text:
alert('start'); var date = new Date(); var curDate = null; do { curDate = new Date(); } while(curDate-date < 5000); alert('finish');Note, I borrowed this horrible example from stack overflow. If you're lucky, that example will not completely crash your browser. A much better solution is something like this:
alert('start'); setTimeout(function() {alert('finish')},5000);The obvious problem is that if you truly want to simply pause for 5 seconds in the middle of a really long method, the anonymous function is not going to help you out very much.. unless you do something like this
alert('start'); //lots of code var a = 'foo'; setTimeout(function(){ alert(a); //lots more code },5000);If you find this solution lacking, you're probably due for some refactoring of your code as you are probably writing highly procedural code and it's likely that javascript is going to cause you other, more serious problems. This is on stack overflow here, upvote if you think it's a good solution.


text:
Readers have reached out and asked how many repositories the index site has. They also wondered why it shows small number of them.
As of today, the index currently tracks over 900+ BigData and Machine Learning repositories and daily updates their status. It isn't, however, exactly obvious from the first look, and the site design does not help either; lack of search, no project count, and/or few broken links here and there.
Although all "issues" cannot be fixed right away, we can definitely spare some time for few low-hanging, easy ones. Among those, infinite scroll issue is looked into today.
The site is to scroll infinitely all the way to end. You do not need to click next pages or indexes to move around. The feature is built in that way so you can scroll through the entire collection of 900+ repositories and pick most appealing ones as quickly as possible.
Nevertheless, it has few glitches. Firstly, it's not easy to see if there are more pages to go.
For that, a spinning indicator is added at the bottom of page when the next page is loading and there are more to see.
Secondly, when you click a project and come back, the site loses its original position and refreshes its content. This is rather irritating, and it would be ideal to keep the context related to old position. Few hours of investigation reveals that it involves a bit more work than expected. (Here's a good link you might like to read about it.)
It will be looked again (promises!). As of now, when a project is clicked, a new tab will be open for further detail and you can keep your old context for further search.
Hope you like the update, and please let me know if you have further question!
Looking into adding your repo? Any suggestion? Comment? Send your feedback to email@example.com or tweet to @stkim1!
Looking for more BigData or Machine Learning repositories? You can find a lot more tools, frameworks and libraries at PocketCluster Index.
Subscribe for upcoming posts!
Join the channel!

subset: commoncrawl


text: Geronimo! began when balloonatic Jihan Zencirli starting gifting oversize, perfectly round balloons with long, flashy tails to her friends and family as markers of birthdays or important events. It didn't take long before intrigued strangers approached the woman-about-LA with a massive cluster of balloons trailing out her car window. "Where did you get those fabulous balloons?" they'd ask. Jihan's answer: "I made them." And so a business was born that custom designs balloons as gifts or festive additions to party décor. Geronimo! offers the 36-inch round latex balloons in a range of styles that differ in tail length and variety of ribbon, hand-cut paper, and tissue used. Single-order prices span between $55 and $125. So whether you give a Geronimo! balloon as a shower present or incorporate a grouping into your reception design, either way the balloons will have your eyes and your spirits looking up. —Phillip B. Crook


text:
Every electronic circuit will work as good as its power supply. However this is often overlooked. There are two main families of power supplies: the linear power supply and switch mode power supplies. Linear power supplies are easier to manufacture and to troubleshoot. But they have the disadvantage (especially for high power), the circuit is more complicated with the results of less power and heat faster than the switch mode power supply.
For a manufacturer, the choice of type of power supply will obviously influence the cost of manufacturing or integration. It should be noted that any hardware vendor does not make the most of its own power supply, and often will buy from a specialist. Creating a switching power supply by amateur is relatively difficult, even with the advent of integrated components provide most of the work.
In any case, strongly suggest a beginner in electronics to start with the realization of linearpower supply. Similarly, you are advised not to solve the problem of switch mode powersupply if you do not fully understand. However, in some articles that this will be discussed about the linear power supply.
Tags: linear power supply switch mode power supplies type of power supply


text:
Ahmed Bakr
Research & Consumer Insight Professional
- Toronto, Canada (April, 2018)
- +971526623833
- [email protected]
"One of the world's leading independent providers for strategic market research and analysis, with over 40 years' experience in publishing market reports, bespoke consulting projects, business reference books, and an integrated online database "Passport", covering various consumer goods industries"
Implemented a thorough research methodology to gather insights over the home & tech vertical and publish market reports and data analysis showcasing industry trends & insights.
"To consistently provide innovative premiums RESEARCH Human Capital Solutions that help organizations capitalize on their most important asset...People".
Used different assessment methods and tools to conduct assessments (evaluations) for seniors and managers, with a development plan for every individual. Worked with the marketing team on several projects on branding and developing marketing plans.
"Consortium of organizations and individuals who explore emerging issues in talent management, staffing, recruiting, employee development, retention and leadership development"
Completed an insight research project on two talent disruptors, Co-creativity and entrepreneurship, to recognize possible trends that would keep firms ahead of competition.
"Global independent market research company ranking third worldwide among research firms"
Designed profitable research proposals, handled presentations with clients, conducted meetings that liaised project status between clients and internal departments.


text:
"Ingmun" Surname
Frequency of "Ingmun" Surname in the US
2000 US Census
The surname "Ingmun" is not included in the US Census Bureau's ranking of surnames with 100 or more people. Since fewer than 100 people with this surname were included in the 2000 Census, it is relatively uncommon.
Source: "Frequently Occurring Surnames from the Census 2000", US Census Bureau.
"Ingmun" Graves on Histopolis
Histopolis currently has 1 grave(s) with the surname "Ingmun".
Search the Histopols Grave Index for the surname "Ingmun".
Resource Links for "Ingmun"
Sorry, there are currently no resource links for the surname "Ingmun".
Do you know of a web page containing information about this surname that would be useful to genealogy or history researchers? Please add it now! (Free registration required)
Surnames that Sound Like "Ingmun"
The surname "Ingmun" has a Soundex code of I525. The following 90 surname(s) may sound similar to "Ingmun" since they share the same Soundex code.
Unconfirmed Surname
Histopolis has an entry for this surname but it might be the result of an error in the source information, since:
- It was not listed in the US Census Bureau list of most frequent surnames for either 1990 or 2000,
- Histopolis currently has few entries (1) for this surname in the Histopolis Grave Index, and
- There are no currently no links to other sites with this surname.
To confirm this surname, add a link (above) to a page with information about this surname or that references individuals with this surname.


text:
Real Football Manager Edition
|Publisher: Gameloft :: Developer: Gameloft
|Download Game
Discover the most extensive football management simulator for your mobile. Choose your club from 8 leagues and more than 200 teams. All transfers have been updated for the 2007/2008 season for more realism.
Plan your players' training schedule up to the finest details to improve their stats and ensure they last a full 90 minutes. Discover the newest rising football star in the world with the help of your scouts who scour the globe looking for young talent. Annalyze your opponents' performances and aim for the championship!
|Showing 0 to 10 of 20, most recent first.
|Add Comment
The new edition of real football is just announced and if you want to download it then get hurry to download it. You may get some help from the writing paper review to know about this latest edition in detail.
The game have 8 leagues and more than 200 teams in which you can use.- Gary McClure
This version is something that will make your nerves tight. The game gets so much into your head that you would think about it all the time whether you go. It is much better you do not play this If you don't want all those stress. cable tv alternatives
This is a concept that is really hard to relate with. But for those who have understood it, things shall be really easy. I hope to see a much more exciting updates that shall make usefulness much more easier than now. directv family package
This is an awesome game. I have been playing this game for more than six months and I found that it is very much addictive. We have the responsibility to choose the proper players for our team. Really interesting!
Amazing post
Amazing post
I coached my dream club
i kind of need cheat to be able to upgrade my stadium, traninig pitches, and so on
Any idea why the game would crash on 01/02/2010? Currently playing in the championship in england and just can't get past this date when playing on my phone.
Have tried saving it then progressing but nothing works, the loading screen just stays there for hours on end. Quite frustrated as I'm horsing my way to the league with 15 points ahead of 2nd placed side.
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We will not show your email on the site, but if we needed to contact you we find them useful. Also we will not pass on any personal information to third parties. For more information please see our privacy policy.


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(630) 385-2646
Much of your anxiety stems from your reaction to any given situation. Control your emotions with assistance from Yorkville's Ron Ahlberg and Associates, LCSW, CADC. Our therapists coach you to change your internal dialogue and combat those feelings. Through the use of cognitive behavioral techniques and behavioral exposure therapy, our therapists help you to identify and confront your feelings of anxiety at the source.
Keep in mind that anxiety is rarely the fault of a particular event. It's actually our thoughts about the event that determine the physiological response. Ron Ahlberg helps you to identify those thoughts, embrace them and flip the script. You'll learn to automatically defend yourself against negative thinking through the use of an internal dialogue. Together, we can change your situation and eliminate unhelpful thinking and behavior. Choose to take back your life and way of thinking. Schedule a consultation with Ron Ahlberg and Associates, LCSW, CADC today.


