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Mar 01, 2019
Spring Cleaning Tips!
Winter is almost over and it's time to take out the gloves, mop and go to...
Spring Cleaning Tips!
Mar 01, 2019
Winter is almost over and it's time to take out the gloves, mop and go to business! Here are some great tips for Spring Cleaning that you can do before or after your Spring Break trip.
No one likes to clean windows, but it definitely makes the view better outside when you have a clean pane to look through. In addition to cleaning your windows with a glass cleaner, also dust blinds, and consider dry cleaning drapers.
Stainless Steel
Fingerprints and grime builds up on stainless steel, but what is the best way to clean it? Spray with a bit of vinegar and using a soft cloth or paper towel, wipe off in the direction of the grain.
Clear clutter off of countertops daily as it will help to keep them clean. The less clutter you have, the easier it is to spray and wipe away bacteria.
To deep clean, spray with a cleaner that is okay for your particular countertop type and wipe away with a soft cloth that will eliminate streaks. For hard-to-reach places, use a clean toothbrush.
Open your dishwasher and make sure all food debris is gone, wipe it clean, and run the dishwasher (without anything in it) through a cleaning phase.
We suggest removing everything from your refrigerator and freezer and giving it a deep clean. This will deter bacteria from growing and keep your food and your family healthier.
For carpets and rugs, either rent or buy a steam cleaner or hire a professional to come in and clean them. For wool carpets and rugs or anything delicate, enlist a professional. For low traffic areas, this can be done every few years.
For hardwood floors regular cleaning should be done, but this time of year give them a deep clean and wax.
Give your oven a good scrub, and utilize the self-cleaning function.
Tiled Surfaces
Spray tiles with a tile cleaner and let set while you clean other areas of the home. Rinse with water and then go back with a soft cloth to wipe away grime and dry.
Your Closets
Go through each of your closets and get rid of anything you (or you family) has not worn for at least a year. Donate what is in your "give away" pile then go through and tidy up what is left.
Washing Machine
After every dry cycle or every few wash cycles, you should be cleaning the lint trap and running the self clean function, but ensure you do both and have a repair man out to do an annual check up to keep machines running smoothly.
Although we should wash our bedding every one to two weeks, our mattress is oftentimes overlooked. Now is the time to strip down the bed and break out the vacuum cleaner to ensure your mattress is as clean as can be. Now is also a good time to turn and flip your mattress to ensure it lasts as long as possible.
Walls, Cabinets, Baseboards, and Woodwork
Grime can build up on these surfaces over time and they are often overlooked. Wipe down and clean painted walls, cabinets, especially in the kitchen where grease can build up, and baseboards and woodwork that can collect dust and are often overlooked.
Just as you did with the refrigerator, clear off all of the contents from your bookshelves and give them a good dusting and cleaning.
Your Cleaning Supplies
You may not think that the very supplies and tools that help you clean need their own cleaning, but they do! Take a vacuum to your broom and mop (or replace both if it is time), and give your vacuum a good cleaning as well. Clean out the filter and wipe down all of the accessories. Also, now is a good time to restock your cleaning supplies if you are running low.
Patio Furniture and Grill
As the weather is warming up, you will want to get outside and enjoy the new season, but before you do, give your outdoor furniture and grill a good cleaning. Wipe everything down, burn off and clean any residue on your grill, and get ready to enjoy the spring and summer months.
Ceiling and Light Fixtures
Ceiling fans and light fixtures collect a lot of dust over time. Get up on a step stool or ladder to dust ceiling fans, bulbs, and fixtures. If need-be remove the light fixture and wash with soap and water to get rid of residue that has built up.
Full article http://www.hvmag.com/16-Fast-and-Easy-Spring-Cleaning-Tips/
Feb 04, 2019
Fun Ways to Celebrate Galentines!
Valentine's Day is almost here, and whether you're single or happily taken, you...
Fun Ways to Celebrate Galentines!
Feb 04, 2019
Valentine's Day is almost here, and whether you're single or happily taken, you can always have an amazing time celebrating with your friends. That's why we're channeling our inner #girlboss Leslie Knope of Parks and Recreation this Galentine's Day. If you can't decide between gathering your girlfriends for a Galentine's Day party or embarking on an epic road trip, here are 15 ideas for how to spend it with your squad.
1. Karaoke Party: What better way to start off your Galentine's Day shindig than by belting out a few tunes with your favorite ladies? Yep, we didn't think we'd have to tell you twice. (Photo via Brit + Co)
2. Nail Decorating Party: Pamper yourself this holiday with a nail design party. Pass a bottle of their favorite polish and this free printable out to your gals as they arrive. (via Studio DIY)
3. Galentine's Day Bake-off: BFFs stand together even when they're single or happily taken. Host a delicious bake-off with your gals to see who can get the most creative in the kitchen. (via Studio DIY)
4. Means Girls Movie Night: Prepare for a cozy night in with your ladies by catching up on one of the most classic flicks. Set the tone for the night with a DIY burn book vase that doubles as a hilarious take-home treat. (via Aww Sam)
5. Puppy-Love Play Date: Galentine's Day isn't just for humans. Invite your BFFs and their furry friends over for a puppy-love playdate. If you want to keep things a bit more relaxed, host a movie night and ask your friends to bring their dog beds too. (Photo via Brit + Co)
6. Beach Day Galentine: If you live on the coast, host your Galentine's Day gathering at the beach. Make sure you bring your favorite beach bag, a pair of sunglasses, and perhaps some bubbly — duh! (Photo via Brit + Co)
7. Road Trip With Your Gals: You don't have to spend the holiday inside. If you're not near the beach, you can take a day trip instead. Some ideas include visiting a ski lodge, going for a hike, or exploring that small town you've been meaning to hit. (Photo via Getty)
8. Wine Tasting Soiree: You and your girls love your wine (the proof is in your Instagram feeds!), so use the day as an excuse to host your very own rosé tasting party. (Photo via Brit + Co)
9. Host a Cooking Party: If you're the known chef in your group, spend the day hosting a cooking party. You can show off your culinary skills and teach your friends a new recipe. (Photo via Brit + Co)
10. Play in the Snow: An adventurous way to get outdoors and spend time with your ladies is to take advantage of the snow. Go skiing or snowboarding, or build a snow(wo)man. (Photo via Brit + Co)
11. Pool Party: If you live in a warmer climate (or have a heated pool), throw a fun G-Day pool party complete with all of the essentials like vino, floaties, and sunhats. (Photo via Brit + Co)
12. Parks & Rec Marathon: The best way to celebrate Galentine's Day is by honoring the creator, Leslie Knope, with a Parks and Rec marathon viewing party. Ask your ladies to supply the booze, and you can serve up the snacks and the show. (Photo via Getty)
13. DIY Party With Planters: Get a jump on spring decor with an easy DIY planter party. With a few basic steps, you and your girls can create living wall decor to decorate your space for February and even all the way through the summer months. (via Brit + Co)
14. The Bachelor Marathon: Calling all Bachelor nation gals! Love is in the air on this ABC reality show. Catch up on past episodes by hosting a marathon viewing party for your favorite rose ceremony show. (Photo via Brit + Co)
15. Outdoor Movie + Dinner: Your ladies deserve a get-together that's sure to give each gal the much-needed fun she deserves. Host an outdoor movie and dinner for your ladies. Step up your party with favors, swag bags, and a night of unlimited drinks. (Photo via Brit + Co)
Check out the original article here: https://www.brit.co/ways-to-celebrate-galentines-day-with-friends/
Jan 03, 2019
New Year, New You!
How to Eat Healthy in College Eat breakfast, even if you wake up at...
New Year, New You!
Jan 03, 2019
How to Eat Healthy in College
Eat breakfast, even if you wake up at lunchtime. Even if you wake up at noon, begin every day with a healthy breakfast that includes a high fiber starch (or fruit) and lean protein or healthy fat.
Not only will it help to help rev your metabolism, stabilize your blood sugar, control your hunger, and boost your energy, but studies have shown (over and over) that students who eat a healthy breakfast do better in their classes. Even if you don't have time to have a "sit down" breakfast, keep healthy items in your dorm room for on-the-go breakfasts. For example:
- Oatmeal packets
- Individual containers of milk (you can even get individual containers of almond milk!)
- Natural peanut butter (also try individual packets of nut butters)
- Fruit (grab a piece from the dining hall to save as a snack later)
- Healthy hot cereal cups such as Earnest Eats (you can always find some hot water!)
