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Copyright © 2017 ALM Media Properties, LLC. All Rights Reserved.
Data privacy concept. (Rawpixel.com/Shutterstock.com)
Though the debate over absolute encryption rages on, encryption is fast becoming a must-have security item in a growing number of law firms and legal departments. But as encryption usage grows, many are finding that it can cause more problems than it solves. End-to-end encryption, for example, can often cripple a legal team's compliance and internal e-discovery efforts.
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Boutt, LA: Sept. 18, 2015 – The Great American Rivalry Series is proud to announce that it will feature the brutal Battle on the River as cross-river rivals in St. Charles Parish the Hahnville Tigers host the Destrehan Wildcats in the Series' sixth weekend of games for 2015. This game will be presented by the United States Marine Corps and played on Friday, September 25 at Hahnville High School. This year's game will also be one of two Game of the Week Broadcasts on the Great American Rivalry Broadcast Network and will be LIVE from Boutte, LA. It is being presented by the Press Play Production team of St. Charles Parish in partnership with BattleOnTheRiver.com and will begin broadcasting on Friday, Sept. 25 at 6:00 PM CST (7:00 PM EST). (The link for the broadcast is here). Featuring some of the longest running rivalries in high school football, the 2015 Great American Rivalry Series will feature 100 showdowns nationwide. All of these great games come with great football, hard earned sweat and a heavy dose of hometown pride.
For the 12th year, the Great American Rivalry Series has selected an exclusive collection of top-notch rivalries across the country, covering 40 states and over 100 cities throughout the country. Wrapped in tradition, these annual battles excite entire communities, while celebrating the schools, their teams, and their fans. We Know Friday NightsTM
In Louisiana's St. Charles Parish the East Bank vs. West Bank rivalry sizzles as one of Louisiana's most heated football rivalries takes the field. The rivalry has escalated over many years of great success at both schools in football. Destrehan has won four state championships, including in 1949 (1A), 1973, 2007 and 2008. They won a six-on-six state title in 1941 and are the two-time reigning 5A state champions. They were state runner-ups in 1993. Hahnville won state championships in 1949 (Class B), 1968, 1972, 1992, 1994 and 2003. They were state runner-ups in 1995. In this series, Destrehan holds a 37-23 lead.
The winning team earns year-long bragging rights and will take home the Great American Rivalry Series Champions trophy, presented by the Marines. The game's Most Valuable Player and the top student-athlete from each school are also recognized as part of the Series. Along with these awards, an alumnus from each school will be inducted into the 12th class of the Great American Rivalry Series Hall of Fame.
"We are excited to partner once again with the Great American Rivalry Series," said Leroy Pennington, Marketing Specialist for the United States Marine Corps "The elite status of this Series fits perfectly with the Elite Warrior brand of the Marine Corps and we are proud to stand alongside these outstanding young athletes."
You can find more information at GreatAmericanRivalry.com or through the Series' Twitter (@AmericanRivalry), Facebook, YouTube and Instagram accounts. Previews and past results will be posted throughout the season with details of the teams and the history of the rivalries, and after the game. Fans can relive the excitement with photo galleries and game recaps.
Fans can add to the intensity of the rivalry via Twitter, Facebook, Instagram using #GreatAmericanRivalry. The Rivalry can also be found on Pinterest (AmericanRivalry) and YouTube. Fan photos posted with this hashtag may be featured on the Great American Rivalry Series website's new Fan Photo Gallery, Pinterest Page and promoted via our other social media outlets.

subset: commoncrawl


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Basin United Soccer Club
4500 Foothills Blvd
Mike's Fieldhouse
Klamath Falls, OR 97603
Mon-Fri: 4PM-8PM
Phone: +1 541 2731152
Email: email@example.com
Use our CONTACT FORM.
Like our Facebook page to keep up-to-date on club news, events and more.
Basin United is always looking to recruit new and experienced referees to join our staff. We have opportunities for those who wish to referee Recreation level games all the way to High School matches.
Those interested in becoming a Referee with Basin United need to fill out an application, which can be obtained from Mike's Fieldhouse. Any questions should be directed to our Referee Coordinator at email@example.com or by calling 541-273-1152.
Effective Spring of 2014 all games assignments and game reports are managed through the online program ArbiterSports. This site allowed for integrated assigning capabilities with referee evaluations, game reports, and payroll. All referees are required to have a unique email address for this system and have to accept games through this system.
As of APRIL 2015 all referee payroll will be processed using ArbiterPay. A FREE online service that allows QUICK and EASY access to your money. You have the option to have your money DIRECT DEPOSITED to your bank account, have a check sent to you, OR sign-up for the ArbiterPay Debit Card.
Sign-up is a breeze and MUST be completed BEFORE you can be paid. You can access the ACCOUNT CREATION PAGE by clicking the link below.


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Join Our Mailing List
About Our Portal_Site
The Herat Portal is located on the campus of Hariwa University, in the center of town, and run by the Afghan Development and Inspiration Bureau (ADIB). ADIB is dedicated to empowering young Afghan men and women through the promotion of entrepreneurship.
Herat_Portal Director & Portal Ambassador
Khaled was born in the Badghis province of Afghanistan and raised in Herat. He is an alumni of the Youth Exchange and Study program in the U.S. Khaled is currently studying medicine at Herat University. He will be a Medical Doctor in a year. He is also a professional simultaneous translator and holds a diploma in business and entrepreneurship.
Herat_Portal Curator
Saied Habibi was born and raised in Tehran. At age 19 he moved to the Herat province of Afghanistan. Saied is currently studying Science and Technology at Herat University. He is an assistant professor at Herat University, and will soon be a full professor.
Chris Wallace visits a Portal in College Park at the University of Maryland and speaks to a stranger in Mexico City.
JUDY WOODRUFF: Now: an art project attempting to build individual connections across the globe.
It's called Portals, and is the brainchild of artist and former television news producer Amar Bakshi, who told us that, in his former profession, the most meaningful conversations often came once the cameras were turned off.
Four months ago, inside a gold-painted shipping container on the Georgetown University campus, Beth Flores stood face-to-face with someone 7,000 miles away.
A global art project recently came to D.C., bringing visitors face-to-face with strangers in Afghanistan, Iran and Cuba. Hundreds of people signed up online to book 20-minute-long one-on-one conversations in the Portal, a shipping container outfitted with video, audio and Internet.
Nick Meeker made a new Facebook friend last week.
Her name is Mahsa Biglow, and she is a 25-year-old Iranian graduate student in photography at the University of Tehran. They met on the Internet — but not in any of the usual ways.
As students rushed to and from their classes in the Edmund A. Walsh Memorial building this week, they passed by a nondescript gold shipping container on N Street — unaware that, inside, unprecedented conversations were taking place.
Participants who stepped into the box, titled the "Portal to Afghanistan," were digitally transported thousands of miles away to have a full-body video chat with a complete stranger in Herat, Afghanistan.


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Other writers may be fortunate to have as their inspiration a muse that more closely conforms to one of the nine sister goddesses of learning and the arts in Greek myth. Me, I've got a reptile. It wasn't as if I chose the creature to be the power that guides my writing, but it happened.
One of the first stories I wrote when I settled in rural Florida during the winter was the one that won me the Sleuthfest short story contest. In it, alligators loom large. You could say one even rescued my protagonist. Of course I entitled it "Gator Aid". I gave a copy of it to a close friend who commented that I seemed to be hung up on alligators. Well, duh. They're all over the place here. We often comment in this county that there are more cattle here than there are people. I'm convinced there are more alligators than people too.
It seemed perfectly normal to me to use the alligator as a source of inspiration because what drives my creativity when I'm in cowboy country is the setting. My protagonists are usually women who are winter visitors, otherwise known as snowbirds. If one snowbirds on the two coasts or in the Keys, the setting is all about the beautiful people, the sand, the sun, the waves, the color of the water, the perfect waving palm trees. It takes some kind of crazy juxtaposition to make murder out of that, but there are plenty of writers who manage it and manage it well.
Here on the range, set foot beyond your manicured lawn and you may be in for trouble. You could step on any manner of awful thing: horse poop, cow pies, reptiles without legs, sticky, prickly stuff like palmetto, or into swampy water inhabited by animals you don't want to know about, and some of them are alligators. Of course, on the good side you could walk into the arms of a handsome cowboy who might pull you onto his horse and the two of you would ride off. . ., well, you understand what I'm saying. It's quite simple here. The gators make for danger, the cowboys for romance. It's the perfect blend of dark and light, murder and passion.
The place is so imposing that it's hard to imagine writing here without writing the rural Florida setting. If I want to work on a manuscript set in upstate New York while I'm here, I have to consciously transport myself to that other world. I find it's not done easily, and that's where the muse comes in, because, really, I'm fortunate to have two muses. I have to. One for up north and the alligator for down here.
My northern muse? Oh, just a boring old ghost named Fred who likes to play tricks on me. His humor inspires my own as I've written on my blog, and his knowledge of my home on the trout stream inspires stories of tornados, drought, floods, hardwood forests, pine topped mountains, microbreweries housed in old dairy barns, a few curious bears, deep snow, craggy-faced hunters and farmers, and, of course, the occasional murder.
Think alligator, and I'm wrestling with humidity, hurricanes, burning cane fields, the citrus aroma from orange trucks, a pink and magenta sunset across the western side of the big lake, the neon sign in the form of a cross proclaiming "Jesus Saves", the yelping of coyotes when the train comes through, air boats howling down the canals, and anhingas grunting to one another across the swamp. How could all of that not make one write setting, beautiful and possibly deadly?
So why am I not inspired by something less, well, ugly than an alligator? I do write murder mysteries, not romance, you know. An alligator is never the villain. I reserve that for humans. Usually when my reptilian muse is personified in my writing doing gatorish things like death rolls and snapping arms off, these unfortunate happenings are to bad people who deserve every toothy bite delivered.
Yeah, I'm hung up on gators because I love where I live, and I write what I love. What's your muse like? Is setting important in your writing? Do you like to read authors whose setting is strong? Who?
My muse is an alligator and his name is George. Life is hard here. Sometime in his earlier life George lost one of his front legs, but he gets around, also an inspiration to me.
Dumpster Dying by Leslie Diehl will be released by Oak Tree Press soon. Ah, the golden years of retirement in the sunshine state. They're more like pot metal to Emily Rhodes, who discovers the body of the county's wealthiest rancher in the Big Lake Country Club dumpster. With her close friend accused of the murder, Emily sets aside her grief at her life partner's death to find the real killer. She underestimates the obstacles rural Florida can set up for a winter visitor and runs afoul of a local judge with his own version of justice, hires a lawyer who works out of a retirement home, and flees wild fires, hand-in-hand with the man she believes to be the killer. Visit the website at www.lesleydiehl.com and blog http://anotherdraught.blogspot.com


