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|yuankui <kui.y...@fraudmetrix.cn>
|Re: limit the size of data type LIST
|Fri, 14 Aug 2015 01:24:41 GMT
Sorry for not making myself clear and thank you for your reply. -------------- I want to know if there is a way to automatically remove old items in the list in SERVER SIDE if the size() of the list reached a certain limit(say 1000). client does not need to care about this, and just do insert and get. and he will get the latest 1000 messages of a user? can I? 在 2015年8月14日,上午12:55,<email@example.com> <email@example.com> 写道: This sounds like something you do on the client side BEFORE you insert. Or are you wanting to limit the size of the list coming out to the client? Sean Durity Lead Cassandra Admin, Big Data Team From: yuankui [mailto:email@example.com] Sent: Thursday, August 13, 2015 9:06 AM To: firstname.lastname@example.com Subject: limit the size of data type LIST hi, friends I am design a message history table CREATE TABLE message_history ( user_name text PRIMARY KEY, time timestamp, message_details list<text>, ); so that I can query a user's message via primary key `user_name` at once. but the item in `message_details` list may be very long so that I want to limit the list size of the message_details list. is there a way to solve this? like a redis operation `LTRIM` - http://redis.readthedocs.org/en/latest/list/ltrim.html <http://redis.readthedocs.org/en/latest/list/ltrim.html> The information in this Internet Email is confidential and may be legally privileged. It is intended solely for the addressee. Access to this Email by anyone else is unauthorized. If you are not the intended recipient, any disclosure, copying, distribution or any action taken or omitted to be taken in reliance on it, is prohibited and may be unlawful. When addressed to our clients any opinions or advice contained in this Email are subject to the terms and conditions expressed in any applicable governing The Home Depot terms of business or client engagement letter. The Home Depot disclaims all responsibility and liability for the accuracy and content of this attachment and for any damages or losses arising from any inaccuracies, errors, viruses, e.g., worms, trojan horses, etc., or other items of a destructive nature, which may be contained in this attachment and shall not be liable for direct, indirect, consequential or special damages in connection with this e-mail message or its attachment.
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Maintaining a Healthy Heart as You Age
As you get older, there are some changes you can expect in your cardiovascular system overall, and specifically in your heart. It's simply a normal part of aging for most people. In fact, it is estimated that more than 85 million adults in the U.S. have some form of cardiovascular disease, and of those cases, […]Read More
5 Stay-cation Ideas for Seniors
As we get older, taking vacations can get more and more difficult. For many seniors, long road trips in a car can be very uncomfortable, even painful. Flying can also be difficult because of how cramped the seating is. Plus, once you've arrived at your destination, activities that require a lot of walking around may […]Read More
4 Vaccines Every Senior Should Discuss with Their Physician
Now that it's Fall – and with winter on the horizon – we are officially in the cold and flu season, even in Florida. Indeed, even though the temperatures in the Sunshine State don't get as cold as they do further north, people are constantly traveling to the Central Florida area and bringing their germs […]Read More
Diagnosing Alzheimer's Disease with An Eye Test
According to the Alzheimer's Association, there are currently about 5.8 million people with Alzheimer's disease in the United States, making it the sixth leading cause of death. It's the most common form of dementia with up to 80 percent of all diagnosed cases. People who have Alzheimer's disease typically have problems with memory and thinking, […]Read More
Though Rare Among Seniors, Spinal Muscular Atrophy Can Be Devastating
It is estimated that, throughout the world, spinal muscular atrophy (SMA) affects as many as one out of every 8,000 to 10,000 people. For the most part, this devastating disease affects babies and toddlers. However, the variation commonly known as Type 4 SMA or proximal spinal atrophy, though extremely rare, can strike at any time […]Read More
Preventing Bug Bites from Ruining Your Summer
In the first 10 years of the new millennia – the decade between 2000 and 2010 – millions of people in the U.S. were victims of a problem that has plagued mankind for thousands of years: insect and spider bites. According to a 2014 study, more than 6.8 million people were treated in emergency rooms […]Read More
Hepatitis C: A Hidden Danger for Seniors
One of the more serious health issues confronting an aging U.S. population is the rate of hepatitis C infection among people born between 1945 and 1965. Statistically, it is estimated that 75% of all people with the disease were born in that window, making them five times more likely to be infected than other age […]Read More
Osteoarthritis and Aging
For decades, scientists believed that osteoarthritis was caused by long-term wear and tear on the joints. While there is a connection to usage injuries that happen throughout a lifetime, doctors now perceive osteoarthritis as a disease of the joints, most commonly the spine, hands, feet, knees, and hips. Also referred to as degenerative joint disease […]Read More
The Benefits of Music Therapy for Seniors
Most people would agree that listening to music can affect one's emotional state. Many people listen to upbeat music when they want to raise their energy level, either for fun or motivation. Or, when they want to relax after a stressful day, soft classical music or light jazz may do the trick. In addition to […]Read More
Understanding How Epilepsy Affects Seniors
When most people think of epilepsy, they think it's a disease that starts in childhood or early adulthood, or they assume it's a genetic disorder and can't be caused by outside factors. Among seniors, however, epilepsy can develop even during their advanced years. In fact, among patients older than 75 years old, one in 10 […]Read More
Living Younger Blog
- Diet and Healthy Eating for Seniors (8)
- Exercise for Seniors (7)
- General Health (64)
- Living Younger (72)
- Men's Health (16)
- Mental Health for Seniors (7)
- MetroHealth News (66)
- Press (2)
- Uncategorized (2)
- Women's Health (17)

subset: commoncrawl


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Thegiftgetter was founded in 2013 to fulfill my very own need for a gift suggestion site. I was looking for a one-stop shop that offered curated gift options so I would not have to jump from site to site for the ideal gift. I did not find one, so here we are.
Here at thegiftgetter, we pride ourselves in being excellent gift givers in our personal lives. We are selective in our product choices and our featured gifts are from our very own personal experiences.
On a monthly basis, we will share tips and articles from partners and contributors. Please contribute as well by following us on Instagram. And, to be one of our featured writers, email us at firstname.lastname@example.com
Ariscielle, founder
Nail polish is that one thing that makes me feel ready to face the world. This spring I recommend we all gift our good friends the gift of great nails. I think this spring has the most perfect collection of colors to complement anyone's style and mood.
There truly is nothing like Christmas time. There is such love and joy all around; it's impossible for me not to get wrapped up in all the holiday cheer! One of the best parts of Christmas is the giving; there is a pure joy that a gift, any size, can bring someone and I just can't get enough of it. These are the things I'm loving this holiday season.
I have a mix of favorites this year. As I learn more about myself I feel more comfortable with my choices. Some are luxury items, some are inexpensive things that others may seem to corny but nevertheless they are my favorite things I will gift and ask for this Christmas.
Be right back, saying goodbye to all of our other bracelets because this is the only one we need. This Mark & Graham bracelet does more than serve as arm candy — it charges your iPhone too! We're loving the Rose Gold, and to really put this gift over the top, add a monogram.
Airport gifts used to make me think that the gift is an after thought but I don't think that's true anymore. Luxury brands have taken over the airport; local boutiques and artisan gifts have also taken space which make airports a great place to find gifts when traveling. This week I am in London, and this is what I am eyeing to take back home and send through the post office.
Glasses may just be the most underrated accessory of them all. In fact, I think glasses have gotten an undeserved bad rep, and, it's about time it gets turned around.
This fall, gift frames!
It's crazy just how many different types of glasses there are out there, and let's face it — it's not always easy to decide what style is the best for ourselves, let alone others. That's where I come in to help.
My favorite things to gift and get this summer all have to do with how I plan to live this summer. Farmers market days are all about comfort, healthy living, and a great summer dress. My look will be completed by a so-much-in-style and eco-friendly market bag. I imagine I would have a really cool beach cruiser — that's not really happening :), but for someone you know, maybe it does.
When I am not prepping for meals in style, I am outdoors swimming or kayaking. To get ready for this, all I need is a red bikini and very carefully picked beach towel.
But anytime this summer, farmers market or beach, I will always have my sunglasses.
That's all for now. I hope you like what we like. I hope you gift what we gift.
This summer, I am all about color! I am spending my summer traveling, spending time with family and friends, and preparing for the next big step in my life after college — my move to New York City.
When you're always on the go, it's important to have staple items that can take your entire look from casual days to fun nights. Since I have been coming and going so much this summer, I thought there would be no better way to chat about my summer list than to do it while packing for my next trip to the city.
We are hitting the home stretch of getting more gifting features available to you, but I had to take a break for this, Les Petits Joueurs Ginny Beaded Heart Clutch Bag. If there is one thing you ask for or gift to that special someone, let it be this.
Go back to swimwear basics this season with this simply perfect LANA Bakini Set. We can't get enough of the blush.
I really, really believe every single girl needs these shoes in her life. And I really, really think you should be the one to give them to her.
This is a gift of a lifetime and for many generations to come. I imagine family get-togethers for a lifetime with this utensil set by Christofle as one of the stars.
Tell your mom she's your world this Mother's Day with this Levtex accent pillow.
In the next few days, I am going to feature gifts to get your mom summer ready. I think Mother's Day should be every day, but that's just me. I love flowers; somehow they make me just feel full of love. Gift a flower bouquet on the regular. So sweet.
