text:
Rambling when I remembered to stop
A limb broken and I heard it drop
Methodically stripping naked
A consciously wrapped envelope
Placed at the trunk of a mighty old oak
Who waved back and forth
Silly gamblers marching about
To the tune of engines
Sputtering confusion
And lay raindrops upon folded corners
Which shall leak kava upon this ancient floor
Upon which I rest upon
To humbly assess the treasure chest.
Treasure Chest (day 2131)
Rambling when I remembered to stop
No One on My Mind (day 1645)
I am a lonely gambler
I've got chips in every pocket
Two cards up my sleeves
But with all my kings and aces
I've got no one on my mind
I visit all the dark rooms
All known enemies of state
Keep me company into the night
But with all my kings and aces
I've got no one on my mind
The rounds at half past midnight
Two cigarettes in my hand
I've been the gambler, I've been the loser
But with all my kings and aces
I've got no one on my mind
Smoking Gun Saloon (day 1098)
I was gambling at the Big Gun Saloon.
A lone mescalito biting the fat end,
Lookin' for a chance.
I gambled often, too often.
A riverboat of floating luck
With legalized six shooters
Yelling my splashing soul goodbye.
I danced with happy Jacks
In a smoking gun saloon.
Ladies with rumpled tushes
Blowin' kisses on good nights.
Everywhere: adultery in bedackled gems,
Fishnet in blackened stockings,
And a room awaiting cowboy's boots.
I loved her like a riverman,
Steady and full of piss
Navigating curls running this stream long.
And then she whispered:
"If you ain't out by noon,
I'll be throwin' in yer boots,
Or if you fancy another ride
I'll be you're shining star all night.
But either way, it's been two days
And I ain't seen you high stack all ride.
So if it's all the same to you, Sir,
I'll have mine paid: fair share.
So I can find myself a meal,
I'm hungry and loveless,
Waiting on this ordeal.
Now I trust you, and don't be puttin on,
This was the deal we made, my trust.
Now do me kind and have it out,
And I'll be on my way."
Those were the words I heard sung again
As my splashing soul went out,
And the legalese was spitting up
Lead tipped water kisses into my eyes.


text:
The aim of this route is to offer you the opportunity to get to know the Costa Blanca from a completely different point of view: l'Alcoià.
We will visit the Natural Reserve of Font Roja located in the north of the province an enclave with an excellent representation of forest where we can observe its diverse array of deciduous trees.
We shall also enter into its history to discover how its inhabitants took advantage of its unique natural resources throughout the centuries. We are able to visit 18th century snow wells used for the production of ice.
After 3 hours of being completely immersed in mother nature we will recharge our batteries in a specially selected restaurant perfectly combining tradition and excellence. You will enjoy dishes typical of this area prepared with elaborate care, such as homemade bread filled with wild mushrooms and chestnuts, salads with seasonal vegetables fruits and flowers to name but a few.
To round off your journey you might wish to extend your day by booking a visit to an excellent bodega in order to sample its cultivated delights and to appreciate the importance of the area's labour. This is available at a small extra charge.
This excursion allows you to lose yourself in the forest of Alcoi enriched with incredible environment and sample its gastronomy steeped in tradition and unique produce.
- 08:30-09:30 Pick up from your accommodation *
- 10:00 Start guided hike by our Tour leader
- 11:30 Break for snack
- 14:00 Lunch at our selected restaurant
- 18:00 Transfer to accommodation of client and end of the journey
- * Exact time to be determined depending of your location
Prices by number of people
- 100 € by person. (VAT incl.)(4 to 8 people groups). Check price for a different number of participants.
Price includes
- Transfers from your accommodation (*) to the area to visit
- Full guide service throughout
- Personal insurance
- Mid morning snack
- 5 course traditional lunch (set menu).
Price not includes
- Visit the Bodega (Additional extra. Booking optional)
(*) Check here for pickup locations included in the price. For other locations please contact us.

subset: commoncrawl


text:
A Simple Plan: Services
Helpful Tips For People Who Need Transmission Repairs in Huntsville, AL Of the many things you do not want to deal with breaking on your car, the transmission is probably at the top of the list. Not only can it be terrifying if your transmission goes out while you're driving, but it is among the most costly repairs that can be made on any vehicle. If, however, you don't have any choice other than paying for the repair your car requires, you're probably reading this guide because you're trying to track down a reputable transmission repair shop in Huntsville, AL. Huntsville is populous enough that there a fairly large selection of garages that have transmission repair services available. Your job is to narrow these down to the best fit for your situation. As you read the rest of this guide, you'll see some helpful information that should make it easier for you to make your final decision. If you finish reading and are left with questions that are particular to your situation, contact the garages you're interested in by phone or email to get the answers you need. Determine If You Have to Take Your Vehicle to a Specialty Garage
The Path To Finding Better Options
Particular vehicles, especially those that were designed and produced in foreign countries, are unable to be fixed by any random transmission repair garage you find in Huntsville, AL. Most of the time, these need to be fixed by technicians who have gone through special certification processes with those vehicle manufacturers. If, for example, you drive an Italian sports car, you will probably need to take it to either a dealership that sells those cars or to a transmission repair service that specializes in exotics.
The Path To Finding Better Options
Determine If It's Worth Having the Repair Done From time to time, people discover that having their vehicles' transmissions repaired actually isn't worth the amount of money they would have to spend. If, for example, the vehicle that had its transmission go out is fairly old and you wanted to get a replacement for it soon, you'd probably end up spending more than the auto is worth to get a new transmission put in. It is, of course, up to you if paying for a transmission is worth it; if you're having money problems, a new transmission is still going to be cheaper than a new car. Do Your Research Before You Select a Garage You should refrain from telling any Huntsville, AL transmission repair service that its technicians can work on your car until you know you've found one you can trust. Reading online reviews of companies like this is the simplest way to research them. It should be quite simple for you to determine which businesses enjoy impeccable public reputations and which ones are rather more dubious. Make sure the garage you pick has a long history of doing great work.


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Cannabis Blog
New technique could more accurately measure cannabinoid dosage in marijuana munchies
As more states decriminalize recreational use of marijuana and expand its medical applications, concern is growing about inconsistent and inaccurate dosage information listed on many products, including brownies and other edibles. But now scientists report that they have developed a technique that can more precisely measure cannabis compounds in gummy bears, chocolates and other foods made with marijuana. They say this new method could help ensure product safety in the rapidly expanding cannabis retail market.
The researchers present their work today at the 251st National Meeting & Exposition of the American Chemical Society (ACS).
Read More:http://www.acs.org/content/acs/en/pressroom/newsreleases/2016/march/edible-cannabis.html
Super Weed Brothers - Deluxe Weed Edition
Super Silver Haze (Sativa)
Ohio Medical Marijuana Initiative Resubmitted To AG's Office
Backers of a proposed 2016 ballot measure to establish a comprehensive medical marijuana program in Ohio resubmitted their initiative petition to the Ohio Attorney General on Tuesday with more than 2,800 signatures.
The office has 10 days to examine the official summary of the initiative and confirm the petition contains at least 1,000 valid signatures of Ohio voters. The petition will then be sent to the Ohio Ballot Board, which will have 10 days to review the measure and confirm it complies with Ohio initiative laws. Initiative backers will then need to collect an additional 305,591 valid signatures of Ohio voters by early July in order to qualify for the November ballot.
Attorney General Mike DeWine notified Ohioans for Medical Marijuana on Friday that he had rejected the group's initial submission because he found three deficiencies in the official summary of the initiative.
In addition to addressing the attorney general's comments in the summary, initiative backers amended the initiative to include additional debilitating medical conditions that would be covered by the program. Specifically, the new version includes autism with aggressive or self-injurious behaviors, sickle cell anemia, severe fibromyalgia, spinal cord disease, spinal cord injury, traumatic brain injury or post-concussion syndrome, Parkinson's disease, muscular dystrophy, and Huntington's disease. It would also make Ohio the first state to allow medical marijuana as a treatment for chronic traumatic encephalopathy, also known as CTE, a degenerative brain disease found in professional football players and others who have experienced repetitive brain trauma.
Read More:http://www.theweedblog.com/ohio-medical-marijuana-initiative-resubmitted-to-ags-office/
Mango (Indica)
Mangos parentage is part KC 33 and part Mango from a hippie fellow who grew it for 30 years, then let KC use it as a cross in 1991. It is a 100 percent indica strain that grows large and produces prodigiously. Dont let Mangos slow start fool you - this variety will eventually rival the impressive size of many KC varieties, which are especially intended for outdoor grows. If you want Mango to stay at an average size in an outdoor garden, allow only 3 weeks of vegetative time and transplant outdoors as late as July in Northern latitudes. Mango can also be grown in an indoor garden, but needs a lot of space.
Pennsylvania House Approves Medical Marijuana, Final Vote Coming Soon
After an excruciating year of political sandbag tactics, it seems that Pennsylvania is finally on the verge of legalizing a decent medical marijuana program.
On Monday evening, the House of Representatives gathered for several hours to discuss a number of proposed amendments to Senate Bill 3, a measure intended to give patients suffering from a handful of qualified conditions access to cannabis products. In a vote of 152-38, the House managed to reach an agreement on an updated version of the bill. It is expected to go before the House for a final vote later this week.
The latest draft of Senate Bill 3 would allow patients with conditions such as post-traumatic stress disorder, cancer, chronic pain, AIDS and epilepsy to get their hands on non-smokable forms of marijuana. It was previously suggested that the pot products manufactured and sold in Pennsylvania would contain no more than 10 percent THC, but an amendment approved in a vote of 97-91 eliminated this restriction altogether.
Read More:http://www.hightimes.com/read/pennsylvania-house-approves-medical-marijuana-final-vote-coming-soon
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|Flight distance:
|It is detected that flight distance between Gadsden, AL and Gulfport, MS, with normal modern plane will be 125 km. Approximate flight time also with plane is 2 hour(s) 59 minute(s)
|About Airports:
|Users reported on our website that near Gadsden, AL and Gulfport, MS is airports near the road about 1 kms around.
|Carbon footprints:
|Based on user reviews and average calculating CO2 release in the air, with normal, modern car during the route from Gadsden, AL to Gulfport, MS will be 399.44 pounds.


