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Brendan Cole; All Night Long
Saint Petersburg Classic Ballet
Artistic Director Marina Medvetskaya
Dancers from SPCB have often been guests with Moscow City Ballet Europe, and this season we are delighted to introduce the entire Saint Petersburg company to the UK. Under the direction of Marina Medvetskaya, Saint Petersburg Classic Ballet was formed in 1996 immediately embarking on tours of North America. They will bring their own training, technique and interpretation to the productions of The Nutcracker, Swan Lake and Giselle, all of which are hugely popular with UK audiences.
An exciting new generation of outstanding young dancers, including the cream of the Saint Petersburg graduates have been specially chosen by Medvetskaya for this first UK tour.
Coliseum 2014
Moscow City Ballet
Former Bolshoi Ballet soloist and choreographer, the late Victor Smirnov-Golovanov founded Moscow City Ballet in 1988. This exquisite company has become one of Russia's most successful touring companies throughout the world. They regularly play shows in Poland and Belgium as part of their European touring schedule. More recent invitations have been to the Far East and the Middle East. Having celebrated a 25th anniversary in 2014, the company is as resh as the day it was formed. MCB is brimming with some of Moscow's best young dancers, outstanding principals, stunning soloists and a sublime corps de ballet, all underpinned by the dynamic vitality of the Hungarian Sinfonietta live orchestra. Artistic direction is in the hands of Ludmila Nerubashenko, who ensures the quality of performance remains first class.
'The dancers are elegant, superbly rehearsed and very savvy. The ensemble pieces are a delightful triumph for the company dancers. The principals finished with a dazzling flourish and brought the house down.' The Sunday Express
*all photos actual production shots
Coliseum 2013
St Petersburg Ballet Theatre
SPBT is a touring troupe brought to the UK by PMB Presentations for regional touring in 1997 when the company was newly formed with accomplished soloists including Sabina Yapparova and Andrei Yakhnyuk who subsequently joined the Mikhailovsky Ballet
"St Petersburg Ballet Theatre continues PMB Presentations tradition of bringing high art to far flung places." The Mail on Sunday
Schedule-Book Online>> Biographical material>> Press reviews>> Photos>>
*all photos actual production shots
Kiev Classical Ballet
SWAN LAKE : THE NUTCRACKER : PETER & THE WOLF / CARNIVAL OF THE ANIMALS : SNOW WHITE The Ballet
The word elegance is much repeated when critics review the productions from Kiev. The company's hallmark is a superb orchestra, outstanding dancers, and works that include enchanting ballets for children.
"As a sumptuous visual feast is cooked up onstage, the accompanying ensemble, Kiev orchestra brings to life equally lavish scores." Metro
Schedule-Book Online>> Biographical material>> Photos>> Press reviews>>
*all photos actual production shots
© PMB Presentations, 2015 Website by UKTW


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Located at the foot of the beautiful Flatirons in Boulder, CO - PintShop.com was founded when we noticed a lack of high quality, affordable brewery items online. We figured there had to be a better way! Be assured we will do what ever it takes to keep you coming back time and again.
From the Daily Camera:
They're folks who live in the Boulder area and work normal jobs, but chase the American dream of running their own business on the side.
No, they're not suckers for television commercials touting Internet terminals as the road to wealth-filled, self employment. Instead, they pour hours outside regular jobs running their own e-commerce Web sites.
They've found markets for anything from shark hats to African safaris, swimsuits to pint glasses. They're finding ways to generate some revenue, too.
"We're doing business how it used to be done — and it's online," says Brian Young, a 26-year-old who moonlights at Louisville-based Raindance Communications Inc. while hawking brewery memorabilia on his 7-month-old PintShop.com. "It's been profitable since day one."
Some, like Young, have visions of eventually making the sites their full-time jobs, maybe even recognizing a windfall someday from their hard-earned creations.
- Log in to post comments

subset: commoncrawl


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Sand sculptor Carl Jara works on "Night Swallows Sun" during Headlands Beachfest on Saturday at Headlands Beach State Park.
Despite gray skies, the first Beachfest in Mentor brought people in from all over Saturday.
A family from Michigan worked together at Headlands Beach State Park to build a sand castle shaped like a turtle, using stones to write "Ohio" on the shell.
A mother and son traveled from Lyndhurst to walk down the beach after the rain cleared up.
And families from Akron, Canada and Buenos Aires took the chance to paddle board with Lake Metroparks staff.
The first-time event brought lots of entertainment to the sand, too, including live music, games and food.
A couple roasted kettle corn under a tent while nearby, a sculptor carved out a detailed, cloaked woman out of sand.
Further down the beach, children chased hundreds of seagulls while others attempted to jump over waves bigger than themselves.
Kites flew high in the cool breeze that likely left those in the water a bit chilly.
The sun came out toward the end of the evening as the sky turned blue.
Branden Edwards of Chagrin Falls brought his kids to the beach and stumbled upon the paddle board area.
He said he couldn't pass it up once he saw it, and that he had a blast out on the water.
Tyler Majors, 7, of Lyndhurst threw a Frisbee around after getting his face painted and making a few crafts.
Brian Fowler of Lake Metroparks said the event has the potential to continue to grow and draw people in from near and far in the future, especially if the sun shines next time.


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Jello no bake crust directions
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text:
Nighmare foxes have attacked pets and left one elderly resident so petrified, she feels she has no option but to move out of Maldon.
Kelly Hall, of Maldon, has called for the return of the hunt amid claims foxes are making her life a misery and have attacked residents' pets.
See this week's MBS for the full story.


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Ruger 10/22 Sporter Contest
FirearmsTalk is proud to announce the release of the Ruger 10/22 Sporter Contest.
Up for grabs is a brand new Ruger 10/22 Sporter that features:
1. Refer someone to FirearmsTalk. Make sure they enter your user name into the referral box upon registration. Only referals after 7/12 will count.
2. Like us on Facebook. (https://www.facebook.com/firearmstalk)
3. Follow us on Twitter. (Twitter)
4. Post a link to the contest on another website.
Contest Details
On August 15th we will close this Giveaway, put all the names in a box and draw one winner on August 16th in a live drawing.
The winner will have 24 hours from the time the winner is posted to this thread to claim their Ruger Sporter 10/22.
In order to claim you must send Notdku a private message with your information. If you do not respond with your
address within that time a new winner will be chosen. The same rules apply to the next winner.
No purchase necessary. To enter by mail send full name, address, phone number to FirearmsTalk PO BOX 911 San Marcos, Texas 78667. Entries must be received by August 15th, 2011. All contests are void where prohibited or restricted by law. Winners are responsible for all taxes or customs fees. You must be 21 or older to enter and win. Free shipping to anywhere in USA that allows this firearm. FirearmsTalk will ship to a registered FFL in your State, if your State allows this firearm.
Liked on Facebook and followed on Twitter! Count me in!
I liked the page on Facebook. PM me if you need details, I use my real name on Facebook.
Posted a link to the contest at another favorite website.
Thanks for the opportunity.
Followed on twitter.
I really need a new 10/22 as I was stupid a long time ago and sold mine.
Liked on Facebook as MyGunDB and I follow you on Twitter as @MyGunDB.
Liked on facebook, following on twitter, posted on another website. I will have someone sign up by the end of the week!
I liked on Facebook, I would follow on twitter but I don't have a twitter account.
I liked the page on Facebook.
I linked on Twitter
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Eco Torque Ltd have received 16 customer reviews.
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"Very impressed with Eco Torque! Professional and courteous service. Got the job done!"


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GAINESVILLE - An autopsy performed by the Medical Examiner on the body of Holly Fox Strickland of Oakwood has determined that she was the victim of a homicide.
Hall County Sheriff's Deputy John Marshall said Tuesday the office was not releasing further details.
"Right now the investigation is still ongoing by the Detective Division, and as we get information that we can release, we'll release it," said Marshall.
The 32-year-old Strickland was last seen alive on Monday, February 3. Her body was found near her home on Rain Tree Trace Saturday morning around 10:45. Initially, authorities said it did not appear foul play was involved in the young mother's death.


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Show Highlights
Wednesday September 20, 2017
The Current 'I was not in the best place in my life': Manitoba NDP leader Wab Kinew confronts abuse allegations
"I've always understood that as long as I put my name on a ballot that people are going to want to talk about my past ... there are legitimate questions that people have about the person that I was."videoListen 23:21
Thursday September 21, 2017
30 years after Man in Motion tour, Rick Hansen still fighting for accessibility
"Right now there's over a billion people on the planet living with a disability according to the World Health Organization — that is one in seven people."
Monday September 18, 2017
The Current 'Decades of grief': Carol Off's long journey to save Afghan man who risked his life to talk to her
What happens when a journalist used to keeping herself out of the story realizes her reporting has put an entire family in danger? For the CBC's Carol Off, it would be life-changing.
Featured Episode
Friday September 22, 2017Listen to Full Episode 1:14:22 Listen 19:09
'High-risk' B.C. schools not ready for an earthquake, says parent
"Some of our most dangerous buildings in earthquakes are our schools and that's where our children are." More
more stories from this episode
Iraqi Kurds set to vote on independence referendum
"The idea of the referendum is for Kurdistan to be a force for good." MoreListen 25:07
Equifax hack: How safe is your personal information?
"We have built a house on sand with respect to the digital economy and we didn't properly architect how we do digital identity ... and now we're paying for it." MoreListen 22:57
September 22, 2017 Full Episode Transcript
Full text transcript for September 22nd episode. More


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TexasOutlaw wrote:How long does carbonation usually take? Or do you think it needed more sugar?
It can depend on the type of yeast and how active the yeast is. 2-3 weeks is probibly about average, my belgian dubbel took like 6-8 weeks.
As for the sugars, don't ramp up the sugar. 1/2 - 3/4 cup of corn sugar OR 1.2-1.5 cups of DME for a 5 gal. batch. Never confuse the two, corn sugar is much more potent than DME.