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WARD, S.C. - Harriett Jean Bell, wife of Don Wagner and daughter of Mr. & Mrs. Harry S. Bell of Ward passed away July 7, 2009. She and her husband lived at 4770 Ivy Ridge Drive, Smyrna, Ga. 30080. Funeral services for Ms. Bell will be held Saturday, July 11, 2009 at 11 am at Johnston Presbyterian Church with interment to follow in Mt of Olives Cemetery. The family is at the home of Ms. Bell's parents, 1829 Hwy 23, Ward, SC and will receive friends at Bland Funeral Home, Johnston, Friday evening, July 10, 2009 from 6-8 pm. Harriett attended Darlington School, Rome, Ga., Converse College and graduated from the University of SC. She worked in Washington, DC for the Senate Judiciary Committee for several years; however, returned to SC and worked for Bank of America. Later she graduated from Midland Technical College, School of Nursing and moved to Atlanta and worked for Shephard Spinal Clinic. Harriett had been in declining health for several months. She was a member of Johnston Presbyterian Church. In addition to her husband and parents, she is survived by brothers: William Bell (Jenny), Ward; Harry Bell, Jr. (Carol), Columbia; and James A. Bell ( Dolores), Canal-Winchester, Ohio; niece: Catherine Bell; nephews: Satcher and Corey Bell, and Tyler and David Mercer. Memorials may be made to Johnston Presbyterian Church, 700 Mims St, Johnston, SC 29832 or National Alliance on Mental Illness (NAMI) of South Carolina, P. O. Box 1267, Columbia, SC 29202. Sign the guestbook at AugustaChronicle.com
The Augusta Chronicle-July 9, 2009


