text:
Bonfire of the Varieties: I'm doing a "Twitter Chat" about Digital Media Models (Now!)
This whole "future of media" thing is pretty damn brutal.
This week, Variety, the Washington Examiner and Time Out Chicago have all announced plans to close their print editions. At the same time, super-blogger Andrew Sullivan has decided to half the number of free "read more"s his visitors get before hitting his paywall. And the Washington Post -- the second-to-last paywall holdout amongst major newspapers (only the Guardian remains) -- has made plans to start charging its most frequent visitors for access.
None of this should be surprising, at least not to people who have been listening to a word I've been saying. In print, the only publications who will survive in print are those which focus on excellent, long-form writing for an audience willing to pay for it (you don't see many book publishers abandoning print). Online, media will bifurcate into two camps: the everything for free / linkbait headline / aggregated content / pageview-jacker crowd, and the we actually do real journalism and charge for it crowd. The two will co-exist perfectly: the linkbaiters directing new subscribers to the journalists and the journalists providing content to the linkbaiters.
Viewed in that context, there's nothing surprising about how things are shaking out this week. Variety is a breaking news paper, and as such has no business being a print daily. Time Out Chicago is listings and reviews. Again, no point in print. Fuck only knows that the Examiner thinks it is. Sullivan and the Post both have real journalists, and real readers making their switch to paid subscriptions absolutely right and proper.
The only odd thing is how slowly and timidly the change is happening. Right now, we're still in the denial stage where print newspapers continue to try to push breaking news on paper and while listings and classifieds magazines still attempt to justify their place on the newsstand. Online, the linkbaiters continue to waste money on real reporters, while the journalists dick around with porous paywalls and metering and stupid grabby headlines to try to compete on traffic.
Each side will learn soon enough: Unnecessary print will continue to die, Buzzfeed will lay off its political staff to double down on cat videos, and people like Sullivan and the New York Times will lock down even more of their work for those who are willing to pay for it.
In about ten minutes, I'll be Right now, I'm the guest on a #Muckrack "Twitter chat" where I'll be talking more about the future of journalism business models. I'll also be gratuitously plugging the upcoming Print Edition of NSFWCORP, despite the fact that issue one is totally sold out.
I'm not entirely sure how Twitter chats work, but I suppose following me or the hashtag #muckrack is a good start. After the chat is over, I'll try to figure out how to embed the best of it here.
Update: join the chat here.
(Image credit: Wikipedia)


text: Hello everyone. I have an Acer Iconia 7" A100 8GB Tablet with version 4.0.3 and a new Motorola Razar M from Verizon. I have attempted to try and tether using Pdanet Tablet and FoxFi with both Wifi and Bluetooth, but they both do not work. The Wifi says it is Obtaining the IP Address, but never receives it. I also recieve an error when I attempt to connect to my phone via Bluetooth with Pdane Tablet, saying it doesn't run the service starter. My phone also recieves an error when I startup the Bluetooth DUN on FoxFi, saying it failed to launch. Any help would be nice.

subset: commoncrawl


text:
i use clockworkmod recovery and when i installed the rom and rebooted it.
It showed only the htc screen that shows up when u boot the phone normally!!!
i am ready to try any rom right now.
this was my first rom that i tried to flash on my htc amaze 4G
my phone is rooted and the bootloader is unlocked.
also i didnt make any backup of the original rom!!!


text:
Mila Kunis, threatened once again her stalker escaped from mental facility
Mila Kunis' stalker is out there. According to various reports, on Saturday evening, a man named Stuart Lynn Dunn escaped from a psychiatric centre in Pomona. He was convicted of stalking Mila Kunis
By: ebolism | Comments: 70 | Views: 2893 | Votes: 0 | Shared: 0
Location: Los Angeles, California, United States | Leaked: Jun-2-2015 in Other News
Man twice convicted of stalking actress Mila Kunis escapes from Pomona mental health facility
Investigators are searching for a man who was twice convicted of stalking actress Mila Kunis after he escaped from a Pomona mental health facility. Stuart Lynn Dunn left a secure mental health faci
By: ebolism | Comments: 71 | Views: 2788 | Votes: 2 | Shared: 0
Location: Pomona, California, United States | Leaked: Jun-1-2015 in Other News, WTF
Interview Mila Kunis on finding herself
Markovna Milena "Mila" Kunis (born August 14, 1983) is an American actress. In 1991, at the age of seven, she moved from the Soviet Union to Los Angeles with her family. Once enrolled in an acting cla
By: renatamanis | Comments: 5 | Views: 511 | Votes: 0 | Shared: 0
Leaked: Jun-20-2014 in Other
'The Sexiest Woman Alive' Mila Kunis Without Make-Up
Try not to laugh! Mila Kunis Is Esquire's Sexiest Woman Alive. Will It Help Her Career? http://www.forbes.com/sites/dorothypomerantz/2012/10/09/mila-kunis-is-esquires-sexiest-woman-alive-wil
By: The Judge | Comments: 12 | Views: 2313 | Votes: 1 | Shared: 0
Location: United States | Leaked: Dec-30-2012 in Other
Actress Mila Kunis Accepts Afghanistan Deployed Marine's YouTube Invite to The Marine Ball in November
Sgt. Scott Moore, of the 3rd Battalion 2nd Marines in Musa Qala, Afghanistan. (FOX News) — And when FOX411 asked Kunis about the invitation over the weekend, her "Benefits" co-star, Justin Timb
By: star53 | Comments: 60 | Views: 8549 | Votes: 4 | Shared: 0
Leaked: Jul-11-2011 in Afghanistan
Mila Kunis On Jesus And Republicans
By: Big_Bang | Comments: 192 | Views: 2272 | Votes: 1 | Shared: 0
Location: United States | Leaked: Jun-27-2014 in Other News
Spaz Interviews Mila Kunis
what more can be said? She could've ripped him but instead played along...
By: toastt21a | Comments: 17 | Views: 1463 | Votes: 0 | Shared: 0
Location: United States | Leaked: Mar-5-2013 in Other Entertainment
Nervous kid interviews Mila Kunis, asks her out for drinks
This is great! The kid had me when he started naming off his buddies - The Convict, Django the beast...LOL!
By: j_ramone | Comments: 37 | Views: 2396 | Votes: 6 | Shared: 0
Location: United Kingdom (UK/GB) | Leaked: Mar-5-2013 in Other Entertainment
Mila Kunis Smacks Down A Reporter In Russian
You tell em' girl
By: tubezzz | Comments: 39 | Views: 6069 | Votes: 4 | Shared: 8
Location: Russia | Leaked: Aug-17-2012 in Other Entertainment
Ted: Movie Trailer (Uncensored Scenes)
A story centered on a man and his teddy bear, who comes to life as the result of a childhood wish. Rated R. Director: Seth MacFarlane Writers: Seth MacFarlane (screenplay), Alec Sulkin (screenplay),
By: gorillabiscuits | Comments: 5 | Views: 25905 | Votes: 0 | Shared: 126
Location: United States | Leaked: Jun-8-2012 in Other Entertainment
YouTube clip lands Marine dream date with famous actress
07/11/2011 09:06 PM YouTube clip lands Marine dream date with famous actress By: Ashley White WILMINGTON – A dream will come true for one U.S. Marine, all thanks to social media. According to publ
By: menderman | Comments: 15 | Views: 3833 | Votes: 1 | Shared: 0
Location: Greenville, North Carolina, United States | Leaked: Jul-12-2011 in Other News
YS: Simpson got mail from across the Atlantic
(Volume Warning - (on some computers)) A note about a box, from the states, addressed to Simpson - came in the mail. So I went on a trip around Stockholm to our Postal Office to get the mysterious th
By: DukeBrazi | Comments: 98 | Views: 2469 | Votes: 19 | Shared: 0
Location: Södermalm, Stockholm, Sweden | Leaked: Oct-26-2012 in Your Say


text:
Tables that provide student pass rates on the Praxis national examination; program completion rates; and job placement rates can be found by clicking on the appropriate documents below. Links are also provided below for the department's strategic and action plans, the program's student assessment and assistance plans, and the department's and ASHA's complaint policies.
If you would like more information about our program and its accreditation by the Council on Academic Accreditation in Audiology and Speech-Language Pathology (CAA), please contact the department chair, Lesli Cleveland by phone at 509.828.1328 or email email@example.com
email: email@example.com
phone: 509.828.1376
fax: 509.828.1386
View the desktop version of this page.


