text:
Human Choline Kinase beta / CKB type Protein
Human CKB, His Tag (CKB-H5143) is expressed from E.coli cells. It contains AA Pro 2 - Lys 381 (Accession # AAH19259).
Predicted N-terminus: Met
Molecular Characterization
This protein carries a polyhistidine tag at the N-terminus.
The protein has a calculated MW of 43.5 kDa. The protein migrates as 45 kDa under reducing (R) condition (SDS-PAGE).
Less than 1.0 EU per μg by the LAL method.
>95% as determined by SDS-PAGE.
Lyophilized from 0.22 μm filtered solution in PBS, pH7.4. Normally trehalose is added as protectant before lyophilization.
Contact us for customized product form or formulation.
Please see Certificate of Analysis for specific instructions.
For best performance, we strongly recommend you to follow the reconstitution protocol provided in the CoA.
For long term storage, the product should be stored at lyophilized state at -20°C or lower.
Please avoid repeated freeze-thaw cycles.
No activity loss is observed after storage at:
- 4-8°C for 12 months in lyophilized state;
- -70°C for 3 months under sterile conditions after reconstitution.
Human CKB, His Tag on SDS-PAGE under reducing (R) condition. The gel was stained overnight with Coomassie Blue. The purity of the protein is greater than 95%.
Creatine kinase B-type (CKB) is also known as Creatine kinase B chain, B-CK and CKBB. CKB consists of a homodimer of two identical brain-type CK-B subunits. CKB reversibly catalyzes the transfer of phosphate between ATP and various phosphogens (e.g. creatine phosphate). Creatine kinase isoenzymes play a central role in energy transduction in tissues with large, fluctuating energy demands, such as skeletal muscle, heart, brain and spermatozoa. Furthermore, ectopic expression of the CKB in red cells and platelets is a rare, benign anomaly detected during a newborn screening program for Duchenne muscular dystrophy.
Please contact us via email@example.com if you have any question on this product.


text:
packing materials in Kensington, MD
Best Choice Moving And StorageServing Gaithersburg, MD
Four Star Plastics6733 Mid Cities Ave, Beltsville, MD map
Pack N Roll Movers12154 Darnestown Rd #359, Gaithersburg, MD map
By far the worst business I have ever dealt with. I had 2 moves scheduled 1 at 10am and the other at 2pm ... more
Move-Rite Inc3704 Lottsford Vista Rd, Bowie, MD map
Penske Truck RentalCapitol Hi, Washington, DC map
Move For LessServing Rockville, MD
On The Road Movers267 Kentlands Blvd, Gaithersburg, MD map
Latino's Moving IncServing Falls Church, VA
Top Trending Articles
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subset: commoncrawl


text:
If an item you ordered from TextbookRush does not meet your expectations due to an error on our part, simply fill out a return request and then return it by mail within 30 days of ordering it for a full refund of item cost.
Learn more about our returns policy
Limited time offer:
Get the first one free! (?)
All the information you need in one place! Each Study Brief is a summary of one specific subject; facts, figures, and explanations to help you learn faster.
List price: $14.95Copyright year: 2007Publisher: Franciscan MediaBinding: PaperbackPages: 232Size: 5.50" wide x 8.25" long x 0.75" tall Weight: 0.748Language: English
JOANNE TURPIN wrote Twelve Apostolic Women and Catholic Traditions: Treasures New and Old (St. Anthony Messenger Press). She received a journalism degree from the University of Washington and is a resident of Seattle.
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text:
So i might have converted an Acura driver to a Subaru driver!
View Single Post
Scooby Guru
Join Date:
Mar 2002
Denver CO
1999 White Mock 22B
2014 Plasma Blue GR STi
So i might have converted an Acura driver to a Subaru driver!
So last night i was down at Sheetz getting the cheapest gas you can find ($1.57) down on rt 50 and this Acura MDX? (i think thats the SUV) pulls up next too me when im pumping gas. This older African American gentleman gets out and starts looking at my car. At first i think he was just looking at the CF hood and whatnot and i didn't notice. All of a sudden, he comes up to me and is like "whats the hp on that." So i tell him stock about 170 bc its not the non-turbo'd Scoob and the WRX is around 230...but the STi is comming out and its going to be stock 300 hp. So he looked back at my car and starts talking about racing and whatnot and asking if i did it. So i tell him that i dont street race, its stupid but im going to get into auto-x and track this year, just hadnt done it before bc i just got my car in the winter. So he starts to talking to me about the STi and whatnot, and he was all about it. He says he has an Acura CL, but he had been looking at Subaru's and saw the STi and was impressed but never talked to anyone about how Subarus drove in general. So of course i gave praise to Subaru's and was raving about how great the drive was and whatnot. Well this went on for about 10 min or so, but to make a long story a little bit shorter, he left with this huge smile on his face saying that he was defintly going to sell his CL and buy a Subaru.
So there could be another converted one soon enough!
+1-Subaru -1-Acura!
Registered users
of the site do not see these ads.
View Public Profile
Visit SpamMansXe's homepage!
Find More Posts by SpamMansXe


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Report Locally – Connect Globally
Have you ever wondered what it's like to be a reporter?
You can learn the fundamentals of journalism and become a reporter for Daniel Pearl Youth News when you successfully complete a free four-part online course to become certified as a Daniel Pearl Reporter.
Sign up for the online course, read the course materials, watch the accompanying videos, and take a short quiz on each of the first three segments. Upon successful completion of the three quizzes, you write an article and submit it to a mentor/editor for posting on Daniel Pearl Youth News. Mentors guide you in the process of gathering information, interviewing, checking facts, and editing your articles.
Daniel Pearl Youth News is open to all secondary school students. It prepares aspiring reporters to write news stories with emphasis on integrity, ethics, and truth in reporting by following the footsteps of journalist Daniel Pearl, who revealed the human element behind the story.
Please fill out the following information. If you are a teacher, please fill out the Teacher Sign-Up form.
* Required Field


text:
Two movie buffs invent a memory-erasing machine that allows them to watch their favorite movies over and over again like the first time.
Watch Movie Mind Machine online free at OnlineMovieMonster.com. No registration, no credit card needed.
If you are wondering where can I watch Movie Mind Machine online, you have come to the right website.
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best website to watch free movies online without downloading.
To watch Movie Mind Machine free online movie, just click the red play button above and
your free Movie Mind Machine movie stream will start after an age verification. We don't require you to sign up for Movie Mind Machine online stream.
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Watch free movies at OnlineMovieMonster.com in HD, we add free movies everyday. Our online movies database is updated daily, so stream free movies online unlimited. Enjoy.


text:
- Home
- Demos
- Betas
- Free to Play
- Patches
- Game Mods
- Game Genres
- Browse Game Genres
- Top Games
- Browse Top Games
Auto Cross Racing Demo
Download Auto Cross Racing Demo
File Info: Auto Cross Racing Demo
Auto Cross Racing Demo
KJM Software
Description: Auto Cross Racing DemoAuto Cross Racing is a racing car game, viewed from above the cars for Windows PC Computers. The aim of the game is to unlock each successive track by finishing in the top 3 places of the previous race.
Once all 10 tracks are unlocked, ( 5 tracks in easy mode ) you will be able to race for the championship trophy on the last track.Cash is earned as each race is completed and new cars will become available for purchase as each new track is unlocked.
Game Features
Advanced Pacejka based car physics allowing for controlled skids, powerslides and drifting.
Unlock new tracks and cars by winning races.
Earn cash by winning races and purchase any of the 20 available cars.
Easy and Normal modes of play.
20 AI controlled opponents to race against often allowing for multiple pile ups.
Different road surfaces ranging from tarmac, mud, snow and ice to desert.
Many tracks contain multiple routes, so choosing the best route is vital in shaving seconds off your lap times.
Nitro boost in order to accelerate past your opponent's.
Each car has its own unique sound effects.
Real-time car damage.
In game pitstops for both AI and player cars, allowing for repairs and nitro top ups.
Toggable in game mini map.
Choose the number of laps to race. 1 - 99.
6 camera types ( fixed and rotational ) with variable height adjustment and auto zoom.
Play your own in game music by placing your favorite music files into the in game MUSIC folder ( mp3, wav ).
Variable detail levels allowing for 3 different screen resolutions (1024 x 768) (800 x 600) (640 x 480) , 16 / 32 color depth display as well as fullscreen and windowed modes.
Redefinable keys and Digital Joystick support.
Save game feature.
System Requirements
1Ghz processor
DirectX V7.0 or higher and compatible graphics card with 64mb video ram.
39mb hard drive space for the demo.
113mb hard drive space for the Full Game.
Windows 95/98/ME/2000/XP/Vista.
You can download the latest version of DirectX at http://www.microsoft.com/download/
Less Information
More Information »


text:
Welcome to TipTopShop!
You have no items in your shopping cart.
For years our customers had to choose between the handy little pliers on the original Squirt P4 or the scissors on the Squirt S4. Now you can have both in one lightweight mini-tool that comes in handy for everything from snipping fishing line to wrapping a birthday present on the go.
The Brewzer has been designed to pop the top on your favorite beverage in just one try, making it the only keychain bottle opener you'll ever need. The multi-purpose flat tip also acts as a mini pry tool. Made from high-grade, heat-treated stainless steel, the Brewzer fits perfectly on your keychain split ring (through the beer-bottle-shaped hole) or in your pocket.
1) Bottle Opener
2) Mini Pry


