text:
Sample, 4 links of inserted block conveyor chain N
Sample, 4 links of inserted block conveyor chain No. 123 083, 1" pitch, brass blocks drilled and tapped vertically
- Category:
- Lifting & Mechanical Handling
- Object Number:
- 1935-178
- credit:
- Renold and Coventry Chain Co. Ltd.
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All Set for the 2016 RS:X Youth World Windsurfing Championship
The International RS:X Olympic Windsurfing Class Association, A step ahead of all Olympic Sailing Classes with its first Major event after the successful 2016 RIO Olympics, now looking forward to a week of exciting racing, where the Youths- the next generation, our future Windsurfing Olympic sailors from around the World take on the challenge in waters of the Eastern Mediterranean on this scheduled World Championship event at the Limassol Nautical Club on the island of Cyprus. All is set and ready for the upcoming races from the 21st trough the 25th of November.
The Opening Ceremony took place on the 19th of November at the Evagoras & Kathleen Lanitis Foundation – Carob Mill Limassol after a Flag parade with marching competitors trough the streets near the Limassol Marina.
The event was officially announced open with Opening speeches from:
The evening continued with Traditional Cyprus dance performances and a cozy dinner for all invited guests.
The Numbers:
A total of 98 Sailors registered from 19 Countries and 5 Continents
25 Women
73 Men
The Defending Champions
Women Division:
Men Division:
Text: worldofwindsurf
IMAGES:Robert Hajduk - ShutterSail.com || All Rights Reserved || Event GALLERY
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subset: commoncrawl


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Interview: David Krsevan, Infigen
The decision to buy Weather Risk Transfer can be based on a number of different drivers, many of which will be tied to project or portfolio-specific concerns. In this interview, taken from GCube's February report, Gone with the Wind, David Krsevan, Energy Markets Manager at Infigen, explores the factors behind the firm's choice to purchase cover for its Australian portfolio.
Gone with the Wind: An Asset Manager's Guide to Mitigating Wind Power Resource is available to all insureds and brokers in a digital format. To request a copy, please click here.
Who are Infigen and what do you do?
Infigen is a developer, owner and operator of renewable energy assets in Australia. We currently own six wind farms and a solar farm with a combined capacity of 557 MW, located across New South Wales, South Australia and Western Australia. In addition, we have interests in an extensive pipeline of renewable energy projects – 1200 MW in total – covering large-scale wind and solar across five states in Australia.
We were formed in June 2003, but listed on the stock exchange in October 2005. In terms of size, we are still a modest company, and employ about 60 people at the Sydney head office.
Who and what were the key drivers that led to the decision to buy weather risk cover in 2015?
One of our core mantras is to constantly look for opportunities to make our revenue more predictable and stable across our portfolio, but at the same time also retaining the upside of good production or market conditions. So we had been looking at a number of different products for a few years.
At the end of the day, the contract that we actually executed was one that we co-developed with a large reinsurer – and that was the result of a lengthy process of developing an appropriate mechanism that delivered the desired outcome for us. In the end, it was a portfolio product, across all of our wind assets.
You might expect this kind of product to be of more interest to asset owners who don't have a portfolio, but actually we've been seeing demand from companies with significant portfolios like yourself – why might this be?
I would say there are probably a few reasons why demand is not exclusive to young companies that generally don't have a portfolio. Australia is already a pretty big market and our portfolio extends across multiple regions, so we do already benefit to some extent from geographical diversification.
However, having access to in-house skill sets and resources enables larger companies to assess and identify opportunities to participate in weather risk transfer products more readily. A company with just one wind farm for example, may not be able to commit the intellectual capital, time or resources to assess products and make an informed decision to participate. For larger companies with a portfolio of assets, they are more likely to be able to do this.
That said, if smaller companies did acquire this sort cover, it may mean that they don't have the same motivation to expand for the purposes of portfolio diversity; they can operate a portfolio of just one or two assets, secure in the knowledge that their revenue flows will stay predictable within the limits of the product entered into.
Was your motivation for buying this cover purely risk aversion, or were you hoping to achieve better terms on (re)financing?
While we understand that there could be some benefit to people that want to refinance – or perhaps even want to finance a new asset – in this case our need to underpin a minimum level of cash flows from the portfolio was the primary reason. The decision was based on a desire to manage risks within the wind portfolio. It was a short-term (12-month) deal. Obviously if you're looking at refinancing you would probably seek a longer-term product. On both sides it involved 'road-testing' the product to see if the structure in question worked and was viable to look at again for a longer period in future.
What advice would you give to one of your peers considering their exposures to sustained low wind speeds and resource risk?
The important part is really investing the time to drill down into and assess the risk profile. That's the first point. The second point, once you've reached that stage, is actually working with your insurer to find a structure that works for both sides.
You must also know how resource variations are actually affecting your revenue streams. This will involve looking at both interactions with market prices, with other contracts you have in place, and also other operational risks you have in your portfolio. Another important point that we spoke a lot about when formulating the product was basis risk; making sure that the product didn't expose us to underinsured basis risk in terms of the contract pay out versus our revenue structures.
We spoke to a number of insurers and underwriters, enabling us to work through the high- level concept with a number of players. Once we had filtered the shortlist, there was a lot of work still to be done after the initial structure had been agreed to actually document and agree the delivery mechanisms.
We understand you based the structure on actual energy production rather than synthetic wind speeds or other potential settlement methods. Why?
The contract was settled off a measure closely related to actual energy production. Obviously the structure itself includes some specific contract mechanisms, so it is not as simple as just looking at the naked energy production. There were several layers of structure within the product itself. But, as you say, it was not based on a wind index like a satellite measurement or a separately measured wind index from somewhere off-site.
How could the market make procuring lack of wind cover easier?
We found that you have to build up a fair amount of understanding in terms of what your risk profile is and what types of low wind risk might manifest as issues for a particular company. You could probably identify 4 or 5 different ways that low wind might be an issue for your assets, and a number of different ways you might want to look at protecting them.
For smaller companies with limited resources it may be difficult to achieve a thorough initial understanding of what those risks are and what sorts of cover might be available. So, for the insurance market, making procuring risk transfer easier will, to a large extent, come down to helping clients reach that thorough understanding.
This could involve standardising some of the terminology, or the ways these risks are talked about. In addition, this would help to build that knowledge of what the risk types are, what type of product structures are available, and what sort of indices can be used to settle them.
From our perspective, having gone through the process of talking to a number of underwriters and looking in depth into what sorts of areas we would like to cover for our business, it's evident that the product will always need to be bespoke in nature. If you go back a couple of years, the Chicago exchange weather derivatives had a very high amount of basis risk because they were highly standardised. As such, it is likely to be a balancing act of standardising concepts where possible, while retaining the ability to tailor products for each client.
Would you say weather risk transfer products are more complex than traditional insurance products covering property damage?
It can be argued, especially from the clients' side, that the complexity is probably a bit higher for weather risk products. The way they interact with revenue streams is different from outage protection, for example. Outage protection is pretty standard and fairly simple to understand, including how it might affect your revenue, whereas, with these kinds of resource-based products, it can be a bit more complicated for the client to get an accurate picture of how they will impact revenue flows and therefore the precise value they offer.
Looking at the traditional insurance industry, there is standardised language, and standardised types of risks that are put into various baskets. With weather risk, there is always going to be a bit more complexity, but the sector could move further towards having common descriptions of similar types of risks.
Would you agree that one advantage of weather risk products is that the settlement process itself is much more simple?
I agree. The documentation is quite detailed, so the process of settlement becomes relatively straightforward because a lot of time has already gone into documenting mechanisms and actually building the spreadsheets. It's probably fair to say that we spent roughly as much time analysing how to create a settlement structure that functions correctly as we did analysing the underlying risk itself. But you don't have concerns like bringing in independent engineers, and, once you have the structure set up, the settlement process is pretty simple.
What benefits have there been to transferring weather risk, and, more broadly, what benefits could there be to yourselves or others in the future?
Once you have a product in place, you will have greater revenue certainty, so that you can focus more on actually running the business and making best use of the available resources within it. That means having to allocate fewer contingencies or buffers in forecasts, particularly related to concerns about low production periods. It also leads to greater efficiencies with capital, which allows companies to focus on other areas that create more value. So you can deploy capital to value creation or growth areas with the knowledge that wind risk has been kept in check.
What impact does this, and could this have on (re)financing?
We do think there is potential down the track for weather risk transfer to positively impact financing, because it reduces the effect of resource variations. This is still fairly new ground in Australia – we haven't seen this play out in discussions with financiers or live projects yet, but it's certainly something we'll look at for future projects.
We have, however, heard of smaller, single-asset companies that have secured better financing terms and higher gearing as a result of having similar products. Also if these players are looking at contracting off the offtake for a new asset, this allows them to provide a bit more certainty to the off-taker on the levels of energy they'll be able to contract to. Our understanding is that this has happened for a few operational assets internationally – but it's very new ground globally and to our knowledge, hasn't been done on a portfolio level, only for single assets.
Do you see weather risk transfer becoming an increasingly popular risk mitigation strategy among renewable asset owners?
Absolutely, yes. Looking at the Australian market, the country is geographically diverse, so assets do have diverse resource risk profiles. However, we also have a market that is fairly well integrated across the entire country – the NEM (National Electricity Market) connects a good portion of the country into a single electricity market. Furthermore, national green certificates ensure similar value can be extracted from wind resource across the country. There is already an advantage in being able to spread the resource risk across a nation where the market as a whole is well put together and connected. Initially, when you've got targets of higher renewable penetration and a large number of the players are looking to build one or two assets at a time, it can be helpful to get those first projects underway with a degree of resource protection in place, rather than deploying capital to achieve geographic diversification by building multiple wind farms. You can gain some of these advantages through the use of WRT products.
Why do you think that the product is starting to take off now and hasn't done so before?
I think the origination process is improving. Several products have been successfully implemented, and we're getting a lot of insurers approaching us with different ideas now. This is part of the evolution of the weather risk management market. On top of that, as you get more build of renewables, you have a more liquid market – you are not trading exactly the same thing, but, if insurers are pricing and selling products to a large number of potential customers, there are efficiencies to be gained in terms of how insurers can price the product with regards to margining on their side – which in turn leads to more effective products for customers. It has a snowball effect.
In addition, with more capital going into the market, you need more risk management products to protect that capital. And, with the regulatory space improving, particularly in Australia, the uncertainty surrounding renewable energy targets has settled in the last year or so – translating into more demand for renewable energy assets and as a consequence more demand for these kinds of products.