text:
14 October 2009
The Answers: installment II
What surprising thing made you smile this week?
This is a great question, which is why I didn't want to just stick it on the end of last night's post. My first thought was ugh, nothing made me smile this week (glass half full I am not right now), but then I pulled myself together and it became a rewarding and worthy exercise. Kind of like that choosing joy attitude. So, some surprising things that made me smile recently:
• An email exchange with my boss (not my immediate boss of whom I have spoken fondly on the blog before, but our bigger boss). I tend to be quite formal with the big boss, and I had politely requested some information, to which he replied he and his colleague would wrestle for it, which made me smile. I responded "Thumb wrestle?" and one replied "No mud!" while the other said "Jelly, thanks". His next email even included a smiley emoticon. Gosh, they're human after all.
• Clue no. 2 of the Through the Loops Mystery Sock turned out beautifully, not that I expected any less. It's pleasingly similar to the Artichokes socks I am still plodding along with three times a week during my work lunch hour.
• Son #3 announcing that he thought instead of Celebrity MasterChef (with which he and I have become slightly obsessed), they should hold a Celebrity MasterDoc in which famous doctors battle it out in front of Ministers for Health (ok I came up with that last bit). Ladies and gentlemen, you have one and a half hours to cure this patient; your time begins ... NOW. [...] Tick tick tick, etc. Bzzzzz! Step away from the slab. Sorry Karl, your patient carked it, and Cindy yours is scarred for life but Fiona! You go through to the next round!
• The sunrises this week. Silver and blue and soft apricot, with ribbons of mist in the valleys.
• The totem tennis spiral thingie that always manages to show up in the photos I take of our view, but which I never notice until they're already uploaded (hint: bottom right of shot).
There were several other lovely things that made me smile I know, because I was mulling this over on my drive home last night and I remember that I thought of a plethora of smile-inducing moments. However they're gone from my head.
Anyway, I might try and do this on a regular basis. As I said, it's an exercise that deserves a regular airing.
What's your next big knitting project you're longing to get started on?
That one's easy. I am hanging out, yet simultaneously terrified, to cast on for a Gail (aka Nightsongs) Shawl (Ravelry link).
10 comments:
- Louisa said...
Hi Suse,
Your "things that have made me smile" responses reminded me of "Three Beautiful Things" - http://threebeautifulthings.blogspot.com/ - taking joy in the small yet wonderful bits of life.
I've noticed that since I've been reading Three Beautiful Things, I've been constantly constructing my own in my head and it's made me notice them more in my everyday life. :)
- Wednesday, 14 October 2009 at 23:03:00 GMT+11
- fifi said...
such a beautiful landscape you have there. I love it.
and , as for the other stuff, oh I am lolling. Jelly indeed. Doc wars....bahahaha
- Wednesday, 14 October 2009 at 23:20:00 GMT+11
- peppermintpatcher said...
Interesting work place. I am inagining that you have a mud pit and an enormous jelly bowl on hand should they be required.
- Wednesday, 14 October 2009 at 23:26:00 GMT+11
- pinry said...
your answers made me smile! particularly the totem tennis one - from the sublime to the prosaic. Oh, Gail is a lovely shawl - you can do it! (thanks to your encouragement I managed to do the cast on for the shetland! now up to third repeat!!)
- Wednesday, 14 October 2009 at 23:43:00 GMT+11
- Fioleta said...
:-) I used to be addicted to MasterChef, the Doc version would be hilarious, but a bit scary at the same time.
Gail Shawl looks beautiful, you can do it! :-)
Universities are funny places, I suspect that you are working in the same one I used to few years ago.
- Wednesday, 14 October 2009 at 23:49:00 GMT+11
- craftydabbler said...
The Gail Shawl is lovely.
- Thursday, 15 October 2009 at 08:04:00 GMT+11
- Bells said...
It is an excellent exercise. Pinry asked me the same in my QandA and I like your responses very much.
jelly wrestling. *giggle*
And Gail! Oh yes! She is on my horizon!
- Thursday, 15 October 2009 at 08:49:00 GMT+11
- Stomper Girl said...
My friend tells me her 9yo is bitterly disappointed with celebrity Masterchef because he was expecting all the guest chefs like Matt Moran et al to be duking it out for the cameras, and was rather miffed to find a while heap of people he knew nothing about.
- Thursday, 15 October 2009 at 12:35:00 GMT+11
- vaguelyspecific said...
Things that make us smile are always good! ;-)
- Thursday, 15 October 2009 at 12:49:00 GMT+11
- dillpickle said...
Can't wait to see the finished shawl - it looks amazing on Ravelry! I understand the terror factor though - I think you're brave just planning to attempt it ;-)
- Thursday, 15 October 2009 at 16:32:00 GMT+11


text:
Ask a Mechanic Online and Get Answers to Your Car Questions
HEADLAMP ASSEMBLY OR HEADLAMP BULB REPLACEMENT REMOVAL PROCEDURE CAUTION: Halogen bulbs contain a gas under pressure. Handling a bulb improperly could cause it to shatter into flying glass fragments. To help avoid personal injury:Turn off the headlamp switch and allow the bulb to cool before changing bulbs. Leave the headlamp switch off until the bulb change is complete. Always wear eye protection when changing a halogen bulb. Handle the bulb only by its base. Avoid touching the glass. Do not drop or scratch the bulb. Keep moisture away. Place the used bulb in the new bulb's carton and dispose of it properly. Keep halogen bulbs out of the reach of children.
Here is the diagram to remove the fuse box:
According to the Saturn manual, you need to remove the headlamp assy. to replace the headlight bulb. I would suggest following the recommended procedure. You may try to remove the fusebox to access the bulb, but I believe that would take more work than is necessary.
Please let me know if there is anything else I can do to assist you.


text:
Frangipane (almond cream)
1/4 cup granulated white sugar
3 tablespoons unsalted butter
1 large egg
1/2 teaspoon pure vanilla extract
1/2 cup (42 grams) almond meal (flour)
1 tablespoon (12 grams) all purpose flour
Pate Brisee (Short Crust Pastry):
1 1/4 cups all-purpose flour
1/2 teaspoon salt1 tablespoon granulated white sugar
1/2 cup unsalted butter, chilled, and cut into pieces1/4 cup (30 - 60 ml) ice water
5 cups apples, peeled, cored, and sliced into slices
2 tablespoons granulated white sugar
1 tablespoon unsalted butter, melted
Pate Brisee:
Add the butter and process until the mixture resembles coarse meal (about 15 seconds). Pour 1/8 cup (30 ml) water in a slow, steady stream through the feed tube until the pastry just holds together when pinched. Add remaining water, if necessary.
Do not process more than about 30 seconds. Turn the pastry out onto your work surface, gather it into a ball, cover with plastic wrap, and refrigerate for about one hour to chill the butter and allow the gluten in the flour to relax.
Once the pastry has chilled sufficiently, remove from refrigerator and place on a lightly floured surface. Roll the pastry into a 13 inch (33 cm) circle. Lift and turn the dough when rolling so it does not stick to the counter. If it gets too soft and starts to fall apart when rolling, put dough back in the refrigerator for 10 minutes to firm up. Transfer the circle to a baking sheet that has been lined with parchment paper, cover with plastic wrap, and return it to the refrigerator for about 15 minutes.
Then remove the chilled pastry from the refrigerator and spread the frangipane evenly over the dough, leaving a 2 inch (5 cm) border. Cover and return to the refrigerator while you prepare the apples.
In the bowl of your electric mixer, or with a hand mixer, cream the sugar and butter. Beat in the egg and vanilla extract until smooth. Add the almond meal and flour and beat until it forms a smooth paste.Transfer to a small ball.Refrigerate.
Assemble Tart:
Preheat oven to (190 degrees C) and place the rack in the center of the oven.
Arrange the apple slices evenly over the frangipane layer (concentrically or free form), again leaving a 2 inch (5 cm) border. Fold the (5 cm) border of pastry up and over the apples (sealing any cracks) and brush the pastry crust with the melted butter. Pour any leftover butter over the apples. Sprinkle about 2 tablespoons of sugar (depending on the tartness of the apples) over the crust and apples.
Bake the tart for 50 to 60 minutes or until the apples are cooked and the crust is golden brown. (The apples should be soft, but not mushy, when pierced with a knife.) Remove from oven and place on a wire rack to cool.
Serve warm or at room temperature with vanilla ice cream or softly whipped cream.