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cloth diapers
View Single Post
March 22nd, 2012, 07:36 AM
Mega Super Mommy
Join Date: Sep 2010
Posts: 1,172
I also use econobum and prefolds with a snappi. I like them but they can be exhausting to do in the middle of the night if he's wiggling around (and we still do night fun times
). They haven't leaked pee but his poop sometimes gets out of control and leaks onto the cover. Although, any diaper would leak with the amount of poop he saves up for us! Hehe.
I also have Smartipants pockets and I use them more lately. You don't have to pull the inserts out; they come out in the wash! I use the basic inserts and add a newborn one from when I had bum genius. They occasionally leak if I don't get them in the right spot on his legs. I have to make sure there isn't a gap near his butt or the pee will drip out if he's on his side (while nursing).
Bum genius worked okay for my first son but after a while the velcro stopped sticking and the elastic wore out on the legs. I think I washed them wrong!
I had my rainbow baby on December 14, 2011. Now my family is complete!
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HALO investment, construction and providing cemetery service joint stock company.
Environment analysis
Corporate strategy
Business plan
Halo cemetery park
1. Trading name
HALO investment, construction and providing cemetery service joint stock company.
VND 1,000 billion
- Headquarter: Hanoi
- Location of cemetery:
Ba Vi district, Hanoi.
- Cemetery square: 17ha
Providing Hanoi metropolitan and the surrounding areas with many services for people to celebrate the life of the individual when it ends and for the living to gather social support to bring closure and move ahead with their lives.
In the short-term :
Mobilize capital from the investors.
Complete essential infrastructure to attract the attention of customers.
Approach the targeted market.
In the long-term :
Tie the company into the community of the living and develop positive associations.
Develop a strong position by creating unique and high standard services. These efforts will be supported by a publicity campaign in order to be the first in people's minds with this new category of cemetery services provider.
Serve the owner's interest in helping others and improving the community.
The mission of Halo is fulfilling customers' expectations which is supreme in our objectives, demands responsibility, accountability and commitment of highest order and providing new burial space and other services; orienting people towards a new image of cemetery and eliminate taboo notions or fear referring to the cemetery.
External analysis
The political and the legal environment provide our company with some advantages and requirements as well.
(Government decree 35/2008/NĐ-CP).
The Vietnam macroeconomic is developing. The GDP per capital is growing rapidly ( GDP per capital is USD 1,596 in 2012). The life standard is improved result in the spiritual life is more concerned.
On the other hand, public cemeteries are more and more degraded and the cemetery services are not diversified.
Forms of handling with human remains :
- Traditional internment : buried three years or more then reburied, without any environment protection method.
- Cremation
Status of cemetery and technical infrastructure
All the examined cemeteries had no drainage system and water repellent treatment. In many cemeteries, internal road systems are almost none, also the lack of support facilities such as fences, the spirit, the site management, parking, water supply systems, etc.
Environment concern :
The cemetery environment is one of the most concerned problems of the investors and the society as well.
The Vietnamese consider the death ritual as one of the most important things in spirit life. Cemetery is a scared place that contain cultural, civilization of nation that is organized by a certain social order, with regulations about religion, custom and habits. . We need to consider cemetery as a part of nation's architecture.
Industry development stage
Embryonic stage
Overview about the industry
Threat of new entrants
The growth of the industry is slow and standstill.
Existing companies in the sector are strategically expanding into new geographic market area.
Power of suppliers
Conduct strategic cooperation with some suppliers in raw materials to reduce supplying costs, improve the quality of materials and tighten cost saving for both parties.
Intensity of rivalry
Staging advertising battle.
Increasing customer guaranties or services.
Strategic group: closet competitors: An Lac Vien, Lac Hong Vien.
The customers can choose many kinds of ways to deal with the human remains with variety level of prices and quality.
Weight table for
external factors
The evaluation of resources and capabilities
Competitive advantages
We determine that the Halo's competencies are :
● Diversified and unique services
● Great location, landscape and architecture
● Using high technology to provide the best services and protect the environment
● Understanding on culture and traditional rites
Weight table for internal factors
Tangible resources
Finance: stable capital and borrowing capacity
Physical resources:
A nice location is very important.
We invest extensive land with beautiful scenery and convenient transportation. We also focus on the Feng Shui factors and the architecture.
High quality standard: follow strictly procedure in construction and use good quality material in order to make durable grave
Technological resources: Applying latest technology to provide unique cemetery services and to help protecting environment.
Intangible resource
Human resources:
Manager team are people who not only have knowledge of
management and finance to invest and construct cemetery but also have enthusiasm towards work and motivate employee.
Staff team are hard-working, creative and always enthusiastic to serve customers.
Innovation resources: We are always acquire and learn experiences and ready to innovate the services . We always try to understand to meet customer requirements for the services.
Design creativity
Research what customer needs and offer many options of design for customer to choose. We hire design team to create unique design.
Supply chain
Use the just-in-time inventory and establish long-term relationship with supplier. Cemeteries using materials such as brick, stone, gravel that are susceptible to mold so should use JIT technology. Reduce construction time.
Company understand Feng Shui, culture and customer needs to bring out the best advice to customers
Business level
Business level
Burial spaces
Pre burial services
Rites of spirit
High market segmentation approach
A different product is offered to a market segment
to determine which customers we should serve in this industry.
WHO (targeted customers)
Those who need high standard burial spaces
Targeted customers will be those who mostly are living Hanoi and nearby, have the high income
WHAT (Customers' needs)
- Good location with convenient facilities.
- Good landscape
- High standard services after the interment
- Meet other special requirements such as: spirit reasons, the social position, etc,.
Diversified products
Location, landscape and architecture
- Located in the West
-The grave's architecture and the space surrounding will be modern and traditional.
-Including an artificial scream and some penjings and many green trees and flowers
- Meeting the Feng Shui requirements but also try to bring fresh air, beautiful sightseeing for the relatives and visitors.
-Divided into 5 areas (each area will include a hill)
Technology will specialize in solving water and soil contamination. The grave will be built following the "Kim Tinh" method, waterproofing technology. The bottom and 4 sides of the grave are built in bricks and cement , 20 centimeters thick. All the coffins will be heaped by the tea and the chemicals to deodorize, etc. We also strictly follow the procedure in construction and use good quality material in order to make durable graves.
Supply Chain
Just-in-time inventory and establish long-term relationship with supplier.
Faster Procedures
All the referendums faster and more efficient. The land and the grave will be provided as soon as they need.
Attentive management services
Those who need a different way from interment. We determine that our targeted customers are also customers who have the high income
What (customer's needs):
- Faster services
- Prefer the body to be returned quickly and cleanly to the elements
- Having environmental concerns
- Having a special way, which can help them to store the ashes of the relatives beside them.
Unique products
The cemetery will use the latest technology in cremation, for example, our company will use imported the crematory equipment from developed country; To offer unique services in this segment, we also use some special chemical or method, which are also imported from other country.
Location and spaces
We also provide the cremation services in the cemetery's campus. So the location also very convenient to travel there to get the services. Our company will also offer the grave to store ashes like the interment services. The customer can choose to store the ashes in a box, which are placed in a modern building. So the customer can get all the convenience as the interment services.
Those who need pre- burial services for their relatives and want to have the best services for them .
Targeted customers are also customers who have the high income
-The relatives want the most well preparation for the dead before they leaving this world.
-They want the dead have the best thing in other world ( has the best casket, the best image, etc.)
Like other services, our company will also
use the differentiation strategy for this service. We will offer many special services such as "make- up" for the dead, and wash the body with scent liquid, etc. We will also to offer the best coffins with special design and other services as the customers' requirements.
Those who are using the burial space of our cemetery.
The relatives of the dead are living far away or aboard.
Targeted customers are also customers who have the high income.
-We have a human resource, who have great knowledge on the Vietnamese traditional rites for the dead
-Provide the best rites of services. (memorial services, and other special events)
-Film all the process we make the rites and send to the customers.
-Carry out the rites online – the customers can easily watch what we do through internet.
The customer's needs : Have the best traditional rites to the dead as they were living in the country.
Strategic alliances
Advantages of strategic alliances
Facilitates investment in specialized
assets: In order to create a meaningful
landscape, we need to buy unique
statue representing for a specificmeanings.
Just-in-time inventory systems:
Integrating with tombs suppliers enables us the ability to
response specific order quickly. Moreover, tropical weather in Vietnam depreciate our inventory.
Disadvantages of strategic alliances
Loss of control: The business that we team up with is going to want at least some control of the product demand and leads to potential alliances fall apart. To overcome this difficulty, it is vital that we enter into a written agreement when entering a strategic alliance
Reduce price of input
Control the quality of input so that
final products are more durable and suit with unique customer's order
develop long-term relation because
our products take a long time to
be sold
Horizontal integration
Achieve the competitive advantages from larger size and scope of operation
Opportunity to serve a number of new, complement areas, while enable us to capitalize on significant synergies and operational efficiency.
Advantages of
Horizontal integration
Reduces rivalry within the industry
Increasing bargaining power over
suppliers and buyers.
Apply successful approach in solving water ground contamination. This action strengthens our brand and allow us charge a higher price as well as boost demand of our products.
Disadvantages of
Horizontal integration
Overestimate the benefits and underestimate
the problems:The pollution in these cemeteries may be serious that requires a huge investment in environment protection and can results in reducing value of our company.
Functional Strategy
Marketting Strategy
Our company will carry out the marketing strategy following some rules:
Market research:
We carry out the market research to understand deeply the needs of the customers and catch up with the customers' trends.
Official website of the company
Seminars , organizing cemetery tours
Concentrating on consultant services
for potential customers.
Financial Strategy
From the owners
Get loan from bank
Capital contribution from
Main sources of funding
Message from
After analyzing the macro-environment, industry environment, and our orientation and objects of our future business plan. We has determined our competencies and the strategies to develop the business entity. We believe that these strategies will work out in reality.
We hope that the establishment of HALO will contribute a great effort in resolving the lack of high standard cemetery services of Hanoi in particular and surrounding areas in general. HALO will not only focus on profit but also try to have a great effect on the customs of Vietnamese in dealing with human remains. HALO also ensures that we will operate well to protect the environment
We, the co founders of HALO company believe that we can success and gain profit in this plan. We hope that the authority and the citizens will support this plan and the investors, the banks will realize the feasibility of this project and consider to invest in .
Sincerely yours
Co founders
Thanks for listenning!
2. Capital
3. Location
4. Aims of the company
5. Objectives of the company
Meet current and future needs in respect
of burial options, while providing accessible green space and encouraging biodiversity and sustainable environmental practices.
Provide a supportive and inclusive service that fully reflects the religion and culture of the area and supports the wellbeing of the bereaved.
7. Vision
6. Mission
Cemetery services industry is "the industry which includes
establishments that operate sites or structures reserved for the interment of human or animal remains. The industry also includes companies that offer cremation services.
Type of cemetery in Vietnam :
Services in Vietnamese cemetery
Almost the cemeteries do not have cemetery services. Every task related to the graves has to been done by the owners. Only some cemeteries provide some set of services but they are not diversified do not meet the needs of almost customers.
" Death is not the end but is the final stage of one life to be transfered into another."
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Although copper is an essential metal, it is capable of catalyzing the formation of reactive oxygen species that can cause intracellular oxidative damage. We investigated the hypothesis that metal- and oxidative stress-responsive signal transduction pathways mediate the cellular and molecular responses associated with copper exposure. Transient transfection assays using COS-7 cells and mouse metallothionein-I (MT-I) or rat NAD(P)H:oxidoreductase 1-based reporter genes demonstrate that copper activates transcription via metal and antioxidant response elements. Concomitant with copper exposures is a decrease in the level of total glutathione and an increase in oxidized glutathione. Depletion of glutathione, before copper exposure, increases metal- and oxidative stress-inducible transcription and cytotoxicity. Pretreatment with the reactive oxygen scavengers aspirin or vitamin E provides partial protection against copper toxicity and reduces inducible transcription. Experiments using signal transduction inhibitors and a metal transcription factor (MTF)-1 null cell line demonstrate that copper-inducible MT-I transcription is regulated by protein kinase C and mitogen-activated protein kinase signaling pathways and requires MTF-1. The results of these studies indicate that copper activates transcription through both metal- and oxidative stress-responsive signal transduction pathways.
- metallothionein
- signal transduction
the transition metal copper serves an essential role in biological processes because of its catalytic and structural properties. It is critical for cells to maintain homeostatic concentrations of this metal, because abnormally high or low levels can lead to pathological conditions. Pathologies associated with copper toxicity include gastrointestinal disorders and liver cirrhosis (3). On a cellular level, the pathological effects associated with the accumulation of excess copper are consistent with oxidative damage of lipids, proteins, and nucleic acids.
Copper is a redox-active metal that is able to catalyze the formation of hydroxyl radicals via a Haber-Weiss or Fenton-like reaction (19, 44, 50). In addition, copper can be toxic by directly binding to sulfhydryl groups in proteins, which results in enzyme inactivation or altered protein conformation (26). The wide variety of adverse effects attributed to excess copper is likely to be due to the various mechanisms by which this metal can induce intracellular damage.
Exposure to toxic concentrations of copper will produce an intracellular stress response. The copper-induced stress response involves altered transcription of multiple genes, which are responsible for maintaining metal homeostasis and protecting cellular components from damage (55). To help maintain copper homeostasis and scavenge toxic by-products of copper exposure, cells express metallothioneins (MTs). Metallothioneins are small, cysteine-rich proteins that are ubiquitous among eukaryotes (21, 27). Proposed functions of MTs include maintaining homeostasis for essential metals such as zinc and copper, cellular detoxification, and scavenging free radicals. Elevated concentrations of many transition metals have been shown to elicit rapid induction of MT mRNAs and proteins (21, 23, 27, 35, 42). In addition, a variety of nonmetal stressors such as heat shock, alkylating agents, oxidative stress, and UV radiation induce MT transcription (9, 38, 40, 43, 48). This suggests that MT may function as a general stress-response protein.
Metal-inducible MT transcription is regulated primarily through the interaction between metal response elements (MREs) and metal transcription factor (MTF)-1 (22, 37). MREs are 13- to 15-bp upstream regulatory elements with the core consensus sequence CTNTGCRCNCGG that are found in the promoter region of most metal-inducible MT genes (47). MTF-1 specifically binds to the MRE and has been shown to be essential for the metal-inducible transcription of MT (22, 37). Although MTF-1 is essential for copper-inducible MT transcription, the interaction among MREs, MTF-1, and copper has not been defined.
Additional cis regulatory elements that also regulate inducible MT transcription are the antioxidant response elements (AREs), which have a core consensus sequence of TGACNNNGC (7). AREs regulate oxidative stress-inducible transcription of many genes, including glutathione S-transferase (GST) and NAD(P)H:quinone oxidoreductase (NQO1), and several MTs (7, 24). The ARE of the GST Ya gene is activated in response to a variety of chemical agents, including metals, β-naphthoflavone, t-butylhydroquinone (tBHQ), butylhydroxyanisol, and hydrogen peroxide (11, 31–33, 49, 52). The ARE-mediated induction of detoxification genes is thought to be a critical mechanism involved in protecting cells from challenges by electrophiles and reactive oxygen species (ROS). The ability of redox cycling transition metals to activate MT transcription through the ARE has not been fully addressed.
One mechanism by which the transcription of detoxification genes are activated is via mitogen-activated protein kinase (MAPK) pathways, which include extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK), and p38. Many forms of cellular stress preferentially activate the JNK/SAPK and p38 pathways (16, 29). In human bronchial epithelial cell line BEAS 2B cells, exposure to copper, vanadium, arsenic, zinc, or chromium results in the phosphorylation and subsequent activation of JNK, p38, and ERK (39). Once activated, ERK, JNK, and p38 can phosphorylate a number of proteins and transcription factors. This results in the enhancement of the transcriptional activity of a multitude of genes (28).
Copper has the potential to mediate several important biological processes through multiple signal transduction pathways. However, the cellular and molecular responses underlying copper-regulated gene expression and toxicity are poorly understood. In this study, we report that copper increases the transcription of MRE- and ARE-containing genes. Transcriptional activation by copper involves MAPK pathways and changes in cellular glutathione status. Results from this study suggest that copper is capable of activating transcription through both metal- and oxidative stress-mediated mechanisms.
Cell culture. COS-7 (ATCC CRL-1651) and MTF-1 null cells (dko7) (37) were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 5% nonessential amino acids, 2 mM l-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. Cells were maintained at 37°C with 5% CO2.
Reporter gene constructs. The level of MT transcription was established by measuring the amount of chloramphenicol acetyltransferase (CAT) produced in cells transfected with –42CAT, which contains the minimal mouse MT-I promoter (–42 to +62); –153CAT, which contains the region of the mouse MT-I gene from –153 to +62; MREd′5CAT, which contains five tandem copies of MRE-d′ inserted upstream of TATA box in –42CAT; and ARE4CAT, which contains four tandem copies of the upstream stimulatory factor (USF)/ARE from the mouse MT-I gene inserted into –42CAT. The –42CAT plasmid contains a TATA box but lacks metal- or stress-responsive regulatory elements (i.e., negative control). In contrast, –153CAT contains 153 bp of the mouse MT-I regulatory region and contains four functional MREs, as well as an ARE. Complete descriptions of the MT-CAT reporter genes can be found in Dalton et al. (7).
The level of oxidative stress-responsive transcription was measured by using a CAT reporter gene that contains the ARE from the rat NQO1 gene (pARE). pARE consists of a single copy of the ARE from NQO1 fused to a rat GST minimal promoter. As a negative control, a two-nucleotide mutant form of the ARE, which is unresponsive to oxidative stress, was used (pM1) (10, 11). The pM1 plasmid is identical to pARE with the exception of a mutation of the GC box in the consensus ARE sequence (GTGACNNNGC).