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Brent Central MP hosts bash in recognition of Harlesden school's placement scheme
PUBLISHED: 17:57 17 October 2014 | UPDATED: 17:57 17 October 2014
The MP for Brent Central hosted a celebratory bash in the House of Commons to mark the successes of a work experience scheme at a secondary school in Harlesden.
Sarah Teather welcomed a number of pupils from Newman Catholic College, in Harlesden Road, who took part in a programme, dubbed 'Work Ready.'
Speaking at the event, the Liberal Democrat politician, who launched the programme in 2012, said: "It is giving the opportunity for students to develop their talents to ensure that they will be able to get a good job that they deserve.
"I would like to thank the employers. Sometimes it's a risk but from what I've heard, everyone involved seem to have loved it.
In its third year, the scheme is aimed at giving the school's disadvantaged teenagers who have a smaller chance of achieving higher education grades a taste into working life by arranging placement at some of the most reputable business in London.
Clive Nyawo, 20, who took part in the programme, was offered a permanent job from bespoke interiors firm, Wagstaff, after impressing his employers during his placement at the company.
Danny Coyle, head teacher at the school, said: "There is a view that young people have to go to university in order to progress and move on in life which is fine. But it is only one pathway available for our young people.
He continued: "We are going to develop the program because the more we work together the stronger we become. We urge employers out there to get involved and help develop our youngsters."
Companies participating in the scheme includes Barclay's Bank, the Metropolitan Police Service and the Brent & Kilburn Times.
For more information on how to get involved contact the school on 0208 965 3947.
If you value what this story gives you, please consider supporting the Brent & Kilburn Times. Click the link in the orange box above for details.


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Our electrical engineering division services range from Electrical Infrastructure for industrial developments and substations to Electrical and Electronic Building Services for luxury residential and commercial developments.
Balancing the effective supply and use of energy, and managing the interface between energy use and the environment, are the greatest challenges facing our modern world. Electricity is vital for economic growth and development. While efficient energy must be provided for industries and commercial use, there is also a critical need to address energy poverty and provide low-cost energy sources in both urban and rural areas.


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This is where is all about internet marketing and using the power of the internet.
What is your dream job? ... mine would be blogging for profits and earning a residual income from my blog online every single month! There are many ways to make money in this world, and on the internet, but my favorite one is blogging for profits.
Are you one of those people who really wants to build a successful money making blog online? ... If you are, then the following blogging statistics can really help you! Information is really important when you are building a blog online.
If you have ever had your WordPress site hacked, then you know that it is not any fun. If you are fortunate enough to never have experienced such an attack, then you should still be aware of the potential dangers out there. The fact is that most WordPress sites are almost defenseless against malicious hackers.
There are many ways to drive free traffic to your blog online and to any website online, pretty much. Today I'm going to teach you about getting free traffic from free solo ads that work. There are so many free solo ads sites online and you can really choose which ones you want to use for your marketing online.
There is a new powerful marketing system people are using to earn a lot of money online called "Total Funnel System". The funny thing is, this new marketing system allows you to earn commissions without having to spend a penny to get started! Here is what you will learn in this Total Funnel System Review...
Building a Blog's Subscribers List is as important as building an email list. You can and should build both lists for your blog online. If you have a little bit of experience online then you know the importance of building an email list. You will need to invest in an Email Marketing Software like Aweber.com.
When you are building a blog online, one of the things you should really work on is the average visit duration time from each visitor. I'm going to share with you how a simple sticky navigation bar can help you increase the duration time.As a new blogger online, you want to test things out with your blog's design and navigation to make people stick around as long as possible.
Today you can learn how to build an HTML Page from scratch and start creating your own Mini Capture Pages and Google Hangout Pages and any simple, static HTML Page you want. This can save you some money when you are getting started online. This is a very simple process for the newbies online.
Today I'm going to share with you 17 Mobile Apps to earn money with. You can use these mobile apps to earn a little bit of extra money every month. I know there are many apps to help you earn a little bit of money by doing different things for the app.


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New Berlin — Gov. Jim Doyle said Wednesday his signing of a wind farm siting bill should serve as a call to wind developers who have been avoiding building projects in the state.
"It signals to the world that Wisconsin is in the wind business, and that we intend to be one of the leading states in production of wind energy and components that are necessary to harness that energy," Doyle said as he signed the bill into law.
Though Doyle and other political leaders have been supporters of renewable energy, wind-industry developers have been hesitant to be active in the state, in part because of a patchwork of local ordinances governing any wind farm under 100 megawatts, and because of past state Department of Transportation permitting problems that made it cumbersome to transport wind-turbine components across the state.
"The wind industry - some felt that it was just a difficult place to do business," Doyle said after the signing ceremony. "But this is a huge step forward."
The siting law will require the state to develop specific standards that would be enforced statewide. That process could take up to a year.
Groups concerned about the impact of wind turbines on property values were disappointed that the bill passed. They preferred local regulations that governed issues such as how far wind turbines could be placed from homes.
The bill signing took place in New Berlin, where ABB makes drives and motors for use in industrial and commercial applications including wind and solar power.
"We are seeing interests in the alternative energy sector skyrocket," said Rick Hepperla, regional manager of automation projects, North America, for ABB.
Jeff Anthony of the American Wind Energy Association said the bill was a needed step to attract the wind industry to the state.
"It restores common sense and consistency to wind project siting in Wisconsin," he said. "Wind project developers will now view Wisconsin as a viable place for considering wind power development."


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Ashish is an aircraft maintenance cum mechanical engineer from the Indian Institute of Aeronautics. Since graduating in 2014, he worked as production engineer with Dan Block Brake India Private Limited for a year.
Meet Ashish Sharma, a twenty-nine-year-old techie-turned-child rights activist, who left his job at a multi-national corporation to walk 17 thousand Km on foot for achieving 'child begging-free India'.
Ashish is an aircraft maintenance cum mechanical engineer from the Indian Institute of Aeronautics. Since graduating in 2014, he worked as production engineer with Dan Block Brake India Private Limited for a year.
A Delhite since childhood, Ashish was highly inspired by Dr APJ Abdul Kalam, Swami Vivekananda, Gautam Buddha, Hazrat Muhammed and great men who worked for upliftment of humanity. But on his way back from office, Ashish often saw children begging on the streets of Delhi.
"It is pathetic to see little children begging for a living. They have no idea of parents. I rescued nine such children and rehabilitated them in juvenile homes. Most of them were victims of trafficking and organized begging", Ashish told indianexpress.com here, after he reached Tripura through Mizoram as part of his nationwide walk.
After he rescued the last child, Ashish decided that he would walk across the country to raise awareness on child rights and put an end to child begging.
He started his walk from Udhampur in Jammu and Kashmir and has covered 9,139 Km across 21 states including Jammu and Kashmir, Delhi, Himachal, Haryana, Punjab, Rajasthan, Gujarat, Daman-Diu, Silvasa, Maharashtra, Goa, Madhya Pradesh, Uttarakhand, Bihar, Sikkim, Assam, Mizoram, Nagaland, Manipur and Tripura.
Ashish walks with a backpack, a mobile phone, some cash and an Indian flag. He wishes 'Jai Hind' to everyone he talks to.
Speaking on how the comfort-accustomed technocrat has managed on the streets, he says, "I am covering the entire distance solely on foot. It started with troubles and a lot of dehydration. Now I have a backpack filled with all I need".
Ashish feels though governments have their responsibilities but demanding rights from governments haven't yielded much till now.
"Seventy two years after Independence, we still have to raise voice against child begging. It says enough. I feel the society has as much responsibility as the government does. It's time we started taking them up", Ashish says.
Two of his friends who are Indian Administrative Service (IAS) officers are helping him with the finances of his mission. "I don't beg from anyone. It would sound ridiculous to fight begging with begging", he said with a wink.
Ashish's father is a Public Sector Undertaking (PSU) employee, his brother is an advocate and his mother is a homemaker. When he first told his idea of quitting a job of Rs. 6 lakh per annum, the entire family went berserk.
"I even got beaten at the beginning. But gradually, they understood my genuine hope for a child begging-free India. Now they support me unequivocally", the man on a mission said.
He wishes to meet senior government officials during his mission on road. The 17 thousand Km long walk is expected to end in October, 2019.
Ashish wants to complete his mission in the burial place of former President Late APJ Abdul Kalam at Rameswaram in Tamil Nadu.
"It was his vision that all children should go to school by 2020. I shall not rest till then", he said.
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Source: Read Full Article


