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- Emergency Service Available
- Non-Invasive Detection Methods
- Residential and Commercial Service
If you are experiencing a water leak in your home or business, finding the source must be done quickly to avoid long term damage. Luckily, Cork'd leak detection in Woodcliff Lake helps you easily compare pricing and get a specialist to your door as soon as possible. Woodcliff Lake leak detection services will range from acoustic to infrared tests of your walls, floors, and major appliances to determine the exact nature of your leak. Armed with high quality equipment, Cork'd professionals are available 24/7 to supply you with:
- Water Leak Detection in Woodcliff Lake, NJ
- Slab Leak Detection in Woodcliff Lake, New Jersey
- Underground Leak Detection in Woodcliff Lake
Contact us today to speak with one of our experts and get that leak stopped before it causes more damage.
Professional Leak Detection in Woodcliff Lake
Non-Verified Leak Detection Companies
Force Plumbing and Heating, LLC
974 Livingston Avenue
North Brunswick, NJ 8902
Abat Plumbing And Heating
99 Davidson Mill Road
North Brunswick, NJ 8902
Abat Plumbing and Heating
99 Davidson Mill Road
North Brunswick, NJ 8902
Joe Kjersgaard Plumbing & Heating
454 Kenbury Road
Somerville, NJ 8876
Master Tech, LLC
20 Mills Street
Raritan, NJ 8869
Leacy's Plumbing & Heating, LLC
538 Black Point Road
Hillsborough, NJ 8844
Five Star Plumbing & Heating, LLC
14 Sturgis Road
Kendall Park, NJ 8824
ClearFlow Drain Cleaning
226 Us Highway 1
Edison, NJ 8817
Amrowski Plumbing & Heating, Inc.
560 Garretson Road
Bridgewater, NJ 8807
KBK Plumbing, LLC
667 East Main Street
Bridgewater, NJ 8807
Exactly how much is leak detection in Woodcliff Lake, NJ?
Jason F.
Hillsdale, NJ, 7642
$ 211
Leakage detection and repair in Woodcliff Lake.
Courtney W.
Montvale, NJ, 7645
$ 310
The hot water heater inside my house has water around it.
Angelo H.
Westwood, NJ, 7675
$ 192
How much do you charge for leak detection in Woodcliff Lake, NJ?
Jake S.
Township Of Washington, NJ, 7676
$ 276
Get Started
What zip code do you need leak detection in?


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Tu sei qui
Rivista HydroResearch
Hydro Research (http://www.keaipublishing.com/en/journals/hydroresearch/) is an international Open Access journal publishing original research papers, short communications, review articles and book reviews in the broader field of hydrology and hydrogeology.
The Editor-in-Chief is Prof. Elango Lakshmanan, Department of Geology, Anna University of Chennai, India, Vice President of the IAHS.
The journal welcomes both regional and global studies on:
· all aspects related to the hydrological cycle;
· interactions and inter-relationship between groundwater, surface water, snow and ice;
· all hydrological processes including physical, chemical and biological;
· groundwater flow and transport processes;
· hydrological extremes and its impact;
· natural and artificial water treatment (physical, chemical and biological);
· impact of climate extremes on hydrological processes;
· effects of land-use changes on water resources;
· hydrogeochemistry, controlling processes and modelling;
· geological and anthropogenic contamination of water resources;
· urban hydrology;
· water management and policy issues;
· application of statistical and remote sensing methods in water resources;
· social and economic aspects of water resources management.
Manuscripts providing new insights on the hydrological processes in a region, the development of new methods, and/or instrumentation and field-based research on pollution detection and treatment are welcome.
The journal encourages multi-disciplinary and inter-disciplinary research.

subset: commoncrawl


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Paperell's Casing Look at Composing Service plan Positive aspects
Paperell's Casing Look at Composing Service plan Positive aspects
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- There are many impressive authors who may have written and published several performs of different different types and offered the top quality, and are generally set to get results for you. Read more →


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Available as Framed Prints, Photos, Wall Art and Gift Items
Discover our great selection of the weird and the wonderful of the natural world, get animal Posters & Prints on WorldInPrint.
Choose from 11 pictures in our Animals collection for your Wall Art or Photo Gift. Popular choices include Framed Prints, Canvas Prints, Posters and Jigsaw Puzzles. All professionally made for quick delivery.
Christmas Wall Art & Decor
Nursery Wall Art
Religion & Spirituality
Images Dated
Emperor penguin (Aptenodytes forsteri), Snow Hill Island, Weddell Sea, Antarctica
Emperor penguin (Aptenodytes forsteri), Snow Hill Island, Weddell Sea, Antarctica, Polar Regions The Emperor Penguin (Aptenodytes forsteri) is the tallest and heaviest of all living penguin species. It is the only penguin that breeds during the winter in Antarctica. Emperor Penguins eat mainly crustaceans (such as krill) but also occasionally indulge in small fish and squid. In the wild, Emperor Penguins typically live for 20 years, but some records indicate a maximum lifespan of around 40 years. The Emperor Penguin should not be confused with the closely related King Penguin or the Royal Penguin
© Thorsten Milse


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Availability date:
Hanover Entertainment Wall SystemThe Hanover Entertainment Wall features a beautiful Cherry Spice finish over warm Cherry veneers with a clean, transitional design. The center unit has satin nickel bar pull hardware, two drawers with a spacious component shelf between them, and four doors with more shelving behind each. The left and right pier units have adjustable glass shelves, a drawer, and a wood door. Connecting all three pieces is light bridge that provides even more shelving space.
Enter Your Information
* Required fields
|Home Entertainment
|TV Console
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Next Steps
Next Steps at Lakeside
At Lakeside, we're committed to helping people discover and fully follow Jesus. Whether you're new or you've been around for years, take a step, then another one, then another one. The Christian life is a journey and we're here to walk alongside you. If you don't know what your next step is yet, check out some of the most popular next steps that people around Lakeside take when they're looking to start or grow their faith, get connected, or find healing.
Sign Up For Alpha Online
Alpha is a wonderful opportunity to ask and explore questions that may be new to you about the Christian faith, and questions that you may have avoided in the past. Click here to sign up.
Come To Celebrate Recovery
Celebrate Recovery (CR) is a Christ-centered, 12-step recovery program for anyone struggling with hurt, pain or addiction of any kind (shame, control, people-pleasing, anxiety, abuse, fear, anger or anything else). Click here to learn more.
Become a Partner
Partnership is our word for "membership." We believe that it best reflects the relationship between an individual and their church community. Click here to find out more.
Join a Group
God created us to live in community. We were created to have a relationship with Him and with others. Genuine life change occurs within the context of relationships Search for a group and sign up online today.
Get Baptized
One of the first steps in following Jesus is being baptized — getting wet. It sounds weird but it's a very powerful spiritual experience – a living symbol of the change that happens to us as we live each day with Jesus. Click here to explore this further.
Volunteering at Lakeside means you're not just filling a position or meeting a need, but you're saying YES to what God has in store for you. Click here to find an opportunity today.


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Call Us Today!
07 4068 1466
| Address:
3/19 Bruce Highway, Tully QLD, 4854
Logbook Servicing
Wheel Alignment
Logbook Servicing
Logbook Servicing on the Cassowary Coast
Dore's Goodyear Autocare Tully is a fully licensed and insured auto mechanic that can perform log book servicing for clients in Tully and other parts of the Cassowary Coast. If you bring in your vehicle to our shop, our mechanics can look over it and perform routine maintenance and repairs as per the terms of your warranty.
What is Logbook Servicing?
Many clients are confused about the terminology of logbook servicing. It is an understandable confusion, even among car owners. A log book is also called a handbook, which is the booklet that you receive when you buy your car from a dealership. It details the checking and replacing of components that must be done periodically to ensure that your car is performing optimally.
The key to logbook servicing is to ensure that you are having your car serviced by a mechanic who knows what they are doing. Many customers assume that if they are getting these checks done as per the terms of the warranty, they must take it to the place where they bought the car. That's not necessarily true.
According to Australian law, you are free to have your car serviced and maintained from any certified auto mechanic. We can perform these routine maintenance checks and component replacements as per your warranty and we can carry out all other work you require as well without voiding your warranty.
Check Your Log Book
If you need routine maintenance on your car, it may be a good idea to check your vehicle's log book. You should have it with the rest of the paperwork that you got when you bought the car. The logbook will go into great detail about the checks that must be done and when they must be done.
Examples of the work includes replacing the oil, replacing your battery, rotating your tyres, conducting a wheel alignment, checking the engine, suspension and other components and performing numerous other checks to ensure that everything is running as it should.
The great news is that if you have your log book you can bring it into our shop along with your car. We will happily take a look at your book, see when you last had particular checks done and carry out the work for you. We will only do the work that is specified in your log book.
If our mechanics find any other problem or potential problem while we are working, we will come back to you and ask if you want us to perform quick repairs to fix or prevent the issue. That way you can decide whether to nip a small problem in the bud or wait until it's a much bigger and potentially more expensive problem later.
Customers in Tully and other parts of the Cassowary Coast that require logbook servicing can bring in their car and log book to Dore's Goodyear Autocare Tully.


