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Pastor Hale Saw Obama Re-elected A Year Ago in a Prophetic Dream
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[quote:Anonymous Coward 27245028:MV8yMDQzMjI3XzM0MzM4MDAyXzVGNUJBNDQw] Thank you Lisa for posting this. This is so true. I can feel the Spirit of The Lord when I was reading this. Also I need to thank you for opening my eyes to a pastor that you said you did not care for. I was getting deeply entrenched in his teachings. Teaching that I now see is false. Many blessings to you for being God's vessel and leading me out of harms way. [/quote]
Original Message
The Bad and Good News for America
Urgent Warnings to America
Pastor T. D. Hale - 12/28/11
I had a dream last night: In my dream, I saw myself going across America, as if I was floating but with no fear. The land looked like it was bombed, totally destroyed. I saw people standing out around their homes, weeping, holding onto each other; there were a few who laid dead. I heard someone say, "This should never have happened; this should never have happened."
National destruction had hit America: no food, no water, babies crying, grownups, men all crying, holding onto their families, begging God for mercy. As I moved along the way, I saw people running, looking for loved ones missing, totally out of their minds.
I quickly came over a large city; it looked like it was Columbus, Ohio. As I came close, there it was -- the riots we have all heard about. Store windows busted, grabbing what they could get but I could tell they were not too concerned about TVs iPads, etc.; they were grabbing food, water, chips, things to survive on. There were riots and fighting. I even saw one man shot to death.
As I left that place, going at the speed of light I was standing on the back side of the White House. I looked up and there was President Barack Obama on the balcony, holding a shotgun. I heard a loud scream, real loud. I turned my head to see where the scream came from. Flying high in the air was an eagle -- majestic, flying around Washington. I saw Barack point that shotgun toward that eagle and then shoot it dead and it fell to the ground. I looked up at him and he just had a smile on his face, a smirk. These were the words I heard: "I've done it and I won't have to deal with this in my administration." It was dead silent. At that moment, I heard a voice that said, "Tell the people this is My will; this is My hand, both upon the generation of the righteous and upon the cursed. The righteous will find their way and know what to do; the cursed will wonder around with no compass. The cup is full!"
From that point on, I knew that we were coming to a showdown; it was going to be between us and them -- good versus evil. I saw people gathering into some homes that were not destroyed. I saw people having prayer meetings, just praying in the Spirit.
And then I heard in the dream: "To My servants and handmaids, a special anointing will reside on you in these last days. Hold not back thy voice but speak your hearts, for out of them are the issues of life. Pick up the mantle of prayer, cover yourself with it and find you a secret place to cover My servants, who stand for truth in prayer. I see their hearts and the desires to understand and know more. Their eyes have been anointed with a special anointing to see. Others are blinded to My Word. All things will be revealed in their due course. A supernatural wave of my Spirit will come over this generation soon; the FINAL voices are in the land to speak one last time. Think it not strange the happenings around you; they must and will come to pass but I have placed in your hands the food to sustain you in the months ahead. I have spoken and shown all that matter. Tell my servants, saith the Lord, there is coming harsh days ahead!"
As I stood there hearing the voice of God, I saw a table, very old as if it was a table that had many documents signed in times past of importance. I saw a voting ballot lying there.
As I looked, I saw two names on that ballot: Barack Obama and Mitt Romney. It was check-marked beside Obama's name.
I looked at the bottom of the ballot and these words were written: "For this is the will of the Lord." Then I woke up.
50% rule observed, read the rest here [
link to www.ubm1.org
Listen to his 1 hour interview where he goes into full detail about what's coming to america, and it isn't good.
link to www.trunews.com
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I Was Reading This write-up yesterday which talked about exactly what people can look forward to as they age, in terms of their health, that is. (Anyone can easily read the full info here.) Sound judgment tells folks that they are going to likely have an elevated quantity of issues to their well being the older they get. The truth is, there are a selection of sorts of sickness as well as disease that get more regular as soon as individuals cross the mid-century mark and turn 50. Exactly what may well they end up being? Heart disease, for one. A lifetime associated with harmful eating and inactive behavior incorporates a strategy for catching up with everyone. Look for elevated blood pressure, as it's often a pre-cursor to cardiovascular problems.
A variety of different kinds of cancer is actually one other. Men and women will need to get scanned often for skin cancer (in particular those who are fair, blue-eyed, or who may have invested significant amounts of time within the sun's rays), colon cancer, breast cancer (women) plus cancer of prostate (guys). Cancers will be survivable in the event that discovered very early. While not really life-threatening, arthritis is another unwelcomed trouble that gets to be rather common to many individuals over time. Joint inflammation frequently possesses both innate and chosen lifestyle related components. This means that to a point, it may be avoided yet to one more it has to be managed. People tend to try this and that, often a blend of pharmaceutical drugs and nutritional aid with regard to pain alleviation. In the event you check over here, you'll find some useful and established suggestions.
Yet another change that happens simply because folks grow older is the way in which many of them have an extremely dry mouth. In addition to being unpleasant, this has a tendency to result in difficulty with one's teeth and gums, which actually, can lead to more severe health concerns, like having diabetes, heart disease, or even cancer. Even though people may suffer dry mouth as the result of aging, it is actually commonly detailed as a unwanted effect of countless of the drug treatments which are typically approved to older people pertaining to problems that tend to affect older people. Thankfully, it's a affliction that is manageable. In the event that sipping supplemental water does not help, visit your dentist to get a prescription mouthwash that will keep your mouth moistened.

subset: commoncrawl


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Throw Pillow
Evil Bug Gives Flowerby Boriana Giormova
Throw Pillow made from 100% spun polyester poplin fabric, a stylish statement that will liven up any room. Individually cut and sewn by hand, each pillow features a double-sided print and is finished with a concealed zipper for ease of care. Sold with or without faux down pillow insert.
Dark textured illustration with an evil black bug who gives a flower to someone, on dark vintage texture.
See all characters from the Evil Flower Bug family here: http://society6.com/borianagiormova/collection/my-art-evil-flower-bug
This evil bug was initially drawn for my collaboration with Ganech Joe - http://society6.com/BorianaGiormova/Meeting-At-Moonlight-WhE_Print
bug, bugs, dark texture, evil, evil character, evil creature, goth, gothic, halloween, texture, evil bug, character, vintage texture, dark, vintage, evil flower, evil flower bug


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ARTIST LOFT+OFFICE in PRIME WILLIAMSBURG!United StatesNew YorkBrooklyn
About this listing
A clean, well-designed room, with a comfy bed for 2. It's Artistic and a Home at the same time.
PRIME W'burg!
Safe & secure location. Central to both Manhattan & Brooklyn. Easy to get anywhere in the city!
The Space
We are full-time professional visual artists. We've lived in Williamsburg for 10+ years and can help you with all kinds of tips :)
We are officially registered with the state as "Bridge Seal" B&B; we have hosted over 400 guests since 2012, so you can know that we help you in any way!
We have everything you'll need to make yourselves comfortable and enjoy the space! We have for you:
--Fresh and clean towels and sheets.
--A lofted Queen size bed.
--A work desk
--Plenty of storage: A hanging closet and plenty of shelving
--By some architectural design, it feels incredibly separated from the rest of the house, as if you are in your own world.
--Amazingly soft Northeast light!
--Newly installed light & sound blocking window drapery for the light sleepers and people suffering jet-lag.
--Your own Air Conditioning and/or Heater.
--Your bedroom has secure keyed locks.
--A healthy selection of plants & flowers.
Breakfast is available for an additional $5 per guest, per day:
It is a continental breakfast (REALLY good coffee, breads, fruit, cereals & milk, anything you need to make a great start to the day). *Please request this in advance so that I can have things ready for you, upon your arrival.*
**Early check-ins (before 2pm) and late check-outs (after 12 noon) must be arranged ahead of time and approved on a case by case situation. 95% of the time, it's fine.
Who WE are:
I am a full-time visual artist. I am also a 3rd generation ceramic artist. There's hand-made pottery all over the space! In a not-so-distant past, I was also a Sommelier at one of the best and most highly rated "farm-to-table" restaurants in Manhattan. True Hospitality and knowing how to make guests feel welcome are things I know so very well :)
My girlfriend, Kara, is a professional artist as well. Both of us have separate studio practices, so we can keep the mess out...! She is also a curator, art critic and an editor, so the conversations are always exciting!
And we have a new puppy! :) He's a Samoyed/Eskimo mix, so he is very hypoallergenic. I have Asthma, and Kara has bad allergies, and he does not affect us at all. He lives in our bedroom or his crate. He has NO access to the guest room! We do encourage guests to interact and play with him, just please do NOT feed him human food!
Whether you are traveling partner, a couple on vacation, someone extending a business trip, or someone looking to host your family while they are visiting you-- we get you :)
During your stay, look forward to pleasant chat around the kitchen table. We can always help you find the best shops, the best food, the best art exhibitions, the best walks, the best cocktails, the quickest subway route, and best insider deals around!
We care about with whom we share our home . A good fit makes your stay amazing and our experience as hosts equally rewarding! We always give preference to civilized, pleasant and respectful people. We will not accept inquiries from partiers looking to pound beers at the kitchen table until 4am... We do our fair share of entertaining, but we keep it classy! ;)
All together, everyone here is pretty awesome! Every person who's ever stayed here has left with great memories.
All the best and we hope to meet you soon!
Jsun & Kara
Guest Access
--Extra fast Internet.
