text:
Yesterday I converted my ISO-8859-1 WP database to UTF-8 and everything looks good (I'm located in Sweden that uses å, ä and ö)
However the email notifications and all emails sent out by WP looks like gibberish: "Hej Åse! Vet inte om du har bytt lösenord under "Livet i övrigt"
How do I solve this?
Thank you in advance!


text:
When installing PGP Universal Server 3.x or Symantec Encryption Management 3.3.0 (formerly PGP Universal Server) and above on VMware you receive the following error and setup cannot continue.
Could not allocate requested partitions:
Partitioning failed: Could not allocate partitions as primary partition for /boot.
This error occurs when the VMware instance is configured to use the BusLogic SCSI Adapter for the virtual machine. PGP Universal Server 3.x and Symantec Encryption Management Server 3.3.0 and above do NOT support using the BusLogic SCSI Adapter in a VMware virtual machine.
Best Practices for Creating a New Virtual Machine for Symantec Encryption Management Server (formerly PGP Universal Server) version 3.0 and newer.
Use the following procedure to use a LSI Logic adapter and install the Symantec Encryption Management Server (formerly PGP Universal Server) on a VMware virtual machine.
Applies To
VMware Hypervisor ESXi
VMware Vsphere 5+
VMware ESX 3.5+
Did this article resolve your issue?
Did this article save you the trouble of contacting technical support?
How can we make this article more helpful?
Email Address (Optional)
Terms of use for this information are found in Legal Notices.
Terms of use for this information are found in Legal Notices.
Login to Subscribe
Please login to set up your subscription.
Create and manage cases, manage licensing and renewals, submit threats, and enroll with Symantec Rewards.
Customer and Technical Support phone numbers and hours of operation.
User-to-user forums, blogs, videos, and other community resources on Symantec Connect.
Set default language
Do you wish to save this as your future site?

subset: commoncrawl


text:
Alexander Wang
Product Actions
Smooth leather taper toe ankle bootie designed with a lace-up front and leather pull tab at back. Detailed with a thick platform and block heel.
Style # 502731721
- Approximately 3.75" heel (95mm), 25mm platform
- Textured rubber sole
- Available in Black
- Imported
Style # 502731721
American designer Alexander Wang founded his signature label in 2005, quickly becoming known for his use of luxe textiles in his edgy, sophisticated designs. His men's and women's collections, handbags, shoes and accessories are consistent must-haves for the smart, urban crowd.
Need Help? Have Questions? Contact Us.
Please call 1.888.222.7639 to speak with a Customer Service Representative.
We're here to help you:
Monday - Friday, 9 AM - 9 PM (ET)
Saturday - Sunday, 10:30 AM - 7 PM (ET)
If you prefer email, we can be reached anytime at firstname.lastname@example.com.
Free Shipping & Returns on all U.S. Orders. See Details.


text: 000 FXUS63 KAPX 270143 AFDAPX Area Forecast Discussion National Weather Service Gaylord MI 943 PM EDT TUE JUL 26 2016 .UPDATE... Issued at 943 PM EDT Tue Jul 26 2016 Weak frontal boundary combined with lingering low level convergence via this afternoon`s lake breeze continues to produce a line of showers and thunderstorms across Upper Michigan late this evening. Overall intensity has diminished with sunset/decreasing diurnal instability. Latest RAP13 does suggest some weak widely scattered convection will continue to fire along this boundary as it sinks southward thru our CWA overnight. This seems reasonable given MUCAPES of 500-1000 J/kg and some 850 mb theta E ridging holding on along the front thru the night. Have slightly increased POPs overnight for much of our CWA...and better defined timing of these POPs. Still do not expect severe storms for the balance of the night thanks to weak wind fields and diminishing instability. && .SHORT TERM...(This evening and overnight) Issued at 250 PM EDT Tue Jul 26 2016 Overview...A cold front in Northern Minnesota/Western Lake Superior is slowly moving toward the forecast area. It is connected to a low pressure system near James Bay. South of the front is the High pressure system that has been producing mostly sunny skies over the forecast area. Tonight/Wednesday...Main concern over the next 24 hours is the cold front in northern Minnesota as it sags south into the forecast area. The models continue to try and kick off thunderstorms that are supposed to move into E Upper this afternoon/early evening. As noted in previous update AFD, closest precipitation is well west in Minnesota with the front. As the front approaches the forecast area, we get more and more stable, and with a weak front like this, less of a chance for showers, let alone thunder. GFS is the wettest of the models with a cluster that moves into the region. The ECMWF, on this run, has something with the front. Hires models and NAM, have brief showers that pass through. So have kept the pops low as it moves through the region. The better chance will be on Wednesday as the low slows to a crawl near M-72 and produces scattered showers and thunderstorms through the day, especially in the afternoon with the heat of the day. .LONG TERM...(Wednesday through Tuesday) Issued at 250 PM EDT Tue Jul 26 2016 Wednesday night through Friday... High impact weather potential: Scattered thunderstorms possible across parts of the area at various times throughout the period. Pattern synopsis/expected evolution: Weak mid-upper level troughing is present at the start of the period with a more pronounced shortwave trekking through the upper Mississippi Valley and into the Great Lakes Wednesday night-Thursday. Another, more subtle wave, approaches the region from the west late in the day Friday. Both of these features will ultimately result in increased precip chances through the period. Forecast concerns/challenges: PoPs and thunder chances. Wednesday night - Thursday: Aforementioned shortwave approaches from the northwest Wed night into the day Thursday, ultimately leading to low end chance PoPs, mainly along and south of M-72 as dry surface high pressure is anchored to the north and the greatest forcing for ascent lies from there south. As hinted at by the aforementioned PoPs, would expect that any scattered precip able to develop or survive the trip across Lake Michigan would be fairly scattered in nature. While instability remains marginal, there certainly could be a rumble of thunder or two so have no reason to remove inherited thunder chances. Thursday night - Friday night: Very similar feel during this portion of the forecast with another, although more subtle, shortwave rotating through mid-level flow. High pressure centered to the north is expected to limit the overall moisture and associated precip across northern Michigan. Latest trends suggest much, if not all, showers and storms will remain south of M-55 through Friday night. Run-to-run model inconsistencies preclude removing PoPs completely south of M-55, but did decrease somewhat for this forecast package and if trends continue, would find it hard to see more than just a widely scattered shower or storm prevail. Little in the way of temperature concerns with near-normal temperatures expected Thursday and Friday as highs ranging from the upper 70s to lower 80s. Lows in the middle to upper 50s. Gillen Extended (Saturday through Tuesday) issued at 300 PM EST TUES Jul 26 2016 At 500mb... A broad trough will position itself over the Great Lakes basin for the remainder of the weekend. The troughing is weak and should not result in any significant weather as the the model suite has converged on a weaker, larger trough that lacks any vorticity. Ridging will takeover for the rest of the forecast period. At the surface... Saturday through Tuesday looks to remain dry with a surface high located over Ontario keeping our flow from the northeast. The air associated with that flow will be dry limiting any precip chances. The period looks to be dominated by that surface high keeping our temps in the upper 70s to mid 80s. && .AVIATION...(For the 00Z TAFS through 00Z Wednesday evening) Issued at 729 PM EDT Tue Jul 26 2016 A weak cool front will slide thru Northern Michigan tonight into Wednesday...producing small chances of showers and thunderstorms. However...chances of convection are too small to include in the TAFs for now. Overall conditions will remain VFR thru Wednesday evening away from any showers/storms that do form. West winds AOB 10 kts tonight will become N/NW on Wednesday behind the front. && .MARINE... Issued at 250 PM EDT Tue Jul 26 2016 Tonight through Thursday...A weak pressure gradient is expected to settle in over the Upper Great Lakes for the next 60 hours. this will leave the winds at 10 knots or less, with gusts no higher than 20 knots, and the worst would be on Thursday as the sfc trough forms in the Ohio Valley and high pressure builds into the Upper Great Lakes from the north. Waves for the most part will remain 3 feet or less through the period. && .APX Watches/Warnings/Advisories... MI...NONE. LH...NONE. LM...NONE. LS...NONE. && $$ UPDATE...MLR SHORT TERM...JSL LONG TERM...Gillen/BPH/MSB AVIATION...MLR MARINE...JSL


text:
SUMMARY: Between website design, email campaigns, social media and more, it can be challenging to determine which marketing tactics will maximize your limited budget.
In this weeks chart, we review the 2011 B2B budget allocations of more than 900 marketers. How do you compare?
Get the latest research on email, lead gen, and social media along with MarketingSherpa updates and promotions.
Content Marketing Generated a 5,100% ROI for Health Care Innovator Optum
Read More »


text:
Definition of triskaidekaphobia in English:
Extreme superstition regarding the number thirteen.
Example sentences
- That test included words to spell such as 'revelry' and 'triskaidekaphobia.'
- Surely Stoica could not have been suffering from triskaidekaphobia (that's fear of the number 13, for the trivia fans among you).
- I've below gathered a collection of passages concerning the folklore and superstition regarding both Friday the 13 th and triskaidekaphobia - the fear of the number 13.
Early 20th century: from Greek treiskaideka 'thirteen' + -phobia.
For editors and proofreaders
Syllabification: tris·kai·dek·a·pho·bi·a
Definition of triskaidekaphobia in:
What do you find interesting about this word or phrase?
Comments that don't adhere to our Community Guidelines may be moderated or removed.
= trending


