text:
The Onion, a satirical paper famous for headlines like "I'm Like a Chocoholic, But for Booze" now helps brands create funny content. We interviewed its president, Mike McAvoy, about The Onion's custom content agency, Onion Labs.Continue Reading
The Onion's Custom Content Agency Makes Brands Seriously Funny
Confessions From a Reddit Junkie and Why You Should Care
As a backwoods hiker, I have a morbid interest in bear attacks, and that's how I first heard about reddit. I stumbled on excerpts from an account by Allena Hansen who was mauled by a bear and managed to drive herself down a rutted mountain road with, as she put it, her "face hanging off." (Apologies for the goriest opener in the history of CMI.)
Reddit is a little hard to describe to the unfamiliar, but Wikipedia does it well: "Reddit is an entertainment, social networking, and news website where registered community members can submit content, such as text posts or direct links."Continue Reading
Society of Grownups Designed to Delight and Educate Audience
A few months ago, MassMutual quietly opened up the most ambitious, exciting content project we've seen in a long time. The Society of Grownups teaches classes, hosts supper clubs, and offers one-on-one financial advice … all with a bohemian vibe.
On a Saturday evening in November, I walked into an airy retail space in a hip neighborhood outside Boston. Inside, the converted storefront is part library, part bar, and part modern classroom outfitted with the latest technologies. The interior would make any design junkie swoon.Continue Reading
The Wizard of Moz Talks SEO and Shares Tips for 2015
Rand Fishkin, founder of Moz, a leading inbound marketing software company, and host of the popular video series, Whiteboard Fridays, answers questions about what he wishes marketers understood about search, and tips for upping your game this year.
Marketers seem ambivalent about search these days. As Google focuses more and more on quality content, there's a growing sense among marketers that search isn't as important as it once was. What do you wish marketers understood about SEO, and what are their blind spots?Continue Reading
19 CCO Articles in 2014 That Every Marketer Should Read
Over the last 12 months, CCO magazine doubled down on content marketing strategy for executives. While we still publish some how-to, tactical information, we are more focused on publishing content that helps marketers refine their existing systems, and prepare for future disruptions. Picking my favorite articles from 2014 is a little like asking me to choose my favorite child, but I did it. (Note: The titles are from the print version.) If you have a print issue, look these up, as we often feature sidebar content that doesn't make it online. (If you don't subscribe to the print version of CCO, make sure you sign up today – it's free.)Continue Reading
Star Shares What Content Marketers Can Learn From The Entertainment Industry
I had the pleasure of interviewing Academy Award winner Kevin Spacey at this year's Content Marketing World. Spacey opens up in this interview about nurturing creatives in the entertainment industry, collaborating with non-media brands, and crafting binge-worthy stories.
McDermott: Do you think non-media brands can pull off the kind of rich, nuanced storytelling we see coming out of the entertainment industry?Continue Reading
Choosing the Right Marketing Technology: The Yelp of Software Reviews
How do you make the right choice when buying content marketing technology? You can start the review process with G2 Crowd, which offers crowd-sourced enterprise software reviews. But unlike some review platforms under fire for spam, G2 authenticates each review by connecting members to their LinkedIn profiles.
G2 disrupts a market dominated by big market research firms like Gartner and Forrester because it covers smaller tech startups and highly specialized verticals – something the big research firms can't duplicate cost effectively. And the results of G2's lengthy surveys (as many as 60 questions) offer extremely thorough, robust information about individual products, as well as aggregated information about how users are deploying and optimizing software.Continue Reading
7 Inspiring Lessons from 2014's Top Content Marketers
One of the best ways to learn about the content marketing industry is to study what others are doing. Over the past three years, it's been our pleasure to observe the field and, more specifically, identify those who are making us think about successful content marketing in new ways.Continue Reading
The Artist's Perspective: Collaborating on Content Creation
Thomas Hawk's hauntingly beautiful — and often gritty — photography is admired across the world. He calls himself a "photography factory" and aims to capture 1 million photos before he dies. Among his favorites are images of American cities, particularly those in various states of decay: "I think people will want to go back in the future and remember a different type of America; a place that was unique to their timeframe," he says.
But to say Hawk is revered for his art is only partially true. He's also an avid blogger who writes about a wide range of issues, including new media, technology, creative commons, and copyright law. With a massive fan base across Flickr and Google+ in particular (more than 6.5 million followers), Hawk is sought by brands to photograph events and locations, and share this visual content with his audience. Yet, Hawk cautions: "Don't ask artists to shill your story." Continue Reading
Why Podcasting is a Huge Content Marketing Opportunity
Eight years ago, podcasting was held up as the "Next Big Thing." Chris Brogan, author of The Impact Equation and longtime podcasting fan, thinks it's finally ready to take off as a viable channel in your content marketing plan. Continue Reading


text:
On Tuesday, the Indian Government gave a tough reaction against the
five countries ‑ New Zealand, United States, United Kingdom, Canada and
Australia ‑ who had issued an advisory for their citizens asking them not to
travel to India during the holiday season on account of terror threats.
security of various tourist locations in India has been increased fourfold in
recent past as the reports of possible militant attacks have been doing rounds
within the media circuit. It is believed that the Indian Government's reaction
came in a bit too late as the number trail has been already hitting a low in
many of the tourism spots in terms of bookings and hotel reservations.
If sources are to be believed, the sense of negation that has come in
from the governments of these five countries seems to have affected the inbound
tourism. A small talk with the managing director of Le Passage to India, Arjun
Sharma, reveals that the possibilities of acquiring any fresh bookings from
these five countries has become almost negligible but thankfully there has been
no cancellations for the bookings already made.
Arjun was also quoted as saying
that 'Our season starts post-Diwali and we hope
things get sorted out at the start of the season. If the situation continues as
it is, new bookings will take a huge hit'.
Alarming Reports of Possible Terrorist
Attacks in India
On October 18 2011, the local consulates of New Zealand,
United States, United Kingdom, Canada and Australia issued a travel advisory that
held a statement of pure caution for their citizens, alarming them against the
possible terror strikes during the festival season in India. The consulates
were expected to continue this advisory until January 2012.
The US Consulate released a statement regarding this issue,
which read 'The Department of State reminds US citizens of the continued
possibility of terrorist attacks throughout India and urges citizens to pay
particular attention to their personal security during the Indian holiday
season, which includes Hindu, Islamic, and other religious and secular holidays
between October and January'.
Dispelling the Fears
However, the Indian set of ministers seems to a have a
different view on this issue. According to the Tourism minister Subodh Kant
Sahay, the advisory is nothing more than scaremongering, which will hit the
tourism industry hard during the peak of overseas business season. In a
statement to a media agency, Mr. Sahay said 'I have taken
it up with the External Affairs Ministry and asked them to persuade these
countries to withdraw the travel advisories immediately'.
The facts that the tourism ministry data reveals add to the
already built-up tension levels. According to the recorded data, of all the
tourists that visit India every year, 4.3% come from Canada, 13.5% hail from
Britain, 16% from the US, and 4.9% come from Australia. If the advisories are
not lifted soon, then the Indian tourism industry might face its darkest days
S.M. Krishna, External Affairs Minister, India, is in talks with his
counterparts in Canada, Australia and New Zealand. He has been attending the
CHOGAM Foreign Ministers' meeting and has been discussing this issue in detail on
the sidelines.
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subset: commoncrawl


text:
"Free client for P2P file exchange, based on the popular LimeWire."
FrostWire is a new P2P file exchange client based on the popular LimeWire.
The objective of FrostWire is to improve and amplify some of LimeWire`s functions. In FrostWire you can find a totally rennovated interface (which you can personalize with various visual themes), and it works similar to LimeWire. It is especially oriented to music and music files, but of course it will also download other file types.
FrostWire is an absolutely free P2P client, and is free of spyware and adware.


text:
Town Lodge Roodepoort
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Egoli the city of gold
Friday 14th August
Emotional farewells over we headed to Krugersdorp to drop bags at Jason and Pauline. After a quick cuppa with them we hit the road again past Soweto to spend the day at Gold Reef City. What a fabulous day out, as it isn't school holidays we had the run of the park
and fitted in the mine tour and gold pouring without waiting. The kids could go
on rides without queueing, sadly for Oli he is too short for many of ...
Ant's Hill
... then went to our large suite which was sort of one room split into three sections. The first was a massive 7foot bed for my mum and dad and then I had bunk beds. The third section was a large bathroom with a big bath and a shower that was more like a waterfall!!! This section also had a shower outside so you could watch the wildlife. The front of this was a massive window so you ...
The Flight
Clare and I made a conscious decision on this trip we would not fly "Staff Travel". Was that a mistake? We paid for Premium Economy seats so that we would not get bumped and our travel was guaranteed. Well there were 30 free seats in business on our flight so there was no doubt that wee would have flown but was there any great advantage? We had the same food, we had the same wine and the service was business by all means. The seats were not up to ...
The Itenerary
After careful thought and planning we have come up with the following game plan. Date and Activity Day 1 // November 27th ------ JHB-DBN drive down ------ DBN overnight Day 2 // November 28th ------ Flight DBN-CTN | Flight JE133 Mango: Airport 5:30am / Depart 6:45 / Arrive 9am CTN. ------ Book into hotel & car rental ------ Two Oceans Aquarium ------ Dinner in Bloubos strand ------ CTN overnight Day 3 // ...
Random Thoughts on Ultimate Africa 2013, Circuit 3
... hotels accommodated that need without any hesitation, and he was pleased at the consistent efforts to provide him with satisfactory alternatives to the routine menus.
The table service at the camps is quite elaborate -- large tables, cloth napkins, sometimes artfully folded, placemats or table cloths, candles, various decorations; soup and dessert are served at the table; the main course is buffet style, often on heated plates. The quality of the food ...