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Jungle camping is adventurous, there is no doubt it. Most of us get excited just on listening the name of a lion, tiger and other wild animals and imagine during jungle camping you see these animals in real just at a distance of few meters.
Jungle camping is great but remember that it is different from camping at hill stations and Gardens. For a successful jungle camping, you should have all the practical information how to stay in a jungle, what kind of clothes you need to take with you. How to ensure the maximum cleanness and comfort during Jungle camping? And, how you will arrange the food and water in the jungle for yourself?
In Dandeli in addition to wild life, you also enjoy beautiful Mullangi Falls. To make your Dandeli Jungle camping most beautiful and memorable moment of your life, follow the below- given tips for safe and secure Jungle Stay in Dandeli
- There are running and standing waters. There may be bugs, suck and stings which can make your uncomfortable. So you should make all the necessary arrangements for a comfortable jungle stay in Dandeli.
- You should know how to set a campsite or build a tent in a jungle for sleeping. It is safe to use a tent for sleeping in the jungle if a resort is not included in your safari package.
- It can rain anytime in Jungle so you should not forget to take a rain gear and umbrella with you. You can wear tightly woven or light clothes in a jungle. It depends on your choice.
- Don't forget to take Emergency gear with you like a blanket, duct tape, fire starters, compass and cordage.
- Don't forget to take a toothbrush, batteries, camera and other odds and ends to ensure maximum comfort and cleanliness in Jungle.
Sum and Substance
If you avail Safari package for a Jungle Stay in Dandeli you will not need to worry about food, water and accommodation in Jungle. Because these facilities are included in the safari package provided by the Travel Company for Camping. In Dandeli you are also able enjoy beautiful Mullangi Falls.
Follow the above tips for a safe, secure and complete jungle stay in Dandeli.


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"The indications of ongoing hybrid warfare are becoming stronger in Central Europe and Scandinavia too, and there's always the risk of it developing into actual warfare," Janis Sarts said Wednesday, as quoted by the Latvian Public Broadcasting English-language service.
The head of the NATO center pointed out that Sputnik's news coverage is one of "the signs of hybrid-warfare directed at us in Latvia."
He admitted that blocking the media in the country would be "counterproductive" and unable to stop people interested in alternative information sources since "there are simple solutions to bypass the block."
The Baltic states have been consistent in its information warfare against Russian media. In September 2015, the Estonian border agency denied entry to Rossiya Segodnya's Multimedia Programs Director Marina Perekrуstova. In May, Lithuanian President Dalia Grybauskaite announced amendments to the country's media law to counter the information it deemed as Russian propaganda.
Sputnik is a news agency and radio network with multimedia news hubs in dozens of countries. Sputnik broadcasts through its websites, analog and digital radio, mobile apps and social media. Sputnik newswires run around the clock in English, Arabic, Spanish and Chinese.
The concept behind Sputnik is one that combines cutting-edge technology, high professionalism and close proximity to readers/listeners, which helps Sputnik develop and win over foreign audiences. Its motto "Telling the Untold" underpins the content Sputnik news services produces, with the aim of giving audiences an opportunity to compare alternative opinions.
A survey conducted by ICM Research exclusively for Sputnik and released in May 2015, showed that over half of those people polled in the United Kingdom, France, Germany, and Greece would like to get global news from sources other than mainstream Western media.


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When Old Man Winter starts getting on your nerves, bundle up your babes and battle the brr's with a cup of hot chocolate at one of our favorite (hot chocolate and coffee) bars. From exotic to the everyday, nothing beats a little cocoa to cajole a smile from under all those layers. Here are eight hot spots to sit down and enjoy a cup with your cuties.
ChocoLate Coffee
Kids will go choco-crazy for this spot that boasts a decadent dessert counter, outside patio for warm weather and a giant blackboard for doodling. Order up their house blend of dark and milk chocolate or the hazelnut variety for a heart-warming sweet treat after you do your shopping at Publix.
2558 Shallowford Rd. NE, Ste 201
Online: mychocolatecoffee.com
Photo courtesy of ChocoLate Coffee via Facebook
Did we miss a spot? Where do you go for hot chocolate in Hotlanta? Spill below!
—Phebe Wahl & Shelley Massey


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Study Finds High Incidence Of Bowel Disease In People With Lung Conditions
People suffering from airway disease, i.e., asthma, chronic obstructive pulmonary disease (COPD), have higher incidence of inflammatory bowel disease such as Crohn's disease and ulcerative colitis, a new study has found.
It is the first population-based study to examine the association between airway disease and the incidence of bowel disease.
Findings of the study showed that the incidence of Crohn's disease was 27% higher in people with asthma and 55% higher in people with COPD, compared to the general population. The incidence of ulcerative colitis was 30% higher in people with COPD compared to the general population, the press release added.
Earlier, studied had suggested a link between the two conditions, which could be a result of common genetic and environmental factors, or similar inflammatory responses seen in the immune system. If the link is confirmed, it would have the key implications for clinicians treating people with airway diseases, according to the press release.
"These findings have important implications for the early detection of inflammatory bowel disease in airway disease patients. Although a link has previously been suggested, this is the first study to find significantly increased rates of inflammatory bowel disease incidence in people with asthma and COPD. If we can confirm a link between the two conditions it will help diagnose and treat people sooner, reducing their symptoms and improving their quality of life," said Dr Paul Brassard, lead author of the study from the Lady Davis Institute for Medical Research of the Jewish General Hospital in Montreal, Canada, in the press release.
The study was published in the European Respiratory Journal.
See Now: What Republicans Don't Want You To Know About Obamacare


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Demi Lovato talks about not caring if she would have died
HollywoodNews.com: Demi Lovato is being very open about her personal struggles and now she is revealing how little she cared about her life.
The actress and singer revealed in a '20/20′ interview that she was never trying to kill herself when she started cutting, but she also wasn't concerned about living, states Hollyscoop. "I knew that if I had ever gone too far that I wouldn't care," Lovato said about her self-mutilation.
However, she eventually checked into rehab and has been working on herself ever since.
Do you think Demi Lovato's openness will help others?
Follow Hollywood News on Twitter for up-to-date news information.
Hollywood News, Hollywood Awards, Awards, Movies, News, Award News, Breaking News, Entertainment News, Movie News, Music News
Image by PR Photos


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Once you register as a Buyer you will be able to access all the phone details and many more features
371 , Macarthur Avenue, Hamilton, QLDPrice:
$414pw | 3 Person Workspace | Private OfficeEnquire Now
Type: FOR LEASEProperty Features
PROPERTY OVERVIEW >> $414pw - Price incl. This gateway office space, situated midway between Sydney Airport and the CBD, makes it a high-in-demand rental, especially those constantly on the move. Additionally, housed in a newly constructed business and tech precinct makes this an attractive flex space for those looking to grow their companies through networking and collaborating with other like-minded enterprises.
The advantages to working here don't stop at a desirable location and the opportunity to forge business relationships, but also comprise fully-equipped private offices, the complimentary use of large boardrooms, as well as a spacious kitchen and communal breakout areas.
Office features that will enhance your renting experience, include working in light-filled rooms and having unlimited, fast internet that will never let you down. Around the clock access to the building is yet another perk to being here, affording you the chance to set your own hours.
The briefly mentioned kitchen deserves some praise too due to its ample size. Replete with an oven, microwave, toaster and other kitchen essentials and several seating options, your lunch hour and coffee breaks will be more enjoyable.
Conveniently located within the new Business and Technology Precinct, this space is a great opportunity for businesses ready to network! Located only seven kilometres from the Brisbane CBD and five kilometres from the Brisbane Airport, tenants will have easy access to major roads and highways.
The listing below is only $414 per week ($1800 p/month), perfect for a team of 3 and comes fully furnished with flexible rental terms. This Private Office is located in Hamilton and if you book a tour Business & Technology Precincts can show you 3 available office spaces ranging in size from 1 to 4 desks.
Did you know our team offer a free personalised service to help you shortlist, book and negotiate the best rate on your ideal workspace. From a 1 person hot desk to an enterprise team of 1000+ the Office Hub team can customise a flexible furnished office solution for your team.
What you get for your weekly rent
> Fully furnished office with desks, chairs & storage
> Electricity usage included
> Unlimited fast internet connection**
> Complimentary use of meeting rooms**
> Use of modern kitchen facilities
> Weekly cleaning
> Flexible terms
> 24 x 7 access
Office Features
> Air Conditioning
> Modern fit out
> Showers
> Unlimited fast internet connection**
> Professional environment
> Dedicated property manager
Building Features
> Car parking - on-site and in surrounding areas
> Lift access
> Landscaped open shared spaces
> Modern commercial building
> Quality finishes and amenities
> Bike racks
Office Hub is Australia & New Zealands No 1 small office space commercial real estate agency.
We have the largest collection of shared spaces, coworking and serviced offices in Australia & New Zealand, perfect for 1 - 100 employees. Let us take you on a tour of all the spaces that fit your needs and budget. Our service is complimentary and we work for you and the landlord to negotiate the most competitive deal in the market. Let us show you how shared and serviced offices can be the perfect solution for your business.
[Web Link Censored]
[Web Link Censored]
** Conditions apply - A portion of the square meterage factors in access to shared space. Price includes any incentives offered. This is for new tenants only.
Advert Id:88429
Name: Sean O\'Connor
Phone: 130077 ****


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Senior Member
Last Activity: 27th November 2018 06:37 AM
Most Thanked
Post Summary
Work on my device perfect,,mksh om adi,, Sent from my C5503
Glad to see you come back,,thanks to airlessproject for developing our device dogo,,,
Guys, now I official maintainer this ROM for ZR. New builds are now on the official RessurectionRemix hosting. Also you can check the availability of new builds via OTA (settings->configuration->updates). Cheers! Thanks Chippa you are the bes...
Awesome location to put it :thumbup: Sent from my LG-D722 using XDA Free mobile app Yeah,,I put in navbar,so i can see music effect visualizer,when i open any app,even when pulling expanded and lock screen wake up,, Sent from my C5503...
It's a known issue and has already been fixed by Rajeev here Thanks,,its work now,, ---------- Post added at 03:33 PM ---------- Previous post was at 03:32 PM ---------- will it work for xperia z purex 5.1.1? MM only bro...