text:
Intensity Modulated Radiation Therapy (IMRT) has allowed a large degree of healthy tissue sparing while delivering therapeutic dose to tumors. However, the treatment planning process for IMRT is iterative and time consuming and the resultant plan quality is dependent on the skill and experience of the planner.
Following the work of Chanyavanich, a knowledge-based approach to IMRT treatment planning was used to generate high quality IMRT plans for patients from another hospital, using previously treated Duke plans as a reference library. An image-similarity metric was used to identify the patient from our database with the most similar anatomy to each new patient. Parameters from the Duke plan were then modified and applied to the new patient, resulting in quality dose distributions.
In conclusion, the treatment planning time was reduced to approximately ten minutes for all cases, and the resultant plans were frequently of higher quality than the original, manually produced plans. The quality of the Duke treatment plans was preserved as the plans were adapted to new patient anatomy.
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text:
Reexamination of the Akp3−/− mouse intestine showed that, despite the lack of intestinal alkaline phosphatase (IAP), the Akp3−/− gut still had considerable alkaline phosphatase (AP) activity in the duodenum and ileum. This activity is due to the expression of a novel murine Akp6 gene that encodes an IAP isozyme expressed in the gut in a global manner (gIAP) as opposed to duodenum-specific IAP (dIAP) isozyme encoded by the Akp3 gene. Phylogenetically, gIAP is similar to the rat IAP I isozyme. Kinetically, gIAP displays a 5.7-fold reduction in catalytic rate constant (kcat) and a 30% drop in Km, leading to a 4-fold reduction kcat/Km compared with dIAP, and these changes in enzymatic properties can all be attributed to a crucial R317Q substitution. Western and Northern blot analyses document the expression of Akp6 in the gut, from the duodenum to the ileum, and it is upregulated in the jejunum and ileum of Akp3−/− mice. Developmentally, Akp3 expression is turned on during postnatal days 13–15 and exclusively in the duodenum, whereas Akp6 and Akp5 are expressed from birth throughout the gut with enhanced expression at weaning. Posttranslational modifications of gIAP have a pronounced effect on its catalytic properties. Given the low catalytic efficiency of gIAP, its upregulation during fat feeding, its sequence similarity with rat IAP I, and the fact that rat IAP I has been implicated in the upregulation of surfactant-like particles during fat intake, it appears likely that gIAP may have a role in mediating the accelerated fatty acid intake observed in Akp3−/− mice fed a high-fat diet.
- alkaline phosphatases
- site-directed mutagenesis
- computer modeling
- fat absorption
- intestinal physiology
intestinal alkaline phosphatase (IAP), as the name implies, is expressed in the small intestine of many species. Lymph and serum levels of IAP increase after a fatty meal and the lymphatic triglyceride output is directly correlated with the lymphatic IAP output (13, 23), whereas IAP levels are dramatically decreased upon starvation (16). IAP is found associated with the brush border of the intestinal epithelium and enriched in surfactant-like particles (SLP) (1, 2, 8, 9, 14). After fat feeding, an IAP-containing membrane surrounds the lipid droplet in the enterocyte (36, 37). These findings suggest a role for SLP in fat absorption involving the induction of IAP by fat, which in turn results in IAP-induced SLP secretion.
In humans, alkaline phosphatases (APs) are encoded by four genes traditionally named after the tissues where they are predominantly expressed; the tissue-nonspecific AP (TNAP) gene (ALPL), located on chromosome 1, is expressed at highest levels in liver, bone, and kidney (hence the alternative name "L/B/K" AP), in the placenta during the first trimester of pregnancy, and at lower levels in numerous other tissues. The other three isozymes, i.e., placental (PLAP), placental-like or germ cell (GCAP), and IAP, show a much more restricted tissue expression; hence the general term tissue-specific APs. These isozymes are encoded by three genes (ALPP, ALPP2, and ALPI, respectively) clustered on human chromosome 2, bands q34–q37 (for review, see Ref. 24) and are closely related to one another, showing 90 and 87% identical nucleotide and amino acid sequences, respectively. The orthologous TNAP gene in mice is called Akp2 and is located on mouse chromosome 4. The mouse Akp3 gene encodes the IAP isozyme and the mouse Akp5 gene encodes the embryonic AP (EAP) isozyme that appears to be related to both the human PLAP and GCAP isozymes. In addition, mice have an inactive pseudo-AP gene Akp-ps1 (21). Although the human and mouse genomes appear to harbor a single IAP gene, the rat genome encodes two IAP genes, i.e., Alpi1 and Alpi2 (11, 20, 29), that produce mRNAs of 2.7 kb (IAP I) and 3.0 kb (IAP II) (10). Bos taurus in turn have an unprecedented level of complexity in their IAP genes since the bovine gut has been shown to coexpress up to seven IAP-like genes (22, 33).
Inactivation of the mouse IAP gene (Akp3) gives rise to apparently healthy mice under routine laboratory conditions. However, challenging these Akp3−/− mice with a long-term high-fat diet or with short-term administration of corn oil leads to an accelerated rate of fat absorption and elevation of plasma triglycerides, and these mice gain weight faster than wild-type (WT) littermate controls (26). These data indicate that IAP has a functional role in a rate-limiting step during fat absorption. Interestingly, we found residual AP activity in the gut of Akp3−/− mice (25). This activity was not due to expression of either the Akp2 or Akp5 genes. A search for homologous genes in the Mouse Genome databases revealed the presence of a new locus that appears to encode an IAP-like gene that we have named Akp6. The mouse Akp-ps1, Akp5, Akp6, and Akp3 loci, in this spatial order, are clustered in the same region of mouse chromosome 1. Here, we report that the Akp6 encodes an AP isozyme expressed through the small intestine, which we have named global IAP (gIAP) to distinguish it from the product of the Akp3 gene that is expressed in a duodenum-specific manner (dIAP). Although gIAP is catalytically fivefold less efficient that dIAP, it is upregulated in the gut of Akp3−/− mice during high-fat feeding.
cDNA cloning.
To obtain a full length Akp6 cDNA, total RNA isolated from the small intestine of Akp3−/− mice was reverse transcribed, and a 2.3-kb fragment was amplified by PCR using primers (forward primer: 5′-ATGCAGGGAGACTGGGTGCTGCTGTTGT-3′, reverse primer: 5′-TAGCCTTCTGGAAATCAGTTGGTACCTCC-3′) chosen from the sequence XM_129951, which was predicted from the annotated genomic sequence (NT_039173) by the GNOMON gene prediction algorithm. The PCR reactions were carried out using platinum Pfx DNA polymerase (Invitrogen, Carlsbad, CA) and Taq Extender PCR additive (Stratagene, La Jolla, CA). After confirming the entire sequence, we subcloned the 1.6-kb coding region of the 2.0 kb into an mammalian expression vector pCMV Script to express native Akp6 protein (gIAP) in vitro. To express a FLAG-tagged soluble form of gIAP, a DNA sequence encoding the FLAG peptide and a stop codon was added after the corresponding site of codon 525 (7). We used a forward (5′-CTCAGGCACACCTGGTGCACG G-3′) and a reverse primer (5′-TGCAAGGAGGGCCCTTA CTT GTC ATC GTC GTC CTT GTA GTC TGTGGTGGTGG-3′) to introduce an ApaI restriction site (italic), a stop codon, and the FLAG epitope (underlined).
To obtain a coding region of Akp3 cDNA, total RNA isolated from small intestine of WT mice was reverse transcribed, and a 1.6-kb fragment was amplified by PCR using primers chosen form the cDNA sequence NM_007432, which is derived from a genomic sequence, M61705 (forward primer: 5′-CCAGCCATGCAGGGACCCTGGGTGCTGC-3′; reverse primer: 5′-TAGATGTGCTGGAGTTTAGGACTCCGCA-3′). The sequence of the amplified 1.6-kb fragment was confirmed and the fragment was subcloned into the pCMV Script to express native Akp3 protein (dIAP). A DNA sequence encoding the FLAG peptide and a stop codon was added after the corresponding site of codon 532 by using the 5′-GAGCCAGCGGGCCCTACTTATCGTCATCGTCTTTGTAGTCGTTATGGACC-3′ oligonucleotide as forward primer and the 5′-GCCAGCCAGCTCACTAGTGAACGC-3′ as reverse primer to introduce an ApaI site (italic), a stop codon, and a FLAG epitope (underlined).
The full-length Akp5 cDNA, BC052874, was available as an IMAGE clone, 30033693, and was purchased from ATCC (Manassas, VA). The 2.1-kb insert was subcloned into the pCMV Script to express native Akp5 protein (EAP), and a DNA sequence encoding the FLAG peptide and a stop codon was added after the corresponding site of codon 506 using 5′-TGCCAACAGGTACCGCAAGGTTCACTTGTCGTCATCGTCCTTGTAATCGCTCTGGCCA-3′ as forward primer and 5′-GGCTGATGTCACTGAGGAAGAGAG-3′a 3′ as reverse primer to introduce a KpnI site (italic), a stop codon, and a FLAG epitope (underlined).
Expression of recombinant enzymes.
Each expression vector was transfected into COS-1 cells by standard electroporation, and after 24 h the FCS-supplemented culture medium was replaced by serum-free medium, OptiMax I (Invitrogen, Carlsbad, CA). Supernatant was collected after 60 h of further culture and concentrated ∼15 times with Centriplus (Millipore, Bedford, MA). We also transfected Chinese hamster ovary cells with the expression vectors and obtained clones stably expressing each of the mouse AP genes after 2 wk of selection with 400 μg/ml G418.
Kinetic analysis.
All kinetic measurements were performed in triplicate at 25°C. For the measurement of relative catalytic activities, microtiter plates were coated with the M2 antibody (depending on the goals, ranging from 0.2 to 1 μg/ml). For the calculation of catalytic rate constants kcat, we used PLAP-FLAG with known kcat = 460 s−1 as a reference for each microtiter plate. Recombinant APs were incubated with the plates for 2 h at room temperature, after which plates were washed with Tris-buffered saline, containing 0.008% Tween 80. The AP activity of bound enzymes or enzyme mutants was then measured at 405 nm as a function of time, using p-nitrophenylphosphate (pNPP, 0.05. 20 mM) as a substrate, either at pH 9.8 in 1.0 M diethanolamine buffer, containing 1 mM MgCl2 and 20 μM ZnCl2 or at pH 7.5 in 1 M Tris·HCl, containing 1 mM MgCl2 and 20 μM ZnCl2, as indicated. Recordings for kinetic computation were selected from those parts of the curve, where absorbance at 405 nm (A405 nm) vs. time was linear and reaction rates were recorded as A405 nm/time, taking into account an extinction coefficient of 10.08 × 103 M−1 cm−1 for the reaction product p-nitrophenol. Thus initial rates were calculated over a time interval up to 2 h, excluding the first 5 min from evaluation, because of non-steady-state conditions. Data were linearized in 1/velocity vs. substrate. Michaelis-Menten concentration plots and linear regression were performed, calculating slopes ± SD and intercepts ± SD in GraphPad Prism version 3.0a (GraphPad Software, San Diego, CA).
Antibody production.
Protein structure analysis and alignment using Laser Gene software indicated that peptide 208–231 from the deduced Akp6 sequence was potentially antigenic due primarily to its location on the 3D model of the enzyme and also based on the number of dissimilarities with the corresponding sequences of the Akp3 and Akp5 cDNAs. Two rabbits were immunized with the corresponding gIAP synthetic peptide, and the antiserum was affinity purified on an affinity column using solid-phase peptide. Peptide synthesis, immunization, and affinity purification were processed by QCB (Hopkinton, MA).
Tissue collection, histology, and immunohistochemistry.
All animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the Burnham Institute for Medical Research (Animal Usage Form # 06–106, approved on 6/23/2006). The production and characterization of Akp3−/− mice has been described previously (26). The intestine was fixed with 10% buffered formalin, washed with increasing sucrose concentrations, and embedded in optimal cutting temperature compound as described (26). Serial sections (8 μm-thick) were used for immunohistochemistry. Alternatively, some of the formalin-fixed tissues were processed through standard protocols for paraffin sections and used for hematoxylin and eosin staining. To examine IAP expression, a Vectastain ABC Kit (Vector Laboratories, Burlingame, CA) was used according to the manufacturers' instruction. The antisera were diluted in blocking solution and incubated with the tissue for 2 h at room temperature. Normal rabbit serum was used as the negative control.
Western blot analysis.
