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$ 44.99 $ 70.00Size Guide
Bridesmaid Gifts, African Jewelry set, African Print Bangle Bracelet And Earring Set, African Fabric Earring And Bangle Set,African Ankara Fabric jewelry gift set is beautiful.
♥ Are you looking for a one of a kind interesting gift for your special bridesmaids? Or something different for them to wear at your wedding? You chose your African Fabric and sizes (small, medium or large) and we will create a custom set just for you
These hand-wrapped bangles are made with fabrics from Liberia. They feature traditional designs, as well as modern interpretations of traditional motifs. Many different colors and designs available, see photos for examples. They are great to mix and match, both large and small, modern and traditional....
Slip-on (no closure)MEDIUM/LARGE BANGLES -
Diameter: about 3"
Circumference: about 11''
Because fabric is cut by hand, pattern will be similar to those pictured but not exact. All earrings are made to order and therefore each will be unique and different from the last.
This set includes 2 bangles, a set of dangling earrings and a necklace, It is sure to please the most discerning buyer.
For more styles please visit....Please visit my shop at Etsy.com/shop/ZabbaDesigns
For more photos, updates and special offers please visit Zabba Designs on Facebook
For styling suggestions follow Zabba Designs
WAX & African Print Fabric
Wax is the name for a factory-made fabric produced using a mechanical wax resist (batik) during the cloth dying process; each color is dyed into the cloth. African Print Fabrics are also made by rolling ink across a printing plate and rolling the ink onto the cloth.
Matching earrings and bangles available
$ 64.99Size Guide
Aqua African Print HeadWrap And Jewelry Set., African Ankara print headwrap and jewelry set includes a fabric cover necklace design complete with matching bracelet and earrings this set is guaranteed to bring compliments. African Fabric jewelry bridal party jewelry.These hand-wrapped bangles are made with fabrics from Liberia. They feature traditional designs, as well as modern interpretations of traditional motifs. Many different colors and designs available, see photos for examples. They are great to mix and match, both large and small, modern and traditional....
Item Description:
- A gorgeous and exotic 3 piece set of traditional and authentic women's fabric jewelry.
- Set includes matching necklace and earrings.
- Beautiful tones of green and yellow
- The necklace is a choker with green stones. A stunning beautiful piece which can be worn on bare skin with a low cut dress/blouse/top or on top of the fabric of a high cut dress/blouse/top.
Perfect holiday gift set. Your purchase will arrive beautifully packaged for immediate gift giving (or as a splendid present for yourself!). If this purchase is a gift and you would like me to include a handwritten message, please leave me a note in the "message to Zabba Designs" box at checkout to let me know!
$ 24.99Size Guide
$ 24.99Size Guide
If you are not sure of your size go a size up from the size your normally go for.


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Our diffuser is intended to be used only with 100% pure, high quality essential oils. The oils should be used full strength, not diluted with water or any carrier. Resinous oils like frankincense and myrrh will clog the system and should never be used. The diffuser is small and light weight, making it easy to move from room to room to inhibit mold growth and reduce pathogens in the air. However, prolonged inhalation is not recommended. It is better to sit six feet away from the diffuser and operate the diffuser for 3-5 minutes at a time. If the room is unoccupied, the diffuser can run for hours. May we suggest putting the diffuser in the basement, attic, and any other rooms that have poor air circulation and/or high moisture. Over a period of weeks, the quality of the air should improve.
Diffuser Blend, organic, 30 ml.
This blend of 100% pure, steam distilled organic essential oils is intended for use in rooms that have a history of mold. When used over a period of weeks, the mist creates an invisible shield that inhibits mold growth. The spores may still be there, but the oils discourage the mold from becoming vegetative. Of course, the oils smell very nice so this blend can be used to improve the smell in a room. The oils can be inhaled but the recommended amount is 3-5 minutes of inhalation. This length of time is not adequate for mold protection so the rooms should be vacant most of the time when diffusing for hours or days on end.
Contents: proprietary blend of organic essential oils lemon, rosemary cineole, eucalyptus globulus, cinnamon bark, and clove bud essential oils. All oils are steam distilled and 100% organic.
Professional Ultrasonic Aromatherapy Nebulizer with Organic Essential Oil Blend
This ultrasonic nebulizer is used with water. It has an automatic shut off in case the water level is too low. It can be used with high quality, pure essential oils as an air purifier, disinfectant, or humidifier. Since we specialize in therapeutic use of essential oils, we recommend two to five short inhalation sessions per day. Sit three to six feet from the unit and inhale some of the aromatic vapor for not more than five minutes. It is safe to run the nebulizer in an empty room, no pets or people, for days at a time to inhbiit mold growth and/or purify the air. We are willing to help you to choose the right oils but will ship this unit with two small samples.
For pdf on the Diffuser, click here click here.
Contents: Contains one high quality nebulizing diffuser and two 5 ml. bottles of essential oil as samples.
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David Thurman, of the Free Town Road Church of Christ in Grand Prairie, delivers a powerful message on the Gospel in the real world. Titled "God, Bible, Heaven: What Does It Mean In The Real World?", this lesson has quickly become the most requested lesson of all time at Bridgewood. Check it out for yourself by clicking on the video link above.
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I can and do listen to this lesson over and over because it refreshes my soul!
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text: Are you to blame for his affair? I think to some extent yes you are and women need to start paying attention to the little details. Looking back at that time I do admit I feel some responsibility for my husband's wandering eye. It happened years ago when I was pregnant with our first child. While many will argue that no matter what the circumstances are no man should … [Read more...]