text:
NaBloPoMo Day 16: Birthday trip to @Nemacolin
So this is really yesterday's post, but I was laying in bed last night and realized I forgot to post, and just was not going to get out of bed for the sake of blogging. So 2 posts tonight and I say we're on track. My birthday is tomorrow, and to celebrate my birthday we went on a family trip up to Nemacolin Woodlands Resort. I've never been there before and I always love spa treatments and excellent food.
We started our visit with brunch at Lautrec. It's a lovely buffet with all kinds of pastries, fish, pancakes & waffles, desserts and even prime rib. Baby Beez's behavior has generally been getting better as she has gotten older, but I wanted to make sure she was on her best behavior. To encourage fancy manners, we wore fancy clothes. She loves fancy clothes so this was a thrill in and of itself. She also likes getting to choose her own foods out of a buffet. She was able to show her best manners for the meal and we had a very nice brunch.
Mr. Beez and I went to the spa while Baby Beez spent the day at the Kidz Klub. She was literally the only kid there, but that didn't bother her any. In typical only child fashion, she made up stories with her imaginary Harry Potter friends, went swimming, watched movies and played with toys. It did not bother her any that there were no other kids there. Although I venture to guess she was a little bit lonely, because by the time we left the resort, she was saying that she missed her friends.
After having an amaaaaazing time at the spa, Mr. Beez and I spent a good part of the afternoon sitting around, drinking wine and reading the whole internet. It's kind of a silly thing to do, but we don't get to just sit and relax very often so it was very nice.
We had dinner at Autumn, which included scallops with bacon jam. Yes, Please. The meal was lovely. We do get to go out to eat quite a lot, but it was nice to be able to just wander down and pick up our kid afterward, and then wander back to our room. Because we are total party animals, we were all in bed and asleep by 9:30. But that's a perfect birthday treat for me. I like sleep.


text:
Euro heading to parity
Tatiana Norkina, FBS analyst
Having tested the four-hour Ichimoku cloud lower border resistance and almost reached 1.1000, the rate of the major currency pair on Forex collapsed again yesterday, into the 1.0860 area. Both short-term and long-term trends on the four-hour timeframe have changed dramatically. Thus, the Tenkan and Kijun lines have formed a dead cross, and the Senkou Span A line drastic decline has led to intensifiaction of the Ichimoku cloud bearish character.
However, there is quite a strong support of 1.0840/60 at the current levels that could keep the pair from further tumbling. But a break through these levels would mean the downtrend strengthening.
Technical levels: support – 1.0840/60; resistance – 1.0920.
Trade recommendations:
1. Sell — 1.0920; SL — 1.0940; TP1 — 1.0840; TP2 — 1.0770.


text:
Check out samples here
For more info, or to set something up, email me
A collection of 22 of my best
artist advice articles
Pick it up for just $1
w/ Michael Imperioli ('14)
w/ Millionaires ('12)
w/ Adam Duritz, iLLspoKinN & Notar ('10)
w/ Kevin Pereira on the old set of
Attack of the Show ('09)
w/ Kevin Pereira on the old set of
Attack of the Show ('09)
At 10:10 PM, Lisa said…
Aww,I can't believe I missed Cap'n Crunk.Oh well.And I was all over that BET Fresh Prince Marathon too.I love that show.From reading this it seems like interviewing a big time rapper is just one of those everyday things,huh?Cool,cool.I still can't believe you don't have any friends buggin your for an autograph.Your blog should have more readers-it's interesting.
At 10:13 PM, Lisa said…
Oh,and have you heard of Raheem Devaughn(R&B singer)?
At 10:41 PM, Adam said…
I guess not too many people have surfed on over to my little place on the web. Hey, to each his (or her) own, right?In answer to your question about Raheem, I was actually at an event of his a couple years ago when he first signed to Jive. He can SING! Check that, he can SANG! Dude is seriously talented.And yes, you are correct, interviews are a part of my daily life, no matter how big or small the name is.
At 3:20 PM, Lisa said…
Cool.I'm waitin on his debut to drop.
Ten Secrets for Making a Show a Success Despite a Low Turnout
What Happens to an Artist When Their Record Label Folds


text:
The United States Congress is currently debating a major immigration reform and Britain's prime minister David Cameron wants to create the toughest rules in Europe for migrants. Everyone from the Queen to former US President George W Bush is weighing in on the immigration proposals for their countries.
The US and UK are hardly alone. Japan, Australia and Germany, among other nations, are also in ongoing political conversations about who should be allowed in their borders, who should be allowed to stay, and who should get what benefits.
Yet no one is asking: what country would you want to live in if immigration and visa rules weren't an issue?
Government data (pdf) show that legal immigration to the US is down slightly compared to its peak in the early 1990s, although the US clearly remains a top destination with over a million people getting permanent resident status a year. It's a similar story in the UK where migration is on the rise.
What country would be your top destination to live in and why?


text: About KEMETKEMET's common stock is listed on the NYSE under the symbol "KEM." At the Investor Relations section of our web site at http://ir.kemet.com/, users may subscribe to KEMET news releases and find additional information about our Company. KEMET applies world class service and quality to deliver industry leading, high performance capacitance solutions to its customers around the world and offers the world's most complete line of surface mount and through-hole capacitor technologies across tantalum, ceramic, film, aluminum, electrolytic, and paper dielectrics. Additional information about KEMET can be found at http://www.kemet.com. About NEC TOKIN NEC TOKIN was established in 1938 to commercialize the innovative material technology developed by the TOHOKU University. Taking advantage of its most advanced material technologies, NEC TOKIN is proud to offer tantalum capacitors, electric double layer capacitors, electro-magnetic devices, miniature relays, IC cards and IC tags, piezoelectric devices and sensors. For further information, please visit the NEC TOKIN home page at: http://www.nec-tokin.com/english/. Cautionary Statement on Forward-Looking Statements Certain statements included herein contain forward-looking statements within the meaning of federal securities laws about KEMET Corporation's (the "Company") financial condition and results of operations that are based on management's current expectations, estimates and projections about the markets in which the Company operates, as well as management's beliefs and assumptions. Words such as "expects," "anticipates," "believes," "estimates," variations of such words and other similar expressions are intended to identify such forward-looking statements. These statements are not guarantees of future performance and involve certain risks, uncertainties and assumptions, which are difficult to predict. Therefore, actual outcomes and results may differ materially from what is expressed or forecasted in, or implied by, such forward-looking statements. Readers are cautioned not to place undue reliance on these forward-looking statements, which reflect management's judgment only as of the date hereof. The Company undertakes no obligation to update publicly any of these forward-looking statements to reflect new information, future events or otherwise.


text:
The Hugh O'Brien Youth Leadership credit card is not a student credit card, but an affinity rewards credit card that benefits this nonprofit group. Hugh O'Brien Youth Leadership Program (HOBY) is designed to help youth find their leadership skills, develop interests in their community and simply make a difference in the lives of others. HOBY allows schools to pick out promising children and teens from different schools to attend training and motivational classes.
Compare this with other affinity reward credit card offers now!
HOBY has been providing similar programs since 1958 because they understand the need of inspiring youth to make a difference. If the children of this country are the future of this country, then HOBY wants to ensure that they understand the importance of this future and the impact that these youths will have on the world.
HOBY is a non-profit organization and as such depend on membership fees, donations and grants in order to continue providing their programs. Banks such as UMB have also stepped in to expand the ways that people can help HOBY by creating the Hugh O'Brien Youth Leadership Visa Credit Card.
Hugh O'Brien Youth Leadership Credit Card Overview
The HOBY credit card offers a 0% APR for the first 6 months of use for balance transfers and new purchases; as is the case with many balance transfer credit cards. After the introductory period is over, the APR increases to 12.99%. This percentage rate suggests that the HOBY credit card can be used as a good credit credit card or an excellent credit credit card.
UMB will donate $50 to HOBY for each person who applies and qualifies for the HOBY Visa. In addition, UMB will donate a small amount of money for every purchase that you make when you use your credit card. You will be provided the details of the percentage amount when you receive your HOBY credit card. You can also choose from five HOBY designs for the face of your credit card.
Hugh O'Brien Youth Leadership Credit Card Benefits
- No annual fee
- Low annual interest rate
- Benefits an excellent youth program
- 100% fraud protection, warranty management and purchase security
Hugh O'Brien Youth Leadership Credit Card Bottom Line
This is a credit card that is beneficial to the community and can make a difference in the lives of others. You do, however, have to have great credit to qualify. Compare other online credit card offers to the HOBY credit card now!


text:
Panasonic Digital Camera SV-AV100 / SV-AV20 / SV-AV30 Battery Charger
Item No.: Dekcell - CB-1021
Ratings: (0 review).
Availability: Out of Stock
Ships from U.S Warehouse. Most customers receive within one week.
Price: $8.95
Note: Order placed before 12:30PM Pacific Standard Time will normally be processed in the same day.
Related Items
- This Panasonic camera battery charger supports AC adapter input voltage: 90-250V 50/60Hz
- Car adapter input voltage: 12/24V
- It is compatible with these Panasonic cameras: SV-AV10, SV-AV100, SV-AV10-A, SV-AV10-R, SV-AV10-S, SV-AV20, SV-AV25, SV-AV30, SV-AV35
- Color: Black
- Brand new, one year warranty!
- Package contents: 1 x Compact ultra fast battery charger with built-in foldable AC plug, 1 x car adapter.
Product Reviews
Be the first to write a review for Panasonic Digital Camera SV-AV100 / SV-AV20 / SV-AV30 Battery Charger


text:
News By Tag
Industry News
News By Place
Industry News
Optimum Prime Products Introduces Optimum Mesh Screen Door with Money Back Guarantee
Optimum Prime Products has announced the release of the Optimum mesh screen door that incorporates 26 invisible powerful magnets and keeps bugs out.
Optimum Mesh Screen Door incorporates 26 invisible powerful magnets to ensure instant magnetic closing. It means when anyone walks through the mesh screen door, it will be closed instantly to prevent insects entering the house. The high-quality mesh screen door comes in a variety of designs to enable any homeowner buying screen door that fits interior décor as well.
Unlike other contemporary screen doors, Optimum Mesh screen door seals every possible gap effectively. Its screen size measures body dimensions of 36"x 83", therefore, it will be an ideal fit for doors with up to 31" x 82". Homeowners do not require any tools to install this mesh screen door as heavy-duty Velcro sewn in around edges to ensure it would not fall apart. There are 12 pairs of Velcro strips sewn into the mesh.
Due to 26 powerful invisible magnets, Optimum Mesh screen door quickly closes and seals the door automatically every time anyone walks through to keep bugs out. It does not only keep home pests & bug-free, but appropriate for pets as well as pets can come in and move out without any help. Optimum mesh screen door comes with risk-free 100% money-back guarantee that reflects company's trust and confidence in the product.
Powerful magnets of the Optimum Mesh screen door ensure that both panels of door seal quickly. It won't fall apart like any ordinary meshes shown on television. In bottom line, Optimum Mesh screen is durable, portable and storable, and capable of keeping your home free from irksome insects and pests.
Interested buyers can buy this product online and enjoy the benefits of this premium quality mesh door. The product will reach the shipping address within 3-5 business days. To place an order online, visit- http://www.amazon.com/
Olivia Williams
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text:
Broccolini with Sesame and Ginger
Broccolini is delicious when used in place of broccoli or asparagus in recipes. This side dish goes well with broiled fish or roasted or sauteed chicken.
- Servings: 4
Source: Everyday Food, October 2010
- 2 teaspoons white sesame seeds
- 2 teaspoons vegetable oil
- 2 bunches broccolini (about 1 pound), stalks halved lengthwise if large
- 2 garlic cloves, thinly sliced
- 1 small piece peeled fresh ginger, thinly sliced
- 1/2 cup water
- 2 teaspoons soy sauce
- 1 teaspoon toasted sesame oil
Heat a large skillet over medium. Add sesame seeds and toast, tossing, until golden, about 3 minutes. Transfer to a small bowl.
Return skillet to heat and add vegetable oil. Add broccolini, garlic, ginger, and water. Cover and cook, stirring occasionally, until broccolini is crisp-tender, about 12 minutes. Add soy sauce and cook until it evaporates, 30 seconds. Top with sesame oil and sesame seeds.


