text:
Is Merrion Business Development incorrect or missing key business information? Update your Hotfrog listing today.
Contact details
Are these details correct?
Hotfrog Ireland provides information regarding Merrion Business Development in Dublin. Merrion Business Development is located at 79 Merrion Square, Dublin 2 and provides marketing services. Contact them on 353 1 685 3450.
Is Merrion Business Development in 79 Merrion Square, Dublin 2 your business? Claim your listing and attract more leads by adding more content, photos and other business details.
We have more marketing services in Dublin available on Hotfrog Ireland. You can update your search for marketing by location, keyword or service options.
4Th Floor Prosperity Chambers 5-7 Upper Oconnell St 4Th Floor , Dublin, Dublin , Dublin 10, Dublin St 4Th Floor , Dublin, Dublin , Dublin 10, Dublin Dublin City Dubiin Dublin DUBLIN 10
15 CASTLEKNOCK MEADOWS Dublin
Unit 6 Bayside S C Sutton Unit 6 Dublin DUBLIN 13
67 Harcourt St 2 Dublin
Small Business Hub
Sharing tips, ideas and advice to help grow your small business.
Create your free Hotfrog business profile
Follow Hotfrog on:
Share on:
Hotfrog Small Business Hub


text:
She gets on top of her partner who lies down naked, and spreads her legs wide open and proudly shows her clean shaved pink pussy getting penetrated. more info 1782337 Mens Golden State Warriors Mitchell & Ness Camo Cuffed Knit Hat With Special Pom£. Add to this low insurance, competitive pricing and a five year warranty, and the i30 gives the likes of the Volkswagen Golf and the Ford Focus a run for their money.
Learn how to protect your baby Perfect Human Playground That's what this mom looks like when trying to do yoga near her kids. research form the University of Oklahoma found that women who drink 2 cans a day are more likely to develop fat around their stomach due to the amount sugar content in the drinks. Shyla likes Mike so much that she even lets him give her a little anal appreciation and bang that perfect ass of hers.
??00 (Mon Fri)09:00 18:00 (Sat)10:00 17:30 (Sun) Call us Call Centre 0333 321 0099* *Free within inclusive minutes package on mobile otherwise standard rates apply Opening hours: 09:00 22. Gérard Musy's Kaleidoscope series display a Share on Facebook Filed Under: Galleries, Magazine, Photography DEPARTMENT H TOKYO September 2, 2013 DEPARTMENT H TOKYO JAPAN The Japanese fetish scene has always been somewhat shrouded in mystery. Get your ISIC discount cards before you travel to save money abroad and sort out your travel visas and vaccinations.

subset: commoncrawl


text:
Filter Results:
Publication Year
Publication Type
Key Phrase
Publication Venue
Learn More
In forensic investigations, the recovering of latent fingerprints is one of the most essential issues. Driven by human experts, today this process is very time consuming. An automation of both examination of suspicious areas and acquisition of fingerprints lead on the one hand to the covering of larger surfaces and on the other hand to significant speed up… (More)
- ‹
- 1
- ›


text:
Diamond Shirts Hong Kong
Diamond Shirts are usually sold for HK$ 30.00 - HK$ 305.00 on iprice. You can either choose between a Dresses, Shirts and Tops. Diamond Shirts today are mostly sold in White, Red and Purple depending on your preference. Many people prefer Fashion Women Ruffles Sleeveless Dress, Korean Style Ruffles Chiffon Patchwork Wrap Long Sleeve Dress and Charming Lady Pure Color Backless Low-cut Black Sleeveless Dress models from Diamond Shirts. Look out for other reputable brands like ASOS, SheIn and Aglini if you think Diamond Shirts is not your cup of tea.


text:
I spent my first rotation at the Gatsby computational neuroscience unit, and now I'm at the Neural Encoding Lab.babyliss curl secret bon de reduction 5€ I suggest that those people happy to issue forth expletives about the nasty Tories look in the mirror occasionally. We've got all you need to boost your conception chances, from our ovulation calculator and the most effective sex positions, to the simple lifestyle swaps that will increase your odds of getting pregnant.
Show More 13 stylish saddlepads to suit your saddle Cathy Comerford 09:30 28 December, 2015 Don't miss our pick of the best saddlepads.babyliss pro perfect curl kullananlar blog Why Democrats didn't feel the Bern lowkell March 2, 2016 Support Blue Virginia Recent CommentsBruce Hedrick on Fairfax County Tax Vote a Case of Bad Policy and Bad Politics. Council leader Warren Morgan said the consultation showed public backing for his administration's plans to cut the £5.babyliss pro perfect curl lowest price list
She is wearing black corset paired with black panties, and her long finger nails have white french tip. In partnership with Bainbridge makers of the world's finest mounting boards we now offer through our MicroChamber technology a range of proactive museum and conservation quality mounting boards. Our focus is on Children & Young People, Coaching and Workforce, Competition and Events and Club Development.babyliss curling wand thick
Again, whether you fully believe Donaghy or not, he does provide a fascinating perspective into the world of refereeing that simply no one else can provide.


text:
If you log in, you can comment on buildings, submit new photos or update photos that you've already submitted.
We need to upgrade the server that this website runs on. Can you spare a quid to help?.
Description: Foxenden Manor
Grade: II*
Date Listed: 27 August 1952
English Heritage Building ID: 176435
OS Grid Reference: TQ9445156554
OS Grid Coordinates: 594451, 156554
Latitude/Longitude: 51.2749, 0.7865
Location: Newnham, Kent ME9 0NE
Locality: Newnham
Local Authority: Swale Borough Council
County: Kent
Country: England
Postcode: ME9 0NE
Incorrect location/postcode? Submit a correction!
Explore more of the area around Newnham, Kent at Explore Britain.
3/31 Foxenden
27.8.52 listed as
House. C16, 1604 and 1948. Timber framed and exposed with
plaster infill, on flint base, with plain tiled roof. C20
extension to left of rendered brick with applied timbers. End
right framed bays 2 storeys and jettied, with ogee braced frame,
and returned hipped roof. Central gable of 2 storeys and attic,
jettied at first floor and to gable, with carved gable bresummer,
bargeboard and pendant, and dated 1604 in framed surround in
gable. Stacks to centre left and at end right. Irregular
fenestration of 1 wooden casement to left and right on each floor,
and brick and wooden mullioned bay window below gable of 5 lights,
alternating with and without transoms. Boarded door in right side
of C20 hipped porch to centre right. Interior: internal frame
complete. (See BOE KENT II 1983 404).
Listing NGR: TQ9445156554
This text is a legacy record and has not been updated since the building was originally listed. Details of the building may have changed in the intervening time. You should not rely on this listing as an accurate description of the building.
Source: English Heritage
Listed building text is © Crown Copyright. Reproduced under licence.


text: Home > Experts > Question Question Posted by: Clark | 2010-08-16 Bloeddruk Bloeddruk systolic verhoog, maar diastolic verlaag. Moet ek steeds Zaik 5mg gebruik in die geval? Not what you were looking for? Try searching again, or ask your own question Our expert says: CyberDoc Hallo Clark Ja, jy moet dit gebruik as jou sistoliese bloeddruk hoog is. Dr Bets The information provided does not constitute a diagnosis of your condition. You should consult a medical practitioner or other appropriate health care professional for a physical exmanication, diagnosis and formal advice. Health24 and the expert accept no responsibility or liability for any damage or personal harm you may suffer resulting from making use of this content. 1 Our users say: Posted by: Cyberdoc | 2010-08-16 Hallo Clark Ja, jy moet dit gebruik as jou sistoliese bloeddruk hoog is. Dr Bets Reply to Cyberdoc Have your say Your name (optional) Your comment Security × Thanks for commenting! Your comment will appear on the site shortly. Search health advice Find an answer × Thanks for commenting! Your comment will appear on the site shortly. Reply to Your name (optional) Your comment Security Close Submit comment Recently: Asked Questions Answered Questions Commented Questions All I want to do is run Foot is blue with swelling Crazy or some other form of psychosis Divorce Divorce Crazy or some other form of psychosis All I want to do is run Advice please Highly risky insertive oral with cuts n blood ongoing ars symptoms since 4 months I'm losing interest in Sex and my wife is starting to notice it. Is revenge really sweet? I only want to date married older men Teenager behaviour Vindictive, spiteful no life MIL advertisement From our sponsors Keep an eye on your vision Which skin products are better, 'medical grade' or 'over-the-counter'? Win 1 of 6 R5000 cash prizes Win Skin Renewal voucher


text: iPhone 4 Multitasking Use #385: GPS Navigation AppsBrian Barrett6/18/10 4:00pmFiled to: MultitaskingNavigon appIphone 4NavigoniPhoneIphone 4 multitaskingAppleGPSNavigationNavigation appsappsSoftwareIphone 4 apps155EditPromoteDismissUndismissHideShare to KinjaToggle Conversation toolsGo to permalink Navigon—long one of our preferred navigation apps—is showing off how their latest update will take advantage of iPhone 4 multitasking. Sure, it'll be fun to have Pandora on while I browse, but this I can actually use. It's not a huge revelation as to what the Navigon app will do on iPhone 4; basically, you won't have to quit out of the application to surf the web or take a call. But seeing it in action is a reminder of how welcome multitasking will be, and how annoying it's been not to have it already. Oh, and speaking of annoying: I hope that having Navigon narrate directions while you're on a phone call is an optional feature. Because that would drive me totally up the wall. [MacRumors]Gear from Kinja DealsHow To Get Started With Philips HueLogitech G502 Proteus Core: The Gaming Mouse To BeatBecome a (Part Time) Standing Desk User With This Discounted Monitor RiserReply155 repliesLeave a reply


text:
Incanto Royale Necklaces Philippines
For colors, Gold are amongst the popular shades when it comes to Incanto Royale Necklaces. Many people prefer Diamond and Ruby Drop 18K Gold Necklace, Diamond and Emerald Drop 18K Gold Necklace and Diamond and Emerald Drop 18K Gold Pendant Necklace models from Incanto Royale Necklaces. You can check the products of JewelryAffairs, SheIn and ROMWE if you're not sure about purchasing Incanto Royale Necklaces. iprice provides Incanto Royale Necklaces from ₱ 47,356.00 - ₱ 612,986.00. You can find two types of Incanto Royale Necklaces online, particularly Necklaces, Rings and Earrings; you can choose the one that suits your need.


