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Monday, April 16, 2012
Practical Tips for Incense
The choice of incense is a big topic. A good start for planets are my planetary rulership pages. If a planet or fixed star, for that matter, rule a substance you can use it as incense. Just watch out that it isn't poisonous or creates noxious smoke when burned.
Let me talk about a bit about practical use of incense. The key with incense is first USE IT! Making sure that you do invocations with incense at all, is more important than what kind you use. I find that when I recommend incense that clients can be so worried about getting the wrong kind or making some kind of mistake that they don't do the invocation at all. You don't have to get mastic gum resin for Jupiter or rosehips for Venus, though the planets appreciate the effort, you can use standard stick incense.
Now, if you are allergic to incense, don't feel like this is a disaster either. I've had clients use a vaporizer or aroma therapy style oils instead. Or punch up the candles, use a number of them, rather than just one, if you can't use incense. Another option is using a tiny amount of incense. That's what I tend to do, just one or two beads of frankincense or mastic. My sinuses can't handle the huge clouds of incense that seem to the norm among ceremonial magicians. A third option is smokeless or rather more realistically low smoke incense.
Here's the smokeless incense page at Scents of Earth, a company which a client just turned me on to
I got a box of the Baiedo Bikou Kobunboku Smokeless and I really like this. I use this for my Buddhist altar, half a stick and we don't even notice the smoke or incense at all.
This does look like a good source for traditional incense, here's the
Scents of Earth main page One thing I am particularly into is the special Japanese charcoal
Here's the big size of Japanese charcoal, but I like the small size charcoal, which I cut in two for my super minimal incense use.
You have to use charcoal for authentic resin incense like frankincense (it comes in these little beads). You need an incense burner, charcoal, tongs (charcoal is HOT!!!) and the incense beads. Pick up the charcoal with the tongs, light the edges in a candle till they are glowing red and then place the charcoal in the burner. Then put the beads of incense on the charcoal. I put the incense burner on a glass plate and I NEVER LEAVE BURNING CANDLES or INCENSE UNATTENDED!!! I don't care how magical it is, I don't care if people have left candles in their bathtub or oven unattended, this is a VERY BAD IDEA!!!!
The great thing about the Japanese charcoal, as opposed to the standard round version you get at hoodoo shops or botanicas is that the Japanese charcoal doesn't spark like crazy.
Another excellent source for incense is Harry at Alchemy Works.
2 comments:
- Gary (NJ) said...
I'm an Acolyte in an Anglo-Catholic (Episcopal) Church and love the High Magic quality and symbolism of the High Mass; especially clouds of incense. I heard that some of the other altar servers had complained to the priest about the amount of incense smoke they were using. I felt annoyed and wanted to say, if you can't stand the smoke, get out of the sanctuary. ;)
But I know what you mean about the dangers of red hot charcoal. The thurifer is one of the last positions that an acolyte is allowed to learn and perform due to the dangers of getting burned or starting a fire. Even lighting the upper candles on the High Altar can be precarious. I've had burning tapers drop off and hit my hand and been splattered with hot wax on more than one occasion.
Gary (NJ)
- April 17, 2012 at 10:50 AM
- Christopher Warnock, Esq said...
Incense smoke can be a real problem for people. We have Zen practitioners and priests who cannot participate in zazen or services if incense is used due to respiratory illness. This is something of a dilemma since incense is traditional, but when push comes to shove, we drop the incense, rather than exclude people.
- April 17, 2012 at 10:54 AM


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The Golden Cross
The Golden Cross (Stranraer)
Going A77 southward by the firth of Clyde and you'll find port town Stranraer. This town is only 2 hours away from Belfast and Ireland atmosphere remains. The pub is in the middle of the town and it was built in 1732. This old pub has object d'art of thistle on the front part of the roof and rose and shamlock on the side. Inside the pub is not that wide but you can meet many frequenters there.
COPYRIGHT©2004 Pub Mahorobi. All rights reserved.

subset: commoncrawl


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Editor's note: If you have questions, we'll answer as many as we can, but please remember that Rob Giannangeli's comments aren't intended as a substitute for advice from a professional familiar with your individual circumstances. Also check out our Tax discussion group and Tax Library for more guidance.
Dear Tax Man:
Please explain how I can legitimately deduct the cost of buying or leasing an automobile. I'm a physician partner in a medical practice organized as a limited liability corporation. What is the "6,000 lbs vehicle weight" issue? Thank you. --BW
CBS.MW: Expenses incurred in operating a vehicle in the conduct of a trade or business are deductible. The same can be said of a transportation expense incurred by an employee if they are required to incur the expenses as a condition of employment. As an employee of the corporation, you can deduct the costs of operating a vehicle for the benefit of the corporation so long as you are required to incur them.
Expenses incurred in operating a vehicle in the conduct of a trade or business are deductible
Business use of the vehicle consists of such things as travel between your office and hospitals, making house calls and attending medical conferences. The expense of driving from your residence to your first stop each day is a non-deductible commuting expense. Expense incurred on the last trip home each day is also considered a non-deductible commuting expense.
To figure your deduction, total up expenses for gas, insurance, repairs, depreciation or lease, vehicle registration, taxes, etc., and then claim the business portion as a business expense. Any reimbursements received from the corporation must be used to reduce the allowable business expenses.
If you prefer, in lieu of itemizing all the operational expenses, you may claim the standard mileage rate based on your business miles. The standard mileage rate changes from year to year and is published annually by the IRS.
The "6000 lb. vehicle weight" issue is a reference to the definition of an automobile found in Internal Revenue Code Section 4064 -- the section of the law that deals with the gas-guzzler tax.
That section defines an automobile as "a foul-wheeled vehicle propelled by fuel which is manufactured primarily for use on public streets, and highways (except any vehicle operated exclusively on a rail or rails), and which is rated at 6,000 pounds unloaded gross vehicle weight or less." --The Tax Man
Whose money is it?
Dear Tax Man:
I invested $20,000 in my daughter's name under the Uniform Gift to Minors Act and made some money from this investment. After paying tax, we still have some money left. Does this money belong to her, or to me? If it belongs to her, can she invest this money into her IRA? --Wei
CBS.MW: If you made a gift to your child under the Uniform Gift to Minors Act, those funds now are hers, not yours. You can still invest the fund for her. If investment income is her only source of income, she may not open and contribute to an IRA. She must have earned income or compensation such as wages, salaries, commissions and self-employment earnings in order to contribute to an IRA. --The Tax Man
Estimated tax payments
Dear Tax Man:
I sell naked put options every month. Do I have to pay quarterly taxes? -- GG
CBS.MW: If you will have income taxes due for any year, you have an obligation to pay those taxes as you go, either through withholding at the source or through quarterly estimated tax payments. --The Tax Man
Day Trading with an IRA
Dear Tax Man:
Can one engage in day trading futures contracts in an IRA account? If so, can one make withdrawals as frequently as desired, subject to the IRA early withdrawal penalty rules? Also, can one make partial rollovers between IRAs as frequently as desired? --BW
CBS.MW: With a self-directed IRA, you can make trades within the IRA. You also can make withdrawals from the IRA if you are willing to pay the tax and penalty that goes with premature withdrawals. You are limited to one rollover per year per IRA. --The Tax Man
Prize money
Dear Tax Man:
I recently won a lump sum of cash from a radio station. The winnings can be claimed in 2 to 3 months when I receive a check for the total sum. What amount of the winnings should I set aside for my tax liability this year? Also, the amount I set aside for taxes, should I put the money in a six-month CD to gain additional earnings? What is the best way to take advantage of my winnings? I plan to use the winnings to pay off credit-card balances to reduce my monthly spending and get out of debt. - BR
CBS.MW: If the radio station follows the law, they should withhold 30 percent of the prize money and turn that over to Uncle Sam. You'll get a check for 70 percent and a Form 1099 at the end of the year showing the gross prize amount and your withholding credit. If they don't withhold, then you most likely have the responsibility of paying in estimated taxes, spread over the remaining quarters. Your best bet is to consult a tax professional to see if you really are required to make the estimated payments. If not, then by all means, put the balance of the unpaid tax in some sort of interest bearing instrument that matures about the time your tax return is due.
As for what to do with the money, well, that's really your call. You should look at your life style and see what's best for you. It never hurts to pay off things like credit-card debt, since interest charges are not only very high and but also not deductible. --The Tax Man
Sale of residence not a one-time thing
Dear Tax Man:
I am wondering if you can answer a question for me that relates to capital gains on real estate. With the new law, can I avoid paying the tax on the first $500,000 of profit or can I do this several times over my lifetime? -- MK
CBS.MW: If you own and live in your personal residence for two years out of the five years immediately preceding the date of sale, you can exclude up to $250,000 of the gain made on the sale of the residence if you're a single person; $500,000 if you are married and file a joint return with your spouse. This treatment is available to you every two years, if you've owned and lived in the personal residence for the two years. It is not a once-in-a-lifetime situation. --The Tax Man
Is it a rental or a residence?
Dear Tax Man:
I am single and have a house that I have lived in two of the last five years. I then rented it for 3 years. Can treatment of the capital gain (probably $400,000) of a straight sell be mitigated first by the $250,000 deduction for sale of a primary residence, and then put the remaining $150,000 unprotected capital gain into a 1031 exchange for a "like" property? -GBS
CBS.MW: You're mixing apples and oranges. Either the property is personal residential property, or it's property used in a trade or business. Pick one. If you lived in the house as your personal residence for two out of five years prior to the sale, you can exclude up to $250,000 gain that is not the result of depreciation deductions claimed since May 1997. Any gain attributable to the depreciation allowance claimed while rented is fully taxable. In the alternative, if the property is deemed rental property, you can exchange the property for another of equal or more value and defer all the gain realized. -The Tax Man.