text:
ratchanok intanon
Ratchanok Intanon !!!
- By toppinkul
- On 14/08/2013
- In Top Variety
Ratchanok Intanon !!! In August 2013, Ratchanok won the BWF world championships, beating Olympic Gold medallist Li Xuerui 22-20 18-21 21-14 in the final. At the age of 18, she is the youngest world champion ever !!!
"Thai badminton as RATCHANOK INTANON, the history of the new page to grab the Championship "world champion nachip" China. After the fall of # 1 Li Rui always hand the world local 1-2-game owner khwanjai assuming a sport first Thai woman and youngest world champion at 18 years of age in.
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text: Wolfeboro Branch Recreational Rail Trail Activities and Amenities Activities and Amenities The Wolfeboro Rail Trail is 11 miles long and begins at Depot Street in Wolfeboro and ends at Route 16 in Wakefield. This trail is a great four season trail is not only exceptional for walking and biking, but it is one of only three trails under DRED management that have rails intact and are used recreationally by local rail car clubs. The trail begins in downtown Wolfeboro just across the street from Lake Winnipesaukee's Back Bay and the trail's usable surface ends at Fernald Station, Rt. 109, Wolfeboro, NH. The Wolfeboro Rail Trail and has beautiful scenery, especially along the two causeways along Lake Wentworth and Crescent Lake. Permitted Uses Hike, Bike, Horse, Snowshoe, Nordic, Snowmobile, and Mushing. ATV and Trailbike are permitted with snowcover only. Parking Areas Wolfeboro –Parking at the old railroad Depot on Depot Street. Take Route 28 to Route 109 (North Main Street) and turn right on to Railroad Ave. Just ahead on Depot Street is the old Wolfeboro Depot. Wolfeboro – Parking at Fernald Station off Route Rt. 28/109 North (3.2 miles from Wolfeboro Center ) to the flashing yellow light intersection with Rt. 109 and take a right. Fernald Station will be 1/4 mile on your left. Organizations that work with the Bureau of Trails to help maintain this trail are: Trails Rails Action Committee (TRAC) Cotton Valley Rail Trail Club (CVRTC) Wolfeboro Snowmobile Club Seven Lakes Snowmobile Club


text:
Mr. Driller
This article may need to be rewritten entirely to comply with Wikipedia's quality standards. (April 2016)
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|Mr. Driller
North American PlayStation cover art
SCEE (EU, PlayStation)
Virgin Interactive (EU, Dreamcast and GBC)
|Yasuhito Nagaoka
|Hideo Yoshizawa
|Kaori Shinozaki
Taro Hasegawa
Yasuko Kusakabe
|Go Shiina
Tomoko Tatsuta
|Arcade, Various
October, 1999[1]
|single player
|Arcade system
|Mr. Driller: Namco System 12; Mr. Driller 2, Mr. Driller G: Namco System 10
Vertical orientation
Mr. Driller[a] is a series of video games developed and published by Namco. Mr. Driller puts the player in the role of a driller moving down through screens of blocks, having to keep his air supply from running out while avoiding being squashed by falling blocks. Games in this series have been released for PlayStation, Dreamcast, WonderSwan, Game Boy Color, Game Boy Advance, Nintendo GameCube, Nintendo DS, WiiWare, Xbox Live, Windows, and iOS in addition to the arcade versions. The main character of these games is Susumu Hori, the son of Taizou Hori, who was the star of the original Dig Dug game.
The project that would become Mr. Driller was originally intended to be a third game in the Dig Dug series, under the working title of Dig Dug 3. Hexagonal blocks were briefly considered, but this idea was abandoned when chain construction proved to be too complicated. Dig Dug 3 was initially developed unofficially by a few Namco staff; however, work on the game was suspended while R4: Ridge Racer Type 4 was being completed. However, Namco executives were impressed with the game and decided to support it as an official project.
In Mr. Driller, after a character is selected and a country is selected, the player then drills a seemingly endless onslaught of colored blocks, ending with that country's given mileage (given in meters). The levels are randomly generated, and the blocks can merge with other similarly colored blocks, therefore disppearing after 4 or more blocks merge. There are 4 main colors of blocks: red, blue, green, and yellow. There are also 3 special kinds of blocks: white blocks, which are pale blocks that don't merge with other similarly colored ones; crystal blocks, blocks that last for around a few seconds before disappearing; and X-blocks, brown crate-like boxes that take 5 drill hits to disintegrate, and also take off 20% of the player's air supply. The player has an air supply that acts as his vitality gauge, and when it is under 30%, the player's complexion will turn blue and a bubble with an air capsule will appear over his head. To keep the air from running out, the player can collect air capsules that replenish the driller's health by 20%. A life is lost when the player's air runs out, or if he/she is squished by falling blocks. The gameplay has been described as "Dig Dug meets Tetris."
- Susumu Hori - the series's main protagonist and the highest-ranked Driller in the world, and the son of legendary Driller, Taizo Hori (of Dig Dug fame). He is a little crazy, but is still a good-natured, courageous young boy with a strong sense of justice. He is Japanese, has brown hair and eyes, and wears a pink and light blue jumpsuit and helmet. His performance in the game is all-around, with normal walking speed and a normal air-consumption rate. He is usually recommended for intermediate players. Susumu is voiced by Mitsuko Horie.
- Puchi - a genius talking wonder dog and companion to Susumu. He has been Susumu's closest confidant ever since Susumu found him abandoned as a little puppy and nursed him back to health. He is a white dog with brown spots. He was first playable in Mr. Driller G. In the game, Puchi moves slower than Susumu, but his air consumption is slower as well. He also has the ability to jump up two blocks high. In the Scenario Driller mode of Mr. Driller G, he follows Susumu in one of the stages and barks when he is near a hidden Dristone. He is usually recommended for beginning players. Puchi is voiced by Mitsuko Horie.
- Toby Masuyo- Susumu's mother and star of Baraduke, an ex-soldier of the United Galactic Space Force (UGSF) and took part in the battle of Alien Sector in 1985. Masuyo is the mother of Susumu and Ataru. She used to be married to Taizo Hori, but for unknown reasons, they are separated. She is Japanese and has short light brown hair with dark brown eyes. She wears a yellow space bio-suit with orange gloves, blue/green boots and white belts going around her body that connect to a jet pack. She is a support character and assists her son in the Mr. Dig games.
- Taizo Hori - Susumu's father and star of Dig Dug, a legendary Driller and chairman of the Driller Council. He is the hero of the 1982 Dig Dug Incident and is a globetrotting adventurer. He is Japanese, has brown eyes and a goatee, wears a white suit with red chevrons and a blue belt, red boots, blue gloves, and a white-and-blue helmet. He was first playable in Mr. Driller G. In the game his walking speed is slow and his air consumption rate is fast. However, he can make blocks disappear much faster, making his descent speed quicker as well. He is usually recommended for expert players.
- Anna Hottenmeyer - Susumu's friend and fierce rival and the second-highest-ranked Driller in the world. She is German, and has blue eyes, blonde hair, and wears a white jumpsuit with a pink star on the front, pink sleeves, and blue goggles. She was first playable in Mr. Driller 2. In the game, her performance is only slightly different from Susumu, with a slightly faster walking speed and a slightly faster air consumption rate. She is usually recommended for expert players. Anna is slightly older than Susumu by two months. Anna is voiced by Mitsuko Horie.
- Ataru Hori - Susumu's older brother. He works as an unlicensed Driller ever since he left home after an argument with his father, Taizo, and has been trying to find his purpose in life ever since. He is Japanese, has brown eyes, has gray hair, and wears a gray suit with a white star on the front, a gray helmet, and a red scarf. Normally has a black rabbit named Usagi (or Rabbit, in English-speaking countries) that follows him on his travels. He was first playable in Mr. Driller G. In the game, he is able to move very fast, but has a fast air consumption rate. He is usually recommended for expert players.
- Holinger-Z - A robot created by Dr. Z to take on jobs that are too dangerous for humans, and serves as Anna's butler-robot. He is a gray robot who can morph into the form of a drill. He was first playable in Mr. Driller G. In the game, Holinger-Z moves fairly slow and has a slow air consumption rate. His special feature is that he can withstand a fallen block once before dying; when a rock falls on him, his head will act as a "barrier" and the rest of the body continues to drill. He is usually recommended for beginning players. Holinger-Z's name is based on Mazinger Z.
- Usagi - Also known as Rabbit in English-speaking countries. The companion of Ataru Hori. He came from outer space and crashed to earth. Ataru saved him from the wreckage, and has since befriended him. He has normally been an unlockable character in the game since the introduction of the six standard playable characters in Mr. Driller G. In the game, he is able to move very fast, but has a fast air consumption rate. He also can jump up two blocks akin to Puchi, make blocks disappear much faster akin to Taizo, and destroy three blocks instead of one. Rather than talking, he makes unusual sounds. In a secret Scenario Driller stage in Mr. Driller G, he follows Ataru and notifies when he is near a hidden Dristone. It is debated whether his skills make gameplay easier or harder, but the former is more likely because of his numerous skills.
- Dr. Z - a brilliant scientist who sends the Driller team out on various missions around the world. He has gray hair, a round cucumber-shaped nose, and wears a lab coat and tie. He bears a slight resemblance to Dr. Ochanomizu from Astro Boy.
- Dr. Manhole - a drill scientist who has been mocked by fellow scientists, and since then has made attempts to destroy the world with giant drills. He sometimes works with the Ankoku Drillers. He was the main antagonist in the Scenario Driller mode of Mr. Driller G and Mr. Driller Drill Land (disguised as the founder of the amusement park), and appears in the Pressure Driller mode of Mr. Driller Drill Spirits, possibly the one who made the giant drill that closes in on the player.
- Ankoku Drillers - Sometimes called Team Ankoku, these are three drillers who work with Dr. Manhole to stop Susumu and his friends. The leader is Keel Sark, who is fast and is said to have ninja skills. The other members are Eguri Hatakeyama, a mean-tempered girl who uses a whip-like drill and has a crush on Ataru Hori, and Kowasu, an android stolen from the Driller laboratories who holds respect towards Taizo Hori, and uses a three-direction drill. They have so far only appeared in Mr. Driller G for the PlayStation and Mr. Driller Drill Land for the Nintendo GameCube; they are secretly playable in the former's Versus mode after certain conditions are met.
- Undergrounders - strange blue creatures that inhabit the underground world. They are the ones usually responsible for the numerous blocks that often invade the Earth. They are said to be very intelligent but a little careless. They are ruled over by a king. In the game, they occasionally appear behind drilled blocks and offer a small additional bonus. They also appear when a certain score amount is reached and another block is drilled; the undergrounder holds up a sign that says "1-Up," giving the player another life. There are apparently also undergrounders in space, except they have been colored as either purple or yellow, depending on the game.
List of games[edit]
- Mr. Driller (Arcade, 1999); ported to: Microsoft Windows, Game Boy Color, Dreamcast, PlayStation, WonderSwan Color, Mobile phones, PlayStation Store, PlayStation Classic, and iOS
- Mr. Driller 2 (Arcade, 2000); ported to: Microsoft Windows, Game Boy Advance, Wii U Virtual Console (GBA version)
- Mr. Driller G (Great) (Arcade, 2001); ported to: PlayStation, PlayStation Store
- Mr. Driller A (Ace): Fushigi na Pacteria (Game Boy Advance, 2002); ported to: Wii U Virtual Console
- Mr. Driller: Drill Land (Nintendo GameCube, 2002)
- Mr. Driller: Driller Puzzle (Mobile phones, 2003)
- Mr. Driller Drill Spirits (Nintendo DS, 2004)
- Mr. Driller Aqua (Mobile phones, Android, 2006, 2011 for Android version)
- Mr. Driller: Driller Puzzle S (Mobile phones, 2006)
- Mr. Driller Online (Xbox Live Arcade, 2008)
- Mr. Driller W (World) (WiiWare, 2009)
- Mr. Driller: Drill Till You Drop (DSiWare, 2009)
- Mr. Driller (iOS, 2009)
- Mr. Driller VS (Mobile phones, 2010)
- Mr. Driller for Kakao (iOS, Android, 2015)
- ^ a b Mr. Driller at Arcade-History
- ^ http://www.esrb.org/ratings/search.jsp
External links[edit]
- 1999 video games
- Arcade games
- Bandai Namco Entertainment franchises
- Dreamcast games
- DSiWare games
- Game Boy Color games
- Game Boy Advance games
- IOS games
- Mobile games
- Namco games
- Namco arcade games
- PlayStation (console) games
- PlayStation Network games
- Puzzle video games
- Video game franchises
- Windows games
- WonderSwan Color games
- Video game franchises introduced in 1999
- Video games developed in Japan
- Single-player video games


text:
Forrest Gump
American Dreamz
Red Eye
View from the Top
Memoirs of an Invisible Man
The Flintstones
Eye for an Eye
Cast Away
What Lies Beneath
All Elizabeth Lapp films
Movies.com, the ultimate source for everything movies, is your destination for new movie trailers, reviews, photos, times, tickets + more! Stay in the know with the latest movie news and cast interviews at Movies.com.
Copyright © 2019 FANDANGO MOVIES.