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Ask Baba Yaga: I Feel Tired and Ill and Powerless When I Spend Time With My Family
Transcript after the jump.
Dear Baba Yaga,
I feel tired and ill and powerless when I spend time with my family. They love me very much and I love them, but I still feel like an alien around them. How can I learn to spend time with them and not feel exhausted and depressed afterwards?
With your family, you are as planet — fixed & solitary in your orbits, & you; the coldest loneliest one,. But this is not:the you-yourself. Neither is it the truth of yr family that they are such. ;Remember the animal of you with its heat & hunger, pacing the stars & tearing them open. ;So is yr family made of need&itching bones. Angry as it is, the stew of all yr bones together is better & truer than All yr mutely orbiting. Feel yr own meat & howl out to theirs when the fire draws you close.
Previously: "One Morning I Am Emboldened and On Fire, the Next I Am Choking in the Smoke"
Taisia Kitaiskaia is a poet, writer, and Michener Center for Writers fellow. She's taking questions on behalf of Baba Yaga at email@example.com.


text: Hello! I am a lifelong PC user, now wrestling with my new Nexus 7. I have two major issues with the e-mail function supplied with the device. I have amended the settings on my PC's Outlook Express POP e-mail accounts to ensure mail is left on the server for a minimum of 15 days. I have created four email accounts on the tablet, and successfully receive e-mail. However, here come the problems: 1. Email messages I want to keep are routinely removed from the in-boxes, and cannot be found anywhere. When this happens, I have to reload all the messages from the past 4/5 days, delete all the rubbish (again) and leave the ones I want to refer to back in the inbox. However, in just one or two days, they've gone again! I have just returned from a two week holiday, when this problem occurred repeatedly. My PC was obviously not active during this time, so cannot be the culprit. I have tried 'starring' the selected e-mails (whatever that means) but it doesn't stop them disappearing. I have read that someone deletes them, but saves them in the Recycle Bin - which is ludicrous! And so to the second problem/issue: 2. On my PC there is a simple option in Outlook Express to 'Empty Deleted Items Folder' to remove all the rubbish in one click. With the tablet, I have to go through each individual message and click on it, before I can delete them. Surely there is an easier way to delete the entire contents of the Recycle Bin in one go? Can anyone provide answers/solutions to these problems/issues? Thanks.


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She's just three-years-old and already Katherine Heigl's daughter Naleigh is a scene stealer!
Sitting in the audience Monday while Heigl, 33, promoted her new flick One for the Money on The View, Naleigh couldn't help but wave to her mommy when cohost Sherri Shepherd pointed her out.
Later, to the delight of The View's cohosts, the tot ran onstage and into her mother's lap as she finished out the interview.
"You know, forget this interview," Barbara Walters told Heigl, who adopted Naleigh with husband Josh Kelley from Korea in 2009. "She has completely stolen the show!"
Agreed Heigl: "She is completely upstaging me!"
If it's up to Heigl and her husband of four years Kelley, Naleigh won't be an only child much longer.
"We have talked about having biological children as well, but we decided to adopt first," Heigl has said. "I just wanted to be a mom. So however we did that, it's fine with me. I'd like to adopt again."


