text:
By Arshad Mohammed WASHINGTON (Reuters) – A compromise allowing the U.S. Congress to vote on a nuclear deal with Tehran may prompt Iranian negotiators to drive a harder bargain, but does not drastically weaken President Barack Obama's ability to deliver on a final agreement. For months Obama resisted attempts led by Republicans and some Democrats to open an agreement with Iran to congressional approval. On Tuesday he backed down in the face of mounting bipartisan support for the bill, which gives Congress at least 30 days to review a final deal during which time Obama would be unable to waive or suspend many U.S. sanctions. Negotiators for Iran and six major powers are trying to ensure Iran does not acquire an atomic bomb by securing an agreement by June 30 under which Iran would curb its nuclear program in exchange for relief from international sanctions.
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Pet and animal games online - play free pet games.
Exotic pets for sale - sell / buy exotic pets online.
Learn about Grooming Your Cats
No cat wants to be dirty, and it's up to you to ensure that your cat stays clean. Grooming is very important, as it helps your cat remain clean and healthy. Grooming starts with brushing, as brushing helps to keep the cats hair from becoming hairballs. Brushing will remove loose hair, and help prevent the risk of hairballs. A lot of cats have trouble dislodging hairballs. If the cat isn't able to get a hairball out, it could result in a blockage of the intestines. Blockages are very serious, and can quickly become life threatening for your pet.
Cats that have long hair need to be brushed and combed on a daily basis, while cats with short hair need to be groomed on a weekly basis. When you brush your cat, you should always watch for lumps in the coat and skin irritations. You should start brushing along the cat's back, going from his head to his tail. Do this a few times on one side, and then switch to the other. Each area should be brushed a few times in the direction of the way your cat's hair is growing.
When you brush your cat, you should always avoid brushing his face and paws. If a cat doesn't like to be brushed, you can still groom him using another tool. The grooming glove is an excellent alternative to the brush, as most cats don't mind it at all. Starting out young is the key to grooming, as it will get your cat used to brushing and grooming. Some cats that have started grooming when they were kittens grow to love it, many of which often look forward to their grooming time.
When brushing your cat, make sure that you check his ears, eyes, teeth, and claws. His eyes should be bright and clear with nothing residing in the corners. His ears should be clean, pink in color, and no sign of ear mites. Ear mites result from dirt built up in a cat's ears, and can result in the ears shriveling up and your cat losing his hearing. Ear mites are very annoying for the cat, and hard for you as an owner to get rid of. The best way to get rid of them - is to ensure that your cat's ears stay clean and healthy.
Each time you groom your cat, you should always aim to go a bit further with your examinations. When your cat remains still, you should always praise him and give him a treat. If your cat doesn't cooperate and starts to struggle, you shouldn't fight with him, but instead let him go and try the next day. Once your cat gets used to grooming he will look forward to it each and every day.About the Author
Article source: Article-Dashboard.com
More ArticlesGuide to Grooming Your Cat
Grooming is also a big part of having a cat. Just as you get a shower every day, do your hair, and whatever else your grooming entails, your cat needs to be groomed as well. (more)All About Grooming Your Cats
No cat wants to be dirty, and it's up to you to ensure that your cat stays clean. Some cats that have started grooming when they were kittens grow to love it, many of which often look forward to their grooming time. (more)Related Articles
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When you decide to add a new cat to your home, it's typically more exiting for you and your family than it is for the cat you already own. Although most cats are lone by type, most will also come to accept or eventually tolerate any additions. (more)The Different Kinds of Toys Cat Will Play With
Do you walk through the pet store, see the many toys available for your cat, and wonder if the expense will be worth it? Cats can be fickle creatures, but a good time can be found with almost any toy. (more)How To Stop Your Cat From Urinating Everywhere
Anytime your cat backs himself up to a door or other object in your house, lifts his tail, and releases urine - you have a problem. This problem is known as spraying, and is very common with cats kept indoors. Even though it is a very annoying problem, it's a problem that can be solved. (more)Learn More about Breeding Cats
Cat breeding is not as relaxed as just leasing cats mate. Breeding is not just about choosing cats to create adorable kittens to earn money. To breed cats is not the same as to give them. (more)Good Treatments for Cats
As most pet owners already know, heartworm treatment for cats and dogs isn't the same. Never, under any circumstances, should you give your cat heartworm treatment that is designed for a dog - or vice versa. (more)
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subset: commoncrawl


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Trip Planner : Europe / Norway / Eastern Norway / Buskerud / Hol Municipality / Geilo / Winter Sports / Geilo Skiheiser
Geilo Skiheiser, Geilo
Categories: Ski Areas, Nature & Parks, Outdoor Activities
Geilo Skiheiser is located in Geilo. Plan your visit to Geilo Skiheiser and a wealth of other attractions, well-known and undiscovered, using our Geilo travel itinerary maker.
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Eerlijk gezegd had ik altijd een aversie tegen wintersport-centrums. Te toeristisch, veel uiterlijk vertoon. Hier is het anders. Iedereen voelt zich er thuis, dat is voelbaar en zichtbaar. Er is ruimt... read more »Honestly I always had an aversion to winter sports centers. Too touristy, lots of shows. Here it's different. Everyone feels at home there, that is palpable and visible. There is space. Everywhere. All you need is there. What appealed to me personally was the separate space for even very small children and see how much fun they had with their parents. I was also in Austria and have seen the huge tourist adjustments there. This is also Norway, touristy, but different. Norway is from its traditions very much a family-country, sports is as breathing, nothing aparts: just. Norwegian Forest cats are sober, calm, friendly, not interfere with you, help you when you need them, and their natural itself inspires others. This is still more than enough to your winter holiday experience?show original
Flotte alpinbakker for hele familien med lite køer og varierende løyper. Her treffer både store og små bekjente.Great Alpine slopes for the whole family with little queues and varying trails. Here hit both young and old acquaintances.show original
Anine Olsbø H
Helt greit ski anlegg. Ikke for bratte bakker og alle kan være med. Flott for de minste barna med skibånd! Men om du søker utfordringer er ikke dette plassen!Perfectly fine resorts. Not for the steep hills and everyone can join, great for the smaller children with skibånd! But if you seek challenges, this is not the place!show original
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"In Jason we are getting a solid young catcher that knows how to handle the pitching staff," Dodger Executive Vice President and General Manager Paul DePodesta said. "Offensively, he has proven to be a patient hitter that can drive the ball to all fields as well as hit the ball out of the ballpark. He is someone we have targeted for quite some time."
Phillips, 28, has a lifetime .262 average with 19 home runs and 95 RBI over parts of four seasons with the Mets. Over 88 games behind the plate, his .998 fielding percentage was tied for second in the Majors among catchers. He threw out 19 of 61 (31%) base runners attempting to steal, fourth best in the National league among catchers with at least 50 stolen base attempts.
Phillips batted .218 last seasn in 362 at-bats with seven homers and 34 RBI. In 2003, he batted .298 with 11 home runs and 58 RBI over 119 games and 403 at-bats. He was second among national League rookies with a .373 OBP, third in RBI (58) and slugging (.442).
Ishii went 13-8 last season for the Dodgers with a 4.71 ERA in 172 innings. He gave up 155 hits, struck out 99 and walked 98. He has a 36-25 record and a 4.30 ERA over 86 starts in three seasons for the Dodgers. The native of Japan signed a free agent contract with the Dodgers before the 2002 season after spending 10 years with the Yakult Swallows of the Japanese Central League.
The Dodgers played a split-squad doubleheader Sunday afternoon, meeting Washington in Dodgertown meeting the Mets in St. Lucie. It wasn't a good day in either city with Los Angeles losing both games.
Nationals win 9-3--Grand slam home run in the eighth inning broke open a 4-3 game and gave the Washington National a 9-3 victory at Dodgertown. Scott Erickson worked six strong innings and could have been unscored upon but did give up three earned runs.
Washington scored twice in the second inning. Erickson gave up a single and a drive to left field that was misjudged into a double, then nearly got out of the inning when JD Drew, playing center field, made a strong throw to the plate to hold the runner at third.
Erickson got the second out with a strikeout but a two-run single broke the 0-0 tie. A passed ball by rookie catcher Dioner Navarro moved the runner to second and the National's pitcher singled to left. In a close play at the plate, Navarro couldn't get a hold the throw and the Dodgers were quickly in a 3-0 hole.
Erickson gave up only one other hit during his six innings and it resulted in a run of the unearned variety. An error at third base by Antonio Perez was followed by a single to right that ran through Milton Bradley, allowing the fourth run to cross the plate. He finished his six innings of work having allowed seven hits and three unearned runs.
Los Angeles broke through in the seventh inning. Joe Thurston dropped a single into center and with one out Norihiro Nakamura walked. Mike Edwards singled to left, scoring Thurston and pinch-hitter Henri Stanley singled to center to give the Dodgers their second run. Tony Schrager lined a single to center to close the gap to 4-3 but Perez fanned to end the inning.
Mike Venafro opened he eighth by walking the first batter and giving up a double. He walked the next batter to load the bases and was excused for the rest of the afternoon.
Harold Eckert took over with the bases FON (full of Nationals). His first pitch was lined over the left field fence to clear the bases and a run-scoring single followed to put the Dodgers in a 9-3 hole.
Yhency Brazoban worked a scoreless ninth and Los Angeles went quietly in the ninth.
A couple of players who have had less than normal success at the plate perked up. Cesar Izturis neatly stroked a couple of hits to boost his average to .241 and Bradley was two-for-three and his now hitting .259.
Catcher David Ross was 0-for-2 and is hitless in 17 times at-bat this spring. With the trade for catcher Jason Phillips of New York announced during the game, Ross' future with the Dodgers is cloudy. Rookies Dioner Navarro (Las Vegas) and Russell Martin (Jacksonville) are the future of the team's catching corps so Ross, who turned 28 yesterday, may be on the trading block.
ST. LUCIE, Florida--The Dodgers rallied from a 6-1 deficit to take a 9-6 lead into the last of the ninth but the Mets pounced on Aquilino Lopez for four runs to take a 10-9 decision.
DJ Houlton started for Los Angeles but was unusually wild, walking six, throwing a wild pitch and allowing four hits. He left the game in the fourth inning trailing 5-1.
Ryan Rupe got the Dodgers out of trouble in the fourth but allowed a run in the sixth and left the game trailing 6-1.
Kelly Wunsch and rookie Jeff Schmoll kept the Mets at bay, handing he game to Lopez in the ninth. He struggled, allowing four hits and four runs, including a one out, three-run, game-winning homer.
Olmedo Sanenz, who played both first and third base, led the Dodger offense, banging a single and his first spring homer run, knocking in three. Jose Flores added a homer and Ricky Ledee and Mike Rose chipped in with doubles.
Jason Repko continued to batter the ball, lining a pair of singles in five trips, and is leading the Dodger batters with a .415 average (17-for-41) and in slugging percentage (.610).
Dodger Blue Notes--The Dodgers sent RHP Orlando Rodriguez to Jacksonville and OF Cody Ross to Las Vegas. … Jayson Werth is playing catch and swinging a bat but tentatively and is still some time away from seeing action. … Stopper Eric Gagne wants to see his first action Monday against Boston but officials are not guaranteeing his appearance. Odalis Perez will make his first pitching start Tuesday against the Florida Marlins in Viera. … Brad Penny pitched batting practice for the first time this spring but pitching coach Jim Colburn noted that it didn't indicate he was ready to see action in a game. … Manager Jim Tracy left the club Sunday evening to attend the funeral service for Don Schaly, his coach at Marietta (Ohio) College and will miss the game with Boston. …
Dodgers Make Ishii for Phillips Deal
LA Dodgers Insider Top Stories
Kershaw VS. Hendricks NLCS Game 6Kershaw VS. Hendricks NLCS Game 6
LA Dodgers InsiderSaturday at 1:01 PM


