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Kerry Francis Bullmore Packer, AC (17 December 1937 – 26 December 2005) was an Australian media tycoon. The Packer family company owned a controlling interest in ...Kerry Francis Bullmore Packer AC (17 December 1937 – 26 December 2005) was an Australian billionaire media magnate. He owned the Nine Network, and is famous for ...The Australian business tycoon Kerry Packer, who has died aged 68 following a long period of ill health, loved gambling, whether in the boardroom or the casino.Enjoy this look at Kerry Packer, media tycoon and business figure who, at the time of his death, was one of the richest men in Australia, on Biography.com.And in a new book by Michael Stahl called Kerry Packer — Tall tales & true stories the larger-than-life fables of Mr Packer come to life. Mr Stahl was a journalist ...While most famous blackjack players accumulate their wealth on the tables and through books, Kerry Packer is an exception to the norm. Packer built a media empire ...1/12/2006 · If there is one story which sums up the legend of Kerry Packer better than any other, it's the tale of the big man saying "toss you for it" to a ...TELEVISION executive David Leckie has inflamed his bitter rift with James Packer by claiming the mogul's late father, Kerry, was "overrated" and knew less about ...It is fairly safe to assume no-one has referred to the late Kerry Packer as a hippy before, but that is how former daughter-in-law Jodhi Meares fondly remembers him.11/2/2013 · MEDIA mogul Kerry Packer not only knew the thief who stole $5.4 million in gold bars from his office, they dined at the same restaurants, at the same time ...
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Conference theme: Equitation Science - The Road Ahead - will showcase how equitation science has developed as a discipline and how new innovations in technology can be used to improve practice.
ISES UK 2012 took place in Olympic year in the UK - and marked the tenth anniversary since Equitation Science was first recognised as a field of study. The three day international conference was held from July 18th to 20th, hosted by the University of Edinburgh's Royal (Dick) School of Veterinary Studies in bonny Scotland.
Prof Natalie Waran - Chair of the Local Organising Committee of the ISES 2012 Conference
Mike Steele - Event Organiser (British Society of Animal Science, BSAS)
For more information contact: ISES President Andrew McLean PhD email@example.com
Photos: Jane Barr

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ARLINGTON, Texas -- Pitcher C.J. Wilson said Tuesday that the Texas Rangers made him a long-term contract offer a few weeks ago, but that it's too late in the process to get anything done before Opening Day.
"They did eventually make an offer," Wilson said during an appearance on the Ben & Skin Show on ESPN 103.3 FM in Dallas. "But it didn't really make any sense for me to bite at the first offer. I was like, 'Well, maybe if you guys would have made that offer in January we would have had time to work it out.' It will take a little longer than two or three days or a week."
Wilson has said this spring that he doesn't want any contract talks distracting from his preparation and the team's goals this season. But he added Tuesday that it doesn't mean his agent might not talk with the team at some point. Rangers assistant general manager Thad Levine said the club doesn't want to distract from anything, either, but is open to talking with Wilson's agent during the season.
Wilson, who is making $7 million in 2011 just one season removed from joining the starting rotation, will be a free agent at the end of the season. Wilson was 15-8 with a 3.35 ERA in 33 starts (204 innings) in 2011. He had 170 strikeouts and 93 walks and heads into 2011 as the No. 1 starter on the staff. He'll toe the rubber for Opening Day on Friday.
"We have a good relationship with his agent and we've had productive dialogue," said Levine, who happened to sit by Wilson on a flight from Phoenix to Dallas on Monday afternoon. "We certainly don't feel it's dead in the water."
Levine said the team typically doesn't complete many long-term deals during the season, but added that this was an unusual case in that Wilson will become a free agent at the end of the season.
"Things were trying to come together too late for it to really get worked out," Wilson said. "A multi-year contract is a very complicated scenario."


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Posted by francoud on
I posted this in the "general" forum:
I think that a recently added feature in the user.module can give some problem: when admin activates a newly user-created account, the "last access" field is set to time()--> so now it's not possible to understand if that user had really logged in, or never...
Please use...
AjK commented
Please use http://drupal.org/node/add/project-issue/drupal to create a bug report rather than the forums. And it's wise to avoid cross posting forums like this.
This is a duplicate of
BioALIEN commented
This is a duplicate of #295839: Bug in user.module "last access"
iScene.eu :: UK Drupal Consultancy


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The Mighty Walzer
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In 1950s Manchester, England, Oliver Walzer, a shy Jewish boy, comes of age in a household dominated by women and finds that he has a natural talent for ping-pong, but things do not go as smoothly for him when it comes to women and sex.


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Location:This is a small farm set in the heart of the Chianti hills, surrounded by forests, vineyards and olive groves.
Relax, peace and a wonderful panorama view in a pleasant and comfortable homely atmosphere will give you the possibility to enjoy your holiday in a real Tuscan farmhouse.
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Two big panoramic terraces, pool, relax area with hammocks, wi-fi area, restaurant, mountain bikes and more more get your holiday in Tuscany unforgettable.
The owners, Fabio and Ilenia, live and work here and they will help you during the stay for any need , suggestion or question giving to your holiday an authentic feeling.
Price includes: -Sheets for bed - electricity-water-gas* consumption - final cleaning (if the apartment is left in decent condition) - one wi-fi card for the Internet connection in the wi-fi area - use of all entertainment- in our farmhouse (swimming pool, common areas, playgrounds, mini volley pitch, mini football pitch,…) Price doesn't include: - Towels ( except our offer) euro 8,00 per person - Heating (extra charge based on energy consumption); we remind you that according to Italian law, heating is permitted from the 1stNovember to the 15th of April every year - Tourist tax: euro 1,50 per person per day (up to 5 days per calendar month; children under 12 years free) CAUTION DEPOSIT Euro 150,00 to be paid in cash at the check-in.
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Right now, your pet is wondering, "What can I do to reduce my carbon pawprint?" Here at EnviroCitizen.org, we know that natural pet care is easy and affordable — just take a look at these simple tips below, and help your pets stay healthy while reducing their impact on the environment.
Choose a Recycled Pet
Each year, between 3 and 4 million dogs and cats are put to death in shelters throughout the United States. Despite this tragic fact, people still fork over thousands of dollars for a purebreed pet, ignoring the realities of inbreeding, factory-like conditions, overbreeding and other perils. The greenest and kindest way to get a dog or cat is to skip the overpriced breeders and get a pet from a pet shelter.
Spay or Neuter Your Pets
Here's another rather unfortunate fact: Each hour in the United States, about 5,500 puppies and kittens are born. Yes, that's right, every hour. Compare that number to the 415 humans born each hour, and you can see why it's important to spay or neuter your pet. If you think the procedure is unnatural, perhaps you can find something natural about a puppy being killed by a car, or a kitten starving to death — I can't.
Pets Are Not Wildlife
On the contrary, pets eat wildlife. An estimated 39 million birds are eaten each year by domestic cats — and that's just in the state of Wisconsin! Multiply that by all 50 states, and you can see how big the problem is. Fido and Fluffy may seem cute to you, but to birds and wildlife, they're just another hungry predator. Keep your cats indoors, and keep dogs on a leash when you're out hiking through the woods.
Choose Organic Pet Foods
If you think the standards for labeling and ingredients in human processed foods are low — and they are — just imagine what's allowed in the pet food industry. Some of the animals used in pet foods are diseased or already dead when they're made into pet food. By purchasing organic or natural pet foods that contain healthier ingredients, you're doing your pet and the planet a big favor, as well as sending a message to the pet food industry that they need to clean up their act.
Natural Pet Care and the Poop Patrol
All responsible pet owners want to clean up after their pets. Cat litter, however, comes with a host of environmental and health problems. The clay for kitty litter is usually strip-mined — an environmental nightmare — and contains silica dust that's been implicated in serious health problems. Green cat litters are made of recycled newspaper, pine, corn or wood chips. And your dog's poop bag is probably made of petroleum-based plastics. Instead, try biodegradable bags, available from most pet supply stores. But if you really want to go whole-hog, try a pet waste septic system.
Safer, Natural Pet Toys
Some pet toys are made of petroleum-based plastics or contain harmful ingredients like lead or BPA (bisphenol A, a synthetic compound suspected of causing cancer, nerve damage and other health problems). But many manufacturers are now making chew toys and other items with organic or natural materials like hemp, cotton, plant-based dyes and safer forms of plastic.
What a Glossy Coat!
Just like many low-end human soaps and shampoos, pet products like shampoo and conditioners are often made with some cheap or possibly unhealthy ingredients. Instead, try safer alternatives to give your pet a clean, shiny coat.
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Dogs, I'm told, spend about 12 hours a day sleeping. That's about the best argument I've ever heard for bedding made of organic cotton and other natural materials. And some bedding manufacturers fill bedding with recycled content. And, of course, there are dozens of makers of all-natural, organic or recycled pet collars, leashes, scratch posts and other accessories.