I can have deviled eggs everyday. Bring your Easter hostess a tray of deviled eggs and let her keep the tray! This jade deviled eggs tray is my absolute pic and at $30 you can still afford a dozen eggs to go with it. Best Deviled Eggs Recipe on Food52
Extra cute and extra special. This personalized book will make for the greatest Easter morning.
Invite Peter Rabbit for Easter by bringing this beautiful cake to wherever you end up. Hop to it, gift it.
We're not sure there is a cuter gift than this Levtex accent pillow for this Easter season.
If she's not already obsessed with BeautyBlender, this limited edition set of micro-minis will make that happen. Oh, and their colors just happen to scream Easter!
This takes the cake! I always wonder why not more people have cake stands. I love these by imm designs.
It's been tradition in my family to give something up for Lent; I think it's because we want to indulge when we celebrate Easter. Starting today, we will drop hints weekly on what to gift for Easter. First up, give the gift of Dana's Bakery macarons.
Say goodnight to boring gifts. AROS just took the hoodie to a whole new level — with an inflatable hood. A piece of clothing that makes sleeping comfortably anywhere possible.
Mid snow I decided to gift myself a new tote bag for spring. My choice, a classic from Valextra. I can't wait for spring! ⚘
I guess I am more sentimental than I thought! This New York Times birthday will keep her or him nostalgic for years to come.
Trend Alert! Contrast Denim is seriously in and you will be seriously amazing to bring the Adrian Denim Skirt her way.
Personalize this vintage looking street sign to create the perfect housewarming gift.
It could be for a new homeowner, a host, a newlywed or the hostess of the mostest. This solid copper Leaf Wedding Cake Set by Beautifully Served by Jill is so unique, it's sure to be a keeper.
We all wake up pretty but there's no harm in having a bag full of tricks just in case. Gift a beauty bag by Katie Loxton.
Trish McEvoy has given women everywhere the gift of working toward beauty security, and you can help spread it.
Wicked good cupcakes: Unique and delicious. Cupcakes... in a jar! Need I say more?
The perfect gift for that out-of-state friend who is always referencing his or her home state. These homesick candles have scents for all 50 states, providing the comfort of home away from home.
I live by this book. All my best gal pals get gifted A Short Guide to a Happy Life at some point. Welcome her to your inner circle with this book.
Taking beauty sleep to a whole new level, the Slip Silk Pillowcase will have you waking up with better skin and smoother hair. Goodbye, cottton!
Home is where your heart is. This pillow by Jonathan Adler is a great addition to the sofa or bed you snuggle in. We love it.
Find the rights words, with a card by Matt Butler. Don't forget the card!
A gift that represents two of his favorite things: his team and wine, in a seriously cool way. Don't worry, it comes for almost all MLB teams; we won't be too sad if the New York Yankees aren't your pick.
Complete the man cave theme with these whiskey accessories:
Mini bags are all the rage nowadays and you would be SO COOL as to purchase her something to get her on trend this spring. Alexander Wang's Roxy Mini Bucket in Black Suede with Studs is to love (die) for.
1atelier offers customizable luxe bags that any woman will fall in love with. I am surely in love! My choice is the 1atelier clutch; I customized everything about it including the price!
Fashionable and philanthropic! This cute clutch is not only perfect for a night out, but also a great Valentine's Day gift that benefits a cause. With every purchase of a Rosa clutch, 50 school meals are donated around the world. While sporting your clutch, you'll be reminded of the impact you made on the lives of 50 children.
To kick off our Valentine's Gift Guide, I want to start with a gift suggestion for your SO. Sprucing up his man cave is a good way to support his independence — inside the house ;)
Give the gift of beautiful lips with Koko K's perfect shade of pale pink. The kit includes a matte liquid lipstick along with the matching liner to create a flawless, long-lasting lip. Pucker up, ladies!
For the friend who has everything and a pet. Give the the gift of Barkbox. This was the perfect thank you gift to my friend Amy for doing me a wonderful favor.
Baby it's cold outside! Warm up your loved ones with the gift of high-tech warmth. The Ravean self-heating jacket is the best fashionable tech-wear for all seasons.
The classic style of music paired with the latest technology will give him the opportunity to start the vinyl collection he will keep forever while having the option to stream the music he has now.
Because keys deserve to be pretty, too.
Customize this kids book, The Little Boy Who Lost His Name or The Little Girl Who Lost Her Name by entering a child's name, then choose the hero and characters based on the letters of the child's name. Write a dedication to make the book feel even more personal.
What's better than a bottle of wine? A pretty bottle of wine with a little inspiration. Give New Year's cheers with "The best is yet to come" 2015 Torront�s bottle.
Why sacrifice style for quality sound? will.i.am's exclusive i.am+ BUTTONS have made headphones a necessary accessory to keeping him looking fresh with the intense sound he deserves. We love the black.
Luxury sweets in a blue box. Elegant, grown up candy that's worth every bite. Sugarfina's bento box will be a huge hit with everyone. No re-gifting here.
Keep your drinks cold for 24 hours and hot for 12 with this stylish water bottle and give back while doing so. S'well is partnered with many charities to help provide clean water to children in need all around the world. Our favorite is the Elements Collection 17oz White Marble.
Decorate your kitchen with the Mackenzie Child's Courtly Check Enamel Tea Kettle for a modern touch on classic charm.
Gifts can have extraordinary power. They have the potential to hold great meaning and memories, and they represent so much more than what they may seem to be. My most cherished belongings are gifts that I have received from others because of why, how, or by whom they were given.
Three years ago, my godmother was faced with a health battle that required immense strength and faith. Before she was entering surgery, she asked if I could hold on to her bracelet. Having never noticed it before, I was taken aback by its simplistic beauty — a quaint silver chain made up of small hoops connecting a petite dove. I immediately complimented her; she explained it was a gift to her, and that the dove represented the holy spirit.
Months later, I was back from college for the summer and one of my first stops was to my godmother's house. She handed me a box and inside was the bracelet.
I wear this bracelet as a reminder of the love I have for my family, the strength that life requires, and the faith that can help guide us through even the worst of times.
Give her a gift that she can hand down to the next generation. A luxe item, the Saint Laurent Monogram Kate Clutch is a keeper.
The YETI Tundra Roadie lets him get back to the basics of ice chests with the best of 'em. With its selection of natural colors and undeniable masculinity, this is a great choice. Our favorite is blue.
The HIGH KEY modernized aviator features oversized frames with mirrored lenses to keep you (and me) looking fashion forward and effortlessly fierce. With three colors to choose from, we love the High Key Gold.
Heaven in a jar. Need I say more?
We believe in the wonder of books. "The Day the Crayons Quit" is whimsical and funny for young readers.
I love Eleni's Color Me! Christmas Gift Set. Keeps the kids busy after dinner. It's a built-in babysitter and a special treat for the young artist.
In fashion velour but with a sexy twist. Gift her this 3 piece velour set from Free People that shows just how intimate your relationship is.
Sitting at home with friends and family is the best time to shake it up. For mixologists all over, glam up with this Hobnail Glass Cocktail Shaker.
Put your money where your heart is. I love these initial stud earrings. They're subtle but yet it speaks volumes.
Pretty box for pretty things. This one is our pick: feminine, modern and affordable. Magic.
We imagine your best girlfriends lounging in this every opportunity she gets. She will love wearing this wide neck off-shoulder sweatshirt the morning after every holiday feast ❤.
For a little angel. Moma's Cloud Light is the perfect addition to a baby's room. With two price options, this is the perfect gift for a new parent.
He is worth the price tag! The Urban bike is sleek, cool and sophisticated. It's a guarantee that your man will travel in style and stay in shape. Not a bad trade:).
I have committed to a classic look this fall, and I plan to take others with me. For those who are on the same quest, the best gift to get and give is a brooch. I can simply pin it on a coat or on an accessory of my choice: belt, scarf, or bag. The Diora Brooch is a must and this week's price is unbeatable at $22.00.
Just like the old days but better and more stylish. The Fujifilm Instax Mini 8 is my pick this week. It comes in a variety of colors at a good, kid-friendly gift price. My little one would love the blue camera.
The most coveted float at the pool party? We've found it. Shaped like a giant red gummy bear, this sugary treat lets you have your sweets and sun in them too.
I always start summer with a treat to my family. This year, it's a beach tent. It promises fun in the sun and time together.
Gift this to your favorite family. For me, it's my family and my cousin Victoria.


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Are you currently looking for a senior living facility for a loved one? Most people want to live in their homes for as long as possible. However, health and safety concerns often necessitate specialized care that can best be provided at an assisted living facility. Though this is always a difficult decision, knowing what to look for can make it a little bit easier. All great care facilities share a few characteristics in common. Read on to learn more about the top qualities you should look for when seeking out assisted living in Long Island.
Respect for Residents
The most important trait to look for in an assisted living facility is respect for the residents. The seniors living in the facility should be treated with kindness and respect, regardless of their physical or mental condition. The staff should be kind, caring, and supportive when they are interacting with the residents. They should also show respect for the residents' privacy by doing things like knocking before entering a room or clearly communicating their actions. Additionally, guests, family members, and friends should be welcome to visit at any time.
Safety and Cleanliness
When you go to visit the facility, take note of how clean it looks. The grounds, doorways, and hallways should all be well-lit and free of blockages that could potentially cause harm. The rest of the facility should also be hygienic, odor-free, and tidy. The residents should be well-groomed, as well.
