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Susan Kuchinskas | September 20, 2012 | Comments
ProTexting, provider of a suite of mobile marketing tools, launched the first of four videos starring comedian Walli Collins.
Today, ProTexting, provider of a suite of mobile marketing tools, launched the first of four videos starring comedian Walli Collins. "Goat," in which Collins uses the company's mass SMS-delivery tool to text a joke to a nightclub audience, posted to ProTexting's Facebook page. New videos starring Collins will post on the next three Thursdays, followed by voting for the best video on Facebook.
"The campaign was sparked by some of our clients being comedy clubs," said Kalin Kassabov, CEO of ProTexting. "It was designed to be a unique way of presenting the interactions that we provide. We also wanted to reach out to businesses so they can get the idea of how it can be used in real time."
The videos will be posted on social media channels, as well as comedy-oriented Facebook pages like Comedy Central and StandUpNY, as well as those of television stations.
ProTexting lets businesses create, deliver, and monitor mobile engagement campaigns including not only mobile coupons but also mobile surveys and voting, mobile sweepstakes, mobile virtual business cards, and QR codes.
For example, ProTexting client Sodexo USA, a food service company, uses the tools to increase sales and customer loyalty at student dining facilities. It sends coupons and deal alerts at times when traffic is slow, sends updates on menus, and asks for student feedback. Kassabov said that when one Sodexo dining hall used the tools to send out an SMS promoting a hamburger special, burger sales were 10 times higher than the previous day.
While more comedy clubs will likely take notice of the campaign, Kassabov said that there is no special targeting for this campaign. "We didn't want these videos to be in your face advertisements. We wanted people through the comedy angle to see the technology in action in a more indirect way," he said.
Susan Kuchinskas has covered interactive advertising since its invention. The former staff writer for Adweek, Business 2.0, and M-Business covers technology, business and culture from Berkeley, CA.
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text:
Sierra Madre Jungle Tour
We had a great time on the Sierra Madre' jungle expedition. The ride alone to the jungle was interesting with a stop in some small town so that we could get that experience (as well as quickly see hand-made tortillas being made).
The jungle tour was very interesting as our guide pointed-out all kinds of information on wildlife, insect, and trees/plants. The free lunch was basic sandwiches with drinks.
The tourguides did a superb job informing us as well as keeping us entertained.
The excursion was well-worth the money... I believe about $500 pesos each.Related to:
- Jungle and Rain Forest
- National/State Park
- Hiking and Walking
Folklore Tequila Tour
Well I have been to Puerto Vallarta a number of times and didn't want to spend what little time we had in PV on a tour, so we decided to head straight to the tequila factory. I had called ahead of time to get information on the location and how to quickly get from the pier to the factory.
So we got off the tender and headed straight to the taxi stand. 5 minutes (and $5 USD) later, we arrived at Aquí Jalisco. And wouldn't you know it, a tour was going on at the same time...so we tagged along.
Received information on the history ot tequila, how it's grown and the different versions made from the agave plant. After that, we went into the tasting room to sample different tequilas. 10 - 12 shots later, we staggered to the counter to purchase some for taking home.Related to:
- Historical Travel
- Budget Travel
Day Tour Excursion
We went on a day trip offered by Orbitz.com and found it to be a great time. For $25 a person we got a little taste of everything in PV. A nice description of the history of PV and the major landmarks. We had an opportunity to do a little shopping, treated to open bar, and visited a Tequila Distillery. It was a full day.
Jungle tour
A day-long bus tour, which included walking around Puerta Vallerta with a charming guide talking about history and such is something I would really recommend.
We also drove out into the jungle a ways where you could go hiking, swimming or have lunch. It was just after the rainy season and pretty muddy to go hiking, and I had a great seafood plate (everything seemed so fresh) lunch under a thatched restaurant there. (I can't remember the name)Related to:
- Hiking and Walking
- Beaches
I was there in Sept. and I had the gumption to try an adventure tour and naturally I did some research and found that the number one tour operator in PV is Vallarta Adventures and so I set up to do a few tours with them and they were all fantastic. The hardest part is deciding which tours to do because they offer so many amazing things...from swimming with dolphins to jungle hiking to flight seeing to anything you could imagine...they do it...and professionally...I was blown away.....check out their website if you´re planning on coming here. THANK ME LATER!
San Blas /Boat tour thru mangroves
Visit the small town of San Blas, where you can see beautiful 16 th century forts and ruins. There are a lot of interesting Mexican arts/crafts not seen in Puerto Vallarta. I was kicking myself later for not having bought these unique tara -cotta beads that, I , of-course could not find anywhere else! The trip takes about 2 hrs by bus, north into the hills, lots of good sight seening along the way... tobacco fields, banana tree's, tequila cactus, etc. Also many small Mexican villages along the way as well. After visiting San Blas the highlight of the trip is the boat ride up the mangroves,but not for those who are afraid of crocadilles, as you see many out in the wild and they are huge! Also, a lot of wild birds, large turtles, and interesting bugs. Once at the top of the mangroves you can dock and take a tour through the wild life preserve again, lots of croc's, quotimundo's(not sure of spelling), colourful birds, etc... they are all caged of-course. It was definitly the highlight of our trip, we had our 3 teenagers and they loved it, something they will never forget!Related to:
- Birdwatching
- Historical Travel
- Family Travel
Tequilla Factory Tour
You will learn about the manufacturing process, You will go on to a traditional tequila factory where the old-fashioned methods of producing tequila will be demonstrated, and of course samples will be given. They will teach you how to properly drink tequilla: ..Breathe in with your nose, hold your breath... drink it and then exhale through your mouth
ONE TEQUILLA!, TWO TEQUILLA, THREE TEQUILLA, FLOOR!!!!!Related to:
- Family Travel
ATV Tours
This was a lot of fun. A group of us took an ATV tour that lasted a better part of the day. We started out from town and our to the desert . We traveled through lush river beds, dry river beds, through an old town and then to the old tequila factory where, yes, we tasted different tequilas.
Going through the small town was quite humbling. Not only is this old Mexico, but so many of these people still live without running water or electric. We stopped for a soda and to use the restroom. Outside the restroom sat a little boy taking tips for handing us paper. Tony gave the little boy $2 and the little boy was so excited. Makes you really think about how lucky you are.
At the tequila factory there is an authentic local Mexican restaruant where they cook everything in front of you on fire stoves. GET SOMETHING TO EAT. It was wonderful.Related to:
- Desert
- Adventure Travel
Go for a Drive
We rented a car and decided to do our own sightseeing, which if you really want to see outside of town, you must rent a vehicle and explore. This is Los Arcos, we snorkeled around these rocks, and that particular day, the currents were shifting and the water was murkey and there were lots of jellyfish in the water.
Playa Los Animas
South of Mismaloya, just past Le Kliff restaurant, there is a beach (Boca de Tomatlan) where you can grab a beer and then a water taxi to Las Animas.
The ride is short (5-10 minutes) and the beach is small and charming. Just hanging out for an hour is fun. Last trip I tried parasailing which was easy and an amazing view of the area.
For those who are not so adventerous or who do not want to get stuck on an all day adventure, I highly recommend.Related to:
- Luxury Travel
- Food and Dining
Trekking a Creek
As we drove by on our way south out of Puerto Vallarta after Mismaloya Beach, we trekked up the mtns, stopped at road side restaurant, walked around, saw some hill fires, and then I spotted a cross on the side of the road, blue glitter shrine, (there are many of these on the side of the road), and though we'd look over the edge on our way back, and discovered there was a beautiful water fall, which it took 2hrs to climb down to, it was nice to dip our feet in the pool to cool off.
Elizabeth Taylor's former viila
Elizabeth Taylor is possibly the most famous of all celebrities to live in Puerto Vallarta. Vallarta was put on the world scene when she accompanied her lover Richard Burton, while still married to Eddie Fisher, to a small fishing village on the coast of Mexico where he was filming "Night of the Iguana."
Their subsequent decades long torrid romance still echoes through the narrow alleys of Gringo Gulch where they bought houses across the street from one another and linked them with a pink bridge. Casa Kimberly still exists and is now open to tours.
Source: VirtualVallarta.com
Great Tours, Sightseeing, Restaurants, etc.
We've gone on many tours, geat finds on restarants, nightlife, and visited small towns outside the city.
We've gone on Jungle tours on 4 wheelers, Horseback riding through the mountains, City Tours, and Boat tours to private islands.
Great restaurants in the Marina, go to the top of the Marina lighthouse for a cocktail at sunset, eat at a restaurant on Los Muertos Beach, and party at many of the clubs on the Malecon. Go to Le Kliff restaurant for a special dinner. Lazy Lizard palapa restaurant on Los Muertos beach for a cheap bucket of beer and great food. For a secluded beach visit Mismaloya Beach, Nuevo Vallarta, or the small town of Sayulita.
Ocean from Condo for rent.Related to:
- Beaches
- Road Trip
- Romantic Travel and Honeymoons
Explore villages and beaches out of town
One day I took the bus all the way south of town to Boca de Tomatlan. It is a small, mostly untouristed, fishing village. There is not a whole lot to do there, but it is a nice place to relax, wander around, and take a few pictures. The views are very nice. There is an open air restaurant / bar on the waterfront.
From here you can take the water taxi to remote beaches further south. I went to Yelapa which is the most popular, but is somewhat of a tourist trap although I enjoyed it. At Yelapa I kayaked in the inlet, strolled the village, and went to see the waterfall. There are also several other beaches that are much less crowded to deserted along the way. See my travelogues on "Boca de Tomatlan" and "Afternoon in Yelapa".