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The triglycerides in chylomicrons are hydrolyzed by lipoprotein lipase (LpL) along the luminal surface of the capillaries. However, the endothelial cell molecule that facilitates chylomicron processing by LpL has not yet been defined. Here, we show that glycosylphosphatidylinositol-anchored high density lipoprotein–binding protein 1 (GPIHBP1) plays a critical role in the lipolytic processing of chylomicrons. Gpihbp1-deficient mice exhibit a striking accumulation of chylomicrons in the plasma, even on a low-fat diet, resulting in milky plasma and plasma triglyceride levels as high as 5,000 mg/dl. Normally, Gpihbp1 is expressed highly in heart and adipose tissue, the same tissues that express high levels of LpL. In these tissues, GPIHBP1 is located on the luminal face of the capillary endothelium. Expression of GPIHBP1 in cultured cells confers the ability to bind both LpL and chylomicrons. These studies strongly suggest that GPIHBP1 is an important platform for the LpL-mediated processing of chylomicrons in capillaries.
Dietary fats in mammals are packaged by the intestine into chylomicrons, which are large, triglyceride-rich lipoproteins (Havel and Kane, 2001). After reaching the bloodstream, the triglycerides within chylomicrons are hydrolyzed by lipoprotein lipase (LpL) along the luminal surface of capillaries, mainly in heart, skeletal muscle, and adipose tissue (Havel and Kane, 2001; Nestel et al., 1962). In those tissues, LpL is synthesized and secreted by the parenchymal cells (i.e., myocytes and adipocytes) and then translocated to the lumen of capillaries, where lipolytic processing of chylomicrons occurs (Goldberg, 1996). The liver also secretes triglyceride-rich lipoproteins called very low density lipoproteins (VLDL). The initial phase of VLDL metabolism closely resembles that of chylomicrons, with LpL-mediated hydrolysis of triglycerides in the heart, skeletal muscle, and adipose tissue (Havel and Kane, 2001; Nestel et al., 1962).
LpL is thought to bind to the surface of capillaries through the interaction of its positively charged heparin-binding domains (Sendak and Bensadoun, 1998) with negatively charged heparan sulfate proteoglycans (HSPGs) expressed on the surface of endothelial cells (Goldberg, 1996; Parthasarathy et al., 1994). This concept stems from the observation that LpL can be released from the capillary endothelium with an injection of heparin (Korn, 1955), and also from the fact that normal endothelial cells bind more LpL than endothelial cells depleted of HSPGs (Shimada et al., 1981). However, the precise endothelial cell molecules required for the lipolytic processing of chylomicrons have remained obscure (Goldberg, 1996).
A deficiency in LpL causes familial chylomicronemia, which is characterized by extremely high levels of triglycerides in the plasma (>2,000 mg/dl) (Brunzell and Deeb, 2001). Familial chylomicronemia can also be caused by a deficiency in apolipoprotein CII, a cofactor for LpL (Bengtsson and Olivecrona, 1979; Breckenridge et al., 1978). No cases of chylomicronemia have yet been ascribed to defects in endothelial cells affecting the binding of either chylomicrons or LpL.
In the current study, we examined the functional importance of glycosylphosphatidylinositol-anchored high density lipoprotein–binding protein 1 (GPIHBP1) in plasma lipid metabolism. GPIHBP1 was initially identified, by expression cloning in Chinese hamster ovary (CHO) cells, as a glycosylphosphatidylinositol (GPI)-anchored cell-surface protein that conferred the ability to bind high density lipoproteins (Ioka et al., 2003). GPIHBP1 contains a highly negatively charged amino-terminal domain (with 17 of 25 consecutive residues in the mouse sequence being glutamate or aspartate) followed by a Ly-6 motif containing multiple cysteines (Ioka et al., 2003).
Because GPIHBP1 was identified as an HDL-binding protein, Ioka and coworkers (Ioka et al., 2003) speculated that it might be involved in reverse cholesterol transport. Here, we show that GPIHBP1 is a protein within the lumen of capillaries that plays a critical role in the lipolytic processing of triglyceride-rich lipoproteins.
To investigate the biological role of GPIHBP1 in plasma lipid metabolism, we examined Gpihbp1-deficient mice (Gpihbp1−/− mice). Gpihbp1−/− mice were produced by replacing all of the exons and introns of Gpihbp1 with lacZ and neo cassettes (Figure 1A). Southern blots showing gene-targeting events are shown in Supplementary Online Figure 1. Northern blots confirmed a complete absence of Gpihbp1 transcripts in Gpihbp1−/− mice (Figure 1B). By western blot, GPIHBP1 could be detected in the adipose tissue of wild-type mice but not of Gpihbp1−/− mice (Figure 1C). Although the targeted Gpihbp1 allele contained a lacZ reporter, lacZ transcripts were undetectable in knockout mice (Supplementary Online Figure 2), and no β-galactosidase activity could be detected. The reason for the absence of lacZ expression in the Gpihbp1 knockout mice is not known with certainty, but presumably relates to the removal of important regulatory elements contained within the gene's introns.
Both male and female Gpihbp1−/− mice had milky plasma on a normal chow diet (Figure 2A). This phenotype appeared as early as four to five weeks of age, and was invariably present by 14 weeks of age. The plasma triglyceride levels were significantly increased at all ages, with nearly all mice having plasma triglyceride levels over 1,000 mg/dl by 7–10 weeks of age, and some having triglyceride levels as high as 5,000 mg/dl (Figure 2B). Prior to weaning, the plasma triglyceride levels in Gpihbp1−/− pups were significantly elevated compared with wild-type mice, but not markedly so (120 ± 12 vs. 23 ± 16 mg/dl; P = 0.0023). The plasma triglyceride levels in Gpihbp1+/− mice were indistinguishable from those in Gpihbp1+/+ mice (Figure 2B). Plasma cholesterol levels were also elevated in Gpihbp1−/− mice (Supplementary Online Figure 3A).
The plasma lipoproteins of adult Gpihbp1−/− mice contained increased amounts of apo-B48, as judged by a Coomassie blue–stained SDS-polyacrylamide gel (Figure 2C) and by a western blot with a mouse apo-B-specific monoclonal antibody (Nguyen et al., 2006) (Figure 2D). Fractionation of the lipoproteins by FPLC revealed that the vast majority of the triglycerides in the plasma of Gpihbp1−/− mice was in large lipoproteins (i.e., in the "chylomicron/VLDL" peak) (Figure 2E). Most of the cholesterol in the plasma was also located in the chylomicron/VLDL peak (Supplementary Online Figure 3B). The HDL cholesterol levels in Gpihbp1−/− mice were low (Supplementary Online Figure 3B), as is generally the case with hypertriglyceridemia (Brunzell and Deeb, 2001). The diameters of the triglyceride-rich lipoproteins in the plasma of Gpihbp1−/− mice were extremely large, much larger than in wild-type mice, as judged by laser light scattering (Figure 2F) and electron microscopy (Figure 2G).
The milky plasma in Gpihbp1−/− mice, the increased plasma levels of apoB48, and the large lipoproteins suggested a defect in the lipolytic processing of chylomicrons. To determine if the processing of chylomicrons were abnormal, a retinyl palmitate clearance study was performed. Following the oral administration of retinyl palmitate, retinyl esters are packaged into chylomicrons in the intestine and enter the circulation; the disappearance of these retinyl esters from the plasma indicates the rate of chylomicron clearance. In wild-type mice, plasma retinyl ester levels peaked after 1–3 h and had largely disappeared by 10 h (Figure 2H). In the Gpihbp1−/− mice, the peak retinyl ester levels were more than 10-fold higher than those in Gpihbp1+/+ mice, and very high levels persisted for 24 h (Figure 2H).
The finding of chylomicronemia in mice lacking a GPI-anchored protein led us to hypothesize that GPIHBP1 might be involved in the processing of triglyceride-rich lipoproteins by LpL at the surface of capillary endothelial cells. In support of this idea, the tissue pattern of GPIHBP1 expression closely resembled that of LpL and CD36 [a putative fatty acid transporter (Greenwalt et al., 1995)]. By northern blot analysis, all three genes were expressed at particularly high levels in adipose tissue and heart (Supplementary Online Figure 4). In addition, immunofluorescence microscopy revealed that GPIHBP1 is expressed exclusively in endothelial cells, colocalizing with the endothelial cell marker CD31 (Figure 3A). Confocal immunofluorescence microscopy showed that GPIHBP1 was located on the luminal face of capillaries, both in brown adipose tissue and heart (Figures (Figures3B3B and and3C).3C). GPIHBP1 was also expressed on the capillaries of skeletal muscle, but in multiple experiments was undetectable in the capillaries of the brain (Supplementary Online Figure 5), a tissue that relies heavily on glucose uptake rather than on lipolysis of triglyceride-rich lipoproteins.
We considered the hypothesis that GPIHBP1 might be the sole binding site for LpL in capillaries and that the amount of LpL bound to capillaries might be dramatically reduced in Gpihbp1−/− mice. To examine this possibility, we measured the levels of LpL and hepatic lipase (HL) in the plasma after an injection of heparin (which releases LpL from capillaries into the circulation). To avoid methodological problems associated with measuring lipase activity in lipemic serum, we separated LpL and HL in post-heparin plasma by heparin-Sepharose chromatography and then quantified the HL and LpL activities (Figure 4). In four independent experiments with age and sex-matched plasma samples, LpL activity was reduced by 53.3, 27.2, 16.6 and 68.4% in Gpihbp1−/− mice when compared to wild-type mice, and the LpL to HL ratio in the Gpihbp1−/− mice was reduced by 71.3, 36.1, 56.6, and 90.4% (P = 0.03). The HL activities tended to be higher in the Gpihbp1−/− mice, but these changes did not achieve statistical significance (P > 0.05).