text:
- Preferences are only preferences. Personality preferences help us to understand our tendencies. Some of our tendencies may be quite pronounced and may color the lenses through which we view our surroundings. My experience is that rather than introduce constraints, understanding this creates options. Better understanding how I interpret and respond to my environment offers me options for growth and development. Understanding how others interpret their environment gives me the opportunity for adaptability and flexibility.
- Preferences do not equal behaviors. Preference is about my most natural inclinations for reacting and responding. It also colors how I interpret the behaviors of others. My behavior is only dictated by my preferences when I am blind to those preferences. Knowledge is power.
- The focus of assessment should be developmental. Leadership assessment should be about improving leadership effectiveness so when assessment is used for hiring or promotion, don't try dressing it up as leadership assessment. Assessments that are validated for developmental purposes may not be validated for hiring and promoting. In addition to ethical concerns, this can also have serious legal implications.
- Don't be lazy. People are complex so don't use personality assessment as a substitute for listening and asking good questions. People are not labels so don't use them.
- Confidentiality is supreme. Leadership assessment should be confidential. If I choose to share an insight that I have learned about my natural preferences or someone else's perceptions about my effectiveness then it is my choice.
Contact Discovery Learning, Inc. to learn more about incorporating Leadership Assessment into your Leadership Development Program.


text:
|Some books by Don Blanding, a prolific writer in Hawaii.
|The upper photo shows the braiding of the haku lei. The raffia fiber is almost in the middle of the plant sprigs, meaning there is a lot of overhang of the plant material that will later be trimmed.The upper lei is hala pepe (the yellow leaves) and a'ali'i native plants.
This is the finished, braided style haku lei by Kiana of the Maui Native Nursery,
which specializes in native Hawaiian plants.
After my lesson in lei making, she gave me the lei. I felt honored. The lei was also fragrant, not like a strong floral perfume, but with a very gentle and sweet fragrance.
|Flowers and leaves everywhere!
|I'm pretty sure this is hala pepe.
Photo taken at Native Intelligence in Wailuku.
|It surprised me to think that people have been signing petitions for centuries, for causes they believed to be important. Despite collecting hundreds or thousands of signatures, Hawaii was still annexed by the US government. I'm reminded of how 20,000 Maui signatures were collected for a ballot initiative on GMOs last year. I guess petitions are nothing new in Hawaii.
|Taro anatomy poster.
Mamaki plants sold by Maui Native Nursery. Mamaki tea is considered a healing tea. Mamaki likes cooler, damp places to live and will supposedly thrive in Haiku.
|The treasured maile plant. My first time seeing a living maile plant. Maile is used in maile leaf leis, often in weddings or as part of blessing ceremonies for a new home, building, or grand opening of a store or event. Maile used to grow wild freely, but is rarer and harder to find these days. It has fragrant leaves.
|Kauna'oa - a stringy yellow vine found along some beaches and used in lei making.
|A haku lei using kauna'oa. The other plant material is unknown.
|The upper lei is the haku lei I finished making at the May Day Lei Heritage Festival at the Bailey House Museum. It's definitely a beginner lei. The plant bunches are sticking out everywhere and it's not tightly braided. The raffia gets tangled in the plant stems so it has to be constantly smoothed out and untangled while holding two or three plant bunches from falling out! The lower lei is the one made by Kiana, who makes the braiding look deceptively easy. Her lei is very tightly woven and neat! I tried unsuccessfully to give away my haku lei in the spirit of May Day, but perhaps it was just as well..
my beginner haku lei was gently falling apart.
Making Haku Leis With the Native Hawaiian Plant Society
Biography of Don Blanding and list of books
Information for research assistance for Ku'e Petition Descendants
Lei Heritage Festival, May Day event guide book:


text:
Are All S&P 500 ETFs the Same?
The S&P 500 is the benchmark by which thousands of money managers overseeing trillions of dollars in assets are judged. For passive index investors who believe that active management doesn't add value over the long run, choosing index-tracking exchange-traded funds tied to the S&P 500 helps them meet their goal of matching the market's overall return.
But many different ETFs track the S&P 500. Which one makes the most sense for you? Let's look at some of the more popular S&P 500-tracking ETFs to pinpoint their differences.
A look at three top S&P ETFs
The biggest S&P-tracking ETF is the SPDR S&P 500 , affectionately known as the Spiders. It was the first exchange-traded fund product to gain widespread use, and it's now a giant in the industry, with $140 billion under management. Its expense ratio is 0.09%, and with average volume of about 133 million shares daily, the Spiders trade $22 billion worth of shares each day -- meaning it takes less than seven days for the entire outstanding share-count to turn over.
Much smaller is the iShares Core S&P 500 ETF . With about $43 billion under management, the iShares ETF comes with lower expenses of 0.07%, but much lower average daily volume of just 3.8 million shares. Like the Spiders, its share price roughly corresponds to a tenth of the value of the S&P 500 index.
Finally, the Vanguard S&P 500 ETF rounds out the major S&P ETFs. Unlike the other two ETFs, its share price bears no resemblance to the index's actual value. With less than $10 billion under management and barely 1.1 million shares trading hands each day, it's also the least liquid of the three ETFs. Yet its expense ratio of 0.05% is also the lowest of the three.
Does an S&P 500 ETF by any other name perform as well?
From a performance standpoint, all three of these ETFs perform the way you'd expect, with returns that trail the S&P 500 index by roughly the amount of their respective expense ratios over the long run. From year to year, ETFs sometimes manage to do better or worse than that expected return, as trading strategies and handling special events like additions and deletions from the index introduce some variables.
For investors, though, you also have to incorporate trading costs, both in the form of commissions and bid-ask spreads. With the largest number of shares and greatest trading volume, the SPDR ETF has the narrowest spreads and the most consistent liquidity. But the iShares ETF also does a relatively good job of keeping bid-ask spreads relatively narrow. Spreads for the Vanguard ETF tend to be higher, making it a more costly choice if you trade frequently, as a $0.01 greater spread amounts to about 0.13% -- or more than two years' worth of expenses for that ETF.
Finally, in considering costs, keep in mind that some companies offer commission-free trading for certain ETFs. Vanguard offers its ETF at no commission, while Fidelity and TD Ameritrade both offer the iShares ETF commission-free.
Choosing the right ETF
In the end, which S&P ETF you pick isn't likely to make a huge difference to your total return. Nevertheless, understanding the differences between similar ETFs is helpful not just in evaluating these ETFs but also in identifying the issues you should look at when you're looking to invest in any ETF.
To learn more about a few ETFs that have great promise for delivering profits to shareholders, check out The Motley Fool's special free report "3 ETFs Set to Soar." Just click here to access it now.
The article Are All S&P 500 ETFs the Same? originally appeared on Fool.com.Fool contributor Dan Caplinger owns shares of iShares S&P 500 ETF. You can follow him on Twitter: @DanCaplinger. The Motley Fool has no position in any of the stocks mentioned. Try any of our Foolish newsletter services free for 30 days. We Fools don't all hold the same opinions, but we all believe that considering a diverse range of insights makes us better investors. The Motley Fool has a disclosure policy.
Copyright © 1995 - 2013 The Motley Fool, LLC. All rights reserved. The Motley Fool has a disclosure policy.


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- what conditions are covered by the policy
- how long is the period covered by the policy for a given condition
- what financial limits, if any, are in place for a given condition
- what options are available for customization to ensure that the plan fits you and your pet specifically
Wednesday, 1 May 2013
Travel insurance for your pet?
5 comments:
They provided me a phone number to register at- but it's a bit hectic. Its easier to just find a computer and register that online. It saves you 20 minutes.ReplyDelete
compare travel insurance
Travel and tourism - the two enchanting fields, contrive their pattern of services from time to time.Rentals car dealsThey are an indispensable part of both professional and personal lifeDiscount on Rental cars. Assuring services to the point of ultimate subtlety and due reference, Blue Bird Travels proposes itself as a travel agent of the 21st century.Traveling Dreams Our output is calmly in itself and between the implications and performance - there is a satisfying equipoise of response.Tourism Research ,states that our vision sees shinning in and through the competition - the partition lines of excellence and dedicated teamwork leading to the actuality of success.ReplyDelete
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What do you know about keyloggers? You know that person can find this software on their device? But what if this is a remote keylogger? Click on a link, guys, you won't regret itReplyDelete


