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About this Show
News/Business. Interviews, news stories, reviews. (CC) (Stereo)
San Francisco, CA, USA
Comcast Cable
Channel 88 (609 MHz)
Susan Boyle
Nicole Jordan
Ka Ching
Alicia Ball
Borrow a DVD of this show
News/Business. Interviews, news
stories, reviews. (CC) (Stereo)
November 24, 2012
2:05 - 3:00am PST
"today" with kathie lee gifford and hoda kotb from studio 1-a in rockefeller plaza. hey, everybody, it is try day friday november 23rd and we are so stuffed. i don't think we want to try anything today, except for maybe these, which is, you know, a little pretzel with some peanut butter that's what everybody wants right after thanksgiving. >> get to that in a minute, our friday thing. it is time for christmas shopping it is official. >> too soon. >> black friday is the day. the question is when do people stop the christmas shopping? there are people -- >> obnoxious, it done, had it done in august. >> not friends with those people. >> no. we don't like those people. almost a fifth start shopping in the back-to-school season, there are lots of great sales and things. 10% start when the christmas decorations begin to go up. >> which is now. >> some up before halloween here. blown away by hurricane sandy but they put them back up.
3% wait until two weeks before christmas, like hody. >> i wait because i like to feel in the mood. i'm not in the mood to buy christmas presents in july, august or even september. then you have thanksgiving and all the other holidays that you are enjoying. >> we live in -- excuse me, i just spit. why can't we live in the now, in the presents, the present, a present, here and gone before you appreciate it. i think we are always projecting the future, we are missing what's happening right now. and i -- i'm upset that some of the stores opened at 8:00 last night to get a jump on it. you have hardly furnished dinner with your family. and everyone is going out. >> all the sales are there. i guess people are trying to take advantage of what they can get. >> and the heart of the economy is for people i understand it. but do you really need that stuff so much? it seems to me that the retailers need need the sales. but we don't need more stuff.
>> i remember very few presents that i was given. i don't really remember them, sure i loved them. i love the card. i get more of a kick reading what someone wrote than the other part. but here's a much more -- >> a sign of getting old, by the way. welcome to my world. >> so, here is a pressing question. what do you wear to bed? >> which has nothing to do with anything. >> nothing. what do you wear to bed? do you wear nothing, like some people? >> i wear my wedding ring. excuse me, that is something. >> do you wear a t-shirt and underwear, like a lot of people. >> what do you wear? >> that's what i wear. do you wear a full-on pajama outfit with pants and the top? do you wear -- >> like bobby, hello kitty pjs? >> jockey did a survey, said just under half of us preferred to wear nothing to bed. according to esquire.com, most women want their husbands to come to bed in their underwear. >> why? >> because it's better. i like underwear, too.
you want them naked? you want frank naked? >> i have many times. i want him to be comfortable. whatever -- sometimes he wears -- he wears what he is comfortable in. if he's cold he puts on something. >> talk sexy for a something, that is not what this is about. let's talk about that. to me, a man is much sexier wearing something than he is standing there stark naked, i think. i think a guy is sexier in a cool t-shirt, boxers or jeans than standing there full-on naked. what do you think? do you think he is sexy naked? >> a long time i have seen anybody other than my husband, honey. i don't remember. that's my story and i'm sticking to it. >> i remember watching -- an ex vacuuming without clothes on. >> now, that would be the biggest turnoff in the world, unless he is a really good vacuumer. >> there's something about just takes all the allure away. they are getting things. >> eww. >> not sexy at all. we will tell but what your bedtime says about you, from
dangerous lee biz, if you wear girly pajamas. which i guess is like the tops -- >> us, not your husband. >> your bed sometime is about comfort as well as fun. you don't mind your man seeing in the morning because you look kind of cute. >> short, silky little number, you are confident, sassy, sexual and you know what you want in the bedroom. bed sometime about sensuality than sleeper. >> here's what bothers me, there i wear a little sexy short -- i'm in my new york apartment because of the show. i'm alone and putting on -- there i think -- this building and what if an alarm goes off? >> you have to have something on. it is not like being home. >> i will wear something. >> if you wear a never ending nightgown -- all tangled up now, sensible, practical and reliable. >> if you're naked, yes, you are
uninhibited and comfortable with your body. it's dark. i am. you enjoy the sensation of like in bed in the buff. i don't lay around thinking i'm enjoying this sensation. >> but you must remember in the morning who are we talking to, total -- body parts will shift with travel. >> what? >> you move around. roll around. >> where did you go in the middle of the night. might need to put them back in place before your partner wakes up. what the heck does that mean? >> you know what that means, laying on your back. >> they are not going -- >> they are here. >> they may go to the side but they are not actually leaving the same zip code. >> you never know. just line them up. >> dumbest thing i have ever heard. go to the next dumbest thing. >> social cues. a woman has been writing about -- she has had family members come to her house because of the hurricane and things. this applies for people no matter why you are having guests but it's when the guests sit and don't contribute. in other words, you are doing dishes, sitting. >> they eat, they drink. >> they sit. what do you do?
should you say something if they're sitting there not doing anything, or should you do it yourself? and stew about it later? >> philip says about each letter about slothful guests who don't lift a finger he receives as many about guests who won't back of off. >> that do too much? >> seems real. also no problem asking somebody, would you mind helping me a little bit? would you -- you know what i usually do the first time you're a guest, second time you're family and you get up and help. >> pitch in. i think you have to ask, people treat you the way you let them. if you sit there and stew and do everything and complain about it. they are treating you a certain way, you are allowing it. say to them listen -- >> the only power people have is power you give them, hoda woman. >> lots of wisdom in this program, don't you think? >> mm-hmm. >> another, what is it? >> study. receiving a compliment has the same positive effect as receiving cash. forbes.com. >> compliments don't pay the rent but improve performance a
similar way to get a cash award. everybody wants to be valued. everybody wants to know that they have been noticed and appreciated. >> no i agree with that. >> unless you are a waiter and someone says that was really great. thank you so much. >> and then don't leave -- like we talked about. >> the tip. i think it depends, i think it is nice to receive a compliment from your boss. >> has to be sincere. you known somebody's not -- >> you know what, i think bosses can tell you they love you, which is important, and show you, showing you means they give you a raise. >> ka ching. >> i think there's a light. nice when a boss says that was a great story, hey, good job on that you worked well on this project but i think ultimately, if -- >> so you're saying keep the compliments, show me the money? >> show me the money. >> you know what it's time for? your friday funny. from our facebook -- marsha braun yungle, i hope i produced that right, dorothy and edna
two senior widows were talking. dorothy says that nice george johnson asked me out for a date. i know you went out with him last week and i wanted to talk to you about him before i give you my answer. edna replies, dorothy, he shows up at my apartment at 7:00 p.m. dressed like such a fine gentleman that suit and brings me such beautiful flowers. then he takes me downstairs and what's there? a limousine. uniformed chauffeur and all. marvelous dinner, lobster, champagne and the works. then we go see a show. "scandalous" i enjoyed it so much i could have died from pleasure. >> she added that. >> so we go back to my apartment. and he turns into an animal. completely crazy. he tears off my expensive new dress. and has his way with me three times. dorothy says" goodness gracious, are you telling me i shouldn't go?" and edna said "no no no i'm just saying wear an old dress."
>> that's ridiculous. >> he tore off the expensive dress. you win some lose some. >> it is time for our girl's bobbie buzz. hey, girl. >> adorable. >> so cute. >> a lace overlay. i just called myself out. so, it is the day after turkey day and shopping is going to be in full swing. so i have to point out these really cute fashionable accessories from post it. these adorable shoe dispensers for the girl on the go. >> what are they? >> the shoe has tape. >> a shoe tape dispenser. >> this, amazing, for your purse, a compact, with a mirror for your lipstick and your post-it note, keep on the go then sara is going to help me out with our next -- this is the best idea ever. okay. >> this one is mine and this one is sara.
>> some people can't get enough of themselves. >> getting a glimpse of the closet. this stand in says "i wish i could be there today." you send this where you can't go. it's so cute. you care about grandma or someone else. i thought this was hysterical. >> you'lling celebrating christmas with this. >> what does that cost? >> not too expensive, i think around 30ish dollars. we have the information online and a really great thing, one king's lane is a web grate website to find home furnishings but teamed up with designers, rachel roy, holiday paper, all the proceeds go to charity. feel good about ripping that paper. >> so nice. finally a way to give back. thank you. >> good job. >> all right. wait until you see the new guys, these ambush makeovers are so worth waiting for. >> going to be great. and singer susan boyle takes us somewhere over the rainbow. first, these messages.
sfx- "sounds of african drum and flute" look who's back. again? it's embarrassing it's embarrassing! we can see you carl. we can totally see you. come on you're better than this...all that prowling around. yeah, you're the king of the jungle. have you thought about going vegan carl? hahaha!! you know folks who save hundreds of dollars by switching to geico sure are happy. how happy are they jimmy? happier than antelope with night-vision goggles. nice! get happy. get geico. fifteen minutes could save you fifteen percent or more. starts with arthritis pain and a choice. take tylenol or take aleve,
the #1 recommended pain reliever by orthopedic doctors. just two aleve can keep pain away all day. back to the news. just two aleve can keep pain away all day. new pink lemonade 5-hour energy? 5-hour energy supports the avon foundation for women breast cancer crusade. so i can get the energized feeling i need and support a great cause? i'm sold. pink lemonade 5-hour energy? yeah and a portion of every sale goes to the avon foundation for women breast cancer crusade. i'm sold. new pink lemonade 5-hour energy.
get the alert, energized feeling you need and support breast cancer research and access to care. we are back with a special installment of our plaza ambush makeovers, when we pluck two turkey-stuffed ladies off the plaza >> that's terrible. who said that? >> a little stuffing, too and surprise them with a brand-new look. >> here to show us the result is our resident makeover team, "today" contributor and stylist to the stars, louis licari and "us" contributor and author, jill martin. >> how was it today? >> it was great. everyone does feel a little full. >> i have to admit. >> a festive mood. >> exactly. festive. so we just thought thin. that's all.
