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Submit Artwork for Art in Found Spaces
Use the form below to submit artwork
What you need before you start:
- Your artist statement and your bio, in one file saved as a PDF — the database will not accept Word Processor or text files. If you do not have a utility on your computer for making PDFs out of your documents, please use this online MS-Word-to-PDF converter to do the job for you!
- NOTE: for incognito only a very brief description is required
- TWO images of your artwork for judging/exhibition catalogue purposes, not larger than 2 Megabytes in size and/or less than 2000 pixels per side at 300 dpi (For help on re-sizing, see our instructions on how to use Ribbet.com for image editing)
The submission process:
You will receive confirmation that your submission was received successfully immediately via email if you have submitted online. Note that any other type of submission requires manual processing which may take a few days.
If you have trouble uploading your application, please check to be sure that all required fields are completed (those marked * are required and may not be left blank) and that your photos are sized appropriately. Should you be unable to upload your images after following the resizing instructions, please contact us for assistance.
The Exhibition Process:
- For our incognito show, the maximum number of pieces that an artist can show is three (3). In the majority of cases, all artwork submitted is accepted unless it contains content that would be considered inappropriate for show in a public place
- Please note that the price of all the works for Incognito is $100 and there is no need for a full artist biography
- For the regular art in found spaces, submit samples and we will determine if we will be able to show your group's artwork


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The past few days have been full of emotion for me. As you (may) know, I recently became a Doula. I have been teaching Hypnobirthing for almost 2 years, so I decided to expand my services. I really want to be with my Moms in their labors as both support and advocate. My first client (we'll call her Ann) was due on March 22. Most first time moms go an average of 41.5 weeks, so I figured I was safe that she wouldn't need me during Sectionals.
Oh Universe. How funny you are!
She texted me on Thursday night at midnight saying she was having contractions. The first thing I thought was, "Oh no! I don't have my doula bag all packed!" So I got up and got some clothes and food ready in case I needed to jet out the door in the middle of the night. I tossed and turned most of the night waking up at 7am to text her to see what was up.
Ann sounded like she was still in Early labor (which could go on for a while) so I got up, got the kids ready and sent them off to Preschool. This was my big rest day before Sectionals, and I didn't really think I would just be laying around all day anyway. I had a private class to teach in the morning, last minute running around to do, etc. So "rest" was a bit iffy.
At 11am after my private, I called Ann's sister to check in. She said Ann was resting but still having irregular contractions. We decided it was time to head over to the Birthing Center since it was Friday- Spring Break and SXSW traffic was already getting bad!
As I was gathering my things, I began to feel the enormity of this situation I was about to enter. I was about to be a part of a huge, life-changing experience in this woman's life. I could go on and on about the experience of childbirth. You emerge as a different person after having a baby. It is a rite of passage that women before you have been going through since the dawn of time.
Here is a quote that sums it up quite nicely:
"The moment a child is born, a mother is born. She never existed before. The woman existed, but the mother. Never. A mother is something absolutely new." ---Rajneesh
I have been through two births myself. But watching another woman go through this process was something completely different. It was amazing. It was heart-breaking. It was beautiful. It was transformative.
When I arrived, Ann was sitting on a chair moaning. Not in a painful way, but in a good way. Birth is a primitive act and the key to a successful natural birth is to surrender to that primitive part of yourself. I'm not going to re-enact the entire thing, but it was a cooperative effort on the part of all three of us. Her husband would support her for a while then I would take over. Sometimes we would both lay hands on her to help her relax. It was hard for me to know when to back off. I definitely didn't want to be in the way of their intimacy. Birth is very intimate, and you can see a stronger bond form between husband and wife in the process of labor.
As I looked at my watch, I realized I was missing the packet pickup for Sectionals. It was 6pm. But it really didn't matter. More on that later. I was content to be where I was and I was focused on the present.
Ann began to push around 6:45. The next 30 minutes were by far the most intense and miraculous part of the whole birth. I am not a shy person so I watched in amazement as she pushed that baby out!! I got to see the first bits of head as it emerged with each contraction then went back in a bit. 2 steps forward, 1 step back. The baby's head slowly began to crown- there was so much hair on his little head! Ann was doing great. This is by far the most intense part of labor. The head emerged- we could see his little ear, then his cheek. Finally the rest of his body slipped out and holy shit! there was a baby!
The midwife placed him on Ann's chest covered by a towel. They didn't know the sex of the baby, and I was so worried that I was going to spoil it by accident! I didn't even see the "parts" since the towel was over it. But of course I was like "did you look?!" "what is it?!"
It was a boy! A beautiful little boy.
I am so grateful and honored that I got to be a part of this birth. I still can't even put into words what it was like to be there, to be part of it, and to witness this amazing feat of strength and beauty that was so transformative for this woman. I look forward to the next birth with bated breath. I hope I can continue to serve women in this way. To be a calming presence. To be a loving guide, and to be of humble assistance to their Partner as well.
And that was just on Friday!!
Next up, Sectionals.... a whole other kind of intensity!