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Phosphorylation/dephosphorylation events in human blood platelets were investigated during their adhesion to collagen under flow conditions. Using 32P-labeled platelets and one-dimensional gel electrophoresis, we found that adhesion to collagen mediated primarily by the α2β1 integrin resulted in a strong dephosphorylation of several protein bands. Neither adhesion to polylysine nor thrombin-induced aggregation caused similar protein dephosphorylation. In addition, treatment with okadaic acid (OA), an inhibitor of serine/threonine protein phosphatases type 1 (PP1) and 2A (PP2A), caused significant inhibition of adhesion, suggesting that adhesion is regulated by OA-sensitive phosphatases. Recent studies indicate that phosphatases may be associated with the heat-shock proteins. Immunoprecipitations with antibodies against either the heat-shock cognate protein 70 (hsc70) or heat-shock protein 90 (hsp90) showed the presence of a phosphoprotein complex in 32P-labeled, resting human platelets. Antibody probing of this complex detected hsc70, hsp90, two isoforms of the catalytic subunit of PP1, PP1Cα and PP1Cδ, as well as the M regulatory subunit of PP1 (PP1M). OA, at concentrations that markedly blocked platelet adhesion to collagen, caused hyperphosphorylation of the hsc70 complex. In platelets adhering to collagen, hsc70 was completely dephosphorylated and hsp90, PP1α, and PP1M were dissociated from the complex, suggesting involvement of heat-shock proteins and protein phosphatases in platelet adhesion.
INTEGRIN-MEDIATED adhesion events are involved in many biologic functions such as development, tumor cell growth and metastasis, apoptosis, hemostasis, and the response of cells to mechanical stress.1 Binding of extracellular matrix proteins to integrins initiates cell signaling and the formation of focal adhesions that link integrins to cytoskeletal complexes and actin filaments.2 Heat-shock proteins (hsp) are a group of abundant and highly conserved molecules with important roles in the response of cells to environmental stress. Members of the 70- and 90-kD hsp families can associate with the cytoskeleton, bind to actin and tubulin, and participate in intracellular protein transport.3-9 There is evidence that these hsps are involved in regulating the activities of signaling molecules such as the steroid hormone receptors and several protein kinases, including pp60v-src, Raf, the initiation factor (eIF )-2α kinase, and casein kinase II.10 In humans, the hsp70 family consists of at least four members: the heat-shock cognate protein 70 (hsc70), hsp70, the glucose-regulated protein 78 (GRP78; also known as BiP), and hsp75.11 The Na+/H+ exchanger, which responds to integrin-activated signaling, has been shown to form a complex with hsp70.12 Tyrosine phosphorylation of hsc70 in response to ligation of T-cell antigen receptors and of a 75-kD protein, a member of the hsp70 family, in response to oxidative stress has been reported.13 14 Integrin-mediated signaling may also involve phosphoprotein phosphatases that are essential to preserve focal adhesions, to regulate cytoskeletal structure, and to maintain the structural integrity of intermediate filaments and microtubules.15-17 A critical role for the okadaic acid (OA)-sensitive protein phosphatases type 1 (PP1) and 2A (PP2A) in binding of the αLβ2 integrin to intercellular adhesion molecule-1 and in adhesion via the α4 integrins to vascular cell adhesion molecule-1 has been proposed.18 19 Dedhar and Hannigan20 have suggested that the association of signaling molecules, such as calreticulin, with the cytosolic tail of the β1 integrin and integrin activation may depend on dephosphorylation/phosphorylation events involving serine/threonine protein phosphatases and kinases. However, there have been no investigations of possible interactions between heat-shock proteins and protein phosphatases during integrin-activated signal transduction after cell adhesion to the extracellular matrix.
Platelet adhesion to collagen is one of the first steps in hemostasis when blood vessels are injured.21 Several membrane proteins have been proposed as collagen receptors, including glycoprotein (GP) Ia/IIa (α2β1 integrin), GPIV, and GPVI.22-25 However, it is uncertain which receptor serves as the major mediator of platelet adhesion to collagen or which one plays a signaling role. We previously described an approach for studying the initial events of adhesion to collagen under flow conditions.26 Immuno-inhibition experiments with the antibodies directed against different platelet receptors showed that adhesion to collagen in a plasma-free medium is primarily mediated by the α2β1 integrin.27 Use of a continuous-flow microcolumn adhesion approach26 enables investigating adhesion under arterial-flow conditions in the absence of secretion and αIIbβ3 integrin-mediated aggregation. 32P-labeled human platelets are pumped over a microcolumn of collagen-coated Sepharose beads to permit analysis of sub-second adhesion kinetics and rapid biochemical events.28 29 In the present study, we have focussed on protein phosphorylation/dephosphorylation changes and the involvement of hsc70, hsp90, and serine/threonine phosphoprotein phosphatases during adhesion to collagen.
Materials and antibodies.Native, salt-soluble type I collagen from rat skin was donated by Dr Gary Balian (University of Virginia, Charlottesville, VA). A purified rat monoclonal antibody (MoAb) 1B5 against constitutively expressed heat-shock cognate protein 70 (hsc70), a mouse MoAb N27 (N27F3-4) against both the constitutive and inducible forms of human hsp70, and a mouse MoAb AC88 against hsp90 isolated from the water mold Achlya ambisexualis and cross-reacting with hsp90 from a variety of vertebrate species were all purchased from StressGen Biotechnologies Corp (Victoria, British Columbia, Canada). A mouse MoAb 13D3 recognizing hsc70 was obtained from Affinity BioReagents, Inc (Golden, CO). Purified bovine hsc70 and hsp90 were from StressGen Biotechnologies Corp. Nonimmune rat IgG was from Sigma Chemical Co (St Louis, MO). Nonimmune mouse IgG was from Vector Laboratories (Burlingame, CA). A polyclonal antibody recognizing the α catalytic subunit of PP1 was obtained from UBI (Lake Placid, NY). A polyclonal antibody to the δ catalytic subunit of PP1 was a gift from Dr John Lawrence (University of Virginia) and a polyclonal antibody recognizing the M regulatory subunit of PP1 was a gift from Dr David Hartshorne (University of Arizona, Tucson, AZ). [32P]-orthophosphate (carrier-free) was from Amersham Corp (Arlington Heights, IL). OA was from Biomol (Plymouth Meeting, PA). The Immun-Lite chemiluminescent assay was from Bio-Rad (Hercules, CA), and other specialty chemicals used were from Sigma Chemical Co.
Preparation of human platelets.Human venous blood was anticoagulated with acid-citrate-dextrose (ACD) buffer to a final citrate concentration of 11.5 mmol/L in whole blood and centrifuged at 350g twice for 3 minutes and once for 5 minutes, all at room temperature. The resultant platelet-rich plasma was mixed with 0.05 vol of ACD and with inhibitors of platelet activation (apyrase [7.5 U/mL ADPase activity], indomethacin [1 μg/mL], and prostacyclin I2 [0.3 μg/mL]) and centrifuged at 620g for 20 minutes. The platelet pellet was then resuspended in phosphate-free buffer (140 mmol/L NaCl, 5 mmol/L KCl, 0.05 mmol/L CaCl2 , 0.1 mmol/L MgCl2 , 0.01 g/mL bovine serum albumin [BSA], 16.5 mmol/L glucose, 15 mmol/L HEPES, pH 7.35) containing 1 μg/mL indomethacin and left for 20 minutes before incubation with 0.1 mCi/mL carrier-free [32P]-orthophosphate for 90 minutes at 37°C. After a further wash, the platelets were resuspended at 6 × 108 cells/mL in a modified BSA-free Tyrode's-HEPES buffer (140 mmol/L NaCl, 0.34 mmol/L Na2HPO4 , 2.9 mmol/L KCl, 10 mmol/L HEPES, 12 mmol/L NaHCO3 , 5 mmol/L glucose, 2 mmol/L MgCl2 , pH 7.4). Because collagen activation may lead to the formation of thromboxane A2 from arachidonate via cyclooxygenase and release of ADP from platelet dense granules, we added indomethacin (10 μmol/L) and apyrase (7.5 U/mL) to the platelet suspension to block any possibility of secondary stimulation by these platelet agonists. To prevent thrombin generation, hirudin (0.01 U/mL) was also included in the suspension buffer. Neither inhibitor affected adhesion.27
Platelet adhesion and aggregation assay.The continuous-flow adhesion approach was essentially as described.26 29 CNBr-activated Sepharose 4B beads were coated either with native soluble collagen type I from rat skin or with polylysine. Platelets and isotonic saline were pumped through a microcolumn of 50 μL of beads at a pumping speed of 3.4 μL/s, giving a shear rate of about 1,700 s−1 and a contact or adhesion time of 1.35 seconds. Adhesion was determined by counting single platelets in the suspension before and after exposure to the beads and is expressed as the percentage of platelets bound to collagen. To assess the changes in protein phosphorylation during adhesion, usually 250 μL of 32P-labeled platelets (1.5 × 108) suspended in a Tyrode's-HEPES buffer were pumped through a column at 37°C for 90 seconds. Knowing the percentage of platelet adhesion under the above flow conditions, usually set to give approximately 50%, identical amounts of resting (unstimulated), effluent (flow-through), and adherent platelets were solubilized for the electrophoresis and immunoprecipitation experiments described below. In some experiments, platelets were treated with 0.1 and 1 μmol/L OA dissolved in 0.1% (vol/vol) dimethyl sulfoxide (DMSO) for 10 minutes at 37°C. Control platelets were incubated with 0.1% (vol/vol) DMSO.
For aggregation experiment, platelets were stirred with 1 U/mL thrombin at about 1,000 rpm for 90 seconds at 37°C and aggregation was assessed by following the decrease in numbers of single platelets. A 10-fold excess of hirudin was used to neutralize thrombin before treating with lysis buffer.
Electrophoresis, immunoblotting, and immunoprecipitation.Collagen- and polylysine-adherent, thrombin-aggregated, and control (resting and effluent) platelets were all solubilized at 4°C with a low-salt lysis buffer containing 1% (vol/vol) Nonidet P-40, 0.1% (vol/vol) Triton X-100, 0.25% sodium deoxycholate, and protease and phosphatase inhibitors29 followed by the addition of sodium dodecyl sulfate (SDS)-containing buffer (2% SDS [wt/vol], 5% 2-mercaptoethanol [vol/vol], 10% glycerol [vol/vol], 0.002% bromophenol blue [vol/vol], and 62.5 mmol/L Tris, pH 6.8). Proteins corresponding to 1.5 × 107 platelets were applied to each lane and were separated by vertical electrophoresis on 1.5-mm–thick and 20-cm–long SDS-polyacrylamide (8% and 12%) slab gels, stained with Coomassie blue, destained overnight, and dried. The dried gels were exposed to x-ray film at −80°C.
For immunoprecipitation and immunoblotting experiments, proteins were separated and electro-transferred using the Mini-Protean II system (Bio-Rad). Briefly, lysates from 5 × 108 platelets in the low-salt lysis buffer29 were first usually cleared of debris by centrifugation at 15,000g for 10 minutes at 4°C and then incubated overnight at 4°C with different antibodies, followed by the addition of a rabbit antirat or rabbit antimouse IgG bound to protein A-Sepharose beads. The low-salt lysis buffer was chosen to minimize dissociation of protein complexes. The immune complexes bound to protein A beads were washed twice in the low-salt lysis buffer and twice in phosphate-buffered saline, pH 7.2. After final washing, platelet proteins were solubilized off the beads by heating in SDS-containing buffer and separated on minigels usually by 8% or 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE). When required, gels were stained by either Coomassie Brilliant blue or silver nitrate using the Bio-Rad silver stain kit as described by the manufacturer. For autoradiography, the gels were dried and exposed to x-ray film. When Western blots were required, the resolved proteins were electro-transferred to nitrocellulose membranes, followed by blocking with 5% BSA (wt/vol) in a buffer containing 50 mmol/L Tris-HCl, 150 mmol/L NaCl, and 0.1% Tween (vol/vol), pH 7.5. Western blotting and detection were by enhanced chemiluminescence techniques.
Platelet fractionation.Adherent and control (resting) cells were first lysed in ice-cold lysis buffer as described above. Subcellular platelet fractions were obtained according to Fox et al,30 but with slight modifications. Samples (1 × 109 platelets/mL) were vigorously mixed and left on ice for 30 minutes. Platelet cytoskeletons were isolated by centrifugation at 15,000g for 10 minutes at 4°C. The supernatant (detergent-soluble fraction) was removed and centrifuged at 100,000g for 3 hours at 4°C to obtain a pellet (membrane-skeleton fraction) and supernatant (cytosolic fraction). The cytoskeletal and membrane-skeletal pellets were resuspended in 200 μL of SDS-containing buffer. The detergent-soluble supernatants were mixed at a ratio of 1:3 with 4 times concentrated (4×) SDS-containing buffer. Thus, the final detergent-insoluble samples (both cytoskeleton and membrane-skeleton fractions) were concentrated approximately 7 times relative to the detergent soluble fractions. The platelet fractions were boiled, subjected to 8% SDS-PAGE, electro-transferred to nitrocellulose membrane, and immunoblotted with the anti-hsp90 antibody, AC88.
Peptide sequencing.The 32P-labeled 67-kD protein band obtained by gel electrophoresis was transferred to a nitrocellulose membrane and digested in situ with trypsin.31 Sequencing of an aliquot of the digest was performed using microcapillary high-pressure liquid chromatography followed by electrospray ionization tandem mass spectrometry.32
Platelet protein phosphorylation/dephosphorylation changes induced by adhesion to collagen.One-dimensional gel electrophoresis of 32P-labeled human platelet proteins shows that platelet adhesion to collagen caused marked phosphorylation of pleckstrin, a 47-kD protein (p47; Fig 1) usually used as an index of protein kinase C (PKC) activation33 and the dephosphorylation of unknown protein bands of apparent molecular weights of 235, 220, 120, 100, 95, 67, and 40 kD, which were the major phosphorylated proteins in unstimulated platelets (Fig 1). Moreover, adhesion to collagen also increased phosphorylation of a 26-kD protein band, unlike polylysine or thrombin-activated platelets. In contrast to adhesion to the nonspecific substrate polylysine or thrombin-induced aggregation, adhesion to collagen did not induce significant phosphorylation of the 20-kD (p20) myosin light chains (Fig 1). Pleckstrin phosphorylation occurred in platelets stimulated with thrombin, as expected, but was also found in platelets adherent to polylysine. Analysis of platelets emerging in the effluent from the microcolumn show that shear forces acting alone26 29 or brief contact with collagen during flow through the beads caused little change in overall phosphorylation patterns (effluent; Figs 1 and 2). The dephosphorylation of the protein bands mentioned above was specific for collagen-induced adhesion, because it was not induced by adhesion to polylysine or by thrombin-induced aggregation. Although very similar labeling patterns were observed for 6 donors in 10 separate gels, there was some donor variability in the relative intensity of dephosphorylation observed in adherent platelets. Thus, dephosphorylation of a 40-kD protein band (Fig 1) was less apparent in some experiments (Fig 2). Nevertheless, we consistently observed dephosphorylation of the various bands in 10 of 10 experiments.
Effect of OA on adhesion and adhesion induced dephosphorylation.Significant evidence exists for the involvement of protein phosphatases in platelet function, both serine/threonine linked34 and tyrosine linked. 35 Most reports on serine/threonine phosphatases have concerned platelet aggregation and were often based on the use of OA and calyculin A, which are the strong inhibitors of PP1 and PP2A.36 37 Thus, Chiang37 suggested that PP1 is involved in collagen-induced aggregation. However, there are few studies on the role of protein phosphatases in platelet adhesion, and because protein dephosphorylation during adhesion could result from phosphatase activation, it is possible that their inhibition would block adhesion.
The involvement of protein phosphatases was therefore studied by using OA at concentrations of 0.1 and 1 μmol/L, respectively. We found that 10 minutes of preincubation with 0.1 μmol/L OA had little influence on platelet adhesion to collagen, causing only 2.6% inhibition compared with controls studied at the same adhesion time (Table 1). Treatment of resting platelets with 1 μmol/L OA significantly blocked subsequent adhesion (60%; Table 1). The extent and pattern of protein phosphorylation at 0.1 μmol/L OA were similar to those that were observed for untreated cells (Fig 2), whereas 1 μmol/L OA caused a marked phosphorylation of a 50-kD protein and a moderate increase in phosphorylation of 67-kD and 95-kD protein bands (Fig 2). Those platelets that actually managed to adhere to collagen in the presence of OA were still able to stimulate phosphatase activity as indicated by dephosphorylation of the 67-kD protein band and by comparison with OA-treated nonadherent (both resting and effluent) platelets. A 50-kD protein band that was phosphorylated during OA treatment was also dephosphorylated in adherent platelets compared with control OA-treated cells. Interestingly, a new unknown phosphorylated protein band slightly below 67 kD appeared in adherent cells.
Presence of hsc70 in the 67-kD protein band.An initial aim of our study was to identify proteins present in the 67-kD protein band that was dephosphorylated during adhesion to collagen and that was sensitive to OA treatment (Figs 1 and 2). Microsequencing of proteins in this band conclusively identified the presence of hsc70 based on detection of the following sequences: VEXXANDQGNR, TTPSYVAFTDTER, FEEXNADLFR, and DAGTXAGXNVXR (in which X stands for either Leu or Ile that cannot be distinguished under the low-energy collision conditions that were used). Using a database, exact matches were found for all sequences to a human 71-kD heat-shock cognate protein (hsc70).38
Immunoprecipitation of human platelet hsc70.It is known that small hsps are present in platelets and that their phosphorylation state can change during platelet activation.39-41 Recent observations suggest regulation of protein phosphatase activity not only by small hsps, but also by a group of hsps of higher molecular weight.42-44 Our finding that hsc70 was present in the 67-kD protein band helped stimulate us to study the association of hsc70 with platelet protein phosphatases and their possible involvement during cell adhesion to collagen. The hsc70 represents a constitutive member of the hsp70 family of stress proteins, which usually act as molecular chaperones in protein folding45 and recently have been proposed as important signaling molecules capable of associating with protein kinases, transcription factors, and receptors.46 We immunoprecipitated hsc70 from 32P-labeled, resting platelets using a purified anti-hsc70 rat MoAb (1B5) or a nonimmune rat IgG as nonspecific control. Immunoblotting with anti-hsc70 1B5 antibody of either whole-platelet lysates or of anti-hsc70 immunoprecipitates showed that these resting platelets contained a protein recognized by the anti-hsc70 MoAb having an apparent molecular weight of 67-71 kD and that migrated identically to purified bovine brain hsc70 (Fig 3A). Nonspecific binding of control rat IgG to this platelet protein was not observed (Fig 3A). Based on these observations, we suggest that the 67-71–kD protein seen in the immunoblots contains platelet hsc70. We wish to note that the molecular weight of this protein when determined from full-size 20-cm–long slab gels was about 67 kD. However, when the molecular weight analysis was performed using minigels, its apparent molecular weight was about 71 kD. Consequently, we have termed this protein a 67-71–kD protein.
Coimmunoprecipitation of 32P-labeled hsc70 with other phosphoproteins in resting platelets.Autoradiograms of 32P-labeled platelet proteins immunoprecipitated with the anti-hsc70 1B5 antibody in seven separate immunoprecipitation experiments consistently showed 10 phosphoprotein bands possessing apparent molecular weights of 197, 167, 128, 102, 95, 82, 62, 52, 45, and 38 kD (Fig 3B), in addition to the 67-71–kD protein recognized by anti-hsc70 antibody. Because hsc70 is an abundant protein in cells, it is important to note that these 10 phosphoproteins might associate with hsc70 in a nonspecific manner. To clarify this issue, we immunoprecipitated hsc70 and associated phosphoproteins from 32P-labeled platelets by using two additional MoAbs against hsc70: a purified mouse MoAb (N27) and a mouse MoAb (13D3). Nonimmune mouse and rat IgGs served as controls (Fig 3C). Autoradiography of SDS-polyacrylamide gels showed a very similar pattern of distinct bands in platelets immunoprecipitated with the three anti-hsc70 MoAbs (1B5, N27, and 13D3), but not with the control antibodies. The fact that the different anti-hsc70 MoAbs immunoprecipitated essentially the same amount of protein (Coomassie blue stain; data not presented) and phosphoproteins (Fig 3C), which migrated similarly, suggests specific interactions between hsc70 and the other phosphoproteins. As just noted, the phosphorylation patterns obtained with the different antibodies (Fig 3C) were very similar for the same donor. However, we have observed that there was some variation in the extent of phosphorylation of the lower molecular weight components in the hsc70 complex for different donors.