We also measured LpL levels in whole plasma (i.e., not fractionated on a heparin-Sepharose column) following an injection of heparin. The post-heparin LpL protein mass levels were lower in Gpibhp1 knockout mice than in control mice (175 ± 111 ng/ml vs. 581 ± 98 ng/ml; n = 5 in each group; P = 0.00028). Levels of LpL mass in the pre-heparin plasma of wild-type and Gpihbp1−/− mice were very low and not different.
The fact that there was still some heparin-releasable LpL in Gpihbp1−/− mice suggested that other proteins, perhaps HSPGs, provide a reservoir for LpL binding. Nevertheless, the presence of GPIHBP1 within capillaries, along with the chylomicronemia in Gpihbp1−/− mice, strongly suggested that GPIHBP1 is likely an important "platform" for lipolysis. We hypothesized that GPIHBP1 might be capable of binding LpL or chylomicrons or both. This hypothesis seemed plausible given that GPIHBP1 contains a strongly negatively charged domain, and that both LpL and several apolipoproteins within chylomicrons contain positively charged domains that are known to mediate protein–protein interactions (Brown and Goldstein, 1986; Cardin et al., 1989).
To test the hypothesis that GPIHBP1 binds to LpL and/or chylomicrons, we constructed a full-length mouse Gpihbp1 cDNA expression vector. Transient transfection of CHO ldlA7 cells with a Gpihbp1 expression vector yielded high levels of GPIHBP1 expression at the surface of cells, as judged by confocal fluorescence microscopy (Figure 5A). This GPIHBP1 could be released from the surface of cells with a phosphatidylinositol-specific phospholipase C (PIPLC) (Low, 1992) (Figure 5B). To assess the ability of GPIHBP1 to bind LpL, we examined the binding of LpL to a mutant CHO cell line (pgsA-745) that lacks the ability to synthesize HSPGs (Lugemwa and Esko, 1991). CHO pgsA-745 cells stably transfected with Gpihbp1 bound 10–20-fold more LpL than cells transfected with empty vector, and the binding of LpL to cells was saturable, with nonspecific binding being negligible (Figure 5C). An analysis of LpL binding indicated that GPIHBP1 binds LpL tightly (calculated Kd = 3.6 × □10−8 M). The increased binding of LpL to Gpihbp1-transfected cells was eliminated by treating the cells with PIPLC (Figure 5D). Heparin was effective in releasing LpL from GPIHBP1 expressed on the surface of pgsA-745 cells (Figures (Figures5E5E and and5F5F).
To determine if GPIHBP1 binds to triglyceride-rich lipoproteins, we isolated the d < 1.006 g/ml lipoproteins ("chylomicrons") from the plasma of Gpihbp1−/− mice, labeled them with the fluorescent dye 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI), and incubated them with CHO ldlA7 cells stably transfected with the mouse Gpihbp1 cDNA or empty vector. The Gpihbp1-transfected cells bound chylomicrons avidly, whereas the cells transfected with the empty vector did not (Figure 6A). The binding of chylomicrons was dramatically reduced by treating the transfected cells with PIPLC (Figure 6A). In transient transfection experiments, ~10% of the cells expressed GPIHBP1, as judged by immunofluorescence microscopy, and only those cells bound chylomicrons (Figure 6B). In similar experiments, we found that Gpihbp1-transfected cells also bound apo-AV-phospholipid disks (Supplementary Online Figure 6).
Lipolysis of triglyceride-rich lipoproteins is controlled at multiple steps, according to metabolic need. For example, LpL activity levels in muscle are controlled by feeding status (Lithell et al., 1978). We therefore tested whether Gpihbp1 expression in muscle changes with feeding status. Indeed, Gpihbp1 expression increased twofold in muscle during fasting (P < 0.001) (Figure 7).
In this study, we show that adult Gpihbp1−/− mice have severe hypertriglyceridemia, associated with a marked delay in the clearance of retinyl palmitate from the plasma. Gpihbp1 is expressed at high levels in heart, adipose tissue, and skeletal muscle—the same tissues that express high levels of LpL. In these tissues, GPIHBP1 is located on the luminal face of the capillary endothelium. Expression of GPIHBP1 in cultured cells confers the ability to bind both LpL and chylomicrons. Together, these findings strongly suggest that GPIHBP1 is a key platform for the LpL-mediated processing of chylomicrons in capillaries.
We found a large difference in GPIHBP1 expression in different capillary beds. While GPIHBP1 was expressed highly in the capillaries of lipolytic tissues such as adipose tissue, heart, and skeletal muscle, it was virtually absent in the brain. These findings raise the possibility that differences in endothelial cell gene expression could play an active role in regulating the delivery of lipid nutrients to different tissues.
The ability of GPIHBP1 to bind both to LpL and chylomicrons favors the concept that GPIHBP1 serves as an important "platform" for lipolysis by drawing chylomicrons and LpL into close proximity. However, it is unlikely that Gpihbp1 deficiency completely eliminates LpL-mediated processing of lipoproteins. Lpl-deficient mice die shortly after birth with plasma triglyceride levels as high as 20,000 mg/dl (Weinstock et al., 1995), whereas Gpihbp1−/− mice survive and have far lower triglyceride levels. These differences between Gpihbp1- and Lpl-deficient mice strongly suggest that some LpL-mediated lipolysis occurs in the absence of GPIHBP1. Without question, LpL is present in Gpihbp1−/− mice; we clearly identified mouse LpL—with normal enzymatic activity—in the plasma of Gpihbp1−/− mice after an injection of heparin.
The fact that LpL was identified in the post-heparin plasma of Gpihbp1−/− mice indicates that, in vivo, there are additional binding sites for LpL—aside from GPIHBP1. Cell culture experiments have shown, quite convincingly, that LpL binds to HSPGs on the surface of endothelial cells (Cheng et al., 1981). However, at this point, we do not know with certainty whether the heparin-releasable LpL in Gpihbp1−/− mice is attached to HSPGs, and indeed whether the LpL in Gpihbp1−/− mice that is mobilized by heparin is actually located within the lumen of capillaries. At this time, is not known with certainty how much of the LpL found into the bloodstream after an injection of heparin is actually released from the luminal surface of capillaries versus other sites (e.g., parenchymal cells, basement membranes, and subendothelial spaces). Further, the percentage of post-heparin LpL that arises from endothelial cells versus those other sites could differ in Gpihbp1−/− and Gpihbp1+/+ mice. In any case, since the post-heparin plasma LpL levels are higher in wild-type mice than in Gpihbp1−/− mice, it seems possible that a significant fraction of the heparin-releasable LpL in wild-type mice is bound by GPIHBP1. In future studies, it will be of interest to determine the fraction of the total LpL pool on endothelial cells that is bound to GPIHBP1 and HSPGs, and whether LpL bound to GPIHBP1 is catalytically more efficient than LpL bound to HSPGs.
The degree of hypertriglyceridemia in Gpihbp1−/− pups before weaning was milder than that in 4—6 week-old Gpihbp1−/− mice, which in turn was milder than that in 7–16-week-old Gpihbp1−/− mice. The explanation for this finding is not known with certainty, but we suspect that it relates to the fact that Lpl mRNA levels are much higher in the liver during the suckling phase (Langner et al., 1989). We have confirmed higher Lpl expression levels by quantitative RT-PCR: suckling C57BL/6 mice have hepatic Lpl mRNA levels that are 50-times greater than those of 10-week-old mice, and hepatic Lpl mRNA levels in 4-week-old mice are approximately double those in 10-week-old mice (Supplementary Online Figure 7). Although we have not yet proven that the differences in hepatic expression of Lpl are responsible for the lower triglyceride levels in the younger mice, the time course of Lpl expression would be consistent with this possibility. In any case, we emphasize that the defect in LpL activity in adult Gpihbp1−/− mice is severe. When newborn Lpl knockout mice were injected with an LpL adenovirus, some survived the suckling period, owing to the adenoviral-mediated LpL expression in the liver (Strauss et al., 2001). However, this adenoviral-mediated LpL expression disappeared by 30 days of age. After the extinction of LpL expression, the plasma triglyceride levels in the rescued mice were 2,000–5,000 mg/dl—very similar to those in chow-fed Gpihbp1−/− mice in this study.
The plasma levels of apo-B48 were markedly elevated in Gpihbp1−/− mice. In the mouse, apo-B48 is synthesized by both the liver and the intestine (Hirano et al., 1996). Given the markedly delayed clearance of the retinyl palmitate in Gpihbp1−/− mice, we suspect that much of the apo-B48 accumulation is due to delayed clearance of intestinal lipoproteins. However, the lipolytic processing of chylomicrons and hepatic VLDL are similar, and there is no reason to believe that the accumulation of apo-B48 is exclusively due to an accumulation of intestinal lipoproteins.