Mouse tissues were homogenized in lysis buffer (150 mM NaCl, 1% NP-40, 0.1% SDS, 50 mM Tris·HCl, pH 7.4), supplemented with 1 mM PMSF and 1% proteinase inhibitor cocktail (Sigma, St. Louis, MO). Protein concentration was determined by using the BCA assay kit (Pierce, Rockford, IL). Protein samples were denatured in 2% SDS and 0.025% β-mercaptoethanol and loaded on 8–16% acrylamide Tris-glycine gel (Invitrogen, Carlsbad, CA). Gels were transferred to Optitran nitrocellulose membranes (Schleicher & Shuell, Dassel, Germany), and blotted membranes were stained with Ponceau S to ensure efficiency of transfer. Washed membranes were blocked with SuperBlock reagent (Pierce) prior to incubation with primary antibodies, which were raised against peptide 208–231 of Akp5 or Akp6 in rabbits. Peroxidase-labeled goat anti-rabbit IgG antibody (Santa Cruz Biotechnology, Santa Cruz, CA) was used as secondary antibody, and a positive reaction was visualized by ECL+ plus kit (Amersham Pharmacia, Piscataway, NJ). Since standard control genes, such as β-actin, GAPDH, or cytochrome c, showed a segment-specific variability of expression in the small intestine, we used endogenous mouse IgG as internal control for protein loading. In case of recombinant enzyme samples derived from COS-1 or CHO cells, a mouse monoclonal anti-FLAG antibody (Sigma) was used as primary antibody and peroxidase-labeled goat anti-mouse IgG as second antibody (Calbiochem, San Diego, CA).
Enzyme immunoassay.
Postnatal mouse small intestines were divided into four segments and homogenized in Tris·HCl buffer (pH 8.9) containing 0.1% Triton X-100. the homogenates were emulsified with an equal volume of n-butyl alcohol, and the water phase was separated from the butyl alcohol phase after repeated centrifugation. Protein concentration of the extracts was measured by BCA assay kit, and samples containing 1.5 mg/ml protein were incubated on 96-well ELISA plates coated with anti-gIAP antibody. The wells were saturated with recombinant gIAP protein and incubated with serial concentrations of pNPP (20, 10, 5, 2.5, 1.25, and 0 mM), and the kinetic reaction was measured at A405 nm via a microplate reader (Molecular Devices, Sunnyvale, CA) to calculate Km values. For endo-β-galactosidase treatment, butanol extracts of intestinal segment 1 from 2-day-old mice (1.5 mg/ml protein × 50 μl per well) were incubated on 96-well plate coated with the anti-gIAP antibody to trap gIAP. After washing, the wells were treated with 0.003 units of endo-β-galactosidase from Escherichia freundii (Seikagaku) overnight, followed by washing and enzyme immunoassay.
Low- and high-fat diets.
Gender-, age-, and genotype-matched mice (3–5 in each group) were fed either a 4.5% or an 11% fat diet (mouse chow no. 5015, Purina Mills, St. Louis, MO) as previously reported (26). The high-fat diet was given to the Akp3−/− mice and littermate WT control mice for 2 mo. Adult mice were also fed 500 μl of corn oil by gavage following an overnight fast. The intestines were immediately immersed in RNAlater (Ambion, Austin, TX), divided into segments, which were opened up to remove the ingesta, and processed for RNA isolation.
Northern blot analysis.
Total RNA was isolated by a standard protocol (5). Ten micrograms of RNA was loaded in each lane. One set of samples was used for ethidium bromide staining. The other set of samples was used for blotting. Specific probes were prepared from 3′ UTR region of Akp6: a 284-bp ApaI-KpnI fragment and Akp3: a 236-bp BamHI fragment. The entire Akp5 cDNA was used as a probe owing to relatively low level of Akp5 expression compared with Akp3 and Akp6. The specificity of each probe was tested by Southern blot hybridization.
Identification of Akp6 at the genomic and mRNA level.
Previous data (26) had shown that Akp3−/− mice had a normal birth rate, normal life span, and no obvious abnormalities unless challenged with a high-fat diet, either via forced fat loading by gavage or long-term high-fat feeding, in which case the mice became obese as a consequence of an accelerated transport of fatty acids through the gut. Reexamination of the Akp3−/− mouse intestine showed that, despite the lack of IAP, the Akp3−/− gut still had considerable AP activity in duodenum and ileum (25). Upon examining the expression of the known AP genes along the length of the small intestine by RT-PCR, we found no evidence of upregulation of the Akp2 or Akp5 genes or of expression of the Akp-ps1 pseudogene (21). Similarly, we examined the intestines of the Akp5−/− mice and found no upregulation of Akp2, Akp3, or the Akp-ps1 pseudogene (not shown).
Akp6 and Akp3 sequence comparison.
Therefore, we searched for the possible existence of a novel AP gene that might explain the persistent AP activity in duodenum and the upregulated activity in the ileum. We used the BLAT tool at the http://genome.ucsc.edu to query the mouse genome database, since this software is designed to quickly find sequences of 95% and greater similarity of length 40 bases or more and DNA BLAT works by keeping an index of the entire genome in memory. Using the mouse Akp3-derived protein sequence as query, this search revealed the presence of a predicted new locus, based on spliced mouse ESTs deposited by the RIKEN Consortium (XM_129951 RIKEN Database) and also by The Genome Institute of Novartis Research Foundation using the Affimetrix U74B chip. We have named this new locus Akp6 and the isozyme it encodes gIAP to reflect its nonsegmental, generalized expression in the gut and to distinguish it from the dIAP encoded by the Akp3 gene and expressed exclusively in the duodenum, as we demonstrate in this paper. As shown on Fig. 1, all four mouse tissue-specific AP loci (Akp3, Akp5, Akp6, and the Akp-ps1 pseudogene) are closely linked at the 1C5 site in chromosome 1, with each gene occupying ∼3.5 kb and composed of 11 exons and 10 introns. In contrast, the Akp2 gene that is located at 4D3 in chromosome 4 and stretches for 55 kb and consists of 12 exons and 11 introns including an alternative exon (exon 1b), located ∼30 kb downstream of exon 1a (30, 31). The direction of the Akp3 gene and the Akp-ps1 pseudogene is opposite to that of Akp5 and Akp6 genes. We also extended our search for novel AP genes by probing the databases, this time with the less stringent BLAST search, using the Akp2, Akp3, Akp5, and Akp-ps1 DNA sequences as queries and found no further evidence of additional loci besides the Akp6 locus. Furthermore, with the caveat that negative data are seldom conclusive, to date we have found no confirmation in the Mouse Genome Databases that the Akp1 or Akp4 loci actually exist. These loci were postulated based on hybrid maps and biochemical data (34) but no sequences exist for either of these two proposed loci.
We then proceeded to cloning and sequencing the full-length Akp6 cDNA from Akp3−/− intestine RNA. The 2.2 kb obtained by PCR was sequenced and its coding sequence was identical to the predicted cDNA, XM_129951. Table 1 shows all amino acid differences between gIAP and dIAP. Notably absent from that table are all the active site residues, i.e., D42, H153, S155, E311, D316, H320, D357, H358, H360, H432, that are perfectly conserved between gIAP and dIAP. However, the Zn1 and Mg neighboring H317, a residue involved in the indirect control of enzyme catalysis in human PLAP (18) and preserved in EAP, was differentially substituted for Gln in gIAP and for Arg in dIAP (Table 1). gIAP and dIAP display 87% sequence identity, with the largest degree of substitution in gIAP occurring in the crown domain of the molecule, which consistently displays the largest degree of dissimilarity among AP isozymes and that confer unique biological properties to the isozymes (3). The NH2 terminus of the molecule, recently shown to be involved in a structural stabilization of APs (17), is very well conserved, showing two amino acid substitutions only.
Structure of the gIAP protein.
Phylogenetic comparison of the gIAP sequence with those of the human, mouse, rat, and bovine AP genes showed that the gIAP sequence is closely related to mouse dIAP and also to rat IAP I (Fig. 2). This is significant since in the rat IAP I mRNA can be found in both the duodenal and jejunal mucosa, but IAP II mRNA is only expressed in the duodenum (4). It would appear that gIAP corresponds in expression and perhaps function to that of rat IAP I, whereas dIAP corresponds more to rat IAP II (15).
We then modeled the structure of gIAP on the basis of the crystallographic coordinates of human PLAP (1EW2; http://pdbbeta.rcsb.org/pdb/) (19). Our a priori hypothesis was that this murine isozyme would have the same fold, the same quaternary structure, and nearly all the same amino acid orientations as the human PLAP structure. We did the modeling using the Swiss Model server via Deep View as the local interface. Waters and nonamino acids were removed from the template structure. The returned model had only 0.09 Å RMS on backbone atoms. These were observed to be due to mostly very small shifts (∼0.15 Å, up to 0.26 Å) in the backbone positions around amino acid differences. There is 77% sequence identity and 87% sequence similarity in the mature gIAP protein with respect to PLAP across the modeled domain. The quaternary structure is based on notes in PDB submission and also from the EBI PQS server analysis which states that the "oligomer state appears to be correct" (4,168.7 Å2 of surface area is buried via the formation of the dimer in the template structure). Superposing the gIAP model and the crystal structure of PLAP showed complete preservation of the backbone structure. Figure 3A shows the modeled gIAP structure with active site metal atoms displayed. Equally shown is Q317 in the vicinity of the active site. Figure 3B shows the microenvironment of Q317 in larger detail and superimposed onto the detailed microenvironment in the PLAP structure, harboring H317. In PLAP, H317 is positioned so as to be a direct ligand to an active site water molecule and an indirect ligand to the Mg2+ ion and the noncovalently bound phosphate group (18). As can be observed in Fig. 3B, the H317Q substitution brings residue 317 closer to the Zn1 and away from the Mg2+ ion and this movement opens space to accommodate the D273E substitution.
Kinetic characterization of the gIAP isozyme.
In view of potentially important structural differences between gIAP and dIAP, we characterized the enzymatic properties of this novel murine AP isozyme in more detail. To that effect we introduced a FLAG epitope at the COOH terminus of the Akp3, Akp5, and Akp6 cDNAs (at the location predicted to harbor the GPI anchor based on its homology to PLAP) and we expressed each plasmid and recovered and purified the recombinant EAP, gIAP, and dIAP isozymes. The flagged enzymes were bound to anti-FLAG antibody-coated microtiter plates and the bound activity was measured kinetically to determine kcat and Km at pH 9.8 by using pNPP as a substrate. The data shown in Table 2 demonstrate that gIAP has kinetic characteristics quite distinct from those of dIAP and EAP. Thus gIAP displays a 5.7-fold reduction in kcat and a 30% drop in Km. These changes lead to a 4-fold reduction in catalytic efficiency (kcat/Km) of gIAP compared with dIAP. The catalytic efficiency of gIAP is comparable to that of EAP, although EAP displays a 40-fold lower kcat. However, the 8-fold drop in Km leads to a kcat/Km ratio equivalent in bothgIAP and EAP.
Next we investigated whether differences in catalytic efficiencies (Table 2) could be associated with amino acid substitutions of critical active site residues. Since all amino acid residues involved in active site metal coordination were identical, with only residue 317 in the active site vicinity differing between gIAP, dIAP, and EAP (Table 1), this residue was mutagenized. In PLAP, H317 is indirectly involved in Mg2+ ion stabilization through coordination with a water molecule (18), and mutations at this position have been shown to affect kcat and to modulate the enzyme activity. Mutagenizing R317A, R317H, and R317Q in dIAP leads to different effects: whereas the former two substitutions enhance kcat more than threefold, the R317Q substitution reduced the turnover rate to that equivalent of the gIAP isozyme (Fig. 4). The catalytic upregulation induced by the R317A and R317H mutations indicates that it is not the introduction of a His residue at this position that is responsible for the enhanced catalysis, but rather the loss of the Arg and its coordination. The poor EAP enzymatic efficiency therefore depends on residues other than H317, conserved between PLAP and mouse EAP.
Expression of gIAP in the gut.
Using the amino acid sequence information (Table 1) and the three-dimensional model (Fig. 3), we could identify three putative regions useful for the generation of anti-peptide antibodies, specific to gIAP. Because of its favorable location in the easily accessible shoulder region of the gIAP modeled structure (Fig. 3A), we chose as epitope the peptide C208FPKGTPDPEYPSDSNQSGTRLDD231Q of the mature gIAP polypeptide to generate an antiserum. This peptide, with a Cys residue added at the NH2 terminus to facilitate conjugation was used to produce rabbit polyclonal anti-peptide antisera to gIAP. The antiserum reacted strongly with gIAP but also cross-reacted with dIAP, but not with EAP. Immunohistochemical staining of the small intestine of Akp3−/− (devoid of dIAP) mice reveals strong expression of gIAP in the intestinal mucosa and also of what appear to be neuroendocrine cells scattered at low frequency throughout the length of the intestine (Fig. 5). The antiserum was also useful in Western blot analyses to confirm the presence of gIAP in WT and Akp3−/− intestines, extending from the duodenum to the ileum of WT but especially Akp3−/− intestines. In addition, whereas gIAP is expressed along the intestinal tract, this new isozyme is upregulated in the jejunum and ileum of Akp3−/− mice (Fig. 6).