- Greek yogurt
- Individual hummus containers
- Individual portions of cheese such as cheese sticks
- Hardboiled eggs (can buy pre-cooked or hard boil a big batch in your dorm's kitchen at once)
- Individual chia seed packs
- Nuts (such as raw cashews, almonds and walnuts)
- For makeshift breakfast ideas, try:
- Hard boiled eggs, cheese stick and a piece of fruit
- Oatmeal, packet of nut butter
- Apple, piece of cheese
- Yogurt, nuts and chia seeds
Navigate the dining hall with healthy finesse. On your way or before you go to the dining hall, think of a "meal game plan". Are you headed to the salad bar or the sandwich line? Are going for the hot entree or the soup station? Whichever you choose, prep yourself before you walk in and stick to the game plan. Treat this like you are prepping to write a paper or studying for your chemistry test. It's just as important and definitely more fun! Some tips for whichever way you go:
- Take the grilled chicken (or burger or veggie burger) off of its bun, and place it on top of vegetables from the salad bar to make a grilled chicken salad.
- Add extra vegetables like beets or roasted peppers from the salad bar to an open faced turkey sandwich or wrap to "beef" it up and make more filling without loading up on more cals.
- If the hot "main" appears uber loaded with some buttery sauce, then skip it, grab the hot veggies only and add lean protein from the sandwich station to your plate.
- Pour cereal in a small coffee cup instead of those family sized bowls and couple it with protein. It's hard to judge how much to pour when cereal is served in huge self-serve dispensers. This will help. Better yet, go for hot cereal like oatmeal which is a better choice than most of the cold cereals in those dispensers.
- Always choose seltzer instead of sodas and juices from the drink dispensers. Those sugary drinks are loaded with calories, which are commonly underestimated. Plus, when free refills are available, one easily turns into two or three, which can easily add an extra 300 sugary calories to your day.
- Don't hang in the dining hall. Use it to eat. Lingering can cause you to eat more than you need just because you are there. Move the social gathering to another spot.
- Don't forget to look around and assess what is really available. Often there are many options that are baked, broiled, steamed, roasted, or grilled. This will make it easier to avoid foods that are fried, sautéed, breaded, crispy, creamed, batter-dipped, or buttered.
Make sure you take exercise 101. When you plan your semester and know your schedule, plug in where and when you will exercise, just as if it were a class you couldn't miss. That's right, treat physical activity in your schedule with the same importance as your classes, study sessions, group meetings, social events, and other important appointments.
Whether you go to the gym, walk to class, join an intramural sports team, or take a fitness class for credit, daily exercise can improve your mood, help relieve stress and anxiety, increase your energy, promote better sleep, and help you maintain your weight.
Keep your caffeine in check. While 1-2 cups of coffee per day are fine, don't depend on soda, coffee or energy drinks as a substitute for sleep. This can actually backfire and cause insomnia, disrupt concentration, and cause restlessness and anxiety!
Instead, try to get adequate sleep (8 hours a night), and take mental breaks while studying to refresh yourself. A good, consistent exercise regimen and consuming nutrient dense snacks throughout the day will help keep your energy up too.
If you're rolling your eyes, then listen to this. Research shows the less sleep you get, the more weight you gain. Yep, the freshman 15 we always blame on late night pizza and beer could actually have a little something to do with college students' crazy sleep habits, too!
Never leave home without healthy snacks. Keep healthy snacks on hand to avoid going to the vending machines. You need snacks to fuel your brain and stabilize your blood sugar.
Set yourself up for success by stocking your 'pantry' and make sure there is always a healthful and non perishable snack in your bag. Ask loved ones to send you healthy care packages and write down the snacks you'd like to receive. They'll love to do it. Promise. Some examples of non perishables that you can keep in your dorm room (in addition to the breakfast items above) are:
- Trail mix (ask mom to pull together your favorite nuts, shredded coconut, dried apricots, sunflower seeds, and dried cranberries, and then portion into ¼ cup containers
- No sugar added dried fruit and dehydrated vegetables
- High fiber crackers
- Seaweed snacks
- Popcorn (invest in an air-popper – even easier than having a microwave and much healthier!)
- Bars (go for nut based bars such as Kind)
- Tea (chamomile, green and mint)
- Beef, tuna or salmon healthy jerky (you can find grass fed beef and wild salmon)
I hope you have a great semester! Prepare yourself to be successful and you will be cum laude in the healthy diet department in no time. Practice the grown up habit of making healthful choices now and they'll set the foundation for when you hit the working world.
But, all that said, enjoy sleeping late, having Fridays off and living with friends as much as possible right now, because you'll miss those collegiate perks after graduation.


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The Premiere Site For Celebrity Plastic Surgery By A Real Plastic Surgeon
I'm a Michigan-based Board Certified Plastic Surgeon who has been featured on Dr. 90210. The info here is my opinion alone and should not be taken as fact or as medical advice. I've not treated any of the celebrities presented here.
Wednesday, June 13, 2007
Mickey Rourke and Plastic Surgery
Our friends at Celebrity Smack blog have done it again. A week or so ago they posted a very interesting photo chronology of Joan Rivers and her plastic surgery. Now they have done the same thing with Mickey Rourke. Where did things go wrong? I'd say around 1995?
Click here for the Evolution of Mickey Rourke.
Click here for my previous posts on Mickey Rourke.
Photo credit: getty images and photorazzi.com
Thanks for reading.
Michigan-based Plastic Surgeon
Anthony Youn, M.D.:
Posted by Dr. Tony Youn at 2:13 PM
Labels: Mickey Rourke
1 comment:
- health watch center said...
Joan Rivers looked best in 2003!
Skin Care Beauty Zone
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subset: commoncrawl


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For no apparent reason I was thinking the other day of one of my former staff from years gone by. A noted procrastinator, he would always respond to my question of when something would be ready with "Wednesday". He did this automatically without even bothering to think what the question had been! I recall telling him one day that there were three great lies in the world:-
The cheque's in the post;
Ready Wednesday;
And ….
I can't for the life of me recall what the third one was.
Thoughts that spring to mind and / or were suggested by Googling the issue are:-
I'm working late tonight, Darling;
Of course, I love you;
Of course, I'll respect you in the morning;
..But we can still be friends;
It's not the money, it's the principle of the thing;
I'm from the government, and I am here to help you;
(One for our mechanic) It's supposed to make that noise;
(for the weatherman) Tomorrow will be bright and sunny;
(for Tesco) This fish is fresh;
(and one for Jo) Then take a left. You can't miss it.
But none of those was my original third greatest lie. Any suggestions?
Black Snow by TheMrKraken
2 hours ago


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Everyone ends up wishing and wanting something in their lives. Whether it be a piece of cake after dinner or a holiday abroad. Everyone wants something on life.
So at Bedroom Taboo, we are forever getting messages from our followers saying how they would love a certain object in their drawer or to try something new.
Well, why not create a Bedroom Taboo Wish List.
- Join Delivery Code.
- Create your profile and Bedroom Taboo Wish List.
- Go to our website and click on an item you would like to add to your list.
- Copy the Web Address and add it to the wish list.
- Share your wish list with someone you want to drop a hint to, or maybe publically for anonymous admirers.
- Someone can purchase an item on your wish list and have it sent to you directly.
How easy is that!??
And if you work in the Adult Industry, email us about promoting your Bedroom Taboo Wish List.
And if you work in the Adult Industry, email us about promoting your Bedroom Taboo Wish List.
Now it is up to you to encourage someone to buy that product you have had your eye on for too long now.


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Welcome to the library blog. You'll be able to see what is happening in this busy place, find resources to help with study and homework, view the books we love, find careers information and have some library fun. The blog is a mix of the interesting, the helpful, the quirky and the fun! Comments are welcome, staff and students can submit reviews and suggestions to Bridget Schaumann, the librarian at firstname.lastname@example.com Our school is located in Dunedin, Otago, New Zealand.
Thursday, March 12, 2015
Sam and Dave Dig A Hole by Mac Barnett and Jon Klassen
This is a book for everyone, a picture book which works for grownups as much as it works for little kids and teenagers of every age. It is the story of Sam and Dave who go digging for something spectacular. A long hard day ensues. And that is about it, but that is not really it. There is lots of funny stuff in the book, lots of quirky little jokes and you have to be clever to see them. I'm a big fan of these authors, they have a special niche in the picture book world, capturing ideas that appeal to kids are logical to kids but spectacularly funny to adults. Below is the book trailer but also a TED talk by Mac Barnett about what it is to write for kids. It is really inspiring and funny and well worth the 20 minutes watch. I'm pretty sure you will enjoy it.