A HUGE Celebration in a Small Town
The first 2 weeks f January there is a HUGE party honoring Our Lady of Guadalupe in a small town 45 minutes South of Vallarta.
It includes the towns people dressing in various costumes marching to the church, carnival games in the Town Square as well as cockfights too boot.
Vallarta has theirs in the beginning of December but many have said that it is a show for the gringo's. Go and visit El Tuito's and you are sure to see the real deal.
It is a safe little village and you are garneted to have a blast there. For more info regarding El Tuito, see my travelogues.
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Marival Residences & World Spa Puerto...
We had a DREAM VACATION at the Marival Resort. We will be going back once or twice a year. The...more
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text:
Still Locked in Storm's Horror, Hondurans Are Fearful of Future
By JAMES C. McKINLEY Jr.
Published: January 17, 1999
SANTA ROSA DE AGUAN, Honduras— Mirtha Martinez Arriola returned to her home on a recent Sunday and wept when she saw her husband's hat floating in brackish water beneath the roofless frame house where they once lived.
She wept not for the things they had lost, not for the years they had worked to build their home and found a family, but for him and for three of her children, who were all swept out to sea in October and drowned in the worst hurricane of the century.
''I would like to go far from here, to forget all this,'' she said. ''Somewhere where there is a future.''
Around her lay the detritus of broken houses and derailed lives, left there by the wrath of Hurricane Mitch. Half of this coastal village of 6,000 people -- more than 80 houses -- was swept out to sea or submerged under the engorged River Aguan during three days of floods, gales and rain so hard it felt like a thousand whips. Forty-one people perished in a desperate attempt to reach higher ground.
Where once there was a road into the village, there is now only a wide expanse of dirt-brown water, with the wreckage of two school buses peeking from the waves. The grade school and high school have been wrecked, along with the bus station, the library and several businesses. Dozens of houses not swallowed up entirely by the sea have been reduced to piles of rubble.
The storm even changed this sleepy village's geography. More than 200 yards of land disappeared under the relentless pounding of the surf. The narrow river that once ran through the village has been moved a quarter of a mile from its original course and has expanded to four times its normal size. Part of the community has been turned into a sad island of devastated homes that will never recover.
More than two months after the hurricane struck, little has improved. Power and phone lines are still lying on the ground. There is no potable water. Many children are sick with diarrhea and unexplained fevers.
Although some shipments of food and medicine have been ferried in by charities and the Government, two large containers of aid from a New York City group are being held up in port by customs officials.
And though it was always remote and impoverished, the village is more isolated than ever. To reach Santa Rosa, one must travel more than two miles on a dirt road, cross a swollen river in a canoe where there was once a ford, then travel 11 more miles to the edge of the village, where a canoe or launch can carry the traveler to the town's center a mile across the water on the other side of the Aguan River.
Founded in 1887, Santa Rosa de Aguan has never had much political influence in the capital, Tegucigalpa. Only in the last few years was electricity brought in, and requests for a paved road and a bridge to link the town to the outside world have been bogged down for decades.
The Heartache
Sad Songs Are Sung For a Lost Family
One reason is cultural. Like many coastal communities in Honduras, the village is dominated by the Garifuni, descendants of slaves and Carib Indians from the island of St. Vincent who were exiled to Central America in 1795 after a revolt there. The Garifuni, of African descent, have never wielded as much political power as lighter-skinned Hondurans of Spanish and Indian ancestry.
Many of the people appear to be still in shock from the hurricane. Like shipwrecked sailors, some spend their days scavenging wood from the wreckage to build temporary shelters. Others who lost family members have fled to live with other relatives in nearby towns like Trujillo and La Ceiba, unable to bear the place any longer. But most of the villagers were born here and say they want to rebuild their small town.
One is Robert Blanco, who spent 23 years working on tracks for the Metro North railroad in New York before returning four years ago to retire on a pension. Mr. Blanco's house is under the river, along with all his belongings.
''So many people say they are going away, and others say they will rebuild in a different spot,'' he said. ''I'm going to build me a little house over here because I don't want to leave the place. I was born and raised here.''
Others say they would leave if they could, but they have nowhere to go.
Ms. Martinez Arriola's mother, Hilda Arriola Arouz, runs a soda shop and outdoor restaurant on the side of town that was largely swept away by the river. She used to live with her youngest daughter, Nori Esperanza, but the 32-year-old woman has lost her composure completely and can no longer bear to be in the village. She becomes hysterical each time she returns, Mrs. Arriola Arouz said.
Mrs. Arriola Arouz is 67, and a widow. So many people have abandoned the village there is no business for her restaurant. Her son-in-law and four grandchildren are dead. Her surviving daughters are both having trouble coping with the tragedy. The twisted roof of her house lets in the rain now and she has no way to fix it. The house seems dark and empty to her, bereft of children, who are the light of the world, she says. Although she puts on a brave face, she too says it is hard to confront each new day.
''Most days I pass in sadness,'' she said, ''because all the grandchildren that I lost lived most of the time in my house. Now I'm sad and there is nothing but this sadness.''


text:
Citigroup cutting jobs in Phoenix
HAGERSTOWN, Md. — Citigroup Inc. said it's cutting 950 jobs across the country, including positions in Phoenix and Las Vegas.
Spokesman Mark Rodger said Friday that the jobs will be cut by the end of
March as the company sells the rights to service 64,000 defaulted mortgages,
mostly on behalf of Fannie Mae.


text:
Email this article from PlasticsNews.com
Dynisco's Vertex sensors measure melt pressure directly
Dynisco LLC says its Vertex melt-pressure sensor is robust, faster and significantly friendlier to the environment than other sensors. The breakthrough lies at the tip of the sensor, where melt pressure is registered directly through a single diaphragm.
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Come visit me, TAZ-.Thanks for the great honors..., SCOOBER AS A WARLOCK, CHICA ... HALLOWEEN, ROSIE....HALLOWEEN, NAPOLEON...GO HAWKS!, SUZIQ...Happy Halloween, THE GREY'T GREY WINGS - THANKS TEAM, The Proctors...GO HAWKS!, SIMON...HALLOWEEN, Chloe - HALLOWEEN & JEWELS-- HALLOWEEN.
Come visit me, Paddy Cake & Piddy Girl, CORI vote STORMY BOTM 212678 & BOO BOO vote STORMY BOTM 212678.
Come visit me, Hulk Vote Stormy For BOTM.
Come visit me, Derby Farms, Chyna & Bailey ♥ RIP.
Come visit me, ANGEL LOVES CORI & PEPPER LOVES ZIPPY--BOTD 9/2/15.
Come visit me, Remington, Athena ~ Thank you all for Bird of the Day Honors! & Mackenzie - Thank you for BOTD!.
Ronnie Lee, Charlotte, NC.
Cynthia, Costa Mesa, CA.
Come visit me, 10K Hall of Fame~THANK YOU FOR THE BOTD HONOR, BOTD ARCHIVES, Sugar and Pippo~VOTE STORMY 212678 BOTM & The Calendar Page~Some Figs for October.
Ron & Dee Ann, Bellevue, NE.
Come visit me, PARKER, SILVER, APACHE MARRIED HIS LOVE SNOWFLAKE ON 09/20/14, OZ (MY MOMMY'S DREAM BIRD) & CHEYENNE MARRIED HER JEDI ON 09/20/14.
Come visit me, Gizmo and Divo, mom loves you both, Willie, Stormy for BOTM, Peppino, vote forStormy & Falco, vote Stormy for BOTM.
Come visit me, Pearl & Silver.
Come visit me, Silver Sluggers Page Where Greyt Things Happen & Sam & Miranda at the Milonga Fair!!!.
Come visit me, Sunny(RIP) and Haley, Romeo and Julie(RIP) and loving memory of Maxi & Tahoe and Pearl(RIP).
Come visit me, Angel Bertie's Rainbow Gym: Thanks for BOTD!, Angel Bertie: Happy New Year!, Chirps: Happy New Year!!!, The Happy Couple!!!, Spot: Vote Mikey D for May BOTM,276082 & BonBon and Parker:Vote for BOTM!.
Come visit me, Screech & Roof Top - One In A Million.
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The Australian All Ordinaries closed slightly lower again at 5,235 points after the index wavered in a tight 60-point range throughout the session.
Wolf Minerals is one the great success stories in the resources sector, transitioning from explorer to producer of tungsten in the U.K. and generating first revenues.
MD Russell Clark will present at Proactive's Spotlight Investor Luncheons - Oct 20th & 21st. Register now to Attend!
What makes the Keysbrook so compelling is that it will be a high margin long life project with strong growth potential. Commissioning and production in Q4 2015, first sales Q1 2016. First full year forecast EBITDA of $39.9M.
Cauldron Energy is set to extend its exploration rights on the Bennet Well uranium project in Western Australia after identifying as much as 7.4 additional kilometres of potentially economic mineralisation at the site.
Drill logs from Chapman West indicate the presence of a series of sub-vertical epithermal veins trending north-south and north-northwest with associated cross linking flat-lying arsenopyrite rich epithermal veins that dip northerly.
Nexus is a $4M market-capped company with $6M in cash, and has entered a JV with gold producer Saracen Mineral Holdings.
Here is your chance to meet and greet CEO, Andy Tudor, at Proactive's Spotlight Investor Luncheons in October. Register now.