--We provide fresh and clean towels & linens.
--Soap, shampoo, and conditioner in shower.
--Hair dryer, iron & board in your room.
--Have full access to Kitchen, Living Room, (email hidden)l cleaned and maintained consistently.
Interaction with Guests
We've hosted over 400+ people. Come and go as you please. Enjoy your privacy or conversation with us. You will typically see us in the morning at breakfast (a good time to get tips on the day's planning).
We do our fair share of entertaining, but we keep it classy! ;)
The Neighborhood
Williamsburg is a safe and well-lit neighborhood, now. It is THE center of cultural and artistic Brooklyn! 20 years ago, used to be one of the roughest neighborhoods in all of New York. Now, the most beautiful people live & play here!
You are right around the corner from a lively and thriving local music & nightlife scene, great vintage boutiques and cool Internet/Coffee shops.
Getting Around
Steps away from:
--L/J/M/Z subway
--East River Ferry
--Williamsburg Bridge Pedestrian Walkway
--Citibikes are around the corner
--B62 bus (to Queens & MoMA P.S.1)
Other Things to Note
Neighborhood highlights:
FOOD: (W'burg is a foodie MECCA!)
--Amazing grocery 2 blocks away.
--Diner: By far the BEST burger in NYC! (I do not exaggerate!). Next door.
--Peter Luger: Best steak restaurant in NYC for 30 years. 3 blocks away.
--Pies 'n Thighs: Best brunch for the southern comfort food. Insane doughnuts. 5 minutes walk.
--Maison Premiere: Oysters & Absinthe... enough said! 5 minute walk.
--Cafe Mogador: Best healthy Mediterranean food and a gorgeous garden. 10 minute walk.
--Baby's All Right: Local music is alive and well! See the coolest new bands before your friends do. 3 blocks away.
--Bembe: World famous for dancing to afro-caribean beat. 1 block away.
--The Knitting Factory: World famous stage for starting big careers. 5 minutes walk.
--Sideshow Gallery: Put your finger on the pulse of the NY and Brooklyn art scene, here. 5 minutes walk.
--Journal Gallery: Always great exhibitions of the COOLEST of the cool artists, who are hot right now. 5 Minutes walk.
--Brooklyn Bowl: Live music & bowling under the same roof! 10 minutes walk.
--Brooklyn Brewery: Tasting room is a 10 minute walk there, and a 15 minute walk back :)
--Night Hawk Cinema: Dinner theater! Have your mac 'n cheese, brooklyn beer, and comfy seats while at the movies!
We both do a lot of running, so if you are a runner, we have a lot of great routes for you to explore the city and neighborhoods with.
Kara has been a long-time yogi. If you are into yoga, she has a lot of experience with several studios in the neighborhood and Manhattan.
Be considerate.
Come and go as you like.
Feel free to say "hi" to neighbors.
Flexible Check-ins & outs are OK, just coordinate with us.
Your Host
Other than being an AirBnB host, I am a full-time visual artist. I create large-scale, colorful, abstract paintings. My paintings seldom get installed at the house because of their size. I am also a 3rd generation ceramic artist. Much of the cups you will drink from have been made by me or my family!
My girlfriend, Kara, is a professional artist as well. Both of us have separate studio practices, so we can keep the mess out...! Kara is also a curator, art critic and an editor, so the art can change from time to time :)
We both do a lot of running, so if you are a runner, we have a lot of great routes for you to explore the city and neighborhoods with. Kara has been a long-time yogi. If you are into yoga, she has a lot of experience with several studios in the neighborhood and Manhattan.
We both have a wealth of overall knowledge of the neighborhood and the city!
We have a new puppy. His name is Lono. He's a special Eskimo/Samoyed mix. He has a hair undercoat, so he is very hypoallergenic. I have Asthma, Kara has bad allergies, and he does not affect us at all. He lives in our bedroom or his crate. He has NO access to the guest room. He loves all people, such a mushy, just please do NOT feed him human food. Everyone falls in love with this little guy.


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Breaking the church's silence on mental health
Read about how you and your church can engage in the mental health conversation and help those in your area suffering from mental illness. Click below to read stories spotlighted in the summer edition of TX Baptists, including features on Mental Health and the Church, a minister's view point, a church's model and a first-hand account of a ministry family's struggle with mental illness and their road to recovery.
Read spotlighted stories…
5th Sunday Hunger Offering
Childhood & Family Ministry Summit
Special Friends Retreat
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A campground committed to the Lord: How Go Now students are sharing the Gospel in Spain
by Kalie Lowrie on July 28, 2016 in news
Priscille Murphy and Naraim Enriquez are serving as Go Now missionaries at Alfa & Omega camp in Denia this summer, watching the Lord at work in mighty ways in a region where Christianity is scarce.
Almost 60 years ago, American missionaries established Alfa & Omega camp on the southern coast of Spain, in the town of Denia. At the time of its establishment, Christians in the region were under persecution for their faith and the Iglesia Evangelica Bautista La Trinidad connected with the camp only had a/p>... [continue]
We've Moved
We value your partnership and continued giving! Please send all future payments and gifts – including Cooperative Program giving – to our new finance and accounting address:
Texas Baptists
7557 Rambler Road
Suite 1100
Dallas, TX 75231-2310
All other mail may be sent to our new primary mailing address at Texas Baptists, 7557 Rambler Road, Suite 1200, Dallas, TX 75231-2388.
Ministry Safe
Texas Baptists is partnering with an organization known as Ministry Safe to better address the issue of child sexual abuse in the local Church. We are concerned about the wellbeing of children and greatly desire to see that suffering, especially at the hands of child predators, be prevented. Through this partnership, we are offering training opportunities for your church leaders, as well as other resources to help your church become aware and informed about how to prevent child sexual abuse.
TX Baptists Magazine
In this edition of TX Baptists magazine, we are spotlighting how you and your church can help break the silence on mental health. Also, read stories about the power of partnerships in planting a new church, why singing is integral in worship, why ministers should have an annual mental health checkup and more!
In the Summer edition of txb.life, see how Texas Baptists are on the go. From mission trip preparation, to sharing your faith in a digital space, you will find great insight into missional living at home and around the globe.


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November 22, 2013 - Products
Waytek Inc., a distributor of electrical wiring supplies and electrical connectors, now carries Eaton Rocker Switches to meet OEM manufacturing requirements.
The NGR, SVR, X Series, and Euro SR switch lines from Eaton are currently in stock and ready to ship from Waytek's 100,000 square foot facility.Waytek has added the field-proven New Generation Rocker switch line of fullsized rocker switches from Eaton. Eaton initially developed this line of switches for the heavy-duty truck industry. These quality switches are now found in a variety of vehiclerelated applications.
The Sealed Vehicle Rocker switch from Eaton's electrical business is nowstocked by Waytek and offers an above panel actuator style in addition to the below panel and paddle actuators designed to meet the severe environmental requirements of the construction and agricultural vehicle markets.
The SVR is sealed at the front and back of the switch and meets the rigorous sealing requirements of IP68.
Also stocked by Waytek are the X Series Rockers from Eaton for use in outdoor applications to meet almost every market need. The X Series rockers are available in single- and double-pole with various circuit options, LED illumination and laser etching.
The Euro SR switches are available in a complete range of rugged single- and double-pole, illuminated or non-illuminated circuits.
Tags: Eaton, Waytek
Go directly to this company's home page
We offer e-newsletters that deliver targeted news and information for the entire fleet industry.
The expert, Brian Fletcher from Eaton, will answer your questions
Gates Offers 6 Heavy-Duty Water Pumps
Reyco Granning DM400e is Lighter Than Predecessor
Baldwin Filters Offers Aftermarket Filter Designed for Kenworth Models
ARI Introduces Driver Scorecard for Simple Analysis


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Binding of the catalytic divalent ion to the ternary DNA polymerase β/gapped DNA/dNTP complex is thought to represent the final step in the assembly of the catalytic complex and is consequently a critical determinant of replicative fidelity. We have analyzed the effects of Mg2+ and Zn2+ on the conformational activation process based on NMR measurements of [methyl-13C]methionine DNA polymerase β. Unexpectedly, both divalent metals were able to produce a template base-dependent conformational activation of the polymerase/1-nt gapped DNA complex in the absence of a complementary incoming nucleotide, albeit with different temperature thresholds. This conformational activation is abolished by substituting Glu295 with lysine, thereby interrupting key hydrogen bonds necessary to stabilize the closed conformation. These and other results indicate that metal-binding can promote: translocation of the primer terminus base pair into the active site; expulsion of an unpaired pyrimidine, but not purine, base from the template-binding pocket; and motions of polymerase subdomains that close the active site. We also have performed pyrophosphorolysis studies that are consistent with predictions based on these results. These findings provide new insight into the relationships between conformational activation, enzyme activity and polymerase fidelity.
It is generally accepted that DNA polymerases utilize a 'two-metal–ion' mechanism for nucleotidyl transfer (1). For DNA synthesis, the catalytic 'metal A' is thought to lower the pKa of the 3′-OH of the growing primer terminus, while the nucleotide-binding 'metal B' coordinates the triphosphate moiety, facilitating binding of the incoming dNTP. The crystallographic structure of a pre-catalytic complex of DNA polymerase (pol) β with two Mg2+ ions provides compelling evidence for this mechanism (2,3). Both metals are coordinated by conserved aspartates (Asp190 and Asp192), and the catalytic metal is coordinated by an additional aspartate (Asp256). Since the 'metal A' binding site includes oxygen-ligands contributed by the incoming dNTP and primer terminus 3′-OH, the catalytic metal may be the last ligand to bind. The presence of the catalytic metal results in an altered sugar pucker of the primer terminus that has O3′ in a favorable position to attack the Pα of the incoming dNTP (2). The natural metal cofactor is Mg2+, but other transition metal ions, including Zn2+, have been shown to support catalysis by some polymerases (4), and studies involving other metal ions have proven useful to probe the polymerase catalytic mechanism (5–7).