>> let's start off with our friend denise hasn't, 49 years old from clewiston, florida. she has her husband pick out her clothes because he has better taste than she does. so she was thrilled to get the chance to get a brand-new style. let's listen to her story. >> denise told me outside she is very conservative, of course, we picked you. get a little sexy, right? >> this will be incredible because i would never do this on my own. absolutely never. >> so roger, who is at home, you say he hasn't seen you different in how long? >> well, we have been married, be 20 years in january and i have always had longer hair, i seldom wear makeup, i'm just -- that's just not who i normally am. i'm afraid to try something new this is going to give me something really out there i'm excited. >> denise has brought a posse with her. among her friends, sarah, ashley, brandy and a whole big group. >> that's called a tribe. >> everybody, put your blindfolds on, cover up your eyes, keep them on. i will tell you when to take them off. here is denise before. all right, denise, let's see the new you.
oh, wow. all righty, are you guys ready? all right, girls, take them off. denise, are you ready, hottie? i can't wait to see this. [ screaming ] >> you look amazing. >> you look hot. >> oh, the kleenex. >> i need you to look right here at this dam ra if you wopt mind. you look -- your hair is gorgeous. tell us, louis. >> dab your eyes, sweetheart. >> what we did to denise is, of course, a great haircut. side-swept bang, we love and adore it is so sexy. and then i -- >> look at the difference. >> -- i did her hair.
which is turning a little bit gray in the front, i made her a slightly warmer dark blond with those highlights. >> unbelievable. >> accent the focal point of the bangs. >> look at the posse. are you guys busting? >> what do you guys think? >> we always knew her glow inside and so now everybody, the whole world gets to see the shine that we see every day through this incredible woman. >> you have such great friends. jill you the outfit is perfect. >> gorgeous figure. all about peplum going into winter and fall, a great dress by mark by andrew mark, the earrings and bracelet are kendra scott. >> beautiful. big round of applause. denise, you can join your friends. >> and our second lady is nicole jordan, 37 from lexington, kentucky. she has had the same look for, as she says, way too long. she jumped at the opportunity to get a whole new look. let's listen to her story. >> okay. first time in new york city. >> right. >> so, what kind of look are you going for here? >> what kind of look? i guess anything different than what i'm looking now. >> and you're single.
we have a lot of men watching here. what are you looking for? >> i just want a man with a job and doesn't live with his mom. >> i think we will find that after this makeover. okay. are you ready to get a little glam? >> i am. >> how hard can it be to find one of those? she is here with her sister, kim, friends angie and mark. keep on the blindfolds, one last look at nicole before and bring out nicole jordan. my god, wow. >> you guys take off -- stay right there. and take them off. >> she's gonna freak. >> turn around, nicole, take one look back here in the mirror, hon. >> oh, my god. >> you look beautiful. >> she is getting kleenexes. you look beautiful.
>> jiminy cricket. >> we don't cry in kentucky. >> sweetie, spin right around and look right there -- oh, you look beautiful. >> what a job. >> i tell you. >> this is a layered haircut and played with the highlights, accent the layers to make everything more -- just more alive and you do you look young, more vibrant and a lot sexier. >> you sure do look at her people. >> i don't recognize her. >> gorgeous. >> extra day and hit the town. >> jill, tell us about this color-blocking thing. >> color blocking for winter, she picked this dress, we tried on a few, felt most comfortable, booties again booties big for
winter. these are available at macy's. >> big round of applause for both. [ applause ] >> good job, guys. >> after revealing these brand new looks, we are getting ready to show pictures some people may want to hide. >> those quirky, funny photos that make you say what the what? right after this. [ male announcer ] years ago, when the inventors of twix unveiled their bar, the tension between them reached a breaking point, literally. so they divided the production between two separate factories. each factory took a vastly different approach. left twix flowed caramel on cookie, while right twix cascaded caramel on cookie. left twix bathed in chocolate, while right twix cloaked in chocolate. both bars as different as the vastly distinct men who invented them. to this day, sharing nothing but a wrapper and an ill-designed driveway. try both and pick a side.
i have a cold, and i took nyquil, but i'm still "stubbed" up. [ male announcer ] truth is, nyquil doesn't unstuff your nose. what? [ male announcer ] it doesn't have a decongestant. no way. [ male announcer ] sorry. alka-seltzer plus fights your worst cold symptoms plus has a fast acting decongestant to relieve your stuffy nose. [ sighs ] thanks! [ male announcer ] you're welcome. that's the cold truth! [ male announcer ] alka-seltzer plus. ♪ oh what a relief it is! ♪ [ male announcer ] to learn more about the cold truth and save $1 visit alka-seltzer on facebook.
time to look at all those photos that make you say "what the what." >> sara sifted through the photos, all four of them, and here with the top picks. >> they are on the edge of their seat waiting for the first photo from linda from iron, michigan, corn maze for blondes. i don't get it. >> that's funny. >> i love it. >> we'd be lost in there forever.
>> so would you, sara. >> genius. >> leah submitted this photo. no personal dumping. you can do any kind other than that. but not the personal stuff. >> we are slow. >> next up, a photo from valerie nicholson from pennsylvania. you want to read this? >> faster, faster, oh, yes! yes! yes! i'll have what she's having. >> on a broadband truck in england. i think that's brilliant. >> michelle woods from shelton connecticut, sent us this photo. if it's not in stock, it's free. >> in stock or free. how can you take it home if it's not in stock? >> back to the corn maze. >> there you go. >> says one per customer for two for $3. >> hoda just joined the corn maze. >> mary submitted this photo, three triplets going the first
day of school and little 2-year-old brother was upset he wasn't going. what about me? >> how cute. >> can you see -- that's like so -- excited. >> go anyway. >> thank you, sara. >> those were actually charming. >> if you have something you think is photo worthy of "what the what" send it to klgandhoda.com. tech that will take the trouble out of travel. >> and chef english will transform a feast for us from entirely a new meal. 2@
we are back with today's tech and taking trouble out of the holiday travel. >> apps to make your trip smooth is the founder of digitwirl.com. carly block. >> hi, guys. >> perfect time for this segment. >> hello. >> tech stuff.
>> heading to the airport. there's some great apps that can help you. this is my absolute favorite app for the airport, called gate guru. >> what does it do? >> a bird's eye view of every terminal in the airport, all the shops, all the restaurants and all the service. if you have a little bit of a layover or trying to figure out a better place to eat. >> ratings and everything. >> i once used this to discover a little manicure kiosk on a layover a little bit better. >> is it all just the big airports, carly? >> all the different larger airports. metropolitan? >> i love that gate guru. . now, you have to see this app to believe it. called world lens. you are in a foreign country, signs everywhere, instead of fumbling with a translation book, check this out. >> these are our signs. we don't know what they say. >> it magically translates the text -- >> my gosh. >> right in front of your eyes. >> look what's happening. clothes optional. >> do the next one. >> danger, sharks in the water. >> are you kidding me?
>> wait we have to check out the last one. >> apology -- >> sold all of our wine. apology. >> is that not amazing. >> what is that called? >> world lens. $4.99 per language pack, once buy it down the need a wi-fi connection. >> that's unbelievable. tell you only one bag or burn bag and a carry-on. >> usually carry a purse you are going to use when you're traveling and a laptop bag, this is both in one, from alicia ball, 289, a little bit pricey but two bags in one, the front panel. >> your computer. >> up to 15-inch computer. >> it is stylish. >> gorgeous. in this gray and also black leather. really nice. >> i love that. >> now, some people have disorganized bags. just saying. >> that's rude. that's rude. >> doesn't mean they are not good people. >> some people might want to get more organized. this is the grid it system from cocoon, 25 to $50.
all different shapes and sizes, strap your cords, headphones. gum. >> i would never put anything in there, but nice for certain people. this is genius, i have left cable and cords in the those. >> makes them stand out, it stays on the cord the whole time, from nice you $16, sort of use it to charge your phone anywhere. >> bright color, to remind to grab it. >> don't leave the cord. >> that last look in a hotel and -- >> that is brilliant. >> so cute, right? so you guys, have you heard of noise canceling technology? if you are on an airplane, there is a crying baby, a noisy neighbor, you flip a little switch on these head phones and the world goes away, amazing. >> what do you get instead? >> like a combatting frequency that cancels out all the ambient noise. >> wear them at home, too? >> yes, you can. >> brand new from beats, the executive, really, really comfy fit for the airplane. >> are they comfy, light? >> what? are you talking?