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If you've ever harbored a certain kind of Brooklyn townhouse fantasy–but aren't ready for the responsibility (or the mortgage)–this is about as close to the dream as it gets. And though it might require a tiny bit of imagination (picture it with furniture!) this just-renovated triplex rental at 198 Warren Street checks all the boxes–charming restored original details, five bedrooms (though one is tiny) if you've got a big family or just want to share the rent, 2,500 square feet of living space, a brand-new kitchen, central A/C, and outdoor space. And you're in one of Brooklyn's most desirable enclaves in Cobble Hill on a picture-postcard block near, as the brokers say, all.
The parlor floor has a classic layout with high ceilings, marble mantels, wedding-cake molding, perfectly-restored wood floors, and the standard front and back parlor configuration. The formal dining room has more than enough room for guests and their guests, or it can be a casual everyday gathering spot.
At the back of the home, a sunny renovated kitchen (according to the listing the gut renovation is "just finished and never lived in") offers a wall of windows overlooking the charming back gardens of Cobble Hill. Stainless steel appliances and plenty of cabinet space await lazy breakfasts and late-night snacking.
Upstairs, bedrooms also have the standard row house layout, which means large rooms (except that one third-floor bedroom, though it'll make a perfect nursery, dressing room, or office) and plenty of closet space. There's at least one working fireplace and the same warmth and restored historic details throughout with the added convenience of laundry.
Also freshly updated, baths (one full bath on each of the upper floors) are clean and spacious; this one features a glass-walled rain shower.
Though there's no backyard access–a slight blemish on this picture of South Brooklyn perfection–but this large, 180-square-foot deck off the second floor is the perfect addition to a master suite. It offers more than enough space for entertaining, sunbathing, or dining al fresco (though you'll have to trudge upstairs with the dishes).
As previously mentioned, this prime spot in a covetable historic neighborhood is the picture of Brooklyn charm, with the F and G subways two blocks away, great restaurants in every direction, Atlantic Avenue, Smith and Court Street shops, the Columbia Street Waterfront, Cobble Hill Park, Trader Joe's and several other great neighborhoods nearby.
[Listing: 198 Warren Street, by Jeb Hart for Corcoran Group]
- Beastie Boy Mike D's Brilliant Brooklyn Townhouse Can Be Yours for $5.7M
- 'Eat, Pray, Love' Firehouse in Cobble Hill Sells for $6.25M
- Supermodel Freja Beha Erichsen Snags a Stunning $3M Carroll Gardens Townhouse
Images courtesy of Corcoran Group.
Tags : 198 Warren Street, townhouses
Neighborhoods : Cobble Hill


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Hi i tried to run vxrail initial setup but finaly popup below error.
am using dedicated HP 1950 10Gig switch for this vxrail.
i created 3 vlans.
Vlan 55 vmotion
vlan 56 vsan
vlan 32 for vmnetwork.
all interfaces tag with above vlan and last interface trunk.
this trunk port connected to the cisco switch.vlan 32 gateway cisco switch.
so DNS server in vlan 31.but vlan 31 and 32 connectivity ok.
please help to resolve this issue.
Check /var/log/marvin/tomcat/logs/marvin.log.
ssh into VxRail Manager and verify DNS accessibility:
# nslookup
server 10.10.10.Y
Another way:
add the DNS IP in /etc/resolv.conf
and verify it:
# nslookup google.com
If this is OK, check if the Default Gateway in JSON/VxRailManager UI is correct.
Thanks for your support.
what is the default password vxrail manager?
i tried
root Passw0rd!
admin admin
See EMC KB 458852 for default password.
If you cannot see KB above, you don't have right to login to VxRail Manager by SSH
Point a browser to ESXi and open VxRailManager console. You should be able to login with the credentials you tried above.
5,6 times tried to run initial setup but DNS unable to reach.
but dns server restart and the i tried so last moment popup "BUILD VXRAIL".am happy with this and continues.
take 45 min and without error completed this task.
after login to vcenter and i checked DNS server details.but its showing VCENTER SERVER IP.
Can we change this dens server ip to our external dns server ip?
Good to hear that
In the How To field in the SolveDesktop, you can find the procedure for changing DNS ip address for internal VCSA
Also, you can find the KB#492132
I need to totaly down internal VC DNS and add to my external DNS server.because my internal vcenter dns FQDN and my external DNS FQDN same.
When using embedded vCenter, the ESXi servers will point the DNS to vCenter. It is not supported to change that.
Hello ajacobsen,
So what you are saying is that when you look at the web client for the hosts within the VxRail Cluster, the DNS IP address should actually be the IP address of the Vcenter Server and NOT the actual DNS IP address?


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Throughout the year, Crackerjack Training raise money for a variety of charities. Here we will show you who we have been supporting and how we have raised money for charity.
Comic Relief 2017
To raise money for Comic Relief 2017, we had an 80s themed day and held a bake sale. We raised an amazing £85.00. Thank you to everyone who dressed up, baked and ate cake!
Pajama Day 2016
To raise money for Children in Need, Crackerjack staff and students dressed in their pajamas for the day.
Macmillan Coffee Morning 2016
Every year we hold a Coffee Morning to raise money and awareness for Macmillan. This year we invited a volunteer from Macmillan to come in and talk to our students about what Macmillan do and talk about some personal experiences. The talk was followed by a Q&A session and lots of cake!
Fun Day 2016 for the Simon Richards Award
During July 2016, our Traineeship learners organised a fun day to raise money for the Simon Richards Award for Young Violinists.


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Jesus in John 21, tells us of his interactions with the disciples. What results is a miraculous parable, a calling, a future, and a mission.
1. Go with what Jesus says. Hear and obey……yes, Listen for Jesus and obey!
2. We meet God when we come to the end of our resources. My way often produces little or nothing: his way results in bursting nets and 153 fish!
3. You have to put the net where the fish are.
'……a long obedience in the same direction.'
Jesus is our primary business and in our following Him we are called to do things a different way. Left to myself its survival & scarcity, but if I'll trust the Saviour, His way is abundance.
Anyhow……here's some questions following……
1. Why may the disciples have had a difficult time doing what Jesus asked?
2. Why do we sometimes find it hard to obey?
3. What happens when we listen to Jesus and do what he says?
4. Eugene Peterson talks about "a long obedience in the same direction." Why is this important, and how does this apply to the story in John 21?
5. Why do we often have to come to the end of our resources before we will give God control?
6. How does humility and a teachable spirit help us as we go through difficult situations in life?
7. Many times we overlook the answer because it is too obvious. How can we get better at listening to God so we don't miss the answer?
8. Has God ever given you an answer that you did not like? Why is it important to obey in those times?
9. We are sometimes willing to do something great, but not something small. Why is that?
10. Read Psalm 51:12 How can you develop a willing spirit?


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Paul R. Brown EPIC Center Community Rooms
Zane State College Cambridge Campus
9900 Brick Church Road
Cambridge, OH 43725
Ohio Department of Higher Education (ODHE) Chancellor John Carey; Zane State College President Dr. Chad Brown; Belmont College President Dr. Paul Gasparro; Washington State Community College Vice President for Finance and Operations Jess Raines; ODHE staff; officials from Zane State College, Eastern Gateway Community College, Belmont College and Washington State Community College; students; and business and industry partners.
Chancellor Carey will visit the Zane State College Cambridge campus to discuss the $1 million Regionally Aligned Priorities in Delivering Skills (RAPIDS) grant awarded to the region. The roundtable discussion with regional education and business officials will highlight the benefits of the grant as they pertain to preparing students for in-demand jobs in the region. The RAPIDS grant was received by the Southeast Ohio Oil & Gas Training Collaborative, an initiative consisting of Zane State College, Washington State Community College, Belmont College and Eastern Gateway Community College.
Ohio Department of Higher Education Contact:
Jeff Robinson
Office: 614.752.9487
Email: firstname.lastname@example.com