Although the role of the metals in catalysis has been described, their role in the conformational activation that couples correct base pairing with catalysis is not well understood. We are particularly interested in characterizing the activated state of pol β, since correct nucleotide insertion inherently dominates DNA polymerase fidelity (8). Crystallographic structures of binary DNA–polymerase complexes from several polymerase families indicate that a subdomain repositioning occurs upon binding a correct nucleotide (9). For right-handed DNA polymerases, the fingers subdomain closes to sandwich the nascent base pair between the protein sensor domain residues and the primer-terminal base pair. For pol β, the thumb subdomain plays an equivalent role and is referred to as the N-subdomain (10). Consequently, correct base pairing is signaled through an open to closed N-subdomain transition that results in a series of protein side-chain rearrangements that alter hydrogen-bonding interactions (Figure 1). This signaling cascade connects sensor residues of α-helix N (e.g. Arg283) to the catalytic site >10Å away. These conformational changes facilitate formation of the metal–ion complex necessary to activate the primer 3′-OH and position the Pα of the incoming dNTP. The importance of this conformational activation is illustrated by the observation that mutagenesis of residues which participate in this signaling reduces nucleotide insertion (11,12).
In a crystallographic study of pol β complexes in globally open conformations using various metals, Pelletier et al. (13) found that Mg2+ was unusual. All of the other metals evaluated led to an active conformation of the Asp192 side-chain without subdomain closing. Furthermore, transition metals that form tighter complexes with enzyme and substrate might induce activation without correct nascent base pairing. Thus, substitution of Mn2+ for Mg2+ has permitted crystallographic observation of pre-insertion structures of ternary complexes containing mutagenic intermediates (5,14). In this way, these ions could obviate the signal transduction pathway that connects the catalytic C-subdomain and the nascent base pair N-subdomain of pol β. The present studies were undertaken to characterize the role that the divalent metal ion plays in the conformational activation process.
It is of fundamental importance to identify factors that affect metal binding and the structural, thermodynamic and kinetic consequences. However, the physical constraints imposed by crystallography limit the ability of the enzyme to respond to various stimuli and significantly populate alternative conformers. We have shown that DNA binding and catalytic activation in pol β can be conveniently monitored in solution with the NMR signals of 13C-labeled intrinsic methionine residues (15,16); the sensitivity of these resonances to conformational activation of the enzyme allows us to probe in solution the various factors that influence substrate binding and specificity (i.e. fidelity). Furthermore, conformational activation is also fundamental to the enzyme's reverse reaction, pyrophosphorolysis. This reaction plays an important role in removing blocked primer termini (e.g. 3′-azidothymidine) by HIV-1 reverse transcriptase (17) and pol γ (18), the mitochondrial replicative DNA polymerase. Mitochondrial toxicity represents a significant complication of long-term anti-HIV therapy with antiviral nucleosides (19).
In order to understand the effects of transition metal ions while avoiding the paramagnetic broadening caused by Mn2+, we performed NMR studies to probe the effect of divalent metals (Mg2+ or Zn2+), DNA sequence, and temperature on formation of the closed activated-state of a series of pol β/DNA complexes. This evaluation of the role of metal ions in conformational activation is central to an understanding of their effects on catalysis, fidelity and inhibitor sensitivity.
The [methyl-13C]methionine-labeled pol β was prepared as described previously (15) by growth of the plasmid-containing Escherichia coli on a medium containing [methyl-13C]methionine (CIL, Cambridge, MA). The only modification of the protocol was the use of HiTrap Heparin HP (GE Healthcare) chromatography instead of ssDNA cellulose chromatography. The C267A, C239A and E295K pol β mutant enzymes were produced using the Quikchange kit (Stratagene). NMR samples contained 0.3–0.6mM pol β in a D2O buffer consisting of 100mM potassium phosphate (pH 6.7), 1mM DTT, 0.1mM AEBSF, 5mM NaN3 and 50µM DSS as an internal chemical shift standard. Oligonucleotides (Oligos Etc., Wilsonville, OR) were dissolved in D2O to make a stock solution of ~10mM. Non-hydrolyzable deoxynucleoside triphosphates were obtained from Jena Bioscience. The double-hairpin, 1-nt gap DNA substrates have the following sequences: 5′-PGGCGAAGCCTGGTGCGAAGCACC-3′ or 5′-PGGCGAAGCCGCGTGCGAAGCACG-3′ (underlined nucleotide is in the gap). The double-hairpin, 2-nt gap DNA substrate has the sequence: 5′-PGGCGAAGCCTGGTGCGAAGCAC-3′ (underlined nucleotides are in the gap).
The protein concentration was determined using an extinction coefficient of 20088M−1cm−1 at 280nm, and the DNA concentrations were determined using their 260nm extinction coefficients.
NMR experiments were performed on a Varian UNITY INOVA 600MHz NMR spectrometer, using a 5mm Varian (600MHz) 1H{13C,15N} triple-resonance probe, equipped with actively shielded Z-gradients. The 1H-13C HSQC spectra were acquired using Varian's gChsqc sequence (20). The data were acquired as a 230 (t1) × 512 (t2) complex matrix, with acquisition times of 63.9 (t1) and 64.0ms (t2), 128 scans per increment, and a 1.0s delay between scans. The spectra were processed using NMRPipe version 2.1 (21) and analyzed using NMRView version 5.0.4 (22). All spectra were processed using squared cosine bell apodization functions in all dimensions and forward-backward linear prediction in the indirect 13C-dimension (23).
A 34-mer oligonucleotide DNA substrate containing a 1-nt gap was prepared by annealing three gel-purified oligonucleotides (IDT, Coralville, IA) to create a 1-nt gap at position 16. Each oligonucleotide was resuspended in 10mM Tris–HCl, pH 7.4 and 1mM EDTA, and the concentration was determined from their UV absorbance at 260nm. The annealing reactions were carried out in a PCR thermocycler by incubating a solution of 10µM primer with 12µM each of downstream and template oligonucleotides at 95°C for 5min followed by 30min at 65°C, and then slow cooling (1°C/min) to 10°C. The sequence of the gapped DNA substrate was: primer, 5′-CTGCAGCTGATGCGC-3′; downstream oligonucleotide, 5′-GTACGGATCCCCGGGTAC-3′; and template, 3′-GACGTCGACTACGCGXCATGCCTAGGGGCCCATG-5′ (X is the template residue in the gap). The primer was 5′-labeled with [γ-32P]ATP using Optikinase (USB Corp.) and the free radioactive ATP was removed with either MicroSpin G-25 or Biospin 6 columns. The downstream oligonucleotide was synthesized with a 5′-phosphate.
Steady-state kinetic parameters for dCTP insertion opposite guanine in a 1-nt DNA gap were determined by initial velocity measurements as described previously (10). Enzyme activities were determined using a standard reaction mixture containing 50mM Tris–HCl, pH 7.4 (37°C), 100mM KCl, 5mM MgCl2 and 200nM 1-nt gapped DNA. Enzyme concentrations and reaction time intervals were chosen so that substrate depletion or product inhibition did not influence initial velocity measurements. Reactions were stopped with EDTA and mixed with an equal volume of formamide dye, and the products separated on 16% denaturing polyacrylamide gels. The dried gels were analyzed by phosphorimagery to quantify product formation. Steady-state kinetic parameters for Zn2+-dependent DNA synthesis were determined at room temperature as described above, except that ZnCl2 replaced MgCl2. Additionally, the divalent metal concentrations for the activities tabulated in Supplementary Table S1 were 2.5mM.
Pyrophosphorolysis was quantified by following the removal of the primer terminus upon addition of 150µM PPi under steady-state reaction conditions. Unless noted otherwise, enzyme activities were determined using a standard reaction mixture (30µl) containing 50mM Tris–HCl, pH 7.4 (room temperature or 37°C), 100mM KCl, 5mM MgCl2 and 200nM 1-nt gapped DNA. Assays were initiated by mixing enzyme with DNA/PPi/metal. Enzyme concentrations (2–5nM) and reaction time intervals were chosen so that substrate depletion or product inhibition did not influence product formation. Reactions were stopped with 15µl of 0.5M EDTA and mixed with an equal volume of formamide dye. The substrates and products were separated on 16–18% denaturing (8M urea) polyacrylamide gels and quantified in the dried gels by phosphorimagery.