Coimmunoprecipitation of platelet hsp90 and PP1 with hsc70.Based on evidence in other cells that hsp70 may associate with hsp9045 and interact with phosphoprotein phosphatases,44 we performed additional experiments in which either anti-hsc70 (1B5) or nonimmune IgG immunoprecipitates were blotted with antibodies against hsp90 (AC88) and the catalytic and targeting subunits of PP1. This approach showed that the hsc70-associated 95-kD band (Fig 3B) corresponded to hsp90 (Fig 3D), the 38-kD band (Fig 3B) to the α and δ catalytic subunits of PP1 (Fig 3E), and the 128-kD band (Fig 3B) to PP1M (Fig 3E). Nonspecific binding of hsp90, PP1Cα, PP1Cδ, and PP1M to control immunoprecipitates with nonimmune rat IgG was not observed (Fig 3D and E).
Dephosphorylation of hsc70 during platelet adhesion to collagen.We then assessed whether platelet adhesion to collagen influenced the phosphorylation state of the 67-71–kD protein, presumably hsc70. Lysates of 32P-labeled platelets from either control or adherent platelets were immunoprecipitated with anti-hsc70 MoAb. Because the pattern and extent of phosphorylation of hsc70 immunoprecipitates were very similar for effluent and resting platelets (data not shown), in most experiments we used resting (unstimulated) platelets as controls. Autoradiography of the hsc70 immunoprecipitates showed complete dephosphorylation of the 67-71–kD protein and dephosphorylation or dissociation of the other phosphoproteins after adhesion (Fig 4A, two left lanes). Both silver stain and immonoblots of immunoprecipitates with anti-hsc70 antibody showed similar amounts of 67-71–kD protein in control and adherent cells (Figs 4B and 5A, respectively). In sharp contrast to adhesion, thrombin-induced aggregation performed for the same length of time did not initiate dephosphorylation of platelet proteins (Fig 1). When hsc70 was immunoprecipitated from thrombin-aggregated 32P-labeled cells, it was even more highly phosphorylated after thrombin stimulation than in resting platelets and the complex remained intact (data not shown), indicating different signaling pathways induced by thrombin and collagen.
Adhesion-induced dissociation of hsc70/hsp90/PP1 complex.We subsequently assessed whether platelet adhesion to collagen influenced the association of the 67-71–kD protein with other proteins. To evaluate whether these proteins may have dissociated from the complex, immunoprecipitation experiments with the anti-hsc70 MoAb were performed on control and adherent platelets, followed by immunoblotting with anti-hsc70, anti-hsp90, anti-PP1Cα, and anti-PP1M. Immunoblotting with anti-hsc70 showed that comparable amounts of 67-71–kD protein were precipitated from the adherent and control platelets (Fig 5A). However, the 95-kD protein (hsp90; Fig 5B), PP1Cα (Fig 5C), and PP1M (Fig 5D) were no longer associated with the hsc70 complex, indicating that adhesion had caused their release.
Immunoprecipitation of platelet hsp90.hsp90 is a major phosphoprotein that can interact with various proteins such as actin, tubulin, calmodulin, steroid-hormone receptors, and a number of protein kinases.46 We therefore assessed the presence and phosphoprotein associations of hsp90 in unstimulated, 32P-labeled platelets by means of immunoblots and immunoprecipitations with both anti-hsp90 and control antibodies. We used the AC88 antibody that recognizes free hsp90 or hsp90 not complexed with steroid hormone receptors47 and that was previously used to immunoprecipitate proteins that react with antibodies against α and β tubulin.9 This antibody recognized a 95-kD phosphoprotein that migrated identically to purified bovine hsp90 (data not shown). Immunoprecipitations of control 32P-labeled platelets showed a pattern of coimmunoprecipitating phosphoproteins similar to that observed for hsc70 with the 1B5 antibody (Fig 4A, two right lanes). As observed before (Fig 3C), control nonimmune mouse IgG did not precipitate significant amounts of 32P-labeled platelet proteins.
hsp90 is translocated to the cytoskeleton during adhesion.Autoradiograms of 32P-labeled hsp90 immunoprecipitates of adherent platelets showed a complete loss of radioactive phosphate (Fig 4A, two right lanes). However, in contrast to our observations for hsc70, the amount of hsp 90 immunoprecipitated with the AC88 MoAb in adherent cells was almost undetectable compared with control or nonadherent platelets as determined by silver stain of the hsp90 immunoprecipitates (Fig 4C). Because our methods involved preclearing platelet lysates by centrifugation at 15,000g for 10 minutes before incubation with precipitating antibody, the 95-kD protein could have been translocated to the platelet cytoskeleton and thus been cleared during the 15,000g centrifugation. To examine the subcellular localization of hsp90 in resting and adherent platelets, differential centrifugation methods30 were used with lysates of control and adherent cells to obtain the cytoskeleton (csk; 15,000g pellet), the membrane-skeleton (msk; 100,000g pellet), and solubilized cytosolic proteins (cyt; 100,000g supernatant). Western blot analysis with anti-hsp90 MoAb AC88 showed that the majority of hsp90 in resting platelets was present in solubilized cytosol and that trace quantities of hsp90 were detectable in the membrane-skeleton. After adhesion, the amount of hsp90 was greatly reduced in the cytosol of adherent platelets and more hsp90 became associated with the cytoskeleton and membrane-skeleton (Fig 6).
Effect of OA on hsc70 complex.Because OA blocked adhesion (Table 1) and is known to cause marked changes in protein phosphorylation of resting platelets,34 we also examined the effects of OA on the phosphorylation of the hsc70 protein complex. Immunoprecipitation with anti-hsc70 of 32P-labeled resting platelets showed that OA significantly increased 32P incorporation into hsc70 (p71) and hsp90 (p95) as well as into other unknown proteins that coimmunoprecipitated with hsc70, particularly p62, p52-50, p45, and p38 (Fig 7).
In the present study, we have shown that platelet adhesion to collagen under flow conditions induced distinct changes in protein phosphorylation compared with adhesion to the nonspecific substrate polylysine or to aggregation induced by thrombin. In particular, adhesion to collagen caused significant dephosphorylation of several protein bands, which were highly phosphorylated in resting platelets (Fig 1). We also observed that OA strongly inhibited adhesion. These new findings suggest that activation of serine/threonine protein phosphatases could be involved in the initiation of adhesion as well as occur as a result of adhesion. We have now found that resting platelets contain a phosphoprotein complex of hsc70 with hsp90, PP1Cα, PP1Cδ, PP1M, and 7 unidentified phosphoproteins. The association of hsc70 with these proteins was modified during α2β1 integrin-mediated adhesion to collagen such that hsc70 became dephosphorylated and hsc70-associated proteins were released from the complex. Stimulation with thrombin did not cause dephosphorylation of hsc70 and complex dissociation, showing that this behavior was specific for activation by collagen.
In addition to the dephosphorylation events noted above, adhesion to collagen caused phosphorylation of a 47-kD protein (p47, pleckstrin) indicating PKC activation.33 PKC activation also occurred in platelets stimulated by thrombin or in platelets adherent to polylysine (Fig 1). However, in contrast to thrombin-and polylysine-stimulated platelets, we observed minimal phosphorylation of a 20-kD protein (p20; Fig 1), which is a substrate for the Ca2+/calmodulin dependent myosin light-chain kinase,48 indicating that adhesion did not induce a significant increase in [Ca2+]i. This observation parallels other studies showing that either adhesion to collagen or stimulation with collagen in the presence of indomethacin causes only a small degree of phospholipase C activation and IP3 formation, without a corresponding increase in [Ca2+]i.49 50 In contrast, Smith et al51 found that direct activation by collagen involves an increase in platelet cytosolic free Ca2+ but also noted that this increase is not required for platelet adhesion to collagen. Poole and Watson52 observed an increase in cytosolic calcium in single human platelets adherent to collagen fibers by using dynamic fluorescence ratio imaging. Under the labeling conditions used in our studies, a 67-71–kD protein band was the most highly phosphorylated protein band in resting platelets, and adhesion to collagen was correlated with significant dephosphorylation of this band (Fig 1). This finding indicates that in unstimulated platelets these constitutively phosphorylated proteins have a potential role for initiating signaling and that there is a need to identify the proteins that are dephosphorylated to help establish the significance and mechanisms of activation of phosphatases during adhesion. Interestingly, a correlation has been described between aggregation induced by either phorbol myristate acetate or by U46619 and the dephosphorylation of a 68-kD protein band sensitive to OA.53
Treatment of platelets with 1 μmol/L OA, a potent inhibitor of PP1 and PP2A, increased the phosphorylation of several proteins, including the 67-71–kD protein band; in general, the increases were similar to changes reported previously.54 This finding parallels other results indicating that constitutive protein kinases and phosphatases are actively cycling and are responsible for the observed pattern of phosphorylation seen in unstimulated cells.34 Our observation that OA increased phosphorylation of the 67-71–kD band in resting cells suggests that OA-sensitive phosphatases are involved in dephosphorylating proteins in this band under basal conditions. It is also possible that the phosphorylation state is controlled by both OA-sensitive phosphatases and by unknown kinases. Inhibition of PP1 and/or PP2A by OA blocked adhesion (Table 1), indicating either a direct or indirect role for dephosphorylation in adhesion, and this corresponds to other studies with human B cells.18 19 Those platelets that actually managed to adhere still exhibited strong dephosphorylation of the 67-71–kD protein band. These OA-treated adherent platelets were concentrated such that the same amount of platelet protein was then added to each electrophoresis lane. This result could be interpreted that, under these conditions, the phosphatase(s) involved is insensitive to OA or that OA is poorly penetrant into platelets. Alternatively, a subpopulation of platelets insensitive to OA could exist. In this context, it is well known that a significant pool of platelets (15% to 20%) can adhere to collagen even in the presence of high levels of cAMP.27 The identity of the new small band just below the 67-kD band (Fig 2) is unknown. The importance of phosphatases in platelet activation has been suggested by the transient phosphorylation of proteins such as pleckstrin (p47) and myosin light chain (p20) that occurs after thrombin- or collagen-induced aggregation.34 In addition, prior studies with OA and calyculin A showed that, in thrombin-stimulated platelets, inhibition of PP1 and PP2A blocked aggregation, phosphatidyl inositol (PI) metabolism, Ca2+ entry, and secretion from dense granules.37 54-56 In all of these cases, the inhibition of protein phosphatases reversed the phosphorylation changes that occur during activation of feedback pathways involving protein kinase A and C. However, our understanding about signal transduction events during adhesion is still limited, including protein phosphorylation changes and how these relate to the establishment of stable irreversible adhesion.
The finding that hsc70 was present in the 67-kD protein band encouraged us to study the association of hsc70 with platelet phosphoprotein phosphatases. In many cell types, hsc70 and hsp 90 are the most abundant proteins in cytosol and both can exist as phosphoproteins.57 58 There is considerable evidence that these hsps are involved in regulating protein folding, serve as chaperones, and assist protein-protein interactions.10 11 45 46 There is some evidence that hsp70s can interact with protein phosphatases and alter their activities in vitro.44 59 We have now shown that resting platelets possess phosphorylated hsc70 that associated with a number of other proteins (Fig 3B), using immunoprecipitations with antibodies directed against hsc70, followed by immunobloting and autoradiography. Some of these proteins have been identified: hsp90, PP1Cα, PP1Cδ, and PP1M. The association of hsp70 with hsp90 has been previously described in other cell types such the human mammary tumor T47D cell line, the hepatoma cell line 1c1c7, and chicken oviduct,60-62 but not in human platelets. It is possible that the proteins described here could have become associated with hsc70 nonspecifically after platelet lysis. However, specific associations and interactions are likely, because a limited number of proteins coimmunoprecipitated with hsc70 in a highly reproducible manner, even using different antibodies. Whether these proteins interact directly with hsc70 or bind to one of the other proteins complexed with hsc70 requires further analysis. Recent reports support our new findings on the association of hsc70 with protein phosphatases. The 78-kD glucose-regulated protein, a member of the hsp70 family, has been shown to associate with the PP1γ2 catalytic subunit.44 In addition, purified hsp70 when added to rabbit reticulocyte lysates may activate phosphoprotein phosphatases.59 In the studies reported here, some of the proteins associated with hsc70 have been identified. Other proteins bound to hsc70 that were not detected by 32P-labeling may well exist. For example, tyrosine-phosphorylated proteins, nonphosphorylated proteins, or proteins only weakly bound to hsc70 that could have been washed off the immunoprecipitates, even by the low-salt conditions, were used.
Little is presently known about how collagen binding to the α2β1 integrin might initiate intracellular signaling. Keely and Parise63 found that the α2β1 integrin may be required as coreceptor for activation of pp72syk and subsequent phosphorylation of phospholipase C-γ2 in aggregating platelets stimulated with collagen fibers. In addition, GPVI appeared to be stongly involved in pp72syk activation and tyrosine phosphorylation of phospholipase C-γ2, pp95vav, and of focal adhesion kinase, pp125FAK.25 We previously observed that adhesion caused rapid tyrosine phosphorylation of pp125FAK that was blocked by the anti-α2β1 antibody and associated with changes in cyclic nucleotides.27 29 In the present study, we have shown that adhesion to collagen was correlated with dephosphorylation of hsc70. Some of the proteins which coimmunoprecipitated with hsc70 in resting platelets, such as hsp90, PP1α, and PP1M were released from the phosphoprotein complex after adhesion and moved to new cellular compartments. For example, hsp90 was partially translocated to the cytoskeleton and to the membrane skeleton. The localization and phosphorylation changes of the other complex components after adhesion are currently under study. Recently, Uzawa et al64 reported the presence of a centrin/hsp70/hsp90 complex in CSF-arrested Xenopus oocytes that dissociates after treatments known to activate the oocyte for embryologic development. At the moment we do not know if or how the platelet complex may be associated with the α2β1 integrin or another collagen receptor. It has been recently shown that the α2β1 integrin can directly bind to actin65 that might thus form a bridge to heat-shock proteins and the collagen receptor. In addition, hsp70 may cross-link intracellular molecules to actin microfilaments3 and indirectly modulate adhesive receptors.
The 95-kD protein present in the phosphoprotein complex of resting platelets and sensitive to OA was shown to be hsp90 (Figs 1, 2, and 6). It may correspond to the unknown 90-kD protein whose phosphorylation was increased by OA and calyculin A and that correlated with dramatic changes in the morphology of resting platelets.66 This unknown 90-kD protein was shown to coimmunoprecipitate with tubulin. Previously, colocalization of hsp90 along tubulin-containing fibrillar structures was reported in epithelial cells.9 In addition, Fostinis et al67 showed colocalization of hsp90 with microtubules and the presence of hsp90 in the Triton X-100 insoluble cytoskeleton of a human endometrial adenocarcinoma cell line. Thus, there is good evidence that hsp90 may associate with the cytoskeleton of cells, an association supported by our localization studies.
A number of platelet proteins become hyperphosphorylated in the presence of OA, in particular, a 50-kD protein whose phosphorylation correlates with inhibition of platelet function and that is markedly potentiated by prostacyclin.68 This 50-kD protein is also a major substrate for cyclic nucleotide-dependent kinases in platelets and is known as the VASP protein.69 We showed earlier that adhesion is blocked by both prostacyclin and sodium nitroprusside.27 In the present study, we found that platelet adhesion was inhibited by OA, correlating with enhanced phosphorylation of the 50- and 67-kD protein bands. OA also caused hyperphosphorylation of hsc70 and other complex components (Fig 7), such an unknown protein of molecular mass of 52-50 kD, which on one-dimensional SDS-PAGE ran similarly to phosphorylated VASP. This finding would fit in with the general theme that the 50-kD protein is VASP in its hyperphosphorylated state69 and is associated with inhibition of platelet function, plus the observation that the heat-shock protein complex is phosphorylated in the resting or inactive state of platelets (Fig 3). The theme that heat-shock protein complexes are phosphorylated in resting cells and are poised to initiate and regulate cellular signaling pathways has been developed by Rutherford and Zucker.46
We wish to emphasize that we do not have direct evidence that the 67-71–kD protein band dephosphorylated during adhesion (Fig 1) and that migrates at the same molecular weight of hsc70 in whole-cell lysates is the same hsc70 that was immunoprecipitated with the anti-hsc70 antibody and shown to be dephosphorylated in immunoprecipitates of adherent cells. It is possible that hsc70 itself was not phosphorylated in unstimulated platelets, but rather was associated with another phosphoprotein(s) of similar molecular weight that was immunoprecipitated with the anti-hsc70 MoAb. Although the present study shows an association of hsc70 with hsp90, PP1, and other unidentified phosphoproteins in resting platelets, the functional significance of these interactions is still unknown. The interaction of hsc70, hsp90, and PP1 could have little relevance in platelets, possibly due to the high intracellular levels of these proteins that may lead to nonspecific associations. However, the finding that α2β1 integrin-mediated adhesion to collagen was always correlated with complex disassembly and dephosphorylation of some components supports the thesis that the complex plays an important role in cell adhesion. Recent studies describing tyrosine phosphorylation of hsc70 in response to ligation of the T-cell antigen receptors13 and of hsp75 in response to oxidative stress14 indicate that this modification might regulate hsp function and thus cell adhesion. There is strong evidence linking hsp90 and hsp70 to the signaling functions of the glucocorticoid receptor complex70 and hsp90 is known to form complexes with a number of signaling molecules, including protein kinases, such as pp60v-src and Raf.46 Our findings now show that heat-shock proteins possess the ability to bind other critical signaling enzymes, such as protein phosphatases.
We thank J. Lawrence and D. Hartshorne for providing antibodies; D. Brautigan and A. DePaoli-Roach for advice about phosphatase antibodies; T. Haystead, T. Sturgill, G. Balian, and J. Hockensmith for critical review of the manuscript and helpful discussions; and V. Gordon and L. Beggerly for technical assistance.
Research supported by the Carman Trust and initially by National Institutes of Health (NIH) Grant No. HL-27014. The mass spectrometry studies were supported by the NIH Grant No. GM-37537.
Presented in part in abstract form at the American Society for Cell Biology Thirty-fifth Annual Meeting, Washington, DC, December 1995 (Mol Biol Cell 6:384a, 1995 [suppl]).
Address reprint requests to Renata Polanowska-Grabowska, PhD, Department of Biochemistry, University of Virginia, Charlottesville, VA 22908.
- Submitted July 31, 1996.
- Accepted April 10, 1997.
- Copyright © 1997 American Society of Hematology


