text:
By Felicity Business articles, Wine December 21, 2015
Monks, spas and luxury wines
The spa sommelier is going through the options. On the table, three bottles of Abadía Retuerta wines. Next to each, a bottle of massage oil. Each oil has been blended with scents that match the wines. I choose the Sauvignon Blanc and then have a massage which starts with a symbolic washing of the feet, done in memory of the monks who once lived and worked within these massive walls.
If the monks were somehow whistled up from the 12th century, they would easily recognise their monastery, restored to its original beauty and topped with stork nests. But their jaws would drop at the opulence of Abadía Retuerta. Everything about this spa, the hotel it's in, and the nearby winery, is luxurious, combining 21st century innovation with a back-to-nature ethos. It's an approach that's seen Abadía Retuerta's wines gather awards and acclaim wherever they go.
Yet less than a decade ago, the company was a Spanish winery making well respected, but not world-beating wines. There's no doubt being backed by a wealthy company helped change things. But so did the business strategy.
Created to wow
Enrique Valero Quintana, director general of Abadía Retuerta, stops the car in front of an oak tree. "This is one of our 'wow' moments," he says.
The ancient tree is impressive. We're doing a circuit of the property, which is organised into various trails. There are viewing points, wetlands, and an old lime oven to look at, along with 140 species of birds to watch. Quintana says the trails have been devised to give a 'wow' moment every 15 minutes, a principle he discovered in the Napa Valley. Quintana's got a deep background in branding; he started at Diageo, moved to United Wineries, and then on to Gonzalez Byass. He came to Abadía Retuerta in September 2009, to implement a new strategy.
Abadía Retuerta is owned by Swiss pharmaceutical company Novartis; it was originally part of Syngenta, their crop division. By the late 1980s, Novartis no longer needed the land for seeds, so decided to develop it. They called in Pascal Delbeck from Château Ausone, who analysed the soils and made recommendations about what to plant. The winery was founded in 1996, and began to produce a big range of wines trying to be all things to all people. "When you have a new vineyard, you just have to produce, to have something to sell," says Quintana. "We had up to 1m bottles per year and the portfolio focused on different price levels and wines. We were finding our model."
Álvaro Pérez Navazo, director of marketing, says the early days were about experimenting. "The vines were recently planted and we, the team, were also young." They did everything from carbonic maceration to Joven Roble – wines with three to six months in oak. But as the vines matured, the viticulturalists came to understand the terroir better and how it needed to be expressed. The carbonic maceration was the first to go.
"You cannot be selling wines at €4.00 ($4.24) and at €100.00, because the consumer associates your brand with one positioning," says Quintana. "We decided to analyse where we should be playing in the next 20 years." The startling conclusion was to enter the super-premium end of the market. Startling, because Spain was convulsing after the financial crisis. Quintana says the middle of the market had become a dangerous place, because there were no buyers there. Indeed, many other Spanish wineries came to the same conclusion, and headed for the cheap end of the market. But Abadía Retuerta went the other way, setting themselves the goal of becoming one of Spain's top-ten brands by 2016, by "focusing on terroir, low yields and a wine range that's not complicated".
It was a risky thing to do, not least because the winery has to be profitable under its own steam; it took from 2010 to 2013 to make the changes. Production dropped from 1m bottles to 550,000 and the change to a brand-oriented approach required intensive education of both staff and distributors and importers. "All distributors love to have pallets of cheap wine," says Quintana wryly.
Marco Serra, a Swiss-Italian architect, was brought in to refurbish the abbey, a Romanesque building founded in 1146. The €370.00-a-night Abadía Retuerta Le Domaine hotel opened in 2012. It's certainly luxurious, from the mobile phone every guest is given so they can summon their butler at any time, to the personalized newspaper in the morning. "Swiss quality," says Quintana proudly.
The hotel does more than simply accommodate tourists. Having a luxury destination on tap has allowed Abadía Retuerta to enter into partnerships with other luxury companies, like Porsche and Montblanc. "Wine, gastronomy and top hotels are very attractive for luxury brands," says Quintana. Abadía Retuerta has also created a private club, whose members have access to a luxurious flat in central Madrid, where they can relax, work or host dinners and presentations, among other benefits.
Enrique Valero Quintana, CEO
The grapes coming into the winery are harvested from 54 different sites, from vines that live on soils as varied as riverbank clay, pebbles and sand, gravel or limestone, from what is the coolest region of Spain, because the altitudes can reach 850 metres.
To make the winemaking process transparent, there are glass walls everywhere. One can easily watch the tanks being lifted and seamlessly filled with grapes; wine.com has named this one of Europe's most modern wineries.
A tasting has been prepared: the Abadía Retuerta Selección Especial, a blend, is on the table, as are the single-vineyard, or 'Pago', wines: Pago Negralada, Valdebellón, Garduña and Petit Verdot. The wines, made under the supervision of consultant Angel Anocíbar Beloqui, win consistently high scores from prestigious magazines, including being named in the top 100 wines of the world.
It is, of course, always nice to make wines that the critics admire – but have these changes paid their own way?
"Yes," says Pérez Navazo, who says the winery turnover had previously remained static at around €4m per year, but now reaches €6m per year.
One thing that Abadía Retuerta can't buy is the name of a famous appellation, because they lie just outside the borders of the Ribera del Duero. When they tried to join the appellation, their application was "gently declined" as Pérez Navazo puts it. However, they have applied to Brussels for the appellation Vino de Pago Abadía Retuerta; a Pago is a single vineyard with a distinguishing characteristic of some kind, such as unique soil composition. Abadía is also a member of the Grandes Pagos de España (GPE), an association of 30 producers with high-quality Pagos, formed in 2000. To become a member, a winery has to demonstrate a track record of five years of recognition from international magazines and wine guides. Quintana says members come together to share knowledge and communicate in international markets. "It's having a real difference at the premium end of Spanish wineries," he says.
As for their own plans: "Our focus is now on developing international markets and strengthening our direct sales. We're opening an online shop," says Quintana. There's also the biodiversity project. "We keep eagles and falcons to stop small birds from eating the grapes," says Quintana. He pauses. "But then we needed rabbits to feed the eagles." Quintana says the more they plant and solve problems naturally, the more they find themselves recreating a version of what the monks did before them – though with the added appeal of massages, butlers and some of Spain's most desirable wines.
This article originally appeared in Meininger's Wine Business International.
abadia retuertamonasterywine
Previous articleThe taste of McLaren Vale terroir
Next articleCrimea and the game of wines


They were the murders that gripped the nation. Eight years back Aarushi Talwar was found murdered in her bedroom in Noida. A day later the body of the prime suspect-the family servant Hemraj was discovered. The case rocked the media, with practically every news channel lapping up every bit of information. And with a press conference immediately following, that maligned the character of the young girl and the parents; all hell broke loose in the case. Over the next few years, the case went underway, with multiple twists and turns, eventually the Ghaziabad trial court convicting the parents of the crime. But are they really guilty or merely victims? Aarushi- the book is the account of this crime, by journalist turned author Avirook Sen. A sharp and detailed narration of the Noida double murder case.
Right at the outset, Avirook maintains his stand. He didn't know the Talwars. But after going through the case, having attended every trial session in the lower court, and covering the proceedings with due diligence, he is convinced that there has been a mis-carriage of justice. He considers them to be victims of a system- with premier investigating agencies in the country having botched up investigations. From the day the crime was discovered -16 May 2008, till the conviction in 2013, Avirook traces the important events and milestones, that leaves almost all intrigued.
The book is divided into these three parts that constructs the case sequentially. The writing is simple and keeps you hooked. As a reader you do conjure up images of the entire crime scene and I did get a sense of witnessing it all first hand. Yes- that's Avirook's impeccable writing skill. He digs further and deep inside, bringing out minute details- the way our investigative agencies work, the forensic teams and the larger Central Bureau of Investigation (CBI).
Lines from the book: The CBI conducted a "scientific" test in Aarushi's bedroom using Shalimar Superlac paint as blood, and when a judge asked if the paint had the same consistency as blood, a forensic 'expert' said, "Woh khoon jaisa tha" ("it was like blood").
Apparently Shalimar paint has none of the properties of blood, except probably the colour. Avirook uses court documents, eyewitness accounts, and in-depth insight into how exactly the Indian Judicial system works.
Yes, Avirook does believe in the parents' innocence and he brings this out with great conviction. As a reader when you do look at the case with an open mind, you cannot but agree that there have been shortcomings in the judgment, the investigations that have been botched up, prejudices/pre-judgment strewn in the trial process and justice has probably not been served. We hardly have a forensic team that is efficient. The competence of our premier investigation agencies is questionable and the media's increasing propensity to play judge and jury.
Why did the key witnesses change their stance at court?
How could a verdict be written well before the defence could conclude its argument, a month prior to announcement of the sentence?
The crucial evidence of the pillow cover- tampering of the seal and was there really a typographical error?
Aarushi- The book has been impeccably written is well researched and perfectly constructed with diligence. The book is a must read for those who follow the case, and for those who don't too. It gives you a grim picture of our State, our Judicial System and the thought processes which are strongly prevalent in society. Avirook Sen has done a brilliant job trying to fit in the missing pieces of a greatly jumbled murder puzzle.
I've got the book yet I don't think I have the mental strength to read the book. Which is also why Talvar remains unwatched. Maybe some day soon.
I followed the case only a little. It is heartbreaking to see the dramas in real life scenario.Haven't read the book or watched the movie either. One of my friends told the same as you have written. The unanswered questions will stay with you.
Arushi's a case is actually a simple one. I believe in the first theory of Noida poice. It was definitely committed by Her father out of a sudden raise. After that they cover it up shifting the blame towards many…yes there are unanswered questions as it was a high profile case…so it got a strong media hype. But the basics remain same….
I watched the Irfan Khan film which suggests the same idea. I had this unbreakable faith in our judiciary – that ultimately truth prevails. The idea that that doesn't happen still shocks me after so many days since the conviction. And the saddest part is nothing can be done. It's heartbreaking.
I can imagine when you said it was a difficult read, i have always been a believer in the judiciary system of our country but this case which i followed every bit does have a lot unanswered. would be a great read indeed.


text:
Les aminotransferases (o transaminases) són un conjunt d'enzims del grup de les transferases, ja que transfereixen grups amino des d'un metabòlit a un altre, generalment aminoàcids. Aquests enzims són induïbles, perquè la seva activitat pot augmentar per l'acció de diverses hormones com la tiroxina o els glucocorticoides, la seva reacció és lliurement reversible i la seva constant d'equilibri és a prop a la unitat.

La transaminació és molt important per:

 La síntesi d'aminoàcids no essencials.
 La degradació de la majoria dels aminoàcids. La transaminació no és possible amb els aminoàcids lisina i treonina.
 Intercanvi de grups amino entre molècules que no són aminoàcids.