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Reunion Weekend Update
Wegesegum turns 90 this summer. We're planning a weekend anniversary family camp in celebration from July 12th to 14th. Lori Perley has volunteered to lead the music for the weekend. We hope all of you can make it for all or part of the camp! Registration details will follow in the new year.
90th Aniversary Reunnion 2013
Wegesegum turns 90 in 2013. Here is nineteen months notice of a reunion weekend on August 16 to 18th, 2013. We would love to see past campers and leaders from every decade there to catch up and celebrate Wegesegum. Many of us haven't been to camp since our youth or childhood, but the expereinces we had and friends we met made a lasting impression. Come back for a weekend of fun! This weekend is being planned so far by Yan Storr, Shirley Hunt, and Taunya Pynn. If you are interested in helping out, let us know!
March 2015
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Mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1) is a dual-specificity phosphatase that is involved in the regulation of cell survival, differentiation and apoptosis through inactivating MAPKs by dephosphorylation. Here, we provide evidence for a role of MKP-1 in the glutamate-induced cell death of HT22 hippocampal cells and primary mouse cortical neurons. We suggest that, during glutamate-induced oxidative stress, protein kinase C (PKC) δ becomes activated and induces sustained activation of extracellular signal-regulated kinase 1/2 (ERK1/2) through a mechanism that involves degradation of MKP-1. Glutamate-induced activation of ERK1/2 was blocked by inhibition of PKCδ, confirming that ERK1/2 is regulated by PKCδ. Prolonged exposure to glutamate caused reduction in the protein level of MKP-1, which correlated with the sustained activation of ERK1/2. Furthermore, knockdown of endogenous MKP-1 by small interfering (si)RNA resulted in pronounced enhancement of ERK1/2 phosphorylation accompanied by increased cytotoxicity under glutamate exposure. In glutamate-treated cells, MKP-1 was polyubiquitylated and proteasome inhibitors markedly blocked the degradation of MKP-1. Moreover, inhibition of glutamate-induced PKCδ activation suppressed the downregulation and ubiquitylation of MKP-1. Taken together, these results demonstrate that activation of PKCδ triggers degradation of MKP-1 through the ubiquitin-proteasome pathway, thereby contributing to persistent activation of ERK1/2 under glutamate-induced oxidative toxicity.
Glutamate toxicity has been implicated in a variety of neurological disorders such as Parkinson's disease, Alzheimer's disease, seizures, stroke and trauma (Coyle and Puttfarcken, 1993). Glutamate has been shown to induce neuronal cell death through both receptor-mediated excitotoxicity and non-receptor-mediated oxidative stress. Glutamate-induced oxidative toxicity provides a model for studying the mechanism of oxidative stress in neurons and neuronal cells. In this model, glutamate induces oxidative stress by promoting cystine efflux and/or competitively blocking cystine uptake by inhibiting a glutamate-cystine antiporter. This results in the loss of intracellular cystine and glutathione, thereby inducing oxidative cell death (Murphy et al., 1989).
Mitogen-activated protein kinases (MAPKs) become phosphorylated and activated in response to a wide variety of external stimuli, resulting in the regulation of cell proliferation, differentiation, survival and apoptosis (Chen et al., 1995; Bhat and Zhang, 1999). They can be subdivided into at least three classes based on sequence homology: these include the extracellular signal-regulated kinases (ERK1/2), the Jun N-terminal kinases (JNKs) and the p38 kinases. MAPKs are activated by dual phosphorylation on Ser/Thr and Tyr residues in the TEY sites within the activation loop, whereas dephosphorylation of these residues by a family comprising about 11 dual-specificity phosphatases or MAP kinase phosphatases (MKPs) terminates such activation (Keyse, 2000). These phosphatases exhibit differential specificity towards MAPK substrates, show distinct subcellular localizations, and have different modes of regulation. Among these phosphatases, MKP-1 was originally identified as an ERK-specific phosphatase (Sun et al., 1993). However, it has also been shown to dephosphorylate and inactivate both JNK and p38 in a cell-type-specific and context-specific manner (Franklin and Kraft, 1997; Lasa et al., 2002). MKP-1 is overexpressed in many human tumors, and its overexpression has been shown to protect cells from apoptosis induced by the anticancer agent cisplatin, as well as ultraviolet (UV) irradiation and proteasome inhibitors (Franklin et al., 1998; Sanchez-Perez et al., 2000; Small et al., 2004). However, the role of MKP-1 in controlling cell survival, proliferation and apoptosis has been suggested through use of overexpression approaches in non-neuronal cells, and it has not been clearly demonstrated whether MKP-1 plays a role in the regulation of neuronal cell death.
Members of the protein kinase C (PKC) family of Ser/Thr protein kinases have been suggested to have important roles in the regulation of cell survival and programmed cell death (Whelan and parker, 1998; Maher, 2001). Several studies suggest that PKCδ is involved in apoptosis by acting as a pro-apoptotic signal. Many studies have shown that a selective inhibitor of PKCδ, rottlerin, and a dominant-negative mutant of PKCδ attenuate apoptosis induced by phorbol ester, H2O2, UV radiation, taxol and etoposide (Konishi et al., 1997; Denning et al., 1998; Majumder et al., 2000; Matassa et al., 2001). A previous study demonstrated that an apoptotic stimulus resulted in proteolytic activation of PKCδ, which was inhibited by treatment with caspase inhibitors and transfection with anti-apoptotic proteins such as Bcl-2 or Bcl-xL (Emoto et al., 1995). This observation is in agreement with previous results demonstrating that expression of the catalytic fragment of PKCδ in HeLa cells was sufficient to induce cell death (Ghayur et al., 1996). Thus, PKCδ activity seems to have an important role in the regulation of apoptosis in many different cells. However, the mechanism by which activation of PKCδ drives cell death still remains largely unknown.
Recent studies have shown that persistent activation of ERK1/2 is strongly associated with glutamate-induced cell death in HT22 mouse hippocampal cells and immature primary cortical neurons (Stanciu et al., 2000). However, the mechanism by which prolonged activation of ERK1/2 occurs remains largely elusive. On the other hand, inhibition of PKCδ by rottlerin blocked glutamate-induced cell death in primary cortical neurons (Maher, 2001), suggesting that activation of PKCδ is implicated in oxidative stress. In this study, we investigated the roles of PKCδ and MKP-1 in glutamate-induced death of HT22 cells and immature primary cortical neurons. Our results show that glutamate treatment triggers activation of PKCδ, which causes degradation of MKP-1 through the ubiquitin-proteasome pathway, thereby contributing to the sustained activation of ERK1/2. Moreover, we show that downregulation of MKP-1 promotes glutamate-induced cell death, through an ERK1/2-mediated mechanism. Collectively, we suggest that MKP-1 is an important mediator in controlling neuronal cell death.
ERK1/2 contributes to glutamate-induced cell death in HT22 cells
The role of ERK1/2 during glutamate-induced oxidative toxicity in HT22 cells has been subject to controversy (Stanciu et al., 2000; Maher, 2001). We thus first examined the effect of glutamate on activation of MAPKs in our culture system. As shown in Fig. 1A, phosphorylation of ERK1/2 was observed at 3 hours after glutamate treatment, and was sustained up to 9 hours. By contrast, phosphorylation of p38 and JNK kinases was detected only at 9 hours after glutamate treatment, and was undetectable at 6 hours. Next, we examined the involvement of MAPKs in glutamate-induced cell death by using specific inhibitors of MEK1/2 (U0126), p38 (SB202190) and JNK (SP600125). Using the MTT assay [which measures the amount of yellow 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide oxidized to purple formazan], we found that more than 80% of the cells underwent cell death in response to glutamate after 12 hours. U0126 substantially protected the cells from death (Fig. 1B), and similar results were obtained from terminal transferase dUTP nick-end labeling (TUNEL) assay (Fig. 1D), measurement of propidium iodide staining and fluorescence activated cell sorter (FACS) analysis (data not shown). The effect of U0126 was confirmed by its ability to reduce the level of phospho-ERK1/2 elevated by glutamate treatment (Fig. 1C). By contrast, SB202190- or SP600125-treated cells did not exhibit significant recovery from the glutamate-induced cell toxicity as compared with control cells (Fig. 1B). Neither SB202190 nor SP600125 had any effects in unstressed cells when treated alone (data not shown). The inhibitory effect of SP600125 on JNK was confirmed by its ability to block the glutamate-induced phosphorylation of c-Jun, which is a substrate of JNK (Fig. S1, supplementary material). To confirm further that p38 is not involved in glutamate-induced cell death, we used another specific inhibitor of p38, SB203580. MTT assay revealed that SB203580-treated cells did not exhibit any significant recovery from the glutamate-induced cell toxicity (data not shown) as in SB202190-treated cells. The inhibitory effect of SB203580 on activation of the p38 pathway was independently confirmed (data not shown). Taken together, these results suggest that activation of ERK by glutamate has a crucial role in regulating cell death, whereas JNK and p38 kinases do not.
Sustained activation of ERK1/2 might have resulted from elevated Ras-Raf-MEK signaling, but prolonged activation of ERK1/2 in some cases has been reported to be independent of MEK1/2 (Grammer and Blenis, 1997). We therefore examined the effect of glutamate on the activation of MEK1/2. As described above, glutamate increased phosphorylation of ERK1/2 in a time-dependent manner. Phosphorylation of MEK1/2 also increased up to 3 hours of glutamate treatment, but declined afterwards (Fig. 1E), implying that activation of MEK1/2 might not play a predominant role in the sustained phosphorylation of ERK1/2, which continued to increase up to 9 hours of glutamate treatment. To investigate the involvement of MEK1/2 further, we examined whether U0126 still had a protective effect when treated after glutamate had triggered cell death. The protective effect of U0126 started to decrease when added 4 hours after glutamate treatment, and a substantial amount of cell death was observed afterwards (Fig. 1F), supporting the notion that activation of MEK1/2 might not be the major cause for the sustained activation of ERK1/2 that was observed even after 9 hours of glutamate treatment.
Glutamate induces activation and tyrosine phosphorylation of PKCδ in HT22 cells
A recent study demonstrated that rottlerin, a selective inhibitor of PKCδ, blocked glutamate-induced oxidative toxicity (Maher, 2001). We thus examined whether PKCδ was involved in our system. For this purpose, we first examined the activity of PKCδ. As shown in Fig. 2A, PKCδ became highly activated by glutamate, and its activity reached maximum at 6 hours. The activity declined afterwards, but was sustained up to 9 hours. PKCδ can be activated by tyrosine phosphorylation or by enzymatic cleavage to a constitutively active catalytic fragment (Li et al., 1994; Emoto et al., 1995). To assess the mode of activation during glutamate-induced oxidative toxicity, we examined whether PKCδ was phosphorylated on tyrosine residues. As shown in Fig. 2B, a low basal level of tyrosine phosphorylation was detected in untreated cells. Maximal phosphorylation by glutamate was observed at 6 hours, and the phosphorylation decreased thereafter. The time course of tyrosine phosphorylation of PKCδ correlated with the window of time in which PKCδ was activated. We also examined whether glutamate induced proteolytic cleavage of PKCδ. We found that the full-length form of PKCδ decreased in a time-dependent manner, which was accompanied by a concomitant increase in a 40 kDa fragment (Fig. 2C). Although the 40 kDa fragment slightly increased, the time course of PKCδ cleavage did not match the mode of PKCδ activation. On the basis of these results, we concluded that the cleavage of PKCδ is unlikely to be the major mode of activation during glutamate-induced cell death. However, we cannot entirely exclude the possibility that this slight increase might have contributed to the activation of PKCδ.
Inhibition of PKCδ activity suppresses glutamate-induced ERK1/2 phosphorylation in HT22 cells
To elucidate whether PKCδ mediates the activation of ERK1/2 in response to glutamate, we first examined the effect of rottlerin. As shown in Fig. 3A, 5 μM rottlerin completely blocked the phosphorylation of ERK1/2 in response to glutamate. Since the inhibitory effect of rottlerin on PKCδ has been subject to controversy (Gschwendt et al., 1994; Soltoff, 2001), we further investigated the role of PKCδ in the glutamate-induced phosphorylation of ERK by using a dominant-negative mutant (DN) of PKCδ (K376R). Exogenous expression of PKCδ DN did not have any significant effects on the basal phosphorylation of ERK (Fig. 3B, lanes 1 and 2). However, it significantly inhibited the glutamate-induced phosphorylation of ERK (Fig. 3B, lanes 3 and 4). HT22 cells cultured at high densities (∼80% or higher) were less sensitive to glutamate, i.e. glutamate-induced cell death was not as prominent as compared with cells cultured at lower densities (50-60%) (data not shown). For this reason, cells were transfected when the density reached 50-60%. This resulted in rather lower transfection efficiency (30-40%), which provides an explanation for the relatively less prominent inhibitory effect of PKCδ DN on ERK phosphorylation (Fig. 3B) as compared with the dramatic effect of rottlerin (Fig. 3A). Effects of rottlerin and exogenous expression of PKCδ DN on glutamate-induced activation of PKCδ were confirmed (Fig. 3A,B, lower panels). Together, these results suggest that activation of PKCδ mediates the glutamate-induced phosphorylation of ERK.