Transient transfection and reporter gene assays. For transient transfection studies, cells were grown to ∼60% confluence in complete DMEM in 24-well dishes. The medium was removed, and cells were washed with Opti-MEM and then transfected using Lipofectin (COS-7) or LipofectAMINE (dko7), according to the manufacturer's instructions (Life Technologies). Cells were transfected for 3–6 h with 650 ng/well of one of the CAT reporter plasmids and 160 ng/well of the control plasmid pSV-βgal (Promega). To inhibit JNK activity, cells were cotransfected with pcDNA3-Flag-MKK4 (Ala), which expresses a dominant negative form of the JNK-activating kinase MEK-4/SEK-1, thereby inhibiting JNK phosphorylation and activation (51). In experiments utilizing the dominant negative SEK plasmid, cells were transfected with 650 ng/well of CAT reporter plasmid, 160 ng/well of pSV-βgal, and 300 ng/well of pcDNA3-Flag-MKK4 (Ala).
After transfection, the medium was replaced with complete DMEM, and the cells were allowed to recover for ∼12 h. Copper (as CuSO4), ROS modulators, or signal transduction inhibitors were then added as described above. CAT concentrations and β-galactosidase activities were determined by sandwich ELISA, using a CAT-ELISA kit (Roche Biotechnologies) and the β-Galactosidase Enzyme Assay System (Promega), respectively. All assays were performed in triplicate, and CAT protein levels were normalized to β-galactosidase activity.
Cytotoxicity. Cytotoxicity assays were performed as described by Shokri et al. (45). Briefly, cells were seeded in a 48-well tissue culture dish in complete DMEM and incubated for 24 h. Copper sulfate was then added, and incubation continued for an additional 24 h. In experiments in which glutathione was depleted, cells were exposed to 1 mM buthionine sulfoximine (BSO) for 8 h before the addition of copper. After copper exposure, the medium was removed and the cells were then incubated with neutral red solution (40 μg/ml) in DMEM for 3 h at 37°C. The cells were subsequently washed and then fixed with calcium chloride (1%, wt/vol) in formaldehyde (0.5%, vol/vol). To extract the dye from viable cells, cells were lysed with acetic acid (1%, vol/vol) in 50% (vol/vol) ethanol. Optical density was then measured at 540 nm (Abs540). Positive controls, representing 100% cytotoxicity, consisted of cells exposed to distilled water. Cells incubated with complete DMEM in the absence of copper represented 100% viability and were included as a negative control. The level of cytotoxicity was calculated by using the following calculation:
Glutathione measurements. Cells were grown for 72 h and then incubated in the presence of copper for an additional 24 h. Extracts were prepared from phosphate-buffered saline-washed cells by sonicating cell pellets suspended in 0.5 ml of 20 mM sulfosalicyclic acid. The lysate was centrifuged at 10,000 g for 2 min, and the supernatants were collected. Total glutathione (GSH + GSSG) and GSSG concentrations were determined by using the enzymatic recycling assay for plate readers (1, 2).
Statistical analysis. All statistical analysis was performed using StatView software (SAS Institute). Results are presented as means ± SE. The significance of mean differences was detected by analysis of variance (ANOVA) followed by Fisher's protected least-squares difference post hoc test for individual comparisons. The criterion for statistical significance was set at P < 0.05.
Effect of copper and glutathione depletion on cell viability. The effects of copper exposure and the depletion of glutathione on COS-7 cell viability were determined. Significant decreases in cell viability were observed at copper concentrations of 400 μM and above (Fig. 1). The viability of cells exposed to 400 μM copper and depleted of GSH was significantly lower than cells treated with 400 μM copper alone (40 vs. 71% viable cells, Fig. 1). This finding suggests that glutathione protects the cell from copper-inducible stress. Depletion of GSH in the absence of copper did not significantly affect cell viability.
Effect of copper on glutathione levels. Exposure of COS-7 cells to 400 μM copper for 24 h caused a significant decrease (35%) in total glutathione (oxidized plus reduced) (Table 1). Treatment of cells with BSO resulted in a 93% decrease in total glutathione. In addition, treatment with BSO and 400 μM copper caused a 97% decrease in total glutathione. This level of depletion was significantly greater than that observed with BSO alone.
The ratio of reduced to oxidized glutathione is commonly used as an indicator of intracellular oxidative stress. In untreated cells, oxidized glutathione accounted for 1.5% of the total glutathione. This value significantly changed when cells were treated with 400 μM copper for 24 h or when they were exposed to BSO for 8 h (Table 1). Likewise, cells exposed to 400 μM copper following GSH depletion showed a significant, synergistic increase of the level of oxidized glutathione (Table 1). These results suggest that exposure to copper alters the intracellular redox potential. It should be noted, however, that this observation is confounded by the high degree of toxicity observed in cells exposed to both copper and BSO (Fig. 1).
Effect of copper on metal- and oxidative stress-responsive transcription. The ability of copper to induce MT transcription (i.e., MT transcriptional activity) was investigated by using CAT reporter genes whose expression is regulated by portions of the mouse MT-I promoter, or concatenated copies of MT upstream regulatory elements (7). COS-7 cells, transfected with MT-I-based reporter plasmids, were exposed to 0–600 μM copper for 24 h. Copper at any concentration did not significantly induce transcription when assayed using the –42CAT reporter gene (i.e., negative control; Fig. 2A). In contrast, significant increases in transcriptional activity were observed at copper concentrations of 400 and 600 μM in cells that contained the –153CAT, MREd′5CAT, and ARE4CAT reporters. The maximum response (3.5-fold increase) was observed at 400 μM in cells transfected with ARE4CAT. The decrease in reporter gene activity that was observed at higher concentrations may be attributed to the cytotoxicity observed at these metal concentrations (Fig. 1).
The increase in ARE4CAT transcriptional activity reported above suggests that copper may activate transcription by an oxidative stress-responsive mechanism. The ARE in the mouse MT-I promoter, however, overlaps with a USF sequence. The USF element may influence the responsiveness to copper exposure. To further define the role of oxidative stress in copper-inducible transcription, plasmids containing an ARE from the rat quinone reductase gene were used. A 24-h exposure to 400 μM copper caused a 5.5-fold increase in reporter gene expression in cells transfected with pARE (Fig. 2B). The addition of copper did not cause an increase in the level of reporter gene expression in cells transfected with the negative control plasmid pM1 (Fig. 2B).
MTF-I involvement in copper-induced MT transcription. To further investigate the role of metal-responsive gene regulation in copper-induced transcription, the effect of copper exposure on reporter gene activity was examined in the MTF-1 knockout cell line dko7 (18). Cells were transfected with –42CAT, –153CAT, MREd′5CAT, or ARE4CAT plasmids and then exposed to 600 μM copper for 24 h. There was no significant induction of –153CAT and MREd′5CAT expression in dko7 cells, which indicates an essential role for MTF-1 in copper-inducible transcription, via the MRE (Fig. 3). In contrast, there was a significant (5.3-fold) increase in the level of expression when ARE4CAT was used. This finding suggests that copper also activates transcription by an oxidative stress-response mechanism. To further investigate the roles of AREs and oxidative stress in copper-inducible transcription, dko7 cells were transfected with pARE and pM1. Consistent with the earlier results, copper exposure resulted in a significant (4.6-fold) increase in pARE reporter gene expression. These results indicate that both metal- and oxidative stress-responsive mechanisms contribute to copper-inducible transcription. Furthermore, these pathways can function independently.
Effects of GSH depletion on copper-inducible transcription. Treatment of COS-7 cells with BSO caused a >90% depletion of total glutathione (Table 1). A significant increase in the level of copper-inducible expression was observed when glutathione levels were reduced before copper addition, compared with copper-exposed cells that were not pretreated with BSO (Fig. 4A). The depletion of glutathione alone caused a significant increase in the level of reporter gene expression (Fig. 4A). Thus glutathione depletion did not significantly increase the level of-153CAT and MREd′5CAT reporter gene expression. Statistical analysis shows that there was not a synergistic interaction between copper and BSO for any of the reporter genes.
In cells transfected with ARE4CAT (5.2–6.8 fold), a significant increase in transcription was observed, which suggests that an ROS-mediated mechanism may be involved in copper-activated transcription. To confirm the involvement of ROS, cells were transfected with pARE and pM1 plasmids. Consistent with the above results, glutathione depletion increased copper-inducible pARE expression compared with cells exposed to copper alone (Fig. 4B). However, the degree of induction for the pARE plasmid (1.5-fold) was much less than was seen for the ARE4CAT plasmid. In addition, reporter gene expression in the absence of added copper was higher in BSO-treated cells for all four MT plasmids compared with cells not treated with BSO. These results suggest that the levels of ROS generated in the absence of glutathione are sufficient to activate MT transcription via the ARE-mediated pathway.
In cells transfected with the –42CAT reporter, gene expression was significantly higher than in cells exposed to BSO and copper compared with cells treated with copper alone (4.1- to 4.4-fold). The same effect, however, was not observed in cells transfected with the pM1 plasmid. This finding indicates that GSH depletion increases the basal level of transcription.
Effects of ROS scavengers and antioxidants on copper-induced transcription. Previous experiments demonstrated that pretreatment of cells with the ROS scavengers aspirin and vitamin E partially protects cells from the cytotoxic effects of transition metals (34). Aspirin and/or vitamin E had no effect on levels of expression observed using the –42CAT reporter (Table 2). Aspirin or vitamin E treatment had no significant effect on the level of –153CAT expression, relative to the level of expression observed with copper alone. In combination, however, aspirin and vitamin E caused a 10.6% increase that was significantly higher than treatment with copper alone.
Pretreatment of cells with aspirin, vitamin E, or the combination of the two before copper addition significantly decreased MREd5 ′CAT expression (∼12.5% for each). Similar results were obtained in cells transfected with ARE4CAT. Aspirin caused a 36.3% decrease in copper-inducible transcription, vitamin E caused a 26.5% decrease, and in combination, aspirin and vitamin E caused a 26.5% decrease. The combination of aspirin and vitamin E was not additive.
Effects of signal transduction inhibitors on copper-induced transcription. COS-7 cells were treated with kinase inhibitors to identify potential signal transduction pathways that may regulate copper-inducible transcription. The protein kinase C (PKC) inhibitor H-7 was used to determine the role of PKC in the transcriptional response to copper. Treatment with 100 μM H-7 completely blocked copper-induced –153CAT, MREd5 ′CAT, and pARE expression (Fig. 5). Copper (400 μM) did not significantly change the level of transcription of ARE4CAT during the 4-h incubation used in this and subsequent inhibitor studies. Time-course studies (results not shown) have shown that a 4-h exposure to 400 μM copper is insufficient to induce ARE4CAT reporter gene expression, whereas a 24-h exposure as used above induces significant levels of expression.
MAPK inhibitors were used to investigate the role of ERK1/2, p38, and JNK signaling pathways on copper-inducible gene expression. ERK1/2 and p38 were inhibited by the chemicals PD-98059 and SB-203580, respectively. JNK inhibition was achieved by transfecting the cells with a plasmid expressing a dominant negative form of SEK-1 that prevents the phosphorylation and activation of JNK (51). Kinase inhibitors were added individually (Fig. 6) or in combinations (Fig. 7). Inhibition of p38 kinase caused a significant increase in copper-inducible expression of –153CAT and MREd5′CAT. Similarly, inhibition of JNK activity caused an increase in –153CAT. The inhibition of ERK1/2 activity did not significantly affect copper-inducible transcription of any of the reporter genes.
Exposure to the combination of ERK and p38 inhibitors caused a significant decrease in the level of copper-inducible expression of –153CAT and MREd′5CAT. Similarly, inhibition of ERK in combination with either p38 or JNK inhibition caused a significant decrease in copper-inducible expression of –153CAT and MREd5′CAT. In contrast, the combination of JNK and p38 inhibitors caused a small but significant increase in –153CAT and MREd5′CAT. Simultaneous inhibition of the three MAPKs caused a significant decrease in copper-inducible expression of –153CAT and MREd5′CAT. In all of the inhibitor studies, treatment with inhibitors alone did not significantly affect reporter gene expression (data not shown).
The ability of several transition metals and oxidative stress to induce MT transcription has been well established (4, 7, 8). Studies of MT transcription, however, have primarily focused on the ability of zinc and cadmium to active gene expression through interactions between MTF-1 and MREs. In addition, the mechanism by which MT expression is induced by ROS has also been examined. Oxidative stress, produced by exposing cells to hydrogen peroxide or tBHQ, activates transcription through MREs. The USF/ARE sequence in mouse MT-I mediates induction by hydrogen peroxide and cadmium but not by zinc and tBHQ (7, 8). Unlike zinc and cadmium, copper can directly catalyze the formation of ROS. Thus copper has the potential to affect transcription by two separate mechanisms: a metal-responsive pathway and an oxidative stress-responsive pathway. The results presented in the current study provide evidence that copper is able to induce transcription via both pathways.
Copper activation of transcription via a metal-responsive pathway was demonstrated using reporter genes in wild-type and MTF-1 null cells. Copper activates transcription via the MRE in a concentration-dependent manner (Fig. 2). In addition, copper did not induce MRE-mediated transcription in the absence of MTF-1 (Fig. 3.). These results are consistent with those previously observed in BHK cells containing a MRE-βGeo reporter gene (36). Copper has also been shown to increase transcription factor binding to the MRE in the human MT-2A gene (20).
Several experiments indicate that copper activates transcription by inducing oxidative stress and that the oxidative stress response is independent of the metal response. First, exposure to copper induces transcription of reporter genes containing AREs from MT-I and NQO1 (Fig. 2). The magnitude of the response is attenuated when cells are exposed to chemicals that reduce ROS (Table 2) and increases following glutathione depletion (Fig. 4). The observation that ROS-mediated gene expression occurs in the absence of MTF-1 suggests that the metal-responsive transcription machinery is not required for ROS-activated transcription.
Copper can induce oxidative stress by two mechanisms. First, it can directly catalyze the formation of ROS via a Fenton-like reaction (19, 44, 50). Second, exposure to elevated levels of copper significantly decreases glutathione levels (Table 1). An underlying mechanism of copper- and cadmium-induced oxidative damage is the depletion of glutathione. Depletion of intracellular GSH has also been shown to increase the cytotoxic effects of other classes of xenobiotics (5, 15, 46). A decrease in glutathione would allow the endogenous levels of ROS to become cytotoxic. The large increase in copper toxicity due to GSH depletion (Fig. 1) demonstrates that GSH contributes to the cellular defense against copper toxicity.
Depletion of glutathione by BSO treatment causes an increase in oxidative stress responsive MT transcription (Fig. 4). Glutathione has multiple functions in intracellular copper metabolism and detoxification. It has been proposed that glutathione quickly binds the metal following uptake. Once copper is bound, the metal can then be transferred to other proteins (13, 14). Glutathione also has several roles in copper detoxification. It can ameliorate copper toxicity by directly chelating the metal. Binding by the thiol group can protect cells by maintaining copper in a reduced state, making it unavailable for redox cycling. Glutathione is also a substrate for several enzymes that remove ROS as well as a hydroxyl radical scavenger. The depletion of glutathione may allow the metal to remain in a state that is 1) a stronger inducer of metalresponsive transcription and 2) more catalytically active, thus producing higher levels of ROS, which activates oxidative stress-responsive transcription.
Additional evidence that copper activates transcription via ROS was demonstrated in studies in which cells were treated with aspirin or vitamin E. Treatment with aspirin or vitamin E led to decreases in copper-induced transcription, using MREd′5CAT or ARE4CAT reporters (Table 2). The decrease was greater for ARE4CAT, which is consistent with reduction in oxidative stress. In addition, the level of reporter gene expression did not change when aspirin or vitamin E was added simultaneously with the metal, suggesting that the effects of copper were primarily intracellular (results not shown). The mechanisms by which aspirin and vitamin E treatment reduce copper-inducible transcription are currently unclear. Vitamin E is a lipid soluble vitamin that acts as an antioxidant in cells to interrupt the propagation of lipid peroxidation in the plasma membrane (6). Treatment with vitamin E may reduce the formation of secondary products of lipid peroxidation that can potentially trigger signal transduction pathways. The effects of aspirin are likely due to a different mechanism. Salicylic acid is commonly used as a trap for hydroxyl radicals, which suggests that aspirin may act as a radical scavenger. Aspirin also possesses some degree of metal-binding ability and may thus withdraw copper ions from the site of ROS formation.
To identify potential signal transduction pathways that may mediate copper-activated transcription, the effects of kinase inhibitors on metal-activated transcription were examined. Inhibition of PKC activity prevents both ARE- and MRE-mediated transcription (Fig. 5). This observation suggests that copper activates both metal- and oxidative stress-responsive cascades through signaling pathways that are regulated by PKC. This is similar to previous results that demonstrated the involvement of PKC in metal-inducible transcription, which is activated by zinc and cadmium (30, 41). In addition, it has been shown that inhibition of PKC in Chinese hamster ovary cells blocks MT mRNA transcription, suggesting that PKC is essential for MT expression (53). The inhibition of ARE-mediated, copper-inducible transcription is consistent with PKC regulation of the oxidative stress response (17).
MAPK inhibitors were used individually and in combinations to identify the MAPK pathways that may be involved in regulating copper-inducible transcription. Copper is capable of activating the three MAPK pathways (ERK, p38, and JNK), often in a cell type-specific manner (25, 39). Inhibition of ERK1/2 did not have a significant effect on copper activation of –153CAT and MREd5′CAT reporter genes (Fig. 6). Inhibition of JNK/SAPK or p38 activity led to an increase in copper-inducible transcription, which suggests that JNK/SAPK and p38 may be negative regulators of the response to copper. Whereas ERK1/2 inhibition alone had little effect, in combination with p38 inhibition there was a significant decrease in reporter gene activity (Fig. 7). A similar response was observed when ERK1/2 and JNK/SAPK were both inhibited. Inhibition of both JNK/SAPK and p38 led to a significant increase in copper activation, consistent with inhibition of either pathway alone. Inhibition of all three kinases led to a strong reduction in copper-inducible transcription, which suggests that ERK1/2 has a dominant effect over both p38 and JNK (Fig. 7). Comparable results were obtained when the three MAPK pathways and zinc- and cadmium-inducible transcription were examined (unpublished data). It has been suggested that ERK is capable of attenuating both p38 and JNK activity through its activation of MAP kinase phosphatase-1 (MKP-1). ERK phosphorylation of MKP-1 thereby activates it to dephosphorylate p38 and JNK, resulting in an attenuation of their activity (12). These findings may explain the decreases in copper-induced MT transcription in response to the inhibitor combinations (Fig. 7).
The kinase inhibitor studies are consistent with a model in which copper activates a MAPK signal transduction cascade to ultimately affect the level of MTF-1 phosphorylation to induce transcription. This model is similar to that previously reported for cadmium and zinc (41). It is unlikely that copper is directly binding to MTF-1 to activate transcription; however, an alternative model suggests that copper may displace zinc from MT (54). The "free" zinc would then bind to a zinc finger in MTF-1 that would allow the transcription factor to bind to the MRE and activate transcription.
The results presented in this report support a model in which copper activates transcription via both metal- and oxidative stress-responsive mechanisms. Copper is able to activate transcription through MREs and AREs. In addition, glutathione modulates the level of metal- and oxidative stress-inducible transcription. In COS-7 cells, the ERK1/2 pathway exerts a dominant effect relative to p38 and JNK in regulating metal-inducible MT transcription. Both metals and ROS can independently affect the activity of members of the three MAPK signaling pathways. Thus the roles of specific kinases in regulating transcription in response to these activators may be overlapping. Furthermore, there are additional signaling pathways (casein kinase II, calcium-activated kinase) that participate in the regulation of metal-responsive MT activation (41). Additional studies will be required to more adequately assess copper activation of signal transduction and transcription.
We thank Dr. Glen Andrews (University of Kansas Medical Center) for providing the mouse MT-1-based CAT reporter plasmids and dko7 cells, Drs. Truyen Nguyen and Cecil Pickett (Schering Plough Research Institute) for the NQO1 plasmids, and Dr. Roger Davis (Howard Hughes Medical Institute, University of Massachusetts Medical Center) for the pcDNA3-Flag-MKK4 (Ala) plasmid.
This work was supported in part by National Institute of Environmental Health Sciences Grants ES-10356 and 5T32-ES-07031-22.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Copyright © 2004 the American Physiological Society