Les transaminases necessiten un coenzim anomenat piridoxal fosfat (derivat de la piridoxina o vitamina B6) per exercir la seva funció; actua com a transportador del grup amino entre els substrats, alternant la seva estructura entre la forma aldehídica (piridoxal) i la forma amines (piridoxamina). El piridoxal fosfat s'uneix a les transaminases a través de l'amino èpsilon d'un residu de lisina, i durant la reacció és transferit a l'aminoàcid amb formació d'una base de Schiff, a partir d'aquest compost es produeixen les modificacions químiques que condueixen a la transaminación.

Les principals aminotransferases són les hepàtiques com l'AST (en anglès: aspartate transaminase, també anomenada serum glutamic oxaloacetic transaminase -SGOT-) i l'ALT (en anglès: alanine transaminase, també anomenada serum glutamate-pyruvate transaminase -SGPT-), que augmenten en associació a diverses malalties. De vegades, el tipus específic d'aminotransferasa elevada suggereix l'òrgan afectat per la seva relativa abundància en ell.

Nivell de transaminases en sang

Els nivells de transaminases en sang s'utilitzen com a indicador per detectar possibles patologies en les funcions del fetge. Les malalties hepàtiques, com l'hepatitis, la cirrosi, el fetge gras (esteatopatia alcohòlica i no alcohòlica), quists o tumors al fetge o obstruccions greus de la via biliar poden provocar una elevació important dels nivells de les transaminases en sang.

Malalties hepàtiques -hepatitis vírica o no vírica, cirrosi- provoquen un augment notable de l'aspartat-aminotransferasa (AST) al plasma sanguini, mentre que en un infart de miocardi es produiria llavors un increment més marcat de l'alanina-aminotransferasa (ALT). També alguns fàrmacs, la diabetis o alteracions tiroidals poden provocar unes ALT i/o AST augmentades.

Referències

Enllaços externs
 Transaminasas.com Artícles sobre les transaminases.
 Elevated transaminases. Audiopedia. 2014
 Overview & The Aminotransferase Reaction. Biochem CAM. 2015
 Transaminasas: Valoración y significación clínica. Protocolos diagnóstico-terapéuticos de Gastroenterología, Hepatología y Nutrición Pediátrica SEGHNP-AEP. 2010

Transferases

redpajama_set_name: RedPajamaWikipedia


JOHANNESBURG – Dr Johan van Zyl, Chairman of Toyota South Africa Motors (TSAM) and President and CEO of Toyota Motor Europe (TME), has been promoted to Senior Managing Officer at Toyota Motor Corporation (TMC).
The appointment will be effective April 1, becoming the first African to be appointed to the position at TMC.
Van Zyl started his career with TSAM in 1993 as the Director: Vehicles Sales and Dealer Network. This was the starting point of a great career with the company. Prior to Toyota South Africa, Dr van Zyl was mainly involved in education both as a lecturer and a student.
He was a lecturer before entering the motoring industry.
He was promoted into the position of President and Chief Executive Officer of Toyota South Africa Motors in 2002. In 2009, Dr van Zyl was appointed as Managing Officer of Toyota Motor Corporation in Japan and in 2013, he added Chief Executive Officer for Toyota in Africa to his responsibilities.
His exceptional business acumen and knowledge of the industry have been strengthened over the years with travels to Japan, the United States and into Africa. The latest promotion comes exactly two years after Dr van Zyl was appointed CEO of TME.


text:
Mashonaland is a region in northern Zimbabwe.It is home to nearly half of the population of Zimbabwe. The majority of the Mashonaland people are from the Shona tribe while the Zezuru and Korekore dialects are most common. Harare is the largest city followed by Chitungwiza.

Currently, Mashonaland is divided into four provinces,
 Mashonaland West
 Mashonaland Central
 Mashonaland East
 Harare
The Zimbabwean capital of Harare, a province unto itself, lies entirely in Mashonaland.

Provincial history

It was originally one of the regions that the country was divided into following occupation by the Pioneer Column in 1890 and designated the extent of territory under administration of the British South Africa Company as distinct from the remainder of the territory that was directly under the control of the Matabele king, Lobengula, which was named Matabeleland when it was occupied in 1893. The two had separate administrations for part of the BSA Company colonial period.

Revolt broke out against the British South Africa Company in 1896, led by priests of the Mwari religion.  The British prevailed, executed some leaders, and tried to reform the system.

In 1923, the territory became part of the self-governing colony of Southern Rhodesia and Mashonaland became one of the five provinces. In 1970, an administrative reform led to Mashonaland being divided into a northern and a southern half. Most recently, in 1983, it was divided into the current three sectors and the capital city of Harare was given its own provincial status as well. Since the constitutional amendments that took effect in 1988, each is run by a governor appointed by the president.

Geographical features
The territory is composed of a broad plateau that slopes gradually to the north and north-west. The lowest land is on its northern border, which is formed by the Zambezi River, with Zambia beyond. A small part straddles the plateau at its south-eastern edge and here the land drains into the Save River but the rest of Mashonaland is part of the Zambezi drainage basin. To the south, the Munyati River forms the border with the current and former province of Midlands. The Nyangadzi river forms the border with Manicaland to the east.

Much of the landform is rolling low hills divided by river valleys. About half the land is over  altitude and the central watershed in the south and centre is at . Only a few isolated mountains and the spine of the Umvukwes Range in the west rise higher. The highest point is in the Wedza Mountains in the south east at .

Economy
The region's economy consists of the mining, agriculture and the service industry. Its facilities for tourism, fertile lands and mineral filled regions such as Bindura could improve the economy, but due to poor leadership and policies, the economy is lagging behind. Many Mashonaland citizens are farmers, and gain their source of income from agriculture.

References

External links
 James Anta: African Missionary to Mashonaland
 Provinces of Zimbabwe

Regions of Africa
British South Africa Company
Former British colonies and protectorates in Africa
Geography of Zimbabwe
Subdivisions of Zimbabwe


This is the twelfth post in a twelve part series debating the Twelve points of Reform for a New Christianity proposed by John Shelby Spong. This series of posts was written to deepen my understanding of the conflicts inside of Christianity between the Fundamentalists and the more "liberal" practitioners of the Christian community. This project began as research for a novel/screenplay – The Hero Science Project.
clearly unbiblical actions simply because society today accepts them, and he personally does not think them wrong.
Foundation is different. Christianity is founded upon the Bible. It bases upon the Bible both its thinking and its life. Liberalism on the other hand is founded upon the shifting emotions of sinful men."Spong"s reliance on preferences rather than absolutes is logically untenable.
advocating that anything is right or wrong, not even the discrimination he so violently hates. Christian moral absolutes are the only standard that transcends human opinions, and it is only from a standard like this that a person can consistently condemn evil and praise good.
"It is no wonder, then, that liberalism is totally different from Christianity, for the Foundation is different. Christianity is founded upon the Bible. It bases upon the Bible both its thinking and its life. Liberalism on the other hand is founded upon the shifting emotions of sinful men. Spong's reliance on preferences rather than absolutes is logically untenable.
It is no wonder that Fundamentalist Christianity is totally different from the liberalism of Jesus, for the Foundation is different. Fundamentalism is founded upon the arrogance of its divine infallibility. A Fundamentalist Christian bases upon their belief that they are the Word of God, and carries this arrogance into both their thinking and their life. The followers of the Liberal Jesus, on the other hand, are grounded to a humility that grows from an awareness of the limits of their humanity. The liberalism of Jesus is a search for fairness, and searching is something that Fundamentalist Christians have lost the capacity and courage to comprehend.
This transcendent realm is just what Spong rejects, so he has no foundation for advocating that anything is right or wrong, not even the discrimination he so violently hates. Christian moral absolutes are the only standard that transcends human opinions, and it is only from a standard like this that a person can consistently condemn evil and praise good.
This arrogant self-worship is just what Spong rejects, so he has no foundation for pretending that he is superior to others because he has swallowed the Words of God, and does not bestow upon himself the divine right to discriminate. Fundamentalist Christians, on the other hand, love to discriminate, because this allows them to exercise their egotistical fantasies of superiority. They believe they are the only ones who know for certain what God thinks, so they consider themselves to be the only ones who have the right to tell us what we can and cannot do.
Fundamentalism is an illness that can be cured, but only if the Fundamentalist wants to be cured. Unfortunately very few of them ever will, because egotistical arrogance is addictive, and they have long since lost the courage it takes to deal honestly with uncertainty. Given that it is rarely cured, and that it spreads ferociously, the main effort to deal with this illness should be focused on preventing the spread. It is not too late to save the young from contracting this illness, and becoming trapped inside of it for their entire lives.
We must be compassionate when dealing with those who are trapped inside this mental illness, for truly, they know not what they do. Their minds have been suffocated by a need to block out any contradiction to their Truth, and this has driven them deep into an egotistical fantasy of their divine infallibility, and rage at a reality that does not accept their moral supremacy. We need to be aware that the progression of this illness can make those who suffer from it extremely dangerous, as they have no doubts about the righteousness of their actions, and all too frequently, a ravenous hunger to punish those who do not accept their divinity overcomes them, with tragic consequences. As a society, we can not allow our sacred oath in support of religious freedom to include the freedom to harm others in the name of God. This includes the fundamentalist love for discrimination.
This is exactly the freedom that fundamentalism demands.


text:
Q: Android HTTP exception I am using following code to log in to Dropbox, but it throws an exception.
Code:
    HttpClient client = new DefaultHttpClient();
    HttpPost post = new HttpPost("https://www.dropbox.com/login");

    // set values you'd like to send
    List pairs = new ArrayList();
    pairs.add(new BasicNameValuePair("login_email", "my email id"));
    pairs.add(new BasicNameValuePair("login_password", "******"));
    /*'login_email' and 'login_password' i found html form fields name by viewing the page source of https://www.dropbox.com/login */

    try {
        post.setEntity(new UrlEncodedFormEntity(pairs));
        // set ResponseHandler to handle String responses
        ResponseHandler<String> responseHandler = new BasicResponseHandler();
        String response = client.execute(post, responseHandler);
        Log.v("HttpPost", "Response: " + response);
        if (response.contains("SUCCESS")){
            // express your joy here!
        } else {
            // pop a sad Toast message here...
        }
    } catch (Exception e) {
        Log.e("", String.valueOf(e));
    }
}

Logcat
         11-14 23:17:35.734: E/Trace(900): error opening trace file: No such file or directory (2)
         11-14 23:17:38.793: D/dalvikvm(900): GC_CONCURRENT freed 251K, 4% free 8194K/8519K, paused 126ms+9ms, total 234ms
         11-14 23:17:39.895: E/(900): org.apache.http.client.HttpResponseException: Forbidden
         11-14 23:17:40.364: I/Choreographer(900): Skipped 75 frames!  The application may be doing too much work on its main thread.
         11-14 23:17:40.394: D/gralloc_goldfish(900): Emulator without GPU emulation detected.
         11-14 23:17:40.784: I/Choreographer(900): Skipped 62 frames!  The application may be doing too much work on its main thread.
         11-14 23:17:43.614: W/chromium(900): external/chromium/net/disk_cache/backend_impl.cc:1829: [1114/231743:WARNING:backend_impl.cc(1829)] Destroying invalid entry.