>> just kidding. >> they are not plugged in. >> excuse me. they are not plugged in. i was expecting something. got to plug it into something. >> so these are $300, a little prieszy -- cheaper option. >> peace of mind. >> seriously, invaluable. these are urban ears, $60, they have an on-ear fit, nice and curby and drowns the sound out, doesn't have that noise-canceling technology, $60, bright colors. >> great stuff. thanks, carly. >> welcome. >> bon voyage. now we have taken the headache out of travel what are going to get them for the holidays? >> gifts that give back, right after this. >> excuse me, who is singing? i believe that's barbara streisand.
♪ now, today's holiday gift guide, gifts that give back. >> from saving the planet, from plastic waste to putting money toward college scholarships, companies doing great things when you purchase their products. >> here to share ten -- i was going off listening to my -- it is chef and author kathleen
dellmans. we love her, every time you come, good stuff is released into the universe. we love you very much. >> thanks to you guys and everybody at the today show. >> happy to do t. >> start with vino, shall we? >> start the way we always end. >> something for mommy and hoda. they -- this is a swell bottle. swellbottle.com. these great water bottles. however, they created one that's just for wine. keep your wine cool. their gift this season is two bottles, one for mom and kids or your purse, hoda. >> hello. >> i know. >> $45. >> okay. i like these classic tees. >> this is out-of-print clothing. it's books on tees and they are on a mission for every shirt that you purchase, they give a book to someone in need. >> oh, that's great. beautiful. >> yeah. and their deal in honor of leo dicaprio's new movie with toby mcgwire "the great gatsby," free
shipping, $38. for two tees. >> how are they going to stop the other movie with robert redford? mia farrow? stop it. >> all these bags, messenger bags and what not. >> project repat. you turn in your favorite tees, gently used, they will turn it into something. for moms this holiday season, baby onesies you don't want to part with turn them into a baby stroller blanket. $65, free shipping. they are doing some amazing things. they work with americans who have special abilities. they are also helping to revitalize some of our manufacturing plants. >> okay. >> we need that. >> what about these messenger bags? >> united by blue. for every purchase on their website, they remove a pound of trash from our oceans. >> yeah. >> these are beautiful bags. high quality. >> land's end or something. >> wax cotton, regular $100, $65 with free shipping. >> love the free shipping. >> i love this. >> this is the man tree. this is inspired with men in mind, but a lot of women do the shopping.
so, this is a way for people to get familiar some of our artisan and crafts vendors across the company -- the country. wouldn't get necessarily to west virginia or louisiana or something, but you can taste what they are doing over there $75. free shipping. then we have mighty wallets, these are mighty great, a photographer working at moma, a brooklyn guy, they are made out of the same thing things that shipping envelopes are made out of, $13.99, ross.com, free shipping. >> how about the shades? >> these were designed by two college kids in california. couldn't make money, stay cool, looked cool, started their own company, 14, 21. >> these watches pop. >> flex watches, regular 40, 30 with free shipping, ten colors and pick your charity and this
deal is great, $65 for a built skateboard and it's usually about $120 or $130 and free shipping. these guys give away skateboards to kids in orphanages across the world. >> for the pooch? >> and then for the pooch, better buddies. pet place mats, i don't know if your pets are slobs, mine are 12.99, free shipping and they have pet beds. >> so cute. >> and all the rest are going to be on our website. >> ten more wonderful ideas go to klgandhoda.com. >> thank you. >> thank you. >> find more go about these gifts on our website, klgandhoda.com. >> what are you going to do with thanksgiving leftovers? >> they are gone. chef english will show you how to turn last night's feast into a new meal.
turkey day leftovers, did you love the flavors of last night's feast but like to enjoy them an entirely different way today? >> to transform your turkey into meatballs is todd english, chef and restauranteur of olives, food hall and cava. >> a challenge what will you come up with, something fun, new, innovate at this time. i figure, meatballs are in now. who doesn't like a good meatball. >> start with a little wine. most importantly, get the cooking going, the creative juices flowing. very important. cheers. all right. so while you are doing that i'm going to have you stir here is. >> i will stir. >> what's in there again? >> turkey, i see. ground beef. >> ground turkey. so, these the only extra ingredient you need to have. not buying turkey, have that around. i have diced, leftover turkey. >> cornbread. >> drop some eggs.
>> two eggs. >> some sage. a little garlic. >> fresh sage or -- >> fresh sage, yeah. >> mustard. >> this is cornbread. >> grey poupon? >> it is. whole grain mustard. a little bit of milk. use dark meat, light meat, use your stuffing leftover as well. >> dump it all in. >> a little green onion in. >> now you spoiled it with the onions. >> we do. >> make little balls. >> sear them in the pan like that. >> look what's happening down there. >> see the chunks of meat. >> you cook those until brown and cooked through. >> nice and brown and finish them in the leftover gravy. >> if there isn't, make another batch. >> so good. could use barbecue sauce, you have some of that in the cupboard, that works well. >> the point is to take
everything and mix everything up. >> make a little extra so you have -- i'm even thinking of making these for the main meal, turkey and turkey meatballs. >> let's live here, look how beautiful. >> these are beautiful. >> mashed potatoes, of course, the cranberry to go with it. how delicious is that? >> oh, my god. oh, my god. >> delicious. >> todd. >> yes? >> what else did you make, honey? >> this is just turkey and gravy over biscuits with a fried egg. >> brunch. >> sara, get in, baby. >> and you know, if you want to spice it up a little bit you got turkey quesadillas. whatever cheese you have left over from your cheese board, if you have one. between two piece of flour tortilla, saute it, cran berry, avocado. how delicious is that? almost better than the turkey, right? >> delicious. >> this is heavenly. todd, thank you. >> welcome. >> find the recipe for turkey cornball meatballs on our website.
>> a lot of balls. >> get ready for another thanksgiving after treat coming up. >> susan boyle sings a beautiful all-time classic song. >> my god. >> this is "today" on nbc. ♪ you can help othersink along the way. ♪ ♪ a portion of every bottle that they sell goes to fight ♪ ♪ breast cancer and i think that's swell. ♪ ♪ the more you take, the more they'll pay, ♪ ♪ so make them write a big check today. ♪ ♪ and if you're feeling a little slow, ♪ ♪ then 5-hour energy will help you go. ♪ ♪ so buy a bottle of pink lemonade and ♪ ♪ you can help fight breast cancer today. ♪ but i still have a runny nose. [ male announcer ] dayquil doesn't treat that. huh? [ male announcer ] alka-seltzer plus rushes relief to all your worst cold symptoms, plus it relieves your runny nose.
[ sighs ] thank you! [ male announcer ] you're welcome. that's the cold truth! [ male announcer ] you're welcome. anyone have occasional constipation, diarrhea, gas, bloating? yeah. one phillips' colon health probiotic cap each day helps defend against these digestive issues with three strains of good bacteria. approved! [ phillips' lady ] live the regular life. phillips'. [ phillips' lady ] live the regular life. well that was uncalled for. folks who save hundreds of dollars switching to geico sure are happy. how happy, ronny?
happier than gallagher at a farmers' market. get happy. get geico. chances are, you're not made of money, so don't overpay for motorcycle insurance. geico, see how much you could save.
>> the toyota concert series on "today" brought to you by toyota. the world first fell in love with susan boyle in 2009 when she appeared on the tv show "britain's got talent." since then sold over 18 million albums and topping the charts in more than 30 countries. now out with her fourth album "standing ovation" a collection of songs from musical theater. here is susan boyle singing "somewhere over the rainbow" by the fabulous harold arlen. have a great weekend, everyone. ♪ somewhere over the rainbow way up high ♪
♪ in a land that i heard of once, once in a lullaby ♪ ♪ somewhere over the rainbow skies are blue ♪ ♪ and the dreams that you dare to dream really do come true ♪
♪ someday i'll wish upon a star and wake up where the clouds are far behind me ♪ ♪ where troubles melt like lemon drop drops way above the chimney tops that's where you'll find me ♪ ♪ somewhere over the rainbow
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text:
The invention provides a DNA encoding at least one part of genetic information of protein which has biochemical and immunological properties of a major allergen of Dermatophagoides farinae (Derf II).
1. An isolated DNA comprising a DNA segment encoding the major mite allergen Derf II of Dermatophagoides farinae, wherein said allergen has the amino acid sequence selected from the group consisting of amino acid residues 14-142 of SEQ ID NO: 2 and amino acid residues 14-142 of SEQ ID NO:4.
2. The isolated DNA of claim 1, wherein said DNA segment comprises a nucleic acid sequence selected from the group consisting of nucleotides 40-426 of SEQ ID NO:1 and nucleotides 40-426 of SEQ ID NO:3.
3. A replication vector comprising the nucleic acid sequence of the isolated DNA of claim 1 and at least one selective marker and at least one restriction site outside of said DNA segment.
4. A plasmid comprising the nucleic acid sequence of the isolated DNA of claim 1 and an expression cassette, wherein said cassette comprises said DNA segment.
5. A host cell of a species other than Dermatophagoides farinae, comprising the vector of claim 3.
6. The host cell of claim 5, wherein said host cell is a prokaryotic cell.
7. The host cell of claim 6, wherein said prokaryotic cell is Escherichia coli.
8. A replication vector comprising the nucleic acid sequence of the isolated DNA of claim 2 and at least one selective marker and at least one restriction site outside of said DNA segment.