The molecular basis for chylomicron binding to GPIHBP1 requires further study. Chylomicrons contain several apolipoproteins (e.g., apo-B48, apo-E, apo-AV) that have positively charged domains that bind to heparin sulfate and/or HSPGs (Cardin et al., 1984; Cardin et al., 1986; Lookene et al., 2005), so it is easy to imagine that one or more of these apolipoproteins could mediate the binding of chylomicrons to the strongly negatively charged domain of GPIHBP1. In particular, apo-AV would make a good candidate as a ligand for GPIHBP1, given that Apoav deficiency causes hypertriglyceridemia associated with decreased LpL-mediated lipolysis (Calandra et al., 2006; Grosskopf et al., 2005; Pennacchio et al., 2001). In support of this idea, our studies showed that apo-AV-phospholipid disks bind avidly to Gpihbp1-transfected proteoglycan-deficient cells (Supplementary Online Figure 6). Another interesting hypothesis would be that GPIHBP1 could act as a receptor for apo-B48, a key structural protein of large, triglyceride-rich lipoproteins. It would make sense that a receptor for apo-B48 would be located in the capillary endothelium of muscle and fat, where lipolysis occurs. Also, this hypothesis is in line with evolutionary considerations. Both apo-B48 and chylomicrons (defined as intestinal lipoproteins that are secreted into the lymph) are unique to mammals (Teng and Davidson, 1992). Similarly, GPIHBP1—defined as a GPI-anchored Ly-6-motif protein with a very strong acidic domain—is present in all mammals but is absent in fish, amphibians, and birds.
Transfection of a Gpihbp1 cDNA into cultured cells promotes the binding of both LpL and chylomicrons, but it is not yet clear whether a single GPIHBP1 molecule is capable of binding chylomicrons and LpL simultaneously. If a single GPIHBP1 molecule binds only a single ligand, chylomicrons and LpL could still be drawn together if the GPIHBP1 molecules were clustered together on the cell surface. This possibility seems plausible given that other GPI-anchored proteins are concentrated within lipid rafts on the plasma membrane (Varma and Mayor, 1998). Also, GPIHBP1 could exist as a homodimer on the cell surface and therefore bind to more than one ligand. Of note, urokinase-type plasminogen activator receptor, a related GPI-anchored Ly-6-motif protein, exists as a dimer on the cell surface (Cunningham et al., 2003).
In summary, GPIHBP1 is crucial for the lipolytic processing of triglyceride-rich lipoproteins. It is located on the luminal surface of the capillary endothelium and binds both LpL and chylomicrons. It likely forms a platform for lipolysis and plays an important role in the delivery of lipid nutrients to cells.
Gpihbp1−/− mice were created with a sequence-replacement gene-targeting vector designed to replace all of the exons of Gpihbp1 with a lacZ reporter and a PGK-neo cassette. Targeted cells were identified by Southern blotting of genomic DNA with 5′- and 3′-flanking probes. The 5′ probe was amplified from genomic DNA with oligonucleotides 5′–GGTCTGTAGCAAGCCTTCC–3′ and 5′–GTGTCCTTCCTGCCAAATCC–3′ (after cleaving genomic DNA with HpaI, the targeted band is 11.8 kb and the wild-type band is 17.1 kb). The 3′ probe was amplified with oligonucleotides 5′–CATCTGTACAGCCCTTGTCC–3′ and 5′–CCAGAAGCACAACAGCCAG–3′ (after cleaving genomic DNA with EcoRI, the targeted band is 7.1 kb and the wild-type band is 8.5 kb). Routine genotyping was performed by PCR; the mutant allele was detected by PCR with oligonucleotides 5′–GCAGCGCATCGCCTTCTATC–3′ and 5′–GGAGGCC TACACTAGAAGG–3′ (amplifying a 655-bp PCR product between the neo cassette and sequences 3′ to Gpihbp1). The wild-type allele was detected by PCR with oligonucleotides 5′–GGTGATGCGGACCCGGAG–3′ and 5′–GTGTCT GATTGCAGCTCTCC–3′ (amplifying a 519-bp PCR product spanning intron 2). All mice had a mixed genetic background (<10% 129/Sv and >90% C57BL/6); the mice were fed a chow diet and housed in a virus-free barrier facility with a 12-h light-dark cycle. All studies were approved by UCLA's Committee on Animal Research.
Total RNA was prepared from mouse tissues with Tri Reagent (Sigma, St. Louis, MO), size-fractionated on a 1% agarose/formaldehyde gel, and transferred to a sheet of Nytran SuPerCharge membrane (Schleicher and Schuell, Keene, NH). The blot was hybridized to 32P-labeled cDNAs for mouse Gpihbp1, human LPL, mouse Cd36, and mouse 18S ribosomal RNA.
Total RNA was prepared from mouse tissues with Tri Reagent (Sigma), treated with DNase I (Ambion, Austin, TX), and reversed transcribed into cDNA with a mixture of random primers and oligo dT and Superscript III (Invitrogen). Primers for each gene were designed with the Primer 3 software (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi), and were assessed by DART-PCR (Peirson et al., 2003) to validate equivalent efficiencies between genes and the endogenous control gene (β2-microglobulin). Quantitative PCR analysis was performed on 50 ng cDNA, 200 nM of each primer, and 10 μl of SYBR green PCR master mix (QIAGEN, Valencia, CA). PCR reactions were performed in triplicate on a 7500 Fast Real-Time PCR system from Applied Biosystems (Foster City, CA). The relative expression of all gene-specific mRNA was calculated by the comparative CT method.
A segment of the mouse Gpihbp1 cDNA (spanning nucleotides 88–636, corresponding to amino acids 23–205) (NM_026730) was cloned into the BamHI and SalI sites of pQE-30 (Qiagen, Valencia, CA) to create a bacterial expression vector for 6His-tagged GPIHBP1. This fragment was amplified from mouse adipose tissue cDNA with forward primer 5′–CCATCACGGATCCGCACAAGAAG ATGGTGATGCGGA–3′ and reverse primer 5′–GCTGCAGGTCGACGCCTGAAG GGTATCCTGCCCCAC–3′ (underlined sequences indicate restriction endonuclease sites). The plasmid was transformed into Escherichia coli Max 5αF′ cells; bacteria from 1.0-liter cultures were resuspended with 40 ml of lysis buffer (20 mM Na2HPO4, pH 7.0, 250 mM NaCl, 6M guanidinium chloride) and lysed by fifteen successive 10-min periods of sonication (at a power of 50 watts) alternating with 10-min incubations on ice. The lysate was stirred at room temperature for 30 min and then centrifuged at 4000 × g for 30 min in an SS34 rotor. The supernatant fluid was then incubated with 2 ml of TALON metal affinity resin (Clontech, Mountain View, CA) with continuous stirring for 60 min at 4° C. The resin was sedimented by low-speed centrifugation, loaded onto a column, and washed overnight with one liter of wash buffer containing 20 mM Na2HPO4 (pH 7.0), 250 mM NaCl, 8 M urea, and 5 mM imidazole. The recombinant GPIHBP1 fragment was eluted with a 30-ml linear gradient of imidazole (5–300 mM) in wash buffer. Three-ml fractions were collected, and aliquots were size-fractionated on SDS-polyacrylamide gels. Fractions containing the 6His–GPIHBP1 fragment (identified by Coomassie-blue staining) were pooled and dialyzed against PBS containing 0.02% sodium azide. Two rabbits were immunized with the GPIHBP1 fragment (75 μg/rabbit) in complete Freund's adjuvant and then boosted with the GPIHBP1 fragment (150 μg/rabbit) in incomplete Freund's adjuvant 3 and 6 weeks later.
Plasma cholesterol and triglyceride levels were measured on nonfasted plasma samples with the serum triglyceride determination kit (Sigma) and cholesterol E kit (Wako, Richmond, VA). Fractionation of mouse plasma lipoproteins was performed by fast phase liquid chromatography (FPLC) (Cole et al., 1988). The diameters of lipoprotein particles in the d < 1.022 g/ml fraction of plasma were measured with a Nanotrac 250 particle analyzer (Microtrac, Clearwater, FL) fitted with a flexible conduit–sheathed probe (Walzem et al., 1994). Lipoprotein particle diameters were recorded as frequency distributions of particles within a given diameter interval. Negatively stained lipoproteins (d < 1.006 g/ml fraction) from the plasma of Gpihbp1−/− and Gpihbp1+/+ mice were imaged by electron microscopy (Hamilton et al., 1980). The protein composition of lipoproteins was analyzed by SDS-PAGE followed by Coomassie-blue staining.
Proteins from tissue or cell culture extracts (20 μg) were size-fractionated on 4–12% Bis-Tris gradient SDS-polyacrylamide gels and then transferred to nitrocellulose membranes for western blots. The antibody dilutions were 1:500 for a mouse monoclonal antibody against mouse apo-B48 and apo-B100 (Nguyen et al., 2006); 1:500 for a rabbit antiserum against GPIHBP1; 1:500 for a mouse antibody against V5 tag (Invitrogen, Carlsbad, CA); 1:500 for a mouse monoclonal antibody against β-actin (Abcam, Cambridge, MA); 1:5,000 for an HRP-labeled anti-mouse IgG (NA931V, GE Healthcare, Piscataway, NJ); 1:5,000 for an HRP-labeled anti-rabbit IgG (NA934V, GE Healthcare); 1:2,000 for an IRdye680-conjugated goat anti-rabbit IgG (Li-Cor, Lincoln, NE); 1:2,000 for an IRdye800-conjugated goat anti-mouse IgG (Li-Cor). Antibody binding was detected with the ECL Plus chemiluminescence system (GE Healthcare) and exposure to X-ray film. When the IRdye-conjugated secondary antibodies were used, binding was detected with the Odyssey infrared scanner (Li-Cor).