A: I/Choreographer(900): Skipped 75 frames!  The application may be doing too much work on its main thread.

It is always suggested to do Network activity on separate thread instead of main thread by using AsyncTask.
Here is android tutorial on Connecting to the Network

A: i tried your request from rest plug in and got the following response from server:
Status
403 Forbidden

<title>Dropbox - 403</title>
<link href="https://www.dropbox.com/static/css/main.css" rel="stylesheet" type="text/css">
<link rel="shortcut icon" href="/static/images/favicon.ico"/>

Error (403)
It seems you tried to do something we can't verify.  Did you log into a different Dropbox account in a different window?  Try clicking here to go back to the page you came from, or just go home.

where is the dropbox API definitions that you are using for login?
you should try their rest API for the integration defined at following link:
https://www.dropbox.com/developers/reference/api
EDIT1: dropbx also have sdk for mobile platform that you can use. check this link for SDK's.
EDIT2: just checked the login page of dropbox. Following is the relevant HTML from login form:
<form action="https://www.dropbox.com/login" method="post"><input type="hidden" name="t" value="Hch4dxqGBwUN024pIuQbsxUQ" />
Email<input type="text" id="login_email_elm" name="login_email" tabindex="1" />

Password<input type="password" id="login_password_elm" name="login_password" tabindex="2" />

<input style="vertical-align: middle; margin-left: 0;" name="remember_me" class="no-border" type="checkbox" id="remember_me" tabindex="3" />Remember me<input tabindex="4" type="submit" class="freshbutton-blue" value="Sign in" id="toplogin-submit" /><input type="hidden" name="cont" value="https://www.dropbox.com/" />

Create an account
</form>

it has two hidden parameter t with auto-generated value which i guess is used by the Dropbox for stoping people to use it the why you are using. Another one is a cont whoch has hardcorded value https://www.dropbox.com/
Any way I tried the request with these two parameter and was able to get the success response. Following is the detail of my request and response using the rest plug in for chrome:
Request URI: https://www.dropbox.com/login
Request content type: application/x-www-form-urlencoded
Request post data:

login_email=bscscomsats@gmail.com&login_password=yourpassword&t=Hch4dxqGBwUN024pIuQbsxUQ&cont=https://www.dropbox.com/

Reponse: To:https://www.dropbox.com/ with status: 302
Response Header:
Server: nginx/1.2.3
Date: Wed, 14 Nov 2012 18:29:36 GMT
Content-Type: text/html; charset=utf-8
Transfer-Encoding: chunked
Connection: keep-alive
set-cookie: lid=AACt_twZtlN8Tz7TDINxpJFUFvG44oy3YAKZ7ht0umJMxA; Domain=www.dropbox.com; Path=/; secure; httponly
set-cookie: forumjar=eyJ2ZXJpZmllZCI6IDAsICJ1aWQiOiAxMjEwMjcwODYsICJleHBpcmVzIjogMTM1MzAwNDE3NiwgInBhaWQiOiAwLCAibG5hbWUiOiAiQ2xvdWRldHMiLCAiZm5hbWUiOiAiUG9ja2V0IiwgImRpc3BsYXlfbmFtZSI6ICJQb2NrZXQgQ2xvdWRldHMiLCAiZW1haWwiOiAiYnNjc2NvbXNhdHNAZ21haWwuY29tIn0%3D; Domain=dropbox.com; Path=/; httponly
set-cookie: blid=AACUWWo4_oPc00S7cf-xtgZ8dkkXXIkiyP8uQazNqQXjXw; Domain=dropbox.com; Path=/; secure; httponly
set-cookie: oscar=; Domain=www.dropbox.com; expires=Wed, 14-Nov-2012 18:29:36 GMT; Path=/; httponly
set-cookie: jar=W3sidWlkIjogMTIxMDI3MDg2LCAiaCI6ICJBQUNiSHJrUElzeWtiYUltak5MZWVnWE52OGYtRmxtZXJubXVXc0xnbnRlallRIiwgImV4cGlyZXMiOiAxMzUzMDA0MTc2LCAibnMiOiAxOTU2NzYxNjcsICJyZW1lbWJlciI6IGZhbHNlfV0%3D; Domain=www.dropbox.com; Path=/; secure; httponly
set-cookie: checkpoint=; Domain=www.dropbox.com; expires=Wed, 14-Nov-2012 18:29:36 GMT; Max-Age=0; Path=/; httponly
set-cookie: t=vmmVuSRbO6xqVJTJ9meSJVkf; Domain=dropbox.com; expires=Fri, 14-Dec-2012 18:29:36 GMT; Path=/; secure; httponly
set-cookie: grouch=; Domain=www.dropbox.com; expires=Wed, 14-Nov-2012 18:29:36 GMT; Path=/; httponly
set-cookie: bjar=W3sic2Vzc19pZCI6IDY3MDc1MTI0NDU3NDk5NzkwMTQ3OTg2NzExOTY1Njc4MDQ1NTA1LCAiZXhwaXJlcyI6IDEzNTMwMDQxNzYsICJ1aWQiOiAxMjEwMjcwODZ9XQ%3D%3D; Domain=dropbox.com; Path=/; secure; httponly
set-cookie: forumlid=NTM1NmJmYTU0NzQ2ZjgzMTNhZDI0YWE5ZjljZjZjNzViY2IxMzliYw%3D%3D; Domain=dropbox.com; Path=/; httponly
strict-transport-security: max-age=2592000; includeSubDomains
location: https://www.dropbox.com/
pragma: no-cache
cache-control: no-cache
x-frame-options: SAMEORIGIN

Redirection response:
Status
200 OK

Response header:
User-Agent: Mozilla/5.0 (Windows NT 6.1) AppleWebKit/537.11 (KHTML, like Gecko) Chrome/23.0.1271.64 Safari/537.11
Content-Type: application/x-www-form-urlencoded
Accept: */*
Accept-Encoding: gzip,deflate,sdch
Accept-Language: en-US
Accept-Charset: UTF-8,*;q=0.5
Cookie: gvc=NjcwNzUxMjQ0NTc0OTk3OTAxNDc5ODY3MTE5NjU2NzgwNDU1MDU%3D; t=Hch4dxqGBwUN024pIuQbsxUQ; __utma=74146836.1139928866.1352917086.1352917086.1352917086.1; __utmb=74146836.4.10.1352917086; __utmc=74146836; __utmz=74146836.1352917086.1.1.utmcsr=google|utmccn=(organic)|utmcmd=organic|utmctr=(not%20provided)

the response data can be found at this pastebin link.
So since the vale of t parameter in login form is auto generated by Dropbox and which i am sure expire after few minutes so I would suggest not to use this approach for login..

redpajama_set_name: RedPajamaStackExchange


Romanian military purchases are not intended to put a strain on relations with nearby Russia but to strengthen the country's defences, President Klaus Iohannis said on Saturday (15 July).
Earlier this month, the US State Department said it has approved the possible sale of seven Patriot missile defence systems worth $3.9 billion (€3.4 billion) to Romania.
A member of NATO since 2004 and of European Union since 2007, Romania has committed to boosting its defence spending to two percent of gross domestic product this year.
Russia, whose annexation of the Black Sea peninsula of Crimea from Ukraine in 2014 prompted concern among NATO's eastern members such as Poland and the Baltic states, has said NATO was threatening stability in Eastern Europe by building up its military presence there and staging war games.
A final decision by Romania to buy the missile defence systems, whose prime contractors would be Raytheon Co and Lockheed Martin Corp, has yet to be announced by the country's defence ministry.
Moscow says the shield's aim is to neutralise Moscow's nuclear arsenal long enough for the United States to strike Russia in the event of war. Washington and NATO deny that.


text:
How to Hunt Big Game
Selling to enterprise companies can be a long process, but there's a big payoff.
By Adam Bluestein, Freelance writer@AdamBluestein
David Axelbank/Gallery Stock
His business cards were barely dry when Ben Werther, CEO and founder of Platfora, a Big Data analytics company, got a phone call from a large media corporation. The caller had read about the startup in a trade journal. "Within months of forming the company, we were in their offices pitching them a technology we hadn't even finished building yet," says Werther. Since its 2011 founding, Platfora has signed on many large customers, including Disney, Comcast, Citigroup, and Hewlett-Packard.
Of course, not all enterprise startups have new customers lining up so soon after launch, but Platfora's story illustrates how much the once stodgy world of enterprise technology is changing. "Enterprise CTOs are sitting on huge budgets, and they're now willing to talk with emerging, innovative companies," says Ravi Belani, managing director of the Alchemist Accelerator, a program for enterprise tech startups. "That used to be impossible."
In the first half of 2014, VCs poured $5.4 billion into these startups, according to a TechCrunch database. That's just short of the $5.5 billion invested in all of 2013. "What I like about enterprise is that the customers have plenty of financial resources," says Josh Stein, a partner at DFJ, which has invested in Box, Yammer, and Twilio. "If you can solve a problem for them, they'll happily pay you for that. While consumer tech tends to be more winner-take-all, I think the batting average is higher in enterprise." Still, landing a big corporate customer requires plenty of homework and legwork.
1. Find the Decision Makers
A growing number of enterprise-focused accelerators--including Acceleprise, Alchemist, and Work-Bench--tout their ability to connect startups with top executives of large companies. But you can hack your way to these decision makers on your own. "Lots of larger companies have really innovative people actively seeking out new technology," says Cameron Campbell, business development director of the New York City-based Work-Bench. He suggests looking at Twitter, tech conferences, and your LinkedIn network to find forward-thinking corporate techies. (Hint: They often have the word innovation in their titles.) "You want to try to bypass the gatekeepers," says Campbell.
Often, decisions aren't made at the top. Jeff Lawson, co-founder and CEO of Twilio, focused from the start on a grassroots sales approach, winning over developers--not just suits--to his cloud communications platform. "We built the best platform for developers and reached out to them where they were," he says. "Developers have a strong say in what gets built in their companies."
2. Keep Innovation in Context
Unlike consumers, large corporations don't generally buy new stuff just to be the first on the block to have it. They want to solve real, well-defined problems in areas like security, workflow, process automation, and Big Data analytics. "There are lots of problems that haven't been solved for businesses," says Justin Hyde, an entrepreneur and senior associate at Acceleprise, which has programs in San Francisco and Washington, D.C. "There's a huge amount of value there."
Rather than merely trying to prove how disruptive you are, you need to show enterprise customers how well you understand their problems--and how you can solve them faster, better, and more cheaply than your competitors. Enterprise customers also want to know how seamlessly your solution fits into their way of doing things. By focusing on the right selling points, "startups can unseat established competitors," says Campbell.
3. De-Risk Your Business
While big corporations are increasingly willing to take chances on startups, they still want assurances that you'll be around a few years down the road. A big company could spend more than a year doing due diligence on a new tech vendor. "We always say they're not buying the product, they're buying the company," says Campbell. "And that's really about being comfortable with the leadership of the startup and their vision." Having someone on the management team with a history at large corporations isn't a must, but it can certainly be a big plus.
An enterprise customer may also want to check some numbers--mainly, whether you have revenue coming in from other customers or adequate venture funding. Much more so than in consumer tech, a VC stamp of approval carries a lot of weight with potential enterprise customers.
Once you get the green light, these clients are supportive of your continuing success. "If a Fortune 500 company is taking a risk on you," says Campbell, "they want you to grow to ensure you'll be around for a while."
4. Play the Long Game
Courting large companies is a lengthy process. It can take 30 to 60 days just to get on an executive's calendar, and timelines for making decisions can be much longer than you might expect. In heavily regulated industries such as financial services, it can take 12 to 18 months from the time you talk with someone to get a final deal. Many startups need to raise bridge funding or start with some midlevel clients to provide cash flow while they chase one or two marquee accounts to build brand recognition, says Campbell.
But it's probably best to have more than one big customer, anyway. It helps you be certain you're creating a product that serves a larger market, and not just one company. "Of course, it's important to get GE or some other big account up on your webpage," says Campbell. "But you can't sell your soul for that." If you make a product tailored to just one company, and the deal goes south, he says, "you're left holding a bag of crap."
How to Break In
These four founders snagged large clients by being more attentive--and more nimble--than the competition.
Listen to the customer. "Product development and selling go hand in hand. Don't build a product in a vacuum. Instead, keep your feet on the street and interact with all kinds of would-be buyers. In the end, that will make your product stronger and future customers happier." --Ben Werther, CEO and founder of Big Data analytics startup Platfora
Speed it up. "The implementation cycle for enterprise used to be long and complex. It's much easier now. In one of our first big sales, our developer had a prototype up and running days after we met with the client. They thought it would be a nine-month process." --Jeff Lawson, CEO and co-founder of Twilio, a cloud communications platform
Break it down. "Your message has to be about real business value. We mostly deliver software to the IT department, but we explain why our product matters outside of the data center. Retailers want better customer engagement and loyalty. Manufacturers need more predictable outputs from their supply chains and factories." --Mike Olson, co-founder and chief strategy officer of Cloudera, a Big Data analytics company
Give it away. "Most businesses we're talking to already have pockets of users who like our free product, so the conversation naturally starts at a much higher level, around topics like enterprise-grade security and workflow. If freemium makes sense for your product, it's an amazing way to build awareness and demand." --Aaron Levie, co-founder and CEO of cloud storage company Box, and Inc.'s 2013 Entrepreneur of the Year
From the March 2015 issue of Inc. magazine