text:
European Stocks Fall After U.S. Factory Data; Repsol Jumps
European stocks fell after a report showed growth in U.S. manufacturing slowed in September, prompting speculation that last month's rally in equities has overshot the prospects for economic growth.
Banco Santander SA retreated after European Union officials warned that the fallout from the debt crisis could hurt Europe's lenders. Swatch Group AG fell 4.1 percent as its Chief Executive Officer Nick Hayek Jr. said the weakness of the dollar is hurting profit. Repsol led energy producers higher, jumping 5.3 percent after saying China Petrochemical Corp. will invest $7.1 billion in its Brazilian unit.
The benchmark Stoxx Europe 600 Index fell 0.3 percent to 258.95 at 3:47 p.m. in London, extending its biggest weekly drop in three months. Stocks declined as a report from the U.S. Institute for Supply Management showed manufacturing expanded in September at the slowest pace in 10 months, while measures of orders and production fell to the lowest level since June 2009.
"We've had a superbly strong September and people were waiting for ISM to see if this rally can be carried forward," said Alberto Espelosin, who helps manage about $12 billion at Zaragoza, Spain-based Ibercaja Gestion. "With a report on manufacturing that's just in line, and within normal levels of expansion, it will stress the case for some consolidation or profit taking in indexes. Only a very positive number would have helped markets in what is a very volatile and range-bound session."
Derail Recovery
The Stoxx 600 is heading for a 1.4 percent drop this week amid lingering concern that the region's sovereign-debt will derail the economic recovery. Still, the gauge yesterday completed its biggest quarterly gain in a year.
The ISM's factory index dropped to 54.4 from 56.3 in August, the Tempe, Arizona-based group said today. Readings greater than 50 signal growth and economists forecast a decline to 54.5, according to the median estimate in a Bloomberg News survey.
Stocks rallied earlier today after a report showed China's manufacturing expanded at the fastest pace in four months in September, adding to signs that economic growth is stabilizing.
"There is something of a paradox going on," said Manoj Ladwa, a London-based senior trader at ETX Capital. "Traders are split between two camps -- either we have seen the worst of the economic crisis and we are on a path to recovery, or we going to see a double dip."
Brussels Meeting
Banks declined after the EU said the region's lenders may be at risk of contagion from the region's debt crisis, just one day after Ireland said it will take majority ownership of Allied Irish Banks Plc, its second bailout of the lender.
A statement issued after a meeting in Brussels today said that finance ministers and central bank governors had "pointed out the risks related to the sovereign debt market that could have a contagious effect on the banking sector."
Santander, Spain's largest bank, fell 2.5 percent to 9.08 euros, while rival Banco Bilbao Vizcaya Argentaria SA lost 2 percent to 9.71 euros. Moody's Investors Service yesterday cut Spain's top credit rating by one notch, citing a "weak" economic outlook and doubts that the nation will reach deficit- reduction targets. Swatch fell 4.1 percent to 354.4 Swiss francs, the most in three months, after Hayek told Blick newspaper that every 1 centime appreciation of the franc against the dollar cuts 20 million francs ($20.5 million) to 30 million francs from earnings. If the franc gains 10 centimes against the dollar, it would cost the company about 280 million francs, he said. Beatrice Howald, a company spokeswoman, confirmed the comments.
Brazilian Unit
LVMH Moet Hennessy Louis Vuitton SA dropped 2.3 percent to 105.15 euros. The maker of Louis Vuitton bags gets more than half of sales outside of Europe.
Schmolz & Bickenbach AG plunged 14 percent to 24.60 Swiss francs after the steelmaker announced plans to raise 297 million francs ($303 million) selling shares.
Repsol jumped 5.3 percent to 19.90 euros, the highest level in two years. China's Sinopec will buy new shares in the company's Brazilian unit and will hold 40 percent of that division after the capital increase. Repsol will also hold the remaining 60 percent stake.
U.K. partner BG Group Plc climbed 3.7 percent to 1,159.5 pence while Sacyr Vallehermoso SA, which owns 20 percent of Repsol, surged 12 percent to 4.93 euros. Galp Energia SGPS SA, Portugal's biggest oil company, increased 6.6 percent to 13.50 euros.
Accenture Forecast
Cap Gemini SA, Europe's largest computer services provider, rallied 3.6 percent to 38.14 euros after Dublin-based Accenture Plc, the world's second-largest technology-consulting firm, forecast net revenue of $5.6 billion to $5.8 billion for the quarter ending November. Analysts had projected $5.62 billion, according to the average of estimates compiled by Bloomberg.
Celesio AG advanced 3.2 percent to 16.47 euros after Exane BNP Paribas raised its recommendation for Europe's biggest publicly traded drug wholesaler to "outperform" from "neutral."
Subsea 7 Inc. rallied 3.1 percent to 118.5 kroner after the oil-services company that operates in the North Sea was awarded a $250 million contract by Total E&P UK Ltd. for the Laggan Tormore deepwater gas field development.
---With assistance by Maryam Nemazee in London. Editors: David Merritt, Andrew Rummer.


text:
Email to a Friend
Be the first to review this product
Sign up to get notified when this product is back in stock
Availability: Out of stock
Double click on above image to view full picture
Use spaces to separate tags. Use single quotes (') for phrases.
Check items to add to the cart or select all
Mark Twain Was Right: The 2001 Cincinnati Riots
Girls Don't Poop
Poo Log: A Record Keeper
What's Your Poo Telling You? 2013 Daily Calendar
The Truth About Poop
Everyone Poops
You have no items in your shopping cart.


text:
A soft and lightweight jersey blend forms the Wildfox Women's Loose T-Shirt in romantic pink. Featuring a crew neck and short sleeves, the top is adorned with a 'Do you want to know a secret? (I'm in love with...)' print to the chest and is made with a loose, fit and an asymmetric hem. K.D.
50% Cotton, 50% Polyester.
|Clothing Style:
|Size / Fit:
Please remember to add postal time (2-3 working days) to obtain a complete estimate of delivery to your door.
You will receive an email to confirm when your item has been sent.
You can also check the status of your order and individual items by accessing My Account details and following the prompts.
Please allow 10 working days from dispatch of your order before notifying us of any late deliveries.
It may be advisable to check with your neighbours to see if a parcel has been left with them, check any outhouses you might have where it may be left if it cannot fit through your letterbox and contact your local sorting office to see if the item has been returned to the depot as undelivered and awaiting collection.
Please see our returns policy.
There are currently no reviews.


text:
How highly do you rank Roberto Duran as a welterweight?
View Single Post
ESB Senior Member
Join Date: Oct 2011
Posts: 1,214
vCash: 500
Re: How highly do you rank Roberto Duran as a welterweight?
Originally Posted by
The problem with Duran was that he used to beat himself up with the balooning in weight, so every match was his to lose really.
Nevertheless, at welter he beat HOF Palomino, Sugar Ray Leonard 1 which is a colossal win. This win did wonders to his greatness and confirmed that this beast who is beating many hall of famers and monstrous fighters at lightweight really can be devestating against the best of the best.
It was a short lived welterweight career that's the problem. He's a VERY great fighter at welter, but can we say he's an ATG when he's only really fought 2 fighters? We can't forget his *****ing out in SRL 2 either.
Palamino is in Hall of Fame? I did not know that. I don't think he beloings. Plus he did not make effort against Duran, he held and ran, and already knew he was going to retire after their bout, for his acting career (Yeah, that went great
) Palomino vs Duran was bad. Duran in first Leanard fight was good, and if he stayed in shape and kpt it up he may have earned greatness at welter.
If 140 poiund division was as big then as now, he would have ben great there, but he had few fights there then went to welter. He was great at 140.
View Public Profile
Find More Posts by Danmann