text:
Cry How To...
Set system properties logo and information
On the 'general' tab of the 'System Properties' dialog (right click 'My Computer' and select 'Properties') will normally be displayed the manufacturer's logo, company and support information (assuming these have bee set up). This note describes how to change this or set net information up from scratch.
There are two parts to updating that panel - the graphic and the text information.
To create the text information, create a text file named '
and place it in '
c:\winnt\system32' (or presumably '
for a Windows 9x variant). The format of the file is as follows:
Manufacturer=Some Manufacturer Name
[Support Information]
Line1=For Technical Support contact:
Line3=Manufacturer service dept.
The first section, "[general]", lists the information shown on the System Properties panel. This can take an optional additional line:
The second section, "[Support Information]", is optional. If added, then the "Support Information" button will appear. Clicking that button brings up a panel with the text listed.
Be aware that leading spaces are ignored, so if you want to indent some text then be sure to enclose it in quotes, for example:
Line1=For Technical Support contact:
Line3=Service dept.
Line4=" Tel: NNN..."
Comments can be added to the
OEMINFO.INF file, and these
should be preceded by a semicolon ';' for example:
; Information displayed in System Properties
To add the graphic, create a '.BMP' file containing the graphic, name
it '
OEMLOGO.BMP', and place it in the '
directory. Note that the logo will not appear unless the '
file exists and has a 'Manufacturer' entry. The size of the graphic may
need to be altered to get it to fit correctly in the allocated space. The
BMP file should be an RGB BMP file sized about:
96 x 96 Windows 95/98 small fonts 120 x 120 Windows 95/98 large fonts 176 x 110 Windows 2000 172 x 100 Windows XP
These sizes are taken from the Microsoft documentation, but there seems to be some degree of variation even on the same operating system so be prepared to tweak the size of the logo to fit best with your target system.
If all you want to do is to simply remove the manufacturer's logo and
information then you can do this by simply deleting or renaming the
OEMLOGO.BMP and
OEMINFO.INI files.
These notes have been tested with Windows NT4 and Windows 2000.
About the author: Brian Cryer is a dedicated software developer and webmaster. For his day job he develops websites and desktop applications as well as providing IT services. He moonlights as a technical author and consultant.


text:
Nike Men's Hyperdunk 2015 Premium Basketball Shoe - Black/Green Glow/Anthracite
Bring your ball game up to a whole new level with the Nike Hyperdunk 2015. Offering responsive, low-profile cushioning, flexible support with Zoom Air technology and a solid herringbone rubber outsole for superior traction on and off the court, you'll be dribbling, dunking and nailing 3-pointers in undeniable style.
Available in men's US sizing - see size chart for correct fit!
- Nike footwear
- For men
- Style: Hyperdunk 2015 Premium
- Model no: 749567030
- Colour: Black/Green Glow/Anthracite
- Shoe width: N/A
- Zoom Air heel & forefoot cushioning
- Dynamic foam collar for sock-like fit
- Flywire cables wrap foot for lockdown
- Solid herringbone rubber outsole for traction
- Materials:
- Synthetic upper
- Textile lining
- Rubber outsole
- Country of origin: Vietnam
Helping serious athletes and casual trainers alike reach their true potential for over 50 years, Nike is one of the world's most trusted makers of sportswear. Maximise your body to its fullest and go far beyond what you thought possible.


text:
Hello guys. We have come up with a new free recharge offer. Now just complete a survey and get Rs 10 free recharge. This is an amazing offer. Completing this survey will take around 2-3 minutes and you will get Rs 10 free recharge within few hours. Offer is valid for limited time so grab this offer as soon as possible.
Thanks to Akash Jain for sharing this offer with us. Keep supporting bro.. 🙂
Steps To Complete A Survey And Get Rs 10 Free Recharge :-
1) Go to Offer Page
2) Enter your name, mobile number and email
3) Click on "Next"
4) Fill the survey
5) Click on "Submit". You will see success message.
That's it. You will get Rs 10 free recharge within 24-48 hours.
Note : We don't know how much time they take to recharge your mobile. So, try this offer at your own risk.
Limited period offer.


text:
Show your friends that you've got enough class to drink from a flask instead of a glass! This cute little container allows you yo carry your favorite beverages with you everywhere you go.
(based on 1 review)
Reviewed by 1 customer
Displaying review 1
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In love
By April
from undisclosed
Comments about Spencer's Girls With Class Don't Need A Glass Flask 6 oz:
I love mine and gifted 2 of them. My friends love them!
Was this review helpful? Yes / No
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text:
'He was groomed' Family defends Yorkshire teen who is UK's YOUNGEST suicide bomber
A YORKSHIRE teenager has become Britain's youngest-ever suicide bomber after blowing himself up in Iraq.
Talha Asmal, 17, is thought to have detonated a car bomb in Iraq, similar to the one pictured
Talha Asmal, from Dewsbury, West Yorkshire, was believed to be one of four suicide bombers who targeted security forces near an oil refinery in the town of Baiji.
The family of the 17-year-old said they were "utterly devastated and heartbroken" after he detonated a vehicle fitted with explosives in the north of the country.
They claimed ISIS militants had preyed on his "innocence and vulnerability".
Talha was a loving, kind, caring and affable teenager
Asmal, who went by the name of Abu Yusuf al-Britani while fighting with the fanatical islamists, travelled to the region via Turkey in April, his family said.
In a statement, they added: "Talha was a loving, kind, caring and affable teenager.
"He never harboured any ill-will against anybody nor did he ever exhibit any violent, extreme or radical views of any kind."
The family said he came "from a close-knit, hard-working, peace-loving and law-abiding British Muslim family" which unreservedly "condemns and abhors all acts of violence wherever perpetrated".
They said despite him never exhibiting any extreme or radical views, he had been exploited by extremists on the internet "in a process of deliberate and calculated grooming of him".
Unbeknown to them and completely against their will, they said he travelled to Iraq via Turkey and fell under the spell of Isis handlers who are "too cowardly to do their own dirty work".
"We are all naturally utterly devastated and heartbroken by the unspeakable tragedy that now appears to have befallen us," they said.
"We need time and understanding to come to terms with our unimaginable and painful loss.
"As a family we would like to take this opportunity to unequivocally state that 'Isis' are not Islam.
"They do not represent in any way, shape or form Islam and Muslims and we are no longer prepared to allow a barbaric group like 'Isis' to hijack our faith.
"'Isis' - not and never in our name."
"Talha's tender years and naivety were, it seems however, exploited by persons unknown, who, hiding behind the anonymity of the worldwide web, targeted and befriended Talha and engaged in a process of deliberate and calculated grooming of him."
"Whilst there it appears that Talha fell under the spell of individuals who continued to prey on his innocence and vulnerability to the point where if the press reports are accurate he was ordered to his death by so-called Isis handlers and leaders too cowardly to do their own dirty work.
"We are all naturally utterly devastated and heartbroken by the unspeakable tragedy that now appears to have befallen us."
Hasib Hussein, also from West Yorkshire, was 19 when he detonated a bomb on a bus in London in the 7 July 2005 attacks.


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The DuPont Brothers
Tuesday March 28, 2017
Doors: 6:00 pm / Show: 7:30 pmJammin Java
GA $15 | Premier $18 | Premier Plus $20
This event is all ages
• Full dinner and drink menu available
• The Premier Plus section is a raised area with great views and reserved seats and tables. There is a dedicated server for faster servicehttps://www.jamminjava.com/event/1444531/
The DuPont Brothers
Two brothers living on the indie circuit, driving around sharing their stories through songwriting. That's life for Sam and Zack DuPont. Based out of Burlington, VT the sibling indie-folk-rock duo has kept busy on the road since 2013. Their two records, Heavy as Lead (2015) and A Riddle For You (2016), have taken them all over the Northeast, Mid Atlantic, South and Midwest. The DuPont Brothers have made festival appearances at Grand Point North & SXSW and have supported a wide array of national acts including grammy award winning artists like Sturgill Simpson, Jerry Douglas and Blake Mills. There's an EP in the works for 2017. Stay tuned for new music and tour dates in the spring and summer.
Venue Information:
Jammin Java
227 Maple Ave E
Vienna, VA, 22180
Jammin Java
227 Maple Ave E
Vienna, VA, 22180


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Fun quick painting practice. Personal Sith character design in a storyline I'm working on for myself right now. Set just over 1,000 years before Episode I, right before the Sith "extinction" for a millenium.
Pretty sweet stuff, I was actually thinking of making my sith lord a former bouty hunter or trooper myself.. loving the style btw.
EPIC SAUCE! with a side of bacon, and fried chicken
Nice design...just curious as to why a Sith is wearing clone trooper armour.
Because he isn't. While design changes have been made since then to further separate it from established Star Wars design tropes a bit, it's been, since the beginning, based off of Mandalorian armor, just like the clone troopers were. My story also takes place centuries before Episode II. I'm still making tweaks every now and then, so thanks for speaking up.
HK!!! NOOOOOO!!!! (nice work though ^_^ )
I always liked the idea of armored Sith using blaster(s) as well as lightsaber(s). Nice job.
Soooo... about the era of "The Old Republic" (the upcoming MMO)?
is he a mandalorian sweet
Nice design. I see he's equipped with a vibroblade. As normal knives are uncommon in the Star Wars universe, I'm guessing it's either what I said, a vibroblade, or a lightknife. Either way, very nice, smooth.
Probably closer to a hybrid. It's a like a regular machete, but with an energized plasma cutting edge. Metal blade with energy edge. That's about the best I can describe it until I have a visual for it.
I see. Did you even look into it before I mentioned it? Was it meant to be just for show?
I love this guy's design. The teeth on the helmet are brilliant.
So the New Sith Wars?
But wait, if it's around that time, why is he a Darth?
He's a part of the remaining Sith that held with the many Sith philosophy of those that followed the path of Darth Revan and Malak in the KotOR games (and the new MMO). It's a new faction I'm calling the Sith Remnant. (They are separate from Dath Bane's group and philosophy of only two. I still haven't chosen a specific time frame, except for that it happens after the new Bioware MMO and before the "extinction". Remember, the Brotherhood of Darkness only lasted for 6 years from 1006 BBY to 1000 BBY. This would be before 1006 BBY
Aaaah, alright. Didn't think there would still be any Revanchists out there so late in the galaxy's old history.
WIN! (as a starwars nerd myself, I would be happy to help with the story )
Denied. Pretty kewl muchacho!