text: Module Name: src Committed By: christos Date: Wed Mar 21 23:20:58 UTC 2012 Modified Files: src/external/gpl3/gdb/dist/gdb: nbsd-thread.c Log Message: Use tid - 1 for the thread id, so that thread 1 matches the main thread before the threaded code gets loaded. fixes spurious breakpoints. Is there a better way? To generate a diff of this commit: cvs rdiff -u -r1.9 -r1.10 src/external/gpl3/gdb/dist/gdb/nbsd-thread.c Please note that diffs are not public domain; they are subject to the copyright notices on the relevant files.


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Flash Draw Created Jun 07, 2008 470 x 338px Unless noted Copyright 2008 BlueHearts.
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WestSidePhat Jun 14, 2008
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Back to the Future
Those features were introduced back in the 1980s under the News's last editor, Ed Petykiewicz, when local newspapers saw their future in imitating USA Today. Without them, AnnArbor.com, the paper, has a pleasantly old-fashioned feel--yet another step back to the future.
[Originally published in April, 2012.]
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Mukta Sharma
Affiliation: World Health Organization
Country: Switzerland
Detail Information
- Substance dependence treatment interventions: why we continue to fail people who use drugs in AsiaMukta Sharma
HIV AIDS Asia Regional Program
Subst Use Misuse 47:1617-23. 2012..Substance use disorders contribute to a large number of preventable deaths in Asia; a majority of the negative health consequences are associated with opioid use. User friendly and effective drug dependence treatment is limited...
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World Health Organization, Regional Office for South East Asia, New Delhi, India
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Harm Reduction HIV AIDS Unit, WHO SEARO, New Delhi, India
Subst Use Misuse 43:1062-77. 2008..We urge for focused HIV prevention interventions targeting substance-using women and female sex partners of male substance users to reduce vulnerability...