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( 1960 1961 1962 1963 1964 1965 1966 )
2,426 Classics and Exotics For Sale Today - Buy One From Us
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Top Linksmain detailsbiographyby votesphoto galleryquotes
Filmographiesby yearby typeby ratingsby votesby TV seriesby genreby keyword
Did You Know?photo galleryquotes
Fiery Blaze (Character)
from "The Tick" (2001)
FilmographyEdit Credits
- "The Tick"
- Couples (2001) TV episode, Played by Ron Perlman
Additional Details
Fun Stuff
Fiery Blaze: So, eh... What does Arthur do?
The Tick: He flies, once in a while. Mostly he yells, like when I leave the cap off the toothpaste.
Fiery Blaze: Wow, there... Hey, that's no good for you, man. You're the superhero! You gotta keep your head clear to wage war against the wicked. Sidekicks are supposed to put the cap back on the toothpaste.
The Tick: I hear that!
Fiery Blaze: Hey, you're the starplayer on this team. He's supposed to take care of the little stuff - bills, laundry, sending out Christmas cards.
The Tick: Go tell it on the mountain!
The Tick: Dude... I'm *on* the mountain!
See more »
You may correct errors and omissions on this page directly. Clicking the 'Update' button will take you through a step-by-step process.


text:
Current address: Tata Institute of Fundamental Research, Department of Biological Sciences, Colaba, Mumbai, India
Conceived and designed the experiments: SR MS. Performed the experiments: SR MPL. Analyzed the data: SR MPL MS. Wrote the paper: SR CNV MS.
The authors have declared that no competing interests exist.
Changes in tissue homeostasis, acquisition of invasive cell characteristics, and tumor formation can often be linked to the loss of epithelial cell polarity. In carcinogenesis, the grade of neoplasia correlates with impaired cell polarity. In Drosophila,
In metazoans, the body surface and linings of several organs are formed from membranous tissue called epithelia. The functions of epithelia include secretion, absorption, and protection. Epithelial cells exhibit polarized distribution of several proteins, which is essential for their function. In carcinomas, which are cancers of epithelial origin, this epithelial cell polarity is impaired. Intriguingly, defects in cell polarization can also lead to tumorigenesis in some animal model systems. It is thus important to understand how cell polarization and epithelial growth control are linked so as to treat the carcinomas better by identifying new drug targets. Here we show that in zebrafish a gene named
Tumor suppression is a concept first formulated in
The establishment as well as the maintenance of apico-basal cell polarity and eventually the depolarization of a cell is a complex process, involving several factors. In recent years, a conserved mechanism for the establishment and maintenance of apico-basal cell polarization has emerged, which mainly involves two pathways. Accordingly, the formation of the apical domain is controlled by the Par (partitioning defective) pathway, which consists of the PDZ domain containing proteins Par3, Par6, and atypical protein kinase C (aPKC). In contrast, a pathway consisting of
The two vertebrate orthologs of the
The forward genetic approach in zebrafish has revealed a novel function for the second
From the analysis of several cancer models it is evident that autocrine self-stimulation with growth factors is one of the hallmarks of tumorigenicity
We have previously shown that zebrafish
DIC images of 5-day-old wild-type (A) and
Along with uncontrolled cell proliferation, migratory behavior mediated by EMT is another hallmark of cancer cells
Basal epidermal cells of wild type and
To observe the behavior of basal epidermal cells of
We conclude that in absence of
To understand if
Graphical representation of expression data of some of the relevant genes obtained by microarray (A) and quantitative RT-PCR (B). RNA In situ hybridization analysis in wild-type (C, E, G, I, K) and
The canonical way to achieve EMT is to down-regulate
We confirmed the tissue specificity of differential expression of the genes
Our analyses indicate that basal epidermal cells in
As human carcinomas are often a consequence of massive over activation of growth factor signaling
Western blot analysis of Erk1/2 phosphorylation in untreated wild-type and
Our data indicate that loss of functional
In mammals four ErbB receptors are known, ErbB1 to ErbB4, which get activated upon the binding of ligands such as EGF, HB-EGF, neuregulins, betacellulin
(A) Phylogenetic analysis of
In zebrafish,
DIC images of the morphology of wild-type (A),
Our data suggest that
Impaired cell polarity is one of the hallmarks of carcinoma but was considered to be a secondary effect for a long time. Recent findings however suggest it can also be a cause rather than a consequence of tumor progression
The basal epidermal cells in
How does deficiency in components of the baso-lateral pathway lead to epithelial cell proliferation and EMT? It has been suggested that the protein Scrib stabilizes the coupling between E-cad and the Catenins and thus behaves as a regulator of epithelial cell adhesion and migration
We show that inhibition of
The primary function of Lgl2 in hemidesmosome formation
ErbB signaling plays an important role in the development of human carcinomas, as it is able to induce proliferation and EMT and further is able to suppress apoptosis
The ErbB signaling pathway includes multiple ligands and receptors in vertebrates. ErbB1 and ErbB4 represent discrete RTKs, as they contain ligand binding sites as well as a kinase activity, essential for auto-phosphorylation and signal transduction. In contrast, ErbB2 and ErbB3 do not independently transduce extracellular signals as homodimers since ErbB2 has no ligand-binding domain and ErbB3 has no kinase domain. Thus, ErbB2 and ErbB3 must interact with each other or with other ErbB receptors to transduce signals
To summarize,
The morphological and immuno-histological analysis of
Protein sequences for ErbB zebrafish paralogues and human orthologues were obtained from Genebank (
The ΔNp63 promoter was cloned by enzymatic restriction of BAC dkey-13d19 with HhaI (NEB) and subsequent blunt-end cloning of a resulting 4.96 kb fragment into a plasmid containing a Gal4,UAS::GFP expression cassette and mini-TOL2 sites (based on
5-day-old wild type and
For IHC procedures the following antibodies were used: Anti-BrdU antibody ab6326 (abcam); anti Cytokeratin antibody Ks pan 1–8 (Progen Biotechnik) and anti E-cad antibody (BD Transduction Laboratory). Embryos were either fixed in 4% PFA (E-cad, BrdU) or in Dent's fixative (cytokeratin). After downgrading the larvae to 0.1 M phosphate buffer (PB), they were washed with PBT (PB+0.8% Triton X-100) five times and blocked in 10% normal goat serum. For BrdU staining, larvae were treated with 4 N HCl for 20 min., washed in PB and blocked in 1% BSA for 1–3 hours. Antibodies were diluted as: Ks pan 1–8 (1∶10), anti-E-cad (1∶250), anti-BrdU (1∶50) and samples were incubated at room temperature for 4 hours or overnight at 6–8°C. Afterwards, larvae were washed five times in PBT, incubated with Cy3 or Alexa 488 anti-mouse or anti-rat antibodies, post fixed in 4% PFA and upgraded in 70% glycerol for fluorescence/light microscopy.
DIG-labeled RNA probes for
Probe templates have been amplified with the following primer combinations by PCR reaction and subsequently cloned into pGEM-T Easy Vector (Promega). DIG-labeled probes were synthesized using T7 and Sp6 RNA polymerases (Roche).
Transplantations were carried out at blastula stage. After transplantations, donor and corresponding recipients embryos were cultured together (3–5 in number) in a 24 well plate. The
For screening, all inhibitors (SU5402; AG1024; PD168393; AG1478 all Calbiochem) were used at 10–50 µM concentration in 1% DMSO in E3 Medium starting at 96hpf. For BrdU, IHC and ISH experiments, PD168393 and AG1478 were used at a concentration of 10 µM starting at 96hpf.
For western blots, mutants were identified by the development of the characteristic
The microarray experiment was conducted using tissues (tail) posterior of the anal opening of wild type (Tuebingen) and
As for the microarray experiment, larval tails posterior to the anal opening were used as tissue sample (3 technical replicates of each biological replicate; 3 biological replicates). Total RNA was isolated using TRIZOL reagent (Invitrogen) and cDNA was synthesized using AMV cDNA Synthesis Kit (Invitrogen) with oligo-dT15 primers (Promega). SYBR Green (BioRad) was used for quantitative real-time PCR. Time emission readings were recorded with DNA Engine Opticon 2 (MJ Research), and analyzed as described
Genotyping to indentify mutants was done by a PCR based restriction fragment length polymorphism (RFLP) method for
Time-lapse movies were acquired using AxioVision 4.6 (Zeiss) and compressed in the Codec H264. For encoding in H264, the OSS Virtual Dub was used. Cell area (
Expression of GFP under the ΔNp63 promoter. Antibody staining of 5.5dpf tg(ΔNp63::Gal4,UAS::GFP) zebrafish larvae using anti Cytokeratin (red) and anti GFP antibody (green). The co-labeling of both antibodies reveals activity of the 4.96 kb upstream promoter element of ΔNp63 exclusively in basal epidermal cells in the skin.
Knockdown of
Genotyping of
Inhibition of
Expression profile of
Comparison of phenotypes and genotypes in
Phenotype of basal epidermal cells in the wild-type larvae. A 90-minute timelapse movie of tg(
Phenotype of basal epidermal cells in the
Phenotype of basal epidermal cells in wild-type larvae. A 240-minute timelapse movie of tg(Δ
Phenotype of basal epidermal cells in
We thank all members of our lab, in particular Nadine Wittkop, Jana Krauss, and Kellee Siegfried-Harris for the helpful discussions and comments on the manuscript. Our special thanks go to Matthew Harris, Martina Konantz, Hans-Martin Maischein, Heinz Schwarz, and Sören Alsheimer for help with some of the experiments. We further want to thank William Talbot and Matthew Voas for sending us the st61 allele of


text:
Stop! Thief!
5 comments:
- belinda said...
Good on ya if you can catch that one.
Kind Regards
- October 26, 2009 at 11:27 PM
- Gavin said...
Heck, not a chance down here in Melton. I still haven't got the tomatoes in the ground coz it is far too cold! Better get them in by Cup week or none for the Christmas table.
- October 27, 2009 at 4:24 AM
- Slices of Beauty... said...
Ha, nice one!
Love the question you pose on your profile, sure you are beautifully balancing already.
- October 27, 2009 at 10:37 AM
- Michelle said...
I have the same thieves here!! But they are so lovely that I can't bring myself to shoo them away :)
Luckily I have a gazillion tomatoes and I can spare a few for my beautiful feathered friends.
- October 27, 2009 at 9:57 PM
- Caroline said...
I think his reply would be ....
"Just doing my job lady just doing my job!!"
- November 6, 2009 at 9:59 PM
Subscribe to: Post Comments (Atom)