text:
Trickster Online
Trickster Online is a casual game in the sense that it emphasizes steady progression alone rather than the competitive PvP style gameplay found it many other free to play MMORPGs. With a level cap of 400, 8 playable classes and 12 stats, there's plenty to keep fans hooked. The way Trickster handles character progression is a bit different than most games. During character creation, players chose how they want their 4 primary stat types (Power, Magic, Sense, Charm) to grow each level. In addition to this, players are rewarded 4 'pips' each level to distribute among the 12 stats. It takes 4 'pips' to increase any stat by 1. Each stat type has two character classes associated with it, one male and the other female for a total of eight classes:
Power Type:
Buffalo (male) – Melee class that advances to Warrior and specializes in hitting multiple targets. Third job options include Gladiator and Mercenary.
Bunny (female) – Melee class that advances to Boxer and specializes in damaging single targets. Third job options include Champion and Duelist.
Magic Type:
Dragon (male) – Caster class that advances to Magician and gains light/dark based spells. Third job options include Dark Lord, Priest, and Wizard.
Sheep (female) – Caster class that advances to Bard and gains elemental spells. Third job options include Soul Master and Witch.
Sense Type:
Lion (male) – Long ranged class that advances to Inventor who specializes in guns. Third job options include Scientist and Cyber Hunter.
Fox (female) – Long ranged class that advances to Explorer who specializes in throwing items. Third job options include Thief Master and Hunter Lord.
Charm Type:
Raccoon (male) – Evasive class that advances to Card Master who uses special cards to deal damage. Third job options include Gambler and Duke.
Cat (female) – Evasive class that advances to Entertainer who has even greater evasive abilities. Third job options include Primadonna and Diva.
- Status: Shut Down
- Business Model: Free to Play
- Developer: Ntreev
- Publisher: Gamerage
- Category: Stand-alone
- Genre(s): 2D, MMO, RPG, Fantasy
- Platforms: PC
- +Cute anime/manga style graphics.
- +Quick and rewarding tutorial.
- +Plenty of classes.
- +High level cap (400.)
- -Complicated stat growth.
- -Repetitive scenery during early levels.
- -Limited appearance customization.
- Recommended System Requirements:
- OS: Windows System 98/2000/XP
- CPU: Intel Pentium III 1GHZ or better
- RAM: 512 MB or more
- GPU: 3D Display
- HDD: 3.5 GB or more
Available On
Articles You May Enjoy
- Hi-Rez Studios announces the SMITE World Championship presented by Xbox One
- Today Hi-Rez Studios revealed the first details for the SMITE World Championship presented by Xbox One (SWC).
- Cabal 2 Brings Black Wave: Gray Canyon Update and Reindeer Rampage Event
- ESTsoft, Inc. is bringing the holiday cheer tomorrow with a new content update and a limited-time holiday event.
- Lunar New Year celebrations arrive in Dragon Saga
- Dragon Saga is beginning its Lunar New Year festivities, which will run as late as February 14.


text:
Used Chevrolet Malibu Maxx for Sale in Johnson City, TN
New Search
Filter Results
|- Loading...
|- Loading...
Days on Market
|- Loading...
Accidents Reported
Text Search
Add Similar Cars
Choose Other Models to Add
Listings for these models will be added to the listings you are currently viewing.
CarGurus Quick Poll
|by Barry
"Our Malibu Maxx"
This car has plenty of power for meging with traffic. We have ample room for the kids in back along with the DVD Player for long trips. I enjoy the steeing wheel controls for the radio. This car handles well and is very solid! On trips we get close to 30 MPG!.
Used Cars in Louisville, TN 37777
Price: $2,950
Mileage: 193,854
Location: Louisville, TN 37777
Used Cars in Louisville, TN 37777
Price: $2,950
Mileage: 190,231
Location: Louisville, TN 37777
Used Cars in Hickory, NC 28602
Price: $3,500
Mileage: 81,202
Location: Hickory, NC 28602
Used Cars in Mooresville, NC 28115
Price: $3,788
Mileage: 178,815
Location: Mooresville, NC 28115
Used Cars in Warrensville, NC 28693
Price: $4,495
Mileage: 110,000
Location: Warrensville, NC 28693
Used Cars in Rural Retreat, VA 24368
Price: $4,995
Mileage: 115,520
Location: Rural Retreat, VA 24368
Used Cars in Shelby, NC 28151
Price: Negotiable
Mileage: 116,032
Location: Shelby, NC 28151
Used Cars in Asheville, NC 28806
Price: $8,995
Mileage: 120,225
Location: Asheville, NC 28806
Used Cars in Boiling Springs, SC 29316
Price: $7,500
Mileage: 165,122
Location: Boiling Springs, SC 29316
Used Cars in Spartanburg, SC 29303
Price: $8,995
Mileage: 99,499
Location: Spartanburg, SC 29303
Filter your search criteria!
A large number of listings matched your criteria. You are seeing the top 2,000 listings.
Used Chevrolet Malibu Maxx By Year
|Starting Price
|Total Available
|2007 Chevrolet Malibu Maxx
|5 Great Deals
|78 listings
|2006 Chevrolet Malibu Maxx
|1 Great Deal
|166 listings
|2005 Chevrolet Malibu Maxx
|230 listings
|2004 Chevrolet Malibu Maxx
|126 listings
Similar Cars
Chevrolet Malibu
1,169 Great Deals out of 72,682 listings starting at $695
Chevrolet Impala
949 Great Deals out of 35,738 listings starting at $700
Chevrolet Malibu Hybrid
1,891 listings starting at $20,228
Used Chevrolet Malibu Maxx By City
Chevrolet Malibu Maxx in Charleston WV
4 listings
Chevrolet Malibu Maxx in Charlotte NC
12 listings starting at $6,995
Chevrolet Malibu Maxx in Chattanooga TN
2 listings starting at $3,049
Chevrolet Malibu Maxx in Columbia SC
4 listings
Chevrolet Malibu Maxx in Greensboro NC
10 listings starting at $4,850
Chevrolet Malibu Maxx in Greenville SC
6 listings starting at $5,500
Chevrolet Malibu Maxx in Knoxville TN
8 listings
Chevrolet Malibu Maxx in Lexington KY
12 listings starting at $3,000
Chevrolet Malibu Maxx in Roanoke VA
4 listings


text:
Recruiting is never ending and is as unpredictable as ever for the University of Michigan.
In March, after falling victim to an NCAA tournament upset at the claws of the Ohio Bobcats, the Wolverines received some other setbacks in the form of three scholarship players announcing their plans to transfer away from Ann Arbor. To top it off the possibility arose that Co-Big Ten Freshman of The Year Trey Burke could be bolting for the NBA after a break out campaign.
What were the Wolverines to do?
The Michigan coaching staff kept their options open in the 2012 class and when it became apparent they would need to add a player or two to an already star studded bunch, the Wolverines quickly earned commitments from guards Caris LeVert and Spike Albrecht.
Many observers have long believed the class of 2013 has been wrapped up for quite as well. Meanwhile, the Michigan coaching staff has worked hard to keep options as evidenced by the ongoing courtship of Reggie Cameron and Vitto Brown. Their determination to keep open the 2013 posibilities became even clearer when another 2013 rose to the fore last weekend. Ontario SF Donnavan Hastings knocked down three after three after three for the Northern Kings, in a huge victory over 2014 Indiana forward and Michigan target, Trevon Bluiett.
"I saw the Michigan coaches sitting court side and that was an amazing feeling to see them really have interest in me," Hastings told GoBlueWolverine. "Seeing them there along side all the other coaches I think really gave my teammates and I a boost to perform and show them our talents."
"I think nobody really had hope in us that game but we were able to prove everyone wrong and give the Michigan coaches and others a show."
With several strong performances throughout the five day tournament, including the impressive showing in front of Michigan coaches, interest in the off-the-radar Hastings grew immensely over night.
"My coach was contacted by some schools that have been offering me and that have interest in me, but I have never spoke to any myself," said Hastings. "In that tournament I picked up New Mexico State, Tennessee Tech, St. Bonaventure and I'm being heavily recruited by Michigan."
At 6-6 and a slender 195 pounds, the sharp shooting Hastings has the size to play small forward but the quickness and scoring ability to slide over to the two as well. Versatility is a major priority for the Wolverines when evaluating talent and Hastings falls right into that category on the floor.
Although Hastings calls Canada home, the young man has a tremendous understanding of some of the Wolverines history and definitely sees the progress the program has made under Head Coach John Beilein.
"I know that the Wolverines have a great basketball program from their great success with the fab five in past history, and still have an outstanding program until this day with Coach Beilein leading them to success year after year," said Hastings of Michigan.
GoBlueWolverine will have more from Hastings head coach in the coming days.


text:
Join Now
Member Log in
Follow TradersCity
TradersCity Twitter
TradersCity Facebook
Trade Leads
Suppliers wanted
Agents wanted
Joint Ventures
Submit Quick Buy/Import Lead
Trade Services
B2B Directories
Products Category
Members Directory
Business Cards
Products Photos
B2B Videos
Country Targeted Leads
Trade Connections
B2B Marketplace Chat
Weekly Newsletter
Free Trade Alerts
Free B2B Magazines
Free B2B Magazines:
Weekly Trade News
Search Data
Contact Us
Privacy Policy
Terms & Conditions
Copyright Infringement Information (DMCA)
Contact TradersCity
Hello visitor from
visiting from
15 China
12 France
1 Japan
Sell Black Rice Pigment
Posted at: Offers to Sell and Export | Posted on: Wed 08 Jul, 2009 1:30 pm |
Rice Pigment Photos Catalog
Rice Pigment
Product Category
Rice Pigment
Photos Catalog
Rice Pigment
Business Cards
Rice Pigment
Members Directory
Rice Pigment
Rice Pigment
email alerts
Offers to Sell and Export
Free Business Magazines
Black Rice Pigment
Description:Cyanidin-3-glucoside(75%), methylcyanidinglucoside(13%), Cyanidin-3-rhamnoglucoside
are the main parts of the black rice pigment. We can see red or purple powder with our naked eyes. It is pure water-soluble. It is absolute a natural pigment abstracted from the episperm of murrey rice by modern biotechnology. Widely used in foodstuff, medicine, cosmetics industry etc as coloring(high-temperature coloring).
Action: in addition to the normal treatment can improve the antioxidative capacity and decrease some cardiovascular risk factors in CHD patients, which is probably related to the antioxidative and anti-inflammatory properties of anthocyanins rich in BRF.
Packing: 25kg/paper drum, Two sealed plastic-bags inside.
Company Contact:
Contact Name: Cherry
Company Name: Baoji Herbest Co., Ltd
Tel: 0086-917-3591456
Fax: 0086-917-3322701
Street Address: No. 1912 Meilun
Building , Huoju Road, Baoji City,
Shanxi Province, China
Member name: Herbest
Member Since: 15 April 2008
Total Leads:
10 Herbest Import Export Business Leads
Business focus: Herbal Extract, Green Tea Extract, Bilberry Extract
Safe Import Export Tips
Similar Suppliers And Manufacturers Import Export Trade Leads
Black Rice Pigment
- By cherrysaler - On Thu 16 Jun, 2011 9:02 am: rice by modern biotechnology widely used in foodstuff medicine cosmetics industry etc as coloring high-temperature coloring a blueberry anthocyan....
Sell Black Rice Pigment
- By Koralle1 - On Wed 28 Nov, 2007 5:52 am: rice pigment we can see red or purple powder with our naked eyes it is pure water-soluble it is absolute a natural pigment abstracted from the epispe....
Black Rice Pigment Anthocyanin
- By DSLHerbBio - On Sat 02 Jul, 2011 6:21 am: rice by modern biotechnology widely used in foodstuff medicine cosmetics industry etc as coloring high-temperature coloring ps we have other p....
Black Rice Pigment Anthocyanin
- By snowlotus1 - On Tue 09 Nov, 2010 1:50 am: rice by modern biotechnology widely used in foodstuff medicine cosmetics industry etc as coloring high-temperature coloring furthermore we have ....
Sell Black Glutinous Rice Pigment
- By Koralle1 - On Wed 28 Nov, 2007 5:51 am: rice pigment it is ethylether-insoluble it is absolute a natural pigment abstracted from o sativo l subsp hiesn amp #12288 hiesn ting by modern biot....
Thai Jasmine Rice / Thai Fragrant Rice / Thai Hom Mali Rice / Glutinuous Rice / Parboiled Rice
- By Majorthaiproducts - On Thu 21 Jun, 2007 6:08 pm: rice or thai hom mali rice we also export glutinuous rice parboiled rice white rice and others please feel free to contact us for quotation at ka....
Multifunctional Laser Machine For Skin Rejuvenation, Endogenous Pigment, Exgenous Pigment
- By BeijingStarlight - On Thu 10 Mar, 2011 6:05 am: pigment particles releasing them from their encapsulation and breaking them into fragments small enough for removal by the body these tiny particles....
Rice, Long Grain White Rice, Basmati, Yasmin, In A One Kilo Bag With Our Logo On
- By sonyko - On Sat 18 Jul, 2009 2:03 am: rice in a one kilo bag with our logo on and in a different size bags please offer us what you have in consumer pack pls offer your best export pr....
Import Export Startup Stories
Share Your Story & Get Listed
at StartImportExport.com
Onward International Co Ltd
: Naoko Takahashi Of Onward International Offering Shipping Services and Promoting Sale Of Agricultural Products From Thailand
Datta Global Traders
: Mukund Nambiar Of Datta Global Traders Exporters Of Food Products And Agro Commodities From India
KR Exports and Imports
: Raguram Of KR Exports and Imports From India
: Michael Wendt owner of Asiaservice operating in Thailand, Germany, and China markets
TradersCity.com shall not be held liable for any user posted/submitted content including but not limited to trade leads, profiles, images, and any other data. TradersCity.com does not and did not verify any of users posted/submitted data nor is implicitly or explicitly recommending these business offers. TradersCity does not verify truthfulness, accuracy, completeness, nor legality of any businesses, services, and leads posted here. TradersCity does not represent Sellers or Buyers in any transaction between users of the website and is unable to make any opinion in regard to their performance in any transaction. TradersCity neither guarantees nor undertakes in any dispute between sellers and buyers. Protect your business from fraud by
trading safely