To determine whether PKCδ participated in the regulation of glutamate-induced neuronal cell death, we examined the effects of rottlerin and siRNA against PKCδ. The large number of apoptotic nuclei revealed by TUNEL assay, which was observed in cells 12 hours after glutamate treatment, did not appear when co-treated with rottlerin and glutamate (Fig. 3C). Since rottlerin has also been reported to inhibit other kinases such as calmodulin (CaM) kinase III (Parmer et al., 1997), we confirmed the effect of PKCδ on glutamate-induced cell death by using siRNA against PKCδ. As shown in Fig. 3C, glutamate-induced cell death was significantly recovered when cells were transfected with siRNA against PKCδ, suggesting that PKCδ plays an important role in the regulation of cell death in glutamate-treated cells.
Glutamate-induced ROS generation is involved in the activation of PKCδ and ERK1/2 in HT22 cells
Since exposure of HT22 cells to glutamate has been shown to increase the level of reactive oxygen species (ROS) (Tan et al., 1998) (Fig. S2A, supplementary material), we compared the kinetics between intracellular accumulation of ROS and PKCδ activation under glutamate treatment. The level of ROS slightly increased for the first 3 hours under glutamate exposure, followed by a much higher level. The time course of ROS production correlated with the window of time in which PKCδ was activated in response to glutamate treatment (Fig. S2A, supplementary material), suggesting that ROS might play a role in the activation of PKCδ. To test this possibility, cells were treated with N-acetyl cysteine (NAC), a scavenger of reactive oxygen intermediates. NAC treatment inhibited glutamate-induced activation of PKCδ and reduced the level of phospho-ERK1/2 elevated by glutamate treatment (Fig. S2B, supplementary material), suggesting that ROS generation is upstream of the activation of PKCδ and ERK1/2 in glutamate-treated HT22 cells.
MKP-1 is involved in the regulation of ERK1/2 phosphorylation
We then investigated the mechanism by which PKCδ induced sustained activation of ERK1/2. Dual-specificity phosphatases are an emerging subclass of the protein phosphatase gene superfamily, which appears to be selective for dephosphorylating the phosphotyrosine and phosphothreonine/serine residues within MAPKs (Keyse, 2000). To determine whether sustained activation of ERK1/2 induced by glutamate was associated with downregulation of MKPs, we examined the protein levels of MKPs in HT22 cells exposed to glutamate in a time-dependent manner. We observed that MKP-1 increased slightly during glutamate exposure until 1 hour, after which it substantially decreased (Fig. 4A). However, MKP-2 and -3 were not detected in unstressed and glutamate-treated cells up to 9 hours (data not shown), excluding the involvement of MKP-2 and -3 in glutamate-induced cell death.
To examine the role of MKP-1 in the regulation of ERK1/2 in HT22 cells, we transiently transfected the cells with either wild-type MKP-1 or its catalytically inactive mutant (MKP-1CS). Overexpression of MKP-1 reduced the level of phospho-ERK1/2 elevated by glutamate (Fig. 4B, lane 5), whereas overexpression of MKP-1CS significantly enhanced the glutamate-induced ERK1/2 phosphorylation (Fig. 4B, lane 6). Similarly, the basal level of phospho-ERK1/2 was reduced when cells were transfected with MKP-1, but was enhanced when cells were transfected with MKP-1CS (Fig. 4B, lanes 2 and 3).
We also applied siRNA against MKP-1. As shown in Fig. 4C, depletion of MKP-1 caused a pronounced enhancement in the basal level of phospho-ERK1/2. Moreover, glutamate-induced ERK1/2 phosphorylation was also increased in cells transfected with siRNA against MKP-1. Similar results were obtained when we used another siRNA that was designed against a different region of MKP-1 (Fig. 4D). We found that phosphorylation of both p38 and JNK in unstressed and glutamate-treated cells was not altered by siRNA against MKP-1 (Fig. 4D). These results suggest that MKP-1 plays an essential role in the regulation of both basal and glutamate-induced activation of ERK1/2.
Glutamate induces ubiquitylation and downregulation of MKP-1 through activation of PKCδ
We next examined whether PKCδ was involved in the downregulation of MKP-1 under glutamate exposure. Glutamate-induced downregulation of MKP-1 was not observed when cells were transfected with PKCδ DN (Fig. 5, lanes 3 and 4), and similar results were obtained when cells were treated with rottlerin (data not shown). These results suggest that activation of PKCδ significantly contributes to the downregulation of MKP-1 under long-term glutamate exposure.
MKP-1 has been reported to be a labile protein that is targeted for degradation by the ubiquitin-proteasome pathway (Brondello et al., 1999). To investigate whether the ubiquitin-mediated degradation pathway was involved in the glutamate-induced downregulation of MKP-1, we examined the effects of MG132, ALLN and lactacystin, which are common proteasome inhibitors. As shown in Fig. 6A, the level of MKP-1 in cells treated with proteasome inhibitors was dramatically higher as compared with untreated cells. Furthermore, a glutamate-induced decrease of MKP-1 was not observed in ALLN- or lactacystin-treated cells, indicating that proteasomal degradation of MKP-1 was involved in the downregulation of MKP-1 under glutamate exposure. We then examined whether MKP-1 was ubiquitylated in response to glutamate treatment. As shown in Fig. 6B, glutamate induced polyubiquitylation of MKP-1, suggesting that glutamate triggers degradation of MKP-1 through the ubiquitin-proteasomal pathway.
Since PKCδ contributed to the downregulation of MKP-1 during glutamate exposure (Fig. 5A,B), we examined whether ubiquitylation of MKP-1 was also regulated by PKCδ. As shown in Fig. 6C, rottlerin significantly prevented the polyubiquitylation of MKP-1 in glutamate-treated cells. Similar results were obtained by exogenous expression of PKCδ DN (data not shown). Since activation of PKCδ was regulated by ROS production (Fig. S2B, supplementary material), we examined whether generation of ROS was involved in the glutamate-induced downregulation of MKP-1. NAC blocked the glutamate-induced downregulation of MKP-1 as expected (Fig. S2B, supplementary material). Collectively, these results indicate that glutamate-induced activation of PKCδ contributes to the ubiquitylation and downregulation of MKP-1.
Inhibition of PKCδ activity suppressed the ubiquitylation-mediated degradation of MKP-1 in primary immature cortical neurons
In agreement with a previous study (Stanciu et al., 2000), primary cultures of rat immature cortical neurons underwent apoptotic cell death within 24 hours of exposure to glutamate, which was prevented by administration of U0126 (Fig. 7A). Similar results were obtained from the TUNEL assay (Fig. 7A). As observed in HT22 cells (Fig. 1A), treatment of mouse immature cortical cells with glutamate led to the activation of ERK1/2 (Fig. 7C, lane 4), and U0126 reduced both the basal and glutamate-induced elevation of phospho-ERK1/2 (Fig. 7C, lanes 3 and 6). Moreover, MKP-1 was significantly increased at 30 minutes after glutamate exposure, followed by a substantial decrease as observed in HT22 cells (data not shown and Fig. 4A).
To investigate the role of PKCδ during glutamate-induced toxicity in immature cortical cells, we examined the effect of rottlerin and siRNA against PKCδ. As shown in Fig. 7, both rottlerin and siRNA against PKCδ conferred significant neuroprotection against glutamate-induced toxicity. Rottlerin caused a reduction in the level of phospho-ERK1/2 elevated by glutamate (Fig. 7C), and it also caused an increase in the level of MKP-1 (Fig. 7D). Glutamate evoked polyubiquitylation of MKP-1, which was significantly blocked by rottlerin (Fig. 7D), supporting the notion that the decrease of MKP-1 is strongly associated with the glutamate-induced activation of PKCδ in immature mouse cortical neurons as in HT22 cells.
Role of MKP-1 in the regulation of glutamate-induced cell death
Since downregulation of MKP-1 by glutamate was associated with persistent activation of ERK1/2, which contributes to oxidative cell death, we examined whether cell viability was affected by directly regulating the level of MKP-1. Glutamate-induced cell death was rescued by overexpression of MKP-1 (Fig. 8A), but was significantly increased when cells were transfected with siRNA against MKP-1 (Fig. 8B). The effect of siRNA against MKP-1 on glutamate-induced cell death was reversed by U0126, which prevents activation of ERK1/2 (Fig. 8D), supporting the notion that downregulation of MKP-1 is involved in glutamate-induced cell death through activation of ERK1/2. Cell viability was substantially decreased when cells were transfected with siRNA against MKP-1 even in the absence of glutamate, suggesting that MKP-1 plays an essential role in the regulation of cell survival both in unstressed and glutamate-treated cells.
To confirm the role of MKP-1 in glutamate-induced cell death, immature cortical cells were transfected with siRNA against MKP-1. As expected, cell viability was significantly decreased when cells were transfected with siRNA against MKP-1, both in the absence or presence of glutamate (Fig. 8C). Moreover, depletion of MKP-1 caused a pronounced enhancement in the basal level of phosho-ERK1/2 in immature cortical cells as in HT22 cells. Furthermore, glutamate-induced ERK1/2 phosphorylation was also increased in cells transfected with siRNA against MKP-1 (Fig. 8C), indicating that MKP-1 contributes to the glutamate-induced neuronal cell death through regulation of ERK1/2 phosphorylation.
In the present study, we suggest that downregulation of MKP-1 is responsible for the sustained activation of ERK1/2, which plays an important role during glutamate-induced neuronal cell death. This study also provides evidence that downregulation of MKP-1 occurs through its ubiquitylation by activation of PKCδ.
ERK has been shown to be activated in response to oxidative toxicity. A previous study provided evidence for a protective role of ERK during oxidative stress, suggesting that H2O2-stimulated activation of ERK2 has a crucial role in preventing apoptosis (Guyton et al., 1996). By contrast, other studies demonstrated that the MEK/ERK pathway has a pro-apoptotic effect on neuronal cell death. For example, several neurological insults, such as ischemia and kainate-induced seizure, induce excessive release of excitatory amino acids and the subsequent neuronal cell death resulted in the activation of ERK1/2 in vivo (Hu and Wieloch, 1994; Kim et al., 1994), and inhibition of the MEK/ERK pathway protected the brain from ischemic injury (Alessandrini et al., 1999). A recent study has demonstrated that oxidative toxicity in HT22 cells and immature embryonic rat cortical neurons was associated with the delayed activation of ERK1/2 and that inhibition of ERK1/2 activation protected the cells against glutamate-induced cell death (Stanciu et al., 2000). In agreement with that report, we found that glutamate led to persistent activation of ERK1/2, which was associated with neuronal cell death (Figs 1, 7). The mechanisms that underlie such diametric effects of ERK are unclear, but several lines of evidence suggest that cell death and survival might be related to the kinetics of its activation, i.e. rapid and transient activation of ERK1/2 has been shown to be associated with enhanced survival response, whereas delayed and sustained activation of ERK1/2 has a tendency to trigger cell death (Guyton et al., 1996; Wang et al., 2000). Therefore, regulating activation of ERK appears to be one of the key events that control cell death and survival.
Protein phosphatases have an important role in controlling the magnitude and duration of ERK1/2 activation. Thus, we focused on the possible role of MKP family members, which are known to be important for dephosphorylating specific MAPKs (Keyse, 2000). MKP-1, which was initially identified as an oxidative stress-induced protein phosphatase, was shown to dephosphorylate ERK, p38 MAPK and JNK (Sun et al., 1993; Franklin and Kraft, 1997; Lasa et al., 2002), depending on the cell type as well as the cellular context. Studies have demonstrated that the anti-apoptotic effect of MKP-1 was mainly associated with its ability to dephosphorylate p38 or JNK (Guo et al., 1998; Wu and Bennett, 2005). By contrast, it has also been reported that MKP-1 inactivates ERK, which resulted in inhibition of re-entry into the cell cycle (Brondello et al., 1995). Here, we suggest that glutamate-induced oxidative toxicity leads to downregulation of MKP-1, which results in persistent activation of ERK1/2. Overexpression of MKP-1 decreased the level of phospho-ERK1/2 (Fig. 4), whereas knockdown of MKP-1 using siRNA resulted in an enhanced phosphorylation of ERK1/2 (Fig. 4). MKP-1 has also been reported to regulate p38 MAPK and JNK activation (Lasa et al., 2002), and both kinases were previously shown to have crucial roles during oxidative stress in many cell types (Maher, 2001). However, we could not detect phosphorylation of p38 and JNK during glutamate exposure until 9 hours, in agreement with a previous report (Levinthal and DeFranco, 2005). Moreover, treatment with SB203580 and SP600125, specific inhibitors of p38 MAPK and JNK, respectively, failed to block the cell death induced by glutamate, excluding p38 MAPK and JNK in glutamate-induced cell death.
