text:
To embed, copy and paste the code into your website or blog:
Brazil's long-debated "Internet Bill of Rights" has finally become law. The legislation, which passed the Brazilian Senate unanimously in April, is intended to secure equality of access to the Internet in Brazil—i.e., Net Neutrality—and provide privacy protections for Brazilian users of the Internet. Experts hailed the law "for balancing the rights and duties of users, government and corporations while ensuring the Internet continues to be an open and decentralized network."
The law, known as the Marco Civil da Internet or "Marco Civil" (in English, the Civil Internet Regulatory Framework) was first proposed in the Brazilian Congress in 2011, but received new significance in late 2013 after revelations that the U.S. National Security Agency had spied on the communications of persons across the world—including Brazilian President Dilma Rousseff. Rousseff signed the Marco Civil into law on April 23, 2014. The law goes into effect in July.
Commonly referred to in English as an "Internet Bill of Rights" or "Internet Constitution", Brazil's new Marco Civil provides for the freedom of expression and of content on the internet while also limiting the amount of metadata that can be gathered on Brazilian Internet users.
The legislation also includes broadly worded protections for privacy on the internet. Disclosure of personal data to third parties generally requires the informed consent of an Internet user, except under a valid court order.
Companies that collect personal data from residents of Brazil will be subject to Brazil's laws and courts in cases involving information on Brazilians, even if the data is stored on servers abroad. In order to ensure its passage, a controversial provision was dropped from the law that would have required companies to store data related to Brazilian Internet users on servers within country.
In addition, ISPs will be required to retain Internet access logs for one year.
The Marco Civil may not be the end of legislation related to the Internet and personal data protection for Brazil. The Brazilian Congress was also working on law to protect personal data protection bill at the same time that the Marco Civil was under consideration. The personal data protection bill was described as containing "an EU-style adequacy standard that would prohibit data transfers outside of Brazil unless the recipient country ensures an adequate level of data protection."
The Brazilian Congress was expected to take up this data protection bill after the Marco Civil was passed, though it is not clear whether the Marco Civil's final form has effectively tabled this issue.
DISCLAIMER: Because of the generality of this update, the information provided herein may not be applicable in all situations and should not be acted upon without specific legal advice based on particular situations.
© Davis Wright Tremaine LLP
Read fresh new writing on compliance, cybersecurity, Dodd-Frank, whistleblowers, social media, hiring & firing, patent reform, the NLRB, Obamacare, the SEC…
…or whatever matters the most to you. Follow authors, firms, and topics on JD Supra.
Create your news brief now - it's free and easy »
Davis Wright Tremaine is a national business and litigation law firm representing businesses based...
View Profile »
Sign up for your custom alerts now, using LinkedIn ›
Back to Top
Copyright ©


text:
Powerful Storms Leave Damage Behind In Allegheny County
Strong storms ripped through the area on Monday night, leaving some damage behind.
Man Arrested For String Of Allegheny Co. Robberies
Tarentum police arrested a man accused in a string of robberies in Allegheny County.
Crews Work To Rescue Elderly Man From Embankment
Emergency responders are working to rescue an elderly man who went over an embankment.
Verona Man Killed In Maryland I-70 Crash
Maryland state police say a 59-year-old man died after he ran his car across the median and into the wrong lanes of Interstate 70.
Animal Rescue League's Wildlife Center Gets Special Thank You
Spring is the busy season for Jill Argall, director of the Animal Rescue League Wildlife Center in Verona.
Firefighters Save Wedding, Prom Dresses From Verona Fire
Firefighters saved some very special dresses from the scene of an apartment fire in Verona.
Security Guard Headed To Trial After Shooting, Injuring Robbery Suspect
A security guard charged after chasing down and shooting a robbery suspect will go to trial.
2nd-Floor Bedroom Damaged In Verona Fire
A home in Verona was damaged by fire this morning.
Penn Hills Kindergarten Students Collecting For A Pajama Drive
With Thanksgiving less than a week away, a lot of organizations are gathering food and donations for those in need. But in Penn Hills, some students are collecting something else.
Verona Home Damaged By Fire
A home was scorched by flames in Verona overnight.
Young Fan Stuns Parents With His Penguins Knowledge
Four-year-old Macin DeFazio is both player and announcer as he scores another goal in his Verona basement. Like any play-by-play man worth his salt, he knows his players.
Woman Mourns Another Family Tragedy
A Verona man is behind bars after being accused of killing his own brother. The fatal shooting happened in East Oittsburgh this weekend and family members are devastated.
1 Fatally Shot In East Pittsburgh, Victim's Brother In Custody
Authorities are now identifying the victim in a deadly shooting this morning in East Pittsburgh.
Wounded Officer Grew Up In Verona, Described As A Very Private Person
Eight years with Pittsburgh Police and now the force is rallying around Officer Morgan Jenkins who was shot in the line of duty early Thursday morning.
Masked Man Robs Verona Dunkin' Donuts
Police are investigating a bold early morning robbery at a local Dunkin' Donuts shop.

subset: commoncrawl


text:
Similar Products:
Average rating from 5 reviews.
Average rating from 7 reviews.
Average rating from 7 reviews.
Editor's Best Lists:
Products We Are Testing:
Wide Range Thermometer
Average rating from 6 reviews.
(4.5 out of 5, based on 6 reviews)
Photosadd photo
Reader's Reviews (6)
Good thermomter for quick checks
posted 6 years ago
This stick-to-the-side-of-the-tank thermometer is a good one for the average aquarium owner. I like it because it can be placed where you will see it any time you walk past the tank. If there's a change, you'll see it if you are paying attention. I've heard the floating kinds may be more accurate, but for me this is all I need.
Pros: easy to read, handy
Cons: none
0 of 0 people found this review helpful.
Did you?
Want to review a product, but not finding it? Just add it and review it!
Recent Product Reviews
November 15, 2014 | By Robyn
July 14, 2013 | By Xiaoxiao111
Pink is my favorite color these are wonderful.I will recommend a good website fou you: www.9footballshirt.org
Freshpet Select Roasted Meals for Cat
June 28, 2013 | By Kittymom314
I introduced this food to my two cats, one is a senior at 13 years and the other is about 7 years. Both females. The 7 year old is a new addition, recently adopted :) Because of their age differenc… more ›


text:
- Exchange: -
- Sector: Technology
- Industry: Hardware
- Sub-Industry: Electronics Components
DVS Korea Co Ltd
046400:KSTicker Delisted
Company Profile for DVS Korea Co Ltd (046400)
DVS Korea Co., Limited manufactures and markets digital video disk (DVD) loaders. The Company mainly exports its products to overseas markets.
Contact Information
DVS Korea Co Ltd
497-3 Sangha-dong
Yongin-si Giheung-gu
Gyeonggi, 451-833
Korea, Republic of (South)
Phone: 82-31-546-3000
Key Executives for DVS Korea Co Ltd (046400)
Quotes delayed, except where indicated otherwise. All prices in local currency. Time is ET.
Recommended Symbols:
- 1
- 2
- 3
- 4
- 5


text:
21st Theater Sustainment Command
|This article needs additional citations for verification. (January 2011)
|21st Theater Sustainment Command
21st Theater Sustainment Command Shoulder Sleeve Insignia
|United States
|U.S. Army
|Part of
|US Army Europe
|Kaiserslautern, Germany
|21st TSC
|First In Support
|Operation Iraqi Freedom
Operation Enduring Freedom
|Major General John O'Connor
The 21st Theater Sustainment Command (21st TSC) provides theater sustainment throughout EUCOM and AFRICOM Areas of Responsibility in support of USAREUR and 7th Army.[1] On order, deploys to support theater opening, distribution, and Reception, Staging, Onward Movement & enable Integration (RSO&I) functions. Be prepared to support Joint and Coalition forces.
The heritage of 21st Theater Sustainment Command began on 23 June 1965 with the activation of the 1st Support Brigade. The 21st TSC originated as a maintenance support headquarters under Seventh Army Support Command, the brigade's first home was Taylor Barracks, Mannheim, Germany.
After the United States military withdrawal from France in 1967 and the organization of the U.S. Theater Army Support Command, Europe, informally known as TASCOM, the brigade became the rear-area counterpart of the newly formed corps support commands. Assigned to TASCOM in 1969, 1st Support Brigade was responsible for Prepositioning of Materiel Configured to Unit Sets (POMCUS) and provided combat service support for U.S. units in the area west of the Rhein River.
In 1974, TASCOM was merged with Headquarters, United States Army Europe, and the missions of 1st Support Brigade were expanded to include base operations support for eight military communities as well as the management of regional area support. The brigade was upgraded to a general officer command and the headquarters moved to Panzer Kaserne, Kaiserslautern, Germany.
By mid-1976 the 1st Support Brigade's personnel strength was approximately 16,000 – the largest brigade in the U.S. Army. Consequently, the Department of the Army approved the elevation of the commander's position to major general and on 19 August 1976 the brigade was re-designated 21st Support Command. The command came to include the 47th Area Support Group with a major depot located at RAF Burtonwood, Lancashire, in the United Kingdom, stockpiling POMCUS equipment.[2] Other units included Combat Equipment Group Europe, 29th Area Support Group, and 60th Ordnance Group.
During the next several years, important missions and units were assigned to the 21st and, in June 1981, the President of the United States elevated the commander's position to lieutenant general, reflecting the organization's status as a Theater Army Area Command. Accordingly, on 18 October 1988, 21st Support Command was officially re-designated the 21st Theater Army Area Command.
After the dissolution of the Soviet Union, U.S. forces in Europe began a reduction in personnel to accompany the reduced threat. Authorizations for both military and civilian positions were decreased substantially and, in 1993, the grade of the 21st TAACOM commander was returned to major general.
On 18 October 2000 the 21st TAACOM structure was replaced by the Army and U.S. Army Europe and transformed into the 21st Theater Sustainment Command (TSC). In 2006, the 21st TSC HQ further changed by absorbing some functions of some of its former units, such as the 200th MMC (Material Management) and 1st TMCA (Transportation Movement Control) because these units deactivated. This restructuring was needed to meet the needs of the Army and the nation in the 21st century, by becoming a deploy able force. This change made the 21st the Army's largest forward deployed unit and a major forward presence, in theater logistics organization in support of Central Region, Europe.
Throughout this evolution the 21st has retained the lineage and honors, distinguishing flag (colors), and distinctive unit badge and shoulder sleeve insignia of the "FIRST IN SUPPORT!"
Units and Command Group c.2012[edit]
Subordinate Units[edit]
- 21st Sustainment Command (Theater)
Command Group[edit]
- Major General MG John R. O'Connor
- Command Sergeant Major CSM Rodney J. Rhoades
- Deputy Commanding General BG Paul M. Benenati
- Deputy Commanding Officer COL Michael Snyder
- Chief of Staff COL Redding
- Deputy Chief of Staff Mr. Daniel Vargas


text:
Silver and cream accents combined with sculpted elements make this four-arm chandelier unique. The shades also feature a gray and white crackle effect finish to complement the accents.
Looking for stunning elegance? The silver distressing on this six-arm chandelier embodies a subtle design adorned with crystal that will bring a palace-like feel to any room.
Organic design inspired by the delicate beauty of a thriving garden Traditional style of decorative lighting Earthy Rose Gold finish adds subtle brilliance to the natural theme Faceted crystal beads and leaves are amber-tinted and glisten when lit UL listed Damp location for use in bathroom Swirled Mist glass emits a dawn-like glow Provides a well diffused light over a vanity or mirror for grooming 6 Feet of chain and 10 Feet of wire are ...
Organic branches sculpted from steel combine with crystal to create a forest canopy Cascading crystals are used to romantically diffuse the light Faceted crystal beads and leaves are amber-tinted and glisten when lit Mystic Gold finish with layers of antiquing Bulb Included A chandelier creates a stylish focal point Dramatically sized for prominent living and dining rooms or lobbies 6.875 Inch canopy included Cable: 4 x 8' of chain and 2 x 10 Feet of ...
Graceful sweeps are draped with crystal-clear glass beads Sheer, hand-wrapped Pearl Chiffon shades add elegance Golden Aura finish layers soft gold over silver undertones Shades are optional on chandeliers Graciously sized for taller dining and living areas Includes metal candlesticks, Pearl Chiffon Shade Bathroom light fixtures feature Opal glass, providing a pure white light A chandelier creates a stylish focal point 5.5 Inch Diameter canopy included 6 Feet of chain and 10 Feet of wire ...
Montego Bay Silver-Gold Twelve-Light Chandelier
Belluno Silver-Gold Five-Light Chandelier
Belluno Silver-Gold Twelve-Light Chandelier
Dress your room fabulously with this petite chandelier. It is remarkably styled with elegant, draping crystal and a lighted center bowl to create a divine glow. The new tortoise shell finish and its silver gold accents complement the crystal details beautifully. Cream drip candle covers and chic bobeches complete the piece with grace. An exquisite choice for a smaller space, this piece is one to cherish. Please note that it includes 10' of chain.
Give your room a romantic makeover with this darling mini chandelier. It is an impressive showcase of fine details with impressive crystal accents that glimmer against the new tortoise shell finish. This deep finish is enhanced by silver gold hues, and cream drip candle covers lend additional charm. You will instantly fall for this sparkling work of art. Please note that it includes 10' of chain.