Mice were given 5,000 IU of retinyl palmitate (all trans) or vehicle alone (soybean oil) in 50 μl by oral gavage. A blood sample was taken immediately after gavage and 1, 2, 4, 10, and 24 hours later. Plasma levels of retinyl esters were measured by HPLC on a 250 × 4.6 mm Beckman Ultrasphere C18 column (Palo Alto, CA) (Mills et al., 1992; Redlich et al., 1996). Retinyl esters were separated in a mobile phase consisting of acetonitrile:methanol:dichloromethane (70:15:15 v/v) at a flow rate of 1.8 ml/min and detected by UV absorbance at 325 nm with a Waters 996 photodiode array detector (Milford, MA). Retinyl acetate was used as an internal standard. Retinyl ester standards were synthesized from authentic all-trans retinol and the corresponding fatty acyl chloride (Blaner et al., 1994). The reported plasma retinyl ester concentrations represent the sum of individual retinyl ester concentrations (retinyl linoleate, retinyl oleate, retinyl palmitate, and retinyl stearate). All extraction and HPLC procedures were carried out under N2 and reduced light to prevent oxidation of the compounds. Plasma samples (25–200 μl) were denatured with an equal volume of absolute ethanol containing known amounts of retinyl acetate and then extracted into hexane. Following phase separation, the hexane extract was evaporated under a stream of N2 and the residue resuspended in benzene for injection onto the HPLC column. The lower limit of detection for retinyl esters was 2 ng/ml (Burger et al., 1997).
Two independent strategies were used to express a mouse Gpihbp1 cDNA in cultured cells. Strategy 1: the complete coding sequence of Gpihbp1 (nucleotides 1–711) was amplified from a mouse adipose cDNA library with primer 5′–CTCGAGCGGCCGCGACAGAACAGCCTGAGCCAGGAT–3′ (underlined segment is a NotI site) and 5′–TTAAGCTTGGTACCTCTTCAGGCC CCTGACGCCCAC–3′ (underlined segment is a KpnI site). The fragment was ligated into the NotI and KpnI sites of pcDNA3.1(−) (Invitrogen, Carlsbad, CA) to produce pcDNA3-Gpihbp1. CHO-K1 cells, Ldlr-deficient CHO cells (ldlA7 cells, a generous gift from Dr. Monty Krieger) (Kingsley and Krieger, 1984), and CHO cells deficient in the ability to synthesize heparan-sulfate proteoglycans (pgsA-745 and pgsB-761 cells, a generous gift from Dr. Jeff Esko) (Esko, 1991) were transfected with pcDNA3-Gpihbp1 or empty vector using FuGENE 6 Transfection Reagent (Roche, Indianapolis, IN) according to the manufacturer's instructions. Stably transfected cells were obtained following selection with G418 (0.5 mg/ml). The cells were then subjected to a second round of transfection with pcDNA3-Gpihbp1 and pCMV/Bsd (Invitrogen) and selected with blasticidin (5 μg/ml). Strategy 2: an IMAGE clone (ID #30298145) containing the complete open reading frame of mouse Gpihbp1 in pDNR-LIB was purchased from Open Biosystems (Huntsville, AL). An NcoI restriction site immediately upstream to the translational start site was created by site-directed mutagenesis (QuickChange, Stratagene, La Jolla, CA). An NcoI–HindIII fragment containing the complete open reading frame was subcloned into the mammalian expression vector pTriEx-4 (Novagen, San Diego, CA). To facilitate subsequent cloning steps, the ApaI site in pTriEx-4 was replaced with an SfoI restriction site by site-directed mutagenesis. An ApaI site was subsequently created by silent mutation at nucleotide 66 of the Gpihbp1 cDNA (between the signal peptide and the acidic domain). A Gpihbp1 construct with an amino-terminal S-protein tag was created by inserting the S-protein coding sequence (AAAGAAACCGCTGCTGCGAAATT TGAACGCCAGCACATGGACTCG) into the newly created ApaI site. The integrity of all constructs was verified by DNA sequencing.
For the detection of GPIHBP1 in cultured cells, cells were plated on coverslips at ~25,000 cells per well in 24-well plates, fixed in 3% paraformaldehyde, blocked with 10% sheep serum, and incubated with the rabbit anti-GPIHBP1 antiserum diluted in blocking buffer (1:800). Detergents were omitted to facilitate the detection of only cell surface GPIHBP1 protein. Bound rabbit immunoglobulin (IgG) was detected with a FITC-conjugated donkey anti-rabbit IgG (1:800; Jackson ImmunoResearch). After washing, cells were stained with DAPI to visualize DNA. For cells transfected with the S-protein–tagged GPIHBP1 construct, the fusion protein was detected with a FITC-conjugated goat antibody against the S-protein tag (Abcam, Cambridge, MA; 1:400). Images were obtained with an Axiovert 200 MOT microscope (Zeiss, Germany) with a 63×/1.25 oil-immersion objective and processed with AxoVision 4.2 software (Zeiss). Confocal fluorescence microscopy was performed with a Leica TCS-SP MP confocal inverted microscope (Heidelberg, Germany) equipped with an argon laser (488 nm blue excitation), diode laser (561 nm green excitation), and a two-photon laser tuned at 768 nm for UV excitation. Individual images were captured sequentially with a 63×/1.32 planapo objective and merged images were generated with Leica confocal software (version 2.5).
For the detection of GPIHBP1 in mouse tissues, 10-μm thick frozen sections were fixed with 4% formaldehyde and blocked with 10% donkey serum. The samples were incubated overnight with the rabbit antiserum against mouse GPIHBP1 (1:200) and a rat monoclonal antibody against mouse CD31 (1:50, BD Pharmigen, San Diego, CA). Samples were subsequently washed and incubated for one hour with a FITC-conjugated antibody against rabbit IgG (1:200, Jackson ImmunoResearch, West Grove, PA) and an Alexa Flour 568-conjugated antibody against rat IgG (1:800, Molecular Probes, Eugene, OR). After washing, the sections were stained with DAPI to visualize DNA. Microscopy was performed as described above.
CHO pgsA-745 cells transfected with pcDNA3-Gpihbp1 or empty vector were grown to confluence in Ham's F12 medium containing 5% fetal bovine serum. The cells were placed on ice, washed twice with ice-cold PBS, and incubated for 2 h at 4°C with 1.0 ml of Ham's F12 medium supplemented with 0.5% BSA and highly purified chicken LpL (Bensadoun et al., 1999). After this incubation, the cells were washed once with PBS supplemented with 0.5% BSA and lysed with 400 μl of CHAPS lysis buffer (4 mM CHAPS, 50 mM NH4Cl, and 3 U heparin/ml). The amount of LpL in the medium and cell lysates was measured by ELISA (Cisar et al., 1989).
Experiments were also performed to determine if the binding of LpL to cells could be prevented by treating cells with a phosphatidylinositol-specific phospholipase C (PIPLC). CHO-pgsA-745 cells transfected with either pcDNA3-Gpihbp1 or empty vector were grown to confluence in 35-mm dishes. The cells were incubated in 2 ml of Ham's F12 in the absence or presence of 10 U of PIPLC at 37°C for 1 h. Cells were then placed on ice and incubated in Ham's F12 supplemented with 0.5% BSA and 1.5 μg of avian LpL for 2 h. Cells were then washed once with PBS/0.5% BSA and lysed with CHAPS buffer. Bound LpL was determined by ELISA (Cisar et al., 1989). Recombinant PIPLC was purified from the media of Bacillus subtilis transfected with a PIPLC gene from B. thuringiensis (a generous gift from Dr. Martin Low at Columbia University) (Low, 1992). The enzyme was purified from media by ion-exchange chromatography on Amberlite CG-50, followed by ammonium sulfate precipitation, gel filtration on Sephadex G-75, and a final anion-exchange chromatography step on MonoQ. PIPLC activity was documented with l-3-phosphatidyl[2-3H]inositol (GE Healthcare) (Low, 1992). The final PIPLC preparation had an activity of 199 units/ml.
To estimate LpL and HL activities in post-heparin plasma, mice were injected intraperitoneally with 0.5 U heparin sodium/g body weight, and blood was collected under anesthesia from the retro-orbital sinus 15 min later. To avoid the possibility of the elevated plasma triglyceride levels interfering with the lipase assays, the lipases were partially purified from the plasma samples by heparin-Sepharose chromatography. Post-heparin plasma (200 μl) was loaded onto 1.0-ml heparin-Sepharose HiTrap columns (GE Healthcare) equilibrated with 0.25 M NaCl, 20% glycerol, 1% BSA, 10 mM sodium phosphate, pH 6.5. The column was washed with 10 ml of the equilibration buffer and eluted with a 30 ml NaCl gradient (0.25–1.5 M in 20% glycerol, 1% BSA, 10 mM sodium phosphate, pH 6.5). Fractions were assayed for lipolytic activity with [3H]triolein as described previously (Hocquette et al., 1998). The activities in fractions corresponding to the HL and LpL peaks were added together to calculate the lipase activities per ml of plasma.