The suggested roles of MKP-1 in the regulation of cell death and survival have been controversial. A recent study showed that blocking dexamethasone-induced MKP-1 expression by siRNA protected cells from chemotherapy-induced apoptosis (Wu et al., 2005). By contrast, MKP-1 was also implicated in the anti-apoptotic effect of retinoids in mesangial cells incubated with H2O2 (Xu et al., 2002), and a recent report showed that MKP-1+/- and MKP-1-/- mouse embryonic fibroblasts (MEFs) proliferate at a slower rate compared with wild-type cells, owing to increased cell death (Wu and Bennett, 2005). Our results show that glutamate-induced cell death was rescued by overexpression of MKP-1 (Fig. 8A), but was significantly increased when cells were transfected with siRNA against MKP-1 (Fig. 8B,C). These results suggest that MKP-1 has a protective effect during oxidative toxicity.
It has been shown that degradation of MKP-1 protein is attenuated by inhibitors of the ubiquitin-directed proteasome complex (Brondello et al., 1999). Recently, Lin et al. suggested that degradation of MKP-1 was triggered by ERK signaling through activation of the ubiquitin-proteasome pathway, which contributes to the sustained activation of ERK1/2, thereby providing a positive-feedback mechanism (Lin et al., 2003). In the present study, we found that MKP-1 was downregulated after prolonged exposure to glutamate (Fig. 4), which was accompanied by its polyubiquitylation (Fig. 6). On the basis of these results, we concluded that long-term glutamate exposure induced degradation of MKP-1 through the ubiquitin-proteasome pathway. MKP-1 mRNA is an early response gene, and induction of MKP-1 was shown to be associated with activation of ERK1/2 and PKCϵ (Brondello et al., 1997; Valledor et al., 2000). In HT22 cells, we observed an increase in the protein level of MKP-1 as well as transient activation of ERK1/2 within 1 hour of glutamate treatment (data not shown). Moreover, the induction of MKP-1 was blocked by U0126 (data not shown), suggesting that activation of ERK1/2 is involved in the induction of MKP-1 observed within 1 hour after glutamate treatment.
Recent studies have reported that the level of PKCδ increased during kainate-induced neuronal cell death (Kaasinen et al., 2002) and after transient global brain ischemia (Koponen et al., 2000). In addition, dieldrin-induced and 1-methyl-4-phenylpyridinium (MPP+)-induced oxidative stress also caused persistent activation of PKCδ in dopaminergic neurons (Kaul et al., 2003; Kitazawa et al., 2003). Although several downstream targets of PKCδ have been described, the mechanism by which PKCδ regulates neuronal cell death is poorly understood. PKCδ has been reported to activate MAPK through several mechanisms. For example, a constitutively active mutant of PKCδ was reported to activate MAPK (Ueda et al., 1996), and neurogenic agents and estrogen induced PKCδ-dependent activation of MAPK (Corbit et al., 1999; Keshamouni et al., 2002). However, the molecular mechanism by which PKCδ activates MAPK has not been demonstrated. Here, we show that glutamate treatment resulted in a dramatic and persistent activation of PKCδ (Fig. 2), and that inhibition of PKCδ activity suppressed the glutamate-induced ERK1/2 phosphorylation (Fig. 3), demonstrating that PKCδ-dependent activation of ERK1/2 occurs during glutamate-induced neuronal cell death. Our data also show that glutamate-induced downregulation of MKP-1 was blocked by inhibition of PKCδ activation in HT22 cells (Fig. 5) as well as immature primary cortical neurons (Fig. 7). Furthermore, inhibition of PKCδ suppressed the polyubiquitylation of MKP-1 (Figs 6, 7). Therefore, we suggest that activation of PKCδ under glutamate exposure causes sustained activation of ERK1/2 through degradation of MKP-1.
Further studies are required to determine the exact mechanism by which activation of PKCδ leads to degradation of MKP-1. PKCδ might lead either to a direct or an indirect activation of the ubiquitin-proteasome pathway, targeting MKP-1 for degradation. Interestingly, PKC and ATM protein kinase were shown to phosphorylate Parkin and Mdm2, respectively, and modulation of the phosphorylation state of these proteins has been suggested to have a regulatory role on their E3 ubiquitin ligase activities (Yamamoto et al., 2005; Meulmeester et al., 2005). Therefore, it is plausible to suggest that activation of PKCδ results in phosphorylation of an E3 ubiquitin ligase and/or its upstream compartments involved in activating E3 ubiquitin ligase, which might participate in targeting MKP-1 for proteasomal degradation. An alternative mechanism might involve post-translational modification of MKP-1 itself by glutamate-induced PKCδ activation. In fact, p42/44 MAPK-dependent phosphorylation of MKP family members, such as MKP-1, MKP-2 and MKP-3, affect the rate of degradation by the proteasome (Brondello et al., 1999; Marchetti et al., 2005). In particular, phosphorylation of MKP-3 resulted in enhanced degradation by the proteasome (Marchetti et al., 2005). It is thus possible to suggest that activation of PKCδ might induce phosphorylation of MKP-1 directly or through activation of another kinase, which might accelerate the ubiquitylation and degradation of MKP-1. However, it is also possible that activation of PKC might induce degradation of MKP-1 without affecting its phosphorylation status, as illustrated in the case of downregulation of the dopamine transporter, which was rapidly downregulated without being phosphorylated (Granas et al., 2003; Miranda et al., 2005). Thus, further studies are required to clarify whether glutamate-induced PKCδ activation induces the direct phosphorylation of MKP-1, and whether phosphorylation triggers its ubiquitylation and degradation.
Taken together, we conclude that MKP-1 is an important mediator during glutamate-induced oxidative toxicity. Its degradation through the ubiquitin-proteasome pathway is a crucial step regulating persistent activation of ERK1/2, which contributes to glutamate-induced cell death in HT22 mouse hippocampal cells and immature cortical neurons.
Materials and Methods
Plasmids and materials
The plasmids pSG5-MKP-1 and its catalytically inactive mutant, pSG5-MKP-1CS, containing a single copy of the Myc epitope tag were kindly provided by N. Tonks (Sun et al., 1993). The plasmids pHA.CE-PKCδ and its dominant-negative mutant, pHA.CE-PKCδ (K376R) were kindly provided by Y. S. Lee (Lee et al., 2002). L-glutamate was purchased from Sigma. U0126, SB202190, SP600125, rottlerin, lactacystin, MG132 and histone H1 were obtained from Calbiochem. [γ-32P]ATP was purchased from NEN Life Science Products.
HT22 cell cultures and cell transfection
HT22 cells, a mouse hippocampal cell line, were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum and 1% penicillin/streptomycin in a humidified 5% CO2 incubator at 37°C. In a 6-well tissue culture plate (Falcon), HT22 cells were seeded at a density of about 2×105 cells per well. When the density of cells reached 50-60%, cells were transfected with expression plasmids for the dominant-negative mutant of PKCδ (K378R), or wild-type MKP-1, or its catalytically inactive mutant (MKP-1CS) using Lipofectamine™ reagent (Invitrogen). After incubation for 24 hours, the transfected cells were treated as indicated for analysis.
Primary cortical cell cultures
Mixed cortical cell cultures, containing both neuronal and glial elements, were prepared from embryonic day 13-15 ICR (Institute Cancer Research) mice as described previously (Kim et al., 1999). In brief, dissociated cortical cells were plated on poly-L-lysine-coated 24-well dishes and maintained for 24 hours in DMEM (glutamine-free) supplemented with 20 mM glucose, 2 mM glutamine, 5% fetal calf serum and 5% horse serum. When cultured cortical cell preparations were subjected to immunocytochemistry, the astrocyte marker glial fibrillary acidic protein (GFAP) was barely detectable (data not shown).
Cell viability assay
Cell viability was assessed by measuring their ability to metabolize 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Cells were seeded onto 96-well plates at a density of about 5×103 cells per well in growth medium and incubated until about 60-70% confluency, prior to the initiation of experimental treatment. Following the treatments as indicated, 15 μl of MTT solution (5 mg/ml) was added to each well, and cells were maintained for 4 hours at 37°C. 100 μl of solubilizing solution (50% dimethylformamide and 20% SDS, pH 4.8) was then added. After an overnight incubation at room temperature, absorbance at 570 nm was measured.
Visualization of apoptotic cells through TUNEL assay
After fixation in 4% paraformaldehyde, cells were washed with phosphate-buffered saline (PBS) and permeabilized with 0.2% Triton X-100, washed again with PBS, and incubated for 60 minutes at 37°C in the dark with the DeadEnd™ Fluorometric TUNEL System (Promega). In-situ-labeled nuclei were observed and photographed under a fluorescence microscope (Axioplan2, Zeiss).
Preparation of cell homogenate
In a 60 mm culture dish (Falcon), HT22 cells were seeded at a density of about 5×105 cells per dish. When the density of cells reached 60-70%, cells were treated with several stimulants for analysis. Cells were washed and resuspended in serum-free medium. The dishes were placed on ice, scraped with a rubber policeman, and centrifuged at 1000 g for 10 minutes. The supernatant was aspirated, and the cell pellet was resuspended in lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5% Triton X-100, 1 mM EDTA, 1 mM PMSF, 10 μg/ml pepstatin A, 10 μg/ml leupeptin, 10 μg/ml aprotinin, 1 mM NaF and 1 mM Na3VO4) on ice for 40 minutes. The cell lysates were centrifuged for 15 minutes at 22,250 g, supernatants were removed, and 5× sample buffer was added.
Immunoblot analysis
Lysates (40 μg of protein) were resolved by SDS-polyacrylamide gel electrophoresis (PAGE) and were transferred to nitrocellulose membranes. The membranes were blocked with 5% dry milk in PBS-T and subsequently incubated with the primary antibody. The mouse monoclonal antibody to PKCδ was obtained from BD Transduction Laboratories. Rabbit polyclonal antibodies to pERK, ERK and MKP-1 were purchased from Cell Signaling Technology and Santa Cruz Biotechnology, and mouse monoclonal antibody against actin was from NeoMarkers. Specific reactive bands were detected by using a goat anti-rabbit or goat anti-mouse IgG conjugated to horseradish peroxidase, and the immunoreactive bands were visualized by the SUPEX western blotting detection kit (Neuronex).
Briefly, cell lysates were prepared in lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerol phosphate, 10 μg/ml pepstatin A, 10 μg/ml leupeptin, 1 mM PMSF, 10 μg/ml aprotinin, 10 mM NaF and 1 mM Na3VO4). Equal amounts of proteins were immunoprecipitated using anti-PKCδ, anti-MKP-1 and anti-ubiquitin antibodies (Santa Cruz Biotechnology) and collected with protein A-Sepharose beads (Santa Cruz Biotechnology) at 4°C for 16 hours. The immunoprecipitate was then washed four times in cold lysis buffer and subjected to western blot analysis and PKCδ kinase assay.
PKCδ kinase assay
The activity of PKCδ was determined by immune complex kinase assay (Lee et al., 2003). The anti-PKCδ immunoprecipitate was washed in a kinase reaction buffer (25 mM Tris-HCl, pH 7.5, 5 mM β-glycerol phosphate, 2 mM dithiothreitol, 0.1 mM sodium orthovanadate and 10 mM MgCl2). The kinase assay was carried out in a total volume of 30 μl of a kinase reaction buffer containing 50 μM ATP, 1 μCi of [γ-32P]ATP and 200 μg/ml histone H1 for 30 minutes incubation at 30°C. Phosphorylated histone H1 was resolved on 14% SDS-PAGE followed by autoradiography.
siRNA experiments
siRNA duplexes targeting MKP-1 (5′-AACGAGGCTATTGACTTCATA-3′ and 5′-CCGCACAAGATCGACCGACTT-3′) and PKCδ were purchased from Dharmacon. A scrambled non-targeting siRNA was used as a negative control. Transfections of siRNA duplexes at 50 nM of final concentrations were carried out using Metafectene (Biontex) according to the manufacturer's recommendations. After transfection, the cells were processed for western blotting and/or MTT assay as indicated. Effect of PKCδ siRNA in primary cortical cells was confirmed by quantitative RT-PCR, which was performed as described in detail elsewhere (Lee et al., 2005). Primers used in PCR were as follows: GAPDH forward 5′-GCCATCAATGACCCCTTCATT-3′ and reverse 5′-GCTCCTGGAAGATGGTGATGG-3′; PKCδ forward 5′-TCTGGGAGTGACATCCTAGACAACAACGGG-3′ and reverse 5′-CAGATGATCTCAGCTGCATAAAACGTAGCC-3′.
Statistical analysis
All experiments were independently repeated a minimum of three times. All quantitative data are presented as mean ± s.d. Comparisons between two groups were analyzed using Student's t test, and values of P<0.05 were considered to be significant.
This work was supported by the Brain Neurobiology Research Program (M10412000023-05N1200-02310) and Systems Biodynamics NCRC sponsored by the Korean Ministry of Science and Technology and Brain Korea 21 Program of the Ministry of Education. We thank N. Tonks (Cold Spring Harbor, NY) and Y. S. Lee (Korea Cancer Center Hospital, Korea) for offering valuable MKP-1 and PKCδ plasmids, respectively. We also greatly appreciate D. Schubert (The Salk Institute for Biological Studies, CA) for offering HT22 cells and Y. Kang for critical reading of the manuscript.
Supplementary material available online at http://jcs.biologists.org/cgi/content/full/7/1329/DC1
- Accepted December 15, 2005.
- © The Company of Biologists Limited 2006