9. A method for producing the Derf II allergen, comprising culturing a host cell of a species other than Dermatophagoides farinae, wherein said host cell comprises the vector of claim 8, under conditions to permit expression of the Derf II allergen encoded by said DNA segment.
10. In the method for the treatment of an allergic disease by means of desensitization therapy by administering to a subject gradually increasing doses of a causative agent, the improvement wherein said causative agent is the Derf II allergen expressed upon culturing of the host cell of claim 6, under conditions to permit expression of the Derf II allergen encoded by said DNA segment.
11. In the method for the treatment of an allergic disease by means of desensitization therapy by administering to a subject gradually increasing doses of a causative agent, the improvement wherein said causative agent is the Derf II allergen comprising amino acid residues 14-142 of SEQ ID NO:2 or amino acid residues 14-142 of SEQ ID NO:4.
The present application is a continuation of application Ser. No. 08/288,888, filed Aug. 10, 1994, which is a continuation of application Ser. No. 07/658,596, filed Feb. 21, 1991, now abandoned.
The present invention relates to DNAs encoding a major allergen of house dust mites, replication vectors, plasmids and host cells which have a concern in the DNAs, and the use thereof.
Allergic diseases generally have several kinds of symptoms which are developed by sensitization of antigen causing the diseases, in which IgE antibody (reagin antibody) specific for allergen in blood serum and tissue is produced, the antibody is exposed again to the antigen and the antibody reacts with the antigen in each tissue.
There is a desensitization therapy as a method for curing drastically the allergic diseases. In the method, the causative antigen is repeatedly administered to a patient in small doses, while the dose is gradually increased according to the symptoms. It is considered that, by the therapy, blocking antibody is produced, the production of the IgE antibody is controlled and the amount of histamine derived from a mast cell is lowered to give treatment effect.
On the other hand, it appears that allergic diseases such as bronchial asthma, childhood asthma, atopic dermatitis and the like are mainly caused by allergy to mites living in house dust. Several kinds of proteins of major mite allergens have been identified (Platts-Mills et al., J. Allergy Clin. Immunol., 80, 755(1987)). The above desensitization therapy of the allergic diseases which uses the major allergens is very effective. However, it is impossible to prepare a large amount of purified allergens which are required in the therapy.
The inventors of the present invention have found that the above problems are solved by preparing mRNA from Dermatophagoides farinae and making a cDNA gene library corresponding to the mRNA, and by isolating a clone expressing Derf II, one of the major allergens, by a colony-immunoassay and isolating DNA fragments containing the objective gene from the clone.
Accordingly, objects of the present invention is to provide a DNA sequence coding the major allergen of mites in genetic engineering and to provide a method for producing a large amount of the objective allergen by expressing and preparing the major mite allergen encoded by the DNA.
The present invention provides a DNA encoding at least one part of genetic information of protein which has biochemical and immunological properties of a major allergen of Dermatophagoides farinae (Derf II).
To obtain a gene which codes the major mite allergen Derf II, mites are artificially bred with feed or a feed composition for breeding animals such as experimental animals, domestic animals, pet animals and the like, and then the mite bodies can be isolated by a method, for example, a method disclosed in Japanese Patent Unexamined Publication No. 49-69820/1974. To obtain mRNA from the mite bodies, for example, a method disclosed by Chirgwin J. M. et al. is usable (Biochemistry 18, 5294-5299(1979)). Moreover, a commercially available RNA extraction kit (manufactured by Amersham Company, trade name: RPN. 1264) is usable. In general eukaryotic mRNAs having a poly (A) tail at the 3' terminal can be easily purified by an affinity chromatography over oligo (dT) cellulose (Auffray C. et al., Eur. J. Biochem., 107, 303-304(1980)). Further, a commercially available mRNA purification kit (manufactured by Pharmashia Company, trade name: mRNA purification kit 27-9258-01) can be used conveniently.
The mRNA purified is used as a template for synthesizing the first strand of cDNA with reverse transcriptase and primer. Oligo (dT) or synthesized primer may be used. The second strand of cDNA can be synthesized by conventional methods (Huynh, T. V. et al., DNA cloning, Vol. 1, chapter 2, pp49-78(1985), edited by D. M. Glover). A commercially available kit for making cDNA (manufactured by Amersham Company, trade name: RPN. 1256Y) may be also used. The resulting double-stranded cDNA is inserted into a cloning site of a suitable vector, preferably, plasmid pUEX1 (Bressan G. and Stanley K., Nucl. Acids Res., 15, 10056(1987)). Conventional methods are usable for the insertion of the double-stranded cDNA (Maniatis T. M. et al., Molecular Cloning, Cold Spring Harbor Laboratory, (1982)). The cDNA gene library derived from the mites can express a gene product by transformation of a suitable host, preferably Escherichia coli.
Then, a colony which is producing the desired major mite allergen Derf II should be found from the transformed Escherichia coli. Although several other methods are usable, colony immunoassay is suitable for that purpose. For example, the following method can be used with pUEX1 as the plasmid.
After the E. coli transformed was grown in an L broth agar culture (1% bactotrypton, 0.5% yeast extract, 0.5% sodium chloride, 1.5% bactoagar, 50 μg/ml ampicillin, pH 7.4), the resulting colonies were replicated on a nitrocellulose filter to grow them and recombinant protein was allowed to express on the filter. The cells were lysed by exposure to chloroform vapor and the protein expressed was fixed on the filter.
The inserted DNA derived from the mites bound to the down stream of the DNA encoding β-galactosidase, and the corresponding protein was produced in the form of protein fused together with β-galactosidase. The desired Derf II protein was detected on the filter by using an anti Derf II antibody which was prepared from a rabbit, and two positive clones were obtained. The corresponding E. coli colonies were selected from the master agar plate preserved, and they were inoculated to an L broth culture (removing agar from the L broth agar culture). The colonies were cultured with shaking at 30� C. for about 18 hours. The cells were collected from the culture, and plasmids were recovered by a conventional alkali extraction method. The plasmids obtained were digested or not digested. An agarose gel electrophoresis procedure is followed for analyzing DNAs derived from mites which were inserted into the plasmids. About 500 base pairs of DNAs were inserted into the two plasmids pFL1 and pFL11, respectively.
On the other hand, E. coli cells were transformed by using the plasmids and vector pUEX1 into which the DNA derived from mites was not inserted, and they were inoculated into the above L broth culture. The cells were cultured with shaking at 30� C. for about three hours and then at 42� C. for about three hours. The resulting cells were collected and washed in a buffer solution (Tris-HCl buffer, 10 mM, pH 7) and fusion protein was extracted by a method of Marston and Carroll et al. (DNA cloning, IRL Press, Volume 3, Chapter 4(1985)). The proteins extracted were subjected to SDS-polyacrylamide gel electrophoresis. Then, the protein separated on the gel was transferred onto a cellulose filter by a Western blot technique (Towbin H. et al., Proc. Natl. Acad. Sci., U.S.A., 76, 4350(1979)). The filter on which the proteins were transferred was blocked with bovine serum albumin, and it was reacted with anti-Derf II antibody which was prepared in a rabbit. Then, the filter was washed with a buffer solution (Tris-HCl buffer: 10 mM, pH 7.4, sodium chloride: 150 mM, Tween 20:0.05%), and it was reacted with anti-rabbit IgG antibody which was labelled with peroxidase (manufactured by Amersham Company). Again, the filter was washed in the same buffer solution and the peroxidase-labelled antibodies remaining without reaction was removed. Further, the filter was reacted with hydrogen peroxide and a dye of 4-chloro-1-naphthol.
As a result, while the band of protein having a molecular weight of about 130 thousands contained in the supernatant which was obtained from the E. coli transformed with plasmids pFL1 and pFl11 show positive, no band of protein contained in the supernatant which was obtained from the E. coli transformed with the vector without the DNA of ticks show positive. Accordingly, it is apparent that DNA coding Derf II which is a major mite allergen is in the plasmids pFL1 and pFL11, and the gene expresses in E. coli as the fused protein together with β-galactosidase.
The DNA segment derived from the mites which is inserted into said plasmids pFL1 and pFL11 can be cleaved with a suitable restriction enzyme, preferably by a complete cleavage with BamHI or partial cleavage with NcoI. The sequences of the cleaved DNA segment can be determined by a dideoxy chain termination method (Sanger F. et al., J. Mol. Biol., 162, 729-773(1982)) and the like. The determined DNA sequences encoding the major mite allergen are shown by Formula I and Formula II. In these Formula I and Formula II, amino acids corresponding to the sequences are shown. ##STR1##
Further, the underlined parts in these formulas show the same sequences as those of amino acids shown in the following Table I. *** shows a termination codon.