text:
Instant "Members Only" Coupon Codes Emailed NOW When You Sign Up for FREE Membership!
You are already an existing user.
Do you want to opt back in?
Please check your email inbox for a confirmation message.
Just click the link to complete your subscription and receive your coupon codes.
Popular Destinations
- Mexico
- Caribbean
- Las Vegas
- Orlando
- Hawaii
- Florida
- New York City
- Costa Rica
- New Orleans
- San Francisco
Fairfield Inn & Suites Kenner New Orleans Airport
With a stay at Fairfield Inn & Suites Kenner New Orleans Airport in Kenner (Kenner - Louis Armstrong International Airport), you'll be convenient to Esplanade Mall and Susan Park. This hotel is within close proximity of Pontchartrain Center and Treasure Chest Casino. . Read More About Fairfield Inn & Suites Kenner New Orleans Airport
Fairfield Inn & Suites Kenner New Orleans Airport Deals & Specials
- 24-Hour Front Desk
- Complimentary Coffee
- Conference Rooms
- Dry Cleaning Service
- Elevator / Lift
- Express Check-Out
- Fitness Facility
- Free Airport Shuttle
- Free Breakfast
- Free Local Calls
- Jacuzzi/Hot Tub
- Laundry Services/Laundry Facility
- Multilingual Staff
- Outdoor Pool
(+) = Additional fees may apply for the use of these amenities.
(*) = Free, some restrictions may apply.


text:
Gov. Buddy Roemer (R) @BuddyRoemer
Politwoops no longer follows this account.Forbes: "Buddy Roemer and the Possibility of Political Reform" http://t.co/MgCfcBg4 #ows #p2 #tcot #decision2012
An archive of the public statements deleted by U.S. politicians. Explore the tweets they would prefer you couldn't see.
To meet the Twitter API Terms of Service, all deleted tweets shown here since June 22, 2012 have been reviewed and approved by the Sunlight Foundation.


text:
Department > Accessories > Men's Accessories > Backpacks & Bags >
Herschel Supply Little America Backpack
Available Colors:Navy
Additional Views:
Availability: In Stock
Notify Me! The product you are searching for is out of stock. If you wish us to notify you when the product is in stock please submit your name and email.
Little Ameri
© 2000-2012 ASLU LLC. all rights reserved


text:
Obama warns Wall Street not to block tighter regs
He said, "We must be wary of the reality that – in an attempt to address yesterday's failures – Congress will put in place regulatory schemes which will fundamentally undermine risk taking."
Anticipating such criticism, Obama shot back against those pushing for less regulation.
"Do you really believe that the absence of sound regulation one year ago was good for the financial system?" he said. "Do you believe the resulting decline in markets and wealth and unemployment, the wrenching hardship that families are going through all across the country, was somehow good for our economy?"
He also told Wall Street that it had no need to wait for new laws to begin helping consumers with straight talk in the meantime.
Much of Obama's speech amounted to a recap of his proposals, first outlined in June.
He has sought tougher capital requirements for banks, arguing that banks' buying of exotic financial products without keeping enough cash in reserve was a key cause of the crisis. He wants more openness for the markets in which banks trade the most complex products.
Obama's plan also would give the Federal Reserve new oversight powers and impose conditions designed to discourage companies from getting too big. And he proposes a consumer protection agency to make rules for financial products, so people know what they're buying.
The House Financial Services Committee, led by Rep. Barney Frank, D-Mass., who supports much of Obama's plan, is expected in October to take up the first piece of the legislation, one that would establish an agency focused on consumer protections. The panel has already passed legislation intended to curb excessive compensation at financial institutions.
Obama's plan could face significant revisions in the Senate, where Democrats have joined Republicans in questioning whether more power should be given to the Federal Reserve.
Industry is working particularly hard to kill or at least weaken the consumer protection agency idea, which it says will lead to increased costs for consumers, and various corporate interests are fighting the new rules for complex financial transactions, arguing they could stifle legitimate commerce.
Meanwhile, Obama made sure to dole out credit Monday – to his own administration.
He introduced his economic team and said its "outstanding leadership" of a financial stability plan helped the system emerge from crisis.
Obama warned against complacency but said: "We can be confident that the storms of the past two years are beginning to break."
Associated Press writers Anne Flaherty and Julie Hirschfeld Davis contributed to this story from Washington.


text:
Step 1: Preparation
STONES We built this fire pit from landscaping blocks. You can use field stone or other materials too. Do not use stones that have been submerged in water; they can explode with the heat of the fire. Concrete blocks may deteriorate from the heat, but they are cheap to replace.
DRAINAGE In the bottom center of the pit, we dug a fencepost-sized hole 2 feet deep and filled it with gravel. The hole works like a sump, helping to drain rainwater.
ADHESIVES We dry-stacked the stone. It's a quicker way to build the fire pit. If you have to replace cracked or broken stones, dry-stacking makes that job easier as well. If you want to cement the courses, lay cement down only on the outside half of the stones to protect the cement from the heat. Adhesives may melt and give off fumes; we advise against using them.
SAFETY This fire pit is built in a wooded area. Before we started the fire, we soaked the area around the pit with water. We also had 5-gallon buckets of water and a shovel handy to put out any stray fires.
What You'll Need
98 retaining wall blocks
steel pit ring with tabs
metal grate
We bought the ring and grate as specialty items from a garden store. We couldn't find a place to order these pieces from on the Internet, so we'd suggest welding your own or having them produced at a welding shop.
The retaining wall blocks used in this project were 12 inches wide, 4 inches high and 8 inches deep.
We purchased about one-half ton each of sand and gravel.
Total Cost: about $500
In my experience, it's very important to include ventilation
holes when constructing a paving
brick fire pit. Otherwise, it can be a challenge providing sufficient
oxygen to the flame, which makes it hard to get a fire started.
Hi there,
This is a pretty good design and build. I am pleased to see the drainage included, as many people tend to skip this stage. However, I prefer to have a 6" deep layer of gravel over the whole area of the bottom of the fire pit. 1. Its easier than digging a two foot deep hole. 2. It works perfectly for drainage, as it is a large surface area. 3. It forms an ideal base on which to set your fire.
For those people who have commented that they could not find a fire pit liner online, you could visit
As an alternative you can also use the same fire pit liner to make an in-ground fire pit. You can find instructions for how to do it at
We used a washing machine bowl and attached it to an old mowed frame so we could move it around,also put in storage when not needed.
Fire is fun :D


text:
Average rating from 3 reviews.
More Products By Hoegger Supply:
Average rating from 6 reviews.
Average rating from 5 reviews.
Average rating from 4 reviews.
Similar Products:
Average rating from 7 reviews.
Average rating from 35 reviews.
Average rating from 20 reviews.
Products We Are Testing:
Readers' Reviews add review
2 reviews
- Sort by:
- Most Helpful |
- Date
Self explanatory
posted 6 years ago
this method has also used for puppies tails and i guess still is used. it is an effective and cheap for od castration and docking tails. as with anyting of this type of non surgacial you must still keep an eye on it
Pros: does the job
Cons: must keep an eye on
0 of 0 people found this review helpful.
Did you?
No cutting castration
posted 6 years ago
This tool is used to slip a rubber ring over thew scrotum of baby goats and lambs to castrate. There is no cutting involved. The blood supply is stopped so the teticles dry up and fall off.
Pros: Easy to use
Cons: none
0 of 0 people found this review helpful.
Did you?
Want to review a product, but not finding it? Just add it and review it!
Recent Product Reviews
November 15, 2014 | By Robyn
July 14, 2013 | By Xiaoxiao111
Pink is my favorite color these are wonderful.I will recommend a good website fou you: www.9footballshirt.org
Freshpet Select Roasted Meals for Cat
June 28, 2013 | By Kittymom314
I introduced this food to my two cats, one is a senior at 13 years and the other is about 7 years. Both females. The 7 year old is a new addition, recently adopted :) Because of their age differenc… more ›


text:
Comment: How long is your commute? (Score 1) 577
by Andrew Tanenbaum (#20716209) Attached to: What To Do When Broadband is Not An Option?
I bet it's pretty long. When you work in the city, it's pretty damned irresponsible to live far outside of it. You're like a suburbanite, only worse.