text:
Four parades made their way through Terrebonne and Lafourche parishes Saturday, and four more roll today.
Four parades made their way through Terrebonne and Lafourche parishes Saturday, and four more roll today.
Revelers bundled up Saturday night to watch Aquarius in Houma and Ambrosia in Thibodaux.
Kicking off the parades at noon were the Krewe of Petit Caillou in Chauvin and Des T-Cajun in Larose.
Temperatures reached a high 53 degrees in Houma Saturday afternoon but dipped into the high 30s before the parades ended for the night.
Even Francis Tebben, 83, who came to Houma with his family from South Dakota to experience Carnival said it was cold.
"It's a different kind of cold," he said. "The wind is what gets you. It was warm when we got here, and we got acclimated."
The Sioux Falls resident held up a "South Dakota Loves Mardi Gras" sign as Aquarius floats headed down West Park Avenue near Southland Mall.
Bayou Blue resident Laura Chaisson, 39, tied a yellow scarf around her face while she watched the parade. Though she wished it wasn't so cold, nothing would keep her and her family away from Aquarius, she said.
"Rain, snow — nothing's going to stop us," Chaisson said.
Chaisson said Aquarius is a nice change after watching the rowdy Krewe of Hercules the day before.
"It's one of our favorites. ... The women are spectacular, and they're great with the kids," she said, who came with her daughter and nephew.
The all-female krewe, 295 members with 19 floats, rolled at 6:30 p.m. under the theme "Parade of Nations." Denise Boudreaux reigned as queen.
In Thibodaux, the Krewe of Ambrosia rolled about an hour earlier, making the city's first night parade in nearly 100 years. The parade was considered a test to help determine if Thibodaux will hold future night parades, and at 9 p.m., police said there had not been any major incidents.
Choctaw resident Crystal Andras, 37, had wrapped a sleeping bag around herself as she watched Ambrosia roll by. She said she was excited that Thibodaux finally had a night parade.
"Houma had all the good parades before," she said. "A night parade is nicer than a regular day parade."
The parade was themed "Ambrosia's Night Parade Turns a Page in History," and the surprise grand marshals were Louisiana State University football players Chase Clement of Thibodaux and T-Bob Hebert, son of south Lafourche native and former New Orleans Saints quarterback Bobby Hebert.
The parade has more than 400 male and female members and 30 floats.
Peter Verret III and his wife, Katherine Verret, reigned as king and queen. According to Krewe Captain Tracy Westerman, Peter Verret III, longtime volleyball coach at H.L. Bourgeois High School, has advocated for Ambrosia to parade at night ever since the krewe was formed 27 years ago.
"It is such a natural high to be doing this, and the energy is fabulous," Westerman said before her float pulled out of the parade's starting point at Nicholls State University.
All of today's parades roll during the afternoon. Weather is expected to be mostly sunny with the high temperature around 52 degrees.
Katie Urbaszewski can be reached at 448-7617 or firstname.lastname@example.com.