redpajama_set_name: RedPajamaCommonCrawl


Ontario's economy is one of the world's most successful; and most signs – low unemployment rates, solidly performing stock market, and a strong Canadian dollar – reinforce this. But it is not meeting its full potential. Two decades ago, Ontario's Gross Domestic Product (GDP) per capita ranked close to the median of the sixteen most populous North American jurisdictions consisting of 14 US states and Ontario and Quebec. It now stands second to last – Ontario's GDP per capita is now $6,100, or 12.4 percent, behind the median of the 16 jurisdictions.


Buy 3 get one free dozen 2015 Titleist Pro V1 or 2015 Pro V1x golf balls, extended sale date through April 20, 2015. Titleist Pro V1 is the #1 ball for more players and more champions across the worldwide professional tours. To say thank you for trusting your game to Titleist Pro V1 and Pro V1x golf balls, this hot deal will let you start the season with the hot new model.
CEOgolfshop.com offers only factory custom personalized golf balls and logo golf balls. The factory manufacturing process ensures the highest quality imprinting that endures the game. We offer many Titleist products that are not in the online store.
CEOgolfshop specializes in custom executive gifts and active lifestyle apparel. At CEOgolfshop.com we don't offer every brand, we offer the BEST!


The City of Las Cruces seeks to be a customer focused, higher performing organization through PEAK Performance achieving better customer service, continuous improvements, transparency, and better communication.
Click on PEAK Performance for more information and to see department plans.
The mission of the Legal Department is to provide legal, risk, and compliance services to City Council, the City Manager, and all organizational units of the City, so the public will be confident that City government is protecting valuable resources and operating ethically, effectively, efficiently, and transparently.
Welcome to the City Attorney's Office!
Welcome to the City Attorney's Office. The City Attorney serves as the chief legal advisor to the City Manager, the City Council, and all City departments. The City Attorney will discuss the Las Cruces Municipal Code, City Council agenda items or other City-related questions. However, the City Attorney is not permitted to give legal advice to citizens.


text:
Q: AWS cognito authenticate user with number Is it possible to authenticate a user with just their mobile phone like whatsapp does on mobile?
The mobile hub asks for a password, along with phone number.
So should i randomly generate it on the users mobile.
Then store it in keychain for future logins?

A: Cognito provides a custom authentication flow where you can add your own custom challenge to authenticate the user by using lambda functions. Basically you generate a code in your lambda that you send to the mobile phone number and the user enters the code to authenticate.
An example is given in the following AWS presentation (towards the end)
https://www.youtube.com/watch?v=8DDIxqIW1sM


NOTE: With the release of 10.0.3, this tool is obsolete. Use the CU Administration tool instead.
Use the ArcFM System Favorites Manager to create compatible units (CUs) that are available to all Designer users connected to the geodatabase. System favorites using macros and CUs can also be created on this tab.
Click the Favorites icon on the ArcFM Solution toolbar, or right-click and select ArcFM Favorites.
Select the Compatible Units tab.
NOTE: This tab is available only if you have Designer installed and a valid Designer license activated.
Right-click a category and select Create CU.
Select feature class from Select a Target dialog.
In the Create Compatible Unit window, enter a description.
This description is listed on the Compatible Units tab.
Enter a value in the WMS Code field.
This code is required and associates the CU to your CU library.
You can edit the Work Function attributes.
Click Create to add the CU to ArcFM System Favorites Manager display.
Clicking Create again adds a copy of your CU with same attribute values and description.
You can add an existing GIS or non-GIS CU to the compatible unit just created.
Click Close to dismiss the Create Compatible Unit dialog.


Hi everyone, my yellow lab cichlid is getting to aggressive for the last barb I have in the tank with him. I am afraid the barb won't make it much longer. I have contemplated giving the cichlid the flush, but can't pull the trigger. If anyone wants the cichlid I am asking $20 for him. He is about 3.5-4 inches long and is healthy. I want to restart with a tropical tank instead of semi aggressive fish. For pictures pm me. Thank you.
Giving cichlid away for free now. On Saturday if no one contacts me the cichlid dies. Pm for pictures. I will also throw in the barb for free.
If you're anywhere near Crestwood/Midlothian, Animal Island would probably take him off your hands. They specialize in cichlids and might even give you something towards other fish.
I posted an ad on Craigslist and have people interested in the cichlid.
Chitown-Angler was started on February 10, 2003 and has received 207433293 page views in total since that date.


Back zip full wetsuit for women from ROXY. Features include: FN lite neoprene packed with air cells for the lightest of lightweight warmth, Glued & blind stitched (GBS) seams that reduce sew throughs & water entry to keep you warmer, back zip entry system, YKK® #10 back zip, Hydroshield water barrier and Hydrowrap adjustable neck closure.
ROXY™ Womens Pop Surf 3/2mm GBS Steamer Wetsuit – Back zip full wetsuit for women from ROXY. Features include: FN lite neoprene packed with air cells for the lightest of lightweight warmth, Glued & blind stitched (GBS) seams that reduce sew throughs & water entry to keep you warmer, back zip entry system, YKK® #10 back zip, Hydroshield water barrier and Hydrowrap adjustable neck closure.
This is a guide only: weather, sensitivity to the cold and conditions at your local break will affect temperatures and choice of equipment.


text:
from django import template
from django.template.defaultfilters import stringfilter
from django.utils.html import conditional_escape
from django.utils.safestring import mark_safe
from datetime import date
from website.models import Order, OrderProduct
import re

register = template.Library()

@register.filter
def get_item(dictionary, key):
	val = dictionary.get(key)
	if val is None:
		val = ''
	return val

@register.filter
def get_available_stock(product_id):
	return ''

@register.filter
def get_order_total(totals,order):
	totals_filtered = totals.get(order=order)
	return totals_filtered.amount

@register.filter
def get_additional_spaces(name, max):
	spaces = ' ' * (max - len(name))
	return name + spaces
@register.filter
def get_preorder_release_date(release_date):
	actual_date = date.today()
	if release_date is not None and release_date != '' and release_date > actual_date:
		return release_date
	return None

@register.filter
def get_products_on_order(order):
	order_products = OrderProduct.objects.filter(order=order).distinct('product')
	return order_products

@register.filter
def get_price_with_currency_symbol(price):
	price_str = str(price)
	price_str = price_str.split()
	if price_str[1] == 'EUR':
		return '€'+price_str[0]
	elif price_str[1] == 'USD':
		return '$'+price_str[0]
	elif price_str[1] == 'GBP':
		return '£'+price_str[0]
	return price

@stringfilter
def spacify(value, autoescape=None):
	if autoescape:
		esc = conditional_escape
	else:
		esc = lambda x: x
	return mark_safe(re.sub('\s', '&'+'nbsp;', esc(value)))
spacify.needs_autoescape = True
register.filter(spacify)

redpajama_set_name: RedPajamaGithub


text:
https://apnews.com/d757ce57f8e540eb93dce9c72f2ffedd
Reagan rallies for wild win against Johnson
Adam ZuvanichMay 19, 2017 GMT
Josh Killeen thought he might have blown Reagan's chance for a series-opening win by committing consecutive errors at second base in the fifth inning, which allowed Johnson to erase a two-run deficit.
He atoned two innings later, and all he had to do was keep the bat on his shoulders.
Killeen drove in the go-ahead run with a bases-loaded walk in the seventh. The Rattlers blew it open from there, winning 7-2 in Game 1 of a Region IV-6A quarterfinal series Thursday night at Blossom Athletic Center.
"I felt relieved that I got that go-ahead run," Killeen said. "It was awesome."
The unconventional winning play was part of an eventful first playoff meeting between the North East ISD rivals, which both have reached the UIL state tournament within the last three seasons. They committed three errors apiece.
The game also turned on a failed suicide-squeeze bunt by Johnson with one out and the bases loaded in the sixth inning and a catcher's interference play that loaded the bases for Reagan with one out in the seventh. The game was still tied at the time, and then the Rattlers (25-8) erupted for five runs.
"That last inning put a little dagger in our hearts," Johnson shortstop Dalton Shuffield said. "We're just going to have to come out tomorrow with clear heads."
Johnson (25-9), a state finalist last year, must win Game 2 at 7:30 p.m. Friday to extend the series to a decisive third game at 1 p.m. Saturday.
Reagan got to Johnson ace Richie Gilbert early with the help of his first-inning walk and two errors by the Jaguars. Porter Brown reached on the first error, stole second base and scored when Cade Bormet's hard grounder went between the legs of Shuffield.
The Rattlers doubled their lead in the third, when Brown, Ramon Garza and Bormet started the inning with consecutive hits. Brown came around on Bormet's single to center. Garza also tried to score on the play, but Johnson center fielder Jake Sims threw him out at home plate.
The Jaguars tied it with a two-out, error-aided rally in the fifth. Dante Tamez singled, stole second and scored the first run on Shuffield's single to right field.
Sims and Gilbert then reached on consecutive errors by Killeen, with Shuffield racing home from second on the second one.
Johnson threatened to take the lead in the sixth, loading the bases with one out, but could not. Pinch hitter Kai Hendershot could not connect with a low offering from Cal Carver on a suicide squeeze, so Reid Romine was caught stealing home. Carver struck out Hendershot to end the inning on the next pitch.
The Jaguars had to remove Gilbert after six innings because he reached the UIL pitch-count limit of 110. The Rattlers capitalized with their winning rally.
adam.zuvanich@express-news.net
Twitter: @AZuvanich


text:
Back to Beginning – drugi album studyjny polskiej rockowej grupy Cochise. Wydawnictwo ukazało 14 maja 2012 roku nakładem wytwórni Mystic Production. Na płycie znalazło się 16 premierowych kompozycji, a także cover utworu Five to One zespołu The Doors.