text:
Liverpool, UK: Region has new draft bylaw for food trucks - March 5, 2017
HongKong: Hong Kong food bazaar diners back call to allow hawkers to cook with gas -survey - March 5, 2017
Denver, CO: A Food Truck We Love – Moontime Crepes - March 5, 2017
Plano, TX: Hub Streat Food Truck Derby set for Saturday - January 23, 2017
Iowa, IA: City Council Approves Food-Truck Plan Again - January 23, 2017
Pasco, WA: Pasco food truck class helps local community - January 23, 2017
Santurce, PRI: Inside Puerto Rico's Food Truck Boom - January 23, 2017
Monte Vista, TX: New food truck park open near Monte Vista - January 23, 2017
Austin, TX: East Side King opening new trailer location in East Austin - January 10, 2017
Boulder, CO: Roxie's Tacos Brings Indian Flavors to Boulder's University Hill - January 10, 2017
Winnipeg, CAN: Putting food trucks on the map
A new smartphone technology is helping put Winnipeg food trucks on the map.
By David Larkins | Winnipeg Sun
A new smartphone technology is helping put Winnipeg food trucks on the map.
Winnipeg is the third city targeted by the Saskatchewan-based start-up Snack Map, a GPS-driven app that allows users to find where and when their favourite food trucks will be set up on any given day.
Currently nine Winnipeg operators have bought in to the idea, paying a refundable $250 deposit and $15 per month fee.
Users can log onto snackmap.com, or download the app to their smartphone, and a list of Winnipeg food trucks will appear showing their location, their hours of operation and what payment methods are offered.
Snack Map has found success in Saskatoon and Regina, where about 30 different operators combined have bought in to the service.
"(Winnipeg has) such a great food truck scene and food truck culture that we reached out to a lot of the owners to ask them what their thoughts were and drum up some interest," Snack Map's Kit Hillis said. "That was the start of our journey … and they couldn't wait to be on board with Snack Map. It was such a positive response."
Lead developer Evan Taylor said the idea sprung to mind in 2014 when finding food trucks in the upstart Saskatoon market proved challenging.
"We found it was hard to figure out how to find these food trucks when they were out, and the only way you could possibly do that is maybe through their Twitter account, maybe a Facebook post," he said. "We saw a real need and we thought someone should just make an app where you can see where the food trucks are, and we figured why don't we just do that? We launched into it as a side project and it's really taken off."
Derek Collins, who operates the Poutine King in Winnipeg, was one of the first to support the idea locally and has lobbied other owners on Snack Map's behalf.
"It gets us closer to our customers … I think at the beginning we won't see a big difference in our business, but the more that we grow and the more food trucks that have it and more people that see, you build a customer base and data base, then we'll start seeing a difference. It's like anything, you start at the bottom and keep building," said Collins.
The Winnipeg food trucks to be found on Snack Map:
-The Poutine King
-Tot Wheels
-The Red Ember
-The Churro Stop
-Olybee's Kitchen
-Little Bones Wings
-Habanero Sombrero
-Fired Up
-Extreme Fries
Leave a Comment
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text:
Despite a loss to its bitter rival, Tennessee cemented its standing with Jalen Tabor.
The Watch List safety from Washington Friendship told ESPN that Tennessee is firmly in his top five, along with Alabama, Maryland, Ohio State and Florida.
"It was great," Tabor said via text message when asked about the visit. "I couldn't ask for anything more."
Tennessee lost to Alabama 44-13 to drop to 3-4 and 0-4 in the SEC. There has been widespread speculation that there could be a coaching change if third-year coach Derek Dooley doesn't turn things around for the Vols. Tabor said he has a good relationship with Dooley, defensive coordinator Sal Sunseri, offensive coordinator Jim Chaney and cornerbacks coach Derrick Ansley.
"Yeah, that's my only worry about Tennessee," Tabor said when asked of a potential coaching change.
Tabor said he doesn't have more visits planned at this time. The junior has long shown strong interest in the Vols. He has visited the campus several times.
Landing Tabor could provide a windfall for any school. Friendship is one of the most talented schools in the East region.
When asked about Alabama's performance Saturday, Tabor stated, "They are just a great program. There's nothing I can say about UA."


text:
Short animation about a machine that will change your life, except it definitely won't.
TeezMe is a presentation from a retro futurist company whose attempt to build an "intelligent pleasure droid" turns out to be a useless Rube Goldberg machine. Being left and forgotten somewhere it continues to loop the same sales pitch.
This started out as a small modelling excercise in between jobs where I wanted to do something a bit stupid and turned into a kind of love letter to motion graphics tropes. Special thanks to Zelig for some amazing sound design.
Design, direction & animation: Jason Drew
Music & sound design: Zelig Sound


text:
India's reputed company Cyclonewebz provides the comprehensive services and varied styles of the net solutions to meet the client's expectations. Based in Delhi, the company specialize in mobile app development, hosting technology, website designing, and digital marketing.
Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.
India's reputed company Cyclonewebz provides the comprehensive services and varied styles of the net solutions to meet the client's expectations. Based in Delhi, the company specialize in mobile app development, hosting technology, website designing, and digital marketing. The main aim of the experienced team is to help the client throughout the way to achieve the complete objective. Curve of growth rate is increasing continuously by catering the customer's requirements.
A world that we've got created with our patrons by providing them with services that met expectations and addressed their business necessities. We tend to square measure a forthright net solutions and offshore development company in urban center. We provide all our customers with varied styles of net solutions, from net coming up with, net development and code development. The websites developed don't seem to be solely conspicuous and reasonable, however at identical time square measure user friendly and are designed victimisation the newest net coming up with tools, thereby we tend to empower all our customers to represent themselves on-line with confidence within the international arena, to get a lot of revenue.
Online Selling (SEO+SMO+SEM)
Website Development
Ecommerce Solutions
Mobile App Development
Domain & net Hosting
BULK SMS Solutions
CRM Package
ERP Software System
Mobile Apps are now part of the business-life because it can help business owners to connect with customers, vendors and employees, by using mobile apps business can easily accessible from anywhere. When it comes to Android apps, they make business operations simple and entertainment continually available.
A decade-long we are in IT industry with having expertise in website development. We provide cost effective and flexible web development services. A best quality and user friendly websites play a vital role in business success. Beautiful graphic design is an essential part of website to entice visitors. Our experience designers and developers understand the value of solid information architecture design and integrative branding that engages the customer from the first stop at your site.
Electronic commerce, commonly written as e-commerce, is the trading or facilitation of trading in products or services using computer networks, such as the Internet. Electronic commerce draws on technologies such as mobile commerce, electronic funds transfer, supply chain management, Internet marketing, online transaction processing, electronic data interchange (EDI), inventory management systems, and automated data collection systems. Modern electronic commerce typically uses the World Wide Web for at least one part of the transaction's life-cycle, although it may also use other technologies such as e-mail.
Harsh Store 4 U
Ecommerce website + Android App
Harsh Store 4 U is a Kirana & General store which delivers products on request. You can either place your order by Android App or by visiting http://www.harshstore4u.com
Office Commuters
Android Applocation + Backend
OfficeCommuters provides all new way to Home pick and Drop facility for daily commuters.
Shankar Garden, Vikaspuri, New Delhi, India
+91 8826714133


text:
We recommend upgrading to the latest Internet Explorer, Google Chrome, or Firefox.
If you are using IE 9 or later, make sure you turn off "Compatibility View".
Handpicked brands for you
It's Hoonigan gold sticker time! As always, when I win - you win. So because I won #GlobalRallycross round 4 here in North Carolina, every order you place on #hoonigandotcom comes with a free gold sticker that isn't available at any other time. Orders...


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Discussion in 'Android Devices' started by kichoufumetsu, Mar 8, 2010.
ok, so i'm showing
sw version 1.5
application version 2.2
can i still root my cliq?
should i go with the dude's method? is that the best option?
Use the dude method for both just make sure if u have the new radio code u must use the one for 1.3.18 and not 1.3.13
For both? Now I have to go read more...radio code?
you will need the RSD Lite software, the motorola drivers for your phone and this code http://tinyurl.com/yf5yy9l for radio code 1.3.18
if you are still on radio code 1.3.13 you will use this code ModMyMoto - Software Modding Forums and Updates for Motorola Phones ...
use the same method as the dude root... meaning
instead of using the rom code for 1.3.13 use 1.3.18 if you have that respective radio code...
Ok, so I thought if you were on 1.3.18 you couldn't root. I keep getting mixed messages on this.