text:
Creme Pur Equilibre
rebalancing cream for combination skin
contains a sebum regulating complex
Creme pur Equilibre acts to:
- purify oily skin
- combat shine
- tighten pores
- restore balance
- 50ml
creme pur equilibre
Creme pur Equilibre treats shine and dilated pores caused by excess sebum in the skin. So what's the magic ingredient? Guinot's Sebum Regulating Complex! Full of active ingredients Guinot's laboratory created and tested the active complex. The result? Intensive anti-seborrheic activity. Creme pur Equilibre has demonstrated effectiveness on all types of oily skin. Your skin will feel rebalanced. Skin is soothed, purified and oiliness is visibly reduced.
Use all year round, morning and/or evening. To maximise results use with Mousse Microbiotic for in-depth cleansing. Follow with Lotion Microbiotic to complete the cleansing process and to mattify the skin.


text:
Cherry Tree Montessori Nursery & Pre-School
We are a small, close-knit nursery, Ofsted registered for 27 children. We are located adjacent to the Ringwood Forest in the village of Ashley Heath, providing a picturesque and natural setting. We are conveniently located less than 1 mile from the A31/A338 junction close to Ringwood.
- Telephone
01425 477400
Mobile: 07706 660268
- email@example.com
- Website
- http://cherrytreemontessori.com/
- Venue address
1 High Street
Ashley Heath
- Postcode
- Locality
- East Dorset
- Opening times
- Mon: 08:00-18:00
Tue: 08:00-18:00
Wed: 08:00-18:00
Thu: 08:00-18:00
Fri: 08:00-18:00
Service Details
- Vacancy information
Please call for vacancy details.
- Cost(s)
£5.50 per Hour
£22.00 per Session
£49.50 per Day
£247.50 per Week
- Cost variations
- Breakfast 50p. There is also a price variation depending on the child's age, prices quoted above are for babies up to the age of 2. For children up to 3 years old - £5.40 for lunch and morning/afternoon sessions is £21.60 = £48.60/day. Children over 3 years old - £5.35 lunch and morning/afternoon Sessions = £21.40 = £48.15. We also offer an afterschool club and holiday club at £35/day. The nursery also offers the new 30 hour funding alongside the standard 15 hour funding. Funding is offered between 8.30am to 5pm, Monday to Friday.
- Additional Information
We now have a breakfast club between 8am to 9am, please ring for further details. We have Dance and Musical Movement class on a daily basis and French classes on a Wednesday, Pilates on a Tuesday. We do regular cooking, arts and craft activities. We incorporate the Montessori method into the EYFS curriculum to prepare our children for school. We have also now extended the garden and are building an outside classroom to extend the childrens inside learning activities outside, along with a raised garden for the children to learn about the outside environment. Each afternoon we take the children out for a walk into the forest and onto Moors Valley. After 5pm an additional charge will apply - please ask for details. We have been awarded a Level 5 (Very Good) in Food Hygiene'. Provision has an inclusion / equal opportunities policy.
- Age Range
0 years 0 months to 8 years 0 months
Ofsted Registered Details
- Ofsted inspection result
Date of last inspection Inspection result Ofsted report 26/11/2019 Satisfactory/Requires Improvement View here
- Ofsted Report notes
- If a childcare provision is run by a school they may not be separately registered with Ofsted, therefore the link to their report will be for the school.
- Registration details
Registration number Ofsted registration date EY395931 15/06/2009
Early Education
- Free early education places
15 Hours For 2 year olds
No What is this?
15 Hours For 3 & 4 year olds
Yes What is this?
30 Hours For 3 & 4 year olds
Yes What is this?
- Wheelchair accessible
- No
- Parking availability information
- Off road parking. We have a small but secure off road car park where parents can park whilst dropping off their children.
- Toilet facility information
- Ground floor toilet.
- Other Facilities
- Senior Staff members are trained in supporting special needs. We have a dedicated special educational needs co-ordinator, Chris Needham - working with special needs children 0 to 5 years accredited by National Open College S.W.Region.
Special Educational Needs and Disabilities Local Offer
- Support for children and young people with SEN and disabilities
The nursery has two qualified SENCO staff who support all children with special needs, and we a good relationship with outside professionals and CDC.
- Record Last Updated
- 28/08/2020
Record Actions
Journey Planner
Address: 1 High Street Ashley Heath Ringwood Dorset