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Beispielbild für diese ISBN
Bibliografische Details
Titel: A Dead Man in Deptford
Verlag: Carroll & Graf Pub
Erscheinungsdatum: 1995
Einband: Hardcover
Zustand: Very Good
0786701927 Very good. Clean text. Email for further information. Quality, Value, Experience. Buchnummer des Verkäufers BINGX83411676
Inhaltsangabe: The whole of Elizabethan England--from the court and its intrigue to the theatre and its genius to London and its slums--is brilliantly recreated in this joyous celebration of the life of Christopher Marlowe, killed in highly suspicious circumstances in a tavern brawl in Deptford hundreds of years ago.
Über den Autor: Anthony Burgess was born in Manchester in 1917. He served in the army from 1940 to 1954 before becoming a colonial education officer. It was while he held this post that doctors told him he would die, and he decided to try to live by writing. A prolific and respected author, Burgess died in 1993.
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There are millions of Internet-Servers daily hacked from the same hand full of Hackers.
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Should that then go on forever?
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Tue, 25 Oct 2016 19:19:46 GMT - Actual more as 21.000.000 Hackers in our Database. (The newest 100 are displayed on the Globe and Map)
Details for IP 22.214.171.124
WebServer Output: http://126.96.36.199
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☎ Whois 18.104.22.168
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Description of the accommodation:
Rooms, services Take a remarkable trip to Mexico and enjoy the hospitality of Hotel Granada Puebla! The hotel is offering 98 tasfefully furnished, nice rooms for those looking for a place to stay at in Puebla. You may taste delicious, fresh meals in the restaurant of the hotel every day. Parking possibility for your vehicle is available nearby. Car rental service will help you discover the most important attractions. The café and the bar are an ideal location for a personal meeting and a relaxing evening talk. Wireless Internet connection (WiFi) is available at the hotel, ensuring that you can stay up-to-date during your holiday. If you wish to learn more about the exact scope of services provided by Hotel Granada Puebla (including those available for an extra fee) or about the equipment of the rooms, you can find the complete list on the information sheet below.
Payment Advance payment by credit card or providing credit card guarantee may be necessary for finalizing the reservation, depending on the chosen date, room and price.
Arrival, opening hours The earliest check-in time to your room is 07:00, the latest check-out time from your room is 13:00. The personnel can speak Spanish and English languages.
Hotel Granada Puebla foglalás, szoba árak Foglalás, szoba árak
- For accurate prices please enter the date of your trip!
Accommodation services
WiFi in shared spaces
Car rental nearby
Bar, Cafe, Elevator
Own restaurant
Desk in the room


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Satellite tracking could prevent airliner disappearances, developers say
- Top 10 News for 10/17 - 10/21: Merger Rumors Abound; CEOs Depart; Tesla Kicks Autopilot Up A Notch
- Wall Street ends little changed; Microsoft hits record
- AT&T (T) in Advanced Talks to Acquire Time Warner (TWX) - DJ
- Rockwell Automation (ROK) Said to Attract Takeover Interest from Schneider Electric - Source
- British American Tobacco Offers to Acquire Remaining Shares of Reynolds American (RAI) for $56.50/Share
FILE PHOTO: The shadow of a Royal New Zealand Air Force (RNZAF) P3 Orion maritime search aircraft can be seen on low-level clouds as it flies over the southern Indian Ocean looking for missing Malaysian Airlines flight MH370 March 31, 2014. REUTERS/Rob G
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BERLIN (Reuters) - Two U.S. companies have developed an airline tracking system that they say would prevent planes disappearing in the manner of the Malaysia Airlines MH370.
Instead of sending tracking signals to ground stations - which means planes' locations can be lost over oceans or remote areas - the new system would beam them to satellites.
"It doesn't matter if they're flying over the ocean, desert, or North Pole, we'll know where the plane is," said Daniel Baker, CEO of FlightAware, the internet flight tracking service which is working with Aireon LLC, which has developed the satellite technology.
Aireon's system will place ADS-B (automatic dependent surveillance – broadcast) receivers on low orbit satellites operated by Iridium Communications Inc and is due to be operational from 2018. The system was initially conceived to help air traffic controllers route planes more efficiently.
The new tracking system, called GlobalBeacon, will make the location data from the space-based receivers available to airlines so they can track their planes in near real-time on a web-based tool.
After the disappearance of MH370 in March 2014, regulators and airlines were criticized for responding too slowly to French tracking recommendations after the crash of an Air France plane in 2009.
The International Civil Aviation Organization (ICAO) plans to impose a 15-minute standard for normal flight tracking, or more frequently in case of emergency, by November 2018.
FlightAware said it would reveal the first airline customer for the product next week, to coincide with an ICAO meeting in Montreal.
"We only reached out to a few of our dozens of airline customers to discuss this opportunity and although all are interested in using our space-based ADS-B data, only one could hit the tight timeline for our announcement," Baker told Reuters.
(Reporting by Victoria Bryan; Editing by Robin Pomeroy)
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Today's Tarot for Hilary Duff
Three Fates
spread is an exceptionally popular way to gain insight into the emerging arc of the past, present, and future. The
International Icon Tarot
renders traditional occult symbolism in Swiss iconography. It is a humorous and direct approach to divination, and one of our favorite new decks. If you would like your own copy of the International Icon Tarot, you can
buy it now
The left card represents an
important element of the past
Eight of Wands (Swiftness)
: A sudden release of raw power, cutting through confusion and indecision, and setting things in motion. Rapid progress towards a desired goal, brought about by immediate and decisive action. Boldness and daring in love, business, travel, or spiritual growth.
The middle card represents a
deciding element of the present
Nine of Cups (Happiness)
: Contentment and satisfaction in romance, friendship, or other relationships. Achieving your deepest desires and savoring beauty and sensual pleasures. A state of joy and abundance radiating fulfillment and bliss.
The right card represents a
critical element of the future
Page of Wands
: The essence of fire behaving as earth, such as wood or coal: The surprising appearance of a new passion. An adventurer who blazes through life, acting as a catalyst that others may harness. The intense enthusiasm and childlike imagination that fuels any new venture, needing only the application of mind and material to make it a success. Inner fire that can drive away fear and replace it with fury. Can represent a person of some timidity, but whose innate passion can be easily ignited. May indicate the birth of a child.
Copyright 1993-2016
. All rights reserved. Copying any image or other content on Facade is strictly prohibited. The services of Facade are provided for entertainment purposes only. Please view our
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and our
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. Images from The International Icon Tarot are copyright 2003 Robin Ator. All rights reserved. Hosting by
Entertainment Earth