To confirm the sequences of Formula I and Formula II coding Derf II which is originally the major mite allergen, the Derf II protein was purified, the amino acid sequences were determined, and the sequences were compared with the amino acid sequences shown in Formula I and Formula II. The allergen protein Derf II can be purified by a combination of conventional methods (Biochemical experimental lecture, Vol. 1, "Chemistry of protein", edited by Tamio YAMAKAWA and Kazutomo IMABORI, Tokyo Kagaku Dojin), advantageously by a liquid chromatogrpahic technique. The amino acid sequence of the allergen Derf II purified was determined with a peptide sequencer of Type 471A (manufactured by Applied Biosystem Company). The results are shown in Table I. From these results, it was confirmed that the DNA sequences determined by the above method coded Derf II which was the major mite allergen.
TABLE I______________________________________Analysis Amino Analysis Amino Analysis AminoNo. acid No. acid No. acid______________________________________ 1 Asp 16 Val 31 Arg 2 Gln 17 Met 32 Gly 3 Val 18 Val 33 Lys 4 Asp 19 Asp 34 Pro 5 Val 20 Gly 35 Phe 6 Lys 21 X 36 Thr 7 Asp 22 His 37 Leu 8 X 23 Gly 38 Glu 9 Ala 24 Ser 39 Ala10 Asn 25 Asp 40 Leu11 Asn 26 Pro 41 Phe12 Glu 27 X 42 Asp13 Ile 28 Ile 43 Ala14 Lys 29 Ile 44 Asn15 Lys 30 His 45 Gln______________________________________
The DNA segment and a part of the segment can be expressed in yeast with a suitable vector, for example, YEp13 (Broach J. R. et al., Gene, Vol. 8, 121-133(1979)), etc. Suitable yeast cells can be transformed with a yeast vector having an expression casette carrying the mite Derf II gene according to the present invention. For the purpose, the DNA sequences according to the present invention should be placed under controlled conditions of not a E. coli promotor but a strong eukaryotic promotor and the like, for example delta P8 (Otake et al., Agric. Biol. Chem., Vol. 52, 2753-2762(1988)).
The mite Derf II protein prepared by the genetic engineering technique according to the present invention, the fragments of the protein, and the protein fused with the other protein and having biochemical and immunological properties of the major mite allergen Derf II can be used in medical treatment or diagnosis of all sorts of allergic diseases caused by mites.
According to the present invention, a clone which expresses major allergen protein Derf II of Dermatophagoides farinae can be isolated by genetic engineering, and DNA fragments containing the desired gene can be isolated from the clone.
As a result, a mass production method of allergen protein can be provided by expressing and by obtaining the major mite allergen which is coded by DNA, and the protein can be used in medical treatment or diagnosis of all sorts of allergic diseases.
Furthermore, DNA hybridizable with and corresponding to the DNA of Formulae I and II and which codes for proteins having the biochemical and immunological properties of Derf II, can be obtained by synthesis, semisynthesis or from nature.
The following examples illustrate the present invention in details.
Construction of a cDNA gene library
Mites were artificially bred for about 30 days in feed for breeding experimental animals under conditions of a temperature of 25� C. and a humidity of 75%. The mites were separated from the feed with a saturated sodium chloride solution, and mites were filtered with suction (Japanese Patent Unexamined Publication No. 49-69820/1974). The mite bodies (wet weight, about 5 g) obtained were rapidly frozen in liquid nitrogen, and then total RNA was extracted and purified with an RNA extraction kit (manufactured by Amersham Company, RPN. 1264). After purifying mRNA from the total RNA with an oligo (dT) column (manufactured by Pharmacia Company, trade name: mRNA purification kit 27-9258-01), about 3 μg of mRNA was obtained. The cDNA was synthesized from the total amount of the mRNA obtained with a cDNA synthesis system plus (manufactured by Amersham Company, trade name: RPN. 1256Y). The RNA extraction, the mRNA purification and the cDNA synthesis followed manuals annexed to the kits.
pUEX1 reported by Bressan et al. (Nucl. Acids Res., Vol. 15, 10056(1987)) was used as an expression vector. By the insertion of a cDNA fragment into the down stream of a β-galactosidase gene, the expression vector could express the desired gene as a fused protein with β-gal. The expression amount was determined by a λ PR promoter in the upper stream and it was greatly increased by shifting the cultivation temperature to 42� C. The cloning steps follow a method of Haymerle et al. (Nucl. Acids Res., Vol. 14, 8615-8624(1986)) and cDNA cloning system pUEX1 (manufactured by Amersham Company, trade name: RPN. 1282) was used. E. coli MC1061 (gene type: araD139, Δ ara, leu!7697, ΔlacX74, galU-, galK-, hsr-, hsm+, strA, mcrA-, mcrB-) was used for the transformation. The preparation of competent cells follows a method of Hanahan (DNA cloning, A practical approach, edited by Glover D., IRL Press, Vol. 1, 109(1985)).
Detection of pFL1 and pFL11
The clones reacting with anti Derf II antibody derived from a rabbit were detected by a colony immunoassay method from the transformants. The transformants were spread on an L broth agar culture plate (1% tryptone manufactured by Difco Company, 0.5% yeast extract manufactured by Difco Company, 0.5% sodium chloride and 1.5% agar) containing 50 μg/ml of ampicillin and these strains were cultured at 30� C. to obtain colonies having a diameter of 1 mm. Nitrocellulose filters (High bond C, manufactured by Amasham Company, trade name: RPN. 1782C) were carefully placed on the colonies to make a replica of the colonies. The remaining colonies on the plate were cultured again at 30� C.
On the other hand, the replica of the colonies blotted onto the nitrocellulose filters was placed on the surface of a new L broth agar plate as the colonies were upside and the colonies were cultured at 42� C. for two hours. Then the nitrocellulose filters were hung in a tank filled with chloroform and exposed to chloroform vapor for about 25 minutes. Each filter having the colonies at the underside was placed on a petri dish. The dish was immersed into 10 ml of TBS buffer (10 mM Tris hydrochloride buffer (pH 7.4) and 150 mM sodium chloride) containing 4 mg of lysozyme and 100 μg of deoxyribonuclease and it was shaken slowly to lyse the colonies for one hour at room temperature. Washing operation was repeated four times by shaking the dish for five minutes with 20 ml of TBS buffer. Then, the filters were immersed into TBS buffer containing 2% bovine serum albumin for one hour as a blocking operation in order to prevent the nonspecific adsorption.
The filters were immersed and shaken for one hour in a solution containing the anti Derf II antibody derived from the rabbit as a primary antibody which was diluted 1000-3000 times with 10 ml of TBS buffer containing 0.2% bovine serum albumin. Then the primary antibody which was not adsorbed was washed away four times with 20 ml of TBST buffer (0.05%, TBS buffer containing Tween 20).
The same procedure as the primary antibody was repeated with filters having a peroxidase-labelled anti rabbit IgG antibody (manufactured by Biorad Company) as a secondary antibody. After adsorbing and washing the antibody, the filters were immersed in TBS buffer in which 0.4 mg/ml of diaminobenzidine as a color-producing reagent was dissolved and hydrogen peroxide water was added in end concentration of 0.01% to obtain colored filters.
The above operation was repeated with about 1600 transformed strains. Two strains of colonies giving a strong signal were found. The corresponding colonies were picked from the master plate and cultured at 30� C. to provide plasmids to be analyzed. The techniques of plasmid extraction, breakage of restriction enzyme, gel electrophoresis and the like were based on a method of Maniatis et al. (Molecular cloning, Cold Spring Harbor Press (1982)). Two kinds of E. coli thus obtained had plasmids into which segments (about 500 base pairs) derived from cDNA were inserted. These were designated as pFL1 and PFL11. These strains were deposited at the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken 305, JAPAN, on Dec. 26, 1995, as deposit numbers FERM BP-5356 and FERM BP-5357, respectively.
Determination of DNA sequence for Derf II gene (pFL1 and pFL11)
The techniques of restriction enzyme treatment of cDNA fragments of pFL1 and pFL11 and ligation thereof and transformation of E. coli host JM105 (gene type: thi, rpsL, endA, sbcB15, hsdR4, Δ(lac-proAB), F', proAB, lacI9 ΔDM15, traD36!) were carried out by conventional methods (Maniatis et al., Molecular Cloning, Cold Spring Harbor Press, p. 104, 146 and 396 (1982); DNA Cloning, IRL Press, Vol. 1, Chapter 6 (1985)). Vectors (pUC118 and pUC119, Messing et al., Methods in Enzymology, Vol. 101, 20-78 (1978)) suitable for Sanger's sequence determination (Science, Vol. 214, 1205-1210 (1985) was used for the cloning.
Thus isolated Bam HI-cDNA fragments as it was or digested with several restriction enzymes such as Nco I were inserted into vectors which were treated by the same method as described above. The E. coli competent cells were transformed. Each single-stranded DNA of the recombinant plasmids was isolated and purified, and the DNA sequence was determined by the Sanger method. The sequence was analysed by a computer program (DNASIS) manufactured by Hitachi software engineering company.
Determination of N-terminal amino acid sequence for Derf II protein
Amino acid sequence of Derf II protein purified by reverse phase HPLC was determined. The sequence determination was carried out with a gas phase sequencer manufactured by Applied Biosystems company (Model 471A). Initial amount of 10 μg made it possible to determine the sequence from N-terminal to the 45 amino acid residue. The amino acid sequence obtained was coincident with expected sequence, namely the amino acid sequence estimated from the DNA sequence of cDNA as shown in the above Table I.