subset: commoncrawl


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That is "feel" not actually "be." It is a key distinction. And it is that "need to feel" that thing--in this case, safe--which serves as the foundation of so many illusions.
But what exactly is "fear" about? Good question. Bill the Butcher has an answer:
It's a pretty powerful weapon. Maybe the strongest in any man's (or nation's) arsenal. Used wisely, it is a supremely effective agent of control. But to be used wisely, it must be used in concert with something else. In this case, something else is the "need to feel safe."
Let's take, for example, the "war on drugs."
Some people think all drugs are bad, mmmkay? And that's why, I s'pose, we have a war on drugs.
Plenty of people get rich because of drugs. Some of those people are drug dealers. Some are suppliers. Some are attorneys. Some of those attorneys become elected officials. It is a mutually benefical bifurcation. For the most part. Right up to the point where someone gets arrested and sent to prison for a kajillion years. At that point, there's only one side cakin' up.
And how ridiculous is that? In this straight-from-the-gut editorial, one man speaks on it very truthfully:
The truth is, big drug busts do almost nothing to stem the flow of drugs or change the complexion of the culture, save for making a handful of rather uninformed citizens and angry parents feel better for about 10 minutes, and causing the street price of your narcotic du jour to jump 20 percent for a week. Which, I suppose, is a big part of the reason it happens at all, to give the appearance of justice and enforcement and overall safety, to prevent everyone from freaking out and whining to the mayor.The "appearance of justice and enforcement." Rather, the appearance of safety.
He's right. We don't need to watch The Wire to recognize the cyclicality of the capital-G Game. It goes on. It will go on. You can't stop it completely because you can't fully eliminate human desire. Not collectively anyway.
Which leaves you--and by "you," I mean a so-called "civilized society"--to choose between A) perpetuating an illusion and B) governing yourself pragmatically.
Obviously, no balkanized group of people is smart enough to agree on a practical solution. So, here we are. And there we go. Exciting ourselves about the evil scourge of all drugs--which is another way of saying that we're ashamed of our own humanity--and cheering on the occasional "victory" that the rich guys who allegedly do the least harm can take credit for.
And, in the end, some of us feel "safe."
While the rest of us are in "the bottle."
Say, brother, can you spare a dollar nine?


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If you've been reading the New York Times, you may have noticed that Apple is considering coming out with a smaller version of the iPad. This one would be 7.85 inches as opposed to the current ten. As the tablet market grows, Apple is seeing that there may be room for other options. The Amazon Kindle Fire and Barnes and Noble's Nook are both seven inches, and there are more about to join the market. Microsoft will be producing the Surface tablet and Google will soon begin shipping the Nexus 7.
Keep in mind though, that seven inches is only slightly larger than many smartphones. It sounds like Apple is trying to catch those customers who find the iPhone screen too small for comfort but may not want to carry around an iPad. Although Steve Jobs had publicly rejected the idea of a seven inch tablet, Apple has been known to try to corner device markets by offering numerous sizes. The best example was their line of MP3 players, which ranged from the large iPod Touch to the iPod shuffle that was too small to have even the most basic screen.
A 7 inch tablet is still too small for a pocket, but may fit comfortably in a handbag. It's also about the same size as a paperback book and very comfortable for reading. The question is, would you buy this instead of or in addition to a tablet? When you buy a device like this, how much is size a factor? Personally, I consider operating system to be the primary issue. However, I am rarely on-the-go and can't resist the temptation to carry a large backpack everywhere I go, so I might not be the best judge. But I'd love to hear from Consumer-Rankings.com readers. Is size a factor in choosing a different product over an Apple device?


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Girls: Should you make the move?
Date posted: July 1, 2009
i just wanted to ask u what to do with my problem….
i'm 23 years old now ,i have a colleague which i know him for 6 years now, i used to like the guy all this time but it was just "like" i know him yes but we have no contact ,coz i'm a little bit shy i dont deal with any guy in my uni or so,but now my problem is that i think i'm in love with him now i can see no one but him ,i'm really attracted to him and i donno what to do
i donno if he likes me or he's in love with another
i wanna talk to him and tell him but i'm not sure of the results ,i may lose his respect if i did so and even lose the "hi" he says to me every now and then.
my friends says i'm too good for him but i don't think so ,i think i dont even deserve him but my problem is that i donno what to do? should i make a move and take the risk? or just be happy with my life now
Ok … let me ask you this …. why does he not take initiative? I mean if
he knows you, sees you, says hi to you, and is supposedly interested .. why
doesn't he say anything?
actually i don't know if he's interested maybe he just think of me as a friend or something i donno
i mean maybe he's not in love with me
i donno so u think i should do nothing and wait for him to make a move? or should i forget about him and go on my life?


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I have been using PTCL Broadband for 6 years. But now, because of their disappointing service, I need to change to another ISP.
After thoroughly researching, I am down to 2 options: Qubee and Connect.
I live at Numaish (near Soldier Bazaar). Are there any Connect/Qubee users here who can tell me about their service.
I want 4 Mbps connection. I am dependant on torrents (in PTCL i get 400+kbps download speed with 4 Mbps connection) and I stream youtube ALOT! Also i am not much into online gaming.
Thanks in advance!
I have used torrents on Connect but speed is not good while torrents.
[quote="masters, post:2, topic:21625"]
Which connection do you have and what speed do you get in torrents?
[quote="Syed_Muhammad_Maaz, post:3, topic:21625"]
PTCL 8 Mb DSL. on average gets 850-900 KB/s.
Zong Mifi on average gets 1.5-2 MB/s
For wireless networks, it depends on area to area. Lower the users in your area the higher the speed you will attain. I am using Qubee from past 4 years and pretty much satisfied. Reason being that there are very few users of Qubee in my area and most importantly my location from base station is very near and open. No houses or buildings are around so signal strength is like getting 100% direct signals.
Connect is also a good option but never the less I don't know if they provide uninterrupted services in your area or not. (In case of load shedding or power failure)
Zong is also a good option but they have volume issues you only get 50 GB volume in 2000 RS.
Sent from my XT1080 using Tapatalk
Our landlord has connect communication internet and its great, but their download speed is not very good. Whenever I download any file it gets disconnect in the middle, I have to try 2-3 times But for browsing it is fast.
I have never tried Qubee.