text:
Test Your Vocabulary Skills - Places in a Town 2
In this ESL worksheet, students write the names of the given places in a town in their native language. They write the 20 original words in alphabetical order.
12th - Higher Ed English Language Arts 3 Views 7 Downloads
Resource Details
Start Your Free Trial
Save time and discover engaging curriculum for your classroom. Reviewed and rated by trusted, credentialed teachers.Try It Free
Figurative Language Packet
A definitive resource for your figurative language unit includes several worksheets and activities to reinforce writing skills. It addresses poetic elements such as simile and metaphor, personification, hyperbole, and idioms, and...
6th - 12th English Language Arts CCSS: Adaptable
Writing Portfolio: An Autobiography Assignment
An extensive portfolio project encourages your kids to reflect on their personalities with multiple writing assignments. From activities on extended metaphors to writing about an important day in their lives, kids explore the way they...
6th - 12th English Language Arts CCSS: Adaptable
501 Writing Prompts
Never again will you need to worry about coming up with a writing prompt! This packet contains, as it says, 501 prompts that are suitable for fourth graders on up. The prompts are paired into four categories (persuasive, expository,...
4th - 12th English Language Arts CCSS: Adaptable
Their Eyes Were Watching God: Figurative Language
Zora Neale Hurston's beautiful language in Their Eyes Were Watching God is not only a pleasure to read, it helps to further the plot and character development. Once your class has finished Chapter 6 of the novel, have them discuss...
9th - 12th English Language Arts CCSS: Adaptable
A Wizard of Earthsea: Figurative Language
Examine figurative language through the words of Ursula K. Le Guin in her novel A Wizard of Earthsea. The fifth lesson in a series of ten, the steps listed here ask pupils to go back into the text and find a striking sentence to analyze...
7th - 12th English Language Arts CCSS: Adaptable
What Is the Writing Process?
Use this packet to support a narrative writing assignment. The materials included in the packet center around a prompt for writing about a rite of passage. Each part of the writing process is described and demonstrated. After reading the...
7th - 12th English Language Arts CCSS: Designed
New Review Grade 12 ELA Module 4: Literary Analysis
Does identify come from within, or is it shaped by external forces? Explore the complex concept of identify with a two-unit module designed for 12th grade language arts. The first unit uses A Streetcar Named Desire by Tennessee Williams...
12th English Language Arts CCSS: Designed
Let's Go Exploring!
Use a Courbet painting of a cave or tunnel opening to reinforce the importance of descriptive writing. Writers of all ages use sensory details to describe what the scene depicts as they pretend to be in the painting. Then they imagine...
K - 12th English Language Arts CCSS: Adaptable
Picture This - Stars Over Hoke
The classroom becomes a safe and inclusive place for your ELLs as they create documents about their lives. Learners create, read, and present story books based on their own personal experiences. They use digital cameras to take...
5th - 12th English Language Arts CCSS: Adaptable
What Members Say
- Pamela M., Special Education Teacher
- Fremont, OH


text:
Quotation of Jack Canfield
View more random threads:
Look we play the star .
Bad Minute
you may like this.........
Afraid of making mistakes
Don't waste your time
The smaller the company the better the share.
Surrender to Moment
Enjoying the little things
Be kind always
There are currently 1 users browsing this thread. (0 members and 1 guests)
View Tag Cloud
Forum Rules


text:
The Microsoft® Web Platform offers the right tools for the right task. Design, build, debug, and deploy websites withwholesale sterling bangles . And then test your sites in Windows® Internet Explorer® 10 using the built-in developer tools.
??Video from the city shows what appears to be a crocodile slithering down the city streets, as well as a hippopotamus standing around looking confused.wholesale sterling bangles Jan 16 The right conditions for learning Learning 9710 views Jay Cross, many moons ago, likened the job of a learning designer to that of a gardener. , via Wikimedia Commons)Much digital ink has been spread about how Republicans created Donald Trump and now are paying the price for it.
" Alexandra Keen, Operations Manager, Imago "Online exhibitors require internet services on their stand, which is an integral part of their exhibiting presence when interacting with our visitors promoting their business.wholesale sterling bangles ??First, news came out that plans to hold the "Great and Holy Council" of the world's Orthodox churches in Istanbul had been abandoned in favour of a venue in Crete. We know he is based in Cley and becasue of that he was happy to charge us for only a couple of hours of his time, something many other photographers were not open to.wholesale sterling bangles
??*Free To be eligible for a free account you simply have to place on of our banners on your website homepage. ??the not quite fabulous lead vocal sounds like a real person singing, not a studio veteran LikeLiked by 1 person Reply nafalmat said: December 6, 2014 at 9:43 am How can anyone only rate this as a 7. North Warwickshire is home to the historic Atherstone Ball Game, which has taken place every year on Shrove Tuesday since the 12th Century.wholesale sterling bangles


text:
Demi Lovato Shares Rare Kissing Photo With Wilmer Valderrama: "He Is Perfect"
Demi Lovato shared a rare PDA pic with boyfriend Wilmer Valderrama on Monday. She tweeted the shot of Valderrama kissing her cheek and wrote, "I never post personal pictures like this but f*ck.. He is perfect. I miss my @WValderrama." The shot predictably sent fans into a tizzy. See a larger version of the image below!
The two stars have been largely private about their on-and-off romance in recent years. In that time, Gossip Cop has repeatedly busted false rumors about Lovato and Valderrama being engaged. In recent months, however, the couple has been a little bit more open in their public affection.
Last January, Lovato posted a touching birthday tribute to her beau, calling him "the funniest, most inspiring, incredible man I know… You truly are a light to this world and to my life." In April, Lovato lashed out at The Daily Beast for ridiculing Valderrama in an article. The singer tweeted, "@thedailybeast such a classy 'news site' you are. Writing horrible things about people you know NOTHING about. Get a LIFE you ignorant f**ks."
Meanwhile, Valderrama shared his affection for Lovato in August on her birthday in August. He tweeted, "Wishing u happy birthday would never come close 2 all the inspiring things I wish 4 ur life Love u thru all @ddlovato." Check out the new pic of the couple below, and tell us what you think.


text:
Items: 0
Total amount: € 0,00
ISSUES AND ARTICLES MOST READ eTOC
Indexed/Abstracted in: EMBASE, PubMed/MEDLINE, Science Citation Index Expanded (SciSearch), Scopus
Impact Factor 0,877
Frequency: Bi-Monthly
Online ISSN 1827-1626
Romano G., Bianco F., Esposito P.
The treatment of faecal incontinence includes: the education of the patient, medical therapy, biofeedback and sphincteric exercises, surgical therapy. Conservative, non-surgical treatment is almost always the initial therapeutic approach, except in those cases in which an evident defect of the sphincter muscle is present. Surgical treatment has seen a noteworthy increase in the last fifteen years as a consequence of the development of new surgical techniques. These techniques include: external anal sphincter plasty, pelvic floor plasties, sacral neuromodulation, muscular transpositions with or without electrostimulation, artificial anal sphincter. These procedures may be employed as first or second level treatment depending on the type of pathology considered and its aetiology. The 1st results achieved by surgical treatment authorise us to believe that reconversion with artificial sphincter is a valid alternative to graciloplasty, notwithstanding the fact that its costs are higher. Attentive pre- operative assessment of patients is important. Patients must be strongly motivated and able to manage the new condition. Although further studies are necessary, the degree of satisfactory of the 1st patients operated is the best stimulus for pursuing the development of this technique.
language: Italian


text:
what kind of battery can I get for my 2002 nissan altima v6 that doesn't cost much as a temporary/
After an accident I had to buy a new battery for my car, someone offered to buy it for me and is now trying to get it back so I need something that will at least make my car work until I can buy the actual battery type. Is there anything cheap I can use for a few days?
1 Answer
Since it IS the Holiday Season, everyone is having sales on just about everything, including batteries at auto parts stores, like Pep Boys and Auto Zone, etc. I definitely don't recommend getting something cheap "for a few days", just to have to buy ANOTHER battery that's better when you have more money. Makes zero sense. See who has them on sale...
Your Answer
Looking for a Used Altima in your area?
CarGurus has 87,984 nationwide Altima listings starting at $1,195.
Search Nissan Altima Questions
Nissan Altima Experts
Tom Demyan
Reputation 1,760
Reputation 1,120
Cassie Sapphire
Reputation 530
Related Models For Sale
Used Nissan Maxima"
466 Great Deals out of 24,113 listings starting at $800
Used Honda Accord"
1,316 Great Deals out of 78,667 listings starting at $1,395
Used Toyota Camry"
1,733 Great Deals out of 98,538 listings starting at $895
Used Honda Civic"
1,183 Great Deals out of 58,594 listings starting at $1,295
Used Nissan Sentra"
907 Great Deals out of 49,791 listings starting at $500
Used Hyundai Sonata"
1,036 Great Deals out of 51,797 listings starting at $1,200
Used Toyota Corolla"
1,457 Great Deals out of 67,383 listings starting at $1,295
Used Chevrolet Malibu"
1,169 Great Deals out of 72,684 listings starting at $695
Used Kia Optima"
623 Great Deals out of 32,409 listings starting at $1,000
Used Chevrolet Impala"
949 Great Deals out of 35,743 listings starting at $700
Used Nissan Murano"
463 Great Deals out of 22,080 listings starting at $1,295
Used Cars For Sale
2017 Nissan Altima 2.5 S For Sale
5,786 listings starting at $15,999
2016 Nissan Altima 2.5 S For Sale
14 Great Deals out of 9,377 listings starting at $12,900
2015 Nissan Altima 2.5 S For Sale
401 Great Deals out of 6,928 listings starting at $9,899
2014 Nissan Altima 2.5 S For Sale
100 Great Deals out of 2,507 listings starting at $7,999
2013 Nissan Altima 2.5 S For Sale
107 Great Deals out of 2,421 listings starting at $4,980


text:
[Watch] Mayor of Rotterdam Tells Islamists to "F*** Off" and "Pack Your Bags And Leave" On Live Television
The mayor of an ethnically diverse European city has earned the praise of London mayor Boris Johnson after expressing his disapproval with fellow Muslims who failed to appreciate the freedoms enjoyed in the Western world, telling them to "pack your bags".
Mayor Aboutaleb said: "It is incomprehensible that you can turn against freedom… But if you don't like freedom, for heaven's sake pack your bags and leave."
"This is stupid, this so incomprehensible. Vanish from the Netherlands if you cannot find your place here. All those well-meaning Muslims here will now be stared at".
FOR ENTIRE ARTICLE CLICK LINKClick here for the Top 12 Moments in Jewish History...LET THE ADVENTURE BEGIN! »