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Serving customers in and around the region, St. Charles Chrysler, Dodge & Jeep is widely recognized as being the number one Jeep Patriot dealership and known for our exceptional customer services and remarkable deals on all of our vehicles. Further, St. Charles Chrysler, Dodge & Jeep always puts customer satisfaction as the main priority. Regarded as a leading Jeep Patriot dealership, we are thrilled to carry the exceptional 2015 Jeep Patriot 4 x 4. Deemed as the Best Priced SUV in America, the Jeep Cherokee price isn't just the only thing that's extraordinary about it! The Jeep Patriot offers unmatched off-road ability and amazing gas economy. Even better, it is fabulous to drive! Each time our Glen Ellyn neighbors are seriously thinking of investing in a Jeep Patriot SUV, St. Charles Chrysler, Dodge & Jeep is the name not to ever forget!
Glen Ellyn Jeep Patriot For Sale
Glen Ellyn, Illinois is a beautiful affluent village situated in DuPage County, and there is right around 27,500 individuals residing there. Glen Ellyn is only a mere 13.8 miles southeast of St. Charles Chrysler, Dodge & Jeep. With our economical Jeep Patriot MSRP, we tend to quite a bit of Glen Ellyn traffic coming in to test drive our luxurious compact Jeep Patriot SUV. On top of that, countless Glen Ellyn locals are also interested in getting a Jeep Patriot lease, too. No matter what, the Jeep Patriot delivers on every level! It provides a roomy interior that is splendid, although at the very same time, completely Jeep-durable. If actually is a 2015 Jeep Patriot 4 x 4 that our Glen Ellyn friends are wanting, we will have the paperwork ready and waiting for them! Further, whenever Glen Ellyn residents stop by St. Charles Chrysler, Dodge & Jeep, they are totally aware that we will do everything possible to exceed their expectations. Â
Glen Ellyn Jeep Patriot SUV
Purchasing a completely new 2015 Jeep Patriot 4 x 4 should not ever be any kind of ordeal! It should be a fun and exciting experience! You can always depend upon St. Charles Chrysler, Dodge & Jeep's Jeep Patriot sales team to do everything to guarantee that we offer the most dependable, gratifying and efficient solutions. What is even more so, the Jeep Patriot SUV offers superb safety features that are standard - not just options - simply because, as we all can guess, being safe shouldn't ever be optional! Also, the 2015 Jeep Patriot has once again for the third year-in-a-row, the Kelley Blue Book's "5 Year Cost to Own Award!" And, don't stress, St. Charles Chrysler, Dodge & Jeep won't forget about you soon after you drive away from our lot in your brand-new Jeep Patriot. Our service department will be right there for all of your future repair and maintenance requirements when time.Â
Glen Ellyn Jeep Patriot Lease
The Glen Ellyn Park District features residents over 700 programs and special events per year regardless of age or interests. Some of the events include Lake Ellyn Cardboard Regatta, Freedom Four? Four-Mile Run and much more! There Glen Ellyn Park District maintains over 30 parks and includes Ackerman Sports & Fitness Center, Lake Ellyn Boathouse, 23 multipurpose parks and playgrounds, two lakes, four recreation buildings and an outdoor aquatic facility. Some of the events residents take part in is the Holiday Walk, Taste of Glen Ellyn, Shredding Day, Jazz in Glen Ellyn and the Halloween Parade. Glen Ellyn is home to Stacy's Tavern Museum, a stagecoach inn from the 1840s.ÂÂ
Glen Ellyn Jeep Patriot Price
To get to St. Charles Chrysler, Dodge & Jeep from Glen Ellyn, head east on Pennsylvania Avenue toward N Main Street for about 72 feet; turn left at the 1st cross street onto N Main Street about 1.8 miles; turn left onto IL-64 W/North Avenue for about 12 miles to St. Charles Chrysler, Dodge & Jeep. Also, you can phone us at: (877) 768-4955 to see about taking a Jeep Patriot for a test spin.ÂÂ


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1. How do I get tickets?
Tickets can be purchased from our main outlets, Ticketmaster or Skiddle. All e-tickets MUST BE PRINTED before arrival as there are no printing facilities on site. Your ticket will be scanned at the entry gate. Tickets purchased from other outlets (eg. Fatsoma, Shoobs, etc.) are still valid. If you have purchased one of these tickets, please go to the box office on arrival before the entrance gates.
2. What time does it start?
Our festival is open from midday - 9pm on Saturday 30th July. However, last entry is 6pm. If you arrive after this time, you will not be allowed into the festival site and your ticket will not be refunded.
3. What if I lose my ticket?
If you lose your ticket, please contact the website you purchased it from (eg. Ticketmaster, Skiddle, etc.).
4. How do I get there?
The site is just minutes from Wembley Park tube on the Jubilee and Metropolitan lines. For full travel details, please see the Wembley Park website or there is a handy little map on our Contact page.
Looking to stay the night? Check out Cheap Rooms for deals in the area!
5. What kind of pizza will be available?
There will be a range of traditional and unique pizza slices on offer (Keep an eye out on our Instagram for updates).
Everyone loves a margherita every now and then, but our pizza vendors have also come up with some brand new concoctions that we'd love you to try. There will also be a range of vegetarian, gluten-free and vegan options - everyone deserves good pizza!
All served by the slice, so you can't buy a whole one…but hear us out. This was one of the very first things that we decided on when creating the festival. It's so that you can try as many different slices as possible from different vendors.
It is a pizza festival after all!
6. Is there first aid on site?
Yes, first aid and welfare services will be on site throughout the day should you need them - ask one of our stewards or security team, they'll point you in the right direction.
7. Do any age restrictions apply?
Yes, you must 18 or over to enter MySliceFest. ID is required on the entry gate. Suitable forms of valid photographic ID include your passport, driver's licence and any photographic ID that carries the PASS logo. Photocopies will not be accepted.
Please note that Student ticket holders will also need a valid photo student ID - NUS cards will be accepted.
8. What do I do if I lose any personal belongings?
Lost property will be held at the box office on the day. For any assistance after this, please email us.
9. UPDATE: Will I need cash?
Yes - initially we advertised that MySliceFest would be operating a token system throughout the festival, this has now changed.
All pizza and non-food vendors will be accepting cash payments and cash/card payments will be taken on the bars.
Please note: there will be a cash machine on site.
10. Are there any items that I shouldn't bring to MySliceFest?
Please do not bring anything on site that security could see as harmful to yourself or others. Our security team reserve the right to refuse entry (see our full Terms & Conditions for more details).
11. Can I bring my own food and drink?
No, only food and drink purchased on site is allowed.
12. Am I allowed to bring a video and sound recording device?
Only mobile phone cameras and pocket-sized cameras are permitted for personal use. Professional cameras and video/audio equipment are strictly prohibited.
13. Does the festival have disabled access?
Yes, we aim to make sure that everyone can enjoy the event, so we try our best to ensure that the site itself is as accessible as possible. Please email us for more details.
14. Can I leave and return later?
Nope, but why would you want to leave. We've got pizza, we've got drinks, we've got games.
We've got everything you need.
15. What is the refund policy?
Tickets are non-refundable and non-transferable I'm afraid. This is because each ticket is unique to each attendee (see full Terms & Conditions for more information).


text: Management of Large Tongue Schwannoma – A Short ReportJayanta Medhi, Hanifa Akhtar Laskar, +4 authors Rajni ThabahIranian journal of otorhinolaryngology2016Schwannoma is a benign nerve sheath tumor composed of schwann cells. Oral cavity is a rare site for schwannomas, tongue being the most common location. Here we are presenting a case of a young adult who presented with a huge swelling in the tongue which was removed by mandibulotomy approach and pre-operative tracheostomy. A 22-year-old male patient… (More)


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Tips on How to Buy Internationally
In today's world you are putting your business at risk if you are not sourcing internationally. As barriers to international trade are being removed it has now become easier than ever to source product using websites such as this website from any part of the world.
There are a lot of variables in the international sourcing. It is complicated and there are many different parties involved, such as Sellers, Quality Control Inspectors, Freight Companies, Insurance Companies, Customs and finally the customer. Cultural issues, time zone issues and communication barriers can present problems which can slow down your progress .
There is a lot of information out there on the internet which while helpful can overpower the reader. So we have noted th key points and steps and put a simple narrative to each point. Always remember if you have any questions please do not hesitate your local Goodada partner and they will be delighted to provide your with additional information.
The basics steps you need to know before sourcing internationally are as follows:
1. Fully know your product
While it sounds simple it is the most important part. You must know the materials used in your product, what are the national standards including health and safety standards, the packaging required. Think of it as baking a cake – you must know the recipe with all the ingredients before you can start to bake that cake.
2. Search for suitable Sellers and have them independently Inspected and assessed
There are several ways to do this including usings this website, contacting the trade desks of embassies, the internet and attending international trade shows. The key here is to really check out the potential manufacturers before placing any orders and using quality control inspection companies to conduct Factory Audits. You should use the inspection services offered by Goodada.com as they offer inspections for many countries. Tip: Always try to avoid using commission only trading companies as they are not on your side and you will never get the best price. Think of this; Do they want 10% of $100,000 or 10% of $80,000.
You can find Products and Sellers, from many countries on this website.
You can visit Goodada's dedicated Inspection website here http://inspection.goodada.com/
3. Decide the country as it can affect the import taxes and duties
Export/import regulations often differ from country to country. While a certain country might be able to offer a product or material a cheap price other factors could dramatically affect the price. One story comes to my mind about a European company that was importing plastic bags from China into the EU. They had negotiated a fantastic cost price with the manufacturer but did not realise that the European Union applied anti-dumping duties on the product. The plastic bags ended up costing this company a lot more than if they were paying their local supplier! Tip: Find out the products item's Harmonized Tariff Schedule classification number and confirm the import tax rate with your local tax authority.
You can use the Goodada Import tax calculator to estimate the import taxes - click on this link to access the calculator http://www.goodada.com/us/customs-import-and-export-duty-calculator
4. Have a bi-lingual purchase contract drawn up between you and the Seller that is enforceable in the country of your Seller
If you are purchasing more than $50,000 per annum from a seller, you must have a purchase contract drawn up, which is in the local language of your seller and is enforceable in the courts of their country. This is one of the most important things that you should not forget. Sellers often hand a Pro-forma invoice and convince the Buyer that this is a suitable document - It is of little or no value if you have a dispute and go to court. You should always have this view point, I am the buyer and I will issue my buying terms to the seller, not the other way round! Tip: Always think if a judge was looking at the document what language would he speak – this must be the language in your purchase contract
5. Only pay a deposit of maximum 30% of the product value
A lot of Sellers will try to get you to pay full value of the product prior to manufacturing your goods. Do not do this. The reason is that you will still retain a lot of power over the Seller at a 30% deposit. If you walk away they will lose money as they will not any profit on a 30% payment. Only pay the balance AFTER the goods have had a Finished Goods Inspection and a Container Loading Inspection. Once you have these reports, are happy with them and have a copy of the Bill of Lading then you should issue the balance payment.
6. Always get the products inspected at the Factory
This is the most important piece of information that can offered to you. Goodada's Inspection catch phrase is "Its far better to find a fault on the factory floor rather than at your door". How many horror stories have you heard of people opening a container and the goods were faulty, incorrect or not even the goods that they ordered! If you ask them had they the goods independently inspected at the factory before it left you will hear "No". Don't let this happen to you!
7. Use a logistics company from your Country/ State
People often think that they will save money but using a logistics company from the country/ state that they are buying from. That is an incorrect assumption in-fact will may actually cost you more money in terms of cost, time and stress. What you want to do is make life easier for you and the process as stress free as possible. Having a person who you can talk to (no time zone issues) and understands you is vital. You will also save money as there are no additional mark up's added by the sellers for organising the shipping company from their side. You should contact the Freight Companies that are listed for your area on Goodada. They are leaders in their field. Tip: Do not ship your goods last minute. This might cost you more than you need to pay.
8. Strongly Consider Freight Insurance
Some people advise against the needs for freight Insurance. However it is an area that cannot be ignored. You will be gald to have it in the event of any issues happening especially if you are buying expensive products.