As shown previously (15,16), the methyl resonances of [methyl-13C]methionine-labeled pol β provide useful probes of DNA binding (Met18, Met236) and conformational activation (Met155, Met158, Met236, Met282). Met236 is the only methionine residue that makes direct contact with the substrate, and its resonance's shift is highly variable. In contrast, the shift and linewidth characteristics of the other methionine methyl resonances are dependent on indirect, conformationally-mediated processes and exhibit characteristic perturbations upon binding gapped DNA and upon formation of an abortive ternary complex (15,16). Figure 2A shows the changes observed in the HSQC spectrum of labeled pol β upon formation of a ternary pol β/gapped DNA/Mg2+/dNTP complex. Addition of Mg-dATP to a dideoxy-terminated 1-nt gapped DNA substrate with a templating thymine perturbs the resonances of Met155 and Met158, located in the catalytic C-subdomain near the active-site metals, and Met282 of the N-subdomain. The resonance shift of Met236, positioned near the primer terminus, is altered due to changes in the position of the DNA and enzyme conformation that result from dNTP binding. The Met282 residue, positioned at the center of α-helix N, is particularly valuable as an indicator of the helix repositioning that occurs upon formation of a complex with a complementary dNTP (16). Addition of Mg2+ to pol β or to pol β•DNA at 25°C produces negligible shift perturbations (data not shown), and crystallographic data indicate that catalytic metal binding is not necessary for dNTP-induced conformational activation (2). In order to obtain further insight into the role of the metal ion in producing an active conformation, we added ZnCl2 to the binary DNA complex. Unexpectedly, this resulted in spectral perturbations that are very similar to those observed for the ternary abortive complex (Figure 2A and B), suggesting that comparable conformational changes have occurred in response to the addition of Zn2+ alone. Thus, it is possible to induce a nucleotide-free activation of pol β that appears to resemble that induced by the presence of a complementary incoming dNTP (15). Kinetic evaluation of pol β gap-filling DNA synthesis indicates that Zn2+ is a good substitute for Mg2+ (Supplementary Table S1).
We note here that, based on NOE and nudge mutation data summarized in the Supplementary Data, assignments of Met158 and Met191 resonances are now reversed relative to our previous studies (Supplementary Figure S1). As stated in our previous paper (15), 'in the absence of specific assignments for the other proton resonances of the protein, definitive assignment information cannot be obtained'. The methyl groups of methionine residues 155, 158 and 191 are all located within several angstroms of each other and, since we have not as yet developed a specific model for relating changes in these resonances to the conformational activation process, these have been interpreted phenomenologically as a set of conformational activation-dependent resonance perturbations. This is not the case for Met18, Met236 and Met282, for which the resonance perturbations have been interpreted on the basis of specific conformational and ligand-binding effects (15,16). Hence, the interchange of the Met158 and Met191 assignments has no effect on any of our earlier conclusions.
To further understand the basis for the Zn2+-induced activation, we investigated the dependence of the activation on the length and chemistry of the gap. A comparison of the spectra obtained with pol β complexes formed with 2′-deoxy- or 2′,3′-dideoxy-terminated primers showed that coordination of a metal ion with the 3′-OH of the primer terminus, observed in the crystallographic ternary complex (2), was not required for Zn2+-dependent activation (Supplementary Figure S2). It also was found that a 2-nt gap substrate did not support Zn2+-induced activation (Supplementary Figure S3).
The dependence of the Zn2+-induced conformational activation on DNA gap size suggested an explanation for the effect. We hypothesized that the activation might involve substrate translocation such that the base pair involving the primer terminus would occupy the active site and the unpaired templating base would become extra-helical, thereby permitting α-helix N to interact with the primer terminal base pair in the active site. Such an effect could be promoted by Zn2+, which would interact with the bridging, 5′-phosphate group of the primer terminus. This interpretation is also consistent with the Zn2+-induced Met236 resonance shift (Figure 2B, red spectrum), since DNA translocation would be expected to significantly perturb the environment of this residue which is located near the primer terminus. It was further reasoned that the tendency of the templating base to adopt an extra-helical orientation could be dependent on its ability to stack within the DNA helix. Accordingly, pyrimidines should exhibit a greater propensity for adopting an extra-helical conformation than purines. Consistent with this hypothesis, use of a substrate with a templating guanine rather than thymine was found to block Zn2+-induced conformational activation almost completely (Figure 3A, purple spectrum). This finding supports the model in which the conformational activation produced by Zn2+ in the presence of a 1-nt gapped DNA results from a translocation of the primer terminus into the nascent base-pair-binding pocket, displacement of the templating base into an extra-helical position, and repositioning (i.e. closing) of the N-subdomain to interact with the primer terminus and its templating base.
A second implication of the above hypothesis is that a pol β/nicked DNA complex should also be susceptible to Zn2+-induced activation, since the active site is again occupied by a base pair. A crystallographic structure of the binary pol β/nicked DNA complex (PDB ID 1BPZ) (24), shows the enzyme in an inactive open conformation without metals. Consistent with this expectation, the 1H-13C HSQC spectrum of a binary methionine-labeled pol β/nicked DNA complex, with a chelator present to sequester the divalent metals, showed none of the perturbations associated with conformational activation. However, when Zn2+ was not chelated, the spectral perturbations were similar to those observed for the complex containing a 1-nt gapped DNA with templating thymine (Figure 3B, blue spectrum). This result indicates that Zn2+ is able to interact with the active-site aspartyl residues and, in combination with the presence of a base pair in the active site, induce a closed, conformationally activated structure without the need for DNA translocation. Addition of MgCl2 to the nicked-substrate complex produced a different spectral response, suggesting a mixture of slowly inter-converting activated and non-activated structures (Figure 3B, red spectrum), indicating that the differences observed between the responses to Zn2+ and Mg2+ may be more quantitative than qualitative.
A mutation in a signal transduction cascade residue (Glu295) of pol β has been identified in some human gastric carcinomas and was found to disrupt enzyme activity and base excision repair (12,25). The HSQC spectra of the methionine-labeled E295K mutant apoenzyme and its binary complex with gapped DNA indicate that the mutant enzyme is properly folded and demonstrates the expected shifts in Met18 and Met236 resonances upon binding DNA (Supplementary Figure S4). In contrast, the typical spectral changes observed for the resonances of Met155, Met158, Met282 and, to a more variable extent, Met236 were not observed upon formation of either a ternary E295K pol β/1-nt gapped DNA/Mg-dNTP complex or a binary E295K pol β/1-nt gapped DNA/Zn2+ complex (Figure 3c). This demonstrates that Glu295 plays a critical role in the signal transduction pathway that couples base-pair sensing to catalytic activation, and shows that the mechanism of Zn2+ activation also utilizes this pathway.
The NMR evidence for the interaction of Zn2+ and, in some instances, Mg2+ with the 5′-phosphate of the primer terminus in both nicked and 1-nt gapped DNA complexes has direct implications for the reverse 'depolymerization' reaction, pyrophosphorolysis. In this reaction, the catalytic metal ion must interact with the primer terminus 5′-phosphate, which ultimately becomes the α-phosphate of the dNTP product (3). Pyrophosphorolysis would be expected to require conformational changes that occur prior to and following the chemical step (26).
We have previously documented a pyrophosphorolysis reaction for pol β with nicked DNA in kinetic studies performed at 37°C (27), however, attempts to catalyze the reverse reaction in the presence of Zn2+/PPi were unsuccessful, perhaps as a result of the lower ability of Zn2+ (compared with Mg2+) to support the chemistry of the transition state, and/or as a result of the low solubility of Zn2+/PPi. Additionally, pyrophosphorolysis is not readily observed at room temperature with physiological concentrations of Mg2+/PPi (28); this is consistent with the NMR studies indicating that Mg2+ by itself cannot induce the activated complex. Additionally, the lack of pyrophosphorolysis presumably indicates that even in the presence of PPi, Mg2+ cannot induce an active conformation with a 1-nt gapped DNA substrate.
Since the reactions described above were performed at 25°C, we determined whether increasing the reaction temperature to 37°C might promote conformational activation and the ability of the templating base to assume an extra-helical position, presumably required to provide a substrate for pyrophosphorolysis. In contrast with the results at 25°C, the reverse reaction was readily observed with 1-nt gapped DNA as indicated by the shortened primer (n–1 product) (Figure 4). As expected if helical stacking influences conformational activation, the reverse reaction was much weaker with templating purines than with pyrimidines. Also note that a weak n+1 product was produced when guanine was the templating base; this confirms that dCTP was a product of the observed reaction since the primer terminal nucleotide is cytidine. Even though very low concentrations of dCTP are produced over the short time span of the experiment, dCTP can fill the gap on a 1-nt gapped substrate with G as the templating base but not with other templating bases, since these would require an incorrect insertion.
Although the NMR studies were qualitatively consistent with the pyrophosphorolysis results, it was unclear from the NMR studies why this reaction could not be observed at 25°C; therefore, we sought to obtain NMR data at 35°C. However, enzyme stability can be problematic when obtaining NMR spectra over extended time periods at elevated temperatures, and we encountered a significant degree of precipitation and/or the presence of soluble, partially unfolded pol β, depending on the conditions of the study. More specifically, a variable resonance previously suggested as arising from incompletely proteolyzed Met1 (15) is now attributed to partially unfolded but soluble pol β. After a series of studies, we found that replacing Cys267 with alanine permitted longer collection times at elevated temperature (35°C) without significant degradation of the protein, as evidenced by the stability of the NMR spectra and the absence of observable precipitate. Alanine substitution at Cys267 does not dramatically alter the structure or behavior of the enzyme as demonstrated by the similar steady-state kinetic behavior for 1-nt gap filling with the mutant and wild-type enzymes (Supplementary Table S2), as well as the minimal perturbations observed for the methionine 1H-13C HSQC spectrum of labeled pol β.