text:
Let's log you in.
Don't have a StudySoup account? Create one here!
Create a StudySoup account
Be part of our community, it's free to join!
Already have a StudySoup account? Login here
PHIL 1313 Week 3 Notes (Plus Cheat Sheet!)
PHIL 1313 Week 3 Notes (Plus Cheat Sheet!) PHIL 1313-002
OK State
About this Document
Popular in Logic and Critical Thinking
Class Notes
Carly Pate
verified elite notetaker
Popular in PHIL-Philosophy
Class Notes
Dr. Alisha Osinski
verified elite notetaker
Class Notes
psyc 101
Krista de Vera
verified elite notetaker
Class Notes
Michelle Ngo
verified elite notetaker
Study Guide
Jacob Lynn
verified elite notetaker
This 9 page Class Notes was uploaded by Colleen Maher on Thursday January 21, 2016. The Class Notes belongs to PHIL 1313-002 at Oklahoma State University taught by Justin Rice in Spring 2016. Since its upload, it has received 13 views. For similar materials see Logic and Critical Thinking in PHIL-Philosophy at Oklahoma State University.
Popular in PHIL-Philosophy
Reviews for PHIL 1313 Week 3 Notes (Plus Cheat Sheet!)
Report this Material
What is Karma?
Karma is the currency of StudySoup.
You can buy or earn more Karma at anytime and redeem it for class notes, study guides, flashcards, and more!
Date Created: 01/21/16
Operator Name Compound Type Used to translate ~ Tilde Negation it is not the case that . dot Conjunction and; also; moreover v wedge disjunction or; unless ⊃ horseshoe conditional if…then…; only if <> (or triple bar) Triple bar biconditional if and only if NEGATION, CONJUNCTION, DISJUNCTION Not either A or B = ~(A v B) Either not A or not B = ~A v ~B Not both A and B = ~(A ● B) Both not A and not B = ~A ● ~ B Neither A nor B = ~(A v B) Either A or B is not ___ = ~A v ~B Not A, but B = ~A ● B Both A and B are not ___ = ~A V ~B NECESSARY AND SUFFICIENT CONDITIONS 1. A is a sufficient condition for B whenever the occurrence of A is all that is required for B. 2. A is a sufficient condition for B whenever B cannot occur without A. 3. Sufficient introduced the antecedent; Necessary introduces the consequent. TRUTH T T T CHARTS NEGATION T F F p ~p F T T T F F F T DISJUNCTION F T p q p v q CONJUNCTIONS: T T T BICONDITIONAL p q p <-> q p q p . q T F T T T T T T T F T T T F F T F F F F F F T F F T F F F F CONDITIONAL F F T p q p ⊃ q Truth Charts Hey guys! This looks really complicated, but I promise it's not. The truth charts will always look something like this: a b axb T T X F X F F X F T X The lowercase letters stand for statements, and the uppercase letters are for True or False. (The Xs are just placeholders) Let's start with negations: p ~p T F F T The truth table for negation will always look like this, so you don't have to worry. How you read it is "If p is true, ~p is false." You follow the letter down the chart. So, if ~p is true, p is false. Negations are simple like that. EX) Let's just say that it's true that I like cats (and it is). p = I like cats. (true) then we negate it: ~p = I do not like cats. (false (and super sad!)) Likewise, if it was true that I didn't like cats, it would be false to say I did. You follow me? CONJUNCTIONS: p q p . q T T T T F F F T F F F F For conjunctions, F IS A DISEASE! *gasp!* And it makes sense when you think about it. p = April has a three legged dog. (true) q = Andy has a threelegged dog. (Also true) p . q = April and Andy have a threelegged dog. (True to the max) See how this follows the first section of the table? But let's say April has a threelegged cat. Then p would be false, which would make the whole statement false. Easy, right? DISJUNCTION p q p v q T T T T F T F T T F F F As you can see, disjunction is a little different than conjunction. This is because when you say an "or" statement, you're saying "this could be OR this could be." They don't depend on each other to be true. p = Frodo will throw the ring in the volcano. q = Samwise will throw the ring in the volcano. p v q = Frodo or Samwise will throw the ring in the volcano. No matter which of the statements is true, that ring is still getting chucked into a volcano. The only way it wouldn't be is if BOTH statements were false. Then the whole thing would be false and they couldn't defeat Sauron. Just FYI, and exclusive disjunction means that ONLY ONE of the simple statement could be true. These usually have the word either in them EX) Bilbo is either eleventyseven or eleventyeight. CONDITIONAL p q p ⊃ q T T T T F F F T T F F T p = If Elinor marries Edward, (q = ) then Marianne marries Colonel Brandon. Both of these happen to be true, so the conditional is true. If Marianne had married Willoughby, then the antecedent wouldn't be satisfied and the conditional would be false. If Elinor had run away and become a nun, Marianne would still be married to Colonel Brandon, so the result would still be true. If neither p nor q is true, the result is still true. This is difficult to explain here, but if you read page 326 of the reading, it becomes clearer. Alan Rickman! </3 BICONDITIONAL p q p <-> q T T T T F F F T F F F T (The <> stands in for the triple bar) This one is very similar to the conditional, with the exception of the second to last row, which tells us that id the antecedent is false, the conclusion will be false because the conditions have not been met. Well guys, that was fun! I hope you understand it at least slightly now. Have fun on the quiz Friday!
Buy Material
Are you sure you want to buy this material for
25 Karma
BOOM! Enjoy Your Free Notes!
We've added these Notes to your profile, click here to view them now.
You're already Subscribed!
Looks like you've already subscribed to StudySoup, you won't need to purchase another subscription to get this material. To access this material simply click 'View Full Document'
Why people love StudySoup
"I was shooting for a perfect 4.0 GPA this semester. Having StudySoup as a study aid was critical to helping me achieve my goal...and I nailed it!"
"When you're taking detailed notes and trying to help everyone else out in the class, it really helps you learn and understand the material...plus I made $280 on my first study guide!"
"There's no way I would have passed my Organic Chemistry class this semester without the notes and study guides I got from StudySoup."
"Their 'Elite Notetakers' are making over $1,200/month in sales by creating high quality content that helps their classmates in a time of need."
Refund Policy
All subscriptions to StudySoup are paid in full at the time of subscribing. To change your credit card information or to cancel your subscription, go to "Edit Settings". All credit card information will be available there. If you should decide to cancel your subscription, it will continue to be valid until the next payment period, as all payments for the current period were made in advance. For special circumstances, please email email@example.com
StudySoup has more than 1 million course-specific study resources to help students study smarter. If you're having trouble finding what you're looking for, our customer support team can help you find what you need! Feel free to contact them here: firstname.lastname@example.com
Recurring Subscriptions: If you have canceled your recurring subscription on the day of renewal and have not downloaded any documents, you may request a refund by submitting an email to email@example.com
Satisfaction Guarantee: If you're not satisfied with your subscription, you can contact us for further help. Contact must be made within 3 business days of your subscription purchase and your refund request will be subject for review.
Please Note: Refunds can never be provided more than 30 days after the initial purchase date regardless of your activity on the site.


text:
Linz :Former No. 1 Ana Ivanovic was docked a game for taking too long in the toilet but still eased past Barbora Zahlavova Strycova of the Czech Republic 6-3, 6-2 to reach the Generali Ladies quarterfinals.
Second-seeded Daniela Hantuchova, who won the event in 2007 on her last appearance, also advanced after defeating Carla Suarez Navarro of Spain 6-4, 6-3. Hantuchova will face Swiss veteran Patty Schnyder, whom she beat in that 2007 final.


text:
Evacuation order issued as B.C. wildfire grows to 13,000 hectares
VANCOUVER – An evacuation order has been issued for anyone living in the region of the northeast half of Tetachuck Lake to the south side of the Chelaslie River in the Bulkley-Nechako area.
Evacuees are asked to call the Regional District of Bulkley-Nechako at 250-692-3195 or 250-692-9411 to be registered.
Everyone is required to leave the area immediately, taking with them any personal items that are within easy reach, all pets and to help out any neighbour who may need assistance.
This evacuation order has come into effect due to the Chelaslie Arm wildfire.
This fire is estimated to be 13,000 hectares in size and is located about seven kilometres south of the Chelaslie River.
The fire has forced the closure of Entiako Provincial Park.
It was started by lightning and has been growing since Tuesday, July 8.
There are currently 17 firefighters battling the blaze along with three helicopters and two air tankers.
There are currently 70 wildfires across B.C., with seven fires of note.
© 2014 Shaw Media
Editor's Picks
Why some voters will never abandon Donald Trump
Bombardier layoffs a symptom of Canada's failure to stay competitive, says expert
Dennis Oland granted new trial in case of father, Richard Oland's murder
Con man persuaded women to buy him luxury cars, claimed he was U.S. marshal: police
Justin Trudeau campaign promise


text:
The Classic Nintendo Test
Their result for The Classic Nintendo Test ...
Game Over! You scored ###%!
Their Analysis (Vertical line = Average)
They scored 83% on health, higher than 77% of your peers.
All possible test results
In Need of Play
Have you ever seen or heard of a Nintendo before? You can probably pick one of these baby's up on Ebay or a Pawn Shop or maybe someone's basement. Make sure you blow in it a few time and then it'll ... Read more
You know the basics. Read more
Mega Man
You know your Nintendo games enough to call yourself a Nintendo fan. Read more
Your legend was a success! You defeated Ganon and rescued the princess... now for another quest. Read more
Recent Results
FromTheFuture34 / m / Bisexual
_i_give_up_31 / m / Straight
TheFatUnicorn27 / f / Straight
hotspicycougar41 / f / Bisexual
Tests others are taking
The LONG Scientific Personality Test
qalden242630 / f / Straight
5440kev52 / m / Straight
KarpKing18 / m / Straight
Esha_marie260225 / f / Gay
The Commonly Confused Words Test
Ckclines28 / f / Straight
More tests we think you'll like
Taken 12271 times.
The Are you my type Test
So, to be honest, I am just bored and I wanna know if there is anyone out there that matches my criteria for the type of man I want. Yes, I do want a ...
Taken 7319 times.
The Adventuresome Test
In my opinion, being adventurous is one of the best things you can do for yourself. If you try new things every now and then, sure you could run into...
Taken 20641 times.
The Should You Teach High School Test
Find out if I think teaching high school is the right career for you! Keep in mind, it's only my personal opinion. This test will be based on me, basically.
Taken 2189 times.
The Megaman NES Test
Hi! And welcome to my Megaman NES Test. I will ask you questions about the Megaman NES saga! Some questions will be easy, others will be hard! Good l...
Taken 2136 times.
The Does 300 MYA Matter To Me Test
Hi! And welcome to my Does 300 MYA Matter To Me Test. MYA means Millions of Years Ago, so if you don't care, don't know, or don't want to humiliate yo...
Taken 242 times.
Test Bloque III [ITIL]
Bloque III - JL (2)
Taken 1758 times.
O teste das facções
Find out witch of Divergent faction you belong.
Taken 44816 times.
The Which Wife are You Test
This test is to show which kind of wife you are, or will be (if that is your thing). Remember to answer honestly, otherwise any future spouses out th...
Taken 10101 times.
Famous Movie Quotes Test
What is your knowledge of Famous Movie Quotes?
Taken 53234 times.
The dumbest Test
Hi! And welcome to my dumbest Test. I'll be using no logic and little knowledge to determine something.
Connect your existing OkCupid account
Sign in
Welcome Back!
Restore your account to continue meeting new people.
Reset your password
We'll email you a link to reset it.
Email sent!
An email is on its way to .
If you don't see it, try checking your Spam folder.
Enter Authentication Code
Sign in
Not so fast!
You need to complete your basic info on the mobile app before you can use the desktop site.