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Taurine occurs in high concentrations in muscle and is implicated in numerous physiological processes, yet its effects on many aspects of contractility remain unclear. Using mechanically skinned segments of human vastus lateralis muscle fibers, we characterized the effects of taurine on sarcoplasmic reticulum (SR) Ca2+ accumulation and contractile apparatus properties in type I and type II fibers. Prolonged myoplasmic exposure (>10 min) to taurine substantially increased the rate of accumulation of Ca2+ by the SR in both fiber types, with no change in the maximum amount accumulated; no such effect was found with carnosine. SR Ca2+ accumulation was similar with 10 or 20 mM taurine, but was significantly slower at 5 mM taurine. Cytoplasmic taurine (20 mM) had no detectable effects on the responsiveness of the Ca2+ release channels in either fiber type. Taurine caused a small increase in Ca2+ sensitivity of the contractile apparatus in type I fibers, but type II fibers were unaffected; maximum Ca2+-activated force was unchanged in both cases. The effects of taurine on SR Ca2+ accumulation 1) only became apparent after prolonged cytoplasmic exposure, and 2) persisted for some minutes after complete removal of taurine from the cytoplasm, consistent with the hypothesis that the effects were due to an action of taurine from inside the SR. In summary, taurine potentiates the rate of SR Ca2+ uptake in both type I and type II human fibers, possibly via an action from within the SR lumen, with the degree of potentiation being significantly reduced at low physiological taurine levels.
- contractile apparatus
- taurine
- sarcoplasmic reticulum
- single fiber
taurine is a small organic acid found in particularly high concentrations in cardiac and skeletal muscle and is thought to be involved in numerous important processes such as modulating osmotic pressure, cation homeostasis, ion channel activity, proinflammatory mediation, protein stabilization, and acting as an antioxidant, to name a few (6, 19, 21, 40, 42). Given its many roles, it is not surprising that taurine deficiency can lead to pathogenesis in both cardiac and skeletal muscle (6, 21, 40). Furthermore, the pathogenesis that stems from a lack of taurine has been shown to be partially ameliorated with therapeutic oral taurine supplementation (4, 35). Additionally, there is considerable interest in the health consequences of a strict vegan diet, which is devoid of taurine (24, 38), and also in possible ergogenic effects of increasing muscle taurine content (1, 14, 35, 39, 42, 50, 51).
Taurine content in skeletal muscle has been shown to be highly fiber-type dependent, with type I fibers containing approximately twice as much taurine as type II fibers in both rat muscles (22) and in camel gluteal muscles (7). In vastus lateralis muscle of untrained healthy young adults, the taurine level is ∼12 to 14 mM in type I fibers and between ∼3 and 11 mM in type II fibers (15, 17, 46) (amounts given here in units of mmoles per liter cytoplasmic water). Oral taurine supplementation has been found to increase muscle taurine content in rats (14), but in healthy humans such supplementation did not raise muscle taurine content above the normal level (13). However, muscle taurine content can be significantly lower in chronic renal failure patients (3). Furthermore, cardiomyopathies develop in cats with taurine-deficient diets (36), and severe skeletal muscle impairment is observed in taurine transporter knockout mice (48). In both cases, taurine supplementation partially reversed these deleterious effects, suggesting taurine is important for normal muscle function.
In chemically skinned cardiac trabeculae of rats (43), taurine (up to 30 mM) was found to cause a minor increase in the Ca2+ sensitivity of the contractile apparatus without any change to maximum force. In mechanically skinned extensor digitorum longus (EDL) fibers of rats, which are almost exclusively type II fibers, 20 mM taurine seemed instead to cause a small decrease in contractile apparatus Ca2+ sensitivity (2). In contrast to such minor effects on the contractile apparatus, the presence of 5–30 mM taurine markedly potentiated the peak and area of caffeine-induced force responses in both the skinned EDL muscle fibers (2) and cardiac trabeculae (43). Steele et al. (43) attributed this effect to taurine increasing Ca2+ loading into the sarcoplasmic reticulum (SR), while Bakker and Berg (2) proposed the effect could have been due either to increased Ca2+ accumulation by the SR or to sensitization of Ca2+ release through Ca2+ release channel-ryanodine receptors (RyRs). It is presently unknown 1) whether physiological levels of taurine exert comparable effects on SR Ca2+ accumulation in human skeletal muscle fibers, 2) whether the effects of taurine differ between type I and type II muscle fibers, and 3) precisely how taurine exerts its apparent effects on net Ca2+ uptake.
Using both type I and type II fibers from human vastus lateralis muscle, we sought to determine whether the presence of taurine affects 1) the contractile apparatus properties, 2) SR Ca2+ uptake, as suggested by the studies on rat tissue (2, 43), and 3) the sensitivity of SR Ca2+ release and, furthermore, to ascertain if taurine exerts its effects on SR Ca2+ accumulation from an action on the myoplasmic side or from inside the SR lumen. This was achieved using mechanically skinned fibers in which excitation-contraction coupling remains functional (26) and where the myoplasmic taurine concentration could be rapidly and precisely manipulated, with each fiber able to act as its own control. We examined the effects of 0 to 20 mM taurine, more than spanning the physiological range in human skeletal muscle type I and type II fibers. We hypothesized that acutely altering taurine concentrations would have only minor effects on the contractile apparatus, and that increasing the myoplasmic taurine concentration across this range would appreciably affect force responses, primarily by increasing SR uptake in both fiber types.
Muscle biopsy, subject details.
Eleven healthy subjects, 10 men and 1 woman (age 23 ± 5 yr; height, 178 ± 6 cm; body mass, 76 ± 12 kg), gave written, informed consent and participated in this study, which conformed to the standards set by the Declaration of Helsinki. These subjects were all recreationally active [i.e., performed regular physical activities such as jogging and cycling (30–60 min, 2–3 times per week)], but were not specifically trained in any sport. All protocols and procedures performed were approved by the Human Research Ethics Committees at Victoria University and La Trobe University. After injection of a local anesthetic into the skin and fascia (1% lidocaine), a small incision was made in the middle third of the vastus lateralis of each subject, and a muscle sample was taken using a Bergstrom biopsy needle (30). An experienced medical practitioner took all biopsies at approximately constant depth. The excised muscle sample was rapidly blotted on filter paper to remove excess blood and placed in paraffin oil (Ajax Chemicals, Sydney, NSW, Australia) at ∼10°C for 45 min before individual muscle fibers were dissected.
Fiber mounting.
The muscle biopsy was pinned at resting length in a petri dish lined with Sylgard 184 (Dow Corning, Midland, MI) and immersed in paraffin oil and kept cool (∼10°C) on an icepack. Using jeweler's forceps, segments of individual fibers were mechanically skinned (25) and individually mounted at 120% of resting length on a force transducer (AME801, Horten) with a resonance frequency of >2 kHz. The fiber segment was then transferred to a 2-ml Perspex bath containing standard K+-based solution that broadly mimics the myoplasmic environment (see below). All force measurements were made at room temperature (23 ± 2°C).
All chemicals were purchased from Sigma-Aldrich (St. Louis, MO), unless specified otherwise. The standard potassium-hexa-methylene-diamine-tetraacetate (K-HDTA) solution used for examining SR Ca2+ release experiments contained the following (in mM): 50 HDTA2− (Fluka, Buchs, Switzerland); 8 total ATP; 36 Na+; 126 K+; 8.5 total Mg2+ (giving 1 mM free [Mg2+], where brackets denote concentration); 10 phosphocreatine; 0.05 total EGTA; 90 HEPES; pH 7.1 and pCa (= −log10 [Ca2+]) ∼ 7.0, except where stated. Where required, the SR of the skinned fiber was totally depleted of releasable Ca2+ by exposing the fiber to a K-HDTA solution with 30 mM caffeine and low (0.05 mM) free Mg2+ (2.15 mM total Mg2+) and with 0.5 mM EGTA (pCa > 8.0) present to chelate much of the released Ca2+ (12), referred to hereafter as the "full release solution." This caffeine-low [Mg2+] solution is a very potent stimulus, which is insensitive to subtle changes in Ca2+ release, and provides a reliable indication of the total amount of releasable Ca2+ in the SR (12, 33). In contrast, the experiments examining the sensitivity of the RyRs to Ca2+-induced Ca2+ release (CICR) used a submaximal stimulus [8 mM caffeine in the presence of normal (i.e., 1 mM) free Mg2+] with little Ca2+ buffering, made by adding caffeine to the standard K-HDTA solution containing only 0.05 mM EGTA (pCa ∼ 7.0). Examination of the contractile apparatus properties required the use of heavily Ca2+-buffered solutions in which all HDTA was replaced with EGTA (i.e., relaxing solution) or Ca-EGTA (i.e., maximum Ca2+-activating solution), as described previously (9). The pCa of solutions (for pCa < 7.2) was measured with a Ca2+-sensitive electrode (Orion Research, Boston, MA). In addition, a strontium-based solution (at pSr 5.3) was made by mixing relaxing solution (50 mM EGTA) with a Sr-EGTA solution similar to the maximum Ca2+-activating solution; this solution was used in the contraction experiments to indicate the likely fiber type under examination (a pSr 5.3 solution typically elicits <5% of maximum force in type II fibers and >65% of maximum in type I fibers; see Refs. 5, 9, 28).
Taurine is a highly soluble compound (solubility of 838 mM at 25°C; see Ref. 18), and a 500 mM stock was made simply by dissolving it in standard K-HDTA solution. A matching stock with 500 mM sucrose (Ajax, Sydney, NSW, Australia) was also made up in standard K-HDTA solution. The effects of taurine solutions were compared with "control" solutions, with equimolar sucrose replacing taurine, to verify that any observed effects were not due simply to a change in osmolality. The osmolality of experimental solutions containing 20 mM sucrose or taurine was 315 ± 5 mosmol/kgH2O, and for those containing 5 mM sucrose or taurine it was 300 ± 5 mosmol/kgH2O. Both taurine and sucrose negligibly bind either Mg2+ or Ca2+ at pH 7.1. The preequilibration solution with 10 mM carnosine (0.5 mM EGTA, pCa > 8) was made by mixing K-HDTA solution with a 80 mM carnosine stock solution containing similar constituents, made as described previously (9).
Contractile apparatus experiments.
The acute effect of 5, 10, and 20 mM taurine on the contractile apparatus was examined by directly activating the contractile apparatus in a series of heavily Ca2+-buffered solutions of different known pCa, with either sucrose (i.e., control condition) or with taurine (i.e., test condition) present. After a 5-min treatment with Triton X-100 (2% vol/vol in relaxing solution) and two subsequent 1-min wash periods (again in relaxing solution), the skinned fiber segment was exposed to a range of solutions with progressively higher free [Ca2+] (0.1–20 μM, i.e., pCa 7.0 to 4.7). These solutions were made by mixing appropriate amounts of the 50 mM EGTA (relaxing) and the 50 mM Ca-EGTA (maximum activating) solutions. This solution sequence elicited force responses resembling a "staircase" (see Fig. 1). Fibers were preequilibrated in relaxing solution with either taurine or sucrose for >2 min before the effects on Ca2+ activation were examined. For each given condition, the isometric force response elicited at each pCa was expressed as a percentage of the corresponding maximum Ca2+-activated force ("max."), plotted against the pCa value and a Hill curve fitted using GraphPad Prism 4 (GraphPad Software, San Diego, CA).
Ca2+-release experiments.
After we mounted the skinned fiber segment on the force transducer and equilibrating it for 2 min in standard K-HDTA intracellular solution (50 μM EGTA, pCa ∼ 7.0), the SR was depleted of all of its releasable Ca2+ by exposing the fiber to the "full release" solution (see earlier) for 1 min, eliciting a force response that was used as an estimate of the endogenous amount of Ca2+ that had been initially present in the SR (27, 28). Before each exposure to the full release solution, the fiber was first equilibrated for 15 s in K-HDTA solution with 0.5 mM EGTA solution, so as to raise the [EGTA] within the fiber before applying the potent caffeine-low [Mg2+] stimulus. After the full release, the fiber was washed for >1 min in K-HDTA solution with 0.5 mM EGTA to remove the caffeine and to raise the free [Mg2+] back up to 1 mM. The SR was then reloaded with Ca2+ to a particular level by bathing the fiber in "load" solution (K-HDTA solution with 1 mM total Ca-EGTA-EGTA at pCa 6.7) for a set time (5 s to 5 min), and then again depleted of all Ca2+ with the full release solution, and the fiber subsequently washed again. These load-release-wash cycles were performed with either sucrose or taurine present in all solutions (as in Fig. 2) or a combination of both present (as in Fig. 5). When exposed to the Ca2+ loading solution, the depleted SR initially takes up Ca2+ approximately linearly with load time and then at a progressively slower rate as the SR Ca2+ content approaches its maximal level (28, 33, 47).
The sensitivity of the Ca2+ release channels (RyRs) was tested using a submaximal caffeine (8 mM) solution with only very weak Ca2+ buffering (i.e., standard K-HDTA solution, containing 50 μM total EGTA, pCa ∼ 7.0), so as to allow possible self-reinforcement of any CICR. By using such a submaximal stimulus (as in Fig. 4), it was possible to determine whether taurine modified the responsiveness of the RyRs to CICR. The procedure was similar to the cycles above but with additional intermediate steps: 1) the SR was first loaded for a set time in absence of taurine; 2) the fiber was preequilibrated in standard K-HDTA solution for 15 s and then exposed to the same solution with 8 mM caffeine for 15 s, with taurine or sucrose as appropriate present in both solutions; 3) all Ca2+ remaining in the SR was released by preequilibration in K-HDTA solution with 0.5 mM EGTA for 15 s followed by exposure to the full release solution for 1 min, with no taurine present; and, finally, 4) the fiber was washed in K-HDTA solution with 0.5 mM EGTA.
Western blot analysis.
Following the physiological measurements, each fiber segment was placed in an Eppendorf tube containing 10 μl of relaxing solution (50 mM EGTA, pCa > 9, described above) with 5 µl of 3× solubilizing buffer, which contained 0.125 M Tris·HCl, 10% glycerol, 4% SDS, 4 M urea, 10% mercaptoethanol, and 0.001% bromophenol blue, pH 6.8. Fibers were stored at −80°C until analyzed. Western blots were performed to determine the myosin heavy chain (MHC I or II) isoforms present, as described previously (9, 28, 32). Total denatured protein from individual fiber segments was separated on 4–15% Criterion TGX Stain Free gels (BioRad, Hercules, CA) and then transferred to nitrocellulose. Membranes were exposed to mouse primary antibodies diluted in 1% BSA in PBS-Tween 20 (anti-MHC I: 0.19 μg/ml, mouse monoclonal IgM, clone A4.840; anti-MHC II: 0.15 μg/ml, mouse monoclonal IgG, clone A4.74; Developmental Studies Hybridoma Bank, University of Iowa), following which the appropriate secondary antibody (i.e. either goat anti-mouse IgG or IgM) conjugated with horseradish peroxidase was added to the membranes. Bands were visualized using West Femto chemiluminescent substrate (ThermoScientific), and images captured using Quantity One software (BioRad).
All values are presented as means ± SE, with n denoting the number of individual fibers examined. Statistical significance (P < 0.05) was determined using paired Student's one- or two-tailed t-tests, as appropriate. Exponential curves were fitted to mean data of response area for various SR Ca2+ loading times (10 s to 5 min) in presence or absence of taurine (see Fig. 2B), as described previously (28).
Fiber typing.
The fiber type of each fiber segment used in the physiology experiments was later ascertained by Western blot analysis of the MHC isoform(s) present (see materials and methods). Of the 55 fibers from 11 subjects used in this study, 25 were classified as type I (containing only MHC I), 27 were classified as type II (containing only MHC II), and only 3 were considered of "mixed" type, containing appreciable levels of both MHC I and MHC II. The MHC II antibody used here is documented as detecting both MHC IIA and IIX, and so the type II fibers here were not subdivided into IIA, IIA/X, and IIX categories, but, based on other studies in similar untrained subjects, it is expected that the great majority of the fibers studied here were pure IIA fibers (20, 45). As our laboratory has reported previously for other vastus lateralis muscle fibers from young adults (9, 28), the fiber type (i.e., type I or type II) could also be gauged during the course of the physiology measurements by the responsiveness of the fiber to a Sr2+-containing solution at pSr 5.3 (see materials and methods); the response to Sr2+ differs depending on troponin C isoform present (slow or fast), which was found to be consistently in accord with the MHC isoform(s) present.
Effect of taurine on contractile apparatus properties in human type I and type II fibers.