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Penname: bekquai [Contact] Real name: Bekquai
Member Since: 04/30/04
Website: http://www.livejournal.com/users/realistik/
I'm just a random college student in dire need of smokes. I enjoy yaoi greatly, and my OTPs for Saiyuki are Sanzo/Goku and Hakkai/Gojyo, though I definitely enjoy the S/G-ness more.
Progress report:
In the process of writing a couple different fics, but none are ready to post as of yet. ^^;; I'm a-gonna get going on them pretty quickly, but real life is kinda hectic at the moment. I'll post it... eventually. ^^;; In the meantime, please read and review what I've got up so far. Thanks, yo!
Beta-reader: No
Membership status: Member
None None None None


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Writing a section on hiking with kids for my Let Them Be Eaten By Bears book (coming out with Perigee/Penguin Group next spring). So happy to be writing this:
"Pace is also something to consider. If you've ever watched a little kid playing by himself in a backyard, you know that there are no straight lines. The best words to describe the motion are "scattered" or "wandering." Now, if you have a really good trail system, and the young person is highly motivated to get to the end of the hike (say, you've told him that he can swim as soon as he gets there), he might travel in somewhat of a straight line. Somewhat. The young person will still generally zigzag back and forth across the trail, stop to look at the bright orange lichen on the tree, fling mud at his brother, spit off of the bridge, stare at a buzzard, go into the forest after a hiking stick, chase a lizard, stomp in a puddle, try to find a woodpecker, balance on a log, pick flowers, pick his nose, and stare off.
With young people, you will travel slowly."


text: Beware of 16GB and 32GB Transcend JetFlash V30 flash drives being sold on eBay! A victim of a 32GB counterfeit JetFlash V30 flash drive on eBay sold by seller hellopetpet, reported in to SOSFakeflash. eBay and Paypal gave him the run around. He dealt with it. Read the rest of this entry »


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Mercer Bay Loop Walk is located in Karekare regional park
A favourite, all-season track through low coastal scrub, the Mercer Bay Loop Walk is a great spot to clear the cobwebs. Stop and watch the sunset or introduce visiting friends and relations to the wild west coast of Auckland. The track makes its way along the top of the highest sea cliffs in the Auckland region and provides great views north towards Muriwai and south to Whatipu.
There is a great spot for a picnic on calm days about half way around the loop. If you are walking the whole loop, the climb back to the carpark is constant and can be slippery if wet. Elderly or walkers with moderate fitness levels should allow extra time.
Note: This track has a number of natural features that may pose increased risk to visitors. Please stay away from cliff edges and keep to the track. Do not climb barriers and take notice of signs and closure notices.
From the parking area by the site of the World War II radar station (now a navigation beacon) at the end of Log Race Road, the gravel walk passes through regenerating bush and leads around Takatu Head with views over Piha and the Tasman Sea. Walk loops inland then climbs steadily up a small valley to a gate on the southern side of Log Race Road parking area. A short branch tracks joins Te Ahua Point with Ahu Ahu Track.
Short walks are suitable for most people including families with young children and some mountain buggies; however, please check the individual track descriptions for detail. All short walks are part of the path classification of tracks and are suitable for people of most ages, mobility and fitness.
Theft from vehicles is not common but unfortunately can happen.
You should take the same reasonable precautions you take when parking at other locations and remember to lock your vehicle and take your valuables with you.
Help make Auckland the best place inthe world to work, play, study and invest.