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Hamilton hospitals scramble to 'fill gaps' as departure of Saudi medical residents looms
153 Saudi students at McMaster University are completing medical residencies at Hamilton hospitals
Hamilton hospitals are scrambling to put in place contingency plans to deal with the looming departure of 153 Saudi medical residents.
But they are assuring the public that patient care will not be compromised despite the significant role residents play in delivering patient care.
The students are caught up in the diplomatic tussle which took a turn for the worse on Sunday when Saudi Arabia suspended diplomatic ties with Canada and expelled the Canadian ambassador. The moves came after Foreign Affairs Minister Chrystia Freeland and her department issued tweets criticizing the arrest of social activists and asking for their immediate release.
Among the measures taken by the Saudis was recalling students — including medical students and residents it funds here — to Saudi Arabia.
As Saudi medical trainees ordered home, Canada prepares for potential impact on hospitals
Mumps occurring in Hamilton, public health officials confirm
Dr. Michael Stacey, Professor and Surgeon in Chief at Hamilton General Hospital said the departure of the students will have "a big impact" on healthcare delivery here.
"Losing that number of postgraduate residents and fellows has an impact and obviously we have to look at existing resources and then, if needs be, additional resources to help to fill those gaps," Stacey told CBC Hamilton.
"They're very valuable members of our workforce and provide a very good service to both Hamilton Health Sciences and St. Joseph's Hospital here in Hamilton.
They form an integral part of clinical service, delivering service to patients, so definitely we'd be impacted by that."
Losing that number of postgraduate residents and fellows has an impact.- Dr. Michael Stacey
Stacey said the Saudi students represent about 15 per cent of the postgraduate workforce at Hamilton Health Sciences (HHS) and St. Joseph's Hospital.
The residents work a minimum of 40 hours weekly, and often more with their on-call commitments, he said.
"Some of them come for shorter periods but overall it's about the equivalent of 126 fulltime workers," Stacey said, adding that their departure would "have a big impact for sure."
'We remain hopeful that a resolution will be found'
McMaster University says it is supporting the Saudi students during this time of uncertainty.
While they remain hopeful that a resolution will be found, a spokesperson for the university said it also understand this is a time of great uncertainty and stress for students.
"It is a fluid situation and we are working with fairly limited information, but the university is doing its best to provide them with help," communications director Gord Arbeau said in a e-mailed statement.
Stacey agrees that the situation has had a big impact on the students, who were still on the job on Thursday.
"Obviously they're in training and this impacts both their training but also has a big impact on their families as well," he said.
"The concern is should this requirement for them to return to Saudi remain in force, then obviously that is going to take more of a toll on them as this month continues on."
Contingency measures still being worked out
Stacey said should the residents be removed, the biggest concern is to make sure that there is a continuity of care for patients.
"We're trying to gage the impact and the areas that would be most impacted and then we're working on contingency measures to ensure that patients can continue to get good care and that's obviously a key priority at this point," Stacey said.
"In the next couple of days we'll have a clearer picture of where we'll have most impact and then we'll be working with both McMaster University and St. Joseph's Healthcare to work out how we're going to manage that."
Patient care remains a 'key priority'
In the face of the uncertainty, Stacey is assuring that patient care will not be compromised, even though he could not give specifics about how they would make up for the shortfall on medical personnel.
"We definitely will continue to provide care to our patients, so I think we can give patients the reassurance that will happen. How we do it and exactly the process I can't give you specifics at this time, but we absolutely can give that assurance to patients," he said.
We will rise to the challenge
At St. Joseph's Healthcare in Hamilton, a similar situation exists.
Chief executive officer Dr. Tom Stewart said they always have plans in place should there be a crisis, but he admits that the current situation is a unique one.
"This is new to us . . . but there is no doubt we are going to have to come up with some contingency plans which will disrupt our normal workflow, but will not disrupt the good care that we give to patients," he told CBC Hamilton.
"I don't think that there is risk to patients who show up at the hospital in terms of getting great care, but there will be a trickle down effect. Our academic physicians will likely have to do more things that residents were doing. So there will be a ripple down effect across the healthcare workers but other people will step up to the challenge and do the job that these individuals were doing.
We have to be, in healthcare, always planning for the unexpected and this is an example of an unexpected that may be more prolonged than usual. So it will be a challenge but we will rise to it," Stewart added.


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The 3D solution structure of the GCC‐box binding domain of a protein from Arabidopsis thaliana in complex with its target DNA fragment has been determined by heteronuclear multidimensional NMR in combination with simulated annealing and restrained molecular dynamic calculation. The domain consists of a three‐stranded anti‐parallel β‐sheet and an α‐helix packed approximately parallel to the β‐sheet. Arginine and tryptophan residues in the β‐sheet are identified to contact eight of the nine consecutive base pairs in the major groove, and at the same time bind to the sugar phosphate backbones. The target DNA bends slightly at the central CG step, thereby allowing the DNA to follow the curvature of the β‐sheet.
Expression of pathogenesis‐related proteins (Bowles, 1990) is induced in plants by stimuli including UV, salicylic acid and ethylene (Green and Fluhr, 1995), and leads to a defence response by inhibition of bacterial and fungal growth (Hammond‐Kosack et al., 1996; Ryals et al., 1996). A consensus nucleotide sequence, AGCCGCC, known as the GCC‐box, has been identified in the promoter region of the pathogenesis‐related genes (Ohme‐Takagi and Shinshi, 1995). Four cDNAs coding ethylene‐responsive element binding proteins (EREBPs) from tobacco have been isolated, which specifically bind to the GCC‐box (Ohme‐Takagi and Shinshi, 1995). Equivalents of EREBPs, AtERF1‐4 (M.Ohta, H.Shinshi and M.Ohme‐Takagi, unpublished results) and AtEBP (Büttner and Singh, 1997), have been identified in Arabidopsis thaliana.
A region of ∼60 amino acid residues (Figure 1B) is highly conserved among EREBPs (Jofuku et al., 1994; Ohme‐Takagi and Shinshi, 1995). The region is referred to here as the GCC‐box binding domain (GBD). A large number of divergent genes in a wide range of plants contain the GBD domain (Weigel, 1995; Elliott et al., 1996; Klucher et al., 1996; Wilson et al., 1996; Okamuro et al., 1997). No animal or fungal protein has been reported to possess a GBD.
In this paper we describe the solution structure of the AtERF1 GBD in complex with the target DNA, and discuss the mode of the specific interaction of the two molecules. The GBD binds to the DNA via its β‐sheet. This is somewhat unexpected, since this study reveals that GBD has an arrangement of secondary structural elements which resembles that of zinc fingers to some extent, although it is the α‐helices of zinc fingers that are known to bind to DNA. The mode of DNA binding of the GBD β‐sheet is different from that of the β‐sheets of other known DNA‐binding domains in the number and arrangement of β‐strands, in the contacting pattern of amino acid residues and DNA bases, and in the number of base pairs contacted. The solution structure of GBD determined in the absence of DNA is also reported to enable better understanding of the structural requirements imposed on the protein structure for DNA recognition.
Three dimensional (3D) structure of GBD
The solution structure of the AtERF1 GBD, Lys145‐Val206, in the absence of DNA was determined using 1187 unambiguous conformational constraints (Table I) obtained from multidimensional nuclear magnetic resonance (NMR) experiments. Forty‐six structures were selected after simulated annealing, which showed the smallest violations of the constraints (Figure 2A). The average root mean square (r.m.s.) deviation among the selected structures was 0.54 Å for the heavy atoms in the backbone, His146‐Pro203 (Table I). The final structure (Figure 2B) was determined by energy minimization of the mean co‐ordinates of the ensemble. All the residues were identified in the favored or additionally allowed regions in the (φ, ψ) space (Morris et al., 1992).
The determined GBD structure consists of a three‐stranded anti‐parallel β‐sheet comprising strand 1 (Val149‐Arg152), strand 2 (Lys156‐Asp163) and strand 3 (Ala169‐Phe176), packed along an α‐helix (Thr178‐Arg194) (Figure 1A). The structure is stabilized by extensive hydrophobic contacts of the side‐chains of Tyr146, Val149, Phe157, Ala159, Ile161, Val171, Leu173, Phe176, Ala179, Ala182, Ala183, Ala185, Tyr186, Ala189, Ala190, Ala198, Leu200 and Phe202 with each other. The geometry of the α‐helix relative to the β‐sheet appears to be determined by the interaction of the many Ala residues in the α‐helix and the larger hydrophobic residues in the β‐sheet, in particular, Phe157, Phe176, Val171 and Ile161, which clamp the α‐helix at the four corners. The N→C direction of the α‐helix is approximately parallel to that of β‐strand 2. This arrangement is slightly unusual, since an α‐helix is typically tilted slightly with respect to β‐strands (Janin and Chothia, 1980).