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Nestled in Moorestown's exclusive and very private Cobblestone Court enclave, this magnificent custom home features dramatic architecture and high end finishes throughout. The unique character of this imposing residence is obvious from the moment you cross its cobblestone driveway entrance and traverse its circular drive surrounding a landscaped oasis replete with water fountain. Through handsome oak double doors; you enter a soaring 20+ foot double foyer complete with faux painted walls, a curved stairway and marble flooring ? a dramatic introduction to a home that has been masterfully designed for comfortable living and gracious entertaining. To the right off the foyer is a spacious dining room, while straight ahead is the great room, with voluminous double-height ceiling and views of the back patio, entertaining space, and pool through glass doors and large windows. To your left is a beautifully appointed living room, beyond which is located an office/music room featuring floor-to-ceiling wood paneling, a fireplace and bay window. The rich wood paneling continues as you move toward the back of the home, where you'll find a professionally appointed bar, complete with banquette and another fireplace. From this handsome room, you cross back through the great room and into the large, modern kitchen featuring all high-end appliances, granite countertops, and a cozy breakfast room overlooking the pool and grounds. The kitchen is connected to the dining room via a butler's pantry with built-in cabinetry, providing welcome convenience and utility during your private dinner parties. Heading through the hall/butler's pantry toward the attached garage with 3 spaces, you'll find an oversized laundry area, mudroom/drop space, and access to the garage or side yard. Also just off this hall is a second half bath, with another off the foyer/living room. The side door provides access to a detached garage with another 3 spaces, bringing the total to six ? a rare benefit for car enthusiasts. Heading upstairs via either the main or back stairway, you'll find the sumptuous master suite, complete with private balcony, sitting room, huge closet, triple chambered master bath, and coffee room. Privately situated down the hall are four more bedrooms, each with its own private full bath. On the lower level, a huge, lavishly finished basement affords even more living space and ample storage, including a gym, arcade rm, ent. space, TV rm, wine room and tasting room.
|New Value: $2,750,000


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The TU/e Intro day for new colleagues
Feel welcome at the TU/e together with your partner. We frequently organize English spoken 'Welcome@tue days'. These days are specifically designed for the university's new employees and their partners. We warmly invite you and your partner to participate in one of these days. The two of you can participate together, but your partner can also participate independently, for instance if she or he joins you later. Also, if you work with one of the institutes on campus (like FOM, ICMS, ILI) we kindly invite you to attend one of these days.
These are the dates and times
The Welcome@tue days take place on Fridays, from 8.30 am till roughly 6 pm. The next editions are scheduled for:
- 5 April 2019
- 24 May 2019
- 14 June 2019
- 6 September 2019
- 4 October 2019
- 29 November 2019
These are the objectives of the introduction day
We organize these days with several objectives in mind. First, we would like to extend a word of welcome to you. We will provide you with useful information about the TU/e, Eindhoven and The Netherlands. Additionally, we aim to help you and your partner to get to know other new colleagues at the TU/e so you can expand your network. And last but not least, we want to introduce you to the Hub in the Eindhoven city center where you can meet other internationals.
Our ultimate goal of these days is to help you feel welcome and at home on our TU/e campus.
About the program
During these welcome days different speakers will provide you with useful information about our university, the city and The Netherlands. We will guide you on an interesting campus tour with visits to, for example, our sports center.
During your lunchbreak, we will be joined by representatives of the TU/e network of international accompanying spouses and career support initiative. They are there to welcome your partner, to answer questions that she or he may have and to inform your partner about the special programs provided by our university.
Sign up now and don't miss the intro day: Welcome@tue
Approximately one week before the introduction day for which you have enrolled, we will send you a reminder, the program of the day, and the list of participants. Feel welcome and join other colleagues.
Click here for registration.
To enrol your partner, please send an e-mail to: email@example.com
Do you want more information?
Please send an e-mail to: firstname.lastname@example.com
Your hosts of the Intro days are Femke Verheggen and Susan Lambriks.


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I feel bad that I don't spend more time writing and keeping up on post and what is going on in the process of finishing "the book" more regularly. Still the process of this book is filled with a lot of down time, hours spend in front of this old laptop, scheduling shoots, and connecting with people on creative date ideas. I kind of think that people don't want to hear about these simple moments and leg work that is going on. But then again it's a part of the process and there are so many incredible moments that happen when I am just working alone. There are moments while working on the book I wish others could experience the feelings and moments I get to have. But I think that this site is that way for people to stay connect to this project and Sarah's dream, and everything that will and has come out of it.
It's the emotions of making and working on this book that I want to convey to people. It's all of these feelings that I get to feel and have to face while making this book that are so monumental. All of the ups and downs that make every moment perfect, scary, upsetting, and beautiful; that I hope people will get to hear about and understand. I am really writing today to tell you about how editing is going. I am still working on editing the photo shoot that took me out to Owosso. And let me tell you it's moments like these I need, I hope for, and I am thankful for. While sitting and finishing the last few photos I was smiling! Excited! And having an overwhelming excitement as I can see this book still coming to life. Not because of how happy I am with how the photos have come out, but because they caught moments that beautiful and meaningful. They are just another reason of why this book will be special. It's these moments I wish I could share will everyone. This internal joyfulness and excitement; the feelings you get when your heart flutters and you could almost cry because you know deep down in your soul that it is well; It's almost the same feeling I had when I fell in love with Sarah; and it's that understanding of how little I am in the wake of how big and meaningful this book and dream really is.
I want to tell you that everyday editing is like this. The overwhelming of emotions of joyfulness, but in reality it is not. The ups and downs of emotions come quick. The downward emotions may come more, but when there are these uplifting moments I want you to know about them, because of how fantastic they are and how they truly out weight the downs. And I think that not only while working on this book, but also in life, even with all of the dark and downward emotions we feel, that we need them to enjoy the light and better side of life. We need the dark to prove the light. We need sadness to make happiness have more of an importance.
Thank you for reading, and sorry if this seem more of a rambling. But I found myself caught in a moment that I want people to know about, to maybe even feel with me. Still I hope today that you see the good, and feel a deeper happiness and peace within your soul, so that in the dark moments you know what good is really out there.


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Before embarking on the F.A.I.L.S.A.F.E. journey, my family and I loved eating at Chipotle. I was craving it the other day, so I came up with a safe version for us and it was a HUGE hit! I lined up the ingredients on the counter, buffet style, and everyone could "order" whatever they wanted.
Here's our "Chipotle" food line…
Cooked rice with chopped parsley (looked like cilantro rice:)
Cooked hamburger (I sautéed with garlic, green onion and salt)
Beans (I used black & kidney so it was more colorful and looked like more choices:)
Green onion
sour cream (I know some people argue whether or not US sour cream is FAILSAFE, but my family is fine with it)
It was delish and the kids loved it. For lunch the next day (we use a 24 hour period for eating leftovers) we had "taco salad" to get rid of the overage – just used lettuce as a base instead of the rice…


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LISTSERV Archives
Carcinoid neuroendocrine cancers are relatively rare malignancies distinguished by small, slow growing tumors which affect the neuroendocrine system. These tumors typically originate in the gastro-intestinal tract, but sometimes arise in the lungs or other organs. Incidence is around 4 persons in 100,000 - but in many parts of the world the disease is under-diagnosed. A variety of excellent treatment options are available, most of which target specific peptide receptors on the tumor cells. Among the best known treatments are Octreotide, Lanreotide and similar hormonal treatments, which provide symptomatic relief and have anti-proliferative benefits. Other newer therapies use these drugs in combination with radioisotopes, as a targeted radiation treatment. This is an unmoderated forum for patients, caregivers, researchers, and physicians to discuss the clinical and nonclinical aspects of Carcinoid neuroendocrine cancer. Please refer to the house rules before you post any commercial, copyrighted, political or off-topic material. To access this forum, click on the "Subscribe or Unsubscribe" link, enter a name (or nickname), provide a valid email address, select the type of subscription (Index HTML format is recommended), submit the form and acknowledge the email sent to you for verification. For more information, click on the 'help' button (a green question mark in the upper-right corner of this page), or send email to the list owners.
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Meet Charlie
Charlie is Council's innovative approach to engage, educate and help the community, and will be travelling around the region to villages, events and more.
The truck is a unique opportunity for Council to utilise a space outside of the Civic Centre and proactively educate, inform, engage and meet face-to-face with residents.
Charlie stands for:
Keep your eyes peeled for him (you can't miss his bright yellow exterior!) and pop in for a visit and to find out how Charlie can help you.
While you're there you can:
- Get an update on Council projects
- Ask a question
- Provide feedback
- Lodge a request
- Access Council's online services
If you have an idea for how Charlie can be utilised let us know! Put it on the People's Panel forum or email firstname.lastname@example.com
Where can I find Charlie?
Click on the drop pins to find out when and why Charlie will be visiting that location, or check out the schedule below.
The next round of Charlie visits are scheduled to occur in October and November.
Where has Charlie visited?
Community partners
Council would like to thank the following sponsors for their contribution to Charlie:
Charlie's transformation
A former Riverina Regional Library truck, Charlie has been transformed inside and out and now has a customer counter, dedicated work/desk spaces, air conditioning, whiteboard, smart television and even a kitchen sink.One of the unique features of Charlie is a 19-foot expandable pod, meaning there's plenty of space inside.Watch the video below to see Charlie's transformation.


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Latest Announcements
The online application process for September 2019 entry has now closed. In a small number of individual cases late applications, due to extenuating circumstances, are accepted - Please contact the College Admissions Office if you wish to discuss the matter further.
The online application for September 2019 entry has now closed. In a small number of individual cases late applications, due to extenuating circumstances, are accepted - Please contact the College Admissions Office if you wish to discuss the matter further.
Exam Results
Our students achieved outstanding A and AS level results in this summer's examination series. The levels of achievement were excellent and above national average levels. A detailed document outlining the College's excellent student achievements and progression in the 2018 academic year is also still available for download from this website.
We offer over 50 A Level courses - probably the largest range of options available in any college in the country. Options include familiar subjects but also many that are not usually available at GCSE, including the well established International Baccalaureate qualification. A number of Additional Studies courses are also available.
IB Diploma
The International Baccalaureate Diploma programme will be of interest to those students who want to maintain a very broad range of subjects. It is designed for well-motivated students with balanced GCSE qualifications and is highly regarded by employers and higher education, not only in the UK but also internationally.
Your Care
We aim to be a college of sanctuary, a place where everyone feels welcome, safe and supported throughout their studies. We all have difficulties and face challenges in our lives at times and any one of us may find we need to seek help or support. There are a number of ways students can find help. The first port of call should be their personal tutor, but we have a range of places where students can find the support they may need.
Your Progression
The Careers Department has a specialist team of staff and many resources to help you to make decisions about your future. Resources are used in the weekly tutorial programme and special events are planned throughout the year to ensure you make informed choices about your future. You may also book careers appointments regarding advice and guidance, on a one-to-one basis, at any time.
Additional Studies
The College is a vibrant young adult community and there is a great deal going on. A very wide-ranging programme of Additional Studies and extra-curricular activities is available, providing a large number of sporting, creative and cultural opportunities to all students.
College Facilities
At the Sixth Form College, we teach all of our courses in specialist sixth form accommodation on one purpose-built site, located in a very accessible central area of Colchester. There are seven separate buildings, all specifically focused upon the academic teaching, learning and wider experiences appropriate for 16-19 year olds.
Our new 2019 Prospectus is now available for immediate download from our online library of key documents for viewing or printing. Printed copies of our Prospectus are no longer available.
News stories covering the educational, sporting, cultural and charitable activities and achievements of the students at the College. This includes details of ongoing projects to improve our environmental and sustainable position via a variety of tasks and initiatives.
Downloadable documents for parents and students, including the Students Handbook Charter and Diary, Additional Studies Handbook, Prospectus, the 2018 Ofsted Inspection Report, Student Leavers' Notes, Parents' Handbook, occasional Students' Notes, and more.
For Parents
Information and guidance useful to parents/guardians of students currently enrolled at the Sixth Form College, as well as those of students considering making an application, including information regarding transport, student finance and important dates. Updated regularly.