text:
The E-NTPDase family of ectonucleotidases: Structure function relationships and pathophysiological significance
- First Online:
- Received:
- Accepted:
DOI: 10.1007/s11302-006-9003-5
- Cite this article as:
- Robson, S.C., Sévigny, J. & Zimmermann, H. Purinergic Signalling (2006) 2: 409. doi:10.1007/s11302-006-9003-5
- Citations
- 2.3k Downloads
Ectonucleotidases are ectoenzymes that hydrolyze extracellular nucleotides to the respective nucleosides. Within the past decade, ectonucleotidases belonging to several enzyme families have been discovered, cloned and characterized. In this article, we specifically address the cell surface-located members of the ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase/CD39) family (NTPDase1,2,3, and 8). The molecular identification of individual NTPDase subtypes, genetic engineering, mutational analyses, and the generation of subtype-specific antibodies have resulted in considerable insights into enzyme structure and function. These advances also allow definition of physiological and patho-physiological implications of NTPDases in a considerable variety of tissues. Biological actions of NTPDases are a consequence (at least in part) of the regulated phosphohydrolytic activity on extracellular nucleotides and consequent effects on P2-receptor signaling. It further appears that the spatial and temporal expression of NTPDases by various cell types within the vasculature, the nervous tissues and other tissues impacts on several patho-physiological processes. Examples include acute effects on cellular metabolism, adhesion, activation and migration with other protracted impacts upon developmental responses, inclusive of cellular proliferation, differentiation and apoptosis, as seen with atherosclerosis, degenerative neurological diseases and immune rejection of transplanted organs and cells. Future clinical applications are expected to involve the development of new therapeutic strategies for transplantation and various inflammatory cardiovascular, gastrointestinal and neurological diseases.
Keywordsapyrase brain CD39 ecto-ATPase immunology ischemia kidney liver nervous tissue NTPDase platelet vasculature
apyrase conserved regions
adenosine deaminase
- ATPDase
ATP diphosphohydrolase
- bFGF
basic fibroblast growth factor
cholecystokinin octapeptide-8
endothelial cell
- E-NTPDase
ecto-nucleoside triphosphate diphosphohydrolase
extracellular regulated kinase
focal adhesion kinase
mitogen-activated protein
MAP kinase
nitric oxide
nucleotide pyrophosphatase/phosphodiesterase
- NTPDase
nucleoside triphosphate diphosphohydrolase
platelet derived growth factor
phosphatidylinositol 3-kinase
- RanBPM
ran binding protein
tetrachlorodibenzo-p dioxin
Extracellular nucleotides modulate a multiplicity of tissue functions including development, blood flow, secretion, inflammation and immune reactions. Indeed, signaling via extracellular nucleotides has been recognized for over a decade as one of the most ubiquitous intercellular signaling mechanisms [1, 2]. Essentially every cell in a mammalian organism leaks or releases these mediators, and carries receptors for nucleotides of which seven ionotropic (P2X) and at least eight metabotropic (P2Y) receptor subtypes have been identified and characterized to date. Whereas P2X receptors respond to ATP, P2Y receptors can be activated by ATP, ADP, UTP, UDP, ITP, and nucleotide sugars, albeit agonist specificity varies between subtypes and the multiple animal species [3]. Depending on the P2 receptor subtype and signaling pathways involved, these receptors trigger and mediate short-term (acute) processes that affect cellular metabolism, adhesion, activation or migration. In addition, purinergic signaling also has profound impacts upon other more protracted responses, including cell proliferation, differentiation and apoptosis, such as seen in atherosclerosis, degenerative neurological diseases and in several inflammatory conditions [2, 4, 5].
The effects of extracellular nucleotides appear to overlap, at least in part, with those of vascular growth factors, cytokines (inflammatory), adhesion molecules and nitric oxide (NO). Nucleotide-mediated activation may be also synergistic with polypeptide growth factors (PDGF, bFGF) and insulin, the signaling being mediated via phospholipase C and D, diacylglycerol, protein kinase C, ERKs, phosphatidylinositol 3-kinases (PI3K), MAP kinases (MAPK) and Rho [6, 7, 8]. The situation concerning extracellular nucleotide-signaling can be suitably contrasted with the unique specificity of peptide hormones or vasoactive factors for often single, defined receptors [9, 10]. Within purinergic/pyrimidinergic signaling events specificity is dictated by three essential modulatory components: 1) The derivation or source of the extracellular nucleotides [1, 11, 12]; 2) the expression of specific receptors for these molecular transmitters (and for the nucleotide and nucleoside derivatives) [13, 14, 15, 16] (See also Molecular Recognition Section of National Institutes of Health, http://mgddk1.niddk.nih.gov/ also http://www.ensembl.org/index.html and http://www.geocities.com/bioinformaticsweb/speciesspecificdatabases.htm), and, 3) select ectonucleotidases that dictate the cellular responses by the stepwise degradation of extracellular nucleotides to nucleosides [17, 18, 19, 20].
Ensembles of ectonucleotidases, associated receptors and signaling molecules
Within the past decade, ectonucleotidases belonging to several enzyme families have been discovered, cloned and functionally characterized by pharmacological means. Specifically, we refer here to members of the ecto-nucleoside triphosphate diphosphohydrolase (ENTPDase) family (EC 188.8.131.52) as ectoenzymes that hydrolyze extracellular nucleoside tri-and diphosphates and have a defined pharmacological profile. Most notably, in many tissues and cells, NTPDases comprise dominant parts of a complex cell surfacelocated nucleotide hydrolyzing and interconverting machinery. This ensemble includes the ecto-nucleotide pyrophosphatase phosphodiesterases (E-NPPs), NADglycohydrolases, CD38/NADase, alkaline phosphatases, dinucleoside polyphosphate hydrolases, adenylate kinase, nucleoside diphosphate kinase, and potentially ecto-F1-Fo ATP synthases [21, 22, 23, 24, 25] that may interact in various tissues and cellular systems.
The ectonucleotidase chain or cascade, as initiated by NTPDases can be terminated by ecto-5′-nucleotidase (CD73; EC 220.127.116.11) with hydrolysis of nucleoside monophosphates [26]. Together, ecto-5′-nucleotidase and adenosine deaminase (ADA; EC 220.127.116.11), another ectoenzyme that is involved in purine salvage pathways and converts adenosine to inosine, closely regulate local and pericellular extracellular and plasma concentrations of adenosine [10, 27].
Several of these ectonucleotidase families and additional functions of NTPDases [28, 29, 30] are addressed elsewhere in this issue in detail. This review focuses on the surface-located mammalian members of the E-NTPDase protein family. It starts with a brief introduction of molecular structure and functional properties, followed by an analysis of the physiological and pathophysiological roles at various sites with an emphasis on vasculature and neural tissues.
Molecular identities unraveled
The literature on the molecular and functional characterization of the E-NTPDase family has been intensively reviewed [18, 19, 20, 21, 22, 31, 32, 33, 34, 35, 36] and will not be repeated here in detail. Our intent is to summarize principal properties of the enzymes that will be of use for the reader new to this field.
Nomenclature of mammalian members of the E-NTPDase family and chromosomal localization
Protein name
Additional names
Gene name human, mouse
Chromosome location human, mouse
Accession number human, mouse
ENTPD1, Entpdl
10q24, 19C3
ENTPD2, Entpdl
9q34, 2A3
ENTPD3, Entpd3
3p21.3, 9F4
ENTPD4, Entpd4
8p21, 14D1
ENTPD5, Entpd5
14q24, 12E (12D1)a
ENTPD6, Entpd6
20p11.2, 2G3
ENTPD7, Entpd7
10q24, 19D1 (19C3)a
ENTPD8, Entpd8
9q34, 2A3
The presence of ATP and/or ADP hydrolyzing activity at the surface of many cell types had been recognized for several decades [17, 37, 38, 39, 40]. However, the molecular identity of the first member of the ENTPDase family (NTPDase1) was not unraveled and determined until the mid-1990s. The prototypic member of the enzyme family had first been cloned and sequenced as a lymphocyte cell activation (CD39) antigen of undetermined function [41]. Final success came from three independent approaches. Handa and Guidotti [42] purified and cloned a soluble ATP diphosphohydrolase (apyrase) from potato tubers and noted that this protein was related not only to similar enzymes of some protozoans, plants and yeast but also to human CD39. They also recognized conserved sequence domains and the relation to members of the actin-hsp70-hexokinase superfamily. This was then followed by the functional expression of human CD39 and the demonstration that this protein was in fact an ecto-apyrase [43]. In parallel, ectonucleotidases (termed ATP diphosphohydrolases) from porcine pancreas and bovine aorta were purified. The partial amino acid sequences for both ATP diphosphohydrolases revealed identity with the cloned cDNA sequence of CD39 [44]. The cDNA was isolated from human endothelial cells and functional, thromboregulatory studies confirmed that the dominant vascular ectonucleotidase (ATP diphosphohydrolase) activity was identical to the previously described and cloned human CD39 [44]. Several internal peptide sequences obtained from the purified human placental ATP diphosphohydrolase [45] revealed that in retrospect this protein was also identical to CD39.
It was originally thought that there existed a single ectonucleotidase of the NTPDase type with potential post-translational modifications [46]. However, a close molecular relative was soon cloned that revealed functional properties of an ecto-ATPase (now NTPDase2) rather than of an ecto-ATP diphosphohydrolase [47, 48]. Further human genomic analysis of expressed sequence tags (ESTs) allowed the identifi-cation of additional members of the gene family [49, 50, 51]. These genes were originally named CD39L(ike)1 to CD39L4. Then followed the identification, cloning and functional expression of all members of the ENTPDase family, the last to date being NTPDase8 [52].
Potential splice variants have been isolated for the surface-located NTPDase1 and NTPDase2 [for references see 34, 53]. It should be further noted that heterologous expression of potential splice variants does not necessarily result in the formation of a functional protein [54].
The initially proposed nomenclature [50] has been somewhat confusing as it did not meet with generally accepted norms for human cell differentiation molecules [55]. While CD39 (now NTPDase1) indeed belongs to the cluster of differentiation antigens, CD39L1 (NTPDase2), CD39L3 (NTPDase3), CD39L4 (NTPDase5) and CD39L2 (NTPDase6) do not. Scientists at the Second International Workshop on Ecto-ATPases proposed that all E-NTPDase family members be termed as NTPDase proteins and classi-fied in order of discovery and characterization [34, 56]. The CD39 nomenclature should fall away for all but the prototypic member NTPDase1 that already has a long history of use in the Immunology and Oncology fields. Further revisions are however inevitable.
Catalytic properties
Membrane-bound NTPDase1 hydrolyzes ATP almost directly to AMP with the transient production of minor amounts of free ADP (Fig. 2). This functional property largely circumvents activation of P2Y-receptors for nucleoside diphosphates. Interestingly, significant amounts of UDP are accumulated when UTP is hydrolyzed by NTPDase1 [57]. In contrast, ADP is released upon ATP hydrolysis by NTPDase2, then accumulates and is slowly dephosphorylated to AMP. On the one hand, this results in the removal of agonists for nucleoside triphosphate-sensitive P2Y-receptors (Fig. 2). On the other hand, it generates agonists for nucleoside diphosphate-sensitive receptors such as platelet P2Y1 and P2Y12 receptors [62]. The actions of NTPDase3 and NTPDase8 result in intermediate patterns of product formation, leading to a transient accumulation of diphosphonucleosides with the simultaneous presence of triphosphonucleosides.
Principal structural features
The hallmarks of all NTPDases are the five highly conserved sequence domains known as 'apyrase conserved regions', abbreviated and termed ACR1 to ACR5 [42, 63, 64] that are involved in the catalytic cycle. This notion is supported by a considerable variety of deletion and mutation experiments [for reviews see 30, 34, 64, 65, 66, 67, 68].
NTPDases readily form homo-oligomeric assemblies. NTPDase1 to NTPDase3 were found as dimers to tetramers [29, 64, 73, 74, 75, 76, 77, 78]. In contrast to the P2X receptors that share a similar membrane topography, hetero-oligomeric complexes between NTPDases have not been reported, to date. Oligomeric forms reveal increased catalytic activity [73, 75, 76] and the state of oligomerization can affect catalytic properties [77, 78].
NTPDase1,2,3, and 8 are firmly anchored to the membrane via two transmembrane domains that in the instance of NTPDase1 are important for maintaining catalytic activity and substrate specificity [29, 64, 79]. The two transmembrane domains interact both within and between monomers. They may also undergo coordinated motions during the process of nucleotide binding and hydrolysis [29, 61]. This could in turn induce conformational changes [80] involving movement of the two major domains (I and II) relative to each other (Fig. 3). Alterations in quarternary structure and subunit interactions may thus affect the impact or interaction of ACRs involved in substrate binding and hydrolysis. Whether posttranslational modifications such as protein phosphorylation contribute to this dynamic behavior remains to be investigated.
Functional modifications
Biologically active NTPDase1 is subject to differential forms of surface modification under conditions of oxidative stress that inhibit enzymatic activity, as influenced by unsaturated fatty acids [81, 82]. It also undergoes limited proteolysis that increases enzyme activity and differential glycosylation reactions that appear to be required for membrane expression [64]. Since the surface-located ATP-hydrolyzing members of the NTPDase family pass through the endoplasmic reticulum and Golgi apparatus, the associated catalytic activity might abrogate ATP-dependent luminal functional processes. NTPDase1 becomes catalytically active on reaching the cell surface and glycosylation reactions appear crucial in this respect [83].
The N-terminal intracytoplasmic domain of NTPDase1 is palmitoylated. Truncated forms of NTPDase1 lacking the N-terminal intracytoplasmic region and the associated Cys13 residue, are not subject to palmitoylation. This post-translational modification appears to be constitutive and to contribute to the integral membrane association of this ectonucleotidase in lipid rafts [84, 85, 86]. This raises the possibility that NTPDase1 may be recycled to and from cell membranes via sequential actions of putative palmitoyltransferases and palmitoyl-protein thioesterases [87], in order to fine tune and modulate purinergic signaling responses. In contrast to NTPDase1 and NTPDase3, NTPDase2 does not have the required intra-cytoplasmic Cys to undergo this post-translational modification. The potential multimerization of NTPDase1 [35] may be facilitated by acylation with intermolecular interactions within the cholesterol and sphingolipid-rich microdomains of the plasma membrane [88]. Experiments using endothelial cells from caveolin-1 deficient mice suggest that caveolae are not essential for the enzymatic activity or for the targeting to the plasma membrane of NTPDase1. However, cholesterol depletion results in a strong inhibition of the enzyme [86]. The targeting of palmitoylated NTPDase1 to lipid rafts could influence defined G-protein coupled receptors within this plasmalemmal microenvironment and thus regulate cellular signal transduction pathways. Furthermore, the caveolar co-localization of ecto-5′-nucleotidase, P2 receptors, and NTPDase1 could serve to modulate signaling via both ATP and adenosine at the cell surface and possibly also within endosomal compartments [20].
Transcriptional regulation of expression
Members of the E-NTPDase family are constitutively expressed in many tissues. To date, there is only scattered evidence on promoters and the factors controlling NTPDase expression [22]. The transcription of NTPDase1/CD39 is constitutive in venous, arterial and certain non-fenestrated microvascular endothelium and certain immune cells e.g., B cells, dendritic cells and defined T-cell subsets [20, 89]. The modulated expression of NTPDase1 has been closely associated with inflammatory cytokines, oxidative stress and hypoxia in vitro and in vivo [19, 90].
Expression of NTPDase1 is increased in differentiating melanomas followed by a gradual decrease with tumor progression [91] and enhanced NTPDase1 activity of stimulated endothelial and mesangial cells is downregulated by glucocorticosteroids [92]. Activity of 'ecto-ATP diphosphohydrolase' in human endothelial cells in-vitro is increased by aspirin [93] and glomerular 'ecto-ATP diphosphohydrolase' immunoreactivity might well be modulated by estradiol [94].