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During the testing phase, which will last around 3 months, the app will be used initially by 50 officers under careful supervision prior to sharing the results with senior policing leaders and partners.
Deputy Chief Constable Richard Lewis said: "This new app means that, with a single photo, officers can easily and quickly answer the question of 'Are you really the person we are looking for?' When dealing with a person of interest during their patrols in our communities, officers will be able to access instant, actionable data, allowing to them to identify whether the person stopped is, or is not, the person they need to speak to, without having to return to a police station.
"Officers will also be able to verify the identity of a vulnerable person in seconds, rather than hours, resulting in less time digging for information and more time keeping the peace.
"I want to stress that our police officers will only be using the new technology in instances where it is both necessary and proportionate to do so and always with the end goal of keeping that particular individual, or the wider public, safe. We have given additional training to the officers who are part of the trial and will closely monitor the use of the app to assess its effectiveness."
Facial recognition is just one way in which technological advancements are being used across South Wales Police as part of joint work with Gwent Police with the aim of making the work of police officers easier and faster so that they can have more time to work at keeping our communities safe.
Police and Crime Commissioner Alun Michael added: "I maintain close scrutiny of the operational decisions on the introduction of technology, and with the Chief Constable Matt Jukes QPM, subject each new step to independent oversight and scrutiny because of the ethical and social concerns that have been expressed over the use of facial recognition technology. People want to know that members of the public who have done nothing wrong are not being subjected to inappropriate surveillance and their privacy and anonymity will be respected and protected. However people also want us to keep them safe and to use the technology to apprehend people who have committed serious offences and take them off the streets. As a result of the robust systems of scrutiny and challenge that I and the Chief Constable have put in place I can reassure the public that we are getting that balance right. We are committed to protecting human rights as well as keeping the public safe.
"That is why I can say that in South Wales the use of this technology is both proportionate and legitimate and South Wales Police are required to adhere to rigorous frameworks in terms of data protection, including the General Data Protection Regulation (GPDR).
"Police officers have always been able to spot a person who is wanted for a crime and stop them in the street. The difference with the use of this technology is simply the speed and accuracy with which the individual can be identified and arrested, and the speed with which a mistaken identity can be cleared up. This is about doing more with less after we have lost a third of the money to employ police officers that used to come from the Home Office and it is absolutely not about Big Brother taking over. Our determination to police the communities of South Wales effectively is under the challenge as never before due to the severity of the cuts, increased demand and the complexity of calls on the police – so we need to harness technology to increase effectiveness, but we will only do so when we can at the same time respect the spirit as well as the letter of the law as we enhance the ability of our officers and staff to prevent crime and create a safer environment in which everyone can be safe and confident."
© Copyright Heddlu South Wales Police


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About Us
OTAG MUSIC CENTER LTD. which is an affiliate of TUMATA (ENSEMBLE) aims to serve the activities of TUMATA. The manufacturing, publishing and the sale of CDs, cassettes and books and musical instruments which are the products of TUMATA are executed through OTAG MUSIC CENTER channel. Apart from these, concert, seminar, exhibition, tour and course organizations are being implemented. At OTAG MUSIC CENTER, apart from TUMATA CDs, cassettes and books, the manufacturing and the sale of other books and Turkish Classical Instruments, as well as Central Asian Musical instruments, are being carried out. Authentic Central Asian costumes, hats (doppa or tepetei), covers, carpets, and rugs are also available.
The manager of the OTAG MUSIC CENTER is retired Agricultural Engineer Yasar Guvenc.
Address: Alemdar Mah. Alaykosku Cad. Civan Han 20/B, Fatih - Istanbul
Phone: 0212 511 15 38 Fax: 0212 514 40 47 E-mail: email@example.com
Web site: www.otagmusic.com


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DescriptionHeavy, fleshy and versatile, these tangy, sturdy, almost smoky-flavored tomatoes are a popular favorite. In Italy, they serve layers of sliced beefsteaks, fresh mozzarella and whole basil leaves, with a drizzle of good olive oil and a little balsamic vinegar. Top your burger or roast beef sandwich with a thick, juicy slice of beefsteak for extra-meaty flavor.Claims
Lancaster Farm Fresh Cooperative (LFFC) is a non-profit organic farmers cooperative of over 75 farmers in Lancaster County, Pennsylvania. We focus on creating healthy, high quality foods from our highly maintained and enriched soils on our small scale family farms. When you purchase LFFC products you can be confident that you will receive the freshest products, farmed with consideration for the land, animals and people. (from Lancaster Farm Fresh Cooperative)
- Organic
- Nutrition
Explanatory Information
Lancaster Farm Fresh Cooperative Local Organic Beefsteak Tomatoes
Availability Note
Item is temporarily unavailable.


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Are you an entrepreneur or a small scale company who does not have enough time or resources to build a new payroll system? Is the size of your company increasing? If yes then you have landed at the right article because improving payroll management is a crucial task for you.
So, here are 4 tips to build a better payroll management for your company.
Have One Pay Schedule
Many companies pay their employees following different schedules. For example,:
Monthly payment
Bi-weekly payment
Weekly payment
However, having a single pay schedule can do wonders to quicken your payroll management. Which pay schedule you choose, depends on your business and employees hired. Weekly is a perfect system to have for hourly staff, while bi-weekly is perfect for salaried workers, and monthly is perfect for well-paying corporate employees.
Paperless Payroll Process is the Need of the Hour
Technology comes to our help at every step. Eliminating physical checks and using various software's like NolaPro, Quickbooks, etc. can come in handy for recording payroll services. A paperless payroll system is beneficial for several reasons. Managing paper documents and salary slips is a hassle. With everything accessible on the digital medium, you are able to perform better and don't waste time hunting for documents. Additionally, you and your employees can access the system remotely and are not limited to the location.
Outsourcing Your Payroll Operation
Outsourcing is a viable option if you are a small business owner who does not have enough resources at hand. It has been proven to be a cost-effective option for the majority of small-Scale enterprises and entrepreneurs who have outsourced it to professional firms like Roth Schroder.
When you outsource your payroll operation, it is taken who by professionals who streamline the process in a highly efficient manner.
Increase Involvement of Your Employees
The employees are the most important asset of any organization. Many studies have found out that employees work to their full potential and deliver great results when companies entrust responsibilities upon them. The best way for improving payroll management is to allow the employees to record their attendance hours and to verify their expenses.
When you have an employee's payroll policy which they are:
They are aware of the payroll process clearly
Their job classification
They are aware of their salary break up
Armed with this information, you will discover that employees can contribute and make your system more efficient.
Thus, payroll management in an organization not only increases efficiency but also decreases error rates and it is advisable to reach out to a professional payroll management service provider.


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Customers can leave tidying their cars to professionals with this interior or exterior clean and polish that includes detailing
What You'll Get
Choose from Three Options
- AED 179 for a car interior clean and polish with detailing
- AED 229 for a car exterior clean and polish with detailing
- AED 349 for a car interior and exterior clean and polish with detailing
The Fine Print
Expires 90 days after purchase. Limit 2 per person, may buy 2 additional as gifts. Booking required by phone, contact 054 4949049 or email email@example.com. Prior booking (subject to availability) at least 24 hours in advance. 12-hour cancellation policy. Valid only for option purchased. AED 100 surcharge for SUVs. Valid Sat-Thu 9am-1pm and 3pm-8pm. Printed voucher must be presented. See the rules that apply to all deals.
About Fem Auto Upholstery & Polishing
Fem Auto Upholstery & Polishing is an Abu Dhabi-based car cleaning service. Customers can drop their vehicles off and return two hours later with their automobile looking good as new inside and out.


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Stéphanie PERES holds a Ph.D. from the University of Bordeaux (France) in Economics, and a degree in tasting from the University of Bordeaux (ISVV). Her current research topic focuses on the evolution of the wine consumer demand and the consumption context in wine quality perception.
Wines Consumers, Experimental Auction, Sensorial Analysis ; Land Economics, Viticulture
Wines, Experimental Auction, consumption
Research unit and labs
Institut des Sciences de la Vigne et du Vin
Ecole Doctorale: Entreprise, Economie et Société (E.D.42)


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Welcome at the GAUGHOF
The Arabian stud farm in Wangen/Ritten in the Tyrolean Alps (Italy)
More than twentyfive years ago, the Bolzano furniture maker Peppi Selva began with two Haflinger mares: a leisure-time horse breeder. He then discovered his love for Arabian thoroughbreds and educated himself to the point of becoming a specialist in the complicated pedigrees and heredity lines of purebred Arabian horses.
Selva purchased stallions and mares throughout all of Europe from famous and successful breeding families and founded the "Gaughof" Arabian stud farm in Wangen/Ritten in the Tyrolean Alps, one of the first breeding operations in Italy where thoroughbred Arabians were bred in the grand style. Steeply climbing paths, blossoming meadows, a man-made lake in the vicinity, abundant sunshine, and in winter an orderly frost – the bracing climate of the mountains is apparently precisely the right one for these horses of the desert who are accustomed to challenging conditions.


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The Jerusalem Portfolio Launched Wednesday At The Waldorf Astoria Beverly Hills
Posted Thursday, May 30, 2019 - 5:00 pm
By John L. Seitz
Beverly Hills was the site Wednesday for the launching of The Jerusalem Portfolio (TJP), a new investment vehicle empowering individuals, foundations, and institutions to carve out a portion of their portfolios to own a stake in Israel's thrieving economy.
Century City-based RVW Wealth hosted the event at the Waldorf Astoria Beverly Hills, which included speakers like Stanley P. Gold, one of the earliest major private investors in Israel, who is chairman of the board of the late Roy Disney's private investment firm, Shamrock Holdings.
For full story see the print edition of The Beverly Hills Courier, or download the e-edition.