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*Orthomolecular Medicine News Service, August 10, 2012*
Let's All Do Exactly What Consumer Reports Tells Us To
*Commentary by Andrew W. Saul, Editor*
(OMNS Aug 10, 2012) Chicken Little was in the library one day when a copy
of *Consumer Reports* for September 2012 fell on her head. She read their
"10 Hidden Dangers of Vitamins" and it scared her so much she trembled all
over. Why? Because Chicken Little had read other negative articles on
nutrition supplements. . . and now this! She became so afraid that half her
feathers fell right out.
"Help! Help!" she said. "Vitamins are killing us! I have to go
tell the President!"So she ran in great fright to tell the President. Along
the way she met Henny Penny
. "Where are you going, Chicken Little?" said Henny Penny.
"Oh, help! Vitamins are killing us!" said Chicken Little.
"How do you know?" said Henny Penny.
"I read it with my own eyes," said Chicken Little, "It was in *Consumer
Reports*! And part of it fell on my head!"
"Vitamins are killing us? This is terrible, just terrible!" said Henny
Penny. "We'd better hurry up."
So they both ran away as fast as they could. Soon they met Ducky Lucky.
"Where are you going, Chicken Little and Henny Penny?"
"Vitamins are killing us! Vitamins are killing us!" said Chicken Little and
Henny Penny. "We're going to tell the President!"
"How do you know vitamins are killing people?" said Ducky Lucky.
"I read it with my own eyes in *Consumer Reports*," said Chicken Little,
"And heard it with my own ears on the news, saw it on the internet, and the
magazine fell on my head."
"Oh dear, oh dear!" Ducky Lucky. "We'd better run!"
So they all ran down the road as fast as they could. Soon they met Goosey
Loosey walking down the roadside.
"Hello there. Where are you all going in such a hurry?"
"We're running for our lives!" said Chicken Little.
"Vitamins are killing us!" said Henny Penny.
"And we're running to tell the President!" said Ducky Lucky.
How do you know that vitamins are killing people?" said Goosey Loosey.
"I read it in *Consumer Reports*," said Chicken Little, "And part of it
fell on my head!"
"Goodness!" said Goosey Loosey. "Then I'd better run with you."
And they all ran in great fright across a field. Before long they met
Turkey Lurkey strutting back and forth.
"Hello there, Chicken Little, Henny Penny, Ducky Lucky, and Goosey Loosey.
Where are you all going in such a hurry?"
"Help! Help!" said Chicken Little. "Vitamins are killing us!"
"We're running for our lives!" said Henny Penny.
"And by the way, the sky is falling!" added Ducky Lucky.
"So we're running to tell the President!" said Goosey Loosey.
"How do you know that vitamins are killing us?" asked Turkey Lurkey.
"*CONSUMER REPORTS* SAID SO!" they all answered together.
"Oh dear!" said Turkey Lurkey. "I always suspected those damned supplements
were dangerous! I'd better run with you."
So they ran with all their might, until they met Pharma Fred the Fox.
"Well, well, well," said Pharma Fred. "Where are all of you rushing off to
on such a fine day?"
"Help! Help!" cried Chicken Little, Henny Penny, Ducky Lucky, Goosey
Loosey, and Turkey Lurkey all together. "It's not a fine day at all.
Vitamins are killing us, the sky is falling, and we're running to tell the
"But of course vitamins are killing you," cooed Pharma Fred the Fox. "Now
all of you calm down. Here, have a Prozac."
Chicken Little, Henny Penny, Ducky Lucky, Goosey Loosey, and Turkey Lurkey
each swallowed a few Prozacs, and some Valium for good measure.
"Well then," said Pharma Fred the Fox. "How did you learn of how dangerous
those vitamin supplements really are?"
"I read it with my own eyes in *Consumer Reports*," said Chicken Little.
"I see," said Pharma Fred the Fox. "Well then, come with me. I'll take you
straight to the President. Yes, indeed I will!"
So Pharma Fred the Fox led Chicken Little, Henny Penny, Ducky Lucky, Goosey
Loosey, and Turkey Lurkey away. He led them across a field and through the
woods, but took them nowhere at all near 1600 Pennsylvania Avenue. Pharma
Fred led them straight to his den, and none of them ever saw the President.
However, Foxy Pharma Fred had a most delicious dinner.
The moral of the story? Vitamin therapy pioneer Abram Hoffer, M.D., once
told me that the worst fate vitamin bashers could experience would be to
never, ever have use of the nutrition supplements they so often criticize.
As for the rest of us, the sky is not falling. There is not even one death
from vitamins over the last 28 years. [1] We are a nation of sick,
undernourished, and overmedicated people. Half the population is on
prescription drugs. [2] Pharmaceuticals, taken as directed, kill over
100,000 per year just in the US. [3] Antibiotics cause 700,000 emergency
room visits per year, just in the US. [3] Modern drug-and-cut medicine is
at least the third leading cause of death in the USA. Some estimates place
medicine as the number one cause of death. [3] Over 1.5 million Americans
are injured every year by drug errors in hospitals, doctors' offices, and
nursing homes. [3] If in a hospital, a patient can expect at least one
medication error every single day. [3]
Vitamins are not the problem; they are the solution. Taking supplemental
B-vitamins, vitamin E and vitamin D each *individually* reduces lung cancer
by 50%. [4-7] Vitamin C stops cancer more effectively than chemotherapy.
[8-10] HIV-positive persons taking vitamins are 50% less likely to develop
AIDS. [11] And if they do, AIDS patients taking supplements have a 27%
lower death rate. [3]
*Consumer Reports* should stick to testing laundry appliances. That's what
they are good at.
*Alternate Health Weekly Digest*
*From Natural News:*
USDA conspires with factory chicken producers to reduce inspections, speed
factory output
http://www.naturalnews.com/ 036772_chicken_factories_
Bananas aren't just for monkeys - here are 25 reasons to eat more bananas!
http://www.naturalnews.com/ 036771_bananas_digestion_
Is there fluoride in your grapes?
http://www.naturalnews.com/ 036753_fluoride_pesticides_
Thimerosal (Mercury) in vaccines continues to be linked to neurological
http://www.naturalnews.com/ 036748_thimerosal_vaccines_