Comfortable Surroundings
In addition to cleanliness, the facility should also be comfortable. For the residents, the facility should feel as home-like as possible. This means allowing seniors to bring their own furnishings, decorate their rooms how they want, and hang family photos from home. The garden and yard should also be inviting for the residents, making them feel comfortable and encouraging them to spend some time outdoors.
Since your loved one will be spending the majority of their time at the assisted living facility, it's crucial that they provide the amenities needed to lead a normal life. Your senior will feel more at home and will be able to lead a more fulfilling life. Some amenities to look for include a hair salon or barbershop, library, gym, and more. Also, consider trying to find a facility that is close to shopping areas, so the residents can easily get something if they need it.
Assortment of Activities
Along with the amenities, the facility should also offer a number of activities that their residents can take part in. They should provide a wide assortment of educational, social, creative, and physical activities to appeal to all of their residents. The residents should be able to lead full lives with lots of things to do and places to go within the facility. Try to find a facility that offered activities organized by outside groups, too. This includes live music or community groups that bring pets to the facility.
Tasty and Healthy Food
Another characteristic to look for is balanced, attractive, and delicious meals. This is not only necessary for your loved one's health, but it can also provide social benefits when the meals are served in a pleasant communal dining space. The facility should also be accommodating of any dietary restrictions based on health problems, religious needs, or ethical concerns.
If you are looking for a Long Island assisted living facility, reach out to us at Oyster Bay Senior Campus.


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BURIED REALM Debut "The Wonder And Terror Of Fortune" Lyric Video
June 27, 2020, 7 days ago
Buried Realm is a solo studio project, hatched from the mind of Josh Dummer. It is the second album he has put out under that moniker and once again he has written an epic, creative record filled not only with his own talent but also with that of a smorgasbord of special guests from around the world. Dummer comments on the album:
"After nearly three years of creating Embodiment Of The Divine, I am very pleased to present and share this album with everyone. I'm honored to be joined by a number of guests that have influenced my writing and playing over the years. Their contributions have truly helped bring the album to another level."
"Embodiment Of The Divine features guest performances from Andy Gillion, Benjamin Ellis, Bob Katsionis, Dean Arnold, Lee McKinney, Peter Wichers, Rafael Trujillo, and Ryan Strain. Listeners can expect to hear a whole lot of riffs, solos, and melody. The album has its traditional arrangements at times but is also built on technical and chaotic grounds. I think the overall atmosphere of this album is darker compared to the previous one."
The second single off the album "The Wonder And Terror Of Fortune" features guests Rafael Trujillo (ex-Obscura) and Ryan Strain (Chaos For The Masses).
Dummer adds: "Over the years, music has been a very important part of my life. It's always been there for me in any situation, it became a lifestyle and many opportunities have come from it. For that I am grateful. With that comes the unfortunate part of losing sight of friendships, relationships, other opportunities, etc. These lyrics are about the interesting blend of excitement and sorrow that music brings to me and the constant reminder of how I've had to choose what's most important at the time."
Recommended for fans of Soilwork, Nevermore, and Children Of Bodom, Buried Realm is progressive melodic death metal. Embodiment Of The Divine is out on July 24. Pre-orders can be placed now at this location.
"The Burning Remnants" ft. guests Dean Arnold (Operus, ex-Vital Remains)
"Overlord" ft. guest Andy Gillion (Mors Principium Est)
"Master Psychosis" ft. guests Benjamin Ellis (Scar Symmetry), Ryan Strain (Chaos For The Masses)
"Scales Of Queen Dragon" ft. guests Andy Gillion (Mors Principium Est)
"Silver Tongue" ft. guests Bob Katsionis (ex-Firewind), Dean Arnold (Operus, ex-Vital Remains)
"In The Shallow Light" ft. guests Peter Wichers (ex-Soilwork), Ryan Strain (Chaos For The Masses)
"Embodiment Of The Divine" ft. guests Benjamin Ellis (Scar Symmetry), Peter Wichers (ex-Soilwork)
"Infinite Mutation" ft. guests Lee McKinney (Born of Osiris), Ryan Strain (Chaos For The Masses)
"The Wonder And Terror Of Fortune" ft. guests Rafael Trujillo (ex-Obscura), Ryan Strain (Chaos For The Masses)


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Muffy writes without the use of a surrogate
Thursday. 10.1.09 4:45 pm
The verdict: 6.5/10
It wasn't a BAD movie. Just not very inventive. I feel like it's a big combo of The Matrix, I, Robot, and the Minority Report. All of which are very good movies in themselves, but Surrogates spreads itself too thin trying to cover themes in all three films. I feel like it just came up short from what the trailers were putting out...the premise was great, but when the mystery cleared, there wasn't really much to say other than an unenthusiastic "oh." And the ending was horrid! I'm all about good endings. A good ending can just about cinch any movie, and this was one was definitely a disappointer.
OK I just remembered the best way to turn a mood around: music. Duh. I hopped onto the piano today and played some tunes...felt so good. Some guy came up to me and interrupted me to ask if I could play some song. Now normally interrupting my playing does a BAD THING for my mood...but he was nice enough and I wasn't about to give a complete stranger the cold shoulder. I don't think I'll be seeing him again but it was good to see someone appreciated my sounds (again with the appreciation, eh Stevo?
And now I'm in my room moving to the beat of my iPod...but not moving TOO much because me dancing in public would just be weird.
Oh and I think I need to come up with a codename for the individual mentioned in my last private entry. How about we say...Irrelevant-Man!
Time for practice...Muffy over and out!
Does anybody really look at profiles and galleries anymore? I feel like they're rarely-used gems.
Do not try to bend the spoon. That's impossible. Instead, try to realize the truth. There is no spoon. Then you will see that it is not the spoon that bends, it is only yourself.
7 Comments.
Woo, dancing in private. :P
I don't often look at galleries, but if I'm bored then I'll skim the profiles. People don't seem to update them that often, though...
» randomjunk on 2009-10-01 08:56:16
I look at profiles ^_^...even though they aren't current , most of the time.
Hm, my movie choices for tomorrow are probably going to be Surrogates or Cloudy with a Chance of Meatballs.
I'm leaning towards Meatballs after reading this.
» Mockiller on 2009-10-02 01:33:39
I only dance behind locked doors.
My gallery.... you might find weird things in there =)
» Nuttz on 2009-10-02 07:36:36
Well, regardless of whether or not they're real, the freakout videos are funny. :P
And yeah..... I have nothing to do here, so it just shows what I do when I'm desperate for something to distract me and my camera is sitting out. D:
» randomjunk on 2009-10-02 08:18:43
you can dance if you want to. you can leave your friends behind.
i barely ever look at galleries. i do visit profiles sometimes, but i feel like facebook edges out nutang a bit in those instances.
anyone here want a facebook friend? *crickets* anyone?
» thaitanic on 2009-10-02 09:27:02
Yup! I'm founder and president ^__^.
» Mockiller on 2009-10-03 01:36:28
You know, I completely forgot to take pictures when we met. If the meeting had been more exciting I probably would've remembered... Next time, 'kay? :P
» randomjunk on 2009-10-05 07:09:38
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South Coast Secret – Out And About – February
Experience the natural beauty of the south coast in Pennington, bordered almost entirely by wonderful nature trails…
Pennington is home to one of the most beautiful natural forestry preserves on the south coast. Once a dump site, the Nkomba Reserve is now a thriving sub-tropical coastal forest under the protection and care of the Pennington Conservancy in Cherry Lane, Pennington.
Carefully marked trails allow locals and visitors alike to meander through the coastal forest and truly take in the beauty of their surrounds. Remember to take your cameras along with you as the area is abundant with small wildlife and birds!


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What Is Hecm Loan What is a HECM to HECM Refinance? – Understanding Reverse – A HECM, or Home Equity Conversion Mortgage, is the technical term for the federally-insured reverse mortgage. Therefore a HECM to HECM refinance (also known as a H2H Refi), occurs when the borrower is paying off an existing HECM with a new HECM.
HECM Senior Home Financing was founded by Tim and Tiffany Linger, two real estate professionals who set out to help as many individuals as possible. Tim possesses over 16 years of experience in the home equity conversion mortgage (hecm) industry. He is one of only 106 people in the world to have achieved the title of "CRMP".