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Through the largest British banks are almost 740 million dollars. from funds laundered by Russian cyber criminals with ties to the government and the FSB as part of a major international scheme under the code name "global Laundry" ("The Global Laundromat"), The Guardian reports, citing data from the international organization of investigative journalists (Organized Crime and Corruption Reporting Project – OCCRP) Novaya Gazeta and Süddeutsche Zeitung.
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According to documents leaked to the Guardian has, not less than 20 billion dollars. was transferred from Russia in the four-year period between 2010 and 2014, but the actual amount could reach $ 80 billion.
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Related news: the FCO has published an investigation on "corruption schemes," Medvedev
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Notably, the data referenced in the publication, received within the three-year investigation conducted by the police of Latvia and Moldova. They were provided by the sources, who requested anonymity.
In General, the documents contain information on about 70 thousand of banking transactions, including 1 920 operations that passed through UK banks and 373 through US banks.
It was noted earlier that the largest British banks until 2017 can come out of the UK in respect of concerns in relation to the consequences of Brexit.
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FOR years Scotland has been marketed around the world as a tax haven, as jurisdiction with all the corporate secrecy of a Belize or a Panama but also the respectability of a Sweden or a Germany.
At the centre of such advertising is a single kind of corporate entity, a Scottish limited partnership or SLP, a firm whose owners, provided they hide behind shell companies in a more traditional fiscal paradise, can get away with both being secret and paying no taxes.
Herald View: Scandal of our tax haven status
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Nobody noticed this booming little industry until two years ago when it emerged such SLPs were among shell companies used to loot $1 billion from banks in Moldova, one of Europe's poorest countries.
Now Scottish firms routinely form part of money-laundering and tax evasion kits openly offered online. Customers can choose between a menu of, say, Scottish, English or Canadian firms with fake owners, usually corporate entities in traditional fiscal paradises.
For an extra fee you will get power of attorney over all the firms concerned and their patsy directors and the choice of what amounts to an anonymous bank account in Latvia or Estonia to boot. Such a kit can cost just £1600. The registration fee for an SLP is £20.
Herald View: Scandal of our tax haven status
Those manufacturing off-the-peg shell companies are not responsible, in law, for what their customers do with the entities.
But Scottish politicians are alarmed enough to ask counterparts in Westminster to plug the loophole that makes SLPs so attractive to those wishing to hide their wealth.
Today's news that 120 Scottish firms, all but four SLPs, were used to flush billions out of Russia using the now infamous "Laundromat" will add to those worries.
We may never know exactly how much dirty money was drained through the accounts of nominally Scottish businesses that probably only ever existed on paper.
Herald View: Scandal of our tax haven status
Our data comes from several years worth of investigation by the the Organised Crime and Corruption Reporting Project (OCCRP), an alliance of journalists across the Continent that first uncovered the Laundromat in 2014.
Banking records obtained by OCCRP suggest just four Scottish limited companies, now dissolved, accounted for £4bn of laundered dirty money at today's exchange rates. Britain, as The Herald revealed earlier this year, has just six officials responsible for policing its giant register of nearly 4 million companies and other firms.
British "offshore" firms, including SLPs, are marketed overseas as "prestige" products. Criminals seem lured by the UK's mix of soft-touch regulation and a reputation for fair play.
Paul Radu, of OCCRP, who is based in Bucharest, Romania, has seen UK firms crop up time and time again in his investigations.
He said: "For years now, British companies, limited liability partnerships and limited partnerships have been central to some of the biggest fraud cases in Eastern Europe.
"They provided the perfect tool for criminals who used them for stealing billions of dollars and who hid behind the lack of disclosure requirements both in Scotland and England."
Herald View: Scandal of our tax haven status
Until recently, this did not seem to concern UK ministers. One, Simon Kirby, last week in the House of Commons told an SNP MP, Roger Mullin, that he did not believe SLPs were used in the Laundromat.
Mr Mullin said: "This was a quite extraordinary admission that the Government does not realise what has been going on".
The Guardian this weekend first revealed that perhaps half the Laundromat's money had gone through UK corporate entities.
At least two of the UK entities found by The Herald in OCCRP papers were familiar. Now defunct SLPs Sunrise Cotton and MetalForum were also named in the Moldovan fraud probe.