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Catholic Campus Ministry programs at the colleges and universities within the fifteen counties of the diocese are provided resources and support by the Diocesan Office for Youth & Young Adult Ministry.
Campus Ministers and Chaplains gather twice a year to discuss relevant issues, have input on the offerings of the Office, network with one another, and reflect and pray together in shared ministry.
"Catholic Campus Ministry has become my family. They are a gift from God to help those who ask for help."
–Samantha Bailey, '17
Our goal is to help the Priests and Lay Professionals to articulate and fulfill the overall mission and vision of Christ-centered, comprehensive ministry using the six pastoral priorities found in the USCCB document: Empowered by the Spirit.
"Catholic Campus Ministry has had a huge impact on my life, from the friends I've made and the experiences that I have grown from, all in a faith based atmosphere."
–Lucas Liberacki, '16
- Messiah College
- Millersville University
- Penn State- Mont Alto Campus
- Penn State – York Campus
- Shippensburg University
- Susquehanna University
- York College
Bloomsburg University, Bloomsburg
Rev Richard J. Mowery
Director of Catholic Campus Ministry
Office: 570-784-3123
Nina Camaioni
Associate Director of Catholic Campus Ministry
Office: 570-784-3123
Bucknell University, Lewisburg
Dickinson College and School of Law, Carlisle
Rev Joshua R. Cavender
Office: 717-243-4411
Elizabethtown College, Elizabethtown
Franklin and Marshall College, Lancaster
Gettysburg College, Gettysburg
Elizabeth Smith
Assistant Catholic Campus Minister
Office: 717-337-6284
Lebanon Valley College, Annville
Messiah College, Mechanicsburg
Renee Thomas
Assistant Catholic Campus Minister
Office: 717-697-2614
Millersville University, Millersville
Alicia Q Spelfogel
Assistant Catholic Campus Minister
Office: 717-497-5675
Shippensburg University, Shippensburg
Rev Dwight D. Schlaline
Catholic Campus Minister
Office: 717-532-2912
Susquehanna University, Selinsgrove
Rev Tukura Pius Michael O.P.
Catholic Campus Minister
Office: 570-374-4113
York College, York
Ileana Feistritzer
Assistant Catholic Campus Minister
Office: 717-815-6524
Alicia Q Spelfogel
Millersville University, Millersville
Office: 717-497-5675
Nina Camaioni
Bloomsburg University, Bloomsburg
Office: 570-784-3123


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To quickly review parts of a set, the students colored their names to sort the vowels and consonants. They then counted how many vowels were in their name out of the whole set and how many consonants out of the whole set. This mini lesson can be found on my TpT store.
Teaching Art Outside of the Art Room
1 day ago


text: Dejavu is one of my favourite place here in Tallinn, it's a cosy lounge bar with nice atmosphere,low lights,often entertainment during the weekend,possibility to smoke waterpipe in the second floor. It's perfect place to chat with friends and sip a cocktail with comfortable chairs and couchs. But what surprised me some month ago was the food,or to be more exact...the... More


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Facebook Vote Exchange Groups
Various groups on Facebook have been created to help Facebook users ask for votes for the various contests that they may be involved in. You will not have How to get votes on facebook in such groups, but you may be required to return the favor and giving your vote to another person who voted for you. Such groups are not hard to find, and you can easily search them on Facebook. Facebook Contest Forums There are some reliable Facebook contest votes forums that are specially created for issues revolving around exchanging votes. You can place your request for votes here, get the votes and in turn, vote for other people in that forum. There are guidelines and rules that govern the vote request and it is advisable that you first go through them. The best thing about these forums is you get fb votes for free but if you want to win a big contest, you need votes very fast and as such, you must have how to get votes on facebook from reliable providers. Bulk Online Contest Votes Service If you indubitably need a win in any Facebook contest, then your best bet should only have to buy Facebook votes. You can get as many as over ten thousand in just one day in some bulk online vote services. When you buy Facebook votes, you will find several packages, and you can then choose the package that best meets your preferences. There are various reliable websites that will give you genuine and credible Facebook votes. You, however, must do some background checks on the service that you plan on using before you make a purchase.


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Tiffani Williams
You won't find anyone that likes to have fun as much as Tiffani. She was made to be the life of a party.
" " Tiffani worked with us over the phone and in person before the wedding to have a timeline for all of her introductions, special songs, and the overall vibe of the music (dance party!) and the most important songs to play." Malia and Michael, Morris Center Wedding, Savannah GA
DJ, Karaoke Host, Veteran and an all around good time, Tiffani hails from Shreveport Louisiana. She graduated from Northwestern State University in Natchitoches, La. While in school, she hosted a late night college radio show called "Random Radio". This would fuel here love of music. She was commissioned into the Army upon graduation in 2006 and served two tours in Iraq. While in Iraq, she taught herself to play the drums and would play during her unit's worship service. She decided to change career paths in 2012. She entered into the civilian world as a bartender, karaoke host and wedding DJ and now calls Savannah, GA. home. Her DJ name is TiFi (like WiFi) the Entertainer.