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Isolation and characterization of farnesyl diphosphate synthase from the cotton boll weevil Anthonomus grandis.код для вставкиСкачать
A r t i c l e ISOLATION AND CHARACTERIZATION OF FARNESYL DIPHOSPHATE SYNTHASE FROM THE COTTON BOLL WEEVIL, Anthonomus grandis A. Huma Taban, Claus Tittiger, Gary J. Blomquist, and William H. Welch Department of Biochemistry and Molecular Biology, University of Nevada, Reno, Nevada Farnesyl diphosphate synthase (FPPS) catalyzes the consecutive condensation of two molecules of isopentenyl diphosphate with dimethylallyl diphosphate to form farnesyl diphosphate (FPP). In insects, FPP is used for the synthesis of ubiquinones, dolicols, protein prenyl groups, and juvenile hormone. A full-length cDNA of FPPS was cloned from the cotton boll weevil, Anthonomus grandis (AgFPPS). AgFPPS cDNA consists of 1,835 nucleotides and encodes a protein of 438 amino acids. The deduced amino acid sequence has high similarity to previously isolated insect FPPSs and other known FPPSs. Recombinant AgFPPS expressed in E. coli converted labeled isopentenyl diphosphate in the presence of dimethylallyl diphosphate to FPP. Southern blot analysis indicated the presence of a single copy gene. Using molecular modeling, the three-dimensional structure of coleopteran FPPS was determined and compared to the X-ray crystal structure of avian FPPS. The a-helical fold is conserved in AgFPPS and the size of the active site cavity is C 2009 Wiley Periodicals, Inc. consistent with the enzyme being a FPPS. Keywords: farnesyl diphosphate synthase; cotton boll weevil; isoprenyl transferase; molecular modeling Correspondence to: Gary J. Blomquist, Department of Biochemistry and Molecular Biology, University of Nevada, Reno, NV 89557-0014. E-mail: [email protected] ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY, Vol. 71, No. 2, 88–104 (2009) Published online in Wiley InterScience (www.interscience.wiley.com). & 2009 Wiley Periodicals, Inc. DOI: 10.1002/arch.20302 Boll Weevil Farnesyl Diphosphate Synthase 89 INTRODUCTION Isoprenyl diphosphate synthases, also referred to as prenyltransferases, catalyze the consecutive condensation of isopentenyl diphosphate (IPP) with allylic prenyldiphosphates. These enzymes are classified according to the product chain length and the geometry of the newly formed double bonds (E or Z). The short chain E-prenyltransferases consist of geranyl diphosphate synthase (GPPS), farnesyl diphosphate synthase (FPPS), and geranylgeranyl diphosphate synthase (GGPPS), which produce products with chain lengths C10, C15, and C20, respectively (Ogura and Koyama, 1998). Long chain E-prenyltransferases produce polyprenyl diphosphates ranging in length from C30 to C50, which are involved in respiratory quinone biosynthesis (Okada et al., 1996). The Z-polyprenyl diphosphate synthases are used for synthesis of the long chain dolichols and several other very long chain isoprenoids (Sato et al., 1999). The structural genes for a variety of E-isoprenyl diphosphate synthases have been cloned and characterized (Ogura et al., 1997; Wang and Ohnuma, 1999). Multiple alignments of the deduced amino acid sequences indicate two characteristic aspartate-rich DDXXD motifs (first aspartate-rich motif: FARM, and second aspartaterich motif: SARM) and five highly conserved regions (Koyama et al., 1993; Chen et al., 1994; Kellogg and Poulter, 1997). Mutational analyses (Wang and Ohnuma, 1993; Ohnuma et al., 1998) and X-ray crystallography studies (Tarshis et al., 1996) have revealed the mechanisms of chain elongation and determination of product chain length. Farnesyl diphosphate synthase catalyzes the consecutive condensation of two molecules of IPP with DMAPP to form farnesyl diphosphate (FPP). In insects, FPP is used for the synthesis of ubiquinones, dolicols, prenylated proteins, and juvenile hormone (Schooley and Baker, 1985; Havel et al., 1992). In A. grandis, prenyltransferases have additional importance. A. grandis is among the few insects that use the isoprenoid pathway for pheromone component synthesis (Mitlin and Hedin, 1974). The male-produced pheromone components of A. grandis are monoterpenoids (Tumlinson et al., 1969), which are likely synthesized from geranyl diphosphate. The female boll weevil produces a sesquiterpene pheromone (Hedin et al., 1979), which is presumably synthesized from FPP. FPPS has been sequenced from several insects including, Agrotis ipsilon (Castillo-Gracia and Couillaud, 1999), Bombyx mori (Kikuchi et al., 2001), Drosophila melanogaster (Sen et al., 2007a), and Choristoneura fumiferana (Cusson et al., 2006). The X-ray structures have been obtained for the unliganded avian FPPS at a 2.6-Å resolution (Tarshis et al., 1994), and for a double mutant in the presence of DMAPP, GPP, and FPP (Tarshis et al., 1996). In addition, crystal structures of Staphylococcus aureus and Escherichia coli FPPSs bound with IPP were resolved (Hosfield et al., 2004). More recently, X-ray data have also been reported for Trypanosoma cruzi FPPS (Gabelli et al., 2006), and for human FPPS (Rondeau et al., 2006). Based on these studies, the enzyme is a homodimer. The binding site is composed of 10 a-helices that form a large central cavity and two aspartate-rich motifs are positioned on opposite sides of the cavity. In this report, the molecular cloning and expression of the first coleopteran FPPS is reported. The AgFPPS cDNA was isolated from an A. grandis male gut cDNA library, and a putative three-dimensional structure of A. grandis FPPS was determined using molecular modeling. Archives of Insect Biochemistry and Physiology 90 Archives of Insect Biochemistry and Physiology, June 2009 MATERIALS AND METHODS Insects Adult boll weevils and diet pellets were obtained from the USDA-APHIS Misson Plant Protection Center, Mission, Texas. Adults were segregated by sex according to Burke (1959). PCR Cloning of A. grandis FPPS An AgFPPS gene fragment was amplified by PCR using A. grandis male gut cDNA library (Taban et al., 2006) as template. Two degenerate PCR primers were designed based on consensus sequences of previously isolated cDNAs from A. ipsilon and D. melanogaster. The forward and reverse primers were: YIACNCCNGARAAYAT (I:inosine) and TCYTWIATRTCNGTNCC, which correspond to the amino acid sequences LTPENI (position 151–156) and GTDIQD (position 333–338)in the A. ipsilon protein (AJ009962). PCR amplifications were done in a 50-ml final volume, containing 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 2.5 mM MgCl2, 50 pmol of each primer, 2-ml template, 0.2 mM dNTPs, and 2.5 U Taq DNA polymerase (Life Technologies). The cycling profile was 1 min at 951C, 35 cycles of 40 s at 941C, 1 min at 501C, 1 min at 721C, and a 10-min extension at 721C. The 560-bp PCR product was purified from agarose gel using an Ultrafrees-DA centrifugal filter device (Millipore), cloned into pST-Blue1 (Novagen) and sequenced. The complete cDNA was isolated by PCR using sequence-specific primers and the cDNA library as template. The fulllength sequence was assembled from overlapping clones, and confirmed by amplifying the full-length cDNA with primers that span the open reading frame. All other PCR products were purified with Microcon-PCR centrifugal filter devices (Amicon). Expression and Purification of A. grandis FPPS The coding region of FPPS cDNA was modified to insert BamHI and EcoRI sites at the 50 and 30 ends, respectively, using oligonucleotide primers GCGGATCCGTTTTCGG TGAAATTGTCC and GCGAATTCACACTCCCGTTTGTACAAC by PCR to amplify the cDNA library template. The PCR reaction was carried out in 50 ml containing 2.5 mM MgCl2, 50 pmol of each primer, 2 ml template, 0.2 mM dNTPs, 2.5 U of Pfu DNA polymerase (Stratagene), and 1 Pfu buffer provided by the supplier. The cycling profile was 1 min at 951C, followed by 3 cycles of 40 s at 941C, 1 min at 371C, 2 min ramp to 721C (0.31C/s), 4 min at 721C, 30 cycles of 40 s at 941C, 1 min at 601C, 4 min at 721C, and a 15 min final extension at 721C. The PCR product was digested with BamHI and EcoRI and subcloned into pTrcHis2c (Invitrogen) to produce a polyhistidine-tagged fusion protein. The correct insertion of FPPS in the vector was confirmed by sequencing. Recombinant FPPS was expressed in E. coli Rosetta (DE3) cells (Novagen) and the fusion protein was purified by immobilized metal affinity chromatography (IMAC) using Xpress System Protein Purification Kit according to the supplier's protocol (Invitrogen). Sequence Analysis Sequences were determined using BigDye terminator cycle DNA sequencing kit (Applied Biosystems) and ABI prism 310 DNA sequencer. Amino acid sequences were aligned by the program CLUSTAL W (Thompson et al., 1994). Archives of Insect Biochemistry and Physiology Boll Weevil Farnesyl Diphosphate Synthase 91 Southern Blot Analysis Genomic DNA was isolated from 5 individual males and 5 individual females using MasterPure DNA purification kit (Epicentre Technologies) and digested with EcoRI. After agarose gel electrophoresis and blotting to nylon membrane (Hybond N1, Amersham), the blot was hybridized with 32P-dCTP-labeled AgFPPS cDNA fragment at 421C in 50% formamide, 0.12 M sodium phosphate, pH 7.0, 0.25 M NaCl, and 7% SDS, and washed twice for 10 min at room temperature with 2 SSC, 0.1% SDS, and once for 15 min at 371C with 0.2 SSC, 0.1% SDS. The blot was imaged with a phosphorimager (BioRad). Prenyltrasferase Assay and Product Analysis Prenyltransferase assay was performed according to Fujii et al. (1982) with modifications. The prenyltransferase activity of recombinant FPP was measured in a 200-ml reaction mixture containing 50 mM Tris/HCl, pH 7.4, 2.5 mM MgCl2, 1 mM DTT, 