Turn 1, Arg152‐Lys156, between β‐strands 1 and 2 is classified to type 3:5, and turn 2, Asp163‐Ala169, between strands 2 and 3 is classified to type 5:5 (Sibanda et al., 1989). The N‐terminus of the α‐helix is capped by hydrogen bonds to and from Thr178. The N‐terminal loop, His145‐Gly148, is better defined than the C‐terminal loop, Gly195‐Pro203 (Figure 1A).
DNA recognition by GBD
The solution structure of the GBD–DNA complex (Figure 3B) was determined by simulated annealing by using NOE constraints obtained from NMR spectra (Figure 3A) followed by restrained molecular dynamic (rMD) calculation (see Figure 4D for the nucleotide sequence and the scheme for the numbering of the bases).
The 25 structures selected after the simulated annealing (Figure 3A) had an average r.m.s. deviation of 0.61 Å for the heavy atoms in the protein backbone, His146‐Pro203. The average r.m.s. deviation for all the heavy atoms in the polypeptide, His146‐Pro203, nucleotides 3–11 in the coding strand, and nucleotides 16–24 in the complementary strand, was 1.20 Å.
During the rMD calculation the van der Waals energy term in the AMBER force field decreased by 684 kcal/mol. The average inter‐base pair rise parameter in the GBD–DNA complex before the rMD calculation had a value of 4.02 ± 0.26 Å. In contrast the structure after the rMD calculation possessed a value of 3.21 ± 0.25 Å, which was much closer to the values in the known crystal DNA structures. The improvement was due to consideration of the effects of water molecules, which resulted in the hydrophobic effects of base‐stacking being included in the rMD calculation.
The orientation of the protein surface with respect to the target DNA was determined unambiguously on the basis of 37 intermolecular nuclear Overhauser effect (NOE) connectivities identified in homonuclear two dimensional (2D) NOE spectroscopy (NOESY) spectra: Arg162 to T16 and G17, Arg150 to C19, Trp154 to T3, A4 and C22, Trp172 to A4, G5 and C6, and Thr175 to A4 (Figures 5C and 6). The intermolecular cross‐peaks were observed isolated from the other peaks in two sets of NOESY spectra measured at different temperatures, 298 and 303 K, and were assigned to the same protein–DNA connectivities.
By using 13C, 15N‐labeled GBD in the complex with the unlabeled DNA, the 37 intermolecular NOE connectivites were studied further (Figure 7). Splitting of cross‐peaks was expected in one of the two frequency dimensions only for intermolecular NOE connectivities (see Materials and methods). The splitting was observed for 16 out of 37 connectivities. For the rest (21 connectivities) the intensities of the cross‐peaks were too weak for examination. The splitting of the 16 peaks was terminated by decoupling of the heteronuclear spin connectivities (Figure 7C), which was further evidence that the peaks were those of intermolecular connectivities.
The GBD binds to the major groove of the GCC‐box via the three‐stranded β‐sheet. The N→C direction of strand 2 is approximately parallel to the 5′→3′ direction of the coding strand (Figure 3). Only one residue from the α‐helix, Tyr186, is identified as binding to a phosphate, while no contact is identified to the bases.
The guanidyl groups of four arginine residues are identified to contact five guanine bases through hydrogen bonds (Figure 5B); Arg150 to O6 of G20, Arg152 to O6 and N7 of G5 and O6 of G21, Arg162 to N7 of G17, and Arg170 to N7 of G8. The stems of the arginine side‐chains make hydrophobic interactions with pyrimidine bases; Arg150 to C19, Arg170 to C7, and Arg162 to T16. The side‐chain Oϵ of Glu160 is positioned close to HN4 of C19. Although the distance, 3.11 Å, is slightly larger than would be expected for a standard hydrogen bond, no space remains for accepting a water molecule. The aromatic rings of Trp154 and Trp172 make hydrophobic interactions with T3 and A4, and G5 and C6, respectively. These interactions cover six base pairs in the conserved AGCCGCC sequence and thus serve as the specific recognition of the GCC‐box by GBD.
All the Arg and Trp residues which contact the bases also bind to the sugar phosphate backbones either by ionic interactions to the phosphates or by hydrophobic interactions to the sugars, with the exception of Arg152 which does not bond to the backbone: Arg150 to C19 phosphate; Arg162 to G17 phosphate and T16 sugar; Arg170 to C7 phosphate; Trp154 to T3 sugar and A4 phosphate; and Trp172 to G5 phosphate and G5 sugar. Certain other residues bind to the sugar phosphate backbones: Lys156 to A4 phosphate; Thr175 to G5 phosphate and A4 sugar; Arg147 and Tyr186 to G17 phosphate; and the backbone amide of Gly148 to G18 phosphate. These interactions determine the geometry of GBD relative to the DNA and thereby comprise a framework for the specific base recognition.
Although some of the intermolecular contacts identified after the rMD calculation were not formed correctly before the rMD calculation, i.e. one of the four ionic contacts and eight of the 13 hydrogen bonds, the deviation of the candidates in question from the criteria used for identifying contacts was small. In one of the eight hydrogen bonds the distance criterion was satisfied, while in the other seven hydrogen bonds and in the single ionic contact in question the deviation from the distance criteria was 0.74 Å on average. Only two of the eight hydrogen bonds did not fully satisfy the angle criterion; the excess being ∼44.7–45.6°.
For the final complex structure around the eight hydrogen bonds that were not correctly formed before the rMD calculation 33 intermolecular NOE connectivities are expected, where the intermolecular distances between the protons are <4 Å. Here connectivities that involve protons whose resonances are not expected to be observed at neutral pH are not included in the calculation. In the NOESY spectra 16 of the 33 were identified, i.e. the identification rate was 49%. Comapred with a similar rate of identification of intramolecular NOE connectivities calculated for the well‐determined core of GBD, 58%, the value appears to be acceptable to justify the formation of the hydrogen bonds.
Changes in the two molecules upon binding
The overall structure of the DNA in the complex is close to the standard B‐DNA structure, with the r.m.s. difference of 0.62 Å, but it is kinked by ∼20° around the major groove at the CG step (Figure 4A). The roll value of the CG step is the largest among the steps, while the helical twist value is the smallest (Figure 4C). The width of the major groove around the CG step is slightly narrower than that expected for the standard B‐DNA, while the minor groove is distinctively wider (Figure 4B). These observations are consistent with the previous analysis of the DNA bending at pyrimidine–purine steps in crystal structures (Suzuki and Yagi, 1995a; Suzuki et al., 1997).
The plot of the major groove width reveals a W‐shaped feature around the CG step (Figure 4B). This seems to be a consequence of two contradictory requirements; namely, a primary requirement that the major groove needs to become narrower globally to allow a better fit around the β‐sheet, which is carried out by the rolling of the CG step in the center, and a secondary requirement that the β‐sheet is widest around Ala159, which is close to the CG step (Figure 5D), and thus the groove becomes wider locally and approaches its standard size around the step.
To confirm the DNA bending the rMD calculation was repeated twice more by replacing the initial DNA structure by the straight standard B‐conformation and the standard B‐conformation but modeled to bend to a large degree by inserting an untwisted‐rolled CG step found in a crystal DNA structure (Hegde et al., 1992), respectively. The two rMD calculations resulted in structures very similar to those described above (Figure 4B and C).
The overall GBD structure does not change much upon binding to the target DNA (Figure 1A). The average r.m.s. difference between the mean structure determined in the complex with the target DNA and that determined in the absence of the target DNA is 0.78 Å for the backbone heavy atoms, His146‐Pro203, and 1.38 Å when the side‐chain heavy atoms were included. These values are comparable with the average r.m.s. deviation in each of the two ensembles (Table I). Locally, turn 1 bends slightly towards the DNA, thereby enhancing the concave curvature of the DNA‐binding surface (Figure 1A, inset).
Higher disorder is observed at turn 1, turn 2 and the C‐terminal loop in the GBD structure (blue and red lines in Figure 1A) compared with that observed in the GBD–DNA complex. At turn 2 the difference between the two mean structures is subtle (black line in Figure 1A) with only a slight change in the disorder (compare blue and red lines in Figure 1A). However, at turn 1 disorder decreases upon DNA binding, and the r.m.s. difference between the two mean structures is more substantial. It is likely that the changes observed are induced either for or by the binding of Arg152, Trp154 and Lys156 to the DNA.