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Girls who lack fathers are always searching for that male replacement. A figure to hold on to that shows them what its like to feel protected and loved. Often times, girls seek so hard to find that missing love that they end up pregnant at 15, lost, alone, and mad at the world for dealing them a deadbeat dad and no love for self.
The love of a father is so important because it molds women into who they are to become. I was blessed enough to have figures to step in my life to show me that I was good enough, but I didn't think I was good enough.
This is not a pity party about me though. I have overcame those inner battles with self and look to my Heavenly Father to step in where my earthly father has stepped out.
Nevertheless, girls without fathers have to be careful. There are men out there who take advantage of that information and attempt to use that as leverage to keep you feeling like you are less than.
For the past few years, I was in an abusive relationship. I worked so hard to please and I forgot who I was. The sad part about it was, I did not realize that I was in the abusive relationship until it was too late. I would break my neck trying to please and make them happy, however, I was continually going to be a puppet on their string.
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When you allow someone to tell you that you are nothing for so long, you start to believe it. I would dread waking up in the morning, but loved sleeping because it was an escape. My mental escape. However, I lost who I was.
Sometimes I felt as though I became a robot and only spit out what was wanted or expected to be heard.
Once I broke free, the albatross was immediately released from my shoulders. I never felt so free. I never pictured myself leaving, because I was made to feel like I was not smart enough to leave.
Abusive relationships show themselves in many forms: best friends, boyfriends, girlfriends, coworkers, bosses, husbands, wives…
Look at your life. Examine it. Who are you living for? God should be first. Keep looking at your life… Pay attention to who surrounds you and who is in your ear. Is it positive? Is it negative? Are you being silenced or made to feel less than? Do you find yourself afraid to speak up? Do you think that you'll be criticized or hurt physically, mentally, or emotionally?
Break free.
It will be hard. It is easier said than done. Sometimes you won't have a plan, hell, I didnt have a pl… BUT, I found that upon breaking free, I found my inner strength. I started listening to God and not others.
You allow yourself to be treated by what YOU think you deserve, and I had was in a relationship that made me think I didn't deserve much; but when I broke free… I realized that I am a child of the King.
Grow in your faith. Grow in yourself. Give it to God, and Break Free.


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Modal analysis and damage assessment of cracked plates
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Kolar, Ramesh
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Dynamic characterization of cantilevered cracked plates assumes importance for a variety of applications including aging aerospace, mechanical and ship structural components. In this paper modal characteristics of isotropic plates with damage identified as cracks are investigated. The paper describes results obtained by finite element simulation of cracked plates. The frequency degradation as a function of the crack length is presented for selected aspect ratios. Some damage metrics are developed based on the modal parameters to describe the structural integrity of the plate type structures with root cracks. Frequency response analysis is presented to show the effect of the crack growth on the response of the plates. Some suggestions are developed based on the observations of the simulation for health monitoring of structural components.
Conference: 2003 IMAC-XXI: Conference & Exposition on Structural Dynamics
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Join us for an evening of teachings and meditation with Diamond Way Teacher Cecilia Blasquez. Cecilia will give a lecture entitled "Buddhist Refuge & The Enlightened Attitude".
Cecilia Blasquez Albarrán was born in México City. Cecilia was 19 years old when she first learned about Buddhism at Casa Tibet, a Buddhist Institute of Learning in México City. In 1991, Cecila met her teachers, Lama Ole and Hannah Nydahl, and began practicing the methods of the Diamond Way.
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Since that time, Cecilia has traveled extensively with Lama Ole through Australia, Latin America, Europe and USA. She actively supported the creation and development of new Diamond Way groups and centers in México and founded the Diamond Way Buddhist Center in Metepec, Mexico.
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Mr Dorian Lovell-Pank QC Door Tenant
Appointed Queen's Counsel: 1993
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Mr Lovell-Pank has defended in and prosecuted almost every kind of criminal case from minor theft and assault to contract murder and large scale fraud. Although he practices predominately on the South Eastern Circuit Mr Lovell- Pank has extensive experience in cases in Liverpool, Manchester and Birmingham.
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International Criminal Work
Mr Lovell - Pank has been widely involved with the Bar Council, the Criminal Bar Association, the Foreign and Commonwealth Office and the British Council in programmes of judicial and legal reform and advocacy training of prosecutors and defence lawyers, particualrly in Europe and Latin America. The countries involved include: Bosnia, Holland, Russia, Spain, Argentina, Colombia, Mexico, Peru, Uruguay and Venezuela.
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Mr Lovell-Pank was appointed Queen's Counsel in 1993 and is a leading Criminal Advocate. His areas of practice involves all aspects of criminal work acting for both the prosecution and defence. He is a strong proponent of the adversarial system, has a wealth of practical experience in the Court room and international experience of advising governments in the developement of their criminal justice systems. Fluent in Spanish, he has presented papers and given lectures in Central and Southern America. Mr Lovell-Pank has been a leading participant in the reintroduction of jury trials in Russia and is recognised as one the the United Nations advocacy trainers at the International Criminal Tribunal at the Hague.
Appointments and Memberships:
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- Recorder of the Crown Court 1989 -2006
- A Chairman of Police Appeal Tribunals 1991 - ongoing
- Member of the International Bar Association (IBA) 1993 - ongoing (Council Member 2001 - 2005)
- Member of the IBA Human Rights Institute 1996 - ongoing (Council Member 200-2004)
- Member of the American Bar Association 1997 - ongoing
- Member of the Bar Council 1989 - 2005 (Chairman of the International Relations Committee 2001 -2005)
- Committee Member of the Criminal Bar Association 1989 -2006 (Chairman of the International Relation Sub-Committee 1993 -2006)
- Appointed Queen's Counsel 1993
- Chairman of the Bar Conference 1999
- Member of the Foreign and Commonwealth Pro Bono Lawyer's Panel 2002 - ongoing
- Elected a Bencher of Inner Temple 1998. Member of the Advocacy Training Committee
- Downside School
- Collegio Sarmiento, Buenos Aires
- London School of Economics
Bilingual in Spanish
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Students from Northeast Community College in Norfolk will soon be able to transfer credits and continue their education at the four-year Mount Marty College in Yankton.
The presidents of the two schools have signed a memorandum of understanding to create the path from Northeast to Mount Marty. Northeast President Michael Chipps says it helps expand their outreach.
"Community colleges in Nebraska are highly comprehensive," Chipps says. "About 50% of our students are transfer students and about 50% go right into the career and technical education and then on to the workforce."
Chipps says they already have many students who could qualify to make the switch.
"We carry about 18,000 credit and non-credit students and about 7,000 of those are found to be credit," Chipps says, "so there will be a lot of students who will be looking at different opportunities like these."
Mount Marty President Marc Long says many community college students go in intending to go to a four-year institution.
"And some go to community college who don't really know what the next step is," Long says. "We want to make sure they have that option. Since Northeast is only 60 miles away from Yankton, there are a good number of people in northern Nebraska who could easily travel to Mount Marty. We think this is just a natural collaboration between the two institutions."
Long says the partnership could help keep students in the region. The agreement will be effective this fall.
By Jerry Oster, WNAX, Yankton


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At age 32, this might be Greenway's 10th and final season in the NFL. Drafted in the first round out of Iowa in 2006, he has become the rare player to stick with the same team the whole time.
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EDEN PRAIRIE, Minn. (AP) — Chad Greenway's role with the Minnesota Vikings has been reduced, as has his salary.
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Their past two opponents have sure felt it. Detroit's Matthew Stafford and San Diego's Philip Rivers each took plenty of painful hits, and neither of these prolific quarterbacks had a particularly difficult day. Vikings cornerback Captain Munnerlyn said he heard Rivers pleading to his injury-depleted offensive line at one point Sunday for better protection.
Still the starter at the weak side linebacker spot in the base defense, Greenway comes off the field for the nickel package. With so many pass-first teams now, that means he's on the sideline far more than not.
Against the Chargers, Greenway was on the field for 19 of 72 plays, relegated to mostly running downs. He realized this was coming, after taking a pay cut for the second straight year. So he has become even more of a leader at a position with two rookies and three others who entered the league in 2012, 2013 and 2014.
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Like Sunday, for example, when Greenway grabbed a deflected pass and took the interception 91 yards for a touchdown to seal Minnesota's 31-14 victory over San Diego. He had more than half of the defense accompanying him on the return and the entire sideline exuberantly encouraging him into the end zone.
"'Man, don't let me get hit like that again,'" Munnerlyn said, quoting Rivers.
"I said, 'This guy can be a great mentor to you,'" Zimmer said, later adding: "He's always trying to learn. He's eager to be a great football player all of the time. Those are the kind of things that I really respect about him."
"That was quite the convoy," Greenway said.
His value to the coaches and players, though, has never been greater.
The Vikings (2-1) are fifth in the NFL with an average of 16.7 points allowed per game and tied for fourth with an average of 6.4 yards yielded per pass attempt, two vital statistics for any defense.