Transcription of NTPDase2 in mouse hepatoma cells is inducible by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) [95]. These cells contain both constitutive and TCDD-inducible NTPDase2 activity. The NTPDase2 core promotor reveals constitutive activity that is independent of TCDD [96]. TCDD does not increase expression of NTPDase1, NTPDase3 or other ectonucleotidases and apparently fails to induce NTPDase2 in a variety of other cell lines derived from varying species [97]. In rat Sertoli cells, NTPDase2 is upregulated by follicle-stimulating hormone and cAMP [98] and it is selectively downregulated in biliary cirrhosis [99]. Human epidermoid carcinoma cells increase the cascade for extracellular nucleotide hydrolysis when periodically treated with extracellular ATP, suggesting that the substrate itself may affect the expression of its own hydrolysis chain [100].
A considerable number of compounds alter and inhibit extracellular nucleotide hydrolysis by NTPDases. These include non-hydrolysable nucleotide analogues and inhibitors of P2 receptors. Ideally, NTPDase inhibitors should not be P2 receptor agonists or antagonists and not be subject to dephosphorylation by the ectoenzyme [22, 101, 102]. The only commercially available compound reported to effectively inhibit hydrolysis of ATP in a variety of tissues without significantly acting on purinoceptors is the structural analogue of ATP, ARL 67156 (FPL 67156) (6-N, N-diethyl-D-β,γ-dibromomethylene ATP) [103, 104, 105]. Other potential inhibitors include 8-thiobutyladenosine 5′-triphosphate (8-BuS-ATP) [106] and 1-naphthol-3, 6-disulfonic acid (BG0136) [101]. Periodate-oxidized ATP inhibits ecto-ATPase activity in 1312N1 human astrocytoma cells [107] and Gadolinium ions have been found to effectively inhibit the ecto-nucleoside triphosphate diphosphohydrolase from Torpedo electric organ as well as potato apyrase [108].
It is noteworthy that the potency of inhibitors can vary considerably between individual members of the E-NTPDase family [109, 110, 111]. This necessitates a functional evaluation of each inhibitor for the enzyme investigated in a given tissue or cell type. The failure to develop specific inhibitors remains a major impediment to ongoing discoveries.
Principal functional contexts
Cell surface-located NTPDases are considered to be of major importance for controlling the availability of extracellular nucleotide agonists at P2 receptors. They also contribute to recycling of nucleosides derived from extracellular nucleoside phosphates and metabolic salvage pathways. The number of studies that define a functional impact of individual NTPDases in purinergic signaling in situ is limited and has been dependent to date on global genetic modifications of mice and swine to delete or upregulate the NTPDase or P2 gene of interest [20]. Subtype-specific inhibitors, siRNA approaches, and animals in which the encoding gene can be inactivated or selectively induced in specific tissues will be of major importance.
There is increasing experimental evidence that ectonucleotidases compete with P2 receptors for a limited pool of endogenously released nucleotide [112, 113] and —by hydrolyzing released nucleotide —terminate or modulate the function of P2 receptors [114, 115, 116]. Portal fibroblasts regulate P2Y receptormediated bile duct epithelial proliferation via expression of NTPDase2 [117] (see liver section, below). NTPDases functionally interact with P2Y-receptors [112] and may also co-localize with these G-proteincoupled receptors (GPCR) in lipid rafts and possibly caveolae [118, 119, 120, 121]. The modulatory effects of NTPDases are complex as the enzymes differentially regulate agonist availability in a process that is dependent upon P2 receptor subtype by either degrading ATP/UTP or by generating ADP/UDP (Fig. 2).
Recent experiments suggest that plasma membranebound NTPDases may have functions distinct from their catalytic properties alone. In a yeast two-hybrid system using techniques developed by Zhong for yeast apyrases [122], the N-terminus of human NTPDase1 (used as bait protein) has been shown to interact with truncated Ran Binding Protein M (RanBPM, otherwise known as RanBP9, NM_005493) in the human library screened [122a]. RanBPM contains conserved SPRY (repeats in splA and RyR) domains that appear to be crucial for the interaction with NTPDase1 and is preferentially distributed in human heart tissues [123]. RanBPM is known to interact with Sos and regulate ERK/Ras signaling. NTPDase1 interacts with RanBPM to directly modulate Ras activation and cellular proliferation in liver regeneration following partial hepatectomy [124].
The N-termini of NTPDases also have consensus sequences for protein phosphorylation by protein kinase C [47] that could have additional functional impacts. Furthermore, the C-terminal sequence of NTPDase1 contains a putative PDZ domain (-K-DM-V). This may have utility in determining interactions with select P2Y receptors e.g., the purinergic P2Y1 and P2Y2 receptors that terminate in -D-T-S-L and -D-I-R-L, respectively [125]. PDZ domains are most often found in combination with other protein interaction domains (for instance, SH3, PTB, WW), participating in complexes that facilitate signaling or determine the localization of receptors [126, 127, 128].
Finally, the general membrane topography of NTPDase1 and oligomeric assembly resemble the morphology of channel forming proteins such as P2X nucleotide receptors and members of the epithelial Na+ channel/degenerin gene superfamily [129]. This raises the question whether, in addition to their catalytic activity, NTPDases could function as channels. Release of ATP from Xenopus oocytes induced by hyperpolarizing pulses requires functional ecto-ATPase activity [130]. To what extent this functional property is shared by the structurally related NTPDase2, NTPDase3 and NTPDase8 has not been investigated.
The normal vascular endothelium provides a barrier that separates blood cells and plasma factors from highly reactive elements of the deeper layer of vessel wall. The vessel wall maintains blood fluidity and promotes flow by inhibiting coagulation, platelet activation and promoting fibrinolysis [131]. These properties are governed by important thromboregulatory mechanisms; key biological activities of the vasculature have been already identified and shown to be ecto-nucleotide catalysts that generate the respective nucleosides by phosphohydrolysis [19, 82]. The dominant ectonucleotidases of the vasculature have now more fully been characterized as NTPDases. This important biological property expressed by the endothelium and associated cells is responsible for the regulation of extracellular and plasma levels of nucleotides [20, 44, 82, 132, 133].
NTPDase1 is the major ectonucleotidase in the vasculature [112]. Other NTPDases associated with the vasculature are the cell-associated NTPDase2 and the soluble monocyte expressed NTPDase5 [32, 50, 137]. The phosphohydrolytic reaction of NTPDase1 limits the platelet activation response that is dependent upon the paracrine release of ADP and activation of specific purinergic receptors [81, 132, 138]. In contrast, NTPDase2, a preferential nucleoside triphosphatase, activates platelets by converting the competitive antagonist (ATP) of platelet ADP-receptors to the specific agonist of the P2Y1, and P2Y12 receptors. In keeping with these biochemical properties, NTPDase1 is dominantly expressed by endothelial cells and the associated vascular smooth muscle where it serves as a thromboregulatory factor. In contrast, NTPDase2 is associated with the adventitial surfaces of the muscularized vessels, microvascular pericytes of some tissues and organs as the heart and the stromal cells and would potentially serve as a hemostatic factor [62].
Extracellular nucleotide stimulation of P2 receptors represents components of platelet, endothelial cell and leukocyte activation that culminate in vascular thrombosis and inflammation in vivo [19]. In these inflammatory settings, with oxidant endothelial injury, NTPDase1 biochemical function is substantially, albeit temporarily, decreased because of post-translational changes; reconstitution of vascular NTPDase activity occurs following transcriptional upregulation of CD39 in endothelium [82, 139]. This functional change may relate, at least in part, to alterations in acylation and associated membrane lipid association with consequent disruption of multimer structure. Interestingly, palmitate supplementation may protect against loss of NTPDase activity following cellular activation in vitro [81]. These observations may provide several avenues of research to augment NTPDase activity within the vasculature at sites of injury [134].
Mechanisms of endothelial cell activation by nucleotides
ATP and UTP increases intracellular calcium levels, results in cytoskeletal rearrangements and stimulates phosphorylation of several proteins in human endothelial cells (EC) that are also associated with integrin signaling [140, 141, 142]. These include the focal adhesion kinase (FAK) and paxillin, proline-rich tyrosine kinase 2 (Pyk2) (also named related adhesion focal tyrosine kinase, RAFTK) and p38 MAP kinase. Further, UTP preferentially increases EC migration in a PI3-kinase and ERK-dependent manner. Moreover, extracellular nucleotide-mediated EC activation involves cytoskeletal rearrangements and increases in cell motility, comparable to that seen with ligation of integrins by extracellular matrix proteins [143]. These phenotypic changes (seen in both nucleotide-and matrix-mediated activation) are associated with tyrosine phosphorylation of FAK, paxillin and p130 Crk-associated substrate (p130cas) and down-stream activation of p38 MAP kinases. FAK has been implicated to play an important role in integrin-mediated signal transduction pathways [144], suggesting that P2-receptors are implicated in 'inside-out' integrin signaling in EC, as well as platelets [20, 112].
Therapeutic considerations
To test how extracellular nucleotide-mediated signaling influences pathophysiological events, several techniques have been developed and validated to manipulate NTPDase1 expression in the vasculature and to study conditions of inflammatory stress. The first mutant mouse derived and studied concerned the global deletion of the gene encoding the dominant ectonucleotidase NTPDase1 (Entpd1,cd39). The mutant mice exhibit major perturbations of P2 receptormediated signaling in the vasculature and immune systems [19, 89, 145]. These phenomena manifest as hemostatic defects, thromboregulatory disturbances, heightened acute inflammatory responses with a failure to generate cellular immune responses that are all associated with vascular endothelium, monocyte, dendritic cell and platelet integrin dysfunction [20, 112, 134].
The therapeutic potential of NTPDase1 to regulate P2 receptor function in the vasculature and mitigate against thrombotic/inflammatory stress has been further established by the generation of NTPDase1 transgenic mice and swine [20, 146], the use of adenoviral vectors to upregulate NTPDase1 in cardiac grafts [147] and the use of soluble derivatives of NTPDase1 and apyrases [133, 148]. The beneficial effects of administered NTPDases have been determined in several animal models of vascular inflammation [148, 149]. Exogenous infusions of soluble NTPDases are able to rescue Entpd1-deficient mice from systemic toxicity induced by ischemia reperfusion injury and after stroke induction [145, 150].
Angiogenesis requires the dynamic interaction of endothelial cell proliferation and differentiation with orchestrated interactions between extracellular matrix and surrounding cells (such as vascular smooth muscle and/or pericytes) [151, 152, 153]. NTPDase1 appears crucial in the co-ordination of angiogenic responses in inflammation, organ remodeling and transplantation [20, 134]. For example, in syngeneic pancreatic islet transplantation, the maintenance and revascularization of grafted islets appears dependent upon expression of NTPDase1 by the developing vasculature within the islet (Fig. 4).
In summary, multiple experimental studies largely reveal beneficial effects of over-expression of NTPDases within the vasculature, or by their pharmacological administration [20, 133]. Clinical studies of these soluble thromboregulatory factors are in planning [20, 154, 155].
Immune system
There are multiple P2X and P2Y receptor subtypes expressed by monocytes and dendritic cells, whereas lymphocytes express only P2Y receptors [2]. NTPDase1/CD39 was first described as a B lymphocyte activation marker and also shown to be expressed on activated T cells [156, 157] and dendritic cells [89]. The CD39 enzymatic function on dendritic cells is involved in the recruitment, activation and polarization of naive T cells. ATP is released by CD4+ and CD8+ T cells upon stimulation with Con A or anti-CD3 mAb while CD39 functions as an additional recognition structure on haptenated target immunocytes for HLA-A1-restricted, hapten-specific cytotoxic T cells [156, 157].
In cd39 null mice, there are major defects in dendritic cell function antigen presentation and T-cell responses to haptens (type IV hypersensitivity reactions) [19, 89]. Immunocyte-associated CD39 may play an immunoregulatory role by hydrolyzing ATP (and perhaps ADP) released by T cells during antigen presentation and thereby generating adenosine [19, 89, 158]. Ectoenzymes, including ectonucleotidases, are known to play an important role in leukocyte trafficking (for an excellent review on this topic, see [159]).
Recent work has indicated that regulatory CD4 + ve CD25 + ve T cells (Treg cells) play important roles in the maintenance of immunological reactivity and tolerance [160]. The selective expression of CD39 by Treg and the question whether this ectonucleotidase and/or extracellular nucleotides influence(s) the function of these interesting cells is a focus of current work.
Digestive and renal systems
Released nucleotides are polarized and do not re-enter cells. They have to be transformed into the corresponding nucleosides that enter cells via specific transporters to rebuild nucleoside pools. If this did not occur, they would be lost from the metabolic pool. The same may pertain to dietary ingestion of nucleotides where NTPDases are potential participants in the digestion of exogenous nucleotides and intestinal function. In addition, extracellular nucleotide and adenosine receptors are highly expressed in the digestive and renal systems, so these molecules are likely to have homeostatic functions [2].
An important nucleotide-mediated mechanism that seems common to various epithelia, as well as to hepatocytes, involves the autocrine regulation of cell volume by ATP via P2 receptors [161, 162]. As P2 receptors are expressed by epithelia in a polarized manner and can be linked to several digestive and homeostatic functions [163, 164], the presence of NTPDases in the immediate environment may serve as regulatory switches.
In the liver, extracellular nucleotides are potentially involved in several functional contexts [161]. There is evidence that extracellular nucleotides regulate glycogenolysis through activation of glycogen phosphorylase and inactivation of glycogen synthase by inhibition of the glucagon effect on cAMP and by the activation of phospholipase D [165, 166]. In addition, nucleotides may be involved in the regulation of canalicular contraction and bile flow [167, 168, 169]. Concentrations of canalicular adenine nucleotides in bile samples and effluents from hepatic cell lines are estimated to be around 0.1 to 5 µM [161, 168]. Hepatocytes and bile duct cells have been shown to interact and communicate via local ATP release in vitro [170]. Extracellular ATP acts as a hepatic mitogen and activates JNK signaling and hepatocyte proliferation both in vitro and in vivo [171].
Several ectonucleotidases are expressed in liver. Of the nucleotide pyrophosphatase/phosphodiesterases, NPP1 (PC-1) is expressed on the basolateral membrane of hepatocytes while the closely related NPP3 (B10) has a predominant canalicular in distribution [172, 173]. NTPDase1 is highly expressed on larger vessels and more weakly on sinusoids as well as in Kupffer cells [174]. In the quiescent liver, NTPDase2 is expressed by cells of the subendothelium of veins and adventitial cells of arteries, but not in sinusoids. In addition, NTPDase2 is expressed by portal fibroblasts near basolateral membranes of bile duct epithelia [175]. Activated but not quiescent hepatic stellate cells express NTPDase2 at the protein level [176]. Only low expression of NTPDase3 could be demonstrated at the mRNA level in the liver [50, 177].