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To want to make myself believe in god?(1000 Posts)
I have always been very logical and so despite going to a Christian primary school, having a very religious mum(though not in a pushy way) and reading the bible when I was younger(the story version I was a bit of a book worm) I have never really got my head around how god could be possible.
But I really wish I had the extra "something" that some people seem to find by believing in god. I'm probably not making much sense, but I wish I could get myself to feel like there's somebody watching out, that there's something after death, that everything happens for what'd ultimately a good reason/what's meant to be so on.
AIBU to try going to church for a bit even though I don't believe in god? Or am I just being silly, is it something you can't 'make' yourself feel?
I am like the OP. I want to believe in God. I really want to. I just can't seem to. In my teens I went on an Alpha course. Ds1 has just done his holy communion, so I went to church every week for months.
And still I am no further on. No closer. I sOOOOO want to believe. I am still unsure on what to do next.
shall we pray?
The merest glance at human religious history tells you that people have fervently believed in many things at some time or other. If onevflavour doesn't do it, try another.
Why not believe in multiple Gods, much more interesting and statistically more likely.
PramelaAndherson Well, gosh. If he said it must be true.
HopHopHop There are numerous religions that claim they are the one true religion, that all others religion are false and their followers deceived. The only logical conclusion is that none of them are the one true religion and all of them are deceived.
Religion is a false comfort, based on lies.
But if you really want to explore religion, you don't have to automatically believe in god(s). There are atheistic religions like Pagan Atheism, Buddhism, Confucianism, Taoism and Jainism you can explore.
"btw I don't think you can make yourself believe. That's God's job"
That's what I'm worried about. That it is impossible for me to make myself believe.
If it's impossible to make yourself believe, then maybe that's because you find the god myth impossible to believe, and there's nothing wrong with that. There are plenty of non-religious organizations you can get in touch with that will discuss it with you, churches don't have to be your first port of call, the British Humanist Association would be a great place to start.
You don't have to get a sense of purpose from being part of a church or religion, try clubs, hobbies, charities etc.
There are hundreds of thousands of non-religious people who live driven, successful, purposeful lives, without suspending their disbelief in the supernatural.
Oh and far as the Alpha course goes, don't be taken in by them, as others have said it's there for one reason and one reason only, to convert you to Christianity, that's it.
Religion is a false comfort, based on lies.
In your opinion, Normal . Please do qualify that that is your opinion. Strongly held, yes, but your opinion.
It happens to be my opinion that religion has nothing to do with - or very little to do with - God. Never the twain, usually. ie religious practise usually has practically nothing to do with God at all imo. the opposite spirit.
Oblomov , maybe tell God you're worried about that ? It doesn't have to be an essay. If it's up to him, and he is who he says he is, then he'd find a way to make himself known to you, no?
springtoto I don't see you asking the religious people on this thread to qualify their statements as being purely their opinion, but yes it is my opinion, although at least I can quite easily find proof of some of the lies contained in many of the most popular religions.
The fact that you want to believe means that you have begun a search. If you pursue the search then you will find what you seek and you will receive the gift of belief.
You have to be open and open your mind in order to receive. You can't force it, it will come when you are ready for it. I would continue the search by buying some books on spirituality and spiritual philosophy because this will enhance the spirit of wonder which will open you up to receive belief.
OP: you could visit some sacred sites and sit quietly.
Old churches are built on sacred sites and have lovely atmosphere and energy.
Though if you have DCs the sitting quietly may be difficult!
You could go to a Meditation group and see what that brings to you.
I am so pleasantly surprised that there are so many lovely replies on here.
it is possible to attend church and not believe in God as such, just enjoy the community gathering and hymns, reflection etc., and live by the teachings of Jesus....I know at least one person who does this...(not me btw!)
Oh and OP I've had these same feelings before, I came quite close to joining Islam in my teenage years, after studying it for several years with a close friend of mine, but I couldn't suspend my disbelief, you can't mske yourself believe anything, either you do or you don't.
pamelaandherson you are making the presumption that the "Father" is the only god out there. Even Jehovah admitted there were other gods - he just wanted to be the top dog - "Thou shalt have no other gods before me.".
I'm a panentheist - that means I think the divine is both imminent and transcendent. I express this through Wicca, and contrary to a pp, Wiccans don't think we are right, we just think we are right for those who choose to follow our path.
We all need to find our path to "God" ourselves.
My take on it is that the Alpha course clearly and methodically explains what christianity is about. A surprising number of people don't know what christianity is actually about. imo the evidence is so compelling that a considerable percentage do go on to believe in Jesus - eg what he did, and why. I'd personally have an issue with the (sometimes) churchiness of the follow-up to the Alpha course. There can sometimes be a bums-on-seats strain to the follow-up, but not always - a lot get the drift that it's God's job to forge a relationship, the church's job is to introduce imo. I can see how christians can get so excited about introducing Jesus, the story of it all - it isn't called the good news for nothing - that they can get a bit overzealous and forget where the boundaries lie, forget to keep back.
(Mind you, I am perhaps largely speaking in a cultural context here - in our cultural it is paramount to respect privacy, personal space etc. This is not always, or necessarily, the case in other cultures which are inherently more religious, understand religious reasoning and belief - where eg Pram's presentation of unequivocal beliefs would be appropriate and readily understood.)
OP like you I teetered on the edge of belief for many, many years, and when I first started going to church I didn't believe. Faith came very gradually -- a bit like one day waking up and realising that you 're actually in love with someone you've been friends with for ages. What changed it all for me was not trying to decide if I believe that God exists, but beginning to pray - that changed so much - and beginning to try to live out Jesus's message - love God with all your heart and mind and strength, and love your neighbour as yourself ( still working on that one!).
I suggest just be yourself, you were not born Christian, Jewish or Muslim, you were born a blank slate, free from the shackles of imposed dogma. Make sense of the world using reason, experience and shared human values. Take responsibility for your actions and base your ethics on the goals of human welfare, happiness and fulfillment. You can make the best of the one life you have by creating meaning and purpose for yourself. You don't need sky pixies to find that 'extra something', that's the easy way out. Good luck.
I was filled with doubts, I hoped that it would "hit" me in some way but then one day I just decided to believe. It worked for me and I think my life is richer tbh. I don't attend church every week but when I go I enjoy it and wonder why I don't go more?!
I was baptised catholic but not confirmed which left me feeling a bit left out so I embarked on a course and got confirmed, this was through choice and I enjoyed it. Talking to like minded normal people about God was actually refreshing. Maybe try a course? The catholic one is the RCIA, normally done in someone's home one evening a week for a few weeks (with tea and biscuit) or I have heard good things about the Alpha course.
Pram, you need to open your mind a little. Every religion says the same thing about their God: that it is the one true God etc. Just because Christianity is your religion doesn't mean it is the right one. If you were born and raised in India, you would be saying the same things about a different religion.
I have a few Muslim friends who are very definite in their belief in God (they believe Jesus was a Prophet).
I envy them their certainty of faith but I just don't have that certainty.
Normal - no-one has written a para saying:
Religion is a comfort. It is true.
Some have implied it but not made an unequivocal statement as you have.
Did anyone see Derren Brown's program on fear and faith. It was facinating, he was able to convert a staunch atheist. Her reaction was overwhelming. I'll try and find a link.
I'm sure you all know this but he is an atheist
The interesting thing about most religions is that if you strip out which particular god/s they believe in, and all the worshipful flummery around that, there is a remarkably consistent message of human ethics. Now whether that was revealed by the divine, the great Human subconscious or just hard experience over and again, I leave to you - but it seems you can live the identical ethical life quite easily while enjoying a spiritual life worshipping whatever you please.
Btw re Religion vs Spirituality - anyone read Terry Pratchett's "Small Gods" - says it all IMO.
I am so glad to have found this message as it resonates so strongly with me at the moment. I have been an active member of a high church all my life. My Dad was a vicar until he died 10 years ago. I was very happy in my faith, met my husband through church and we baptised our children. My faith gave me great comfort when my father died suddenly and the power of the church community was a great lifter at that overwhelming time.
Now I am happy and settled with my family we have both come to the conclusion(very strangely how we both reached this at the same time), that there most likely is no God. Religion, ceremony and the lovely music is a nice way of bringing people together but meaningless really for us now. We have always and will always try do the moral thing and instil these values in our children but now feel we do not need to be Christian to do this. I consider myself Humanist now. I do get why people believe but cannot believe it myself now. I have to say I feel extremely liberated.
I am reading 'The God Delusion 'and it makes a lot of sense to me.
As yet I cannot talk to people in real life about it as our close family are strong believers and we don't want to hurt them or shatter their 'delusion' (meant in the kindest way).
Apologies for this being incoherent but a) I'm on an iPhone, b) it's my first ever post and c) I am excited/confused by my feelings and seeing other people have been having a similar journey.
I think it's normal to want to believe in something. Research into the human brain shows that about 80% (IIRC) of us are hardwired into having a faith, although the expression of that faith tends to vary according to the culture you brought up in, etc.
If you find yourself struggling to deal with the logic of it, however, you could also try looking into humanism, etc. There is a great deal in humanism that shows you how to take joy from, and develop faith in, the human spirit in the same way that others have faith in a deity for example.
As long as you are not hurting anyone, you can believe whatsoever you want to believe and no one is entitled to make you feel different.
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Just a few weeks after giving birth to son Finn Davey, Tori Spelling is back in the hospital dealing with a postpartum health scare, her rep tells Us Weekly.
"Tori Spelling underwent emergency surgery over the weekend due to complications from her C-section," her rep shared in a statement Tuesday.
Spelling and her husband Dean McDermott welcomed their fourth child together on August 30. They are already parents to Liam, 5, Stella, 4, Hattie, 10 months, and Jack, 13 (from McDermott's previous marriage to Mary Jo Eustace).
No further details regarding Spelling's hospitalization are available at this time, although her rep adds that the Craft Wars host is still under the care of the medical staff there and "is resting comfortably."


text: My friend called me last night. She said she doesnt know what to do, her dogs wouldnt stop fighting. I did not believe her. She gave me an elaborate story and I laughed. I come into work today and she shows me her arms which are covered in bites and scratches from breaking them up. She tells me the hematomas in the 3 month old puppies ears were ripped open by the 8 month old boxer mix. She tells me the 3 months old puppy has a hole in his neck and the 8 month old is limping. I FEEL TERRIBLE. I didnt believe her and said things like "Well I guess youll have to put one of them down, lol." When she was going through real pain. We talked about it today and apparently the 8 month old got out last night and ran into the apartment complex parking lot and was not hit by, but startled by the security guys golf cart. He is now limping. The fighting started after that. I told her to crate and rotate them, and keep the dog off his leg until she can get him to a vet. He is in pain and thats prob why he is lashing out. She said he doesnt seem hurt and i said dogs hide it better than youd think. She left work early to take him in. But I still feel terrible.


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"Anthroposophy is a path of knowledge, to guide the Spiritual in the human being to the Spiritual in the universe."-Rudolf Steiner
What then is Anthroposophy? A religion, a cult, a set of rules or dogmas, a movement?
None of these encompasses the reality. In fact, Anthroposophy is hard to categorise – it does not fit in any given frame and it is alive.
Rudolf Steiner, who gave it this name, said if he could he would give it a different name every day.
It is possibly best to describe what people involved in Anthroposophy have done and why they have done it
in order to understand its uniqueness.
One thing is obvious:
Anthroposophists often like to do things differently, not because of any principle of their choosing but because they think about things more deeply than a 'business as usual' approach often allows.
To do this you need conscious freedom and it is no surprise that Steiner's seminal work is the "Philosophy of Freedom".
Not an easy book to read, yet a deep exploration of how we think by observing the act of thinking itself.
Observation is a typical tool of science and science is what Rudolf Steiner had studied.
He insisted that the scientific method has to be applied to research in all fields – even the spiritual field of experiences. That was new and still is for many who believe that spiritual things require mysticism and a non-thinking approach.
The object of Steiner's science of the spiritual is in essence man himself because everything in the universe can be found represented in the human being. Hence Anthroposophy, from the Greek Anthroposophia – the knowledge of the nature of man. Man here seen in his fullness, as a thinking, feeling and willing entity, not as any of these faculties in isolation. And man consisting not only of body and soul – which is generally accepted – but also of spirit, out of which his aspiration to work on his and mankind's further evolution is ignited.
Coming back to freedom.
How do we gain it? The answer is difficult and simple at the same time. By knowing oneself we learn to recognise what is the source of everything, what helps us, what hinders us and tries to derail us, what is morally sound and what has to be done to go forward.
Anthroposophy is therefore experienced very individually. It is not limited by dogmas or creeds, it is inclusive and not exclusive. In a way it is the most natural and universal path we can take if we are honest with our selves. We start from the knowledge of our selves and travel to an understanding of the universe as a whole, we observe our own evolution as a human being and grasp the laws of the wider evolution as well.
And how do we know if that which we do is right or wrong?
Steiner calls it ethical individualism and we know if we have acted ethically when we experience the results of our deeds and can see that they are balanced.
The list of fields where Anthroposophy becomes visible and can be experienced is long, impressive and still growing.
Here in Australia we are a bit behind Europe, where the starting point for all these practical endeavours was the end of WWI and its shattering effects. The first Steiner school in Australia was founded in Sydney in the fifties. Now there are more than 45. Australia has a strong section of Biodynamic Agriculture, the products of which are sometimes better known under the brand name of Demeter.
Anthroposophy is a flowing, growing and living being because at its centre are humans who carry it and apply it and expand it based on their own experiences. It is little wonder that the issue of Karma is alive in it, understood as the overlap of sequential lives not as a tool of punishment.
When you join the Anthroposophical Society, you agree to pay a yearly contribution to support the work of Anthroposophy both in Australia and in Dornach (near Basel, Switzerland). You can apply after about two years to become a member of the School of Spiritual Science if you wish to take up more responsibility for Anthroposophy in your own life. Otherwise you can join any of the discussion groups, courses, workshops, lectures and artistic events – or better still you can help with organising them yourself. Being active is part of being an Anthroposophist. And if you like a quiet time, over 300 books by Rudolf Steiner have been published, many of them available in English translation. Other Anthroposophical authors have contributed thousands of books in every imaginable field of human experience.
The Anthroposophical Society is active in 78 countries of the world, but its centre is Dornach. Here in Australia we have branches all over the country.
Anyone who wants to familiarise themselves with the Anthroposophical Society can contact a local group or any of the following members of the Committee of the Anthroposophical Society in Australia:
Jan Baker-Finch
- email@example.com
- 0403 171 651
Ron Vaisey
- firstname.lastname@example.com
- 0487 434 532
Cristina Rubsamen
- firstname.lastname@example.com
- 02 6633 7045
Peter Schirrmeister
- email@example.com
- 0432 944 508
Joan Weir
- firstname.lastname@example.com
Greta Adams
- email@example.com
(Above contacts updated: 6/10/14)
This web site is intended to be another means whereby Anthroposophy and work arising out of Anthroposophy (see articles on various views of what Anthroposophy is about) can be communicated, disseminated and discussed. It is hoped that this will in some way help reduce the isolation of distance if we can share what's going on with each other.
It is intended that articles and reports on activities will be placed up on this site if they are sent in a soft copy format. Discussions and views can be shared and built upon on the messageboard.
This site is also a portal to Australian branches of the Anthroposophical Society. It may take some time for all the branches to update some sections of the site, so please be patient and check back often.
If you have any content for these areas please contact email@example.com
If you have content for specific branch, please contact them directly.
More details of each Branch's activities can be found under Branch News.