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Home > New Mobile Home Models for Sale > Travis by Cavco Industries
Travis by Cavco Industries New Mobile Home Model for sale
$75,000 | | 3 Beds | 2 Baths
Manufactured Home Details
|32ft X 72ft
|3 Beds
|2 Baths
|Located in a mobile home park
|Purchase Method
|Off Market
|Posted on
|Oct 29, 2012
|Updated on
|Jul 28, 2016
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Aaron HuttsellFactory Expo Homes South
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It simply does not get much better than the luxury appointments and lavish floor plan of the Travis mobile home. This 3-bedroom home is complemented by a large family room that can be upgraded with optional in-wall shelving units and an entertainment center. In addition, the spacious living room of the Travis mobile home can also be upgraded with an optional fireplace.The Travis mobile home kitchen and dining room area is designed with a large pantry, raised snack bar and a shelf/serving island combination. The over-sized utility room includes space for a freezer and optional overhead shelving. The master bath includes a double-sink, walk-in closet and large bath. The enormous master bedroom (15???ï¾?-8?????x15???ï¾?-8?????) is enough to satisfy the space requirements of virtually any single individual or married couple.


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If one has ever seen the movie The Longest Day there are a few scenes and stories that stick in one's head. For instance, one probably can recall the clickers that John Wayne's character introduces to his paratroopers. One can find those in souvenir shops here (€3). Many students bought these things. They're a little bit like Overlord's Fidget Spinners©.
More to the point, we did get to Utah beach, which is famously enshrined in The Longest Day when Theodore Roosevelt, Jr., lands to find his unit has landed in the wrong spot. That's okay, "We'll start the war from here." He then proceeded to lead his division in a flanking maneuver that, at relatively low loss of life, neutralized the German defenses there, clearing the way for Utah's successful landing.
|General Roosevelt's grave at Normandy American Cemetery.
Ironically, at Omaha beach, missed landing destinations paved the way for near-catastrophe.
Lauren and I talked a little bit about why Roosevelt won the Medal of Honor. She's a little frustrated: Was it just because he led a flanking maneuver prefaced by a great quotable line? How often do little guys (the grunts) and their heroics get overlooked? Did Sgt. Strezcek win one for finding a way off Omaha?
|Some of our students scale the bluffs at Utah.
|Along Utah beach, looking north (well, the student pictured is actually gazing east, but you get the idea).
|Flags over the bluff at Utah.
Utah Beach is a wonderful site to visit though there's not as much there that catches the eye. A good museum exists there, and I spoke of it and it's B-26 in another post. Also, some good memorials there.
Allow me to pause for a minute about those memorials. There are several spaces there to memorialize the Naval and Coast Guard personnel lost in the invasion. My roommate on this trip is Paul, a retired Coast Guard Commander, so I guess that has made more inclined to look out for such acknowledgements. But I also wanted to offer that this trip has made me much more aware of and curious about these sailors who made the invasion possible. The famous opening battle scene in Saving Private Ryan begins seeing Captain Miller and his unit on a Higgins boat as they are about to land on the beach. In the background, though, though there is an anonymous sailor steering the boat. He never has a line. He's forgotten as soon as the gates lower.
|One view of the sailors memorial at Utah.
|A view of Utah from the approximate location of the sailors memorial.
But I've learned a lot about these sailors. First of all, he might not have been a Navy sailor. He may have been a Coast Guard sailor. They were deployed to a great extent in the invasion, crewing landing craft and searching for survivors. I pause when I think of men who signed on the Coast Guard who might not have had expectations of serving their country in this particular way. But they served.
Higgins boats had crews of four: a coxswain, a mechanic, two anti-aircraft gunners. In addition to taking men to the beach, they brought the wounded and the dead back off the beach. And they would repeat this throughout D-Day.
The invasion armada was the biggest assemblage of ships ever. There were hundreds of warships involved pounding the beaches, sweeping for mines, and defensing against submarines. One destroyer that was sunk, Corry, was brought down when the plane providing it with a smokescreen was shot down, thus exposing it to German coastal batteries. However, it might be that a mine sunk it. Uncertain. The chaos of combat sometimes makes the exact cause uncertain. And there were hundreds and hundreds of sailors in the midst of that chaos, getting the invasion troops to and from the beach at risk of great personal harm.
The Institute has heightened my curiosity about the role of Naval and Coast Guard personnel in the invasion. It's an aspect of the campaign that I have taken for granted, which is a shame, for the Navy exists as a means of projecting national power. And that invasion might have been the greatest expression of how the U.S. and Britain could express that power in 1944.
Whew. I thought I was talking about sites from The Longest Day. It's dawning on me that the men in blue didn't come up much in the movie.
|The original bridge.
|Where the original bridge stood.
|First liberated house.
|Monument at Pegasus Bridge site.
Another famous scene from The Longest Day involves the assault on Pegasus Bridge. An attack by British glider troops, this was meant to secure a crossing over the canal so that British troops wouldn't get caught on the beach. Their mission called for great precision: glider pilots landing at very precise locations, squads that could neutralize and secure the bridge before the Germans could defend it or blow it up, holding the bridge until reinforcements arrive from the beach. These British troops' landing was one of the earliest operations on Overlord. In fact, many say that the house and restaurant on the west bank of the bridge are the first buildings liberated in Occupied France. The bridge, with its distinctive hinge, still exists though it now rests at a museum about 200 meters away from the canal. A sturdier bridge now serves as the means of crossing the canal. This site was our first visit after arriving in Paris.
The irony of us visiting this site first, a site famous from The Longest Day, on our longest day isn't lost on me. We woke around 6 am in College Park on Thursday, June 22. We eventually got to the airport, made our flight easily, and arrived in Paris around 6 am their time (or midnight EDT). We then drove three hours to Pegasus bridge. After visiting it and the museum, we snuck in another site before arriving at Bayeux, where we had dinner (most places don't open until 7 pm) and finally got to bed around 10:00 pm. That's almost 36 hours on the move!
I guess this post on The Longest Day is becoming the longest post. I better get cracking.
|The church at St. Mere Eglese taken at the corner from which Sgt. Steele was hanging.
|Note the dummy hanging from the steeple on which Sgt. Steele was caught.
|Most students I talked with told me that this exhibit at the Airborne Museum in St. Mere Eglese was their favorite museum.
Perhaps the most famous scene from the movie involves an American airborne trooper whose parachute was caught in the steeple of the church at Ste. Mere Eglese. That was our first visit on the morning of our first full day in Normandy. Sergeant Steele was shot in the foot and feigned dead while his unit landed in the square below. His unit fell right into an accidental trap: a fire had broken out in a building before the troopers landed, and the garrison had come out as firefighters did their work. The German soldiers were in the right place at the right time to wipe out the American troopers. I learned recently that most airborne troops land with their rifles unavailable. Their Garands are disassembled and holstered so as to prevent damage to the weapon upon landing. Steele had no choice but to watch helplessly as his buddies were gunned down.
|Small village church used as an aid station.
|Stained glass pays tribute to liberators.
|The grave of one of the American medics who died in 2013.
We saw a few spots not featured in the movie as well. We toured a house that Germans had used as a headquarters and barracks for much of the occupation. We visited a village where three American medics turned a church into an aid station where nearly 100 men were treated, men who were American, German, and French. The three earned the admiration of the village's inhabitants for their fearless work on the fallen throughout a chaotic night in which the church changed hands several times between American and German troops.
One of those medics who survived the war requested that he be buried there. His grave is in the cemetery. It's a relatively new grave, marked very simply with his initials and his identification as a medic.
Part of me wonders if there are more sites we could have visited. Then answer is probably yes. But the vastness of this battlefield makes it impossible to see everything. And I can't take for granted that all on this trip find such sites as interesting as I do. The students came here to honors soldiers, which isn't necessarily the same thing as seeing battle sites. And had we packed our days too full, had we tried to put ten pounds of potatoes in a five-pound sack, they might not have been able to eulogize their soldiers and bond with one another as meaningfully as they did.


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For old-world Manhattan living it would be hard to argue with the Parc Vendome, from its award-winning formal English tea gardens to graciously-configured apartments—like this 2,000 square-foot, two-bed-plus-office beauty—with wood-burning fireplaces, entry galleries and capacious closets. Built in 1931, the elegant two-building complex at 333 West 56th Street was converted to condominium apartments in 1983. In addition to space and charm and a fortunate location blocks from both Lincoln Center and Central Park, you'll have an impossibly lovely south-facing private terrace and open city views, all for $10K a month (gas and electric are included).
Parc Vendome
Cool Listings, Midtown West
Find Prewar Manhattan Glamor in Modern Midtown for $10K a Month, Wed, August 3, 2016
Celebrities, Midtown West, Recent Sales
We don't think the uber-chic, ever-polished Joan Harris would approve of this pretty basic, slightly cluttered midtown apartment that her real-life counterpart Christina Hendricks just picked up, but seeing that the actress is so well known for her timeless fashion sense, we think she'll turn it into her very own glam pad in no time.
The Daily News reports that Hendricks and her husband, actor Geoffrey Arend, bought the one-bedroom unit at the Parc Vendome at 353 West 56th Street for $1.16 million. The timing makes sense, as "Mad Men," which films in Los Angeles, is airing its series finale in just a couple weeks.
Take a look around the home here