10 mM KF, 10% glycerol, 10 mM [14C] IPP (55 mCi/mmol, Amersham Pharmacia Biotech.), 50 mM DMAPP (Sigma), and the indicated amounts of recombinant enzyme. The mixture was incubated for 1 h at 301C and then treated with concentrated HCl for 30 min at the same temperature to hydrolyze products. The hydrolysates were extracted with hexane and the radioactivity of extracts was determined by liquid scintillation counting. The products of the prenyltransferse assay were analyzed by HPLC using a 250- 4.6-mm C18 reversed-phase column and monitored at 214 nm (Zhang and Poulter, 1993). Instead of HCl treatment, samples were boiled for 5 min and mixed 1:1 with 25 mM NH4HCO3, pH 7.0, and the following gradients of 25 mM NH4HCO3, pH 7.0/acetonitrile were applied: 100% 25 mM NH4HCO3 isocratic for 5 min, 100–0% over 30 min at a flow of 1 ml/min. One-milliliter fractions were collected and the radioactivity of the fractions was determined by liquid scintillation counting. Cross-Linking Reaction Cross-linking reactions were performed on purified, recombinant AgFPPS with dimethyl suberimidate (DMS, Pierce Biotechnology) according to Davies and Stark (1970) with some modifications. Each reaction (20 ml) contained 3.5 mg recombinant purified protein in 0.2 M triethanolamine-HCl, pH 8.5, and 5 or 10 mg of DMS freshly dissolved in the same buffer. The reaction was incubated at room temperature for 30 min or 1 h and stopped with the addition of an equal volume of Laemmli sample buffer (BioRad). Samples were denatured at 551C for 30 min and analyzed by 7.5% SDS-PAGE as described by Laemmli (1970). Gels were stained with Coomassie brilliant blue and densitometry analysis was performed using Labworks Analysis software (UVP, Inc.). Molecular Modeling The three-dimensional structure of AgFPPS was determined with a combination of homology modeling (COMPOSER) (Srinivasan and Blundell, 1993) and threading (GENEFOLD (Jaroszewski et al., 1998)) methods as implemented in SYBYL (Tripos Associates, St. Louis, MO). The avian FPPS X-ray structure (Tarshis et al., 1994, 1996; Protein Data Bank entries: 1FPS, 1UBV, 1UBX, 1UBY) was used as a template. Modeling was carried out with SYBYL, versions 6.6 to 8.0 (Tripos Associates, St Louis, Archives of Insect Biochemistry and Physiology 92 Archives of Insect Biochemistry and Physiology, June 2009 MO), on a Silicon Graphics (Mountain View, CA) Indigo2 or Fuel workstation. Energybased methods (AMBER force field) were used to refine side chain positions and to remove bad torsions and bad atomic contacts while retaining the peptide backbone of the structurally conserved regions (SCRs) of the template. The torsion angles of peptide bonds were adjusted to 1807151 with minimal constraints (0.1 kJ) using the AMBER4.1 or AMER7 force field. DMAPP, IPP, and Mg21 were manually docked into the active site and the FPPS reaction was simulated in the active site. The coordinates of allylic substrates were transferred from template models into the AgFPPS structure. The positions of substrates were refined by constrained molecular dynamics using the TRIPOS force field. Substrates were also docked into the wild-type avian FPPS X-ray structure (1FPS) and the reaction was carried out in a similar manner. The dimer of AgFPPS was built using avian FPPS dimer (PDB code: 1FPS) as a template and structures were subjected to energy minimization. RESULTS Isolation of AgFPPS cDNA A PCR cloning strategy was used to isolate the FPPS cDNA from the male boll weevil cDNA library. Sequencing of the initial 560-bp fragment amplified by PCR using two degenerate primers confirmed that it encoded a putative FPPS. Isolation of the fulllength cDNA was performed by PCR using sequence-specific oligonucleotide primers. The full-length cDNA is 1,835 bp and contains a 1,317-bp open reading frame, 160-bp 50 untranslated region (50 -UTR), and 359-bp 30 UTR including a polyA tail (Fig. 1). The open reading frame encodes a 438–amino acid protein with a calculated molecular weight of 50 kDa. Subsequently, FPPS cDNAs have been isolated from two other coleopterans; Dendroctonus jeffreyi (AY966009) and Ips pini (AY953507). The boll weevil FPPS sequence has a 79 and 66% identity to D. jeffreyi and I. pini FPPS sequences. AgFPPS shares 49% amino acid identity with Bombyx mori, 48% with A. ipsilon, and 46% with D. melanogaster and avian FPPSs (Fig. 2). Two aspartate-rich motifs and other conserved regions found in other prenyltransferases also exist in A. grandis FPPS (Figs. 1, 2). Gene Copy Number A Southern blot analysis of genomic DNA yielded a single band indicating the presence of a single FPPS gene in A. grandis (Fig. 3). Two alleles of the gene at sizes 8 and 10 kb were detected in the insects used for the analysis and one of the insects was heterozygous for the gene (Fig. 3, lane 8). Confirmation of the FPPS Activity In order to verify prenyltransferase activity isolated from A. grandis, the enzyme was expressed as a His-tagged fusion protein in E. coli. The protein was purified under native conditions using a His-tag affinity column and assayed for prenyltransferase activity. The A. grandis protein showed significantly higher prenyltranferase activity using substrates IPP and DMAPP compared to IPP as the only substrate (Table 1). Reversed-phase HPLC analysis showed that the major product of the reaction was FPP, and only minor amounts of GPP were detected (Fig. 4). The longer chain length product GGPP was not detected under our experimental conditions. Archives of Insect Biochemistry and Physiology Boll Weevil Farnesyl Diphosphate Synthase 93 Figure 1. Nucleotide and deduced amino acid sequence of A. grandis FPPS. Underlined regions are the sequences from which the degenerate primers were designed for PCR amplification of AgFPPS gene. Two aspartate-rich motifs are shown in bold. Cross-Linking With DMS In order to determine the quaternary structure of AgFPPS, cross-linking was performed with DMS. The densitometry analysis of polyacrylamide gel of products indicated that the monomeric form of enzyme (47 kDa) decreased, whereas the dimeric form of the enzyme (103 kDa) increased both with a higher concentration of DMS and with longer incubation time (Fig. 5). Although SDS-PAGE analysis indicated the presence of additional bands, the only protein bands that were affected by DMS concentration and incubation time are the ones corresponding to the monomeric and dimeric forms of the enzyme, suggesting that AgFPPS is a dimer. Molecular Model of AgFPPS Based on the structure of crystallized avian FPPS, the three-dimensional structure of AgFPPS was determined. Superposition of structure backbone atoms of the avian and AgFPPS structures gave a root mean square (r.m.s.) difference of 0.703 Å, indicating that the overall tertiary structures of both enzymes are very similar. The AgFPPS primary sequence has a 70–amino acid N-terminal extension, which is not present in the avian FPPS sequence. However, since there is not any experimental evidence of cleavage of this sequence in the boll weevil, the whole AgFPPS sequence was used to build a three-dimensional structure. The molecular structure of AgFPPS shows the protein folded as a single domain, which was composed of all-antiparallel a-helices Archives of Insect Biochemistry and Physiology 94 Archives of Insect Biochemistry and Physiology, June 2009 Figure 2. Alignment of the deduced amino acid sequence of FPPSs. AgFPPS is aligned with insect FPPSs from I. pini, D. jeffreyi, B. mori, A. ipsilon, D. melanogaster, and avian FPPS. Identical residues are indicated by an asterisk, and similar residues by a period. The boxes indicate five highly conserved regions. Archives of Insect Biochemistry and Physiology Boll Weevil Farnesyl Diphosphate Synthase males M 1 2 3 4 95 females 5 6 7 8 9 10 M 10 kb 8 kb 6 kb 4 kb Figure 3. Southern blot analysis of genomic DNA from individual male (lanes 1–5) and female (6–10) boll weevils. M: DNA marker. Table 1. Prenyltransferase Activity of the Purified Recombinant AgFPPS Expression vector pTrcHis2c without insert PTrcHis2c with insert IPP1DMAPP IPP only no enzyme Prenyltransferase activity (dpm) 122 23,095 1,943 188 The enzyme reaction was performed using His-tag column purified proteins of E. coli expressing either pTrcHis2c without insert or pTrcHis2c with AgFPPS cDNA insert. connected by loops (Fig. 6). The overall folding pattern of two enzymes is highly conserved except for the N-terminal region of AgFPPS (Fig. 6). The average positional energy of AgFPPS is 0.06 kT, whereas the template crystal structure has an average positional energy of 0.21 kT. The higher positional energy of AgFPPS is mostly due to the N-terminal extension. When the N-terminal extension is omitted, the average positional energy improves (0.12 kT). Two aspartate-rich sequences that are highly conserved among isoprenyl diphosphate synthases and other catalytically important residues are located around the major central solvent-accessible channel analogous to the active site cavity in avian FPPS (Fig. 6). All six aspartate residues in FARM and SARM regions have the same rotomers and the side chains of all the catalytic residues extend into the active site cavity and superimpose with analogous residues in the avian FPPS structure except for the side chain of Lys141 (Lys71 in avian FPPS). When the substrate DMAPP and two Mg21s are docked into the AgFPPS structure, Lys141 side chain changes its position and becomes like the analogous Lys in avian structure (Lys 71) (Fig. 6c). In the AgFPPS and avian structures, DMAPP and two Mg21s occupy the equivalent positions. DMAPP binds to the allylic substrate-binding site (FARM) as it has been shown for avian FPPS crystal structure