text:
WHO: Ebola Death Toll Reaches 932
Posted: 08/06/2014 - The World Health Organization says the death toll from the Ebola outbreak in West Africa has risen to 932.
Posted: 08/06/2014 - The World Health Organization says the death toll from the Ebola outbreak in West Africa has risen to 932.
Posted: 08/05/2014 - Nevada started taking in applications for medical marijuana establishments in Carson City Tuesday.
Posted: 08/05/2014 - Washoe County Health District monitors have detected unhealthy levels of fine particulates (PM2.5) in Reno and Sparks.
Posted: 08/05/2014 - Lagos state health commissioner Jide Idris says seven people have Ebola symptoms and are in quarantine after primary contact with the man who flew to Lagos and died of the disease last month.
Updated: 08/05/2014 - The second American aid worker who was recently diagnosed with Ebola in west Africa has arrived at an Atlanta hospital for treatment.
Posted: 08/04/2014 - Health officials say they've detected the first mosquitoes carrying West Nile virus so far this year in southern Nevada.
Posted: 08/04/2014 - California's highest court says people with Alzheimer's disease are not liable for injuries they may cause their paid in-home caregivers.
Posted: 08/04/2014 - The Liberia airport authority says a chartered evacuation plane that will take home the second American sick with Ebola has landed at the airport in the capital.
Updated: 07/31/2014 - With the first day of school just about a week away, a lot of parents are scrambling to get everything ready. But before kids are sent off to school, there may be one very important thing parents need to add to their to-do list.
Updated: 07/28/2014 - GoMacro is recalling some of its Macrobars because they could be contaminated with salmonella.
Posted: 07/24/2014 - Prosecutors in Central California have obtained an arrest warrant for a tuberculosis patient who they say is contagious and has refused treatment, putting those around him at risk.
Updated: 07/22/2014 - San Francisco voters will be asked this fall to approve a 2-cent-per-ounce tax on sugary drinks sold in the city.
Posted: 07/22/2014 - Medical marijuana advocates in San Jose have failed in its petition drive to let voters decide whether to overturn the city's tough restrictions on pot clubs.
Posted: 07/21/2014 - A "rogue" gynecologist's secret use of tiny cameras to record hundreds of videos and photos of his patients' sex organs has led to a $190 million settlement with some 8,000 women and girls, lawyers said Monday.
Updated: 07/21/2014 - Several varieties of fruit sold at Costco, Trader Joe's and Sam's Club are being recalled because they may be contaminated with listeria.
Posted: 07/21/2014 - A new Reno business offers a simple solution for building muscle strength and bone density.
Posted: 07/18/2014 - The Food and Drug Administration is warning consumers to avoid pure powdered pure caffeine sold on the Internet after the death of an Ohio teen.
Posted: 07/17/2014 - Federal authorities have charged FedEx Corp. with assisting illegal pharmacies by knowingly delivering dangerous drugs to customers without prescriptions.
Updated: 08/19/2014 - To help prepare children for the upcoming school year, Immunize Nevada is holding several clinics during the month of August.
Posted: 07/17/2014 - New details from two studies reveal more side effects from niacin, a drug that many Americans take for cholesterol problems and general heart health. Some doctors say the drug now seems too risky for routine use.
Posted: 07/16/2014 - Renown Health continues to improve its robotic surgery technology, providing major benefits for both doctors and patients.
Updated: 08/19/2014 - People can buy or sell bikes at Idlewild Park while helping raise awareness about one of the deadliest forms of cancer.
Updated: 07/15/2014 - The life of a three-year-old boy was apparently saved at Rock Park in Sparks Sunday when only one person in the sizeable crowd on hand knew CPR.
Updated: 07/15/2014 - A young child is in critical condition Monday night at Renown Medical Center after a near-drowning at Rock Park. The situation could be a lot more dire had it not been for a quick-thinking bystander who knows CPR. Garet Bartholomew talked to KOLO 8 News Now along the shore of the Truckee River where the incident happened.
Posted: 07/14/2014 - A 12-year-old Reno-area girl has won a trip to the White House by submitting a healthy recipe.


text:
T.ED is an application that can be used for the design of 3D outdoor worlds.
doing it! T.Ed is designed ... 'Dynamic/LOD'. T.Ed can help ... of both! T.ED is a complex
Work smarter with this intelligent, power packed programmer's editor and IDE.
its capabilities. ED's intelligent editing ... errors within ED. ED's regular expression
Vector CANeds is a tool for creating and testing EDS files.
and testing EDS files. This ... of creating EDS files manually
It enables you to convert different types of fax files to jpg/jpeg images.
Edmics-mmr ED, black-white


text:
HAZLETON — The ever-changing face of local health care is once again going under the knife after Wednesday's announcement that the Greater Hazleton Health Alliance will soon be part of the Lehigh Valley Health Network.
At an official announcement Wednesday at the hospital, James Edwards, president and chief executive officer of the Greater Hazleton Health Alliance, said "Both organizations value their people and realize that's where the care starts. That's what has made working together a natural fit and what makes merging a logical choice."
The announcement comes four years after a partnership between the two nonprofit health-care organizations was established and follows similar mergers, purchases and affiliations between other hospitals in the region:
* In 2012, Danville-based Geisinger Health System, which operates Geisinger Wyoming Valley in Plains Township and Geisinger South Wilkes-Barre hospitals in Luzerne County, acquired Community Hospital of Scranton.
* In recent years the for-profit Community Health System, of Franklin Tenn., has acquired eight hospitals in the region: Wilkes-Barre General, Tyler Hospital in Tunkhannock, Special Care Hospital in Nanticoke, First Hospital in Kingston and the former Mercy and Moses Taylor hospitals in Scranton, Mid-Valley Hospital in Peckville and Berwick Hospital.
Edwards said that the successful relationship between the two systems led to discussions about how to continually strengthen the relationship.
Benefits explained
"It's really a natural progression," Edwards said. "As we discussed how to move forward, it really made sense."
Dr. Ronald Swinfard, president and CEO of the Lehigh Valley Health Network, said the partnership benefits both systems and he sees the ability to expand offerings available to Hazleton patients. He said he hopes the Hazleton community will not view Lehigh Valley as an interloper.
"We're ready to do that with you, not to you," Swinfard said.
At separate meetings in April, the boards of both organizations signed a formal agreement to allow a full asset merger of the Greater Hazleton Health Alliance into Lehigh Valley Health Network. Brian Downs, a spokesman for the network , said that because the deal is for a full asset merger, there is no price tag attached because there is no sale being made.
Downs said the merger transaction must be reviewed and approved by the Federal Trade Commission, the state Attorney General's Office and the state Department of Health. Once all that is done, the merger date will be determined and so will additional details.
When the merger is complete, the 150-bed Hazleton General Hospital and the Hazleton Health and Wellness Center will remain separate entities under Lehigh Valley Health Network, officials said.
The board of directors will remain in place, but network-appointed members will have a majority of the seats. The Alliance Medical Group will become a separate division of the Lehigh Valley Physician Group.
Edwards said there are no plans for reductions in staff, though the opposite is possible.
"We would anticipate growth," Edwards said. "Growth means more jobs."
Thomas Kennedy, chairman of the Greater Hazleton Health Alliance Board of Directors, made the formal announcement and said the plans have been in the works for more than a year. He called the decision "absolutely the right thing to do."
William Hecht, the chairman of the Lehigh Valley Health Network Board of Directors, also praised the merger and called it "a giant first step in enhancing the health and well being in both communities."
Donna Palermo, the president of the Greater Hazleton Chamber of Commerce, called the proposed merger "a great benefit to our community and our citizens." "People who currently travel out of our area, mostly to Lehigh Valley, can now receive their care here locally," said Palermo who, along with Hazleton Mayor Joe Yannuzzi, was in the audience to hear the announcement.
Mergers, acquisitions and closures have become the norm in health care in recent years as population shift, rising costs and greater demand for certain services have caused many systems to make changes.
The Greater Hazleton Health Alliance, which employs more than 1,000, itself was among those at the forefront of health alliances for the greater good of a community. It was formed in 1996 with the consolidation of Hazleton's two hospitals, Hazleton General and Hazleton-St. Joseph.
The Lehigh Valley Health Network employs 12,000 and includes two full-service hospitals: Lehigh Valley Hospital-Cedar Crest in Salisbury Township — which includes an additional clinical campus, Lehigh Valley Hospital-17th Street in Allentown — and Lehigh Valley Hospital-Muhlenberg in Bethlehem. Lehigh Valley acquired the Muhlenberg hospital 15 years ago.
Pending regulatory approval, officials from the two systems said they plan to do a needs assessment to determine how the merged organization can meet the health care needs of the expanded Lehigh Valley Health Network community.
Until approval is given, both systems will continue operating as they have. The pending sale of the former St. Joseph's Hospital also will move forward, and Edwards said he believes that deal will close before the merger approval is granted.


text:
To embed, copy and paste the code into your website or blog:
Summary of special education law and practice in the State of Maine
Firefox recommends the PDF Plugin for Mac OS X for viewing PDF documents in your browser.
We can also show you Legal Updates using the Google Viewer; however, you will need to be logged into Google Docs to view them.
Please choose one of the above to proceed!
LOADING PDF: If there are any problems, click here to download the file.
DISCLAIMER: Because of the generality of this update, the information provided herein may not be applicable in all situations and should not be acted upon without specific legal advice based on particular situations.
© James Clifford, Law Office of James Clifford, LLC
Read fresh new writing on compliance, cybersecurity, Dodd-Frank, whistleblowers, social media, hiring & firing, patent reform, the NLRB, Obamacare, the SEC…
…or whatever matters the most to you. Follow authors, firms, and topics on JD Supra.
Create your news brief now - it's free and easy »
My practice focuses on employee and civil rights, representing individuals and students with...
View Profile »
See more »
Sign up for your custom alerts now, using LinkedIn ›
Back to Top
Copyright ©


text:
`Avatar' blasts off with $232.2 million worldwide
LOS ANGELES James Cameron launched his science-fiction epic "Avatar" into a safe orbit as the costly film soared to No. 1 with $73 million domestically and $159.2 million overseas, for a $232.2 million worldwide total.
With that big a start, distributor 20th Century Fox was quick to proclaim it made a good investment with the estimated $400 million spent to make and market the film, which is Cameron's first narrative feature since 1997's "Titanic," the king of modern blockbusters.
"Absolutely. No question," said Chris Aronson, head of distribution for the studio, which reported stellar reaction in exit polls from audiences after seeing "Avatar." "The word of mouth is something that I don't know I've ever seen in this business before."
"Avatar" was a test case for the future of digital 3-D projection, which until now has been a hit with audiences mainly on animated family films.
The film fell short of the record for December debuts of $77.2 million set two years ago by Will Smith's "I Am Legend." But it did break the record for a film opening in 3-D, previously held by last summer's "Up" with $68.1 million.
"What they spent on this movie was totally justified, and they're going to more than earn it back," said Paul Dergarabedian, box-office analyst for Hollywood.com.
"Avatar" stars Sam Worthington and Zoe Saldana in a love story amid human-alien conflict on a distant moon in the 22nd century.
Storms on the East Coast kept many people at home, cutting into weekend movie business.
"The weather really, really hurt," said Rory Bruer, head of distribution at Sony, whose comedy "Did You Hear About the Morgans?", opened at a weak No. 4 with $7 million. Sony had expected the movie to debut at about $8 million.
The movie stars Sarah Jessica Parker and Hugh Grant as a couple whose crumbling marriage gets a fresh jolt after they see a murder and are sent into witness protection.
The overall box office shot up on the strength of "Avatar." Hollywood's domestic revenues came in at $134 million, up 51.5 percent from the same weekend last year, when "Yes Man" opened at No. 1 with $18.3 million, according to Hollywood.com.
The previous weekend's No. 1 movie, Disney's animated musical "The Princess and the Frog," slipped to second place with $12.2 million, raising its total to $44.8 million.
Paramount's recession story "Up in the Air," which led last Tuesday's Golden Globes announcement with six nominations, broke into the top 10 as it widened to more theaters ahead of its nationwide expansion Wednesday.
"Up in the Air" came in at No. 8 with $3.1 million. The film stars George Clooney as a man addicted to the frequent-flyer life as he travels the country firing people at downsizing companies.
Two music-themed films had strong starts in limited release as they position themselves for Academy Awards attention. The Weinstein Co. musical "Nine" opened big with $246,933 in four theaters, for an average of $61,733 a cinema, compared to an average of $21,147 in 3,452 theaters for "Avatar."