text:
|By Maureen O'Gara
Article Rating:
|November 13, 2012 10:30 AM EST
Amazon has set up data centers in Australia to supply its cloud services to Australia-based businesses and global companies with customers in Australia.
It'll be Amazon Web Services' ninth region, the Asia Pacific (Sydney) Region, and the third Region in Asia Pacific after the Singapore and Tokyo Regions.
The new Sydney Region consists initially of two separate Availability Zones.
AWS senior VP Andy Jassy said in a statement that AWS had over 10,000 customers in Australia and New Zealand before opening the new centers. "With the ability to achieve single-digit millisecond latency to end users in Sydney, store data locally in Australia, and get to market more quickly and inexpensively by using AWS's infrastructure," he said, the company is expecting to add more customers.
Amazon will be adding a local technical support operation in Australia early next year.
See http://aws.typepad.com/aws/2012/11/asia-pacific-sydney-region-open.html.
The IoT is changing the way enterprises conduct business. In his session at @ThingsExpo, Eric Hoffman, Vice President at EastBanc Technologies, discussed how businesses can gain an edge over competitors by empowering consumers to take control through IoT. He cited examples such as a Washington, D.C.-based sports club that leveraged IoT and the cloud to develop a comprehensive booking system. He also highlighted how IoT can revitalize and restore outdated business models, making them profitable ...
Jul. 29, 2016 10:15 PM EDT Reads: 2,067
Internet of @ThingsExpo, taking place November 1-3, 2016, at the Santa Clara Convention Center in Santa Clara, CA, is co-located with the 19th International Cloud Expo and will feature technical sessions from a rock star conference faculty and the leading industry players in the world and ThingsExpo Silicon Valley Call for Papers is now open.
Jul. 29, 2016 08:00 PM EDT Reads: 2,746
We all know the latest numbers: Gartner, Inc. forecasts that 6.4 billion connected things will be in use worldwide in 2016, up 30 percent from last year, and will reach 20.8 billion by 2020. We're rapidly approaching a data production of 40 zettabytes a day – more than we can every physically store, and exabytes and yottabytes are just around the corner. For many that's a good sign, as data has been proven to equal money – IF it's ingested, integrated, and analyzed fast enough. Without real-ti...
Jul. 29, 2016 07:45 PM EDT Reads: 1,179
SYS-CON Events announced today that 910Telecom will exhibit at the 19th International Cloud Expo, which will take place on November 1–3, 2016, at the Santa Clara Convention Center in Santa Clara, CA. Housed in the classic Denver Gas & Electric Building, 910 15th St., 910Telecom is a carrier-neutral telecom hotel located in the heart of Denver. Adjacent to CenturyLink, AT&T, and Denver Main, 910Telecom offers connectivity to all major carriers, Internet service providers, Internet backbones and ...
Jul. 29, 2016 06:00 PM EDT Reads: 931
Big Data, cloud, analytics, contextual information, wearable tech, sensors, mobility, and WebRTC: together, these advances have created a perfect storm of technologies that are disrupting and transforming classic communications models and ecosystems. In his session at @ThingsExpo, Erik Perotti, Senior Manager of New Ventures on Plantronics' Innovation team, provided an overview of this technological shift, including associated business and consumer communications impacts, and opportunities it ...
Jul. 29, 2016 05:15 PM EDT Reads: 393
You think you know what's in your data. But do you? Most organizations are now aware of the business intelligence represented by their data. Data science stands to take this to a level you never thought of – literally. The techniques of data science, when used with the capabilities of Big Data technologies, can make connections you had not yet imagined, helping you discover new insights and ask new questions of your data. In his session at @ThingsExpo, Sarbjit Sarkaria, data science team lead ...
Jul. 29, 2016 04:45 PM EDT Reads: 1,251
WebRTC is bringing significant change to the communications landscape that will bridge the worlds of web and telephony, making the Internet the new standard for communications. Cloud9 took the road less traveled and used WebRTC to create a downloadable enterprise-grade communications platform that is changing the communication dynamic in the financial sector. In his session at @ThingsExpo, Leo Papadopoulos, CTO of Cloud9, discussed the importance of WebRTC and how it enables companies to focus...
Jul. 29, 2016 04:15 PM EDT Reads: 1,086
ReadyTalk has expanded the capabilities of the FoxDen collaboration platform announced late last year to include FoxDen Connect, an in-room video collaboration experience that launches with a single touch. With FoxDen Connect, users can now not only engage in HD video conferencing between iOS and Android mobile devices or Chrome browsers, but also set up in-person meeting rooms for video interactions. A host's mobile device automatically recognizes the presence of a meeting room via beacon tech...
Jul. 29, 2016 04:15 PM EDT Reads: 430
Manufacturers are embracing the Industrial Internet the same way consumers are leveraging Fitbits – to improve overall health and wellness. Both can provide consistent measurement, visibility, and suggest performance improvements customized to help reach goals. Fitbit users can view real-time data and make adjustments to increase their activity. In his session at @ThingsExpo, Mark Bernardo Professional Services Leader, Americas, at GE Digital, discussed how leveraging the Industrial Internet a...
Jul. 29, 2016 03:15 PM EDT Reads: 675
On Dice.com, the number of job postings asking for skill in Amazon Web Services increased 76 percent between June 2015 and June 2016. Salesforce.com saw its own skill mentions increase 37 percent, while DevOps and Cloud rose 35 percent and 28 percent, respectively. Even as they expand their presence in the cloud, companies are also looking for tech professionals who can manage projects, crunch data, and figure out how to make systems run more autonomously. Mentions of 'data science' as a skill ...
Jul. 29, 2016 03:00 PM EDT Reads: 515
IoT generates lots of temporal data. But how do you unlock its value? You need to discover patterns that are repeatable in vast quantities of data, understand their meaning, and implement scalable monitoring across multiple data streams in order to monetize the discoveries and insights. Motif discovery and deep learning platforms are emerging to visualize sensor data, to search for patterns and to build application that can monitor real time streams efficiently. In his session at @ThingsExpo, ...
Jul. 29, 2016 02:00 PM EDT Reads: 1,208
"delaPlex is a software development company. We do team-based outsourcing development," explained Mark Rivers, COO and Co-founder of delaPlex Software, in this SYS-CON.tv interview at 18th Cloud Expo, held June 7-9, 2016, at the Javits Center in New York City, NY.
Jul. 29, 2016 01:00 PM EDT Reads: 2,130
SYS-CON Events announced today that LeaseWeb USA, a cloud Infrastructure-as-a-Service (IaaS) provider, will exhibit at the 19th International Cloud Expo, which will take place on November 1–3, 2016, at the Santa Clara Convention Center in Santa Clara, CA. LeaseWeb is one of the world's largest hosting brands. The company helps customers define, develop and deploy IT infrastructure tailored to their exact business needs, by combining various kinds cloud solutions.
Jul. 29, 2016 11:15 AM EDT Reads: 1,352
Amazon has gradually rolled out parts of its IoT offerings in the last year, but these are just the tip of the iceberg. In addition to optimizing their back-end AWS offerings, Amazon is laying the ground work to be a major force in IoT – especially in the connected home and office. Amazon is extending its reach by building on its dominant Cloud IoT platform, its Dash Button strategy, recently announced Replenishment Services, the Echo/Alexa voice recognition control platform, the 6-7 strategic...
Jul. 29, 2016 11:15 AM EDT Reads: 696
For basic one-to-one voice or video calling solutions, WebRTC has proven to be a very powerful technology. Although WebRTC's core functionality is to provide secure, real-time p2p media streaming, leveraging native platform features and server-side components brings up new communication capabilities for web and native mobile applications, allowing for advanced multi-user use cases such as video broadcasting, conferencing, and media recording.
Jul. 29, 2016 09:45 AM EDT Reads: 1,041
SYS-CON Events announced today that Venafi, the Immune System for the Internet™ and the leading provider of Next Generation Trust Protection, will exhibit at @DevOpsSummit at 19th International Cloud Expo, which will take place on November 1–3, 2016, at the Santa Clara Convention Center in Santa Clara, CA. Venafi is the Immune System for the Internet™ that protects the foundation of all cybersecurity – cryptographic keys and digital certificates – so they can't be misused by bad guys in attacks...
Jul. 29, 2016 09:45 AM EDT Reads: 1,490
The cloud market growth today is largely in public clouds. While there is a lot of spend in IT departments in virtualization, these aren't yet translating into a true "cloud" experience within the enterprise. What is stopping the growth of the "private cloud" market? In his general session at 18th Cloud Expo, Nara Rajagopalan, CEO of Accelerite, explored the challenges in deploying, managing, and getting adoption for a private cloud within an enterprise. What are the key differences between wh...
Jul. 29, 2016 09:45 AM EDT Reads: 2,159
There will be new vendors providing applications, middleware, and connected devices to support the thriving IoT ecosystem. This essentially means that electronic device manufacturers will also be in the software business. Many will be new to building embedded software or robust software. This creates an increased importance on software quality, particularly within the Industrial Internet of Things where business-critical applications are becoming dependent on products controlled by software. Qua...
Jul. 29, 2016 07:15 AM EDT Reads: 1,657
In addition to all the benefits, IoT is also bringing new kind of customer experience challenges - cars that unlock themselves, thermostats turning houses into saunas and baby video monitors broadcasting over the internet. This list can only increase because while IoT services should be intuitive and simple to use, the delivery ecosystem is a myriad of potential problems as IoT explodes complexity. So finding a performance issue is like finding the proverbial needle in the haystack.
Jul. 29, 2016 06:45 AM EDT Reads: 2,385
Machine Learning helps make complex systems more efficient. By applying advanced Machine Learning techniques such as Cognitive Fingerprinting, wind project operators can utilize these tools to learn from collected data, detect regular patterns, and optimize their own operations. In his session at 18th Cloud Expo, Stuart Gillen, Director of Business Development at SparkCognition, discussed how research has demonstrated the value of Machine Learning in delivering next generation analytics to imp...
Jul. 29, 2016 04:45 AM EDT Reads: 2,585


text:
News By Tag
* Pakistan blasphemy
* More Tags...
Industry News
News By Place
Industry News
Muslims Condemn Blasphemy Charges Against Christian Girl in Pakistan
Muslims together condemn the arrest and imprisonment of a Christian girl, Rimsha Masih, in Pakistan on blasphemy charges. We urge Muslims in Pakistan and around the world to focus on this particular topic and seek to abolish the blasphemy laws.
Contact: Mike Ghouse, America Together Foundation, email@example.com, 214.325.1916
Dallas, Texas - There is a way to find lasting solutions and to rid of abusive practices by a few clergy men. These men, literate or illiterate, claim to follow Prophet Muhammad's (pbuh) life examples; however, they don't. It is our immediate responsibility to pass on Prophet Muhammad's practices to these men who pass judgments without any reverence for the life of the Prophet. Screaming at them or pushing them to the corner is neither Jesus' way nor Muhammad's way.
Prophet Muhammad (pbuh) is considered a blessing to the humanity and rightfully known as Rahmutul Aalameen, the mercy to mankind. It behooves Muslims to protect the integrity of that title and never commit an unmerciful act invoking his name.
There are innumerable examples of forbearance, forgiveness and kindness of the prophet (pbuh) toward those who insulted and even tried to harm him.
On his way to the city of Taif, the Prophet was pelted with rocks by miscreants, his associates wanted to retaliate and go beat up the bad boys. Even the archangel Gabriel offered to crush them and bring relief to the situation. The prophet said no, and instead asked them to join him in forgiving and asked God to bless them with good will. The Prophet preached that the dearest human act to God is forgiveness. As Muslims we need to keep his message of kindness alive and bring to fruition what he was all about: Rahmutul Aalameen. Let the mercy and kindness he taught become a blessing to the universe and continue to shower on humanity.
We appeal to the Ulema, thoughtful people and the Government of Pakistan to give life to the examples of the Prophet. Blasphemy has become a source of harassment and to a few who have nothing better to do than create chaos.
We ask the people of Pakistan to seriously debate the blasphemy laws. Islam is about free will and as Muslims we need to stand against any oppression towards any human being following in the footsteps of the prophet.
Full article at: http://www.huffingtonpost.com/
Mike Ghouse is president of the World Muslim Congress, a think tank and a forum of Muslims together building cohesive societies. MikeGhouse is committed to building a Cohesive America and offers pluralistic solutions on issues of the day. He is a professional speaker, thinker and a writer on pluralism, politics, civic affairs, Islam, India, Israel, peace and justice. Mike is a frequent guest on Sean Hannity Show on Fox TV, and a commentator on national radio networks, he contributes weekly to the Texas Faith Column at Dallas Morning News and regularly at Huffington post, and several other periodicals across the world. The blog www.TheGhouseDiary.com is updated daily.
Follow Mike Ghouse on Twitter: www.twitter.com/
Account Email Address Account Phone Number Disclaimer Report Abuse


text:
Gadgets and Gizmos
This section of our site features all the gadget and gizmo related articles on InventorSpot.com.
Some of our more popular articles include:
- Top 10 Best New Spy Cameras For The Sneaky Surveillance Enthusiast
Subscribe here to our latest "Gadgets and Gizmos" articles.
Rinspeed Squba:Underwater Fun for the Motor Enthusiastby George Delozier
Need This? USB Mouse Typing Speedometerby Luke McKinney
Shake These Salt and Pepper Maracas!by Toby
Yes, I just wrote about salt and pepper grinders, and I promise to get off the subject, but I just had to share these Fukasawa-designed Salt and Pepper Maracas with you, just for quick look at a beautiful design, but impractical concept. What can you do with these? read more »
Exercise While Web Surfing with Springflexby Christine
Need This? Spongebob Squarepants Rectal Thermometerby Luke McKinney
The 6 Innovations Of Chef'n Salt & Pepper Grindersby Toby
I go through two or three salt and pepper grinders a year, a ridiculous number for one who cooks only three or four meals a week. Either the tops break off or the blades get dull, or the turning mechanism fails. Another kitchen tool goes to gadget hell, where nothing is recycled. read more »
Lock Jaw Security Offers Low-Tech Solution to High-Profile Problemby Ian Palmer
Is Ultra-Portable Computing Viable? - The HP UMPC 2133by Ben Arnold
Great Invention Idea? The Most Unremarkable Animal Toy Ever!by Elizabeth Valeri
My recent investigations into the mysteries of the United States Patent Office, well not so mysterious as it is undiscerning and undiscriminating, has unearthed a whole host of opportunistic patents. That is, the patenting of stuff we've all been doing, independent of the patent process, for centuries. And United States patent 6360693 is perhaps the best (worst?) example of this practice. read more »