Extraction of fusion proteins and identification of antigenicity
E. coli having pFL1, which were selected from two kinds of E. coli expressing Derf II as fusion proteins and having pFL1 and pFL11 respectively, were cultured in an L broth culture, and the fusion proteins were extracted to confirm their antigenecity. The cells obtained were inoculated in 5 ml of an L broth culture containing 50 μg/ml of ampicillin (L broth-Ap culture) and cultured with shaking at 30� C. overnight. The cultures were inoculated in a 1% L-broth-Ap culture and cultured with shaking at 30� C. When the value of OD660 nm became 0.6, the same volume of an L-broth-Ap culture which was previously warmed to 54� C. was added. Further they were cultured with shaking for two hours at 42� C. The cells cultured were recovered and extracted by a method of Marston, Carroll et al. (DNA Cloning, IRL Press. Vol. 3, Chapters 4 and 5 (1985)) to obtain fusion proteins. By the observation of a phase-contrast microscope (x 1000), it was confirmed that the fusion proteins were visualised in the cells in the form of inclusion bodies.
The fusion proteins obtained were separated with a SDS-polyacrylamide gel electrophoresis system (manufactured by Daiichi Kagaku company, vertical type, gel concentrations of 4-20%). One part of the cells separated was dyed with Coomassie Brilliant Blue and the other part was transferred on a membrane (manufactured by Imobiron Milipore company) by a Western blot technique (Towbin H. et al., Proc. Natl. Acad. Sci., U.S.A., Vol. 69, 1409-1412(1972)), and then the expression of Derf II antigen proteins was confirmed with anti Derf II antibodies. Proteins showing positive reaction had a molecular weight of about 130 thousands and they were fused proteins with β-galactosidase.
__________________________________________________________________________SEQUENCE LISTING(1) GENERAL INFORMATION:(iii) NUMBER OF SEQUENCES: 4(2) INFORMATION FOR SEQ ID NO:1:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 517 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: cDNA(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 1..426(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:GGTACCATGGTTTCATTGTTGGTAGCAGCCGTTGTTGCCGATCAAGTC48GlyThrMetValSerLeuLeuValAlaAlaValValAlaAspGlnVal151015GATGTTAAAGATTGTGCCAACAATGAAATCAAAAAAGTAATGGTCGAT96AspValLysAspCysAlaAsnAsnGluIleLysLysValMetValAsp202530GGTTGCCATGGTTCTGATCCATGCATCATCCATCGTGGTAAACCATTC144GlyCysHisGlySerAspProCysIleIleHisArgGlyLysProPhe354045ACTTTGGAAGCCTTATTCGATGCCAACCAAAACACTAAAACCGCTAAA192ThrLeuGluAlaLeuPheAspAlaAsnGlnAsnThrLysThrAlaLys505560ATTGAAATCAAAGCCAGCCTCGATGGTCTTGAAATTGATGTTCCCGGT240IleGluIleLysAlaSerLeuAspGlyLeuGluIleAspValProGly65707580ATCGATACCAATGCTTGCCATTTTGTCAAATGTCCATTGGTTAAAGGT288IleAspThrAsnAlaCysHisPheValLysCysProLeuValLysGly859095CAACAATATGATATCAAATATACATGGAATGTGCCGAAAATTGCACCA336GlnGlnTyrAspIleLysTyrThrTrpAsnValProLysIleAlaPro100105110AAATCTGAAAACGTTGTCGTTACAGTCAAACTTATCGGTGATAATGGT384LysSerGluAsnValValValThrValLysLeuIleGlyAspAsnGly115120125GTTTTGGCTTGCGCTATTGCTACCCATGGTAAAATCCGTGAT426ValLeuAlaCysAlaIleAlaThrHisGlyLysIleArgAsp130135140TAAAAAAAAATAAATAAGAAAATTTTCACCAACATCGAACAAAATTCAATAACCAAAATT486TGAATCCAAAAAAAAAAAAAAAAAACCATGG517(2) INFORMATION FOR SEQ ID NO:2:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 142 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:GlyThrMetValSerLeuLeuValAlaAlaValValAlaAspGlnVal151015AspValLysAspCysAlaAsnAsnGluIleLysLysValMetValAsp202530GlyCysHisGlySerAspProCysIleIleHisArgGlyLysProPhe354045ThrLeuGluAlaLeuPheAspAlaAsnGlnAsnThrLysThrAlaLys505560IleGluIleLysAlaSerLeuAspGlyLeuGluIleAspValProGly65707580IleAspThrAsnAlaCysHisPheValLysCysProLeuValLysGly859095GlnGlnTyrAspIleLysTyrThrTrpAsnValProLysIleAlaPro100105110LysSerGluAsnValValValThrValLysLeuIleGlyAspAsnGly115120125ValLeuAlaCysAlaIleAlaThrHisGlyLysIleArgAsp130135140(2) INFORMATION FOR SEQ ID NO:3:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 513 base pairs(B) TYPE: nucleic acid(C) STRANDEDNESS: single(D) TOPOLOGY: linear(ii) MOLECULE TYPE: cDNA(ix) FEATURE:(A) NAME/KEY: CDS(B) LOCATION: 1..426(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:GGTACCATGGTTTCATTGTTGGTAGCAGCCGTTGTTGCCGATCAAGTC48GlyThrMetValSerLeuLeuValAlaAlaValValAlaAspGlnVal145150155GATGTTAAAGATTGTGCCAACAATGAAATCAAAAAAGTAATGGTCGAT96AspValLysAspCysAlaAsnAsnGluIleLysLysValMetValAsp160165170GGTTGCCATGGTTCTGATCCATGCATCATCCATCGTGGTAAACCATTC144GlyCysHisGlySerAspProCysIleIleHisArgGlyLysProPhe175180185190ACTTTGGAAGCCTTATTCGATGCCAACCAAAACACTAAAACCGCTAAA192ThrLeuGluAlaLeuPheAspAlaAsnGlnAsnThrLysThrAlaLys195200205ATTGAAATCAAAGCCAGCCTCGATGGTCTTGAAATTGATGTTCCCGGT240IleGluIleLysAlaSerLeuAspGlyLeuGluIleAspValProGly210215220ATCGATACCAATGCTTGCCATTTTATGAAATGTCCATTGGTTAAAGGT288IleAspThrAsnAlaCysHisPheMetLysCysProLeuValLysGly225230235CAACAATATGATGCCAAATATACATGGAATGTGCCGAAAATTGCACCA336GlnGlnTyrAspAlaLysTyrThrTrpAsnValProLysIleAlaPro240245250AAATCTGAAAACGTTGTCGTTACAGTCAAACTTGTTGGTGATAATGGT384LysSerGluAsnValValValThrValLysLeuValGlyAspAsnGly255260265270GTTTTGGCTTGCGCTATTGCTACCCACGCTAAAATCCGTGAT426ValLeuAlaCysAlaIleAlaThrHisAlaLysIleArgAsp275280TAAAAAAAAAAAATAAATATGAAAATTTTCACCAACATCGAACAAAATTCAATAACCAAA486ATTTGAATCAAAAAAAAAAAACCATGC513(2) INFORMATION FOR SEQ ID NO:4:(i) SEQUENCE CHARACTERISTICS:(A) LENGTH: 142 amino acids(B) TYPE: amino acid(D) TOPOLOGY: linear(ii) MOLECULE TYPE: protein(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:GlyThrMetValSerLeuLeuValAlaAlaValValAlaAspGlnVal151015AspValLysAspCysAlaAsnAsnGluIleLysLysValMetValAsp202530GlyCysHisGlySerAspProCysIleIleHisArgGlyLysProPhe354045ThrLeuGluAlaLeuPheAspAlaAsnGlnAsnThrLysThrAlaLys505560IleGluIleLysAlaSerLeuAspGlyLeuGluIleAspValProGly65707580IleAspThrAsnAlaCysHisPheMetLysCysProLeuValLysGly859095GlnGlnTyrAspAlaLysTyrThrTrpAsnValProLysIleAlaPro100105110LysSerGluAsnValValValThrValLysLeuValGlyAspAsnGly115120125ValLeuAlaCysAlaIleAlaThrHisAlaLysIleArgAsp130135140__________________________________________________________________________
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Space where guests can cook their own meals
Continuous access in the listing
Breakfast is provided.
Space where guests can cook their own meals
Towels, bed sheets, soap, and toilet paper
Central heating or a heater in the listing
In the building, free or for a fee
In the building, free or for a fee
Continuous access in the listing
Breakfast is provided.
The listing can accommodate a gathering of 25 or more attendees
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The Space
In our recently renewed house we rent this double or twin private room for one or two persons with external bathroom shared with just one other room. We have 4 rooms in total (see our other ads if this appears full). Breakfast with fresh local fruits included for stays shorter than a week, wi-fi, share of common areas, access to the kitchen, and our best advices about our city!
'Casa Nuestra' is located in Barranco, the bohemian and traditional district of Lima that offers lots to see and nice art galleries, coffees, restaurants and bars. We are 300 meters from the ocean view, few minutes from Miraflores district and 20 min by direct bus to the historical centre. We are an italian-peruvian couple that loves to travel and share and would like to offer you the possibility of enhance your stay in Lima by giving a familiar and personal attention to our guests. We have a peruvian hailess dog very nice that lives with us in the roof. We are eventually available for going out and show you around Lima and surroundings. We want casa Nuestra to be your home in Lima!