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"A small-scale travelling museum in itself, Greatest Hits is an ongoing work and ever evolving installation which includes a collection of irreverent everyday-life and personal objects but with their names titled originally from wars and military operations. From words to objects and back again, Greatest Hits can be regarded as a three-dimensional dictionary of these operations." Didem Yazici - Curator.
Installation view, 'Afgang' exhibition, Kunsthallen Brandts, Odense, 2010.


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Croatian Post Ltd. Mostar issued four commemorative frogs stamps as a part of special series Fauna 2016. The stamps feature four different species of frog. Frogs belong to the family of amphibians that consist of freshwater and inland vertebrates but …Read More »
UNESCO World Heritage Site Baekje Historic Areas
UNESCO World Heritage Site Baekje Historic Areas is composed of historic sites that reveal the Baekje Kingdom's unique culture as well as its religion and art. The Baekje Historic Areas was recognized for their exceptional value as historic sites and …Read More »
Latvian 19th century Historical Ships stamp – Andreas Weide
Latvian post launched a new series of 19th century Historical Ships stamp with the first stamp dedicated to Andreas Weide. Andreas Weide is the largest transatlantic sailing ship ever built in Latvia. The face value of the new stamp Andreas …Read More »
Oldest Latvian lighthouse stamp – Ovisi Lighthouse
Latvijas Pasts issued the eleventh stamp in the series of Latvian lighthouse stamp dedicated to the oldest lighthouse in Latvia, Ovisi Lighthouse. The print run of the new stamp is 100,000 copies and its face value is € 0.90. The …Read More »
Hindu Festival Diwali commemorated on USPS stamp
The USPS commemorates the joyous Hindu Festival Diwali, also known as Deepavali, a festival of light, knowledge and victory of good over evil. One of the most important annual Hindu events, the holiday celebrates the triumph of good over evil. …Read More »
Special stamp celebrating World Heart Day
Croatian Post Mostar joins in spreading awareness about cardiovascular diseases by issuing a commemorative postage stamp on World Heart Day. The postage stamp is Heart Shaped and very beautifully designed in red colour. World Heart Day The human heart is …Read More »
Sports of Rio 2016 Olympic Games Brazil
The 2016 Summer Olympics, commonly known as Rio 2016 Olympic Games, are the thirty-first Summer Olympic Games to be held at Rio de Janeiro, Brazil. The 2016 Summer Olympics are officially known as the Games of the XXXI Olympiad. The …Read More »