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On the basis of those stereotypes, which are unsuccessfully driven into one's head by school literature programmes, many people of my generation (I will not speak for young people) assume that Germany is a conservative country, not inclined to welcome any innovation. Of course, this is not true, or rather not in general true. The world (and chess is no exception) is in a constant state of dynamics, with Germany at the forefront of the innovation process, so that the traditional picture is largely outdated. But to turn to chess. Changes were made this time to the «Grenke» Festival, which ahs already established itself. Previous festivals were usually run under the formula "an elite round robin + open", but, apparently, the organizers did not wish to be stuck with the established format and are trying to find something new or maybe they wish to implement further the idea of the democratization of the chess community? Or maybe there are other factors. Although last year's tournament with Carlsen, Caruana, Aronian, Anand and Co. was interesting, exciting (won, we recall, by the world champion, with Naiditsch second), this year it was decided to change the format. In any case, this fact cannot but please the broad masses of working grandmasters.
The current «Grenke Chess Open» gathered 940 participants in three sections. The prize fund of the "A" tournament is 40 800 euros, of which 10,000 will go to the winner. The organizers are proud to note that the line-up (26 grandmasters with rating over 2600 and more than 100 players with Elo coefficient above 2400) can compete with their counterparts in Gibraltar and Qatar. By the way, the tournament changed its location - last year it was in Baden-Baden, this year in Karlsruhe. But the shift is negligible, and the event remains within the confines of Bad-Württemberg.
Li Chao is top seed
List of players
The official website «Grenke Chess Open» points out that it is not often that the likes of Gata Kamsky, Alexei Shirov, Arkady Maiditsch and Loek van Wely meet each other in an open tournament. Clearly this refers to round robin events, rather than opens, which are rather more common.
Alexei Shirov. Will there be 'fire on board' again?
Given the size of the field, it is a little early to draw many conclusions after the first five rounds. but the leading group contains a few surprises, with not all the favourites making a great start. Kamsky, for example, has just a modest 3,5 points. Many interesting moments can be seen in the current round, for example:
Kunin - Fedoseev
Vladimir had quickly driven his opponent into a passive position and was looking forward to exploiting his trumps in a quiet endgame. But suddenly he lost concentration 20…Rfc8? – forgetting about his central pawn. 21.f3 Bg6 22.Rb5 b6 23.Rxd5 – and it was a matter of converting the extra pawn, although the Russian player could probably have posed his opponent greater problems (1:0). This allowed Kunin to lead (together with Milos Perunovic) the table.
One of the female players Ekaterina Atalik (Turkey)
The young but already quite experienced Austrian GM realised his advantage convincingly:
Donchenko - Ragger
After 31…Rxf3 White had to play 32.exf5 [On 32.Kxf3 – 32…Qd3 33.Kg2 (33.Kf4?? – 33…h5 mating quickly) 33…Qxe4 34.Kg1 Nd4 etc] 33…Qe2 34.Qxb6 Rxf5 – the heavy-piece ending is not much fun for White but he hasreasonable drawing chnaces. But the German played less than ideally:
There followed 41…Kh5! 42.Qe1 Rd3 43.Kh2? e4 44.Qa5 f5 45.Qa7 h6 46.Rg1 e3 47.Rg2 e2 48.Qe7 Rd4 49.Kh3 Re4 White resigns (0:1).
Scores after 5 rounds
Text: Sergey Kim


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Terry looking strong on the deadlift!
Strength: Front Squat
3 Sets of 3-6 reps at 70-75% 1RM
– Final set for max reps BUT practice ditching before you get under a weight you may fail with
Partner WOD:
3 x 4:00 rounds for max total reps and max total calories
2:00 max effort row (for calories)
1:00 unbroken handstand hold (partner watches the clock) – 10 burpee penalty for breaking HSPU after completion of the workout
1:00 max effort chest to bar pullups
4:00 rest while partner is working
– Total workout time is 24 minutes, each partner will be working for 12 minutes.
– Be sure to be on the rower and ready to go as your partner finishes the C2B pullups to keep on the 4:00 interval
– When 3 rounds are completed move immediately to any burpee penalty owed


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The Kar-Jenner girls are looking pretty in pink!
On Tuesday night, Kylie Jenner shared an adorable snapshot on Instagram of her daughter, Stormi getting cozy with Kim Kardashian and Kayne West's youngest, Chicago. The two had a sleepover, and their mothers Kim Kardashian and Kylie Jenner shared the photo on their social media pages.
The two were in matching pink pyjamas, looking like twins.
Facebook Comments


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Behind The Scenes Of The PS4 Launch Trailer
Sony already released a launch trailer for the PS4 when it landed in North America so instead of rehashing old ground with another launch trailer, they've gone and made a brief behind the scenes video about that launch trailer. Weird? Cool? Random? These are all words. I don't really know why but this video is worth watching even though it will do nothing to get you excited for PS4.
Source: VG247