DiI (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate) was purchased from Invitrogen. DiI was dissolved in DMSO (3 mg/ml) and 10 μl of this solution was added to 70 μl of serum from a Gpihbp1−/− mouse and 250 μl of human lipoprotein-deficient serum. This mixture was then incubated at 37°C overnight. The d < 1.006 g/ml lipoproteins ("chylomicrons") were isolated by centrifugation at 100,000 rpm in a Beckman TLA-100.3 rotor for 6 h at 4°C. The DiI-labeled chylomicrons were harvested and dialyzed against PBS containing 5.0 μM EDTA.
To evaluate the binding of the DiI-labeled chylomicrons to GPIHBP1, transfected CHO ldlA7 cells were incubated with 1 μg/ml of DiI-labeled chylomicrons in PBS containing 1.0 mM CaCl2, 1.0 mM MgCl2, and 0.5% BSA for 2 h at 4°C. The cells were then washed five times in the same buffer, fixed in 3% paraformaldehyde, and chylomicron binding detected by fluorescence microscopy. Cells expressing GPIHBP1 on the cell surface were detected by immunofluorescence microscopy as described above.
An apo-AV-FLAG expression vector was constructed by replacing the apo-AV coding sequence in the apo-AV/pET vector (Beckstead et al., 2003) with sequences encoding apo-AV-FLAG. Recombinant apo-AV with a carboxyl-terminal, 8-residue FLAG extension was expressed and purified as described (Beckstead et al., 2003). To generate apo-AV-FLAG-phospholipid complexes, dimyristoylphosphatidylcholine (DMPC) was dissolved in chloroform:methanol (3:1 v/v) and the solvent evaporated under a stream of nitrogen. The sample was then incubated in vacuo for 16 h to remove residual solvent. DMPC was resuspended in a solution containing 50 mM sodium citrate and 150 mM NaCl (pH 3.0), followed by the addition of apo-AV-FLAG (6:1 DMPC:apoA-V; w/w). The mixture was sonicated until clear, dialyzed against 50 mM phosphate, pH 7.4, 150 mM NaCl and filter-sterilized (0.22 μM). Protein concentration was determined with the BCA Protein Assay Kit (Pierce), and the phospholipid content was determined with an enzyme-based colorimetric assay (Wako). To evaluate the binding of the apo-AV-DMPC disks to GPIHBP1, pgsA-745 CHO cells were transiently transfected with the S-protein-tagged Gpihbp1 cDNA and incubated with 5 μg/ml of apo-AV-DMPC disks in PBS containing 1.0 mM CaCl2, 1.0 mM MgCl2, and 0.5% BSA for 2 h at 4°C. Apoprotein binding was detected with a mouse anti-FLAG antibody (and a Cy5-conjugated goat anti-mouse antibody) and the S-protein–tagged GPIHBP1 was assessed as described above.
This work was supported by a Beginning Grant-in-Aid from the American Heart Association, Western States Affiliate (to A.P.B.), by BayGenomics (HL66621 and HL66600 from the National Heart, Lung, and Blood Institute) (to S.G.Y.), and by HL073061 (to R.O.R.). We thank Jennifer A. Beckstead for discussions and advice.
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About Me
Andrew has been co-founder and CEO of Crittercism since 2011. Prior to starting Crittercism, Andrew Levy was the co-founder of AdThrow, a Y Combinator company that built a data processing pipeline for real-time ad targeting. Before YC, Andrew worked at HP Software where he led teams specializing in agile programming methodologies and advocating rapid product iterations. Andrew also worked for several companies in defense and intelligence, such as Silicon Graphics Federal, Northrop Grumman, and Computer Sciences Corp. He has a B.S. in Computer Science from Johns Hopkins University.
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We recommend consulting your vet when feeding prescription diet foods. Read More
By purchasing Hill's Prescription Diet, you confirm your pet has been examined by a vet who has recommended the use of this food on the basis of examination and diagnosis. You understand it is recommended that while your pet is being fed Hill's Prescription Diet, you seek veterinary advice at least every 6 months regarding the usage of this food and the on-going care of your pet. Should your pet's condition deteriorate in any way while using Hill's Prescription Diet seek immediate veterinary advice.
DescriptionPlease consult your vet for further information and guidance on what is best for your dog.
Hills Prescription Diet Canine j/d Reduced Calorie is clinically proven to improve mobility in just 3 weeks with the right balance of healthy nutrients and a special omega-3 oil. Joint related problems in pets are very common. Cartilage in the joints may wear away faster than it can be replaced and cause discomfort for your pet. Feeding your dog Hills Prescription Diet Canine j/d Reduced Calorie helps your pet walk, run and play better by soothing aching joints and preserving healthy cartilage and is particularly beneficial for those dogs which are large than they should be!
Key benefits:
- Clinically proven to help dogs walk, run, play and climb stairs more easily, see a difference in 21 days
- Helps your dog walk, run and play better by soothing aching joints and preserving healthy cartilage
- Helps maintain joint cartilage thanks to EPA, a special omega-3 fatty acids from fish oil and glucosamine and chondrotin sulphate from natural source
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Customer Ratings & Reviews
Help For Joint Problems posted on 27/09/2016 by Rufus
Our dog has had joint problems most of his life. However, since switching to Hills J/D he moves much more freely without limping.
Fabulous Price posted on 10/07/2014 by lucyseymour2012
Best price for this product that l could find
He is a big fan posted on 02/05/2014 by paulinestamper
My 11 yr old black lab started this food on the recommendation of the Vet as he has arthritis in his elbows and is overweight He has now lost 4kg and he loves this food by ordering from petplanet I saved 13 on the 12kg size I will definitely keep him on this
Brilliant results posted on 29/09/2012 by shirleyharrall484
My border collie cross has been on this food for a couple of years now and since starting on it hes a different dog Hes 16 next month and always likesto come for wales with my 2 young dogs and he keeps up JD is not cheap but Ive found Pet Planet much less expensive than elsewhere and its worth it for the results
Great value works well posted on 27/07/2012 by janemellor3
My lab is recovering from cruciste ligsment surgery and is doing well on Hills jdLack of excercise means keeping her weight down and jd is ideal for this
Works a treat posted on 16/02/2012 by SH
One of my dogs is now in his 16th year hes a border collie X and we also have a young lab and spaniel The old dog wants to come on all of our walks keeps up has the occasional trot Before I began feeding this JD he was very stiff and achy It takes a few weeks to take effect but its worth the spend to see him still enjoying himself at his age
Great Value posted on 04/01/2012 by MHolland
My dog really enjoys this food. Her joints have definitely improved with this product and i'd recommend it to anyone who has a dog suffering from joint disease
Much cheaper than buying it from the vet posted on 22/11/2011 by helandpop
This product has made a huge difference to the quality of our dogs life Thank you to Pet Planet for selling it at a reasonable price in comparison to our local vet
Noticed a difference when I stopped it posted on 31/08/2011 by AbbyP
Ive used it for my 11 year old labrador Lucy who has osteoarthritis, ruptured cruciate ligament and hip dysplasia. I changed it a while ago to a standard pet food and she started limping again. She went straight back on it Its excellent at controlling her weight and good for her bowels she occasionally had colitis on other foods nil on this. Pet planet is one of the cheapest at selling it and really fast delivery
Top product from top retailer posted on 30/12/2010 by iainmed
Pet planet continue to provide excellent value, easy ordering and efficient shipping; product also continues to be loved by the dog...and definitely contributes to his arthritis relief.
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Whole Grain Corn, Chicken By-product Meal, Flaxseed, Soybean Mill Run, Brewer's Rice, Soybean Meal, Pork Fat (preserved With Mixed Tocopherols And Citric Acid), Chicken Liver Flavor, Powdered Cellulose, Fish Oil, Lactic Acid, Potassium Chloride, L-lysine, Calcium Carbonate, Choline Chloride, Iodized Salt, DL-methionine, Vitamin E Supplement, L-ascorbyl-2-polyphosphate (source Of Vitamin C), Vitamin E Supplement, Niacin, Thiamine Mononitrate, Vitamin A Supplement, Calcium Pantothenate, Biotin, Vitamin B12 Supplement, Pyridoxine Hydrochloride, Riboflavin, Folic Acid, Vitamin D3 Supplement, L-threonine, Taurine, Soy Lecithin, Glucosamine Hydrochloride, Ferrous Sulfate, Zinc Oxide, Copper Sulfate, Manganous Oxide, Calcium Iodate, Sodium Selenite, L-tryptophan, L-carnitine (preserved With Mixed Tocopherols And Citric Acid), Chondroitin Sulfate, Phosphoric Acid, Beta Carotene And Rosemary Extract.


text: CYC1 UAS-less Vectors (ATCC® 87539™) General Information Documentation Print Email Share Share this product Facebook Twitter Google+ Type Strain no Biosafety Level 1 Biosafety classification is based on U.S. Public Health Service Guidelines, it is the responsibility of the customer to ensure that their facilities comply with biosafety regulations for their own country. Comments A set of three vectors carrying the beta-galactosidase coding sequence designed to test cloned inserts for promoter enhancer activity. Product Format freeze-dried Basic Documentation Product Sheet Certificate of Analysis SDS


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Do You Know What A Fixie Is?
It seems everywhere I look these days I see these fantastically colored bikes that are not quite a mountain bike or a street bike or a cruiser bike, but are some kind of beautiful hybrid. I'm talking about a type of bike that has exploded in popularity and has so many [more…]
Spring Break Float On: Review Of Flotation Fun
Well it's that time of the year again, when thousands of #SpringBreakers start heading to tropical shores with trim bikini bodies, party attitudes and the desire to cook themselves in the baking sun. So we are here to provide you with some helpful reviews of some essential Flotation Fun. (note: [more…]
Oh, Darling…
Oh, darling.