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CodePlexProject Hosting for Open Source Software
I'm missing
Microsoft.JScript.Compiler, -.-Runtime, -.Scripting, -.Scripting.Core, -.Scripting.ExtensionAttribute, -.Scripting.Silverlight.
do you have a download url?
You need to install Silverlight. You can get the SDK here -
http://www.microsoft.com/downloads/details.aspx?FamilyID=2050e580-f1d5-4040-bb09-e6185591b6b5&displaylang=en. If you have Visual Studio you can get the tools here -
I installed Silverlight, SL-SDK, Visual Studio Tools, but I'm still missing Microsoft.JScript.Compiler, -.-Runtime, -.Scripting, -.Scripting.Core, -.Scripting.ExtensionAttribute, -.Scripting.Silverlight.
Check the readme in the product file. You need to get the DLLs from the URLs described there. Copy and pasted the pertinent sections here:
First off, you'll need version 0.4 of the Dynamic Languages SDK available here:
Secondly, you'll need a few custom DLLs created for Popfly that we weren't able
to release as open source. The first DLL is a color converter DLL used for
converting color strings as described in XAML, such as "#FF000000", "Red", etc.
We didn't release this because it was copied from the WPF source base. If
someone wants to implement this functionality independently we can remove this
requirement. The second DLL contains some modifications to the jscript runtime
to hack around differences between interacting with Silverlight via the native
jscript API and the managed Silverlight API. These changes aren't particularly
interesting and so no legal review was done to ship them under Ms-PL.
Both these and their accompanying license agreement can be found here:
Are you sure you want to delete this post? You will not be able to recover it later.
Are you sure you want to delete this thread? You will not be able to recover it later.


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While I dont think this woman should be recalled I have to say she refuses to clear the record on the Brandye Mells situation. If she lied about that she should resign ..today
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*In stays of more than 2 days, one massage or lymphatic Drainage is included as a GIFT.
*Tour guide of Madrid area and interpreter services available at additional cost.
*Botox and hyaluronic acid filler injections.
Cosmetic Surgeon
Cosmetic and Aesthetic Surgeon
We offer a comprehensive approach to aesthetic problems of our patients. Our plastic surgeons offer the best solution for each case with the most advanced techniques at the best price.
upon request
Sanitas is a hospital group in Spain with two major facilities in Madrid that accommodates all medical specialties.
The Ruber International Hospital is designed as a "whole hospital". thus achieving maximum efficiency in the organization and development of the various medical, welfare, educational and research activities.
Sant Joan de Déu-Barcelona Children's Hospital is one of the leading medical centers in Europe for childhood and adolescence medicine and offers a comprehensive and multidisciplinary approach to health care from birth through 21 years of age.
A modern (opened in 2007), general, private hospital located in Madrid, part of the NISA group of hospitals. The international patients department can assist patients with accomodation and trasportation, and can communicate in English as well as in Spanish.
The surgeons at Cirumed Clinic Marbella all hold academic positions at the department of Plastic, Aesthetic and Reconstructive surgery of Hospital Quiron Malaga - Spain's largest private hospital. The clinic offers the full range of cosmetic surgeries.
Quirón Hospital of Valencia is ranked as one of the best private schools in Spain, winning nine times in the TOP 20 award in recognition of its management and quality of care.
This information is intended for general information only and should not be considered as medical advice on the part of Health-Tourism.com. Any decision on medical treatments, after-care or recovery should be done solely upon proper consultation and advice of a qualified physician.
What is Dermabrasion?
Dermabrasion (or Dermaplaning), is an aesthetic procedure that uses abrasion, or scraping, to wear down the skin's upper layers. Dermabrasion is carried out with special instruments to remove the surface of the skin.
The purpose of dermabrasion is to remove scars, fine lines and wrinkles and to smooth the skin as a new layer grows back.
Dermabrasion can be carried out on small areas of the face or the whole face.
How is Dermabrasion carried out?
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Finishline Powersports in Richland, Washington can help you find the perfect motorcycle for your needs. Buying a motorcycle is about more than just picking out one that looks good. When you shop with the right company, you can learn all sorts of tricks and useful information that will help you find the perfect motorcycle for your needs.
It's important to look for a premium option that is going to be the right fit for you, whether you have recreational use or to competition racing in mind.
Along with a good looks, you also want a bike with features that match your experience level and budget. It's important to work with a salesperson who cares about your satisfaction. The owner of Finishline Powersports is active in the Richland community, sponsors many motorcross races and plays a big role in the racing scene.
You can expect to meet a friend who you will see at local races when you do business with this Finishline. It's nice to be able to take your time picking out a bike without feeling pressured.
Our motorcycle shop cares more about your satisfaction than selling a motorcycle quickly. If you want to take your time searching for the right bike, Finishline Powersports is for you. If you need a product that isn't in stock, you can receive a ten percent discount off it when we place the order. Most parts and products ordered before 12:00 PM are shipped and arrive by the next business day!
If you're looking to sell or trade in your bike look no further! Fill out the form below and we'll get back to you as soon as possible.


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Know more about this business than we do? Cool! Please submit any corrections or missing details you may have.Help us make it right
Light & Medium Towing
Vehicle Storage
Jump Starts
Medium Truck Towing
Motorhome Towing
Boat Trailer Towing
AAA Emergency Road Service
Motorcycle Towing
Insurance Towing
Fuel Service
Auto & Truck Transportation
SE Habla Espanol
Change or Air Flats
Local & Long Distance
Be the first YellowBot user to add photos or videos of East County Towing
I had my car towed from them and they broke it. Then they lied about doing it to the insurance company. Stay away from this company they are not very honest at all.
They broke my car while trying to tow it and blamed me!! Then they lies to AAA about it. They are the worst stay away!!!!!!!!
Vehicle Storage, SE Habla Espanol, Motorhome Towing, Motorcycle Towing, Medium Truck Towing, Lockouts, Local & Long Distance, Light & Medium Towing, Jump Starts, Insurance Towing, Fuel Service, Change or Air Flats, Boat Trailer Towing, Auto & Truck Transportation, AAA Emergency Road Service
East County Towing can be found at W 10th St 590. The following is offered: Towing. The entry is present with us since Sep 9, 2010 and was last updated on Nov 14, 2013. In Pittsburg there are 4 other Towing. An overview can be found here.
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24 comments found.
Where you able to find anything out on my IronAgeCompany.com site? I sent you my login and password via your contact email on your profile page.
For buyers notice: We resolved the problem.
live preview not working!
The live preview does not work – not really trustworthy..
Theme works fine but I had some issues with my server. I will upload it next week and update the theme.
how can I get the website menu icons to work on an iPhone? when I click on about or contact it does not slide on my phone. using ios 9. thanks.
Sorry for the late reply but the theme doesn't have that feature. Send me a mail and I will update it according to your needs free of charge.


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Orange Crunch Yogurt Recipe
5 1 1
It took mere minutes for the home economists in our Test Kitchen to toss together these parfaits. The crunchy granola and cashews contrast nicely with the mandarin oranges and yogurt.
Recommended: 20 Recipes to Make with Fresh Citrus
VERIFIED BY Taste of Home Test Kitchen
- 1/3 cup granola cereal without raisins
- 1/3 cup flaked coconut
- 1/3 cup chopped cashews
- 4 cartons (6 ounces each) orange yogurt
- 1 can (11 ounces) mandarin oranges, drained
- In a bowl, combine the granola, coconut and cashews. In each of four parfait glasses or bowls, layer half of a container of yogurt, 2 tablespoons granola mixture, 5 or 6 orange segments, remaining yogurt and remaining granola mixture. Garnish with remaining oranges. Serve immediately. Yield: 4 servings.
Originally published as Orange Crunch Yogurt in Quick Cooking January/February 2004, p12
Reviews forOrange Crunch Yogurt
My Review
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Average Rating
Reviewed Jun. 16, 2013
"This was delicious, have it often."