text:
Historic Preservation Office
The Historic Preservation Office (HPO) promotes stewardship of the District of Columbia's historic and cultural resources through planning, protection and public education. HPO is an integral component of the Office of Planning and serves as the staff to the Historic Preservation Review Board (HPRB) and Mayor's Agent for historic preservation. HPO also implements federal historic preservation programs as the State Historic Preservation Office (SHPO) for the District of Columbia.
State Historic Preservation Office
Designated by the Mayor, the State Historic Preservation Officer (SHPO) for the District of Columbia, David Maloney, is responsible for protecting the District's unique historical, archaeological, agricultural, and cultural resources. This responsibility is shared with each federal agency that administers properties or undertakesconstruction activities in Washington, DC. Each year the SHPO prepares an Annual Work Plan to help the District meet its long-term historic preservation goals. The District of Columbia Historic Preservation Review Board, a group of private citizens appointed by the Mayor to represent professional and community viewpointsin the historic preservation process, advises the SHPO on matters relating to Section 106 review. The Historic Preservation Office serves as staff to the SHPO and the Historic Preservation Review Board.
The District of Columbia State Historic Preservation Office is located at 1100 4th Street, SW Washington, DC 20024. Telephone: (202) 442-7600 Email: firstname.lastname@example.com
Mayor's Agent
Demolition of a historic landmark or building contributing to the character of a historic district.
Subdivision of a historic landmark property (including division or assembly of land).
In cases where the applicant claims unreasonable economic hardship or proposes to construct a project of "special merit".
Upon request of an applicant having received a recommendation for denial from the Historic Preservation Review Board or Commission of Fine Arts.
In any other case deemed appropriate by the Mayor.
For a permit to be issued after the public hearing, the Mayor's Agent must find that failure to issue the permit would result in unreasonable economic hardship to the owner, or that issuance of the permit is necessary in the public interest.
"Necessary in the public interest" is defined to mean consistent with the purposes of the Historic Landmark and Historic District Protection Act, or necessary to allow the construction of a project of special merit.
A "project of special merit" is defined to mean a plan or building having significant benefits to the District of Columbia or to the community by virtue of exemplary architecture, specific features of land planning, or social or other benefits having a high priority for community services.


text:
To embed, copy and paste the code into your website or blog:
The Massachusetts Supreme Judicial Court has just issued an unusual order in the very important Eaton v. Federal National Mortgage Association case, indicating its deep concern over whether its ruling will have a disastrous impact on foreclosure titles and, if so, whether its ruling should be applied prospectively rather than retroactively.
As outlined in my prior post on the case, the Court is considering the controversial question of whether a foreclosing lender must possess both the promissory note and the mortgage in order to foreclose. If the SJC rules against lenders, it could render the vast majority of securitized mortgage foreclosures defective, thereby creating mass chaos in the Massachusetts land recording and title community. If you thought U.S. Bank v. Ibanez was bad, Eaton v. FNMA could be the Nuclear Option!
Firefox recommends the PDF Plugin for Mac OS X for viewing PDF documents in your browser.
We can also show you Legal Updates using the Google Viewer; however, you will need to be logged into Google Docs to view them.
Please choose one of the above to proceed!
LOADING PDF: If there are any problems, click here to download the file.
DISCLAIMER: Because of the generality of this update, the information provided herein may not be applicable in all situations and should not be acted upon without specific legal advice based on particular situations.
© Richard Vetstein
Read fresh new writing on compliance, cybersecurity, Dodd-Frank, whistleblowers, social media, hiring & firing, patent reform, the NLRB, Obamacare, the SEC…
…or whatever matters the most to you. Follow authors, firms, and topics on JD Supra.
Create your news brief now - it's free and easy »
The Vetstein Law Group, P.C. of Framingham, Massachusetts is a law firm specializing in business,...
View Profile »
See more »
Sign up for your custom alerts now, using LinkedIn ›
Back to Top
Copyright ©


text:
Cheap SMX to San Juan flights
Popular Flights from Santa Maria, California
Top Santa Maria Flights to US Destinations
Top Santa Maria International Destinations
Popular Flights from San Juan, Puerto Rico
Top San Juan Flights to US Destinations
Top San Juan International Destinations
SMX to SIG Route Map
Santa Maria to San Juan Flights, Stats and Airplanes
Route information for flights from Santa Maria Public to Fernando Luis Ribas Dominicci.
3,497.33 miles
SMX - Santa Maria Public Airlines
Browse the most popular airlines servicing the Santa Maria (SMX) airport. Visit their flights pages to learn more about the most popular routes these carriers fly.


text:
(Prishtina, DTT-NET.COM) – An Ethnic Albanian rebel group claiming to be part of illegal Albanian National Army (ANA) has emerged last Friday demanding that territories of
Albanian rebels want Southern Serbia to return to Kosovo
31 October 2005
(Prishtina, DTT-NET.COM) – An Ethnic Albanian rebel group claiming to be part of illegal Albanian National Army (ANA) has emerged last Friday demanding that territories of Southern Serbia (known as Presevo Walley) return to Kosovo, as UN Security Council last week approved talks on the status of UN administrated province to begin.
Access to our news services is payable. You can read/use full content of the stories by subscribing to our daily news packages.
Please sent us your subscription request at: info @ dtt - net . com
This media is intended only for personal use. Any other reproduction, publication or redistribution without the written agreement of the DTT-NET.COM is strictly forbidden and any breach of copyright will be considered actionable.
[ back ]
Related News
Montenegro, Kosovo vow to strengthen ties
Serbia backed Serbs block vote on Kosovo new government
Serbia's Nikolic begs Russia's Putin not to scrap South Stream gas pipeline
Mogherini joins Merkel's calls to Putin over Bosnia, Serbia, Western Balkans
Bosnia, Serbia police arrests 15 Serbs over war crimes
Kosovo, Serbia PMs vow to continue with EU mediated peace talks
Russia tells Serbia to seek money compensation from EU over end of gas pipeline
Russia caressing Serbia with position on Kosovo amid scrapping of South Stream
Serbia's president says Kosovo key condition for EU membership
Kosovo NGO calls on UN prosecutor to file indictment for Serbia war crimes
Copyright © 2005-2015 DTT-NET.COM
powered by: HL


text:
Guild Name - Lion's Arch Irregulars
Guild Age - Est. May 2012 (by people who have been MMO gaming since 1997)
Website - http://lionsarch.org
Multiple Guild Membership? - Absolutely fine with us
Server - TBD among the membership after the next BWE
Alliances - None at this time
Countries Served - US, Australia, NZ and even some Europe
QUICK LINKS - About, Lore, FAQ, Charter, Forum
So you've had a look through our site (particularly those links above) and are wondering, "In a nutshell, what exactly are these people looking for in a potential member?"
Here's a checklist:
-- You're 21+ years of age. (Although you might actually be a lot older and that would be fine; we have numerous 'older gamers' in the guild.)
-- You're the kind of person that other people really like being around, ie: you're pleasant, mature, considerate, friendly, smart and can hold a great conversation without dropping the f-bomb even once.
-- You have a desire to drop past your guild forum and not only 'talk shop' (such as organising a dungeon crawl), but start actual conversations about topics outside the game like films or music.
-- You have a genuine interest in Guild Wars 2, ie: you've ideally played the game in beta and are happy with it. You certainly aren't thinking, "Well, this will be a nice game to tide me over for a little while…"
Why do we ask for these attributes? Because they describe us. This is the kind of person we are, and we'd simply like to meet more people like us.
And for those who are still reading, a little background on why I'm doing this:
Back in the late 1990s and early 2000s, MMORPG guilds used to be groups of good friends who genuinely enjoyed each others company and played their chosen MMORPG as a regular social activity similar to the pen and paper RPGs that preceded them. Maybe you even remember those times?
Somewhere along the line, however, the guild concept became corrupted. Less emphasis was placed on real friendships being developed between members and, sadly, people began to be known more by what level their characters were, and what they could do in the short term for other members of the guild, rather than by who the real people behind the characters were.
I have made a deliberate attempt in recent times to wind the clock back to the days when guilds, and more importantly the people inside those guilds, mattered. If this sounds interesting to you, and you want to step back to a time when your guild felt like an extension of your family, and not some kind of factory job, you're welcome to explore this site and apply to join us.
– Captain Flashman


text: Slideshows Published On: Jun 22 2012 07:04:45 PM EDT Updated On: Jan 25 2013 11:28:12 AM EST Before they were famous: Celebrity commercials 7 pains men should never ignore BELLE ISLE AQUARIUM KOI TRANSFER NFL Deflategate: Tom Brady timeline Celebrities who served in the military Where are 'Blossom' stars today? PHOTOS: De La Salle vs. Mona Shores game MAROON 5 ROCK THE PALACE SIT ON IT DETROIT Slideshow: Famous Michiganders in black history Top 10 U.S. beaches of 2015 10 superfoods for spring Recent non-animated Disney films Celebs wear red noses to combat world hunger On this day: May 23 Who is ISIS? Horse racing's Triple Crown winners Test your Memorial Day knowledge Sci-fi movies of the past 10 years Biggest sports scandals of all time Films of George Clooney Award-winning Disney movies America's most frugal cities On this day: May 24 On this day: May 22 1980s TV stars then and now Test your hair band IQ Leading ladies of 'Pitch Perfect 2' Where are 'Beverly Hills, 90210' stars now? Most valuable athlete brands in sports View More


text:
Play the game, modify it or create a new one. 20 levels, 3.000 enemies, fast. 6 new weapons, 5 new enemies, original ingame music, dood sounds for enemies, weapons, itens and menus.
No news were found matching the criteria specified. We suggest you try the news list with no filter applied, to browse all available articles.
Join now to share news with the community.


text:
Tom Kenny To Voice Act on a New Transformers Series?
Saturday, December 23rd, 2006 7:32PM CSTCategories: Cartoon News, People News, Rumors
Posted by: Hotrod Views: 32,236
Recently Tom Kenny did a radio interview on a radio station in Dubuque, IA. In the interview Kenny revealed some interesting information. According to the voice actor that one of his upcoming shows will be the new Transformers series. Many know Kenny as the voice of Sponge Bob Square Pants. For more information about Tom Kenny click here.
Credit(s): TFW2005
Most Popular Transformers News
Most Recent Transformers News
Rumour: Paramount Transformers Expanded Universe - Transformers One Prequel on CybertronPosted 14 hours ago
News Categories: 3rd Party News, Auctions, Book News, Cartoon News, Collectables, Collector's Club News, Comic Book News, Company News, Contests, Editorials, Event News, Game News, Heavy Metal War, Interviews, Knock Offs, Media, Movie News, People News, Podcast, Press Releases, Reviews, Rumors, Sightings, Site Articles, Site News, Sponsor News, Store News, Toy News, Transtopia