Comparison of GBD with some other DNA‐binding domains
The DNA‐binding domains of MetJ and Arc repressors interact with the target DNA via β‐sheets (Breg et al., 1990; Somers and Phillips, 1992; Raumann et al., 1994). The β‐sheets are composed of two identical strands, one from each subunit, and form the dimerization interface. Their target nucleotide sequences are essentially palindromic. In contrast, GBD is monomeric, its β‐sheet is three‐stranded, and it interacts with the nucleotide of a non‐palindromic sequence. In the β‐sheet of GBD many residues contact both bases and the backbones, while in Arc and MetJ repressor residues are differentiated for the two types of function. Bases in six consecutive base pairs can be recognized by a β‐sheet of the MetJ–Arc type (Suzuki, 1995), while bases in nine consecutive base pairs are recognized by the GBD β‐sheet. Thus, the interaction of GBD with the target DNA reported here represents a novel mode of protein–DNA interaction.
The first zinc finger of SWI5 (Dutnall et al., 1996) has the same arrangement of the secondary structural elements as that in GBD. However, zinc fingers bind to the DNA via the α‐helix and not the β‐sheet. Calculations of electrostatic potentials suggest that the combination of positive charges on the β‐sheet and negative charges on the α‐helix in GBD (Figure 2B), and that of negative charges on the β‐sheet and positive charges at the N‐terminus of the α‐helix in the zinc finger (Figure 2C) are important for creating the different DNA‐binding mechanisms. All zinc fingers of the C2H2 type are substantially smaller than GBD (compare Figure 2B and C). Zinc co‐ordination to the four amino acid residues can stabilize the core of zinc fingers as effectively as the extensive hydrophobic interactions in the core of GBD.
Comparison of the DNA‐binding modes of the β‐sheets
In GBD, β‐strand 2 is twisted slightly at Ala159 to follow strand 3 (Figure 5D), whose biphasic regularity is disturbed by the insertion of Gly174; Ala159 makes hydrogen bonds with Leu173 and Gly174. It has been reported that mutation of Gly174 in another GBD caused malfunction of the protein (Jofuku et al., 1994). In one half of the protein–DNA interaction site, downstream of Ala159, strand 3 contacts the bases in the coding strand, while strand 2 contacts bases in the complementary strand (Figure 5D). In the other half, upstream of Ala159, strand 1 contacts the bases in the both strands, but strand 2 contacts no base. At no point do the three strands fit side by side in the center of the major groove as strands 2 and 3 in the upstream half are separated slightly from the groove (Figure 3D). Consequently, the DNA groove accepting the three‐stranded β‐sheet is not much wider or smaller than that accepting two‐stranded β‐sheets of MetJ–Arc (Suzuki and Yagi, 1995b).
A two‐stranded β‐sheet is not totally flat but has a curvature. Thus, there are essentially two ways of placing a two‐stranded antiparallel β‐sheet in the DNA groove; in one mode the convex side of the β‐sheet faces the DNA, while in the other way it is the concave side (Suzuki, 1995). If the N→C direction of each strand follows the 5′→3′ direction of the nearest nucleotide strand the β‐sheet forms a concave mode, but if the N→C direction of each strand follows the 3′→5′ direction of the nearest nucleotide strand, the β‐sheet forms a convex mode. A convex mode, which is used by the MetJ–Arc family, is suitable for contacting bases since the major groove is concave (Figure 3C), but it is unable to follow the groove for more than six consecutive base pairs as it does not curve around the DNA helix axis (Figure 5E). In contrast, a concave mode is appropriate for following the DNA backbone, which is convex if viewed along the double helix axis (Figure 3D). However, the concave mode prevents a β‐sheet completely entering the groove, and hence residues are unable to contact the DNA bases effectively (Suzuki, 1995). The concave mode has been found in β‐sheets which bind to the minor groove, which is shallower than the major groove, by following the DNA backbones (Vis et al., 1995; Rice et al., 1996).
A convex mode is observed in the downstream half of the GBD β‐sheet (Figure 5D), while the β‐sheet is extended upstream by adopting a concave mode, where strand 2 and irregular strand 3 bind to the DNA via the sugar phosphate backbone, creating a curvature appropriate for following the DNA (Figure 5D). Hence, the GBD β‐sheet represents a better design by combining the different modes in two halves, resulting in contact with a larger number of base pairs. The curvature of the β‐sheet closely follows the DNA helix axis (Figure 3D as well as closely fits into the major groove (Figure 3C).
Implication for GBD–protein interaction
Many amino acid residues are identical among the GBD sequences and most of the changes are conservative (Figure 1B). To confirm the formation of essentially the same 3D structure by other GBDs, GBD of C‐repeat/DRE binding factor 1 (CBF1) protein (Stockinger et al., 1997) was modeled. The side chains in the AtERF1 GBD were replaced with those present in CBF1 followed by molecular dynamic simulation in water (M.Tateno, K.Yamasaki and M.Suzuki, unpublished results). The same arrangement of the secondary structural elements was observed in the modeled CBF1 structure; the average r.m.s. difference between the modeled and original structures was 2.59 Å.
In a previous discussion (Okamuro et al., 1997) the GBD sequence was divided into two halves, the YRG element in the N‐terminal half and the RAYD element in the C‐terminal half, and it was predicted that the RAYD element would fold into an α‐helix. The two elements do not, however, exactly correspond to the β‐sheet and the α‐helix, respectively, as reported in this paper. The YRG element corresponds to the N‐terminal loop and strands 1 and 2, while the RAYD element corresponds to strand 3, the α‐helix and the C‐terminal loop. Two alternative possibilities have been discussed for the function of the predicted α‐helix of the RAYD element, DNA‐binding or interaction with another protein (Okamuro et al., 1997).
Amino acids of the AtERF1 GBD identified to contact DNA bases are well conserved among the GBD sequences. Thus, it is likely that the DNA‐binding specificity of the known GBDs is essentially the same. In the GBD–DNA complex many amino acid residues bind to both bases and the sugar phosphate backbones. Alternation of these residues could cause truncation in the binding geometry. Thus, it seems more difficult to design a DNA‐binding domain of different binding specificity on the basis of the GBD structure than that of some other DNA‐binding domains.
The majority of the conserved residues in the GBD sequences can be classified into two categories: those that stabilize the protein structure, and those that are responsible for DNA recognition. However, a few residues appear to have no attributable function. Three such residues, Asp187, Arg194 and Phe202, are accessible to the solvent. It is conceivable that the α‐helix or the C‐terminal loop might be used for interaction through these residues with another domain, either in the same protein or another protein. In vitro studies have demonstrated the ability of AtEBP to interact with some other transcription factors (Büttner and Singh, 1997).
Materials and methods
Preparation of the protein and the DNA
The GBD region of the AtERF1 gene, corresponding to Gly143‐Glu210, was cloned into vector pAF104 (Fukuoh et al., 1997) and expressed by the Escherichia coli strain BL21(DE3). For isotope‐labeling, K‐MOPS minimal medium (Neiderhardt et al., 1974) containing 10 mM 15N‐labeled NH4Cl and/or 0.2% (w/v) [13C]glucose was used for the culture. The protein was purified by cation exchange chromatography (Pharmacia Resource™ S) and gel filtration (Superdex™ 75). By electrospray mass spectrometry it was indicated that the N‐terminal methionine had been removed, possibly after translation. The GBD and the GBD–DNA complex were dissolved into a 50 mM potassium phosphate buffer (pH 5.0 or 6.0) containing 40 mM KCl in the different D2O/H2O ratio, depending on the type of the NMR measurements to yield 2.5 and 1.5 mM solutions, respectively.