and is in position where diphosphate groups can bind to the aspartate carboxyl oxygens through magnesium bridges. In addition to Asp residues in the FARM, Arg196 (Arg126 in avian FPPS) is also in close proximity to allylic substrate. This residue has been implicated to play an important role during catalysis (Tarshis et al., 1996; Kellogg and Poulter, 1997). Condensation of reactants in the active site was simulated, first forming GPP and then FPP (data not shown). The water-accessible channels of both structures with GPP bound to an allylic binding site indicate that the hydrocarbon tail is oriented down the Archives of Insect Biochemistry and Physiology 96 Archives of Insect Biochemistry and Physiology, June 2009 50000 40000 DPM (+/- s.e.) 10000 5000 0 IPP unused substrate FPP GPP products Figure 4. Product analysis of A. grandis FPPS. Products of the prenyltransferase assay were determined by reversed-phase HPLC using [14C]IPP and DMAPP as substrates as described in Materials and Methods. The retention times were 3–4 min for IPP (C5), 16 min for GPP (C10), and 20–21 min for FPP (C15). (A) 1 2 3 4 5 6 kDa 200 116 97 66 45 31 log MW (B) 2.5 2 1.5 1 0.5 0 y = -1.1044x + 2.4538 R² = 0.9742 0 0.2 0.4 0.6 distance 0.8 1 (C) MW (kDa) 114 103 86 47 40 no DMS 5 µ g/30 min 10 µ g/30 min 23 24 7 14 100 58 12 51 11 5 µ g/1 h 10 µ g/1 h 10 14 18 38 26 27 7 6 39 16 Figure 5. Cross-linking with dimethyl suberimidate. The cross-linking experiment was carried out as described in Materials and Methods. A: SDS-PAGE of expressed AgFPPS before and after reaction with cross-linking reagent, DMS. Lane 1, molecular weight standard; lane 2, untreated enzyme (no DMS); lane 3, enzyme treated with 5 mg DMS for 30 min; lane 4, enzyme treated with 10 mg DMS for 30 min; lane 5, enzyme treated with 5 mg DMS for 1 h; lane 6, enzyme treated with 10 mg DMS for 1 h. B: Semilog plot of molecular weight (MW) versus migration of A. C: Densitometry analysis of monomeric and dimeric forms of AgFPPS in A. Values represent % of total protein. Treatment conditions with DMS are indicated at the top of each column. Archives of Insect Biochemistry and Physiology Boll Weevil Farnesyl Diphosphate Synthase 97 Figure 6. Structures of (A) A. grandis FPPS and (B) Avian FPPS (1FPS). The catalytic residues are shown in space fill representation (AgFPPS: R196, R197, C182, F183, D187, D188, D191, D328, D329, D332, K141, K285, K351, F324, F325; Avian FPPS: R126, R127, F112, F113, D117, D118, D121, D257, D258, D261, K71, K214, K280, F253, F254) are shown in space fill. FARM and SARM regions are labeled. C: Two conformers of side chain of Lys141 in AgFPPS. The amino group of the side chain moves from the left side to the top of the panel upon binding of DMAPP. hydrophobic pocket (Fig. 7a). The hydrocarbon tail of FPP also extends down the hydrophobic pocket where further growth is restricted by the size of the binding pocket (Fig. 7b). Interaction energy calculations with docked FPP ligand using molecular dynamics revealed favorable interaction with both enzymes (25379 kcal/mol and 322717 kcal/mol for avian and boll weevil enzymes, respectively). Radius of gyration (ROG) indicates internal motions of the ligand in the active site and deformation (DEF) indicates the relative position of a ligand with respect to a reference position. In both FPPSs, the ROG and DEF averages and standard deviations are similar (Table 2). ROG measurements indicate that avian FPPS has a little more room for the FPP ligand. Based on these measurements, AgFPPS holds the ligand slightly more tightly. In most FPPSs, the fifth and fourth amino acids before the FARM are Phe Phe or Tyr Phe residues that are indicated to be involved in the product chain length specificity of the enzyme (Tarshis et al., 1996; Ohnuma et al., 1996a–c). In wild type avian enzyme (1FPS), these residues (F112, F113) are in position to prevent elongation of the hydrocarbon tail (Fig. 7b). In AgFPPS, these residues are Cys and Phe (at positions 182 and 183) and although the fifth amino acid before FARM is not an aromatic amino acid residue, these residues are still in position to restrict the growth of the hydrocarbon chain (Fig. 7b). Mutation of these residues in the avian protein (1UBW, F112A, and F113S) with smaller side chain residues makes the hydrophobic pocket larger and allows formation of longer products (Tarshis et al., 1996). Although the presence of Cys residue in AgFPPS in place of Phe residue in avian FPPS makes the pocket larger, it is still not wide enough to allow condensation with another IPP. To show more detail, distances between FPP and these two residues and also conserved Asp residue (Asp117 in avian and Asp197 in boll weevil) were measured (Table 3). It should be noted that the model used has been globally minimized only and no refinement of the active/binding site has been made. The comparison of distances reveals that the AgFPPS pocket is even more restricted than the avian pocket. Active site residues in close proximity to FPP were further examined (Table 4). Differences of 0.1 to 4 Å are seen between the boll weevil and analogous avian residues. Again we note that the boll weevil enzyme has been globally minimized: No refinement of the active/binding site has been made. The alpha carbons of the residues in Table 4 superimpose when two structures are overlaid (Fig. 7C), whereas the side chains are mobile and occupy different locations in the FPP-bound enzymes (for example, Lys280 [avian]/Lys351 [boll weevil]; Fig. 7C). The AgFPPS dimer was built using the avian FPPS dimer (1FPS) as a template. Again, except for the N-terminal segment of the AgFPPS, the structures are very similar (Fig. 8a). The N-terminal extension of the AgFPPS does not interfere with Archives of Insect Biochemistry and Physiology 98 Archives of Insect Biochemistry and Physiology, June 2009 Figure 7. Elongation of hydrocarbon tail in the active site. The substrate-binding site cavity of boll weevil and avian FPPSs is shown with GPP (A) and FPP (B). Water-accessible channel surfaces are shown in green. The enzyme has been sectioned to show the reactants in the binding/active sites of the enzyme. Residues involved in chain length determination are shown (AgFPPS: Cys182, Phe183; avian FPPS: Phe112, Phe113). C: Contains the superimposed structures of avian and boll weevil FPPSs showing active site lysine residues and bound FPP (shown in red). Backbones were displayed as tubes (magenta: avian, orange: boll weevil). Boll weevil residues are colored yellow. Table 2. Radius of Gyration and Deformation Values (A˚) for Ligand FPP in Avian and Boll Weevil FPPSs Avian FPPS AgFPPS ROGf ROGI ROGw DEFf DEFI DEFw 1.5570.20 1.5570.03 1.5470.20 1.5470.03 5.5470.12 5.3770.09 0.8370.10 0.6570.19 0.9170.40 0.5970.24 1.4870.27 1.4270.20 ROGf and DEFf, radius of gyration and deformation of first isoprene unit next to the diphosphate group of FPP, respectively. L, last isoperene unit; w, whole ligand. subunit interaction, as it is located outside of the putative subunit interface. The active sites of each subunit are located close to the dimer interface and open to the outside. The interaction surfaces of monomers in both structures contain complementary hydrophobic patches (Fig. 8b). The total amount of hydrophobic areas buried represents 27 and 26% of the total contact surface areas in avian and AgFPPSs, respectively. Avian enzyme has slightly more hydrophobic contacts than does the boll weevil enzyme. Electrostatic potential calculations of the subunit interfaces indicated Archives of Insect Biochemistry and Physiology Boll Weevil Farnesyl Diphosphate Synthase 99 Table 3. Distances (A˚) Between a-Carbon of Selected Active Site Residues and FPPS Methyl Carbon in Avian and Boll Weevil FPPS Structures FPP FPP F112/C182 F113/F183 D117/D187 8.4/6.5 7.8/5.5 10.3/8.9 F112/C182 F113/F183 D117/D187 8.4/6.5 7.8/5.5 3.8/3.8 10.3/8.9 8.4/8.9 5.8/6.2 3.8/3.8 8.4/8.9 5.8/6.2 Residue numbers in avian/boll weevil enzymes, respectively. Table 4. Distances (A˚) Between a-Carbon of Selected Active Site Residues in Avian and Boll Weevil FPPS Structures R126/R196 D117/D187 D121/D191 D188/D258 K214/K285 K271/K342 K280/K351 R126/R196 D117/D187 D121/D191 D188/D258 K214/K285 K271/K342 K280/K351 10.6/10.7 10.6/10.7 11.9/12.3 19.3/16.9 18.4/15.2 8.2/7.1 16.8/12.9 6.3/5.1 11.2/10.4 11.5/10.9 14.1/17.7 13.7/12.1 11.9/12.3 6.3/5.1 11.6/9.0 15.5/13.1 14.8/18.7 11.4/9.0 19.3/16.9 11.2/10.4 11.6/9.0 8.2/8.2 18.0/22.4 9.1/9.1 18.4/15.2 11.5/10.9 15.5/13.1 8.2/8.2 16.8/20.1 13.8/13.3 8.2/7.1 14.1/17.7 14.8/18.7 18.0/22.4 16.8/20.1 16.8/12.9 13.7/12.1 11.4/9.0 9.1/9.1 13.8/13.3 13.2/16.6 13.2/16.6 Residue numbers in avian FPPS/analogue residue in AgFPPS. Figure 8. Left pair: A comparison of the avian and boll weevil FPPS dimers. The a-helices are shown as tubes. One subunit of the dimer is shown in blue and the other one is in cyan. FARM and SARM are in space fill and located on opposite walls of the binding cavity. Middle and right pairs: Hydrophobic potential of the interacting surfaces of the FPPS homodimer. The monomers of FPPS have been rotated 901 on the y axis in opposite directions to show the monomer interface surfaces. The hydrophobic potential is colored from brown (most hydrophobic) to blue (least hydrophobic). that 49% of the contact surfaces are positively charged in AgFPPS, which suggests complimentary electrostatic forces. On the other hand, in avian FPPS only 9% of the surfaces are positively charged, which indicates repulsive electrostatic forces between subunits. DISCUSSION The isoprenoid pathway has been mostly studied in mammalian systems due to its importance in the regulation of cholesterol production (Goldstein