Lista utworów

Twórcy

 Zespół Cochise w składzie
 Paweł Małaszyński – śpiew
 Wojciech Napora – gitara
 Radosław Jasiński – gitara basowa
 Cezary Mielko – perkusja

 Inni
 Daniel Baranowski – producent
 Basia Witkowska – projekt okładki
 André Kozimor – skład graficzny płyty
 Joanna Chitruszko – skład graficzny płyty

Przypisy

Linki zewnętrzne

Bibliografia

Albumy Mystic Production
Albumy rockowe
Cochise
Albumy muzyczne wydane w roku 2012


text:
Coronavirus: South African woman nears end of extreme self-isolation
For those of you embarking on lengthy periods of self-isolation and social-distancing there is a woman from South Africa who can tell you all about long-term confinement.
Her name is Amy Pittaway. She's a 24-year old language teacher who has been living her life in a series of pokey-looking rooms in China and South Africa for the past 58 days.
Last year she took a job in Wuhan City because she wanted to work – and see a bit of the world – but the experience has proven a far greater test than expected.
"It has affected me mentally, in the sense that before this whole thing I have never had anxiety, I have never been claustrophobic, I never had mental health problems, but now I am experiencing those problems. I feel like the walls are closing in on me sometimes."
Amy was working at a private English tutorial centre in Wuhan when the coronavirus spread through the city. The authorities responded by locking it down and the security team in her apartment building would not let her leave.
"One day it was okay, we were going about our business, getting things done and the next day we couldn't go anywhere and all the bus stops and everything were closed… it was definitely a shock, we were definitely unprepared but we had to pull together for what was to come."
We spoke to Amy on day 48, as she peered into her phone from her compact room in Wuhan.
"You have seen the size of my apartment. I wake up and I can't go back to sleep because I keep having bad dreams that something is happening to my family. I can't get to them because I am stuck here. It is things like this that are a problem because you don't know what is going on, you have all these things swirling through your brain the whole time."
A few days later the South African government organised a plane to pick up Amy and 113 other citizens stuck in China but the rescue mission generated a torrent of criticism back at home.
"I have got a lot of private messages on social media, things on Facebook, Instagram and sometimes even my emails where people say don't come back, we don't want you here. I have got a lot of those emails… a lot of the South African public don't want us to come back home."
Amy weathered the abuse and got a seat on the flight back to South Africa. However, the 24-year old still has not won her freedom. Instead she is mid-way through an additional 21-day spell of quarantine imposed by the government at facility north of Johannesburg.
I asked her how this beleaguered group of evacuees were getting on.
"There are a lot of arguments, there is a lot of talking, there are a lot of jokes… the mood goes up and down, like mine, we are all on a rollercoaster because we all have our ups and downs."
This has not been an easy experience for Amy but the language teacher says she has learnt a lot and she knows the end is in sight.
"I know that there is a light at end of tunnel, I might not have passed that bend, I might not be able to see it yet but it is there and I am just carrying on driving my car."
Tags African Coronavirus South wom
Husband who beat wife to death on cruise ship blames testosterone supplements
Smothering bum in banana to get one like Kim Kardashian not good, warn docs
COVID-19: 'Extremely unlikely' coronavirus came from Wuhan lab as evidence points to 'intermediary species'
US election BREAKTHROUGH as GSA declare Biden 'winner' – Trump 'ready' to begin transition
South China Sea: Philippines stokes tensions with Beijing in newvisit in disputed waters
Man arrested in B.C., charged in connection with deadly 2007 stabbing in Edmonton
China's imported coronavirus cases jump as students, expats flock home
New mum's first Mother's Day turns to horror as dog's heart stops beating
Andreas Seidl thinks there is 'still a lot more to come' from Daniel Ricciardo at McLaren
Cummins Announces $3.7B Deal To Buy Meritor
Telefonica confirms talks to merge Britain's O2 and Virgin Media
Canada to impose retaliatory tariffs on U.S. goods, hopes for resolution


text:
Q: DataSelect causes error DataSelect takes 3 arguments I'm trying to create a webdatabase.
So far so good. I have an issue however I'm trinyg to select the UserID in my dataname from a table tbl_login
I'm getting an error No overload for method dataSelect takes 3 arguments.
Here is the code.
This code is in my Class1.cs
public DataTable DataSelect(string query)
        {
            DataTable dt = new DataTable();
            try
            {
                DataAdapter1.SelectCommand.CommandText = query;
                DataAdapter1.SelectCommand.Connection.Open();
                DataAdapter1.Fill(dt);
                DataAdapter1.SelectCommand.Connection.Close();
                ErrorMessage = "";

            }
            catch (Exception err)
            {
                ErrorMessage = err.Message;
                DataAdapter1.SelectCommand.Connection.Close();
            }

            return dt;

        }

This code is in my default.aspx.cs
        DataLayer.DataConnector dat = new DataLayer.DataConnector("Provider=Microsoft.ACE.OLEDB.12.0; Data Source= '" + Server.MapPath("MainData.accdb") + "'; Persist Security Info=false");

Hope someone is able to assist me

A: There is nothing obviously wrong with the DataSelect method itself, aside from that it takes 1 argument, not 3 (hence the compiler error). Could you please post the line of code where the method is called?