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Cells control their own death through a program termed apoptosis, which is indispensable for development and homeostasis in all metazoans. Lysosomal cysteine proteases are not normally thought of as participating in apoptosis; however, recent reports have shown that the cathepsin proteases can be released from the lysosome during apoptosis, where they can participate in cell death. We report here the development of an activity-based probe that, under optimized conditions, reports on cathepsin B activity only in apoptotic cells by reading out the release of cathepsin B from the lysosomes. Biochemical characterization of apoptosis in cells from cathepsin B null mice shows delayed and suboptimal activation of caspases. Our data further supports a role for cathepsin B in the cytosol as a positive regulator of a cell death feed-forward loop and provides a chemical tool for future investigations.
Apoptosis is a tightly regulated form of programmed cell death that plays an essential role in the development and homeostasis in all metazoans (Danial and Korsmeyer, 2004; Rathmell and Thompson, 2002; Zheng et al., 1999). Deficiency in apoptosis is a feature of cancer and autoimmune diseases, whereas improper overactivation of this death program is linked to neurodegenerative disorders (Green and Evan, 2002; Hanahan and Weinberg, 2000; Vaux and Flavell, 2000; Yuan and Yankner, 2000). Two major apoptotic pathways exist in cells, the intrinsic pathway, which senses cellular stress, and the extrinsic pathway, which is induced by extracellular signals such as Fas binding to its cognate death receptor (Riedl and Shi, 2004). Central to both pathways is the cascading activation of a family of cysteine proteases know as the caspases, which are synthesized as inactive zymogens. Upon activation by cross-autoprocessing through dimer- or oligomerization, initiator caspases (caspase-8, -9, and -10) cleave and thereby activate the effector caspases (caspase-3, -6, and -7), which then go on to deactivate, and in some cases activate, numerous cellular targets by proteolytic processing after key aspartate residues (Dix et al., 2008; Mahrus et al., 2008).
Current methods for visualizing these cascades depend heavily on antibodies to detect full-length and/or cleaved, therefore presumably activated, caspases. However, there is evidence that proteolytic cleavage is not always necessary for activation (Stennicke et al., 1999), and endogenous protein inhibitors exist that control caspase activity through a variety of mechanisms including by binding to the processed forms of the enzymes (Deveraux et al., 1999; Riedl and Shi, 2004). Adding to the complexity, some models of apoptosis are dependent on the release of lysosomal proteases known as cathepsins that may act through caspase-dependent or independent mechanisms (Foghsgaard et al., 2001; Guicciardi et al., 2000, 2001; Salvesen, 2001; Turk et al., 2002; Vancompernolle et al., 1998; Yeung et al., 2006). Because of these complications, small molecule inhibitors and substrates targeted against apoptosis-linked proteases have emerged as attractive alternatives to ascertain function. Although current commercially available reagents lack specificity (Kato et al., 2005a), great progress has been made in this area by the recent development of highly selective activity-based probes (ABPs) for caspases-3, -7, and -8 (Berger et al., 2006). These probes have, for instance, allowed for the identification of a novel pathway for caspase-7 activation (Denault et al., 2006).
Here, we describe the development of a cell-permeable ABP originally designed for the monitoring of caspase activity. We utilized a previously developed acyloxymethyl ketone warhead targeted by the general caspase recognition sequence Glu-Val-Asp (Berger et al., 2006; Kato et al., 2005a, 2005b). The cell permeability of our probe was engendered through the use of a two-step labeling procedure in which a bioorthogonal coupling reaction was used to attach a biotin handle to the APB after cell lysis (Figure 1A). Unexpectedly, we find that the conditions of our labeling protocol allow for either the detection of the effector caspases or the selective monitoring of the cysteine protease cathepsin B (CatB). CatB belongs to an 11 member family of cysteine proteases that are typically localized to lysosomal compartments where it primarily contributes to protein turnover. Dysregulation of the cathepsins is linked to a variety of diseases including cancer (Jedeszko and Sloane, 2004; Mohamed and Sloane, 2006), osteoporosis (Saftig et al., 1998), and immunological defects resulting from inefficient major histocompatibility class II antigen presentation (Shi et al., 1999). In addition to its role in lysosomal protein degradation, CatB is also secreted from certain cancer cells where it has been implicated in their detachment and migration (Gocheva et al., 2006; Roshy et al., 2003; Schraufstatter et al., 2003).
Cytosolic relocalization of CatB has been shown to be important in cell death (Kirkegaard and Jaattela, 2009). Lysosomal release of CatB has previously been observed upon stimulation of the extrinsic pathway of apoptosis (Foghsgaard et al., 2001; Guicciardi et al., 2000, 2001). Moreover, this release is implicated in the cross-activation of caspase-9 by caspase-8, thereby linking the extrinsic and intrinsic apoptotic pathways (Guicciardi et al., 2000). In addition, drugs such as bortezomib and granulysin and exposure to UVA/B light have been shown to permeabilize lysosome membranes and induce cathepsin-dependent cell death (Bivik et al., 2006; Droga-Mazovec et al., 2008; Yeung et al., 2006; Zhang et al., 2009). However, the extent of proteolytic activity CatB retains after this translocation event in living cells is less clear or whether CatB release is also a feature of cell death initiated through the intrinsic apoptotic pathway. Our labeling data show that CatB is active in the cytosol of intact cells following stimulation of both the intrinsic and the extrinsic pathways. In the case of the intrinsic pathway, this change in CatB activity is dependent on caspase-9. In addition, intrinsic caspase activation in CatB null cells was both delayed and suboptimal. CatB has been shown in the examples above to induce the release of cytochrome c from mitochondria, the first step in caspase-9 activation, and our own biochemical studies indicate that CatB can cleave caspase substrate sequences at cytosolic pH. The development of our cell-permeable and selective probe allowed us to uncover a positive regulatory role for CatB in a feed-forward loop during the intrinsically activated cell death program through one of the two mechanisms outlined above.
We synthesized azide-containing probe 1 (see Scheme S1 available online; Figure 1A) and set out to characterize its activity against the family of caspase proteases. We first examined its ability to label recombinant caspases in a complex cell lysate. Recombinant active caspase-3, -6, -7, and -9, as well as inactive zymogen of caspase-3 (p3) were added separately in equal amounts to cell lysate derived from MCF7 cells. MCF7 cells lack caspase-3 and have very low levels of endogenous caspase activity, providing a suitable background (Yang et al., 2001). Probe 1 (300 nM) was then added and lysate was labeled for 30 min. Excess probe was then removed and phosphine-biotin 2 (250 μM; Figure 1A) was added to biotinylate reactive alkyl azides via the Staudinger ligation (Lin et al., 2005; Saxon and Bertozzi, 2000). After 2 hr at 37 °C the secondary labeling reaction was terminated by precipitation and biotinylation was accessed by streptavidin blotting (Figure 1B). All recombinant active caspases were labeled, whereas, as expected, inactive full-length capase-3 remained untouched by the probe (Figure 1B).
Next we analyzed the ability of 1 to label caspases in intact cells undergoing apoptosis. Toward this end, NIH 3T3 cells were treated with staurosporine (1 μM) or DMSO vehicle for 3 hr and were then treated with probe 1 (300 nM) for 30 min. The cells were harvested, washed thoroughly, lysed, and reacted with phosphine-biotin 2 as above. Streptavidin blotting revealed the presence of a single biotinylated protein band at a molecular weight of ~30 kDa in apoptotic cells but not healthy cells (Figure 2A). This selectivity is diminished when the proteome is labeled with 1 after lysis (Figure 2A). Note that when higher concentrations of 1 (5–50 μM) were used, we observed this same, labeled protein band in nonapoptotic cells (Figure S1A). Thus, there is a concentration threshold (~1 μM) below which the visualization of this protein appears to be apoptosis dependent. Surprisingly, the molecular weight of this biotinylated protein was inconsistent with either active caspase-9 (~35 kDa) or active caspases-3, -6, and -7 (~21, 19, and 17 kDa). Affinity enrichment of biotinylated proteins followed by anticaspase blotting also suggested our probe did not label cellular caspases (Figure S2). To ensure that the apoptotic program had in fact been activated, the same precipitated lysates were examined using antibodies against caspase-3, -7, -8, and -9 (Figure 2A). Consistent with activation of the intrinsic pathway, caspases-3, -7, and -9, but not caspase-8, all showed proteolytic processing to their active forms.
We were concerned that the apparent lack of caspase labeling may stem from a low efficiency of the Staudinger ligation step. To explore this, we took advantage of an alternative bioorthogonal coupling reaction, the Huisgen 1,3-dipolar cycloaddition between an organic azide and an alkyne, which has been reported to be quantitative when compared to direct labeling methods (Speers et al., 2003). This reaction can be catalyzed by Cu(I) salts (Wang et al., 2003) and has seen great utility for the secondary labeling of several ABPs (Sieber et al., 2006; Speers et al., 2003; Speers and Cravatt, 2004). Probe 3 (Ak-EVD-AMK; Figure 2B) was synthesized in a similar fashion as 1, bearing an alkyne reactive handle in place of the azide. The alkyne of 3 presents an alternative reactivity profile, when compared to the azide, and will for example be resistant to conditions that may reduce the azide of 1 (Agard et al., 2006). NIH 3T3 cells were treated with staurosporine (1 μM) followed by probe 3 (300 nM) for 30 min. Following cell lysis, alkyne-bearing proteins were reacted with azido-biotin 4 under copper catalyzed cycloaddition conditions. Western blot analysis again showed biotin labeling of only one band at 30 kDa in apoptotic cells, despite that fact that caspases had been activated by the staurosporine treatment (Figure 2B). We also performed a similar labeling experiment with probe 3 in which an azido-rhodamine derivative was used in the secondary labeling step (Charron et al., 2009). In this case the labeled proteins can be visualized by direct fluorescence imaging as opposed to western blotting. This allows us to explore if the problem stems from an inability of streptavidin-HRP to recognize the biotin moiety in the context of the labeled caspases. Once again, however, we observed labeling of a single ~30 kDa protein band in apoptotic cells but not in healthy cells (Figure S3).
Conceivably, steric hindrance within the enzyme active site could impede the secondary labeling reaction. To examine this possibility, NIH 3T3 cells were treated with staurosporine (1 μM) and 3 (300 nM) as above. The cells were collected, washed, lysed, and precipitated to denature the proteome. The precipitated proteins were then resuspended in a 4% SDS buffer and subjected to the cycloaddition conditions with azido biotin 4. Under these labeling conditions we observed two bands at ~17 kDa (in addition to the 30 kDa band) corresponding to the expected molecular weight of the effector caspases-3 and -7 (Figure 2B and Figure S3). To further confirm the identity of these bands, we devised a competition experiment involving 1 and the commercially available biotin-VAD-fluoromethyl ketone, which can be used to label effector caspases following cell lysis. Pretreatment of apoptotic cells with increasing concentrations of 3 led to a dose-dependent block of effector caspase labeling by biotin-VAD-fluoromethyl ketone added after lysis (Figure S1B).
To determine the identity of the protein labeled by our probes under nondenaturing conditions, lysates from apoptotic NIH 3T3 cells prior treated with or without 1 were reacted with biotin probe 2 and incubated with streptavidin beads. Proteins captured on the beads were denatured and subjected to on-bead digestion by trypsin. Tryptic peptides eluted from streptavidin beads were collected and identified by tandem mass spectrometry (LC-MSMS). Assessment of eluted peptides revealed the cysteine protease CatB as the only protein present in probe-treated cells when compared to the control sample (Figure 3A). To confirm this result, streptavidin affinity enrichment was performed and analyzed by western blotting with a CatB-specific antibody (Figure 3B). Consistent with the mass spectrometric analysis, CatB was greatly enriched from apoptotic cells treated with probe 1 compared to healthy cells. As further validation, we found that probe 1 could label recombinant CatB in the presence of cell lysates and that no labeling was observed when CatB null (catB−/−) cells (from the corresponding knockout mouse) were treated with our probes (Figure S4).
Labeling of CatB was observed only in staurosporine-treated cells even though total levels of CatB in apoptotic and nonapoptotic cells remain constant (Figure 4A). This suggests that there is an alteration in the enzymatic activity following activation of the intrinsic apoptotic pathway. To see whether a similar phenomenon occurs upon activation of the extrinsic apoptotic cascade, we treated NIH 3T3 cells with cycloheximide (10 μg/mL) and tumor necrosis factor-α (TNF-α; 10 ng/mL) for 5 hr before addition of 3 to the media for 30 min. The cells were collected, subjected to cycloaddition conditions, and analyzed by western blotting (Figure 4B). We observed labeling of a single band consistent with CatB only in cells treated with TNF-α. Western blotting against caspase-8 indicated that the extrinsic pathway had indeed been initiated under these conditions (Figure 4B).
Although CatB has previously been implicated in TNF-α-induced apoptosis (Foghsgaard et al., 2001; Guicciardi et al., 2000, 2001), our data are the first evidence for a change in cellular CatB activity following initiation of the intrinsic pathway in intact cells. To ascertain whether this is a general phenomenon or one specific to NIH 3T3 cells, a panel of cell lines were treated with staurosporine (1 μM) followed by ABP 3 (300 nM) and biotin labeling with 4. All cell types tested, with the exception of Jurkat cells, showed robust labeling of a protein, consistent with CatB upon treatment with staurosporine (Figure 4C). Thus, an alteration in cellular CatB activity, as reported by our APBs, appears to be a common feature of staurosporine-induced apoptosis. Next we asked whether other activators of the intrinsic pathway would also lead to an alteration in CatB activity. Accordingly, we treated NIH 3T3 cells with the topoisomerase II inhibitor, etoposide, followed by ABP 3 (300 nM) and biotin labeling with 4. Consistent with the staurosporine studies, we observed labeling of a single ~30 kDa protein band in apoptotic cells and no labeling in healthy cells (Figure S5). Thus, we have identified conditions (i.e., treatment with low concentration of probe and secondary labeling under native folding conditions) in which probes 1 or 3 allow selective labeling of CatB upon activation of either the intrinsic or extrinsic apoptotic pathways.
To determine if our activity-based probe is labeling CatB in an apoptosis-dependent manner and not as an indirect result of staurosporine treatment, we took advantage of immortalized mouse embryonic fibroblast (MEF) cells derived from caspase-9 null mice (Masud et al., 2007). Caspase-9 null MEF cells and wild-type MEF cells as a positive control (from knockout mice and wild-type littermates, respectively) were treated with staurosporine (1 μM) for 3 hr followed by treatment with probe 3 (300 nM) for 30 min. The cells were harvested, washed thoroughly, lysed, and labeled with azido-biotin 4 as above. As expected, western blot analysis showed labeling of a band of ~30 kDa in wild-type MEF cells corresponding to CatB (Figure 5). In contrast, little or no labeling of CatB was observed in the caspase-9 null MEF cells, despite equal amounts of protein as determined by anti-CatB blotting.