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To counteract fluctuating nutrient environments, plants have evolved high‐ and low‐affinity uptake systems. These two systems were traditionally thought to be genetically distinct, but, recently, two Arabidopsis transporters, AtKUP1 and CHL1, were shown to have dual affinities. However, little is known about how a dual‐affinity transporter works and the advantages of having a dual‐affinity transporter. This study demonstrates that, in the case of CHL1, switching between the two modes of action is regulated by phosphorylation at threonine residue 101; when phosphorylated, CHL1 functions as a high‐affinity nitrate transporter, whereas, when dephosphorylated, it functions as a low‐affinity nitrate transporter. This regulatory mechanism allows plants to change rapidly between high‐ and low‐affinity nitrate uptake, which may be critical when competing for limited nitrogen. These results demonstrate yet another regulatory role of phosphorylation in plant physiology.
Plants have a remarkable ability to adapt and grow in soils containing widely different levels of ions and nutrients. Since the early studies of Epstein in the 1950s (Epstein, 1972), it has been known that plants have evolved two types of system for nutrient uptake, a high‐affinity system and a low‐affinity system. The high capacity low‐affinity uptake system takes up nutrients when the external substrate concentration is high (e.g. >0.5 mM for nitrate), while the high‐affinity system can scavenge substrate at low concentrations (<0.2 mM for nitrate). The saturation kinetics of ion uptake for the two systems have been interpreted as evidence for at least two transporters with distinct affinities, and this idea was further supported by the identification of distinct genes for the respective systems. For example, a model based on the observed biphasic kinetics of potassium influx proposes that high‐affinity active uptake is mediated by proton/potassium symporters, while low‐affinity passive uptake is mediated by inward‐rectifying potassium channels (Kochian and Lucas, 1988; Maathuis et al., 1997). Indeed, both proton‐coupled potassium transporter genes (HAK/KUP family) and potassium channel genes (AKT/KAT family) have been isolated in higher plants (Anderson et al., 1992; Sentenac et al., 1992; Santa‐Maria et al., 1997). In contrast to potassium uptake, both the high‐ and low‐affinity uptake of negatively charged nitrate are proposed to be mediated by proton‐coupled symporters (Glass and Siddiqi, 1995), and, consistent with this, two families of nitrate transporter genes, NRT1 and NRT2, which share no sequence similarity with each other, have been identified in higher plants (Tsay et al., 1993; Trueman et al., 1996) and have been thought for the past decade to be responsible for low‐ and high‐affinity nitrate uptake, respectively.
In contrast to this model in which high‐ and low‐affinity uptake are mediated by different genes, a knock‐out mutant of the AKT1 Arabidopsis potassium channel, which was originally thought to be responsible for low‐affinity potassium uptake, was also found to be defective in high‐affinity potassium uptake (Hirsch et al., 1998). Likewise, the Arabidopsis potassium transporter, AtKUP1, has also been shown to be a dual‐affinity transporter (Fu and Luan, 1998; Kim et al., 1998); when expressed in yeast, it exhibits a Km of ∼44 μM for the high‐affinity phase of uptake and ∼11 mM for the low‐affinity phase (Fu and Luan, 1998). At almost the same time, it was found that the Arabidopsis low‐affinity nitrate transporter mutant chl1 (nrt1) is also defective in high‐affinity nitrate uptake (Wang et al., 1998; Liu et al., 1999). Using the Xenopus oocyte expression system, it was further demonstrated that CHL1 functions as a dual‐affinity nitrate transporter with a Km of ∼50 μM for the high‐affinity phase of uptake and ∼4 mM for the low‐affinity phase (Liu et al., 1999). These discoveries of dual‐affinity uptake by a single transporter raised the question of whether the kinetic properties of other characterized transporters had been examined over a wide enough range of concentrations to rule out the possibility that they also were dual‐affinity transporters, especially since chl1 had been considered as a low‐affinity nitrate uptake mutant for >20 years (Doddema and Telkamp, 1979; Tsay et al., 1993; Huang et al., 1996; Touraine and Glass, 1997).
Numerous proteins are known to have dual functions. Many use spatially distinct domains for each function. For example, the TRP family uses an N‐terminal domain for its channel activity and a C‐terminal domain for enzyme activity (kinase or ADP‐ribose pyrophosphatase; Cahalan, 2001), the enzyme activity being required for channel activity (Perraud et al., 2001; Runnels et al., 2001). In contrast, in some dual‐function proteins, the mode of action is modulated by post‐translational modifications or ligand binding. For example, depending on which amino acid residue (threonine 34 or threonine 75) is phosphorylated, DARPP‐32, a dopamine‐ and cyclic AMP‐regulated phosphoprotein, functions as either a kinase inhibitor or a phosphatase inhibitor (Bibb et al., 1999). Calmodulin modulation of the P/Q‐type (α1A) calcium channel is another prime example, as binding of calcium to the N‐terminal or C‐terminal lobe of calmodulin results, respectively, in inactivation or activation of the P/Q‐type calcium channel (DeMaria et al., 2001).
Dual‐function proteins may not have evolved for an economic reason, i.e. to minimize the size of the genome, since gene duplication and functional redundancy are quite common in higher organisms, e.g. 60% of the Arabidopsis genome consists of duplicated segments (The Arabidopsis Genome Initiative, 2000), but rather because of some advantage of a regulatory switch and interplay between two distinctive modes of activities. It is therefore important to study the underlying operating mechanisms of dual‐affinity transporters and their physiological significance. In the present study on CHL1, we have elucidated a possible mechanism and identified a novel process of post‐transcriptional regulation for plant nutrient uptake.
High‐ and low‐affinity nitrate uptake activities of CHL1 are differentially regulated by PKA activators or inhibitor
Since phosphorylation is known to modulate ion channel activity (Esguerra et al., 1994; Wang et al., 1996; Beguin et al., 1999; Gong et al., 1999; Schiff et al., 2000), we speculated that it might also play a role in regulating CHL1‐mediated uptake activity. We therefore examined nitrate uptake by CHL1 cRNA‐injected Xenopus oocytes in which cAMP‐dependent protein kinase (PKA) activity was either increased or reduced by cAMP agonists or a PKA inhibitor.
When oocytes were treated with cAMP agonists [a mixture of 8‐bromoadenosine 3′,5′‐cyclic monophosphate (8‐Br‐cAMP), forskolin and 3‐isobutyl‐1‐methylxanthine (IBMX)] to enhance PKA activity, the nitrate uptake activity of CHL1‐injected oocytes in medium containing 150 μM nitrate (high‐affinity concentration) increased to ∼180% of that in the untreated control (Figure 1A), while that measured using 10 mM nitrate (low‐affinity concentration) decreased to ∼60% of untreated control levels (Figure 1B). Conversely, when CHL1‐injected oocytes were treated with the PKA inhibitor H89, the uptake activity measured using 150 μM nitrate decreased by ∼40% (Figure 1C), while that measured using 10 mM nitrate increased by ∼40% compared with control levels (Figure 1D). Similar results were obtained using another two batches of oocytes isolated from two other toads (Table I). These data suggest that phosphorylation activates the high‐affinity nitrate transport activity of CHL1 and inhibits the low‐affinity transport activity.
CHL1 protein can be phosphorylated on threonine residues
To determine whether the CHL1 protein could be phosphorylated, a plasma membrane‐enriched fraction was purified from CHL1‐ or water‐injected oocytes, then part of the sample was subjected to immunoblot analysis using anti‐CHL1 antibodies (Figure 2A), while the rest of the sample was incubated with [γ‐32P]ATP and cAMP‐dependent protein kinase catalytic subunit, immunoprecipitated with anti‐CHL1 antibodies, then electrophoresed and an autoradiogram made from the gel (Figure 2B). The results showed that, compared with the water‐injected control, CHL1‐injected oocytes contained an additional band that could be phosphorylated in vitro (Figure 2B) and had the same mobility as CHL1 detected in immunoblots (Figure 2A).
When this 50 kDa phosphorylated protein was eluted and subjected to phosphoamino acid analysis, the results showed that it was phosphorylated on threonine residues (Figure 2C). The CHL1 protein sequence contains four putative phosphorylation sites (RXXT/S), all of which use threonine as the phospho‐acceptor (Figure 3). In the widely accepted membrane topology model for CHL1 and related transporters (Hagting et al., 1997; Covitz et al., 1998), two of these four putative phosphorylation sites, T28 and T101, face the cytoplasm and can probably be phosphorylated. These two sites were therefore subjected to site‐directed mutagenesis analysis.
Mutation at threonine 101 affects the high‐ and low‐affinity nitrate uptake activities of CHL1
Threonines 28 and 101 were separately mutated to alanine and the mutants tested in nitrate uptake studies. When compared with Xenopus oocytes expressing wild‐type CHL1, oocytes expressing the T28A mutant exhibited normal high‐ and low‐affinity nitrate transport activities (Figure 4A and B; Table I), whereas those expressing the T101A mutant had 9 or 70% of the wild‐type CHL1 activity when measured using 150 μM or 10 mM nitrate, respectively (Figure 4C and D), showing that high‐affinity uptake activity was greatly reduced. Uptake measured using 10 mM nitrate includes contributions from both the low‐ and high‐affinity components; when the contribution of the high‐affinity component (Vmax of the high‐affinity component) was subtracted, the T101A mutant was found to have 138% of the low‐affinity uptake of wild‐type CHL1 (Figure 4D). The Vmax values of the high‐affinity component were calculated assuming that the Km in the high‐affinity phase was 50 μM. Similar results were obtained using another two batches of oocytes isolated from another two toads (Table II). These data suggest that the inability to phosphorylate T101 converts CHL1 into an exclusively low‐affinity nitrate transporter.
To mimic the phosphorylated structure, the threonine residue at 101 was changed to aspartic acid to create the mutant T101D. The uptake activity of T101D‐injected oocytes measured using 150 μM or 10 mM nitrate was 109 or 50% of that in wild‐type injected oocytes, respectively (Figure 4E and F); when the contribution of the high‐affinity component to the activity measured at 10 mM nitrate was taken into account, the low‐affinity uptake of the T101D mutant was close to 0% compared with wild‐type CHL1 (Figure 4F; Table II). Thus, by mimicking a constitutively phosphorylated state, the T101D mutation acted as a pure high‐affinity nitrate transporter.
Oocytes expressing T101A or T101D exhibit a monophasic nitrate uptake pattern
Kinetic studies were performed by measuring the uptake activities of oocytes expressing CHL1, T101A or T101D at nitrate concentrations ranging from 40 μM (high‐affinity range) to 30 mM (low‐affinity range). As shown in Figure 5A and in our previous study (Liu et al., 1999), wild‐type CHL1 exhibited a biphasic pattern, with a Km of 125 ± 35 μM for the high‐affinity phase and 2.2 ± 0.7 mM for the low‐affinity phase. In contrast, nitrate uptake by both the T101A and T101D mutants was monophasic, high‐affinity activity being absent in oocytes expressing the T101A mutant (Figure 5B) and low‐affinity activity being absent in oocytes expressing the T101D mutant (Figure 5C). The Km for T101A‐injected oocytes was ∼5.1 ± 1.1 mM and that for T101D‐injected oocytes was ∼51 ± 19 μM.
Rescue of the high‐ but not the low‐affinity uptake defect in chl1 plants expressing the CHL1 T101A mutant
The studies described above were performed using a Xenopus oocyte expression system. To determine whether the phosphorylation regulatory mechanism of CHL1 was also effective in plants, CHL1 cDNA containing the T101A mutation was fused to the cauliflower mosaic virus 35S promoter and introduced into the chl1 deletion mutant, chl1‐5 (Tsay et al., 1993). This mutant is defective in both high‐ and low‐affinity nitrate uptake, and transgenic chl1‐5 plants expressing wild‐type CHL1 cDNA driven by the 35S promoter can rescue the defect in both activities (Figure 4B and C; Huang et al., 1996; Liu et al., 1999). Consistent with the results obtained using the hetero‐expressing oocyte system, three independent transgenic chl1‐5 plants expressing T101A failed to show recovery of the defect in high‐affinity activity (Figure 6A), but did show recovery of the defect in low‐affinity activity (Figure 6B).
To confirm that the T101A mutant functions as a low‐affinity nitrate transporter, but not as a high‐affinity transporter, a kinetic analysis of nitrate uptake activity was performed on transgenic line 35S‐T101A‐14, nitrate uptake activity being measured at seven different nitrate concentrations ranging from 40 μM to 10 mM to cover both the high‐ and low‐affinity phases of uptake. As shown in Figure 7, in wild‐type plants, nitrate uptake was concentration‐dependent and showed a biphasic pattern. Consistent with previous data (Wang et al., 1998; Liu et al., 1999), the deletion mutant, chl1‐5, showed reduced high‐ and low‐affinity phase uptake activity. Expression of T101A in the chl1‐5 mutant (35S‐T101A‐14) resulted in recovery of the low‐affinity, but not the high‐affinity, uptake activity. The Km values for the low‐affinity phase in the wild‐type and 35S‐T101A‐14 plants were, respectively, 3.7 ± 0.6 and 5.2 ± 1.7 mM, while the corresponding Vmax values were 13.7 ± 0.9 and 10.1 ± 1.5 μmol/g root tissue; in contrast, the high‐affinity phase activity in the 35S‐T101A‐14 plant was as low as that in the chl1‐5 mutant. These results show that, in plants, T101A functions exclusively as a low‐affinity transporter and that the T101 phosphorylation site is required for CHL1 to function as a dual‐affinity transporter.
CHL1 phosphorylation in plants is regulated by the nitrogen concentration
To directly demonstrate that CHL1 protein can be phosphorylated in vivo, metabolic labeling with [32P]orthophosphate was performed, followed by immunoprecipitation. Immunoblot analysis using anti‐CHL1 antibodies raised against the central hydrophobic loop of CHL1 showed that a single protein with a predicted molecular mass of ∼50 kDa on the SDS gel was detected in wild‐type plants, but not in the chl1‐5 mutant, showing that the protein detected was the endogenous CHL1 protein and confirming the specificity of the antiserum (Figure 8A). In Figure 8B, autoradiography of 32P‐labeled membrane proteins immunoprecipitated with anti‐CHL1 antibodies revealed a protein with the same mobility as the CHL1 protein detected in immunoblots, indicating that CHL1 is phosphorylated in plants.
To detect the status of CHL1 phosphorylation at different nitrogen concentrations, anti‐P‐T101 antibodies were raised against phosphorylated synthetic peptide IADTFLGRYLT[p]IAIF, corresponding to residues 91–105 of CHL1, and pre‐cleaned by affinity chromatography on the non‐phosphorylated peptide. As shown in the right panel of Figure 9A, the pre‐cleaned anti‐P‐T101 antibodies also detected an ∼50 kDa protein in wild‐type plants, but not in the chl1‐5 mutant. The specificity of the anti‐P‐T101 antibodies was confirmed by calf alkaline intestinal phosphatase (CIP) treatment of the membrane proteins, which resulted in elimination of the anti‐P‐T101 antibody signal (Figure 9A, right panel, lane 2), but not the anti‐CHL1 antibody signal (left panel, lane 2), confirming that the anti‐P‐T101 antibodies recognize phosphorylated CHL1, but not dephosphorylated CHL1.
To determine whether CHL1 phosphorylation was regulated by the nitrogen status, 10‐day‐old plants were shifted for 2.5 h to low‐nitrogen medium (150 μM NH4NO3) or high‐nitrogen medium (12.5 mM NH4NO3), then membrane proteins were isolated and subjected to immunoblot analysis using anti‐CHL1 antibodies (Figure 9B, top panel) or anti‐P‐T101 antibodies (Figure 9B, bottom panel). As shown, similar amounts of CHL1 were detected in plants grown under these two conditions, but the phosphorylation levels were different, CHL1 from plants grown under low‐nitrogen conditions being highly phosphorylated, while that from plants grown under high‐nitrogen conditions was much less phosphorylated. This result shows that a low nitrogen concentration in the medium stimulates CHL1 phosphorylation in plants.
Regulation of membrane transport by phosphorylation
The transport of ions and substrates across cell membranes by pumps, channels and transporters is critical to the living cell. The activities of certain pumps, channels and transporters are known to be regulated by protein phosphorylation. In the case of pumps, phosphorylation of a threonine residue in plant H+‐ATPase creates a binding site for regulatory 14‐3‐3 protein (Fuglsang et al., 1999; Svennelid et al., 1999), while channel activities are often regulated through such processes as phosphorylation modulation of voltage sensitivity (Esguerra et al., 1994; Kwak et al., 1999), open probability (Wang et al., 1996; Beguin et al., 1999), burst duration (Beguin et al., 1999) and channel complex assembly (Schiff et al., 2000).
In contrast to pumps and channels, except for its effect on the trafficking of transporters to the cell surface, little is known about the detailed mechanism by which phosphorylation affects transporter activity (Blakely and Bauman, 2000). In this study, we provide both in vitro and in vivo evidence that the two action modes of the dual‐affinity nitrate transporter, CHL1, are switched by phosphorylation and dephosphorylation at threonine 101. Namely, CHL1 functions as a high‐affinity nitrate transporter when phosphorylated, and functions as a low‐affinity nitrate transporter when dephosphorylated (Figure 10). Although the only two dual‐affinity transporters so far identified are both plant transporters, the regulatory mechanism discovered here might be quite general and extend beyond plants. Supporting this suggestion is the report of Mehrens et al. (2000), which showed that the IC50 values of the rat organic cation transporter, rOCT1, for transported and non‐transported inhibitors decrease after stimulation of protein kinase C by sn‐1,2‐dioctanoyl glycerol.
Corresponding phosphorylation sites in other CHL1 homologs
Not every nitrate transporter in the NRT1 family exhibits dual‐affinity uptake activity. For example, AtNRT1:2, a CHL1 homolog constitutively expressed in Arabidopsis, is a mono‐affinity (low‐affinity) nitrate transporter (Huang et al., 1999). Consistent with the present finding, AtNRT1:2 lacks the phosphorylation sequence motif (RXXT/S) at the site corresponding to T101 of CHL1. Of the 52 CHL1 homologs in Arabidopsis (The Arabidopsis Genome Initiative, 2000), 36 contain this phosphorylation consensus site, but it remains to be determined whether the uptake activity of any of these is regulated by phosphorylation.
Nitrate transporters in the NRT1 family share sequence similarity with a large family known as proton‐coupled oligopeptide transporters (POT) (Paulsen and Skurray, 1994), found in animals, plants, fungi, and bacteria. The human transporters, PepT1 and PepT2, two of the best characterized POT proteins, are responsible for peptide absorption in the intestine or kidney, respectively (Fei et al., 1994; Liu et al., 1995; Boll et al., 1996; Saito et al., 1996). PepT1 and PepT2 have very similar substrate specificities, recognizing a wide variety of di‐ and tri‐peptides, but differ in their affinities for peptides, PepT1 being a low‐affinity transporter and PepT2 a high‐affinity transporter (Leibach and Ganapathy, 1996). In contrast to the Arabidopsis CHL1 homologs, all of the POT transporters so far identified in animals and bacteria lack the phosphorylation consensus motif (RXXT/S) at the site corresponding to T101 of CHL1 (K.‐H.Liu and Y.‐F.Tsay, unpublished observation). This may indicate that, despite significant homology in the protein sequences, high‐affinity nitrate uptake and high‐affinity peptide uptake by the POT family have evolved by different mechanisms.
Physiological role of the phosphorylation switch mechanism
In higher plants, in addition to CHL1 (AtNRT1), the NRT2 protein family also mediates high‐affinity nitrate uptake (Williams and Miller, 2001), raising the question of why CHL1 should have evolved dual‐uptake activity, given the presence of NRT2. The answer may lie in the regulatory mechanism uncovered in this study. Once seeded, plants cannot move and therefore, in order to survive and sustain growth, must respond rapidly to changes in the environment. In contrast to the turning on and off of the expression of different mono‐affinity transporters, which takes time, the phosphorylation and dephosphorylation of a protein already present in the membrane is a much more efficient way of adjusting high‐ and low‐affinity uptake capacity in response to environmental changes. One particular example is when nutrient levels in the soil are low and diffusion limits nutrient movement to the root surface, resulting in a nutrient‐depleted zone adjacent to the root surface (Mengel and Kirkby, 1987). In this situation, nutrient absorption relies on the high‐affinity uptake system. In order to find a new nutrient source, the roots grow out into fresh soil and, when nutrients are found, the plant can then take advantage of the higher capacity of the low‐affinity uptake system. Having the ability to switch between high‐ and low‐affinity modes simply by phosphorylation/dephosphorylation, CHL1 can rapidly change its affinity as the root grows. In support of this idea are the following observations: (i) CHL1 phosphorylation was high when plants were grown under nitrogen‐limited conditions, but undetectable when plants were exposed to a high nitrogen concentration (Figure 9); (ii) the CHL1 gene is abundantly expressed in the root tip (Huang et al., 1996; Guo et al., 2001); (iii) CHL1 is localized in the plasma membrane (S.‐C.Hou and Y.‐F.Tsay, unpublished data); and (iv) the chl1 mutant exhibits a root growth defect (Guo et al., 2001). Taken together, the dynamic regulatory mechanism described in this study could be a key player in the plant's fight for limited nitrogen.
Materials and methods
Plasmid construction and site‐directed mutagenesis
Point mutations T101A, T101D, and T28A were introduced using the recombinant PCR technique described previously (Higuchi, 1990). Wild‐type CHL1 cDNA was cloned into the pGEMHE vector (Liman et al., 1992) as a template with BamHI, EcoRI and XhoI cloning sites on the 5′ end of the CHL1 cDNA, and HindIII, XbaI, and EcoRI cloning sites on the 3′ end. PCR amplifications were performed using VioTaq DNA polymerase (Viogene, Taiwan). T101A and T101D cDNAs were first prepared by PCR amplification using a T101A primer or T101D primer, respectively, in conjunction with a CHL1C primer, then the amplified DNA fragments were gel purified, digested with KpnI and HindIII, and used to replace the corresponding fragment in wild‐type pGEMHE‐CHL1 to generate the plasmids, pGEMHE‐T101A and pGEMHE‐T101D.
To generate T28A cDNA, two primers, the T28A forward primer and T28A reverse primer, were synthesized. In these, the CHL1 ATCGCT close to the sequence coding for threonine 28 was replaced with ATCGAT to create a ClaI cloning site without changing the amino acid sequence (both CGC and CGA code for arginine). In addition to this newly generated ClaI site, wild‐type CHL1 cDNA has an endogenous ClaI site located 600 bp upstream of its stop codon. Two steps were needed to generate the plasmid pGEMHE‐T28A. In the first, pGEMHE‐CHL1 was digested with BamHI and ClaI, and the 3.6 kb fragment containing the pGEMHE vector and the 0.6 kb 3′ portion of CHL1 cDNA gel were purified. The 109 bp DNA fragment amplified with the CHL1N primer and T28A reverse primer was digested with BamHI and ClaI, and subcloned into the 3.6 kb fragment described above to generate plasmid pGEMHE‐CHL1‐109. In the second step, a 1.1 kb DNA fragment amplified with the T28A forward primer and CHL1C primer was produced by ClaI digestion and cloned into the ClaI site of pGEMHE‐CHL1‐109 to generate plasmid pGEMHE‐T28A.
The sequences of the primers used were: T101A primer: 5′‐GGGCAGGTACCTAGCGATTGC‐3′ (sense, KpnI), T101D primer: 5′‐GGGCAGGTACCTAGACATTGC‐3′ (sense, KpnI), CHL1C primer: 5′‐CCCAAGCTTCATGATCAATGACCCATTGG‐3′ (antisense, HindIII), CHL1N primer: 5′‐CCCGGATCCGATCATTAATCCATCTTCAAG‐3′ (sense, BamHI), T28A reverse primer: 5′‐TTGATCGATCGGCGGG‐3′ (antisense, ClaI) and T28A forward primer: 5′‐CCGATCGATCAAAAGCCG‐3′ (sense, ClaI). The substitution sites for the threonine 101 residue (ACG) and threonine 28 residue (ACC) are underlined; restriction sites are shown in bold.
Xenopus oocyte nitrate uptake assay
Oocytes were isolated and injected with 50 ng of in vitro synthesized CHL1 cRNA, T28A cRNA, T101A cRNA, or T101D cRNA as described previously (Tsay et al., 1993). In PKA activation experiments, oocytes were pre‐incubated for 90 min in ND‐96 medium (Roche and Treistman, 1998), containing 0.5 mM 8‐Br‐cAMP (a cAMP analog), 50 μM forskolin (an activator of adenylate cyclase) and 0.5 mM IBMX (an inhibitor of the cAMP degrading enzyme, phosphodiesterase), in order to increase the cytosolic cAMP concentration (Maurel et al., 1995). In PKA inhibition experiments, oocytes were pre‐incubated for 90 min in ND‐96 medium containing 50 μM H89. In nitrate uptake studies, oocytes were incubated for 90 min in medium containing the indicated nitrate concentration, and the amount of nitrate retained in the oocyte (low‐affinity experiments) or depleted from the solution (high‐affinity experiments) was determined by HPLC (Liu et al., 1999).
In vitro phosphorylation and immunoprecipitation of oocyte membranes
Anti‐CHL1 antibody was raised in rabbits against a purified recombinant protein corresponding to the 94 amino acid central loop of CHL1, which shares least homology with other Arabidopsis CHL1 homologs. Two hundred CHL1‐ or water‐injected oocytes were homogenized in 5 ml of 30 mM Tris–HCl pH 7.6, 100 mM NaCl, 5 mM EDTA, 5 mM EGTA and 1 mM phenylmethylsulfonyl fluoride (PMSF), then membranes were collected as described by Maurel et al. (1995). For immunoblot analysis, membrane protein from 50 oocytes was loaded in each lane. For in vitro phosphorylation, the same amount of protein was incubated for 15 min at 30°C in 100 μl of 500 mM HEPES pH 7.5, 10 mM MgCl2, 0.2% Triton X‐100, 1 mM dithiothreitol and 25 μCi [γ‐32P]ATP (NEN Life Science Products, MA), containing 2.5 U of cAMP‐dependent kinase catalytic subunit (New England Biolabs, MA), then the reaction was stopped by placing the tubes at 0°C and adding a final concentration of 7.2% TCA. The labeled membrane proteins were resuspended in buffer I (50 mM Tris–HCl pH 7.6, 200 mM NaCl, 50 mM NaF, 50 mM EDTA, 5 mM EGTA, 5 mM β‐glycerophosphate, 1 mM PMSF) containing 1.5% SDS, then the solubilized membrane proteins were incubated overnight at 4°C with anti‐CHL1 antiserum in buffer I containing 1% Triton X‐100 and 0.2% SDS. Immune complexes were absorbed onto protein A–Sepharose CL‐4B (Sigma), which was washed as described by Maurel et al. (1995), then the bound proteins were eluted at 100°C for 5 min with 50 μl of SDS sample buffer (12% SDS, 6% mercaptoethanol, 30% glycerol, 0.05% Serva blue G, 150 mM Tris–HCl pH 7) and separated on a 10% SDS–PAGE gel.
Phosphoamino acid analysis
Phosphoamino acid analysis of in vitro phosphorylated CHL1 protein was performed as described by Xu et al. (1996).
Arabidopsis nitrate uptake assay
The T101A CHL1 mutant cDNA was fused to the cauliflower mosaic virus 35S promoter, then cloned into the binary vector, pBIN19, to produce plasmid p35S‐T101A, which was introduced into the CHL1 deletion mutant, chl1‐5, using the in planta vacuum infiltration procedure (Bechtold et al., 1993). Three independent transgenic plants, 35S‐T101A‐8, 35S‐T101A‐10 and 35S‐T101A‐14, were isolated and their inserts confirmed by kanamycin resistance and genomic DNA gel blot analysis; 35S‐T101A‐8 and 35S‐T101A‐14 contained a single copy of the insert, whereas 35S‐T101A‐10 contained two copies. Plants were grown in 50 ml flasks containing 10 ml of liquid medium with 12.5 mM (NH4)2 succinate as the sole nitrogen source at pH 6.5. Nitrate uptake activities of 8‐day‐old plants were determined over a 30 min period in 4 ml of medium containing 250 μM KNO3 for high‐affinity measurements and in 2.5 ml of medium containing 5 mM KNO3 for low‐affinity measurements. For kinetic analysis, nitrate uptake activities of 8‐day‐old plants were determined over a 40 min period in 2–4 ml of medium containing the indicated KNO3 concentration. The kinetic parameters, presented as estimated Km or Vmax ± error, were calculated by fitting the data to the Michaelis–Menten equation using a non‐linear least‐squares method in the program Origin 5.0 (Microcal Software Inc., Northampton, MA).
In vivo phosphorylation study in Arabidopsis
Unless specified, the plants used for immunoblot analysis and in vivo labeling were grown hydroponically for 10 days on mesh in Magenta vessels (Sigma) containing 50 ml of liquid medium with 12.5 mM NH4NO3 as the sole nitrogen source at pH 5.5.
For in vivo labeling experiments, the seedlings were washed three times with phosphate‐free medium containing 12.5 mM NH4NO3, and incubated for 2 h in phosphate‐free medium, then for 3 h in phosphate‐free medium containing 5 mCi [32P]orthophosphate. The roots were homogenized in homogenization buffer [15 mM Tris–HCl pH 7.8, 250 mM sucrose, 1 mM EDTA, 2 mM DTT, 1 mM PMSF, 0.6% (w/v) polyvinylpyrrolidone and 50 mM β‐glycerophosphate] and membranes collected as described by Guo et al. (2001). They were then resuspended in homogenization buffer and diluted 100‐fold in 20 mM Na2HPO4· NaH2PO4 buffer pH 7.0, containing 250 mM NaCl, 5 mM EDTA, 1% Triton X‐100, 1 mM PMSF and 50 mM β‐glycerophosphate. The diluted samples were pre‐cleared by incubation for 15 min with protein A–Sepharose CL‐4B beads and removal of the protein A–Sepharose CL‐4B beads by centrifugation, then anti‐CHL1 antibodies pre‐incubated for 30 min at 4°C with membrane proteins from chl1‐5 to remove non‐specific binding were added and the mixture incubated for 3 h at 4°C. The immune complexes formed were absorbed onto protein A–Sepharose CL‐4B (Sigma), which was then washed four times with wash buffer (20 mM Na2HPO4·NaH2PO4 buffer pH 7.4, 150 mM NaCl, 1% Triton X‐100) and twice with wash buffer without 1% Triton X‐100, as described by Adan et al. (2000) and eluted for 5 min at 100°C with 50 μl of SDS sample buffer. The eluted proteins were then separated on an 8% SDS–PAGE gel.
To determine the phosphorylation status of CHL1, a rabbit antiserum against CHL1 phosphorylated at T101 (anti‐P‐T101 antibodies) was raised by Pro‐e Biotech (Taiwan) using the synthetic peptide, IADTFLGRYLT[p]IAIF (residues 91–105 of T101‐phosphorylated CHL1) as antigen. The antiserum was pre‐cleaned by affinity chromatography using the corresponding non‐phosphorylated CHL1 peptide (IADTFLGRYLTIAIF) coupled to agarose (Pro‐e Biotech). To check the specificity of the antibodies for T101‐phosphorylated CHL1, membrane protein extracts from wild‐type plants and the chl1‐5 mutant were prepared in the absence of 50 mM β‐glycerophosphate, then diluted ∼1:5 in 1× reaction buffer 3 (New England BioLabs), treated for 60 min at 37°C with buffer or CIP (Promega; 20 U of CIP and 40 μg of membrane protein in a volume of 80 μl), then tested for antibody binding on western blots. To test the phosphorylation status of CHL1, seedlings were washed three times and shifted to 150 μM NH4NO3 or 12.5 mM NH4NO3 medium for 2.5 h, then root tissue membrane proteins were extracted (Guo et al., 2001) and 20 μg of the sample separated on SDS–PAGE gels, transferred to PVDF membranes and tested for binding of anti‐CHL1 and anti‐P‐T101 antibodies.
We are grateful to Drs Heven Sze, Tuan‐Hua David Ho and Ming‐Jing Hwang for their comments on this manuscript. This work was supported by the Institute of Molecular Biology, Academia Sinica, Taiwan and by NSC grants (NSC‐90‐2311‐B001‐060 and NSC‐91‐2321‐B001‐007) to Y.‐F.T.
- Copyright © 2003 European Molecular Biology Organization