and Brown, 1990). In insects, juvenile hormone has been the most studied product of the isoprenoid pathway (Koyama et al., 1985). However, the importance of the isoprenoid pathway in general cell function, cell signaling, development, and reproduction is becoming more clear (Havel et al., 1992; Lai et al., 1998; Van Doren et al., 1998). FPP serves as a Archives of Insect Biochemistry and Physiology 100 Archives of Insect Biochemistry and Physiology, June 2009 precursor to non-sterol isoprenoid pathway products of insects including dolichols, ubiquinones, prenyl groups of certain proteins, and juvenile hormones in addition to being the putative precursor to female-produced pheromones (Hedin et al., 1979). Insect FPPSs characterized to date are mostly from lepidopterans and were studied in the context of juvenile hormone biosynthesis (Castillo-Garcia and Couillaud, 1999; Kikuchi et al., 2001; Cusson et al., 2006; Sen et al., 2007b). We isolated the first coleopteran FPPS from A. grandis. The predicted amino acid sequence has high sequence identity to other insect sequences (between 46 to 79% identity) and to avian FPPS (46%) for which the crystal structure is known (Tarshis et al., 1994, Fig. 2). The AgFPPS contains a 70-amino-acid-long N-terminal extension, which is not found in the avian FPPS sequence. Similar N-terminal extensions are found in other insect sequences (Fig. 2, Cusson et al., 2006) and also are observed in plant (Cunillera et al., 1997) and mammalian FPPSs (Martin et al., 2007). The N-terminal extension of AgFPPS displays the characteristics of mitochondrial-targeting peptide and contains RXXS sequence, which is a consensus cleavage motif in mitochondrial targeting peptides (Von Heijne et al., 1989, Fig. 2). Recent work has demonstrated that the N-terminal extension of D. melanogaster FPPS targets the protein to mitochondria (Martin et al., 2007), which suggests that N-terminal sequence of AgFPPS and of other insects may act in a similar way. Southern blot analyses of genomic DNA of A. grandis indicated that AgFPPS is a single copy gene (Fig. 3). However, this does not rule out the possibility that a single gene may encode more than one isoform. A survey of the D. melanogaster genome database indicates that FPPS is a single copy gene that produces two FPPS transcripts (Sen et al., 2007a). In A. thaliana, there are two FPPS genes encoding three isoforms, one of which has an N-terminal mitochondrial targeting peptide (Cunillera et al., 1996, 1997). Similarly, in mammals the single FPPS gene is alternatively spliced giving rise to mitochondrial and cytosolic/peroxisomal isoforms (Martin et al., 2007). Insect sequences also exhibit differences in residues that function in product chain length determination. The AgFPPS has Cys and Phe residues in place of Tyr-Phe or Phe-Phe residues found at the fourth and fifth position before the FARM in animal FPPS. In D. melanogaster, the two aromatic residues are conserved. Interestingly, another coleopteran FPPS, that from D. jeffreyi, has Tyr and Val residues in place of these two aromatic residues. In Lepidoptera, two different types of FPPSs were identified (Cusson et al., 2006). In type-2 lepidopteran FPPSs, only one of the aromatic residues was conserved as in the case of A. grandis. On the other hand, type-1 lepidopteran FPPS sequences have His and Gln residues in these positions. In addition, in type-1 moth sequences, the typical DDxxD motif of FARM is replaced by NDxxE. Although it has been suggested that these active site substitutions in lepidopteran FPPS may be involved in production of ethyl-branched FPP precursors of juvenile hormone (Cusson et al., 2006; Sen et al., 2007b), the exact role of these variations in lepidopteran and in other insect FPPSs is not known. The prenyltransferase isolated from A. grandis was shown to produce FPP as the major product (Fig. 4). Molecular modeling studies also suggested that the final product of the A. grandis enzyme is FPP. Putative fold of boll weevil FPPS indicated that in spite of the evolutionary distance, boll weevil FPPS has a remarkable conformational and functional homology to avian FPPS. The global fold is preserved. Except for the N-terminal extension, the three-dimensional structure of AgFPPS is very similar to the X-ray structure of avian FPPS (Fig. 6). The AgFPPS protein folds as a single domain Archives of Insect Biochemistry and Physiology Boll Weevil Farnesyl Diphosphate Synthase 101 composed of anti-parallel a-helices, which surround a large central cavity. Improvement of the average positional energy of the AgFPPS structure (0.06 kT vs. 0.12 kT) when the N-terminal extension is not included in the model, further suggests that AgFPPS is transcribed as a pro-enzyme and N-terminal extension is cleaved. Critical residues at the binding/active site are also conserved. The AgFPPS sequence contains all five conserved regions found in prenyltransferases, including two Asp-rich motifs (Figs. 1 and 2), and they are located around the hydrophobic central cavity as in the case of avian FPPS (Fig. 6). Molecular dynamics simulations demonstrate that the product FPP interacts in an analogous manner with both insect and avian enzymes. The calculated interaction energies of substrates with enzyme suggest similar binding for both structures. These observations leave little doubt that the insect enzyme will have kinetic properties and product specificity similar to the avian enzyme. The major difference between boll weevil and avian enzyme is the replacement of a phenylalanine by a cysteine in the bottom of the product specificity pocket. The role of this substitution is unknown. However, based on our current model of AgFPPS, the active site cavity is still restricted to prevent formation of longer products (Fig. 7b) and the product specificity of the AgFPPS also results from steric constraints of the binding site, similar to avian FPPS. The location of a reactive cysteine residue provides a unique opportunity to gain insights into isoprenoid-synthesizing enzymes. The physicochemical properties of this site can be probed by thio-reactive reagents of varying polarity and bulk. Under favorable circumstances, the modified enzyme can also be assayed for kinetic properties and product specificity. The cysteine can also be used to insert reporter groups such as fluorophores into the specificity pocket. Short-chain E-prenyltransferases require divalent metal ions such as Mg21 or Mn21 for catalytic activity and they have a homodimeric structure (Ogura and Koyama, 1998). The only known exception to this is the GPPS from peppermint, which functions as a heterodimer (Burke et al., 1999). The large subunit of this GPPS shows a high level of sequence similarity with other prenyltransferases and contains aspartate-rich motifs. The small subunit lacks these motifs and has a lower level of sequence similarity to other prenyltransferases. It has been suggested that a small subunit functions in chain length determination (Burke and Croteau, 2002a). On the other hand, other plant GPPSs are homodimers (Burke and Croteau, 2002b). Insect GPPS from Ips pini also has been shown to be active when no heteromeric subunit is present (Gilg et al., 2005). Recent studies with type-1 and type-2 lepidopteran FPPSs indicated that type-1 FPPSs were catalytically inactive and type-1 and type-2 FPPSs can form a heteromer that enhances the activity (Sen et al., 2007b). The cross-linking experiment with recombinant purified AgFPPS gave strong evidence that the functional form of AgFPPS is a homodimer similar to the majority of the other FPPSs (Fig. 5). The model of the AgFPPS dimer indicates that dimer formation is an energetically favorable process, supporting a homodimeric structure of AgFPPS. In the dimer structure, the active sites are not hindered and open to the outside (Fig. 8) as in the case of avian FPPS. The hydrophobic surface interfaces of monomers are also compatible in both structures (Fig. 8). LITERATURE CITED Burke HR. 1959. Morphology of the reproductive systems of the cotton boll weevil (Coleoptera, Curculionidae). Ann Entomol Soc Am 52:287–294. Archives of Insect Biochemistry and Physiology 102 Archives of Insect Biochemistry and Physiology, June 2009 Burke C, Croteau R. 2002a. 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Gary Paul Cox, 73, of Ardmore passed away April 22, 2020 at the Mercy Hospital in Ardmore. There will be no services at this time.
Gary was born May 30th 1946 in Ardmore, OK to Clyde and Ruth Webb Cox. Gary was born and raised around Lake Murray area. He attended school at Love County Public schools and graduated with the Marietta High School Class of 1965.
Gary managed many Snack Bars at many VA Centers and office buildings around Oklahoma, eventually settling back in Ardmore. He married Annette Cox in Lone Grove March 26, 1983. He joined the United States Army in April of 1968, but was Honorably Discharged in May of 1968 due to medical reasons. He loved to garden, go fishing and work on motors of any sort. He also like to go for walks. He was a member of the Church of Christ on Merrick Drive.
Gary is preceded in death by his parents, wife Annette Cox, sister Lovetta Williams, sister Linda Freeman, and three infant brothers.
He is survived by daughters Melissa and John Kennedy of Ardmore, and Lesley Cox of Healdton, grandchildren April Russell, Mathew Russell, Alex Russell, great grandchildren; Brooklynn, Peyton, MaKayla, Allen, Greyson, Jaxon, Jordan, Dakota, sisters Joanne and her husband Bobby Stinson of Allen, TX and brother in law Alvin Freeman of Marietta, OK.
Gary's spirit will live on in many lives he Blessed through Organ and Tissue Donations.