In contrast with the results obtained at 25°C, HSQC spectra of [methyl-13C]methionine C267A pol β obtained at 35°C in the presence of MgCl2 indicated a significantly greater degree of conformational activation with a templating thymidine as judged by the broadening of Met155 and the shift in Met158 resonances (Figure 5A). These spectral changes are similar to those observed at 25°C with nicked DNA in the presence of Mg2+ (Figure 3B). The elongated shape of the Met282 resonance observed at 35°C for C267A pol β complexed with gapped DNA containing a templating thymine base presumably arises due to intermediate exchange between the open and closed conformational states of the enzyme. Simulations using a range of estimated parameters yields a lifetime for the closed (activated) conformation of ~2–5ms (Supplementary Figure S5). These values are near those expected from observed fluorescence transients that monitor subdomain motions (29).
We further evaluated the effect of temperature by comparing results at 25°C and 35°C for pol β complexes with nicked DNA in the presence of Mg2+ (Figure 5B). Consistent with the above results, the spectra obtained at the higher temperature show a more complete conformational activation of the enzyme. Thus, the intermediate degree of conformational activation observed at 25°C becomes more complete at 35°C; this is consistent with the earlier demonstration of pyrophosphorolysis of nicked DNA at 37°C (27).
High fidelity DNA synthesis requires efficient nucleotide insertion when proper base pairing occurs and the base pairing events are physically separated from the site of chemistry by ~10Å. For pol β, bridging this distance is accomplished through a series of altered side-chain conformations associated with repositioning of the N-subdomain (i.e. fingers) relative to the C-subdomain (palm) upon binding a nucleoside triphosphate. Enzyme conformational activation is globally characterized by an open to closed structural transition inducing many subtle conformational adjustments. A defining step of this signaling cascade is the release of Asp192 from its salt bridge interaction with Arg258, allowing Asp192 to coordinate both the catalytic metal and nucleotide-bindng metal (Figure 1). Metal B binds in complex with dNTP (forward reaction) or PPi (reverse reaction). It has been suggested that, following nucleotide insertion, the ternary product complex undergoes a conformational change that facilitates metal/PPi release (30,31). The NMR studies presented here demonstrate that metal ions can play an active role in the induction of the conformationally-activated state in the absence of nucleotide ligand.
The NMR solution studies described here demonstrate that in the presence of 1-nt gapped DNA, Zn2+ can induce an active closed conformation that requires positioning of the primer terminus into the nascent base-pair-binding pocket (Figure 2B). Furthermore, they suggest that this results not only from the ability of the Zn2+ ion to interact with Asp192, but also from the ability of Zn2+ to interact with the 5′-phosphate group of the primer terminal nucleotide. Crystallographic structures of product complexes (post-insertion complex before translocation) of pol β (32) and pol λ (33)—also an X-family DNA polymerase—indicate that the lone active-site metal (B) coordinates PPi, two active-site aspartates (190 and 192), and the 5′-phosphate of the primer terminus. We suggest that neither the crystallographic structure of the metal-free nicked DNA complex nor the product complexes noted above correspond to the complexes observed in the solution NMR studies presented here, since they lack the catalytic metal A required for chemistry (DNA synthesis or pyrophosphorolysis). Because the metal B site is partially formed by non-bridging oxygens on the incoming dNTP (DNA synthesis) or PPi (pyrophosphorolysis), the metal activation observed here probably reflects binding to the catalytic metal A-site. In this context, primer relocation into the dNTP binding site completes the coordination sphere for the catalytic metal site and suggests a low energy barrier for translocation of the primer terminus when the templating base in the DNA gap is a pyrimidine (Figure 6).
The above observations raise more general questions about the ability of Zn2+ and other ions to promote translocation or stabilization of the DNA in the active site by interacting with the 5′-phosphate group of the primer terminus. We therefore investigated the effect of both Zn2+ and Mg2+ (Figures 3B and and5B)5B) on a nicked DNA substrate. The NMR spectrum of the methionine-labeled pol β in the presence of Zn2+ and nicked DNA showed a pattern characteristic for conformational activation: broadening of the Met155 resonance, and broadening and characteristic shifts of the Met158 and Met282 resonances (Figures 3B and and5B).5B). Thus, the Zn2+ ion stabilizes the active-site structure, resulting in a reversal of the signal transduction cascade so that the N-subdomain favors a closed conformation. The substitution of Mg2+ for Zn2+ produces a similar effect at 25°C, but with multiple resonances distributed between the non-activated and activated shift values (Figure 3B). The Met282 resonance observed in the pol β/nicked DNA/Mg2+ complex was broad, consistent with exchange between open and closed structures. The broadening might result from a conformational mixture characterizing the pol β/DNA/Mg2+ complex, or a mixture of pol β/DNA/Mg2+ and pol β/DNA complexes. Thus, the dynamic process being monitored could arise from slow conformational exchange and/or a slow Mg2+ association/dissociation process. Furthermore, studies utilizing nicked DNA at a higher temperature (35°C, Figure 5B) indicated that, from a conformational standpoint, the differences between Mg2+ and Zn2+ tended to be more quantitative than qualitative. Magnesium ions become more effective at inducing conformational activation at higher temperatures when the nascent base-pair-binding pocket is occupied (i.e. nicked DNA). For both metals, these effects involve reversal of information flow through the signal transduction pathway, which is most clearly supported by the studies with E295K pol β that blocks signal transmission (Figures 1 and and3C).3C). Additionally, interaction with the 5′-phosphate group of the primer terminus appears to be an important component leading to translocation of 1-nt gapped DNA so that the primer terminus occupies the nascent base-pair-binding pocket (N-site, see below).
During catalytic cycling, the primer terminus must translocate from the site of nucleotide incorporation to the primer site, opening the dNTP-binding pocket for insertion of the next nucleotide. These have been referred to as the N- and P-sites, respectively (34). The factors that influence the translocation of the primer-terminus between the P- and N-sites are poorly understood.
In the absence of an incoming nucleotide, the position of the primer terminus of 1-nt gapped DNA is dependent on the presence of a divalent metal and the identity of the template base in the gap. For the primer terminus to occupy the N-site, the DNA gap must either move downstream or collapse (i.e. the nucleotide in the gap is extruded to an extra-helical position). The studies reported here demonstrate that the latter scenario is the most likely explanation since the ability to form a Zn2+-activated binary complex depends on the identity of the base in the gap (Figure 3A). Consistent with this interpretation, 2D NMR experiments on duplex DNA with different nucleotides opposite an abasic site indicate that pyrimidines easily assume an extra-helical position, whereas purines do not (35). The ability to stack within the DNA helix is also a key determinant for DNA polymerase discrimination during non-templated DNA synthesis (14). Thus, when a purine is in the gap the DNA remains in an extended conformation and the nascent base-pair-binding pocket is only partially filled with the templating purine. In contrast, when a pyrimidine is in that position, the primer terminus can position itself in the nascent base-pair-binding pocket to stack with α-helix N in a closed conformation and extrude the unpaired pyrimidine (Figure 6).
It has long been assumed that structures involving extra-helical nucleotides must be feasible as that would explain the ability of X-family polymerases to fill gap lengths longer than a single nucleotide; for such multi-nucleotide gapped substrates, bases in the templating strand downstream from the coding position in the gap must be extruded in order to deter synthesis of deletion intermediates. Crystallographic evidence for such an extra-helical base has been obtained in studies of ternary complex structures of pol λ with 2-nt gapped substrates (36). The templating base hydrogen-bonds with the incoming dNTP, and the downstream unpaired nucleotide is situated outside of the upstream and downstream duplexes, sequestered in a binding pocket formed by residues of α-helix N and the lyase domain. An extra-helical base corresponding to a deletion intermediate was also observed upstream of the active site in pol λ (37). For pol β, an extra-helical templating cytosine base has been observed in a structure containing a primer terminal A-A mismatch where the template A of the mismatch stacks with the primer terminus (38).
The observation of polymerase conformational activation in the absence of an incoming nucleotide was unexpected and appeared to represent an intermediate structure relevant to the reverse reaction, pyrophosphorolysis. This reaction requires several conditions: (i) the primer terminus in the N-site; (ii) substrate PPi; (iii) divalent metal ions; and (iv) conformational activation of the enzyme. The NMR results suggested that Mg2+ should be able to support the reverse reaction for nicked DNA and for 1-nt gapped DNA containing a pyrimidine base in the templating position. Vande Berg et al. (27) previously demonstrated this reaction for a pol β/nicked DNA/Mg2+ complex. Pyrophosphorolysis with a 1-nt pyrimidine gapped DNA at 37°C can readily be observed (Figure 4). Although the NMR data indicates that Zn2+ induced conformational activation for a single nucleotide-gapped DNA substrate with a templating pyrimidine, no pyrophosphorolysis was observed. This may result from the insolubility of Zn2+/PPi at neutral pH, as well as the poorer ability of the electronic configuration of the Zn2+ ion to promote catalysis. We conclude that conformational activation is a necessary, but not sufficient, condition for both the forward and reverse reactions. With adenine in the DNA gap (template position), Mg2+-dependent pyrophosphorolysis was not observed whereas with guanine, weak activity was observed. Alternatively, using identical conditions at room temperature, pyrophosphorolysis is not observed with gapped DNA, consistent with the lack of Mg2+-induced activation at the lower temperature.