Several studies indicate that CatB, which normally resides in lysosomes, is released into the cytosol upon activation of the extrinsic apoptotic pathway (Foghsgaard et al., 2001; Guicciardi et al., 2000, 2001; Vancompernolle et al., 1998). We reasoned that our probe may be reporting on this relocalization of CatB and would therefore provide the first direct measurement of CatB activity in the cytosol during apoptosis. For this to be the case several criteria must be met: CatB must be active at cytosolic pH, our probes must be able to label CatB in the cytosol but not in the lysosome, and CatB must be released into the cytosol following activation of both the intrinsic and the extrinsic apoptotic pathways (since we see CatB labeling in both cases).
To address the first of these criteria, we measured the activity of purified CatB from human liver at pH 5 and 7 using both a cathepsin-directed peptide substrate (Z-Phe-Arg-AMC) as well as a panel of caspase substrates (Figure 6A). Although Z-Phe-Arg-AMC was the preferred substrate in this in vitro assay, CatB was found to cleave all of the caspase substrates tested. Moreover, this activity profile did not change appreciably between pH 4 and 7. These results are in good agreement with previous biochemical studies that indicate that CatB retains significant catalytic activity at pH 7 and can cleave, and be inhibited by, caspase-directed substrates and inhibitors, respectively (Foghsgaard et al., 2001; Khouri et al., 1991; Koga et al., 1991). Collectively, this data suggests that cytosolic CatB is able to cleave proteins with more prototypical cathepsin cleavage sites, as well as substrates containing caspase cleavage sites.
Next we asked whether there is measurable CatB activity in the cytosol of apoptotic cells. NIH 3T3 cells were treated with staurosporine (1 μM) for 3 hr and a cytosolic fraction was then prepared using a digitonin extraction protocol (Foghsgaard et al., 2001). The remaining cell pellet was resuspended in NP-40 lysis buffer and centrifuged to provide a sample, termed NP40 soluble, enriched in organelles. These fractions were then assayed for cathepsin activity using the Z-Phe-Arg-AMC fluorescent substrate described above. An increase in cytosolic cathepsin activity was observed in staurosporine-treated cells compared to nonapoptotic cells (Figure 6B). Furthermore, this activity could be inhibited by the cathepsin inhibitor E64 (Barrett et al., 1982), ruling out caspase activity in our assay. Similar results were obtained using cytosolic fractions derived from NIH 3T3 cells treated with TNF-α (Figure S6).
With evidence of CatB release into the cytoplasm during apoptosis, we designed a competition assay to determine if our probe was indeed reporting on this relocalization. The cathepsin inhibitor E64 is not freely diffusible across cellular membranes and therefore unable to covalently modify cytosolically localized proteases in intact cells (Huang et al., 1992). However, it can access lysosomes through endocytosis and can therefore inhibit lysosomally localized cathepsins (Hang et al., 2006). This being the case, we reasoned that treatment of cells with E64 prior to addition of our probe should result in covalent inhibition of the entire lysosomal pool of CatB, resulting in a complete loss of labeling by 3. Indeed, when NIH 3T3 cells were treated with E64 for 3 hr prior to either a selective concentration of 3 (300 nM) in apoptotic cells or a nonselective concentration of 3 (5 μM) in healthy cells, all CatB labeling was lost (Figure 6C). We interpret this result to mean that prior to the induction of apoptosis, CatB is localized to the lysosomes and covalently inhibited by E64.
This allowed us to perform a similar experiment by co-treating cells with E64 and probe 3. We presumed that treatment of healthy cells with a high concentration of probe 3 (5 μM) leads to labeling of lysosomal CatB. Therefore co-treatment with an excess E64 (50 μM) should result in loss of probe 3 labeling, which was indeed the case (Figure 6C). In contrast, we presumed that treatment of apoptotic cells with a low concentration of probe 3 (300 μM) leads to labeling of mostly cytosolic CatB. This being the case, co-treatment with excess E64 should have little or no effect, which was our observation.
To ensure that E64 uptake is not simply impaired in apoptotic cells, we performed one additional experiment. We supposed that in apoptotic cells CatB resides in both the lysosome and cytosol and treatment with probe 3 at a high concentration (5 μM) will label both of these pools. If apoptotic cells can take up E64, co-treatment with excess E64 (50 μM) should result in loss of lysosomal labeling by probe 3. The end result of this co-treatment would be labeling of solely the cytosolically localized CatB at a level similar to probe 3 at 300 nM, which is again what we observe (Figure 6C). Thus, the labeled CatB observed in apoptotic cells co-treated with probe 3 and E64 represents the cytosolic population of the protein.
CatB has been previously shown to be involved in crosstalk between the extrinsic and intrinsic apoptotic pathways (Guicciardi et al., 2000), where activation of caspase-8 by treatment with TNF-α resulted in the release of CatB from lysosomes. Furthermore, use of a cell-free system indicated that active caspase-8 could release CatB from purified lysosomes and that CatB, in turn, resulted in the activation of the intrinsic pathway through the release of cytochrome c from the mitochondria (Guicciardi et al., 2000). Consistent with this model, cells isolated from CatB null mice also showed diminished cytochrome c release, caspase activation, and apoptosis when treated with TNF-α (Guicciardi et al., 2000, 2001).
Because the intrinsic cell death pathway is typically initiated by the release of cytochrome c, we rationalized that CatB may be acting in a positive feedback loop in staurosporine-induced apoptosis and set out to test that hypothesis. CatB null (catB−/−) and wild-type fibroblasts (from knockout mice and wild-type littermates, respectively) were treated with staurosporine (1 μM) and caspase-3 and -9 activities were measured at different times using an ex vivo caspase-dependent luminescence assay (Figure 6D). Both caspase-3 and -9 activities were diminished and delayed in CatB null cells when compared to wild-type controls. This suggests that CatB plays a role as a positive regulator not only in caspase-8-initiated cell death but also in the timely and efficient activation of the intrinsic apoptotic pathway.
We have described the serendipitous discovery of cell permeable activity-based probes that allow labeling of the lysosomal protease CatB in apoptotic cells. These probes were originally designed to visualize activated caspases via a two-step labeling scheme employing a bioorthogonal coupling reaction, either the Staudinger ligation or copper-catalyzed Huisgen cycloaddition. However, several lines of evidence including mass spectrometry and coimmunoprecipitation analyses (Figure 3) indicate that this labeling strategy instead results in the selective visualization of cytosolic CatB activity. Indeed, selective CatB labeling was observed in several different cell lines, regardless of the stimulus used to induce apoptosis (Figure 4). Activity probes that allow labeling of cathepsins (including CatB) in intact cells are known (Blum et al., 2005, 2007); however, selective visualization of CatB in apoptotic cells has not been reported previously.
That our probes should label CatB in cells is not entirely unexpected considering that previous studies have shown that caspase-targeted substrates and inhibitors can cross-react with CatB in vitro (Foghsgaard et al., 2001; Gray et al., 2001; Guicciardi et al., 2000, 2001; Rozman-Pungercar et al., 2003; Vancompernolle et al., 1998). More surprising was the lack of detectable caspase labeling in our initial cell-based experiments, particularly since probe 1 does label recombinant caspases added to cell lysates (Figure 1B). A likely explanation for this comes from our finding that endogenous effector caspases can be visualized when the secondary labeling reaction is performed under conditions expected to denature the cellular proteome (Figure 2B). This suggests that steric hindrance of the secondary labeling reaction in the caspase active site may be partly responsible for the lack of observable caspase labeling in the absence of denaturants. We propose that this steric problem, perhaps in combination with low levels of the activated caspases to begin with, places caspases below the detection threshold using our standard two-step labeling conditions. Presumably, CatB is above this threshold because it is more abundant and/or because the secondary labeling reaction is more efficient in this context. The net result of this is a labeling protocol for selectively visualizing CatB activity in apoptotic cells.
An ever-growing body of evidence has identified the CatB protease as an important participant in TNF-α-induced apoptosis (Foghsgaard et al., 2001; Guicciardi et al., 2000, 2001), and increase in CatB expression and activity initiates apoptosis in cells treated with an attenuated strain of Mycobacterium bovis, bacillus Calmette-Guérin (Sandes et al., 2007). Some small molecules and UVA/B light have also been shown to cause lysosomal permeabilization resulting in cathepsin-dependent cell death (Bivik et al., 2006; Droga-Mazovec et al., 2008; Yeung et al., 2006; Zhang et al., 2009). Additionally, staurosporine-induced apoptosis has been shown to be diminished in inclusion-cell disease fibroblasts, which have lowered levels of cathepsins B, D, and L (Terman et al., 2002). We have bolstered those results by providing the first direct measurement of CatB activity in the cytosol of intact apoptotic cells. We also provide direct evidence that CatB is released from lysosomes during the cell death pathway initiated by the intrinsic pathway in addition to the extrinsic apoptotic cascade (Figure 4). Interestingly, we did not observe CatB labeling in a MEF cell line lacking caspase-9, suggesting that release of CatB from lysosomes is downstream of the initiator caspase in the intrinsic pathway. Previous studies have indicated that caspase-8 is required for lysosomal release of CatB upon stimulation of the extrinsic pathway (Foghsgaard et al., 2001; Guicciardi et al., 2001). Indeed, it has been shown that recombinant caspase-8 can induce CatB release from purified lysosomes in a cell-free system (Guicciardi et al., 2000). Thus, it is possible that caspase-9 plays a similar role to caspase-8 in this lysosomal release phenomenon. Additional studies will be needed to further test this idea.
Upon induction of the intrinsic apoptotic pathway, we reasoned that CatB could be taking a role as a positive regulator of cell death by participating in a feed-forward activation loop, in addition to findings in previous reports that it can induce apoptosis. Feed-forward and -back loops are a hallmark of many biological signaling cascades whereby cells can tune their sensitivity to intra- and extracellular stimuli. The proteolytic cascade of caspase activation is a well-studied example of this. Caspase-9 is both an upstream activator and substrate of caspase-3, and the presence of caspase-3 has been shown to be essential for efficient activation of caspase-9 (Slee et al., 1999; Zou et al., 2003). We propose that CatB is a previously unidentified positive regulator of this important feedback machinery (Figure 7). One possible mechanism is CatB acting to reinforce the release of additional cytochrome c, thereby activating additional caspase-9. We have also showed that CatB is able to cleave peptides containing canonical caspase recognition sites (Figure 6A). Therefore, cytosolic CatB could also be directly activating caspases through proteolytic cleavage. Consistent with both hypotheses, CatB null cells treated with staurosporine show delayed and reduced activation of both caspase-9 and caspase-3 when compared to wild-type controls (Figure 6D). Further studies will be needed to test these ideas and to better understand the mechanism of the lysosomal release process generally. The ability to detect cytosolic CatB in intact cells using our probes should be helpful in this regard.
The development of a cell-permeable cathepsin B (CatB) probe facilitated the further investigation into the role this important protease plays in cell death. Previous studies have relied upon indirect measurements and in vitro experimentation to visualize the release of CatB from lysosomes. The data presented here are the first direct visualization of this release into the cytosol in intact cells during apoptosis. We have used this chemical advance to explore a role for CatB in the intrinsic apoptotic pathway. Biochemical characterization of caspase-9 and CatB null cell lines places the release of CatB into the cytosol downstream of caspase-9, where it plays a role in the timely activation of the caspase proteolytic cascade. We hypothesize that CatB is acting through two nonmutually exclusive pathways. Mirroring its role in the crosstalk between the extrinsic and intrinsic apoptotic programs, CatB could release cytochrome c from the mitochondria resulting in further activation of caspase-9. Furthermore we have shown that CatB can cleave peptide substrates bearing canonical caspase recognition sequences, implicating CatB in the direct cleavage of caspase substrates. In summary, we propose that in addition to its previously understood role in apoptotic pathway crosstalk, CatB is an important member of the highly controlled feed-forward loop that ensures full caspase activation during intrinsically stimulated cell death.
All chemicals were obtained from commercial suppliers and were used as received unless otherwise noted. Staurosporine, E64, and Z-VAD-FMK were obtained from EMD Biosciences. Recombinant caspases and CatB purified from human liver were obtained from Biomol. Phosphine-biotin (2) was synthesized as previously described (Saxon and Bertozzi, 2000). PEO3-biotin was purchased from Pierce. Streptavidin-agarose and Softlink soft release avidin resin were purchased from Sigma and Promega, respectively. Caspase substrates were purchased from EMD Biosciences.
Probes 1 and 3 were synthesized using commercially available amino acids and standard Fmoc solid phase peptide synthesis using DIC/HOBt coupling chemistry and modifications outlined in Kato et al. (2005a) Probes were then purified by RP-HPLC and identities were confirmed by ESI-MS; calculated for 1 C50H71N9O14 (M+H) 1022.51, found 1022.99; calculated for 3 C51H70N6O14 (M+H) 992.50, found 992.46.
Known azidovaleric acid (Seo et al., 2003) was activated to the corresponding pentafluorophenyl ester as follows. Azidovaleric acid (500 mg, 3.50 mmol) was dissolved in CH2Cl2 (5 mL) and pyridine (310 μL, 3.84 mmol) followed by pentafluorophenyl trifluoroacetate (690 μL, 4.00 mmol). The reaction was stirred for 45 min, washed with 1 N HCl, 10% NaHCO3, and H2O. The organic layer was dried over Na2SO4, filtered, and concentrated. This solid pink material (43 mg, 0.14 mmol) was added to a solution of PEO3-biotin (50 mg, 0.12 mmol) in DMF (2 mL) and the reaction was stirred for 16 hr. At this time the mixture was concentrated and the product purified by silica gel chromatography (7:2:1 ethyl acetate:MeOH:H2O) to yield 53 mg (81%) as an off white powder. 1H NMR (400 MHz, D2O) δ 4.57 (dd, 1 H, J = 5.0, 7.8 Hz), 4.38 (dd, 1 H, J = 4.5, 7.8 Hz), 3.59 (app t, 6 H, J = 5.3 Hz), 3.35 (app t, 6 H, J = 5.2), 3.32–3.27 (m, 4 H), 2.95 (dd, 1 H, J = 5.0, 13.3 Hz), 2.74 (app d, 1 H, J = 13.0 Hz), 2.27–2.22 (m, 4 H), 1.74–1.52 (m, 8 H), 1.41–1.34 (m, 4 H); 13C NMR (125 MHz, D2O) δ 177.28, 176.98, 165.75, 70.11, 69.87, 69.32, 62.52, 60.69, 55.80, 51.81, 40.14, 39.35, 35.91, 35.63, 28.35, 28.15, 27.89, 25.58, 23.07; ESI-MS calculated for C23H41N7O6S (M+H) 544.28, found 544.99.