text:
Contact Us
Enter Your Search:
Search tips
Search criteria
Journal titles
Results 1-25 (790272)
« Previous
Go to page number:
Next »
Clipboard (0)
Related Articles
The Genomics of Disulfide Bonding and Protein Stabilization in Thermophiles
Beeby, Morgan
O'Connor, Brian D
Ryttersgaard, Carsten
Boutz, Daniel R
Perry, L. Jeanne
Yeates, Todd O
PLoS Biology
Thermophilic organisms flourish in varied high-temperature environmental niches that are deadly to other organisms. Recently, genomic evidence has implicated a critical role for disulfide bonds in the structural stabilization of intracellular proteins from certain of these organisms, contrary to the conventional view that structural disulfide bonds are exclusively extracellular. Here both computational and structural data are presented to explore the occurrence of disulfide bonds as a protein-stabilization method across many thermophilic prokaryotes. Based on computational studies, disulfide-bond richness is found to be widespread, with thermophiles containing the highest levels. Interestingly, only a distinct subset of thermophiles exhibit this property. A computational search for proteins matching this target phylogenetic profile singles out a specific protein, known as protein disulfide oxidoreductase, as a potential key player in thermophilic intracellular disulfide-bond formation. Finally, biochemical support in the form of a new crystal structure of a thermophilic protein with three disulfide bonds is presented together with a survey of known structures from the literature. Together, the results provide insight into biochemical specialization and the diversity of methods employed by organisms to stabilize their proteins in exotic environments. The findings also motivate continued efforts to sequence genomes from divergent organisms.
Certain thermophiles are found to stabilize their proteins in extreme environments with additional disulfide bonds. A phylogenetic profile identifies a protein disulfide oxidoreductase critical to the stabilization process.
Related Articles
A pursuit of lineage-specific and niche-specific proteome features in the world of archaea
Roy Chowdhury, Anindya
Dutta, Chitra
BMC Genomics
Archaea evoke interest among researchers for two enigmatic characteristics –a combination of bacterial and eukaryotic components in their molecular architectures and an enormous diversity in their life-style and metabolic capabilities. Despite considerable research efforts, lineage- specific/niche-specific molecular features of the whole archaeal world are yet to be fully unveiled. The study offers the first large-scale in silico proteome analysis of all archaeal species of known genome sequences with a special emphasis on methanogenic and sulphur-metabolising archaea.
Overall amino acid usage in archaea is dominated by GC-bias. But the environmental factors like oxygen requirement or thermal adaptation seem to play important roles in selection of residues with no GC-bias at the codon level. All methanogens, irrespective of their thermal/salt adaptation, show higher usage of Cys and have relatively acidic proteomes, while the proteomes of sulphur-metabolisers have higher aromaticity and more positive charges. Despite of exhibiting thermophilic life-style, korarchaeota possesses an acidic proteome. Among the distinct trends prevailing in COGs (Cluster of Orthologous Groups of proteins) distribution profiles, crenarchaeal organisms display higher intra-order variations in COGs repertoire, especially in the metabolic ones, as compared to euryarchaea. All methanogens are characterised by a presence of 22 exclusive COGs.
Divergences in amino acid usage, aromaticity/charge profiles and COG repertoire among methanogens and sulphur-metabolisers, aerobic and anaerobic archaea or korarchaeota and nanoarchaeota, as elucidated in the present study, point towards the presence of distinct molecular strategies for niche specialization in the archaeal world.
Amino acid usage; Isoelectric point; COG distribution; Methanogen; Sulphur metaboliser; Korarachaeota; Oxygen requirement
Related Articles
GDAP: a web tool for genome-wide protein disulfide bond prediction
O'Connor, Brian D.
Yeates, Todd O.
Nucleic Acids Research
2004;32(Web Server issue):W360-W364.
The Genomic Disulfide Analysis Program (GDAP) provides web access to computationally predicted protein disulfide bonds for over one hundred microbial genomes, including both bacterial and achaeal species. In the GDAP process, sequences of unknown structure are mapped, when possible, to known homologous Protein Data Bank (PDB) structures, after which specific distance criteria are applied to predict disulfide bonds. GDAP also accepts user-supplied protein sequences and subsequently queries the PDB sequence database for the best matches, scans for possible disulfide bonds and returns the results to the client. These predictions are useful for a variety of applications and have previously been used to show a dramatic preference in certain thermophilic archaea and bacteria for disulfide bonds within intracellular proteins. Given the central role these stabilizing, covalent bonds play in such organisms, the predictions available from GDAP provide a rich data source for designing site-directed mutants with more stable thermal profiles. The GDAP web application is a gateway to this information and can be used to understand the role disulfide bonds play in protein stability both in these unusual organisms and in sequences of interest to the individual researcher. The prediction server can be accessed at http://www.doe-mbi.ucla.edu/Services/GDAP.
Related Articles
Close relationship of a novel Flavobacteriaceae α-amylase with archaeal α-amylases and good potentials for industrial applications
Li, Chunfang
Du, Miaofen
Cheng, Bin
Wang, Lushan
Liu, Xinqiang
Ma, Cuiqing
Yang, Chunyu
Xu, Ping
Biotechnology for Biofuels
Bioethanol production from various starchy materials has received much attention in recent years. α-Amylases are key enzymes in the bioconversion process of starchy biomass to biofuels, food or other products. The properties of thermostability, pH stability, and Ca-independency are important in the development of such fermentation process.
A novel Flavobacteriaceae Sinomicrobium α-amylase (FSA) was identified and characterized from genomic analysis of a novel Flavobacteriaceae species. It is closely related with archaeal α-amylases in the GH13_7 subfamily, but is evolutionary distant with other bacterial α-amylases. Based on the conserved sequence alignment and homology modeling, with minor variation, the Zn2+- and Ca2+-binding sites of FSA were predicated to be the same as those of the archaeal thermophilic α-amylases. The recombinant α-amylase was highly expressed and biochemically characterized. It showed optimum activity at pH 6.0, high enzyme stability at pH 6.0 to 11.0, but weak thermostability. A disulfide bond was introduced by site-directed mutagenesis in domain C and resulted in the apparent improvement of the enzyme activity at high temperature and broad pH range. Moreover, about 50% of the enzyme activity was detected under 100°C condition, whereas no activity was observed for the wild type enzyme. Its thermostability was also enhanced to some extent, with the half-life time increasing from 25 to 55 minutes at 50°C. In addition, after the introduction of the disulfide bond, the protein became a Ca-independent enzyme.
The improved stability of FSA suggested that the domain C contributes to the overall stability of the enzyme under extreme conditions. In addition, successfully directed modification and special evolutionary status of FSA imply its directional reconstruction potentials for bioethanol production, as well as for other industrial applications.
α-Amylases; Evolutionary position; Site-directed mutagenesis; Thermostability; Domain C
Related Articles
Hyperthermophilic Enzymes: Sources, Uses, and Molecular Mechanisms for Thermostability
Vieille, Claire
Zeikus, Gregory J.
Microbiology and Molecular Biology Reviews
Enzymes synthesized by hyperthermophiles (bacteria and archaea with optimal growth temperatures of >80°C), also called hyperthermophilic enzymes, are typically thermostable (i.e., resistant to irreversible inactivation at high temperatures) and are optimally active at high temperatures. These enzymes share the same catalytic mechanisms with their mesophilic counterparts. When cloned and expressed in mesophilic hosts, hyperthermophilic enzymes usually retain their thermal properties, indicating that these properties are genetically encoded. Sequence alignments, amino acid content comparisons, crystal structure comparisons, and mutagenesis experiments indicate that hyperthermophilic enzymes are, indeed, very similar to their mesophilic homologues. No single mechanism is responsible for the remarkable stability of hyperthermophilic enzymes. Increased thermostability must be found, instead, in a small number of highly specific alterations that often do not obey any obvious traffic rules. After briefly discussing the diversity of hyperthermophilic organisms, this review concentrates on the remarkable thermostability of their enzymes. The biochemical and molecular properties of hyperthermophilic enzymes are described. Mechanisms responsible for protein inactivation are reviewed. The molecular mechanisms involved in protein thermostabilization are discussed, including ion pairs, hydrogen bonds, hydrophobic interactions, disulfide bridges, packing, decrease of the entropy of unfolding, and intersubunit interactions. Finally, current uses and potential applications of thermophilic and hyperthermophilic enzymes as research reagents and as catalysts for industrial processes are described.
Related Articles
Discovery of a thermophilic protein complex stabilized by topologically interlinked chains
Boutz, Daniel R.
Cascio, Duilio
Whitelegge, Julian
Perry, L. Jeanne
Yeates, Todd O.
Journal of molecular biology
A growing number of organisms have been discovered inhabiting extreme environments, including temperatures in excess of 100 °C. How cellular proteins from such organisms retain their native folds under extreme conditions is still not fully understood. Recent computational and structural studies have identified disulfide bonding as an important mechanism for stabilizing intracellular proteins in certain thermophilic microbes. Here, we present the first proteomic analysis of intracellular disulfide bonding in the hyperthermophilic archaeon Pyrobaculum aerophilum. Our study reveals that the utilization of disulfide bonds extends beyond individual proteins to include many protein-protein complexes. We report the 1.6Å crystal structure of one such complex, a citrate synthase homodimer. The structure contains two intramolecular disulfide bonds, one per subunit, which result in the cyclization of each protein chain in such a way that the two chains are topologically interlinked, rendering them inseparable. This unusual feature emphasizes the variety and sophistication of the molecular mechanisms that can be achieved by evolution.
disulfide bond; protein stability; catenane; citrate synthase; thermophile
Related Articles
Structures and functional implications of an AMP-binding cystathionine β-synthase domain protein from a hyperthermophilic archaeon
King, Neil P.
Lee, Toni M.
Sawaya, Michael R.
Cascio, Duilio
Yeates, Todd O.
Journal of molecular biology
Cystathionine β-synthase (CBS) domains are found in myriad proteins from organisms across the tree of life, and have been hypothesized to function as regulatory modules that sense the energy charge of the cell. Here we characterize the structure and stability of PAE2072, a dimeric, tandem CBS domain protein from the hyperthermophilic crenarchaeon Pyrobaculum aerophilum. Crystal structures of the protein in unliganded and adenosine monophosphate (AMP)-bound forms, determined at resolutions of 2.10 Å and 2.35 Å respectively, reveal a remarkable conservation of key functional features seen in the γ subunit of the eukaryotic AMP-activated protein kinase (AMPK). The structures also confirm the presence of a suspected intermolecular disulfide bond between the two subunits that is shown to stabilize the protein. Our AMP-bound structure represents a first step in investigating the function of a large class of uncharacterized prokaryotic proteins. In addition, this work extends previous studies that have suggested that, in certain thermophilic microbes, disulfide bonds play a key role in stabilizing intracellular proteins and protein-protein complexes.
Cystathionine β-synthase; AMP-activated protein kinase; disulfide bond; protein stabilization; hyperthermophile
Related Articles
Structure and Function of Cold Shock Proteins in Archaea▿
Giaquinto, Laura
Curmi, Paul M. G.
Siddiqui, Khawar S.
Poljak, Anne
DeLong, Ed
DasSarma, Shiladitya
Cavicchioli, Ricardo
Journal of Bacteriology
Archaea are abundant and drive critical microbial processes in the Earth's cold biosphere. Despite this, not enough is known about the molecular mechanisms of cold adaptation and no biochemical studies have been performed on stenopsychrophilic archaea (e.g., Methanogenium frigidum). This study examined the structural and functional properties of cold shock proteins (Csps) from archaea, including biochemical analysis of the Csp from M. frigidum. csp genes are present in most bacteria and some eucarya but absent from most archaeal genome sequences, most notably, those of all archaeal thermophiles and hyperthermophiles. In bacteria, Csps are small, nucleic acid binding proteins involved in a variety of cellular processes, such as transcription. In this study, archaeal Csp function was assessed by examining the ability of csp genes from psychrophilic and mesophilic Euryarchaeota and Crenarchaeota to complement a cold-sensitive growth defect in Escherichia coli. In addition, an archaeal gene with a cold shock domain (CSD) fold but little sequence identity to Csps was also examined. Genes encoding Csps or a CSD structural analog from three psychrophilic archaea rescued the E. coli growth defect. The three proteins were predicted to have a higher content of solvent-exposed basic residues than the noncomplementing proteins, and the basic residues were located on the nucleic acid binding surface, similar to their arrangement in E. coli CspA. The M. frigidum Csp was purified and found to be a single-domain protein that folds by a reversible two-state mechanism and to exhibit a low conformational stability typical of cold-adapted proteins. Moreover, M. frigidum Csp was characterized as binding E. coli single-stranded RNA, consistent with its ability to complement function in E. coli. The studies show that some Csp and CSD fold proteins have retained sufficient similarity throughout evolution in the Archaea to be able to function effectively in the Bacteria and that the function of the archaeal proteins relates to cold adaptation. The initial biochemical analysis of M. frigidum Csp has developed a platform for further characterization and demonstrates the potential for expanding molecular studies of proteins from this important archaeal stenopsychrophile.