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Intaba View is located in the tranquil and majestic Valley of 1000 Hills. Overlooking the Phezulu Nature Reserve, we have a panoramic view of this magnificent valley.
Open Monday to Sunday for Breakfast and Lunch, al fresco dining is available on our patio and grassed areas and during the winter months, a cosy fireplace warms the interior of our restaurant. Evening dinners and functions by arrangement only.
We also cater for weddings and conferences where full function co-ordination is provided for your special day.
No detail is overlooked and every effort is made to make your special day tailor-made and unforgettable.


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A Paris poster for the motion picture "Diana" has been removed following criticism for its close proximity to the site of her fatal crash.
The 4-foot by 6-foot ad was place near the Pont de l'Alma tunnel and just feet away from the the Flame of Liberty, a gold colored sculpture that has become an unofficial memorial for the Princess of Wales.
Diana and her boyfriend Dodi Fayed and their driver were killed in a car crash in the Pont de l'Alma tunnel in 1997.
"As soon as the distributor asked us to remove this specific poster, we decided to take it down," a spokesman for outdoor advertising company JCDecaux told ABC News in an email. The distributing group, Le Pacte, was not available for comment.
The biopic starring Naomi Watts focuses on the last years of Diana's life and many feel the poster's location was a deliberate marketing ploy before the film's release across France on Wednesday.
The JCDecaus spokesman would not be drawn into assigning blame for the choice of the poster's location. "The poster close to the tunnel belongs to an entire network of 730 panels in Paris. The campaign is ending tomorow."
Despite the controversy surrounding the film, "Diana" has struggled to make an impression at the box office. After being widely panned by critics, the film has only limped into the top five. The film will reach theaters in the U.S. on Nov. 1.
Kate Middleton and Princess Diana's Maternity Styles Compared


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Strong weekend expected for beach tourism
Thu, 17 Apr 2014 17:03:25 GMT —
MYRTLE BEACH, S.C. (AP) -- This Easter weekend should be a strong one for tourism along the Grand Strand, the heart of South Carolina's $18 billion tourism industry.
The lodging update released Thursday by the Brittain Center for Resort Tourism at Coastal Carolina University forecasts that occupancy will run about 93 percent Thursday and Friday nights. That is based on a sample of lodgings in Horry and Georgetown counties.
And reservations indicate almost 80 percent occupancy for next week. Researchers say that's an increase of almost 92 percent over the same week a year ago. But the Passover and Easter holidays were earlier last year.
AAA Carolinas reports that, while gas prices have increased 36 cents per gallon since Christmas, they are still about a penny a gallon less than last Easter.


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Monterey County
Get those vote by mail ballots in
Tuesday is the last day to request that a vote by mail ballot be sent to you for the June 3 statewide primary election. After that day, you can pick up ballots at the county elections office, 1370-B S. Main St. in Salinas.
The Elections department also recommends that you mail your ballots by Tuesday; it is best to hand-deliver them after.
To request a ballot, call 796-1499, complete the application on the back of the Voter Guide, or see www.montereycountyelections.us/absentee.htm.
Pacific Grove
Naturalist conference coming to town
Registration is open for the first-ever California Naturalist Conference, to be held at Asilomar Conference Grounds from Oct. 17-19.
Organizers say the event is not just for naturalists but for other individuals who may "share their appreciation for California's unrivaled state and national parks, coastal areas, mountains, wetlands, foothills and forests."
The event is put on by the UC California Naturalist Program and will include discussion of natural history, citizen science, climate change and nature interpretation.
Early discounts last until July 1. The conference is $250 for certified naturalists and $325 for others. A limited number of scholarships are available.
For more information, see calnat.ucanr.edu/2014Conference.
Monterey County
Road safety projects get funding
Two roadway safety projects received funding from the California Transportation Commission, the Transportation Agency for Monterey County announced.
$2.2 million was awarded to install a median barrier to reduce the severity and frequency of collisions on Highway 101 near Soledad from north of Hudson Road to the Salinas River Bridge.
And $109,000 was awarded to install median rumble strips on Highway 1 from Carmel Valley Road to north of Carpenter Street to reduce the potential for collisions.
In all, the commission allocated $1.3 billion to projects across the state.
Carmel Highlands
Sewer pipe going in across Highway 1
A project to install a sewer pipe across Highway 1 at Highland Drive in Carmel Highlands, five miles south of Rio Road in Carmel, will take place Tuesday through Friday, Caltrans announced.
The work will be done from 8 a.m. until 4 p.m. and could cause delays up to 10 minutes.
The contractor for this project under a Caltrans permit is Utility Services of Carmel Valley.
Big Sur
Group holds raffle for nonlead ammo
The Ventana Wildlife Society has started a raffle to provide free nonlead ammunition to big game hunters in Monterey and San Benito counties.
Lead bullets have been banned in the condor range since 2008 because of their potential to poison the birds that eat carcasses left by hunters. But the availability of nonlead ammo has been less than adequate to support a substantial switch, the society said.
The raffle will end after the first 200 qualified entries are selected to receive one box of ammo each. To enter, see www.ventanaws.org. Funding was provided by fines and private donors.
More adobes open for tours
Monterey State Historic Park has expanded adobe tours for the summer and fall.
Custom House, Pacific House Museum and First Brick House will be open to the public from 10 a.m. to 4 p.m. Friday, Saturday, Sunday and Monday holidays.
In addition, special tours will be given at Larkin House, Cooper-Molera Adobe and Robert Louis Stevenson House.
On the last Sunday of each month, all entrances and tours are free. For more information, see www.mshpa.org.
Monterey County
MST offers youth pass for summer
Monterey-Salinas Transit has started selling the Summer Youth GoPass, providing youth 18 and under unlimited rides on all routes from June 1 through Aug. 31 for $38.
The pass can be purchased at www.mst.org and MST customer service locations including the Bus Stop Shop in Monterey, Marina Transit Exchange and Salinas Transit Center.
For information, see the website or call 1-888-MST-BUS1.


text:
Sorry, the DVD that you are looking for is not available right now.
We did a search for other DVDs with a similar title, and found some results for you that may be helpful.
Amelie Poulain (Audrey Tautou) lives in Paris, but spends most of her time lost in her own fantasies. Very shy, but with a very wild imagination, Amelie loves the simple pleasures of life: skimming stones over water, plunging her hand into a sack of grain, and cracking the top of her Creme Brulee with a spoon.
When she finds a small box with an old photo hidden behind a tile in her bathroom, she decides she must find out whose it is. In achieving her quest, she realises the magnificent power one feels when you affect change in other people's lives. As she can't change her own reality, to escape her own sorrow at being alone and without love, Amelie attempts to affect the destinies of people she interacts with by making small changes to the personal 'worlds' they've shown her. She attempts to make people fall in love, punishes a nasty neighbour, and gives new leases on life to both the lonely and the unloved.
However, when it comes to the romantic French idea of finding her own true love, Amelie has much more trouble winning the heart of Nino (Mathieu Kassovitz), a young Parisian who happens to work in a sex shop and whose hobby is collecting other people's discarded passport photos. Then there's the bald guy who takes his own photo in all the express photo booths around town, but leaves his pictures behind . . .
Audio: Dolby Digital 5.1 DTS HD
Barcode: 9339065002331
Classification: M (Moderate sexual references)
Encoding: PAL
Format: Blu-ray
Language: English
Number of Discs: 1
Region: B (Australia & New Zealand)
Released: 4th November 2009
Running Time: 122.0 mins
Subtitles: English


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Will she make the right choice?
Advice columnist Carly Wexler has mapped out her entire life, and she's convinced by marrying her best friend, she'll be able to map out her path to happiness as well. No sexual sparks? No problem. Or so she thinks...until her fiancé's brother arrives for the wedding. Mike Novak ignites a desire inside Carly unlike anything she's ever experienced, shaking up everything she's ever believed in. When Mike becomes determined to stop her mismatched wedding to his brother, will Carly be able to accept that her unexpected love for Mike might be the right choice for her after all?
©2014 Karen Drogin (P)2016 Karen Drogin
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