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Wacom reviews to choose you pen tablet: Bamboo, Intuos, Cintiq...which one?
my best source to get a first glimpse is the Amazon "basin" of comsumers:
NOTE: today, 15 October 2010, wacom introduced the "Special Edition" bamboo tablet which features 4-fingers touch and a bamboo bag.
Wacom Reviews for Bamboo Pen and Touch Fun Small Tablet
Medium size recommended for those who want to draw not only with the hand but with the wrist (longer movements). Also better for following larger pictures or drawing directly laid on the pad. (My two cents)
Wacom Intuos4 Large Pen Tablet
Large size recommended for graphic artists. Why using the small size when the price for the small Intuos 4 is only a little more than the the medium-size bamboo tablet? (my opinion).
The Intuos 4 has a better sensitiviyt and touch, the pad is also softer. This fact (the pad is softer, while the pen is the same) creates some problems at the moment: the quick wearing of the nib. You can avoid this by putting a sheet of paper on the board while using it. Here is a insight from the wacom intuos 4 user forum:
Re: Intuos4 nibs flattening quickly
by s12a » Sun 26. Jul 2009, 06:11
I previously had a Bamboo Fun tablet and I felt its standard plastic nibs were almost eternal. After 10 months of use I was still using the first nib, which probably could have lasted much longer, had I kept the tablet for more time.
I recently purchased an Intuos4 tablet too and I agree that black nibs seem to wear out a bit too fast. Recently I've started using more intensively this tablet and I can already see visible nib wear (not a change in tip shape which is normal, really wear). I don't think this is because of the tablet surface, as it's very similar to that of the Bamboo Fun tablet (matte).
If black plastic nibs wear up so fast, I can only wonder what would happen with other nib types, which seem less durable to me.
By the way, tiny scratches on the surface sheet are, I'm afraid, normal and inevitable since it has a matte texture (I almost rendered completely smooth the surface of my previous Bamboo tablet because of the accumulation of tiny marks due to intense use... mostly in the central area of the tablet though).
As for the drop of quality, yes, unfortunately I can see it too in many aspects (check the links in my signature for example).
What can it be done about it?
Except for buying nib refills, as customers, we can only discuss about this problem(s) and make other people aware about it. Hopefully Wacom will eventually recognize this to be a product quality problem.
by Wacom » Mon 27. Jul 2009, 08:19
We use the same nibs and nib material as for previous tablet models. But the surface sheet on the Intuos4 is more rough and 'paperlike'. Apparently this increases the wear on the nibs. We are looking into different possibilities for improvements. - One option at the moment is to place a sheet of paper, plastic, glass, etc on the tablet, any non-metallic and not too thick material is possible.
Here more Wacom reviews:
Return from Wacom reviews back to wacom graphic tablet webpage


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Jackie Chan Plans To Scale Opera House In New Movie
It's his first time working in the science-fiction genre because, although he's one of the world's highest grossing stars, Jackie Chan admits he's not a young action hero any more.
But the star is making sure he includes a scene where he scales the Sydney Opera House roof, he said at a press conference at the iconic venue on Thursday for his new film, Bleeding Steel.
It's the first film he's shot in Australia since 1996's Mr Nice Guy.
The film, a co-production by Heyi Pictures & Village Roadshow Pictures Asia, is the biggest budget Chinese film to be shot primarily in Australia.
"I love Australia and I'm almost half-Australian ... my parents moved here 40 years ago, so I love working here," Chan said.
The movie which is currently being filmed around several parts of Sydney, stars two Australian actors, Tess Haubrich who has just finished working on Ridley Scott's Alien: Covenant in Sydney, and Callan Mulvey (Underbelly, Rush).
"I'm still young but not as young as I used to be so I wanted to do something different," Chan said about working in science fiction for the first time.
However, there will still be plenty of action in the film which Chan said will feature a scene where he scales the roof of the Opera House.
"Whatever I can do myself, I'll do myself but whatever I can't do I'll find a stunt double. "I'm not young any more," he said.
The action hero has been making films for 54 years but the kung fu master said he didn't find it easy to be accepted around the world when he first started out.
"The Jackie Chan style wasn't acceptable in Hollywood, now the whole world knows Jackie Chan style," he said.
He didn't know how dangerous doing his own stunts would be, as he reeled off a list of the various injuries he's received, including broken arms, ankles and a broken back, that all came from doing his own stunts.
"Accidents just come, boom. I ask the camera man 'did you get the shot?' then I say 'get me to the hospital'," he said.
Bleeding Steel is a futuristic thriller starring Chan as special forces agent (Lin) who winds up involved in a sci-fi conspiracy while protecting a woman who witnessed a sinister criminal gang.
The film's director, Leo Zhang, said the film is the first Chinese production to combine action and science-fiction which evolved from discussions with Chan, who also serves as the movie's producer.


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After being in the works for over a year, Melissa McCarthy's clothing line is finally here. As its September 1 launch approaches, the funny lady is putting all jokes aside and getting serious about making her mark in fashion.
"I was not OK with the [plus-size fashion landscape], so I decided to do what I could to change it," she told Glamour at a preview of her first-ever fashion line, Melissa McCarthy Seven7, which will be sold in Nordstrom, Macy's, and Lane Bryant stores. "A size 14 is the number one size in the country, and we're telling those women something's wrong with them by sticking plus-size clothing with the tires and not in the women's department. We need to stop thinking of plus-size women and just think of women. We need to stop putting everyone in these categories and putting a negative spin on it. Women do not stop at a 12, so why should clothes?"
On that note, McCarthy's collection will be made in an extensive range of sizes, from a 4 to 28—and feature basics, signature prints, and her hand-picked must-haves. "I thought, What do I wear in a week? I designed selfishly with things I want in my own closet," said the actress. "I wanted to do basics with great detail that are easy to mix—all so that the customer can dress for her mood."
Overall, the collection is super wearable and will be easy to integrate into anyone's closet. Our favorite pieces? The long-line knit vest, printed Le Meow sweater (side note: her seven-year-old daughter Vivian drew that graphic!), and uber-flattering pencil skirt. Don't just take our word for it—check out how to wear the collection IRL, right here:
Monday: Sweater
"Needing a little more on your bust does not mean that your wrist is the size of a stop sign!" says McCarthy. "I don't think people have necessarily looked at the clothing on the body. I went to every fitting and got down on my hands and knees pinning to make the clothes proportional. You can't just grade up the cuts of clothing, you have to make adjustments in design."
Tuesday: Checkered Skirt
The designer explained, "I wore all the pieces for a full day and made adjustments after that. At first, the waistband in the skirt was a little too tight. I changed that and some lengths too. With those adjustments and everything that's in there as a base, it's like a built-in foundation. You don't need to double up and wear all the underpinnings [like Spanx]."
Wednesday: Checkered Pants
"Somebody said to me, 'Oh, no one plus-size wants a patterned pant!' But I was making patterned pants for myself all the time—because I can't find them in stores—and people were asking me where I got them," she says.
Thursday: Knit Vest
"The basics are stylish but really versatile," says McCarthy. "With the simpler pieces, I want the woman to be the statement. The long-lines are a little chicer and really forgiving. If you don't like it, nothing's easier than taking up a hem."
Friday: Flannel Shirt
"I think we do so much tearing down of women. In the same amount of time with the same amount of energy, you can lift them up. If me doing a clothing line that's like, 'Yeah, wear the checkered pants! Wear the flannel! Wear the damn cheetah print, I beg you!' helps lift them up, that's amazing. We only get one go around, have a little fun," she says. Amen!
For more on brands that offer a wide size range, see:


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Electronic Software Delivery
Safeguard Your Mobile Life
Stay secure on-the-go with ESET Mobile Security for Android. Specially designed for the Android platform, ESET Mobile Security for Android combines the award-winning ESET antivirus engine with powerful antitheft and antispam to provide real-time protection against known and emerging mobile threats. Secure your Android smartphone against malicious apps, viruses and physical theft while preserving performance and battery life—get ESET Mobile Security for Android.
Get Enhanced Security Features
Antitheft System – Guard against phone loss or theft with multiple layers of security that allow you to maintain control remotely over phone-stored data with simple to use text commands.
- Remote Lock —Prevent unauthorized access to your phone's data
- Find Device —Locate your lost or stolen phone on a map
- Remote Wipe —Wipe all personal information and restore phone to factory settings
- Password Reset —Reset your password in case you forget it
- Trusted Friend —Designate a contact to remotely reset your password if needed and who will be alerted by SMS if an unauthorized SIM card is inserted into your phone.
Advanced On-Access Scanning – Protect your smartphone with ESET's heuristics detection that alerts you to any suspicious activity on your phone. All applications, files, folders and SD memory cards are continually scanned for trojans, viruses, worms, spyware, spam, and other attacks.
SIM Matching – Create a list of trusted SIM cards for your phone. In case of an unauthorized SIM card insertion, you are automatically notified by a text message sent to an alternate phone number.
Security Audit with built-in Task Manager - Monitor all vital phone functions, including battery life, free disk space, running processes, Bluetooth, and device visibility. Take action on the spot, including terminating any processes that increase your security risk.
Settings and Uninstall Protection – Password-protect your settings from being modified and unauthorized uninstallation of ESET Mobile Security for Android (available for Android version 2.2 or higher).
Key Benefits
Proactive Protection from the Unknown – ESET delivers real-time and on-demand protection of all information and communications, including Wi-Fi, Bluetooth, GPRS, EDGE and Infrared.
Secure Your Personal Information – Monitor all incoming and outgoing communication to prevent hackers from stealing your personal data.
Eliminates Spam – Define trustworthy contacts via a customizable blacklist or whitelist, or simply block unwanted text and multimedia files from unknown numbers.
Easy on Your Phone – Low CPU use, low memory requirements and compact updates preserve battery life and minimize data use.
Easy on You – Download updates on-demand or automatically at pre-set intervals: daily, weekly or monthly to preserve battery life.
Call Intercept – Block unwanted incoming and outgoing calls.
Your Security Status At-a-Glance
Activity Log – Scan statistics are stored in a user-friendly format so you can easily review the performance of your phone.
Statistics – Access an at-a-glance summary of scan results, SMS filtering and quarantine activities.
Intuitive User Interface – Easily navigate your ESET Mobile Security for Android.
System Requirements
Operating Systems:
- Ice Cream Sandwich (4.0–4.0.4)
- Jelly Bean (4.1–4.3.1)
- KitKat (4.4–4.4.4)
- Lollipop (5.0–5.0.1)
- Rooted phones are not supported
- Optimized for smartphones. Certain functionality may be lost on Android tablets.