Related Articles
Stress Genes and Proteins in the Archaea
Macario, Alberto J. L.
Lange, Marianne
Ahring, Birgitte K.
De Macario, Everly Conway
Microbiology and Molecular Biology Reviews
The field covered in this review is new; the first sequence of a gene encoding the molecular chaperone Hsp70 and the first description of a chaperonin in the archaea were reported in 1991. These findings boosted research in other areas beyond the archaea that were directly relevant to bacteria and eukaryotes, for example, stress gene regulation, the structure-function relationship of the chaperonin complex, protein-based molecular phylogeny of organisms and eukaryotic-cell organelles, molecular biology and biochemistry of life in extreme environments, and stress tolerance at the cellular and molecular levels. In the last 8 years, archaeal stress genes and proteins belonging to the families Hsp70, Hsp60 (chaperonins), Hsp40(DnaJ), and small heat-shock proteins (sHsp) have been studied. The hsp70(dnaK), hsp40(dnaJ), and grpE genes (the chaperone machine) have been sequenced in seven, four, and two species, respectively, but their expression has been examined in detail only in the mesophilic methanogen Methanosarcina mazei S-6. The proteins possess markers typical of bacterial homologs but none of the signatures distinctive of eukaryotes. In contrast, gene expression and transcription initiation signals and factors are of the eucaryal type, which suggests a hybrid archaeal-bacterial complexion for the Hsp70 system. Another remarkable feature is that several archaeal species in different phylogenetic branches do not have the gene hsp70(dnaK), an evolutionary puzzle that raises the important question of what replaces the product of this gene, Hsp70(DnaK), in protein biogenesis and refolding and for stress resistance. Although archaea are prokaryotes like bacteria, their Hsp60 (chaperonin) family is of type (group) II, similar to that of the eukaryotic cytosol; however, unlike the latter, which has several different members, the archaeal chaperonin system usually includes only two (in some species one and in others possibly three) related subunits of ∼60 kDa. These form, in various combinations depending on the species, a large structure or chaperonin complex sometimes called the thermosome. This multimolecular assembly is similar to the bacterial chaperonin complex GroEL/S, but it is made of only the large, double-ring oligomers each with eight (or nine) subunits instead of seven as in the bacterial complex. Like Hsp70(DnaK), the archaeal chaperonin subunits are remarkable for their evolution, but for a different reason. Ubiquitous among archaea, the chaperonins show a pattern of recurrent gene duplication—hetero-oligomeric chaperonin complexes appear to have evolved several times independently. The stress response and stress tolerance in the archaea involve chaperones, chaperonins, other heat shock (stress) proteins including sHsp, thermoprotectants, the proteasome, as yet incompletely understood thermoresistant features of many molecules, and formation of multicellular structures. The latter structures include single- and mixed-species (bacterial-archaeal) types. Many questions remain unanswered, and the field offers extraordinary opportunities owing to the diversity, genetic makeup, and phylogenetic position of archaea and the variety of ecosystems they inhabit. Specific aspects that deserve investigation are elucidation of the mechanism of action of the chaperonin complex at different temperatures, identification of the partners and substitutes for the Hsp70 chaperone machine, analysis of protein folding and refolding in hyperthermophiles, and determination of the molecular mechanisms involved in stress gene regulation in archaeal species that thrive under widely different conditions (temperature, pH, osmolarity, and barometric pressure). These studies are now possible with uni- and multicellular archaeal models and are relevant to various areas of basic and applied research, including exploration and conquest of ecosystems inhospitable to humans and many mammals and plants.
Related Articles
Structural comparison of tRNA m1A58 methyltransferases revealed different molecular strategies to maintain their oligomeric architecture under extreme conditions
Guelorget, Amandine
Barraud, Pierre
Tisné, Carine
Golinelli-Pimpaneau, Béatrice
BMC Structural Biology
tRNA m1A58 methyltransferases (TrmI) catalyze the transfer of a methyl group from S-adenosyl-L-methionine to nitrogen 1 of adenine 58 in the T-loop of tRNAs from all three domains of life. The m1A58 modification has been shown to be essential for cell growth in yeast and for adaptation to high temperatures in thermophilic organisms. These enzymes were shown to be active as tetramers. The crystal structures of five TrmIs from hyperthermophilic archaea and thermophilic or mesophilic bacteria have previously been determined, the optimal growth temperature of these organisms ranging from 37°C to 100°C. All TrmIs are assembled as tetramers formed by dimers of tightly assembled dimers.
In this study, we present a comparative structural analysis of these TrmIs, which highlights factors that allow them to function over a large range of temperature. The monomers of the five enzymes are structurally highly similar, but the inter-monomer contacts differ strongly. Our analysis shows that bacterial enzymes from thermophilic organisms display additional intermolecular ionic interactions across the dimer interfaces, whereas hyperthermophilic enzymes present additional hydrophobic contacts. Moreover, as an alternative to two bidentate ionic interactions that stabilize the tetrameric interface in all other TrmI proteins, the tetramer of the archaeal P. abyssi enzyme is strengthened by four intersubunit disulfide bridges.
The availability of crystal structures of TrmIs from mesophilic, thermophilic or hyperthermophilic organisms allows a detailed analysis of the architecture of this protein family. Our structural comparisons provide insight into the different molecular strategies used to achieve the tetrameric organization in order to maintain the enzyme activity under extreme conditions.
Related Articles
Denaturation and Unfolding of Human Anaphylatoxin C3a
Chang, Jui-Yoa
Lin, Curtis C.-J.
Salamanca, Silvia
Pangburn, Michael K.
Wetsel, Rick A.
Archives of biochemistry and biophysics
The complement C3a anaphylatoxin is a major molecular mediator of innate immunity. It is a potent activator of mast cells, basophils and eosinophils and causes smooth muscle contraction. Structurally, C3a is a relatively small protein (77 amino acids) comprising a N-terminal domain connected by 3 native disulfide bonds and a helical C-terminal segment. The structural stability of C3a has been investigated here using three different methods: Disulfide scrambling; Differential CD spectroscopy; and Reductive unfolding. Two uncommon features regarding the stability of C3a and the structure of denatured C3a have been observed in this study. (a) There is an unusual disconnection between the conformational stability of C3a and the covalent stability of its three native disulfide bonds that is not seen with other disulfide proteins. As measured by both methods of disulfide scrambling and differential CD spectroscopy, the native C3a exhibits a global conformational stability that is comparable to numerous proteins with similar size and disulfide content, all with mid-point denaturation of [GdmCl]1/2 at 3.4-5M. These proteins include hirudin, tick anticoagulant protein and leech carboxypeptidase inhibitor. However, the native disulfide bonds of C3a is 150-1000 fold less stable than those proteins as evaluated by the method of reductive unfolding. The 3 native disulfide bonds of C3a can be collectively and quantitatively reduced with as low as 1 mM of dithiothreitol within 5 min. The fragility of the native disulfide bonds of C3a has not yet been observed with other native disulfide proteins. (b) Using the method of disulfide scrambling, denatured C3a was shown to consist of diverse isomers adopting varied extent of unfolding. Among them, the most extensively unfolded isomer of denatured C3a is found to assume beads-form disulfide pattern, comprising Cys36-Cys49 and two disulfide bonds formed by two pair of consecutive cysteines, Cys22-Cys23 and Cys56-Cys57, a unique disulfide structure of polypeptide that has not been documented previously.
Conformational stability of C3a; Denaturation of C3a; Unfolding of C3a; Method of disulfide scrambling; Scrambled isomers of C3a; Reductive unfolding of native C3a
Related Articles
Catalysis of Protein Disulfide Bond Isomerization in a Homogeneous Substrate†
Kersteen, Elizabeth A.
Barrows, Seth R.
Raines, Ronald T.
Protein disulfide isomerase (PDI) catalyzes the rearrangement of nonnative disulfide bonds in the endoplasmic reticulum of eukaryotic cells, a process that often limits the rate at which polypeptide chains fold into a native protein conformation. The mechanism of the reaction catalyzed by PDI is unclear. In assays involving protein substrates, the reaction appears to involve the complete reduction of some or all of its nonnative disulfide bonds followed by oxidation of the resulting dithiols. The substrates in these assays are, however, heterogeneous, which complicates mechanistic analyses. Here, we report the first analysis of disulfide bond isomerization in a homogeneous substrate. Our substrate is based on tachyplesin I, a 17-mer peptide that folds into a _-hairpin stabilized by two disulfide bonds. We describe the chemical synthesis of a variant of tachyplesin I in which its two disulfide bonds are in a nonnative state and side chains near its N-and C-terminus contain a fluorescence donor (tryptophan) and acceptor (N_-dansyllysine). Fluorescence resonance energy transfer from 280 to 465 nm increases by 28-fold upon isomerization of the disulfide bonds into their native state (which has a lower E°_ = -0.313 V than does PDI). We use this continuous assay to analyze catalysis by wild-type human PDI and a variant in which the C-terminal cysteine residue within each Cys—Gly—His—Cys active site is replaced with alanine. We find that wild-type PDI catalyzes the isomerization of the substrate with kcat/KM = 1.7 _ 105 M–1M s–1, which is the largest value yet reported for catalysis of disulfide bond isomerization. The variant, which is a poor catalyst of disulfide bond reduction and dithiol oxidation, retains virtually all of the activity of wild-type PDI in catalysis of disulfide bond isomerization. Thus, the C-terminal cysteine residues play an insignificant role in the isomerization of the disulfide bonds in nonnative tachyplesin I. We conclude that catalysis of disulfide bond isomerization by PDI does not necessarily involve a cycle of substrate reduction/oxidation.
Related Articles
The role of disulfide bond in hyperthermophilic endocellulase
Kim, Han-Woo
Ishikawa, Kazuhiko
The hyperthermophilic endocellulase, EGPh (glycosyl hydrolase family 5) from Pyrococcus horikoshii possesses 4 cysteine residues forming 2 disulfide bonds, as identified by structural analysis. One of the disulfide bonds is located at the proximal region of the active site in EGPh, which exhibits a distinct pattern from that of the thermophilic endocellulase EGAc (glycosyl hydrolase family 5) of Acidothermus cellulolyticus despite the structural similarity between the two endocellulases. The structural similarity between EGPh and EGAc suggests that EGPh possesses a structure suitable for changing the position of the disulfide bond corresponding to that in EGAc. Introduction of this alternative disulfide bond in EGPh, while removing the original disulfide bond, did not result in a loss of enzymatic activity but the EGPh was no longer hyperthermostable. These results suggest that the contribution of disulfide bond to hyperthermostability at temperature higher than 100 °C is restrictive, and that its impact is dependent on the specific structural environment of the hyperthermophilic proteins. The data suggest that the structural position and environment of the disulfide bond has a greater effect on high-temperature thermostability of the enzyme than on the potential energy of the dihedral angle that contributes to disulfide bond cleavage.
Disulfide bond; Cellulase; Archaea; Thermostability; Crystal structure; Protein engineering
Related Articles
Disulfide Bond Formation in the Herpes Simplex Virus 1 UL6 Protein Is Required for Portal Ring Formation and Genome Encapsidation▿
Albright, Brandon S.
Nellissery, Jacob
Szczepaniak, Renata
Weller, Sandra K.
Journal of Virology
The herpes simplex virus 1 (HSV-1) UL6 portal protein forms a 12-subunit ring structure at a unique capsid vertex which functions as a conduit for the encapsidation of the viral genome. We have demonstrated previously that the leucine zipper region of UL6 is important for intersubunit interactions and stable ring formation (J. K. Nellissery, R. Szczepaniak, C. Lamberti, and S. K. Weller, J. Virol. 81:8868–8877, 2007). We now demonstrate that intersubunit disulfide bonds exist between monomeric subunits and contribute to portal ring formation and/or stability. Intersubunit disulfide bonds were detected in purified portal rings by SDS-PAGE under nonreducing conditions. Furthermore, the treatment of purified portal rings with dithiothreitol (DTT) resulted in the disruption of the rings, suggesting that disulfide bonds confer stability to this complex structure. The UL6 protein contains nine cysteines that were individually mutated to alanine. Two of these mutants, C166A and C254A, failed to complement a UL6 null mutant in a transient complementation assay. Furthermore, viral mutants bearing the C166A and C254A mutations failed to produce infectious progeny and were unable to cleave or package viral DNA. In cells infected with C166A or C254A, B capsids were produced which contained UL6 at reduced levels compared to those seen in wild-type capsids. In addition, C166A and C254A mutant proteins expressed in insect cells infected with recombinant baculovirus failed to form ring structures. Cysteines at positions 166 and 254 thus appear to be required for intersubunit disulfide bond formation. Taken together, these results indicate that disulfide bond formation is required for portal ring formation and/or stability and for the production of procapsids that are capable of encapsidation.
Related Articles
Characterization of SrgA, a Salmonella enterica Serovar Typhimurium Virulence Plasmid-Encoded Paralogue of the Disulfide Oxidoreductase DsbA, Essential for Biogenesis of Plasmid-Encoded Fimbriae
Bouwman, C. W.
Kohli, M.
Killoran, A.
Touchie, G. A.
Kadner, R. J.
Martin, N. L.
Journal of Bacteriology
