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Bill tackles 'modern-day slavery'
Texas is one of the country's hubs for human trafficking, and proposed state legislation would increase penalties for the crime and make it easier to investigate and prosecute.
Published 8:32 pm, Sunday, November 21, 2010
Texas is one of the country's hubs for human trafficking, and proposed state legislation would increase penalties for the crime and make it easier to investigate and prosecute.
A bill filed by state Sen. Leticia Van de Putte, D-San Antonio, would make a 2009 law against human trafficking more like the state's sex crime laws. The proposed law would require convicted traffickers to serve at least half their sentence before being eligible for parole, and it would increase the minimum for repeat offenders to 25 years in prison. It also gives police new tools for investigating human trafficking.
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One of the most important new tools, Van de Putte said, is a change in the law that would allow law enforcement to detain child victims, allowing police to seek help for the victims and continue their investigation.
"It's modern-day slavery," Van de Putte said. "And the big emphasis is right now if you rescue a teenager and they're a runaway, you can't just send them back" to the home they ran away from.
According to a report Attorney General Greg Abbott submitted to the Legislature in 2009, almost 20 percent of the 800,000 trafficking victims in the U.S. travel through Texas, usually along the Interstate 10 corridor.
The state's biggest human trafficking centers are Houston and El Paso, according to the report.
Human trafficking is not human smuggling. Trafficking refers to someone being coerced into performing labor, sexual or otherwise. Trafficking victims tend to be immigrants who were lured into the country under false promises of employment, said Chris Burchell, president of the advocacy group Texas Anti-Trafficking in Persons.
U.S. citizens or residents, often children who have been introduced to drugs, also can end up as trafficking victims, Burchell said.
In Bexar County, all human trafficking cases taken on by the District Attorney's Office have involved victims who were coerced into prostitution, First Assistant District Attorney Cliff Herberg said. Almost all the victims referred to the District Attorney's Office have been U.S. citizens or residents, he said.


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Barbaric turf war escalates in Afghanistan as ISIS execute three 'defectors' days after the Taliban warned terror group to stay out of the country
- Video shows two prisoners being shot in back of the head by ISIS fanatic
- Other pictures appear to show alleged defector being beheaded with knife
- Comes after Taliban said there was only room for 'one flag, one leadership'
- Rival Islamist groups declared war on each other in Afghanistan in April
Islamic State militants have executed three fighters accused of defecting to the Taliban as a brutal turf war escalates between the rival factions in Afghanistan.
The killings come days after the Taliban sent a letter to ISIS warning them to stay out of the country, saying there is room for only 'one flag, one leadership' in their fight to re-establish strict Islamist rule.
One video posted on pro-ISIS accounts shows a line of armed fighters standing behind two kneeling men who are shot dead by one of the militants with a handgun.
In a separate execution, pictures appeared to show another alleged defector being beheaded.
The groups declared war on one another in April after the Afghan Taliban branded ISIS' self-declared caliphate illegitimate and refused to declare allegiance to its leader Abu Bakr al-Baghdadi.
Scroll down to video
Pictures released online appear to show an Islamic State fighter being beheaded after being accused of defecting to the Taliban in Afghanistan where a bloody turf war has broken out between the two groups
Barbaric: An Islamic State fighter yanks the prisoner's head back as he prepares to execute him with a knife
ISIS responded by launching recruitment drives deep into Taliban territory, allowing them to expand rapidly - even reportedly replacing the Taliban as the dominant controlling force in one district.
The warning letter from the Taliban came amid heavy fighting this week in eastern Afghanistan between the Taliban and breakaway factions who now swear allegiance to Islamic State, which in the past year seized large swaths of Iraq and Syria.
The entry of Islamic State, while its numbers remain small, has complicated Afghanistan's already escalating war following the withdrawal of most foreign troops at the end of last year.
The letter addressed to Islamic State leader Abu Bakr al-Baghdadi said the Taliban 'based on religious brotherhood asks for your goodwill and doesn't want to see interference in its affairs'.
The Taliban have fought to topple Afghanistan's Western-backed government since the U.S.-sponsored military intervention that toppled their own five-year rule in 2001.
Merciless: In a separate video, two other alleged defectors are shot dead by a militant with a handgun
In the past year, however, small groups of disgruntled Taliban commanders have switched allegiance to Islamic State's al-Baghdadi, who aspires to establish a global caliphate with himself at the helm.
Some defectors expressed frustration that the Taliban have not gained significant territory and questioned whether supreme leader Mullah Mohammad Omar is still alive.
The rival jihadists have fought each other in a few provinces, with some of the heaviest clashes coming this week in the eastern province of Nangarhar.
Provincial spokesman Ahmad Zia Abdulzai said that 1,000 families had fled villages to escape the fighting after two local Taliban commanders, called Bakhtyar and Abdul Khaliq, changed sides to join Islamic State and launched attacks against their former leaders.
Afghan and U.S. officials say that so far there appears to be little material support flowing from the Islamic State leadership into Afghanistan, but the rise of its ideology is worrying in a region where radical new ideas can catch on among the region's vast population of young, unemployed men.
Brutal: The killings come days after the Taliban sent a letter to ISIS warning them to stay out of the country, saying there is room for only 'one flag, one leadership' in their fight to re-establish strict Islamist rule
The letter to al-Baghdadi, signed by Taliban political committee chief Mullah Akhtar Mansoor, shows the insurgents also are worried.
'Jihad against American invaders and their slaves in Afghanistan must be under one flag, one leadership and one command,' it said.
The letter also appeared intended to dissuade other Taliban fighters considering switching sides.
Besides Arabic, it was written in Dari, Pashto and Urdu languages that are spoken in Afghanistan and Pakistan and was distributed by an official Taliban spokesman.
Worries about the rise of Islamic State also may be contributing to the Taliban's new receptiveness to a parallel effort to start peace talks with the Afghan government.
Taliban representatives and an Afghan delegation were both attending a peace conference in Oslo on Tuesday, raising the possibility of face-to-face meetings.
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Versatile actor, Tannikella Bharani is one man who can do it all. After playing a plethora of roles, he is going to be seen in a quirky avatar in the remake of the super hit Hindi film Vicky Donor.
Bharani will be seen in Annu Kapoor's role and says that the film has ample entertainment and can be watched by every section of the audience. Adding further, Bharani also said that the way the makers have dealt with the film is so simple and will connect to everyone.
Based on the sensitive issue of sperm donation, this film will showcase Sumanth as the sperm donor.
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Title: Ben Maddow & John Huston's screenplay for 'The Asphalt Jungle ' Author: La Familia Film, Name: asphalt_jungle__the. compressed.
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As one of the first heist films that put the culprits in the center of the audience's attention, John Huston's The Asphalt Jungle served as a source of creative Mangler: docs.


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SPA newswire
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Goldman Sachs (New York), Chicago Mercantile Exchange (Chicago), AG Edwards (St. Louis) - The explosive popularity of commodities indexing continues to leave huge footprints on the floors of the US commodities exchanges. Dow Jones Newswire reported that "live cattle futures traders have spent this week preparing for the movement of at least 45,000 long August positions into October during the week of July 10" as the Goldman Sachs Commodities Index rolls into its next session.
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Costa Rica moves to free-floating currency
Costa Rica announced that it would remove the country's nine-year-old currency band system in favor of a floating exchange against the U.S. dollar, according to a statement from the Central Bank (BCCR) Saturday.
The BCCR had not taken any measures to adjust the value of the colón, Costa Rica's currency, outside the band since Dec. 12, 2013, arguing that the currency had been in a "de facto" float since then.
On Oct. 17, 2006, the BCCR announced that it would implement the currency band, which established a floor and ceiling price between which the colón could float before the Central Bank would take steps to adjust its value. The band system was implemented as a way to gradually move the country from a fixed exchange to a floating one.
The colón has depreciated roughly 8 percent against the U.S. dollar since February 2014, selling at 543 on Saturday, according to Monex. The BCCR said that Costa Rica had more than $7.3 billion in reserves.
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The United Nations have succumbed to pressure from Saudi Arabia by removing the country from a UN blacklist for violating children's rights.
The UN had put the Saudi regime on the list just one week ago, saying the coalition it is leading in Yemen was responsible for 60 percent of child deaths and injuries in 2015, killing 510 and wounding 667.