Time To Think About Warmer Days In The Backyard
We all know that warmer days and nights are just around the corner, and it's time to think about upgrading your social game in the backyard. Become the host/hostess with the best set of patio furniture. We've selected and reviewed some new sets, here are some our favorites. 7pc Teak [more…]
Why the pro-macro beer Budweiser ad is so dangerous
Wow! Budweiser has stirred up some unfavorable buzz in the Craft Beer world. It's funny that they are mocking craft beer drinkers, when they themselves are secretly buying them up in bulk to tap into this growing market. The article below from The Beer Babe sums up some good points… On the [more…]
DIY: Cinder Block Projects
Concrete, or cinder block, is an inexpensive and organic building material for the garden. Easily picked up at your local home improvement store, with a little creativity you can use it to create garden hardscaping that will last. It combines well with other elements such as wood, and is easy [more…]
Beards, The New Aphrodisiac
It's the increasingly new trend in men's fashion, the beard, and it has become the new aphrodisiac for hip women. Some women claim that it's science, bearded men are stronger, happier, healthier, manlier, and better looking than bald-faced men. What do you think ladies? Share your comments below. More: One Thousand Beards: [more…]


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A bright lime green awning atop a matching wood-paneled bar is the perfect spot for lounging around for hours on a sunny day. Blue director's chairs are practically made for wet bathing suits.
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If you own a car, you know how it can be worth the effort of maintaining it. Replacements of parts, brake tests, oil changes, etc. – it seems it is a never ending process if you wish to make sure that your car is operating at its optimal condition. To keep a car functional, so much work is needed to the point that it is very easy to overlook some parts that need fixing. But car owners should keep in mind that there is one task that they should remember – car detailing. It may not that be important to some car owners since this is purely aesthetic, there are benefits that come with car detailing that you never dreamed of. It is not only good for your car, but it will also help you personally.
What is car detailing?
This particular activity will always be a mystery to car owners that are usually only concerned about the inner workings and the optimal function of a car. Not all car owners are knowledgeable about what car detailing is all about. So their common reaction would either be a blank or those activities that have something to do with the details. Some even confuse such an activity with customization, which is a practice that is commonly found among street racers. But in essence, car detailing is a is about thorough cleaning of the car, but focuses more on bringing back its usual look when it was first bought. Every detail of the car must be addressed, which is why it is referred to as detailing. It is not only the exterior of the vehicle that is involved in detailing, it includes the interior as well.
Exterior detailing
The first thing that people notice about car detailing its its exterior. Detailers devote a lot of their attention and time on the car's body, making sure that it will look its best once detailing is done. Their aim is to make sure that the look of the exterior is the same way when it first rolled out from the factories. The glossy look of its body paint is a requirement in every detailing since its usual shine dissipates with the passing of time. Aside from repainting the body, detailers also work on the rims, wheels, tires, and windows, ensuring that each of them is looking its best. Rust, grease and dirt are all taken care of during the exterior phase of detailing with the a little help from detergents and degreasers.
Interior detailing
They don't also spend most of their time with exterior detailing. They also do the same with the interior, too. Isn't its main focus is to bring back its usual look to how it was when it was brand new? They ensure that every detail of the car is well taken care of.
Keep in mind that car detailing is different from car washing. Car wash simply involves "washing" the exterior and interior but does not involve any details. There are particular chemicals that detailers use that is not used in car washing. You can also visit Melbourne paint protection coating for great deals.


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Palm Beach Shores Vacation Packages
Palm Beach Shores is a popular place to head to while you are in Palm Beach - West Palm Beach as a large number of the area's main attractions are nearby. Among the places not to miss are the marvelous Port of Palm Beach and the remarkable John D. MacArthur Beach State Park. Two other worthy points of interest you'll find close by Palm Beach Shores are the North Palm Beach Country Club and the Rapids Water Park.
To gain a deeper understanding of Palm Beach - West Palm Beach, visit one of its museums and theaters. Spend part of the day viewing the exhibits at the Palm Beach Maritime Museum in Palm Beach, the Henry Morrison Flagler Museum and the Society of the Four Arts. Treat your partner to a lively show at the Kravis Center For The Performing Arts in West Palm Beach. There are plenty of popular galleries to visit near here including the Norton Museum of Art in West Palm Beach.
Get your body moving in the great outdoors while you are in the area. Relax and enjoy the fresh air. Breathe in the fresh air, throw a ball and enjoy some sunshine at nearby Mid-Town Beach in Palm Beach.
To stock up on supplies and buy some souvenirs, visit the Gardens Mall in Palm Beach Gardens, CityPlace in West Palm Beach and the Worth Avenue Mall in Palm Beach.
Looming large on the local skyline is Roger Dean Stadium, Jupiter, home to the Jupiter Hammerheads, Palm Beach Cardinals and Gulf Coast League Cardinals. On game days, the stadium and surrounding streets becomes a sea of loyal fans donning blue, gray black and red, the colors of local legends the Jupiter Hammerheads.
Finding somewhere handy to stay in the Palm Beach Shores area is easy. If you're looking for great service and a comfortable bed, it's hard to go past the Springhill Suites by Marriott West Palm Beach, the Hilton Singer Island Oceanfront Resort, and the Courtyard by Marriott West Palm Beach.


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Casey's Sneaky Jackoff
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Kentucky Defeats Auburn, Advances to Quarterfinals in SEC Women's Tennis Tournament
April 17, 2014
COLUMBIA, Mo. – The No. 10 seed University of Kentucky women's tennis team defeated No. 7 seed Auburn University, Thursday, in the second round of the 2014 Southeastern Conference Tournament, hosted by the University of Missouri at the Mizzou Tennis Complex. In a match that lasted nearly four hours, the Wildcats knocked off Auburn, 4-2.
"We are grateful and blessed to be in the position that we're in," Kentucky head coach Carlos Drada said. "This match exemplifies the belief that our players have in their training and on each other. Auburn came with great energy and they dominated the first half of the match. Coming back from a deficit on five out of six courts is a tremendous feat and we're looking forward to playing another great match tomorrow."
In a tight doubles competition, No. 39 Caitlin McGraw and Cece Witten entered into a seesaw affair with No. 13 Pleun Burgmans and Emily Flickinger. The senior duo fought hard for the win, but ultimately fell, 8-6. Jakarta, Indonesia natives, Nadia Ravita and Aldila Sutjiadi, led most of the match with their biggest lead, 5-1. However, Auburn didn't back down and tied the match at 7-all, requiring a tiebreak. In the tiebreak, the Kentucky duo fell, 8-7(3) and the doubles point was claimed for the Tigers.


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Proposal Engineer
- Recruiter
- Rise Technical Recruitment Ltd
- Location
- Honiton
- Salary
- £18000 - £20000 per annum + Overtime + Pension + Holiday
- Posted
- 11 Oct 2016
- Closes
- 20 Oct 2016
- Sector
- Engineering
- Contract Type
- Permanent
Proposal Engineer North Devon £18,000 - £20,000 Overtime Pension 28 Days Holiday Excellent opportunity for a mechanically biased engineer with experience of AutoCAD to kick start a career with a well-established organisation in a varied and challenging role. This company, specialist manufacturers of bulk materials handling equipment requires a person with engineering experience to join a strong sales team. In this role you will be responsible for liaising with customers and the Area Sales Manager to ensure needs are understood and met. You will create site drawings using AutoCAD & produce quotations based on the information given. You will also be responsible for following up on quotations and assisting with back-office sales activity. The ideal candidate will have an engineering qualification, be fully conversant with AutoCAD and have great customer service skills. All in all, a fantastic opportunity to join a well-established and growing organisation that will provide you with the training and progression to establish and further your career. The Role; Interpret manufacture drawings Create site drawings using AutoCAD Produce quotations based on information given Follow up on quotations provided The Person; Engineering qualification Conversant with AutoCAD Good communication skills Technical Sales Engineer, Proposals, Support, Technician, Applications, Internal Sales, Manager, Mechanical, Projects, Industrial, Manufacturing, Engineering, Graduate, Trainee, Junior, Engineering, AutoCAD, To apply for this role or for to be considered for further roles, please click 'Apply Now' or contact Ben Gilbert at Rise Technical Recruitment. This vacancy is being advertised by Rise Technical Recruitment Ltd. The services of Rise Technical Recruitment Ltd are that of an Employment Agency. Rise Technical Recruitment Ltd regrets to inform that our client can only accept applications from engineering candidates who have a valid legal permit or right to work in the United Kingdom. Potential candidates who do not have this right or permit, or are pending an application to obtain this right or permit should not apply as your details will not be processed. This job was originally posted as www.totaljobs.com/job/66855274


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FREE EVENT! Wear your Pjs for story time if you must but keep the kiddies at home, Dr. Mitcheson gives a rather flavorful presentation that may be too strong for your little ones! Have you ever thought about keeping bees and what that might entail? Join us and we explore the idea… Learn more about this class and register: http://herbalacademyofne.com/product/stories-from-a-beekeeper-workshop/ Dr. H. David Mitcheson: urologist, surgeon, herbal enthusiast, beekeeper **REGISTRATION on the Herbal Academy of New England website is required as there are limited seats available. Class is Free! If you are interested in signing up for all of our herbal classes this spring, make sure you register for the Spring Herbs Package and save $5 per class. View the entire spring schedule here: http://herbalacademyofne.com/boston-herbal-workshops-spring-2014/
"Bee" sure to also register on the website: http://herbalacademyofne.com/boston-herbal-workshops-spring-2014/
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Best Of :: People & Places
Having public artworks replace private cars could be the perfect example of Mayor John Hickenlooper's plan to transform Denver from an outpost of the automotive age into the very model of a creative-class city. The empty parking lots of this shuttered John Elway Ford dealership are separated from the City Park neighborhood to the north by an aging wooden security fence hung with lively works of contemporary art. The artwork east of Garfield Street consists of colorful constructions that handily show what a good artist can do with leftover materials and scraps of time; the pieces hanging on the fence east of Jackson Street are assembled from repurposed wooden screen doors and discarded housewares. This gallery is open at all hours; art lovers are encouraged to bring their own wine and cheese.