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The sexual revolution reached a tipping point in 2014 and 2015. It happened suddenly after a long time waitin'. After 30 years of people risking reputation, livelihood, and life and limb by actively and at great risk, coming out of the closet about their sexuality. Suddenly LBGT rights and gay marriage and love between people of all kinds became legitimate, recognized as a right and confirmed by even a conservative Supreme Court. The shocking sudden change in acceptability, no one could have predicted—certainly not from the history of injustice and prejudice against gay people that preceded the turnaround. For lifetimes on end, if you desired or liked or wanted sex with members of your own sex you were supposed to pretend that you didn't and never tell anyone. Then, all of a sudden, that went away and it didn't matter so much anymore. What happened? How did it happen? Suddenly everyone's opinion about what other people can do and had a right to do sexually, changed. Or as Malcolm Gladwell said in his wonderful book "The Tipping Point," something "tipped."
In 1959 when I was 19 years old and went to my first civil rights rally in Austin Texas and helped start the theater stand-in movement and restaurant sit-in movement, none of us knew what to do to bring about changes in the ridiculous segregated social order. We all dreamed of and demanded desegregation after so many hundreds of years of racial injustice…and we got arrested and bombed and shot at and beat up and some of us got killed…but we had no idea that within 5 years there would be a civil rights act to legally end segregation. The justifiability of segregation according to race was ended and the right of all humans to be treated the same as all other humans was established by law and consensus among us. And the way we have been living with each other since then has changed. Really. A lot more of us got to know each other than we would have with the older arrangements.
I've been smoking marijuana and growing it and buying it and selling it and giving it away for 54 years, illegally. The last I bought from a state liscensed store in Denver. Inside of a few more years the whole United States will be pretty much as tipsy as Colorado. The DEA is coming down. The bullshit war on drugs is coming to an end.
O.K. that's enough. If you want more there are many other examples of tipping points in Malcolm Gladwell's book. If you want more than that, just stay alive another five years. What seems to make things tip is information that contradicts erroneous beliefs that comes from people you trust who are honest with you.
The Tipping Point of All Tipping Points
I think we and our allies in honesty all over the world are escalating the speed with which things tip.
Either hold on to or let go of your seat. I'm not sure which…or when…you should hold on or let go. A storm at sea is coming. I think we have the chance to join the storm and eventually calm the waves with honesty.
It's kind of scary though.
Could it suddenly become acceptable to come out of the closet about things even more scary than our sexual proclivities?
What if there is developing a kind of meta tipping point of tipping points?
What happens when we all admit we are all fucking nazi's in our minds?
What about love of people it is taboo to love (You can't love her, she's married!). What about our pettiness, anger, embarrassment, pretentiousness, judgmental condemnation of others (and our selves as well), and our arrogant demands to control the actions and behaviors of others?
What about coming out of a whole bunch of closets at once!? (What are crowds of loveable assholes to do?)
Will there be a tipping point on how much control of others is protected, demanded or permitted or even considered necessary? (We don't need the armies, police forces, the legal system that is completely antiquated and corrupt. When all of us knowing that tips, they all must be exposed and taken apart and put aside and a smaller, less expensive and better system created.)
How about lying? Is there a tipping point for stopping pretending that you are not pretending, when you are…or admitting it and taking it back when you do?
Is there a tipping point for honesty in relationships?
Is there a tipping point for world wide radical honesty?
Yes. There is. It is coming. Get ready for it!
When this "bigger room for personal honesty in the world" tips, the worldwide fascist system of law and order cannot survive. Neither can the worldwide economic order. It will suddenly collapse and be replaced by government by friends who trust and depend on each other.
A world organized around honest sharing instead of money?? What? Am I crazy?
People of the traditional paranoid mindset based on "us versus them" cannot survive this either. The "us" I am referring to in that last paranoid statement are those of us who are honest about our lying and the "them" are all the regular assholes who lie about their lying, i.e. normal folks who maintain their status by maintaining the status quo. How do we become allies with these enemies?
What happens when something tips is that both of the opposing crowds change their minds! All of a sudden, people on both "sides" start listening to each other and speaking honestly about what used to be taboo. What used to be in fashion becomes old fashioned overnight! Enemies become friends. Everyone becomes more interested in what is really going on than in maintaining their position of being right.
My friend Charles Eisenstein calls such things "cracks in the foundation of normalcy," a phrase I just love. Here is and excerpt from his most recent post:
"I was pleased to see HuffPo feature this NYT magazine article on the criminal (in)justice system. It describes the effects of the bail system that forces indigent defendants to plead guilty to trumped-up charges or rot in jail for weeks or months (or years) because they cannot make bail. One man was arrested as he walked out of a store with a soda and straw — the straw was considered "drug paraphernalia." He spent weeks in jail, where he was severely beaten. A woman left the homeless shelter to buy diapers for her baby, whom she left in the care of a friend. She was arrested for recklessly endangering her child; six months later she still hasn't regained custody.
"As the statistics in the article demonstrate, such stories are not exceptional (especially for black people). Nor are they a new phenomenon. What is new in my lifetime though is that finally this sort of injustice is emerging into mass consciousness, no longer invisible to the majority of middle-class white people.
"Relatedly, the upsurge of outrage at the police killings of unarmed black men (and at police brutality in general) reveals another crack in the foundation of normalcy. Police brutality is nothing new; what is new is that it is becoming harder to hide — literally, due to ubiquitous video cameras and social media, and figuratively, because of the disintegration of the racial, political, and class narratives that cloaked it.
"It is tempting to blame the situation on racist cops, callous judges, or opportunistic politicians. Certainly such people exist, but blaming them is a trap, a deflection of anger onto a convenient but superficial target. The article gives us a glimpse instead of a monstrous machinery of injustice: an industrialized, inhuman, nearly paralyzed legal system and courtroom culture, embedded in an equally paralyzed political system, itself beholden to deep cultural myths about human nature, criminality, and punishment. At bottom, I believe, we are seeing the crisis and imminent collapse of the ideology of Control, which in its failing days goes to new and absurd extremes in response to its own failures.
"Fortunately, it isn't necessary to understand the deep causes of the horrid injustice that is becoming increasingly plain to see. The understanding will come with time. What is necessary now is to awaken the "no". That will power reforms (one good one the article mentions is to eliminate the cash bail system entirely) that will ultimately prove insufficient as our tolerance for injustice wears thinner and thinner. Our industrial system of punishment and mass incarceration is fundamentally incompatible with the dignity of each human being. Our consciousness is evolving to the point where that is no longer tolerable. (italics mine…Brad)
While it may seem as though indignity — not to mention horrid injustice — is reaching all-time highs, we might also see their heightened visibility as the beginning of a major shift. Someday, maybe sooner than we have any reason to think, we will live in a world where no one is dehumanized and everyone is treated as a full being. Is anything less tolerable to you?"
What Charles refers to is a personal evolution in which many people coming out of the closet about their real experience in life, that leads to the social revolution. It goes by a lot of names. Around here we call it Radical Honesty. It is the ultimate breakthrough that all the former and later breakthroughs will either live or die from.
The systemic injustice of the well-named Criminal Justice System is just a case in point. The rule of kings was replaced by the rule of prejudicial legalistic mindfuck. We simply have to become more unruly. And when the revolution tips we will undo and replace the old with a new and revised mindfuck, short lived, and reviseable again soon.
I trust comedians. They are willing to be embarrassed and embarrassing and call us out of our closets. They make fun of what used to be serious. Andy B here is funny and wise and full of good tips.
"Sanders Shamelessly Pandering to Voters Who Want to Hear Truth
By Andy Borowitz, The New Yorker
13 August 15
The article below is satire. Andy Borowitz is an American comedian and New York Times-bestselling author who satirizes the news for his column, "The Borowitz Report."
Presidential candidate Bernie Sanders is gaining legions of new admirers by shamelessly pandering to voters who want to hear the truth, critics of the Vermont senator say.
According to those critics, Sanders has cynically targeted so-called "truth-based voters" to build support for his Presidential bid.
"People come to Sanders's rallies expecting to hear the truth, and he serves it up to them on a silver platter," the political strategist Harland Dorrinson said. "It's a very calculated gimmick."
But while Sanders's practice of relentlessly telling the truth might play well in states that are rich in truth-based voters, like the early primary states of Iowa and New Hampshire, critics say that his campaign could stall in states where the truth has historically been less important, like Florida.
"At some point in this campaign, voters are going to get truth fatigue," Dorrinson said. "Right now, the novelty of a politician who doesn't constantly spew lies is grabbing headlines. But after months of Bernie Sanders telling the truth, voters are going to start wondering, Is that all he's got?"
Dorrinson is just one of many critics who are eagerly waiting for the Sanders phenomenon to come down to Earth. "Telling the truth may be working for Bernie Sanders, but it shows a serious lack of respect for the American political system," he said."
The new and improved and upgraded online course "Radical Honesty: How to Get Over Shit and BE Happy" will be launched in October. It is designed to be part of the tipping point of the re-evolution of humanity that we need for our survival. Feeling a bit tipsy are you? We certainly hope so.
I will be 75 years old on September 8 (Come on out Labor Day Weekend (Sept. 5-8) for the party if you can. You may not be surprised to hear that my completely subjective opinion after 75 years on the planet is that Radical Honesty is the most important thing in the world. It is a primary source of tipping points, and when it tips everything changes.
Love, Brad
P.S. Watch this. It is John Denver saying no to the old order and yes to the tipping from being killers to being lovers. It is one of the next big tipping points just around the bend.