Secretary General Ban Ki-moon's decision to reverse the decision has resulted in condemnation from various rights groups who say the UN are guilty of shameful flip-flopping.
Thinkprogress.org reports:
The decision to remove Saudi Arabia from the blacklist has been met with criticism from many human rights organizations.
"Political power and diplomatic clout have been allowed to trump the UN's duty to expose those responsible for the killing and maiming of more than 1,000 of Yemen's children," Sajjad Mohamed Sajid, Oxfam's country director in Yemen, told the Guardian. "The killing of children in their homes, at schools and in hospitals should not be swept under the carpet. When the UN identifies crimes such as these it needs to act, regardless of who the perpetrators are."
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The Saudi-led coalition first began airstrikes on Yemen in March 2015. The campaign was directed primarily against the insurgent Houthis, who marched on Sana'a and drove out the government of Yemeni leader Abd Rabbuh Mansur Hadi. The conflict has led to a humanitarian crisis in the country that has severely deteriorated in the last year and change.
"Yemen after five months looks like Syria after five years," Peter Maurer of the ICRC told AP. Of 26 million Yemenis, at least 21.2 million are in need of humanitarian aid.
While the U.N. has changed course, Saudi Arabia's aggression has bit back at them and placed them on the receiving end of increasing international pressure elsewhere.
In March of this year, the Netherlands' parliament became the first European country to vote to halt selling weapons to Saudi Arabia. Despite the friendly relations between Saudi Arabia and the United States, the Senate has also recently upped pressure on Saudi after it was revealed that they were using American cluster bombs to kill and maim civilians in Yemen.
There's currently a bi-partisan effort in the Senate, led by Sen. Chris Murphy (D-CT) and Sen. Rand Paul (R-KY), that would set conditions for selling Saudi Arabia air-to-ground weapons. U.S. criticism of Saudi Arabia could also intensify upon the release of the 28 pages, a classified section of the 2003 investigation into the 9/11 attacks.
Sean Adl-Tabatabai
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Charlottetown, PE Canada, Adult Contemporary. 64kbps Music Mix, Adult Contemporary, Hits
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Edmonton, AB Canada, College. The Northern Alberta Institute Of Technology, College Alternative Radio
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- Reference: 54494
- Job Type: Permanent
- Location: Dublin South
- Category: Accountancy & Finance
Fantastic opportunity to join leading Global Pharmacuetical company based in South Dublin. Ideal candidate will have 10 years plus in a similar environment and looking for a new challenge.
Our client serves the Irish market with a diverse range of health care products including diagnostics, medical devices, nutritionals and branded generic pharmaceuticals.
They are in the process of setting up a Finance SSC in Ireland to maximize the scope of services. You will be working as part of a dynamic and diverse organisation alongside outstanding people who are forward thinking, collaborative and recognized leaders in their areas.
As a member of the shared services leadership team, you will direct and lead the establishment and growth of the regional shared service Purchase to Pay Director (PTP) operations. Direct the day to day PTP shared service operations for the region across captive and outsourced teams, ensuring effective and efficient service delivery with accurate and timely performance measurement and compliance support.
Essential Functions
1.Migrate processes and services to the regional shared service organization, including:
Plan migration of activities to a regional captive shared service center, satellite center(s) or outsourced partner locations supporting the region
Recruit and select the Source to Pay team for the region
Establish and sustain the agreed performance measures, KPIs, and goal setting processes
As operations mature, migrate additional activities to the outsource partner, while continuing to transition more complex activities into the regional captive shared service organization.
2. Direct the day to day shared service PTP operations for the region across captive and outsourced teams, ensuring effective and efficient delivery of services and accurate and timely month end close and corporate reporting.
3. Partner with shared services leadership to sustain service operations and establish robust stakeholder management communications and feedback mechanisms to assure effective collaboration..
4. Promote understanding of approved process standards.
5. Ensure integrity of internal controls, in partnership with the region Compliance and Control Manager
6. Management of the PTP team to ensure resource and competency needs are aligned with current and future shared service organizational requirements. Annual reviews and competency based development plans.
Position Accountabilities / Scope
The PTP Regional Director will direct the day to day shared service PTP operations across both captive and outsourced teams, with direct management of the captive PTP team. This individual will be responsible for building the PTP organization, including recruitment and selection of the PTP captive team, supporting design of the PTP processes with the GPM, and partnering with IT in the execution of applicable PTP process harmonization. In the longer term, this individual will serve on the Customer and Process Councils to build relationships and ensure finance operations are aligned with the business objectives.
Job Qualifications
BS degree in Business, or Accounting or equivalent.
Minimum Experience
10 years in STP arena including end to end Source to Pay processes (Sourcing and Procurement Buy Support, Accounts Payable, T&E, Vendor Enablement)
Experience of project management and program implementation skills
Multiple ERP landscape expertise required
Experience managing 3rd party service providers/ outsourcers.
Preferred Experience
Experience with E-Invoice and on-line Procurement solutions (e.g. Arriba) a plus
Lean 6 sigma qualification
Language Skills: English required, other languages desirable
For further information on this role, please contact Ita Hodder on 087 6755011 or email email@example.com
For more information on our Recruitment and HR Services, and to see a full list of our available jobs across Ireland. please visit our website at www.collinsmcnicholas.ie
For more information on Collins McNicholas, contact one of our five branches across Ireland in Dublin, Cork, Galway, Sligo, Athlone


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This is a list of abbreviations for different word lists that are or may soon be used ... Internet Dictionary Project using a Basic English Word List put in using how to ...
This is a list of business and finance abbreviations. Contents: Top; 0–9; A; B; C; D ; E; F; G; H; I; J; K; L; M; N; O; P; Q; R; S; T; U; V; W; X; Y; Z ...
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An abbreviation (from Latin brevi³re → brevis meaning "short") is a shortened form of a word or phrase used mainly in writing to represent the complete form.
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Toyota Tacoma Custom
Toyota Tacoma Custom - You're looking for articles on our blog under the title Toyota Tacoma Custom of all collections of articles that we had. If you are looking Toyota Tacoma Custom article does not reside here, you can see from some of the articles that may match your search. We will help you to find articles Toyota Tacoma Custom you're looking at some of our articles that may be suitable as your reading material.
Toyota Engine Firm is usually a Japanese car supplier headquartered in Toyota, Aichi, Japan. In March 2014, the worldwide company was made up of 338,875 staff members globally and, by October 2016, was the ninth-greatest organization on the globe by earnings. As of 2016, Toyota could be the worlds secondly-most significant vehicle manufacturer powering German Volkswagen Team. Toyota was the worlds 1st motor vehicle maker to deliver much more than 10 million automobiles each year which it includes carried out due to the fact 2012, whenever it also noted the production of its 200-millionth car or truck. As of July 2014, Toyota was the biggest detailed firm in China by industry capitalization (really worth more than double the amount as #2-graded SoftBank) and by revenue.
Toyota may be the worlds industry leader in profits of hybrid electrically powered motor vehicles, and one of many greatest corporations to encourage the volume-sector adoption of crossbreed motor vehicles around the world. Cumulative world wide sales of Toyota and Lexus crossbreed person car or truck designs reached the 10 million milestone in Jan 2017. Its Prius family members are the worlds top rated trying to sell crossbreed nameplate with more than 6 thousand devices offered for sale throughout the world since Jan 2017.
The business was built by Kiichiro Toyoda in 1937, as a spinoff from his fathers provider Toyota Market sectors to make automobiles. Three years earlier, in 1934, while still a office of Toyota Industrial sectors, it built its first product, what type A engine, and, in 1936, its primary person automobile, the Toyota AA. Toyota Motor Business provides cars beneath five manufacturers, for example the Toyota brand name, Hino, Lexus, Ranz, and Daihatsu. Additionally, it keeps a 16.66% risk in Subaru Corporation, a 5.9% stake in Isuzu, and joints-undertakings with two in Chinese suppliers (GAC Toyota and Sichuan FAW Toyota Electric motor), one in India (Toyota Kirloskar), one out of the Czech Republic (TPCA), in addition to many "nonautomotive" companies. TMC is part of the Toyota Party, one of the major conglomerates worldwide.