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Heme controls expression of genes involved in the synthesis of globins and heme. The mammalian transcription factor Bach1 functions as a repressor of the Maf recognition element (MARE) by forming antagonizing hetero‐oligomers with the small Maf family proteins. We show here that heme binds specifically to Bach1 and regulates its DNA‐binding activity. Deletion studies demonstrated that a heme‐binding region of Bach1 is confined within its C‐terminal region that possesses four dipeptide cysteine–proline (CP) motifs. Mutations in all of the CP motifs of Bach1 abolished its interaction with heme. The DNA‐binding activity of Bach1 as a MafK hetero‐oligomer was markedly inhibited by heme in gel mobility shift assays. The repressor activity of Bach1 was lost upon addition of hemin in transfected cells. These results suggest that increased levels of heme inactivate the repressor Bach1, resulting in induction of a host of genes with MAREs.
Recent progress has highlighted the role of metabolic products as regulatory molecules in gene regulation. Heme is an essential molecule that plays a central role in reactions involving molecular oxygen, as it is the prosthetic group of heme proteins. In addition, heme is known to participate in gene regulation by functioning as a ligand for transcription factors in prokaryotes (Monson et al., 1992) and yeast (Creusot et al., 1989; Pfeifer et al., 1989; Fytlovich et al., 1993; Zhang et al., 1998; Hon et al., 2000). Heme has been proposed to fulfill similar regulatory roles in higher eukaryotes as well (Sassa and Nagai, 1996). However, to date, there is no known transcription factor of higher eukaryotes that is regulated directly or indirectly by heme.
Mitochondrial transport of the mammalian erythroid‐specific δ‐aminolevulinic acid synthase (ALAS‐E) precursor is regulated by intracellular free heme. ALAS‐E possesses several heme regulatory motifs (HRMs) within its signal sequence for mitochondrial transport whose activity is inhibited by heme (Lathrop and Timko, 1993). Within the HRM, a dipeptide motif of cysteine and proline (CP motif) appeared essential for heme‐mediated regulation. HRM‐like CP motifs have also been found in other proteins such as yeast transcriptional activator Hap1 (Creusot et al., 1989; Pfeifer et al., 1989; Fytlovich et al., 1993; Zhang et al., 1998; Hon et al., 2000), mitochondrial heme lyases (MHLs; Steiner et al., 1996), HRI kinase (Chen et al., 1991a,b; Crosby et al., 2000; Rafie‐Kolpin et al., 2000) and heme oxygenase (HO)‐2 (Rotenberg and Maines, 1991; McCoubrey et al., 1997). In the case of Hap1, heme binds directly to the protein through its several CP motifs, and then activates genes that are inducible by heme and oxygen (Creusot et al., 1989; Pfeifer et al., 1989; Fytlovich et al., 1993; Zhang et al., 1998; Hon et al., 2000).
The transcription factors Bach1 and Bach2 are basic leucine zipper (bZip) proteins that form heterodimers with the Maf‐related oncoprotein family (Oyake et al., 1996). Like the erythroid transcription factor NF‐E2 and its related factors Nrf1, ‐2 and ‐3 (Oyake et al., 1996; Marini et al., 1997; Johnsen et al., 1998; Kobayashi et al., 1999), their heterodimers with Maf proteins bind to the Maf recognition elements (MAREs; Igarashi et al., 1994, 1995; Kataoka et al., 1995; Motohashi et al., 1997; Toki et al., 1997). MAREs are present in the regulatory region of various genes involved in heme metabolism such as oxidative stress‐responsive genes (Ishii et al., 2000), globin genes (Talbot and Grosveld, 1991; Kotkow and Orkin, 1995; Igarashi et al., 1998; Yoshida et al., 1999), the HO‐1 gene (Inamdar et al., 1996; Alam et al., 1999) and the ALAS‐E gene (Kramer et al., 2000). Among these MARE‐binding factors, Bach1 and Bach2 function primarily as repressors (Oyake et al., 1996; Igarashi et al., 1998; Muto et al., 1998; Yoshida et al., 1999; Kobayashi et al., 2000). The presence of a repressor class of molecules suggests that transcription regulation operating through MAREs is based on the balance between activation and repression. However, the physiological role of Bach1 has remained unclear. Interestingly, Bach1 possesses several CP motifs (Oyake et al., 1996). Here we show that heme inhibits the DNA‐binding activity of Bach1–MafK heterodimers by directly interacting with Bach1 through the CP motifs. These results demonstrate that Bach1 is a novel mammalian heme‐responsive transcription factor that regulates gene expression by altering its DNA‐binding activity in response to the intracellular free heme concentration.
Heme‐binding activity of Bach1
We reported previously the expression of Bach1 as a fusion protein with maltose‐binding protein (MBP–Bach1) and its purification using amylose resin affinity chromatography (Oyake et al., 1996). The MBP fusion protein possessed the 453 C‐terminal amino acids of Bach1 (see Figure 1). During its purification, we found that the purified protein samples were a brownish color. This color appeared specific to Bach1, since other MBP fusion proteins, e.g. Nrf2, NF‐E2 p45 and MafK, were not this color. The purified MBP–Bach1 protein displayed a typical hemoprotein spectrum with Soret bands by UV‐visible spectroscopy (data not shown). These observations suggest the presence of heme in the purified MBP–Bach1 protein. To explore the possibility that Bach1 is a heme‐binding protein, we expressed various portions of Bach1 as fusion proteins with glutathione S‐transferase (GST), and carried out various binding assays.
Bach1 possesses at least two functional domains, BTB/POZ and bZip domains, which are involved in protein interaction and DNA binding, respectively. To carry out a binding assay, we first expressed a portion of Bach1 (amino acids 174–739 including the putative heme‐binding region but lacking the BTB/POZ domain, see Figure 1) as a fusion protein with GST. There were several other faint bands in the purified fusion protein BA1G174–739 that appeared to be degradation products of the fusion protein (Figure 1B). The BA1G174–739 preparation was found to contain an ∼0.03–0.15 molar ratio of heme, depending on the culture conditions. Upon incubating BA1G174–739 with hemin, a Soret band with a peak at 421 nm characteristic of a heme‐binding protein was observed (Figure 2A). These results indicate that Bach1 is in fact a heme‐binding protein. To estimate the affinity of Bach1 for heme, BA1G174–739 was immobilized on Sepharose beads and was incubated with 55Fe‐labeled hemin at various concentrations. Binding of hemin to BA1G174–739 was found to occur in a concentration‐dependent and saturable manner (data not shown). GST alone did not show any binding. Scatchard analysis revealed that 1.3 mol of hemin bound to 1 mol of BA1G174–739 with a Kd of 140 nM (Figure 2B).
Identification of heme‐binding motifs in Bach1
To understand the molecular basis of heme binding of Bach1, we first attempted to identify a region of Bach1 responsible for heme binding. Towards this end, we generated several GST fusions of Bach1 derivatives (Figure 3). First, two fragments (174–415 and 417–739) were tested for heme binding. The C‐terminal BA1G417–739 showed a high affinity for heme that was comparable with that of BA1G174–739, whereas the N‐terminal BA1G174–415 did not show any specific binding activity (Figure 3). These results indicate that a putative heme‐binding domain is confined within the C‐terminal region between amino acid residues 417 and 739. To define the binding region(s) more precisely, we prepared two subfragments from the heme‐binding fragment (417–739). To our surprise, both fragments (417–645 and 558–739) showed comparable heme‐binding activities.
CP motifs have been found in HRMs (Creusot et al., 1989; Chen et al., 1991a; Rotenberg and Maines, 1991; Lathrop and Timko, 1993; Steiner et al., 1996; McCoubrey et al., 1997) and play an important role in heme binding in various proteins. Interestingly, Bach1 contains six CP motifs (Figure 1). There are two CP motifs downstream of the BTB domain (CP1 and CP2), three CP motifs upstream of the bZip domain (CP3, CP4 and CP5) and one CP motif downstream of the bZip domain (CP6; Figures 1 and 3). Among these CP motifs, CP3–CP6 are present within the putative heme‐binding region. To test the possibility that these CP motifs are involved in heme binding, we prepared GST fusion proteins with single or multiple mutations within the CP motifs in the context of BA1G417–739 by changing a cysteine residue to alanine (Figure 4A). Single amino acid substitutions (BA1G417–739CP3AP, BA1G417–739CP4AP, BA1G417–739CP5AP and BA1G417–739CP6AP) did not influence the heme binding. In contrast, alteration of the three clustered CPs (BA1G417–739CP3–5AP) significantly decreased the heme‐binding activity. Most importantly, replacement of all four CPs (CP3–CP6, BA1G417–739CP3–6AP) abolished the high affinity binding to heme. These results indicate that Bach1 binding to heme is dependent on the presence of the CP motifs, and rule out the possibility that an impurity in the preparations binds to heme. These results also indicate that there is a cooperativity among the four CP motifs of Bach1 for heme binding. Kd values were 170 and 1220 nM for BA1G417–739 and BA1G417–739CP3‐6AP, respectively (Figure 4B and C).
Inhibition of DNA‐binding activity of Bach1 by heme