text:
Call Manager Low Heartbeat.
I have Call Manager 4.1 running on one publisher and two subscribers. I received the following alert from my publisher.
irCriticalTrap irSeverity=irCriticalSeverity irProblemID=100087 irConditionName=CM.HBcount Event=CM HeartBeat low on \134CM1PUB1-NY. Potential service restart in last 5 minutes.
I used the performance monitor to look at the call manager heartbeat of the server and compared the publisher with the other 2 subscribers.
It does show that its low. What should I do to resolve this and is there a need to be concerned.
The heartbeat stats were:
Pub 2697.000
sub1 24931.000
sub2 25484.000
Thanks in advance,


text:
David Symens -
About David Symens
David Symens and is interested in Electronics, Internet / IT.
Not the person you're looking for?
Find more results for David Symens
Find more results for David Symens
Quick Profile Summary
Name: David Symens
Link: http://www.salespider.com/p-2916371/david-symens
Link: http://www.salespider.com/p-2916371/david-symens
Explore using SaleSpider
David Symens's Business Connections
Find more information on the Company
Member Spotlight
Wesley Kincaid
Pro Travel Network
Business Developer
Business Spotlight
Integrity Home Health Care, Inc.


text:
NCTDialogicVoice ActiveX DLL
NCTDialogicVoice ActiveX DLL is a rapid application development for Dialogic voice boards. With help of NCTDialogicVoice ActiveX DLL, you can create a variety of CTI applications...
USPExpress Math Parser
USPExpress Math Parser is a component aimed to parse and evaluate scientific, engineering and math expressions on-the-fly. There are .NET and COM editions of USPExpress available.
SocketQ ActiveX Network Server SDK for 9
SocketQ is a high performance COM DLL designed exclusively for the purpose of creating high performance network server applications. As part of this design goal, SocketQ was built using multi-threaded overlapped I/O to acheive the highest possibl...


text:
5 Pharmacist Intern Jobs in Cape Town
Supervise and guide Pharmacist Assistants and interns. The Pharmacist will be responsible for the provision of medication and supplementary services to patients...from: PNet Jobs - 11 days ago
Registered Pharmacist Intern with SAPC. We have a number of positions available for a Pharmacist Intern to complete the Pharmacist Internship Programme, which...from: Clicks Group Limited - 26 days ago
To ensure competent and motivated employees by overseeing training and development of Pharmacist Assistants and Pharmacist Interns in line with SAPC...from: Clicks Group Limited - 26 days ago
Of pharmacist assistants and interns. As a Pharmacist. Pharmacist at one of their pharmacies in Kathu. Our client a national healthcare group is seeking to...from: Gumtree ZA - 10 days ago
Supervision of pharmacist assistants and interns. Previous experience as a Pharmacist. Our client a national healthcare group is seeking to employ a Pharmacist...from: Indeed - 10 days ago
jobs by