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N.Y. firefighters' killer left note
Published 11:11 pm, Tuesday, December 25, 2012
Webster, N.Y. --
The felon who lured two firefighters to their deaths in a blaze of gunfire left a rambling typewritten note saying he wanted to burn down the neighborhood and "do what I like doing best, killing people," police said Tuesday as they recovered burned human remains believed to be the gunman's missing sister.
William Spengler, 62, who served 17 years in prison for manslaughter in the 1980 hammer slaying of his grandmother, set his house afire before dawn Christmas Eve and took a revolver, a shotgun and a semiautomatic rifle to a sniper position outside, Police Chief Gerald Pickering said.
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- 2 dead, 2 injured in SJ fireKTVU2
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Authorities say he sprayed bullets at the first responders, killing two firefighters and injuring two others who remained hospitalized Tuesday in stable condition. He then killed himself as seven houses burned. Police revealed that a body believed to be the killer's 67-year-old sister, Cheryl Spengler, was found in his fire-ravaged home.
Officers recovered a military-style .223-caliber semiautomatic Bushmaster rifle with flash suppression, the same make and caliber weapon used in the elementary school massacre in Newtown, Conn., that killed 26, including 20 young children, Pickering said.
'Equipped to go to war'
The chief said it was believed that the firefighters were hit with shots from the rifle given the distance, but the investigation was incomplete.
"He was equipped to go to war - kill innocent people," the chief said.
The rambling, two- to three-page typewritten note that Spengler left did not reveal a motive for the slayings, Pickering said. He called the attack a "clear ambush on first responders."
He declined to reveal the note's full content or where it was found. He read only one chilling line: "I still have to get ready to see how much of the neighborhood I can burn down, and do what I like doing best, killing people."
Pickering said it was unclear whether the person believed to be Spengler's sister died before or during the fire.
A next-door neighbor said Spengler hated his sister and they lived on opposite sides of the house.
Roger Vercruysse said Spengler loved his mother, Arline, who died in October after living with her son and daughter in the house in a neighborhood of seasonal and year-round homes across the road from a lakeshore popular with recreational boaters.
As Pickering described it and as emergency radio communications on the scene showed, the heavily armed Spengler took a position behind a small hill by the house as four firefighters arrived after 5:30 a.m. to extinguish the fire: two on a fire truck; two in their own vehicles.
They were immediately met with bullets from Spengler, who wore dark clothing. Volunteer firefighter and police Lt. Michael Chiapperini, 43, driving the truck, was killed by gunfire as the windshield before him was shattered. Also killed was Tomasz Kaczowka, 19, who worked as a 911 dispatcher.
Trying to shield crew
Several firefighters went beneath the truck to shield themselves as an off-duty police officer passing by pulled his vehicle alongside the truck to try to shield them, authorities said.
Spengler had been charged with murder in his grandmother's death but pleaded guilty to a reduced charge of manslaughter, apparently to spare his family a trial. After he was freed from prison, Spengler - a felon who wasn't allowed to possess weapons - had lived a quiet life.
That ended when he left his burning home Monday morning, armed with his three weapons and a lot of ammunition.
"I'm not sure we'll ever know what was going through his mind," Pickering said.


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Before you start a home business, please be aware that home businesses are jam-packed with scams.
So how do you steer clear of falling for the pledge made by those so-called genuine work at home opportunities?
Look for any out of sight fees, because the genuine work at home opportunities must have no fees implicated. They should not be asking you to give them any payment for the job you are going to do for them.
Here are a few samples of work at home opportunities that can be taken advantage of. But keep in mind, if something sounds too good to be true, it most likely is.
Professional spy.
Professional spy or private investigation. A lot of people can not relax until they know for sure whether or not their spouse would cheat on them. For that reason there is quite a reasonable market for that kind of business out there, but if you take advantage of one of those opportunities, your job must consists of testing potentially unfaithful partner by getting someone to try and chat them up, and some people regard this as unfair, but each person is entitled to there own view.
This type of work can be mentally hard to handle because if you succeed, the individual who hired you is generally extremely unhappy to have their suspicions realized. And if you fail, the person who hired you will in all probability blame you for not doing a very good job. So whatever happens, you are bound to get snarled up in the emotional consequences between the two.
Cleaning is straightforward money, and there is truly a market for it. A lot of people are working all hours to pay for the mortgage of their home, merely to find that their house could do with a good clean, but do not have the time. This is were you can suggest your cleaning services, and take up this type of opportunities, but only advertise for professionals who can not find the time to do their own cleaning.
Assembly Work.
Assembly work is really one of the easiest work at home opportunities out there. This type of work can be done in your own time. This type of work at home opportunity only requires the lowest amount specialist skills.
There are usually training materials and information packets incorporated with this type of work. But bear in mind that this types of work is very much prone to scams, so you need to be extremely careful here. Some companies in fact ask that you send in a certain sum of money first before you can take delivery of the informational material from them. In addition, there is a likely possibility that they will reject your finished products by saying that they do not come "up to standard. " You will then unfortunately have to sell the merchandise yourself if you want to get a little back for your effort.
Tailoring services.
If you are experienced with a needle, countless people find that they buy the perfect outfit only to find out that after they put on or lose weight, it does not fit them any longer. You can help these people with your skills and make a tidy profit from it.
How to find work at home ideas.
The best way to find ideas for starting your own work at home business is to first do a search, type in your preferred work, but you must do an advanced search, or you will just get millions of irrelevant results, you will also need to visit a forum relating to what you are interested in doing, and see what others are saying about it. You can also find lots of home business ideas at http://www.lionel1.com.
Working from home and making a living at it, for some people is just the answer to their dreams, but those dreams could turn into a living nightmare if the proper research is not done.
Lionel Estridge is the publisher of Zero to Hero newsletter. Get tips and trick at: http://www.lionel1.com


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Planning for New Kitchen Remodel Ideas : Cheap L Shaped Kitchen Remodel Design
Uploaded by FATAHILLAH HUTAGALUNG in Chula Vista at Wednesday, November 6, 2013. The Surprising Kitchen Ideas images on this page are the Surprising image that we grabbed for you with a dimension of 900 x 575 pixels. Cheap L Shaped Kitchen Remodel Design above are the same as what I saw when I visited my boss's private residence in Albuquerque, USA. Don't wait; you can also read the full writing in the Planning for New Kitchen Remodel Ideas which contains image that I took during a trip to various states such as Iceland, Croatia, and Peru.
Feel free to use the cheap kitchen remodel image that I served even though you are in tropical or desert states like Nepal, Georgia, and Eritrea. Download by right clicking your mouse right on the Surprising image, then give this high resolution image to your home adviser to be implemented soon.
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Confused by the Cultural Olympiad? Want to take an art break between sporting events? Guardian arts correspondent Mark Brown answers key questions about the cultural strand of the Olympics
Each member of the cast was to find the next. That's where the problems began for Hackney Empire show by Rimini Protokoll
The artist responsible for the Obama 'Hope' posters has made a 10-storey artwork broadcasting the power of free speech over London during 2012, writes Lee Bofkin
Asif Kapadia, director of Senna, tells Sarfraz Manzoor how he spent four days in a chopper making a 'democratic' film about London and the Olympics
Dudamel and Simón Bolívar Symphony Orchestra - live Southbank Centre's Sounds Venezuela festival - in pictures
Gustavo Dudamel and his Simon Bolivar Symphony Orchestra rehearse for their 23 June concert, and the Southbank centre's Festival of the World gets underway.


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Personal Statement
Lybrate.com has a number of highly qualified Ayurvedas in India. You will find Ayurvedas with more than 28 years of experience on Lybrate.com. You can find Ayurvedas online in Bangalore and from across India. View the profile of medical specialists and their reviews from other patients to make an informed decision.
Book Clinic Appointment
D G Hospital274/275, M.K. Puttalingiah Rd, Padmanabha Nagar, Gururaja Layout, Banashankari, Landmark: Opp to Syndicate bank, Bangalore Get Directions
Gastrointestinal Disorders
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Treatment Of Male Sexual Problems
Diet Planning
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Treatment of Migraine Treatment
Treatment Of Female Sexual Problems
Piles Treatment (Non Surgical)
Sexually Transmitted Disease (Std) Treatment
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Chronic Skin Allergy Treatment
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Treatment Of Pregnancy Problems
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Siddha Treatment
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I am 35 yrs old woman I have small patch in the hair in that area no hair is there at all pls suggest me some medicines.