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By Andrew T. Scull
Cultural Sociology of psychological affliction: An A to Z Guide appears to be like at fresh experiences that recommend an mind-blowing upward thrust in psychological affliction and considers such questions as: Are there really extra mentally in poor health humans now or are there simply extra humans being clinically determined and taken care of? What are the jobs of economics and the pharmacological during this controversy? on the center of what's happening with psychological affliction in the United States and world wide, the editors recommend, is cultural sociology: How differing cultures deal with psychological affliction and, in flip, how psychological overall healthiness sufferers are stricken by the culture.
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Extra info for Cultural Sociology of Mental Illness: An A-to-Z Guide
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W. Schroe der 1942). But in a broader sense, the fascination Introduction xxix with deviance that the Chicago school exhibited, and the preoccupation of many of the sociologists it trained with ethnographic approaches to the study of social life, can be traced in many of the works of postwar American sociology, not least many of the classic studies emerging in the 1950s and 1960s that were devoted to the sociology of mental illness. Post–World War II Ideologies World War II and its aftermath marked a turning point for American social science and for American universities more broadly.
The WPA also issues a code of ethics called the Declaration of Hawaii, which specifies, among other things, that patients must be informed of treatment options and must consent to treatment unless they lack the capacity to reason. 1977: Edna Rawlings and Dianne Carter publish Psychotherapy for Women, a book claiming that social and external causes, rather than internal and personal forces, are behind many women's psychological problems and that society should become more just rather than expecting women to cheerfully adapt to the unjust state of society.
It becomes a standard treatment, replacing barbiturates and bromides, to treat manic depressive (bipolar) disease. 1949: The Portuguese neurologist Egas Moniz receives the Nobel Prize in Physiology or Medicine for his work in developing lobotomy (which he calls "leucotomy") as a tool for treating psychotic patients. " 1950: The German American psychoanalyst and psychologist Erik Erikson publishes his book Childhood and Society, popularizing the term identity crisis and developing Freud's concept of infantile sexuality.