text:
Highly Porous Cathodes for Lithium Air Batteries - HiPoCat
The aim of the HiPoCat project (Highly Porous Cathodes for Lithium-Air Batteries) is to evaluate metal-organic frameworks (MOFs) and zeolitic imidazolate frameworks (ZIFs) for use as new precursors for cathode materials for Li-air batteries. Through pyrolysis of the highly microporous MOFs and ZIFs, cathode materials with high electronic conductivities, reactivity, and corrosion resistance will be synthesized. Furthermore, thermal analysis and kinetic modelling approaches will be used to determine the pyrolysis parameters leading to optimal porosities. The cathode materials that are developed in this work will be combined with suitable electrolytes and electrolyte additives to assemble Li-air batteries which display specific capacities, coulombic efficiencies, and rate-capabilities which are significantly higher than those of the conventionally used carbon-based cathodes. Additionally, the porous gas-diffusion cathodes will be produced using a lower number of synthesis steps and environmentally-friendly aqueous solvents, which will be a technological first in Austria.
Start: 06/2018
Duration: 1 Year
More Information: https://projekte.ffg.at/projekt/2929279


text:
I just need to put this somewhere. Why not here?
1. Don't say you are always lonely than you are busy when I ask to do something.
2. Don't ask for adventure and than blanche when I offer it.
3. Don't need someone with a big heart because you got none.


text:
Welcome to the Thanksgiving edition of Writing the World with Miss K!
Since I know that many students and families will be making their way out of town for Thanksgiving, I want to make sure you have the information you need to be able to succeed in my class!.
If a student is absent November 19th, 20th, or 21st, they are still responsible to make up the work. So, please read carefully below to know what we will be completed in class over the next two weeks!
9th Grade
(November 12 – 16)
Day 1: We will read Act 2, Scene 2 in class. For homework, you will read & watch (or listen) Act 3, Scene 1. As you read & watch (or listen), please annotate your text. I will check your annotations in class on Day 2. You will also have a quiz on the scene in class also on Day 2!
Note: I highly recommend watching this scene to help you fully understand it! The link that I have provided you to watch is a snippet of a high school production of A Midsummer Night's Dream. It is not perfect, and there are some line changes due to the nature of theater and human error. But, it does follow the play line for line. I recommend watching it and reading at the same time. Maybe read the play first, then watch and read along, to help with comprehension!
Day 2 – 4: After your quiz, we will read Act 3, Scene 2. This will take us through to Day 4. When we are done reading Scene 2, we will read a CommonLit article together and address how it connects to what we have been reading.
(November 19 – 21)
All Week: We will watch the first three acts of A Midsummer Night's Dream, directed by Michael Hoffman. This film can be found on Amazon Video and Itunes. We will watch the first 50 minutes of the film and will write a compare and contrast response, which will be posted on Google Classroom on November 16th. If not finished in class, the essay will be due when we return from the Thanksgiving Break on November 26, 2018.
12th Grade
(November 12 – 16)
Day 1 & 2: After reviewing your homework from the previous weekend, we will begin to read and discuss Act 3, Scene 3 of Othello. This reading and discussion will take us two days. For homework on Tuesday (due Thursday in class), you will read two CommonLit pieces. These will be passed out in class on Tuesday. You will come to class with one discussion question for class on Thursday in addition.
Day 3: Based on your reading from the homework, we will have a discussion and make connections to the play! Think about what essential questions we have been discussing that could be answered through this discussion.
Day 4: We will finish Act 3 on Friday by reading Act 3, Scene 4.
(November 19 – 21)
All Week: We will be watching Othello Act 3 of the film in class, and again, I will be asking you to compare and contrast the film vs. the play. This time the focus will be on the director's purpose – why do you believe the director chose to make these changes?