As illustrated in Figure 1, several key charged side-chains that mediate the signal transduction between the nascent base pair and the metal–ion-binding site alter their hydrogen-bonding pattern during conformational activation. Due to the important role of Glu295 in mediating the interaction between Arg283 and Arg258, it was expected that this non-conservative change should inhibit conformational activation. Meeting this expectation, Lang et al. (12) have reported E295K pol β to be inactive. Further insight into the molecular basis for Zn2+-induced conformational activation was obtained in our studies of E295K pol β. NMR experiments with the E295K mutant protein indicate that it is fully capable of binding DNA (Figure 3C), but that binding of the dNTP substrate fails to promote conformational activation. Thus, the methionine residues in the C- and N-subdomains exhibit almost no response to formation of the ternary complex. Consistent with this explanation, we have found that the addition of Zn2+ to the pol β/nicked DNA or 1-nt gapped DNA with a templating thymidine (Figure 3c) also failed to induce conformational activation, indicating that residue Glu295 plays a critical role in both the forward and reverse signaling processes.
It remains to be seen if metal-induced activation is specific to gap-filling DNA polymerases that must inherently interact with duplex DNA upstream and downstream of the growing primer terminus. The domain organization of pol β includes an 8-kDa domain that anchors the enzyme to the downstream duplex (39). When the primer 3′-terminus is near, the polymerase domain interacts with the upstream duplex DNA to complete gap-filling DNA synthesis. In contrast, replicative DNA polymerases interact primarily with upstream duplex DNA and only to a lesser extent with downstream single-stranded DNA. The low deletion-frameshift fidelity of polymerases belonging to the X-family is consistent with the suggestion that these polymerases modulate template/primer positioning during DNA synthesis. DNA polymerase β readily utilizes the downstream template base when the coding nucleotide base is absent (as in an abasic site) (40).
Supplementary Data are available at NAR Online: Supplementary Results, Supplementary Figures S1–S5, Supplementary Tables S1 and S2, and Supplementary References [15–16 and 41–42].
Intramural research program of the National Institute of Environmental Health Sciences, National Institutes of Health, under projects (Z01-ES050147 to R.E.L.) and (Z01-ES050158 to S.H.W.), in association with National Institutes of Health (Grant 1U19CA105010). Funding for open access charge: Research Project Numbers (Z01-ES050147 to R.E.L. and Z01-ES050158 to S.H.W.). National Institute of Environmental Health Sciences.
Conflict of interest statement. None declared.
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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Robert Carlyle made his name with tough, violent and damaged characters. But it won't stop him appearing in 24 or playing Leonard Rossiter, he tells Kirsty Scott
Philip French: ... a small picture certainly, but a thoughtful and engaging one
Ken Loach's 1996 film Carla's Song starred Robert Carlyle as a Glaswegian bus driver. For the DVD release, the director made extensive changes to the original. Here he explains why.
Trainspotting author teams with Robert Carlyle for film about club's infamous "Soul Crew" hooligan element
Peter Bradshaw: A mainstream comedy with a lot of laughs, crammed with nice performances and most importantly some genuine and touching emotion
The Full Monty star is to appear in a reworking of the spaghetti western set in the Midlands
As his new film receives its world premiere in Edinburgh, Robert Carlyle tells Libby Brooks about the price of fame, the lure of villainy - and why he wants to stay true to his roots
Peter Bradshaw: Enjoyable Yuletide romps ... [even if TWINE] just still looks so weirdly dated
The film was going nowhere. Two directors had walked out, and Robert Carlyle was in tears. Mark Salisbury finds out how Antonia Bird saved a gruesome comedy about cannibalism
Robert Carlyle/Antonia Bird: sound clips
As Stevie, the building-site worker in the Ken Loach movie Riff Raff, Robert Carlyle loses it badly. His girlfriend is performing an audition, singing her talentless heart out for some Hooray Henry producers, who are laughing at her, and simultaneously gossiping among themselves. It is after several excruciating minutes of this that mild-mannered, slight-framed Stevie stalks over and, with narrow-eyed menace, manages to 'persuade' the guilty parties to shut up and take her seriously. Carlyle's genius is that he is not only totally convincing when he does this (even though his character had previously exuded all the violent tendencies of a sleepy birthday kitten), but also that, after the scene has finished, he does not do the obvious thing and continue tiresomely exuding 'quiet menace' for the rest of the movie.
From thug in Trainspotting to serial killer in Cracker, from sad steelworker in The Full Monty to pin-up TV cop Hamish Macbeth, Robert Carlyle is a man of many faces. But which one is closest to his own?
It is hard to feel sorry for a talented, wealthy movie star, but only the stone-hearted would not make an exception for Robert Carlyle. Here is a man who makes superhuman efforts to stick by his roots in Scotland and dodge tabloid celebrity, only to find that his every move is written up in the papers, usually accompanied by a punning headline derived from The Full Monty.
Here is a story about Robert Carlyle. Daniel Boyle, the scriptwriter for the BBC's 11-million-pulling Hamish Macbeth, met a man who asked what Boyle did for a living. He said he wrote Hamish, and the man said: 'Ach yes, that stars Robert Carlyle, I knew him once. Or at least I thought I knew him. I worked with him for six months at the Metro Bar in Glasgow, and for those six months I thought he was English.'


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Instead of dealing with the clear links I posted between Robert Howard, his lobbyist, the "line-item DOD handout" which the Lobbyist inserted in return for Howard's Political Contributions and the SBIR "program" which facilitates this game, the P & D Artists crawling on this Board attack the person who proves the point.I know your game and most of your new "aliases" are merely weeks old.Have fun with your "marks".I have shown them where to look to uncover this latest crime Robert Howard is involved with.If you can't counter the evidence, play your weak "discredit the Messenger" game.There are enough wise lurkers who will take my messages seriously and AVOID Robert Howard and all the stock crimes he has been associated with.Happy Holidays!
Knut4oe, thanks for the thoughtfulness of moving your non-IOTN discussion to the appropriate message board where it belongs. I hope other�s will follow your example and move their non-IOTN discussions to the appropriate boards. It is really rude for someone to come to a message board and continually pump an unassociated stock. IMHO
Here is the Raging Bull ITUI board:http://ragingbull.lycos.com/mboard/boards.cgi?board=BB%3AITUI&origsymbols=itui
How about we take all ITUI discussion to the Raging Bull message board. I posted there today in the hope that the message board will function again.
Yup, only the beginning. Will be atleast $10 by the end of the year, if not much higher. It is still VERY under the radar. This was a huge volume breakout day though. Pretty soon voicestick will be available in Best Buy and Walmart, then it will really take off with the exposure and with the Nasdaq listing. Good luck to all accumulating there.
Okay, Cast500,I have to admit you have done a good job in promoting Ionatron and Howard. You can see how the stock is moving up fast. There are so many buyers in the wings waiting for a fall in price so they can get in or add more to their portfolio.I understand , you have 2 other handles on Yahoo which you use to bash---you aren't really too adept at it as you are running the stock up.Thank you Robert Howard for bringing your expertise to Ionatron. Happy Holidays to All
The messenger is a fraud.
Checking on me ha? Why is that because you are getting expose all over the place not just in this Board. I'm putting all the names that you use that I discover about you and will post accordingly. Happy holidays to you and good luck. It is going to be fun.


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Bachelor of Commerce / Bachelor of Science
Monash University — Faculty of Information Technology
The Monash community includes more than 62,000 students from over 130 countries. Monash is the only university in Australia's prestigious Group of Eight to have a dedicated Faculty of Information Technology, recognising the importance of information technology as a separate group of disciplines, such as business information systems, multimedia applications and computer science.
Bachelor Degree (Pass)
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Year 12 or equivalent; Units 3 and 4: a study score of at least 30 in English (EAL) or 25 in any other English, and a study score of at least 25 in mathematical methods (CAS) or specialist mathematics
Course Description
This course leads to two separate degrees: Bachelor of Commerce and Bachelor of Science. A Commerce and Science degree gives students the skills to commercialise scientific research or outcomes, or apply commercial knowledge in scientific environments. They will develop technical and research skills required across scientific fields while developing an understanding of commercial realities of research as a business. Students can combine majors to explore existing areas of interest or develop new ones. Students gain all the benefits of each degree and are fully equipped to pursue a career in either field separately or combine them. In the Commerce degree, students will develop broad commercial knowledge and can choose from nine majors. The Science degree gives students the opportunity to choose from a wide variety of majors. Students could pursue majors in Applied Mathematics and Actuarial Studies or Econometrics which could lead to an academic career or the development of new mathematical models and principles. They could blend studies in Environmental Science or Ecology and conservation biology with a major in Sustainability and introduce sustainable practices into the community or across organisations or turn majors in Pharmacology and Marketing science into a career marketing new medications or scientific breakthroughs. This course sits as part of a larger group of courses.
Subjects you can Study
Accountancy; Accounting; Actuarial modelling; Actuarial studies; Applied mathematics; Astrophysics; Atmospheric science; Behavioural commerce; Biochemistry; Botany; Business modelling; Business statistics; Chemistry; Commerce; Computational science; Developmental biology; Earth science; Ecology and conservation biology; Econometrics; Economics; Environmental science; Finance; Financial and insurance mathematics; Genetics; Geographical science; Geosciences; Human pathology; Immunology; Management studies; Marketing; Marketing science; Materials science; Mathematical statistics; Mathematics; Microbiology; Molecular biology; Pharmacology; Physics; Physiology; Plant sciences; Psychology; Pure mathematics; Science; Statistics; Sustainability; Zoology
^ Shows the minimum tertiary entrance ranking needed by Australian school leavers to get into each CSP-based course. Cut-offs are not determined in advance. Course data and cut-off scores published on Good Universities Guide are indicative of the 2016 academic year.