NTPDase2 expression in portal fibroblasts, the primary fibroblastic cell type of the portal area, suggests a role in the regulation of bile ductular signaling and secretion [161, 175]. Jhandier et al. tested the hypothesis that portal fibroblast NTPDase2 regulates epithelial cell proliferation. Using co-cultures of cholangiocytes (Mz-ChA-1 human cholangiocarcinoma cells) and primary portal fibroblasts from rat liver, increased NTPDase2 expression decreased cell proliferation, and knockdown of NTPDase2 by siRNA increased proliferation. P2 receptor blockade also attenuated Mz-ChA-1 proliferation [117]. These experiments defined a novel cross-talk signaling pathway between bile duct epithelial cells and underlying portal fibroblasts, regulated by NTPDase2. Because they are the chief fibrogenic cells of the liver, hepatic stellate cells and portal fibroblasts are important targets of liver disease therapy. Loss of NTPDase2 expression in human biliary cirrhosis, as well as in models of bile duct ligation in rat, has been observed. NTPDase2 expression also shifts from the portal area to bridging fibrous bands in cirrhosis with hepatitis C [99].
Functional ATPases were previously shown to be associated with bile canalicular plasma membranes by histochemical techniques [178]; the corresponding enzyme was subsequently incorrectly identified as cCAM105 [179, 180, 181]. More recent studies revealed that the canalicular ecto-ATPase corresponds to NTPDase8 [52], also referred to as hepatic ATP diphosphohydrolase (ATPDase) [174, 182]. NTPDase8 is the mammalian orthologue of the chicken ecto-ATPDase cloned from oviduct and liver [183, 184]. In tandem with ecto-5′-nucleotidase, NTPDase8 has the potential to regulate the concentration of nucleotides in the hepatic canalicule. The ultimate generation of extracellular adenosine from dephosphorylated ATP not only activates adenosine receptors but also produces the key molecule for purine salvage and consequent replenishment of ATP stores within many cell types [17, 185]. Adenosine transporters are of major importance to organs and cells incapable of de novo nucleotide synthesis such as brain, muscle, intestinal mucosa and bone marrow [167, 186]. As the liver appears to be a major source of purines for these tissues, curtailment of nucleotide loss into the bile may be important to maintain appropriate nucleotide/nucleoside concentrations within hepatocytes [185]. Thus, dephosphorylation of nucleotides by ectonucleotidases may be critical for appropriate systemic purine homeostasis [167]. The presence of NTPDase8, ecto-5′-nucleotidase and nucleoside transporters in the canalicular domain of hepatocytes would be consistent with an important role of NTPDase8 in purine salvage.
The exocrine pancreas
The exocrine pancreas secretes digestive enzymes and a HCO3-rich fluid. Acini release ATP and the excurrent ducts express several types of P2 receptors [187, 188]. Thus ATP may function as a paracrine mediator between pancreatic acini and ducts. Ectonucleotidase activity in pancreatic tissues was first detected in the rat in the 1960s [189, 190], followed by analyses in the pig [191, 192]. Cytochemical and biochemical observations have corroborated the association of ATPase activity with zymogen granules [193]. In other studies of small intercalated/interlobular ducts, NTPDase1 immuno-fluorescence can be localized on the luminal membranes, while in larger ducts it is localized on the basolateral membranes [194]. Upon stimulation with cholecystokinin octapeptide-8 (CCK-8), acinar NTPDase1 relocalized in clusters towards the lumen and is secreted into the pancreatic juice, as an active form associated with particulate fractions [188, 195]. As revealed by electron microscopy, NTPDase2 is located on epithelial cells, myoepithelial cells and the basolateral membrane of acini. Interestingly, NTPDase2 could be also detected at the basolateral surface of endothelial cells [194].
Salivary glands
There are only few studies on the localization of NTPDases in salivary glands. NTPDases might play a role in the transport of electrolytes by modulating the extracellular ATP concentration in the salivary gland ducts. NTPDase1 reveals to be mainly vascular in expression. NTPDase2 was immunodetected on myoepithelial cells and in nerves [194, 196]. The immunolocalization of NTPDases 3 and 8 in salivary glands has not yet been determined.
The kidney reveals a complex cellular profile of expression for P1 and P2 receptors as well as of ectonucleotidases. Both ATP and adenosine have been invoked in the regulation of tubuloglomerular feedback [197, 198]. This feedback system links the salt concentrations in the tubular fluid at the macula densa to the vascular tone of the afferent arteriole of the same nephron. As depicted by their localization, NTPDases may participate in the regulation of several biological functions of the kidney, including vascular perfusion. In mouse, rat and porcine kidneys, NTPDase1 can be detected in vascular structures, including blood vessels of glomerular and peritubular capillaries [174, 199, 200]. NTPDase2 is detected on the Bowman's capsules of mouse and rat [199] and NTPDase8 on the luminal side of porcine renal tubules [174]. More recently, an immunohistochemical analysis of various ectonucleotidases of the rat nephron revealed expression of both NTPDase2 and NTPDase3 in the thick ascending limb, the distal tubule and the inner medullary collecting ducts. In addition, NTPDase3 is located in the cortical and outer medullary collecting ducts [201].
The nervous system
All cell types of the nervous system express nucleotide receptors [2]. It is increasingly apparent that NTPDases are distributed in the nervous system as ubiquitously as are P2 receptors and that these ectoenzymes are directly involved in the control of P2 receptor function in nervous tissues [22, 31, 36]. Signaling via nucleotides is widespread both in the peripheral and central nervous system. Major nucleotide receptormediated functions in the central nervous system include the modulation of synaptic signal transmission [202], the propagation of Ca2+ waves between glial cells [203], or the control and activation of astrocytes and microglia [204, 205]. In addition, ATP can contribute to synaptic signal transmission [36]. In the sympathetic nervous system, ATP acts as a fast neurotransmitter together with catecholamines [206], it is an important mediator of central and peripheral chemosensory transduction, including pain [207] and it is involved in the control of myelination formation of peripheral axons [208].
Central nervous system
ATP can be rapidly hydrolyzed to adenosine at brain synapses that in turn activates pre-or postsynaptic receptors, thereby modulating neuronal transmission. Adenine nucleotides undergo conversion to adenosine within a few hundred milliseconds in the extracellular (synaptic) space of rat brain slices [209, 210]. Complex synaptic interactions in the central nervous system may thus be modulated both by the activation of P2 and (after hydrolysis of the nucleotide) P1 receptors that may be located at identical or different cellular targets [202, 211].
Based on immunoblotting and in situ hybridization, NTPDase1, 2 and 3 are expressed in the mammalian brain [47, 57, 59, 116, 177, 212]. NTPDase1 and 2 have been purified from porcine brain [213, 214]. But the exact cellular allocation of individual subtypes is still a challenge. There is ample evidence from early enzyme histochemical investigations that surface-located catalytic activity for the hydrolysis of nucleoside tri-and diphosphates can be allocated to all cell types of the nervous system [for reviews see 22, 31, 36, 215]. This catalytic activity can be localized to synapses, including the synaptic cleft, at the surface of neurosecretory nerve terminals in the pituitary or at peripheral nerve terminals. These data imply a wide distribution of cell surface-located ATP hydrolyzing activity in the CNS.
Ecto-ATPase activity has been observed in synaptosomal fractions isolated from various sources, implying endogenous ectonucleotidase activity of nerve cells. Biochemical studies on isolated synaptosomes permit the determination of the ratios of ATP to ADP hydrolysis rates as well as the analysis of product formation. Total synaptosome fractions isolated from rat brain cortex and immunopurified cholinergic striatal synaptosomes revealed ratios of 3.4: 1 and 2.1: 1, respectively [216]. ADP was found to transiently accumulate after addition of ATP, and was subsequently metabolized to AMP and adenosine. Similar results were obtained with hippocampal synaptosomes [217]. This strongly argues against a major contribution by NTPDase1 and NTPDase2 and would rather be compatible with a neuronal expression of NTPDase3 (comp. Fig. 2). A recent immunocytochemical study allocates NTPDase3 to neurons including axon-like structures of various brain regions [218].
Astrocytes, oligodendrocytes, and microglia
The ratio of ATP to ADP hydrolysis is clearly different in cultured astrocytes. Astrocytes cultured from cortex or hippocampus display a ratio of 8: 1 [219]. Furthermore, cultured rat cortical astrocytes accumulate ADP from ATP that is only very slowly further degraded to AMP [220]. This would be largely compatible with NTPDase2 as the major ectonucleotidase of cultured astrocytes. Immunocytological investigations of adult rat and mouse brain sections assign NTPDase2 solely to the astrocyte-like stem cells in the subventricular zone of the lateral ventricles and the dentate gyrus of the hippocampus and to astrocytes in few distinct additional brain regions [221, 222]. Thus, cultured astrocytes may reveal functional properties that differ from the in situ situation as they tend to rapidly alter their protein expression profile [223].
Enzyme histochemistry assigns ecto-ATPase activity to both central and peripheral myelin [31], but fully supplementary immunocytochemical data are lacking. Enzyme histochemical staining for surface-located nucleoside diphosphate activity has long been used to identify microglia in tissue sections of the adult and developing brain [224]. The major microglial ectonucleotidase has been identified as NTPDase1 [225].
Stem cells in the adult mammalian brain
Apparently, individual enzyme isoforms govern cell surface-located nucleotide hydrolysis in the various cell types of the central nervous system. This does not exclude however, the possibility that individual cell types express more than one isoform with one of the enzymes predominating. For example, PC12 cells express mRNA for NTPDase1-3. But the ATP/ADP hydrolysis ratio, the pattern of product formation and the immunocytochemical surface staining suggest that NTPDase3 is the major functional isoform [59, 228]. Similarly cultured normal and immortalized pituitary and hypothalamic cells express NTPDase1-3 [116]. The future planned use of transgenic mice expressing fluorescent protein under the promoter of the respective NTPDase isoform will greatly facilitate the identification of the expression pattern of individual enzyme isoforms in the developing and adult nervous system.
Peripheral nervous system
Noradrenaline and ATP are co-released from sympathetic nerve terminals of the guinea pig heart whereby ATP enhances noradrenaline release by a mechanism controlled by ectonucleotidases, possibly NTPDase1 [229]. Interestingly, stimulated sympathetic nerves of the guinea pig vas deferens release not only ATP and noradrenaline but also enzyme activity that degrades ATP to adenosine. The latter exhibits similarities to NTPDases and ecto-5′-nucleotidase but their molecular identity has not been defined [230].
NTPDase2 associates with immature and non-myelinating Schwann cells of peripheral nerves whereas NTPDase1 immunoreactivity is absent [231]. NTPDase2 is also expressed by the satellite glial cells in dorsal root ganglia and sympathetic ganglia and by the enteric glia surrounding cell bodies of ganglionic neurons of the myenteric and submucous plexus [231].
Sensory systems
The most comprehensive investigation of expression of NTPDases within sensory systems concerns the inner ear. Ectonucleotidase activity is associated with the tissues lining the perilymphatic compartment of the cochlea [232, 233]. Immunohistochemical analysis of the murine cochlea has assigned NTPDase1 to the cochlear vasculature and primary auditory neurons in the spiral ganglion, whereas NTPDase2 is associated with synaptic regions of the sensory inner and outer hair cells, supporting cells of the organ of Corti and additional tissue elements [234, 235]. Interestingly, noise exposure induces upregulation of NTPDase1 and NTPDase2 in the rat cochlea [236].
Taste buds transduce chemical signals in the mouth into neural messages. Taste cells and nerve fibers express P2X2 and P2X3 receptors [237] and various P2Y receptors [238, 239]. Genetic elimination of P2X2 and P2X3 receptors revealed that ATP is a key neurotransmitter in this system [240]. NTPDase2 is expressed at the mRNA level in mouse taste papillae [241]. Immunohistochemistry and enzyme histochemical staining allocate NTPDase2 to type I 'glial-like' cells in the tongue, palate and larynx. Furthermore, NTPDase2 immunostaining is associated with nearby nerves, suggestive of Schwann cells, implying that NTPDase2 may be a regulator in defined taste transmission.
Pathological implications
Cerebral ischemia
The interruption of blood flow accompanied by an interrupted supply of oxygen and glucose initiates a sequence of events resulting in structural and functional damage of the nervous tissue, comparable to that seen at other sites of vascular injury [20]. Transient global cerebral ischemia of the rat results in a long-term increase in extracellular nucleotide hydrolysis pathways [242, 243]. Preconditioning delays the postischemic increase in ATP diphosphohydrolase activity [243]. During the days following transient forebrain ischemia, mRNA for NTPDase1 (but not of NTPDase2) and ecto-5′-nucleotidase becomes upregulated in the hippocampus [242], corresponding to the upregulation of the entire ectonucleotidase chain for the hydrolysis of ATP to adenosine. The data suggest that the increased expression of ectonucleotidases in the regions of damaged nerve cells is associated with activated glia, mainly microglia [224].
The upregulation of the ectonucleotidase chain is suggestive of an ischemia-induced increased and sustained cellular release of nucleotides. This could have several functional implications. Since microglial cells express the cytolytic P2X7 receptor [244, 245] the activity of these cells may be particularly endangered by increased levels of extracellular ATP. Enhanced activity of NTPDase1 may prevent activated microglia from overstimulation by ATP released from the injured tissue. Alternatively, microglial expression of NTPDase1 might contribute to preventing receptor desensitization on prolonged exposure to elevated ATP levels. The parallel increase in activity of ecto-5′-nucleotidase would facilitate the formation of the final hydrolysis product adenosine that exerts neuro-modulatory and immunomodulatory actions and contributes to the protection of neurons.
Alterations following plastic changes in the nervous system
Additional experiments, analyzing synaptosome fractions, suggest that changes in neural plasticity can be paralleled by changes in ecto-ATPase activity. Enzyme activity is reduced following avoidance learning [246] and status epilepticus [247, 248]. It is altered in two rat models of temporal lobe epilepsy [249], and on pentylenetetrazol kindling [250]. Changes in synaptosomal ectonucleotidase activity have been implicated by a broad variety of additional treatments including acute caffeine treatment [251]. Taken together, these experiments suggest that expression of ectonucleotidases can be altered following a variety of physiological or pathological stimuli, possibly together with that of purine receptors. Further work needs to define the enzyme subtypes involved and the mechanisms underlying the regulation of ectonucleotidase expression.
This review summarizes components of extracellular nucleotide-mediated signaling pathways that are impacted upon largely by the E-NTPDase family of ectonucleotidases. Modulated, distinct NTPDase expression appears to regulate nucleotide-mediated signaling in essentially every tissue, including the vasculature and of immune and nervous systems. For example, extracellular nucleotide-mediated vascular endothelial and accessory cell stimulation might have important consequences for platelet activation, thrombogenesis, angiogenesis, vascular remodeling and the metabolic milieu of the vasculature, in response to inflammatory stress and/or immune reactions.
Nucleotides are also of significant relevance for the communication of nerve cells and glial cells or in the reciprocal signaling between these cells. These purinergic mechanisms might also dictate pathological processes of the nervous system or following vascular injury, thromboregulatory disturbances, and defective angiogenesis with associated perturbations in tissue remodeling and regeneration.
There is a wide field for future investigations of the role of nucleotides and ectonucleotidases in other tissues. Increasing interest in this field may open up new avenues for investigation and the development of new treatment modalities for a large variety of diseases, including neurological pathological states, vascular thrombotic disorders including stoke, atherosclerosis and the vascular inflammation seen in transplant-graft failure.
This work was supported by the Deutsche Forschungsgemeinschaft (140/17-1; GRK 361; to HZ); NIH HL57307, HL63972 and HL076540 (to SCR), and the Canadian Institutes of Health Research (CIHR; to JS). JS is also a recipient of a New Investigator award from the CIHR.