The power of the Wangle™ technology is to enhance privacy and security, increase internet speeds by up to 100% and lower data consumption by up to 50% across popular applications including Facebook, Netflix and Instagram.
Pioneer has recently hit bonanza grade gold at its strategically located assets within the world-class Eastern Goldfields of Western Australia.
David Crook, managing director for Pioneer Resources, speaks exclusively in Proactive Q&A Sessions™.
Kin's Gwalia South also comprises the same geological sequence and structural regime as the Sons of Gwalia mine, which makes this area such a compelling drill target. Gwalia South has now been elevated to one of Kin's priority gold targets.
The recent high resolution aeromagnetic survey highlighted a number of new, subtle anomalies corresponding to major structures and in some cases, historical mines. Follow-up drill testing will commence later this month.
The CE Mark is the mandatory regulatory designation required to commercially market and sell the OncoSil™ medical device in the European Union. OncoSil now awaits the final outcome of the review as the Notified Body continues its assessment over the coming weeks.
Across a 55 metre section, there were 11 down hole depths with disseminated visible gold. This is a very promising start for the company's first ever drill hole at Juma East. Assays are expected in around 30 days.
"Proactive Spotlight Investor Luncheons in October: Wolf Minerals, White Cliff, Blackham Resources, Nexus Minerals, bluechiip. Don't miss out, register today!"
Proactive Investors
"Wolf Minerals has first tungsten production and revenues, but is there more in store? Hear first hand at Proactive's Spotlight Investor Luncheons - Oct 20th & 21st. Register now!"
Proactive Investors
"Nexus Minerals' CEO Andy Tudor to flesh out the the farm-in and JV with Saracen Minerals and strong cash position at Proactive's Spotlight Investor Luncheons - Oct 20th & 21st. Register today!"
Proactive Investors
"Blackham Resources MD Bryan Dixon will discuss Q2 2016 gold production and high $A profit margins at Proactive's Spotlight Investor Luncheons in October. Register today."
Proactive Investors
"White Cliff Minerals is following up visible gold discovered at Aucu and gold nuggets found at Mt Remarkable. Proactive's Spotlight Investor Luncheons - Oct 20th & 21st. Don't miss out!"
Proactive Investors


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JAMAICAN BLACK CASTOR OIL - AMAZING RESULTS ~ HOW TO GROW YOUR HAIRLINE (pics)
What do you think about this video?
Leona Carter: I found it at Sally last week
Drew Corleone: Hey there,
I noticed I'm having a bit of hair loss at my edges (Hairline) so I did some research on this JBCO Product ... Anyways my question is, Do I have to buy the whole kit to get better results, and if so how do I use it?
Aramaki Phuong: how do you apply it to your hair? Do you wash it after a few hours or just sleep with it till your next hair wash? Does your hair have to be clean when you apply the oil? Do you just use it around your scalp or to the body of your hair too? I'm having one JBCO now but I don't know how to use it. please help
Yasmine Charlow: LOL you say your sides are messed up your's look wayyyyyyyyyyyyyyy better than mines lol BUT I have been using the Castor Oil and OMG Im SUPER HAPPY I can wear my pony tail and high bun again <3 <3 <3 <3 <3 <3 oh and make sure the castor oil has NO SALT (Ladies for perm (relaxed) hair)
James Carter: Taking biotin pills daily also helps
JayleeJ: ayyyy 757 in the house.
Dana Lewis: Sallys has step their game up they now sell the jbco as well they just started last mth. I bought some today from there and it was 6 dollars and some change. So im going to try it and hope and pray I see good results. Ur results are very good!!!!!
Lina S Osei: Pls is the Jamaican black castor oil good for relaxed hair. i just bought the product and im scared to use it coz somebody said is not good for relaxed hair
sburke757: i would stick to jbco i mean normal castol oil may work but i dont want to tell you about something i havent tried..jbco works for me
sburke757: hey sorry for late response!!..but yes it should work the same with men as well..i even have my dad using it. !! also thanks for the compliment!!
sburke757: hey sorry for late response im not really sure of your question..but also unfortunately im not familiar with kiratin so i dont want to give you false info :/
ريوف الشهري: i want ask nobtablem if i used and in my hair kiratin
ريوف الشهري: hi how are u I want know how I use the jbco and nobrablem if I use shamoo fast with oil or need use same oil shampoo ????
adam nur: and does it have to be jbco or can it be normal castor oil
adam nur: hey burke love your vids.. im a guy and have very thin hairline and edges..so will this work for me and revive my hairline..
sburke757: agreed!!
sburke757: hey thanks!!! and yes i love this stuff still using it!
sburke757: for me i started to see result by the end of the month and definitely the end of 2 months!
Iyauna Shaniece: I use this product as well. How long does if take.
FORGIVENESS ISFREEDOM: Thx Lutricia lol I was hoping I didn't waist my money on this... anywho someone was tellin me its important to know your hair type cause using too much oils can cause damage... My hair is the then wavy curly type... I don't kno how to find out my hair type lol
JAMAICAN BLACK CASTOR OIL - AMAZING RESULTS ~ HOW TO GROW YOUR HAIRLINE (pics) 5 out of 5


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Blowout pricing! (While supplies last)
The Garmin Zumo 660 LM GPS motorcycle navigator with lifetime map upgrades features 3-D buildings, lane assist with junction view and a 4.3 sunlight-readable, glove-friendly touch screen display. Both a motorcycle mount with hardware and an automotive mount are included, so your Zumo is ready to navigate right out of the box, no matter how many wheels it has under it.
The Garmin Zumo 590LM incorperates all the featues motorcyclists want in a GPS navigation system. With its huge five-inch glove-friendly screen, Bluetooth capability, rugged waterproof housing, and ability to run TOPO maps and off-road tracks, this GPS is a must-have for adventure riders and motorcycle tourers alike.
The Garmin Zumo 390LM has a wide 4.3" touchscreen that is easy to read, even in sunlight. Its glove-friendly intuitive interface makes entering information as easy as shifting gears. With a rugged body that is waterproof to IPX7 and resistant to fuel sprays and UV rays, Zumo 390LM is built for life on the road.
The Garmin Tire Pressure Monitor Sensor (TPMS) enables you to monitor your motorcycle's tire pressure easily and conveniently, as you ride. The system works with up to 4 tires in any configuration (not intended for automotive use). One TPMS sensor is required for each tire (sold separately; metal valve stems only).


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Hope we all had a good week of DFS NBA. Last weekend the article did just ok. If I remember right, last weekend was weird. The stud players really didn't hit value and the value guys didn't come through. Let's not dwell on the past. Sunday night we have a bigger than normal nine game slate. Think that has to do with what is going on next Sunday. I also have a little piece of DFS news linked here. Good luck in your games and let's win a little coin.Please, blog, may I have some more?
Eric Gordon
Little was known about the severity of LaMarcus Aldridge's thumb injury when he missed Wednesday's game. I was texting with Razzball's own JB about the Blazers and JB voiced his concern that Aldridge would "be out a while." After undergoing another MRI on Thursday, the Trail Blazers announced that Aldridge would be out 6-8 weeks with a torn ligament in his left thumb.
Is JB some sort of psychic? As his college roommate, I can neither confirm nor deny that, but what I do know is that owners will be hard pressed to replace Aldridge in their lineups. He was averaging 23.2 ppg, 10.2 rpg, and 1.2 bpg. Those are some pretty huge shoes to fill.
The Blazers are already shallow in the frontcourt, since Robin Lopez and Joel Freeland are also ailing. As a result, they will use Dorell Wright and Thomas Robinson more at the four. Wright started Wednesday against the Suns (15 points, 5 assists, 3 threes), but then Robinson started Thursday's game against the Celtics (9 points, 12 rebounds, 2 blocks). It's unclear who will start or if coach Terry Stotts will play matchups. Unless someone gets the majority of minutes, Wright and Robinson will eat into each other's playing time, limiting their fantasy appeal.
To make matters worse, Nicolas Batum left early in the third quarter with a wrist injury. An upcoming MRI this weekend will reveal more information, but this could be even more devastating to the Blazers.Please, blog, may I have some more?
Last year, I think Jonas Valanciunas might have won the "most annoying player for JB" award. Eh, Jeff Green was up there too… But we've got a clear front-runner for 14-15!
If you had money on Brandon Jennings racking up the first double dimebag of the season, then all praise be onto you! Great game against the Magic, as he gashed their "defense" for a 24/0/21/0/0 line. Pretty empty to me, only two cats in there! First 20-assist game this season, doubling up the dimebags and making BJ's value as high as Katt Williams. You're probably looking like this as a Jennings owner right now! He's only the 4th guy to put up a 20/20 game since 2000, and I guess all my sell Jennings high hooblah is looking a little dumb now… I still think if you can get one of the higher-tier guys like a Jeff Teague you have to do it, but otherwise I guess it's a strong hold and ride the wave. Upside of two dimebags on any given night?! That's a friend you need to keep for life! Here's what else went down last night in fantasy basketball action:Please, blog, may I have some more?
We all love fantasy basketball, but it's a marathon, not a sprint, and if you've run marathons, you know that you get tired after half of it is done. I mean, I don't have any clue at all, but it stands to reason, right? Doldrums is the running term for that. And doldrums is also a sailing term, and not at all related to running actually. But I get a little frustrated, depressed, let down, etc. this time of the season, realizing the cats who have under performed, or acted just plain goofy (I'm talking to you Kevin Garnett! you used to be so damn beautiful, and you're tainting your legacy a bit with this sucky stuff). But like G.I. Joe said, knowing is half the battle, and we can turn our frowns upside down with a little creative trading and/or acquisition. Take a gander at these booms and busts:Please, blog, may I have some more?