text:
Nitric oxide (NO) is synthesized froml-arginine by the Ca2+/calmodulin-sensitive endothelial NO synthase (NOS) isoform (eNOS). The present study assesses the role of Ca2+/calmodulin-dependent protein kinase II (CaMK II) in endothelium-dependent relaxation and NO synthesis. The effects of three CaMK II inhibitors were investigated in endothelium-intact aortic rings of normotensive rats. NO synthesis was assessed by a NO sensor and chemiluminescence in culture medium of cultured porcine aortic endothelial cells stimulated with the Ca2+ ionophore A23187 and thapsigargin. Rat aortic endothelial NOS activity was measured by the conversion ofl-[3H]arginine tol-[3H]citrulline. Three CaMK II inhibitors, polypeptide 281–302, KN-93, and lavendustin C, attenuated the endothelium-dependent relaxation of endothelium-intact rat aortic rings in response to acetylcholine, A23187, and thapsigargin. None of the CaMK II inhibitors affected the relaxation induced by NO donors. In a porcine aortic endothelial cell line, KN-93 decreased NO synthesis and caused a rightward shift of the concentration-response curves to A23187 and thapsigargin. In rat aortic endothelial cells, KN-93 significantly decreased bradykinin-induced eNOS activity. These results suggest that CaMK II was involved in NO synthesis as a result of Ca2+-dependent activation of eNOS.
- endothelial function
- nitric oxide
- protein phosphorylation
- signal transduction
- thapsigargin
in endothelial cells, stimulation of muscarinic M1 receptors by acetylcholine (ACh) results in the activation of phospholipase C-γ1, followed by a transient increase in the formation of inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] and diacylglycerol (2). The production of Ins(1,4,5)P3 is considered as an initial event leading to Ca2+ release from intracellular stores that precedes a steady or oscillating plateau phase resulting from a more prolonged transmembranous Ca2+ influx (16). A potential target of increased intracellular free Ca2+concentration ([Ca2+]i) is the endothelial nitric oxide (NO) synthase (NOS) isoform (eNOS). Ca2+-mediated activation of eNOS requires the ubiquitous Ca2+-binding protein calmodulin (49, 11) when calmodulin inhibitors do not influence the relaxant response to exogenous NO (42, 37, 50). Furthermore, the endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin, which induces an increase in [Ca2+]i, triggers NO-dependent relaxation in vascular tissue (32). Thus the increase in [Ca2+]i results in the reversible formation of the Ca2+/calmodulin complex, which binds to eNOS, stimulating its activity (19). Unstimulated endothelial cells continuously produce NO, suggesting that the intracellular Ca2+ level under resting conditions is sufficient for basal NO synthesis. In endothelial cells, the main signal transduction pathway of agonist-stimulated eNOS activation depends on Ca2+/calmodulin. However, NO synthesis can be, at least in part, regulated by serine/threonine kinases, including cAMP-dependent protein kinase (9, 7), protein kinase C (36,26), and protein kinase B/Akt (20, 14). Although eNOS contains consensus sequences for phosphorylation by serine/threonine kinases, its regulation by Ca2+-dependent and/or Ca2+-independent phosphorylation remains to be specified. Ca2+/calmodulin-dependent protein kinase II (CaMK II) is a ubiquitous Ca2+/calmodulin-dependent enzyme involved in various Ca2+-mediated mechanisms. Its relationship with neuronal NOS (nNOS) has been previously established by Nakane et al. (35). Although Deli et al. (13) showed that CaMK II was expressed in endothelial cells, the link between CaMK II and NO synthesis in the endothelium remains largely unknown. We hypothesized that CaMK II might modulate Ca2+-mobilizing agent-dependent eNOS activity. The role of CaMK II in rat aorta endothelium-dependent relaxation was investigated by pretreatment with CaMK II inhibitors. Furthermore, we studied the interaction between Ca2+-induced NO production and CaMK II-dependent phosphorylation by measuring NO release and eNOS activity in a cultured porcine aortic endothelial cell (PAEC) line and rat aortic endothelial cells pretreated with CaMK II inhibitors.
Reagents were from Sigma and RBI, distributed by Sigma (Saint-Quentin Fallavier, France), unless otherwise stated. Polypeptide 281–302 (P281–302) bound calmodulin and was a potent inhibitor (IC50 = 80 nM) of exogenous substrate phosphorylation (12). KN-93 was an inhibitor of CaMK II activation (K i = 0.37 μM) (45). Its related compound, KN-92, does not show any CaMK II inhibitory activity. Lavendustin C (Calbiochem; Meudon, France) is a potent inhibitor of CaMK II (IC50 = 200 nM). Lavendustin C has noncompetitive inhibitory action on the tyrosine kinase ATP binding site and uncompetitive inhibitory action on the peptide binding site (3). P281–302 was dissolved in desoxygenated bidistilled water. KN-93, KN-92, and lavendustin C were dissolved in DMSO to prepare a stock solution of 0.01 M. Phenylephrine hydrochloride (α1-adrenoreceptor agonist), acetylcholine chloride (muscarinic agonist), Ca2+ ionophore A23187, sodium nitroprusside (NO donor), andN ω-nitro-l-arginine methyl ester hydrochloride (l-NAME; analog of arginine and competitive isozyme-nonselective NOS inhibitor) were made up in bidistilled deionized water. Thapsigargin (Calbiochem) was dissolved in DMSO. Each was made fresh daily and protected from light. All other drugs were dissolved in bidistilled deionized water except indomethacin, which was dissolved in ethanol. Further dilutions were made in Krebs solution for in vitro experiments. The drug solutions were prepared each day from dry powder.
Tissue preparation and tension measurement.
All animal procedures were applied in accordance with the European Directive for Animal Experiments 86/609 (Centre National de la Recherche Scientifique; Paris, France). At the time of experimentation, adult male Sprague-Dawley rats (251–275 g, Charles River; Saint-Aubin Les Elbeufs, France) were anesthetized with thiopental sodium (Nesdonal; 80 mg/kg ip) and heparinized (100 IU ic). A thoracotomy was performed, and the aorta was excised en bloc in cold Krebs balanced salt solution [composed of (in mM) 118 NaCl, 5.9 KCl, 1.2 MgSO4 · 7H2O, 1.2 NaH2PO4 · 2H2O, 2.5 CaCl2 · 2H2O, 25.5 NaHCO3, and 5.6 d-glucose] containing 1 mM indomethacin. The thoracic aorta of internal diameter 2.5 ± 0.2 mm was isolated clean of adherent fat and connective tissue and cut into rings of 3.0 mm in length. The vessel was gently handled to avoid stretching and endothelial damage. Two L-shaped stainless steel wires were inserted into the arterial lumen, and the rings were suspended in a 20-ml tissue bath. One stainless steel holder was attached to the chamber, and the other holder was attached to an isometric force-displacement transducer (Emka Technologie; Paris, France). The temperature of the organ chambers was kept constant at 37°C in Krebs buffer solution gassed with 95% O2-5% CO2(Air Liquide Santé; Paris, France). The rings were set at an initial resting tension of 1.5 g (the resting tension previously determined to be the optimal tension for length development in response to 60 mM KCl) and allowed to equilibrate for 60 min, the rings being repeatedly washed every 15 min. Verification of endothelium integrity was performed by testing the vascular relaxation produced in phenylephrine (0.1 μM)-precontracted rings by the endothelium-dependent vasodilator ACh (0.1 μM). In experiments with endothelium-denuded rat aortic rings, the endothelial cell layer was removed by rubbing the luminal surface of the vessel with a cotton swab. After equilibration, the vascular rings were submaximally precontracted with phenylephrine (1 μM), and a stepwise pharmacological sequence was initiated.
Characterization of the pharmacological responsiveness of aortic rings.
At the plateau phase, concentration-relaxation curves to ACh (0.001–10 μM), Ca2+ ionophore A23187 (0.01–100 μM), and thapsigargin (0.001–10 μM) were constructed by increasing the concentration in the organ chamber in cumulative increments after a steady-state response had been reached with each increment on precontracted rings. After the highest concentration of ACh, the rings were totally relaxed by sodium nitroprusside (10 μM). In preincubation experiments, CaMK II inhibitors were added to the bath at the plateau phase, and relaxation was proceeded after equilibration with the tissue for 15 min. Two concentrations of P281–302, KN-93, and lavendustin C were administrated, corresponding to 10−6 and 10−7 M in a 20-ml organ chamber for P281–302 and lavendustin C and to 10−5 and 10−6 M for KN-93. KN-93 and lavendustin C were also added at 10−5–10−3 M to assess their effect at high concentration in endothelium-denuded vascular rings. Controls were determined by the addition of vehicle (0.01% DMSO) or KN-92 (1 μM).
Endothelium integrity was assessed by postpharmacological challenge histology analysis. Tissues were harvested, fixed in 10% neutral buffered formaldehyde, embedded in paraffin, and sectioned. Histological sections (5 μm thick) were stained with hematoxylin and eosin (Sigma) and examined.
PAEC culture.
All the reagents used for cell culture were from GIBCO-BRL (Cergy-Pontoise, France) if not otherwise specified. The PAEC line established by Malassagne et al. (27) was a gift from Dr. Bernard Weill (Laboratoire d'Immunologie Biologique, Hôpital Cochin; Paris, France). PAECs were cultured in 25-cm2Primaria dishes (Polylabo; Paris, France) in RPMI 1640 medium-glutamax-1 supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, 50 μg/ml amphotericin B, and 10% fetal bovine serum. The cultures were incubated at 37°C in a humid atmosphere with 5% CO2. When cells reached confluence, they were detached by incubation with 0.05% trypsin-EDTA in RPMI 1640 medium-glutamax-1 for 3 min at 37°C, centrifuged at 300 g for 10 min, suspended in fresh complete medium, and further cultured under the same conditions. Cells were characterized as endothelial cells by their morphology, their ability to take up acetylated low-density lipoproteins (34), the detection of von Willebrand factor (41), and the expression of E-selectin (27).
Cell stimulation.
At least four sets of transfected cells (passages 29 and30) were tested. The cells were subcultured in 24-well flat-bottom culture plates (105 cells/well) during 24 h to reach confluence. The culture medium was removed, and the cells were washed once and equilibrated in isotonic phosphate buffer (pH 7.4) containing (in mM) 8 Na2HPO4, 1.5 KH2PO4, 137 NaCl, 2.7 KCl, 0.9 CaCl2,1 MgCl2, and 1 indomethacin oxygenated with 95% O2-5% CO2 (Air Liquide Santé). For real-time NO measurements, the sensor probe was inserted vertically into a well with confluent cells, the sensor membrane was positioned 50 μM above the monolayer by using a manual micromanipulator, and the well was sealed. To investigate the response to the agonists, the cells were stimulated with either A23187 (5 × 10−8–10−5 M) or thapsigargin (5 × 10−9–10−5 M). The effect of CaMK II was evaluated on the concentration-response curve for A23187 or thapsigargin of cells previously incubated with KN-93 during 30 min. For nitrate and nitrite (NOx) measurements, the cells were incubated with KN-93 (1 μM) or KN-92 (1 μM). After the cells were incubated for 30 min, they were stimulated by the Ca2+ionophore A23187 (0.1, 1, and 10 μM) or thapsigargin (0.1, 1, and 10 μM). The samples of effluent were then withdrawn from the culture well and immediately centrifuged at 300 g and 0°C for 10 min. The NOx concentration was determined in the supernatant by chemiluminescence.
Rat aortic endothelial cell culture.
The isolation of primary rat aortic cells was achieved according to the method of McGuire et al. (29). Cells were placed on a substrate including laminin and cultured in RPMI 1640 medium supplemented with 20% fetal calf serum. They were characterized by the detection of von Willebrand factor (41) and their ability to uptake acetylated low-density lipoproteins (34).
Cell stimulation.
Cells were subcultured in six-well flat-bottom culture plates (4 × 105 cells/well) and incubated with KN-93 (1 μM) before stimulation with bradykinin (0.1 mM). The cells were detached by incubation with 0.05% trypsin-EDTA, centrifuged at 300 gfor 10 min, and suspended in Tris · HCl (50 mM, pH 7.4) with EGTA (0.1 mM), EDTA (0.1 mM), leupeptin (1 μM), aprotinin (1 μM), and PMSF (1 μM) to measure eNOS activity.
NOx measurement.
The NOx content in the culture medium was determined by measuring NO based on a gas phase chemiluminescent reaction between NO and ozone with a NO analyzer (model 280, Sievers Intruments; Boulder, CO) (31). Nitrite and nitrate were reduced by vanadium and hydrochloric acid at 90°C. NO release in the headspace was purged from the solution by an inert gas for subsequent detection by chemiluminescence. Raw data were recorded with a NO analysis liquid program (Sievers Intruments), and the peaks were integrated. The sample concentrations were computed by using a previously determined calibration curve.
Electrochemical detection of NO.
S-nitroso-N-acetyl penicillamine (SNAP), CuCl, EDTA, and NaOH were obtained from Sigma-Aldrich and Fluka. All the solutions were dissolved in desoxygenated bidistilled water. Amperometric measurements of NO released were quantified with a Clark-type electrode (2 mm platinum disk NO sensor, Iso-NOP, World Precision Instruments; Stevenage, UK) connected to a Iso Mark II NO meter (World Precision Instruments). The newly developed electrode has a high selectivity for NO and a detection sensitivity of 1 nM. The principles of measurement have previously been described (44). NO diffusing through the gas-permeable membrane is oxidized at the working platinum electrode. The resulting redox current is proportional to the concentration of NO gas in the aqueous solution. The output current was recorded with the constant laboratory temperature kept constant. Calibration of the electrode was performed daily according to the procedure described by Zhang et al. (52). The NO sensor was immersed in saturated CuCl solution. After stabilization, a known volume of the SNAP solution (final concentration of 10, 20, 50, 100, and 200 nM) was then added, and the response was monitored. Measurements of NO were performed under constant stirring in glass vials sealed with a septum. Linear calibration curves were obtained from the resulting calibration plot.
eNOS activity.
NOS activity was measured by the conversion ofl-[3H]arginine tol-[3H]citrulline according to the methods described by Bredt et al. (8). Enzyme extract (25 μl) was incubated in the buffer [50 mM HEPES (pH 7.4), 0.5 mM NADPH, 5 μM FAD, 5 mM tetrahydrobiopterin, 1.25 mM CaCl2, and 10 μg calmodulin per ml] and 50 nMl-[3H]arginine. The enzymatic assay was terminated by the addition of 2 ml of ice-cold 20 mM HEPES (pH 5.5)-2 mM EDTA and was applied to 1-ml columns of Dowex-50W X8 (Bio-Rad).l-[3H]citrulline was eluted with 2 ml of deionized water and quantified by liquid scintillation spectroscopy.
CaMK II-α immunoblot analysis.
All reagents were from Bio-Rad (Marnes la coquette, France). Whole cell lysate was prepared from nonstimulated cells. Cells were detached as previously described and suspended in PBS. After centrifugation, harvested cells were homogenized in lysis buffer [50 mM Tris (pH 8.1), 1 mM EDTA, 0.2 mM sodium orthovanadate, 1 μM leupeptin and pepstatin, and 7 μM PMSF] and ultrasonicated. Whole cell lysates were added to SDS sample buffer [125 mM Tris (pH 6.8), 4% SDS, 10% glycerol, 0.01% bromophenol blue, and 2% β-mercaptoethanol], boiled for 5 min, and separated on a SDS-PAGE gel according to the method of Laemmli (25). After the migration, the proteins were transferred on nitrocellulose membranes in semidry transfer buffer [25 mM Tris (pH 7.5), 200 mM glycine, and 20% methanol]. The blots were blocked in 1% bovine serum albumin, 25 mM Tris (pH 7.5), 150 mM NaCl, and 0.05% Tween 20 and incubated with CaMK II antibodies (Transduction Laboratories; Lexington, UK) for 30 min at 37°C. After being washed, the membranes were incubated with horseradish peroxidase-conjugated anti-mouse IgG and developed according to the enhanced chemiluminescence immunodetection procedure (Amersham Pharmacia Biotech; Orsay, France).
Data analysis.
All responses to phenylephrine were expressed as force (in g). Responses to vasodilator agents were expressed as a percentage of maximal relaxation. In the endothelium-intact vessels, the effects of CaMK II inhibitors were measured on two rings for each aorta and the values were averaged. For all experiments, n is the number of rat aorta studies. Data are expressed as means ± SE ofn number of experiments. Results were analyzed using two-way ANOVA with repeated measures to compare the effects of inhibitors versus control with increasing concentrations of ACh. When theF-value for an effect was globally significant, comparisons were made using the Mann-Whitney U-test. The NOxconcentration was normalized for cell number, and the results were expressed as the content of NO release (in nM) per 105cells. The cell counts were obtained manually using a hemocytometer (Neubauer type), with viabilities determined by trypan blue dye exclusion. The cells were counted after experimentation. NOx measurements were realized twice and averaged, andn corresponds to the number of sets analyzed. Statistical evaluation of the difference between pretreated PAECs and the control was assessed with Student's two-tailed t-test. All statistical tests were considered significant for an α-level below 0.05.
Effects of CaMK II inhibitors on endothelium-dependent relaxation to ACh.
In precontracted endothelium-intact aortic rings, ACh (10−8–10−5 M) induced concentration-dependent relaxation (EC50 = 2.57 ± 0.13 × 10−7 M, n = 8). CaMK II inhibitors (1 μM P281–302, 1 μM KN-93, and 1 μM lavendustin C) shifted to the right the ACh concentration-relaxation curves (EC50 = 4.80 ± 0.24 × 10−7, 7.67 ± 0.31 × 10−7, and 5.98 ± 0.30 × 10−7 M, respectively, n = 5).
P281–302 (0.1 and 1 μM) partially reversed the ACh-induced relaxation by 14 ± 6% and 20 ± 4% for 0.1 and 1 μM, respectively, at an ACh concentration of 10−6 M and by 14 ± 6% and 19 ± 4% for 0.1 and 1 μM, respectively, at an ACh concentration of 10−5 M (P < 0.05,n = 6; Fig. 1).
Treatment of aortic rings with KN-93 (1 and 10 μM) decreased the ACh-induced relaxation by 23 ± 3% and 29 ± 3% for 1 and 10 μM, respectively, at an ACh concentration of 10−6 M and by 20 ± 2% and 28 ± 6% for 1 and 10 μM, respectively, at an ACh concentration of 10−5 M (P < 0.05, n = 5). Its inactive analog, KN-92 (1 μM), did not affect the ACh-induced relaxation (Fig.2).
Lavendustin C (0.1 and 1 μM) elicited a concentration-dependent reduction of ACh-induced relaxation of aortic rings precontracted with phenylephrine by 17 ± 9% and 28 ± 3% for 0.1 and 1 μM, respectively, at an ACh concentration of 10−6 M and by 14 ± 7% and 14 ± 5% for 0.1 and 1 μM, respectively, at an ACh concentration of 10−5 M (P < 0.05,n = 5; Fig. 3).
Vehicle (0.01% DMSO) had no effect on both phenylephrine-induced contraction and ACh-induced relaxation.
Effects of CaMK II inhibitors on endothelium-dependent relaxation to A23187 and thapsigargin.
In precontracted endothelium-intact aortic rings, A23187 (10−7–10−4 M) induced concentration-dependent relaxation (EC50 = 9.33 ± 0.23 × 10−7 M, n = 6). KN-93 (1 μM) shifted to the right the A23187 concentration-relaxation curves (EC50 = 6.46 ± 0.30 × 10−6 M,n = 5).
Treatment of aortic rings with KN-93 (1 μM) decreased the A23187-induced relaxation by 26 ± 5% at an A23187 concentration of 10−6 M and by 22 ± 3% at an A23187 concentration of 10−5 M (P < 0.05, n = 6). Its inactive analog, KN-92 (1 μM), did not affect the A23187-induced relaxation (Fig. 4).
In precontracted endothelium-intact aortic rings, thapsigargin (10−8–10−5 M) induced concentration-dependent relaxation (EC50 = 1.91 ± 0.30 × 10−7 M, n = 5). KN-93 (1 μM) shifted to the right the thapsigargin concentration-relaxation curves (EC50 = 2.45 ± 0.28 × 10−7 M, n = 5).
Treatment of aortic rings with KN-93 (1 μM) decreased the thapsigargin-induced relaxation by 22 ± 8% at a thapsigargin concentration of 10−6 M and by 13 ± 5% at a thapsigargin concentration of 10−5 M (P < 0.05, n = 6). Its inactive analog, KN-92 (1 μM), did not affect the thapsigargin-induced relaxation (Fig.5).
Effects of CaMK II inhibitors on exogenous NO-induced relaxation.
Sodium nitroprusside (10 μM) totally relaxed the aortic ring (98 ± 2%). Neither CaMK II inhibitors nor the inactive analog KN-92 at all concentrations have inhibitory action on sodium nitroprusside (10 μM) relaxation (data not shown). None of the three CaMK II inhibitors evoked spontaneously relaxation in endothelium-intact or -denuded vascular rings.
Effect of CaMK II inhibitors on endothelium-denuded and -intact aortic ring contraction.
In endothelium-denuded aortic rings, the three CaMK II inhibitors did not have an inhibitory action on phenylephrine (1 μM) contraction. In endothelium-intact aortic rings, both P281–302 (1 μM) and KN-93 (10 μM) did not alter phenylephrine (an α1-adrenoreceptor agonist) contraction. However, lavendustin C at a concentration of 10 μM induced an additional contraction of endothelium-intact aortic rings precontracted with phenylephrine by 18 ± 7% (P < 0.05,n = 5). The lavendustin C-induced increase in vascular tone was inhibited by l-NAME (10 μM).
Immunoblot characterization of CaMK II-α.
After SDS-PAGE and semidry transfer, membrane extracts of PAECs were revealed with anti-CaMK II isotype IgG1. IgG1bound to 52-kDa antigens. The same band was revealed on the control (rat brain lysate; Fig. 6).
Effect of the CaMK II inhibitor KN-93 on NO production.
In cultured endothelial cells, A23187 (5 × 10−8–10−5 M) and thapsigargin (5 × 10−9–10−5 M) induced NO release (EC50 = 9.12 × 10−7 and 7.24 × 10−7 M, respectively). Preincubation withl-NAME (10 μM) inhibited agonist-induced NO release. KN-93 (1 μM) shifted to the right the concentration-response curve of A23187- and thapsigargin-induced NO release (EC50 = 1.26 × 10−6 and 9.77 × 10−7 M, respectively; Fig. 7).
Decreased NOx concentration by KN-93 on cultured endothelial cells stimulated by A23187 or thapsigargin.
Stimulation of endothelial cells with the Ca2+ ionophore A23187 (1 and 10 μM) increased the NOx concentration in PAEC culture medium by 1,477 ± 51 and 2,075 ± 111 nM/105 cells, respectively. l-NAME (10 μM) inhibited the NOx production. The background NOx concentration from the isotonic phosphate buffer was 114 ± 20 nM/105 cells.
KN-93 (1 μM) reduced the NOx concentration in culture medium of PAECs stimulated with A23187 (1 and 10 μM) by 26 ± 5% and 10 ± 4%, respectively (P < 0.05,n = 4; Fig. 7 A). CaMK II inhibitor reduced the NOx concentration in the culture medium of PAECs stimulated with thapsigargin (1 and 10 μM) by 22 ± 4% and 17 ± 5%, respectively (P < 0.05,n = 4; Fig. 7 B).
Decreased rat aortic endothelial cell eNOS activity by KN-93.
Bradykinin-dependent eNOS activity as assessed by the conversion ofl-[3H]arginine tol-[3H]citrulline was reduced to 43 ± 3% by KN-93 (1 μM). Note that eNOS activity in rat aortic endothelial cells was totally inhibited by l-NAME (Fig.8).
Our results provide evidence that three different CaMK II inhibitors decreased endothelium-dependent relaxation elicited by ACh in normotensive rats. None of the CaMK II inhibitors inhibited relaxation induced by NO donors. KN-93 inhibited both receptor-independent and -dependent agonist-induced relaxation. The effects of CaMK II inhibitors were confirmed in both the PAEC line and rat aortic endothelial primary cultured cells. In the former, KN-93 significantly decreased the NO release in response to both the Ca2+ ionophore A23187 and thapsigargin. In the latter, KN-93 markedly reduced bradykinin-stimulated eNOS activity. Our results suggest that NO synthesis is dependent on CaMK II. The involvement of CaMK II in porcine endothelial NO production and rat NO-dependent relaxation characterized by the response to P281–302, KN-93, and lavendustin C on endothelium-dependent relaxation may be due to1) an interference with ACh-induced Ca2+release, 2) a direct effect on eNOS and/or calmodulin phosphorylation, and 3) an inhibition of NO-independent vascular smooth muscle relaxation.
To avoid bias due to nonspecific inhibitory effects of the compounds, our experiments were repeated with three structurally different CaMK II inhibitors. The synthetic polypeptide P281–309 contains the calmodulin-binding site (amino acids 290–309) and the autophosphorylation (Thr286) of CaMK II and therefore inhibits CaMK II by blocking Ca2+/calmodulin activation and the enzyme active site. KN-93 and lavendustin C inhibit CaMK II in a competitive fashion against calmodulin by decreasing the autophosphorylation of CaMK II. The effects of KN-93 on other CaMK isoforms remain unknown. CaMK II and eNOS represent two Ca2+/calmodulin-dependent enzymes. Their activation follows the agonist-induced increase in [Ca2+]i. Our results showing that CaMK II inhibitors decreased ACh-induced relaxation suggest that CaMK II inhibitors may alter the Ca2+ release induced by ACh. However, a specific interference at the level of the muscarinic M1 receptors only may be excluded because KN-93 decreased receptor-independent Ca2+ ionophore A23187-induced rat aorta relaxation, and, in PAECs, the [Ca2+]i increase-dependent eNOS activation elicited by A23187 was attenuated by pretreatment with CaMK II inhibitor. These findings are consistent with previous results showing that, in endothelial cells, KN-93 and lavendustin C did not modify basal [Ca2+]i, unlike the calmodulin antagonist-provoked dose-dependent increases in [Ca2+]i (48). Furthermore, our results with thapsigargin, which induced an increase in the [Ca2+]i by mobilization from Ins(1,4,5)P3 Ca2+ stores (15), suggest that CaMK II was not involved in endoplasmic reticulum Ca2+- ATPase activity and did not inhibit Ca2+ release from internal stores. In concert, our data support the hypothesis that CaMK II is involved downstream of the mobilization of intracellular Ca2+ stores directly on eNOS activation.
As with other NOS isoforms, eNOS might represent a direct protein substrate for CaMK II (43). However, Bredt et al. (7) reported no effect on soluble nNOS, whereas Nakane et al. (35) showed that CaMK II-induced phosphorylation of nNOS decreased its activity. Alternatively, Toda et al. (47) have shown that, in the cerebral artery, NO-mediated relaxation was attenuated by an inhibitor of CaMK II. This different behavior may be explained by the particular localization of eNOS in endothelial cells, where it is initially targeted to the membrane fraction (38) and subsequently translocated from the membrane to the soluble fraction after stimulation of the cells (30). This particular property of eNOS is partly due to the presence of NH2-terminal myristoylation and palmitoylation (10, 39). However, these posttranslational modifications are not sufficient for membrane localization, and phosphorylation of the enzyme is an alternative mechanism for the reversible association of the enzyme with membrane phospholipids (28). Alternatively, in cultured endothelial cells, agonist-induced eNOS phosphorylation increases its sensitivity to activation by Ca2+ (17). Thus pretreatment of endothelial cells with CaMK II inhibitors might either inhibit the translocation of eNOS or enhance the desensitization to Ca2+. Our results showing the decrease of eNOS activity in parallel of the reduction of NO-dependent relaxation suggest that CaMK II directly phosphorylates eNOS protein. This hypothesis was recently confirmed by Fleming et al. (18), who demonstrated that porcine eNOS activity depends on serine (Ser1177) eNOS phosphorylation by CaMK II. Their biochemical approach, together with our results, demonstrate the effect of CaMK II inhibitors on PAEC NO production and rat aorta endothelium-dependent relaxation after stimulation by Ca2+-mobilizing agents.
Our results with sodium nitroprusside suggest that CaMK II did not affect soluble guanylyl cyclase activity. These findings are consistent with those of Toda et al. (47), who showed that the response to exogenous NO was unaffected by CaMK II inhibitors, whereas the agonist-dependent NO-induced increase in cGMP concentration was reduced. Thus ACh-induced relaxation was attenuated by CaMK II inhibitors, indicating their roles in NO synthesis. Furthermore, CaMK II inhibitors did not spontaneously relax endothelium-denuded rat aortic rings, suggesting that an endothelium-insensitive effect may be ruled out.
CaMK II has been implicated in the regulation of tonic vascular smooth muscle contractility mediated by myosin light chain phosphorylation (1, 46, 40). However, the CaMK II inhibitor KN-93 did not impair the contraction in response to the α1-adrenoreceptor agonist phenylephrine (24). Our results with endothelium-denuded aortic rings confirm that vascular smooth muscle was insensitive to KN-93. However, a high concentration of lavendustin C slightly increased endothelium-intact contraction, suggesting that lavendustin C can increase vasoconstrictor tone by inhibiting basal NO activity. This hypothesis was confirmed by the absence of the lavendustin C contractile effect in the presence of l-NAME.
In conclusion, P281–302, KN-93, and lavendustin C, three structurally different CaMK II inhibitors, decreased the endothelium-dependent relaxation of isolated vessels and NO production by endothelial cells. These results suggest that, in addition to protein kinases A, B, and C, CaMK II was involved in NO synthesis. The agonist-induced [Ca2+]i increase leads to Ca2+/calmodulin-mediated eNOS activation but may also potentiate the NO synthesis by CaMK II-dependent phosphorylation of eNOS. Thus CaMK II modulates, at least in part, the activity and/or the intracellular localization of eNOS and may add another level of regulation in endothelium-dependent relaxation, which is of pharmacological interest in cardiovascular therapy.
The authors are grateful to S. Chouzenoux for technical assistance.
This work was supported by a grant from Air Liquide Santé.
Address for reprint requests and other correspondence: A. T. Dinh-Xuan, Service de Physiologie-Explorations Fonctionnelles, Hôpital Cochin, 27, Rue Du Faubourg Saint-Jacques, 75679 Paris cedex 14, France (E-mail:anh-tuan.dinh-xuan{at}cch.ap-hop-paris.fr).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published January 30, 2003;10.1152/ajpheart.00932.2001
- Copyright © 2003 the American Physiological Society