Northern blot analysis (Fig. 7), using Akp3-, Akp5-, and Akp6-specific cDNA probes, detected bands in the range of 2.5–2.3 kb for each of these three mRNAs. The intestines for this analysis were collected and cut into four segments. In agreement with the Western blots, Akp3 expression was only found in the duodenum, whereas, in addition to its presence in duodenum, Akp6 was expressed throughout the gut, and it was particularly upregulated in Akp3−/− mice (Fig. 7). We then examined the expression of Akp3, Akp5, and Akp6 in WT as well as Akp3−/− mice fed a high-fat diet. For the gavage experiment, mice were fasted 16 h and 0.5 ml of corn oil was administered via a feeding needle into the stomach. Intestine samples were collected 5 h later. For high-fat feeding, mice were housed with 11% high-fat diet for 8 wk before analysis of their intestines. Northern blot analysis of the four intestinal segments show that Akp6 is indeed expressed along the entire length of the intestinal tract, after fat feeding, both in WT and Akp3−/− mice, but that its expression is downregulated in the duodenum and upregulated in the ileum of mice when fed a high-fat diet either by gavage or by long-term feeding.
Given the differential expression of Akp3, Akp5, and Akp6 in the adult intestine, it became of interest to probe for the putative differential regulation of these genes at earlier developmental stages, i.e., neonatal and postnatal stages bracketing the time of weaning, since the nutritional status of mice changes dramatically at weaning time. As shown on Fig. 8, Akp3 is not expressed in the early postnatal stages. Akp3 expression only becomes visible at day 15 and full expression is reached after day 17. In contrast, both Akp5 and Akp6 are expressed from birth, but their expression also increases after weaning.
Posttranslational modulation of the catalytic properties of gIAP.
The Western blot data shown both in Figs. 4 and 6 clearly indicated that the recombinant gIAP used as a control for the antibody detection had a lower molecular mass than the native gut-derived gIAP. This is likely the result of underglycosylation at one or more of the four possible sites available for N-linked glycosylation, present in dIAP, gIAP, and EAP, by the cells used to express the recombinant enzymes. Although a single gIAP species is present in the adult intestine, we wanted to confirm that such would also be the case in the neonatal and postnatal gut. To our surprise, intestinal samples from 2-day-old and 10-day-old mice showed a larger band (∼70–75 kDa) and a smaller band (∼55 kDa), which corresponds to the predicted molecular mass of unmodified GPI-anchored gIAP polypeptide (54,526 Da) (Fig. 9). The larger band observed in intestinal segment 4 appears to be the same size as gIAP detected in adult gut. To examine the catalytic properties of these modified gIAP, intestinal segments from 2-day-old WT mice were homogenized in Tris buffer (pH 8.9) containing 0.1% Triton X-100 and extracted with n-butyl alcohol. Extracts (1.5 mg/ml protein concentration) were incubated on 96-well plates coated with the anti-gIAP antibody. Enzymatic activity of the specifically bound gIAP protein was measured with serial concentrations of substrate (20, 10, 5, 2.5, 1.25, and 0 mM pNPP). Intestinal segments 2 and 3 (corresponding to jejunum) showed lower Km values (0.67 ± 0.18 mM and 0.77 ± 0.20 mM, respectively) than gIAP from segment 1 (duodenum; 0.86 ± 0.13 mM) or segment 4 (ileum; 1.00 ± 0.44 mM). Enzyme activity was also lower in segments 2 and 3 (Fig. 9). To assess whether the change in molecular mass was associated with N-linked glycosylation particularly by polylactosamines (12), butanol extracts of intestinal segment 1 from 2-day-old mice were bound onto anti-gIAP-coated 96-well plates. After washing, the wells were treated with 0.003 units of endo-β-galactosidase overnight. Endo-β-galactosidase specifically cleaves β-galactosidic linkage in polylactosamines (12). We found that this enzymatic treatment reduced the activity of gIAP to levels comparable to those present in segments 2 and 3, suggesting that it is a change in polylactosamines that is associated with modulation of catalytic properties of gIAP. A similar change was observed for EAP, indicating that segments 2 and 3 of the neonatal gut are unable to fully glycosylate these glycoproteins in these intestinal segments before weaning, consistent with the developmental expression of galactosyl-transferases in the postnatal gut (27).
The finding that Akp3−/− mouse intestines still express alkaline phosphatase activity with the characteristics of IAP has constituted the basis for our search for the existence of an unknown IAP isozyme in the mouse. The present work confirms the existence in the mouse of an IAP gene, which we denominated Akp6, and which shows homologies but also clear dissimilarities to the previously reported Akp3 gene. As for mouse Akp3, Akp5, and the Akp-ps1 pseudogene, Akp6 is closely linked at the 1C5 site in chromosome 1. Its size equals ∼3.5 kb and it contains 11 exons and 10 introns.
In the presence of normal biliary flow, fat is digested and absorbed primarily in the proximal small intestine (duodenum and jejunum) with minor amounts absorbed in the distal ileum (35). The ileum retains a good capacity for fat absorption but under normal circumstances does not receive much dietary lipid, because the jejunum is so efficient in fat absorption. We found by Western and Northern blot analysis that Akp3 is exclusively expressed in the mouse duodenum, compatible with a partial role for dIAP in fat absorption. Similarly, when analyzed via Western blotting, we found that the novel Akp6 gene is expressed from the duodenum to the ileum of WT and Akp3−/− mice, fed a normal chow. Owing to its lower molecular size, the expression in various parts of the gut could be monitored accurately via Western blotting, an analysis revealing that gIAP was similarly present in the duodenum of WT and Akp3−/− mice, but that the absence of Akp3 triggered upregulation of areas more distal than the duodenum, down to the ileum. Northern blot analysis of various segments of the gut similarly demonstrated that Akp6 was expressed in the duodenum of WT and Akp3−/− mice, but that distally gIAP was expressed at higher levels in the Akp3−/− mice. Our study also revealed that Akp6 is expressed in the entire postnatal small intestine and can be detected as early as embryonic day 18.5, whereas the duodenum-restricted expression of Akp3 starts only prior to weaning.
Our finding that high-fat feeding upregulates the expression of Akp6 even further in Akp3−/− mice, over the entire gut, and that in WT mice, corn oil as well as high-fat feeding induce a strong upregulation of Akp6 expression in all parts of the gut, raises the question of the functional role for both gIAP and dIAP. Akp3−/− mice fed a normal diet show normal growth curves, despite absent dIAP and upregulated gIAP. However, under stressed feeding conditions, Akp3−/− mice show a rapid weight gain, compared with that in WT mice (26). It would be tempting to associate the weight gain in Akp3−/− mice with a role for the upregulated Akp6 in either accelerated fat transport or increased capacity for fat absorption, or both. Recent studies (25) showed that high-fat diet feeding in Akp3−/− mice resulted in liver steatosis but that the fat intake did not differ in these mice. Under conditions of high-fat feeding (15% fat) rodents develop steatorrhea (28). It seems likely that the Akp3−/− animals were able to absorb triacylglycerol fat more efficiently than WT, leading to increased weight and liver fat, whereas dietary intake did not change. Hence, these results confirm an association of dIAP with proximal fat absorption but implicate gIAP with enhanced fat absorption in the distal intestine. The mechanism by which dIAP regulates production of gIAP is not clear. The data imply a modulatory role of dIAP in controlling fat transport or fat absorption, absent in the Akp3−/− mice.
Analysis of the kinetic properties of gIAP and EAP vs. those of dIAP shows that they are very inefficient enzymes, at least when assayed with artificial substrates. In fact, this conclusion is to some extent valid for all mouse and human intestinal-like AP isozymes compared with those in the calf intestine that have the highest turnover number of any AP known to date (∼6,000 s−1) (22). It is tempting to speculate that these distinct kinetic properties could be related to the different substrate specificity, perhaps related to the different dietary habits of cattle vs. mice and humans, i.e., a purely vegetarian diet vs. a fat-containing diet. Alternatively, these data may also indicate that the catalytic activity of the mouse and human enzymes do not necessarily reflect their biological role(s). The highest degree of structural dissimilarity was found in the crown domain of gIAP and dIAP, an area known to vary considerably from one AP isozyme to another. These large differences both kinetically and structurally therefore suggest that the functions of gIAP and dIAP are different, an interpretation further supported by their differential expression regulation in WT and Akp3−/− mice. Other novel observation made in this study are that two molecular forms of EAP and gIAP, which appear to differ in the N-glycosylation state, are found in the neonatal intestine in a segment-restricted manner and that the degree of glycosylation has an effect on the catalytic properties of these isozymes. dIAP is not affected because it is not expressed at these early neonatal stages and also not expressed in those regions of the gut that have differential galactosyltransferase activity (27). Thus, if EAP and gIAP have a catalytic role in the gut, posttranslational modification plays a modulatory role on their function.
Except for Q317, all active site residues were preserved in gIAP compared with dIAP. This residue was shown before to modulate AP catalysis (18). We presently have found that the single mutation in dIAP of R317G sufficed to eradicate most of the enzyme activity, a finding supported by active site modeling, which clearly showed that substitution of H317 in the crystal model of the PLAP active site for Q317 resulted in a microenvironment in which the new Q317 points toward the active site Zn1 metal. It is not hard to imagine that such an exchange interferes with catalysis, thus generating an almost inactive IAP mutant.
In view of the similarities between gIAP and rat IAP I on the one hand and dIAP and rat IAP II on the other, it is tempting to speculate that the function of gIAP and that of rat IAP I are similar. The mechanism whereby an enzyme anchored in the apical membrane of the enterocyte could enter the lymph and serum is not obvious. IAP is anchored to the external surface of the apical brush border via a glycosyl-phosphatidylinositol anchor, and the absence of a transmembrane sequence made especially puzzling the appearance of IAP in the serum. By using tannic acid as such a preservative, membranes were found on the surface of the enterocyte that were enriched in IAP (6). These membranes appeared to have morphological characteristics similar to those of pulmonary surfactant, namely occurring in swirls but without cross-linking features. Indeed, membranes isolated from the rat small intestine were found to contain surfactant proteins B and D, whereas those membranes from rat and human small intestine also contained surfactant protein A (8). In the rat, IAP II mRNA increased much more after fat feeding. Transfection of Caco-2 cells with cDNA encoding rat IAP I increased cellular IAP by twofold, but the increase in secreted IAP was much greater, averaging 32-fold (32). Thus increased IAP production appears to drive the production of SLP in Caco-2 cells. The secreted IAP is membrane associated, and these membranes have the properties of SLP (6). The yield of SLPs is highest in the proximal half of the rat intestine (8), similar to the area of maximal lipid absorption, and coinciding with the mucosa that expresses both IAP I and IAP II. Hence, if a similar function would exist in the mouse, gIAP would be primarily involved in the production of surfactant following high-fat feeding, especially in the absence of dIAP, when gIAP is upregulated.
In conclusion, we describe a novel murine intestinal phosphatase, gIAP, homologous to dIAP, but with structural and enzymatic properties that underscore a functional distinction between both isozymes. Furthermore, their differential expression in the gut and regulation during high-fat feeding is suggestive of different biological functions for gIAP and dIAP.
This work was supported by Grant DE12889 from the National Institute of Dental Research.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Copyright © 2007 the American Physiological Society