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Welcome to the shipper thread of Jung Hae In & Han Ji Min or Yoo Ji Ho & Lee Jung In ("Spring Night") ◇ LeeYoo Couple ◇ Reason Couple 이유 ◇ HaeMin Couple Jung Hae-In Hangul: 정해인 Born: April 1, 1988 Height: 178 cm As Yoo Ji Ho - Pharmacist "One Spring Night" Han Ji-Min Hangul: 한지민 Birthdate: November 5, 1982 Height: 161cm As Lee Jung In - Librarian " One Spring Night" Jung Hae In Interview on Han Ji Min : * On 2015 - He mentioned he want to work with HJM sunbaenim. * On 2019 - He changed his mind and dont want to call her sunbaenim or even noona Credit & details as below. Have a great & beautiful time here... I hope you become happier after your visit here Original Starter : Lawyerh


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Creative Harp Series #3-Streaming
Creative Harp Series #3-Streaming
Creative Harp#3 - Aeolean Mode - Christina Tourin
Playing Harp Effortlessly!
For those who have not studied the Creative Harping Signature Series ~
we are offering you not only the FREE first Lesson but a 60% savings on the heartfelt Aeolian Lesson for harp players from the very beginning stages through the most advanced! This is a "streaming" lesson which you can download on your Phone, iPad, Tablet or Computer. You will be able to download the accompanying support papers! All students in the Therapeutic Harp Course study these lessons which ultimately have them playing for hours on end without depending on written music.
You can now Steam the Creative Harp Series Online, no more costly shipping!
The Signature Method for Learning to Play Music by Ear – Creative Harping is the Basis for Music Development for the International Harp Therapy Program.
# 3 AEOLIAN MODE – Learn how to improvise in the Aeolian Mode and add Little Boats, Mary Young and Faire and Greensleeves to your repertoire. A countryside journey through England opens this pastoral video.
Lessons included:
- Exercise
- Improvisations
- Improvisations in the Aeolian Mode
- 8-Bar Chord Change
- Running Bass Improvisation
- The Trill
- Greensleeves – Beginner
- Greensleeves – Intermediate
- Mary, Young and Faire – Beginner
- Little Boats – Beginner
- Little Boats – Intermediate
- Play Along Improvisation
The Aeolian Mode touches and enters into the inner depths of our Soul. It is often referred to the relative minor of a major key of music. In this lesson you will have the choice of three songs to focus on. In the Int'l Harp Therapy Program (IHTP), students are required to memorize one of the songs and then follow it by an improvisation in the same tempo and time signature. They are to utilize at least three variations of the left hand improvization patterns so that the music does not sound repetitiously 'boring' but lends itself to creative flow. If you are preparing for study in the IHTP, you will want to focus on memorizing one of the songs followed by the short improvisation (not to exceed 4 mins. in length). When you enroll in the program you can then send on these recordings to your mentor and be far ahead of your schedule on music development.
We are excited to offer you the opportunity to save money on shipping by STREAMING the Creative Harping Lessons. We still offer the hard copies and for our International Students, it is most affordable to get them in bulk to save on the shipping costs. But we now offer you the lessons where you can study on-line. They are not available to download onto your computer which means that you will always need to be on-line to view the lesson and practice with it. As with works of a composer, these are copyrighted and when you receive your access to the stream, the code is only intended for you and not to be shared.
Most importantly to remember is that you are getting lessons here that will go on forever. When you go to a teacher and pay $50.00 for one lesson, you will realize that through the Creative Harping Program you are getting many multiple lessons in each DVD/Viewing for the cost of 1 lesson. This is a continuing lesson from the most beginner to the most advanced level of player.
* After purchasing (see 'Add to Cart' button below) and completing checkout you will access a PDF file with complete details of how to access your streaming lessons.


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Last Updated on Tuesday, 30 November 1999 05:00 Written by Administrator Tuesday, 06 August 2013 09:42
A spokesman of All Pakistan Cement Manufacturers Association (APCMA) said the lower consumption could also be the outcome of higher taxes imposed in the budget and increase in transportation charges due to strict compliance of axle load restrictions on the cement carriers.
He said these increases coincided with the advent of Ramazan when pace of construction declines.
Local sales recorded major decline of 10.43 percent while exports reduced by 0.75 percent. The total cement despatches in the first month of this fiscal stood at 2.595 million tonnes against despatch of 2.816 million tonnes in July 2012.
The local sales amounted to 1.846 million tonnes of which, 1.506 million tonnes were despatched by mills located in the Northern region and 0.339 million tonnes from the South.
The industry during the corresponding month of last year despatched 2.060 million tonnes of cement.
The total exports in July 2013 amounted to 0.749 million tonnes against 0.755 million tonnes in July 2012. Exports from the North declined from 0.615 million tonnes in July 2012 to 0.525 million tonnes in the first month of this fiscal. The exports from South however, registered healthy increase from 0.139 million tonnes in July last year to 0.224 million tonnes in the same month of this fiscal.
The spokesman expressed hope sales would pick up in the coming months, however he urged the government to resolve the problem of high transportation cost to the industry.
Courtesy: ĀDaily Times
Forex open Market rates & comments Archive