MCF7 cells, grown to confluency, were collected from a 10 cm dish by gentle scraping and pelleted by centrifugation at 2000 × g. After the cell pellet was washed with PBS (1 mL) two times, the cells were lysed in 200 μl of ice-cold NP lysis buffer (0.5% NP-40, 50 mM Tris, and 5 mM MgCl2 [pH 7.4]) for 20 min and centrifuged at 4 °C for 10 min at 10,000 × g. The supernatant (post-nuclear lysate) was collected and the protein concentration was determined by Bradford assay (Bio-Rad). Probe labeling of recombinant caspases was accomplished by incubation of MCF7 cell lysate (50 μg) with recombinant caspase (10 μL, 1000 U) and 1 μM probe 1 (1 mM in DMSO stock) in a total volume of 100 μl for 90 min at room temperature. The lysates were then concentrated using Vivaspin (Sartorius) 500 columns (10,000 MWCO PES) and washed with lysis buffer (500 μL). The resulting protein solutions were diluted with NP lysis buffer to a volume of 100 μl and subjected to the Staudinger ligation.
NIH 3T3, wild-type MEFs, caspase-9 null MEFs, CatB null MEFs, CHO, Jurkat, HEK293, or HeLa cells at ~80%–90% confluency in a 10 cm dish were treated with serum-free media (10 mL) for 16 hr. At this time staurosporine (10 μL; 1 mM in DMSO) was added to give a final concentration of 1 μM (DMSO vehicle was added to all control plates). After 3 hr the cells were treated with probes 1 or 3 (0.1 to 50 μM; 1000× in DMSO stock) or DMSO for 30 min. Alternatively, cells were activated with etoposide (10 μM) and cycloheximide (10 μg/mL) for 12 hr. The cells were then collected by gentle scraping and pelleted by centrifugation at 2000 × g, followed by washing with PBS (1 mL) two times. Cell pellets from ~2.0 × 107 cells were resuspended in 100 μl of ice-cold NP lysis buffer (0.5% NP-40, 50 mM Tris, and 5 mM MgCl2 [pH 7.4]) with Complete Mini protease inhibitor cocktail (Roche Biosciences) for 30 min and then centrifuged at 4 °C for 10 min at 10,000 × g. The supernatant (total cell lysate) was collected and the protein concentration was determined by Bradford assay.
NIH 3T3 cells at ~80%–90% confluency in a 10 cm dish were treated with cycloheximide (10 μg/mL; 10 mg/mL stock in H2O) and TNF-α (10 ng/mL) for 5 hr. At this time, probe 1 (300 nM) was added for 30 min and the cells were processed as above.
Staudinger ligation labeling of azide-modified proteins was performed by incubation of post-nuclear lysates (100 μg) with 250 μM of phosphine-biotin (2; 5 mM in DMSO stock) and 5 mM dithiothreitol (50 mM in H2O stock) in a total reaction volume of 100 μl for 2 hr at 37 °C. The reactions were terminated by addition of ice-cold acetone (1 mL), incubated at −20°C for 20 min, and centrifuged at 4 °C for 10 min at 18,000 × g to precipitate proteins. The supernatant was careful decanted and the resulting pellet dried at room temperature for 5 min to remove any excess acetone. The protein pellet was subsequently resuspended in SDS-protein loading buffer with 2-mercaptoethanol and boiled for 10 min.
Cells lysates (100 μg) were prepared with modified NP-40 lysis buffer (1% NP-40, 50 mM triethanolamine, and 150 mM NaCl [pH 7.4]) with Complete Mini protease inhibitor cocktail (Roche Biosciences) and cycloaddition reactions were performed as follows. Azido-biotin tag (4; 100 μM; 5 mM stock in DMSO) or azido-rhodamine (100 μM; 5 mM stock in DMSO) was added, followed by 1 mM TCEP (50 mM stock in H2O) and 100 μM triazole ligand (Speers and Cravatt, 2004) (1.7 mM stock in DMSO/t-butanol 1:4). The samples were gently vortexed and 1 mM CuSO4 (50 mM stock in H2O) was added. Samples were vortexed again and allowed to reaction at room temperature for 1 hr. At this time, the reactions were terminated by addition of ice-cold acetone (1 mL), incubated at −20°C for 20 min, and centrifuged at 4°C for 10 min at 18,000 × g to precipitate proteins. The supernatant was careful decanted and the resulting pellet dried at room temperature for 5 min to remove any excess acetone. The protein pellet was subsequently resuspended in SDS-protein loading buffer with 2-mercaptoethanol and boiled for 10 min.
NIH cells, grown to confluency and treated with staurosporine and 3 as above, were collected from a 10 cm dish by gentle scraping and pelleted by centrifugation at 2000 × g. After the cell pellet was washed with PBS (1 mL) two times, the cells were lysed in 200 μl of ice-cold modified NP lysis buffer (1% NP-40, 50 mM triethanolamine, and 150 mM NaCl [pH 7.4]) for 20 min and centrifuged at 4°C for 10 min at 10,000 × g. The supernatant (post-nuclear lysate) was collected and the protein concentration was determined by Bradford assay (Bio-Rad). The 200 μg of total protein were then precipitated by addition of ice-cold acetone (1 mL) and incubation at −20°C for 30 min. After centrifugation at 10,000 × g for 10 min and careful decanting, the precipitated proteins were resuspended in SDS buffer (100 μl; 4% SDS, 50 mM triethanolamine, and 150 mM NaCl [pH 7.4]) and subjected to the cycloaddition conditions.
Cell lysates (10 mg for mass spectroscopic analysis or 1/10 scale for western blot analysis) from NIH 3T3 cells treated with 1 as above were reacted with 25 μM of 2 (5 mM in DMSO stock) and 5 mM dithiothreitol (50 mM in H2O stock) in a total reaction volume of 10 ml at 37°C with rocking. The reactions were terminated by the addition of ice-cold acetone (40 mL), incubated at −20°C for 20 min, and centrifuged at 4°C for 10 min at 10,000 × g to precipitate proteins. The supernatant was decanted and the protein pellet was resuspended in 10 ml of 0.2% SDS and 50 mM Tris (pH 7.4) with sonication. Proteins were precipitated again with ice-cold acetone (40 mL) and resuspended in 0.2% SDS and 50 mM Tris (pH 7.4) with sonication to remove any residual 2. Prewashed streptavidin-agarose beads (250 μL; for mass spectroscopic analysis) or Softlink soft release avidin resin (25 μL; for western blot analysis) were then added to resuspended protein lysates (1 mg/mL) and allowed to incubate at 4°C for 1 hr with rocking. The beads were centrifuged for 5 min at 4000 × g at 4°C and washed with 10 ml of 0.2% SDS and 50 mM Tris (pH 7.4) four times.
Washed streptavidin-agarose beads (~250 μL) were washed twice with 1 ml of 50 mM triethylammonium bicarbonate (pH 7.4). Attached proteins were then denatured and reduced with 500 μl of 6 M urea, 10 mM TCEP, and 50 mM triethylammonium bicarbonate (pH 7.4) for 30 min at room temperature with rocking. Iodoacetamide from a stock solution of 1 M was added to this slurry to give a final concentration of 20 mM iodoacetamide and was allowed to react for 30 min at room temperature with rocking in the dark. The beads were then washed with 1 ml of 50 mM triethylammonium bicarbonate (pH 7.4) three times, resuspended in 500 μl of 50 mM triethylammonium bicarbonate (pH 7.4) with 2 mg of porcine trypsin, and allowed to digest overnight at 37°C with rocking. The beads were centrifuged for 1 min at 4000 × g, and the supernatants were collected and concentrated to a volume of ~15 μl by speedvac. Glacial acetic acid was added to stop digestion and adjust the pH of the solution to 3.5. The sample was pressure loaded directly onto a 360 × 75 μm Pico-Frit column equipped with a 15 μm tip and a 5 cm bed of BioBasic C18 material (New Objective, Inc.). Subsequently the sample was analyzed on a Finnigan LTQ (Thermo Fisher Scientific) in positive electrospray mode using a linear gradient of 0%–100% B over 90 min (A = 0.1 M acetic acid in nanopure water, B = 70% acetonitrile in 0.1 M acetic acid) and a flow rate of 150 nL/min on a Smart System HPLC (Pharmacia). The spectra were acquired with the instrument operating in data-dependent mode. A full scan mass spectrum was followed by 10 MS/MS spectra on the ten most abundant ions. The isolation window for precursor selection was set to ±1.5 Da, the collision energy to 35%, and the activation Q was set to 0.25. The target value for the full scan was set to 3e4 and for the MS/MS scan to 1e4. The selected ions were set on a dynamic exclusion list for 30 s, except predicted tryptic peptides of avidin were set on a static exclusion list. The MS/MS data was analyzed by using the open source search engine, X! Tandem (version 2007.01.01), to search the spectra against the NCBI database using the X! Hunter search algorithm. Searches were limited to the Mus musculus genome, and the parameters were set to a fragment mass error of ±0.4 Da. Four tryptic peptides covering ~25% of CatB were identified from the fragmentation data. The base-10 log of the expectation value was −97.4, signifying a high confidence that CatB was present in the sample. No CatB peptides were identified from a control sample of apoptotic NIH 3T3 cells that had not been treated with probe 1.
Purified CatB (1.5 μg; Biomol) was added to BSA (100 μg) and Jurkat cell lysate (2 mg/mL; modified NP-40 lysis buffer), 100 μl total volume. DMSO vehicle or probe 3 (500 μM stock in DMSO; 1 μM final concentration) was then added. After 1 hr at room temperature, the reaction was terminated by addition of ice-cold acetone followed by incubation at −20°C for 20 min. The resulting precipitated proteins were centrifuged at 18,000 × g and the supernatant was carefully decanted. The protein pellet was washed with ice-cold acetone twice and then resolubilized in 100 μl of 4% SDS, 50 mM triethanolamine, and 150 mM NaCl (pH 7.4). Cycloadditions were then performed as above.
Biotin (~25 mg) labeled proteins were separated by SDS-PAGE and transferred to a PVDF membrane and the membrane was blocked with 5% nonfat dried milk in TBST (50 mM Tris, 150 mM NaCl, and 0.5% Tween 20 [pH 7.6]) over-night at 4°C or 1 hr at room temperature. The membrane was washed with TBST for 10 min (3 × 20 mL) and incubated with streptavidin-HRP (GE Health-care; 1:5000 in TBST) for 1 hr. The membrane was washed again with TBST for 10 min (3 × 20 mL) and developed using ECL reagents (PerkinElmer) and Kodak Biomax MR film. To ascertain levels of endogenous proteins in Figure 2, equal amounts of protein were run and probed with anti-caspase antibodies. In all other figures the above membranes were stripped and blocked as above, and then probed with anti-caspase antibodies (Cell Signaling Technologies; 1:1000), an anti-CatB antibody (Santa Cruz Biotechnology; 1:300), or an anti-PARP antibody (BD PharMingen; 1:1000). After washing three times with TBST, the blots were incubated with HRP-conjugated secondary antibodies (GE Healthcare) for 1 hr in blocking buffer, washed three more times with TBST, and developed using ECL reagents (PerkinElmer) and Kodak Biomax MR film.
AMC derivatized peptide substrates (DMSO stocks) were added to an imidazole-HCl buffer (pH 4.8 or 7.0) (100 μL) containing 8 mM dithiothreitol and 4 mM EDTA. A solution of CatB (200 ng/μL) in 0.1% Brij 35 solution (5 μL) was then added. After incubating for 30 min at 37°C, the reaction was terminated by addition of 100 mM sodium chloroacetate in 100 mM sodium acetate buffer (pH 4.3) (150 μL). Fluorescence (excitation at 380 nM; emission at 460 nM) was then measured.
NIH 3T3 cells were either activated for apoptosis with staurosporine or TNF-α as above or left untreated. The cells were then collected by gentle scraping and pelleted by centrifugation at 2000 × g, followed by washing with PBS (1 mL) two times. Cells were treated with 25 μg/mL digitonin, 20 mM triethanolamine, and 10 mM KCl2 for 10 min. After centrifugation at 10,000 × g for 10 min the supernatant was collected, which contains the cytosolic fraction. The pellet was then suspended in modified NP lysis buffer followed by centrifugation at 10,000 × g for 10 min. The resulting supernatant contains the NP-40 soluble fraction. Total protein from each fraction (30 μg) was normalized and then added to an imidazole-HCl buffer (pH 4.8) (100 μL) containing 8 mM dithiothreitol and 4 mM EDTA and was either treated with the cathepsin inhibitor E64 (200 μM) or with DMSO vehicle for 30 min. At this time Z-Phe-Arg-AMC was added to give a final concentration of 200 μM and the reactions were incubated at 37°C for 30 min. Fluorescence (excitation at 380 nM; emission at 460 nM) was then measured. All experiments were performed in triplicate.
NIH 3T3 cells were either pretreated with E64 (50 μM) for 1 hr prior to activation with staurosporine (1 μM) or DMSO vehicle for 3 hr. Staurosporine-treated cells were then treated with 3 alone (300 nM or 5 μM) or cotreated with 3 (300 nM or 5 μM) and E64 (50 μM) for 30 min prior to lysis and the cycloaddition conditions described above. Nonapoptotic cells were treated with 3 alone (5 μM) or cotreated with 3 (5 μM) and E64 (50 μM) for 30 min prior to lysis and the cycloaddition conditions.
CatB null or the corresponding wild-type MEFs (~25,000 per well) were plated in a white plate, clear bottom 96 well assay plate (Corning Incorporated). After 16 hr the cells were washed with PBS and 100 μl media containing staurosporine (1 μM) was added. At the indicated times, 100 μl of Caspase-Glo 3/7 or Caspase-Glo 9 assay (Promega) was added to the appropriate wells. The mixtures were incubated for 45 min and luminescence was measured according to the manufacturer's directions using a 96 well luminometer (Molecular Biosystems).
We thank M. Bader and E.C. Schwartz for helpful discussions, Howard C. Hang (Rockefeller University) for azido-rhodamine, Bonnie F. Sloane (Wayne State University) for wild-type and CatB null MEFs from the corresponding knockout mice (C57BL/6J genetic background), and Richard Flavell (Yale Medical School) for wild-type and caspase-9 null MEFs from the corresponding knockout mice (C57BL/6J genetic background). We also thank Brian T. Chait (Rockefeller University) for access to mass spectrometers. We acknowledge National Institutes of Health (NIH) grants (EB001991 and GM072015) to T.W.M., NIH grant (RR00862) to B.T. Chait, and American Cancer Society postdoctoral fellowships to M.R.P. and K.P.C. for research support.
Supplemental Data include Supplemental Experimental Procedures, one scheme, and six figures and can be found with this article online at http://www.cell.com/chemistry-biology/supplemental/S1074-5521(09)00243-9.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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Okutadami Maruyama (Uonuma City, Niigata)
Okutadami Maruyama is located deep in the mountains of the eastern region of Uonuma City, close to Niigata-Fukushima Prefecture border. Approach to the resort however is only possible from the Koide area of Uonuma city using the Silver Line road. Much of the second half of the journey is through quite narrow tunnels. Okutadami Maruyama is famous for effectively having two seasons - it actually closes for the peak winter months, usually from early January until mid-March. As such it is popular for early and late skiing and snowboarding. Uonuma city is located just to the north of Minamiuonuma and Yuzawa in Niigata Prefecture. There are 6 ski and snowboard resorts scattered around the mountains of Uonuma and while getting to them may not be as convenient as some other resorts to the south, the region enjoys very heavy snowfall each winter.
Niigata Prefecture is located in the central region of Honshu to the north of Tokyo and has a 250km long coastline facing the Sea of Japan. Convenient transportation links make Niigata very easy to get to from Tokyo and other surrounding regions. There are over 50 ski and snowboard resorts in Niigata Prefecture, ranging from small one-lift hills to some of the most well-known and popular ski resorts in Japan. While they are dotted around the mountains of the prefecture, most are concentrated in the southern regions - in particular the Yuzawa town and Minamiuonuma city area in the Chuetsu region and Myoko city in the Joetsu region.
Okutadami Maruyama Snow Depth Statistics
Official snow depth numbers from the current season and previous season
Okutadami Maruyama Season & Opening Times
Okutadami Maruyama Lift Ticket Charges
Season dates and information
At the time of writing (mid May), almost all ski resorts in Japan have closed for the 2016-2017 season.
The information shown on this page is for the 2016-2017 season. Planned season dates and information for the 2017-2018 season will be updated over the coming months. Data for every ski resort should be updated in time for the start of the 2017-2018 winter season.
Current resort snow depth
Daily updates during snow season
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Approx. distance from Okutadami Maruyama by road (in winter)
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About the information on this page
For more details and explanations about the information presented on this page, please read this.
It is very important to us that the information presented on SnowJapan is correct and up-to-date. Checks and updates are made throughout the year. If you find information that you feel needs checking and updating, please let us know by contacting us. We appreciate your support.