The properties of the contractile apparatus in each skinned fiber segment were examined by directly activating the contractile apparatus in a sequence of solutions with the free [Ca2+] heavily buffered at progressively higher levels, with maximum Ca2+-activated force defined as that produced at pCa 4.7 (i.e., ∼20 μM free Ca2+) (see materials and methods). As shown in Fig. 1, this was performed before, during, and after exposure to taurine, with equimolar sucrose replacing the taurine in the bracketing "control" cases. The presence of taurine caused a small concentration-dependent increase in the Ca2+ sensitivity in type I fibers (mean change in pCa50 being +0.019 pCa units with 5 mM taurine and +0.035 pCa units with 20 mM taurine), but had no effect in type II fibers (see Table 1). The Hill coefficient and maximum Ca2+-activated force were not altered by the presence of taurine in either fiber type.
Effect of taurine on SR Ca2+ accumulation.
Taurine has been shown to augment SR Ca2+ accumulation in mechanically skinned EDL muscle fibers (2) and in chemically skinned cardiac trabeculae (43) of rats. We sought first to determine whether this phenomenon also occurred in human vastus lateralis muscle fibers, and second, if there were any differences between type I and type II fibers, particularly given that they typically contain different amounts of taurine and different SERCA isoforms. Fiber segments were exposed to either 5 mM taurine or to zero taurine for ≥10 min, during which time the SR was loaded with Ca2+ (at pCa 6.7, with 1 mM total EGTA-Ca-EGTA) for a range of set times (i.e., 10–60 s) and then depleted of all releasable Ca2+ by exposing the fiber segment to the full release solution (see materials and methods). This assay of the SR Ca2+ content produces highly reproducible force responses, which increase progressively in peak size and area (i.e., time integral) with longer Ca2+ loading times (see Fig. 2, A and B). The order of examination of different load times was varied to verify that this had no effect on the responses (this was also routinely performed in other experiments in this study). In the four type I fibers examined (from two subjects), the presence of 5 mM taurine potentiated both the peak and area of the force response to the full release solution (see Fig. 2), with the largest degree of potentiation relative to control being seen with the shorter load times (i.e., 10–20 s, see Fig. 2B). Similar results were also found in two type II fibers from two different subjects (not shown). This effect of taurine on Ca2+ uptake could be fully reversed by prolonged washout of the taurine (Fig. 2C). These data indicate that the presence of taurine increased the rate at which the SR accumulated Ca2+ at pCa 6.7 in both fiber types.
We also explicitly examined whether the presence of taurine affected the maximal SR Ca2+ load level attained. In these experiments, the given fiber segment was first preequilibrated with or without 5 mM taurine for 5 min and then placed in a pCa 6.7 load solution at the same taurine concentration (i.e., 0 or 5 mM) for a further 5 min, which should have been ample time to reach maximal SR Ca2+ loading (see Fig. 2B), and then the amount of Ca2+ accumulated by the SR was assayed with the full release solution. The maximal SR Ca2+ content found after loading with 5 mM taurine present was not significantly different from that found in bracketing measurements with loading in the absence of taurine [mean relative force-time integral: 102 ± 6% for type I fibers (n = 6) and 100 ± 7% for type II fibers (n = 6), 3 subjects]. Thus the presence of taurine increased the rate at which the SR took up Ca2+ at pCa 6.7, but did not alter the maximal amount that could be accumulated.
Comparison of effects of brief or prolonged exposure to taurine.
To further verify that taurine's apparent ability to augment SR Ca2+ accumulation was not the result of some direct effect of taurine on the contractile apparatus, in other experiments, fibers were preequilibrated with taurine for 10 min, and then the SR was loaded and emptied of Ca2+ in the absence of taurine. This procedure, which involved prolonged preexposure to taurine and its removal from the myoplasm before loading, still resulted in increased SR Ca2+ accumulation [compare first two traces in Fig. 3B, and mean data (solid bar) in Fig. 3C]. In marked contrast, when the period of exposure to taurine before loading was only relatively brief (e.g., <30 s, Fig. 3A), there was no apparent increase in SR Ca2+ accumulation (e.g., Fig. 3A). In the nine fibers examined that were exposed only briefly to taurine (20 mM for 30 s) and then loaded for 30 s with taurine present, there was no significant change in the peak or area of the force response elicited by the full release solution (mean peak and area of response with taurine, relative to bracketing responses without taurine: 102 ± 3 and 104 ± 4%, respectively, in five type I fibers, and 100 ± 4 and 99 ± 6%, respectively, in four type II fibers). These data strongly suggest that taurine exerts its effect on Ca2+ accumulation from within the SR lumen and requires substantial time to enter and equilibrate inside the SR (see discussion). In apparent accord with this, it was found also that the effects on SR Ca2+ accumulation of a prolonged (>10 min) preexposure to taurine persisted for some minutes after complete removal of taurine from the cytoplasm, as seen in Fig. 3B by the comparatively large size of the third compared with the first full release response and in the mean data in Fig. 3C.
Effect of taurine on submaximal caffeine-induced force responses.
We also specifically examined whether the presence of taurine in the myoplasm affected the responsiveness of the RyRs by testing whether taurine altered the force response elicited by a submaximal caffeine concentration in conditions of weak Ca2+-buffering (see materials and methods), which is a sensitive measure of RyR activation (9, 37). In these experiments, the SR was loaded with Ca2+ for a set time (20–40 s) in the absence of taurine, and then the fiber was equilibrated with either 0 or 20 mM taurine for 15 s before being exposed to a solution at that taurine level (i.e., 0 or 20 mM) containing 8 mM caffeine, which elicited a submaximal force response (e.g., Fig. 4). After exposure to this submaximal stimulus, the SR was depleted of all remaining releasable Ca2+ by exposure to the full release solution before the cycle was repeated. The responses generated by the submaximal caffeine stimulus are sensitive to very small differences in Ca2+ release, and yet the presence of 20 mM taurine had no significant effect on either the peak or force-time integral of the response relative to the control level, or on the amount of Ca2+ remaining in the SR, in either type I or type II fibers (see Fig. 4 and Table 2). Thus there was no evidence to suggest that the presence of cytoplasmic taurine altered either the responsiveness of the RyRs or the extent of any Ca2+ leak through the RyRs.
Effect on SR Ca2+ accumulation of varying taurine concentration over physiological range.
The normal cytoplasmic concentration of taurine in vastus lateralis muscle fibers in healthy young adults appears to be in the range ∼12–14 in type I fibers and ∼3–11 mM in type II (see Introduction). The preceding experiments in this study compared the effects of having taurine present or completely absent, the latter being a more extreme case than would be expected to occur in humans, even with diets almost completely lacking any taurine. To examine the effects on SR Ca2+ accumulation of varying taurine over its likely physiological range, we directly compared the uptake properties with 5, 10, and 20 mM taurine in the same fiber (Fig. 5). In these experiments, the given fiber was always equilibrated with the indicated level of taurine for >10 min before making measurements, and the effect of 10 mM taurine was examined both before and after examining each of the other concentrations (5 and 20 mM taurine). The order of presentation of 5 and 20 mM taurine was randomized in different fibers, and the effects of a given taurine level were highly reproducible, regardless of the order of presentation (as in Fig. 2A). It was found that increasing the taurine concentration from 10 to 20 mM had no significant effect on the rate of SR Ca2+ accumulation in either type I or type II fibers, whereas lowering the concentration from 10 to 5 mM caused a significant reduction in the uptake rate in both fiber types (Fig. 5).
Comparison of the effects of carnosine and taurine on SR Ca2+ accumulation.
Finally, we also examined whether a physiological level of carnosine (10 mM, see Ref. 9) affected SR Ca2+ accumulation in a similar way as taurine. In these experiments, the fiber was preequilibrated for 10 min with either 10 mM carnosine or 10 mM taurine or 10 mM sucrose before loading and releasing Ca2+ in standard conditions without either carnosine or taurine. In the one type I fiber and the one type II fiber examined, such preequilibration with carnosine had no apparent effect on Ca2+ accumulation, whereas taurine had its usual effect of increasing uptake (Fig. 6).
This study demonstrates that the presence of physiological levels of intracellular taurine potentiates the rate of SR Ca2+ accumulation at pCa 6.7 in both type I and type II fibers in vastus lateralis muscle of humans, similar to findings reported in rat EDL fibers (2) and cardiac trabeculae (43). The rate of Ca2+ accumulation was increased without any change to the maximum amount of Ca2+ accumulated by the SR. Furthermore, the increase in Ca2+ uptake only became apparent many minutes after adding the taurine to the cytoplasm and persisted for a number of minutes after complete washout of the taurine from the cytoplasm (Fig. 3). In the skinned fiber preparation, the cytoplasmic concentration of a diffusible, small molecular weight substance such as taurine equilibrates (>95%) with the level present in the bathing solution in <1 s (31), as evident from the very rapid response of the contractile apparatus to bath application/removal of Ca2+, ATP, or other agents (8, 10). Hence, we conclude that taurine was probably not mediating its effect on Ca2+ uptake via a cytoplasmic action, but instead via an action from inside the SR, with it taking some minutes for the taurine concentration inside the SR to come into equilibrium with the concentration in the cytoplasm. However, at present, there is only indirect evidence to support this suggestion, and the issue needs further investigation, including whether taurine is actively transported across the SR membrane by a specific transporter or by other means (e.g., see Ref. 41).
The increase in the rate of net Ca2+ uptake at pCa 6.7 was most likely the result of taurine modifying sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) function, either by increasing its maximum pumping rate or increasing its affinity for Ca2+ (or both). The fact that the effects of taurine were similar in type I and type II fibers might be viewed as somewhat surprising, given that the two fiber types have different SERCA isoforms: type I fibers containing primarily or exclusively SERCA2, and type II fibers containing SERCA1 (28, 29). However, numerous factors influence the Ca2+ affinity and maximum pumping rate of the SERCA, including the regulatory proteins phospholamban and sarcolipin (34), as well as pH (49), oxidation, and other cellular and membrane conditions, and it is clear that some factors (e.g., pH changes) exert broadly similar effects on both SERCA isoforms. It is unlikely that taurine increased the net SR Ca2+ accumulation by reducing some concurrent level of SR Ca2+ leakage because such a decrease in Ca2+ leakage would be expected to have had relatively little effect on the net Ca2+ uptake at the short loading times (when there was little Ca2+ present in the SR to leak out), which is the opposite of the behavior observed (Fig. 2C).
The effect of taurine observed here on the rate of SR Ca2+ accumulation at pCa 6.7 (i.e., 200 nM cytoplasmic free Ca2+) seems likely to be of physiological relevance because muscle fiber relaxation involves the SR lowering the cytoplasmic [Ca2+] from >2 μM during a tetanic contraction down to the normal resting level of ∼50 to 100 nM, and SERCA1 and SERCA2 in human fibers display half-maximal Ca2+ pumping at ∼pCa 6.6 and pCa 6.8, respectively (29). In human vastus lateralis muscle, there is normally ∼12–14 mM taurine in type I fibers and ∼3–11 mM in type II fibers (15, 17, 46). It was found here that there was no difference in SR Ca2+ accumulation at pCa 6.7 in either fiber type when changing the taurine level from 10 to 20 mM, but that lowering the taurine from 10 to 5 mM resulted in significantly slower Ca2+ accumulation in both types (Fig. 5), with even greater effect if taurine was absent entirely (Fig. 2B). Such decreases in the SR Ca2+ accumulation rate would be expected to slow muscle relaxation to some degree, and, if the Ca2+ affinity of the SERCA were decreased, it would likely also result in a reduced resting SR Ca2+ content. Thus muscle taurine levels may have a significant regulatory action on intracellular Ca2+ movements and contractile function. An implication of this is that, if muscle taurine levels were to drop substantially below normal, as might occur in particular diseases, such as chronic renal failure or in individuals adhering to strict taurine deficient diets (24, 44), it could have significant adverse effects on SR Ca2+ uptake properties and muscle function, with this possibly being a greater problem in type II fibers, where the normal taurine level can be comparatively low.
Comparison of effects of taurine and carnosine.
It is of interest to compare the effects of taurine on muscle function with those of carnosine, another major intracellular constituent, particularly given that it has been suggested that the muscle concentrations of taurine and carnosine may vary inversely (23), such as when increasing carnosine levels by chronic β-alanine supplementation (11, 16). Certainly, carnosine levels in humans are lower in type I fibers than in type II fibers, whereas, for taurine, levels show the reverse behavior (15, 17, 46). However, β-alanine supplementation in humans was found to increase carnosine levels in both fiber types by 30–50% without significant change in the taurine levels (15). Our laboratory has reported previously (9) that the presence of intracellular carnosine markedly increases the Ca2+ sensitivity of the contractile apparatus in both type I and type II human fibers (by ∼0.12 and 0.10 pCa units, respectively, with 16 mM carnosine), whereas it was found here that taurine had very much less effect, with the addition of 20 mM taurine causing only a ∼0.035 pCa unit increase in Ca2+ sensitivity in type I fibers and having no significant effect in type II fibers (Table 1). Consequently, the net effect on the contactile Ca2+ sensitivity of increasing carnosine levels by supplementation would be little different even if the muscle taurine levels underwent the opposite change. Furthermore, it was found previously that carnosine enhanced the sensitivity of the RyRs-Ca2+ release channels to caffeine, at least in type I fibers (9), whereas the present study found no such sensitizing effect of cytoplasmic taurine (Fig. 4 and Table 2). Finally, it was found here that, unlike taurine, carnosine did not noticeably potentiate the rate of SR Ca2+ accumulation (Fig. 6). Hence, it is clear that carnosine and taurine have distinctly different effects on both intracellular Ca2+ movements and contractile function in human skeletal muscle.
Speculation on the long-term effect of taurine on SR Ca2+ handling proteins.
In the present study, we examined the acute effects of manipulating muscle taurine concentration. If changes in taurine muscle content persisted for a prolonged period, it is possible that there could be altered expression of SR Ca2+-handling proteins, such as calsequestrin or SERCA. Goodman et al. (14) found that, in rats given taurine supplementation, which increased muscle taurine levels by ∼40%, there was an increase in twitch and submaximal tetanic force in the EDL (type II) fibers and also ∼50% increase in the amount of calsequestrin 1 present, with no change in SERCA1 levels. Assuming that the increased muscle taurine level in the rat EDL fibers resulted in a chronic increase in SR Ca2+ uptake, we speculate that there may have been a compensatory increase in calsequestrin 1 expression to help adequately buffer the free Ca2+ concentration within the SR.
Concluding remarks.
We report here the effects of taurine on the contractile properties and SR Ca2+ movements in human type I and type II fibers using a preparation in which the RyRs and SERCA and other key proteins remain fully functional and in their normal in situ locations. Taurine was found to help facilitate SR Ca2+ accumulation, probably involving either an increase in the maximum SR Ca2+ uptake rate or the SERCA affinity for Ca2+. Indirect evidence was presented indicating that taurine possibly exerts its effects via an action from within the SR. Changing muscle taurine levels within the physiological range modulates SR Ca2+ uptake, and it appears that reducing taurine levels to below normal may adversely affect muscle function.
This project was supported by the National Health and Medical Research Council of Australia (Grant 1051460).
No conflicts of interest, financial or otherwise, are declared by the author(s).
Author contributions: T.L.D. and G.D.L. conception and design of research; T.L.D., C.R.L., and R.M.M. performed experiments; T.L.D., C.R.L., and R.M.M. analyzed data; T.L.D., C.R.L., R.M.M., and G.D.L. interpreted results of experiments; T.L.D. prepared figures; T.L.D. drafted manuscript; T.L.D. and G.D.L. edited and revised manuscript; T.L.D., C.R.L., R.M.M., and G.D.L. approved final version of manuscript.
We thank Heidy Flores and Maria Cellini for technical assistance. The monoclonal antibodies directed against adult human MHC isoforms (A4.840 and A4.74) used in the present study were developed by Dr. Blau, and were obtained from the Development Studies Hybridoma Bank, under the auspices of the National Institute of Child Health and Human Development, and maintained by the University of Iowa, Department of Biological Sciences, Iowa City, IA 52242.
- Copyright © 2014 the American Physiological Society