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Dec 23, 2008
Photo Shoot - Heather Talbert
I had been dieing to shoot with Heather Talbert and finally it happened! Last week we did a really amazing shoot. Heather had a really good team on make up and styling. She is SO talented and really fun to work with!
www.heathertalbert.com (photographer)
http://stylerr.com/ (stylist)
Crystal Cadahia (make up artist)
3 comments:
Is there a better moment in the year to leave my first comment to your blog? :)
I wish you and your loved ones a magnificent Xmas!!!
Oh! By the way, I really like these shots you posted, especially the one with the lights in the background, great atmosphere!
- December 24, 2008 at 4:42 AM
- Ektachrome 160T said...
I love these!
- December 24, 2008 at 5:27 PM
- dax said...
Woah !!
Those are some great great shots!! Congrats !!
- December 28, 2008 at 12:20 AM
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The roles of vitamin A (VA) in the development of metabolic diseases remain unanswered. We have reported that retinoids synergized with insulin to induce the expression of sterol-regulatory element-binding protein 1c gene (Srebp-1c) expression in primary rat hepatocytes. Additionally, the hepatic Srebp-1c expression is elevated in Zucker fatty (ZF) rats, and reduced in those fed a VA deficient diet. VA is metabolized to retinoic acid (RA) for regulating gene expression. We hypothesized that the expression of RA production enzymes contributes to the regulation of the hepatic Srebp-1c expression. Therefore, we analyzed their expression levels in Zucker lean (ZL) and ZF rats. The mRNA levels of retinaldehyde dehydrogenase family 1 gene (Raldh1) were found to be higher in the isolated and cultured primary hepatocytes from ZF rats than that from ZL rats. The RALDH1 protein level was elevated in the liver of ZF rats. Retinol and retinal dose- and time-dependently induced the expression of RA responsive Cyp26a1 gene in hepatocytes and hepatoma cells. INS-1 cells were identified as an ideal tool to study the effects of RA production on the regulation of gene expression because only RA, but not retinal, induced Srebp-1c mRNA expression in them. Recombinant adenovirus containing rat Raldh1 cDNA was made and used to infect INS-1 cells. The over-expression of RALDH1 introduced the retinal-mediated induction of Srebp-1c expression in INS-1 cells. We conclude that the expression levels of the enzymes for RA production may contribute to the regulation of RA responsive genes, and determine the responses of the cells to retinoid treatments. The elevated hepatic expression of Raldh1 in ZF rats may cause the excessive RA production from retinol, and in turn, result in higher Srebp-1c expression. This excessive RA production may be one of the factors contributing to the elevated lipogenesis in the liver of ZF rats.
Citation: Li Y, Zhang Y, Li R, Chen W, Howell M, Zhang R, et al. (2012) The Hepatic Raldh1 Expression Is elevated in Zucker Fatty Rats and Its Over-Expression Introduced the Retinal-Induced Srebp-1c Expression in INS-1 Cells. PLoS ONE 7(9): e45210. https://doi.org/10.1371/journal.pone.0045210
Editor: Partha Mukhopadhyay, National Institutes of Health, United States of America
Received: May 2, 2012; Accepted: August 14, 2012; Published: September 13, 2012
Copyright: © Li et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was financially supported by research grant from Allen Foundation Inc (to GC), startup fund from the University of Tennessee at Knoxville (to GC), and Scientist Development Grant from American Heart Association (09SDG2140003, to GC). The authors also thank China Scholarship Council for the financial support (to Y.L. and R. L.) The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
The high obesity prevalence in the population of the United States [1] predicts the increase of patients with noninsulin-dependent diabetes mellitus (NIDDM) [2], a major public health concern [3]. Genetic mutations, such as mutations of leptin and its receptor, have been shown to cause the development of obesity and diabetes [4]. Currently, the associations of a variety of genes with the development of human obesity or NIDDM have been indicated [5], [6]. Metabolic abnormalities are often associated with profound changes of hepatic glucose and lipid metabolism [7], which is attributed, at least in part, to the expression of genes involved in these processes [8], [9]. On the other hand, overconsumption of nutrients, such as fructose in sweetened beverages, has also been implied to play a role in the rise of obesity [10]. Dietary nutrients provide us with not only energy, but also vitamins and other essential factors with regulatory roles. The effects of individual micronutrients on the development of metabolic diseases remain to be revealed.
As an essential and fat-soluble micronutrient, vitamin A (VA, retinol) plays crucial roles in the general health of an individual, such as vision, tissue differentiation, immunity, etc [11]. The majority of the physiological actions of retinol are mediated by its active metabolite, retinoic acid (RA), which exists in multiple isomeric forms, such as all-trans RA and 9-cis RA [12]. RA regulates gene expression through the activation of two families of nuclear receptors, retinoic acid receptors (RARα, β and γ) activated by all-trans RA, and retinoid X receptors (RXRα, β and γ) activated by 9-cis RA [13].
In an attempt to understand the effects of endogenous lipophilic molecules on the insulin-regulated gene expression in primary rat hepatocytes, we have analyzed the rat liver lipophilic extract and found that retinol and retinal in it dose-dependently induced the mRNA expression levels of Pck1 [14], Gck [15], and Srebp-1c [16]. The proximal one of the two previously identified retinoic acid response elements (RAREs) in Pck1 promoter [17]–[19] mediates the RA effect in primary hepatocytes. The previously identified two liver X receptor (LXR) binding sites mediating the insulin-stimulated Srebp-1c expression [20] were also RAREs in its promoter [16], demonstrating the convergence of hormonal and nutritional responses. The hepatic expression level of cannabinoid receptor 1 (CB1), the target of anti-obesity drug rimonabant [21] can also be induced by the treatment of RA via activation of RAR-γ [22], indicating the connection between the nutritional signals and endocannabinoid pathways. The effects of VA status on glucose and lipid metabolism have been summarized [23].
Retinol is reversibly oxidized into retinal by retinol dehydrogenases (RDHs). Retinal is irreversibly oxidized into RA by retinaldehyde (aldehyde) dehydrogenases (RALDHs) [13], [24], [25]. Currently, four RALDHs have been cloned and thought to be responsible for all-trans or 9-cis RA production in various tissues [26]–[29]. RALDH1-4 proteins can be detected in some but not all hepatocytes of the mouse liver using immunohistochemistry, with RALDH1 being frequently expressed in lipid-engorged cells [27]. Raldh1 (also known as Aldh1a1) mRNA is expressed weakly in the rat liver [26], whereas Raldh4 (also known as Aldh8a1) mRNA is expressed at high level in the mouse liver [27]. The mice with Raldh1 deletion (Raldh1−/−) have reduced RA production in the liver two hours after a challenge of retinol [30]. The Raldh1−/− mice are resistant to diet-induced obesity (DIO) and insulin resistance [31]. The Imprinting Control Region (ICR) mice fed a high cholesterol diet had the elevation of the hepatic expression of RALDH1 and RALDH2 through oxysterol-induced Srebp-1c expression, demonstrating the interaction of cholesterol and RA metabolism [32]. All these observations indicate the potential roles of these enzymes in the development of obesity and insulin resistance, which deserves to be further defined.
A and B. Total RNA was extracted from hepatocytes and subjected to real-time PCR analysis. Results were presented as means ± SD of the −ΔCT (the CT of 36B4– the CT of indicated transcript) from the indicated different numbers (in parenthesis) of hepatocyte isolations (* all p<0.05, for comparing the values of ZL and ZF groups of the indicated transcripts using the independent t-test). C. Whole liver tissue lyastes (100 µg/lane) of ZL (lanes 1–4) and ZF (lanes 5–8) rats, and whole cell lysate of INS-1 cells infected by Ad-Raldh1 (50 µg, lane 9) were separated in 10% SDS PAGE gels, and transferred to the PVDF membranes as described in the Material and Methods. Primary antibodies to RALDH1 (1∶1000 dilution in TBST containing 5% dry milk), and to β-Actin (1∶1000 dilution in TBST containing 5% bovine serum albumin) were recognized by goat anti-rabbit IgG conjugated to horseradish peroxidase, and visualized by chemiluminescence. The ratios of RALDH1/β-Actin were calculated after the films were scanned and analyzed. Data were presented as mean ± SD of the indicated numbers (in parenthesis) of ZL and ZF rats (** p<0.04 for comparing the ratios of ZL rats with that of ZF rats using the independent t-test).
The fact that the levels of the enzymes for the hepatic retinoid metabolism can be dynamically regulated leads us to hypothesize that the change of the expression of these enzymes and the production of RA may contribute to the regulation of the expression of hepatic lipogenic genes. Here, we examined the effects of retinol and retinal on the expression of RA responsive genes in primary rat hepatocytes, rat hepatoma cells and insulinoma INS-1 cells. We observed the elevated expression levels of Raldh1 mRNA in hepatocytes and its protein in the liver of Zucker fatty (ZF) rats. The recombinant adenovirus-mediated over-expression of RALDH1 resulted in retinal-mediated induction of Srebp-1c expression in INS-1 cells, which originally lack the response to retinal.
Primary hepatocytes were isolated from SD rats, and pre-treated as described in the Materials and Methods. Total RNA was isolated, and subjected to real-time PCR analysis. A. A 24-h time course of Cyp26a1 mRNA expression induced by RA (5 µM) in primary rat hepatocytes (* for comparing the vehicle control with the RA groups at the indicated time points using the independent t-test; ** and '' for comparing the different time points of vehicle control or RA group using one way ANOVA; all p<0.05). B. The expression levels of Cyp26a1 mRNA in primary hepatocytes treated with increasing concentrations of ROL or RAL for 6 hours ($ for comparing the fold induction of ROL group with that of RAL group at the indicated concentrations using the independent t-test; c > b > a; h > g > f > e > d for comparing the different dosages of ROL or RAL using one way ANOVA; all p<0.05). C. Inhibition of the RAR activation disrupted the induction of Cyp26a1 by RAL treatment. Primary hepatocytes were incubated in medium A without or with 2 μM RAL in the absence or presence of increasing concentrations of Ro 41–5253 for 6 hours (i/j > l, i'/j' > k' > l' > m' for comparing the different dosages of Ro 41–5253 in the absence or presence of RAL using one way ANOVA, all p<0.05). Results were presented as means ± SD of three independent hepatocyte isolations.
Determination of the mRNA levels of enzymes responsible for the retinoids catabolism in primary hepatocytes, and the elevated expression of Raldh1 and its protein in hepatocytes of ZF rats