Are onions healthier than many superfruits? Learn the fascinating facts:
http://www.naturalnews.com/ 036751_onions_superfood_
Mammography causes cancer! Here's a safer alternative that uses no
radiation: Thermography
http://www.naturalnews.com/ 036745_cancer_risk_
Google complies with FDA demands to secretly disable Adwords accounts of
nutritional detox companies
http://www.naturalnews.com/ 036726_Google_Adwords_FDA_
Chemotherapy backfires - causes healthy cells to feed growth of cancer
http://www.naturalnews.com/ 036725_chemotherapy_cancer_
*From Dr Mercola:*
Mammograms Have 'Limited or No Effect' on Breast Cancer Deaths: Study
http://articles.mercola.com/ sites/articles/archive/2012/
08/06/mammogram-on-breast- cancer-mortality-rates.aspx?e_
Food And Water, Nope. Vaccines, Yep.
http://articles.mercola.com/ sites/articles/archive/2012/
08/06/healthy-foods-not- vaccines.aspx?e_cid=20120806_
Decade-Long Feeding Study Reveals Significant Health Hazards of Genetically
Engineered Foods
http://articles.mercola.com/ sites/articles/archive/2012/
08/07/genetically-engineered- foods-hazards.aspx?e_cid=
Harvard Study Finds Fluoride Lowers IQ
http://articles.mercola.com/ sites/articles/archive/2012/
08/07/effects-of-fluoride-to- children.aspx?e_cid=20120807_
This New Drug Appears to Cause Cancer Cells to Self-Destruct
http://articles.mercola.com/ sites/articles/archive/2012/
08/04/dca-and-turmeric-on- cancer.aspx?e_cid=20120804_
( Not new, I reported on DCA over a year ago in this newslletter, and on my
website - www.drbate.com/content/cancer.
If You See This Google Warning, Act Fast: Big Brother is Watching
http://articles.mercola.com/ sites/articles/archive/2012/
Mounting Evidence Shows Many Vaccines are Ineffective and Contribute to
Rise of Outbreaks Caused by Mutated Viruses
http://articles.mercola.com/ sites/articles/archive/2012/
*From Alliance for Natural Health:*
ANH-USA and Trade Groups Meet with FDA on New Supplement Guidance
Another Hatchet Job from Consumer Reports
http://www.anh-usa.org/ another-hatchet-job-from-
The Legislative Battlefield
From Easy Health Options:
Probiotics: Don't Leave Home Without Them
http://easyhealthoptions.com/ general-health/probiotics-
The Truth About Clot-Dissolving Drugs
http://easyhealthoptions.com/ alternative-medicine/the-
*From Dr Bate:*
*The 50% discount ends tonight at midnight. Act now, or wait many months
until Another sale comes along. Remember that all NT CD's are guaranteed.
Please feel free to pass on any articles or information to friends or
family that is in this newsletter. I only hope that some may help someone
Because of problems on weekends, this newsletter will now be published
Sunday Evening or on Monday.
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We offer flat rate shipping fees for all orders. US Orders shipped via Express (2-3 business days) will cost $20, and US orders shipped via Overnight (1-2 business days) will cost $30. Orders received weekdays before 12pm ET generally ship the same day; orders received after 12pm ET, or on weekends / holidays, generally ship the next business day.
We offer a flat rate shipping fee of $20 on all orders shipped to Alaska, Puerto Rico and Hawaii.
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Saturday morning I met Courtney for a run. The weather was perfecto and we ran for almost an hour so we were both happy with that. I did, however have to use Kroger's facilities along the way in order to be able to make it that long though. =)
Ther rest of Saturday morning was spent cleaning - after Kennedy and I were both sick for a bit this past week, I wanted to just dunk my whole house in Clorox. Too bad it's not that easy. I had to settle for cleaning it top to bottom - whew!
Billie came over around 1 and we worked on some shower prep for Abby. Clearly I can't give it away but let me just say...oh my cuteness. =) We worked until about 5 pm and Tim said to me..."Linds, you have about an hour". Oh.....ok I thought.
You see....I've been slightly unhappy about the fact that Tim and I've not had a date night in FOREVER and I've been communicating that to him. In the latter part of this past week he said he was working on getting something together but there'd been no further mention of it so I just figured it wasn't going to happen this weekend. That is until he gave me the "you have one hour to get ready" thing. I was like "um, where are we going, what do I need to wear, whose coming to watch Kennedy, how late will we be out, etc. etc. etc." OCD Mommy coming through! Thankfully Sara Beth babysat (thanks SB!) so I was totally at ease for the evening ahead.
Much to my surprise and delight it wasn't your ho hum typical date to Applebee's and a movie. There was some thought put into this process. We went to Halls on the river, which is a restaraunt I've been wanting to try for at least a year so I was thrilled that we were finally going to go. Giddy almost truthfully. I just love trying new places. The food was really good and the atmosphere even better. I did feel like I myself had been deep fried by the time I left, but oh well. It's not an everyday thing you know? I did feel the need to detox today but it was worth it! After dinner we went to Oktoberfest at Christ the King to see The Peter Rowan Bluegrass band play. They were really good. Tim and I learned early on in our relationship that we both had a liking for bluegrass music but we've never gone to see a concert aside from Nickel Creek so it was just a well thought out date night. We had a really good time!
We sat out on the patio and the weather was just right.
These, my friends are deep fried oreos. We were so full from our meal we contemplated not getting them but the waiter said they were so amazing that we thought we should go ahead and try em'. Yeah...amazing is an understatement.
We clearly weren't prepared for the amount of food that would be served. You know when Tim and I are taking home three to-go containers it was a TON of food. Ha!
Thursday of this week marked my 16th week in the pregnancy so my big helper and I took some belly pictures after church today. The bump is definitely growing.
6 Weeks 12 Weeks
16 Weeks 16 Weeks with Kennedy