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The Balaji Motion Pictures head honcho, Ekta Kapoor has added a new nudity clause to the contract for her upcoming erotic films. According to reports, in order to avoid any last minute tantrums from refusing to do a nude scene or mouth titillating dialogues, Ekta has introduced a nudity clause to the contract.
However, the reason behind this inclusion of new clause is said to be Sunny Leone as she had rejected from doing certain bold scenes in Ekta Kapoor's 'Ragini MMS 2'.
Ekta Kapoor's next production venture is 'XXX' which is a compilation of five different stories that explore various angles of sexuality. Helmed by Ken Ghosh, the film stars a team of new comers and is expected to hit the screens in 2015.


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The story of the Visions reaches its conclusion. It's been an amazing journey.
There's a new Iron Man in town. What makes Doctor Doom feel he can take on the role?
Captain America's new status quo is having repercussions on Civil War II.
Concept »
Costume Change appears in 323 issues.
Variant cover series
The superhero costume is one of the oldest and most vital things any hero can have, it masks the secret identity and shows the world the real hero.
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Seiko Men's SNKK25 "Seiko 5" White Dial Stainless Steel Automatic Watch
• An exhibition back exposes the mechanisms of this Seiko 5 watch, whose streamlined white dial is enhanced with bright red and gray design accents.
• With its automatic mechanical movement, this analog-display watch never needs a battery.
• Protective Hardlex crystal dial window
• Features push-button deployment clasp, stainless steel construction, day and date window at three o'clock position, sweeping second hand, luminous dial accents
• Water resistant to 99 feet (30 M): withstands rain and splashes of water, but not showering or submersion