Enzymatic processes are involved in the conversions of ROL to RAL and RAL to RA [13], [24], [25]. It has been shown that the short chain dehydrogenase/reductase family 16C member 5 (SDR16C5), RDH2, RDH10, and RALDH1-4 are expressed in the liver [33]. Since RALDH4 is for the biosynthesis of 9-cis RA [27], we have focused on the expression levels of the rest enzymes in this study. The CT numbers of these genes were normalized to those of 36B4 (an invariable control gene) in the same samples. Based on −ΔCT numbers (how close the abundance of the transcripts of those genes to that of 36B4), we found that Rdh2, and Raldh1 were expressed in meaningful levels (compared with the expression level of 36B4) in both primary rat hepatocytes and HL1C cells (data not shown).
The dose-dependent induction of Cyp26a1 by ROL, RAL, and RA. HL1C cells were treated with ROL, RAL, or RA at the doses of 0, 0.002 µM, 0.02 µM, 0.2 µM, 2 µM, and 20 µM for 6 h (a/b < c, d/e < f, g/h < i, j < k/l, m < n/o, a/d/g/j < m, b/e/h < k < n, c/f < i/l/o for comparing ROL, RAL and RA at the indicated concentrations, and for comparing each ligand at the different dosages using one way ANOVA; all p<0.05). B. The expression levels of Cyp26a1 mRNA overtime (3, 6, 9, 12, and 24 h) in HL1C cells treated with 5 µM RAL or RA (* or ** for comparing RAL or RA with vehicle control groups at the indicated time points using the independent t-test; all p<0.05). C. The expression levels of Cyp26a1 mRNA in HL1C cells treated with RAR (TTNPB), RXR (LG268), and LXR (T1317) agonists. HL1C cells were treated with 1 μM TTNPB, LG268, and T1317 for 6 h (q > r>s using one way ANOVA; all p<0.05). Total RNA was extracted and subjected to real-time PCR analysis. Data were presented as mean ± SD of three independent treatments. D. Whole cell lysates (50 µg/sample) of HL1C cells treated the vehicle control, 5 µM RAL or RA for 12 (lanes 1–3) and 24 (lanes 4–6) hours were separated in 8% SDS PAGE gels, and transferred to the PVDF membranes. Primary antibodies to CYP26A1 (1∶1000 dilution in TBST containing 5% dry milk), and to β-Actin (1∶1000 dilution in TBST containing 5% bovine serum albumin) were recognized by goat anti-rabbit IgG conjugated to horseradish peroxidase, and visualized by chemiluminescence. The films were scanned and presented as described in the Material and Methods.
Since RDH2 catalyzes the conversion of ROL to RAL [33], and RALDH1 is reponsible for converting RAL to RA [33], we compared their expression levels in freshly isolated primary hepatocytes of ZL rats with those of ZF rats. The mRNA level of Rdh2 in hepatocytes from ZF rats was similar to that from ZL rats. However, the mRNA level of Raldh1 in hepatocytes from ZF rats was significantly higher than that from ZL rats (Fig. 1A). The results observed in cultured primary hepatocytes of ZL and ZF rats were similar to that in fresh isolated hepatocytes. We observed that the Rdh2 expression level was unchanged, and the Raldh1 expression level was elevated significantly in hepatocytes from ZF rats (Fig. 1B). The mRNA level of Raldh4 was detected, and was at the similar levels in hepatocytes from both ZL and ZF (data not shown).
A. 834/40 INS-1 cells were treated with increasing concentrations of RAL or RA for 6 h. B. 834/40 cells were treated with 2 µM of RAL or RA for indicated time. C. 833/15 cells were treated with 1 µM of specific ligands of RXR (LG268), RAR (TTNPB) or in combination. Total RNA was extracted from INS-1 cells and subjected to real-time PCR analysis. Results were presented as means ± SD of fold inductions (c > b > a for comparing the different dosages of RA-treated groups using one way ANOVA, and e > d > f for comparing control with LG 268 in the absence or presence of TTNPB using one way ANOVA; * for comparing RAL with RA groups at the indicated dosages, and ** for comparing RA with RAL or vehicle control groups at the indicated time points using independent t-test; n = 3, all P<0.05).
Fig. 1C shows that the RALDH1 protein levels in the liver samples of ZF rats were higher than that of ZL rats. Since two protein bands migrating close to each other were detected in the Immuno-blot, the lysate of INS-1 cells infected by recombinant adenovirus Ad-Raldh1 expressing RALDH1 protein was used as the control to determine the correct size of it. Based on the size of the control sample, the lower band detected by the antibody should be the correct one in the liver samples. Their densities were normalized to the densities of the β-Actin in the same samples. The quantified data were presented as the ratios of RALDH1/β-Actin as shown in Fig. 1C. The ratios of ZF rats are significantly higher than that of ZL rats, demonstrating that the elevation of Raldh1 mRNA corresponds with an induction of RALDH1 protein.
The adenovirus-mediated Raldh1 mRNA expression. B. Immuno-blot of the over-expression of RALDH1 protein in INS-1 cells. Whole cell lysates (50 µg/sample) of the control cells (lane 1), cells infected by the indicated pfu of Ad-β-gal (lane 2) or Ad-Raldh1 (lanes 3–5) were separated in 8% SDS protein gels, and transferred to the PVDF membranes. Primary antibodies to RALDH1 (1∶1000 dilution in TBST containing 5% dry milk), and to β-Actin (1∶1000 dilution in TBST containing 5% bovine serum albumin) were recognized by goat anti-rabbit IgG conjugated to horseradish peroxidase, and visualized by chemiluminescence. The films were scanned and presented as described in the Material and Methods. C. RAL only induced Srebp-1c expression in cells over-expressing RALDH1, but not β-gal. D. RA induced Srebp-1c expression in cells over-expressing either β-gal or RALDH1. Results were presented as means ± SD of fold inductions (* for comparing the different dosages of RAL in cells infected by Ad-Raldh1 using one way ANOVA, n = 3, all p<0.05).
Retinoids induced the expression levels of Cyp26a1 mRNA in primary rat hepatocytes
To demonstrate that retinoids catabolism can contribute to the regulation of gene expression in primary rat hepatocytes, the expression levels of Cyp26a1 mRNA (a RA responsive gene) in response to retinol (ROL) and retinal (RAL) treatments were determined using real-time PCR. Fig. 2A shows a 24-h time course of the expression levels of Cyp26a1 mRNA in Sprague-Dawley (SD) rat primary hepatocytes treated with 5 µM RA. More than 1000-fold induction of Cyp26a1 mRNA level was observed at 3 h after the RA treatment, the earliest time point checked. The Cyp26a1 mRNA level was further induced at 6 h and maintained elevated at 12 h after treatment. At 24 h after the treatment, its level dropped comparing to that at 12 h, but still remained significantly higher than that at time 0.
A. In ZL rats, the hepatic expression of Srebp-1c is controlled by the RA production and insulin stimulation. The SREBP-1c also regulates RA production via the induction of Raldh1 expression until a homeostasis is reached. B. The hyperphagia of ZF rats due to the leptin receptor deficiency causes the over-supply of dietary VA, and hyperinsulinemia. The possible elevation of RA production in combination with insulin stimulation leads to higher expression of hepatic Srebp-1c in ZF rats, which disrupts the homeostasis. The expression of Raldh1 mRNA is further induced by SREBP-1c. Therefore, the hepatic expression of Srebp-1c in ZF rats is maintained at a higher level than that in ZL rats. The consequence is the elevated hepatic lipogenesis in ZF rats. The up arrows next to the texts indicate induction. The intensified weight of the lines indicates the induction of that part of the pathway. The question marks indicate the steps remained to be confirmed in this hypothesis.
To determine the effects of ROL and RAL on the induction of Cyp26a1 expression, primary rat hepatocytes from SD rats were treated with increasing concentrations of ROL or RAL for 6 h. As shown in Fig. 2B, the levels of Cyp26a1 mRNA were induced by ROL and RAL in a dosage dependent manner. ROL and RAL started to induce Cyp26a1 expression at 2 μM and 0.02 μM, respectively. When the induction folds of Cyp26a1 expression in response to ROL and RAL treatments were compared, RAL groups had significantly higher values than ROL groups did at concentrations of 0.02 μM, 0.2 μM, 2 μM, and 20 μM. It suggests that RAL is more readily converted to RA than ROL to induce Cyp26al expression.
Inhibition of RAR activation blocked the RAL-mediated induction of Cyp26a1 expression in primary rat hepatocytes
We have shown that the activation of RAR or/and RXR can induce Cyp26a1 expression [16]. To confirm the involvement of RAR activation in the RAL-mediated induction of Cyp26a1 expression, primary SD rat hepatocytes were treated with increasing concentrations of Ro 41–5253, a specific antagonist for RAR activation [34]. As shown in Fig. 2C, RAL at 2 µM dramatically induced the Cyp26a1 expression, further confirming the result shown in Fig. 2B. This induction was dose-dependently inhibited with increasing concentrations of Ro 41–5253. When the concentration of Ro 41–5253 reached 10 μM or higher, the induction of Cyp26a1 expression by RAL was completely suppressed. This reverse association between the concentration of Ro 41–5253 and the induction of Cyp26a1 expression by RAL demonstrated that the inhibition of RAR activation disrupted the retinoid-mediated induction of Cyp26a1 expression in primary hepatocytes, indicating the important role of RAR activation in this induction.
Retinoids induced the expression levels of Cyp26a1 mRNA and CYP26A1 protein in HL1C rat hepatoma cells through the activation of RAR and RXR
To screen for cultured cell lines which we can study the effects of retinoid metabolism on the expression of RA responsive gene, we analyzed the expression of Cyp26a1 mRNA and CYP26A1 protein in response to ROL, RAL and RA treatments in HL1C rat hepatoma cells [35]. Fig. 3A shows the expression levels of Cyp26a1 mRNA in HL1C cells treated with the indicated concentrations of retinoids. ROL, RAL and RA started to induce Cyp26a1 mRNA expression at 20 μM, 2 μM, and 0.2 μM, respectively, demonstrating the different potencies of these retinoids. At 2 μM or 20 μM, the fold induced by RAL was comparable to that by RA. The highest induction fold of Cyp26a1 mRNA mediated by RA in HL1C cells was near 300-fold which was much lower than that in primary hepatocytes (1000-fold) as shown in Fig. 2. The effects of RAL and RA on Cyp26a1 mRNA expression in HL1C cells over a 24-h time period were also examined as shown in Fig. 3B. Both RAL and RA induced Cyp26a1 mRNA expression as early as 3 h. At 6 h after the treatment, the elevated level of Cyp26a1 mRNA began to drop in comparison to its level at 3 h. At 9, 12, and 24 h time points, its level further declined, but remained significantly higher than that at time 0. The induction was still elevated at 24 h after treatment.
To examine whether the activation of RAR or RXR was sufficient for the induction of Cyp26a1 mRNA expression, HL1C cells were treated with TTNPB, LG268, and T1317, agonists for RAR, RXR and LXR activation, respectively. As shown in Fig. 3C, the induction folds of the TTNPB, LG268 and T1317 groups are 183.0±60.0, 8.0±3.8, and 1.0±0.4. These results demonstrated that the activation of RAR and RXR, but not LXR, induced the Cyp26a1 mRNA expression in HL1C cells, indicating that RA activated both RAR and RXR to induce Cyp26a1 mRNA expression. The induction fold of TTNPB group is much higher than that of LG268 group, similar to that observed in primary hepatocytes [16].