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urn:lsid:ibm.com:blogs:entries-b72953ff-245c-4373-909f-7ee0088a541a IMS Exchange - Tags - jcl Welcome to the IBM IMS Exchange , a place for IMS developers and administrators to share examples and best practices. Click here for a list of IMS examples. To submit an example to the Exchange: Create an example that follows the IMS Excha 0 30 1 2017-01-10T15:39:21-05:00 IBM Connections - Blogs urn:lsid:ibm.com:blogs:entry-3672f811-c6bf-4077-a776-1396161c662a A new example is available for IMS Enterprise Suite 2.1 SOAP Gateway: administrative script samples DavidHanson 270002MTAW active false 8a337b1c-3c0c-48a5-b7cc-7f805884dbb9 Comment Entries application/atom+xml;type=entry Likes true 2012-02-01T12:59:17-05:00 2012-02-01T18:52:44-05:00
This sample demonstrates how to perform basic administrative tasks for a SOAP Gateway server on z/OS. Follow the instructions in the sample comments to modify the files for your environment and administrative tasks.

The zip file contains two samples: a JCL job that executes one or more SOAP Gateway management utility (iogmgmt) commands and then copies the utility output to the job log, and a sample shell script that prunes the SOAP Gateway log files according to parameters that you specify. The shell script as provided executes once, but includes basic instructions for the Unix cron job scheduling facility. The JCL can be used to execute iogmgmt commands without starting an OMVS session. These scripts are also provided with the SOAP Gateway installation package.

You can also modify the sample JCL to directly execute the sample shell script if you include the shell script as a CMD argument in the JCL.

Although these samples are intended for IMS Enterprise Suite 2.1 SOAP Gateway, they demonstrate concepts that are generally applicable to products that run under the z/OS Unix System Services environment.

 https://www.ibm.com/developerworks/mydeveloperworks/files/app/person/270002MTAW/file/bfc24e0d-afe3-4de0-8e0e-70b8927a766a

 This sample demonstrates how to perform basic administrative tasks for a SOAP Gateway server on z/OS. Follow the instructions in the sample comments to modify the files for your environment and administrative tasks. The zip file contains two samples: a JCL... 0 0 4454 urn:lsid:ibm.com:blogs:entries-b72953ff-245c-4373-909f-7ee0088a541a IMS Exchange 2017-01-10T15:39:21-05:00


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Providers slow to enact risk-based reimbursement model
The benefits are clear, but physician backing and insurer partnerships take time to secure.
Baptist Health South Florida
The change from a fee-for-service healthcare model to a value-based approach might be inevitable, but that doesn't mean it's happening quickly. In fact, the majority of providers facing a host of preparations for enacting value-based systems remain tied to the old model.
"It's going to be a difficult transition for everyone as we go from pure fee-for-service to a mixture of fee-for-service and value-based contracting," said Jim Riopelle, medical director of care management for St. Louis-based Lumeris, a provider of population health management services and technology to hospitals and health systems. "But I think we're ultimately going to go to almost exclusively value-based contracting."
The new value, or risk-based model, is said to emphasize improved patient care delivered at lower cost since reimbursements are not tied directly to the services patients receive.
This is a significant challenge that all organizations in healthcare are going to have to undertake.
There are a number of hurdles to consider when adopting a risk-based model, according to Ralph Lawson, executive vice president and CFO of Miami-based Baptist Health South Florida. First, you must get buy-in from doctors.
"None of this works without physicians," Lawson said. "The physicians also need to share in the savings, and they need to share in the savings first before the hospital or other providers. If the doctors aren't incentivized and on the same page as the insurer and the other providers, it just doesn't happen."
That's not easy, either. Many physicians who are happy with the way they've been paid in the past may not be interested in change, Lawson said.
[See also: 5 keys to value-based purchasing.]
On the other hand, others just want to do what best serves the patient.
"Most people who go into medicine do so because they love people and they want to do good things in their career," he said. "Most doctors can be convinced to embrace population management. It's a matter of education."
"In a risk environment, the healthier you can make the population, the more profitable you are," Riopelle said. "We've got to compensate providers for providing quality measures. There's very little direct reward for quality in fee-for-service medicine, whereas in a population health environment, you have identified quality measures that are specifically financially rewarded."
Those measures would include colon cancer screening rates and other wellness and preventative procedures designed to keep patients healthy and out of hospitals, which provide the most expensive form of healthcare.
In addition to educating physicians about value-based care, hospital CFOs should establish a timeframe for transitioning to the new model, raise board awareness of value-based payments and build partnerships with insurers to improve healthcare services and outcomes while reducing costs.
That last one is important, Lawson said.
"We just reached out to payers," he said. "Most national payers are interested in discussions on this topic. That has educated me a lot, just sitting down with payers and saying, 'OK, if we did do this, how is this thing going to work? What about this? What about that?' There's nothing like healthy face-to-face dialogue. It really changes the relationship between payers and providers – which in the past has often been combative – in a really healthy way."
Lawson says 8 percent to 9 percent of Baptist Health's business next year will likely come from some form of risk-based contract, a percentage he expects to climb even higher in 2016.
"I used to not get too involved in meetings with the commercial payers, but I do now," he said. "It keeps everyone on the same page about lowering costs and improving care."
Riopelle said the switchover will take a lot of planning, and a lot more effort.
"You need to pick really high-quality, long-term partners. This is a significant challenge that all organizations in healthcare are going to have to undertake."


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American Gothic Press: May 3, 2017 Writer: Laszlo Tamasfi Art: Michael Malatini Cover: Michael Malatini
Cover Price: $3.99 UPC: 75420776661900211 Diamond ID: FEB171114 GCIN: 968600
The stories drained from David Wasp's infected mind are becoming downright bizarre, with one featuring a warped world where farm animals eat humans for breakfast! But something isn't quite right. It turns out David's previous employer might want something back, even if it's implanted in his head.
We have not yet discovered a graded sales history for Invisible Hands #2. Once a reported sale is found it will be automatically included in our comics price guide.×
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there is nothing more important in the world than people you can trust – who are your friends…
people like this dont gossip, slander, argue, run away when it gets hard, or expect "more". they let you be. they let you be the person you are and the person you want to be. they are at peace with what goes on. they are in sync. they encourage, promote, laugh, moderate, meditate, mentor, persuade and carry on like crazee chichadees when the time is right. they are wonderful.
and i love them to bits. xc


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From public broadcasting:
A measure known as the transgender bathroom bill is on its way to the governor's desk. House Bill 1008 survived the State Senate on Tuesday. Fifteen lawmakers in that chamber oppose the legislation, but 20 support it.
Supporters of the so-called transgender bathroom bill say it protects student privacy by securing restrooms for opposite biological sexes. The bill determines sex based on anatomy and birth certificates.
What are your thoughts? Should the Governor sign the bill, or should he veto it?