text:
Methodology article
Conservation and diversity of gene families explored using the CODEHOP strategy in higher plants
Marc Morant1,
Alain Hehn1 &
Danièle Werck-Reichhart1
BMC Plant Biology volume 2, Article number: 7 (2002) Cite this article
Availability of genomewide information on an increasing but still limited number of plants offers the possibility of identifying orthologues, or related genes, in species with major economical impact and complex genomes. In this paper we exploit the recently described CODEHOP primer design and PCR strategy for targeted isolation of homologues in large gene families.
The method was tested with two different objectives. The first was to analyze the evolution of the CYP98 family of cytochrome P450 genes involved in 3-hydroxylation of phenolic compounds and lignification in a broad range of plant species. The second was to isolate an orthologue of the sorghum glucosyl transferase UGT85B1 and to determine the complexity of the UGT85 family in wheat. P450s of the CYP98 family or closely related sequences were found in all vascular plants. No related sequence was found in moss. Neither extensive duplication of the CYP98 genes nor an orthologue of UGT85B1 were found in wheat. The UGT85A subfamily was however found to be highly variable in wheat.
Our data are in agreement with the implication of CYP98s in lignification and the evolution of 3-hydroxylation of lignin precursors with vascular plants. High conservation of the CYP98 family strongly argues in favour of an essential function in plant development. Conversely, high duplication and diversification of the UGT85A gene family in wheat suggests its involvement in adaptative response and provides a valuable pool of genes for biotechnological applications. This work demonstrates the high potential of the CODEHOP strategy for the exploration of large gene families in plants.
Plants have evolved extremely diversified gene families as tools to cope with a harsh environment. Some of these families such as cytochromes P450 and UDP-glycosyltransferases (UGT) reflect the extraordinary biochemical versatility of plants and across plant species, and represent a very valuable source of genes for biotechnologies. Both gene families offer a huge potential for bioremediation and control of crop and weed pesticide tolerance [1–3], but obviously also for industrial applications. P450s, considered as the most versatile catalysts known [4], usually activate dioxygen and transfer one of its atoms into various substrates, but also catalyze a great diversity of reactions ranging from C-C and C=N bond cleavage, phenolic coupling, dehydration, dehydrogenation, isomerizations to reduction [5]. Many of these reactions are important for the biosynthesis of hormones, drugs, pigments, aromas, biopolymer building blocks and defense molecules [6, 7]. Glycosyltransferases are also essential for the production of natural compounds since they control their solubility, stability, transport, storage and sometimes also their bioactivity [8, 9]. Should some of this potential become directly accessible through genomewide sequencing, extensive information is restricted to model plants, usually with a small genome, or to plants with a major economical interest. Exploitation of this knowledge to target genes of other plants that need to be studied or engineered, or to explore gene families in plants with specific biosynthetic capacities is an objective for the next several years.
With the growing availability of gene sequences plus information regarding their diversity and phylogeny, increasingly sophisticated PCR techniques have been developed to target gene families. Plant P450s are low abundant membrane-bound and unstable proteins, usually difficult to purify. For this reason, early on, several groups attempted isolation of P450 genes on the basis of the most conserved consensus regions, after generating probes by conventional PCR at low stringency [10–13]. This approach was later refined and used by several other groups for isolation of P450 genes in various plant species [e.g. [14–18]]. It proved successful in many cases, although only leading to a small number of highly expressed and related P450 families. A significant step forward resulted from coupling degenerate PCR with a heme binding primer and differential display of the amplified fragments, an approach that allowed effective identification of nine P450 genes responsive to elicitor treatment of soybean cell cultures [19] and 21 unique P450 genes in Taxus cells induced for taxol production [20]. A carefully controlled and strongly differential system is however needed for such an approach. Another interesting improvement was recently reported that involves use of nested primers to increase PCR selectivity [21]. However, the major limitation of all the strategies reported so far is that they did not take into account the huge diversity and low conservation of P450s recently revealed by genome sequencing in higher plants, and allowed neither focused gene selection nor isolation of the most divergent P450 clades, i.e. no systematic exploration of the P450 superfamily in highly divergent species.
In this paper we report on the high potential of the recently described COnsensus-DEgenerate Hybrid Oligonucleotide Primers (CODEHOP) strategy [22] of primer design, ensuring optimal match and PCR amplification focused on very short conserved sequences, for the isolation of orthologues in evolutionarily distant species and for the focused or systematic exploration of gene families in plants with a very large genome. The method was tested to analyze both the duplication and conservation of the CYP98 family of P450 genes in many plant species. This family was recently suggested to play an essential role in lignification and plant development [23–25]. The same approach was also used for the analysis of the UGT85 family in wheat.
Chasing the CYP98 genes in wheat
The CYP98 family of cytochrome P450 genes encodes the 3'-hydroxylases of coumaroyl esters, which catalyze an essential step in the synthesis of lignin monomers and chlorogenic acid [23–25]. CYP98 activity is also needed for the biosynthesis of many phenolic flavouring compounds such as eugenol, safrole or vanilin. Engineering the expression of this family of P450s has important agro-industrial applications, including enhancement of plant defense and modification of lignin composition to improve forage digestibility and wood pulping [26, 27]. Access to CYP98 genes from major crop and forage plants and most common woody species is a necessary step for modifying their expression. If some partial sequences are made available by EST sequencing of a limited number of species, they do not reflect the whole range of gene isoforms expressed in a plant or plant tissue, especially for large genome plants.
Our first aim was thus to test if it was possible to detect several genes belonging to the CYP98 family expressed in the seedlings from wheat, a major crop plant with a very large genome. When this work was initiated, few CYP98 sequences were available, some of them only partial sequences arising from ESTs. To optimize primer selection, with a bias in favour of monocot genes, available ESTs from rice and maize were aligned with the full-length sequences from sorghum [17], soybean [28] and Arabidopsis thaliana. In the latter case, A. thaliana genome sequencing had revealed three genes. Two of them (CYP98A8 and CYP98A9: function unknown) were closely related and clearly divergent from the third, CYP98A3, recently shown to encode a coumaroyl esters 3'-hydroxylase. CYP98A3 seemed to be the orthologue of the sequences isolated from sorghum, rice, maize and soybean. Primers were designed using the CODEHOP strategy. Three sense (P98a, d and c) and one reverse (P98cr) primer were selected (Figure 1) so as to avoid the strong consensus regions common to other P450 families such as the highly conserved PERF motif. To further ensure high primer selectivity, touch-down gene selection PCR was conducted starting with a high (70°C) annealing temperature.
Location of the P98 primers on the CYP98 alignment used for primer design. Only the region overlapping available monocots ESTs was used for primer selection in order to introduce a bias for monocot sequences. This overlapping region is shown on the full-length alignment (left) shaded in blue. All sequence alignments were performed using the BioEdit program [40].
BLASTp analysis, performed with the consensus protein sequence corresponding to each primer on a local plant P450 library, indicated that the sense primers were more specific than the reverse, and likely to control amplification selectivity (Table I). As predicted by the BLAST analysis, the P98c/P98cr pair was the most specific, and led to the amplification of two different but closely related CYP98 fragments from wheat seedlings cDNA libraries. A single band of the expected size was obtained on an agarose gel that was eluted and subcloned in a 3'-T overhang vector. Out of 19 sequenced clones, 11 corresponded to CYP98A11, 7 to CYP98A10, and one to a non-CYP sequence (Figure 2). As predicted as well, the P98d/P98cr pair was the second most effective. In addition to CYP98A10 and CYP98A11, it also amplified a clearly divergent CYP98 gene, CYP98A12. Out of 14 amplified fragments sequenced 13 coded for CYP98s. The P98a primer is predicted to match a larger number of P450 families (Table 1). Used together with P98cr, it amplified more non-CYP and non-CYP98 CYP sequences than CYP98s (CYP98A11 and CYP98A10). Two of the CYP sequences however were closely related to CYP98s and CYP76s. Initiation of the touch-down PCR at a lower temperature and analysis of the amplified fragments did not reveal additional CYP98 sequences.
Proportion of CYP98 and other CYP sequences amplified with each couple of primers from the template wheat cDNA libraries. 76/75 and 98–76: BLASTp analysis of the amplified fragment assigns equal scores to CYP76 and CYP75, or CYP98 and CYP76 families. Others: non-CYP sequences.
Table 1 CODEHOP primers used for CYP98s amplification. For each primer the consensus clamp (XXX.XXX) is given in upper case and degenerate core (xxx.xxx) in lower case. y = [C, T], n = [A, G, C, T], s = [G, C], r = [A, G]. BLASTp analysis was performed with the consensus protein sequence corresponding to each primer. Hits: best scoring P450, with expectation values lower than 0.1, are listed in the order of scores.
The CODEHOP strategy thus appears well suited for the very focused to broader exploration of gene families in plants with a large genome, depending on primer selectivity. Selectivity of each set of primers can be predicted by a BLAST analysis. In young wheat seedlings, focused CODEHOP screening allowed the detection of three clearly different CYP98 genes. All three genes are apparently related to A. thaliana CYP98A3.
Isolation of CYP98 ortho/homologues in other plant species
The second step of this investigation was aimed at testing the possibility to isolate CYP98A3 orthologues in a broad range of distantly related species. The most selective primer pair P98c/P98cr, without modification for codon usage, was first assayed with the same amplification programme or after shifting the initial annealing temperature from 70 to 65°C using various cDNA libraries, prepared from Capsicum annuum fruit (Solanaceae), Ceratopteris richardii (fern), Coleus blumei cell culture (Lamiaceae), Eucalyptus globulus xylem (Myrtaceae), Helianthus annuus stem and leaf (Asteraceae), Lycopersicon esculentum shoots (Solanaceae), Picea abies cell culture (Coniferales), Pinus pinaster stem and root (Coniferales), Populus trichocarpa x deltoides stem, root and leaf (Salicaceae), Physcomitrella patens protonemal tissue (moss). CYP98-related sequences were amplified from the libraries of C. blumei, E. globulus, H. annuus, P. abies, and Populus. After optimization of primer codon usage, a CYP98-like fragment was also amplified from the C. richardii cDNA library. No amplicon was obtained however for P. patens, P. pinaster or the Solanaceae, neither after optimizing codon usage, nor after further decreasing the initial temperature of the touch-down PCR.
Out of these ten representative species in a broad range of vascular plants, including fern, conifers, monocots and dicots, the CODEHOP strategy thus provided CYP98-related DNA sequences in seven cases (Figure 3). Due to the short size of the amplicons, it is not possible to unambiguously assign them all to the CYP98 family. Full-length sequences would be needed for such an assignment, if not catalytic activity for the proteins. To date, those were only obtained in the case of wheat, confirming gene identity and function (M. Morant, personal communication). Homology analysis of the amplified fragments is however consistent with evolutionary history of vascular plants (Figure 4). Combined with a BLAST analysis, it suggests that the P. abies and C. richardii amplified fragments could be either representative of ancestral forms of CYP98s or derived from a related A-type P450 family, possibly CYP81. No CYP98-related sequence was detected in the moss P. patens, which would be in agreement with the evolution of CYP98s with vascular plants. CYP98 and related sequences are as yet also absent from P. patens ESTs, among which CYP73 expected to code for cinnamate 4-hydroxylase can be found. Evolution of CYP73, involved in an upstream step in the phenylpropanoid pathway and the biosynthesis of flavonoids, is supposed to have preceded that of lignification [29].