The specific binding of heme to Bach1 strongly suggests that heme regulates certain functions of Bach1. Since the four CP motifs (CP3–CP6) that are involved in heme binding surround the bZip domain (see Figure 1), we examined the effects of hemin on DNA binding using electrophoretic mobility shift assays (EMSAs). In this experiment, we first used full‐length recombinant Bach1 (Igarashi et al., 1998) that was overexpressed in Escherichia coli, purified from inclusion bodies and re‐folded in vitro. This procedure permitted us to obtain recombinant protein that is free of heme. A MARE‐containing 240 bp DNA isolated from the β‐globin locus control region (LCR) was incubated with Bach1 and MafK, and the resulting DNA‐binding complexes were analyzed by EMSA using agarose gels (Figure 5A). Two retarded bands were detected; the upper band contained both Bach1 and MafK whereas the lower band contained only MafK (data not shown; Igarashi et al., 1998). Addition of heme to the binding reactions resulted in a concentration‐dependent inhibition of DNA binding by the Bach1–MafK hetero‐oligomer. Even 0.03 μM heme caused a slight but reproducible inhibition. Heme at 1 μM almost completely inhibited the DNA‐binding activity of Bach1. In contrast, there was little effect of heme on DNA binding by the MafK homodimer. To exclude the possibility that heme inhibited DNA binding by BTB domain‐mediated oligomers, full‐length Bach1 and Bach1ΔBTB (with amino acid residues 1–173 deleted, see Figure 1A) were compared in EMSAs using conventional polyacrylamide gels (Figure 5B). Addition of heme efficiently inhibited the DNA‐binding activity of Bach1ΔBTB–MafK heterodimers. These results suggest that heme's effect is independent of BTB domain‐mediated oligomer formation (Igarashi et al., 1998). Nrf2 is distantly related to Bach1 and binds to similar DNA sequences as a heterodimer with Maf‐related proteins. In contrast to Bach1, however, Nrf2 functions as a transcription activator. Therefore, we compared the role of Bach1 and Nrf2 with respect to heme. The DNA‐binding activity of Nrf2–MafK heterodimers was not inhibited by heme (Figure 5C), suggesting that Bach1 is a specific regulatory target of heme among the MARE‐binding proteins.
We next investigated whether the CP motifs that are essential for heme binding may also be involved in the inhibition of DNA binding by heme. We used the GST–Bach1 fusion that harbored cysteine to alanine substitutions in the four CP motifs (BA1G417–739CP3–6AP, Figure 6). Comparison of the amounts of DNA‐binding complex generated in the presence of various concentrations of heme clearly indicated that the CP3–6AP substitutions rendered Bach1 less sensitive to heme. On the other hand, in contrast to its essential lack of heme binding (see above), the CP3–6AP form still retained some sensitivity toward heme. It should be noted that Nrf2 was insensitive toward heme‐mediated inhibition (see Figure 5). Taken together, these results indicate that the four CP motifs are important for both Bach1–heme interaction and heme‐mediated inhibition of DNA binding. In addition, the results also suggest that other features of Bach1 may also contribute to its heme sensitivity.
Effect of heme on Bach1–MafK interaction
Heme may inhibit DNA binding of Bach1 by influencing heterodimer formation with MafK through the leucine zippers since Bach1 cannot bind to DNA by itself (Oyake et al., 1996; Igarashi et al., 1998). To test this possibility, we investigated the effect of heme on the leucine zipper‐mediated Bach1–MafK interaction. FLAG‐tagged MafK was incubated with or without GST‐fused Bach1 (BA1G417–739) and separated through non‐denaturing polyacrylamide gels. Proteins were transferred onto membranes and were detected using anti‐FLAG antibody (Figure 7, upper panel). In the absence of BA1G417–739, a single high mobility band was observed that appeared to be a monomer or homodimer of MafK (lane 4). Another slower mobility band, shown with an asterisk, may be due to conformational differences in native gels or formation of a MafK homo‐oligomer. In the presence of BA1G417–739, both of the complexes disappeared with the concomitant appearance of the third distinct band (lane 5, indicated with a dot). This band was judged to reflect leucine zipper‐mediated formation of a Bach1–MafK complex since addition of BA1G417–564 lacking the bZip domain did not change the mobility of MafK complexes (compare lanes 4, 5 and 9). On the other hand, similar changes in mobility of MafK complexes were also observed by adding BA1G417–739CP3–6AP (lane 7). To confirm further the formation of a Bach1–MafK complex, the same blot was reacted with anti‐GST antibody to detect Bach1 proteins (lower panel). Single bands were observed when Bach1 proteins were electrophoresed alone (lanes 1–3). In the presence of MafK, slower mobility bands were detected with BA1G417–739 or BA1G417–739CP3–6AP (lanes 5 and 7, indicated with dots). In contrast, the mobility of BA1G417–564 did not change by adding MafK (compare lanes 3 and 9). Using this experimental system, we examined the effects of heme. The amounts of slower mobility complexes in the presence of BA1G417–739 or BA1G417–739CP3–6AP did not change in either the absence or presence of 1 μM heme (compare lanes 5–8). We thus concluded that heme did not inhibit Bach1–MafK interaction through the leucine zippers, but that rather it inhibited the DNA‐binding activity of the resulting heterodimer.
Heme abrogates transcription repression by Bach1 in vivo
To probe the functional consequences of the interaction between heme and Bach1 within a cell, we compared the regulatory activities of Bach1 and a Bach1 derivative with multiple CP mutations. Bach1 mCP1–6 harbors replacement of a cysteine by an alanine in all six CP motifs, i.e. CP1–CP6. Each of the expression plasmids was transfected into 293 cells along with a synthetic MARE‐dependent reporter plasmid, and their effects on reporter gene expression in the presence or absence of 10 μM hemin in the medium were compared (Figure 8). The amount of hemin is similar to that utilized in experiments examining heme‐mediated gene induction in cultured cells (Alam et al., 1999). Without addition of hemin, both Bach1 and Bach1 mCP1–6 repressed expression of the reporter gene. Bach1 mCP1–6 appeared to show more efficient repression than the wild‐type (compare columns 2–5). When hemin was added to the medium, repression by Bach1 was markedly abrogated (columns 6–8). In contrast, Bach1 mCP1–6 remained effective in transcription repression even in the presence of hemin (columns 9 and 10). Taken together with the fact that the CP motifs play an important role in Bach1–heme interaction, these results indicated that the repressor activity of Bach1 within a cell is negatively regulated by heme.
Heme plays various critical roles within cells because of its ability to bind molecular oxygen and to mediate electron transfer. Besides these functions, heme is known to induce expression of ALAS‐E (Fujita et al., 1991), globin (Fukuda et al., 1994; Harigae et al., 1998) and HO‐1 genes (Yoshida et al., 1988; Shibahara et al., 1993) while it decreases the mRNA of non‐specific ALAS (ALAS‐N) (Yamamoto et al., 1988; Fujita et al., 1991). These proteins are involved in metabolism of heme or hemoglobin synthesis, and proper control of their expression is important for survival under various conditions. These observations led to the conjecture that heme plays a role in gene regulation (Sassa and Nagai, 1996). While this hypothesis predicts the presence of a heme‐regulated transcription factor, the yeast transcription factor Hap1 has been the only known example in eukaryotes to date (Creusot et al., 1989; Pfeifer et al., 1989; Fytlovich et al., 1993; Zhang et al., 1998; Hon et al., 2000), with no other factors with such function known in higher eukaryotes. We report here the biochemical characterization of the heme–Bach1 interaction. Most importantly, DNA‐binding and repressor activities of Bach1 are negatively regulated by heme. These results suggest that heme may regulate gene expression through shifting the balance between activators and repressors that bind to MAREs.
Despite the fact that Bach1 is not related to Hap1, there is a resemblance between Hap1 and Bach1 in that they interact with heme through their CP motifs, i.e. both proteins bind to heme with high affinity via their CP motifs. However, no single CP motif of Bach1 is essential for heme binding, indicating their functional redundancy. Consistent with this notion, a single CP motif on Hap1 is known to bind to heme when tested using a synthetic peptide (Zhang and Guarente, 1995). Thus, it is reasonable to assume that each CP motif on Bach1 possesses the potential to interact with heme. This may explain the heme‐binding activity of the C‐terminal fragment containing one CP motif (BA1G417–739CP3–5AP and BA1G558–739). Paradoxically, the results of Scatchard plot analyses indicated that Bach1 possessed roughly one heme‐binding site. While each of the CP motifs may be capable of binding to heme, binding of one molecule of heme to Bach1 is likely to preclude further heme binding in the context of Bach1. Evolutionary comparison also underscores the importance of the Bach1 CP motifs. The amino acid sequences around CP3–CP5 are almost identical between mouse (Oyake et al., 1996) and human (Blouin et al., 1998; Ohira et al., 1998). Furthermore, this domain is also well conserved in Xenopus Bach1 (DDBJ/EMBL/GenBank accession No. AW147501, see Figure 9A). The clustering of the CP motifs are also conserved: there are 24 and 29 animo acid spacers between CP3, CP4 and CP5, respectively. Together with CP6, they form a CP cluster surrounding the bZip domain. Binding of hemin may induce structural changes within or around the bZip domain that inhibit DNA binding but not the leucine zipper interaction. Clustered CP motifs are also found in mammalian ALAS‐E precursor (Lathrop and Timko, 1993). Thus, the clustering of CP motifs may be important for high‐affinity heme binding by increasing the local concentration of binding sites.
There is still a remaining question of whether CP3–6 and their clustering are the sole determinant for heme binding. This question stems from the observation that hemin exerts a regulatory effect on Bach1 in vitro at relatively lower concentrations as compared with other mammalian heme‐regulated proteins. For example, hemin is known to inhibit mitochondrial transport of ALAS‐E precursor at 5–25 μM (Lathrop and Timko, 1993). It should also be noted that there are two other CP motifs, CP1 and CP2, on Bach1. Even though BA1G417–739 and BA1G174–739 showed similar heme‐binding activity and BA1G174–415 showed no detectable heme binding, the possibility that the N‐terminal region of Bach1 also contributes to heme–Bach1 interaction cannot be excluded. Another observation that cannot be explained fully at present is that BA1G417–739CP3‐6AP, with the four CP–AP substitutions, still showed reduced but reproducible sensitivity to heme in the DNA‐binding assays. Because Nrf2 was insensitive to heme, it is likely that there may be an additional mechanism for heme–Bach1 interaction that eluded our analyses. In this respect, it should be noted that the CP motifs of heme lyase are suggested to be involved in increasing the local concentration of heme, providing there is a sufficient substrate concentration at the catalytic center (Steiner et al., 1996). Thus, it remains possible that Bach1 possesses another heme‐binding site(s) that mediates a weak and/or transient protein–heme interaction, and we are currently testing this possibility.