text:
Danshen-Gegen decoction protects against hypoxia/reoxygenation-induced apoptosis by inhibiting mitochondrial permeability transition via the redox-sensitive ERK/Nrf2 and PKC8/mKATP pathways in H9c2 cardiomyocytes.
Danshen-Gegen (DG) Decoction, an herbal formulation containing Radix Salviae miltiorrhizae and Radix Puerariae lobatae, has been used for the treatment of coronary artery disease in Chinese medicine. In the present study, the involvement of ERK- and PKC [epsilon] -mediated pathways in the cytoprotection against apoptosis afforded by DG pretreatment was investigated in H9c2 cardiomyocytcs. Pretreatment with a methanol extract of aqueous DG decoction protected against hypoxia/reoxygenation-induced apoptosis in H9c2 cardiomyocytes. The cytoprotection was associated the enhancement of cellular reduced glutathione and a reduced sensitivity to [Ca.sup.2+] -induced mitochondrial permeability transition. DG extract increased the production of cytochrome P-450 (CYP)-dependent reactive oxygen species (ROS) in H9c2 cardiomyocytes, which was accompanied by the concomitant activation of ERK1/2 and PKC [epsilon]. The DG-induced ERK1/2 activation was followed by the translocation of Nrf2 from the cytosol to the mitochondria accompanied by an increase in the expression of glutathione-related antioxidant proteins. In addition, the increased expression of hemeoxygenase-1 was associated with the activation of Akt and BAD, indicative of anti-apoptotic activity. In conclusion, DG treatment activated both ERK/Nrf2 and PKC [epsilon] pathways, presumably by ROS arising from CYP-catalyzed processes, with resultant inhibition of hypoxia/reoxygenation-induced apoptosis immediately after DG treatment or even after an extended time interval following DG treatment.
[c] 2011 Elsevier GmbH. All rights reserved.
Keywords: Salviae miltiorrhizae Puerariae lobatae Extracellular signal-regulated protein kinase (ERK) Protein kinase C-epsilon (PKC [epsilon]) Mitochondrial permeability transition Apoptosis H9c2
Danshen and Gegen, which are roots of Salviae miltiorrhizae and Puerariae lobatae, respectively, are traditional Chinese medicinal herbs that have been widely used in China, Japan, Korea and Taiwan for the treatment of angina pectoris (Ji et al., 2000) and myocardial infarction (Adams et al., 2006). The herbal formulation made of Danshen and Gegen (Danshen-Gegen decoction) has long been used for the treatment of coronary artery disease (Xie et al., 2003). Raw herbs of Danshen and Gegen, and their isolated compounds, were reported to produce beneficial effects on cardiovascular function in human subjects (Tam et al., 2009), rodents (Ji et al, 2003) and cultured human endothelial cells (Sieveking et al., 2005). Recent studies in our laboratory have demonstrated that both acute and long-term treatment with an aqueous extract prepared from Danshen and Gegen in a ratio of 7:3 (w/w) protected the myocardium against ischemia/reperfusion injury in rats ex vivo (Chiu et al., 201 lb,c). In addition, the treatment with this Danshen-Gegen (DG) aqueous extract immediately after the intraperitoneal administration of isoproterenol was found to reduce the extent of myocardial damage in rats (Wong et al., 2011). The cardioprotection afforded by DG pre-/post-treatment may at least in part be attributed to the reactive oxygen species (ROS)-mediated activation of protein kinase C-epsilon (PKC [epsilon]), which in turn opens a mitochondrial ATP-dependent potassium channel (m [K.sup.ATP]), causing inhibition of a mitochondrial permeability transition (MPT) (Chiu et al., 2011b; Wong et al, 2011). The finding that the cardioprotection afforded by long-term DG pretreatment was paralleled by enhancements in mitochondrial glutathione antioxidant status suggests the activation of a redox-sensitive extracellular signal-regulated protein kinase (ERK) pathway in eliciting the glutathione antioxidant response.
PKC [epsilon] and ERK play an essential role in regulating cell survival and death through ROS-mediated processes (Kabir et al., 2006; Slupsky et al., 2007). In a short time-course within minutes, ROS can activate both PKC [epsilon] and ERK, with the former translocating to the mitochondrion and causing opening of the m[K.sup.ATP] (Costa and Garlid, 2008). In a more protracted time-course within hours, the activated ERK can phosphorylate the nuclear factor erythroid 2-related factor 2 (Nrf2), which then translocates from the cytosol to the nucleus, with subsequent enhancement in the expression of antioxidant defensive genes such as the subunits of [gamma] -glutamyl cysteine ligase (GCL), thioredoxin, glutathione S-transferases and NAD(P)H-quinone oxidoreductase (Jeyapaul and Jaiswal, 2000; Kensler et al., 2007; Liu et al., 2007; Zhang et al., 2006). In the present study, we aimed to investigate the involvement of ERK- and PKC [epsilon] -mecliated pathways in the cytoprotection against hypoxia/reoxygenation-induced apoptosis afforded by DG pretreatment in H9c2 cardiomyocytes, a clonal cell line derived from embryonic rat heart tissue (Hescheler et al., 1991).
Materials and methods
Preparation of DC extract
The Danshen-Gegen aqueous extract with an optimal crude herb ratio of Danshen to Gegen (7:3, w/w), as assessed by cardioprotection against ischemia/reperfusion injury (Chiu and Ko, 2004), was prepared in large-scale for experimental and clinical investigations, as described in Chiu et al. (201l b). To remove the sugars (monosaccharides and disaccharides) that could affect the cultured cells, the DG aqueous extract (100 g) was extracted by heating under reflux in 100 ml of methanol. The procedure was repeated twice. The pooled extract was dried by evaporation under reduced pressure to obtain the DG extract at a yield of 40%, and the DC extract was stored at 4 [degrees]C prior to use. In addition to monosaccharides and dissaccharides, polysaccharides, which are unlikely to produce cardioprotective action, were also removed by methanol extraction. As a result, DG extract produced cytoprotective action similar to that of the aqueous DG extract in H9c2 cardiomyocytes, but with a lower effective concentration (data not shown).
Chemical analysis of the DG extract
DG extract were performed as described in our previous study with minor modifications relating to the instrument and chromatographic conditions (Chang et al., 2008). HPLC analysis indicated that the DG extract contained the following marker compounds ((xg/100 mg; mean[+ or -]SD, n = 3): Danshensu (2406.7 [+ or -]100.5), Salvianolic acid B (1407.3 [+ or -]40.0), Protocate-chuic aldehyde (132.7[+ or -]5.9), Puerarin (2821.4[+ or -]82.4), Daidzein 8-C-apiosyl-gIucoside (644.5[+ or -]31.4), Daidzin (260.9[+ or -]7.1) and Daidzein (260.7 [+ or -] 13.9). The methanol DG extract had higher concentrations of these marker compounds than those in the Danshen-Gegen aqueous extract (Chiu et al., 2011b), but both preparations showed similar relative concentrations of the marker compounds. HPLC fingerprint analysis of the methanol extract prepared from two batches of DG aqueous extract showed substantial variations (2-150%) in the contents of marker compounds (Fig. 1 and Table 1). The two DG extracts inhibited cell apoptosis to varied extents which correlated with the concentrations of daidzein 8-C-apiosyI-glucoside and daidzin in the extracts (Table 2). These two markers may be used for chemical standardization of DG preparation.
Table 1 Content of chemical markers in the methanol extract of two batches of DG. Analytes Content (n = ([micro] 3) g/100mg) Batch As SD Batch SD Mean Inter-batch B variation (%) Danshensu 2406.7 100.5 655.2 45.9 1530.9 114 PCA 132.7 5.9 18.9 3.0 75.8 150 Puerarin 2821.4 82.4 2761.1 128.9 2791.3 2.2 DAG 664.5 31.4 865.6 36.2 765.1 26 Daidzin 260.9 7.1 394.0 25.4 327.5 41 SAB 1407.3 40.0 4997.5 172.9 3202.4 112 Daidzein 260.7 13.9 210.2 13.5 235.5 21 Note: PCA, Protocatechuic aldehyde; DAG, Daidzein 8-C-apiosyl-glucoside; SAB, Salvianolic acid B. Table 2 Bioassay on the methanol extract of 2 batches of DG preparation: effect on hypoxia/reoxygenation-induced apoptosis in H9c2 cardiomyocytes. Yield of % Protection against methanol apoptosis extraction 100 [micro] g/ml 300 [micro] g/ml Batch 53 22 55 A Batch 60 21 65 B H9c2 cardiomyocytes were treated with the methanol extracts of DG (batch A and B)at the indicated equivalent doses (with respective to the dry aqueous DG extracts) for 24 b. The cells were then subjected to 2 h of hypoxia followed by 16 h of reoxy-genation. The extent of hypoxia/reoxygenation-induced apoptosis was estimated by flow cytometry. Data were expressed as percent protection with respect to the untreated control.
Cell culture
H9c2 cardiomyocytes were maintained in DMEM medium supplemented with 10% (v/v) fetal calf serum, l00 IU/ml penicillin and 100 [micro]g/ml streptomycin. All cells were grown under an atmosphere of 5% (v/v) [CO.sup.2] in air at 37 [degrees]C Cells were grown to 80% confluence prior to the exposure to drugs in the various experiments.
In vitro hypoxia/reoxygenation challenge
After the incubation with DG extract at 30 or 100 [micro]g/ml for 24 h, cells were then subjected to 2 h of hypoxia and followed by 16 h of reoxygenation, as previously described (Chiu et al., 2008).
Annexin V and PI staining
Apoptotic cell death was measured by Annexin V-FITC-Fluos/propidium iodide (PI) staining followed by flow cytometry analysis using a COULTER[R] EPICS[R] XL[TM] Flow Cytometer (Beckman Coulter, Fullerton, CA), as described previously (Chiu et al., 2008).
Measurement of mitochondrial cytochrome c release
Mitochondrial cytochrome c release was assessed by measuring cytosolic cytochrome c levels by Western blot analysis, as described in Chiu et al. (2008).
Measurement of cellular reduced glutathione
Cell lysates were prepared as described in Chiu et al. (2006). Reduced glutathione (GSH) levels in cell lysates were measured using an enzymatic method of Griffith (1980).
Measurement of mitochondrial permeability transition (MPT) pore opening with Calcein AM
The opening of the MPT pore was measured by adopting the method of MitoProbe[TM] Transition Pore Assay Kit (Molecular Probes, Eugene, OR) with minor modifications using Calcein AM and CoCl2, as described in Chiu et al. (2008).
Cytochrome P-450 (CYP)-dependent ROS production
The extent of ROS production was measured by using 2'-7'-dichlorofluorescein diacetate (DCFDA) in H9c2 cardiomyocytes, with a modification of the procedure described by Bansal et al. (2010). For the time-course study, cells were incubated with the DG extract at concentrations ranging from 30 to 300[micro]g/ml (dissolved in phosphate-buffered saline, PBS) and DCFDA for increasing periods of time (l-4h). For the CYP inhibitor and antioxidant study, cells were pre-incubated with l-aminobenzotriazole (ABT, a wide-spectrum CYP inhibitor; 10 mM final concentration, 2 h) or dimethylthiouracil (DMTU, a thiol-containing antioxidant; 20 mM final concentration, 1 h) prior to the addition of DG extract at 300[micro]g/ml. Vehicle (i.e. PBS) was added for the appropriate control. Then cells were co-incubated with ABT/DMTU and the DG extract, and the fluorescence intensity of each well was measured at an excitation wavelength of 485 nm and an emission wavelength of 530 nm by Victor [V.sup.2] Multi-label Counter (Perkin Elmer, Turku, Finland) every 15min for 6h at 37 [degrees]C The fluorescence intensity of DG-treated cells was normalized with reference to the respective time-matched vehicle control. The extent of DG-induced ROS production was estimated by computing the area under the curve (AUC) plotting normalized fluorescence intensity against time (min), and expressed in arbitrary units.