CYP98-like sequences amplified from the different plant species.The primer regions are shown in the alignment (delimited by arrows) to illustrate primer usage for each amplicon. In the case of C. richardii, a primer adapted to fern codon usage (P98Cr: 5' CTCTTGCAATTgcyyrnacrtt 3') was used for the amplification. These sequences are available from Genebank under the accessions: C. richardii AJ438346, P. abies AJ438350, T. aestivum CYP98A10 AJ439883, T. aestivum CYP98A11 AJ439884, T. aestivum CYP98A12 AJ439885, Populus AJ438351, E. globulus AJ438348, C. blumei AJ438347, H. annuus AJ438349.
Identity and gap scores of the CYP sequences amplified with the P98c/cr pair in different plant species (A), and dendrogram deduced from their alignment (B). Identities and gaps were calculated using the software Genedoc [39]. 98A10, 98A11 and 98A12 are the three sequences amplified from T. aestivum.
Limits of the method
In a few cases, no amplification was obtained after decreasing the initial annealing temperature of the touch-down PCR and adapting codon usage. This occurred with libraries where CYP98 cDNAs were expected to be present, for example in a library of pine stem and root, and libraries from bellpepper fruit or young tomato plants. In pine stem, CYP98 should be expressed at a high level for the synthesis of lignin monomers, while Solanaceae are described as accumulating large concentrations of hydroxycinnamic esters such as chlorogenic acid. The P98c and P98cr primers, initially designed with codon usage for wheat gene amplification, were compared to those designed with optimal codon usage for the other plant (Figure 5). P98c differed in 2 positions out of 16 in the clamp segment from the primer optimized for pine, and in 4 positions out of 16 from the primer predicted as optimal for Solanaceae. P98cr also differed in 4 positions out of 11 in the clamp for both pine and Solanaceae. Difference in codon usage thus seemed to provide an explanation for the failure of our first experiments. New primers using adapted codons were thus tested but did not lead to amplified fragment under any tested PCR condition.
Deviations from most frequent codon usage and protein consensus sequences in CYP98s from pine and Solanaceae. Comparison of the original CODEHOP sense and reverse primers (CYP98c and CYP98cr) to the primers adapted to the codon usage of pine and Solanaceae (P98n and P98nr) and to the real sequences of recently available EST or cDNAs from P. teada (CYP98Pt: AY064170), or from Solanaceae (CYP98S1-6). Alignment of available CYP98 ESTs from tomato and potato reveals several subgroups with slightly different sequences in the primer regions: CYP98S1: BG598096/BM113871/BE450893/BE451611, CYP98S2: BE436335/BE432077/BE431773/BM535273, CYP98S3: BE451666, CYP98S4: BG594285/BG594451/BG598096/BM113871, and CYP98S5: BE450893 (Solanum tuberosum accessions are shown in bold characters, L. esculentum are italicized). Sequences matching both tested primers are boxed in blue. Divergence from amino acid consensus is shown in red.
To find an explanation, EST and cDNA sequences now available for pine and tomato, another Solanaceae, were examined and aligned with our primers and consensus sequences. This comparison to the authentic sequences revealed significant divergences from most frequent codon usage in addition to the selected consensus sequence. The assumption that the clamp segment of the primers has a very minor impact on the amplification [22] thus probably should be reconsidered. The dissimilarity present in this example is however likely to be local and thus successful amplification should be obtained by using multiple primers distributed along the full sequence.
Exploration of another gene family in wheat: UGT85
Glycosyl transferases (type 1) form another large gene family with important applications in agrochemistry, therefore, we tested the efficiency of the CODEHOP strategy for exploring the diversity of glycosyl transferases in hexaploid wheat. In this case, we decided to focus on UGTs related to the UDP-glucose:p-hydroxymandelonitrile-O-glucosyl transferase (sbHMNGT or UGT85B1) catalyzing the last step in the synthesis of cyanogenic glucosides, recently isolated from S. bicolor[30]. When this work was initiated BLASTp search revealed only 2 sequences, UGT85A2 and UGT85A3, significantly related to sbHMNGT (43% identity) in the genome of A. thaliana. CODEHOP primer design, based on the alignment of the 3 full-length sequences, provided five sense and five reverse primers likely to be selective of this group of UGTs (Table 2). PCR was conducted with all combinations of primers, starting touch-down PCR at 70°C. When a couple of primers amplified products of expected size, they were subcloned and analyzed. The primer couple was then discarded and the library was screened using the other primer combinations but with the initial temperature of the touch-down PCR decreased by 5°C. Successive temperature decreases led to successful amplification from 8 couples of primers. Glucosyl transferases were recently shown to be induced by the herbicide safener cloquintocet-mexyl in wheat [31]. In agreement with this report, a stronger amplification was usually obtained using a cDNA library prepared from safener-treated seedlings as a template (Figure 6). Analysis of 63 subclones led to the isolation of 18 distinct UGT sequences resulting from specific amplifications. Their sequences can be obtained from GeneBank under the accessions AJ438327, AJ438326, AJ438330, AJ438331, AJ438332, AJ438316, AJ438315, AJ438318, AJ438320, AJ438317, AJ438319, AJ438333, AJ438335 AJ438337, AJ438334, AJ438338, AJ438329, and AJ438328.
Result of a decrease in initial annealing temperature of the touch-down PCR on the amplification of UGT genes with different couples of primers. Location of amplicons of the expected size is indicated by an asterisk. The same couples of primers were tested with two cDNA libraries made out wheat seedlings treated (+) or not (-) with cloquintocet-mexyl and phenobarbital for inducing herbicide metabolism. Some couples of primers such as PUGTb/PUGTer did not provide any amplified fragment, even at low annealing temperatures. Some (e.g. PUGTe/PUGTer) were effective using stringent annealing temperature.
Table 2 CODEHOP primers used for UGTs amplification. y = [C, T], n = [A, G, C, T], r = [A, G], w = [A, T], k = [G, T]. BLAST analysis of protein consensus sequences corresponding to each primer was performed as indicated in the legend of Table 1.
Not all amplified fragments are overlapping (Figure 7). It is thus not possible to determine if they correspond to more than 12 different wheat UGT genes or allelic variants. Their alignment and comparison with representative members of the different UGT families indicate that they are all phylogenetically related and derived from the same ancestor as the UGT85A subfamilly from A. thaliana and sbHMNGT. None of the fragments corresponded to an obvious orthologue of sbHMNGT.
Alignment of protein segments deduced from amplified wheat UGT sequences with sbHMNGT (UGT85B1).
In this paper we investigated the potential of the CODEHOP strategy for targeted isolation of genes from organisms not yet submitted to extensive sequencing and for exploration of the complexity of selected gene families in large plant genomes. Special emphasis was given to wheat as representative of plants with a complex genome. The CODEHOP method proved extremely useful for the characterization of gene orthologues or homologues in a broad range of plants species. Focus of the gene search can be controlled by changing the degree of specificity of the primers (easily checked by a BLAST analysis) and by the choice of touch-down PCR temperature. In our hands, the method was successful where cDNA library screening with heterologous probes (e.g. cDNAs from maize for screening wheat libraries) had failed. The main advantage of this method is to rapidly provide a representative sample of either the allelic variants and recently evolved paralogues, or of the homologues of a given gene in complex genomes. Compared to other methods, a very high proportion of useful sequences (more than 90% with some primer couples) is obtained. Besides targeted search for specific expressed sequences, it allows complete exploration of the different P450 clades in a single organism, which was not possible using previously described methods. The main source of failure seems to be local sequence or codon divergence from the consensus or most frequent usage. This problem should be easily circumvented by using several primer couples chosen from different regions of the gene. Using the CODEHOP strategy on subgroups of phylogenetically related genes, families or clades, within large superfamilies such as UGTs or P450s is a powerful approach for exploring their complexity in various genomes. It is a very effective tool for the construction of expression libraries for agrochemical and other industrial applications. It can also be used for identifying genes from a given subgroup expressed at a specific stage of development.
Some plant P450 families result from extensive duplications, some of them forming clusters of up to 13 genes in Arabidopsis[7]. Some P450 families or subfamilies are found in only subsets of plant species. Extensive search for CYP98 genes expressed in wheat seedlings did not reveal more than 3 different sequences, all related to A. thaliana CYP98A3. The bread wheat genome is hexaploid and results of successive hybridizations and rearrangements [32]. This suggests that the three CYP98A10, A11 and A12 genes may correspond each to one of the three wheat genomes, with CYP98A12 which is the most divergent possibly resulting from the most recent genome introduction. This hypothesis is currently investigated. In agreement with the low number of CYP98s found expressed in wheat, no extensive duplication of CYP98 was detected in other plant species. This observation, together with the high conservation of the CYP98As gene across evolution argue for essential functions of the CYP98 genes in higher plant development. Accordingly, strongly impaired growth and fertility are observed in cyp98A3 mutants [[24], S. Goepfert, personal communication]. Similar conservation is observed for other P450 genes participating in the early phenylpropanoid pathway and hormone homeostasis.
No obvious homologue of the sbHMNGT gene was found in wheat. This may be connected to the fact that large amounts of dhurrin are not reported to accumulate in wheat, since sbHMNGT is described as showing a strong preference for mandelonitrile substrates [30]. A large number of related genes, all belonging to the UGT85A subfamily were however detected. This is not surprising considering that 6 UGT85A genes have been reported in the small Arabidopsis genome [33]. Such a duplication of genes in this family probably reflects an adaptative evolution and their implication in some type of stress/defense response rather than a developmentally essential function. Significantly, a large number of UGT85A-related sequences are found among wheat ESTs isolated from wheat challenged with pathogens. Recombinant expression of such genes should provide a valuable library for pharmacological and toxicological investigations, and for studying evolutionary ecology of plant pathogen interactions.
The CODEHOP strategy appears as a powerful method for exploring the complexity of gene families in plants with a large genome, and conservation of genes across evolution. CYP98s are genes evolved early and are highly conserved during evolution, as expected for genes with an essential role in homeostasis and development of vascular plants. Conversely, the UGT85s are more variable. No orthologue of the sorghum UGT85B1 gene was detected in wheat, while UGT85As, found in many plants species, are also present in wheat. The great variability of this subfamily in wheat strongly suggests a role in environmental adaptation and plant defense.
Wheat cDNA libraries were constructed from poly(A)+ mRNA from 3–5 mm Triticum aestivum (L. cv. Darius) seedlings, both control or pre-coated with cloquintocet-mexyl (0.1% seed dry weight) and phenobarbital, as described previously [34], in λ ZipLox (GIBCOBRL) by T. Thomas (College Station, Texas). Other cDNA libraries were kindly provided by B. Camara, IBMP Strasbourg (Capsicum annuum), W. Faigl, MPI Cologne (Ceratopteris richardii), M. Petersen, Philipps Universität Marburg (Coleus blumei), J. Grimma-Pettenati, CNRS Toulouse (Eucalyptus globulus), J. L. Evrard, IBMP Strasbourg (Helianthus annuus), A. Schaller, ETH Zurich (Lycopersicon esculentum), D. Ernst, GSF Munich (Picea abies), J. M. Frigerio, INRA Gazinet (Pinus pinaster), C. Douglas, University of British Columbia Vancouver (Populus trichocarpa x deltoides), and Leeds University (Physcomitrella patens).