1. Kitchen and other common areas available to use
2. Breakfast is included only for stays of less that 7 days
3. Towels and linen are changed once a week for stays longer than a week. Consider earlier if requested
4. All bathrooms are equipped with trash cans. Unfortunately the pipes in Lima are not made to withstand the flushing of toilet paper. To avoid plumbing problems, use the trash cans.
5. Clean after yourself in the common areas (kitchen, living room, rooftop).
The Casa Nuestra b'n'b is even more lovely in real life! its definitely in the best neighbourhood, as it has so much character- ive really enjoyed walking everywhere in barranco and exploring the last few days! perfectly situated near all amenities (shops, bars, restaurants, laundry service..) and walking distance to miraflores (approx 25mins to Ovalo de Miraflores and 15mins to Larcomar). Its a really beautifully furnished home, lovely communal spaces to hang out (great rooftop terrace; living area, large kitchen, even a tiny balcony on the main floor overlooking the street-a great place to sit & have a coffee in the morning). The accommodation is very clean, and very quiet at night (no traffic noise or disturbances-so surprising for a big city!). Francesco has been so lovely and friendly, orienting me to the neighbourhood, giving recommendations on best places to visit, and even driving me to the local organic farmers market in the weekend. Im actually staying on here for a few months as im studying in lima, and am so happy that i chose this place to stay. Most other people in my course are staying in Miraflores, but i believe its definitely worth the slightly longer walk to be living here! I definitely recommend it to any short or long-term travellers.
I highly recommend staying with Carmen and Francesco!! Their place is beautiful, welcoming, and bright. The room we stayed in was very clean, comfortable and cozy. We arrived at the Lima Airport around 11 p.m. and they arranged for a driver to pick us up and take us to their house. When we woke up in the morning breakfast was prepared and set up in the kitchen. Fresh fruit, Quinio pops and yogurt, fresh coffee and a delicious frothy pineapple juice. It was a great way to start the day. The location was outstanding. Walking distance to the beach, great places to eat, shop, museums, and transportation services were readily available. Francesco and Carmen made us feel at home and were very friendly and able to help us out with any questions we had. They had lots of information on places to see and activities to do while in Lima and around Peru.
They would be my first choice for staying in Lima!
Hospedarnos en casa de Carmen y Francesco fue ideal, la casa y espacios comunes son muy bonitos y bien cuidados, esta convenientemente ubicado en el barrio de Barranco , cerca de todo lo que era de mi interés durante la visita a Lima. Definitivamente recomendaría Casa Nuestra.
My mother and I had a fabulous stay at Casa Nuestra! The location was perfectly situated to Miraflores, but most importantly to everything awesome and hip in Barranco! Franco, Carmen, baby Leonardo & the house staff provided tremendous warmth so we didn't feel too far from home during our first trip to Peru :) would HIGHLY recommend this lodging and hope that we can return soon! It was also great to connect with other traveling folks/families from as far as Zimbabwe to Oakland, CA over yummy daily breakfast at Casa Nuestra - so cool!
Francesco and Carmen were lovely and very helpful. They provided a map with directions to great food, coffee and bars. Couldn't have found a better location - Barranco is beautiful. We are vegan and thought it would be difficult for us but Francesco and Carmen were very accommodating and pointed out good vege places to eat. The room was big and house was beautiful. We loved their dog Paco.
Location was good - Barranco was a cute, quiet area with some nice restaurants. Cute rooftop terrace. The hosts provided us with some information on where to go around the area. The accommodations were simple but clean. Our biggest issue was the amount of noise you could hear in the house. We were woken up around 7 every morning with multiple sounds so could never sleep in. Breakfast was basic - toast, jam and fruit. Also we had a shared bathroom which was ok, but for some reason after 2 days the bathmat never got replaced so the floor was constantly wet. Otherwise we had a pleasant stay here in Barranco.
It is always nice to come back to casa nuestra. It is a great place and Carmen and Francesco are lovely people.
This host has 273 reviews for other properties.View Other Reviews
We are an italian-peruvian couple and we live in Lima. We like to travel, enjoy good music and art. We are use to share our house with our guests that come from all around the world.
We are living in Lima since 2011 and we adore to go looking for new, hidden places, not well know but that have an artistic, culinary or just tradition interest.
We have also a dog called Paq'u', a Peruvian year and half old dog, he is our security manager and crazy, lovely puppy of the house.
We adore to spend some weekend with him and guests that want to come with us in the surroundings of Lima to escape from the city and enter in different and amazing reality that this country offer.


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WASHINGTON — Democratic officials say Sen. Barbara Mikulski, the longest serving woman in the history of Congress, is ready to announce her retirement.
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Former Pennsylvania Senator Rick Santorum, who is considering another presidential run, called for putting 10,000 U.S. troops on the ground to fight the growth of the extremist group that calls itself the Islamic State. Continue reading
In 1966, Edward Brooke became the first African-American elected to the Senate since Reconstruction. Known for his bipartisan efforts, the Massachusetts Republican served two terms and helped pave the way for future politicians of color. Gwen Ifill remembers the achievements of the late senator, who died over the weekend at the age of 95. Continue reading
Edward Brooke, a former Massachusetts senator who broke racial barriers, hoped not to be remembered as a "first," but for his real political accomplishments. Presidential historian Richard Norton Smith and Adrian Walker of The Boston Globe join Gwen Ifill to discuss Brooke's legacy and whether his moderate politics could have succeeded today. Continue reading
- Closing the Gap: Sen. Marco Rubio on how more education, fewer broken families can change income inequality
In the debate over how to cure income inequality, talk about poverty and opportunity are increasingly converging. In a series of conversations about the growing divide between rich and poor, Gwen Ifill talks to Sen. Marco Rubio, R-Fla., about the importance of encouraging strong families and improving educational opportunities as keys to restoring the American dream. Continue reading
In our news wrap Friday, James Everett Dutschke of Mississippi pleaded guilty to sending letters laced with the toxic substance ricin to the president, a U.S. senator and a judge. Also, California Gov. Jerry Brown declared a drought emergency in light of the state's worst dry spell in 100 years of record-keeping. Continue reading
Democratic Sen. Mark Pyror's seat is up for re-election in Arkansas and Republicans are hungrily looking to win his seat in order to take control of the U.S. Senate in Fall 2014. Part of Pryor's strategy to remain an incumbent, is to distance himself from President Barack Obama and the National Democratic Party. Continue reading
Protestors interrupted a House hearing on a Republican immigration bill focused entirely on law enforcement. By contrast, the Senate bill combines enforcement and a path to citizenship. Ray Suarez talks to Sen. Rand Paul, R-Ky., about his stance on legalization of undocumented immigrants being contingent on border security. Continue reading
The U.S. Senate lost one of its longest serving members and its last World War II veteran. New Jersey's Sen. Frank Launtenberg, who served five terms, died of pneumonia at the age of 89. Herb Jackson of The Record joins Gwen Ifill to look back at the Democratic senator's legacy and explore who may replace him. Continue reading
Two rockets exploded in an area of Beirut controlled by Hezbollah, raising new concerns about the spread of the Syrian conflict into Lebanon and the region. Jeffrey Brown talks to Margaret Warner from Beirut about Hezbollah's support for the Assad regime and the country's history of strife and its remaining sectarian tensions. Continue reading


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After game thoughts Habs vs Bruins
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Join Date: Feb 2003
Location: Richmond, B.C
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Overall, we played very well tonight. Din't give the bruins much and were a lot better in our own zone than usual. Sheldon Souray really impressed me tonight, He has a lot more poise than some poeple give him credit for.
Donald Audette is getting worse and worse in my opinion. If he's not producing hes completely useless because he can't play defensively and gets knocked off the puck way too easily. I would really like to see him taken out of the lineup when Saku returns although I know that won't happen. I also would like to see Higgins given more of a chance(perhaps replacing hossa in the lineup considering he's been less than impressive...)