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text: Importing of Personal Pets The import of goods, including pets, incurs Goods and Services Tax (GST). The GST for import of personal pets is calculated based on the Cost, Insurance and Freight (CIF) value of the goods. Those importing personal pets are eligible for GST relief if: The pet owner is changing his or her place of residence to Singapore. The owner must meet the criteria for GST relief for importing his or her used household articles and personal effects, including personal pets. An In-Non Payment (GST Relief) permit must be declared for the import of the pets; or The pet(s) is imported by air and the total CIF value does not exceed S$400. No Customs permit is required. All imports of pets are regulated by the Agri-Food & Veterinary Authority of Singapore (AVA). The importer should abide by AVA's import licence and pet quarantine requirements. Procedures & Requirements Procedures & Requirements for Importing Personal Pets Scenarios Mode of entry of the pets Remarks As manifested cargoes As part of owner's accompanied personal baggage (for example, hand-carried) 1) Import of pets for permanent or long term stay in Singapore By Air Total value of the pets does not exceed S$400 GST is not payable. Customs permit is not required. The cost, insurance and freight charges (if applicable) should be included when computing the total value of the pets. The importer should obtain the necessary import licences from AVA and declare the Customs GST payment permit before importing the pets (before removal of the pets out from Changi Animal and Plant Quarantine Station (CAPQ), AVA or any other Customs checkpoints). An AVA officer will escort the traveller to the Customs duty office for GST payment. Customs Duty Offices: Changi Airfreight Centre (Cargo Agents Building 'D' #03-23): 6543 8048 Tuas Checkpoint: 6863 9092 Singapore Cruise Centre: 6274 2707 Tanah Merah Ferry Terminal: 6786 4575 Marina Bay Cruise Centre Singapore: 6604 7857 Total value of the pets exceeds S$400 GST is payable. Customs In-Payment (GST) permit is required. GST is payable. Approach Customs Duty Office at Changi Airfreight Centre for assessment and payment of GST. By Land or Sea *Import of pets via land is only allowed at Tuas Checkpoint. GST is payable. Customs In-Payment (GST) permit is required. GST is payable. Approach Customs Duty Office for assessment and payment of GST. 2) Temporary import of pets for approved purposes with intention to export out of Singapore (for example, breeding, exhibition, or veterinary treatment) GST is not payable. Customs In-Non Payment (Temporary Consignments) permit is required. Customs Out (Temporary Consignments) permit must be declared to account for the re-export of the same pets. The importer/appointed freight forwarding agent is required to write to us for our assessment. Please provide information on the purpose of import, value of the pets, transport mode, and duration of the temporary import period. The maximum period of temporary import is 6 months. Importing Personal Pets for Transfer of Residence Those who are transferring residence to Singapore (including returning Singaporeans, Permanent Residents and foreigners) may be granted GST relief on the import of their personal pet(s), if they fulfil the following criteria: The person is changing his or her place of residence to Singapore; The person is the owner of the pet(s)and has owned the pet(s) for at least 3 months; The pet(s) is imported within 6 months of the owner's first arrival in Singapore; The owner gives an undertaking not to dispose the pet(s) within 3 months from the date of import; and For returning Singaporeans and Permanent Residents, he or she must have resided in a foreign country for more than 6 months to qualify for this GST relief. Procedures for Personal Pets Imported via Manifested Cargoes The pet owner or his freight forwarding agent must submit an online Declaration of Facts to Singapore Customs for an assessment of eligibility for GST relief. When approval is received, the owner should inform his freight forwarding agent to apply for the Customs In-Non-Payment (GST Relief) permit within 5 days. If the owner is not eligible for GST relief, he is required to apply for the Customs In-Payment (GST) permit for the clearance of his pets(s) at the checkpoint. For more details, please refer to this table on the procedures and requirements. Procedures for Hand-Carried Personal Pets Pets may be hand-carried by land via Tuas Checkpoint and by air via Changi Airport. The pet owner must complete and submit this application form, along with the supporting documents stated below for verification of GST relief eligibility. The submission details are included in the form. For returning Singaporeans & Permanent Residents working overseas: Copy of passport or identity card; and Proof of overseas employment (for example, overseas working visa/overseas employment pass/letter of appointment) For returning Singaporeans & Permanent Residents studying overseas: Copy of passport or identity card; and Proof of overseas study (for example, overseas student visa/overseas student pass) For foreign citizens: Copy of passport; and Proof of transfer of residence to Singapore (for example, employment pass/dependent pass/student pass); and / or Other supporting documents (for example, letter of employment, or letter of undertaking from local company or in-principle approval from the Ministry of Manpower, if employment pass status is unavailable) If the owner is eligible for GST relief, please provide the approved application form to the checking officer for clearance on the day the pet(s) is removed from AVA's quarantine station. If the owner is not eligible for the GST relief, an AVA officer will escort the owner to the Customs Duty Office for assessment and payment of GST. Payment can be made by cash, NETS, CashCard or Visa/MasterCard. You may also refer here for a step-by-step guide to pet immigration in Singapore.