text:
When climate and ice sheet models of the past closely match other information, in this case sea-level data, climate scientists gain more precise tools for predicting future trends. "There's a lot of mysterious sea-level variability over the last 25 million years that is difficult to explain with Antarctic ice alone," DeConto says. "But if ice sheets and glaciers were present earlier in the Northern Hemisphere, as we think they might have been, they might provide the answer."
With future CO2 levels expected by the year 2100 to approach levels not seen in the last 25 million years, understanding past conditions is crucial for predicting possible implications for Earth's ice sheets and sea levels. "It's important that we get this right," DeConto says. "If we are correct, we are rewriting the history of the cryosphere over the past 34 million years and calling a lot of things into question. It's a challenge to geologists." The cryosphere is the planet's total amount of snow, ice and frozen ground.
The new model, accounting for atmospheric CO2 and changes in Earth's orbit around the sun among other variables, shows that the threshold of atmospheric CO2 at which large ice sheet development in the Northern Hemisphere is possible, is much lower than for Antarctica. The work, supported by the National Science Foundation, also suggests that climate, ice sheets and sea level may be far more sensitive to CO2 levels than generally accepted.
"The last time CO2 levels were as high as they are expected to reach in coming centuries, there was no big ice sheet on Antarctica because the planet was too warm," DeConto says. "This is not to say that we'll see the great East Antarctic Ice Sheet melt, because its large size and high elevations are self-sustaining. But it is alarming. We are trying to understand exactly what the effect of those high CO2 levels will be. It appears there will be an associated rise in sea level because much of the rest of the world's ice cover could be affected."
In addition to DeConto, the team includes climate researchers from Penn State University and Yale University in the United States and the University of Southampton and Cardiff University in Great Britain. Their paper published today is accompanied by an invited commentary by geologist Stephen Pekar of Queens College, New York, an expert on ancient sea level variation over the same period. He notes that DeConto and colleagues' results not only address the long-standing debate among geologists about the cause of ancient sea level fluctuations, but they are "relevant to today's discussions about climate change."
In an earlier paper, DeConto and colleagues had showed that global cooling which began about 34 million years ago during the "greenhouse to icehouse transition" was probably related to declining greenhouse gas levels and less to ocean currents around Antarctica as once believed.
Robert DeConto | Newswise Science News
Further information:
Further reports about: > Antarctic > Antarctic ice > Antarctica > CO2 > Climate change > Hemisphere > atmospheric CO2 > cryosphere > crystalline > ice sheet > sea level
Receding glaciers in Bolivia leave communities at risk
20.10.2016 | European Geosciences Union
UM researchers study vast carbon residue of ocean life
19.10.2016 | University of Miami Rosenstiel School of Marine & Atmospheric Science
Researchers from the Institute for Quantum Computing (IQC) at the University of Waterloo led the development of a new extensible wiring technique capable of controlling superconducting quantum bits, representing a significant step towards to the realization of a scalable quantum computer.
"The quantum socket is a wiring method that uses three-dimensional wires based on spring-loaded pins to address individual qubits," said Jeremy Béjanin, a PhD...
In a paper in Scientific Reports, a research team at Worcester Polytechnic Institute describes a novel light-activated phenomenon that could become the basis for applications as diverse as microscopic robotic grippers and more efficient solar cells.
A research team at Worcester Polytechnic Institute (WPI) has developed a revolutionary, light-activated semiconductor nanocomposite material that can be used...
By forcefully embedding two silicon atoms in a diamond matrix, Sandia researchers have demonstrated for the first time on a single chip all the components needed to create a quantum bridge to link quantum computers together.
"People have already built small quantum computers," says Sandia researcher Ryan Camacho. "Maybe the first useful one won't be a single giant quantum computer...
COMPAMED has become the leading international marketplace for suppliers of medical manufacturing. The trade fair, which takes place every November and is co-located to MEDICA in Dusseldorf, has been steadily growing over the past years and shows that medical technology remains a rapidly growing market.
In 2016, the joint pavilion by the IVAM Microtechnology Network, the Product Market "High-tech for Medical Devices", will be located in Hall 8a again and will...
'Ferroelectric' materials can switch between different states of electrical polarization in response to an external electric field. This flexibility means they show promise for many applications, for example in electronic devices and computer memory. Current ferroelectric materials are highly valued for their thermal and chemical stability and rapid electro-mechanical responses, but creating a material that is scalable down to the tiny sizes needed for technologies like silicon-based semiconductors (Si-based CMOS) has proven challenging.
Now, Hiroshi Funakubo and co-workers at the Tokyo Institute of Technology, in collaboration with researchers across Japan, have conducted experiments to...
#IC2S2: When Social Science meets Computer Science - GESIS will host the IC2S2 conference 2017
14.10.2016 | Event News
Agricultural Trade Developments and Potentials in Central Asia and the South Caucasus
14.10.2016 | Event News
World Health Summit – Day Three: A Call to Action
12.10.2016 | Event News
Resolving the mystery of preeclampsia
21.10.2016 | Health and Medicine
Stanford researchers create new special-purpose computer that may someday save us billions
21.10.2016 | Information Technology
From ancient fossils to future cars
21.10.2016 | Materials Sciences