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Thursday, 31 March 2016
'Pamper Me' - beautiful duochrome from Amelia Knight
Today I would like to show you a lovely duochrome polish which I found in a local shop in Scotland. This is 'Pamper Me' from Amelia Knight collection. I don't know this brand at all but I am really impressed by the beauty of this polish. I applied it over black for an even better effect.
I have to say that it looks amazing especially in the day light. The formula is acceptable, two coats are enough for a proper duochrome look. The finish is a little bit rough, shiny but no gloss and reminds me a little bit of a foil effect. I left it like that because I quite liked it. And do you know what do I love the most in this polish?- the way it is shifting colours from olive green into dark purple. This is a very eye catching outcome.
And I am leaving you with my pictures. Let me know your opinion
And this is the hero of my mani- 'Pamper Me' from Amelia Knight Collection.
Thank you for stopping by. Have a nice day :)
Fife Fantasi
25 comments:
great color!ReplyDelete
That looks really pretty! :) I have a couple polishes like that and I never get tired of looking at them. :)ReplyDeleteReplies
thank you, I rally like duochrome effect on the nails :)Delete
Wow, świetnie wygląda :)ReplyDeleteReplies
ciesze sie, ze Ci sie podoba :DDelete
Bardzo się udał ten nieznany lakier! Świetny.ReplyDelete
You said it right! The color shift is so eye catching! Love it! :-)ReplyDelete
OMG! I looooove this one!ReplyDelete
What an amazing color!!!!!!
Liebst, Colli
Śliczny jest ten lakier!!ReplyDelete
Ooh it does look lovely!ReplyDelete
I love such shades)ReplyDelete
Love this shade! Great manicure!ReplyDelete
new post on my blog
Udany lakier, trochę jak Orly Space Cadet, ma w sobie coś podobnego.ReplyDelete
Śliczny lakier, a Twoje paznokcie na pewno pięknie mienią się w słońcu :)ReplyDelete
Bardzo ładny :)ReplyDelete
Cudny! mega mi się spodobał, jest taki innyReplyDelete
Gorgeous, that's such a unique polish!ReplyDelete
Cudny refleks :DReplyDelete
Zapraszam http://ispossiblee.blogspot.com Odpowiadam na każdą obserwacje i propozycje wejść w linki :)
Duochromes are always fascinating and appealing, no matter what hue. I really love it!ReplyDelete
Wao :D really beautiful glitter duochrome!ReplyDelete
I liked it very much! cute manicure! you are well done! ;)ReplyDelete
What an interesting nail polish!ReplyDelete
Jaki śliczny, uwielbiam efekt duochrome :)ReplyDelete
I love reading your comments and I always try to respond to every one of them so don't hesitate to leave me a message :)
If you're new on my blog, I always come and check out yours. I am always following back my active followers


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Self-adhesive vinyl numbers
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