Disulfide oxidoreductases are viewed as foldases that help to maintain proteins on productive folding pathways by enhancing the rate of protein folding through the catalytic incorporation of disulfide bonds. SrgA, encoded on the virulence plasmid pStSR100 of Salmonella enterica serovar Typhimurium and located downstream of the plasmid-borne fimbrial operon, is a disulfide oxidoreductase. Sequence analysis indicates that SrgA is similar to DsbA from, for example, Escherichia coli, but not as highly conserved as most of the chromosomally encoded disulfide oxidoreductases from members of the family Enterobacteriaceae. SrgA is localized to the periplasm, and its disulfide oxidoreductase activity is dependent upon the presence of functional DsbB, the protein that is also responsible for reoxidation of the major disulfide oxidoreductase, DsbA. A quantitative analysis of the disulfide oxidoreductase activity of SrgA showed that SrgA was less efficient than DsbA at introducing disulfide bonds into the substrate alkaline phosphatase, suggesting that SrgA is more substrate specific than DsbA. It was also demonstrated that the disulfide oxidoreductase activity of SrgA is necessary for the production of plasmid-encoded fimbriae. The major structural subunit of the plasmid-encoded fimbriae, PefA, contains a disulfide bond that must be oxidized in order for PefA stability to be maintained and for plasmid-encoded fimbriae to be assembled. SrgA efficiently oxidizes the disulfide bond of PefA, while the S. enterica serovar Typhimurium chromosomally encoded disulfide oxidoreductase DsbA does not. pefA and srgA were also specifically expressed at pH 5.1 but not at pH 7.0, suggesting that the regulatory mechanisms involved in pef gene expression are also involved in srgA expression. SrgA therefore appears to be a substrate-specific disulfide oxidoreductase, thus explaining the requirement for an additional catalyst of disulfide bond formation in addition to DsbA of S. enterica serovar Typhimurium.
Related Articles
Consistent mutational paths predict eukaryotic thermostability
van Noort, Vera
Bradatsch, Bettina
Arumugam, Manimozhiyan
Amlacher, Stefan
Bange, Gert
Creevey, Chris
Falk, Sebastian
Mende, Daniel R
Sinning, Irmgard
Hurt, Ed
Bork, Peer
BMC Evolutionary Biology
Proteomes of thermophilic prokaryotes have been instrumental in structural biology and successfully exploited in biotechnology, however many proteins required for eukaryotic cell function are absent from bacteria or archaea. With Chaetomium thermophilum, Thielavia terrestris and Thielavia heterothallica three genome sequences of thermophilic eukaryotes have been published.
Studying the genomes and proteomes of these thermophilic fungi, we found common strategies of thermal adaptation across the different kingdoms of Life, including amino acid biases and a reduced genome size. A phylogenetics-guided comparison of thermophilic proteomes with those of other, mesophilic Sordariomycetes revealed consistent amino acid substitutions associated to thermophily that were also present in an independent lineage of thermophilic fungi. The most consistent pattern is the substitution of lysine by arginine, which we could find in almost all lineages but has not been extensively used in protein stability engineering. By exploiting mutational paths towards the thermophiles, we could predict particular amino acid residues in individual proteins that contribute to thermostability and validated some of them experimentally. By determining the three-dimensional structure of an exemplar protein from C. thermophilum (Arx1), we could also characterise the molecular consequences of some of these mutations.
The comparative analysis of these three genomes not only enhances our understanding of the evolution of thermophily, but also provides new ways to engineer protein stability.
Thermophily; Comparative genomics; Protein engineering; Eukaryotes; Fungi
Related Articles
Dissecting the Machinery That Introduces Disulfide Bonds in Pseudomonas aeruginosa
Arts, Isabelle S.
Ball, Geneviève
Leverrier, Pauline
Garvis, Steven
Nicolaes, Valérie
Vertommen, Didier
Ize, Bérengère
Tamu Dufe, Veronica
Messens, Joris
Voulhoux, Romé
Collet, Jean-François
Disulfide bond formation is required for the folding of many bacterial virulence factors. However, whereas the Escherichia coli disulfide bond-forming system is well characterized, not much is known on the pathways that oxidatively fold proteins in pathogenic bacteria. Here, we report the detailed unraveling of the pathway that introduces disulfide bonds in the periplasm of the human pathogen Pseudomonas aeruginosa. The genome of P. aeruginosa uniquely encodes two DsbA proteins (P. aeruginosa DsbA1 [PaDsbA1] and PaDsbA2) and two DsbB proteins (PaDsbB1 and PaDsbB2). We found that PaDsbA1, the primary donor of disulfide bonds to secreted proteins, is maintained oxidized in vivo by both PaDsbB1 and PaDsbB2. In vitro reconstitution of the pathway confirms that both PaDsbB1 and PaDsbB2 shuttle electrons from PaDsbA1 to membrane-bound quinones. Accordingly, deletion of both P. aeruginosa dsbB1 (PadsbB1) and PadsbB2 is required to prevent the folding of several P. aeruginosa virulence factors and to lead to a significant decrease in pathogenicity. Using a high-throughput proteomic approach, we also analyzed the impact of PadsbA1 deletion on the global periplasmic proteome of P. aeruginosa, which allowed us to identify more than 20 new potential substrates of this major oxidoreductase. Finally, we report the biochemical and structural characterization of PaDsbA2, a highly oxidizing oxidoreductase, which seems to be expressed under specific conditions. By fully dissecting the machinery that introduces disulfide bonds in P. aeruginosa, our work opens the way to the design of novel antibacterial molecules able to disarm this pathogen by preventing the proper assembly of its arsenal of virulence factors.
The human pathogen Pseudomonas aeruginosa causes life-threatening infections in immunodepressed and cystic fibrosis patients. The emergence of P. aeruginosa strains resistant to all of the available antibacterial agents calls for the urgent development of new antibiotics active against this bacterium. The pathogenic power of P. aeruginosa is mediated by an arsenal of extracellular virulence factors, most of which are stabilized by disulfide bonds. Thus, targeting the machinery that introduces disulfide bonds appears to be a promising strategy to combat P. aeruginosa. Here, we unraveled the oxidative protein folding system of P. aeruginosa in full detail. The system uniquely consists of two membrane proteins that generate disulfide bonds de novo to deliver them to P. aeruginosa DsbA1 (PaDsbA1), a soluble oxidoreductase. PaDsbA1 in turn donates disulfide bonds to secreted proteins, including virulence factors. Disruption of the disulfide bond formation machinery dramatically decreases P. aeruginosa virulence, confirming that disulfide formation systems are valid targets for the design of antimicrobial drugs.
Related Articles
Evolution and thermodynamics of the slow unfolding of hyperstable monomeric proteins
Okada, Jun
Okamoto, Tomohiro
Mukaiyama, Atsushi
Tadokoro, Takashi
You, Dong-Ju
Chon, Hyongi
Koga, Yuichi
Takano, Kazufumi
Kanaya, Shigenori
BMC Evolutionary Biology
The unfolding speed of some hyperthermophilic proteins is dramatically lower than that of their mesostable homologs. Ribonuclease HII from the hyperthermophilic archaeon Thermococcus kodakaraensis (Tk-RNase HII) is stabilized by its remarkably slow unfolding rate, whereas RNase HI from the thermophilic bacterium Thermus thermophilus (Tt-RNase HI) unfolds rapidly, comparable with to that of RNase HI from Escherichia coli (Ec-RNase HI).
To clarify whether the difference in the unfolding rate is due to differences in the types of RNase H or differences in proteins from archaea and bacteria, we examined the equilibrium stability and unfolding reaction of RNases HII from the hyperthermophilic bacteria Thermotoga maritima (Tm-RNase HII) and Aquifex aeolicus (Aa-RNase HII) and RNase HI from the hyperthermophilic archaeon Sulfolobus tokodaii (Sto-RNase HI). These proteins from hyperthermophiles are more stable than Ec-RNase HI over all the temperature ranges examined. The observed unfolding speeds of all hyperstable proteins at the different denaturant concentrations studied are much lower than those of Ec-RNase HI, which is in accordance with the familiar slow unfolding of hyperstable proteins. However, the unfolding rate constants of these RNases H in water are dispersed, and the unfolding rate constant of thermophilic archaeal proteins is lower than that of thermophilic bacterial proteins.
These results suggest that the nature of slow unfolding of thermophilic proteins is determined by the evolutionary history of the organisms involved. The unfolding rate constants in water are related to the amount of buried hydrophobic residues in the tertiary structure.
Related Articles
Pre-expression of a sulfhydryl oxidase significantly increases the yields of eukaryotic disulfide bond containing proteins expressed in the cytoplasm of E.coli
Nguyen, Van Dat
Hatahet, Feras
Salo, Kirsi EH
Enlund, Eveliina
Zhang, Chi
Ruddock, Lloyd W
Microbial Cell Factories
Disulfide bonds are one of the most common post-translational modifications found in proteins. The production of proteins that contain native disulfide bonds is challenging, especially on a large scale. Either the protein needs to be targeted to the endoplasmic reticulum in eukaryotes or to the prokaryotic periplasm. These compartments that are specialised for disulfide bond formation have an active catalyst for their formation, along with catalysts for isomerization to the native state. We have recently shown that it is possible to produce large amounts of prokaryotic disulfide bond containing proteins in the cytoplasm of wild-type bacteria such as E. coli by the introduction of catalysts for both of these processes.
Here we show that the introduction of Erv1p, a sulfhydryl oxidase and a disulfide isomerase allows the efficient formation of natively folded eukaryotic proteins with multiple disulfide bonds in the cytoplasm of E. coli. The production of disulfide bonded proteins was also aided by the use of an appropriate fusion protein to keep the folding intermediates soluble and by choice of media. By combining the pre-expression of a sulfhydryl oxidase and a disulfide isomerase with these other factors, high level expression of even complex disulfide bonded eukaryotic proteins is possible
Our results show that the production of eukaryotic proteins with multiple disulfide bonds in the cytoplasm of E. coli is possible. The required exogenous components can be put onto a single plasmid vector allowing facile transfer between different prokaryotic strains. These results open up new avenues for the use of E. coli as a microbial cell factory.
Related Articles
Novel Bacterial and Archaeal Lineages from an In Situ Growth Chamber Deployed at a Mid-Atlantic Ridge Hydrothermal Vent
Reysenbach, Anna-Louise
Longnecker, Krista
Kirshtein, Julie
Applied and Environmental Microbiology
The phylogenetic diversity was determined for a microbial community obtained from an in situ growth chamber placed on a deep-sea hydrothermal vent on the Mid-Atlantic Ridge (23°22′ N, 44°57′ W). The chamber was deployed for 5 days, and the temperature within the chamber gradually decreased from 70 to 20°C. Upon retrieval of the chamber, the DNA was extracted and the small-subunit rRNA genes (16S rDNA) were amplified by PCR using primers specific for the Archaea or Bacteria domain and cloned. Unique rDNA sequences were identified by restriction fragment length polymorphisms, and 38 different archaeal and bacterial phylotypes were identified from the 85 clones screened. The majority of the archaeal sequences were affiliated with the Thermococcales (71%) and Archaeoglobales (22%) orders. A sequence belonging to the Thermoplasmales confirms that thermoacidophiles may have escaped enrichment culturing attempts of deep-sea hydrothermal vent samples. Additional sequences that represented deeply rooted lineages in the low-temperature eurarchaeal (marine group II) and crenarchaeal clades were obtained. The majority of the bacterial sequences obtained were restricted to the Aquificales (18%), the ɛ subclass of the Proteobacteria (ɛ-Proteobacteria) (40%), and the genus Desulfurobacterium (25%). Most of the clones (28%) were confined to a monophyletic clade within the ɛ-Proteobacteria with no known close relatives. The prevalence of clones related to thermophilic microbes that use hydrogen as an electron donor and sulfur compounds (S0, SO4, thiosulfate) indicates the importance of hydrogen oxidation and sulfur metabolism at deep-sea hydrothermal vents. The presence of sequences that are related to sequences from hyperthermophiles, moderate thermophiles, and mesophiles suggests that the diversity obtained from this analysis may reflect the microbial succession that occurred in response to the shift in temperature and possible associated changes in the chemistry of the hydrothermal fluid.
Related Articles
Dinosolve: a protein disulfide bonding prediction server using context-based features to enhance prediction accuracy
Yaseen, Ashraf
Li, Yaohang
BMC Bioinformatics
2013;14(Suppl 13):S9.
Disulfide bonds play an important role in protein folding and structure stability. Accurately predicting disulfide bonds from protein sequences is important for modeling the structural and functional characteristics of many proteins.
In this work, we introduce an approach of enhancing disulfide bonding prediction accuracy by taking advantage of context-based features. We firstly derive the first-order and second-order mean-force potentials according to the amino acid environment around the cysteine residues from large number of cysteine samples. The mean-force potentials are integrated as context-based scores to estimate the favorability of a cysteine residue in disulfide bonding state as well as a cysteine pair in disulfide bond connectivity. These context-based scores are then incorporated as features together with other sequence and evolutionary information to train neural networks for disulfide bonding state prediction and connectivity prediction.
The 10-fold cross validated accuracy is 90.8% at residue-level and 85.6% at protein-level in classifying an individual cysteine residue as bonded or free, which is around 2% accuracy improvement. The average accuracy for disulfide bonding connectivity prediction is also improved, which yields overall sensitivity of 73.42% and specificity of 91.61%.
Our computational results have shown that the context-based scores are effective features to enhance the prediction accuracies of both disulfide bonding state prediction and connectivity prediction. Our disulfide prediction algorithm is implemented on a web server named "Dinosolve" available at: http://hpcr.cs.odu.edu/dinosolve.
Related Articles
Intact protein folding in the glutathione-depleted endoplasmic reticulum implicates alternative protein thiol reductants
Tsunoda, Satoshi
Avezov, Edward
Zyryanova, Alisa
Konno, Tasuku
Mendes-Silva, Leonardo
Pinho Melo, Eduardo
Harding, Heather P
Ron, David