Western blot analysis ofERKl/2
Cells were incubated with the DG extract (300[micro]g/ml) or vehicle for increasing periods of time (l-24h). Cells for ERK1/2 analysis were first incubated with serum-free medium for a 24-h period of starvation, which was followed by incubation with drug-containing serum-free medium. Total and phosphorylated-ERKl/2 levels were measured by Western blot analysis, as described in Chiu et al. (2011a).
Preparation of nuclear extracts and Western blot analysis ofNrf2
Cells were first treated with the DG extract for 3 h, followed by the incubation with fresh medium (without drug) for increasing periods of time (2-18 h). Cells were harvested by trypsinization at the indicated period of time, and the nuclear fraction was prepared as described (Langston et al., 2008). Nuclear Nrf2 level was measured by Western blot analysis, as described in Chiu et al. (2011a).
Western blot analysis of glutathione-related antioxidant proteins
Cells were treated with the DG extract as described in the Nrf2 experiment and measured for levels of GSH and glutathione-related antioxidant proteins at increasing time intervals. Cellular GSH levels were measured as described previously. Glutathione antioxidant response was assessed by measurements of cellular glutathione antioxidant protein levels, as described in Chiu et al. (2011a).
Western blot analysis of HO-1, Akt and BAD
Cells were treated with the DG extract at 300 [micro]g/ml for increasing periods of time (2-18 h). Equal amounts of cell lysates (50[micro]ig protein) were subjected to 10% SDS-PAGE followed by Western blotting using antibodies against inducible hemeoxygenase (HO-1), alpha-serine
threonine kinase (Akt) and Bcl-2 associated death promoter (BAD), which are components involved in HO-1 /Akt pathway. All antibodies were rabbit polyclonal antibodies, which are specific for the HO-1 (1:500; Santa Cruz Biotechnology), phos-phorylated and non-phosphorylated antibodies of Akt (Ser 473) (1:1000; Cell Signal Technology) and BAD (Ser 155) (1:1000; Cell Signal Technology). All immuno-stained protein bands were analyzed by densitometry, and the extents of Akt and BAD activation were estimated by computing the ratio of phosphorylated protein level to non-phosphorylated protein level in DG-treated cells with respect to the time-matched control, and the amounts (arbitrary units) of HO-1 were normalized with reference to [beta]-actin (1:1000; Cell Signal Technology) levels (arbitrary units) in the sample.
Western blot analysis of PKC[epsilon]
Cells were treated with the DG extract at 300[micro]g/ml or vehicle for increasing periods of time (1-8 h). After the incubation, cells were harvested with 2 ml of 0.1 mM PMSF in PBS and then centrifuged at 400 x g for 10 min at 4 [degrees]C. The cells were then resus-pended in 400 [micro]l of lysis buffer, and the lysates were further processed by passing through a syringe needle (21G1/2) repetitively for 10 times. The mitochondrial and cytosolic fractions were prepared by differential centrifugation (Wang et al., 2001). In essence, the homogenates were centrifuged at 1000 x g for 10 min at 4 [degrees]C The resultant supernatants were centrifuged at 100,000 x g for 1 h at 4 [degrees]C. The pellet was washed twice with the lysis buffer and then resuspended into 100 [micro]l of ice-cold of lysis buffer supplemented with 1% Triton X-100. After measuring the protein concentrations, the cytosolic and mitochondrial fractions were mixed with 6 volumes of SDS sample loading buffer. Mitochondrial and cytosolic proteins (40 [micro]g) were resolved using 10% SDS-PAGE gels and transferred onto nitrocellulose membranes. An equal loading of proteins was confirmed by staining with Ponceau-S solution [0.5% (w/v) Ponceau S in 1% (v/v) acetic acid] (Bannur et al., 1999). After blocking with 5% skim milk, the membranes were incubated with anti-PKC[epsilon] antibody (Upstate, Temecula, CA) at 4 C overnight. The membrane was subsequently washed and incubated with peroxidase-conjugated secondary antibody (1:2000; Cell Signaling Technology) for 2 h at room temperature. Immuno-stained bands were quantitatively analyzed. The intensity of immunostained PKC[epsilon] band was normalized to that of the sum of Ponceau S-stained bands in the sample. The ratio of mitochondrial (m) PKC[epsilon] level to cytosolic (c)PKC[epsilon] level indicated the extent of PKC[epsilon] translocation.
Effect of ABT and DMTU on ERK 1/2 activation and PKC[epsilon] translocation
Cells were pre-incubated with ABT (10 mM final concentration, 2h) or DMTU (20 mM final concentration, 1 h) prior to the addition of DG extract at 300 [micro]g/ml. Cells were then co-incubated with ABT/DMTU and the DG extract for 4h. After the incubation, cells were lysed with 300 [micro]l of lysis buffer. Equal amounts of cell lysate (10 [micro]g) were subjected to 12% SDS-PAGE and Western blot analysis of phosphorylated and total ERK 1/2 were performed.
For the study on PKC[epsilon] translocation, cells were pre-incubated with ABT/DMTU and then co-incubated with ABT/DMTU and the DG extract for 3h. The cells were harvested, and the cytosolic and mitochondrial fractions were prepared as described above. Equal amounts of cytosolic and mitochondrial proteins (40 [micro]g) were subjected to 10% SDS-PAGE, respectively, and Western blot analysis using PKC[mu] antibody was performed. The extent of PKC[epsilon] translocation was indicated by the ratio of mPKC[epsilon] level to cPKC[epsilon] level.
Effects of PKC[epsilon]and m[K.sub.ATP] inhibition on cytoprotection against apoptosis
Cells were first treated with the PKC[epsilon] translocation inhibitor (Peptide EAVSLKPT, Calbiochem, Darmstadt, Germany; 10 [micro]M, 1 h) or m[K.sub.ATP] inhibitor [5-hydroxydecanoate (5-HD), Sigma Chemical Co.; 100 [micro]M, 1 h] prior to the DG treatment at 300 fig/ml of DG for 3 h, and the ceils were then subjected the hypoxia/reoxygenation challenge. The cell lysates were prepared for apoptotic cell death analysis by Annexin V/PI staining and cellular GSH levels were measured following the procedures as described above.
Effect of post-drug exposure time on cytoprotection
Cells were treated with the DG extract (300 [micro]g/m)) for 3 h without or with subsequent incubation with fresh medium for 12 h. The cells were then subjected to hypoxia/reoxygenation challenge and measurements of apoptotic cell death and cellular GSH level were performed.
Statistical analysis
Data were analyzed by one-way Analysis of Variance. Post hoc multiple comparisons were performed using Least Significant Difference test. P values < 0.05 were regarded as a statistically significant difference.
Effects of DC pretreatment on hypoxia/reoxygenation-induced apoptosis and associated changes in cellular GSH and mitochondrial permeability transition
DG treatment (30 and 100 [micro]g/ml) for 24 h decreased the extent of apoptosis in cultured H9c2 cardiomyocytes, with the extent of inhibition being 19 and 23%, respectively, when compared with the DG-untreated control (Fig. 2a). A two hour-period of hypoxia followed by 16 h of reoxygenation caused a significant increase in the extent of apoptosis in H9c2 cardiomyocytes, with the extent of apoptotic cell death being increased by 1.1-fold, when compared with the unchallenged control. DG pretreatment (30 and 100 [micro]g/ml) concentration-dependently inhibited the hypoxia/reoxygenation-induced apoptosis, with the degree of protection being 38 and 60%, respectively, when compared with that of the DG-untreated and challenged control.
While the DG treatments caused concentration-dependent increases (14 and 34%) in cellular GSH levels, hypoxia/reoxygenation challenge decreased cellular GSH levels in H9c2 cardiomyocytes (Fig. 2b). The cytoprotection against hypoxia/reoxygenation-induced apoptosis afforded by DG pretreatment was associated with increases in cellular GSH levels (5 and 27%), as compared to that of the DG-untreated and challenged control.
DG treatment caused a reduction in the extent of [Ca.sup.2+]-induced MPT pore opening (Fig. 2c), but hypoxia/reoxygenation caused a significant increase (61 %) in the extent of MPT pore opening in H9c2 cardiomyocytes. The cytoprotective effect of DG pretreatment was associated with decreases in the extent of [Ca.sup.2+]-induced MPT pore opening (9 and 33%), when compared with that of DG-untreated and challenged control
To further investigate the effect of DG treatment on MPT pore opening which can eventually leads to cytochrome c release from the mitochondria, hypoxia/reoxygenation-induced mitochondrial cytochrome c release was also measured (Fig. 2d). DG pretreatment suppressed the hypoxia/reoxygenation-induced release of cytochrome c (by 33 and 44%) in H9c2 cardiomyocytes, relative to that of the DC-untreated and challenged control.
DC induced a CYP-mediated ROS production
To explore whether DG can trigger a redox-sensitive signaling pathway, DG-induced ROS production in H9c2 cardiomyocytes was examined. DG caused a time- (1-4 h) and concentration-(30-300 ([micro]g/ml) dependent increase in ROS production in H9c2 cardiomyocytes under the present experimental conditions (Fig. 3a). When the amount of ROS produced was estimated by computing the area under the curve plotting the percent increase in fluorescence (with respect to the appropriate time-matched PBS control) against incubation time (up to 4h), DG treatment at 300 ([micro]g/ml for 3h increased ROS production by 35% (Fig. 3b). Pre-/co-incubation with ABT (10 mM) or DMTU (20 mM) abrogated the DG-induced ROS production in H9c2 cardiomyocytes, with ABT producing a greater inhibitory effect than that of DMTU (Fig. 3b).
DG caused a time-dependent ERK activation via CYP-mediated ROS production
Fig. 4a shows that DG treatment at 300 ([micro]g/ml caused time-dependent increases in the ratio of phospho-kinase to total kinase level of ERK1/2, indicative of ERK activation. The activation of ERK was maximal at 3h, with the extent of ERK1/2 phosphorylation being increased by 2.5- and 3.17-fold, respectively, when compared with the time-matched vehicle (i.e. PBS) control.
To investigate the involvement of CYP and ROS in DG-induced ERK activation, the effects of ABT and DMTU on ERK 1/2 activation were examined in DG-treated H9c2 cardiomyocytes. As shown in Fig. 4bt pre- and co-treatment of ABT (10mM) or DMTU (20 mM) completely abrogated the DG-induced ERK1/2 activation, and the resultant level of ERK activation was even lower than under unstimulated (i.e. basal) conditions.
DC induced Nrf2 activation and a glutathione antioxidant response