Bachelor Degree (Pass)
- Full-time internal = 4 years
- Part-time internal = 8 years
32 units (192 points). 16 commerce units (96 points) and 16 science units (96 points). These include 8 commerce specified study units (48 points) which provide a broad foundation for commerce study and an 8 unit (48 points) commerce major chosen from those offered at the Clayton campus. The Faculty of Science component comprises of 8 science specified study units (48 points) and 8 units (48 points) in a science listed major.
Entry Requirements
Year 12 or equivalent; Units 3 and 4: a study score of at least 30 in English (EAL) or 25 in any other English, and a study score of at least 25 in mathematical methods (CAS) or specialist mathematics
Enrolment Enquiries
How does this course perform?
How do study fields for Bachelor of Commerce / Bachelor of Science at Monash University perform?
Life after Study
Where are graduates that studied Accounting at Monash University?
|Public sector
|Private practice
|Private industry
|Working overseas
Business and management
Where are graduates that studied Business and management at Monash University?
|Public sector
|Private practice
|Private industry
|Working overseas
Computing and information technology
Where are graduates that studied Computing and information technology at Monash University?
|Public sector
|Private practice
|Private industry
|Working overseas
Where are graduates that studied Economics at Monash University?
|Public sector
|Private practice
|Private industry
|Working overseas
Where are graduates that studied Mathematics at Monash University?
|Public sector
|Private practice
|Private industry
|Working overseas
Where are graduates that studied Psychology at Monash University?
|Public sector
|Private practice
|Private industry
|Working overseas
Where are graduates that studied Sciences at Monash University?
|Public sector
|Private practice
|Private industry
|Working overseas
Related Careers
Accountants analyse, report and give advice about the financial dealings of organisations and individuals, and advise on associated record-keeping and compliance requirements.
Actuaries evaluate risk and opportunity, applying mathematical, statistical, economic and financial analyses to a wide range of business problems.
Biochemists study the chemistry of living systems to increase scientific knowledge and develop ways to apply this knowledge in areas such as medicine, veterinary science, agriculture, environmental science and manufacturing. Biochemistry provides a basis for all the life sciences.
Botanists study the biology of all types of plants to increase scientific knowledge and apply this knowledge in areas such as conservation and management of natural resources, agriculture, forestry, horticulture, medicine and biotechnology.
Chemists study the physical and chemical properties of materials to determine their composition. They use this information to develop new materials and products, to devise more efficient processes for making materials and to increase scientific knowledge. Chemists should not be confused with pharmacists (see separate entry for Pharmacist).
Economists perform economic research and analysis, and develop and apply theories relating to the production and distribution of goods and services and people's spending behaviour. Economists advise and provide forecasts to governments and businesses on matters such as taxation levels, wages and prices, employment and unemployment, imports and exports, and interest and exchange rates. They investigate international or national economic situations, or particular features such as industries or regions.
Environmental Scientist
Environmental scientists measure and record features of the environment and study, assess and develop methods of controlling or minimising the harmful effects of human activity on the environment.
Financial Dealer and Broker
Financial dealers and brokers conduct financial market transactions on behalf of clients and offer advice on financial matters.
Geographic Information Systems Officer
Geographic information systems officers design, develop and customise geographic information systems and provide technical and analytical support to address issues such as environmental management, exploration and mining, land ownership and titles, urban and regional planning, utilities and asset management, and demographic marketing.
Life Scientist
Life scientists examine the anatomy, physiology and biochemistry of humans, animals, plants and other living organisms to better understand how living organisms function and interact with each other and the environment.
Management Consultant
Management consultants provide advisory services to organisations (public, private and not-for-profit) regarding management problems, challenges and opportunities.
Marine Biologist
Marine biologists study the origin, structure, functions and behaviour of all forms of life in the sea and connected estuaries, rivers and lakes, as well as their relationships with each other and how they are affected by environmental factors.
Marketing Officer
Marketing officers promote a company's or client's products or services. This can involve marketing existing products, developing new products to cater for consumer demand, or developing markets for new products or services.
Materials Engineer
Materials engineers investigate the properties of metals, ceramics, polymers, plastics and other materials, and develop and assess their commercial and engineering applications.
Mathematicians apply and develop mathematical principles to solve problems in all areas of the sciences, technology, social sciences, business, industry and commerce.
Science Field Officer
Science field officers perform support functions in the field by making observations, collecting and analysing samples, recording information, and looking after the practical tasks involved in maintaining a remote field operation, or operating a city-based office, laboratory or workshop.
Statisticians design and apply statistical techniques for creating, collecting and analysing data to draw conclusions, inform decision-making and direct policy within areas such as science, technology, medicine, education, business, finance and government.
Sustainability Officer
Sustainability officers work to create enduring communities that have a positive environmental, economical and social impact.
Zoologists are biologists who study the structures, characteristics, functions, ecology and environments of animals to increase scientific knowledge and develop practical applications in wildlife management, conservation, agriculture and medicine.
About Monash University — Faculty of Information Technology
Prestige and global recognition
Monash is ranked number one for computer science in Australia (2012 Academic Rankings of World Universities) and number 45 in the world in computer science and information systems (2013 QS World University Rankings by Subject).
It is also well known for its leadership in the information technology (IT) field. In this field, an international profile is very important because the IT labour force is less restrictive than many other disciplines in terms of nationally based practice requirements. As a result, IT professionals tend to be globally mobile. About 50 per cent of enrolments are from international students, who provide a diversity of input and perspective that enhances the experience for all students.
Excellent facilities and flexible study options
The faculty teaches and conducts research on one Australian campus (Clayton), as well as at two international campuses — Monash Malaysia and Monash South Africa. World-class facilities include more than 1500 work stations networked across the Australian teaching campuses, upgraded every two to three years.
Renowned research excellence
The Faculty of Information Technology is one of the largest research organisations in Australia, with over 80 per cent of the academic staff research-qualified to PhD or masters level, and around 200 students undertaking PhD and masters by research projects.
Our leading researchers' strengths are in computing science, software engineering, information systems, and information and knowledge management.
At Monash University, information technology research has a multidisciplinary and multinational approach, providing Monash with the unique capacity to reach further and deeper than any other educational institution in Australia. Our four research flagships are the focus for our internationally recognised research strengths, which are the hubs of our research networks in:
- computational biology
- IT for resilient communities
- machine learning
- modelling, optimisation and visualisation.
Our 150 plus research students are a valued part of the faculty's research community, benefiting from the stimulating and supportive research training environment, with expert supervision from committed world-class researchers.
Visit it.monash.edu/research for further information.
Diverse, quality programs
Courses are available at all levels, from undergraduate (bachelor and honours) degrees, through to graduate certificates and diplomas and on to masters (coursework and research) and doctoral degrees. Programs are offered across all IT disciplines, from business and information systems management to technical specialisations such as network computing, digital communications and multimedia applications.
1. Bachelor of Information Technology
a. Double degrees offered with:
- Arts
- Business
- Business Specialist
- Commerce
- Commerce Specialist
- Design
- Education (Honours)
- Fine Art
- Science
2. Bachelor of Computer Science
a. Double degrees offered with:
- Commerce
- Commerce Specialist
- Education (Honours)
- Science
3. Bachelor of Computer Science Advanced (Honours)
Postgraduate Study:
- Master of Data Science (NEW)
- Master of Business Information Systems
- Master of Information Technology
- Master of Networks and Security
Industry Experience
International students enrolling into an undergraduate or postgraduate coursework degree at an Australian Monash University campus may complete either an Industry Based Learning (IBL) placement or an Industry Experience (IE) project.
The IBL program partners Monash University with leading global and Australian organisations to offer students outstanding placement opportunities and scholarships valued at up to $34,000.
The IE project gives students the opportunity to work with a real business client. Students will apply the knowledge and skills they have learnt throughout their degree in a genuine professional environment.
For further information, visit:
- infotech.monash.edu/future/courses/get-experience/industry-based-learning
- infotech.monash.edu.au/future/courses/get-experience/industry-experience
Choose to study IT at Monash
Monash offers excellent facilities, a diversity of IT course and location options, as well as the profile and prestige to command the attention of any employer. The range of course offerings, outstanding global profile, high academic standards and research achievements make Monash University the place to study information technology.
Visit it.monash.edu/courses for more information.
Course finder
International students


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Because I'm STILL hyped that they'll be at Warped Tour this year.
Last edited by GlowBear; 02-13-2013 at 04:53 AM.
"Guitar Party" was a band fronted by a first grade girl, one of Micheal "Eyedea" Larsen's many projects. This is their only album in its entirety, and conveniently enough the first track here is the one I was listening to just a moment ago on my iPod; 'Navy Blue Baby':
That's not even the song I meant to post but w/e. Kinda hard to do things correctly when my browser keeps freezing up.
With his birthday being 10th February I've finally revisited some Metallica and listening to Cliff's work.


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Understanding a misunderstood generation
Welcome to the Millennial Series
Universum joined forces with INSEAD Emerging Markets Institute and the HEAD Foundation to investigate and question what has become conventional wisdom about Millennials. By conventional wisdom we mean the statements (even stereotypes) we've all read about, such as Millennials' pursuit of work-life balance over money, or their expectation of rising rapidly in their chosen careers. Are these and other stereotypes really true?
To find answers to such questions and more, sign up now to either receive full access or download just the free summary.