Protein folding homeostasis in the endoplasmic reticulum (ER) requires efficient protein thiol oxidation, but also relies on a parallel reductive process to edit disulfides during the maturation or degradation of secreted proteins. To critically examine the widely held assumption that reduced ER glutathione fuels disulfide reduction, we expressed a modified form of a cytosolic glutathione-degrading enzyme, ChaC1, in the ER lumen. ChaC1CtoS purged the ER of glutathione eliciting the expected kinetic defect in oxidation of an ER-localized glutathione-coupled Grx1-roGFP2 optical probe, but had no effect on the disulfide editing-dependent maturation of the LDL receptor or the reduction-dependent degradation of misfolded alpha-1 antitrypsin. Furthermore, glutathione depletion had no measurable effect on induction of the unfolded protein response (UPR); a sensitive measure of ER protein folding homeostasis. These findings challenge the importance of reduced ER glutathione and suggest the existence of alternative electron donor(s) that maintain the reductive capacity of the ER.
DOI: http://dx.doi.org/10.7554/eLife.03421.001
eLife digest
Proteins are basically strings of amino acids that have folded into a specific three-dimensional shape, and this shape is often important for the protein's function. Some proteins have bonds between pairs of cysteines—an amino acid that contains sulfur—in different parts of the protein to maintain its correct shape.
In eukaryotes, such as plants and animals, these so-called 'disulfide bonds' are formed inside a structure within each cell called the endoplasmic reticulum, which is where many proteins are folded. Occasionally, disulfide bonds form in the wrong place in a protein, so they need to be broken and re-positioned—a process sometimes called editing—for the protein to fold correctly. It was widely assumed that a chemical called 'reduced glutathione' fuels the breaking of disulfide bonds in the endoplasmic reticulum, but to date few researchers have tried to test this assumption.
Tsunoda et al. have now taken an enzyme that degrades glutathione elsewhere in the cell and modified it in a way that allows it to work inside the endoplasmic reticulum. When this modified enzyme was produced in human cells grown in the laboratory, it purged the endoplasmic reticulum of glutathione. However, the lack of glutathione had no effect on the folding of a large protein with 30 disulfide bonds, many of which need to be edited at one time or another for the protein to fold correctly. The destruction of a poorly folded protein, via a process that also needs this protein's disulfide bonds to be broken down, was also not affected by a lack of reduced glutathione in the endoplasmic reticulum.
Furthermore, decreasing these levels of glutathione did not affect the unfolded protein response: a stress response in cells that are experiencing a build-up of unfolded or poorly folded proteins within the endoplasmic reticulum.
As such, the findings of Tsunoda et al. challenge the importance of reduced glutathione in the endoplasmic reticulum and suggest that other chemical processes might be involved in editing disulfide bonds. Further work is now needed to investigate the other known processes that might complete this task instead to see which, if any, are involved.
DOI: http://dx.doi.org/10.7554/eLife.03421.002
protein folding; UPR; redox; glutathione; human
Related Articles
Phylogenomic analysis of proteins that are distinctive of Archaea and its main subgroups and the origin of methanogenesis
Gao, Beile
Gupta, Radhey S
BMC Genomics
The Archaea are highly diverse in terms of their physiology, metabolism and ecology. Presently, very few molecular characteristics are known that are uniquely shared by either all archaea or the different main groups within archaea. The evolutionary relationships among different groups within the Euryarchaeota branch are also not clearly understood.
We have carried out comprehensive analyses on each open reading frame (ORFs) in the genomes of 11 archaea (3 Crenarchaeota – Aeropyrum pernix, Pyrobaculum aerophilum and Sulfolobus acidocaldarius; 8 Euryarchaeota – Pyrococcus abyssi, Methanococcus maripaludis, Methanopyrus kandleri, Methanococcoides burtonii, Halobacterium sp. NCR-1, Haloquadratum walsbyi, Thermoplasma acidophilum and Picrophilus torridus) to search for proteins that are unique to either all Archaea or for its main subgroups. These studies have identified 1448 proteins or ORFs that are distinctive characteristics of Archaea and its various subgroups and whose homologues are not found in other organisms. Six of these proteins are unique to all Archaea, 10 others are only missing in Nanoarchaeum equitans and a large number of other proteins are specific for various main groups within the Archaea (e.g. Crenarchaeota, Euryarchaeota, Sulfolobales and Desulfurococcales, Halobacteriales, Thermococci, Thermoplasmata, all methanogenic archaea or particular groups of methanogens). Of particular importance is the observation that 31 proteins are uniquely present in virtually all methanogens (including M. kandleri) and 10 additional proteins are only found in different methanogens as well as A. fulgidus. In contrast, no protein was exclusively shared by various methanogen and any of the Halobacteriales or Thermoplasmatales. These results strongly indicate that all methanogenic archaea form a monophyletic group exclusive of other archaea and that this lineage likely evolved from Archaeoglobus. In addition, 15 proteins that are uniquely shared by M. kandleri and Methanobacteriales suggest a close evolutionary relationship between them. In contrast to the phylogenomics studies, a monophyletic grouping of archaea is not supported by phylogenetic analyses based on protein sequences.
The identified archaea-specific proteins provide novel molecular markers or signature proteins that are distinctive characteristics of Archaea and all of its major subgroups. The species distributions of these proteins provide novel insights into the evolutionary relationships among different groups within Archaea, particularly regarding the origin of methanogenesis. Most of these proteins are of unknown function and further studies should lead to discovery of novel biochemical and physiological characteristics that are unique to either all archaea or its different subgroups.
Related Articles
A Mechanistic Investigation of the C-Terminal Redox Motif of Thioredoxin Reductase from Plasmodium falciparum†
Snider, Gregg W.
Dustin, Christopher M.
Ruggles, Erik L.
Hondal, Robert J.
High molecular weight thioredoxin reductases (TRs) are pyridine nucleotide disulfide oxidoreductases that catalyze the reduction of the disulfide bond of thioredoxin (Trx). It is Trx that is responsible for reducing multiple protein disulfide targets in the cell. TRs utilize NADPH as the source of reducing equivalents to reduce a bound flavin prosthetic group, which in turn reduces an N-terminal redox center that has the conserved sequence CICVNVGCCT, where CIC is denoted as the interchange thiol and CCT is the thiol involved in charge-transfer complexation. The reduced N-terminal redox center reduces a C-terminal redox center on the opposite subunit of the head-to-tail homodimer. It is the C-terminal redox center that catalyzes the reduction of the Trx-disulfide. Variations in the amino acid sequence of the C-terminal redox center differentiate high molecular weight TRs into different types. Type Ia TRs have tetrapeptide C-terminal redox centers of sequence GCUG, where U is the rare amino acid selenocysteine (Sec), while the tetrapeptide sequence in type Ib TRs replace the Sec residue with a conventional cysteine (Cys) residue and can use small polar amino acids such as serine and threonine in place of the flanking glycine residues. The TR from P. falciparum (PfTR) is similar in structure and mechanism to type Ia and type Ib TRs except that the C-terminal redox center is different in its amino acid sequence. The C-terminal redox center of PfTR has the sequence G534CGGGKCG541 and we classify it as a type II high molecular weight TR. The oxidized type II redox motif will form a 20-membered disulfide ring, while the absence of spacer amino acids in the type I motif results in the formation of a rare 8-membered ring. We used site-directed mutagenesis and protein semisynthesis to investigate features of the distinctive type II C-terminal redox motif that help it perform catalysis. Deletion of Gly541 reduces thioredoxin-reductase activity by ~50-fold, most likely due to disruption of an important hydrogen bond between the amide N-H of Gly541 and the carbonyl of Gly534 that helps to stabilize the β-turn-β motif. Alterations of the 20-membered disulfide ring by amino acid deletion or substitution resulted in impaired catalytic activity. Subtle changes in the ring structure and size via homocysteine for cysteine substitution using semisynthesis also caused significant reductions in catalytic activity, demonstrating the importance of the disulfide ring's geometry in making the C-terminal redox center reactive for thiol/disulfide exchange. The data suggested to us that the transfer of electrons from the N-terminal redox center to the C-terminal redox center may be rate limiting. We propose that the transfer of electrons from the N-terminal redox center in PfTR to the type II C-terminal disulfide is accelerated by the use of an "electrophilic activation" mechanism. In this electrophilic activation mechanism, the type II C-terminal disulfide is polarized, making the sulfur atom of Cys540 electron deficient, highly electrophilic, and activated for thiol/disulfide exchange with the N-terminal redox center. This hypothesis was investigated by constructing chimeric PfTR mutant enzymes containing C-terminal type I sequences GCCG and GCUG, respectively. The PfTR-GCCG chimera had 500-fold less thioredoxin-reductase activity than the native enzyme, but still reduced selenocystine and lipoic acid efficiently. The PfTR-GCUG chimera had higher catalytic activity than the native enzyme with Trx, selenocystine, and lipoic acid as substrates. The results suggested to us that: (i) Sec in the mutant enzyme accelerated the rate of thiol/disulfide exchange between the N- and C-terminal redox centers, (ii) the type II redox center evolved for efficient catalysis utilizing Cys instead of Sec, and the type II redox center of PfTR is partly responsible for substrate recognition of the cognate PfTrx substrate relative to non-cognate thioredoxins.
Related Articles
A Mechanistic Investigation of the C-Terminal Redox Motif of Thioredoxin Reductase from Plasmodium falciparum
Snider, Gregg W.
Dustin, Christopher M.
Ruggles, Erik L.
Hondal, Robert J.
High-molecular mass thioredoxin reductases (TRs) are pyridine nucleotide disulfide oxidoreductases that catalyze the reduction of the disulfide bond of thioredoxin (Trx). Trx is responsible for reducing multiple protein disulfide targets in the cell. TRs utilize reduced β-nicotinamide adenine dinucleotide phosphate to reduce a bound flavin prosthetic group, which in turn reduces an N-terminal redox center that has the conserved sequence CICVNVGCCT, where CIC is denoted as the interchange thiol while the thiol involved in charge-transfer complexation is denoted as CCT. The reduced N-terminal redox center reduces a C-terminal redox center on the opposite subunit of the head-to-tail homodimer, the C-terminal redox center that catalyzes the reduction of the Trx-disulfide. Variations in the amino acid sequence of the C-terminal redox center differentiate high-molecular mass TRs into different types. Type Ia TRs have tetrapeptide C-terminal redox centers of with a GCUG sequence, where U is the rare amino acid selenocysteine (Sec), while the tetrapeptide sequence in type Ib TRs has its Sec residue replaced with a conventional cysteine (Cys) residue and can use small polar amino acids such as serine and threonine in place of the flanking glycine residues. The TR from Plasmodium falciparum (PfTR) is similar in structure and mechanism to type Ia and type Ib TRs except that the C-terminal redox center is different in its amino acid sequence. The C-terminal redox center of PfTR has the sequence G534CGGGKCG541, and we classify it as a type II high-molecular mass TR. The oxidized type II redox motif will form a 20-membered disulfide ring, whereas the absence of spacer amino acids in the type I motif results in the formation of a rare eight-membered ring. We used site-directed mutagenesis and protein semisynthesis to investigate features of the distinctive type II C-terminal redox motif that help it perform catalysis. Deletion of Gly541 reduces thioredoxin reductase activity by ∼50-fold, most likely because of disruption of an important hydrogen bond between the amide NH group of Gly541 and the carbonyl of Gly534 that helps to stabilize the β–turn−β motif. Alterations of the 20-membered disulfide ring either by amino acid deletion or by substitution resulted in impaired catalytic activity. Subtle changes in the ring structure and size caused by using semisynthesis to substitute homocysteine for cysteine also caused significant reductions in catalytic activity, demonstrating the importance of the disulfide ring's geometry in making the C-terminal redox center reactive for thiol–disulfide exchange. The data suggested to us that the transfer of electrons from the N-terminal redox center to the C-terminal redox center may be rate-limiting. We propose that the transfer of electrons from the N-terminal redox center in PfTR to the type II C-terminal disulfide is accelerated by the use of an "electrophilic activation" mechanism. In this mechanism, the type II C-terminal disulfide is polarized, making the sulfur atom of Cys540 electron deficient, highly electrophilic, and activated for thiol–disulfide exchange with the N-terminal redox center. This hypothesis was investigated by constructing chimeric PfTR mutant enzymes containing C-terminal type I sequences GCCG and GCUG, respectively. The PfTR-GCCG chimera had 500-fold less thioredoxin reductase activity than the native enzyme but still reduced selenocystine and lipoic acid efficiently. The PfTR-GCUG chimera had higher catalytic activity than the native enzyme with Trx, selenocystine, and lipoic acid as substrates. The results suggested to us that (i) Sec in the mutant enzyme accelerated the rate of thiol–disulfide exchange between the N- and C-terminal redox centers, (ii) the type II redox center evolved for efficient catalysis utilizing Cys instead of Sec, and (iii) the type II redox center of PfTR is partly responsible for substrate recognition of the cognate PfTrx substrate relative to noncognate thioredoxins.
Related Articles
Results 1-25 (790272)
« Previous
Go to page number:
Next »
Remove citation from clipboard
Add citation to clipboard
This will clear all selections from your clipboard. Do you wish proceed?
Clipboard is full! Please remove an item and try again.
Contact Us
PubMed Central Canada is a service of the
Canadian Institutes of Health Research
(CIHR) working in partnership with the National Research Council's
national science library
in cooperation with the
National Center for Biotechnology Information
at the
U.S. National Library of Medicine
(NCBI/NLM). It includes content provided to the
PubMed Central International archive
by participating publishers.