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Marc Roberge (of O.A.R.) 10/22/19 10pmTuesday, October 22 2019 9:30pm Doors / 10:00pm Start
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MARC ROBERGE is a songwriter/producer and lead vocalist for the American rock band O.A.R.
What began as a group of childhood friends making songs in the drummer's basement after school, O.A.R. became widely accepted as one of the most successful independent bands of all time.
With 13 albums, a string of Madison Square Garden sellouts, USO trips to Iraq and Kuwait, millions of albums sold, shows around the world Roberge and O.A.R. continue to cover new ground creatively.


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Board of Trustees
The Catholic Central Schools' Board of Trustees takes an active role in managing the organization. Their mission statement is as follows:
"Building on the legacy of Catholic education, the Catholic Central Board of Trustees provides leadership in generating and creating an environment that is inclusive of all. In partnership with the Archdiocese of Cincinnati, we develop policies to ensure that Gospel values permeate academic excellence and personal development. We ensure stability, fiscal responsibility and effective governance for the school."
Catholic Central 2019-2020 Board of Trustees:
Dave Simonton – Chairperson
Matt Mullen – Vice Chairperson
Suzanne Woeber – Secretary
Mark Derr
Peggy Hearlihy Johnson
Reverend John MacQuarrie
Jennifer Daniels
Charlie Setterfield
Michael Trempe
Genoveva Escamilla


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Annual rental increases are an unfortunate fact of life for tenants, and a vital tool for landlords when it comes to covering expenses and protecting their investment. Getting both parties to agree on a reasonable annual rental escalation isn't always the easiest conversation to have, but according to Jacqui Savage, National Rentals Business Development Manager for the Rawson Property Group, a fair negotiation is in the best interests of everyone involved.
"A lot of people are still under the impression that a 10% annual rental increase is standard," she says. "In reality, there are no legislated minimums, maximums, or 'standard' figures – annual escalation is set on a case by case basis."
That doesn't mean landlords can simply pick a number and force tenants to sign on the dotted line, however.
"The Rental Housing Tribunal is very clear about the fact that increases need to be reasonable, which means landlords need to be able to back up their escalation figures with properly researched industry trends," says Savage.
"It's not all about statistics either," she says. "You need to take the specific tenant into account as well. A good tenant, who looks after your property, pays on time and communicates well with your managing agent can be worth their weight in gold. The national percentage of tenants in good standing is slowly improving, but it's still definitely worth considering a slightly lower escalation rate if it means keeping a reliable tenant in place and happy."
This, says Savage, is the key to a tenant's bargaining power during negotiations.
"You want to be the kind of tenant your landlord doesn't want to lose," she says. "Yes, that means paying your rent on time, but it also means being proactive when it comes to small, day-to-day maintenance chores and minor fixes. If you call the landlord every time a lightbulb needs changing, they're not going to be too sad to see you go."
According to Savage, losing a tenant because of unreasonable rental escalation is a real possibility, even in traditionally high-demand areas like the Western Cape.
"Availability is increasing, tenants are shopping around, and we're seeing more and more vacancies in properties where the landlords aren't adjusting to current market conditions."
But how are landlords supposed to keep track of what is and isn't reasonable? And what's the best way to weigh up the benefits of retaining an existing tenant against the disadvantages of reduced escalation?
"A lot boils down to experience and having an in-depth understanding of the current market," says Savage. "That can be very difficult achieve as a private individual. It's one of the major benefits of using an experienced managing agent – their expertise can be invaluable during negotiations as well as over the course of the lease. Whether acting on your own or through an agent, however, it's vital to have a conversation about escalation with your tenant, and make sure you're all on the same page when the lease is signed."
For more advice or assistance on renewals and fair escalation negotiations, get in touch with a Rawson Property Group Rental Agent near you or email email@example.com .


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BBQ Passion Steak Plates XL Set 35cm - set of 2 by Villeroy & Boch
The dishwasher-safe collection boasts a sophisticated design. The robust BBQ Passion collection has everything true barbecue enthusiasts need for a great BBQ party. These XL steak plates are perfect for larger portions. A compartment separates the meat from the side dishes and sauces. The perfect barbecue experience on robust Premium Porcelain.


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FEAST: Families Eating Affordably and Sustainably Together
November 20, 2018 @ 5:30 pm - 7:00 pm
A series of free workshops late November-April happening Tuesdays at 5:30pm at the Nelsonville Public Library.
Cook together, share a meal, enjoy door prizes, and inspire healthful eating with hands-on learning and demonstrations. Pre-registration and menus at https://www.myacpl.org/feast/.
A project of the Hunger Prevention Coalition. Presented by Community Food Initiatives, Live Healthy Appalachia, and OSU Extension Snap-Ed. With additional support from Athens County Public Libraries, Integrated Services, Ohio State Alumni of Athens County, Nelsonville Emporium, Saving Hardware, and others.


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By Andy Sambidge
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Former Germany goalkeeper Oliver Kahn in trouble for not declaring nearly $10k of Dubai shopping
Former Germany goalkeeper Oliver Kahn has been fined 125,000 euros for failing to declare more than 6,000 euros ($9,745) of luxury clothing he bought on a trip to Dubai.
The 41-year-old was fined for tax evasion by a regional court in Bavaria, after he failed to declare the clothes on his return to Munich airport from Dubai, German media reported.
The collection of polo shirts, t-shirts, sweaters, shirts, trousers, jackets and cuff-links were from top brands such as Armani and Dolce & Gabbana.
At customs in Munich airport, Kahn used the 'nothing to declare' aisle, but the tax owed on the imported goods was 2,119 euros, which resulted in the fine.
Kahn, who retired in 2008, captained both Bayern Munich and Germany during his career, winning 86 caps for his country and he made 557 appearances in the Bundesliga, as well as 103 in the Champions League.
This is exactly the moment when you need these receipts from Karama...
the fine was 50 days salary of €2,500. The initial fine was €350,000 and took the old Bayern salary of €7,000 per day into account. Madness. Money grabbing Government at its worst and a fine completely out of context with the offense. It also shows that shopping in the UAE is ok when you live there or in countries where the import tax is not levied, but don't even think about it when you have to declare it in places such as the EU or the USA.
It is anyhow silly that he bought all this stuff in Dubai as a lot of things are much more expensive here than in Germany.
He was probably at Outlet Mall ;)
Clearly Revilo has not been anywhere outside Deira. Dubai is comparatively cheaper than Europe but yes not when compared to East Asia or North America. Fact.
MM, nonsense!
Seems you haven't been out much either. Everybody knows that you can easily save up to 30 %, despite taxation, on all kind of goods.
Probably because we actually have a competing market and not a regulated import by a handful of agencies.
would love to hear what is cheaper in Dubai than in Germany or Europe? Also take into account availability of credit facilities in Europe that you get there rather than in Dubai?
Sorry ppl but, these days, Dubai is more expensive. As a result, I no longer shop for clothes in Dubai.


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Studio 23 This is Life with Cory Quirino tackles the Efficacy of Novuhair on Hair Loss
Hair is a living protein, and as with any part of our bodies we must be sure to maintain proper health to optimize the chances of continuing to grow a healthy head of hair. Proper nutrition is vital to hair loss prevention, since the hair is a living and growing part of the body's system. A healthy balanced diet, exercise, vitamins and herbal hair loss supplements will all aid in your hair loss prevention efforts.
Remember, the restoration of hair growth is not an overnight process. The process takes time regardless of the method chosen. Be patient, even the most effective hair loss products will take at least 3 months to show signs of improvement. Don't give up, many people have had great success with their hair loss prevention.
Courtesy of: http://www.keepingyourhair.com/
Photo courtesy of: http://www.hairtx.com
A hair follicle is part of the skin that grows hair by packing old cells together. Attached to the follicle is a sebaceous gland, a tiny sebum-producing gland found everywhere except on the palms, lips and soles of the feet. The thicker density of hair, the more sebaceous glands are found.
Also attached to the follicle is a tiny bundle of muscle fiber called the arrector pili that is responsible for causing the follicle lissis to become more perpendicular to the surface of the skin, and causing the follicle to protrude slightly above the surrounding skin (piloerection). This process results in goose bumps (or goose flesh). Stem cells are located at the junction of the arrector and the follicle, and are principally responsible for the ongoing hair production during a process known as the Anagen Stage.
At the base of the follicle is a large structure that is called the papilla. The papilla is made up mainly of connective tissue and a capillary loop. Cell division in the papilla is either rare or non-existent.
Hair grows in cycles of various phases: Anagen is the growth phase; Catagen is the involuting or regressing phase; and Telogen, the resting or quiescent phase. Each phase has several morphologically and histologically distinguishable sub-phases. Prior to the start of cycling is a phase of follicular morphogenesis (formation of the follicle).
There is also shedding phase, or exogen, that is independent of Anagen and Telogen in which one of several hairs that might arise form a single follicle exits. Normally up to 90% of the hair follicles are in Anagen phase while, 10%-14% are in Telogen and 1%-2% in Catagen.
The cycle's length varies on different parts of the body. For eyebrows, the cycle is completed in around 4 months, while it takes the scalp 3-4 years to finish; this is the reason eyebrow hairs have a fixed length, while hairs on the head seem to have no length limit. Growth cycles are controlled by a chemical signal like epidermal growth factor.
Join Cory Quirino as she interviews one of the users of Novuhair and get to know more about the excellent results using the treatment.
Click here to download and watch the video: This is Life May 29, 2010