Spectroscopic measurements
The NMR spectra were recorded on Bruker DMX‐750 (750.13 MHz for 1H, 188.64 MHz for 13C and 76.02 MHz for 15N) and DMX‐500 (500.13 MHz for 1H, 125.75 MHz for 13C and 50.68 MHz for 15N) spectrometers at 283–303 K; 2D NOESY (Jeener et al., 1979), 2D total correlation spectroscopy (TOCSY) (Braunschwieler and Ernst, 1983), 2D double‐quantum‐filtered (DQF) correlation spectroscopy (COSY) (Braunschwieler et al., 1983), 2D 1H–15N HSQC (Bodenhausen and Ruben, 1980), 3D 1H–15N NOESY‐heteronuclear multiple‐quantum coherence (HMQC) (Marion et al., 1989a), 3D 1H–15N TOCSY–HMQC (Marion et al., 1989b), 3D HNCA (Ikura et al., 1990) and 3D HN(CO)CA (Bax and Ikura, 1991). Quadrature detection in indirect dimensions was carried out by the time‐proportional phase incrementation method (Marion and Wüthrich, 1983). During the detection of amide protons, GARP1 15N‐decoupling (Shaka et al., 1985) was used. The mixing time chosen for TOCSY was 50 ms, and for NOESY, 25–150 ms. Spectra were referenced relative to external sodium 2, 2‐dimethyl‐2‐silapentane‐5‐sulfonate for proton and carbon signals, or liquid ammonium for that of nitrogen.
The resonances were assigned using sequential inter‐unit NOE connectivities by the standard procedures (Wüthrich, 1986). The 13Cα–15N‐coupling connectivities observed in the HNCA and HN(CO)CA spectra were also used for the sequential assignment. In the NOESY spectra of the complex the resonances due to the protein moiety and those due to the DNA moiety were identified and separated from each other using the 13C‐ and 15N‐filtration method (Otting and Wüthrich, 1990; Ikura and Bax, 1992). Approximately half of the Hβ resonances were assigned stereospecifically by analyzing the NOESY and TOCSY spectra (Wüthrich, 1986).
Intermolecular NOE connectivities were identified by homonuclear NOESY spectroscopy (Figures 6 and 7) to the same protein–DNA connectivities at two different temperatures, 298 and 303 K. The intermolecular NOE connectivities were further studied by using 13C, 15N‐labeled GBD in the complex with the unlabeled DNA. Intermolecular NOE cross‐peaks were expected to be split in one of the two frequency dimensions in homonuclear 2D NOESY spectra, since the resonances of protons which were covalently bonded to 13C or 15N in GBD would be split by the coupling effects of 13C or 15N spins, while such splitting was not expected with the resonances of protons in the unlabeled DNA.
Amide hydrogen–deuterium exchange experiments were carried out at 283 K (pD = 5.0) in the absence of the DNA, and at 298 K (pD = 6.0) in the complex with the DNA, in order to identify hydrogen bond donors. Thirty hydrogen bond donors were identified for GBD in the absence of the DNA. For GBD in complex with DNA, 30 hydrogen bond donors were identified, among which 28 were the same as those identified in GBD in the absence of DNA.
Determination of the 3D structure of GBD in the absence of the DNA
All the spectroscopic characteristics suggested that Gly143 and Asn207‐Glu210 in GBD were unfolded. Thus, residues Lys144‐Val206 were included in all calculations.
The distance constraints derived from the NOESY spectra were classified into four categories corresponding to inter‐proton distance constraints of 1.8–2.8, 1.8–3.5, 1.8–5.0, and 1.8–6.0 Å, respectively. To the protons whose resonances were not assigned stereospecifically, i.e. all the protons in the methyl groups, and some protons in the methylene groups and the aromatic rings of Phe and Tyr, the constraints were imposed by using the <r−6> averaged distances from all the identical protons in the groups or rings (Brünger et al., 1986). To maintain a hydrogen bond, a constraint of 1.5–2.5 Å was imposed on the distance between the hydrogen and the acceptor oxygen, while another constraint of 2.5–3.5 Å was imposed on the distance between the donor nitrogen and the acceptor oxygen. A force constant of 50 kcal/mol/Å2 was used in order to impose the distance constraints outside the allowed regions using the method of Brünger (1992).
Five torsion angle constraints were obtained by HMQC‐J (Kuboniwa et al., 1994) and were imposed on φ angles in GBD in the absence of the DNA. The angle constraints were classified into three categories, −120 ± 60°, −120 ± 50°, and −120 ± 40°, corresponding to the 3JαN coupling values, <7.5, 7.5–8.5 and >8.5 Hz, respectively. A force constant of 200 kcal/mol/rad2 was used following the method of Brünger (1992).
Dynamic simulated annealing (Nilges et al., 1988a,b) was carried out using the algorithm implemented in the X‐PLOR package (Brünger, 1992). Eighty initial structures were created in the form of random arrays of atoms (Nilges et al., 1988a). Forty‐six structures were selected, where no distance violation was >0.2 Å and no torsion angle violation was >3°; the rate of acceptance was 57.5%. The mean co‐ordinates of the ensemble was submitted to 400 cycles of energy minimization (Powell, 1977). Differences between the 46 structures were characterized by calculating the average r.m.s. deviations of the co‐ordinates from the unminimized mean structure of the ensemble (Table I).
The simulated annealing was repeated twice. In the first round no hydrogen bond constraints were imposed. Of the 54 structures accepted after the first round, candidates for the acceptors were identified using part of a program NAOMI (Brocklehurst and Perham, 1993) for the hydrogen bond donors that were identified by the H–D exchange experiments. When two or more candidates of acceptors were found for the same donor in different structures, the most frequently occurring candidate was selected. In all such cases the pairs selected possessed the most favorable pseudo‐energy values. A hydrogen bond donor, the δ nitrogen of Asn201, was identified to form two hydrogen bonds by using both of the two hydrogens to the same acceptor atom, producing three constraints. For the 30 hydrogen bond donors identified by the H–D exchange experiments, 61 constraints were made. The hydrogen bond constraints were imposed in the second round of the simulated annealing in order to maintain the selected donor–acceptor pairs. The method used was essentially the same as that of Kalia et al. (1993).
Determination of the 3D structure of the GBD–DNA complex
Nucleotides 1–13 in the coding strand and 14–26 in the complementary strand were included in the simulated annealing of the complex. In the rMD calculation, nucleotides 3–13 in the coding strand and 14–24 in the complementary strand were included, since the upstream end of the DNA was not contacted by the protein and a meaningful experimental constraint was not observed with the end section. Residues Lys144‐Val206 of GBD were included in all the calculations.
The simulated annealing of the GBD–DNA complex was carried out essentially in the same way as in that of GBD in the absence of the DNA. The initial structures of DNA were random arrays of atoms, as were the initial structures of GBD. In addition to the 1632 experimental constraints the following theoretical constraints were imposed: 72 constraints on the intra‐base pair hydrogen bonds in order to maintain the base‐pairing (Werner et al., 1995) with a force constant of 200 kcal/mol/Å2; 65 constraints on the rotation of bases around the intra‐base pair hydrogen bonds in order to ensure good stereochemistry in terms of propeller‐twisting and base‐rolling (Omichinski et al., 1997) with a force constant of 200 kcal/mol/rad2; and 122 constraints on the rotation of the atoms around the covalent bonds in the DNA backbones in order to prevent problems associated with the mirror image (Omichinski et al., 1993) with a force constant of 200 kcal/mol/rad2.
Starting with 200 random structures, 25 structures were refined, where no distance violation in the protein structure was >0.2 Å, no distance violation in the DNA structure was >0.3 Å, no GBD–DNA distance violation was >0.3 Å, and no torsion angle violation was >3°. Although the rate of accepting structures after the simulated annealing of the complex of 12.5% was different from that of GBD in the absence of the DNA (57.5%), the calculation algorithm and the criteria for the selection of structures were kept essentially the same. In fact, the r.m.s. deviation values of GBD structures in the two ensembles are found to be similar, i.e. 1.21 ± 0.09 Å for all the heavy atoms of GBD in the complex with the DNA, and 1.32 ± 0.21 Å for those of GBD in the absence of DNA (Table I). The difference between the two acceptance rate values is likely to be due to the difference in molecular weight.
After energy minimization in the AMBER force field the total energy value of the minimized mean structure became significantly lower than the smallest among the values calculated with the ensemble structures. The minimized mean structure was submitted to the rMD calculation.
The rMD calculation was carried out by using the force field (Cornell et al., 1995) implemented in the AMBER package (Pearlman et al., 1994) by imposing only the NOE distance constraints and not by imposing a torsion angle constraint or a hydrogen bond constraint. A force constant of 30 kcal/mol/Å2 was used in order to impose the NOE constraints. The net charge of the protein and the DNA were neutralized by counterions. Approximately 5000 water molecules were modeled by the TIP3P method (Jorgensen et al., 1983). Approximation of the electrostatic force by truncated Coulombic potentials, which ignores interactions beyond a chosen cut‐off length, can distort the molecular system significantly. In this study, long‐ranged electrostatic forces were taken into consideration by the particle mesh Ewald method (Darden et al., 1993). The lengths of covalent bonds from hydrogens were kept constant using the SHAKE method (van Gunsteren and Berendsen, 1977).
