economic hardship and increasingly cost-conscious consumers,
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Vegan Gingerbread Waffles (A Cheater Recipe!)
So, on a Sunday morning, I saw the Trader Joe's gingerbread box mix on the shelf, I thought, "Hey! Why not! Let's try making it into waffles!" Voila! Worked out perfectly! The mix makes a very gingery, moist and yummy breakfast delight. Just use the appropriate vegan substitutions (which is the equivalent of an egg replacement) and you've got yourself an easy, festive treat for holiday mornings! For serving, I sprinkled mine with organic powdered sugar (available at Whole Foods).
Late Fall
Where does the time go? I have all these recipes that I started to post, but never finished. Since I hate loose ends, I had it in my mind that I couldn't post anything until I posted those recipes. You know what, I probably will never complete them, so let's move on! I've also been super busy with my shop, New Sweden Vintage, and honestly, don't have as much time to spend here. I do, however, have a few ideas for crafts and some cookie recipes for the holidays, so I'll make sure not to ignore Scissors and Spice. I promise!
Here are a handful of random shots from the last few weeks. In between packing orders and 3rd-grade homework, there's been lots of walks in the woods, baking (yay! cold weather!), coffee dates, and trips to New York.
Pumpkin cinnamon rolls from Trader Joe's.
Baked cauliflower curries, pre-baking.
Coffee dates at Philter in Kennet Square, PA. Seriously, the best mocha lattes this side of the Mississippi!
One of my favorite parts of going to New York City is taking the kids to the playgrounds. This one is right on the Hudson in Battery Park City.
My kids exploring the Irish Hunger Memorial.
I don't know why, but I was seriously geeking out over the colors of fall this year (I probably have about a thousand photos of foliage alone). We've been spending lots of time out in nature as a family, exploring the woods, and going for long walks along the Brandywine River.
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Matthew Gilchrist
I've a distinctive English accent and I work in American retail and at least 3 - 4 times a day for the last 13 years I have people telling me how they love the sound of my voice and frequently get asked to continue to speak so they can listen to my accent. So if you have a product that you need selling in the U.S. and want your audience to at least listen to your advert then you should consider letting me sell it for you.
English (British), English (North American)
British, England - Yorkshire & Humber, North American
Child, Middle Aged, Senior, Teen, Young Adult
Special Skills
Audiobooks, Business, Documentaries, Educational, Internet, Jingles, Movie Trailers, Music, Podcasting, Radio, Telephone, Television, Videogames
some public speaking
Payment Terms
Hire Matthew as a voice actor. Payment is made through Voices.com SurePay, an escrow service that guarantees your work is done to your satisfaction. Send payment securely by Visa, Mastercard or PayPal through Voices.com then download your files from the website.
Watch a video to see how to make a payment
Next Steps
To contact Matthew Gilchrist about your project, request a quote today. We'll walk you through the process step-by-step. It's fast and easy.
This talent has not uploaded any demos yet.
Hire Matthew Gilchrist as a voice actor. Working with Matthew Gilchrist is fast and easy. We'll walk you through the process step-by-step.
Request a quote for your project today.