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The Ca2+-sensing receptor (CaR), a G protein-coupled receptor, is expressed in many epithelial tissues including the parathyroid glands, kidney, and GI tract. Although its role in regulating PTH levels and Ca2+ metabolism are best characterized, it may also regulate salt and water transport in the kidney as demonstrated by recent reports showing association of potent gain-of-function mutations in the CaR with a Bartter-like, salt-wasting phenotype. To determine whether this receptor interacts with novel proteins that control ion transport, we screened a human adult kidney cDNA library with the COOH-terminal 219 amino acid cytoplasmic tail of the CaR as bait using the yeast two-hybrid system. We identified two independent clones coding for ∼125 aa from the COOH terminus of the inwardly rectifying K+ channel, Kir4.2. The CaR and Kir4.2 as well as Kir4.1 (another member of Kir4 subfamily) were reciprocally coimmunoprecipitated from HEK-293 cells in which they were expressed, but the receptor did not coimmunoprecipitate with Kir5.1 or Kir1.1. Both Kir4.1 and Kir4.2 were immunoprecipitated from rat kidney extracts with the CaR. In Xenopus laevis oocytes, expression of the CaR with either Kir4.1 or Kir4.2 channels resulted in inactivation of whole cell current as measured by two-electrode voltage clamp, but the nonfunctional CaR mutant CaRR796W, and that does not coimmunoprecipitate with the channels, had no effect. Kir4.1 and the CaR were colocalized in the basolateral membrane of the distal nephron. The CaR interacts directly with Kir4.1 and Kir4.2 and can decrease their currents, which in turn could reduce recycling of K+ for the basolateral Na+-K+-ATPase and thereby contribute to inhibition of Na+ reabsorption.
- inwardly rectifying K+ channel
- HEK 293 cells
- yeast two hybrid
the ca2+-sensing receptor (CaR), a member of the G protein-coupled receptor (GPCR) superfamily, is expressed in the parathyroid glands, kidney, and other epithelial tissues. The primary physiological role of the CaR is to regulate PTH levels and Ca2+ balance, but it may also play an important role in the regulation of salt and water transport in the kidney (4, 14). Activation of the CaR mediates the acute adverse renal effects of hypercalcemia including reducing Na+, Cl−, K+, and H2O reabsorption, the renal response to vasopressin, and aquaporin expression (4, 14, 29). Mutations of the human CaR gene cause disorders of mineral-ion homeostasis and lead to several human diseases. Loss-of-function mutations of the CaR cause familial hypocalciuric hypercalcemia (heterozygous) and neonatal severe hyperparathyroidism (homozygous) (29), and activating mutations cause autosomal dominant hypocalcemia (30). Recently, potent activating mutations of the CaR were identified as the cause of a Bartter-like syndrome characterized by volume depletion, Na+, Cl−, and K+ wasting, hypomagnesemia, hypercalciuria, and elevated rennin and aldosterone levels in addition to autosomal dominant hypocalcemia (40, 43).
Five genetically distinct abnormalities of transport proteins that lead to phenotypes characterized by salt-wasting (Bartter's and Gitelman's syndromes) have been described. Bartter's syndrome can be caused by loss-of-function mutations of the Na+-K+-2Cl− cotransporter (NKCC2), the renal outer medullary K+ channel (ROMK), the basolateral Cl− channel (ClC-Kb) and a variant also characterized by sensorineural deafness is caused by loss of function mutations in an accessory protein for ClC-Kb, BSND, or barttin (2, 26, 38, 40). The hypocalciuric, hypomagnesemic variant of Bartter's syndrome (Gitelman's syndrome) is due to loss of function mutations of the thiazide-sensitive Na+-Cl− cotransporter (NCC) (26). The CaR represents a sixth gene, but with gain of function mutations, that can lead to a Bartter-like phenotype (40, 43).
Like a number of other receptors such as those for angiotensin (ANG) II, endothelin, and vasopressin (V1), the CaR couples to multiple G proteins (Gαi, Gαq, Gβγ, and Gα12/13) involved in distinct signaling pathways that can contribute to regulation of ion transport (14, 16, 17). Inhibition of adenylyl cyclase due to CaR stimulation of Gαi with reduced cAMP accumulation inhibits the activity of NKCC2 and ROMK (22). Phospholipase A2 activated by the CaR via Gαq produces arachidonic acid metabolites that inhibit ROMK activity (10, 41). The activity of ROMK channels can also be regulated by phosphatidylinositol bisphosphate (PIP2) hydrolysis and synthesis that are regulated by CaR-activated phospholipase C and phosphatidylinositol-4 kinase (16, 45). Although the CaR activates cellular signaling systems that can explain the inhibition of NKCC2 and ROMK, other receptors such as those for ANG II, endothelin, and α-adrenergic agonists stimulate similar signaling pathways without causing Na+, Cl−, and K+ wasting to the same degree as the CaR. This difference in effects on salt transport between the CaR and other receptors suggests that the CaR may act via distinct mechanisms to regulate electrolyte metabolism such as direct interaction with channels, pumps, or transporters.
To identify additional proteins that interact with the CaR and that could explain its effects on electrolyte balance, we used the COOH-terminal 219 amino acids of the human CaR representing the cytoplasmic tail as bait to screen a human adult kidney cDNA library using the yeast two-hybrid assay. Using reciprocal immunoprecipitation and measurement of channel activity, we found that the CaR interacts with two inwardly rectifying potassium channels, Kir4.1 and Kir4.2, that are expressed in the distal nephron, and that the CaR inactivates these channels (19, 23). Regulation of this family of channels has not been studied extensively. Our results demonstrate that the CaR interacts directly with Kir4.1 and Kir4.2 and suggest a mechanism by which the CaR could inhibit electrolyte transport in the distal nephron.
All chemicals were purchased from Sigma or Fisher Scientific unless specified otherwise. The monoclonal anti-CaR and anti-HA antibodies and HEK 293 cells that stably overexpress the wild-type CaR were described earlier (10, 11). G418 sulfate and cell culture reagents were purchased from Life Technologies. The SuperSignal West Pico chemiluminescent substrate and BCA protein assay reagent were obtained from Pierce. The polyclonal anti-Myc (A-14) and monoclonal anti-Myc (9E10) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The polyclonal anti-Kir4.1, and anti-ROMK antibodies, and their respective specific peptides were supplied by Alomone Laboratories (Jerusalem, Israel). The polyclonal anti-Kir4.1 antibody used for immunohistochemistry was obtained from Yoshihisa Kurachi, Osaka, Japan. The polyclonal anti-Kir4.2 antibody was provided by Dr. C.E. Hill, Queen's University, Kingston, Ontario, Canada.
Yeast two-hybrid cloning.
A cDNA representing the COOH-terminal 219 amino acids (aa) of the human CaR (CaR861–1078) in the bait plasmid pAS2.1 was used to screen a human adult kidney cDNA library in pACT2 (Invitrogen). The bait plasmid was transformed into S. cereviciae strain AH109 by electroporation, mated with the pretransformed library in S. cereviciae strain Y187, and then plated on medium deficient in leucine, tryptophan, and histidine (37). The plates were replicated on media lacking histidine, tryptophan, leucine, and adenine. The colonies that grew under these conditions were tested for β-galactosidase activity. Yeast expressing the library plasmid pACT2 without an insert and the pACT2 vector with β-galactosidase were used as controls. The positive colonies were analyzed further by purifying the plasmids and sequencing the inserts from either end. The cDNA sequences were analyzed by searching the GenBank database for homology to known DNA sequences. To identify the region(s) of the CaR COOH terminus that interact with Kir4.2, we made the constructs that coded for CaR860–908, CaR907–997, and CaR987–1078 by RT-PCR and subcloned them into pAS2.1. These constructs were mated with the Kir4.2243–375 clone isolated from the cDNA library in pACT2 and plated on medium lacking histidine, leucine, tryptophan, and adenine.
Construction of plasmids.
To investigate the relationship between the Kir proteins (Kir4.2, Kir4.1, and Kir5.1) and the CaR without the limitation of specific antibodies, we made Myc- and HA-tagged constructs of each channel engineering epitope tags into the NH2 terminus of the proteins. The cDNA coding for rat Kir4.2 (Kir4.2) was a generous gift from C. E. Hill (13). The Kir4.2pGem construct was digested with EcoRI and NotI and then ligated into EcoRI and NotI sites of pCruz-HA or pCruz-Myc vector. The cDNAs coding for rat Kir4.1 (Kir4.1) and rat Kir5.1 (Kir5.1) were generously provided by Stephen Tucker at the University Laboratory of Physiology, United Kingdom (28). The Kir4.1pBF construct was digested with EcoRI, blunt-ended, and then digested with KpnI. The Kir5.1pBF construct was digested with ApaI, blunt-ended, and then digested with KpnI. The pCruz-Myc or -HA constructs were digested with NotI, blunt-ended, and then digested with KpnI. The fragments were ligated into pCruz-Myc or pCruz-HA. All cDNA constructs were verified by direct sequencing. The ROMK pcDNA3.1 was generous gift from Wenhui Wang at the New York Medical College, Valhalla, NY.
Transfection, immunoblotting, and immunoprecipitation.
HEK-293 cells or HEK-293 cells that stably express the CaR were transiently transfected with the epitope-tagged channel constructs indicated (the Myc-tagged Kir4.1pCruz, the HA-tagged Kir4.2pCruz, the Myc-tagged Kir4.2pCruz, the HA-tagged Kir5.1pCruz, the Myc-tagged Kir5.1pCruz) or the ROMKpcDNA3.1 using the Fu Gene 6 reagent and incubated at 37°C for 24–48 h. All cells were cultured in medium containing 1.8 mM CaCl2. The medium was removed and washed once with 1× PBS. The cells were lysed with 1× RIPA buffer, and the lysates were centrifuged at 15,000 rpm for 1 h at 4°C. The protein concentrations in the cell lysates were determined using the BCA protein assay reagent with BSA as a standard and then adjusted to the same concentration with buffer. The samples were subjected to 11% SDS-PAGE, processed for immunoblotting, and the levels of protein expression were determined. The cellular lysates were used for coimmunoprecipitation as described (16). Briefly, either a monoclonal anti-CaR antibody or a polyclonal anti-Myc (A-14) antibody was separately loaded onto the Dynabead-protein A complex and slowly rotated for 2 h. The antibody-loaded Dynabead-protein A complex was rinsed twice, the beads were mixed with the various cell lysates, and rotated in the cold room overnight. The supernatants were discarded and the Dynabead-protein A complex was washed. Loading buffer was added and vortexed vigorously. The tubes were placed in Dynal-MPC to collect the sample buffer. The samples were heated at 55°C for 20 min for channel proteins or boiled for 5 min for the CaR and then subjected to SDS-PAGE for immunoblotting using the antibodies indicated. For coimmunoprecipitation studies using rat kidney membrane extracts, rat kidneys were isolated, and the cortex and medulla were separated. The cortex and medulla were then cut into small pieces and homogenized in a buffer containing 20 mM HEPES, pH 8.0, 2 mM MgCl2, 1 mM EDTA, and protease inhibitors, and centrifuged at 2,000 rpm for 10 min. The supernatant was centrifuged at 13,500 rpm for 60 min to obtain crude membrane and cytosol fractions. The samples were used for immunoblotting to confirm the presence of the endogenous proteins. The crude membranes were extracted in 1× RIPA buffer for 60 min, centrifuged at 13,500 rpm for 60 min, the resulting supernatants were used for immunoprecipitation using an anti-CaR antibody, and the immunoprecipitated samples were processed for immunoblotting using antibodies against the CaR, ROMK, Kir4.1, and Kir4.2 proteins.