Trajectories were generated every 0.002 ps under the pressure of 1 atm. For the first 20 ps the co‐ordinates of the atoms in the protein and the DNA, and of the counterions were kept the same, while only the co‐ordinates of the atoms of the water molecules were changed. Temperature was kept constant at 300 K. For the following 6 ps only the co‐ordinates of counterions and water molecule atoms were changed for every 2 ps at 100, 200 and 300 K. For the following 8 ps the calculation was continued by changing the co‐ordinates of all the atoms every 2 ps at 100, 150, 200 and 250 K. The calculation was further continued for 302 ps at 300 K. The co‐ordinates of 200 complex structures were selected, that were produced at each 1 ps during the last 200 ps, and the mean co‐ordinates were subjected to energy minimization.
Analysis of the structures
The secondary structural elements in GBD were identified on the basis of NOE profiles characteristic of the elements and the observed protection of the amide protons from the exchange to deuterons (Wüthrich, 1986). The φ and ψ dihedral angles were analyzed using the Procheck–NMR program (Laskowski et al., 1996). Diagrammatic representations were made using MOLSCRIPT (Kraulis, 1991). Electrostatic potentials were essentially calculated by the method of Guenot et al. (1994). Parameters for describing the geometry of dinucleotide steps in the DNA were calculated using a program from Babcock et al. (1994). The widths of the major and minor grooves were calculated according to the method of Suzuki and Yagi (1996).
The criterion used in this study for identifying an ionic interaction between Arg or Lys and a DNA phosphate was a distance of <5.0 Å from the side‐chain HN to the phosphate oxygen (Billeter et al., 1993). The criteria for an intermolecular hydrogen bond was a donor–acceptor distance of <3.4 Å and a donor–proton–acceptor angle of >110°, while the criterion for a hydrophobic interaction was a C–C distance of <4 Å (Chuprina et al., 1993). The criteria used for identifying hydrogen bonds in the protein were the same as those described by Brocklehurst and Perham (1993).
The co‐ordinates of the structures determined and a list of the experimental constraints have been deposited in the Protein Data Bank (Bernstein et al., 1977). The accession code for the final structure of the GBD–DNA complex is 1GCC, while those for the GBD in the absence of DNA is 2GCC for the minimized mean structure, and 3GCC for the ensemble after the simulated annealing.
We thank Drs M.Iwakura and T.Takenawa for mass spectrometry measurements. This work was supported by the Center of Excellence, COE, program of the Science and Technology Agency, and the Core Research for Evolutional Science and Technology, CREST, program of the Japan Science and Technology Corporation.
NMR spectroscopy
- Copyright © 1998 European Molecular Biology Organization


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Los Angeles County sheriff's deputies shot and killed two men during an early morning confrontation in East Los Angeles, authorities said.
The incident began about 2:30 a.m. when a 911 caller reported that a man in a vehicle occupied by three people had pointed a handgun at him, sheriff's Lt. John Corina told KTLA-TV Channel 5.
Deputies followed the vehicle to the 5300 block of Verona Street, where the vehicle then pulled into a driveway, authorities said.
Deputies approached the car and at some point opened fire, killing two men inside the vehicle, authorities said. At least one other suspect involved in the incident remains at large.
No deputies were injured in the incident.
It was not clear what prompted the shooting. No further details were immediately available.
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After two years of roster purging and payroll slashing, the Knicks got their first big payoff Monday and could hardly contain their optimism — rules, contracts and official signing dates notwithstanding.
Amar'e Stoudemire was on the Madison Square Garden marquee, wearing a white Knicks jersey with a No. 1 on the chest. His image filled electronic billboards across the premises. A large banner inside bore a slogan, "Stat City," that played on his nickname, STAT (Standing Tall and Talented).
Finally, early Monday night, Stoudemire emerged with a blue Knicks cap on his head and proclaimed, "The Knicks are back."
It will be two more days before Stoudemire can sign a contract, after the N.B.A. lifts its annual moratorium on deals. But his handshake agreement — on a five-year contract worth about $99.7 million — was enough for team officials to start a small celebration.
Stoudemire, 27, is a five-time All-Star, a prolific scorer and a supreme athlete who, along with Steve Nash, anchored elite Phoenix Suns teams. He is the most talented player to wear a Knicks uniform in more than a decade, since Patrick Ewing's decline.Continue reading the main story
"The best thing about him is he will accept the challenge, and he will not shy away from it," said Coach Mike D'Antoni, who coached Stoudemire for five seasons in Phoenix.
The other best thing about Stoudemire, Knicks officials hope, is his ability to recruit elite free agents. The Knicks are courting LeBron James and Dwyane Wade, who are expected to make a decision in the next few days.
James made no public statements Monday as he attended a basketball clinic in his hometown, at the University of Akron. He is weighing offers from six teams. Wade met Monday with the Miami Heat owner, Micky Arison.
The Knicks have generally been viewed as a long shot to sign either player, because their roster is young, thin and generally unproven. Stoudemire's presence means that Wade and James would no longer have to worry about lifting the Knicks back to respectability alone.
"This could help get the dominoes falling," D'Antoni said. "He'll be a great teammate for somebody."
Stoudemire is friendly with both James and Wade, and said he would be recruiting both of them.
"Both guys, Dwyane and LeBron, I'm pretty sure would love to play with me," Stoudemire said, "and vice versa. So hopefully, we can get one of those guys to join me here this year."
As Stoudemire spoke, a series of beaming Garden officials circulated through the room, including James L. Dolan, the Garden chairman; Hank Ratner, the Garden chief executive; and Scott O'Neil, the MSG Sports president. Also circulating was the film director Spike Lee, who signaled his approval with raised eyebrows and a big smile.
Three decorated former Knicks — Allan Houston, John Starks and Larry Johnson — were also there for the occasion, unofficial as it might have been.
Technically, there is no binding agreement between Stoudemire and the team until the contract is signed Thursday, and salary figures could change slightly. Everyone present flirted mightily with the league's rules on announcing deals during the moratorium. Team officials spoke in conditional terms about the contract, trying hard to use "ifs."
The deal had effectively been in place since late last week, when the Knicks and Stoudemire's agent agreed to a basic outline, pending in-person discussions. It was first reported by Yahoo Sports.
There was not much to negotiate. Stoudemire wanted a maximum contract, and the Knicks said they were willing to give him one. Once he broke off talks with the Suns, his path became clear.
Despite Stoudemire's proven talent, there is risk in the deal. He had major knee surgery in 2005 and eye surgery in 2009, rendering his contract in Phoenix uninsurable. Donnie Walsh, the Knicks' president, said he was uncertain about insurance for the new deal but said, "I guess because we don't know, we're willing to take that risk."
Chris Bosh, the Toronto Raptors power forward, was slightly ahead of Stoudemire on the Knicks' big-man wish list. But Bosh, like Wade and James, is dithering about a decision and seems determined to join one of those two players somewhere.
Stoudemire made a quick commitment to the Knicks, which made their decision easier.
"I just love the city of New York, the state of New York," Stoudemire said, "and being a part of a great organization is the ultimate goal for a player, and to ultimately win a championship."
Once the Knicks sign Stoudemire, they will have about $17 million of salary-cap room left, enough for one more max contract. If they miss on James and Wade, they could split that sum among several role players and make Stoudemire the centerpiece. They have been aggressively pursuing the sharpshooter Mike Miller and are interested in point guards Raymond Felton and Luke Ridnour.
"It's the first step — it's a big step," said Walsh, who worked feverishly for two years to create the cap room for this heralded free-agent class. "I think we're further along, that's for sure."
Stoudemire was undaunted by the possibility of being the Knicks' sole star player.
"Totally comfortable, totally confident," he said. "And my leadership qualities will uplift all of us."
Stoudemire's arrival probably spells the end of David Lee's career in New York. Although D'Antoni said the two could play together, the Knicks have a much greater need now for a point guard and a true center. Lee is being pursued by Chicago, Miami, Minnesota and the Nets, as well as several teams without cap space that want to acquire him in a sign-and-trade deal.
"I don't really say never to anything," said Lee's agent, Mark Bartelstein, when asked if Lee's tenure had just ended. "Stuff goes on every day. We'll just see what happens."Continue reading the main story


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Thursday, August 9, 2012
14 comments:
Hoping your procedure goes well! Sending you peace and blessings...ReplyDelete
I have everything crossed, and I mean everything and I will be praying hard. I am sure everything will be fine.ReplyDelete
Love hearing about your PL weeks, as I have said before it keeps me inspired.