Awwwwww, yeah! Yeah J-Val. Gimme a little peek. Gimme a little glimmer of hope!
Oh man, was it a tale of two halves for the Raptors last night! They were lobbin' shizz all over the place like they were the Globetrotters against the… Who do they play again? That they always beat? Oh yeah, the Knicks, like they were the Globetrotters against the Knicks. Kyle Lowry had 9 dimes in the first quarter and Jonas Valanciunas put up 14 points on 7-8 shooting. Drummo playing uggo! Kinda a "but his D" lately… The Luminescent Lithuanian ended up going 14-15 for 31/12/0/0/1. The FT% is nice (3-4 last night and right at 80% on the year), but his combined – yes COMBINED – Ast/Stl/Blk this year are under 2. Sigh. I thought he could turn into something so much more special! There are some long-term nice things – per-36s he's cut down a few TO, scoring more, fouls are down – but Casey is playing him lower minutes than last year (and wouldn't consistently feed him after that hot start) and I don't know if he's ever going to be more than a slightly taller Zach Randolph. And with that, J-Val is all insulted, buttoning up his clothes, and not giving me that flash of hope. Dammit, I'm my own cockblocker! Me n Valaciunas are gonna be awkward for a while until safely in the friendzone. Here's what else went down on a light night of Monday action:Please, blog, may I have some more?
Yesterday afternoon was a frenzy! A frenzy because no Mozzy! About as crazy as I was last Saturday about Fozzy! Go Panthers!
Our prayers have been answered with Timofey Mozgov getting shipped out to Cleveland for two first round picks. Two first-rounders?! For Healthcare.Mozgov?! Well, they're like controlled picks from Memphis in some sort of hooblah where all that matters is they're not going to be very high. Not as high as say, Afroman! Or 16th! That's where Jusuf Nurkic went in the first round this Summer, originally picked by the Bulls then traded. Good thing for Nurkic! It originally seemed really muddied there in Denver at C, but JaVale McGee just can't get healthy, and Mozgov couldn't go 3 minutes before getting multiple fouls. So out ya go TinaFey, and huge role ahead for Nurkic! At least 35 minutes every night given how good he's been! Andddddddd, only 22 minutes for 8/4/3/1/2 last night. Still respectable numbers though! Given one of his made FGs was this. To quote my favorite sound from 90s gaming, "Oopsie!" Or whatever he really says… Anyway, J.J. Hickson is still there to give us a headache, as he went 11/10/1/2/1 in 30 solid minutes. Yikes. I still love me some Nurkic, and to the patient man goes the spoils! Something like that… Kinda lost in all of this yesterday was Hickson's deep league value shooting up. He's not a terrible player for some Pts/Reb relief in a very poor man's Zach Randolph kinda way now that minutes should be coming. While many owners are mad they didn't scoop Nurkic up quick enough, shoot out some buy lows with who you would've dropped, and maybe last night's dud is enough to sneak Jamba Jus into your Mini-mall. That sounds so wrong… Here's what else went down on a busy night of NBA action:Please, blog, may I have some more?
Some NBA GMs had a case of the Mondays, that's for sure! "Uhhhh, I'm not ready to be back at work, let's do something nuts just to make it look like we're working…"
In a blockbuster three-team trade, the New York Poppycockers salary dumped Iman Shumpert and J.R. Smith to the Cavs. More pressure on Blatt! Now he has to win with Knicks Outkasts… "I like the way you move!" The last NBA coach who said that to Smith was George Karl. Shumpert is likely going to start at the 2 with Smith becoming a 6th man. Both of them must have had a redonkulous party to be heading to a winner. Don't invite Plaxico or Felton! Then there's Dion Waiters to the Thunder. Meh. Not like most leagues were using Waiters anyway, but he takes a minor hit in deepers. Reggie Jackson should still lead the second unit in shots, so I'm not that excited. The Knicks picked up three guys – Captain Kirk (but not the Hinrich one), some European with a lot of hair, but then an interesting name in Lance Thomas. I almost made a cracker joke, but we're gonna gloss right over that and reminiscence in some games he had earlier in the year on a depleted Thunder front line – 12/8/2 (Nov 1), 14/5/1 (Nov 4), the fluky 7/13/6 (Nov 12). And depleted is baked right into the Knicks funnel cake right now. The Poppycockers might as well see what they have at some point, so in uber deep leagues if you need someone with any sort of upside, I'd be sure and star LT on your wire. Here's what else went down on a crazy Monday for fantasy basketball:Please, blog, may I have some more?
In a jam-packed weekend with all sorts of big news and Anthony Davis putting up a career-high, I figured why not start as off-the-fantasy-radar as possible! Well, that's a lie, my Steve Nash open about him being a malnourished librarian turned out to already be a commercial…
The injury gods were working overtime this weekend, as big ol' Roy Hibbert landed funky on his ankle and left very early in the Pacers' game Saturday. The bad news continues to floweth down the bad news river in Indianapolis. Drown your sorrows in onion rings, Pacers fans! Not gonna have any other rings any time soon. "That was mean, JB!" I'm rooting for Paul George to come back healthy next year and shock the Cavs in the playoffs, don't you worry. But back to the issue at hand, when Howard Cosell made the call "Down goes Hibbert!" I immediately had something bubbling up inside [Mahin]me. Certainly not one of the premiere backup Cs in the NBA, but Ian Mahinmi is passable, and a solid short-term add for some early-week big man upside. Mahinmi carried the torch with the starters for an all-leagues usable 12/10/1/3/1 line in only 26 minutes, without even finishing down the stretch as the Suns went full supernova to the Pacers white/brown dwarves (I don't wanna say one or the other, might get into trouble…). The backup Frenchy could easily go 10/10 with 2 swats in a few starts early this week. Tonight is against a gimpy Tyson Chandler who tweaked his kankle, then Wednesday against a Spurs team who have struggled to handle bigs since Tiago Splitter has been in a siesta. So while I Mahin-Me, I hope while reading this there is time to Mahin-You! Here's what else went down over the weekend in fantasy basketball:Please, blog, may I have some more?
You might as well set up a Civil War style triage on the Thunder bench, because somebody has the team set of Thunder voodoo dolls and isn't wasting any time! Adding to this virtually unprecedented injury wave to hit an NBA team through only week one of the season, Perry Jones knocked knees for the second game in a row; this time in a ton of pain and had to exit. They say a picture is worth a thousand words, so check out the Thunder depth chart on Rotoworld, where the red S means sidelined:
Oklahoma City Red S-es! And while a "knee contusion" usually wouldn't worry me too much, George Hill had the same deal and he's going to be out until late-November is sounds…
On the plus side, Serge Ibaka is absolutely en fuego out there. 25/11/2/0/1 and forced to take contested jumpers the whole night, but hit 11-14 including three treys. And played 46 minutes out of desperation! The blocks will come, so if you got Serge late round 1 (like we had him) or round 2, you've got something of your own surging in your pants.
On the negative side, Reggie Jackson is playing with some ill-advised reckless abandon. I mean, give the kid some props for playing through numerous injuries last night, but he ended up with his butt on the hardwood more often then Paris Hilton. Had a fantastic slash of 13/4/14 but hurt his wrist limiting him to 4-14 FG (0-6 3PTM). Plus the ankle still looks gimpy. I know the upside is immense the next month, but I might try to sell RJax high after watching this game. It was like watching Denard Robinson at Michigan. I'm not a huge college football fan, but my dad went there and I watch parts of most games, and Shoelace is still the most dynamic player I've ever seen in Maize & Blue. But he seemed to leave early almost every game, getting battered and beaten up.
The Thunder need to do everything and anything to get healthy, with three straight off days until Friday. My recommendations include – every player eating a live octopus chasing it with raw eggs, hiring the Wolverine (Jackman, not Denard) for some mutant healing blood, and calling Mrs. Cleo to figure out which guy will be hurt on Friday and benching him. Here's what else went down last night in fantasy action:Please, blog, may I have some more?
So this is why preseason wrap ups are tough. News flutters around willy-nilly like that bag that dumbass had an obsession with in American Beauty. And most of the news has about as much meaning…
First there's Razzball lovebrows Anthony Davis spraining his wrist last night, and Monty Williams held him out as a precaution. Davis wanted to come back into the game, but Monty was like, "nuh uh, girlfriend! Shaniqua don't live here no mo'!" Not to be out-overreactioned, Russell Westbrook also sprained his wrist on Sunday. Neither are serious, and this is why the wrap-ups are eye-roll fests through preseason. A lot of worthless mud to pan through just to get a fleck of gold. (True story – when I was like 8 or 9 years old, we did a class field trip to pan for gold and a portly kid got a tiny mote worth, the only one to get any. Then I traded him some peanut butter-filled chocolate my mom packed me for lunch for it. Talk about knowing your audience!)
Can you believe we're a week away from opening tip?! This is the last preseason wrap until next Tuesday to preview the season, and daily from there. Can't wait for this shizz to start! My final re-rankings will be out tomorrow, we'll get to meet some new writers, and two final preseason Pods (tomorrow and next Tuesday) before we get some real games to talk about on air. Here's what else has gone on in the world of preseason NBA action:Please, blog, may I have some more?