The CaR- and Myc-Kir4.2-coexpressing HEK 293 cells were grown on glass coverslips for 2 days. At 40–60% confluence, the coverslips were fixed with cold 4% paraformaldehyde for 20 min and permeabilized with 0.05% saponin. The samples were processed for double immunofluorescence using our monoclonal anti-CaR antibody (16) and a polyclonal anti-Myc antibody (A-14) as the primary antibodies, and Alexa Fluor 488 goat anti-mouse IgG (green) and Alexa Fluor 594 goat anti-Rabbit IgG (red) as secondary antibodies. The coverslips were observed using fluorescent microscopy (Zeiss, Model LSM-5 Pascal) and images were collected using the Axiovert 200 program (Zeiss).
For immunoperoxidase staining, kidney blocks containing all kidney zones were dehydrated and embedded in paraffin. The paraffin-embedded tissue was cut at 2 μm on a rotary microtome (Micron). Staining was carried out using indirect immunoperoxidase. The sections were dewaxed and rehydrated, and endogenous peroxidase was blocked with 0.5% H2O2 in absolute methanol for 10 min at room temperature. To retrieve antigens, sections were treated with a 1 mM Tris solution (pH 9.0) supplemented with 0.5 mM EGTA and heated in a microwave oven for 10 min. Nonspecific binding of immunoglobulin was prevented by incubating the sections in 50 mM NH4Cl for 30 min followed by PBS containing 1% BSA, 0.05% saponin, and 0.2% gelatin. Sections were incubated overnight at 4°C with anti-Kir4.1 or anti CaR antibodies diluted in PBS supplemented with 0.1% BSA and 0.3% Triton X-100. After being rinsed with PBS supplemented with 0.1% BSA, 0.05% saponin, and 0.2% gelatin for 3× 10 min, the sections were incubated in horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin (DAKO P448) diluted in PBS supplemented with 0.1% BSA and 0.3% Triton X-100. The samples were observed using a Leica DMRE light microscope.
Oocytes were harvested and prepared as previously described (36). Kir4.1 and Kir4.2 were subcloned into the oocyte expression vectors, pBF or pGEMSH respectively. Linearized plasmids were used for in vitro cRNA synthesis. Forty six nanoliters of water or cRNA mixtures were injected into stage VI oocytes (CaR at 10 ng/oocyte, Kir4.1 at 5 ng, Kir4.2 at 5 ng). Oocytes were studied 3–7 days after cRNA injection and were preincubated for 24 h in 0.1 mM Ca2+ to minimize activation of the CaR. We voltage clamped oocytes to 0 mV in a K+ ringer (98 mM KCl, 1.0 mM CaCl2, 1.0 mM MgCl2, 5 mM HEPES, pH 7.5). Since intracellular [K+] is 80–90 mM (Romero and Boron, unpublished observations), this high K+ solution results in a slightly inward chemical gradient for K+ and a reversal potential near 0 mV. From 0 mV oocytes were stepped by 20 mV from −120 to +80 mV using a 75-ms voltage pulses. I-V curves were generated by averaging the final 10 ms of each resulting current responses as previously described (8, 36). We verified the activation of the CaR by monitoring current responses (endogenous oocyte Cl− channel) after the addition of 1 mM GdCl3 (3). Only the wild-type CaR (CaRWT) showed an outward current with Gd3+ addition. Subsequently, we used 20–30 sister oocytes to isolate membranes to document the CaR and Kir4.x protein expression.
Evidence for direct interaction of the CaR with the Kir4.2 channel.
To identify proteins that interact with the CaR and that play important roles in the regulation of distal nephron function, we prepared a cDNA construct that corresponds to the COOH-terminal 219 aa (CaR860–1078) of the human CaR representing the intracellular cytoplasmic tail, to use as bait to screen a human adult kidney cDNA library. After plating on medium that lacked histidine, tryptophan, leucine, and adenine, we obtained 64 β-galactosidase-positive colonies. Two of the clones coded for an inwardly rectifying potassium channel and represented the COOH terminus of Kir4.2 (subfamily J, KCNJ15) from aa 243–375 and aa 254–375 (Fig. 1A). To identify the region(s) of the CaR COOH terminus that interacts with Kir4.2, we subcloned three fragments of the CaR COOH terminus (CaR860–908, CaR907–991, and CaR987–1078) into the pAS2.1 vector, mated them with one of the clones we isolated (Kir4.2243–375) in pACT2, and plated them on medium lacking histidine, tryptophan, leucine, and adenine. The Kir4.2 COOH terminus only interacts with the CaR907–991 fragment (Fig. 1B), and the midregion of the CaR COOH terminus interacted with the COOH-terminal 125 amino acids of Kir4.2.
To determine whether the CaR and Kir4.2 interact in mammalian cells, we transiently coexpressed the CaR and Kir4.2 with a Myc epitope tag at its NH2 terminus in HEK-293 cells and carried out coimmunoprecipitations using our anti-CaR antibody. In Fig. 2A, the bands representing both the CaR and Kir4.2 were present in extracts of the cells where they were expressed, but not in the untransfected cells. Figure 2B shows that the specific bands representing Myc-Kir4.2 are coimmunoprecipitated with the CaR using our anti-CaR antibody as the immunoprecipitating antibody. This finding is consistent with our yeast two hybrid results.
Interaction of the CaR and Kir4 is highly selective.
Kir4.2 and Kir4.1 are the only two known members of the Kir4 family and are both expressed in the distal nephron, potentially in the same region as the CaR (19, 23, 33). To determine whether the CaR interacts with Kir4.1, we transiently transfected Myc-Kir4.2 or Myc-Kir4.1 into HEK 293 cells that stably express the CaR, and the lysates were immunoprecipitated with either our anti-CaR antibody or a polyclonal anti-Myc antibody (A-14). In Fig. 3, left, the anti-CaR antibody was used for immunoprecipitation, and immunoprecipitation of the CaR was verified by immunoblotting the immunoprecipitate with the anti-CaR antibody (Fig. 3, left top). Coimmunoprecipitated Myc-Kir4.2 and Myc-Kir4.1 were identified by immunoblotting the same immunoprecipitate with an anti-Myc antibody. In the Fig. 3, bottom left, bands representing the Myc-tagged channel proteins are present only in lanes 3 and 4 where they were coexpressed with the CaR but not in lanes where no immunoprecipitating antibody was used (lane 1) or where no channels were expressed (lane 2). The top band seen in all lanes is a nonspecific band. In Fig. 3, right, the polyclonal anti-Myc antibody (A-14) was used for immunoprecipitation. Bands representing the immunoprecipitated channels (Kir4.1 in lane 3 and Kir4.2 in lane 4) are present, but not in the lanes where no immunoprecipitating antibody was used (lane 1) or where no channel was expressed (lane 2, vector). In Fig. 3, top, a band corresponding to the CaR is present in the third and fourth lanes where the CaR was coimmunoprecipitated with the channels by the anti-Myc antibody.
The next series of experiments demonstrate the selectivity of the interaction between the CaR and Kir4.1 and Kir4.2 based on the failure of the CaR to coimmunoprecipitate with ROMK or Kir5.1. ROMK is predominantly expressed in the kidney where it plays a major role in maintenance of K+ homeostasis. Although ROMK activity can be regulated by the CaR (42), it is located in the apical plasma membrane of the thick ascending limb of Henle (TAL) (41, 42) while the CaR is in the basolateral membrane (33). We transiently transfected either Myc-Kir4.2 or ROMK into HEK 293 cells that stably express the CaR, and coimmunoprecipitation was performed using our anti-CaR antibody. Figure 4A demonstrates that both Myc-Kir4.2 and ROMK are expressed at detectable levels in the cell lysates. Figure 4B shows that only Kir4.2 was coimmunoprecipitated with the CaR, although ROMK was strongly expressed in these cells (Fig. 4A). Kir5.1 is expressed in the kidney (23) and may contribute to the basolateral K+ conductance by forming heterotetrameric channels with Kir4.1 or Kir4.2 (23, 39, 44). To test for interactions of the CaR with Kir5.1 and interactions of Kir4.2 with Kir5.1, we transiently transfected combinations of different cDNAs (CaR and HA-Kir5.1, CaR and Myc-Kir5.1, CaR and HA-Kir4.2, CaR and Myc-Kir4.2, Myc-Kir4.2 and HA-Kir5.1 or HA-Kir4.2 and Myc-Kir5.1) into HEK 293 cells and then performed coimmunoprecipitation using either our anti-CaR antibody or the polyclonal anti-Myc antibody. In the first two lanes in Fig. 5 where the CaR and Myc-Kir5.1 or the CaR and Myc-Kir4.2 were expressed, no immunoprecipitating antibody was used and no bands are seen when antibodies to the CaR, Myc, or HA were used for Western blotting. In the next four lanes, the anti-CaR antibody was used for immunoprecipitation, and the CaR is present in all four of these lanes (Fig. 5, top). Kir4.2 (HA-Kir4.2 lane 5, and Myc-Kir4.2 lane 6) coimmunoprecipitated with the CaR but Kir5.1 did not (neither HA-Kir5.1 lane 3 nor Myc-Kir5.1 lane 4) (Fig. 5). In Fig. 5, middle and bottom, an IgG band is also present near the top of the panels. In lanes 7–10, the last four lanes, the polyclonal anti-Myc antibody was used for immunoprecipitation. In lane 7, Myc-Kir5.1 is immunoprecipitated (middle), but the CaR is not present (top) because they do not interact. In lane 8, the CaR is coimmunoprecipitated with Myc-Kir4.2 as seen previously. In lane 9, Myc-Kir4.2 and HA-Kir5.1 are both present (middle and bottom, respectively), and conversely in lane 10, Myc-Kir5.1 and HA-Kir4.2 are both present (middle and bottom, respectively), indicating that these two proteins interact. These results demonstrate that the CaR selectively interacts with Kir4.1 and Kir4.2, but not ROMK or Kir5.1, and that Kir4.2 and Kir5.1 interact with each other.
Coexpression of the CaR inactivates Kir4.1 and Kir4.2 channels.
Members of the Kir4 subfamily, Kir4.1 and Kir4.2, are expressed in the kidney and may contribute to K+ transport in the distal nephron (19, 23). To determine whether the interaction of the CaR with Kir4.1 and Kir4.2 affects channel function, we coexpressed the channels and the CaRWT or the nonfunctional point mutant CaRR796W in Xenopus laevis oocytes. Experiments were done 3–7 days after cRNA injections. We tested CaRWT and CaRR796W function and expression using Gd3+ to activate the endogenous oocyte Cl− channels (transient outward current) (3). Oocytes expressing the CaRWT alone or with the channels had typical CaR-induced transient outward currents, but the oocytes expressing the CaRR796W mutant did not (representative tracings Fig. 6A, top).
Sweeps of basal currents (98 K+ ringer) are shown for water, each of the channels (Kir4.1 and Kir4.2), and each of the receptors (CaRWT and CaRR796W) as well as combinations of receptors and channels (CaRWT + Kir4.1, CaRWT + Kir4.2, CaRR796W + Kir4.1 and CaRR796W + Kir4.2). The currents in water-injected oocytes were indistinguishable from the vector-injected oocytes. Currents measured in oocytes expressing K+ channels show modest rectification. Coexpression of the CaRWT with either Kir4.1 or Kir4.2 significantly reduced currents, while coexpression with CaRR796W had no effect on these currents (see figure matrix in Fig. 6A). The magnitude of inactivation was similar in the absence and presence of CaR activation by Gd3+ indicating that the effect of the CaR on the channels is tonic in oocytes under these conditions. Figure 6B shows the current-voltage relationships (I-V curves) for the channels alone and coexpressed with CaRWT or CaRR796W. Basal currents for Kir4.1 and Kir4.2 at −120 mV were 8.8 ± 0.8 μA (n = 9) and 9.4 ± 2.3 μA (n = 12), respectively. At −120 mV, coexpression of Kir4.1, and Kir4.2 with CaRWT leads to current inhibition of 36 and 84%, respectively. Oocyte membranes for these same batches of oocytes were isolated, electrophoresed, blotted, and probed for Kir4.1, Kir4.2 and CaR protein expression. The expression levels of neither Kir4.1 nor Kir4.2 varied with CaR-coexpression (Fig. 6C). Thus a functional CaR protein is necessary for Kir4.1 and Kir4.2 channel inactivation. These results demonstrate that interaction of the CaRWT with Kir4.1 or Kir4.2 results in tonic partial inactivation of both channels but that a signaling-defective mutant CaR has no effect on channel activity.