The womb or the uterus is a muscular structure that is fixed in its place by the ligaments and the pelvic muscles. If these ligaments or muscles become weak or stretch, they no longer will be capable of holding the uterus, resulting in a prolapse. Uterine prolapse happens when the uterine slips or sags from its usual position into the birth canal or vagina. The main indications of uterine prolapse are recurrent bladder infections, constipation, a tug at the pelvic area, the cervix or uterus protruding out of the vagina, problems during sexual intercourse, increased discharge and vaginal bleeding.
What causes it?
1. Age is the most important reason for this condition to manifest itself.
2. A dip in the estrogen level as this hormone keeps the pelvic muscles strong.
3. Damage to the pelvic tissues and muscles due to pregnancy or during childbirth can also contribute to this condition.
4. A woman who has undergone multiple vaginal births is at an increased risk of this condition.
5. Any physical activity that exerts pressure on the pelvic muscles can also result in this disorder.
6. Chronic constipation and obesity, over time, can lead to this disorder.
How it can be treated?
1. Nonsurgical methods:
- Shedding the extra kilos helps reduce stress from the pelvic structures. This helps to avert this disorder.
- Avoid lifting heavy objects throughout the course of the treatment.
- Pelvic floor exercises or Kegel exercises help build up the vaginal muscles.
- Estrogen replacement therapy or Hormone Replacement Therapy can help relieve the symptoms of this disease.
- Wearing a pessary (an instrument that is placed into the vagina and fits under the cervix) helps to push up and stabilize the cervix and the uterus.
2. Surgical treatments:
- Uterine suspension- In this case, the surgeon inserts the uterus back into its former position by reattaching the pelvic ligaments by the use of surgical techniques.
- Hysterectomy- Here, the surgeon removes either the whole or just a part of the womb.
However, such procedures should not be opted by women who plan on getting pregnant. If you wish to discuss about any specific problem, you can consult a Gynaecologist.
Sir I have a diabetes from last 6 months what's can I do for clear the diabetes. How can I control please sugest me.
Hello I have a question to ask. Is there any medicines to improve my body color. Sunlight make my face and forearms are looking black.
Hii I have a high blood pressure which type of dite should take wat type of oil. My weight is 93 is this good weight or bad pls give me a good suggestion.
My mother is 50 years old and suffering from diabetes from last 13 years, currently get sugar result is 315 PP and 391 and treatment is going through Air Force hospital since beginning they changed her medicine but you need diet plan a per her condition.
I have a sexual problem. I have a lot of vaginal pain during sexual intercourse. And till few hours my vagina hurts alot and sometimes also burns. But then it gets normal in a day. Please help. Thank you.
What are the causes of high level microsomal antibody and thyroglobulin antibody 230 IU/ML and 345 U/ML respectively. Also tell how to recover this?
Liver Tonic:
Drink one glass of bitter melon juice daily to heal liver problems. Keep consuming this continuously for a week to see results
Immune System:
Boil bitter melon leaves or fruits in water and consume it every day to fight against infections. This also helps to build your immunity
Bitter melon juice benefits include helping to overcome type 2 diabetes. It has been a part of the Chinese and Indian ancient medicine for a long time but only recent research has proven that it is no folklore. Type 2 diabetes is caused partially due to the inability of a cell to absorb the sugar in the blood due to insufficient insulin or due to the development of resistance to insulin. In both cases, the cells are unable to absorb the sugar due to the ineffectiveness of the insulin produced. The absorption of sugar occurs due to the activation of AMP-activated protein kinase in the cells. Bitter gourd activates these kinases due to which the absorption of sugar increases and hence, aids in bringing diabetes under control. Green juice for diabetics: cucumbers, green apples, bitter melon, celery, half green capsicum (bell pepper). Bitter melon contains certain chemicals that are like insulin which help to reduce the blood sugar levels.
Kidney And Bladder:
Bitter melon helps to maintain a healthy liver and bladder. It is also useful in curing kidney stones
Bitter melon can prevent cancer cells from multiplying .
Weight Loss:
Bitter melon contains antioxidants that help to flush out your system. This improves your metabolism and digestive systems, thus helping you lose weight quickly. The two other major factors that aid in weight loss are the calorie control and filling components. Calorie control happens as such because vegetables are low in calories that allow you to consume more quantities of them.


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Upcoming Webinars
PS Best Practices: Efficient Administration For Your PSA Software
Time: 1:00 – 1:30pm EST
You have invested in a PSA tool - now what? One of the essential keys to getting the most out of your investment is the efficient administration of your PSA solution. In this webinar we will discuss:
- What are typical responsibilities of a PSA administrator?
- Who owns the PSA tool?
- What skills should your PSA administrator have?
Following the webinar, we will keep the lines open for 15 minutes for open discussion and Q&A.
NetSuite OpenAir Administration Best Practices
Time: 1:00 – 1:30pm EST
The end of the year is upon us and this is a good time to review NetSuite OpenAir administration best practices. In this webinar you will learn about:
- End of year considerations
- Annual, Quarterly, Monthly checklists
- Tips to support your end users
Following the webinar, we will keep the lines open for 15 minutes for open discussion and Q&A.


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November 22, 2017 - Latest: Pensions – perhaps Nanny really does know best by Steve Herbert
2nd December 2011
However the stakes remain high. Yahoo Voices carries the opinion of a senior Belgium politician.
Former Prime Minister and leader of the Alliance of Liberals and Democrats in the European Parliament, Guy Verhofstadt says: "International companies have started preparations for the breakup of the Eurozone. The EU is fast approaching the breaking point of the euro and it is not because of Greece or Italy, but because of the incapacity of the two main players today in European politics – the incapacity of Merkel and Sarkozy to deal with this crisis."
The Guardian suggests that Merkel is still reluctant to bail out the eurozone periphery. She told the German Parliament: "The ECB's role is different from that of the Federal Reserve and Bank of England. Its role is stabilise the value of the currency." However in the same speech, Merkel did suggest that the ECB may need to buy up the bonds of periphery countries in the "short term".


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See Details on eBay
Seller: kimmndavid (1,002) 99.4%,
Location: York, Pennsylvania,
Ships to: Worldwide,
Item: 152619029670
You get all 4 packs of gum that are pictured with this auction.
Condition: New
See More
High amount of bids. 0 views, 0 views per day, 138 days on eBay. 1 Sold, 0 Available.
Over 1,002 items sold. 0.6% negative feedback. Great seller with very good positive feedback and over 50 ratings.


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What exactly all the different emojis actually mean. The emoji chart!
Disney Lilo and Stitch favourite movie Ohana means family, family means nobody gets left behind or forgotten
I am intentional with my language whilst impacting the thoughts, emotions & words of the people in my life as a great leader to greater leaders.


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Denmark attacks: Copenhagen comes together for candlelit vigil
Tens of thousands of people have gathered in towns and cities across Denmark to commemorate the victims of weekend gun attacks in the capital.
The Prime Minister, Helle Thorning-Schmidt, attended the main rally in Copenhagen, which began with a minute's silence.
The suspected gunman, named by local media as Omar El-Hussein, was shot dead by police on Sunday.
Lucy Manning reports.
16 Feb 2015
- From the section Europe


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Arrestin-1 preferentially binds active phosphorylated rhodopsin. Previously, a mutant with enhanced binding to unphosphorylated active rhodopsin (Rh*) was shown to partially compensate for lack of rhodopsin phosphorylation in vivo. Here we showed that reengineering of the receptor binding surface of arrestin-1 further improves the binding to Rh* while preserving protein stability. In mammals, arrestin-1 readily self-associates at physiological concentrations. The biological role of this phenomenon can only be elucidated by replacing wild type arrestin-1 in living animals with a non-oligomerizing mutant retaining all other functions. We demonstrate that constitutively monomeric forms of arrestin-1 are sufficiently stable for in vivo expression. We also tested the idea that individual functions of arrestin-1 can be independently manipulated to generate mutants with the desired combinations of functional characteristics. Here we showed that this approach is feasible; stable forms of arrestin-1 with high Rh* binding can be generated with or without the ability to self-associate. These novel molecular tools open the possibility of testing of the biological role of arrestin-1 self-association and pave the way to elucidation of full potential of compensational approach to gene therapy of gain-of-function receptor mutations.