Leadville doesn't get a lot of attention these days — so the adopted home town of Jihad Jamie Paulin-Ramirez really didn't appreciate being labeled "nothing to write home about" and a "dirtball town" by a local reporter characterizing the place for Fox. Local lawmakers fought back by offering to host "A Day in a Dirtball Town" that would include tours of Leadville's art galleries and historic sites — including spots visited by Oscar Wilde in 1882. And he did write home about the town, reporting at one saloon, "I saw the only rational method of art criticism I have ever come across. Over the piano was printed a notice — 'Please do not shoot the pianist. He is doing his best.'"
When America voted Lilly Scott off this season's American Idol, our hearts sank. Scott, a Littleton resident who previously fronted the band Varlet, was a breath of fresh air on a show otherwise known for churning out (with a very few exceptions) a steady parade of unmemorable artists who, in turn, produce mainstream pabulum. And while we were being unrealistically optimistic to think she'd make the finals, we couldn't help pulling for Scott. For the first time in years, she made Idol worth watching. We couldn't wait to see what songs she'd pick next: From "Lullaby of Birdland" to "Fixing a Hole" to "A Change Is Gonna Come" to "I Fall to Pieces," she kept things interesting. But that last song eventually proved to be her undoing; Scott's voice, which recalls Joanna Newsom's, was a bit too much for most folks. Your loss, America.
Now that texting while driving is illegal in Colorado, the legislature really should do something about drivers who listen to Caplis & Silverman while behind the wheel between 3 and 6 p.m. This odd couple of the airwaves can be dangerously distracting. While you're wondering just why former DA Craig Silverman doesn't execute a citizen's arrest for Caplis's shameless boosting of the University of Colorado, for example, you could wipe out a fleet of RTD buses. Still, it's almost impossible not to tune into their show during rush hour, and the impressive lineup of guests they book apparently all feel the same inevitable pull, because just about every politician and newsmaker in the state winds up yakking with these talk-show hosts.
He's still finding his voice on morning radio, and his attempt to click with players sometimes comes off a little shmoozy. Still, Channel 4 sports anchor Vic Lombardi overcomes these and any other faults you might be muttering about with his undeniable natural knowledge and feel for Denver sports. He was born with it: He's a native of Denver's north side and interned with the station as a student. But he balances his obvious fandom with honest, astute observations and a geniality that oozes from your flat-screen.
Colorado's real capitol dome is in trouble. It is rusting, rotting and cracking, and fixing it will cost millions. But a scale model of the building, made entirely of canned goods, is doing just fine. Located inside the dome museum — known as Mr. Brown's Attic — the replica is a version of one originally created for the 2004 People's Fair by Intergroup Architects of Littleton as part of Canstruction, a yearly contest held by the Society for Design Administration. Made from a variety of containers, including soup and tomato sauce cans and sardine tins, it's topped by a plastic water bottle. Canstruction projects are typically built as fundraisers, and the food is then donated to food banks, but this one is on permanent display — which may be more than we can say for the real thing.


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Role play interviews can come in handy whether you're a job seeker looking to brush up on your skills or an employer or manager who wants to help your staff learn something new. As a job seeker, you might use a role play interview to prepare for the actual job interview. As an employer, use role play to act out scenarios your employees might encounter.
Play the Part of the Interviewee
Before a job interview, role play the questions you might be asked. Get a friend or colleague to help you. Have your friend play the part of the interviewer, and give her a set of questions. The questions you come up with should pertain to the job. For example, a job in sales might include questions about your sales style or how you handle rejection. To add value to the exercise, get your friend to play a tough prospect who responds negatively to your sales pitch, forcing you to come up with ways to convince her that your product is worth buying.
Then Switch Roles
Another helpful exercise is to play the part of the interviewer, suggests Alison Green in a column for U.S. News & World Report. Playing the role of the hiring manager helps you get insight into the mind of the interviewer and can help settle your nerves or allay your fears. Like the role play interview in which you played the interviewee, ask standard questions such as, "Explain how your greatest strength fits with the needs of our company." Ask the interviewee for more detail on answers that sound too generic or canned. Also, play the part of a tough customer who does not agree with the other person's point of view or is not satisfied with her explanations.
Practicing Your Delivery
Use your research into the job and the company to anticipate the types of conflicts, decisions, skills or persuasive arguments you might have to make, and then practice role plays that deal with those issues. Also important: how you deliver your responses. Practice using open body language, keeping your legs crossed at the ankles and not at the knees, and keeping your arms uncrossed. During your role play interview, sit up straight and lean in a little, showing that you're interested and attentive. Practice making eye contact. Smile and try to appear enthusiastic about the position. When you answer questions, practice lowering the pitch of your voice a little, as it can help you avoid the high-pitched voice that so often comes with being nervous, suggests Carol Kinsey Goman in Forbes.
Job Training Role Play
If you're already employed and you're tasked with training other employees, role play interviews can also come in handy. By simulating situations workers might encounter, you'll help them become more confident and capable. If you're creating a role playing scenario, resist the urge to create the most difficult situation you can imagine -- at least at first. For example, you might not want to start off tackling what someone would do if he suspected an employer was doing something fraudulent. Instead, start with a relatively easy scenario, such as handling a customer who doesn't want to buy a product, and then make the scenarios more difficult as you go along. After each round of role play, talk to employees about what worked and what didn't, and then repeat the role playing again.
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I am brasilian, living at brasil, searching a friend in INDIA.
WHY INDIA??? Because I want peoples from India. No everybody, but many interesting peoples come.
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Alcoholism Procedure Options
Alcoholism is really a sickness that hasn't modified a lot within the U . s . throughout the last numerous a long time. It can be nevertheless one among one of the most major challenges that we face nowadays and is particularly dependable for your death and destruction of a huge selection of countless life every year. Even so, what has adjusted will be the remedy alternatives which might be readily available for alcoholics. Whilst during the the latest previous these alternatives have been really grim and offered minor probability of achievements, present day alcoholic beverages treatment method options are compassionate, progressive and provide an incredible opportunity to interrupt the chains of alcoholism and start a life span of productive sobriety. The subsequent is often a description of these treatment method alternatives and the way they're able to enable any one struggling with alcoholism. iboga plant
The initial step in alcoholic beverages therapy is always to detox. This is actually the physical procedure of ridding your body of liquor as well as symptoms which have been sometimes skilled for the duration of detox can be pretty not comfortable as well as in exceptional cases, fatal. This is why folks experiencing alcoholism should always find support from the experienced detox middle and never attempt to detox by themselves. Soon after completing a detox method long lasting anywhere from 7 to 14 times, the following stage is a lot more intense long-term treatment.
For most people a household inpatient method will supply the very best prospect of achievements. These programs are rigorous and therefore are focused on reality-based treatment method. Consequently purchasers the two live and acquire procedure while in the exact same supervised specialist treatment center. This type of plan frequently lasts for 28 to 30 times and is also probably the most extensive variety of alcoholic beverages remedy readily available. Purchasers with this system will prepare dinner, cleanse, show up at groups and appointments and essentially be needed to just take obligation for his or her have therapy with the thorough advice of dependancy counselors.
The following step down from a residential program is a partial hospitalization or day/night application. This contains intensive therapies throughout the day in a structured therapy center followed by night teams and transport into a supervised cleanse and sober living atmosphere. PHP or day/night plans tend to be the most effective option for men and women who may have a short while ago relapsed or for many who have just finished a residential inpatient system. PHP applications are custom-made to the client and might previous anyplace from 30 to 90 days or simply extended.
Outpatient plans are generally used by individuals who may have relapsed a lot of periods or for those with less extreme addictions, this kind of as marijuana dependence. Outpatient plans usually present remedy quite a few instances each week, plus some far more extensive programs deliver semi-supervised clean up and sober living while in the evening.
These procedure selections for alcoholism make use of several types of remedy in an effort to assistance a recovering alcoholic to acquire and benefit from capabilities and support techniques that could enjoy a critical part in protecting sobriety for life. When you or somebody you like is suffering from alcoholism and you'd choose to get more facts about these remedy plans and exactly how they can perform for you personally, then all you should do is decide up the phone and acquire that very first step. It really is a great deal less complicated than you think!