text:
ODT Online
ODT /directory
The Outram Charitable Trust project to build a BMX track has
been put on hold as its focus shifts to horses.
The reason the project stalled was revealed at the Mosgiel
Taieri Community Board meeting in Mosgiel last week.
In a letter to the board, trust chairman Greg McSkimming said
the BMX project was postponed because the trust was focusing
its ''resources'' on the hosting of the 2015 Cavalcade in
The board was concerned over the announcement because it had
given the trust a grant towards building the BMX track in
Board member Cr Kate Wilson said the funding was ''tagged''
and had to be spent on the BMX track project.
It could not be transferred to another project.
Board chairman Bill Feather said the board needed reassurance
the money would be used for its intended purpose.
Mr McSkimming told the Taieri Times on Thursday the grant of
about $5000 would be used to build a BMX track.
The ''resources'' mentioned in the letter was a reference to
human resources and not monetary resources, Mr McSkimming
The board intended to continue the BMX track project, when
the trust had the time and resources, which Mr McSkimming
forecast would be in spring next year.
A name, residential address, and (preferably residential) telephone number is required from readers who comment on ODT Online. These details will not be visible to site visitors.
Login or Register
to add your business to the ODT/directory. Quick link to
Submit your news & photos - Login or
Opinion? Have your say - Login or Register
Submit your
event or notice
Add a photo
Bookmark ODT
Make ODT my
Otago Daily Times print and digital edition, The
Star - Dunedin, The News - Central Otago, The
Ensign - Gore, Southland Express -
Invercargill, Southern Rural Life - Otago and
Southland, The Courier - Timaru, Courier Country - Canterbury and
West Coast, Queenstown Times - Queenstown, Mountain Scene -
Queenstown, The Oamaru Mail - Oamaru, The
Greymouth Star - Greymouth, Hokitika Guardian - Hokitika,
West Coast Messenger - West Coast, The Ashburton Courier -
Ashburton, The News - North Canterbury, Extra! schools
publication, Property
Times South Canterbury