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HOLLYWOOD - God bless America, and how's everybody?
"Man, this government shutdown is making America look foolish."
Which is harder to believe? The ludicrous shenanigans going down in Washington or the fact that nobody seems particularly interested in doing anything about them? Good neighbors -- it looks like we got ourselves one heck of a bumper crop of official dysfunction this year. Near as high as Manute Bol's eye.
"Republicans are terrorists!" "Arsonists!"
Predictably, my column last week, which expressed my extreme displeasure at the images of World War II vets being barricaded from visiting their memorial on the National Mall, produced a flurry of reactions, both positive and negative.
"Waaaaa! You're not playing fair!"
"DEAR COLUMNIST: I am 8 years old. Some of my little friends say the government shutdown will last a long time, America will default on its debt and our economy and the world economy will nosedive. They say conservative Republicans are bullying the America political system and economy in way that doesn't fit with how democracy is defined in my history books. My father says President Barack Obama and the Democrats started it and want the government shut down and a default. Papa says 'If you see it on Fox News, read it on a conservative website, or if ...
It was Cory Booker's constant sneering attack in the first New Jersey U.S. Senate debate - Steve Lonegan is a "tea party leader." What does that mean?
Reasonable people compromise. Unreasonable people do not. Instead, they trample over those around them to selfishly get their way. And the government is shut down this chilly October day in 2013 because unreasonable Democrats behaving like spoiled children would rather refuse treatment to terminally ill children, ravage the economy, disparage veterans and make the rest of our lives miserable -- than compromise.
Dear Editor,
I remember when a billion used to be a number so big nobody could comprehend it, though it is still a massive number.
The federal government shutdown has made me sick of politics. For a political columnist, this can be tricky, but taking a break from politics also offers an opportunity. This week, I thought I would write a column about what I've learned as a father of two spirited boys, and offer my advice on how to handle tantrums.
Government's closed, everybody! Go home. Except Congress, that is, whose members are still getting paid, classified as "essential workers." Although right now, neither one of those words seems very apt or ept. Unapt and inept is more like it. Inapt? Unept?
Page 66 of 133
What's a teacher to do if he sees a wired briefcase that looks like a homemade bomb in the classroom?
To the one thirteenth of all Americans who watched the latest GOP debate, congratulations on surviving the political equivalent of the 24 hours of Le ...
Is the current refugee crisis gripping the European Union "all America's fault"? That is how my critique of U.S. foreign policy was characterized ...
The Marine Corps just spent $36 million to learn that an average-sized woman is not as big and strong as an average-sized man. I'm ...
I didn't watch CNN's telecast of the Republican debate because I was there in person.
Democrats are bailing on scandal-plagued Hillary Clinton, and the big Democratic Party money can't possibly be enthusiastic about socialist Bernie Sanders.
Apologies are a first world problem. Sometimes, the apology is pro-forma, as in "I am sorry if anyone was offended by my words." Sometimes, an ...
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"No group of birds in the world is more beautiful and more intriguing to science than the birds of paradise. Surprisingly, no book showing them displaying in the wild has been done before. But to gain such perspectives and develop such a wonderful collection of pictures has taken years. The result is truly breathtaking."
--Sir David Attenborough, filmmaker and creator of Life on Earth
"You'll initially find it hard to believe that these pictures are real—but they are. These are the most beautiful photographs of the world's most beautiful birds. The birds' behavior, richly described in the text, is as remarkable as their appearance. This is a book like no other book."
--Jared Diamond, ornithologist and author of Guns, Germs, and Steel
"The resulting extremes of color, form and behavior are beautifully documented in Schole's new book, co-authored with photographer Tim Laman." –Sciencefriday.com
"The exhibit "Birds of Paradise" at the National Geographic museum in Washington, along with a book by Tim Laman and Edwin Scholes, offers research, photographs and video of the 39 species of the bird…" –The Wall Street Journal
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This is an absolutely gorgeous book! Almost all photos are actually beautiful photographs. Many full size richly colored photos. Large book made with heavy stock quality paper. Read morePublished 9 days ago by Amazon Customer
I found out about this book when visiting a museum where an exhibit about birds of paradise was displayed. Read morePublished 2 months ago by Precious
Simply one of the most beautiful books done on a bird subject. And the stories about the making of this amazing are incredible all on their own. Read morePublished 5 months ago by Improv bass 4
Loved it!! The full page close ups of the birds are incredible!!Published 8 months ago by Linda Eakins
Our photographer nephew was very pleased to receive this as a holiday gift. It is indeed beautiful.Published 8 months ago by Bernice Eubank
Amazing photographs. I gifted this book to my sister who studied zoology and absolutely loved how the book covers the intimate lives of these incredible birds. Read morePublished 9 months ago by Srivats Ram
What a wonderful book on these fascinating birds! The video produced by the same authors along side with this book is a good adjunct, but the book provides more in depth in... Read morePublished 10 months ago by Kitty kat


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Kids Clothes Coupon – July 2013 The Childrens Place Outlet Printable Coupon
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Page 1
Purification and immunohistochemical analysis of calcium-binding
proteins expressed in the chick pineal gland
The vertebrate pineal gland is a main source of circulating
melatonin, which is secreted in a daily rhythmic manner
with a nocturnal peak. Especially in the case of the chick,
the pineal gland retains a circadian clock that drives the
daily rhythm in melatonin production even in dissociated
cells [1, 2]. Anatomical studies on the chick pineal gland
have described two types of morphologically distinct
pinealocytes, the follicular and the parafollicular pinealo-
cytes [3]. The follicular pinealocytes are radially oriented
within each follicle, and protrude their rudimentary outer
segments into the follicular lumen, whereas the parafolli-
cular pinealocytes are located at the periphery of the
follicles. These two types of cells express several enzymes
constituting the melatonin synthesis pathway, including
N-acetyltransferase [5] and tryptophan hydroxylase [6].
Pinopsin, a pineal photoreceptive molecule [7], is expressed
in both types of cells: in the outer segments of the follicular
pinealocytes and in the string- and bulb-shaped cellular
structures of the parafollicular pinealocytes [8]. These
observations, together with the light-dependent phase shift
of the daily rhythmic melatonin production in dissociated
cell culture [9–11], suggest that at least a subset of the
melatonin-secreting pinealocytes retains a circadian clock
system equipped with a photic-entrainment pathway.
Crucial roles of intracellular calcium ions (Ca2+) in the
function of the chick pineal cells have been demonstrated in
dissociated cell cultures. In chick pineal cells, Ca2+influx
increases at night through a cationic channel which is
regulated by the endogenous clock [12], and the nocturnal
increase in melatonin secretion is facilitated by intracellular
Ca2+[13, 14]. Pharmacological studies support the contri-
bution of calmodulin to the regulation of melatonin
production in dissociated pineal cell cultures [14–16]. In
contrast, immunohistochemical studies point to the absence
of calmodulin immunoreactivity in chick pinealocytes [17,
18]. This is a typical example of issues possibly based on the
lack of information at the protein level, and therefore we set
out to investigate the molecular identity of the chick pineal
Ca2+-binding proteins (CaBPs). Ca2+-binding proteins
having regulatory roles are termed ?Ca2+-sensor? proteins,
whereas those involved in stabilization of intracellular
concentration and/or its transport are termed
?Ca2+-buffer? proteins [19].
Generally, the Ca2+-sensor proteins are subjected to
large conformational changes upon Ca2+-binding to
expose the hydrophobic domain responsible for interaction
with their target proteins [20]. This allows efficient and
comprehensive purification of a series of Ca2+-sensor
proteins by hydrophobic chromatography on a phenyl-
Sepharose column [21]. Taking advantage of this proce-
dure, we were able to isolate five kinds of chick pineal
proteins which exhibited Ca2+-dependent elution from the
column, and they were identified by micro-sequencing
analysis as calmodulin, neurocalcin, sorcin, annexin II
and annexin V. Together with the protein level study,
Abstract: The pineal gland is a site of melatonin production, of which
intracellular calcium ions (Ca2+) are likely involved in various aspects. To
investigate the identity of molecules responsible for the Ca2+-dependent
processes in the pineal cells, we prepared a cellular extract from 2000 chick
pineal glands and isolated a series of Ca2+-binding proteins by taking
advantage of their Ca2+-dependent hydrophobic interaction with phenyl-
Sepharose beads. The proteins identified by micro-sequencing analysis
included calmodulin, neurocalcin, sorcin, annexin II and annexin V.
Immunohistochemical analysis of the chick pineal sections revealed that both
calmodulin and sorcin are expressed in the follicular and parafollicular
pinealocytes. On the other hand, neurocalcin was expressed in a few neuron-
like cells located predominantly in the parafollicular layer of the pineal
follicle. These results suggest that calmodulin and sorcin may contribute to
cellular functions in the chick pinealocytes.
Fumiko Shimizu, Kamon Sanada*
and Yoshitaka Fukada
Department of Biophysics and Biochemistry,
Graduate School of Science, The University of
Tokyo, Tokyo 113-0033, Japan
*Current address: Department of Pathology,
Harvard Medical School, 200 Longwood
Avenue, Boston, MA 02115, USA
Key words: calmodulin, chick pinealocytes,
circadian rhythm, melatonin, neurocalcin,
protein sequence analysis, sorcin
Address reprint request to Yoshitaka Fukada,
Department of Biophysics and Biochemistry,
Graduate School of Science, The University of
Tokyo, Hongo 7-3-1, Bunkyo-Ku, Tokyo 113-
0033, Japan.