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Those words flew out of my mouth, as sneaky and surprising as little birds that had been waiting behind my teeth to get out. Apparently, they did the trick. I could see my announcement making a fist around his heart. I was so full of myself, so confident. One thing I knowed I could do was charm a man until he couldnt hardly stand it.
I wanted Saul Sullivan, plain and simple. That was all there was to it. I didnt love him—that came later—but I thought that I did. I mistook lust for love, I guess. I knowed that I could fill up some hole that he had inside of himself and hadnt even been aware of until laying eyes on me. Saul looked to me like he needed to lay his head down in somebodys lap and let them run their hand in a circle on his back until he was lulled off to sleep. I knowed that I was the person to do it. I had been waiting a long time for such a feeling to come to me.
That whole summer, I kept one eye on the road as I went about my chores. I throwed corn to the chickens without even watching them, bent over to pick beans and looked upside down at the road, where I might see his horse come trotting down foamy mouthed and big eyed. At first, when I caught sight of Saul heading down into Redbud Camp, I would turn back to the task at hand and make him think I hadnt seen him coming. Hed have to stop at the gate and yell out for me. I did this just to hear him holler. I loved his full-throated cry: "Vine! Come here to me!" I loved to hear my name on his tongue. But as summer steamed on, I couldnt bring myself to continue such games, and Id rush out to the road as soon as I seen him coming. Id throw down the hoe or the bucket of blackberries or whatever I was packing. Id leave one of my little cousins that I was supposed to be tending to, would rush off the porch even though Mama had ordered me to peel potatoes. The more he come by, the harder it was to stay away from him.
Mama frowned on all of this. Every time Id get back from being with him, shed wear a long, dark face and not meet my eyes. "Its not fitting," she said. "People ought to court their own kind."
"There aint no Cherokee boys to court," I said. "Theyve left here."
"Just the same," Mama said, and dashed water out onto the yard. Her face was square and unmovable. "Them Irish are all drunks."
I couldnt help but laugh at her, even though I knew this would make her furious. "Good Lord, Mama, thats what they say about Cherokees, too."
Daddy made no objections. Him and Saul went hunting together and stood around in the yard kicking at the dust while they talked about guns and dogs. Saul brought him quarts of moonshine and sacks of ginseng. We were kin to everybody in Redbud Camp, and when they seen that Daddy had warmed to Saul, they started speaking to me again. Everybody looked up to Daddy, and if he approved of Saul, they felt required to do the same. My aunts Hazel and Zelda and Tressy even seemed to be taken with him. They talked about him while they hung clothes on the line, while they canned kraut in the shade, when everyone gathered to hear Daddys hunting tales at dusk.
"Wonder if hes freckled all over," Hazel whispered. She was much older than me but had been widowed at a young age, and we had always been like sisters. She laughed behind cupped hands. "You know, down there."
"You dont know, do you, Vine?" Tressy asked, jabbing her elbow into my ribs.
"They say the Irish are akin to horses," Zelda said, "if you know what I mean."
I had been around horses enough to know what this meant, so when they all collapsed in laughter, I had to join in.
I couldnt have cared less if they loved him or if they had all hated him and met him at the bridge with snarls and shotguns. I had decided that I was going to have him.
Our courting never took us past the mouth of Redbud. Even though Daddy thought a lot of Saul, he wouldnt allow it. Daddy had said that I was his most precious stone. "Ill let you trail from my fingers, but not be plucked," Daddy told me one evening when Saul came calling.
I didnt care where we went, as long as he come to see me, but I would have liked to ride off on that fine horse with him a time or two without worrying how far we went. I thought a lot about how it would feel to just slip away, to just wrap my arms around Sauls waist and take off. We never got to do that, though. We always went down to the confluence of Redbud Creek and the Black Banks River. There was a great big rock there, round as an unbaked biscuit. It had a crooked nose that jutted out over the water. This was our spot.
Summer was barely gone before he asked me to marry him. I remember the way the air smelled that day—like blackberries ripe and about to bust on the vines. The sky was without one stain of cloud, and there didnt seem to be a sound besides that of his horse scratching its neck against a scaly-barked hickory and the pretty racket of the falls. We sat there where we always did, watching the creek fall into the river. The creek was so fast and loud that you couldnt do much talking there. This wall of noise gave us the chance to sit there and study each other. I spent hours looking at the veins in his arms, the calluses on his hands. He had taken a job at the sawmill and this had made his arms firm, his hands much bigger. When we wanted to speak, wed have to either holler or lean over to each others ears. It was a good courting place on this account. Any two people can set and jaw all day long, but it takes two people right for each other to set together and just be quiet. And its good to have to talk close to somebodys ear. Sometimes when he did this, his hot breath would send a shudder all through me.
That day, he run his rough hand down the whole length of my hair and smoothed the ends out onto the rock behind me. I closed my eyes and savored the feeling of him touching me in such a way. I have always believed that somebody touching your head is a sign of love, and his doing so got to me so badly that I felt like crying out. It seemed better to me than if he had leaned me back onto the rock and set into kissing. I knowed exactly how cool my hair was beneath his fingers, how his big palm could have fit my head just like a cap if he had taken the notion to position it in such a way, and I closed my eyes.
The closer it got to dark, the louder the water seemed to be. The sky was red at the horizon, and the moon drifted like a white melon rind in the purple sky opposite.
"Vine?" I heard him yell.
I turned to face him. "What?"
"We ought to just get married," he hollered.
I nodded. "Well," I mouthed. I didnt want to scream out my acceptance, but I sure felt like it. I turned back to the creek and was aware of my shoulders arching up in the smile that just about cut my face in half.
I stood within the shadows of the porch when Saul took Daddy out in the yard to ask for my hand. I had told Saul that it was customary to ask the mother of a Cherokee girl first, but he felt it would be a betrayal of Daddy if he did not tell him before anyone else. They were friends, after all.
Daddy leaned against the gate, his face made darker and older by the dying light. I knowed Daddy would say it was all right, but that hed tell Saul to ask for Mamas permission. I seen Daddy nod his head and put his finger to the touch-me-not bush that hung on the fence. All of the flowers were gone from it now, for summer was beginning to die. For some reason, I felt sick to my stomach.
Mamas voice was hot beside my ear. "Its been decided, then."
"Not unless you say so."
"What do you expect me to do? Mash out what you want so bad?" She stood there in the doorway, folding a sheet with such force that I thought the creases might never come out. She worked it into a neat square, then snapped it out onto the still air and folded it again.
"Ill tell him to go ahead with it, but you know it aint what I want. Its not right. Your daddys great-great-granny was killed by white men. My people bout starved to death hiding in them mountains when they moved everbody out. I cant forgive that."
"That was a long time ago," I said. "Eighty years, almost."
"Might as well been yesterday."
"Daddy says were Americans now," I said, searching for something to say.
Mamas eyes were small and black and her skin seemed to be stretched tightly on her skull. I turned away, as I couldnt look at her. "Tetsalagia," Mama said. I am Cherokee. I knew this much of our old language, as Mama said it to Daddy when they got into fights about how their children ought to be raised up. "Thats his way," she said. "Not mine."
"Dont do me thisaway, Mama. Your own sister married a white man."
"And I aint heard tell of her since. Shes forgot everything about herself."
"I never knowed much to begin with," I said, more hateful than I intended. "You all act like the past is a secret."
"Well, thats your Daddys fault. Not mine."
In the yard, Saul and Daddy stood with their hands in their pockets. I realized that their friendship was gone. Theyd never go hunting together or go on with their notion of butchering a hog together this winter. Now they would only be father and son-in-law, one dodging the other. Saul would take me away from this creek, and Daddy would hold it against him, whether he intended to or not. They looked like they were searching for something else to talk about.
"You know youll have to leave this place," she said, like she could read my thoughts. She whispered, as if they might hear us. "Leave Redbud Camp. All the people youve knowed your whole life."
"I know it, Mama. Im eighteen year old, though. Most girls my age has babies," I said, but this didnt make a bit of difference to her. She put her hand on my arm, and I turned to face her.
"I dont want you to leave me," she said. I knowed this had been hard for her to put into words; she was not the kind of woman who said what her heart needed to announce. I listened for tears in her voice but could hear none. She was too stubborn to cry for me, but her words just about killed me. "Im afraid Ill never see you again."
"Thats foolishness," I said. "You know Id never let that happen."
There was movement down on the yard, and I watched as Daddy headed up the road. I could see that he was hurt over my leaving. He was walking up on the mountain to think awhile. Most of my uncles got drunk when they were tore up, but Daddy always just went up on Redbud and listened to the wind whistle in the rocks.
Saul strode across the yard, as deliberate and broad shouldered as a man plowing a field. I eased past Mama. I didnt want to be out there when he asked her for my hand. I didnt want to remember the way her face would look when she agreed to it.
I lit a lamp and made the wick long so that I could see good by it. I carried the lamp through each little room, trying to memorize the house I had knowed all my life. I made a list of two or three things I wanted to take: one of the quilts Mama and her sisters had made, the cedar box my granddaddy had carved, the walnut bushel basket I had always gathered my beans in. I was homesick already and hadnt even left. I sucked in the smell of the place, memorized the squeaks in the floor. I run my hands over Mamas enamel dishpan, wrapped my fingers about the barrel of the shotgun Daddy kept by the door.
When I walked back into the front room, I knowed Saul would be standing there in the door. I didnt run to him. I set the lamp down on a low table so that my face would be lost to the grayness. I didnt want him to see the hesitation on my face. He was so happy he was breathing hard. "Its decided," he said.
Still I stood in the center of the room, although I knowed he wanted me to come be folded up in his big arms.
"I know well have to live with your people," I said, "so I want to marry amongst mine."
"All right," he said, and then he come to me and picked me up. I cried into the nape of his neck, not knowing if it was from grief or happiness, for both gave me wild stirrings in my gut.
It is the early 1900s in rural Kentucky, and young Saul Sullivan is heading up to Redbud Camp to look for work. He is wary but unafraid of the Cherokee girl there whose beauty is said to cause the death of all men who see her. But the minute Saul lays eyes on Vine, he knows she is meant to be his wife. Vines mother disapproves of the mixed marriage; Sauls mother, Esme, has always been ill at ease around the Cherokee people. But once Vine walks into Gods Creek, Sauls mother and brother Aaron take to her immediately. It quickly becomes clear to Vine, though, that Aaron is obsessed with her. And when Saul leaves Gods Creek for a year to work in another county, the wife he leaves behind will never be the same again. The violence that lies ahead for Vine, will not only test her spirit, but also her ability to forgive—both others and herself. . . .
2. Explain why the title of Part I of the novel, "Confluence," is an appropriate label for this section of the story.
3. By the end of Part I, Vine seems conflicted: lonely, yet at peace; happy, but restless; homesick, but able to make her own home with Saul. Vine also seems to have a more heightened sensory awareness than the other characters, always noting the smells, sights, tastes, sounds, and feelings around her. How do these character traits serve her during the story?
4. Why is Saul Sullivan such a poor communicator in person but such an articulate letter-writer?
5. How does music operate within this story? Are you familiar with the song references and lyrics? Does the banjo have symbolic meaning in the story?
6. Purple colors are often referred to in the story. What does the color purple signify for you?
7. How does nature serve as a main character in this novel? Consider references to landscapes, creeks, mountains, birds, wildflowers, trees, snakes, etc.
8. Discuss Silas Houses use of vernacular speech in this story. What words or phrases spoken by the characters are unfamiliar to you? How do the characters dialects affect your interpretation of the story? What do you learn about the characters and the place where they live through their speaking styles?
9. Are you familiar with the mountain traditions described in the book such as a "house raising" or a "hog killing"? What about the many folklore beliefs and rituals practiced by Esme? What are some of the traditions or rituals you learned from your family or community, and how do they compare or contrast to the practices of Appalachians at the turn of the 20th century?
10. Even though Vine would not know the modern word "feminist," would you label her as a feminist? Why or why not? What are your personal connotations for the word "feminist" and the notion of "feminism?"
11. Vines friend Serena serves traditional feminine roles in the community such as midwife, caregiver, and mother, yet she is described as being rough as a man; she chain smokes and wears mens clothes. How do these androgynous characteristics affect your perception of Serena?
12. Discuss Vines awareness of the rigidity of mens and womens roles in her time and community. For example, she notes: "Men and women never sat beside one another at the table;" "A woman had never offered to shake my hand before-it was something that only men did;" and "In a place where men had once made things so busy, now there was only women…. Sometimes it seemed like we would do just fine without any men at all."
13. Describe the relationship Vine has with the women in her community who are not related to her: Serena, Esme, and Aidia. What is the significance of both Vine and Aidia being "outsiders?"
14. Discuss the prejudices that the characters in A Parchment of Leaves either endure or participate in. Consider: Native American vs. European American, masculine roles vs. feminine roles, townspeople vs. "creekers," church goers vs. free thinkers, etc.
15. With which character(s) do you most closely identify? Why?
16. By the end of the novel, Vine Sullivan has a complete, complex, and conflicted cultural identity. Discuss how her heritage, region, and gender impact her self-awareness and shape her various roles as mother, wife, daughter-in-law, farmer, and community member.


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DeWalt DW872 - Heavy-Duty 14" Multi-Cutter Saw
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I bought this saw a month ago and it has lived up to the reviews I had previously read about it. A little more expensive but well worth the price in the time saved as it slices through the metal like a knife through butter.


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The potted plant was sitting in an office when Gustave Chiumbo barrelled through and knocked it off the desk in his rush to rescue Rudyard Shelton and Mitchell Crota. After this, Shelton learned that Nixx's syringe had given him the power to talk to flora when the plant asked him to fix his pot.
The plant probably died when the oil rig sank (Disruptive Selection). However, a similar plant was rescued by Shelton, but it's unclear if they are the same plant.


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Make your own body oil
I'm not a big fan of body creams, simply because oils are so much more nourishing and leave your skin with more gleam and shine than the most expensive body shimmer. They also hold a scent well all over your body, so that you smell gorgeous all day – and night.
They are very simple to make by adding some essential oils to a carrier oil, such as sweet almond or apricot kernel oil. And when you grow tired of one scent, you can simply whip up another or have several: one for day, one for night and one for weekends. The other advantage of essential oils is their ability to alter your moods. Feeling down? Why not mix up an oil using ylang-ylang, geranium and sandalwood. Never combine more than five essential oils as the scent will be lost – a bit like mixing too many paint colours and ending up with that dull brown. Combine 1 drop of essential oil for every 1 ml of carrier oil.
Here are some great blends I have used, but if you have one favourite smell you like, then just use that one essential oil. I love the scent of rose so will always default to my rose body oil. And if you're not sure what you want just sniff a few of your essential oil bottles and use the ones you like. Your body can tell you what scent you need.
- Weepy, sad, depressed – use a blend of ylang-ylang, rose geranium and sandalwood.
- In the mood for romance – ylang-ylang is the key here. Just use that or combine with rose and jasmine.
- Stressed out – use a blend of eucalyptus, rose geranium and lavender.
- A bit of a cold – use a blend of peppermint, eucalyptus and lavender.
- Need energy – use a blend of rosemary, orange and peppermint.
- Need to concentrate – use a blend of bergamot, peppermint and rosemary.
- Want to be the life and soul of the party – use a blend of bergamot, clary sage and jasmine.
- A good night's sleep – lavender is the key, but if used in too much concentration it can actually perk you up so blend it with rose geranium and sandalwood.