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Saturday, June 25, 2016
SitcomsOnline Digest: Hulu Sets Premiere Date for Season 5 of The Mindy Project; Sanford & Son Spinoff Grady Coming to DVD
- Season 5 of The Mindy Project is set to debut on October 4 on Hulu. (TVLine)
- Amy Poehler's production company has a pilot production commitment from NBC, as part of a renewed deal with Universal. (Variety)
- PETA wants CBS to stop using wild animals for their series Zoo, and to try to achieve that goal, they've brought out their biggest weapon: Bob Barker. (Entertainment Weekly)
- The Late Show is going to be broadcast live during the two major political party conventions this year. (CNNMoney)
- If you like to stream and binge watch series from The CW, the way you do it will be changing with a new Netflix deal. (Deadline)
- Not only was the series Archer renewed by FX, but it even got three more seasons. (SitcomsOnline)
- This is perhaps one of the oddest (but very much encouraging) pieces of news that we've seen in the past five years: TVShowsOnDVD.com is reporting that Sony (which has mostly been out of the TV on DVD business for years now) has scheduled a July 26 release of the complete series (one season) of the Sanford & Son spinoff Grady as a MOD release.
- Two Guys, a Girl, and a Pizza Place - The Complete Series (DVD, $79.97): One of the most requested sitcoms of the 90s arrives on DVD for the first time in the United States with this release.
New on DVD/Blu-ray (September)
09/06 - Coach - Season One (Mill Creek) / Season Two / Seasons One and Two
09/06 - South Park - The Complete Ninteenth Season (Blu-ray)
09/13 - The Big Bang Theory - The Complete Ninth Season (Blu-ray)
09/13 - Newhart - The Complete Sixth Season
09/20 - 2 Broke Girls - The Complete Fifth Season
09/20 - Family Matters - The Complete Eighth Season
09/20 - Modern Family - The Complete Season 7
09/20 - Veep - Season 5
09/27 - The Wonder Years - Season Six
More TV DVD Releases / DVD Reviews Archive / SitcomsOnline Digest
Sitcoms Airing Tonight
Friday, October 21
Last Man Standing - "Trick or Treat" (ABC, 8:00PM ET/PT)
It's Halloween and the Baxter family is excited for their annual party, except for Mike who's reluctantly tasked with choosing the theme. In hopes of turning everyone against the holiday, Mike decides that maybe if the whole family has to dress up like each other they'll end up making fun of one another. Eve dresses up like Mandy and Mandy dresses down like Eve, Kristin and Ryan swap roles, and Mike shows up to the party as… Donald Trump!? The impersonating turns into bickering but in the end, the Baxters come together to support Vanessa as she learns that she's being laid off.
Dr. Ken - "D.K.'s Korean Ghost Story" (ABC, 8:30PM ET/PT)
On Halloween, Allison is disappointed the holiday doesn't seem to scare Dave anymore, meaning her little boy is growing up, perhaps too fast. But that might change after D.K. tells a scary Korean gwishin (ghost) story. The Park family, as well as the gang at Welltopia, bring the story to life playing the various gwishins.
Tyler Perry's For Better or Worse - "Jack" (OWN, 9:00PM ET/PT)
Marcus deals with the loss of one of his former teammates.
Sitcom Stars on Talk Shows (Week of October 17)
Friday, October 21
- Matthew Perry (The Odd Couple/Go On/Mr. Sunshine) - Watch Matthew on a repeat of Jimmy Kimmel Live! at 11:35pm on ABC.
- Jerry Seinfeld (Seinfeld) - Jerry appears on a repeat of The Tonight Show Starring Jimmy Fallon at 11:34pm on NBC.
- Matt LeBlanc (Man with a Plan/Episodes/Joey/Friends) - Matt is a guest on The Late Show with Stephen Colbert at 11:35pm on CBS.
- Elijah Wood (Wilfred) - Elijah talks about Dirk Gently's Holistic Detective Agency on LIVE! with Kelly, so check your listings. He will also be on the AOL Build Show at 1:15pm.
- Lindsay Sloane (The Odd Couple/The Stones/Mr. Rhodes) - Lindsay visits Home & Family on Hallmark Channel at 10am ET/PT.
- Tom Hanks (Bosom Buddies) and Ron Howard (Happy Days/The Andy Griffith Show) - Tom and Ron will be on NBC's Today sometime between 7-9am.
- Christian Slater (Breaking In) - Christian talks about King Cobra on NBC's Today in the 9am hour.
- Anna Camp (The Mindy Project) - Anna talks about Netflix's Good Girls Revolt on NBC's Today in the 10am hour. She will also be on New York Live on WNBC in New York at 12:30pm ET.
- Jennifer Grey (Red Oaks/It's Like, You Know...) - Jennifer joins the Chew Crew on ABC's The Chew at 1pm ET/12pm PT/CT.
- Thomas Lennon (The Odd Couple/Sean Saves the World/Reno 911!) and Jason Biggs (Mad Love/Drexell's Class) - Thomas and Jason are guests on Chelsea on Netflix.
- Marisa Tomei (A Different World) - Marisa appears on Access Hollywood Live, so check your local listings.
- Paul Reiser (Red Oaks/Mad About You/The Paul Reiser Show) - Paul is a guest on New York Live on WNBC in New York at 12:30pm ET.