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- Equitable: Universal design features should be usable and beneficial to all residents, not just some–older parents or grandparents, for example.
- Flexible: Universal design elements should be flexible to use. For example, right-handed or left-handed persons should be able to use the same feature. A home's spaces should be usable by individuals in wheelchairs, on crutches, or with normal mobility.
- Simple: Universal design features should be easy to use without causing confusion or unnecessary complexity.
- Error-resistant: Features should have as little potential for hazards or errors as possible. The more frequently used a feature is, the safer, more accessible, and error-tolerant it should be.
- Physically easy to use: Universal design features should require little physical effort to use. Residents should be able to activate or use these features from a neutral, unstressed body position.
- Perceptible: It should be easy to tell when a task has been completed or when a universal design feature has done its job. Information about the feature should be easily identified and distinct from its surroundings.
- Easy to approach and use: There should be plenty of space to approach, reach, and use any universal design features. Line of sight should be clear and reach comfortable. Personal assistive devices or assistance should be accommodated.
Universal Design: 7 Principles That Ensure Elements Accommodate All Ages and Functions
By applying concepts of universal design to your home renovation or construction project, you can ensure that the residence will be usable for most family members for the longest period of time. Here are seven principles of universal design that can help keep a home functional for years.


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Day 2
Today you will be trekking all the way up to Namche Bazaar (3440m), the journey taking around 6-7 hours. The full day trekking will be rewarded by the majestic forests containing fir trees and rhododendrons (blooms during spring), along with crossing 6 suspension bridges and amazing waterfalls.
After an hour walk from Phakding, you will be able to see the spectacular Thamserku peak (6608m). At Monjo, the trek enters Sagarmatha (Everest) National Park, which covers approximately 1200 square kilometres of the Khumbu region. The park has also been declared a UNESCO World Heritage Site Since1979. After 3 hours of walking, lunch will be taken at the base of Jorsalle, from where a climb of 600m awaits to reach Namche Bazaar. The first glimpse of Mt. Everest (8848m), Nuptse (7861m) and Lhotse (8414m) are generally seen upon reaching Toap Hill. From this point, Namche is 2 hours walk. Upon reaching Namche, trekkers can enjoy this vibrant village which features numerous restaurants, bakeries, shops and hotels.


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Wandering Busker Mic and Amp Package - 10W Battery Powered Amp
Portable Practice Pack for Singers and Buskers
By popular demand, SWAMP has put together this microphone package for the budding singer. A simple and easy to use set up that allows anyone to have their voice amplified. As well as being great for novice's starting out, its also a compact option for singers to take to band practice to jam with the rest of the band, eliminating the need to plug in to a full blown PA system.
Great Amp for Singer Songwriters
The Beta Aivin BM6 features a standard guitar input with all the usual controls, as well as an additional dedicated microphone input, meaning you can play amplify guitar and vocals at the same time! With all the fun of inbuilt effects (chorus and delay), and all the control of a 3-Band EQ with sweepable mid frequency, this amp really gives a lot of bang for buck.
Great For Busking
The BM6 can run off 10x AA rechargable batteries, and when a shoulder strap is attached, you can take it traveling with you anywhere (shoulder strap and batteries not included). It has a 3.5mm auxiliary input for iPod, iPhone, Laptop, or MP3 players, so you can jam along to a backing track. Become a wandering minstrel and busk for your bread! And at the end of your wandering day, a built in charger lets you recharge you're batteries ready for the next days minstrel wandering.
SWAMP PM05 Dynamic Vocal Microphone
The PM05 is a hyper-cardioid dynamic microphone, designed for vocal use in live performance and general speech applications. With a solid body construction and metal grille, the PM05 is a robust microphone with excellent sound clarity and tone rivaling some of the top dynamic vocal mics.
Package Includes
- SWAMP PM05 Dynamic Microphone
- 5m XLR to 6.35mm Jack Microphone Cable
- BM6 10W Guitar Amplifier with Microphone input
- 10W Guitar Amplifier
- Battery Powered: 10x AA Batteries (not included)
- Batteries Rechargeable with Power Supply
- Inbuilt Effects
- 3-Band EQ
- Sweepable Mid Frequency
- Dedicated Microphone Input
- 3.5mm Auxiliary Input
- Dynamic Microphone
- 5m Cable Included
- Shoulder strap attachable (not included)
- Great for Solo Singer Songwriters
- Play Guitar and Sing Simultaneously!
By: Bill Powell on 6 October 2013Just purchased the "Wandering Busker Mic and Amp Package - 10W". I could not be more pleased with this product. The price is exceptional value and the performance is just great with the good quality microphone. Very pleased!
5 (5)


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In the episode "Ultrawoman", Clark insists that he wears briefs, but while playing strip poker we see that he also wears boxers on occasion.
Nitpick: Why did Superman use his potentially unstable heat vision to disable the low-frequency generator? Why not just use his strength to crush it, throw it into orbit, or similar? You can't throw and evil device TOO far away...
Thug: Mr Gadget! I watched you on TV when I was a kid! You were my idol!
Landry: Great, great, thank you, thank you. Take a hike.
Thug: Hey, I'm talking to the kid, here.
Landry: Mr Gadget has a death-ray that will make all your internal organs explode!
Dr. Klein: Picture in yor mind's eye a white beach... gentle surf... clear blue skies caressed by graceful clouds... the songs of chimps afire with the mating urge...
Superman: Dr. Klein?
Dr. Klein: Oh, sorry.
Dr. Klein: Look, I'm going to run every test I can, then I want you to go home and relax. Soak in a tub and do nothing.
Superman: But I'm Superman.
Dr. Klein: So? You can't play a little golf?
Dr. Klein: Often we find that true power comes to those who first agree to be powerless.
Superman: Rumors are far more dangerous than the truth, and I never hide from the truth.
Goof: Lois calls Clark "Dean" when he is trying to stop cooking the meat with his unstable heat vision.
Jimmy: As I was saying before the "boy wonder" breezed in...
Jimmy is comparing Jerry to the super sidekick Robin.
Lethal Weapon is the title of a popular series of action movies starring Mel Gibson as a cop with a death wish.


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Thursday, March 06, 2008
Is a faked autobiography any less literature for being faked?
There have been several scandals recently when the author of an "autobiography" turns out to have made it all up. It is a novel rather than fact. Now does the artist have to be ethical to be talented? The same question was asked about Leni Reifenstahl, the Nazi filmmaker. In reality art does not change according to the views or behavior of the creator. So if a book is outstanding should it really be removed from shelves if it turns out to be fiction?
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I didn't get the chance to see too much of the Scotland under-17s in action during the European Championships over the last few days but from the reports it was a really good tournament for us.
The boys did exceptionally well to get to the semi-finals and while they will be disappointed not to have reached the final, they can take a lot of pride from their performances. It would have been good to test ourselves against England, who beat the Netherlands in the final, but it just wasn't to be unfortunately.
It will be a fantastic experience for all the players that were there and to beat the likes of Germany and Switzerland to get to the last four says a lot about them.
From Celtic's point of view, they had seven young players there in the squad and that is good to see.
A lot of time and money is put into youth development at Lennoxtown and it is encouraging to see so many players doing well and representing their country.
Chris McCart and his coaches do a fine job and the boys getting capped is recognition for that.
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LinkedIn – Intermediate
Registration is now closed (this event already took place).
Everyone has heard of people talk about the value of leveraging LinkedIn for your job search, but few people know how to really maximize the effectiveness of the tool. Join us for a comprehensive presentation on leveraging LinkedIn to support success in your off-campus job search. Attendees will learn:
- How to utilize LinkedIn's various search features to find and connect with Johnson alumni
- Direction on how to leverage the various tracking and statistics options LinkedIn offers
- Fine tuning your profile to make yourself more easily searchable and accessible
- Comparison of various LinkedIn packages to see which package may make the most sense for your recruiting needs
141 Sage, Sage Hall, East Avenue, Ithaca, NY 14853, United States
Wednesday, February 22, 4:30pm – 5:30pm
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We have been making these hats for about twenty years and are about as close to the old low veins from the past. Each hat is cut down and reformed into a hat that is close to the size and weight of the old low vein hats. We offer a 30 day money back guarantee on the hats and a 6 month guarantee on the hats if the hat is defective because of construction. We make every effort to make these hats so they will last for a long time. These hat do not come apart.
Due to new mining laws, please check with your superiors at the place where you work to make sure you can ware one of these hats because there are no returns on hats unless we get the wrong color.
Shipping is free USPS. If for some reason it charges you shipping call 304-752-0657
Sometimes you won't be able to order on line with a cell phone for security concerns, give us a call and we can take your order over the phone.
choose color for hat from drop down menu.
You don't need a paypal account to order, just go to bottom of form and click on , pay with credit card, debit card or pay pal or call me at 304-752-0657 and I can take your order by phone