Rod photoreceptors mediate night vision in vertebrates. Rods demonstrate remarkable single photon sensitivity (1) and unusual reproducibility of a single photon response (2–4). These features require biochemically perfect signaling cascade with virtually no noise, enormous amplification, and extremely rapid shutoff. Light activates rhodopsin, which then sequentially catalyzes GDP/GTP exchange on numerous molecules of its cognate G protein transducin (5). Within 30–60 ms, active rhodopsin (Rh*)2 is phosphorylated by GRK1, whereupon visual arrestin-13 specifically binds light-activated phosphorylated rhodopsin (P-Rh*), ensuring timely signaling shutoff (6, 7).
Genetic defects in this rapid and efficient two-step shutoff mechanism invariably result in visual disorders. Loss-of-function mutations in arrestin-1 (8) or GRK1 (9) cause Oguchi disease, a form of stationary night blindness. Mutations in rhodopsin that lead to the loss of GRK1 phosphorylation sites result in retinal degeneration (10). Although in principle loss-of-function mutations can be cured by gene replacement therapy, in the case of gain-of-function defects, such as the loss of phosphorylation sites resulting in unquenchable signaling, viable therapeutic strategies remain to be developed.
We have recently proposed a compensational approach using enhanced arrestin-1 mutant that binds Rh* with much higher affinity than wild type (WT) arrestin-1 to reduce excessive signaling by unphosphorylated rhodopsin (11, 12). Although, the first proof-of-principle study showed that compensational gene therapy works in vivo, it also revealed the limitations of existing phosphorylation-independent arrestin-1 mutants (11). Enhanced arrestin-1 improved functional performance of phosphorylation-deficient rods, facilitated their recovery, and prolonged their survival, but "compensated" shutoff was still significantly slower than in WT animals with normal Rh* phosphorylation (11). Thus, improved enhanced arrestin-1 mutants with higher affinity binding to Rh* and sufficient stability for in vivo expression have to be designed.
Mammalian arrestin-1 at physiological concentrations found in rods (13–15) robustly self-associates, forming dimers and tetramers (16–18). Only monomeric arrestin-1 binds rhodopsin (18), because receptor binding surfaces in the tetramer and both possible dimers are shielded by sister subunits (19). Arrestin-1 is the only signaling protein in the visual system that forms inactive "storage" oligomers, and the biological significance of this phenomenon is unclear (20). To elucidate the role of arrestin-1 self-association in photoreceptors, the consequences of the replacement of WT arrestin-1 with a constitutively monomeric mutant in vivo must be determined. In addition, it is unknown whether oligomerization needs to be preserved in a therapeutically effective enhanced mutant with high Rh* affinity. These in vivo experiments require a stable constitutively monomeric version of arrestin-1 as well as enhanced mutants both with normal and absent ability to oligomerize.
Here we report extensive redesign of the rhodopsin binding surface of arrestin-1 that yielded mutants with significantly higher affinity for unphosphorylated Rh*. We identified further enhanced arrestins, constitutively monomeric forms, and a mutant that combines inability to self-associate with high Rh* binding, all of which are stable. These proteins are promising candidates for in vivo evaluation of the potential of compensational gene therapy and for elucidation of the role of arrestin-1 oligomerization.
[γ-32P]ATP, [14C]leucine, and [3H]leucine were from PerkinElmer Life Sciences. All restriction and DNA modifying enzymes were from New England Biolabs (Ipswich, MA). Rabbit reticulocyte lysate was from Ambion (Austin, TX), and SP6 RNA polymerase was prepared as described (21). Cell culture reagents and media were from Mediatech (Manassas, VA) or Invitrogen. All other reagents were from Sigma.
For in vitro transcription mouse arrestin-1 (generous gift of Dr. Cheryl Craft, University of California) was subcloned into pGEM2 (Promega, Madison, WI) with "idealized" 5-UTR (21) between NcoI and HindIII sites as described (70). All mutations were introduced in transcription construct by PCR using the strategy previously described (23). All constructs were confirmed by dideoxy sequencing. For the expression in HEK293 cells, coding sequences with 5′-UTR were excised from pGEM2 constructs using EcoRI and HindIII restriction sites and subcloned into pcDNA3 vector (Invitrogen) with a modified multiple cloning site as described previously (24, 25). In vitro transcription, translation, and preparation of phosphorylated and unphosphorylated rhodopsin were performed as described recently (26, 27).
A direct binding assay was performed as described (26). Briefly, 1 nm arrestin-1 (50 fmol) was incubated with 0.3 μg of P-Rh* in 50 μl of 50 mm Tris-HCL, pH 7.4, 100 mm potassium acetate, 1 mm EDTA, 1 mm DTT for 5 min at 37 °C under room light. Samples were cooled on ice, and bound and free arrestin-1 was separated at 4 °C by gel filtration on a 2-ml column of Sepharose 2B-CL. Arrestin-1 eluting with rhodopsin-containing membranes was quantified by liquid scintillation counting. Nonspecific "binding" was determined in samples where rhodopsin was omitted and subtracted.
Translated radiolabeled arrestin-1 was incubated for 0.5–4 h at the indicated temperatures and cooled on ice. The binding of arrestin-1 in these samples to P-Rh* was compared with that of control sample kept on ice as described above except that 2 nm arrestin-1 (100 fmol/sample) was used.
HEK293 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% serum as described (28). Cells were transfected with 3 μg of DNA per well of a 6-well plate using Lipofectamine according to manufacturer's instructions. After 36 h cells were serum-starved overnight and then kept at 37 °C (control) or at 40 °C for 3 h. Cells were washed with PBS and non-enzymatically detached from plates with 2 × 500 μl of Versene. Cells were pelleted by centrifugation at 4 °C for 10 min at 300 rpm (Eppendorf centrifuge 5417R) and resuspended in 400 μl of 50 mm Tris-HCl, pH 7.4, 100 mm NaCl, 1 mm EDTA, 1 mm DTT supplemented with Complete protease inhibitors mixture (Roche Applied Science) and 1 mm PMSF. Cells were lysed by sonication (Sonic Dismembrator Model 500, Fisher Scientific) 3 × 5 s at 30% power. Unbroken cells and debris were pelleted by centrifugation at 4 °C for 10 min at 14,000 rpm (Eppendorf centrifuge 5417R), and the supernatant was frozen in aliquots and stored at −80 °C until used. Total protein was measured using Bradford reagent (Bio-Rad), and the expression of different forms of arrestin-1 was determined by Western blot with anti-arrestin rabbit polyclonal antibody (14). To determine functionality of soluble arrestins, equal volumes of lysates (10 μl) were incubated with or without (nonspecific) 1 μg of P-Rh* in 50 μl of 50 mm Tris-HCL, pH 7.4, 100 mm potassium acetate, 1 mm EDTA, 1 mm DTT for 5 min at 37 °C in room light. The samples were cooled on ice, loaded onto 0.1 ml of 0.2 m sucrose in binding buffer, and centrifuged 20 min at 100,000 rpm in Beckman TL-120 tabletop ultracentrifuge (rotor TLA120.1). Supernatants were removed, and pellets were dissolved in 50 μl of SDS sample buffer. One-tenth of each sample along with one-tenth of the input were subjected to SDS-PAGE and analyzed by Western blot with anti-arrestin antibody (14) followed by HRP-conjugated anti-rabbit secondary antibody, with detection by SuperSignal Pico (Pierce). Nonspecific binding (the amount of arrestin-1 pelleted in the absence of rhodopsin, likely due to aggregation, was subtracted from binding to P-Rh*. Protein survival was calculated as the ratio of the fraction of arrestin-1 in lysate of cells incubated at 40 °C that specifically bound to P-Rh* to the bound fraction in lysates of control cells kept at 37 °C.