text: I am planning a trip to Las Vegas in January/09. I enjoy solo travel and would like to experience a 6-15 course tasting menu. Since I travel light (casual pants and running shoes) I do not bring dressy clothes. Does anyone have a suggestion of where I can get a reservation for a tasting menu for one person that does not require dressy attire?


text:
FAYETTEVILLE — Over the last three years, Maumelle has tried to go deeper into the Class 4A soccer tournament than the previous year. This season, the Lady Hornets advanced the farthest it has ever been by reaching the state finals. However, they suffered a 4-1 setback Saturday to Valley View at Razorback Field in Fayetteville.
"Each year in state, we've tried to get a little farther," Maumelle Coach Jessica Beavers said. "In three years, we made the state championship. Our senior leadership is the reason, and they always came ready to practice."
The Lady Hornets (11-8) reached the state championship contest with several one-point victories. They defeated Arkadelphia (1-0), Gentry (2-1) and St. Joseph (1-0, penalty kick 4-2). They even took the first lead Saturday at 1-0 on a goal by Lyndsey Palanca at the 8:40 mark in the first half. Valley View knotted the score at 1-all right before the half and added three more goals in the second period.
"I think the heat got to them," Beavers said. "Last week, we played in the 50's and this week the 90's, but I told them Valley View was experiencing the same thing. We scored first but couldn't keep the composure like we usually did."
Early in the year, Maumelle experienced injuries it had to overcome. Finally around conference tournament time, the Lady Hornets were healthy. In the meantime, they played tough non-conference competition like Cabot and North Little Rock to prepare them for a run in state.
"We struggled a little bit early to mid-season," Beavers said. "We switched some formats around state tournament time and made it harder for teams to prepare for us."
With five seniors, the Lady Hornets return most everyone. At times, six freshmen also saw action. After Saturday's contest, the five seniors hurried back to Central Arkansas to make their graduation as it was that night.
"Everyone chose state, and their heart was in the game and in the right spot," Beavers said. "We played tough and fought hard."