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This salad has the perfect combination of sweet, savoury and tang. Watermelon is tossed with rocket, red onion and crumbled feta in a balsamic vinaigrette. Serve as a refreshing side salad or starter.
This recipee is really delicious!
- 14 Jan 2015
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text: World Market Desks September 15, 2016· General Furniture Gallery of World Market Desks KEEP CALM AND CARRY ON: World Market Josephine Desk Get The Look Archives « Page 2 Of 13 « THE FRUGAL MATERIALIST THE Myles Counter Desk World Market Green Josephine Desk | Desks, World Market And Green Wood And Metal Shelved Asher Desk | Wood And Metal, World Market World Market Sourav Collection Desk, Hutch, Chair In 5miles Home Office Furniture Chairs, Desks, Bookshelves & More I Finally Got My Dream Campaign Desk! Decorchick! ® Drafting Desk From World Market. Bargain Price, Pricey Looks. Http Lipstick Is A Life: Putting Together A Workspace Gallery of World Market Desks


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George Takei Responds To "Traditional" Marriage Fans
The legendary George Takei responds in the best way possible to the protesters who gathered during March Prop 8/DOMA hearings outside the Supreme Court. I went there to ask them to express their opinions on a pad of paper; now George is weighing in. Can he be any more amazing?
- The FBI will reopen its investigation into Hillary Clinton's private email server after discovering new emails.
- Former employees of Palantir, one of Silicon Valley's most valuable startups, are struggling to cash out of stock options 📉
- Russia has been blocked from serving on the UN Human Rights Council in a surprising vote.
- Here are some of the best Vines to watch before Twitter kills it 💀


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the food is great, the sauce makes the bbg tastes great I love it both ways, with or without sauce. the polish taste great, smothered with onions and peppers, great. all the sandwiches are too too good. on days when dinners are served, thats the best way and days to have a complete meal. Just monthwatering food.I Love Smoke House
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Best brisket I have ever had. The sides are good and the service was friendly. Do yourself a favor and order online, you can walk right past the line and they will fill your order right away.
Best food you will every taste and their sauce too good to describe!