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Circle City Blues has received several great reviews in the last few weeks. If you'd like to read them, I recommend visiting Smashwords or Amazon.com. As a sort of mini-celebration of this happy news, I'd like to share a 500-word excerpt so that you can get a taste of this funny novel. To set the scene, Mac -- our hero and narrator -- is a truck driver whose wife has recently abandoned him:
New Orleans is one of my least favorite cities. Even though it has recovered considerably since the hurricane that nearly destroyed it, it's still a dirty place that seems perpetually on the verge of collapse, whether from outside forces or interior rot. In addition, it's the murder capital of the country. Intellectually, I know that most murder victims are killed by people whom they know personally. Still, there are random acts of violence and I can't help thinking that there are more of those incidents in New Orleans than anywhere else, simply because their overall murder rate is so high. Therefore, the only thing that made deadheading into Mardi Gras worthwhile was the ultimate destination of Amarillo.
Even though there were plenty of dockworkers at the warehouse, I jumped out of the cab and helped them. I was antsy and anxious to be on my way as soon as possible. Besides, I could use the exercise – my jeans felt a little tight that morning as I pulled them on. I'd been to that warehouse many times in the last couple years, and the manager, a muscular woman called Bess, greeted me by name. "Where's the little woman?" she asked after peeking around the outside of the rig. "Watching TV?"
"No, I'm afraid not. She decided she liked Indianapolis a whole lot better than me."
Bess frowned and she clapped me on the shoulder. "Sorry to hear that, man. Lonely on the road alone."
"Don't worry too much about me, Bess. I have a new partner – he's just on his honeymoon. Next time I come through, you'll probably meet him."
"Good to hear. I've got the bill of lading ready in the office. Come see me when you're loaded up."
"You got it." I helped the guys finish up and pulled the door closed, pad-locking it. Then I trotted over to the square glass cage Bess occupied and stuck my head around the corner of her door. "Knock knock."
"Come on in. Have a seat. Would you like a bottle of water?"
"Wow. What's with the cordiality?" I smiled lopsidedly at her.
"I'm trying to improve your opinion of my native city," she said with a chuckle.
"I don't know if that's possible," I answered honestly.
She took a deep breath, tensing and relaxing her hands. Bess was the shipping and receiving manager for the largest import company in New Orleans; I knew that had to be a stressful job. She'd been using Flo as her primary freight broker for fifteen years, long before I was out of college. "Mac," she said; she sighed. "NOW is a huge company, did you know that?"
NOW was the acronym for New Orleans Worldwide. "Yes, I know."
She cracked her knuckles in a distracted manner and sighed again.
"Spit it out, will you? I need to get on the road."
She laughed and relaxed a bit. "I'm sorry. I can honestly say I've never wanted anything as badly as I want this, and I have a terrible suspicion I'm not going to get it."