text:
Is Ink All-You-Can incorrect or missing key business information? Update your Hotfrog listing today.
Contact details
Are these details correct?
Hotfrog Philippines provides information regarding Ink All-You-Can in Manila Metro Manila. Ink All-You-Can is located at 3/F, SM City San Lazaro, Francis P. Yuseco, Manila Metro Manila and provides requested URL services. Contact them on (02) 392 2918 or by visiting them on their website http://www.ink.com.ph/pages/index.php.
Is Ink All-You-Can in 3/F, SM City San Lazaro, Francis P. Yuseco, Manila Metro Manila your business? Claim your listing and attract more leads by adding more content, photos and other business details.
We have more requested URL services in Manila Metro Manila available on Hotfrog Philippines. You can update your search for Manila requested URL by location, keyword or service options.
Small Business Hub
Sharing tips, ideas and advice to help grow your small business.
Create your free Hotfrog business profile
Follow Hotfrog on:
Share on:
Hotfrog Small Business Hub


text:
This is an index of melodies Abc On Wiener Huma ideal that any of us tell and present for your requirements. Many of us obtain a lot of melodies Abc On Wiener Huma although we all only exhibit the tracks that people feel will be the finest tunes.
The particular music Abc On Wiener Huma is just with regard to test so if you like the song please buy the first mp3 format. Support the vocalist simply by purchasing the initial compact disk Abc On Wiener Huma hence the artist can provide the most beneficial song as well as keep on operating.


text:
Quick Reference
Name for a loose federation of seventeen clans in Scotland that claimed Pictish origin, mostly in Inverness, beginning in the 11th century. The clan takes its name from the title of the chief, Gilliechattan Mòr [Great Servant of St Catan], at Kilchattan Abbey, Isle of Bute.
Subjects: Religion.


text:
Six Signs You Made A Great Impact On xfinity Home Page
4 Stunning Examples Of Beautiful My Yahoo Mail
that permits users to store appointments and schedule meetings from a web based environment. Yahoo Messenger was originally released in 1998 yahoomail as an instantaneous messaging program that enabled Yahoo users to message friends in addition to share photos and also other media with friends.
Five Myths About My Paypal Account
What Is So Fascinating About Netflix Member Sign In?
How To Find Out Everything There Is To Know About Instagram Sign Up In Eight Simple Steps
Give Me 15 Minutes, I'll Give You The Truth About Msn.com Hotmail
Your Amazon Kindle device is linked for a Amazon. Other indications of unauthorized access include mail being deleted, deleted contacts, your set of safe senders being erased, your email signature being set to a spam message, your auto-response being set to some spam message as well as your junk mail being set to "Exclusive," preventing from receiving any mail.
Hotmail.com Log In Is Your Worst Enemy. Seven Ways To Defeat It
Hotmail can be a free web-based email service supplied by Microsoft. Add another email for password recovery, or click the hyperlink to set a security question instead. Hotmail is just one of the several online for free email providers that may be accessed from any computer with Internet access.
Top Www.gmail.com Login Choices
It seamlessly copies on the tags which has been a huge help for me. However, they abandoned that no less than a decade ago now. Polenta is thesubject of my most recentinfatuation. Finally, Mimecast's Chief Scientist, Nathaniel Borenstein and Cloud Strategist, Justin Pirie; and CEO of First Base Technologies ISACA, Peter Wood will host a webinar at 10 a.
What To Do About Login Facebook Before It's Too Late
Nine Simple Facts About E Trade Log On Explained
There are good things about swing trading on e - Trade, particularly should you're a lively trader. Although derivatives are riskier than individual stocks or bonds,. You will still pay a commission, but typical online investment fees less complicated less. The company supports trading in stocks, bonds, options.


text:
British govt. confirms debt pledge ahead of Scottish referendum
January 13, 2014 - 14:04 AMT
PanARMENIAN.Net - The British government confirmed on Monday, Jan 13, that it will take responsibility for all British government debt should Scotland vote for independence in September, a move it hopes will avoid jitters in bond markets ahead of the referendum, Reuters reports.
"In the event of Scottish independence from the United Kingdom, the continuing UK government would in all circumstances honor the contractual terms of the debt issued by the UK government," the Treasury said in a statement.
An independent Scotland would be responsible for "a fair and proportionate share" of Britain's liabilities but a share of the outstanding debt would not be transferred to Scotland, it said, adding the terms of repayment would be subject to negotiation.
Britain's net debt stood at nearly 1.2 trillion pounds at the end of the 2012-13 fiscal year.
Top stories
Ccampaign staffers have been discussing the legality of accepting donations from foreign governments, such as Azerbaijan.
Residents in Aleppo have said many won't go since there are no guarantees that the evacuees won't be arrested by government forces.
The spokeswoman for Kardashian West said the reality TV star was "badly shaken but physically unharmed" after being held up at gunpoint.
Michael Carpenter "hailed the level of cooperation in defense sector between Azerbaijan and the United States," Azeri media reported.
Partner news
Latest news
LG abandoning its modular phone strategy: report Instead ET reports LG will be "applying demands from its customers and markets rather than being buried in creating innovations."
NBC rebooting computer hacker movie "Sneakers" "Sneakers" arrives as USA Network has found success with hacker drama "Mr. Robot," which scored leading man Rami Malek an Emmy.
Momentum nabs Dakota Fanning, Guy Pearce's Western "Brimstone" The film tells the tale of a frontier woman who turns fugitive when she is wrongly accused of a crime she didn't commit.
Gorillaz tease their comeback with "The Book Of Murdoc" The animated band made their online return last month across their social media accounts.


text:
My service engine and service vehicle and trac lights are all on how do I turn them off and what if my ets button doesn't work
4 Answers
You will need to scan the car for the codes since the lights are on. You can only turn them off if you clear them with the code scanner, but if nothing is replaced or repaired they will come back on. A local auto parts will scan the car for you for free, post the 4 digit codes back here.
What will the code look like
Like the one you posted...http://www.engine-codes.com/P0128.html
1 out of 1 people think this is helpful.
Your Answer
Looking for a Used Grand Am in your area?
CarGurus has 1,960 nationwide Grand Am listings starting at $1,995.
Search Pontiac Grand Am Questions
Pontiac Grand Am Experts
Reputation 7,080
John Saffrahn
Reputation 1,480
Tom Demyan
Reputation 1,240
Related Models For Sale
Used Pontiac Grand Prix"
27 Great Deals out of 4,365 listings starting at $1,500
Used Chevrolet Impala"
879 Great Deals out of 35,448 listings starting at $795
Used Pontiac Firebird"
12 Great Deals out of 893 listings starting at $2,995
Used Ford Mustang"
723 Great Deals out of 41,162 listings starting at $900
Used Chevrolet Malibu"
1,085 Great Deals out of 71,910 listings starting at $1,300
Used Chevrolet Camaro"
798 Great Deals out of 39,007 listings starting at $2,650
Used Pontiac Bonneville"
867 listings starting at $2,995
Used Nissan Altima"
1,360 Great Deals out of 87,114 listings starting at $1,195
Used Honda Civic"
1,107 Great Deals out of 57,777 listings starting at $1,295
Used Chevrolet Cavalier"
1,602 listings
Used Honda Accord"
1,250 Great Deals out of 78,336 listings starting at $1,395
Used Cars For Sale
2005 Pontiac Grand Am SE For Sale
149 listings
2004 Pontiac Grand Am SE For Sale
89 listings
2003 Pontiac Grand Am SE For Sale
43 listings
2002 Pontiac Grand Am SE For Sale
13 listings