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Showing1-13 of 13results


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Relevant offers
It would be pretty easy for diligent Press readers to conclude that alcohol is the worst scourge of humanity.
OPINION: Doug Sellman featured several times over the last fortnight warning us all of alcohol's dangers; The Press ran two articles on a new estimate of alcohol's cost to the Canterbury health budget.
And the University of Otago's Professor Jennie Connor told us that minimum prices are remarkably effective in reducing alcohol consumption, pointing to a Canadian study "that showed a 10 per cent increase in the minimum price of alcohol reduced consumption by 16 per cent relative to other drinks".
Connor was quoted in a Mainlander feature on Doug Sellman explaining how Professor Sellman is not in fact a wowser; presumably it's only free-spirited libertines who write op-eds in the New Zealand Herald worrying about how Woodstock commercials encourage boys "to have sex with their best mates' mothers" and that advertisements "encouraging middle-aged women to get their teenage clothes back on and flirt with their son's best mates" do not contribute to a healthy society. (October 30, 2009)
I do not particularly care what the jury decides on who is or is not a wowser. But I work with and care about the numbers around alcohol policy. And the impression most readers would get from the latest reporting in The Press is a bit at odds with, well, reality.
Let us begin perhaps with Jennie Connor's citing of "a Canadian study" on the effects of minimum pricing. Can a 10 per cent increase in the minimum price of alcohol really reduce total alcohol consumption by 16 per cent? No.
The number is so far out of line with the vast consensus of the literature that I got in touch with Chris Auld, one of the authors of what had to have been the study Connor had read, a piece in the May 2012 issue of Addiction.
And my read of his study was right. If you increase the price of beer by 10 per cent, you could get a reduction in beer consumption of 16 per cent, but that's because beer drinkers move over to other alcoholic drinks.
Across-the-board increases in the minimum price of alcohol have far smaller effects: a 10 per cent price increase reduces aggregate consumption by only about 3.4 per cent, as is made reasonably clear in Auld's paper.
That the 16 per cent figure is screamingly wrong should have been obvious to anybody who is familiar with the literature.
The largest estimate of the responsiveness of alcohol consumption to price measures in Wagenaar's 2009 survey of 112 different studies suggested that a 10 per cent price hike reduced consumption by 8.4 per cent; Wagenaar concluded that 4.4 per cent is the best consensus estimate. And for heavy drinkers, it was 2.8 per cent. Auld's 3.4 per cent is then right where a reasonable person would have expected an estimate on the effects of minimum prices might fall. Sixteen per cent - that's right out.
The Auld study does come out in favour of minimum prices, but in conjunction with a decrease in alcohol excise taxes: it's then a way of increasing the cost facing heavier drinkers while doing less harm to moderate drinkers if heavier drinkers drink cheaper alcohol. I worry more that the policy does disproportionate harm to moderate-drinking poorer people.
I was a bit more surprised to read of the new commissioned BERL report on the health costs of alcohol in Canterbury.
Their prior work in the field, a 2009 study that included a measure of costs to the health system among other costs of alcohol, seemed to put a pretty heavy thumb on the scales in estimating the costs borne by the health system.
My article in today's issue of the New Zealand Medical Journal lists a few of those problems. While alcohol greatly increases the burden of disorders like liver cirrhosis, it also reduces the costs of cardiovascular disease.
BERL here replicated work done in Australia by Collins and Lapsley (2008). But where Collins and Lapsley added up all the costs imposed by those disorders where alcohol makes things worse and subtracted from that total all the cost savings from those disorders where alcohol reduces costs, BERL simply erased any beneficial effects of alcohol for disorders including ischaemic heart disease, cholelithiasis, heart failure, stroke and hypertension.
As I had warned BERL against this kind of method when I served as discussant on their paper at the 2009 New Zealand Economic Association Meetings, I was curious whether they'd revised things. But, the new BERL paper wasn't available.
Two separate stories in The Press, with lots of reaction quotes from doctors, were based on a paper that the reporter did not have and was not yet available for public critique.
I received the paper Wednesday courtesy of the CDHB. And BERL, at footnote 14, reports they've done the same thing again: "The Collins and Lapsley fractions indicate some alcohol use may be beneficial for some conditions. We concentrate on harmful drug use, and assume zero fractions for such conditions."
So their measure of the costs of alcohol to the Canterbury health system relies on an assumption that there can be no health benefits from alcohol - an assumption that runs contrary to the weight of international evidence. Assuming one's conclusions is hardly proper method.
Unfortunately, alcohol policy is one of those areas where a lot of people's convictions about the right answer put a pretty strong lens on how they assess the literature.
How often do you read that problem drinking among 15-24 year olds was no different in 2006/2007 than in 1996/1997 before the change in the alcohol purchase age?
Or that per capita alcohol consumption is down substantially since 1991? Or that light drinkers have about a 14% reduction in their chance of dying from any cause than people who never drink, correcting for the host of other health-related behaviours that are usually given as reasons for ignoring the health benefits of moderate drinking?
Be skeptical of the moral crisis around alcohol.
Dr Eric Crampton is Senior Lecturer in Economics at the University of Canterbury. He blogs at Offsetting Behaviour.
- The Press
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Miracle on the mountain: 15-year-old skier survives 8m crevasse plunge
Stabbing in Christchurch leaves two in hospital
Court action on 'shonky' steel mesh creates pressure for government inquiry
Backlog of defective buildings and shoddy workmanship sparks calls for building warranties
Traps lure, 'whack', kill rodents to help make NZ 'predator-free' by 2050
Search under way for diver missing off Canterbury coast
The All Blacks machine is no model for business or life
Tramper in hospital following accident near Queenstown
Thousands of Kiwi kids waiting for mental health treatment
Hundreds get cheap tattoos for suicide awareness in Christchurch
Retirement commissioner Diane Maxwell has another go at pushing for age raise
Ewen Macdonald, man acquitted of Scott Guy murder, finds love


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QR Code Link to This Post Contents of home must sell any reasonable offer will be accepted. Rooms of furniture, tools, household items, too much to mention. The house is also for sale. Saturday Oct 29th 8am to 3 7377 Silver Creek Rd. Off Zion. Between Wesselman and Bridgetown… → Read More
Furniture, holiday decorations, clothing, some tools and vintage items, household items, etc. Everything must go. NO EARLY BIRDS!!… → Read More
Estate Moving Sale-house also for sale
House for sale. Corner Table: $45 3pcs Tea cup set: $8 1 pc 4FT folding Low table: $25 Full size Firm Bed set +1 extra full size mattress+comforter set $150 Queen size air mattress + stand: $35 3PCS Bookshelf: $ 150 Coats : $5 each Clothes : $1 each VHS tapes, CD's, Christmas decorations, Clothes, Shoes and much much more!!! Starting from 10/21 will… → Read More
YardSales.net is the fastest growing yard sale site, with 39 sale photos and 6 garage sales in Sunman, Indiana. YardSales.net offers you the best list of Sunman yard sales every week.


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Electro or species of Acid techno, you intent be ad! All footpath mastered by: For Twisted Minds but! Lineage, IDM. For this 4th manumit we would like to offer to you some new feeling of Gonroga
This Ep is something that includes all that what Gonroga likes in music. All earliest tracks by Gonroga.
- Release date: 2016-08-26
- Label: GonRoGa
- Genre: Indie Dance / Nu Disco
- Quality: 320kbps / 44.1kHz / Full Stereo
Gonroga - The House (Original Mix)
Gonroga - Women With A Child (Original Mix)download


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Apple has reportedly set up a new company called Apple Technology Services (Shanghai) to bring Apple Pay to China. The company "will launch new major commercial activities — Apple payment services, including adding a new mobile payment feature on Apple's iPhone and other end products," Yicai reports. Apple told Agence France-Presse this week that CEO Tim Cook hopes to launch Apple Pay in the country "as quickly as possible".
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- Face recognition ticketing system unveiled