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Some other youtube user invited me as a friend, so since I didn't know them I decided to see what kind of things they like to see whether we would have anything in common. This is how I found headachehealer. This particular user had commented positively on headachehealer's videos and rated them highly.
I just posted a "HELP where do you begin!?" regarding headachehealer over at the new Twin City Fellowship forums.
see the post here
by the way, for some dumb reason, youtube's messaging didn't save all my outgoing messages, so I can't copy and paste them like I did his and just one of mine. Irritating. youtube can be a fun way to share but sure is buggy.


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Happy Friday! How has everyone's week been going? This outfit is from Wednesday but I didn't get a chance to put it up then, so alas, here it is! I'm starting to gravitate towards a more simplistic approach to my wardrobe in terms of color (so black, gray, white, and beige), but it is not to say that I don't enjoy my share of floral prints and flannel. I could never give up wearing those!
Wearing: Thrifted t-shirt ; American Apparel Chiffon Oversized Button-Up + Side Zip Shorts ; Dr. Marten boots


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Sol Beach House Menorca opposite Santo Tomás Beach and next to Adeodato and Binigaus Beaches enjoys a privileged location right next to the sea. Enjoy the crystal clear waters and the fine sand at the beaches of Santo Tomss and San Adeodato. The location together with our specialized and per...
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Broken tooth
I have a front tooth broken since many years. Can someone guide me and suggest me what will be the best option and also the cost details?
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Firstly it has to be seen whether any internal changes have taken place in the nerve tissue of the broken tooth. If the tooth has become dead because of the fracture then root canal treatment has to be done. Then depending on how much the tooth structure is broken there are different methods of restoring it. Direct composit build up or ceramic veneer or full crown are the options available. After seeing your tooth your dentist can give the exact line of treatment.
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Mayor's Community Youth Support Network (MCYSN) - Grant Application Now Open
The City of Tracy is now accepting applications for its Mayor's Community Youth Support Network (MCYSN), Reconnecting Our Youth Grant Program.
Corral Hollow Widening and Sewer Upgrade Project
In order to relieve traffic congestion on one of Tracy's major thoroughfares, the City of Tracy will be widening a stretch Corral Hollow Road between Grant Line Road and Byron Road, from four lanes to six lanes starting this May. The project also includes an upgrade of existing sanitary sewer pipelines between Fieldview Drive and I-205.
Get the latest news and updates from the City of Tracy. We encourage you to join in the conversation and the spread the word too. Check out our social media links below.
Tracy has had a great year so far, and the events calendar is still jam-packed. Make the most of your time and enjoy the diverse activities and events that Tracy has to offer.
Council Meetings
We invite you to participate in your City government. We've made it easy by posting the City Council Calendar, Agendas, Meeting Minutes, streaming video, and more.
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UPDATE 2: All 10 codes have been awarded - congrats to Twitter winners @SseeaannK, @cgalla2008, @Killa91643678, @TheRealMoB21, and @TylerNocks for answering our PlayStation trivia questions correctly and before anyone else. Thanks to everyone for playing, and a special thanks to SuperBot Entertainment and Sony for providing giveaway codes!
UPDATE: We were amazed by the number of character entries to receive a PlayStation All-Stars beta key last night and this morning. PSU community members submitted some truly awesome picks, including plenty that we had never thought of as possibilities. It was very difficult to pick just five winners, but thankfully, we've still got five MORE codes to give out.
'I missed my first chance!' you say? 'How can I get one of the remaining codes?' Great quesion, fine reader. We wanted to mix up the proceedings a bit, so we're gonna play a little game. It's called...
PlayStation Trivia!
Starting at approximately 5 PM ET this afternoon, we're gonna take to the Twitter-sphere and begin tweeting a series of PlayStation trivia questions. The first follower to reply with a correct answer will score a code! It's that simple.
Remember, the trivia questions won't be posted here. You'll need to be following our official Twitter account - @PSUdotcom - to see the questions and get your chance at a code. We'll space them out a bit, but don't be late! There's only five more left to give.
Our first five keys have been awarded to the following participants: reypol6, Husachi, Zach Barry, Jet_Cobra, and Beta_Beast. Congratulations! Check your forum inboxes for your code.
ORIGINAL: Sony and SuperBot Entertainment's upcoming crossover brawler PlayStation All-Stars Battle Royale is going to be one of the year's biggest games, and a very lucky select few are experiencing the game's closed beta right now.
Who's ready to be one of them?
At PSU Towers, we love our readers, community members, and superfans, and we simply couldn't stand to see so many of you go without beta-testing this epic game for yourselves. So, courtesy of our friends at SuperBot Entertainment, PSU has TEN (!) beta codes to give away!
So, how can you get your hands on one of these codes and jump into the PlayStation All-Stars beta tonight? It's simple! (Kinda.) Drop a post in this forum thread and give us your very best answer to the following question:
"What playable fighter (not including announced or leaked characters) do YOU want to see in PlayStation All-Stars Battle Royale? AND describe for us what you envision their Level 1, Level 2, and Level 3 Super Attacks to be!"
Sounds easy enough, right? We'll pick five of the very best answers - thoughtfulness and creativity count! - and shoot you a code if you're one of the special few.
'But, wait!' you ask. 'Didn't you say TEN codes?'
Why yes, we did! That's very astute of you. But we've got an extra special challenge lined up for the other five. Stay tuned to PSU.com tomorrow for information on how you can get your hands on a code if you miss one tonight.
So, fire away! Give us your best character design in this thread, and you just might be playing PlayStation All-Stars tonight. And who knows... maybe SuperBot is watching and waiting to put your ideas in the game?
-Thanks to Sony and SuperBot Entertainment for providing giveaway keys!
-Limit one key per person. Don't cheat. We don't like that. At all.
-Codes are valid for North American PSN accounts only. Sorry to our UK readers and others around the globe!