Mouse arrestin-1 mutants were expressed in Escherichia coli and purified essentially as described (29). All light-scattering measurements were made with a DAWN EOS detector coupled to an Optilab T-rEX refractometer (Wyatt Technologies) after gel filtration on a QC-PAK GFC 300 column (7.8 mm inner diameter × 15 cm) (Tosoh Bioscience). The arrestin samples (100 μl) at different concentrations were incubated in fresh 5 mm DTT for 30 min at room temperature to disrupt covalent inter-arrestin disulfide bonds and injected onto the column at 25 °C at a flow rate of 0.6 ml/min in 50 mm MOPS, 100 mm NaCl, pH 7.2. The column did not resolve oligomeric species but simply acted as a filter to remove highly scattering particulates. Light scattering at 7 angles (72–126°), absorbance at 280 nm, and refractive index (at 658 nm) for each sample were taken for a narrow slice centered at the peak of the elution profile (18). The experimental weight-averaged molecular weight values were obtained from the protein concentration and light scattering data using ASTRA 126.96.36.199 software (Wyatt Technologies). The weight-averaged molecular weight data were analyzed using the two-step monomer-dimer-tetramer (MDT) model,
where M, D, and T are monomer, dimer, and tetramer, respectively. The data for arrestin-1 carrying F86A/F198A mutations did not fit the MDT model and was fitted using the monomer-dimer model instead. Details of the analysis have been previously described (30, 70). Except where noted, the equilibrium constants are given in terms of the corresponding dissociation constants, KD,dimer (KD,dim) and KD,tetramer (KD,tet). The errors in equilibrium constants were determined from least-squares fitting of the data to MDT or monomer-dimer model taking into account an estimated error of ± 1 kDa in the computed values of the average molecular weight (70).
The first enhanced arrestin-1 mutant used in vivo to compensate for the defect in rhodopsin phosphorylation was preactivated simply by the elimination of one of the conformational constraints that holds arrestin-1 in its basal state (11). Triple alanine substitution of the bulky hydrophobic residues anchoring the C-tail (yielding arrestin-1-3A mutant) promotes its detachment, thereby facilitating arrestin-1 transition into a high affinity rhodopsin binding state and arrestin-1 binding to Rh* (31). The replacement of WT arrestin-1 in GRK1-deficient mice with the arrestin-1-3A mutant resulted in an ~3-fold acceleration of Rh* shutoff but did not bring the recovery rate to the WT level (11). Single cell recording from rods expressing the arrestin-1-3A mutant suggested that it allowed Rh* reactivation (11). Thus, arrestin-1-3A complexes with Rh* were not stable enough to survive longer than it takes Rh* to decay to opsin.
Multiple positive charges in bovine arrestin-1, including Lys-14, Lys-15 (32), Arg-18 (33), Lys-163, Lys-166, Lys-167, Arg-171, and Arg-175 (34) were shown to bind rhodopsin-attached phosphates. Mouse arrestin-1 also carries all of these positive charges: Lys-15, Lys-16, Arg-19, Lys-164, Lys-167, Lys-168, Arg-172, and Arg-176. Therefore, we hypothesized that a mismatch between the presence of these positive charges on the receptor binding surface of arrestin-1 and the absence of negatively charged phosphates on the C terminus of Rh* destabilized the complex. To test this idea, we replaced phosphate binding lysines and arginines (Fig. 1A) with neutral hydrophilic glutamine with hydrogen-bonding capability, introducing K15Q, K16Q, R19Q, R172Q, K164Q, K167Q, and K168Q mutations. Although R176E mutation per se in mouse arrestin-1 significantly increases the binding to Rh*, it was not introduced because previously we found that it greatly reduces protein stability (11). Neutralizing mutations were introduced, singly or in groups, in the context of the two arrestin-1 mutants that were successfully expressed in transgenic mice, arrestin-1-3A and arrestin-1-(1–377) (11, 13, 14, 35). As expected, on both backgrounds, mutations eliminating phosphate binding charges significantly reduced arrestin-1 ability to interact with dark P-Rh (Fig. 1). Combinations K15Q/K16Q and K15Q/K16Q/R19Q also reduced the binding to P-Rh*, but only R172Q mutation marginally enhanced Rh* binding (Fig. 1, B, C, and D). These data suggest that the presence of phosphate binding-positive charges is not the main factor destabilizing the complex of enhanced arrestin-1 with Rh*.
Several mutations on the receptor binding surface of the C-domain, such as K257E and E346K, were previously shown to increase bovine arrestin-1 binding to Rh* in the context of WT protein (36). Therefore, we introduced homologous mutations K258E and E347K in mouse arrestin-1 (Fig. 2A). These substitutions proved to act additively with the 3A mutation and the deletion of the C-tail, enhancing arrestin-1 binding to P-Rh* (Fig. 2A), and especially to Rh* well beyond the level achieved by the 3A mutation or C-terminal deletion alone. Because the effects of all mutations tested were essentially the same on the background of arrestin-1-3A and arrestin-1-(1–377) (Figs. 1, B, C, and D, and and2A),2A), we focused on arrestin-1-3A, which showed higher expression levels in transgenic mice (14). On arrestin-1-3A background the addition of R172Q mutation to K258E, E347K, or K258E/E347K combination further increased Rh* binding without appreciably affecting the interaction with P-Rh* (Fig. 2B). The best combination (3A+K258E/E347K/R172Q) yielded the level of Rh* binding virtually indistinguishable from that of WT arrestin-1 to P-Rh* (Fig. 2B).
Because many activating mutations reduce arrestin-1 stability (11, 31) and only sufficiently stable proteins can be successfully expressed in vivo at physiologically relevant levels (11), next we tested the stability of these mutants (Fig. 2C). To this end, radiolabeled WT mouse arrestin-1 and mutants produced in cell-free translation were incubated at elevated temperature for varying periods of time, and then their ability to specifically bind P-Rh* was compared with that of the control samples kept on ice. WT arrestin-1 as well as the 3A and truncated (1–377) mutants did not lose any activity after 60 min of incubation at 39 °C (Fig. 2C). Mutations E347K, K258E, and their combination somewhat reduced arrestin-1 stability on both backgrounds, and the addition of R172Q to any mutation or combination further reduced arrestin-1 stability (Fig. 2C). Thus, the most potent ligands of Rh* in this series are less stable than the 3A mutant successfully used in vivo (11).
Surface residues in soluble proteins often stabilize the structure via intramolecular interactions with neighboring side chains. Therefore, we analyzed the available crystal structure of highly homologous bovine arrestin-1 (37) and then ascertained that the residues of interest are conserved in the mouse protein (Fig. 3A shows mouse residue numbers). R172Q reduced the stability of all arrestin-1 mutants tested (Fig. 2). Arg-172 appears to form a salt bridge with Glu-149, which in turn contacts Lys-151. The R172Q mutation is likely to weaken or destroy the interaction of this residue with Glu-149, which would in this case become fully engaged by Lys-151. We reasoned that replacing Lys-151 with aspartic acid would likely prevent that, making the interaction via H-bond between Glu-149 and Gln-172 more likely. Importantly, the homologous K150E mutation in bovine arrestin-1 did not significantly affect the binding to P-Rh* or other forms of rhodopsin (36). Another reason the R172Q mutation could reduce the stability is inappropriate H-bonding of Gln with some of the neighboring residues. We reasoned that replacing Arg-172 with an alanine should solve this problem. The K258E and E347K mutations also appear to be detrimental for arrestin-1 stability (Fig. 2). Judging by bovine arrestin-1 structure, Lys-258 forms a salt bridge with Asp-139 (Fig. 3A), which would be destroyed by K258E mutation introducing a negative charge that repulses Asp-139. The replacement of Lys-258 with a neutral glutamine would prevent the repulsion and likely create an opportunity for H-bonding to preserve the stabilizing effect of the salt bridge in the WT protein. Glu-347 forms a salt bridge with Lys-333 and H-bond with Thr-335 (Fig. 3A). Lys-347, introduced by E347K mutation, would repulse Lys-333; in addition, its longer side chain allows the end amino group to reach all the way to Ser-345. The modeling shows that replacement of Glu-347 with glutamine, which has exactly the same geometry and no charge, would prevent both repulsion of Lys-333 and unnatural H-bonding to Ser-345. The replacement of Glu-347 with histidine, which has only a partial positive charge at neutral pH and shorter side chain, would moderate the repulsion of Lys-333 and also prevent H-bonding with Ser-345.