Comparison of various heme‐related molecules revealed that hemin and Fe mesoporphyrin IX (FeMePIX) bound to Bach1 and inhibited its DNA binding, whereas porphyrin derivatives without Fe at the center, including protoporphyrin IX (PPIX) and MePIX, did not (data not shown). Furthermore, other derivatives such as octaethylporphine did not show Bach1 binding or inhibitory effects on DNA binding (data not shown). These results suggest that only heme and porphyrin derivatives with chelated iron can bind to Bach1 specifically. Since FeMPIX, which does not have vinyl groups, bound to Bach1, the binding is likely to be non‐covalent, like the iron–imidazole interaction at the histidyl site, unlike the covalent binding of cytochrome c to heme via two cysteine residues.
Based on our observation and previous reports, we propose a hypothetical mechanism by which heme functions as an inducer of gene expression by inhibiting the repressor molecule Bach1 (Figure 9B). In this model, Bach1 represses genes with MAREs in their regulatory regions in the presence of lower levels of heme by competing with activators such as Nrf2 (Alam et al., 1999; Ishii et al., 2000) and NF‐E2 p45 (Igarashi et al., 1998) for binding to MAREs. Higher levels of heme cause inhibition of the DNA‐binding activity of Bach1, resulting in dissociation of Bach1 from the enhancers and derepression of the target genes. The next step will be identification of physiological target genes for Bach1. Bach1 targets will be found among genes that possess MAREs in their regulatory regions and that are induced by heme. It may be worth noting that the LCR of the β‐globin genes (Talbot and Grosveld, 1991; Kotkow and Orkin, 1995; Igarashi et al., 1998; Yoshida et al., 1999) and the heme‐responsive enhancers of HO‐1 (Inamdar et al., 1996; Alam et al., 1999) contain multiple MAREs. Since Bach1 forms mutlivalent DNA‐binding complexes through its BTB domain that can bind cooperatively to reiterated MARE motifs (Yoshida et al., 1999), β‐globin and HO‐1 enhancers may allow effective binding by Bach1. Together with the fact that these genes are directly or indirectly regulated by heme (Shibahara et al., 1993; Fukuda et al., 1994; Harigae et al., 1998; Yoshida et al., 1988), β‐globin and HO‐1 genes are prime candidates. Further studies obviously are necessary to test this hypothesis. Nevertheless, our observations provide, for the first time, a molecular mechanism for the long‐standing contention that intracellular free heme regulates gene expression in mammalian cells.
Materials and methods
Recombinant proteins
FLAG‐tagged MafK, His‐tagged Bach1, and His‐tagged Bach1ΔBTB, a Bach1 variant lacking the N‐terminal 173 amino acids including the BTB domain, were prepared as described previously (Igarashi et al., 1998). To construct GST fusion protein expression plasmids, portions of mouse Bach1 cDNA were amplified by PCR and the resulting fragments were ligated with pGEX‐2T vector (Amersham Pharmacia Biotech AB, Uppsala, Sweden). The plasmids thus constructed were pGEX2/Ba1G693–2393, pGEX2/Ba1G693–1418, pGEX2/Ba1G1422–1865, pGEX2/Ba1G1422–2108, pGEX2/Ba1G1422–2393 and pGEX2/Ba1G1845–2393 (the numbers indicate the base positions of mouse bach1 cDNA; Oyake et al., 1996). These plasmids encoded Bach1 subfragments fused with GST (BA1G174–739, BA1G174–415, BA1G417–564, BA1G417–645, BA1G417–739 and BA1G558–739, respectively; the numbers indicate the amino acid positions of mouse Bach1 protein). Cysteine to alanine substitutions in the CP motifs were introduced into BA1G417–739 cDNA using the Transformer™ Site‐Directed Mutagenesis Kit (Clontech, Palo Alto, CA) to express BA1G417–739CP3AP, BA1G417–739CP4AP, BA1G417–739CP5AP and BA1G417–739CP6AP, bearing mutations at amino acids 438, 464, 495 and 649, respectively. These proteins were expressed in E.coli (strains SG12036 or BL21) either at 37°C for 2 h or at 25°C for 12 h, followed by purification with glutathione–Sepharose 4B (Amersham Pharmacia Biotech AB, Uppsala, Sweden) according to the manufacturer's protocols. Purified GST fusion proteins were eluted from glutathione–Sepharose beads with 10 mM glutathione, 50 mM Tris–HCl pH 8.0 and dialyzed with Slide‐A‐Lyzer 10K Dialysis Cassettes (Pierce) in 10 mM Tris–HCl pH 8.0 containing 0.1 mM EDTA, 0.1 mM dithiothiothreitol (DTT) and 4% glycerol. The purity and integrity of the prepared proteins were checked by SDS–PAGE.
Spectral analysis
Hemin (2.7 μM) was incubated with or without 2.7 μM BA1G174–739 in 10 mM Tris–HCl pH 8.0, 150 mM NaCl (Tris‐buffered saline, TBS). The resulting mixtures were scanned using a UV‐1200 UV‐VIS spectrophotometer (Shimidazu, Kyoto, Japan).
Hemin‐binding assays
[55Fe]Hemin (5.5 × 104 d.p.m./nmol) was synthesized from PPIX and 55Fe (>3 Ci/g, NEN Life Science Products) by the non‐enzymatic method (Taketani and Tokunaga, 1984). GST fusion proteins (1 nmol) immobilized on glutathione–Sepharose beads were suspended in 1 ml of TBS–0.1% Tween‐20 (TBS‐T). [55Fe]hemin dissolved in dimethysulfoxide (DMSO) was added to the mixture and incubated at room temperature for 30 min. After washing four times with 500 μl of TBS‐T, the radioactivity of the beads was counted. Alternatively, non‐labeled porphyrin derivatives including hemin were used as ligands in the same binding conditions described above. In this case, after washing, beads were suspended in 2 ml of 2 M oxalic acid and heated at 110°C for 10 min to remove the metal from metalloporphyrins. The amounts of bound porphyrins were determined from the peak values of fluorescence emission scanning 550–700 nm with excitation at 400 nm.
Electrophoretic mobility shift assay (EMSA)
Chicken β‐globin enhancer oligonucleotide (CβE; Igarashi et al., 1994) or 240 bp DNA from the β‐globin LCR containing the tandem MARE (HS‐2; Igarashi et al., 1998) was labeled with 32P using polynucleotidyl kinase. Alternatively, DNA was labeled with digoxigenin (DIG) using the DIG Oligonucleotide 3′‐End Labeling Kit (Boehringer Mannheim). A 40 ng aliquot of GST fusion or His‐tagged Bach1 proteins (BA1G417–739, BA1G417–739CP3‐6AP, Bach1 or Bach1ΔBTB) and 10 ng of MafK‐FLAG were incubated on ice for 20 min in 10 or 20 μl of gel shift buffer containing 20 mM HEPES pH 7.9, 0.05 μg/μl dIdC, 0.05 μg/μl dAdT, 0.1 μg/μl bovine serum albumin (BSA), 5 mM DTT, 5 mM MgCl2, 1 mM EDTA, 20 mM KCl and 4% glycerol. After addition of hemin or porphyrin derivatives, the reaction mixtures were left on ice for 10 min. Labeled CβE or HS‐2 was then added to the mixture and incubated at 37°C for 10 min. The resulting solutions were separated through 4% polyacrylamide gels or 1% agarose gels. When 32P‐labeled probe was used, gels were dried and exposed to X‐ray films. When DIG‐labeled probe was used, protein–DNA complexes were transferred onto nylon membranes (Boehringer Mannheim) and were detected using the DIG Luminescent Detection Kit (Boehringer Mannheim).
Native gel western blotting
BA1G417–739 (90 ng), BA1G417–739CP3‐6AP (90 ng) or BA1G417–563 (60 ng) were incubated with MafK‐FLAG (15 ng) in 20 μl of the gel shift buffer containing 0.2 μg/μl BSA and left at room temperature for 30 min. After adding hemin to 1 μM, the reaction mixtures were incubated further at room temperature for 10 min, and were then separated through 6% polyacrylamide gels. Proteins were transferred onto Immobilon‐P membrane (Millipore). Membranes were reacted with anti‐FLAG M2 monoclonal antibody (Sigma) and anti‐mouse IgG–horseradish peroxidase (HRP) conjugate (Promega) to detect MafK‐FLAG or with anti‐GST antibody B‐14 (Santa Cruz Biotechnology) to detect GST–Bach1 fusion proteins. Immune complexes were detected using ECL Western Blotting Detection Reagents (Amersham Pharmacia Biotech).
Transfection reporter assays
The synthetic reporter plasmid pRBGP2 was reported previously (Igarashi et al., 1994) and contained three copies of MARE in front of a minimal TATA box promoter and the firefly luciferase gene. Bach1 mCP1–6 cDNA, containing multiple mutations that replace all of the CP motifs (CP1–CP6) by AP, was constructed by two‐step site‐directed mutagenesis using the Transformer™ Site‐Directed Mutagenesis Kit (Clontech). First, CP3–CP6 were mutagenized simultaneously. After verifying the mutations, CP1 and CP2 were then converted simultaneously to AP. To express wild‐type mouse Bach1 cDNA or Bach1 mCP1–6, each cDNA was cloned into pcDNA3.1/Myc‐His A vector (Invitrogen). 293 cells were seeded in 6‐well dishes 24 h before transfection. The cells were transfected with 500 ng of the reporter and 300 or 900 ng of the effector plasmids using FuGENE 6 transfection reagent (Roche Diagnostic, Corp). After incubating cells with the plasmids for 18 h, hemin was added to the medium to 10 μM by supplying 2 μl of 10 mM hemin dissolved in DMSO. As a control, 2 μl of DMSO were added to the wells that did not receive hemin. After further incubation for 4 h, cell lysates were prepared using the Luciferase Assay System (Promega) following the supplier's protocol. Luciferase activities were measured with a Biolumat Luminometer (Berthold). Firefly luciferase activity was normalized for transfection efficiency as determined by a control sea pansy luciferase activity (Muto et al., 1998). Three independent experiments, each carried out in duplicate, were performed, and the results were averaged and diagrammed with the standard errors.
We thank Makoto Kobayashi and Tetsuro Ishii (University of Tsukuba) for continuing and stimulating discussions and critical comments on the manuscript. This work was supported by Grants‐in‐aid from the Ministry of Education, Science, Sport and Culture, the Ministry of Health and Welfare, Japan and an RFTF grant from the Japanese Society for the Promotion of Science, as well as grants from the Uehara Memorial Foundation, the Inamori Foundation, Yamanouchi Foundation for Research on Metabolic Disorders, Japan Foundation of Cardiovascular Research and a USPHS grant (DK‐32890).
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