E-mail: email@example.com
Received October 11, 2002;
accepted December 10, 2002.
J. Pineal Res. 2003; 34:208–216
Copyright ? Blackwell Munksgaard, 2003
Journal of Pineal Research
Page 2
immunohistochemical analysis revealed that calmodulin
and sorcin are expressed in most of the follicular and
parafollicular pinealocytes, supporting their active roles in
pineal function.
Materials and methods
Isolation of calcium-binding proteins
Animals used in this study were treated in accordance with
the guidelines of The University of Tokyo. Newly hatched
male chicks (Gallus gallus) were purchased from local
suppliers and killed by decapitation on day 1–3 during the
daytime. Their pineal glands were isolated, frozen with
liquid nitrogen, and stored at )80?C until use. After
thawing quickly at 37?C, the pineal glands (2,120 glands,
4.75 g wet weight in total) were homogenized with 40 mL
of an extraction buffer (10 mm Tris–HCl, 2 mm ethylene-
glycol bis(2-aminoethylether)tetraacetic
5 mm MgCl2, 1 mm DTT, 4 lg/mL leupeptin, 4 lg/mL
aprotinin, 0.5 mm benzamidine; pH 8.0) using a glass–glass
homogenizer. The homogenate
125,000 g for 30 min at 4?C and the supernatant was
stored. The precipitate was homogenized again with 50 mL
of the extraction buffer and the homogenate was centri-
fuged. The supernatants obtained from the two centrifuga-
tionswerecombined (termed
supplemented with CaCl2 so as to yield a free Ca2+
concentration of 2 mm.
Calcium-dependent hydrophobic-interaction chromatog-
raphy on phenyl-Sepharose column was performed accord-
ing to the method of Gopalakrishna and Anderson (1982)
[21] with some modifications as described previously [22].
The phenyl-Sepharose CL-4B (Amersham Biosciences
Corp., NJ, USA) column (7 mm ID · 52 mm) was pre-
equilibrated with a high-Ca2+buffer (10 mm Tris–HCl,
2 mm EGTA, 4 mm CaCl2, 1 mm DTT, 4 lg/mL leupeptin,
4 lg/mL aprotinin, 0.5 mm benzamidine; pH 8.0), and then
the pineal extract was applied to the column at a flow rate of
28 mL/hr. After washing with the high-Ca2+
proteins were eluted from the column with a low-Ca2+
buffer(10 mmTris–HCl,2 mmEGTA,1 mmDTT,4 lg/mL
leupeptin,4 lg/mLaprotinin,0.5 mmbenzamidine;pH 8.0).
acid (EGTA),
was centrifugedat
?pinealextract?) and
Amino acid sequence analysis of calcium-binding
A phenyl-Sepharose fraction (Fig. 1, fraction 3) containing
the protein with an apparent molecular weight of 20,000
(20K protein) was subjected to an anion-exchanger column
Mono Q FPLC (fast protein liquid chromatography;
Amersham Biosciences Corp.), and proteins were eluted
with a linear gradient of 0–1 m NaCl (25 mm/min) in
10 mm Tris–HCl buffer (pH 8.0) at a flow rate of 2 mL/
min. The 20K protein eluted from the column forming a
peak at a position of 420 mm NaCl. This peak fraction was
then subjected to reversed-phase high performance liquid
chromatography (HPLC) (model 600E, Waters) on a
Cosmocil 5C18-P300 column (Nacalai Tesque, Inc., Kyoto,
Japan), and proteins were eluted with a linear gradient of
10–70% acetonitrile (1%/min) in 0.1% trifluoroacetic acid
at a flow rate of 1 mL/min. The 20K protein that eluted at a
position of 47% acetonitrile was collected and lyophilized.
Because the purified 20K protein was resistant to Edman
degradation, theprotein (approximately
denatured with 8 m urea in 110 lL of 50 mm Tris–HCl
buffer (pH 9.0) for 30 min at 37?C, and then digested with
40 lg) was
Fig. 1. Isolation of the chick pineal calcium-binding proteins.
Upper panel, Elution profile of the pineal proteins from the phenyl-
Sepharose column. Soluble proteins were extracted from 2,120
chick pineal glands and applied to the column in the presence of
2 mm free Ca2+. After washing the column with a high Ca2+buffer
(open bar in the horizontal axis), the bound proteins were eluted
with a low-Ca2+buffer containing 2 mm EGTA (solid bar). Bot-
tom panel, SDS–PAGE of the phenyl-Sepharose fractions. Aliquots
(1/50 volume) of the flow-through fraction (FT), and eluate frac-
tions (1–7) were electrophoresed in a SDS-polyacrylamide gel
(13%) and stained with Coomassie Brilliant Blue. The partial
amino acid sequences of the proteins (indicated with arrowheads)
were determined by peptide sequencing, and the protein band
indicated with an arrow was immunostained with an anti-visinin
Chick pineal calcium-binding proteins
Page 3
lysyl endopeptidase (Wako Pure Chemical Industries, Ltd.,
Osaka, Japan) at an enzyme to substrate ratio of approxi-
mately 1:100 (in weight) in 270 lL of 50 mm Tris–HCl
buffer (pH 9.0) containing 3 m urea for 24 hr at 37?C. The
mixture was further incubated with freshly added protease
(final enzyme to substrate ratio of approximately 1:50 in
weight) for 8 hr at 37?C. The proteolytic fragments were
subjected to reversed-phase HPLC on a Develosil ODS-
UG-5 column (Nomura Kagaku, Aichi, Japan) and were
eluted with a linear gradient of 10–70% acetonitrile (1%/
min) in 0.1% trifluoroacetic acid at a flow rate of 1 mL/min
and collected. The amino acid sequences of the fragments
were determined by automated Edman degradation in an
Applied Biosystems model 491 protein sequencer equipped
with an on-line phenylthiohydantoin-amino acid analyser
(gradient programmer 140C, detector 785; Applied Biosys-
tems, Japan Ltd., Tokyo, Japan).
The internal amino acid sequences of 21K, 22K, 34K and
35K proteins were determined by a micro-sequencing
technique [23]. Briefly, the proteins in the phenyl-Sepharose
fractions (Fig. 1, fractions 3 and 5) were separated on a
sodium dodecyl sulphate (SDS)-polyacrylamide gel (8.5%),
and transferred to a nitrocellulose membrane (0.45-lm pore
size; Schleicher & Shuell, Dassel, Germany). After staining
with ponceau S, the membrane strip containing the protein
band was excised and incubated in 100 mm acetic acid
containing 0.5% polyvinylpyrrolidone-40 for 1 hr at 37?C,
followed by washing with water. The protein on the
membrane strip was digested with lysyl endopeptidase
(Wako Pure Chemical Industries, Ltd.) at an enzyme to
substrate ratio of approximately 1:25 (in weight) in 50 lL
of 50 mm Tris–HCl buffer (pH 9.0) containing 10% aceto-
nitrile for 24 hr at 37?C. The digest was then diluted with
50 mm Tris–HCl buffer (pH 9.0) to decrease the acetonitrile
concentration (<5%), and subjected to the reversed-phase
HPLC and Edman degradation as described above.
Calmodulin, neurocalcin and sorcin were purified also from
the chick brains for production and characterization of
antibodies. Briefly, the chick brains (254 g wet weight in
total) were homogenized with 500 mL of the extraction
buffer, and the CaBPs were partially purified by the phenyl-
Sepharose column in a way similar to that described for the
pineal glands (see the previous section). Fractions contain-
ing the brain CaBPs were subjected to MonoQ FPLC
(Amersham Biosciences Corp.), and proteins were eluted
with a linear gradient of 0–1 m NaCl (25 mm/min) in 10 mm
Tris–HCl buffer (pH 8.0) at a flow rate of 2 mL/min.
Calmodulin, neurocalcin and sorcin were eluted from the
column at positions 420, 140 and 60 mm NaCl, respectively.
After dialysis against phosphate buffered saline (PBS)
(10 mm Na-phosphate buffer, 140 mm NaCl, 1 mm MgCl2;
pH 7.4), each purified protein solution (50 lg each) was
emulsified with an equal volume of complete Freund's
adjuvant (Difco Laboratories, MI, USA) and injected
intraperitoneally to female Balb/c mice (8-wk old Clea
Japan, Inc., Tokyo, Japan). Each mouse was given 50 lg of
the purified protein as a booster 2 wk after the initial
injection, and was bled after 1 wk. For purification of anti-
sorcin antibody, purified chick brain sorcin (100 lg) was
immobilized to a HiTrap NHS-activated Sepharose column
(1-mL bed; Amersham Biosciences Corp.), to which the
antiserum was applied for affinity purification. As a
negative control for anti-sorcin antibody, the whole IgG
fraction was purified from mouse preimmune serum by
using a HiTrap protein G Sepharose column (1-mL bed;
Amersham Biosciences Corp.). Anti-visinin rabbit anti-
serum was a kind gift from Drs Naomasa Miki and Kuo
Che-Hui (Osaka University, Medical School, Osaka,
Japan). The protein concentration was estimated by the
NanoOrange protein quantification system (Molecular
Probes, Inc., OR, USA) using bovine serum albumin as a
Immunoblot analysis
Proteins were separated on a SDS-polyacrylamide gel
(13%), and transferred to a polyvinylidene fluoride mem-
brane (Immobilon P; Millipore). After rinsing with Tris-
buffered saline (TBS) (50 mm Tris–HCl, 145 mm NaCl,
1 mm MgCl2; pH 7.4), the blot was incubated with a
blocking solution (1% skim milk in TBS) for 1 hr at 37?C,
and then with a primary antibody solution diluted in the
blocking solution for 24 hr at 4?C.