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Summary: Roland was exactly where M-21 thought he would be: outside on the balcony.
Spoilers for the identity of the Death Knight for LSK and mild spoilers for what happens at the end of the first novel. Spoilers up to chapter 90 for Noblesse.
Crack crossover. Don't think too hard about how it happened. XD; LSK character in Noblesse world.
For the 'Roland & M-21, Autumn leaves all around' prompt for No True Pair. Didn't really fit it but…
Prodded into existance by Lucathia Rykatu :3
Seroiusly, how do you describe Roland's eyes? XD;;
By Dark Ice Dragon
Roland was exactly where M-21 thought he would be: outside on the balcony, watching the leaves drift down from the trees. The sun had set a few hours ago, so M-21 wasn't worried about Roland being seen. Much. The fact that Roland walked around without a shirt on (his 'armour' didn't count) would be enough to draw attention, especially as the days started to get colder. The wings that spread out from his back, leathery and huge, would gain anyone'sattention. It didn't help that Roland's eyes were completely black and literally burned. Even though Roland had a ring to make him look more human, he didn't leave the house that often.
M-21 held out the tops he'd been carrying towards Roland. "Here."
Though M-21 couldn't see Roland's eyes, from his head tilting down a little, he knew Roland was looking at them.
"I don't need them; I don't feel the elements like you do."
"I know," M-21 said, still holding them out. "But if you visit us at the school, the school's reputation will go down because people saw you, and then Frankenstein will kill you." M-21 paused, realising what he'd said. "Again."
Roland didn't seem that convinced, not making a move towards him.
M-21 sighed – he had a feeling he would have to make this ultimatum. "I won't spar with you if you don't wear one."
And apart from being a master with a sword, Roland's best weapon was looking like a confused puppy. "But…"
M-21 waited.
Roland took the tops without a word.
If only most problems could be solved that easily…
What tops did M-21 give Roland? One each from Frankenstein, M-21, Tao, and Takeo. XDDD