To test these predictions, on the arrestin-1-3A background we added K151D mutation to the R172Q/E347K and R172Q/K258E combinations, made the R172A/E347K combination to compare with R172Q/E347K, and generated three "milder" versions of the K258E/E347K combination: K258E/E347Q, K258E/E347H, and K258Q (Fig. 3B). Because two additional mutations, R291E and Q328K, increased the binding of bovine arrestin-1 to Rh* (36), we constructed homologous mutants of mouse arrestin-1: R292E, its less drastic version R292Q, and H329K (note that in WT mouse protein bovine Gln-328 is replaced with His-329). The data show that the H329K and R292E mutations increase Rh* binding, whereas the R292Q does not; the R172A mutation is less effective than the R172Q; the K151D mutation decreases Rh* binding of any combination it is added to; the combinations K258Q/E347K and K258E/E347H work as well as the original K258E/E347K (Fig. 3B). Based on these data, we generated several combinations of the most effective mutations and constructed the K258Q/E347H, K258Q/E347H/R292E, K258Q/E347H /H329K, and K258Q/E347H/R292E/H329K combination mutants on the arrestin-1-3A background (Fig. 3C). We found that R292E does not act additively with the K258Q/E347H combination, whereas in the context of the K258Q/E347H/H329K mutant it actually reduces Rh* binding (Fig. 3C). Interestingly, the H329K mutation significantly increased the binding of the K258Q/E347H mutant to both P-Rh* and Rh*. Moreover, the absolute level of the K258Q/E347H/H329K mutant binding to Rh* is even higher than achieved with P-Rh* by WT arrestin-1, making it by far the most potent Rh* ligand constructed (Fig. 3C).
Next, we tested the stability of the combination mutants yielding high Rh* binding, extending the incubation time to 2 h to make the test more rigorous (Fig. 3D). The results showed that the R172Q mutation reduces the stability of the K258E/E347H combination to the same extent as that of K258E/E347K (compare Figs. 2C and and33D). We also confirmed that the K258E/E347K combination is significantly less stable than parental arrestin-1-3A and found that both the K258Q/E347H and K258Q/E347H/H329K mutants essentially retain the stability of arrestin-1-3A. Considering that the arrestin-1-3A was successfully expressed in transgenic mice at fairly high levels (11), these data suggest that both the K258Q/E347H and K258Q/E347H/H329K mutants might be stable enough for in vivo expression.
To closer mimic the intracellular environment that these proteins will be facing in rod photoreceptors, we expressed several arrestins in HEK293 cells. We chose the K258Q/E347H+3A mutant, the K258Q/E347H/H329K+3A mutant, with stable WT arrestin-1 and the arrestin-1-3A mutant as positive controls as well as the much less stable K258E/E347H/R172Q+3A mutant for comparison. Three hours before lysis one plate with cells expressing each mutant was kept at 37 °C (control), whereas a second plate was incubated at 40 °C. The cells were lysed and centrifuged to remove debris and aggregated proteins. To measure possible loss due to aggregation during the 3-h incubation at 40 °C, 2 μg of total protein from each cell lysate was analyzed by Western blot with rabbit polyclonal anti-arrestin antibodies (14). We found a slight reduction of soluble arrestin-1 level in the lysates of cells incubated at 40 °C, which in no case exceeded that observed for WT protein (Fig. 4A). Next, we tested the ability of these proteins to bind P-Rh* in our standard extensively characterized direct binding assay (26, 27, 31–36, 38–43) using lysates of cells expressing various mutants (Fig. 4B). Arrestin-containing lysates were incubated for 5 min at 37 °C with or without 1 μg of P-Rh in room light, and rhodopsin-containing membranes were pelleted by centrifugation. We compared the total amount of arrestin-1 added (input (I)) with that bound to P-Rh* (Rh) or non-specifically aggregated (ns) in samples containing no rhodopsin. In all cases at least half of the arrestin added bound P-Rh*, and very little was pelleted in the absence of rhodopsin (Fig. 4B). All mutants performed similarly to WT, with the exception of the K258E/E347H/R172Q+3A mutant, which demonstrated increased nonspecific aggregation and lower specific binding, in agreement with its reduced stability in the in vitro survival test (Fig. 3D). The ratio of specifically bound fraction of each arrestin from cells incubated at 40 and 37 °C was used to estimate protein survival (Fig. 4B). This parameter was found to be ~100% for WT arrestin-1 and 3A mutant, >80% for the K258Q/E347H+3A mutant, and lower for the other two mutants (Fig. 4B). These results match in vitro survival test (Fig. 3D) remarkably well. Both tests suggest that the K258Q/E347H+3A mutant, with significantly higher Rh* binding than 3A (Fig. 3C), demonstrates stability comparable to the 3A mutant (Figs. 3D and and44B).
Arrestin-1 that retains normal ability to bind P-Rh* but does not self-associate is necessary to test the biological role of its oligomerization in photoreceptors. Therefore, we performed both cell-based and in vitro stability tests with the two previously designed constitutively monomeric mouse arrestin-1 mutants, F86A/F198A and F86A/F198A/A349V (19, 70). We found that these mutants express at least as well as WT arrestin-1 in HEK293 cells, and both survive 3 h of incubation of cells at 40 °C at least as well as WT (Fig. 5A). The fraction of the two mutants in cell lysates that specifically bound P-Rh* was similar to that of WT arrestin-1 (Fig. 5B). Both constitutively monomeric mutants showed essentially the same ~100% survival, estimated as the ratio of specifically bound fraction of WT and mutant arrestin-1 from cells incubated at 40 and 37 °C (Fig. 5B). In full agreement with these data, the in vitro testing at 39 °C of both constitutive monomers demonstrated even higher stability than 3A mutant (Fig. 5C). Thus, in contrast to activating mutations, substitutions disabling self-association of arrestin-1 do not appear to adversely affect protein stability (Figs. 33–5).
Mutations preventing oligomerization of arrestin-1 introduced on WT background were shown not to affect the binding to rhodopsin and microtubules (70). On the basis of the multifunctionality of arrestin proteins (44–46) and demonstrated feasibility of changing individual functions without affecting others (28, 47–49), it was suggested that combinatorial mutagenesis can generate arrestins with essentially any desired set of functional characteristics (12, 46). To test this idea, we combined the double mutation F86A/F198A that renders arrestin-1 constitutively monomeric with several mutations enhancing binding to Rh*, including 3A, K258Q/E347H+3A and K258Q/E347H/H329K+3A, and compared these proteins with parental mutants where only a single function was changed (Fig. 6). We found that although the addition of F86A/F198A to enhancing mutations does not appreciably affect P-Rh* interaction, the binding to Rh* of constitutively monomeric-enhanced arrestins was somewhat reduced compared with the parental forms where the same activating mutations were introduced on WT background (Fig. 6A). However, arrestin-1 carrying combinations of K258Q/E347H+3A and K258Q/E347H/H329K+3A with F86A/F198A largely retained its high binding to unphosphorylated Rh* (Fig. 6A). Finally, to determine the suitability of these mutants for in vivo testing, we performed harsher in vitro stability test, measuring the survival of these proteins at 42 °C (Fig. 6B). At this temperature even WT arrestin-1 shows some inactivation, whereas the arrestin-1-3A mutant, which was successfully expressed in rods of transgenic mice, lost ~50% of activity by 4 h (Fig. 6B). These conditions also revealed the difference between the two constitutive monomers: F86A/F198A is almost as stable as WT, whereas F86A/F198A/A349V demonstrated ~50% survival, similar to 3A (Fig. 6B). The F86A/F198A double mutation somewhat reduced the stability of the less robust K258Q/E347H/H329K+3A, and it did not significantly affect the survival of K258Q/E347H+3A (Fig. 6B). This test revealed that the stability of K258Q/E347H+3A on both WT and constitutively monomeric background is comparable to that of 3A mutant. Thus, both forms of enhanced K258Q/E347H+3A as well as constitutively monomeric mutants are suitable candidates for in vivo expression.