To further define the effect of CaRWT-Kir4 interaction on the activity of channel, we performed coimmunoprecipitation using an anti-Myc antibody and HEK 293 cell lysates that stably coexpressed CaRWT and Myc-Kir4.2 or CaRR796W and Myc-Kir4.2. As shown in Fig. 7A, expression and membrane distribution of Myc-Kir4.2 were not significantly different in the cells that coexpressed with the CaRWT or CaRR796W. In Fig. 7B, right, the first two lanes are from samples without immunoprecipitating antibody (negative controls). The anti-Myc antibody isolated similar amounts of Myc-Kir4.2 in the cell lysates that coexpressed CaRR796W and Myc-Kir4.2 (lanes 3 and 4) or CaRWT and Myc-Kir4.2 (lanes 5 and 6), while a band representing the CaR was detectable only in the lanes with the cells that expressed CaRWT with Kir4.2. These results demonstrate that the nonfunctional mutant CaR, CaRR796W, does not interact with Kir4.2 with sufficient affinity to permit coimmunoprecipitation, and with Fig. 6 demonstrate that interaction of the receptor and channel is required for inactivation of Kir4.1/4.2 by the CaR. The presence of high (5.0 mM) Cao2+ or low (0.5 mM) Cao2+ did not alter the efficiency of immunopreciptation of the CaRWT of CaRR797W with the channels (not shown).
Cellular distribution and interaction of the CaR and Kir4 channel in rat kidney.
In HEK-293 cells that stably express both the CaR and Myc-Kir4.2, the two proteins were colocalized using our monoclonal anti-CaR antibody (16) and the polyclonal anti-Myc antibody (A-14) as primary antibodies (Fig. 8, A–F). Because both proteins are overexpressed, we observed some cytoplasmic staining. However, the staining is most intense in the plasma membrane and the staining for the CaR and Kir4.2 overlap in this region. Figure 8, G and H, shows staining for the CaR and Kir4.1 in rat kidney sections. Both proteins are localized to the basolateral membrane of the distal convoluted tubule (DCT).
To demonstrate interaction of the endogenous CaR and endogenous channels from kidney and to verify the specificity of the receptor-channel interactions in renal tissue, we prepared crude membrane and cytosol fractions from rat kidney and processed them for immunoblotting and immunoprecipitation using antibodies against the CaR, Kir4.1, Kir4.2, and ROMK. Specific peptide blocking shows significant levels of CaR, Kir4.2, Kir4.1, and ROMK (Kir1.1) in rat kidney membranes, but little channel or receptor protein in the cytosolic fraction (Fig. 9A). To confirm the interaction of the CaR with Kir4.1 and Kir4.2, we performed coimmunoprecipitation using our anti-CaR antibody for immunoprecipitation with duplicate samples of kidney membrane extracts (Fig. 9B). The first lane of each panel where no immunoprecipitating antibody was present was used as a negative control. A strong band at 130 kDa representing the CaR was immunoprecipitated by our anti-CaR antibody. The bands representing Kir4.2 and Kir4.1 were easily detected, whereas a band representing ROMK was not present (Fig. 9B). These results demonstrate that the endogenous CaR interacts with endogenous Kir4.1 and Kir4.2 in the kidney and that these interactions are selective.
The goal of the present study was to identify proteins that interact with the CaR in addition to heterotrimeric G proteins and that could contribute to its distinctive effects on transport in the distal nephron. Screening a human adult kidney cDNA library with the COOH terminus of the CaR as bait, we identified Kir4.2, an inwardly rectifying K+ channel that like the CaR, is expressed in the distal nephron. Since Kir4.1, a closely related channel is also expressed in the distal nephron, we chose to study it along with Kir4.2. We demonstrated that the CaR interacts with Kir4.1 and Kir4.2 when expressed in mammalian cells, but that it does not interact with Kir5.1 or ROMK (Kir1.1), more distantly related channels that are also present in the distal nephron. Both channels are expressed along the basolateral membrane of the distal nephron, and endogenous Kir4.1 and Kir4.2 coimmunoprecipitate with the endogenous CaR from rat kidney membrane extracts. Kir4.1 is present in the DCT and cortical collecting duct (CCD), but not the TAL, and Kir4.2 is also expressed in the DCT (19, 23). When expressed in X. laevis oocytes, the CaRWT reduces the currents from Kir4.1 and Kir4.2 regardless of its apparent state of activation, but the CaRR796W does not.
Kir channels are divided into seven subfamilies, Kir1–7, and all tissues express some subfamilies (12). Five subfamilies (Kir1.1, Kir2.3, Kir4, Kir5.1, and Kir7.1) are expressed in the kidney, but the function of only Kir1.1 or ROMK is defined to a significant degree (12, 24, 31, 44). ROMK, the apical small- conductance K+ channel, is located in the apical membrane of TAL and other segments and participates in K+ recycling across the apical membrane of the TAL for Na+ and Cl− reabsorption through the apical NKCC2 transporter. Loss-of-function mutations in this channel result in one form of Bartter's syndrome. The other Kir channel subfamilies are present in the basolateral membrane (19, 23, 25) where the CaR is also expressed (33). Here, we demonstrate that the CaR interacts with Kir4.1 and Kir4.2 and that they are coexpressed on the basolateral membrane of the DCT, and possibly other segments of the distal nephron. Other investigators have studied the K+ conductance of the basolateral membrane of the TAL and DCT (44) and showed that the conductance is ∼35–40 pS, pHi sensitive, and ATP independent (23), characteristics that are most consistent with homomeric Kir4.1, Kir4.2, or heteromeric Kir4-Kir5.1 channels (28). Since Kir7.1 is also expressed in this region, there are undoubtedly many different conductances composed of a range of proteins, and the molecular identities of the basolateral K+ conductances in the distal nephron are not known with certainty.
In addition to controlling second messenger levels, G protein-dependent signaling systems regulate Kir channels by mechanisms that involve protein-protein interactions. Stimulated GPCRs activate Gαβγ heterotrimers to release Gβγ subunits that can interact directly with Kir3 channels (designated as GIRK) to activate them, but this mechanism has not been shown to apply to other Kir channels (1, 5, 15, 27, 32, 34). The role of Gα subunits in the regulation of GIRK signaling has been controversial for a number of years (12). Recently, the NH2 terminus of GIRK2 and the COOH terminus of GIRK1 and GIRK2 were found to interact with the Gαi-GDP binding sites (18). RGS proteins that attenuate G protein signaling also inhibit GIRK channel activity, but the molecular basis for this effect is not defined (6, 9). Lavine et al. (21) reported that β2 adrenergic and dopamine D2 and D4 receptors interact directly with heteromeric GIRK channels (Kir3.1/3.4 and Kir3.1/3.2), but the physiological consequences of these interactions are not defined. Our results demonstrate a novel mechanism in that the CaR, a GPCR, interacts directly with Kir4.1 and Kir4.2 and inhibits channel function.
Previous studies have shown that the CaR couples to multiple G proteins (Gαi, Gαq, Gβγ, and Gα12/13) involved in distinct signaling pathways (14). Some of these signaling pathways play an important role in the regulation of ion homeostasis. CaR-activated Gαi inhibits the activity of adenylyl cyclase and reduces cAMP accumulation that abolishes hormone-stimulated Na+, Cl−, and K+ transport via the NKCC2 transporter (12). Two other signaling pathways are also involved in the regulation of ROMK functions by the CaR: 1) CaR-stimulated phospholipase A2 hydrolyzes cellular phospholipids and produces arachidonic acid metabolites which can inhibit ROMK activity (42) and 2) the activity of ROMK is regulated by membrane PIP2, the hydrolysis and synthesis of which can be regulated by CaR-activated phospholipase C and PI-4 kinase (16, 45). Many other GPCRs such as the ANG II receptor, the endothelin receptor, and the adrenergic receptor stimulate similar signaling pathways but do not lead to Na+, Cl−, and K+ wasting as does the CaR. Our finding provides a novel mechanism to explain how the CaR may regulate salt and water balance in the distal nephron.
We do not fully understand how activating mutations of the CaR lead to the Bartter-like syndrome because our understanding of the mechanism of the effects of the CaR on Kir channel activity is incomplete. The inhibitory effect of the CaR on Kir channel activity requires a functional receptor because the CaRR796W mutant has no effect. The inhibitory effect of the CaR appears to require interaction of the receptor with the channels because in addition to its lack of effect on channel activity, the CaRR796W mutant does not coimmunoprecipitate with the channels. The R796W mutation is in the third intracellular loop, a region of G protein-coupled receptors that interacts with G proteins and so is responsible for second messenger regulation via G proteins. However, this domain also interacts with other signaling proteins such as calmodulin, RGS2, arrestin, receptor kinases, 14–3-3 proteins, Src, and filamin. We do not know which, if any, of these proteins interacts with the third intracellular loop of the CaR, or if the R796W mutation affects more than G protein signaling. Many G protein-coupled receptors are leaky in that they catalyze guanine nucleotide exchange in the absence of agonist. If this were the case for the CaR, persistent PLC activity in the vicinity of the CaR-channel complex could result in reduced levels of PIP2 and channel inactivation. If this basal rate of guanine nucleotide exchange were of sufficient magnitude, further activation of the receptor with the addition of agonist could have no additional effect on channel activity. Alternatively, the interaction of the CaRWT and the channels could reduce the affinity of the channels for PIP2, having a similar effect. In both of these scenarios, the CaRR796W would not be expected to affect channel activity. If in fact the CaR regulates the channels in a tonic, ligand-independent manner, the CaR could have effects of different magnitude if it were expressed at varying levels or if its stoichiometry with respect to the channels varied for any reason. Our functional studies of the CaR-Kir4.1/4.2 channel activity were performed in X. laevis oocytes. Although we expect that in other experimental systems and the intact distal nephron, the CaR would inactivate these channels, other proteins may be present in the CaR-Kir4.1/4.2 complex that would result in a ligand-dependent pattern of regulation of the channels by the CaR. These questions will be addressed in future studies.
Our studies demonstrate that the CaR selectively interacts with Kir4.1 and Kir4.2 in heterologous systems and renal tissue, that they colocalize to the basolateral membrane in the distal nephron, and that this interaction results in the inactivation of Kir4.1 and Kir4.2 currents. These basolateral K+ channels are probably involved in K+ recycling for the Na+-K+-ATPase. Inhibition of the basolateral K+ conductance in the cortical collecting duct with Ba2+ inhibits vasopressin-stimulated Na+ transport, so inhibition of basolateral K+ channels by a different mechanism should have the same effect (35). The effects of inhibition of basolateral K+ channels in the TAL may be more complicated due to the importance of paracellular transport in this segment of the nephron but should reduce Na+ transport in the DCT and CCD where both Kir4.1 and Kir4.2 are clearly expressed. By this mechanism in addition to conventional second messenger generation, activation of the CaR which is the combination of CaR-Kir4 interaction and CaR-signaling could significantly inhibit salt and water transport in the distal nephron, consistent with the effects of hypercalcemia and the phenotype of patients with potent activating mutation of the CaR.
This work was supported by grants from the American Heart Association to C. F. Huang and R. T. Miller, National Institutes of Health (NIH) Grants DK-59985 and HL-41618, a VA Merit Review to R. T. Miller, and NIH Grant DK-56218 to M. F. Romero, and the Leonard Rosenberg Research Foundation.
Present address of A. Sindic and M. F. Romero: Dept. of Physiology and Biomedical Engineering, Mayo Clinic College of Medicine, Gu 9–210, 200 First St. SW, Rochester, MN 55905.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Copyright © 2007 the American Physiological Society