CODEHOP primers are designed as to ensure a very high probability of annealing on the gene of interest with 11–12 completely degenerate core nucleotides at their 3'-end and efficient amplification with a consensus 18–25 nucleotide clamp sequence at the 5'-end. Design of the clamp is based on an alignment of available related sequences and codon usage of the target organism. Primers completely degenerate at their 3'-end and ensuring high probability of annealing to CYP98s or UGTs specific sequences were designed using the CODEHOP strategy [22] based on the multiply-aligned sequences: AF029856, AF022458, AA86449, C74921, D47937, AI881302, AI734373, AAG52369, AAG52373 for the P450 family CYP98, and AAF17077, AAF18537, BAA34687 for UGTs. The multiple alignment was first generated using ClustalW [35], then cut into blocks using the BlockMaker server [36]. Primers were designed using the default parameters of the CODEHOP server [37]. It was assumed that barley codon usage proposed by the server was close enough to that of wheat to obtain effective primer design.
From the primer solutions proposed by the server, five P450 primers were selected, which both provided the largest PCR fragments and avoided the most conserved consensus regions common to a large number of P450s. In the case of UGTs, a broader choice was offered by the server, thus 5 sense and 5 reverse primers could be selected on the same basis as for P450s.
Probe amplification and analysis
In the present work, the CODEHOP approach was not used with genomic DNA but with cDNA libraries, our main objective being to identify genes expressed in specific plant tissues. Screening can be directly performed on cDNA libraries but better results were obtained after preliminary extraction of cDNA from phages. For extraction, an aliquot of the cDNA library was heated 10 min at 70°C, then extracted with one volume of phenol-chloroform. PCR screening was then performed on 50 ng of this template using 2.6 U of HiFi (Expand High Fidelity, Roche), 1.5 mM Mg2+, 0.3 mM dNTP and 0.5 μM primers in the polymerase manufacturer's buffer. The PCR program was designed according to the CODEHOP server's tips, including successively a touch down (A) and a classical (B) PCR as follows: first 3 min initial denaturation at 94°C, then (A) 20 cycles of 1 min at 94°C, 2 min at 70°C (-1°C/cycle), and 2 min at 72°C, then (B) 20 cycles of 1 min at 94°C, 2 min at 58°C, and 2 min at 72°C, and finally a 5 min extension. Elongation time was adapted to the largest expected fragment in each screening. When no amplified fragment was obtained with a primer pair, the touch-down starting temperature of annealing was decreased by 5°C until a successful amplification was achieved.
Analysis and cloning of PCR products
PCR products were analyzed on 1% agarose gels, and fragments of expected size were eluted by centrifugation on Ultrafree-DA columns (Millipore), precipitated and cloned into the pGEM-T vector (Promega). E. Coli XL1blue (Stratagene) was electroporated with 1/10 of the ligation volume. Inserts from five white colonies were sequenced.
Sequences were analyzed by BLASTx against a local database generated from the data found [33, 38]. Non-P450 sequences were xblasted on the NCBI site. The CYP98A10, CYP98A11 and CYP98A12 names were assigned on the basis of the full-length coding sequences subsequently isolated from the cDNA library.
Werck-Reichhart D, Hehn A, Didierjean L: Cytochromes P450 for engineering herbicide tolerance. Trends Plant Sci. 2000, 5: 116-123. 10.1016/S1360-1385(00)01567-3.
Cole D, Edwards R: Secondary metabolism of agrochemicals in plants. In Metabolism of Agrochemicals in Plants. Edited by: Roberts T. 2000, Wiley, 107-154.
Chaudry Q, Schröder PP, Werck-Reichhart D, Grajek W, Marecik R: Prospects and limitations of phytoremediation for the removal of persistent pesticides in the environment. Environ Sci Pollut Res. 2002, 9: 4-17.
Coon MJ, Vaz AD, Bestervelt LL: Cytochrome P450 2: peroxidative reactions of diversozymes. FASEB J. 1996, 10: 428-434.
Mansuy D: The great diversity of reactions catalyzed by cytochromes P450. Comp Biochem Physiol C Pharmacol Toxicol Endocrinol. 1998, 121: 5-14. 10.1016/S0742-8413(98)10026-9.
Kahn R, Durst F: Function and evolution of plant cytochrome P450. Recent Adv Phytochem. 2000, 34: 151-189.
Werck-Reichhart D, Bak S, Paquette S: Cytochromes P450. In: The Arabidopsis Book. Edited by: Somerville CR, Meyerowitz EM. 2002, American Society of Plant Biologists, Rockville, MD, doi/10.1199/tab.0028, [http://www.aspb.org/publications/arabidopsis/].
Vogt T, Jones P: Glycosyltransferases in plant natural product synthesis: characterization of a supergene family. Trends Plant Sci. 2000, 5: 380-386. 10.1016/S1360-1385(00)01720-9.
Ross J, Li Y, Lim E, Bowles DJ: Higher plant glycosyltransferases. Genome Biol. 2001, 2: REVIEWS3004-10.1186/gb-2001-2-2-reviews3004.
Meijer AH, Souer E, Verpoorte R, Hoge JH: Isolation of cytochrome P-450 cDNA clones from the higher plant Catharanthus roseus by a PCR strategy. Plant Mol Biol. 1993, 22: 379-83.
Holton TA, Brugliera F, Lester DR, Tanaka Y, Hyland CD, Menting JG, CY Lu, Farcy E, Stevenson TW, Cornish EC: Cloning and expression of cytochrome P450 genes controlling flower colour. Nature. 1993, 366: 276-279. 10.1038/366276a0.
Toguri T, Tokugawa K: Cloning of eggplant hypocotyl cDNAs encoding cytochromes P450 belonging to a novel family (CYP77). FEBS Lett. 1994, 338: 290-294. 10.1016/0014-5793(94)80286-6.
Urvardi MK, Metzger JD, Krishnapillai V, Peacock WJ, Dennis E: Cloning and sequencing of a full-length cDNA from Thlaspi arvense L. that encodes a cytochrome P450. Plant Physiol. 1994, 105: 755-756. 10.1104/pp.105.2.755.
Frank MR, Deneyneka JM, Schuler MA: Cloning of wound-induced cytochrome P450 monooxygenases expressed in pea. Plant Physiol. 1996, 110: 1035-1046. 10.1104/pp.110.3.1035.
Akashi T, Aoki T, Takahashi T, Kameya N, Nakamura I, Ayabe S-I: Cloning of cytochrome P450 cDNAs from cultured Glycyrrhiza echinata L. cells and their transcriptional activation by elicitor treatment. Plant Sci. 1997, 126: 39-47. 10.1016/S0168-9452(97)00091-5.
Mizutani M, Ward E, Ohta D: Cytochrome P450 superfamily in Arabidopsis thaliana: isolation of cDNAs, differential expression, and RFLP mapping of multiple cytochromes P450. Plant Mol Biol. 1998, 37: 39-52. 10.1023/A:1005921406884.
Bak S, Kahn RA, Nielsen HL, Møller BL, Halkier BA: Cloning of three A-type cytochromes P450, CYP71E1, CYP98, and CYP99 from Sorghum bicolor (L.) Moench by a PCR approach and identification by expression in Escherichia coli of CYP71E1 as a multifunctional cytochrome P450 in the biosynthesis of the cyanogenic glucoside dhurrin. Plant Mol Biol. 1998, 36: 393-405. 10.1023/A:1005915507497.
Ralston L, Kwon ST, Schoenbeck M, Ralston J, Schenk DJ, Coates RM, Chappell J: Cloning, heterologous expression, and functional characterization of 5-epi-aristolochene-1,3-dihydroxylase from tobacco (Nicotiana tabacum). Arch Biochem Biophys. 2001, 393: 222-235. 10.1006/abbi.2001.2483.
Schopfer CR, Ebel J: Identification of elicitor-induced cytochromes P450 of soybean (Glycine max L.) using differential display of mRNA. Mol Gen Genet. 1998, 258: 315-322. 10.1007/s004380050736.
Schoendorf A, Rithner C, Williams RM, Croteau RB: Molecular cloning of a cytochrome P450 taxane 10β-hydroxylase cDNA from Taxus and functional expression in yeast. Proc Natl Acad Sci USA. 2001, 98: 1501-1506. 10.1073/pnas.98.4.1501.
Fischer TC, Klattig JT, Gierl A: A general cloning strategy for divergent plant cytochrome P450 genes and its application in Lolium rigidum and Ocimum basilicum. Theor Appl Genet. 2001, 103: 1014-1021. 10.1007/s001220100620.
Rose TM, Schultz ER, Henikoff JG, Pietrovski S, McCallum C, Henikoff S: Consensus-degenerate hybrid oligonucleotide primers for amplification of distantly related sequences. Nucleic Acids Res. 1998, 26: 1628-1635. 10.1093/nar/26.7.1628.
Schoch G, Goepfert S, Morant M, Hehn A, Meyer D, Ullmann P, Werck-Reichhart D: CYP98A3 from Arabidopsis thaliana is a 3'-hydroxylase of phenolic esters, a missing link in the phenylpropanoid pathway. J Biol Chem. 2001, 276: 36566-36574. 10.1074/jbc.M104047200.
Franke R, Humphreys JM, Hemm MR, Denault JW, Ruegger MO, Cusumano JC, Chapple C: The Arabidopsis REF8 gene encodes the 3-hydroxylase of phenylpropanoid metabolism. Plant J. 2002, 30: 33-45. 10.1046/j.1365-313X.2002.01266.x.
Franke R, Hemm MR, Denault JW, Ruegger MO, Humphreys JM, Chapple C: Changes in secondary metabolism and deposition of an unusual lignin in the ref8 mutant of Arabidopsis. Plant J. 2002, 30: 47-59. 10.1046/j.1365-313X.2002.01267.x.
Baucher M, Monties B, Van Montaigu M, Boerjean W: Biosynthesis and genetic engineering of lignin. Crit Rev Plant Sci. 1998, 17: 125-197. 10.1016/S0735-2689(98)00360-8.
Boudet AM: Lignins and lignification: selected issues. Plant Physiol Biochem. 2000, 38: 1-16. 10.1016/S0981-9428(00)00166-2.
Siminsky B, Corbin FT, Ward ER, Fleischmann TJ, Dewey RE: Expression of a soybean cytochrome P450 monooxygenase cDNA in yeast and tobacco enhances the metabolism of phenylurea herbicides. Proc Natl Acad Sci USA. 1999, 96: 1750-1755. 10.1073/pnas.96.4.1750.
Cooper-Driver GA, Bhattacharya M: Role of phenolics in plant evolution. Phytochemistry. 1998, 49: 1165-1174. 10.1016/S0031-9422(98)00054-5.
Jones PR, Møller BL, Høj PB: The UDP-glucose:p-hydroxymandelonitrile-O-glucosyltransferase that catalyzes the last step in synthesis of the cyanogenic glucoside dhurrin in Sorghum bicolor. Isolation, cloning, heterologous expression, and substrate specificity. J Biol Chem. 1999, 274: 35483-35491. 10.1074/jbc.274.50.35483.
Brazier M, Cole DJ, Edwards R: O-Glucosyltransferase activities toward phenolic natural products and xenobiotics in wheat and herbicide-resistant and herbicide susceptible black-grass (Alopecurus myosuroides). Phytochemistry. 2002, 59: 149-156. 10.1016/S0031-9422(01)00458-7.
Rieseberg LH: Polyploid evolution: Keeping peace at genomic reunions. Current Biology. 2001, 11: R925-R928. 10.1016/S0960-9822(01)00556-5.
The Arabidopsis P450, Cytochrome b5, P450 Reductase, and Glycosyl Transferase Family 1 Database at PLACE. [http://www.biobase.dk/P450/].
Cabello-Hurtado F, Zimmerlin A, Rahier A, Taton M, DeRose R, Nedelkina S, Batard Y, Durst F, Pallet KE, Werck-Reichhart D: Cloning and functional expression in yeast of a cDNA coding for an obtusifoliol 14α-demethylase (CYP51) in wheat. Biochem Biophys Res Commun. 1997, 230: 381-385. 10.1006/bbrc.1996.5873.
Thompson JD, Higgins DG, Gibson TJ: CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994, 22: 4673-4680.
Blocks WWW Server. [http://blocks.fhcrc.org/blocks/].
CODEHOP: COnsensus-DEgenerate Hybrid Oligonucleotide Primers. [http://blocks.fhcrc.org/codehop.html].
David Nelson's Cytochrome P450 Homepage. [http://drnelson.utmem.edu/CytochromeP450.html].
Genedoc: a tool for editing and annoting multiple sequence alignments. [http://www.psc.edu/biomed/genedoc].
Hall TA: BioEdit: an user friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucl Acids Symp Ser. 1999, 41: 95-98.
The support of Aventis Crops Science and of the Association Nationale de la Recherche Technique to M. M. is gratefully acknowledged. We thank D. Little for critical reading of the manuscript and B. Camara, W. Faigl, M. Petersen, J. Grimma-Pettenati, J. L. Evrard, A. Schaller, D. Ernst, J. M. Frigerio, C. Douglas and Leeds University for providing cDNA libraries.
Department of Plant Stress Response, Institute of Plant Molecular Biology, CNRS-UPR 2357, Université Louis Pasteur, 28 rue Goethe, Strasbourg, 67083, France
Marc Morant, Alain Hehn & Danièle Werck-Reichhart
Marc Morant
Alain Hehn
Danièle Werck-Reichhart
Correspondence to Danièle Werck-Reichhart.
M. M. adapted and optimized the method, carried out sequence alignments, primers design and PCR fragments analysis. A. H. participated in the analysis of the UGT family. D. W.-R. was involved in the design and coordination, and drafted the manuscript.
Morant, M., Hehn, A. & Werck-Reichhart, D. Conservation and diversity of gene families explored using the CODEHOP strategy in higher plants. BMC Plant Biol 2, 7 (2002). https://doi.org/10.1186/1471-2229-2-7
DOI: https://doi.org/10.1186/1471-2229-2-7
cDNA Library
Codon Usage
CYP98 Gene
P450 Family
Mandelonitrile


I thought the Hitch-N-Ditch was a pretty sweet product and the price is not bad either at $285.00 plus shipping. That's probably around what your going to pay to rent a gas powered trencher for the day. It also has a lifetime warranty. You can use it to put in underground wiring or run conduit/PVC up to 2.5 inches diameter. Owner Chris Montgomery said he's pulled PVC well over 100 yards underground with this product with no problems.
This entry was posted on Monday, July 9th, 2012 at 3:54 PM	and is filed under Misc.. Both comments and pings are currently closed.


The Greater Building Society has closed the following branches for Thursday April 23rd.
The Greater's internet banking, mobile banking, phone banking services are all fully operational. The ATM network is operational but ATMs within closed branches or areas without power are unavailable.
The Greater's customer service centre on 1300 651 400 and social media channels are also operational to assist customers with their banking needs.