Because the effect of the F86A/F198A double mutation on arrestin-1 self-association was only tested on the WT background (70), we determined the ability to oligomerize of the enhanced K258Q/E347H+3A mutant and the combination of K258Q/E347H+3A with F86A/F198A. To this end both proteins were expressed in E. coli and purified, and their self-association was quantitatively tested by multiangle light scattering (18, 19, 70). We found that the K258Q/E347H+3A mutant retained the ability to form dimers and tetramers (Fig. 7). However, its self-association was reduced relative to WT mouse arrestin-1 (70). Although the data were readily fitted to the MDT model, log Kdim and log Ktet were 3.35 ± 0.05 and 3.85 ± 0.28, which translates into KD,dim and KD,tet of 447 ± 7 and 141 ± 10 μm, respectively. Similar to what we previously found for mouse arrestin-1-F86A/F198A, the data for the K258Q/E347H/H329K+3A + F86A/F198A combination mutant (Fig. 7) could not be fitted by the MDT model but were fitted by a simpler monomer-dimer model, with log Kdim = 2.43 + 0.16, which corresponds to KD,dim = 3,715 ± 245 μm. Thus, on both WT (70) and the K258Q/E347H+3A background (Fig. 7), the F86A/F198A double mutation reduced dimerization about 10-fold and totally eliminated tetramerization.
The data (Figs. 6 and and7)7) show that self-association and binding to unphosphorylated Rh* are not completely independent of each other, which could be expected as the mutations necessary to change both functions localize to the concave sides of the two arrestin-1 domains (Fig. 7A). Nonetheless, targeted reengineering of mouse arrestin-1 yielded new forms where two of its functional characteristics, the level of Rh* binding, and the ability to self-associate, were differentially affected, generating expected combinations.
Rhodopsin is a prototypical class A G protein-coupled receptor (GPCR). GPCRs are the largest and arguably the most structurally and functionally diverse family of signaling proteins in mammals (50, 51), with ~800 subtypes in humans and other primates and much larger numbers in most mammals (SEVENS database). Mutations in different GPCRs underlie a variety of genetic disorders (52). In particular, about a dozen gain-of-function mutations resulting in excessive receptor signaling underlie disorders ranging from retinal degeneration (10) to dwarfism, hypo- and hyper-thyroidism, and several forms of cancer (52). In some cases, constitutive activity of the receptor results in its excessive phosphorylation and association with arrestin. Constitutive desensitization of human vasopressin receptor mutant underlies nephrogenic diabetes insipidus (53), whereas in the case of rhodopsin it results in night blindness or retinal degeneration (54).
Despite technical challenges, the treatment strategy for loss-of-function mutations is conceptually straightforward; gene replacement therapy introducing a functional version of the affected protein is the most logical approach, as was recently demonstrated in the case of Leber congenital amaurosis associated with deficit of RPE65 (55–58). However, gain-of-function mutations pose a conceptual problem; these mutations are dominant, which means that the second perfectly normal allele does not help. Theoretically, mutant mRNA can be targeted by appropriately designed ribozyme that spares normal mRNA. The ribozyme must be extremely efficient, as even low expression of non-phosphorylatable rhodopsin induces retinal degeneration (59). In addition, it has to be very selective, because mutant and normal mRNA sometimes differ by as little as a single nucleotide, as in case of frameshift mutations (10). The combination of these characteristics may not be practical.
The only alternative proposed so far is compensation: balancing a receptor that signals too much with hyperactive arrestin or a receptor that cannot be phosphorylated with a phosphorylation-independent arrestin mutant (for review, see Refs. 12 and 46). However logical this sounds, a recent proof-of-principle study testing this approach in the visual system in vivo demonstrated that existing enhanced arrestins are not effective enough to achieve full compensation (11). Here we describe extensive targeted redesign of the rhodopsin-binding surface of arrestin-1 to generate mutant forms with much higher affinity for unphosphorylated Rh* that have a potential to achieve more effective phosphorylation-independent shutoff. We found that the combination of a triple alanine substitution that forcibly detached the arrestin C-tail from the body of the molecule and facilitated arrestin transition into the active high affinity receptor binding state (31, 60–62) with a re-engineered rhodopsin binding surface yields mutants that bind Rh* better than WT arrestin-1 binds its natural target, P-Rh* (Fig. 2). However, many activating mutations reduce thermal stability of the protein (Figs. 2, ,3,3, ,4,4, and and6).6). We found that less drastic changes are not as detrimental for protein stability (Figs. 2 and and3).3). In particular, replacement of charge reversal with charge neutralization, such as changing K258E to K258Q or E347K to E347H, preserves most of the gain in Rh* binding while improving protein survival at elevated temperatures (Figs. 3 and and4).4). Similarly, the H329K mutation, which moderately increases the positive charge in this position, does not adversely affect arrestin-1 stability (Fig. 3). Moreover, the K258Q/E347H+3A mutant shows equally impressive stability in vitro and in cells subjected to elevated temperatures (Figs. 3, ,4,4, and and6),6), which makes it a good candidate for in vivo testing and a promising tool for gene therapy of genetic disorders associated with defects in rhodopsin phosphorylation. It should be noted that we only tested the binding of these mutants to unphosphorylated light-activated WT rhodopsin. Additional tests with actual disease-causing rhodopsin mutants, particularly the truncated form lacking the C terminus (10), are necessary to fully evaluate therapeutic potential of enhanced phosphorylation-independent arrestins.
Bovine, mouse, and human arrestin-1 robustly self-associate at physiological concentrations, forming dimers and tetramers (70). Yet only monomeric arrestin-1 can bind P-Rh* (18). Arrestin-1 is present in rods and cones at very high concentrations (13–15), exceeding those achieved by non-visual arrestins in any cell (63, 64) by 4 orders of magnitude. Its propensity to oligomerize in different mammalian species suggests that this phenomenon must be biologically important. The most straightforward approach to elucidation of the role of arrestin-1 self-association in photoreceptor physiology is the replacement of WT protein with a constitutively monomeric mutant that retains all other normal arrestin-1 functions. We showed that mouse arrestin-1 with mutations disabling its self-association is stable enough for this purpose. Moreover, we showed that deficient oligomerization can be successfully combined with increased ability to bind unphosphorylated Rh*, identifying stable enhanced mutants that can be generated with or without the ability to self-associate. These are the first proof-of-principle results demonstrating the feasibility of engineering proteins with desired combinations of functional characteristics. Depending on the role of arrestin-1 self-association in photoreceptors, the most therapeutically efficient enhanced mutants could be those that either self-associate or lack this ability. Molecular tools described here will help to answer these important biological questions.
Remarkable sequence similarity in the arrestin family (45) along with conservation of the three-dimensional structure among all four vertebrate subtypes (33, 37, 65, 66) suggests that homologous substitutions in non-visual arrestins will likely similarly enhance the binding of arrestin-2 and -3 to unphosphorylated forms of their cognate GPCRs. Enhanced forms of arrestin-2 and -3 proved to be useful tools to study GPCR signaling (60, 61, 67) and trafficking (47). The versions of inherently promiscuous non-visual arrestins with increased specificity for individual receptor subtypes (23, 27, 68) have therapeutic potential in disorders associated with excessive GPCR signaling (12, 20, 46).
Targeted redesign of signaling proteins and regulatory pathways (11, 12, 22, 28, 46, 48, 69) has been proposed as a novel therapeutic approach for diseases where signaling imbalance underlies the pathology. However, activating mutations often destabilize the basal conformation, thereby reducing protein stability. This is a critical difficulty in developing more active mutants engineered to rebalance cell signaling. Here we demonstrate that protein reengineering guided by high resolution structure allows one to create mutants with desired functional characteristics that retain a significant proportion of the stability of parental WT protein perfected over millions of years by evolution.
We are grateful to Dr. Cheryl M. Craft for mouse arrestin-1 cDNA and Dr. Rosalie K. Crouch and NEI, National Institutes of Health, for 11-cis-retinal.
*This work was supported, in whole or in part, by National Institutes of Health Grants EY011500, GM077561, and GM081756 (to V. V. G.), and EY05216 (to W. L. H.), and GM079419 and GM095633 (to T. M. I.). This work was also supported by and the Jules Stein Professorship Endowment (to W. L. H.).
3We use systematic names of arrestin proteins: arrestin-1 (historic names S-antigen, 48-kDa protein, visual, or rod arrestin), arrestin-2 (β-arrestin or β-arrestin1), arrestin-3 (β-arrestin2 or hTHY-ARRX), and arrestin-4 (cone or X-arrestin; for unclear reasons its gene is called arrestin 3 in the HUGO database).
2The abbreviations used are:
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