I know big changes are on the way for you guys but I know all will be well. Remember change, although not always welcome, turns out okay in the end. I am sure home schooling is the right decision for now and when the time is right you will re-assess the situation and make decisions right for then. Trying new things takes courage and I applaud you for that.
Did my first day as a relief Teachers Assistant (education assistant) today and loved every minute of it.
love me :-)
Good luck with your procedure. Wishing you a speedy recovery! Homeschooling is a big step (I did with two of my children in their younger years). It will be a learning experience for all of you. You will learn their strengths and weakness, and they yours. ;) Sounds like you won't be doing the teaching so you will probably have better success than I did.ReplyDeleteReplies
Stephanie, thanks so much for the visit and helpful comment. I did homeschool while they were young also, it was so much fun teaching them. You are right, online teachers this time instead of me doing it all. We are excited and nervous. Thanks again!Delete
My thoughts are with you for a safe procedure and a quick recovery!! I don't like drs either so I understand your nerves but I'm sure everything will go wonderfully!! Good luck with all the major changes in your family, I willbe thinking of you!!ReplyDelete
Good to see the PL posts Dawn. Even I get photo issues. I was not happy with my previous PL post.The pics didn't come out well.ReplyDelete
Will be keeping you in my prayers dear friend. Hope everything goes well with your check-up.
Online homeschooling must be both a big challenge and excitement for you and your family. I'm sure everything will go well with their studies and the best part is you are around to help them in anything they want.
Hope this coming year will be a truly blessed one for you. I did remember that your b'day was this month-:)
Hey Dawn, meant to check in on your blog yesterday to see the update but things just got so busy. Then I see you actually updated a day early, whoops. Heehee. The parade and the outing with your mom look like a lot of fun. Love all the special memories you guys make and record here. Don't worry about being late with updates though. Take all the time you need to recover from your procedure! Best of luck and get well soon :)ReplyDelete
Wow Dawn... you have a lot going on in your life right now! I wish you all the best for your op... they are never pleasant, but hopefully it will see you in a better place. You are a braver woman than me... I think home schooling would do my head in totally! LOL... seriously though, it sounds like it is the best decision right now, and online teachers sound awesome! Hope it all goes well, and as Tracey said, you can reassess when the time is right!ReplyDelete
Inspiring Project life as always!
great pages.. yes your bday is coming up.. wohoooooo! One good thing.. you'll never catch up to me.. hahaReplyDelete
Prayers that all is going to be great for you (health wise) and the homeschooling. Wow, you have a lot in your life now and I pray that you'd be given strength & courage :). I love the bright sunny flowers in one of the PL photos, is it from your garden?. Hugs.ReplyDelete
Ohh so much going on, take some "me time", lol. Home schooling will be a challenge, getting them to do it and stick with it. Good luckReplyDelete


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We just arrived in Sicily today. However my bag did not seem to leave Rome so I am currently without it which is driving me mad. It should be in Sicily tonight & delivered to hotel tomorrow but still makes me crazy especially as my friend's bag made it. However I'm determined to be chill about it & not let it make me more upset than the initial melt in the airport.
So instead of moaning, I bring you pictures from our dinner tonight.
Our starter: ricotta bruschetta topped with pistachio cream with sliced pears
Primi: vegetable risotto with smoked cheese
Seconde: filet steak in a Marsala orange sauce. Sorry no pic of this one as we killed it in about a minute.
Dolci: some kind if chocolate torte we saw them walk by with but wasn't on the menu so we asked about. Deliciousness


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I was hurried out the door with nothing but a pain pill prescription and told to get rest. Now, I am not one to accept just covering the pain with pills and I sure don't have the time to rest. Steve suggested that next I see a Chiropractor.
I am trying really hard to be the girl that thinks the glass is half full but the worrier in me expects
that the remaining half will evaporate by next morning!


text: On Mon, 05 Oct 2009 12:59 -0400, "Kent Johnson" <kent37 at tds.net> wrote: > On Mon, Oct 5, 2009 at 10:22 AM, Oleg Oltar <oltarasenko at gmail.com> > wrote: > > > os.popen4("application -parameter1 -file temp.txt") > > > > I wonder if that possible to execute this script (called application) > > without writing the file with initial data to the hard disk? > > If "application" can take its input from stdin then you can use the > subprocess module to launch it and pipe data to it. If "application" > requires a file for input then I think you have to write a file. Matt Harrison have some notes and a video of a talk about good python scripting practices. He describes some patterns that enable you to write python scripts that can be chained together as they process data from one and send it to the next. See the following links for more info. http://panela.blog-city.com/oscon_scripting_with_python_handout.htm http://en.oreilly.com/oscon2009/public/schedule/detail/8317 He created a tool called poachplate so that it is easier to create these kind of scripts. http://pypi.python.org/pypi?%3Aaction=search&term=poachplate Regards Rudiger


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Agnes "Aggie" LaMore, 87
A gnes R. "Aggie" LaMore, 87, of Elizabeth Street, passed away at the Glens Falls Hospital on Monday, June 23, 2014, following a brief illness.
Born on July 20, 1926 in Whitehall, she was the sixth of 15 children of the late Roland and Louise (Lewis) Burke.
She was a lifelong resident of Whitehall, attended Whitehall High School and was a communicant of Our Lady of Hope Roman Catholic Church.
On June 30, 1946, she married Lawrence "Mutt" R. LaMore. Her husband passed away on Aug. 3, 1978, and they had two children together, Gary R. LaMore, who passed away on March 17, 2013 and their daughter, Joann Titus, who now lives in Oneonta.
Agnes started in the work force in housekeeping, then on to factory work at the Silk Mill; she was a waitress at the Dairy Bar and Duce's Restaurant; E. B. Metal as a shop seward; Jumbo's IGA as a store clerk where she was affectionately known as "Homerand was honored as one of the first clerks of the week by the Whitehall Times; and crossed the finish line of her career with the Hudson Falls-Whitehall office of O.T.B. as a shop seward.
She was a hard worker and was well known by everyone she worked with and the public she served. She was strong willed and opinionated and you knew where you stood with her. She took pride in providing good customer service.
She was an avid bowler, serving 21 years as secretary for two leagues and going with her teams to many bowling tournaments. She also enjoyed golfing with her friend, Joe, at the Skene Valley Country Club. She loved music and loved to dance. She tapped danced in several minstrel shows in Whitehall. She proudly served two terms as president of American Legion Post No.83 Auxiliary. She and her son, Gary, were voracious readers and members of the Isaac Griswold Library.
Besides her parents, her husband and her son, she is predeceased by 11 brothers and sisters.
She is survived by her daughter, Joann Titus of Oneonta; her granddaughter, Gunnery Sgt. Melanie Contreni and her husband, Michael of New Berne, N.C.; sister, Ann Brown of Queensbury; brother, Jim Burke and his wife, Elizabeth of Alplaus, N.Y.; and many nieces, nephews, near and far.
A special thank you to her niece, Pat Manor, for all her help last year and this past week.
Family and friends may call at the Jillson Funeral Home, Inc., 46 Williams Street, Whitehall on Friday, June 27, 2014 from 9:30 to 10:30 a.m. followed by a Mass of Christian Burial at 11 a.m. at Our Lady of Hope Roman Catholic Church, Skene Street, Whitehall with Rev. Liam Condon, officiating.
Interment will be at a later date in Our Lady of Angels Cemetery.
In lieu of flowers, expressions of sympathy may take the form of a donation in Agnes' name to the Skenesborough Emergency Squad, P. O. Box 333, Whitehall, NY 12887, Our Lady of Hope Roman Catholic Church, 9 Wheeler Avenue, Whitehall, NY 12887 or the Isaac Griswold Library, 12 Williams Street, Whitehall, NY 12887.


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Office of Public Affairs
January 31, 2017
FBI Announces Arrest Made in Connection with a Bank Robbery in Plantation
The FBI announces the arrest Tuesday, January 31, 2017, of Stephen Michael Gordon, 26, of Plantation, FL, for his alleged involvement in the robbery of a TD Bank branch at 1800 N. Pine Island Road, Plantation, FL, on January 31, 2017.
The arrest was made without incident.
Gordon is in federal custody and is facing federal charges.
No picture(s)/mug shots are available.
The Plantation PD, Sunrise PD, the South Florida Violent Crime Task Force and the FBI continue their investigation.
No further information will be released at this time.