Souray has impressed me in every game I have seen him. Unfortunately, I could not see the habs-bruins game today as I live in B.C ......but from what I have heard, the habs played a really sound game on the road and peppered Potvin with 38 shouts. I was happy to see Juneau get the goal in o.t and Theodore got his 3rd shutout in 11 games....which is great
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text: New stories Upcoming Incoming 10° Axiom Verge Review - Gaming Nexus 11m ago - From the review: "Old school merely in looks, Axiom Verge is the definition of a great action and... | PS4 Comments (1) GamingNexus Source 8.5/10 gamingnexus.com 0° Life is Strange Episode 2 Review | Gamer's Palace 11m ago - Gamer's Palace: "Out of Time has many pros, but also many weaknesses. The rather strong story suf... | PS4 Comments (0) thomary Source 70/100 translate.goo... Ad Filmwatch Contest Details Now - Check out here how you can win awesome prizes with us over at Filmwatch. | Promoted post Read More SiteBot 0° IndieGames: Axiom Verge Review - A New Standard 11m ago - With incredible storytelling, clever tools for exploration, powerful visuals and sound, and its r... | PS4 Comments (0) JoelCouture Source - indiegames.com 20° From The Hip Men: Let's play Slender: The Arrival and win the game on Xbox One 11m ago - @FromTheHipMen have booted up their Xbox One, this time to bring us their first play of Blue Isle... | Xbox One Comments (0) FromTheHipMen Source 10° Star Wars Rebels Pinball Is Here 12m ago - Skewed and Reviewed have posted information about the new Star Wars Rebels pinball game coming fr... | PC Comments (0) Garethvk Source


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Microwave diagnosis of antennas is considered as a viable tool for the determination of reflector surface distortions and location of defective radiating elements of array antennas. A hybrid technique based on the combination of the spherical near-field measurements and holographic metrology reconstruction is presented. The measured spherical near-field data are first used to construct the far-field amplitude and phase patterns of the antenna on specified regularized


text: Web exclusive! Made of heavyweight carbon steel to encourage even browning, this pan features a nonstick coating, so you don't have to grease it before making a batch of your favorite cookies. Original: $28.00 For Sale price, add to bag Pan features silicone-enhanced handles for a slip-free grip Nonstick bakeware releases food easily and cleans up fast Shallow lip around edge makes pan more versatile Heavyweight carbon steel Imported More


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COBIAN Women's Aqua Bounce Flip-Flops
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Free your feet from the confines of cold-weather shoes, and slip into these adorable COBIAN® women's Aqua Bounce flip-flops. They boast a squishy-soft foam footbed for unrivaled comfort; pop-color straps promise beachy-worthy style.
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Remove meat to platter; keep warm.
Skim excess fat from meat broth.
Pour off broth; measure amount needed; return to kettle.
Shake water and flour together in covered jar until all lumps are gone.
For a smooth mixture, put water in first, flour on top.
Stir flour and water slowly into hot broth.
Bring to boil, stirring constantly.
Boil 1 min.


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Bosnia and Herzegovina
Alberta, Halifax, Hamilton, Montreal, Ottawa, Toronto, Vancouver, Victoria,
Czech Republic
Bengaluru, Chandigarh, Chennai, Delhi, Mumbai, Pathankot,
New Zealand
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United Kingdom
Belfast, Birmingham, Bristol, Edinburgh, Glasgow, Hull University, London, Nottingham, Sheffield, University of Leicester, University of London,
United States
Appalachian Ohio, Athens GA, Atlanta, Baltimore, Chicago, Cleveland, Columbia MO, Columbus, Denver, Des Moines, Durham & Chapel Hill, East Lansing, Fredericksburgh VA, Houston, Las Vegas, Los Angeles, Lubbock TX, Manhattan KS, Muncie IN, New Orleans, New York City, NYU, Pittsburgh, Plattsburgh, Richmond VA, San Fernando Valley, San Francisco, SUNY Oneonta, Tucson, Twin Cities
This morning I got on an elevator with a female colleague on our way to a meeting. A man joined us and as the door closed said "You have beautiful eyes" in a very dirty voice. I said a muffled "thank you", avoiding eye contact. He then had time to say "you're pretty and "I want to talk to you" all in a very disturbing voice. He even said hello to my collegaue, and asked her to tell "her friend" that she had beautiful eyes.
I was only going to the 2nd floor but it felt like the 20th. After my colleague and I got off, I was utterly perturbed. I felt like he had violated me. Looking back, this would have been a perfect time to "hollaback"- I had my cellphone camera easily accessible in my pocket, and a good 30 seconds of captivity with him to get a good shot. But I was so mortified I didnt think of it- I wasn't thinking at all. All I could do was stare ahead at the door, waiting for my floor, hoping he would stop. On one hand I am glad I wasn't alone with him- god only knows what could have happened. On the other hand, it was embarrassing to be treated like that in front of a colleague, without defending myself or speaking out against what was happening to me. I fret that I may have lost her respect- or more, that he took my dignity from me in front of her.
I hope next time I will find the strength and the power to take action. To speak up and to hollaback.
Submitted by Kate
This horrific story reminds us that without a trained and sensitive staff at the MTA, those beautiful subway ads that we worked so hard to get up are useless. The Subway Safety Coalition, of which HollabackNYC is a part of, will be drafting letters to the heads of the MTA, the transporation committees in the New York State Senate and Assembly, as well as to the Public Safety Committee in the City Council.
To do your part, send a letter to the editor of the Daily News at [email protected]. Remember to include your full name, address and phone number.
The driver called "Hey Sexy" and then pulled over in his SUV. He smiled for me when I asked if I could take his picture.
Submitted by Elizabeth


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Tent caterpillars
Is dormant oil effective on Tent Caterpillars and is it safe to use at this time of year in full sun? It's usually in the upper 80's - 90's.
No and no.
Your best option is to try to disturb the nests with a sharp stream of water or an long handled tool of some sort. You could even clip off the ends of the branches where the nests are (they will be Fall Webworms at this time of year).
Fall Webworm caterpillars are a favorite food for many, many different kinds of predators. Breaking open that webby nest makes them easy prey.
This appears to be a bad year for the webworms in much of the country. This, too, shall pass.
If you have nests of leaf eating thingys in your trees and shrubs it will be a cousin ot the Eastern Tentworm, the Fall Webworm. While having the webs in trees may be somewhat of an eyesore there is no good reason to prune the webs out of the trees, something that can cause more damage to the tree then the webworms will. Spraying a dormant oil now is not a good idea since it will cause more damage to the tree then will the webworms.
If the webworms are too old for Bacillus thuringiensis - Kurstaki to control simply poking large holes in the webs will allow access for the many predators, both birds and other insects.
Is this web worm the thing that is in my pecan trees? We bought a tiny strip of land from the neighbor behind us to extend our city lot gardening options. The area has three Mississippi Giant pecan trees, one already bearing. And it is the one with a web on it.
Will spraying with a strong blast of water help get rid of that? I was wondering how to get up there to deal with it.
This is a picture of my "back forty" as we call it...still fairly wild with two large sasanquas and plantings of old fashioned azaleas. The fence gate is there to allow visiting with the neighbor lady. I have purchased 35 star jasmines to plant along the chainlink fence as a hedge, like we saw the plants used in Tuscany.
Probably the fall webworm; they seem to love oaks, hickories, pecans, and the like. And there are LOTS of them this years! Again, your two best options are to snip the tips of the branches where the nests are if you can reach them, or to disturb the nests so that predators can get to them.
Oh, third option is to simply not worry about them!
Thanks... but I really doubt the squirrels are going to eat them. And our coons and opossums are on diets that we had experts in the field specially formulate for us with excellent results. I also doubt they'd eat them when they have access to more palatable and substantial meat.
Keep in mind that pruning off the branches where these webs are is more destructive then the Fall Webworm is. While they may eat the leaves off those branches the leaves will grow back next spring. The Fall Webworm will not kill the branches as pruning them off will.
What happens when a pruning cut is made at the tip of a branch? (This is for Kimmsr)
Dormant buds behind the cut are triggered to activity by the redirection of hormones that exist in those tips. In a matter of a few days, new foliage may grow to replace any that were lost. That is why pruning is a growth generating activity.
We prune to shape a plant, to remove dead, diseased, dieing, or infested parts. We also prune to redirect growth and to stimulate growth.
Excessive pruning can cause a flush of growth that hasn't had time to harden off in preparation of winter. However, snipping of branch tips here and there over the plant body is not harmful. Rather, it is much better for the tree, in terms of physiological response to damage, to have a bit of pruning done as opposed to the feeding of hundreds of caterpillars.


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text: Steve Everts/ThinkStock Free Farm Fun! It's Ag Day at the Washington Pavilion By Karla Brown 2 weeks ago Have you even wondered how cheese is made? Would you like to see how to spin fiber and card wool? Check out Ag Day at the Washington Pavilion this Saturday. Read More|Comment Category: News Tags: Washington Pavilion Send to a friend! Share on Facebook Share on Twitter Pin it! Reddit This! Share on Tumblr!


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38 year old woman | Seattle, WA, USA | Seeking men 30-38 within 50 miles
Looking for a true connection
Im looking for someone who believes in family first.Someone who has patience, loyal,& loves to laugh (Drama free).Communication is important in any relationship, being that noone is perfect, & there will be disagreements, I believe if 2 people can talk and work it out then you will both have longevity.I love music, drawing, cooking, & would love to share my talents with someone who may share the same interests.I have a daughter and she is and has been number 1.I would love to meet someone who is understanding of that & then maybe one day we could all be a family.Right now im just interested in dating & taking my time in getting to know someone, building up that trust between 2 people in order to move forward.And if I dont meet someone and start a relationship, and just meet a friend, im good with that too.Ive been single for about 4yrs now, so im ready to start dating.Looking forward to meeting a real genuine, honest person.If your looking for a 1 night stand, or to play with someones emotions, keep looking, because I wont be that woman,Peace
Camping , Cooking , Dining out , Hobbies and crafts , Movies/Videos , Museums and art , Music and concerts , Playing cards , Playing sports , Religion/Spiritual , Travel/Sightseeing , Volunteering , Watching sports
Basketball, Dancing, Martial arts, Other types of exercise, Walking / Hiking, Yoga
I exercise 1-2 times per week.
I like Birds, Dogs, Fish, Horses
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|5'6" (167cms)
|5'8" (172cms) to 6'4" (193cms)
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|Dark brown
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