Fig. 3D shows the expression levels of CYP26A1 protein in HL1C cells treated with RAL or RA for 12 and 24 hours. The equal loading of protein samples was confirmed by the similar levels of β-Actin protein among the samples. This result demonstrated that the induction of Cyp26a1 mRNA resulted in significant elevation of CYP26A1 protein at both time points. All these results suggest that ROL and RAL may be converted to RA, which causes the RAR activation and the subsequent induction of Cyp26a1 mRNA and CYP26A1 protein levels in HL1C cells.
RA, but not RAL, induced the Srebp-1c expression through the activation of RXR, but not RAR, in INS-1 rat insulinoma cells
We have screened the cultured cell lines in which RAL does not induce Srebp-1c expression for a tool to study the conversion of RAL to RA, and shown that RA, but not RAL, induced the Srebp-1c expression dose-dependently in 833/15 INS-1 cells [16]. To further confirm that INS-1 cells are an ideal tool to study the conversion of RAL to RA, we analyzed the effects of RAL and RA on Srebp-1c expression in 834/40 INS-1 cells, another clonal INS-1 cells [36]. Fig. 4A shows that RA started to significantly induce the Srebp-1c expression at 0.1 μM while RAL did not change its expression at any concentration tested. Fig. 4B shows the induction of the Srebp-1c expression by RA in 834/40 INS-1 cells over a 24-h time period. RA, but not RAL, induced the Srebp-1c expression as early as 3 h and peaked at 6 h. At 9, 12, and 24 h, the induction folds declined in comparison to that at 3 and 6 h, but still maintained significantly higher than that at 0 h. These results and the results shown previously [16] indicate that INS-1 cells may lack the enzymatic activities that convert RAL to RA.
To determine whether the activation of RAR or RXR mediates the effects of RA on the Srebp-1c expression, INS-1 cells were treated with TTNPB (1 μM), LG268 (1 μM), or TTNPB + LG268 for 6 h. Fig. 4C shows that LG268 and LG268 + TTNPB induced the Srebp-1c expression while TTNPB alone did not induce it. Thus, RXR activation mediates the RA-induced Srebp-1c expression in INS-1 cells. All these results demonstrated that the RA-mediated activation of RXR caused the induction of Srebp-1c in INS-1 cells, which makes INS-1 cell lines an ideal tool to study the role of RA production in the regulation of lipogenic gene expression.
The over-expression of RALDH1 resulted in the RAL-mediated induction of Srebp-1c in INS-1 cells
To further study the effects of the elevated Raldh1 mRNA expression in ZF hepatocytes, its full length cDNA was cloned and inserted it into pACCMV5 for the generation of recombinant adenovirus Ad-Raldh1. As shown in Fig. 5, INS-1 cells infected with Ad-Raldh1 had the over-expression of Raldh1 mRNA (Fig. 5A) and RALDH1 protein (Fig. 5B). The equal loading of the protein samples was confirmed by the similar levels of β-Actin protein among the samples. In INS-1 cells infected with Ad-Raldh1 for overnight (∼18 h), but not in those infected with Ad-β-gal, RAL started to significantly induce the Srebp-1c expression at both 0.3 μM and 1 μM (Fig. 5C). On the other hand, RA induced the Srebp-1c expression in cells infected by either Ad-β-gal or Ad-Raldh1 (Fig. 5D). In those cells with the over-expression of RALDH1, the induction folds of Srebp-1c by RAL treatment were comparable to that by RA. These results indicate that the over-expression of RALDH1 introduced the RAL-mediated induction of Srebp-1c in INS-1 cells, indicating the successful conversion of RAL to RA.
In insulin resistant animals, hyperinsulinemia is associated with the elevated hepatic gluconeogenesis and lipogenesis, which should have been respectively suppressed and stimulated by insulin [37]. Our lab has shown that retinoids regulate the expression levels of genes involved in hepatic glucose and fatty acid metabolism. These regulations contributed to the modulation of the insulin-mediated expression of these genes in primary hepatocytes [14]–[16]. The implication of these observations is that the dynamic regulation of RA production may be responsible for the hepatic insulin resistance.
To confirm that the treatments of ROL and RAL can regulate gene expression probably through the production of RA, we analyzed the expression levels of Cyp26a1, an RA responsive gene [38], in primary hepatocytes (Fig. 2). Retinoids induced the expression of Cyp26a1 in a dose- and time-dependent manner. This induction can be blocked in the presence of RAR antagonist, indicating the requirement of RAR activation. This result and the fact that RAL induced the Cyp26a1 expression indicate that RA production occurs.
To find the cultured cell lines that can be used as tools for further studies of the retinoid catabolism, we have analyzed the retinoid-mediated gene expression in HL1C rat hepatoma cells (Fig. 3). ROL, RAL, and RAL also induced the expression levels of Cyp26a1 mRNA in HL1C cells in a dose- and time-dependent manner, which is similar to the results obtained in primary hepatocytes. This induction is mediated by the activation of RAR and RXR. The induction of Cyp26a1 mRNA results in the elevated levels of CYP26A1 proteins in HL1C cells. However, the elevated levels of CYP26A1 protein are not as high as that of Cyp26a1 mRNA at 12 and 24 hours (Fig. 3B and 3D), indicating the potential roles of protein translation and/or stability in the determination of the CYP26A1 protein levels in HL1C cells. The maximal induction folds by reintoids are higher in primary hepatocytes than in HL1C cells. ROL and RAL respectively induce the expression of Cyp26a1 mRNA at 2 and 0.02 µM in primary hepatocytes and at 20 and 2 µM in HL1C cells. These results indicate that primary hepatocytes seem to be more readily to convert ROL to RAL and then to RA. Alternatively, HL1C cells are more readily to convert RAL to ROL so that less RA is generated. Whether it is true or not deserves further investigation. It also indicates that HL1C cells can be used as a tool to study the metabolism of retinoids and its effects on the regulation of the expression of RA responsive genes.
It has been shown that the short chain dehydrogenase/reductase family 16C member 5 (SDR16C5), RDH2, RDH10, RALDH1-4 are expressed in the liver [33]. Since Raldh4 mRNA (encoding RALDH4) is for the production of 9-cis RA [27], we focused on Rdh2 and Raldh1. Their mRNA levels in both freshly isolated and cultured primary hepatocytes of ZL and ZF rats were compared. The mRNA level of Raldh1, but not that of Rdh2, is higher in ZF than that in ZL rat hepatocytes. In addition, the hepatic RALDH1 protein level is also higher in ZF rats than in ZL rats (Fig. 1). The elevated expression levels of Raldh1 mRNA and RALDH1 protein levels may be resposible for the altered hepatic lipogenesis in ZF rats. To test the hypothesis, we made recombinant adenovirus Ad-Raldh1 to over-express RALDH1 protein in INS-1 rat insulinoma cells. We have shown previously [16] and here (Fig. 4) that RA, but not RAL, induced Srebp-1c expression in INS-1 cells. It seems that INS-1 cells lack the enzymatic activities converting RAL to RA. This makes INS-1 cells an ideal tool to test our hypothesis that the elevated expression of RALDH1 in ZF rats causes the increase of RA production, and in turn, the induction of RA responsive gene expression. After the over-expression of Raldh1 mRNA and RALDH1 protein in INS-1 cells, RAL started to induce the Srebp-1c expression as RA did. This indicates that RAL was oxidized to RA by RALDH1 before the induction of Srebp-1c. The elevated levels of hepatic Raldh1 mRNA and RALDH1 protein in ZF rats may contribute to the regulation of hepatic lipogenesis.
It has been shown that the mRNA level of Raldh1 is elevated in the kidney of db/db mice [39]. Both db/db mice and ZF rats develop obesity due to the mutation of leptin receptor [4]. It has been shown that ICR mice fed high cholesterol diet have the elevated expression levels of Raldh1 and Raldh2. This is attributed to the oxysterol-induced expression of SREBP-1c which directly binds to the sterol regulatory response elements (SREs) at the proximal promoter regions of Raldh1 and Raldh2, and induces their expression [32]. Since the expression of Srebp-1c is induced by insulin [40] and RA [16] in primary hepatocytes, the regulation of Raldh1 expression becomes a converge point for a feed-forward mechanism by which VA status regulates lipogenesis in the liver. It is reasonable to hypothesiz that in hepatocytes of ZL rats, RA derived from ROL synergizes with insulin to induce the expression of Srebp-1c mRNA (Fig. 6A). The mature SREBP-1c protein supports the expression of RALDH1, which promotes the production of RA to maintain the homeostasis of Srebp-1c expression in the liver of ZL rats (Fig. 6A). ZF rats are hyperphagic due to the defect of leptin receptor, which leads to the development of obesity, insulin resistance, hyperinsulinemia and hyperlipidemia [41], [42]. We have shown the ZF rat hepatocytes have elevated expression levels of Srebp-1c and Fas mRNA [43]. We think that the excessive supply of dietary VA caused by hyperphagia and hyperinsulinemia in ZF rats probably work together to trigger an elevation of RA production and an induction of Srebp-1c expression in their hepatocytes, respectively. The elevation of the mature SREBP-1c protein induces the expression of Raldh1 mRNA, which leads to more RA production, and further enhances the expression levels of Srebp-1c and its down-stream lipogenic genes. This creates a feed-forward mechanism by which the Srebp-1c expression is maintained at a higher level in the liver of ZF rats (Fig. 6B). Whether the proposed feed-forward mechanism is true or not, and what mechanism is responsible for the up-regulation of Raldh1 expression in the ZF rat liver deserve further investigation.
In summary, the results shown here suggest that ROL and RAL are metabolized into RA to regulate gene expression in rat primary hepatocytes and hepatoma cells. In primary rat hepatocytes, the responsible enzymes are most likely RDH2 and RALDH1. The expression of Raldh1 mRNA is higher in primary hepatocytes from ZF rats than that from ZL rats, which leads to the elevated RALDH1 protein levels in the liver of ZF rats. Over-expression of RALDH1 introduces the RAL-mediated induction of Srebp-1c in INS-1 cells. Thus, we hypothesize that the change of RA production from the over-supply of dietary VA due to the hyperphagia of ZF rats results in higher Srebp-1c expression in ZF hepatocytes. The elevated SREBP-1c expression can further induce Raldh1 expression to create a feed-forward mechanism that could be one of the reasons responsible for the increased lipogenesis in the liver of ZF rats.
Materials and Methods
The reagents for primary hepatocyte isolation and culture have been published previously [44]. Source of LG268 was reported previously as well [15]. All other compounds were purchased from Sigma (Saint Louis, MO) unless described otherwise. The reagents for cDNA synthesis and real-time PCR were obtained from Applied Biosystems (Foster city, CA). Medium 199, liver perfusion medium and liver digest buffer were obtained from Invitrogen (now Life Technologies, Grand Island, NY 14072). Dulbecco's Modification of Eagle Medium (DMEM), RPI-1640 medium, fetal bovine serum, antibiotics, and trypsin-EDTA solution were obtained from Fisher Scientific (Pittsburgh, PA 15275).