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Would You Get A 3D Printout Of Your Head?
A 3D printing company has opened a brick-and-mortar store in Manhattan, featuring an unusual take on the photo booth: instead of producing a strip of silly pictures, the booth churns out a 3D replica of the customer's head.
TechCrunch reports on the opening of the MakerBot store:
MakerBot CEO Bre Pettis unveiled a new 3D photo booth powered by ShapeShot. The photo takes a couple of minutes and costs $5 for three reusable shots. Then you can order prints of your head. In 3D. Depending on the size, they run $20, $40 or $60. The most important part remains the fact that there is now a physical address to experience 3D printing.
Three-dimensional prints of your head? Am I the only one who thinks that sounds kind of creepy?
Even so, maybe the bigger point is publicity. Pettis explains that he wants to show people that 3D printing is not "science fiction." And I'm sure that these models will… turn a few heads.
In addition to the photo booth and demonstrations, the store also sells the actual 3D printers and supplies, as well as small objects produced by the printers.
What do you think? Would you want a giant model of your head in the living room?
[Image via Roman Dillet/TechCrunch]


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Lust always looks beautiful in the night
at two am
when you should be home in bed
but can't.
when the music changes the beating of the heart
and the alcohol makes everything look pretty.
Nothing changes unless you will it
Who let me Adult?... I can't Adult
Living Life to the Fullest by Embracing My Inner Demons
All that I love, all that I see - in all its colours, in all its beauty
Musings on poetry, language, perception, numbers, food, and anything else that slips through the cracks.
Some things you just dont talk about, you Write.
The Physician Wellness Movement and Illegitimate Authority: The Need for Revolt and Reconstruction
Expressive and Emotional word content... poetic justice... my thoughts are perceptive, occasionally subjective but always dimensional. My career lies in psychology and my mind lies in philosophy. To question and ponder is to reflect. I am both reflective and directive. I never walk with caution as our steps need their footprints. I love this journey we call life.
Wanderlust Through Words
The world as I see it, according to myself.
"Where Business Meets Humanity"
take a journey into my mind, misconstrued,misjudge.
FACT: mdawaffes are mammals
Great reads from across WordPress.com, updated daily.
Think outside the cubicle.
Musings of an ex-pat in France.
"I inhale words and exhale poetry ..."


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I take care of this man. He is 26 years old and his name is Sebastian. Sebastian have heh a medical problem.. H..he's smart in some ways and sweet it's hard to take him out in public when people gives him stares. He sometimes twitch and talks to himself sometime but I could careless. Once you get to know him he's really sweet,kind,loving and caring.
- bwwm
- drama
- kindhearted
- lovestory
- mafia
- mentalillness
- romance