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This is a set of two replacement rear swing shafts for the Kyosho Lazer ZX-5 4WD off-road buggy. These are the main dog bone drive shafts that transfer power from the rear differential to the rear wheels.
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text: Nicklaus Children's Brain Institute is home to the largest number of pediatric neuroscience subspecialists in the nation. Many of these experts have achieved international renown. The institute serves as a beacon to families confronting the uncertainty of a child with a brain disorder, offering the latest treatments and assessment techniques available at few other centers in the nation. In fact, many of the institute's most notable successes are in children who could not be helped elsewhere. Mathew was one of them.


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|QUOTE (lordjubblydave @ Aug 9 2008, 23:17)
|i think i might be in as there has been no word from joe if i am my plan is to run around and get killed lots
Just don't do that in the TDM
Damn it,he's a top TDM player...sh*t
Jump to content
Posted 09 August 2008 - 09:48 PM
|QUOTE (lordjubblydave @ Aug 9 2008, 23:17)
|i think i might be in as there has been no word from joe if i am my plan is to run around and get killed lots
Posted 09 August 2008 - 09:58 PM
Posted 09 August 2008 - 10:10 PM
Posted 09 August 2008 - 11:13 PM
Posted 09 August 2008 - 11:17 PM
Posted 09 August 2008 - 11:20 PM
|QUOTE (EL84man @ Aug 10 2008, 01:17)
|At least I wasn't negative in the TDM... That's all I can say. We gave it a really good go though.
Posted 09 August 2008 - 11:21 PM
Posted 09 August 2008 - 11:24 PM
Posted 09 August 2008 - 11:25 PM
|QUOTE (joec1987 @ Aug 10 2008, 01:21)
| Wtf!
Anywayz im sorry bout missing the first game, like i said my internet was down since 3 so i couldn't get on here or psn to tell u guys sorry
And in that TDM im sure they were cheating. this happened a few times in the game right, i went to kill 1 of their team and after i killed them its said ( Died) not LFC-JC9 killed .....! WTF? i also saw the likes of global killing some 1 and then after he killed 1 its said DIED not global killed .....! WTF is going on? sorry if im ranting like but its was either they were cheating or it was my internet. but i can't see this bin the problem to do with the connection i got, its was them im sure of it.
again im sorry for bin late and not getting in touch with you, i just couldn't get the internet going, i rang my mates house and he said his auntie's internet wasn't working (she lives near me) and then my lil bro rang his friend and he also didn't have any internet!! i dunno wtf was going on. and then wen it finally came on at 11pm i was playing tony hawk with my mate, then i had to cancel that game and get str8 to the CnC, 10mins later my mate left lol! ohwell, wot could i do?
and im sorry i was sh*t, i haven't bin on gta all day. had no practice at all. i nearly got away with you riko on the boat but they got me! same with tjb on the bridge, it was close. we nearly won that, they was right up our asses tho. im sorry guys
And then i just got invited to a game with them again with jubbly and i got D.C. im sooo pissed off at the min.
Sorry Guys!
Posted 09 August 2008 - 11:25 PM
Posted 09 August 2008 - 11:28 PM
|QUOTE (Globalklaus @ Aug 10 2008, 00:25)
| Yep, they used the mouse thingy. I asked them.
Man. In that TDM I tried to keep my casuality low. Managed pretty good with that. Just 13 deaths against that wild bunch of superaimers. But where were the fookin choppers? I was there like three times, just waiting for them to spawn. Wasnt until the final 5 minutes that I finally got a chopper and could go to my sniper roof and start getting some kills (because at close range I knew it would be a buh-bye experience for me).
Had 8 kills in the end. Of which 6 were in the last 3 minutes. Go figure &%¤#&%¤" helicopters.
Posted 09 August 2008 - 11:31 PM
|QUOTE (docrikowski @ Aug 9 2008, 23:28)
Anyway the chopper sucks in GTA IV. I got one in that TDM, the bullets just do not hit anyone, no matter what u do.
@ LFC: don't worry about the internet, u did a great job and I was happy u got in TDM.
Too bad for CnC... CnC=pointless MP mode
Posted 09 August 2008 - 11:31 PM
Posted 09 August 2008 - 11:33 PM
|QUOTE (docrikowski @ Aug 10 2008, 01:24)
| Guys, first of all sorry if I left earlier the TDM.... I did it because, first: we lhad already lost the CnC and TW anyway, therefore we lost the match; second: I don't enjoy being a sitting duck against a bunch of kids that use lag switches and fragfx mouses, it's plain stupid; third: we were 7 against 6 of them.
Yes, they use lag switches: I blew up one of them in TW and he was on the floor, I was ready to blow him up and finish him but next thing I see he's on my side and kills me. He just vanished in front of my eyes. We should officially complain to GB about this.
TW: I don't know why we didn't stick to the strategy. 4 turfs, 6 players.
Two stay in defense, two go for a turf, two go for another turf. Gain and defend.
Instead: me alone in the home turf and 5 of you go to one turf. Then we gain the second turf and everybody leaves it ( I guess just one stayed and defended it against 3 of them). WTF?
CnC: I'll never believe that the best strategy is to split. That sounds like a stupid tactic to me.
As cops we had no idea where to go (Anti crashed again ), as crooks why the hell did we split up (or swam)???? I was driving and I went straight to the boat and guess what, I got there with basically no cops around and left. If we all did this simple thing we would easily win.
Last thoughts: NEVER EVER AGAIN PLAY AGAINST THESE KIND OF CHEATERS. Let's blacklist them.
@ lord: if u want, report the loss... I'm too upset to do it. I would send a ticket if I were in you...
@ Blaz: if u have the TDM pics, post them. I wanna know the score just because I saw that LFC was ruling and he deserves his score to be counted in our gang stats
Nothing else beside that if we keep on accepting matches against a bunch of cheaters we are STUPID.
Wasn't it clear last time u played this guys that they use lag switches and fragfx? (I wasn't there)
Posted 09 August 2008 - 11:36 PM
Posted 09 August 2008 - 11:39 PM Edited by joec1987, 09 August 2008 - 11:43 PM.
Posted 09 August 2008 - 11:40 PM
|QUOTE (lordjubblydave @ Aug 10 2008, 01:36)
| did you know if i didnt get killed 38 times we would have won the tdm i kept spawning in the open right in front of them
they will have reported scores by now im not gonna dispute i wasn't in the whole match
Posted 09 August 2008 - 11:41 PM
Posted 09 August 2008 - 11:42 PM
Posted 09 August 2008 - 11:43 PM
Posted 09 August 2008 - 11:44 PM
|QUOTE (explicitmo @ Aug 10 2008, 01:41)
| After we both got a truely beat down against RAW now... I think the NDA wasn't a bad thought for the US-ladder at all!!
We were much better united... but I think it was 8 vs. 8 back then anyway.
They just don't get our ammount of teriffic palyers.. but unfortunately the get a lot of people with FragFx.
Posted 09 August 2008 - 11:44 PM Edited by explicitmo, 09 August 2008 - 11:47 PM.
|QUOTE (Globalklaus @ Aug 10 2008, 01:42)
|Well. On the upside. Now I'm really looking forward to tomorrows DTW. No cheaters. No uberness. Just tons of fun and excitement.
Posted 09 August 2008 - 11:46 PM
Posted 09 August 2008 - 11:47 PM
|QUOTE (EL84man @ Aug 9 2008, 23:46)
| I hate FagFX and gaybattles, they go hand in hand.
See what I did there
Posted 09 August 2008 - 11:50 PM
|I hate FagFX and gaybattles, they go hand in hand.
See what I did there
Posted 09 August 2008 - 11:54 PM
|QUOTE (explicitmo @ Aug 9 2008, 23:44)
Wait till I get my FragFX controller! MUHAHAHAHA
J/K, I would never biy that thing caus it's loosing the GTA feeling for me. I even don't like it too much for PC caus I'm so with the PS-analog controllers.
EDIT: Yeah, Blaz. I get ur point... but I would love to own those cats with a crew of analog users. That would be something to be proud of. It's a "mission impossible", but I guess I'm STILL stupid enough to try it.
Hopeless without mics though...
Posted 09 August 2008 - 11:55 PM
Posted 09 August 2008 - 11:56 PM
Posted 10 August 2008 - 12:01 AM
|QUOTE (joec1987 @ Aug 10 2008, 01:56)
|i owned that Souljah (if thats what he's called) loads of times even if he was cheating, i got the better of him by miles he only killed me twice, and thats when he had 3 other ppl behind him shooting at me all at the same time (must ov told his teamates to back him up ). like i said their all sh*t!!!!!!!!!! without those mouses and their tricks up their sleeves!!!!!
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Eishy Hayata is a young Japanese American who travels to Columbia in search of adventure in the seventies, and decides that he wants to be an emerald cowboy, or esmeraldero. Esmeralderos are emerald brokers, sometimes mine owners, speculators and salesmen. Emerald territory butts up against rebel territory, and the trade is very dangerous, full of cut throats and cheats, ready to kill a naïve esmeraldero at the drop of a hat. Hayata is helped along at first by the lovely Susana, played by the actual daughter of Hayata, Patricia. Hayata soon leaves Susana behind, and starts a partnership with an American and a couple of Columbians.
We watch as Hayata learns the ropes, and scrambles his way to the top from poverty, using his native cunning, courage and sheer force of personality. In the first half of the film, Hayata is played by Luis Velasco who, though he only looks Japanese in the vaguest way, is quite a talented actor. This half of the film deals mostly with the hardscrabble life of the esmeralderos as they struggle to become successful, fall in love and out of love and deal with the violence and uncertainty of life in Columbia. This section ends with the death of two of Hayata's old friends and partners, as the result of an argument over a cock fight.
In the second half of the film, Hayata plays himself, which is a bit jarring as he looks nothing at all like Velasco. This half of the film portrays Hayata's life as a big time emerald broker, perhaps the largest in the world, and his struggles with nativist Columbian unions, trumped up charges from the government and attacks on his mines from rebels. The film is definitely less compelling in the second half, mostly because the self indulgent and vanity aspects are much more pronounced with Hayata playing himself. He is always leading the charge against the invading rebels, bravely refusing to engage in the illegal drug trade, standing front and center when confronting the violent union mob. Who's to say that any of these things are untrue, but by this account Hayata seems to have spent his entire life unswerving from his ideals of honor and integrity. When one considers that Hayata wrote, co-directed and starred in the film a few niggling suspicions cannot help but arise.
There are other problems with the film other than the hagiographic elements. Clocking in at around ninety minutes, there is too much story crammed into too little time. Hayata is certainly a fascinating person, and the story (even if it does seem to ignore anything negative about its subject) is compelling. The film feels more like a series of vignettes than a full biography, however. Characters drift in and out of the film, often with little effort or time spent to flesh them out. Esmeraldero could have easily been a two plus hour film, and the absence of that missing material is sorely felt. This is too bad, as the film has a gritty feel and generally good performances. It was filmed on location in Columbia, often in the actual places that the events portrayed took place.
There are a few problems with the DVD itself. The menu is in Spanish, which presents a few minor difficulties for us mono-linguists, but nothing terribly difficult. There are what appear to be two language track options listed, for Spanish and English. When watching the film, however, there appears to be no difference whatsoever between the two. The film includes spoken English, Spanish and some Japanese. Two subtitle selections are available, one for English and one that does nothing. These are not accessible from the DVD menu, but from the remote. They are only listed as "subtitle - other" on the on-screen display, so it is unclear what the non-working subtitle selection was intended to be.
These problems are quickly overcome, but are momentarily confusing. They detract little from enjoyment of the film. The flaws in the film itself detract enough from its good points to make it merely a good rental opportunity.
The DVD
The video is presented in 2.35:1 widescreen. The quality is mediocre, but generally does the job. There are occasional dust specs or scratches visible on the image, and at times murkiness and shadows overwhelm the action. Colors are pale, but during daylight scenes the action is visible.
The sound is presented in Dolby 2 channel, and also does the job but is nothing special. Dialogue is audible without trouble. The English subtitles are easy to read, but there are a few rare problems with translation, such as rebels yelling "retrieve" instead of "retreat" when running away from a battle with Hayata's miners. Spanish and English language tracks are available on the DVD menu, but do not appear to be any different when watching the film.
There are quite a number of extras, some quite interesting, included on the disc. They are:
Comerciales De Television (2:23)
Four television spots for the film, which are relatively uninteresting.
US Previo (2:51)
The US preview for the film, billing it as an "action documentary". Fairly standard.
Clip De Promocion Americana (1:08)
Another preview highlighting the "real" aspects of the film
Prensa Electronica (3:35)
A short view of behind the scenes footage, discussing the fact that real people and locations were used, and detailing some of the hazards of filming in Columbia.
Diario En Video (30:18)
The video diary is the most substantial, and interesting, extra included on the disc. Narrated by co-director Andrew Molina, it shows a substantial amount of behind the scenes footage. Molina discusses difficulties of the shooting, overcoming rockslides, threats of violence from esmeralderos, dealing with rebel checkpoints, car accidents and riots. The American crew members, including Molina himself, apparently took on significant risk to film in the country, as Americans are frequent targets of kidnapping. All of the guns featured in the film were apparently real, and used for security when not filming. The producers used a lot of non-actors, including many people portraying themselves. In one scene, of a large fight between union members and Hayata's employees, most of the people playing Hayata's men were present at the actual event. The video diary goes a long way to explaining how the producers achieved the realistic, gritty feel of the film. This extra is interesting in its own right.
Escenas Eliminadas (6:06)
A few deleted scenes, mostly in Spanish with no subtitles. Some additional haggling over emeralds, of which there was plenty in the film, an extended scene of Hayata meeting his wife for the first time, and chumming around with his friends. Nothing special here.
Inspecciones Previoes
Previews for other films from Indican: Pariah, Moonlight, El Inmigrante, Screen Door Jesus, Dead in the Water and Little Erin Merryweather.
Final Thoughts:
Esmeraldero is an occasionally compelling, and more occasionally self serving, portrait of a fascinating man. It has good performances and an interesting story, but skips over too much too quickly. One can't help but feel that Hayata would have been better served allowing others to write, direct and star in a film that was more honest and unbiased about his admittedly compelling life. As it is, Esmeraldero strikes the viewer as an indulgent vanity project that is more interested in flattering its subject than accurately portraying him. Rent this one.


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He had a daughter Cordelia A. FULLER who was born 2 Jul 1849 and died 27 Apr 1918. She is buried at the Middletown Lutheran Church in Frederick County with her husband. She married James Valentine BAER, (24 Jan 1846-20 May 1936) James Valentine Baer was the s/o Philip BAER (1807-1871) and Serena JOHNSON (1807-1895).
Cordelia and James had : Eva Earl (1875-1932) md. John Sylvester HOOPER (1878-1945) , Lillie P. (1876-1962) md George W. CULLER (1876-1951), and James Howard (1883-1930) md. Clara Stella KEFAUVER (188-1957)
Does anyone have any information on the ancestors of Elijah FULLER and where he came from?
I finally found an obit in the 1902 Cumberland Times excerpts for a James/John (not sure of first name it said) Riley, one of the oldest trainmen of the area. He died in February 1902 at the age of 70 at the home of a son Harry. He had six grown children, but didn't list their names. He was buried in Rose Hill Cemetery. I called Rose Hill Cemetery and was told that a Jane, his wife's name, was not buried in his plot but that a Mary Riley who died at age 49 in 1915 was buried there as well as her stillborn infant in 1907. Also, there was a William Riley, Lottie Fisher, John P. Fisher, Carl Thomas Stevens buried in 1953 and William Stevens buried in 1956 were located in his plot. Does anyone know of these persons and their connection to James Riley?
Our Ott is James William (b. 2/15/1894 d. 12/22/1968) son of John and Annie Fern OTT. Our FISHER is Cecelia Catherine (b. 2/15/1894 and d. 6/22/1964) daughter of Abraham Lincoln and Anna Mary FISHER. You might know one of their sons George OTT of Cumberland. The family lived for a time in Cumberland and then moved to Ridgely, WV. Any help is gratefully appreciated.
|How to Post Queries/Surnames/Researcher Data
|All In One Posting Form
For questions about this page contact Carol Askey.
This page modified Tuesday, 11-Sep-2007 13:55:44 MDT