A 3-h treatment of the H9c2 cardiomyocytcs with DG extract (300 fxg/ml) caused a time-dependent change in nuclear Nrf2 level, an indirect measure of Nrf2 activation, during the post-drug treatment period, with the degree of stimulation being maximal (46%, compared to the value obtained immediately after drug exposure, i.e. at time 0) at 2h post-DG exposure (Fig. 5a). The Nrf2 level had decreased gradually to the time 0 value by 12-18 h following the exposure to DG. The increase in nuclear Nrf2 level was followed later by an increase in the expression of enzymes related to glutathione redox cycling and synthesis, namely, glutathione reductase (GR), glucose-6-phosphate dehydrogenase (G6DPH) and modulatory subunit of GCL (GCLm), with the maximum degree of stimulation occurring at 8-10 h post-DG exposure for G6DPH (28%) and GCLm (45%) and GR (31%) (Fig. 5b). While G6PDH and GCLm declined from the peak to values obtained at time 0 following 10 h of exposure to DG, the GR level decreased rapidly from the peak at 12 h post-DG exposure (Fig. 5b). Time-dependent increases in cellular GSH levels were also observed after DG treatment, with the degree of stimulation being maximal (56%) at 12 h post-DG treatment (Fig. 5b). GSH levels then declined gradually after reaching peak values.
DC caused a time-dependent induction ofHO-1 and activation of Akt/BAD
The activation of Nrf2 was associated with an enhanced expression of HO-1, with the degree of stimulation being maximal (31 %) at 4h post-DG exposure (Fig. 6). The increase in the expression of HO-1 was followed by a later increase in the ratio of phospho-kinase to total-kinase of Akt and BAD, indicative of Akt and BAD activation, respectively, with the degrees of activation of Akt (1.26-fold) and BAD (1.05-fold) being maximal at 6 and 8 h post-DG treatment, respectively. The activation of both Akt and BAD then subsided, with the ratio of phosho-kinase to total kinas falling below the time 0 value at lOh post-DG exposure (Fig. 6).
DC induced a time-dependent PKC[epsilon] activation via CYP-mediated ROS production
To investigate the signaling pathway triggered by ROS produced in DG-treated cells, the translocation of PKC[epsilon] from the cytosol to the mitochondria was examined. As shown in Fig. 7a, exposure to DG (300|xg/ml) caused a time-dependent increase in the ratio of mitochondrial to cytosolic PKC[epsilon], indicative of PKC[epsilon] activation. The maximal degree of PKC[epsilon] translocation was observed at 3 h post-DG treatment, at which point the ratio mPKC[epsilon] to cPKC[epsilon] was increased 2.8-fold, when compared with the respective time-matched and DG-untreated control.
To confirm the involvement of CYP and ROS in the activation of PKC[epsilon], the effects of ABT and DMTU on DG-induced PKC[epsilon] translocation were examined. As shown in Fig. 7b, pre- and co-incubation with ABT (10 mM) or DMTU (20 mM) largely suppressed DG-induced PKC[epsilon] translocation, with PKC[epsilon] translocation being inhibited by 54 or 46%, respectively, when compared with the respective DG-treated control group.
PKC[epsilon] and m[K.sub.ATP] inhibition suppressed DC-induced protection against apoptosis in hypoxic/reoxygenated cardiomyocytes
To investigate the possible involvement of a PKC[epsilon]-mediated signaling pathway in the cardioprotective action of DG, inhibitors of PKC[epsilon] translocation (Peptide EAVSLKPT) and m[K.sub.ATP] (5-HD) were used in the study. While the susceptibility of H9c2 cardiomyocytes to hypoxia/reoxygenation-induced apoptotic cell death was increased by the inhibition of PKC[epsilon] or m[K.sub.ATP], preincubation with DG (300[micro]g/ml for 3h) did not protect against hypoxia/reoxygenation induced-apoptosis in the presence of PKC[epsilon] or m[K.sub.ATP] inhibition (Fig. 8a). The abolition of DG-induced cardioprotection corresponded with the failure in reversing the hypoxia/reoxygenation-induced depletion in cellular GSH (Fig. 8b).
DG caused both acute and long-term protection against hypoxia/reoxygenation-induced apoptosis
To investigate the relative contribution of PKC[epsilon]/m[K.sub.ATP] (early onset) and ERK/Nrf2 (late onset) pathways to DG-induced cyto protection, the effect of DG treatment on hypoxia/reoxygenation-induced apoptosis in H9c2 cardiomyocytes was examined immediately after a 3-h incubation with DG (300 [micro]g/ml) and at 12-h post-DG exposure. As shown in Fig, 9a, the extent of protection against apoptosis assessed at 12-h post-DG exposure was greater than that observed at time zero, i.e. immediately after the addition of DG (47% versus 28%). The increased extent of cytoprotection was associated with a corresponding increase in the levels of cellular GSH in both unchallenged (45% versus 23%) and challenged (31% versus 17%) cardiomyocytes, when compared with the respective DG-untreated control (Fig. 9b).
Re-introduction of oxygen to previously hypoxic cardiomy-ocytes can cause necrotic and apoptotic cell death (Robin et al.( 2007). In the present study, hypoxia/reoxygenation-induced apoptosis in H9c2 cardiomyocytes was found to be suppressed by incubation with DG extract. The hypoxia/reoxygenation-induced cell apoptosis was associated with a depletion of cellular GSH and an increased sensitivity to [Ca.sup.2+]-induced MPT pore opening, both of which were partially reversed by prior incubation with DG. Consistent with this, recent in vivo studies in rats have shown that both acute and long-term exposure to an aqueous DG extract afforded protection against myocardial ischemia/reperfusion injury which was associated with increased mitochondrial GSH levels and decreased the sensitivity of mitochondria to [Ca.sup.2+]-induced permeability transition (Chiu et al., 201lb,c).
The DC-induced enhancement of cellular GSH and inhibition of MPT are likely mediated by the redox-sensitive ERK/Nrf2 and/or PKC[epsilon]/m[K.sup.ATP] signaling pathway (Jaburek et al., 2006; Trachootham et al., 2008). Consistent with this, DG extract was found to stimulate ROS production in H9c2 cardiomyocytes, with a concomitant activation of ERK1/2 and PKC[epsilon]. The inhibition of DG-induced ROS production as well as ERK/1/2 and PKC[epsilon] activation by ABT and/or DMTU support the involvement of CYP-catalyzed, oxidant-generating reactions in the process. The DG-induced activation of ERK1/2 was followed by a later activation of Nrf2 during the post-DG exposure period, with subsequent increases in the expression of glutathione-related antioxidant proteins and HO-1. While GCLm is responsible for increasing the de novo synthesis of GSH, GR and G6DPH likely play a crucial role in the cellular glutathione redox cycling, and both processes are important for GSH recovery following oxidant challenge (Franklin et al., 2009; Han et al., 2006; Jain et al, 2003). The increased expression of these enzymes is therefore likely to be instrumental in preventing cell apoptosis -a process mediated by oxidants that can cause acute glutathione redox imbalance and thereby trigger the apoptotic signaling (Circu and Aw, 2010). The increased expression of HO-1 induced by the DG extract was followed by the activation of Akt/BAD pathway. It has been shown that BAD phosphorylation by Akt causes the formation of the BAD protein homodimer and thus leaves Bcl-2 free to inhibit the BAX-triggered apoptosis (Datta et al., 2000; Tan et al., 2000). The DG-induced phosphorylation of BAD is therefore anti-apoptotic.
The cytoprotection against hypoxia/reoxygenation-induced apoptosis afforded by the incubation with the DG extract for 3h was completely abrogated by the inhibition of PKC [epsilon] translocation or m[K.sup.ATP] activation. This observation suggests a crucial role of the PKC [epsilon]/ m[K.sup.ATP] pathway in eliciting the DG-induced cytoprotection in H9c2 cardiomyocytes. Activation of PKC [epsilon] prior to ischemia has been demonstrated to afford cardioprotection by mimicking ischemic preconditioning by brief episodes of ischemia and reperfusion (Dorn et at, 1999). While the role of m[K.sup.ATP] in ischemic preconditioning is not well understood (Haiestrap et al., 2007), the prolongation of onset time for MPT by ischemic preconditioning was found to be accompanied by the translocation of PKC [epsilon] from the cytoplasm to mitochondria in isolated adult rat cardiomyocytes (Juhaszova et al., 2004). Conceivably, ROS generated from the biotransformation of DG-derived components in cardiomyocytes may activate both ERK/Nrf2 and PKC [epsilon] pathways. Both cellular responses elicited by DG extract pretreatment render cardiomyocytes more resistant to hypoxia/reoxygenation-induced apoptosis. In this connection, mitochondrial ERK and PKC [epsilon] may form a functional signaling module, which in turn leads to the phosphorylation and thus inactivation of BAD (Baines et al., 2002). It should, however, be noted that while many chemical constituents in the DG extract likely produce ROS through CYP-catalyzed biotransformation resulting in cytoprotection, it is also possible that components such as puerarin and diadzein can act directly by opening the [Ca.sup.2+]-activated potassium channel and activating PKCe or inhibiting nuclear factor-kappa B activation, respectively (Gao et al., 2006; Kim et al., 2009).
Interestingly, the DG-induced increase in cellular GSH was also observed after 3-h of DG exposure, and this effect was largely or completely suppressed by PKC [epsilon] or m[K.sup.ATP] inhibition, respectively, in non-hypoxic/reoxygenated H9c2 cardiomyocytes. While the mechanism underlying the GSH stimulatory effect produced by DG in such a short time interval remains to be investigated, it is unlikely to be due to an increase in GSH synthesis because the Nrf2-mediated increase in GCLm expression in H9c2 cardiomyocytes requires a longer lag time after the DG exposure. Alternatively, the stimulatory action on GSHt which was completely inhibited by 5-HD, may be related to the opening of m[K.sup.ATP], presumably through increasing intra-mitochondrial ROS production (Garlid et al., 2009). The finding that the 3-h DG exposure followed by a 12-h incubation in the absence of DG caused a larger extent of protection against cell apoptosis than that of a 3-h drug exposure perse suggests an important role of the ERKl/2/Nrf2 signaling pathway in affording cytoprotection over an extended time interval following the exposure to DG extract.
In conclusion, preincubation with the DG extract protected against hypoxia/reoxygenation-induced apoptosis in H9c2 cardiomyocytes. This cytoprotection was associated an enhancement of cellular GSH levels and a reduced sensitivity to [Ca.sup.2+] -induced MPT. DG activated both ERK/Nrf2 and PKC [epsilon] pathways, presumably by ROS arising from CYP-catalyzed reactions, with resultant inhibition of hypoxia/reoxygenation-induced apoptosis immediately after DG treatment or even after an extended time interval following DG treatment.
Conflict of interest
All authors have no conflict of interest to disclose.
The work described in this paper was partially supported by a grant from the University Grants Committee of the Hong Kong Special Administrative Region, China (Project No. AoE/B-10/01).
Adams, J.D., Wang, R., Yang, J., Lien, E.J., 2006. Preclinical and clinical examinations of Salvia miltiorrhiza and its tanshinones in ischemic conditions. Chin. Med. 1, 3.
Baines, CP., Zhang, J., Wang, G.W., Zheng, Y.T., Xiu, JX, Cardwell, E.M., Bolli, R., Ping, P., 2002. Mitochondrial PKCepsilon and MAPK form signaling modules in the murine heart: enhanced mitochondrial PKCepsilon-MAPK interactions and differential MAPK activation in PKCepsilon-induced cardioprotection. Circ. Res. 90, 390-397.