Male Sprague-Dawley (SD) rats were purchased from Harlan Breeders (Indianapolis, IN). Male Zucker lean (ZL) and ZF rats were bred at UTK. Rats were housed in colony cages, and fed a standard rodent diet before isolation of primary hepatocytes. All procedures were approved by the Institutional Animal Care and Use Committee at the University of Tennessee at Knoxville (Protocol numbers 1642 and 1582).
Cultures of primary rat hepatocytes, HL1C rat hepatoma cells, 293 human embryonic kidney cells, and INS-1 rat insulinoma cells
Methods for the primary rat hepatocyte preparation and analysis of RNA were described previously [44]. For the hepatocyte isolation and pretreatment, the rat was euthanized with carbon dioxide. A catheter was inserted into portal vein and connected to a peristaltic pump with liver perfusion medium and liver digestive buffer. The inferior vena cava was cut open to allow the outflow of the media at 10 ml/minute. After completion of the digestion, the liver was excised and put into a tissue culture dish containing liver digest buffer for removing the connection tissues and allowing the release of hepatocytes. The medium containing hepatocytes was filtered through a cell strainer (100 µm), and precipitated by 50× g centrifugation for 3 minutes (min). The cell pellet was washed twice with DMEM containing 5% fetal bovine serum (FBS), 100 U/ml sodium penicillin, and 100 µg/ml streptomycin sulfate after re-suspension and precipitation. After the wash, the isolated hepatocytes were plated onto 60-mm collagen type I coated dishes at 3×106 cells/ per dish and incubated in 4 ml of the same medium at 37°C and 5% CO2 for 3 to 4 hours (h). The attached cells were washed once with 4 ml of PBS, and incubated in medium A (Medium 199 supplemented with 100 nM dexamethasone, 100 nM 3, 3′, 5-triiodo-l-thyronine (T3), 100 units/ml penicillin, and 100 μg/ml streptomycin sulfate) containing 1 nM insulin for overnight (14–16 h) until being used for the indicated experiments.
The HL1C cells [35] which was stably transfected with a reporter gene construct containing a fragment of Pck1 promoter sequence were kindly provided by Dr. Donald K. Scott at University of Pittsburgh. They were maintained in DMEM containing 4.5 g/L glucose, 4% FBS, 100 U/ml of penicillin and 100 μg/ml streptomycin sulfate. They were incubated in 60 mm dishes with serum free DMEM containing the indicated reagents shown in the figure legends for the indicated length of time before being subjected to analysis.
The 833/15 and 834/40 clonal insulinoma INS-1 cells [36] were cultured in medium B (RPMI 1640, 2 mM L-glutamine, 1 mM Na-pyruvate, 50 μM β-mercaptoethanol, 100 U/ml of penicillin and 100 μg/ml streptomycin sulfate) containing 10% FBS as described previously. For the treatment, INS-1 cells in 60 mm dishes were incubated in medium B containing the indicated reagents shown in the figure legends for the indicated length of time before being subjected to analysis.
The HEK 293 cells were cultured in DMEM containing 4.5 g/L glucose, 8% FBS, 100 U/ml of penicillin and 100 μg/ml streptomycin sulfate at 37°C and 5% CO2.
RNA extraction and quantitative real-time PCR
Total RNA was extracted from the treated cells on one 60 mm dish using 1 ml of RNA STAT 60 reagent (TEL-TEST, Inc, Friendswood, TX) according to the manufacture's protocol. The contaminated DNA was removed using the DNA-freeTM kit (Applied Biosystems). First strand cDNA was synthesized from 2 µg of DNA-free RNA with random hexamer primers using cDNA synthesis kit (Applied Biosystems). The real-time PCR primer sequences for detecting Cyp26a1 [45], Srebp-1c [16], and Raldh1 [46] have been reported previously. The primers for detecting Rdh2 (sense 5′-CAAGTTCTTCTACCTCCCCATGA-3′, and anti-sense 5′-TCCAGTAGAAAAGGGCATCCA-3′) were designed using software Primer Express 3.0 (Applied Biosystems). Each SYBR green based real-time PCR reaction contains, in a final volume of 14 µl, cDNA from 14 ng of reverse transcribed total RNA, 2.33 pmol primers, and 7 µl of 2× SYBR Green PCR Master Mix (Applied Biosystems). Triplicate PCR reactions were carried out in 96-well plates using 7300 Real-Time PCR System. The conditions are 50°C for 2 min, 95°C for 10 min, followed by 40 cycles of 95°C for 15 seconds (s) and 60°C for 1 min. The gene expression level was normalized to that of invariable control gene, 36B4. Data are presented as either minus Δ cycle threshold CT [43] or the induction fold for which the control group is arbitrarily assigned a value of 1 using ΔΔCT method as previously described [44].
Cloning of the rat Raldh1 cDNA and generating Ad-Raldh1 recombinant adenovirus for the over-expression of RALDH1
To clone the rat Raldh1 cNDA, sense 5′-AGCCAAACCAGCAATGTCTTC-3′, and anti-sense 5′-CACTCTGCTTCTTAGGAGTTC-3′ primers were designed based on the its mRNA sequence (reference number NM_022407.3) from the National Center for Biotechnology Information. The Raldh1 cDNA containing the entire coding sequence was amplified using SD rat primary hepatocyte cDNA as a template. The amplicon was ligated into pCR®2.1 vector through TA Cloning® Kit (Invitrogen) and processed according to the manufacture's protocol. The full length cDNA with the correct sequence confirmed by DNA sequencing was inserted to pACCMV5 for making pACCMV5-Raldh1.
To generate Ad-Raldh1 recombinant adenovirus, plasmids pACCMV5-Raldh1 and JM17 were co-transfected into 293 cells using FuGENE®6 Transfection Reagent (Roche Applied Science, Indianapolis, IN). Transfected 293 cells were incubated in DMEM containing 2% FBS at 37°C and 5% CO2 until the formation of the plaques due to the lysis of the cells. The crude lyaste was collected and stored at −80°C. The presence of Raldh1 cDNA sequence in the viral DNA was confirmed by PCR.
Preparation and purification of recombinant adenoviruses
To generate the crude lysate, HEK 293 cells grew in 100 mm tissue culture plates at 80% confluence were infected by the original crude lysate containing Ad-Raldh1. The ratio of the medium to crude lysate is 10 to 1. After the lysis of the cells at around 48 h post infection, the cell culture medium (the crude lysate) was collected, and stored at −80°C until being used.
For purification of the recombinant adenovirus, NP-40 was first added into the crude lysate to reach the final concentration at 0.5%. The mixture was shaken gently at room temperature for 30 min and subjected to centrifugation at 8,000 rpm and 4°C for 15 min. The supernatant was transferred to a clean bottle, and 0.5× volume of 20% PEG8000/2.5 M NaCl was added. The preparation was shaken gently at 4°C overnight. The resulting mixture was transferred to centrifuge bottles and spun at 13,000 rpm at 4°C for 15 min. The precipitated pellet was re-suspended in a small volume of PBS (2–3 ml), and spun at 8,000 rpm and 4°C for 10 min to remove insoluble matters. Solid CsCl was added to the supernatant until its final density reached 1.34 g/ml. The mixture was spun at 90,000 rpm and 25°C for 3 h using OptimaTM MAX-XP Ultracentrifuge (Beckman Coulter, Inc.). The corresponding band containing pure viral particles was collected in a total volume less than 1 ml for desalting. The PD-10 column SephadexTM G-25 M (Amersham Pharmacia Biotech AB, Sweden) was equilibrated with 5 ml PBS. The purified virus in CsCl solution was loaded onto the column and eluted with 5 ml PBS. The flow through was collected into ten fractions. The optical densities (OD) at 260–nm of these fractions were determined using Spectronic® GENESYSTM 5 Spectrophotometer (Thermo Scientific). The fractions containing significant values of absorbance (usually at around fractions 7–9) were collected and pooled. Bovine serum albumin and glycerol were added to the pooled solution to make the stock viral solution with the final concentrations of them at 0.2% and 10%, respectively. After the filtration of the stock solution for sterilization, its OD was determined to estimate the plaque forming units (pfu). We used that 1 OD equals to 1×1012 pfu/ml. The final purified virus stock was frozen at −80°C until being used in the indicated experiments.
Immuno-blot and quantification of the protein bands
INS-1 cells were infected with purified Ad-Raldh1 for 24 h. The cells in 60 mm dishes were washed once with 3 ml PBS and scrapped from the dish into 400 μl of whole-cell lysis buffer (1% Triton X-100, 10% glycerol, 1% IGEPAL CA-630, 50 mM Hepes, 100 mM NaF, 10 mM EDTA, 1 mM sodium molybdate, 1 mM sodium β-glycerophsphate, 5 mM sodium orthovanadate, 1.9 mg/ml aprotinin, 5 μg/ml leupeptin, 1 mM benzamide, 2.5 mM PMSF, pH 8.0). The lysates were placed on ice for at least 20 min before they were subjected to centrifugation at 13,000 rpm for 20 min. The protein concentration in the supernatant was determined with PIERCE BCA protein assay kit (Rockford, IL). Proteins (40 μg/lane) in whole cell lysates were separated on SDS-PAGE, transferred to BIO-RAD Immuno-Blot PVDF membrane (Hercules, CA) and detected with primary antibodies to RALDH1 (for Fig. 5, catalog #2052-1, Epitomics, Burlingame, CA 94010), RALDH1 (for Fig. 1, catalog #AP1465a, Abgent, San Diego, CA 92121), CYP26A1 (catalog # CYP26A11-A, Alpha Diagnostics International, TX 78244), and β-Actin (#4970 s, Cell Signaling Technology, Danvers, MA 01923) according to the protocols provided by the manufacturers. Bound primary antibodies were visualized by chemiluminescence (ECL Western Blotting Substrate, Thermo Scientific) using a 1∶2,000 dilution of goat anti-rabbit IgG (#7074P2, Cell signaling Technology) conjugated to horseradish peroxidase. Membranes were exposed to X-ray films (Phenix Research Products, Candler, NC) for protein band detection. The films were scanned using an HP Scanjet 3970 (Palo Alto, CA 94304) and stored as Tagged Image File Format (TIFF). The ImageJ Software (http://rsbweb.nih.gov/ij/) was used to determine the density of a protein band by subtracting the background in an area of the same size immediately in front of protein band. The ratio of the densities of RALDH1 and β-Actin in the same sample was calculated and used for statistical analysis.
Data are presented as means ± S.D. The number of experiments represents the independent experiments using hepatocytes isolated from different animals or indicated cells cultured in different days. Levene's test was used to determined homogeneity of variance among groups using SPSS 19.0 statistical software and where necessary natural log transformation was performed before analysis. An independent-samples t-test was used to compare two conditions. Multiple comparisons were analyzed by one-way analysis of variance (ANOVA) using least significant different (LSD) when equal variance was assumed, and Games-Howell test was used when equal variance was not assumed. Differences were considered statistically significant at P<0.05.
The authors thank Dr. Hollie Raynor for checking the spell and grammar of this manuscript.
Author Contributions
Conceived and designed the experiments: YL GC. Performed the experiments: YL YZ RL WC MH RZ GC. Analyzed the data: YL GC. Contributed reagents/materials/analysis tools: YL YZ RL WC MH RZ GC. Wrote the paper: YL GC.
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