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In Egypt, wine was considered a drink for the gods; this saintly drink wasn't just made with grapes in Egypt, but also with dates, pomegranates, leads and other fruits. Here are 8 fun historical facts about the best godly drink in the world !
1) EGYPT : When Tutankhamen's tomb was opened there were jars of wine buried with him that were labeled with the name of the Estate, location, type of wine, date of vintage, name , comment of it's quality.
2) GREECE : You know those age restriction laws for alcohol guess who invented that ?! Blame Plato. He stated that the minimum drinking age should be 18.
3) FRANCE : the word 'champagne' is named after a province in France, the English translation is 'open country'.
4) SPAIN : During the Roman rule in Spain, a wine called the 'Saguntum' was known especially for being good to get your mistress drunk. After the Roman rule, Queen Elizabeth I defeated the Spanish Armada causing them a lot of debt therefore making them very dependant on the money made from Spanish wine exports to USA.
5) ITALY : ancient Romans thought seasoning was more important than the principal palate of wine and often added ingredients the wine e.g fish sauce, garlic, absinthe & other flavours.
6) AMERICA : The earliest wine produced in America were made by French settlers. Guess the French and the Americans have more in common than just the Statue of Liberty. LOL!
7) JAPAN : The first documented consumption of wine in Japan was in the 16th century, due to the arrival of Jesuit missionaries from Portugal. They called Portuguese wine Chintashu. For a long period of time they hated the taste of wine therefore they made it sweeter with e.g honey.
8) AUSTRALIA : Some of the first Australian wine connoisseurs found it difficult to produce wine due to the Australian climate. Wine from the Adelaide Hills was sent to Queen Victoria in 1844.
Remember – Keep loving history cause that is what brought us to this day & age. Without it I think we'd still be putting fish oil or garlic into our wines. Thank goodness for evolution!
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Why am I writing One Moore In The Oven? It started way back in 2011, when we wanted to share news of our growing baby. I was pregnant, thus the name of the blog, and we lived far away from all of our family and many dear friends. So I created One Moore In The Oven in April 2011, so they could follow along. I've have been blogging continuously since then with a few short breaks here and there.
I love to write, and this blog helped to remind me of that truth. Previously, I wrote mostly technical stuff — scientific papers and the like — and hadn't done much non-technical writing in ages. Now I write here. And why not share it with the world?
Happy Reading!
Stephanie (December 2015)


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Antique Heart Pine (Hit Skip) flooring has been reclaimed from the oldest factories and mills that once powered our nation's manufacturing base. This product is commonly used in vital support beams, verifying its support and dependability. This unique product is sure to add character and individuality to your one-of-a-kind space.
We also offer a full line of STAIR PARTS and other ACCESSORIES for this flooring option. CLICK HERE to learn more about how we can provide treads, risers, nosing, vents and more to match your custom wide plank flooring.


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SYNQ brings you state of the art security technology that is easy to use, with competitive pricing. From retail, to construction, to your own home, we bring peace of mind at unbeatable prices.
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We bring effective products to the industry that our competitors haven't even touched. From the highest quality cameras to specialized servers, to our 100% Canadian-made EAS tags, we're proud to offer what no one else has.
SYNQ is committed to building a close relationship between smartphones and security. We use smartphones to take advantage of immediate notification, and provide convenience for CCTV monitoring.
Lock out distracted driving for good. Commercial & consumer to come.
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Subscriptions & servers to record up to 1 year at full resolution.
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SYNQ has worked with several camera manufacturers to hand pick a small collection of cameras that suit any application.
From dark parking lots, to harsh, outdoor remote locations, to large-scale retail, our cameras offer superior picture to our competition.
All of our cameras run 32-bit Linux "System On Chip" (SOC) layouts for compact size and durability. They all support up to a 128GB onboard SD card to record full time, on motion, or when triggered by an external alarm.
General-purpose 2MP IP camera for full HD recording. Compact size and 2-way positioning makes setup easy.More Info Coming Soon
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2007 57 VAUXHALL CORSA 1.2 SXI A/C 16V 5d* IDEAL FIRST CAR * FULL SERVICE HISTORY * 12 MONTHS MOT *
- 103000 miles
- £POA 6m Tax
- 146 CO² (g/km)
- Petrol
- £POA 12m Tax
- Manual
- 46.3 Avg. MPG
- Ins. Group: 4
- 103000 miles
- £POA 6m Tax
- 146 CO² (g/km)
- Petrol
- £POA 12m Tax
- Manual
- 46.3 Avg. MPG
- Ins. Group: 4
- Details
- Features
- Finance
- Gallery
- Price£1,820
- Fuel TypePetrol
- Body StyleHatchback
- Engine Size1229
- Number of Doors5
- Exterior ColourSilver
- Registered2007
- TransmissionManual
- Mileage103000 *
- Owners3
- Emissions146 CO² (g/km)
- Road Fund£POA 6m Tax
- Road Fund£POA 12m Tax
- Max Power80 bhp
- Max Torque81 lbft
- Max Speed104 mph
- 0-60 Time14.1 secs
- MPG Extra56.5 mpg
- MPG Combined46.3 mpg
- MPG Urban35.3 mpg
For more info on this vehicle call our showroom on 01614873000
Every effort has been made to ensure the accuracy of the above information but errors may occur. Please check with a salesperson.
For more info on this vehicle call our showroom on 01614873000
5 door HATCHBACK with 2 KEYS, FULL SERVICE HISTORY, 12 MONTHS MOT and 12 MONTHS FREE AA MEMBERSHIP. This car benefits from being in a LOW INSURANCE GROUP and having an average MPG of 46.3 making this the ideal car for a FIRST TIME DRIVER or someone wanting LOW COST MOTORING. The front WINDOWS and MIRRORS are ELECTRIC and AIR CONDITIONING is present. ISOFIX ANCHORAGE POINTS are located in the rear seats and are easily accessible on this 5 door model. The AUDIO comprises of RADIO/CD/MP3 all of which can be controlled via the MULTIFUNCTION STEERING WHEEL keeping safety at the forefront whilst driving. This vehicle is in GOOD CONDITION. For further information or to ARRANGE a TEST DRIVE please contact our friendly helpful sales team. NATIONWIDE DELIVERY AVAILABLE.
WE ARE AN AUTOTRADER TOP 50 INDEPENDENT RETAILER,WE ARE APPROVED BY THE AA TO OFFER INDEPENDENTLY INSPECTED AA APPROVED USED VEHICLES,12 MONTH AA MEMBERSHIP AND A COMPREHENSIVE HISTORY CHECK. AS A SUCCESSFUL BUSINESS WE HAVE MAINTAINED OUR FAMILY VALUES AND NOW HAVE 3 SITES AND OVER 300 VEHICLES. EVERY VEHICLE IS PREPARED TO A VERY HIGH STANDARD BUT,IN THE RARE EVENT OF ANY CUSTOMER DISSATISFACTION WE ALWAYS STRIVE TO OFFER THE HIGHEST LEVELS OF CUSTOMER SERVICE. CHECK OUR AUTOTRADER REVIEWS. Air Bag Driver, Air Bag Side - Front Side & Front Curtain, Alloy Wheels, Central Door Locking - Remote, Deadlocks, In Car Entertainment - Radio/CD/MP3, Power-Assisted Steering, Seat - ISOFIX Anchorage Point - Two Seats - Rear, Steering Wheel Mounted Controls - Audio, Front Fog Lights, Electric Windows - Front, Mirrors External - Electric/Heated, Seating Capacity - Five Seats, Air Bag Passenger, Anti-Lock Brakes, Side Protection Mouldings, Body Coloured Bumpers, Adjustable Steering Column/Wheel - Rake/Reach, Immobiliser, Seat Height Adjustment - Driver
Every effort has been made to ensure the accuracy of the above information but errors may occur. Please check with a salesperson.
The following documents are available for this vehicle:


text:
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Staphylocoagulase (SC) is a potent nonproteolytic prothrombin (ProT) activator and the prototype of a newly established zymogen activator and adhesion protein family. The staphylocoagulase fragment containing residues 1–325 (SC-(1–325)) represents a new type of nonproteolytic activator with a unique fold consisting of two three-helix bundle domains. The N-terminal, domain 1 of SC (D1, residues 1–146) interacts with the 148 loop of thrombin and prethrombin 2 and the south rim of the catalytic site, whereas domain 2 of SC (D2, residues 147–325) occupies (pro)exosite I, the fibrinogen (Fbg) recognition exosite. Reversible conformational activation of ProT by SC-(1–325) was used to create novel analogs of ProT covalently labeled at the catalytic site with fluorescence probes. Analogs selected from screening 10 such derivatives were used to characterize quantitatively equilibrium binding of SC-(1–325) to ProT, competitive binding with native ProT, and SC domain interactions. The results support the conclusion that SC-(1–325) binds to a single site on fluorescein-labeled and native ProT with indistinguishable dissociation constants of 17–72 pM. The results obtained for isolated SC domains indicate that D2 binds ProT with ~130-fold greater affinity than D1, yet D1 binding accounts for the majority of the fluorescence enhancement that accompanies SC-(1–325) binding. The SC-(1–325)·(pro)thrombin complexes and free thrombin showed little difference in substrate specificity for tri-peptide substrates or with their natural substrate, Fbg. Lack of a significant effect of blockage of (pro)exosite I of (pro)thrombin by SC-(1–325) on Fbg cleavage indicates that a new Fbg substrate recognition exosite is expressed on the SC-(1–325)·(pro)thrombin complexes. Our results provide new insight into the mechanism that mediates zymogen activation by this prototypical bacterial activator.
Staphylocoagulase (SC)3 is the prototype of a newly established zymogen activator and adhesion protein family (1). SC nonproteolytically activates the blood coagulation zymogen, prothrombin (ProT) through interactions localized to the N-terminal 324 residues, and the SC-(1–325)·(pro)thrombin catalytic complex recognizes and cleaves fibrinogen (Fbg) as a specific substrate (2–4). The C-terminal region of SC contains five to eight 27-residue repeat sequences that mediate distinctly different Fbg binding interactions (5). Binding of C-terminal regions of SC to Fbg may localize the active SC·ProT complex to the platelet surfaces through Fbg bound to αIIbβ3 integrin (6).
The recently determined crystal structure of a functional N-terminal fragment of SC (SC-(1–325)) bound to the catalytic domain of ProT, prethrombin 2 (Pre 2) (1) provided the first structural proof of the "molecular sexuality" mechanism of zymogen activation (7). The N terminus of SC is bound to the "Ile16 pocket" (chymotrypsinogen numbering) of Pre 2, presenting Ile1-Val2 residues of SC to Asp194 of Pre 2 and resulting in formation of the salt bridge necessary for proper serine proteinase folding and catalytic activity. SC-(1–325) is a boomerang-shaped molecule that consists primarily of two three-helix bundle domains. The N-terminal domain (D1) of SC-(1–325) interacts with the 148 loop of Pre 2, positioning the N terminus for insertion into the Ile16 pocket, whereas the second domain (D2) binds to (pro)exosite I on thrombin, Pre 2, and ProT (1). The SC·(pro)thrombin complex cleaves Fbg specifically to generate fibrin clots in a process that is thought to contribute to the pathology of Staphylococcus aureus in human diseases such as pulmonary infections (8) and acute bacterial endocarditis (9). Fbg recognition and cleavage by the SC-(1–325)·(pro)thrombin complexes and studies of full-length SC (10) or C-terminal regions of SC (6) indicate that there are two distinct modes of Fbg binding. The two distinct modes of interaction with Fbg may localize the SC·(pro)thrombin complexes pathophysiologically and mediate specific Fbg cleavage, although the relationship between these interactions is unknown.
The procoagulant and anticoagulant specificity of α-thrombin is linked to the expression of exosite I (11–15), which directs specific recognition of numerous macromolecular substrates (factor V (16–18), factor VIII (16), Fbg (19), and proteinase-activated receptors (20, 21)), inhibitors (hirudin (15, 22) and heparin cofactor II (23, 24)), and regulatory proteins (thrombomodulin (25) and fibrin (19, 26)). Substrate specificity studies of SC·(pro)thrombin complexes indicate a highly restricted specificity for Fbg. The SC complexes do not cleave other physiological thrombin substrates, including factor V, factor VIII, or proteinase-activated receptors (3, 27). SC binding to (pro)thrombin imparts resistance to the serpin inhibitors of thrombin, antithrombin-heparin (3, 28), and heparin cofactor II.4 Because SC·(pro)thrombin complexes cleave Fbg specifically with D2 of SC occupying (pro)exosite I, Fbg substrate recognition must occur through a different site. A detailed structural analysis of the interfaces of D1 and D2 of SC-(1–325) with ProT and prediction of the mode of Fbg recognition by the SC-(1–325)·(pre)thrombin complexes are presented in the accompanying paper (29).
To quantitate binding of SC-(1–325) to ProT and binding of Fbg (29), we developed novel active site-labeled fluorescent analogs of the ProT zymogen as probes of the interactions.5 The analogs are based on the reversibility of SC-induced ProT activation, which allows irreversible inactivation of the catalytic site of the zymogen by hemiketal formation with Ser195 and alkylation of His57 with the inhibitor Nα-[(acetylthio)acetyl]-D-Phe-Pro-Arg-CH2Cl (ATA-FPR-CH2Cl), followed by specific attachment of various probes, and separation of the fluorescent zymogen derivatives from SC-(1–325). Equilibrium binding of SC-(1–325) to ProT was characterized for the first time by the use of fluorescent ProT analogs selected by screening a panel of 10 derivatives for reporters of the interaction. Our results demonstrate that SC-(1–325) binds to active site-labeled ProT analogs and native ProT with a 1:1 stoichiometry and extremely high affinity (KD = 17–72 pM). SC-(1–325)·ProT complexes formed with native ProT were cleaved slowly at both thrombin-sensitive sites, Arg155-Ser156 and Arg284-Thr285, generating prethrombin 1 (Pre 1) and fragment 1 and generating Pre 2′ and fragment 2, respectively. D1 binding to labeled ProT accounted primarily for the fluorescence enhancement observed upon SC-(1–325) binding, whereas the affinity of the isolated D2 for ProT was ~130-fold greater than that of D1. SC-(1–325) complexes with thrombin and ProT exhibited equivalent Fbg clotting activities, only small differences in specificity for tripeptide chromogenic substrates compared with free thrombin, and no preference for the sequences preceding the cleavage sites for release of fibrinopeptides A and B. The blockage of exosite I-dependent Fbg clotting activity by SC-(1–325) and the small changes in tripeptide substrate specificity suggest that Fbg recognition is mediated by an exosite on the complex. Further studies of the Fbg interaction are presented in the companion paper (29).
ProT was purified from human plasma (30). α-Thrombin was active site-titrated by previously described methods (30). The proteins were pure as determined by SDS gel electrophoresis. The protein concentrations were determined by absorbance at 280 nm with the following absorption coefficients ((mg/ml)−1cm−1) and molecular weights (31, 32): ProT, 1.47, 71,600; thrombin, 1.74, 36,600; Fbg, 1.54, 340,000; Met-SC-(1–325) and SC-(1–325), 1.00, 38,000; D1, 1.42, 17,562 (calculated); D2, 0.64, 20,398 (calculated); and Met-SC-(1–325)-His6, 0.96, 39,461 (calculated). Absorption coefficients for SC species were calculated from the aromatic amino acid composition (33).
Met-SC-(1–325) and SC-(1–325) were expressed, purified, and characterized as described previously (1). Met-SC-(1–325) fused to a C-terminal His6 tag (Met-SC-(1–325)-His6) was prepared by deleting a stop codon, which allowed translation of additional Lys-Leu-Ala-Ala-Ala-Leu-Glu-His6 residues. Met-SC-(1–325)-His6 was expressed in Escherichia coli strain BL21(DE3) plysS. The cells were grown to an optical density of 0.4–0.6 at 600 nm and induced by the addition of 500 μM isopropyl-β-D-thiogalactopyranoside. The cells were induced for 4–6 h and lysed by three rounds of consecutive freeze-thaw cycles or sonication, and Met-SC-(1–325)-His6 was purified from the soluble fraction after centrifugation, by Ni2+-iminodiacetic acid-Sepharose chromatography. Met-SC-(1–325)-His6 activated ProT, as measured by H-D-Phe-Pip-Arg-p-nitroanilide assay, with comparable activity as determined previously for Met-SC-(1–325) (1). Met-SC-(1–325)-His6 was used instead of Met-SC-(1–325) in ProT labeling to facilitate separation of the activator and labeled zymogen.
The thrombin-selective thioester-peptide chloromethyl ketone, ATA-FPR-CH2Cl, was prepared as described previously (30, 34), and labeling followed a protocol similar to that described previously for streptokinase (SK)-mediated fluorescence labeling of plasminogen (35). The Met-SC-(1–325)·ProT complex was inactivated by adding 145–200 μM ATA-FPR-CH2Cl to 45–60 μM ProT in 50 mM Hepes, 110 mM NaCl, 5 mM CaCl2, 1 mg/ml polyethylene glycol 8000, pH 7.4 buffer (Buffer A) at 25 °C. Inhibition reactions were initiated by the addition of 90–120 μM Met-SC-(1–325) and were monitored by the decrease in the rate of hydrolysis of H-D-Phe-Pip-Arg-pNA. The reactions were typically complete (<0.1% activity) within 1 h. To ensure complete inhibition, 200 μM FPR-CH2Cl was added. Excess inhibitor was removed by Sephadex G-25 chromatography in 0.1 M Hepes, 0.1 M NaCl, 5 mM CaCl2, pH 7.0 buffer. Met-SC-(1–325)·ATA-FPR-ProT (10–25 μM) was labeled by the addition of a 10-fold molar excess of the desired thiol-reactive probe derivative and subsequent addition of 0.1 M NH2OH, followed by incubation for 1–2 h in the dark at 25 °C. Excess label was removed by Sephadex G-25 chromatography. Met-SC-(1–325)-labeled ProT complex was concentrated and dissociated by dialysis against Buffer A with 3 M NaSCN but without polyethylene glycol 8000. The dissociated proteins were separated by fast protein liquid chromatography gel filtration chromatography on 2–3 tandem Superdex 200 HR 10/30 columns equilibrated with the same buffer. Fractions containing the first of two overlapping peaks, consisting primarily of labeled ProT, were pooled and dialyzed against 50 mM Hepes, 125 mM NaCl buffer, pH 7.4 (Buffer B). The pooled protein was rechromatographed on Superdex 200 HR 10/30 in Buffer B under the same conditions. Fractions containing the second of two peaks, consisting of labeled ProT, were pooled, quick-frozen, and stored at −70 °C.