text:
"From her cookbook!"
Grandma's Peanut Brittle
0 recipe photos
READY IN:30mins
Ingredients Nutrition
- 354.88 ml sugar
- 473.19 ml water
- 453.59 g peanuts
- 14.79 ml baking soda
- 14.79 ml vanilla
- In a heavy 2 quart saucepan, over medium heat, bring to a boil sugar, vanilla and water.
- Stir until sugar is dissolved.
- Stir in peanuts.
- Set candy thermometer in place, and continue cooking. Stir frequently until temperature reaches 300 degrees F (150 degrees C), or until hardens when dropped in water.
- Remove from heat; immediately stir in baking soda.
- Pour immediately onto cookie sheet.
- Lift and pull peanut mixture( I do this with 2 forks) into rectangle about 14x12 inches.
- Let cool.
- Snap candy into pieces.
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text:
Newton's Laws of Motion
Newtons Second Law
Newton's second law states that a force acting on a object has the same amount of mass as the other object times its acceleration. Acceleration depends on two main things the objects mass and net force.
Newton's 3rd Law
Newton's third law states that for every action there has to be an equal reaction, but also there needs to be an opposite reaction. What that means is that on every object there has to be two forces acting on it.
Newtons First Law
"An object at rest remains at rest
and an object in motion maintains
it's velocity unless it experiences
an unbalanced force"
Johana Lopez & Fatima Guillen
7th Hour
April 1, 2015
As show on video number one that show every acting has an equal and opposite reaction. The act in this video was the ball falling and the opposite reaction is the ball coming back as soon as it touche the earth.the ball was the first object and the earth was the second object. Object number one gives force to the second object and the second object gives the same force but with unbalance force.
Video number two show a ball at rest and a opposite reaction the kick of a football player.As in video number one object number one is the the ball, but the opposite reaction dose not come form a object at rest it comes form object in motion . The foot gives the ball a force, but also the ball gives the foot the opposite reaction and the same amont of force. Over all the ball is go to air because it ill have more acceleration and has less mass.
Some force on two objects of different mass
will get a different accelerations changes in
motion. Picture number one are two person with the
same mass push tower each other with the same
force so there no accelerations . picture number
two the person has a less mass then the elephant
so there less acceleration. Picture number 3 is different form all pictures because now the object that has more mass is the one push the object with less mass which cause a bigger acceleration and a greater move. this happens because the elephant has more force the the person.
The box accelerations bigger because of the forces acting on it. If the object is bigger then the acceleration will be less, but if the box is smiler then the acceleration will be bigger. in the cause one of the boxes was bigger so if the person mass did not match the mass of the box the acceleration will be less. If the mass was the same then there will be a bigger acceleration. To find the acceleration you may used the formal F=ma.
The van is traveling at constant speed until an unbalanced force or outside force acts upon it. The outside force was the person driving the van. He/she suddenly pushes the breaks and causes the van to come to a sudden stop.
At rest
Outside force
The car has no motion until an unbalanced force acts upon it. The unbalanced force is a person pushing the car from behind. The person pushed the car hard enough that the car started moving.
Physical science book
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Newtons Laws of Motion
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Johana Lopezon 3 April 2015
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