Calmodulin-specific monoclonal antibody (CaM 85;
Zymed Laboratories, Inc., CA, USA) was used at 1 lg/
mL protein concentration, and antisera against neurocalcin
or sorcin were used at 1:5000 or 1:2000 dilution, respect-
ively. After washing with the blocking solution, the blot
was incubated for 24 hr at 4?C with 0.2 lg/mL of peroxi-
dase-labelled anti-mouse IgG or anti-rabbit IgG antibody
(Kirkegaard & Perry Laboratories, Inc., MD, USA) diluted
in the blocking solution. Then the blot was washed with
TBS containing 0.05% Tween 20, and positive signals were
visualized by using the Renaissance chemiluminescence
reagent (Perkin Elmer Life Science Japan Co. Ltd., Tokyo,
Immunohistochemical analysis
The pineal glands isolated from 1–3-day-old male chicks
were fixed with a solution containing 0.2% picric acid and
2% paraformaldehyde in 111 mm Na-phosphate buffer
(pH 7.3) orwith4% paraformaldehyde
Na-phosphate buffer (pH 7.4) for 4 hr at 4?C. The fixed
tissues were cryoprotected successively in 10, 20 and 30%
sucrose in 0.1 m Na-phosphate buffer (pH 7.4) at 4?C,
embedded in Optimal Cutting Temperature (OCT) com-
pound, frozen with liquid nitrogen, and sectioned at 7- or
10-lm thickness. The sections were mounted onto poly-L-
lysine-coated glass slides, air-dried, and rinsed with PBS.
These tissue sections were first treated with a blocking
solution of 10% normal horse serum (Vector Laboratories,
CA, USA) in PBS for 1 hr at room temperature, and then
incubated for 2 days at 4?C with a primary antibody
solution of calmodulin-specific monoclonal antibody (1 lg/
mL), anti-sorcin affinity purified antibody (2 lg/mL) or
anti-neurocalcin antiserum (1:5000) diluted in the blocking
solution. After washing with PBS, the sections were
incubated with a biotin-conjugated horse anti-mouse IgG
in0.1 m
Shimizu et al.
Page 4
antibody (7.5 lg/mL; Vector Laboratories) for 24 hr at
4?C, and then with avidin-biotin-peroxidase complex
(Vectastain Elite ABC kit, Vector Laboratories) in PBS
for 30 min at room temperature, followed by washing with
PBS. The immunoreactive sites were developed by incuba-
tion with 0.4 mg/mL diaminobenzidine and 0.01% H2O2in
50 mm Tris–HCl buffer (pH 7.6) for 5 min at room
In order to isolate CaBPs, 2,120 pineal glands were isolated
from newly hatched male chicks. The glands were homo-
genized initially in the presence of 2 mm EGTA (low Ca2+
condition), which reduces the hydrophobicity of CaBPs [20]
and possibly promotes extraction of these proteins into the
soluble fraction. The pineal extract was then supplemented
with a high concentration of Ca2+(2 mm at final free Ca2+
concentration), and applied to a phenyl-Sepharose column
which traps proteins with hydrophobic nature. After
washing with a high Ca2+buffer, proteins were eluted with
a low Ca2+buffer containing 2 mm EGTA (Fig. 1,
fractions 2–7). SDS–polyacrylamide gel electrophoresis
(PAGE) analysis of the fractions revealed that proteins
with apparent molecular weights of 20K, 21K, 22K, 24K,
34K and 35K (Fig. 1, arrow and arrowheads) were eluted
sharply upon Ca2+depletion, suggesting their Ca2+-
dependent conformational changes.
To uncover the molecular identity of the 20K protein, it
was purified to near homogeneity by MonoQ FPLC and
reversed-phase HPLC, and digested with a lysyl endopept-
idase. The resultant proteolytic fragments were separated
by reversed-phase HPLC, and the amino acid sequence of
the four fragments spanning a total of 92 amino acid
residues were determined by automated Edman degrada-
tion (Table 1, c1–4). All the sequences exactly coincided
with internal amino acid sequences of chicken calmodulin
[24], a CaBP that exhibits an apparent molecular weight of
20,000 on a polyacrylamide gel [21]. Thus the chick pineal
20K protein was identified as calmodulin.
The 21K, 22K, 34K and 35K proteins were characterized
by using micro-sequencing technique (see Materials and
methods). For the 21K protein, the amino acid sequences of
seven proteolytic fragments spanning a total of 101 amino
acid residues were determined (Table 1, n1–7). Except for
two residues in fragment n2 and n4 (Table 1), the amino
acid sequences of all the fragments were identical to the
internal sequences of bovine neurocalcin d, a CaBP with a
calculated molecular weight of 22,144 [25]. We concluded
that the 21K pineal protein is a chicken homologue of
neurocalcin d. For the 22K protein, we were only able to
determine two partial sequences (Table 1, s1 and s2) of 31
amino acid residues in total. Both were most similar to the
internal sequences of hamster sorcin, a CaBP with a
calculated molecular weight of 21,746 [26]. In spite of six
residues divergence between the determined sequence and
that reported for hamster sorcin (Table 1), their close
similarities in sequence and molecular weight led us to
assign the 22K protein as a chicken homolog of sorcin. This
assignment was substantiated by purifying the chick brain
sorcin and comparing it with the pineal 22K protein (see
below). For the 34K and 35K protein, micro-sequencing of
five and three fragments (Table 1, af1–5 and at1–3) resulted
in determination of a total of 49 and 24 amino acid
Table 1. Partial amino acid sequences of the chick pineal CaBPs
Protein Amino acid sequence of the proteolytic fragmentIdentity
molecular weight (species)
20K protein c1 : EAFSLFDKDGDGTITTK
c2 : ELGTVMRSLGQNPTEAELQXXXNEVDXDGNGT
c3 : MKDTDSEEEIREAFRVFDK
c4 : DGNGYISAAELRHVMTNLGXXXTDEEXDEMIR
Calmodulin 16,706 (chicken)
21K protein n1 : GFLRDXPSGHLSMEEFKK
n2a: IYANFFPYGDASK
n3 : WAFSMYDLDGNGYISK
n4a: SEMLEIVQAIYK
n5 : IFRQMDTNRDGK
n6 : LSLEEFIRGAKK
n7 : SDPSIVRLLQXDPSSAGQF
Neurocalcin d
22,144 (bovine)
22K proteins1a: ELWAVVNGWR
s2 : QHFVNFDSDRSGAVDRQELEK
Sorcin21,746 (hamster)
34K proteinaf1 : TRGTVTAF
af2 : GMGTDEETI
af3 : ILTSRNNAQRQEIASAF
af4 : QVYMQEYEANLED
af5 : ITGETSGHFQ
Annexin V 36,198 (chicken)
35K proteinat1 : XXNFDADXDAAA
at2 : TPSQYDASEL
at3 : QLYF
Annexin II 38,509 (chicken)
Underlined, amino acid residue that is different from that in the reported sequences [25, 26]; X, amino acid residue that was not determined.
aFragments which recovered from two distinct HPLC peaks with a slightly different retention time.
Chick pineal calcium-binding proteins
Page 5
residues, respectively. All the sequences derived from the
34K and 35K protein exactly coincided with the internal
sequences of chicken annexin V [27] and chicken annexin II
[28], which are both CaBPs with molecular weight of 36,198
and 38,509, respectively. The 34K and 35K protein were
thereby identified as annexin V and annexin II, respectively.
Our micro-sequencing analysis failed to determine the
sequence of the 24K protein (Fig. 1, indicated by an arrow),
possibly because of the low protein level in the pineal
extract. However, this protein band was clearly immuno-
stained by an antiserum raised against chicken visinin (data
not shown), a CaBP that migrates at 24,000 on a
polyacrylamide gel [29], suggesting that the 24K protein is
visinin, which is expressed in the pineal cells [30].
To verify the molecular identities and electrophoretic
properties of the pineal CaBPs, calmodulin, neurocalcin
and sorcin were purified from the chick brains to near
homogeneity.In SDS–PAGE
apparent molecularweights
22,000, respectively (Fig. 2, lanes 1–3), which coincided
with those isolated from the chick pineal gland (Fig. 1).