A second, improved method was subsequently used for separation of labeled ProT from the Met-SC-(1–325)-labeled ProT complex using Met-SC-(1–325)-His6. The Met-SC-(1–325)-His6·ProT complex was formed, inactivated, and labeled, and excess probe was removed as described above. Met-SC-(1–325)-His6·labeled ProT was chromatographed on Ni2+-iminodiacetic acid-Sepharose in Buffer B. Labeled ProT was step-eluted with 3 M NaSCN, and the majority of Met-SC-(1–325)-His6 was eluted with a gradient of imidazole up to 500 mM. Trace amounts of Met-SC-(1–325)-His6-labeled ProT complex were removed by fast protein liquid chromatography gel filtration on Superdex 200 HR 10/30 in Buffer B. Two peaks corresponding to Met-SC-(1–325)-His6-labeled ProT complex and free labeled ProT were eluted, and the labeled analog was pooled, dialyzed against Buffer B, quick-frozen, and stored at −70 °C.
The stoichiometry of fluorescence probe incorporation into ProT for 5-(iodoacetamido)fluorescein (5F), 2-((4′-iodoacetamido)anilino)naphthalene-6-sulfonic acid (ANS), and Oregon Green 488 iodoacetamide or 5-(and 6)-iodoacetamido-2′,7′-difluorofluorescein (OG) was determined from the absorbance at 280 nm and the corresponding probe absorbance in 0.1 M Tris-HCl, 1 mM EDTA, 6 M guanidine, pH 8.5. For 5F-labeled ProT, 84,000 M−1 cm−1 at 498 nm and an absorbance ratio A280 nm/A498 nm of 0.19 were used (36). Incorporation of ANS was measured using 26,000 M−1 cm−1 at 328 nm and A280 nm/A328 nm of 0.78 (30). Incorporation of OG was measured using 55,800 M−1 cm−1 at 491 nm and A280 nm/A491 nm of 0.29 (37). The concentration of residual active ProT was determined by chromogenic substrate assay of 25 nM labeled ProT after incubation with a 5-fold excess of SC-(1–325) for 20 min at 25 °C, relative to a known concentration of the native SC-(1–325)·ProT complex.
Fluorescence was measured with an SLM 8100 fluorometer, in acrylic cuvettes coated with polyethylene glycol 20,000. All of the experiments were performed in Buffer A at 25 °C. Corrections for background were made from parallel measurements on blanks lacking the labeled species. Binding of SC-(1–325) (or when applicable isolated SC domains) to various labeled ProT analogs was measured in titrations of labeled ProT monitoring the change in fluorescence at probe emission wavelengths (nm) of ProT labeled with: dansylaziridine (DAN), 506; ANS, 438; 6-acryloyl-2-dimethylaminonaphthalene (ACR), 502; 6-bromoacetyl-2-dimethyl-aminonaphthalene (BAD), 510; AF350 (the sulfonated coumarin derivative called AlexaFluor 350 C5 maleimide), 436; BODIPY® 507/545 iodoacetamide or N-(4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene-2-yl)iodoacetamide (BD), 535; tetramethylrhodamine-5-iodoacetamide dihydroiodide (TMR), 580; 4′-(iodoacetamido)fluorescein (4′F), 521; and 5F and OG, 520. The probes were excited at wavelengths (nm): DAN, 343; ANS, 335; ACR, 398; BAD, 400; AF350, 352; BD, 506; TMR, 550; and 4′F, 5F, and OG, 490. The fluorescence changes measured as a function of total SC-(1–325) concentration and expressed as (Fobs − Fo)/Fo = ΔF/Fo were fit by the quadratic binding equation to obtain the maximum fluorescence change ((Fmax − Fo)/Fo = ΔFmax/Fo), the dissociation constant (KD), and the stoichiometric factor (n). Nonlinear least squares fitting was performed with SCIENTIST software (MicroMath). The error estimates represent ± 2 S.D.
[OG]FPR-ProT was excited at 490 nm in the absence and presence of 500 nM SC-(1–325), 8 μM D1, and 8 μM D2, and the spectra were collected (8-nm band pass) in 1 nM steps with two consecutive scans averaged. Emission spectra of D1 binding to [OG]FPR-ProT were recorded at D1 concentrations of 0.5, 1.6, 3.8, 5.6, and 8 μM.
To characterize cleavage of the SC-(1–325)·ProT complex, mixtures of 2 μM [OG]FPR-ProT and 0.05–12 μM SC-(1–325) were incubated for 18 h in the dark at 25 °C in the presence of 10 μM FPR-CH2Cl. Similar reactions were performed in the presence of 2 μM native ProT without inhibitor and in the presence of 100 μM FPR-CH2Cl and 4-(2-aminoethyl)-benzenesulfonyl fluoride (AEBSF). The cleavage products were identified by SDS gel electrophoresis in comparison with purified ProT fragments. The gels were stained for protein with GelCode Blue (Pierce), and fluorescence was visualized with a 300-nm transilluminator.
The effect of native ProT on SC-(1–325) binding to 0.12 nM [OG]FPR-ProT was measured in titrations with native ProT, at fixed concentrations of SC-(1–325) (0.5, 5, and 10 nM). The fluorescence changes for individual reactions were calculated from 30–40-min time course measurements for a reaction blank, [OG]FPR-ProT alone, and mixtures of [OG]FPR-ProT and native ProT after the addition of SC-(1–325). Together with the direct titration data of [OG]FPR-ProT with SC-(1–325), the data were fit simultaneously by the cubic equation for tight competitive binding of SC-(1–325) to labeled and native ProT to determine the dissociation constant and stoichiometry for the native SC-(1–325)·ProT interaction (38, 39).
To determine whether the isolated SC-(147–325) fragment containing D2 and excluding D1 bound (pro)exosite I of ProT, competitive binding experiments were performed using the fluorescein-labeled C-terminal hirudin peptide, Gly54-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr( )-Leu-Gln65 ([5F]Hir-(54–65)( )) (15). The titrations were performed and analyzed with the cubic equation as described previously for Met-SC-(1–325) binding to proexosite I on ProT (1).
Fbg clotting assays were performed at 37 °C with a fibrometer (40). Relative clotting activity was determined by comparison with an α-thrombin standard from the slopes of plots of clotting time against the reciprocal of the enzyme concentration. Hir-(54–65)( ) was used at 20 μM as an exosite I-specific inhibitor (19).
Kinetic parameters for hydrolysis of chromogenic substrates by α-thrombin and SC-(1–325)·(pro)thrombin were determined by monitoring the absorbance increase at 405 nm in Buffer A containing 1 mg/ml bovine serum albumin and 0.1 mg/ml soybean trypsin inhibitor at 25 °C, using polyethylene glycol 20,000-coated cuvettes. For substrates with low Km, H-D-Phe-Pip-Arg-pNA, H-D-Ile-Pro-Arg-pNA, and H-D-Phe-Pro-Arg-pNA, the kinetic parameters, kcat, Km and when applicable, Ki, were determined by simultaneous fits of the integrated Michaelis-Menten equation (including competitive product inhibition) to the full reaction progress curves for conversion of substrate at concentration ranges of 0.9–1, 4–5, 8–10, and 17–20 μM by 0.5 nM enzyme and 60 μM by 5 nM enzyme for each substrate. For Gly-Val-Arg-pNA, Ser-Ala-Arg-pNA, and CH3-Gly-Gly-Arg-pNA, the kinetic parameters were determined by fitting the Michaelis-Menten equation to initial rates collected over a substrate concentration range of 0–6 mM. In all of the assays, SC-(1–325) and (pro)thrombin were preincubated for at least 20 min, and the reactions were initiated by the addition of substrate. Because the complex-dependent proteolysis described below is minimized at the concentrations used in the kinetic studies, there was no variation of the initial rates beyond the 20-min preincubation time.
Active site-labeled fluorescent analogs of ProT were prepared as novel probes of SC-induced ProT activation and Fbg substrate recognition (Fig. 1A and the accompanying paper (29)). The catalytic site induced by Met-SC-(1–325) binding to ProT was inactivated by hemiketal formation with Ser195 and His57 alkylation with the thioester-peptide chloromethyl ketone, ATA-FPR-CH2Cl (30, 34, 35, 41). Subsequent generation of the free thiol by treatment with NH2OH, and probe incorporation by reaction with a thiol-reactive fluorescence probe yielded the labeled ProT zymogen. Specificity of ProT labeling was examined by determining the dependence of fluorescence probe incorporation on each of the steps in the reaction scheme (Fig. 1A), as seen by fluorescence (Fig. 1B) and protein staining (Fig. 1C) on SDS gels. The protein bands of SC-(1–325) and ProT are seen as individual bands on the gels because the complex dissociates in SDS. The reaction in lane 1 showed that without SC-(1–325) no probe was incorporated because of the lack of zymogen activation. The reaction in lane 6 showed that the absence of NH2OH prevented probe incorporation, indicating the need for the free thiol. Blocking of the catalytic site in the Met-SC-(1–325)·ProT complex with FPR-CH2Cl before reaction with ATA-FPR-CH2Cl also blocked probe incorporation, demonstrating the requirement for reaction of the inhibitor with the activated zymogen (Fig. 1, B and C, lane 7). The results demonstrated specific labeling of the SC-activated catalytic site of the ProT zymogen. Preparation of a Met-SC-(1–325)-His6 construct allowed separation of labeled ProT from SC-(1–325) based on the high affinity of His6 for Ni2+-iminodiacetic acid under dissociating conditions (3 M NaSCN) and purification of the labeled analog (Fig. 1, B and C, lane 8). Stoichiometries of probe incorporation into the isolated zymogen were 0.82–0.98 mol probe/mol of ProT for ANS, 5F, and OG. Preparations of labeled ProT had residual activities of 1–3%, determined by the activity generated with excess SC-(1–325).
An array of ProT analogs containing 10 fluorescence probes with various fluorescence spectral properties was prepared. The results of fluorescence titrations with SC-(1–325) are grouped by similar probe structures in Fig. 2. SC-(1–325) titrations of 50–285 nM labeled ProT indicated that SC-(1–325) bound ProT with a 0.84–1.00 stoichiometry for those that could be determined (ACR, AF350, BD, 4′F, 5F, OG, and TMR). The KD values were poorly defined in these single titrations because of the apparent, very high affinity in relation to the labeled ProT concentrations. The signs and amplitudes of the fluorescence changes accompanying SC-(1–325) binding were dependent on the structures and spectral properties of the probes with no obvious correlation with polarity sensitivity (Fig. 2). Interestingly, the polarity-sensitive probes, ACR and BAD, the adducts of which differ only by a methylene group in the linker, showed a 62% enhancement and essentially no change, respectively (Fig. 2A).
All of the high quantum yield fluorescein-ProT derivatives, OG, 5F, and 4′F, showed large fluorescence enhancements of 95, 38, and 50%, respectively, at saturating SC-(1–325) (Fig. 2B). [OG]FPR-ProT and [5F]FPR-ProT were selected for quantitating SC-(1–325) binding to labeled ProT. Simultaneous fits of 0.11, 1, and 50 nM [OG]FPR-ProT titrations with SC-(1–325) showed that SC-(1–325) bound to 0.95 ± 0.05 mol sites/mol of ProT with a dissociation constant (KD) of 17 ± 6 pM, and a maximal fluorescence enhancement (ΔFmax/Fo) of 98 ± 2% (Fig. 3A). These results were confirmed by analysis of similar titrations of 0.075, 0.25, 2.5, 10, and 100 nM [5F]FPR-ProT, indicating binding of SC-(1–325) to 0.96 ± 0.05 mol sites/mol of ProT with a KD of 37 ± 1 pM and a maximal fluorescence enhancement of 39 ± 1% (Fig. 3B).