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when you shit and instead of coming out in one nice big happy block, it comes out in a bunch of little pieces reminiscent of rabbit turds, and then they float and appear to mock your inability to produce a proper crap.
by Demon Phoenix 1337 September 07, 2004
by Grant June 18, 2006
a midget who slips drugs in girls' drinks, drags them to his room at the halfway house, licks her ass for half an hour, waits for her to wake up, then forces her to lick his ass for half an hour.
by dick April 13, 2004
A gnomelike creature to whom anal violations, especially in humans, are attributed.
See also Manganaro, Sprod, Pananian Marsh Beaver and Mangonator.
See also Manganaro, Sprod, Pananian Marsh Beaver and Mangonator.
by D.E March 18, 2004
by Juan Valdez December 19, 2002


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U.S. oks restart of Keystone pipeline
By Tom Doggett and Jeffrey Jones
WASHINGTON/CALGARY (Reuters) - TransCanada Corp can restart its Keystone oil pipeline on Sunday, after the company satisfied a series of safety conditions following leaks that idled the key export line twice in less than a month, the U.S. pipeline safety regulator said on Saturday.
The U.S. Pipeline and Hazardous Materials Safety Administration said it approved resumption of crude flows from Canada on the pipeline after the agency evaluated the company's restart plan and was satisfied safety requirements were met.
PHMSA issued a corrective action order on Friday, demanding the company agree to a series of immediate and long-term measures following an outage last Sunday on the 591,000 barrel-a-day pipeline that runs to the oil pricing hub of Cushing, Oklahoma, from Alberta.
In that incident, 10 barrels spilled due to a faulty fitting at a Kansas pump station.
The outage raised oil prices as it limited oil shipments from Canada, the largest crude supplier to the United States. Canadian cash oil prices weakened as supplies backed up in Alberta.
In early May, Keystone was shut down for nearly a week after a 500-barrel spill at a pump station in North Dakota, which TransCanada blamed on a different piece of equipment.
In its corrective action order, PHMSA revealed a leak in South Dakota on May 25, but said it was below the threshold for reporting it.
With immediate repairs completed, PHMSA had said TransCanada must perform metallurgical testing, analysis of the failures and review other parts of the year-old system. Continued...


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Originally posted on Mets Merized Online | Last updated 1/31/13
Mets season ticket holders were gathered at the Caesar's Club at Citi Field tonight, where Sandy Alderson and his front office fielded questions about a variety of issues. Adam Rubin of ESPN New York did a nice job of covering the event and retweeting some of the quotes from those who were on hand. Regarding the lack of spending this offseason, Sandy Alderson insisted this was his decision and not ownership imposed. "The reason we haven't spent the money is not because of Fred Wilpon," he said. "It's because of me." "Since last year the Wilpons have had nothing but good news regarding their finances. Their financial capacity is far more than adequate and from a baseball perspective." When asked how he will navigate future offseasons when there will always be high priced free agents and over-aggressive bidding, the Mets GM replied: "We understand what the market dictates and we have to be prepared to exist in that market. Am I going to recommended that we sit here in New York City an...
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MMA Forum - UFC Forums - UFC Results - MMA Videos (http://www.mmaforum.com/forumindex.php)
- Previous Sports Wagers (http://www.mmaforum.com/previous-sports-wagers/)
- - NFL: Dallas vs Carolina (http://www.mmaforum.com/previous-sports-wagers/106070-nfl-dallas-vs-carolina.html)
NFL: Dallas vs Carolina
Not really interested in this game. You have Dallas with a crap o-line and Demarco Murray injured. Romo having an off year. And then on the other side you have the Panthers where Cam Newton is a fraction of what he was last year, Olsen is doing nothing and who knows what is going on with Smith. And don't even get me started on their running game.
Dallas won it 19 to 14.
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Reset My Password
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This is the third Jeep i've owned. I had a 2008 2 door wrangler, then a 2010 wrangler unlimited, and now the 2013 oscar mike edition. I am a jeep girl through and through!! Eventually when I win the lottery, I would love to lift it, get some nice big honking tires, and add a few little touches like new fenders, a new front and rear bumper, etc. I have big plans for my girl!


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ADERANT Matter Planning Application Now Available
Enables firms to more confidently respond to and manage requests for alternative fee arrangements
In response to an ever-changing market, Expert Matter Planning enables firms to more easily and confidently respond to and manage requests for both standard and alternative fee arrangements. Firms can search and collate prior or existing matters and design preconfigured templates to build matter profiles that are modeled after prior work experiences. Matter Planning enables firms to review work performed on prior matters to ascertain the duration, effort, and expertise required for the new engagement. It also brings together hours and roles to produce a fee estimate and a projected P&L so that firms can ensure each new matter meets their profitability thresholds. The innovative, flexible design handles all types of alternative fee agreements and monitors the matter through the entire life cycle.
One of the key advantages of Expert Matter Planning is its full integration with ADERANT Expert. Integration allows the use of Expert Rates, Ranks, Timekeepers, Phases, and Tasks all to be shared, ensuring a seamless movement from the matter planning phase into file opening, time recording, and billing. The tight integration with ADERANT Expert also provides accurate monitoring of the estimates created in the planning phase against actual time entries and reports on any variances.
"At Sheppard Mullin we understand that in today's marketplace, a law firm must be flexible and value-driven when pricing client engagements,"
"With Expert Matter Planning, firms can calculate the profitability of their alternative fee arrangements to ensure viability prior to the engagement,"
The release of Expert Matter Planning is in conjunction with the general availability of ADERANT Expert "Golden Gate" Release SP2, which was issued on November 30, 2010. The release introduces Expert Matter Planning as well as enhancements to Found Time, an innovative application that enables timekeepers to automatically reconstruct their day to capture lost or unaccounted for time to increase billable hours and revenue. This release of Found Time includes an active desktop agent that tracks all active application windows on the user's desktop with a full listing of all system interaction. Users can also view changes more clearly and export their activities to Microsoft Excel for further manipulation and printing.
# # #
ADERANT, headquartered in Atlanta, GA, is a leading provider of integrated business management software for law firms and other services organizations. The global company offers a comprehensive suite of solutions, consisting of financial, resource, practice, document, and information management software that enables services organizations of all sizes to maximize operational efficiencies. ADERANT provides solutions to the largest and most prominent firms in the world, including Clifford Chance, CSC, Greenberg Traurig, Clayton Utz, Deloitte Consulting, Allens Arthur Robinson, Herbert Smith, Mayer Brown, Skadden Arps, and Winston & Strawn. ADERANT also has offices in Asia Pacific and Europe. More information is available at www.aderant.com.
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