Access Insights on Millennials
Our greatest fears
Examining Millennials' concerns about career, retirement, and quality of life―And the steps you should take to address them
As real wages have stagnated or even decreased over the last decades – at least in the West – and youth unemployment is at record levels in many regions, you might expect Millennials to hold a dismal view of their economic prospects. Yet while there are certainly some pessimists among them, they are easily outnumbered by those who expect to be doing much better than their parents and even to be able to retire before the age of 60.
Support me but don't tell me what to do
Who and what influences Millennials' career choices? The answers aren't as clear-cut as one might think
It's becoming harder to reach target audiences with career-related messages. When most communication was paper-based, getting your message out was straightforward. Yet, as online channels and social media gradually emerge from hype to standard tools in marketing, the resulting fragmentation and complexity is challenging for employer brand promotion.
You got us wrong
Millennials prove they are diverse in their career aspirations and desire for work-life balance
There has been much debate about the lack of leadership ambition among younger generations. If this were indeed the case, it would need to be taken seriously for several reasons. Firstly, it takes strong leadership to drive innovation in large organizations. Secondly, although technological developments are replacing many jobs, leadership is one of the few areas where people still have a clear advantage.
We are more different than you think
A look at the diversity of Millennial ideas and attitudes within regions, and the implications for employers
Not surprisingly, country-level economics and culture shape how Millennials think about critical career issues. While studies often talk about regions like Asia-Pacific or Western Europe as a unified whole, in truth individual countries within regions often are as different as one region to the next.
Our evolution…how experience changes Millennials
We look in-depth at how age and gender influence Millennial attitudes to work.
In this instalment, we look more closely at older versus younger Millennials. How do Millennials change with a few years of work behind them? Are they more or less secure about their career prospects? Also, we examine the contrasting attitudes of men and women related to work-life balance and career goals.
2020 Outlook
2000+ HR professionals and CEOs give us their take on talent recruitment and employer branding.
In this unparalleled study on the employer perspective of talent acquisition, Universum delves into the world of employer preferences on talent attributes, the state of employer branding to the development of brand activation…and looks at the trends five years ahead.
Webinar presentation on Understanding a misunderstood generation
Jacob Andelius, a senior consultant at Universum, presents some of the key findings around the research on Millennials and provides some key recommendations to employers on how to work with this generation. See the webinar recording to get an excellent overview of what we discovered about this generation and more.
Tomorrow's thinking. Available today.
Thought leadership from Universum industry experts to intrigue and challenge current employer branding professionals and inspire those who are just beginning their talent branding journey.
Most attractive companies among business students
Among business students, financial services employers were hardest hit. Ten out of 12 WMAEs in financial services lost ground in the rankings. Only Goldman Sachs and Morgan Stanley held on […]
WMAE16 Ranking by Industry
On the 29th of June CNNMoney announced the findings of Universum's annual, world's most attractive employers ranking. Universum's Talent Survey takes in to account the opinions of over 265,000 millennials […]
The Importance of Inspiring Purpose
Take a look at your workplace. Do the employees look ready to seize the day? Or are they just pushing through? If they don't seem enthusiastic, you have a […]
Global Locations
With locations around the world, Universum is a global firm with central offices in Stockholm, Sweden.


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Elsa Garmire on Her Groundbreaking Career in Engineering
February 17, 2016 | Dartmouth Now
The Thayer professor discusses connections between physics and God, lasers and graffiti, and more.
5 Questions to Molly Herron (composer)
February 17, 2016 | I CARE IF YOU LISTEN
Brooklyn-based composer Molly Herron finds inspiration in both science and newly-invented instruments. Upcoming projects include collaborations with Dartmouth.
Center Receives $6.2m to Develop Rehabilitation and Assistive Technologies
February 4, 2016 | Center for the Translation of Rehabilitation Engineering Advances and Technology
The Center for the Translation of Rehabilitation Engineering Advances and Technology (TREAT) was recently awarded five years of additional funding from the National Institutes of Health (NIH).
Nation's Top MEM Schools Compete in "Business War Games"
February 1, 2016
Members of the Master of Engineering Management Programs Consortium (MEMPC) will go head-to-head in the PriSim Business War Games Competition starting today.
Five New DartmouthX Courses Announced
January 28, 2016 | Dartmouth Now
Dartmouth engineering professor Rachel Obbard will teach "Materials in Gear"—sports gear, that is—as one of five new DartmouthX courses.
Trustees Establish School of Graduate and Advanced Studies
January 27, 2016 | Dartmouth Now
The school is Dartmouth's first new school in more than a century.
Dartmouth Professor Brian Pogue Inducted into Medical and Biological Engineering Elite
January 26, 2016 | AIMBE
The American Institute for Medical and Biological Engineering (AIMBE) has announced the induction of Dartmouth engineering professor Brian Pogue to its College of Fellows.
Busted knees and broken legs: Why ACL ski injuries have skyrocketed and what you can do about it
January 24, 2016 | Pique
Professor Rick Greenwald weighs in on the importance of bindings that can better release your foot during a twist.
BESC study seeks nature's best biocatalysts for biofuel production
January 14, 2016 | Oak Ridge National Laboratory
A team of researchers from five institutions, including Dartmouth engineering professor Lee Lynd, found that unconventional bacteria could help boost the efficiency of cellulosic biofuel production.
Thayer's Board of Overseers Welcomes Two New Members
January 13, 2016
Thayer School is pleased to announce two additions to the Board of Overseers: Catherine Sellman '93 and Andrew Silvernail '94.


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From the Archive: Twin myths undergird the claim by Israeli hardliners that they own the land of Palestine: the Biblical stories about the Exodus and the ancient kings of Israel and the claim that the Romans forced the Diaspora of Jews…
Tag: Zionism
The Dying Israeli-Palestinian 'Peace Process'
Even before Israel's founding in 1948, some Zionists had plans to seize all of Palestine and purge the native Palestinian population, but global politics required a pretense of "peace talks." So the charade has gone on for decades though it…
Powerful Lobbies v. Public Interest
Some American lobbies are so powerful that U.S. politicians cringe in fear, knowing that standing up for the broader national interest would be career-threatening, a reality most notable on issues of Israel and guns, as Lawrence Davidson explains.
The Battle for Palestine
From the Archive: You can't understand the worsening Mideast violence without knowing the modern history of Palestine, a story that begins with European anti-Semitism causing Zionists to claim Palestine for the Jews and to expel the Arabs, wrote retired U.S. diplomat William…
How Money Silences Criticism of Israel
Israel's never-ending persecution of Palestinians is opening a chasm between the world's public, which is growing disgusted by Israeli behavior, and Western elites who shy from criticism because of career fears and financial dependence, observes Lawrence Davidson.
The Liberal Zionist Dilemma
Several decades ago, liberal Jews and African-Americans were at the forefront for the U.S. fight for civil rights, but the demands from Israel for Jews to support Zionism to the detriment of Palestinian rights created a rift that grows wider even…
The Battle for Palestine
Special Report: Americans often focus on the Israeli-Palestinian conflict based on the latest atrocity and which side is to blame. But there is a long and important back story to this conflict which continues to stir up unrest across the…
The Brutal Failure of Zionism
Israel's renewed slaughter of Palestinians in Gaza after failed peace talks and ethnic slayings by both sides is further proof that the Zionist experiment has failed and that the only reasonable way forward is to recognize the equal rights of…
Selective Sympathy in Israel/Palestine
The mainstream U.S. media often reveals its bias by selecting some personal tragedies for saturation coverage while downplaying or ignoring similar horrors to "others," such as the massive attention given to the search for three kidnapped Israeli teens, as Lawrence Davidson observes.
Changing Israel from Without
For decades, one-sided support for Israel distorted U.S. perceptions and policies in the Middle East. Only recently has the pro-Zionist narrative faced significant challenge, including protests against the abuse of Palestinians from Israeli dissidents like Ilan Pappe, who spoke with Dennis J Bernstein.


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Lakers' Ron Artest Picks Heat to Win In 4 or 5 or 6 or Not At All
Ron Artest, the NBA's favorite halftime-boozing, riot-inducing maniac, has gotten quite interested in this year's NBA Finals -- unencumbered by playoff obligations of his own, he's been making predictions for a week despite not knowing what day the games are on or what city they're being played in.
Memorial day was banging in Miami
It's time to party now
Now I see a tiger skin blouse
Let's party
I see an elephant in a thong
With such an indelibly positive opinion of the city, it was inevitable that he picked the Heat to possibly sweep the Mavs:
I'm extremely proud of Dallas
Not a hater
But I think Miami in four or five
Can Dallas sweep the heat?
I don't care who wins
But I got heat in 5
Oh, wait. No. Four games, plus a thesaurus:
Miami in four not five if dirk is hurt
But j Terry will have big games
I like the mavs
And coach Rick
But I just think the three headed cerebellum us too much for dirk and his perks
But then he seems to think a Mavericks win isn't entirely impossible...
If Dallas wins four one I'll run down Santa monica with electrical shocks on my chest nipples
I was wrong I got mavs in six
I mis calculated
If lebron gets this win n Dallas
I'm going on record as saying he is great!!
But let me say this
I can't see Miami winning now
But I will still say Miami in six
But I don't believe
So we've got the support of the baller/rap producer behind the internationally acclaimed (and inexplicably sneaker-themed) "Ball'n Mixtape," put together by the "World Wide Wariers."
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