To determine the proteolytic stability of the SC-(1–325)·ProT complex, we incubated [OG]FPR-ProT with fixed or varied levels of SC-(1–325), in the presence of inhibitors, and with native ProT in the presence and absence of inhibitors and analyzed the products by fluorescence and protein staining on SDS gels. Fig. 4 shows the time course of ProT conversion to Pre 1 and Pre 2 in a mixture of labeled and native ProT (4 μM each) and SC-(1–325) (8 μM). Although cleavage was not complete, there was significant production of Pre 2 over the 4-h incubation. Fig. 5 (A and B) shows the results for mixtures of a preparation of [OG]FPR-ProT that had <2% residual native ProT activity, incubated with increasing concentrations of SC-(1–325) in the presence of 10 μM FPR-CH2Cl. Labeled ProT in the absence and presence of SC-(1–325) was stable for up to 18 h in the dark at 25 °C (Fig. 5, A and B). In the presence of native ProT and in the absence of inhibitors, however, both native and labeled ProT were increasingly cleaved as a function of the SC-(1–325)·native ProT complex concentration (Fig. 5, C and D), as evidenced by the similar staining pattern seen by fluorescence (Fig. 5C) and protein staining (Fig. 5D). Cleavage was essentially complete in mixtures of 3–6 μM SC-(1–325), 2 μM native ProT, and 2 μM [OG]FPR-ProT (Fig. 5, C and D, lanes 6 and 7). It was also evident that the SC-(1–325)·native ProT complex cleaved native and [OG]FPR-ProT bound to SC-(1–325) because in mixtures incubated for >16 h at concentrations of SC-(1–325) saturating labeled and native ProT (Fig. 5C, lanes 7 and 8), essentially complete cleavage was observed. ProT cleavage in a mixture of labeled and native ProT (2 μM each) and saturating SC-(1–325) (12 μM) was completely inhibited by 100 μM FPR-CH2Cl and 100 μM AEBSF (Fig. 5, C and D, lane 9). Free ProT was also cleaved at a much slower rate by the SC-(1–325)·native ProT complex than the ProT molecules bound to SC-(1–325) (data not shown). Two major fluorescent reaction product bands were identified as Pre 1 and Pre 2′ by comparison of similar reactions with purified ProT activation species (not shown). Pre 2′ is formed by thrombin cleavage of ProT at Arg284-Thr285, resulting in a form of Pre 2 that is 13 residues shorter (42). N-terminal sequencing of the polyvinylidene difluoride membrane-blotted reaction product confirmed the N-terminal sequence of Thr-Phe-Gly-Ser-Gly, corresponding to Pre 2′.
To characterize binding of SC-(1–325) to native ProT, [OG]FPR-ProT was used as a probe of the competitive SC-(1–325) binding equilibria in the absence of proteinase inhibitors. To resolve the rapid binding equilibrium from slower proteolytic cleavage, individual fluorescence intensity measurements were made as a function of time after the addition of SC-(1–325) to 0.5, 5, or 10 nM final concentrations to mixtures of [OG]FPR-ProT (0.12 nM) and varying native ProT. Three fluorescence intensity values were obtained for additions corresponding to buffer, [OG]FPR-ProT, and a mixture of [OG]FPR-ProT and native ProT to which SC-(1–325) was added. The fluorescence intensities stabilized within 2–3 min after the addition of SC-(1–325). The addition of native ProT had no effect on the fluorescence of [OG]FPR-ProT. Simultaneous fitting of the competitive cubic binding equation to the titrations (Figs. 3A and and6)6) gave a dissociation constant for SC-(1–325) binding to [OG]FPR-ProT of 17 ± 6 pM with a stoichiometric factor of 1.2 ± 0.3, and a dissociation constant of 72 ± 36 pM and stoichiometric factor of 0.7 ± 0.2 for SC-(1–325) binding to native ProT (Fig. 6). There were no significant differences between the curves generated by the cubic binding equation when the stoichiometric factor was fit (Fig. 6, solid lines) or fixed at 1 (dashed lines). These results were in good agreement with the dissociation constants of 37 ± 1 and 17 ± 6 pM determined for direct binding of SC-(1–325) to [5F]FPR-ProT and [OG]FPR-ProT (Fig. 3) and indicated that native and labeled ProT bound SC-(1–325) with indistinguishable affinity.
To determine whether the lack of SC cofactor activity exhibited by D2 (1) was a direct result of a loss of proexosite I affinity, a competitive binding experiment was performed with [5F]Hir-(54–65)( ) used as an exosite I-specific competitor (15). Direct binding of [5F]Hir-(54–65)( ) to proexosite I on ProT (KD = 4.4 ± 1.4 μM) resulted in a maximum fluorescence quench of 18 ± 2%, in good agreement with previous studies (15). Analysis of competitive binding titrations (Fig. 7) of D2 to ProT gave a dissociation constant of 40 ± 90 nM and a 1.0 ± 0.1 mol D2/mol of ProT stoichiometry. These results indicated that D2 folds independently of D1 and that the isolated D2 domain binds to proexosite I on ProT in a similar fashion as SC-(1–325). To our knowledge, this is the highest affinity proexosite I interaction for a protein ligand to date.
To determine the relative contribution of SC domains to the fluorescence enhancement that resulted from SC-(1–325) binding to [OG]FPR-ProT, emission spectra were collected for [OG]FPR-ProT alone or in the presence of 8 μM D1 or D2 and 0.5 μM SC-(1–325) (Fig. 8A). D1 binding to [OG]FPR-ProT caused a ~10-fold larger fluorescence enhancement than binding of D2, suggesting that interactions of D1 residues with ProT were the primary source of the fluorescence change that resulted from SC-(1–325) binding. Emission spectra were also collected to characterize the concentration dependence of D1 binding to [OG]FPR-ProT (Fig. 8B). The results suggested that D1 binding was necessary and sufficient to cause a fluorescence signal comparable with SC-(1–325) binding with a modest red shift (~5 nm) in the emission maximum. Quantitation of D1 and D2 binding to [OG]FPR-ProT gave dissociation constants of 3.5 ± 1.1 μM and 27 ± 30 nM for D1 and D2 binding labeled ProT with fluorescence enhancements of 130 ± 14 and 10 ± 2%, respectively. These results in combination with those obtained above by [5F]Hir-(54–65)( ) competition indicated that D2 binds [OG]FPR-ProT and native ProT with similar affinity.
Thrombin in the presence of saturating SC-(1–325) or Met-SC-(1–325) had 120% of the Fbg clotting activity of free thrombin, and the complexes with ProT had 78% clotting activity (Table 1). Although thrombin was almost totally inhibited (88%) by Hir-(54–65)( ) binding to exosite I, the peptide had a negligible effect (5%) on the clotting activities of SC-(1–325)·α-thrombin and Met-SC-(1–325)·α-thrombin, and a small (22%) effect on the corresponding ProT complexes.
To assess differences in substrate specificity of α-thrombin and SC-(1–325)·(pro)thrombin complexes, Michaelis-Menten kinetic parameters were determined for a panel of six tripeptide-pNA substrates. The SC-(1–325)·α-thrombin complex showed a ~2-fold decrease in kcat compared with free α-thrombin for hydrolysis of the tight binding substrates, H-D-Phe-Pip-Arg-pNA, H-D-Ile-Pro-Arg-pNA, and D-Phe-Pro-Arg-pNA (Table 2). Kinetic parameters Km and Ki for product were remarkably similar for α-thrombin, SC-(1–325)·α-thrombin, and SC-(1–325)·ProT complexes for these tight substrates with the exception of a 40-fold higher affinity for the product of D-Phe-Pro-Arg-pNA cleavage for SC-(1–325)·α-thrombin compared with α-thrombin or SC-(1–325)·ProT. To determine whether SC-(1–325) binding affected hydrolysis of substrates based on the P1-P36 cleavage sequences for fibrinopeptides A and B, Ser-Ala-Arg-pNA and Gly-Val-Arg-pNA were examined. SC-(1–325)·α-thrombin complex showed a ~2-fold increase in kcat for both substrates compared with free α-thrombin, whereas there were no consistent changes for SC-(1–325)·ProT complex (Table 2). The combined kinetic data indicated that the SC-(1–325)·α-thrombin complex displayed only small ~2-fold increases or decreases in kcat compared with α-thrombin. In the cases of H-D-Phe-Pip-Arg-pNA and H-D-Ile-Pro-Arg-pNA, titrations of thrombin activity as a function of SC-(1–325) verified the decreases in kcat accompanying tight binding of SC-(1–325) to α-thrombin (not shown). Taken together, these results indicated that SC-(1–325) binding to ProT generated an α-thrombin-like substrate recognition site for tripeptide-pNA substrates, with small but reproducible effects on kcat for hydrolysis of certain substrates, and no significant preference for substrates based on the sequences preceding the fibrinopeptide cleavage sites.
Reversible activation of the ProT catalytic site induced by SC-(1–325) allowed the development of novel, active site-specifically labeled ProT analogs as tools for quantifying SC interactions for the first time. Various thiol-reactive fluorescence probes were covalently linked to the catalytic Ser195 and His57 residues of the SC-(1–325)·ProT complex by a thioester derivative of FPR-CH2Cl. Characterization of the labeling reactions demonstrated that probe incorporation was covalent, active site-specific, and stoichiometric. Screening of 10 fluorescent ProT analogs identified the best quantitative reporters of SC-(1–325) binding. Previous studies indicated that SC bound to ProT with a 1:1 stoichiometry (1, 4, 43, 44), but because of the apparent high affinity of the interaction, it was not possible to obtain accurate estimates of the dissociation constant (3). The results for SC-(1–325) binding to [OG]FPR-ProT and [5F]FPR-ProT studied over a 500- or 1300-fold range of protein concentration, respectively, support the finding that SC-(1–325) binds to 0.95–0.96 sites on labeled ProT with a KD of 17–37 pM.
Stability studies of mixtures of ProT and SC previously found that these mixtures converted Pro to Pre 1, releasing fragment 1 (3, 28, 45). Our studies confirm that Pre 1 is generated initially, but in addition demonstrate that Pre 2′ is generated more slowly. Reactions of ProT with saturating concentrations of SC-(1–325) resulted in conversion of ProT to Pre 1 and Pre 2′ by cleavage of the two thrombin-sensitive bonds at Arg155-Ser156 and Arg284-Thr285 (ProT numbering), respectively. Pre 2′ generation as a result of SC-(1–325)·ProT binding has not been previously reported. At saturating levels of SC-(1–325), the SC-(1–325)·native and labeled ProT complexes were completely cleaved, suggesting that the active complex catalyzed cleavage of other complexes. The mechanism underlying this observation is not known, but conversion of ProT to Pre 1 and Pre 2′ may play a role in the mechanism of fibrin formation by SC. Although sedimentation equilibrium studies indicate that the SC-(1–325)·Pre 2 and SC-(1–325)·α-thrombin complexes were not detectably dimeric under these experimental conditions (29), there is apparently some interaction between SC-(1–325)·ProT complexes.
Because of the complex nature of ProT cleavage at saturating concentrations of SC-(1–325), competitive binding of native and labeled ProT was assessed under conditions of subsaturating concentrations of SC-(1–325) and within a time window that minimized proteolysis. SC-(1–325) bound to native ProT with KD of 72 ± 36 pM and stoichiometry of 0.7 ± 0.2 mol SC-(1–325)/mol ProT. These results were in good agreement with the KD of 17 ± 6 pM determined for the direct binding of SC-(1–325) to the [OG]FPR-ProT analog and KD of 37 ± 1 pM determined for [5F]FPR-ProT. Although there is significant experimental error in the determination of such high affinities, the results demonstrated that the ProT derivatives reported quantitatively indistinguishable binding of SC-(1–325) to labeled and native ProT. This indicates that the presence of the probe and the tripeptide inhibitor in the active site had a ≤2-fold effect on SC-(1–325) binding affinity.