Bannur, S.V., Kulgod, S.V., Metkar, S.S., Mahajan, S.K., Sainis, J.K., 1999. Protein determination by ponceau S using digital color image analysis of protein spots on nitrocellulose membranes. Anal. Biochem. 267, 382-389,
Bansal, S., Liu, C.P., Sepuri, N.B., Anandatheerthavarada, H.K., Selvaraj, V.T Hoek, J., Milne, G.L, Guengerich, F.P., Avadhani, N.G., 2010. Mitochondria-targeted cytochrome P450 2E1 induces oxidative damage and augments alcohol-mediated oxidative stress. J. Biol. Chem. 285, 24609-24619.
Chang, Q., Sun, L, Zhao, R.H., Chow, M.S., Zuo, Z., 2008. Simultaneous determination of ten active components in traditional Chinese medicinal products containing both Gegen (Pueraria lobata) and Danshen (Salvia miltiorrhiza) by high-performance liquid chromatography. Phytochem. Anal. 19, 368-375.
Chiu, P.Y., Chen, N., Leong, P.K., Leung, H.Y., Ko, K.M., 2011a. Schisandrin B elicits a glutathione antioxidant response and protects against apoptosis via the redox-sensitive ERK/Nrf2 pathway in H9c2 cells. Mol. Cell. Biochem. 350, 237-250.
Chiu, P.Y., Ko, K.M., 2004. Schisandrin B protects myocardial ischemia-reperfusion injury partly by inducing Hsp25 and Hsp70 expression in rats. Mol. Cell. Biochem. 266, 139-144.
Chiu, P.Y., Leung, H.Y., Poon, M.K., Mak, D.H., Ko, K.M., 2006. (-)Schisandrin B is more potent than its enantiomer in enhancing cellular glutathione and heat shock protein production as well as protecting against oxidant injury in H9c2 cardiomyocytes. Mol. Cell. Biochem. 289, 185-191.
Chiu, P.Y., Luk, K.F., Leung, H.Y., Ng, K.M., Ko, K.M., 2008. Schisandrin B stereoisomers protect against hypoxia/reoxygenation-induced apoptosis and inhibit associated changes in Ca2+-induced mitochondrial permeability transition and mitochondrial membrane potential in H9c2 cardiomyocytes. Life Sci. 82, 1092-1101.
Chiu, P.Y., Wong, S.M., Leung, H.Y., Chen, N., Zhou, L, Zuo, Z., Lam, P.Y., Ko, K.M., 2011b. Acute treatment with Danshen-Gegen decoction protects the myocardium against ischemia/reperfusion injury via the redox-sensitive PKCe/mKATP pathway in rats. Phytomedicine, doi:10.1016/j.phymed.2011.03.006.
Chiu, P.Y., Wong, S.M., Leung, H.Y., Leong, P.K., Chen, N., Zhou, L, Zuo, Z., Urn, P.Y., Ko, K.M.. 2011 c. Long-term treatment with Danshen-Gegen decoction protects the myocardium against ischemia/reperfusion injury via the redox-sensitive protein kinase C-epsilon/mK(ATP) pathway in rats. Rejuven. Res. 14,173-184.
Circu, M.L, Aw, T.Y., 2010. Reactive oxygen species, cellular redox systems, and apoptosis. Free Radical Biol. Med. 48,749-762.
Costa, A.D., Garlid, K.D., 2008. Intramitochondrial signaling: interactions among mitoKATP, PKCepsilon, ROS, and MPT. Am. J. Physiol Heart Circ. Physiol. 295, H874-H882.
Datta, S.R., Katsov, A., Hu, L, Petros, A., Fesik, S.W., Yaffe, M.B., Greenberg, M.E., 2000. 14-3-3 proteins and survival kinases cooperate to inactivate BAD by BH3 domain phosphorylation. Mol. Cell 6,41-51.
Dorn, C.W., Souroujon, M.C., Liron, T., Chen, C.H., Gray, M.O., Zhou, H.Z., Csukai, M., Wu, C Lorenz.J.N., Mochly-Rosen, D., 1999. Sustained in vivo cardiac protection by a rationally designed peptide that causes epsilon protein kinase C translocation. Proc. Natl. Acad. Sci. U. S. A. 96,12798-12803.
Franklin, C.C., Backos, D.S., Mohar, I., White, C.C., Forman, H.J., Kavanagh, T.J., 2009. Structure, function, and post-translational regulation of the catalytic and modifier subunits of glutamate cysteine ligase. Mol. Aspects Med. 30,86-98.
Gao, Q.. Pan, H.Y., Qiu, S., Lu, Y., Bruce, I.C.t Luo, J.H., Xia, Q.,2006. Atractyloside and 5-hyclroxydecanoate block the protective effect of puerarin in isolated rat heart. Life Sci. 79, 217-224.
Garlid, K.D., Costa, A.D., Quinlan, C.L, Pierre, S.V., Dos, S.P., 2009. Cardioprotective signaling to mitochondria. J. Mol. Cell. Cardiol. 46, 858-866.
Griffith, O.W., 1980. Determination of glutathione and glutathione disulfide using glutathione reductase and 2-vinylpyridine. Anal. Biochem. 106, 207-212.
Halestrap, A.P., Clarke, S.J., Khaliulin, 1, 2007. The role of mitochondria in protection of the heart by preconditioning. Biochim. Biophys. Acta 1767, 1007-1031.
Han, D., Hanawa, N., Saberi, B., Kaplowitz, N., 2006. Mechanisms of liver injury, 111. Role of glutathione redox status in liver injury. Am. J. Physiol Gastrointest. Liver Physiol. 291, G1-G7
Hcscheer. J., Meyer, R.. Plant, S., Xrautwurst, D., Rosenthal, W., Schultz, C.. 1991. Morphological, biochemical and electrophysiological characterization of a clonal cell (H9c2) line from rat heart. Circ. Res. 69.1476-1486.
Jaburek. M., Costa, A.D., Burton, J.R., Costa. C.L, Garlid. K.D., 2006. Mitochondrial PKC epsilon and mitochondrial ATP-sensitive 1< + channel copurify and coreconsti-tute to form a functioning signaling module in proteoliposomes. Circ. Res. 99, 878-883.
Jain, M., Brenner, D.A., Cui, L, Lim, C.C., Wang. B., Pimentel, D.R., Koh, $., Sawyer, D.B., Leopold, J A. Handy, D.E., Loscalzo, J.. Apstein, CS., Liao, R., 2003. Clucose-6-phosphatc dehydrogenase modulates cytosolic redox status and contractile phenotype in adult cardiomyocytes. Circ. Res. 93, e9-el6.
Jeyapaut.J., Jaiswal, A.K., 2000. Nrf2 and c-Jun regulation of antioxidant response element (ARF)-mediated expression and induction of gamma-glutamylcysteine synthetase heavy subunit gene. Biochem. Pharmacol. 59, 1433-1439.
Ji, X., Tan, B.K., Zhu, Y.C, Linz, W., Zhu, Y.Z., 2003. Comparison of cardioprotective effects using ramipril and DanShen for the treatment of acute myocardial infarction in rats. LifeSci. 73, 1413-1426.
Ji, X.Y., Tan, B.K., Zhu, Y.Z., 2000. Salvia miltiorrhiza and ischemic diseases. Acta Pharmacol. Sin. 21,1089-! 094.
Juhaszova, M., Zorov. D.B., Kim, S.H., Pepe, S., Fu, Q., Fishbein, K.W., Ziman, B.D., Wang, S., Ytrehus. K., Antos, C.L. Olson. E.N., Sollott, S.J., 2004. Glycogen synthase kinase-3beta mediates convergence of protection signaling to inhibit the mitochondrial permeability transition pore. J. Clin. Invest. 113. 1535-1549.
Kabir, A.M., Clark, j.B.,Tanno, M., Cao, X.. Hothersall, J.S., Dashnyam. S., Gorog. DA. Bellahcene, M., Shattock, M.J., Marber, M.S., 2006. Cardioprotection initiated by reactive oxygen species is dependent on activation of PKCepsilon. Am. J. Physiol Heart Circ. Physiol. 291, H1893-H1899.
Kensler, T.W., Wakabayashi, N., Biswal, S., 2007. Cell survival responses to environmental stresses via the Keapl-Nrf2-ARE pathway. Annu. Rev. Pharmacol. Toxicol. 47, 89-116.
Kim, J.W.Jin, Y.C, Kim, Y.M., Rhie, SM Kim, H.J., Seo. H.G., Lee, J.H.. Ha, Y.L, Chang, K.C., 2009. Daidzein administration in vivo reduces myocardial injury in a rat ischemia/reperfusion model by inhibiting NF-kappaB activation. Life Sci. 84. 227-234.
Langston, W., Circu, M.L, Aw, T.Y., 2008. Insulin stimulation of gamma-glutamylcysteine ligase catalytic subunit expression increases endothelial GSH
during oxidative stress: influence of low glucose. Free Radic. Biol. Med. 45, 1591-1599.
Liu, X.P., Goldring, CL, Copple, LM., Wang, H.Y.. Wei, W., Kitteringham, N.R., Park, B.K., 2007. Extract of Ginkgo biloba induces phase 2 genes through Keapl -Nrf2-ARE signaling pathway. Life Sci. 80, 1586-1591.
Robin, E., Guzy, R.D., Loor. Ci., Iwase, H., Waypa, G.B., Marks, J.D., Hoek, T.L, Schumacker, P.T., 2007. Oxidant stress during simulated ischemia primes car-diomyocytes for cell death during reperfusion. J. Biol. Chem. 282,19133-19143.
Sieveking, D.P., Woo, K.S., Fung, K.P., Lundman, P., Nakhla, S., Celermajer, D.S.. 2005. Chinese herbs Danshen and Gegen modulate key early atherogenic events in vitro, int. J. Cardiol. 105,40-45.
Slupsky, J.R., Kamiguti, A.S., Harris, R.J., Cawley, J.C., Zuzei. M., 2007. Central role of protein kinase Cepsilon in constitutive activation of KRK'1/2 and Racl in the malignant cells of hairy cell leukemia. Am. J. Pathol. 170, 745-754.
Tarn, W.Y.. Chook, P., Qiao, M., Chan, L.T., Chan,T.Y., Poon. Y.K., Fung, K.P., Leung, P.O. Woo, K.S., 2009. The efficacy and tolerability of adjunctive alternative herbal medicine (Salvia miltiorrhiza and Pueraria lobata) on vascular function and structure in coronary patients. J. Altera Complement. Med. 15,415-421..
Tan, [yen]., Demeter, M.R., Ruan, H., Comb, MJ., 2000. BAD Ser-155 phosphorylation regulates BAD/Bcl-XLinteraction and cell survival. J. Biol. Chem. 275,25865-25869.
Trachootham, D.r Lu, W., Ogasawara, M.A., Nilsa, R.D.. Huang, P., 2008. Redox regulation of cell survival. Antioxid. Redox Signal. 10,1343-1374.
Wang, G.Y., Zhou, J.J., Shan, J., Wong. T.M., 2001. Protein kinase C-epsilon is a trigger of delayed cavdioprotecuon against myocardial ischemia of kappa-opioid receptor stimulation in rat ventricular myocytes. J. Pharmacol, Exp. Ther. 299, 603-610.
Wong, S.M.. Chiu, P.Y.,Leung, H.Y., Zhou, L, Zuo, Z., Urn, P.Y., Ko, K.M., 2011. Myocardial post-conditioning with Danshen-Gegen decoction protects against isoproterenol-induced myocardial injury via a PKCe/mKATP-mediated pathway in rats. Chinas Med. 6, 7.
Xie, R.Q., Du, J., Hao, Y.M., 2003. Myocardial protection and mechanism of Puer-arin Injection on patients of coronary heart disease with ischemia/reperfusion. Zhongguo Zhong. XL Yi.Jie. He. Za Zhi. 23. 895-897 (in Chinese).
Zhang, Y., Munday, R., Jobson, H.E., Munday, CM., Lister, C, Wilson, P., Fahey.J.W., Mhawech-Fauceglia, P., 2006. Induction of GST and NQOl in cultured bladder cells and in the urinary bladders of rats by an extract of broccoli (Brassica oleracea italica) sprouts. J. Agric. Food Chem. 54, 9370-9376
Abbreviations: ABT, 1-aminotriazole; Akt, alpha-serine threonine kinase; BAD, Bcl-2 associated death promoter; CYP, cytochrome P-450; ERK, extracellular signal regulated kinase; DCFDA, 2'-7'-dichlorofluorescein diacetate; DG, Danshen-Gegen; DMTU, dimethylthiouracil; GCL, [gamma] -glutamyl cysteine ligase; CCLm, modulatory subunit of GCL; G6DPH, glucose 6-phosphate dehydrogenase; GR, glutathione reductase; GSH, reduced glutathione; 5-HD, 5-hydvoxydecarioate; HOl.hemeoxygenase 1; mKAn>, mitochondrial ATP-dependent potassium channel; MPT, mitochondrial permeability transition; Nrf2, nuclear factor eryhtroid 2-related factor 2; PBS, phosphate-buffered saline; PI, propidium iodide; PKC [epsilon], protein kinase C-epsilon; ROS, Reactive oxygen species.
* Corresponding author. E-mail address:firstname.lastname@example.com (K.M. Ko).
0944-7113/$ - see front matter [c] 2011 Elsevier GmbH. All rights reserved. doi:10.1016/j.phymed.2011.07.002
Po Yee Chiu (a), Hoi Yan Leung (a), Pou Kuan Leong (a), Na Chen (a), Limin Zhou (b), Zhong Zuo (b), Philip Y. Lam (a), Kam Ming Ko (a),(*)
(a) Division of Life Science. The Hong Kong University of Science & Technology, Clear Water Bay, China
(b) School of Pharmacy, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong SAR, China
Reader Opinion
|Printer friendly Cite/link Email Feedback
|Chin, Po Yee; Leung, Hoi Yan; Leong, Pou Kuan; Chen, Na; Zhou, Limin; Zuo, Zhong; Lam, Philip Y.; Ko
|Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
|Article Type:
|Geographic Code:
|Feb 15, 2012
|Previous Article:
|Protective and ameliorative effects of mate (Ilex paraguariensis) on metabolic syndrome in TSOD mice.
|Next Article:
|In vitro anti-influenza virus activity of a cardiotonic glycoside from Adenium obesum (Forssk.).