text:
The documents reveal the Britons will be presumed to be enemy combatants by the handpicked US military officers hearing their cases before they start, have limited rights to call witnesses, have no lawyer, and that hearsay evidence can be used against them.
The decision comes despite the US agreeing last year with the attorney general Lord Goldsmith that the Britons would not face military commissions. The detainees' lawyers said some of the tribunal's features were even worse than the military commissions, which human rights groups condemned.
It represents a second snub by the US to Tony Blair over Guantánamo, as the prime minister today prepares to address Labour's conference, with the Iraq war and his closeness to George Bush concerns for delegates.
In June the Guardian revealed the prime minister had directly asked Mr Bush to return the four Britons, a request that was rejected.
The Pentagon documents come from US court hearings where lawyers for the Britons are trying to challenge their detention before civilian courts, a move the Bush administration is resisting.
US military documents outline the rules for the combatant status review tribunals, a new process announced by the Pentagon in July after the Supreme Court ruled Guantánamo Bay was subject to the jurisdiction of US courts.
In a letter Thomas Lee, US deputy assistant attorney gen eral, says the tribunals for Britons taking legal action in the US will be completed by mid-October.
The documents reveal that prisoners have no right to a lawyer, only to a US military representative, who can inform his superiors of what the prisoner tells him.
Furthermore the tribunal will start with the assumption that the US government case against the Britons is correct, the documents reveal: "There is a rebuttable presumption that the government evidence ... is genuine and accurate."
The CSRT rules describe the three presiding members of the panel as "neutral", despite the fact they are all senior serving US military officers.
The documents also reveal that secondhand testimony will be allowed as evidence of guilt: "The tribunal is not bound by the rules of evidence such as would apply in a court of law. Instead the tribunal shall be free to consider any information it deems relevant and helpful ... it may consider hearsay evidence, taking into account the reliability of such evidence in the circumstances."
Gitanjail Gutierrez, the US lawyer for British prisoners Moazzam Begg and Feroz Abbasi, is in London to press the British government over the tribunals. She will meet Lord Goldsmith and Foreign Office officials tomorrow. Ms Gutierrez said: "It's as much of a kangaroo court as the military commissions."
Brent Mickum, the US lawyer for Briton Martin Mubanga, who he will see in Guantánamo on Wednesday, has told his clients not to take part: "Findings from the CSRT would be used against my clients at trial. They are backdoor trials. The personal representative is an agent of the government who can be compelled to testify against you at the tribunal and at any subsequent trial."
Last night a Foreign Office spokeswoman repeated demands for the British detainees to be sent home because they could not get a fair trial at Guantánamo. She could not say whether Britain had made any protest over the tribunals.
Menzies Campbell, the Liberal Democrat foreign affairs spokesman, said: "What is going on here? Not a squeak, not a complaint by the British government so far as we're aware. Great store was set by the agreement the attorney general apparently reached, but it does not seem to have any effect in preventing these proceedings."
Captain Becki Brenton, spokesperson at the Pentagon for the CSRT, said: "It is not a backdoor trial. The CSRT is modelled and built on army regulations, it is very fair. It is a non-adversarial administrative process, established to allow each detainee an opportunity to contest their designation as an enemy combatant."
Detainees can refuse to take part, but their case would be heard in their absence. Their lawyers say the tribunals were set up by the US military to avoid the cases being heard by civilian courts.
If found to be enemy combatants, the Britons could continue to be held without charge or trial.
Out of 53 cases decided, only one person, a Pakistani, has been released.
Five Britons were released from Guantánamo in March, alleging they had been beaten and humiliated by their US captors.
Four have been held for up to two-and-a-half years without charge or trial. Two of the four still incarcerated, Moazzam Begg and Feroz Abbasi, have been designated by Mr Bush to face military commissions. The other two Britons are Martin Mubanga and Richard Belmar.


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Photo Flash: ARTHUR Flick with Mirren, Brand et al. Premieres in NYC
Arthur, the comedy film and remake of the 1981 film of the same name is directed by Jason Winer and written by Peter Baynham. It opened last night at the Ziegfeld Theater in New York City and Broadway brings you red carpet coverage below!
Arthur stars Russell Brand as Arthur, a drunken playboy who stands to lose a wealthy inheritance when he falls for a woman his family doesn't like. Jennifer Garner, Greta Gerwig, Helen Mirren, and Nick Nolte also star.
Photo Credit: Ken Babolocsay/Globe Photos/ZUMAPress.com
Greta Gerwig
Billy Chrystal
Jennifer Garner, Greta Gerwig, Russell Brand and Helen Mirren
Nigel Barker and Wife
Gerladine James
Zoe Buchman and David Schwimmer
Taylor Hackford and Helen Mirren
Tamsen Fadel


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First off the I still believe the O-line is the only major weak point on the offense and if we can get them in gear, I believe we can make a run for the ship. Anyway i still think if Phil costa is going to be a backup at C we need to go sign a vetern guard. Which I would like to see Brandon Moore come to Dallas for a cheap 2 year deal
LT: Smith
LG: Leary
C. Frederick
RG: Moore
RT: Free
Or if We want Costa in the starting line up
LT: Smith
LG: Leary
C: Costa
RG: Frederick
RT: Free
Livings and Bernadu should be nothing more than backups and if the line plays good I promise the Dallas Cowboys will be in the SUper Bowl or very close.
We have a top 10 RB when healthy
Top 5 WR core
Top 10 QB
Bottom 5 OL
Top 10 OL
Top 5 LB core
Top 10 secondary
IMO we just need the OL to step up and it would have helped if JJ would have drafted a OL in the 2nd instead of a TE


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TAPPER: If you don't - if you don't support the Wyden-Ryan plan, then what is the president proposing a plan to solve the problem of Medicare and funding in the future?
CARNEY: Well, what I mentioned at the top is the president has in fact proposed that as part of his comprehensive deficit reduction and debt control plan, and he looks forward to working with Congress on that. And his whole point during -
TAPPER: That just nibbles around the edges, doesn't it?
CARNEY: No, the whole point that the president made during the negotiations this summer on the debt ceiling, and again when he put forward his proposal for the supercommittee, is that if we approach this in a balanced way - the way that bipartisan commissions recommend that we approach it, the way that the vast majority of Americans want us to approach it - we don't have to privatize Medicare.
We don't have to, you know, severely constrain Medicaid. We don't have to slice or slash programs that fund education or innovation or clean energy, because if we do it in a balanced way, we can reform entitlements in a way that preserves Medicare for seniors and doesn't stick them with a $6,000-a-year cost hike, as the original Ryan plan would.
TAPPER: I just want to make sure I understand. You're saying that what the president proposed is all that needs to be done to contain the growth of health care and Medicare and whatever problems Medicare created? Because that's what the Ryan-Wyden plan is attempting to do.
CARNEY: It was attempting to do, although within it, as I understand it, there's no - even in their own proposal, there's only the hope of cost savings. So what the president has proposed, as I just laid out, would result in $320 billion of savings over the next 10 years, would reform the entitlement programs in a way that would continue to allow them to provide the essential services that, in the case of Medicare, seniors deserve and must have and would not require the kind of radical privatization or ending of Medicare as we know it that the Ryan proposal suggests and that the Wyden-Ryan plan gets you to eventually.
TAPPER: But - so that's a yes, that all that would need to be done to Medicare is what the president -
CARNEY: Look, I'm not saying that five year, 10 years down the road that - I mean, we are always having to, you know, push forward with reforms in different parts of our government. But the plan - the president's proposal, as you know, dealt, you know, in an expansive way - $4 trillion, if you include the trillion in savings through the Budget Control Act - dealt with our need to reduce our deficit and get our debt under control at the same level of magnitude as the Ryan plan.
And it did it without asking seniors to bear the burden of the - of the - of the costs. So this goes back to the need for balance.
You don't have to do something that's radical. As the president has said, our problems are not as great as they are in some countries that we would have to do something that radical if we were only willing to take a balanced approach, which is what the president -
TAPPER: I'm not advocating for the Wyden-Ryan plan. I'm just - I'm just saying - you're actually saying that that's all that needs to be -
CARNEY: Well, no, I know you're not. No, and I'm just - that's the president's plan.
TAPPER: OK. The other question I have is Speaker Boehner today said there's no need to tie the omnibus bill to the payroll tax because he is willing to keep Congress here, to keep the House here, so that as soon as the Senate passes a payroll tax fix, the House will reconvene within 24 hours and act on whatever the Senate passes. Your response?
CARNEY: Well, I'm not going to negotiate on behalf of the Senate or the president here. These are ongoing conversations. What is unacceptable is the idea that we should take a promise of future action on behalf of 160 million Americans when, as you know, once Congress passes a spending bill, they can go home. And it sounds like the speaker would let them go home. He might call them back, or he might not. I mean, again, it's a promise. What we want is -
TAPPER: So you're saying you don't believe his promise?
CARNEY: Well, I'm saying I don't know. What is essential here - the absolute top priority that this president has is that Congress makes sure it does not go home on its vacation until it has taken care of the payroll tax cut extension and unemployment insurance extension because it would be unacceptable, he believes, for Congress to hike taxes on 160 million Americans as it's heading out the door here.
TAPPER: He - OK, he's saying you don't need to tie the two, right? You don't need to do this - and he's trying - he says he's trying to avoid the government shutdown tomorrow night. Congressman Moran has said the deal has been done and the Congress is ready to act.
CARNEY: Well, the deal is not done until some of these issues that Senator Reid and others have mentioned, I've mentioned, need - are - need to be resolved. It is - they are resolvable, no question. There's no need to shut down the government. There is time to get all of it done. And if more time is needed, Congress should do what it's done seven times already in this year and pass a CR - a short-term CR, just to ensure that they get their work done. There's no reason to talk about government shutdown.
TAPPER: I guess I'm just surprised that the Speaker of the House is basically saying "You don't need to do this; I'll keep the Congress here. We'll reconvene as soon as they're ready - "
CARNEY: But we need to make sure -
TAPPER: You said that "we don't believe you." Basically said -
CARNEY: No, look, I'm just saying -
TAPPER - a promise is meaningless.
CARNEY: Jake, I'm just saying that there are negotiations going on. As Ben noted in his questions, there have been some signs that - of a willingness to find some bipartisan compromises here on these big remaining issues.
But the president's priority is - you know, this is not a question where 50 percent is OK, because if you take these two issues - the omnibus and the payroll tax cut - it is not OK just to get one of them. It simply isn't, because it - because that means 160 million Americans are stuck with a bill of congressional inaction. So that's not OK.
-Jake Tapper


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