Contrary to previous estimates of the Fbg clotting activity of SC·thrombin, SC·Pre 1, and SC·ProT complexes ranging from 20 to 90% (3, 46), we found no significant difference in the Fbg clotting activity of SC-(1–325) complexes with ProT or thrombin compared with free α-thrombin. Occupancy of exosite I by D2 (1) imparted resistance of SC-(1–325)·(pro)thrombin complexes to inhibition by the exosite I-specific ligand, Hir-(54–65)( ). Chromogenic substrate kinetics results indicate that binding of SC-(1–325) to ProT induces expression of a functional active site with similar P1-P3 substrate specificity as thrombin alone and no detectable preference for cleavage of peptides corresponding to P1-P3 of fibrinopeptides A and B.
The results presented here also assessed the binding of D2 to (pro)exosite I on ProT. Our previous studies indicated that D2 binding did not result in conformational activation of ProT at concentrations up to 43 μM (1). A possible interpretation of these results could have been that D2 does not fold independently of D1, resulting in the loss of binding affinity. Another interpretation could have been that D2 binds to a nonproductive site on the zymogen, eliminating its potential ProT activation ability, similar to a result seen for the isolated α-domain of SK binding to μ-plasminogen (the catalytic domain of the plasminogen zymogen (47)). The results indicate that D2 binds to (pro)exosite I on ProT with rather high affinity but does not induce detectable ProT activation. This finding is contrary to results reported previously for a chymotryptic fragment of SC containing D2, which indicated that the fragment activated ProT with an apparent KD of 120 nM (43).
Fluorescence measurements with the isolated domains of SC-(1–325) allowed us to dissect further their individual contribution to the fluorescence enhancement that accompanies SC-(1–325) binding. The fluorescence enhancement accompanying D1 binding (~130%) is primarily responsible for the enhancement seen with SC-(1–325) (98%) binding [OG]FPR-ProT, whereas D2 makes only a small contribution. The affinity of D1 binding to labeled ProT was 3.5 ± 1.1 μM, whereas the affinity of D1 determined for native ProT activation was ~4.5-fold higher (780 ± 150 nM). By contrast, the affinity of D2 binding to [OG]FPR-ProT was 27 ± 30 nM, similar to the affinity determined for native ProT obtained by competitive displacement of [5F]Hir-(54–65)( ) (40 ± 90 nM). The presence of the probe thus decreased the affinity of D1 binding while having no detectable effect on the D2 interaction. On this basis, the change in binding energy for D1 caused by the presence of the probe is ~1 kcal/mol. This represents a small fraction of the total binding energy of SC-(1–325) for labeled and native ProT, both about −14 kcal/mol, and is within the experimental error of the determinations. Having estimates of the affinity of SC-(1–325) and the separate domains for ProT allows estimation of the free energy advantage or disadvantage for binding of the two-domain fragment compared with the individual domains, represented by the coupling free energy (48). The estimated coupling free energies, for the labeled and native ProT interactions, are −4.5 and −3.5 kcal/mol. These negative values indicate that, surprisingly, binding of the two-domain SC-(1–325) fragment is less favorable than the isolated domains. The source of this effect is not clear from the present results, and further studies will be required to address this question.
The crystal structures of the SC-(1–325)·(pre)thrombin complexes labeled with p-hydroxymercuribenzoate attached to the thioacetyl-peptide chloromethyl ketone provide some insight into the sources of the diverse fluorescence changes seen with different probes. In the SC-(1–325)·(pre)thrombin complexes, the probe probably binds into the shallow depression formed by the side chains of thrombin residues Arg221–Glu146 and Glu217, together with the SCAsp106 side chain (and maybe SCThr104 and SCGlu107) of the cognate SC D1. Before SC-(1–325) binding, the probe could well make contact with the side chain of Trp148 of thrombin, which in some previous FPR-thrombin structures is defined (11–13, 49) but which in several other thrombin structures is (together with the whole 148 loop) flexible. The 148 loop in the SC-(1–325)·(pre)thrombin complexes is trapped in the SC-(1–325) groove of D1, and the mercury ion is only 2.9 Å away from one of both SCAsp106 carboxylate oxygens, thus in ion bond distance. From these observations and the weakening of the affinity for D1 by the presence of the probe, it is apparent that direct contact of SC-(1–325) with the probe and/or the possible interaction and movement of the 148 loop likely account for the fluorescence changes observed for the fluorescein derivatives and some of the other probes. It is important to note that this is not the case for all of the probes, however, because, for example, [ANS]FPR-ProT shows large spectral changes accompanying proteolytic activation to thrombin, whereas the fluorescein-ProT derivative does not.7 A very similar probe-specific effect is observed for active site fluorescein-labeled plasminogen, which reports SK binding but not activation to plasmin, whereas the ANS derivative reports proteolytic activation and SK binding with similar, large spectral changes (35).8 This effect is not limited to cofactor-zymogen binding, because interactions for which arrays of probes have been examined, including SK with plasmin (37) and thrombin with exosite I and II ligands (41, 50), show similarly diverse behavior. For the interactions studied thus far with multiple probes, there is no significant correlation of their signaling with bulk solution properties of the probes, such as the dependence on dielectric constant. The highly structured microenvironment of the probes and the structures of the probes themselves appear to place them in slightly different locations, resulting in idiosyncratic fluorescence changes on SC or SK binding to their respective zymogens, which allows them to report different molecular events. This is evident by the results for the highly polarity-sensitive probes, ACR and BAD, which showed a large fluorescence enhancement and essentially no change, respectively, accompanying SC-(1–325) binding. These probes differ only in an additional methylene group in the linker connecting the probe to the thiol group of the inhibitor peptide. On this basis, it can be expected that subtle changes in the probe microenvironment, established in part by the probe itself, the intrinsic spectral properties of the probes, and the reporting of different structural changes mediated by direct contact and transmitted by conformational changes dictate the observed spectral changes for various interactions. At present, there is no clear basis from the structures or spectral properties of the probes to support a detailed explanation of the differences observed or their predictability.
Development of fluorescent ProT analogs enabled quantitative analysis of SC-(1–325) interactions reported by changes in probe fluorescence intensity because of changes in the microenvironment of the active site-bound probes. These probe fluorescence changes showed a complex and sensitive dependence on probe structure as well as spectral properties. The versatility of the labeling approach allows selecting the best derivatives for characterizing interactions of ProT by fluorescence intensity, resonance energy transfer, and anisotropy changes. Although the purpose here was to characterize SC-(1–325) interactions, [ANS]FPR-ProT and likely other fluorescent derivatives report activation of ProT by the factor Xa·factor Va, prothrombinase complex,7 which should enable fluorescence studies of prothrombinase complex interactions and prothrombin activation. Finally, the use of expression of a unique thiol group as the basis for active site labeling allows specific labeling with other types of spectroscopic probes, heavy atom labeling (1), active site-specific immobilization (18), and affinity ligand labeling. These derivatives should provide valuable new tools for investigating ProT interactions and the mechanism of activation of ProT by both physiologic and pathologic activators.
We thank Dr. Ingrid Verhamme for assistance in the analysis of the kinetic data and comments on the manuscript and Elena Consacro and Sarah Stuart for performing the clotting assays.
*This work was supported by National Institutes of Health Grants HL038779 and HL071544 (to P. E. B.) and by the SFB 469 of the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie (to W. B.).
3The abbreviations used are: SC, staphylocoagulase; SC-(1–325), staphylocoagulase fragment, residues 1–325; D1, domain 1 of SC, residues 1–146; D2, domain 2 of SC, residues 147–325; Fbg, fibrinogen; SK, streptokinase; FPR-CH2Cl, D-Phe-Pro-Arg-CH2Cl; ATA-FPR-CH2Cl, Nα-[(acetylthio)acetyl]-(D-Phe)-Pro-Arg-CH2Cl; ANS, 2-((4′-iodoacetamido)anilino)naphthalene-6-sulfonic acid; ACR, 6- acryloyl-2-dimethylam-inonaphthalene; BAD, 6-bromoacetyl-2-dimethyl-aminonaphthalene; AF350, the sulfonated coumarin derivative called AlexaFluor 350 C5 maleimide; BD, BODIPY® 507/545 iodoacetamide or N-(4,4-difluoro-1,3,5,7- tetramethyl-4-bora-3a,4a-diaza-s-indacene-2-yl)iodoacetamide; TMR, tetramethylrhodamine-5-iodoacetamide dihydroiodide; 4′F, 4′-(iodoacetamido)fluorescein; 5F, 5-(iodoacetamido)fluorescein; OG, Oregon Green 488 iodoacetamide or 5- (and 6)-iodoacetamido-2′,7′-difluorofluorescein; pNA, p-nitroaniline; AEBSF, 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride; ProT, prothrombin; Pre 1, the product of cleavage of ProT at Arg155-Ser156; Pre 2, the product of cleavage of ProT at Arg271-Thr272; Pre 2′, the product of cleavage of ProT at Arg284-Thr285; DAN, dansylaziridine.
4I. M. A. Verhamme, P. Panizzi, and P. E. Bock, unpublished results.
5Fluorescent analogs of ProT are represented by: [probe abbreviation]connecting thioester peptide chloromethyl ketone-ProT.
6Schechter-Berger (51) notation referring to the residues of a substrate (from the N-terminal end) as …-P4-P3-P2-P1-P1′-P2′- … with the scissile bond at P1-P1′, which interact with complementary specificity sites -S4-S3-S2-S1-S1′-S2′ … on the proteinase.
7P. Panizzi and P. E. Bock, unpublished observations.
8P. E. Bock and P. D. Boxrud, unpublished observations.
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2974Making a "Box" Using a Style Sheet (CSS)
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- Jul 30, 2002Hi All,
I need to make some boxes around some text and want it to always
fit correctly for a lack of better terms. The boxes are used for
links with names, always in the same place, but the link
descriptions will change size. I want it to do like  and
have a few pixels spacing between the text and border. This is
what I have, but it is not what I want. I understand why it is
doing what it is as far as the box size goes. (I think I will
be all set after this - I hope.)
A small part of my funnies.css:
font-size: 100%;
text-align: center;
/*width: 100%;*/
border-style: solid;
border-width: 1px;
border-color: #00CCFF;
background-color: #FFCCCC;
margin-left: 60%;
margin-right: 20px;