Then the purified preparations were used for production
of specific antibodies (see Materials and methods). These
antibodies, together with the commercial monoclonal
antibody against calmodulin (CaM 85, Zymed), were
subjected to immunoblot analysis, in which the superna-
tant and precipitated membrane fractions of the pineal
homogenate prepared under low Ca2+concentration were
electrophorsed. Each antibody recognized a major band
showing the mobility identical to the CaBP purified from
the chick brain (Fig. 2, lanes 6–14). Calmodulin was
distributed not only in the supernatant but also in the
membrane (plus nucleus) fraction of the chick pineal
gland (Fig. 2, lanes 6, 7), whereas neurocalcin and sorcin
were enriched in the supernatant (Fig. 2, lanes 9, 10, 12
and 13). By quantifying the immunoreactive bands in
Fig. 2. Characterization of calmodulin, neurocalcin, and sorcin,
and their specific antibodies. Upper panel, Coomassie Brilliant
Blue staining pattern of proteins separated in a SDS-poly-
acrylamide gel (13%). Calmodulin (CaM), neurocalcin (Neu)
and sorcin (Sor) were purified from the chick brain, and 1 lg of
each protein (lanes 1–3) was loaded onto the gel. Pineal glands
from ten one-day-old chicks were homogenized in 200 lL of
Tris–HCl buffer (pH 8.0) containing 2 mm EGTA, ultracentri-
fuged at 105,000 g, and 20 lL of the resulting supernatant (Sup;
lane 4) or precipitate (Ppt; lane 5) fraction was loaded onto the
gel. Bottom Panels, Immunoblot analyses. The Sup (lanes 6, 9,
12) or Ppt (lanes 7, 10, 13) fraction was electrophoresed (20 lL/
lane) withtheCa2+-binding
chick brain (lane 8, 400 ng CaM; lane 11, 10 ng Neu; lane 14,
65 ng Sor).
proteins purifiedfrom the
Fig. 3. Localization of calmodulin, neurocalcin and sorcin in the
chick pineal gland. The isolated chick pineal glands were fixed with
a solution containing 0.2% picric acid and 2% paraformaldehyde
(a–k) or 4% paraformaldehyde (l–p), and the cryostat sections
(10-lm thickness for g–h, and 7-lm for the others) were prepared.
a, hematoxylin-eosin staining of a midsagittal section of the whole
gland; PS, pineal stalk, Scale bar, 200 lm. Inset, a magnified view
of a single pineal follicle. L, follicular lumen; IF, interfollicular
space. Scale bar, 20 lm. b-p, Immunohistochemical analyses. The
pineal sections were incubated with a calmodulin-specific mono-
clonal antibody (b, d–f), anti-neurocalcin mouse antiserum (g, i–k),
or anti-sorcin mouse affinity-purified antibody (l, n–p), and positive
signals were visualized by immuno-peroxidase reaction. In a series
of negative control experiments (c, h, m), the sections were incu-
bated with a monoclonal antibody to a non-pineal protein (c),
mouse normal serum (h), or purified mouse whole IgG (m), in place
of the primary antibodies (at the same concentration or dilution).
In another series of control sections (e, f, j, k, o, p), the primary
antibodies to calmodulin, neurocalcin and sorcin were pre-incu-
bated with 20 lg/mL of corresponding antigen (e, j and o,
respectively) or with the same concentration of neurocalcin (f),
calmodulin (k) and calmodulin (p), respectively, to demonstrate
specificities of the effect of pre-adsorption. F, follicular layer; PF,
parafollicular layer. Asterisks, the immunoreactive outer segments
of the follicular pinealocytes. Scale bars, 15 lm.
Shimizu et al.


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Reclamation Releases a Draft Environmental Assessment for Piping the Slack and Patterson Laterals
GRAND JUNCTION, Colo. - Reclamation announced today that it has released a draft environmental assessment on piping Roger's Mesa Water Distribution Association's Slack and Patterson Laterals off the Fire Mountain Canal, located in Delta County, Colo. The project involves replacing approximately 9.4 miles of unlined earthen laterals with buried water pipeline. The purpose of the project is to improve the efficiency of water delivery to ditch users and reduce salinity loading in the Colorado River Basin.
The draft environmental assessment is available our website or a copy can be received by contacting Reclamation.
Reclamation will consider all comments received prior to preparing a final environmental assessment. Comments can be submitted to the email address above or to: Ed Warner, Area Manager, Bureau of Reclamation, 445 West Gunnison Ave, Suite 221, Grand Junction, CO 81506. Comments are due by Friday, March 14, 2014.
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- Reclamation Releases Draft Environmental Documents for Del Puerto Water District Warren Act Contract to Convey Supplemental Irrigation Water Due to the Drought
- Bureau of Reclamation releases Draft Environmental Assessment for Planet Ranch Lease
- Reclamation Issues Draft Environmental Assessment for Shoreline Protection Project at Lake Roosevelt
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"If we are disconnected from our food and where our sustenance comes from, it's a very dangerous thing for humanity," says Natalie Bogwalker, founder of Wild Abundance. In November, Bogwalker teaches a two-day workshop that focuses on humane, reverent and conscious slaughtering and butchery practices.
Smart bets: Resonate Asheville
New Mountain hosts the meeting of minds Sunday, Oct. 4, from 11 a.m. to 8 p.m.
Southern Appalachia's first craftspeople
Discoveries at Garden Creek, an archeological site near Canton, suggest it served as a home for native craftspeople who produced artifacts for a religious and cultural movement that swept ancient North America nearly 2,000 years ago.
Conscious party: Tim Freke's spirituality talk to benefit nonprofits fighting hunger
A portion of proceeds from the spirituality event will go to Jubilee Community Church's Hunger Fund, which benefits local nonprofits MANNA FoodBank, Meals on Wheels and Loving Food Resources.
Conscious party: getting Zen with Poetry and Pie
Poetry and Pie pairs sweets for the mind and body in support of Great Tree Zen Temple. The fundraiser takes place at Urban Dharma, 29 Page Ave., on Sunday, Sept. 27, from 6-7:30 p.m.
Faces in the crowd: WNC crowdfunding initiatives
Each week, Xpress highlights notable WNC crowdsourcing initiatives that may inspire readers to become new faces in the crowd. This week features the second edition of Appalachian Trail-inspired board game Thru Hike plus nonprofit literary press Orison Books' fundraising efforts for three new spiritually-inclined books.
Smart bets: Jim Bizer and Jan Krist
The duo opens Mountain Spirit Coffehouse's eleventh season at the Unitarian Universalist Congregation of Asheville on Sunday, Sept. 13, at 7 p.m.
Smart bets: Three Days of Light Gathering
The annual Three Days of Light Gathering — now in its fourth year — takes much of what's great about summer festivals (camping, music, arts and craft vendors, nature) and enhances all of that with an element of spirituality and learning. The festival is held at Camp Grier in Old Fort, from Friday, Aug. 28, to Sunday, Aug. 30.
Faces in the crowd: WNC crowdfunding initiatives
Each week, Xpress highlights notable WNC crowdsourcing initiatives that may inspire readers to become new faces in the crowd. This week features one artist's random, magnetic acts of kindness and a filmmaker's depiction of his spiritual journey in and out of India.
Small Bites: Mountain Jewish Festival
The Mountain Jewish Festival kicks off in Hendersonville, workshops are offered on fermented foods and foraging for mushrooms, Black Mountain Ciderworks celebrates its second year, ASAP hosts its Local Summer Cookout and food writer Jonathan Ammons talks about Nightbell's unique take on the classic deviled egg.
Two women plan Asheville stop on cross-country bike tour and fundraiser
Two women bicycling across the country — from Maine to Seattle — will be making a pitstop in Asheville this weekend, giving a short presentation on their cause at both Sunday morning Jubilee! services.
Activist Bree Newsome and other 'faith-driven agitators' lead a charge for social justice at Wild Goose
The third annual Goose Festival brought together more than 2,000 people from various spiritual persuasions that fall under a very inclusive Christian umbrella. Featured speakers at this year's event included Bree Newsome, the activist who gained national attention for removing the Confederate flag from the South Carolina statehouse.
JCC to host ecology, food and permaculture panel, July 19
Asheville Jewish Community Center will host a panel discussion on Sunday, July 19, from 4-6:30 p.m. focused on helping help people connect with and better understand Jewish values that encourage environmental stewardship.
Faces in the crowd: WNC crowdfunding campaigns
Each week, Xpress highlights notable WNC crowdsourcing initiatives that may inspire readers to become new faces in the crowd. This week features a local artist's mask-making residency in Mexico, a new dance school called Terpsicorps, a foam party to follow the cyclocross nationals in Asheville in 2016 and upgrades to an astrology podcast.
In photos: Firestorm Books & Coffee serves up some anarchy on Haywood Road
Firestorm Books & Coffee is back. And though the doors are currently open — the coffee flowing and the pages turning, the 7-year-old cooperative will host its official grand opening celebration on Sunday, July 12.
Smart bets: Wild Goose Festival
Wild Goose Festival is a spiritually-charged four-day gathering held in Hot Springs from Thursday to Sunday, July 9-12.
Quirky studio The Little Volcano brings cat yoga to Asheville
"Yoga with cats was an idea we had because we love Brother Wolf and would like to help kitties find homes," says studio co-owner Rosie Volcano, "and because we just love cats and yoga."
Tricia Fogle explains the inspiration behind her new business, Life of Energy Retreats
Tricia Fogle was inspired to create her up-and-coming business, Life of Energy Retreats, in order to share her knowledge of alternative and holistic health methods and provide a refreshing, full-service retreat space where participants can relax their bodies and refresh their minds.
Day of mindfulness to be held at Asheville Insight Meditation Saturday, June 27
A day-long mindfulness retreat will be held at Asheville Insight Meditation on Saturday, June 27. The retreat will explore mindfulness for "health, wellness, recovery and freedom from suffering," says clinical psychologist Larry Cammarata, co-host of the retreat with Eddie LeShure, owner of Mindful Emergence.
Celebrate land preservation on Land Trust Day, this June 6
Conservation groups like the Southern Appalachian Highlands Conservancy need the support of residents and local businesses in order to achieve their work. Building on this co-dependence, the nonprofit will hold its annual "Land Trust Day" celebration Saturday, June 6.
Local action for Nepal: update
Since Mountain Xpress' last report on people and businesses around Asheville working to aid those in need in the wake of Nepal's devastating earthquake, efforts have redoubled, and more of those involved have become visible.