Air Farce, Yep, it's Spelled Right Air Force One

Top of Page

Oh, also, I would like to have a vertical line the same size as
the border in between the links. Do I use a 1x1 image and is
there a way to repeat it inside the box, or what? :)
Would it be best to use .r and then ,
, or
something else than the way I have it now? (Geesh, thank God for
RegExp, else I would have quit this a long time ago. ;)
Say, what does "float" mean?
I do not use any headers and footers in the .css file, correct,
or can it be used, is it acceptable both ways? CSE Validator 5.00
thinks  is CSS code, or it did the way I had it.
Thanks again guys and gals!
The Subject: "... Using a Style Sheet (CSS)" is for archive purposes
to help us that need it to find old posts.
Happy HTML'n!
The NoteTab and Html List...
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text:
New Member
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- Hi, My name is Kim James.
We recently moved to the Las Cruces from Las Vegas. I have been homeschooling my 17 y/o daughter (w/Down Syndrome) for about 2 years. I recently embarked on homeschooling my youngest daughter, age 4. I am still fairly new to homeschooling as my older daughter education is fairly fluid. However, my youngest needs structure so any guidance would be great.
Kim James
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October 20, 2014 Total Views: 6096 Today: 1
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text:
Links, html, images etc & external application problem
Hi all, I have a just started to use WP for a personal site and have two issues, I will list them below…
1. I can't seem to post any kind of 'dynamic' content within my WP posts. when I post any type of link, image or similar I find that it does not post and one I try to 'publish' the post it forwards me to the parent website and the post is not published, so basically one I have pressed the publish button it will forward me to http://www.[mydomain].com/ rather than back to the 'write' panel within the WP admin pages.
2. this issue is me trying to post with an external application like, mars edit, or MacJurnal or any other application (I have tried pretty much all of them), all do exactly the same thing, I can enter my login details and when I think it tries to communicate with the WP server (on my host) and doesn't 'connect' I have a feeling it is a problem with the 'xmlrpc.php' file, but I might be wrong.
If anyone can help me out with these issue I would really appreciate it. My blog is pretty private but if a Mod would like the details I can PM this to get to the bottom of it.
I am using 2.6.2 (this even happened with 2.5.x or 2.6.x) on a Mac, have tried Firefox, Camino, IE (PC) Safari all do the same with the posting issue.
Thanks guys in advance.
- The topic 'Links, html, images etc & external application problem' is closed to new replies.


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A beautifully photograph book which aims to capture the many mysteries that surround nature's few remaining sanctuaries.
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|1,614 sq. ft.
|Property type
|Minimum Stay
If you are looking for a Sardinian villa that puts you as close to the sea as you can possibly get, Villa Elios will tick all the boxes. You'll only have a 50 metres walk until you hit the sand, so if you forget to take anything to the beach you can simply pop back to the villa to get it!
However, Villa Elios has plenty more to offer than this. It also takes full advantage of a cosy secluded and grassed garden with shady spots and plenty of comfortable garden furniture. With large sliding doors opening up the interior dining room to the exterior dining area, you can effortlessly transfer your meal outside to enjoy the long pleasant evenings to the full.
There are three bedrooms here ��� two doubles and one twin. They are generously sized and pleasantly decorated. Two bathrooms provide all the facilities you could need and these are both well appointed. Bright tiling and plenty of space – those are the signature features of these rooms. Make the most of the modern kitchen with all the equipment you require, and come back to the villa after a day's exploring to enjoy a family meal together.
With the use of a veranda and a terrace, not to mention the garden area, you'll have plenty of room to relax and enjoy the good things in life. The sprawling nature of Villa Elios is its most appealing feature, providing you with an impressive use of space while not trying to cram too much in. If a calming, secluded and relaxing holiday is in your itinerary, add Villa Elios to make your dreams come true in Sardinia this year.
Services: Bedlinen, towels, final cleaning and electricity usage are an extra cost, payable locally. Please ask for details on booking.
Property URL:


text: Parodies Dexter's Laboratory/Toy Story Dexter's Laboratory/Toy Story 2 Dexter's Laboratory/Toy Story 3 Retrieved from "http://scratchpad.wikia.com/wiki/Dexter%27s_Laboratory/Toy_Story_Movies?oldid=1736047" Ad blocker interference detected! Wikia is a free-to-use site that makes money from advertising. We have a modified experience for viewers using ad blockers Wikia is not accessible if you've made further modifications. Remove the custom ad blocker rule(s) and the page will load as expected. Categories: Dexter's Laboratory Toy Story Movies Dexter's Laboratory parodies


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Tolkien wrote:Tsuneo wrote:Ryuuchi wrote:]Tsuneo wrote:Mandated duality!
For every "good" character you have, you must make an evil one! And vice versa!
Automatically rank-approved for your dual-twin!
If only this were true...though not everyone can play a good bad guy.
Practice makes perfect.
I think if everyone were forced to make one good guy and one bad guy, eventually the players here would have the skills necessary to pull off what Isuzu is talking about.
Currently, they do not.
I don't agree with that. There is talent here just needs refocus.
EDIT: But I do understand where you're coming from.
I didn't say talent. I said skill.
Really what people lack is detachment to their characters.
EDIT: I suppose I should elaborate.
Ryuuchi is misunderstanding what I'm saying. What I'm saying is that rather than making a bad guy counterpart to the good guy character, you're forced to make one of a more "evil" alignment. They don't have to be connected.
You make spunky Konoha guy who wants to be Hokage and believes in friendship.
You then have to make something else. I dunno, cold assassin who likes the taste of blood. From Yuki.
Every time one goes up, you have to rank the other too, which means you're thinking about the story. Maybe you don't like your assassin that much, but you have to make him stronger cause you made your good guy stronger.
So then you have to make an antagonist. Well, you happen to have this cold assassin who likes blood that you can use. And you don't mind killing him off, cause you don't care.
"But Tsuneo, I don't fall in love with that character, it's not as good as two direct opposition stories where you don't know where to cheer, etc etc etc."
That's the point.
If you get attached, you won't kill anyone. You won't let them lose. You love them too much.
What the people on this site need to learn is the ability to detach. This would teach that. Once you see how great a story can be when killing someone off you don't care about, you're more willing to do it with characters you /do/ care about.
That's what I mean.


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Back into the amber yearning, all my soul within me curling, soon again I felt a knotting somehwat lowered than before. "Truly," said I, "Truly that is… Read more "The Whispered Word II"
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Deep into the darkness freezing, long I stood there wandering, healing,
Doubting, gleaming feels no mortal ever dared to beat before;
Choices III
Where, my heart, hast thou forlorn thy song, thy beat, thy sense, thy soil? Lost my feet floating flushed by thy meth 't can't but love thee,… Read more "Choices III"
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Where, my wit, hast thou forthgone in this very matter, in this affair? Lift my spirit, left my breath I can't choose or change the depth of… Read more "Choices II"
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What, my love, hast thou forthgone in the black and misty shades of dawn Where, my love, hast thou forlorn my heart, my neck, my sunny morn?… Read more "Choices I"
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You'll find me at the end of the rainbow… talking to my cigarette where doubled colours bend, and golden puddles flow… A frown – for all those… Read more "Bent Colours"
All those lines are simply too much too full of I's, too much fuss that cannot touch the heat of my heart or the warmth of my… Read more "Reflections"