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A Mutant Tropomyosin That Causes Hypertrophic Cardiomyopathy Is Expressed In Vivo and Associated With an Increased Calcium Sensitivity
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Abstract—Mutant contractile protein genes that cause familial hypertrophic cardiomyopathy (FHC) are presumed to encode mutant proteins that interfere with contractile function. However, it has generally not been possible to show mutant protein expression and incorporation into the sarcomere in vivo. This study aimed to assess whether a mutant α-fast tropomyosin (TM) responsible for FHC is actually expressed and determines abnormal contractile function. Since α-fast TM is expressed in heart and skeletal muscle, samples from vastus lateralis muscles were studied from two FHC patients carrying an Asp175Asn α-fast TM mutation and two healthy control subjects. TM isoforms from whole biopsy samples and single fibers were identified by gel electrophoresis and Western blot analysis. An additional faster-migrating TM band was observed in both FHC patients. The aberrant TM was identified as the Asp175Asn α-fast TM by comigration with purified recombinant human Asp175Asn α-fast TM. Densitometric quantification of mutant and wild-type α-fast TMs suggested equal expression of the two proteins. Contractile parameters of single skinned muscle fibers from FHC patients and healthy control subjects were compared. Calcium sensitivity was significantly increased in muscle fibers containing Asp175Asn α-fast Tm compared with fibers lacking the mutant TM. No discernible difference was found regarding cooperativity, maximum force, and maximum shortening velocity. This is the first demonstration that the mutant TM that causes FHC is indeed expressed and almost certainly incorporated into muscle in vivo and does result in altered contractile function; this confirms a dominant-negative, rather than null allele, action. Since the mutant TM was associated with increased calcium sensitivity, this mutation might be associated with an enhancement and not a depression of cardiac contractile performance. If so, this contrasts with the hypothesis that FHC mutations induce contractile impairment followed by compensatory hypertrophy.
Familial hypertrophic cardiomyopathy is an autosomal dominant inherited heart disorder characterized by cardiac hypertrophy in the absence of increased workload. FHC has been linked to point mutations in certain cardiac contractile proteins: more frequently to >40 β-MHC and cardiac MyBP-C mutations,1 2 less frequently to troponin T and α-fast TM,3 4 and very rarely to cardiac MLC mutations.5 It has been suggested that cardiac hypertrophy is actually caused by such mutations through a dominant-negative effect; ie, all mutant proteins would in some way interfere with the function of WT proteins impairing cardiac performance. Such impairment would give rise to compensatory cardiac hypertrophy.3
The above hypothesis has been mainly derived from studies of β-MHC mutations. For one such β-MHC mutation (Arg403Gln), it has been shown that in FHC patients the mutant gene is transcribed in the heart and in skeletal muscle6 7 and that the mutant protein is expressed in skeletal muscle.8 Functional studies8 9 have shown that several mutant MHCs have abnormal function; ie, these studies have determined a slowing of actin-myosin kinetics and decrease in force. Studies of mouse models of FHC have supported such findings.10
Mutations in troponin T, α-fast TM, and MyBP-C have been less studied, and their pathogenetic role is much less understood. This is mostly due to the fact that, apart from the Arg403Gln β-MHC mutation, the identification of the mutant form of the protein in skeletal and/or cardiac samples of affected individuals has not been possible. A possible role for troponin T mutations in the pathogenesis of FHC has been suggested by the demonstration of abnormal function in two mutations expressed in vitro.11 12 One such mutation (equivalent to Ile79Aln in human troponin T) has been shown to result in an increase, not a decrease, in the velocity of actin-troponin-TM filaments in an in vitro motility assay; thus, it has been suggested that not all mutations necessarily act through a common mechanism of functional impairment and compensatory hypertrophy.11 Certain mutations might determine a "hypercontractile" state, which would directly induce cardiac hypertrophy.11 Interestingly, the very rare mutations in cardiac MLCs have also been associated with an increase in the velocity of actin-myosin interaction.5
Very little is known of the mechanism underlying the rare cases of FHC linked to α-fast TM mutations. Five kindreds carrying one of the three known TM mutations (Asp175Asn, Glu180Gly, and Ala63Val) have been identified, and their genetic, clinical, and histopathological features have been studied.13 14 15 Since three of the five kindreds carried the same Asp175Asn mutation, nucleotide residue 579 in the α-TM gene was considered a "hot spot" for mutation.15 No information is available on whether mutant TM is actually expressed in human skeletal and cardiac muscle.
The present study aimed to assess whether the mutant TM is actually expressed in FHC patients and has abnormal function. Two subjects belonging to two different kindreds carrying the same Asp175Asn TM mutation15 and two healthy control subjects were enrolled in the study. Because of the ethical reasons that prevent cardiac biopsy and the favorable prognosis of this form of FHC, analysis was performed on needle-biopsy samples of the vastus lateralis muscle. This approach, previously used to study function of mutant β-MHC,9 is well suited to the functional study of mutant α-fast TM; of the three main TM isoforms, α-fast, α-slow, and β, the α-fast isoform is expressed both in skeletal and in cardiac muscle.
Because TM is believed to be involved not only in modulating calcium sensitivity and cooperativity in propagating the calcium signal along the thin filament16 17 18 but also in conferring rigidity to the thin filament itself,19 alterations not only in the force-pCa relationship but also in Po and Vo might result from α-fast TM mutation. Force-pCa curves, Po, and Vo of skinned fibers from FHC patients and from the two control subjects were compared. TM composition of single fibers and whole-muscle samples was studied by SDS-PAGE and subsequent Western blot analysis. Purified recombinant Asp175Asn and WT human α-fast TMs were prepared and used to identify the expression of the mutant TM by coelectrophoresis. The findings show that mutant TM is expressed and very likely incorporated in skeletal muscle of FHC patients carrying the Asp175Asn α-fast TM mutation and that this results in an increase in calcium sensitivity.
Materials and Methods
Two subjects, one male (aged 55 years) from an Italian family and one female (aged 40 years) from an American family, carrying the Asp175Asn α-fast TM mutation and two healthy male control subjects (aged 40 and 55 years) were enrolled in the study. The clinical, genetic, and histopathological features of the two FHC patients have been previously described in detail (Coviello et al15 in 1997). Both patients had virtually normal functional capacity, exercise levels, and peripheral circulation; ie, skeletal muscle changes from disuse were not expected. The study was approved by the ethics committee of the Department of Medicine of the University of Genoa (Italy), and informed consent was obtained.
Muscle Biopsy and Fiber Dissection
Needle biopsy samples (≈100 mg), taken from the vastus lateralis muscle,20 were divided in two portions of similar size: one was put in sample buffer21 and stored at −20°C for subsequent TM isoform identification, and the other was further divided in small bundles of ≈50 fibers. From such bundles, which were stored at −20°C in skinning solution with 50% glycerol added for up to 4 weeks before experiment, single fiber segments 2 to 4 mm long were dissected. To ensure complete removal of the plasma membrane, fibers were bathed in skinning solution with Triton X-100 1% (Sigma Chemical Co) for 1 hour. Light aluminum clips were used to attach the fiber segments to the beams of the force transducer (AE 801 SensoNor) and of the isotonic lever (model 101 vibrator, Ling Dynamic System) in the experimental setup.
Experimental Setup for Single Muscle Fiber Analysis
The experimental setup used was very similar to that previously described in detail20 ; however, there were seven instead of three muscle chambers. The setup allowed quick transfer of the muscle fibers from the first (larger) chamber to the other six (smaller) chambers (70 μL) containing different solutions (relaxing, preactivating, and activating solutions at maximal or submaximal calcium concentrations). The electromagnetic puller could either keep the length of the fiber segment constant to elicit isometric contractions or impose on the specimen quick releases of preset amplitude completed in 2 milliseconds. A stereomicroscope was fitted over the apparatus to view the fiber at ×20 to ×60 magnification during the mounting procedure and during the experiment. The setup was placed on the stage of an inverted microscope (Axiovert 10, Zeiss). Since the floors of the muscle chambers were made with coverslips, specimens could be viewed at ×320 magnification through the eyepieces of the microscope. A video camera (MICAM HRS, System Sud), fitted to the camera tube of the microscope and connected through an analog-to digital converter (Cyclope, System Sud) to a computer (Olivetti M24), allowed viewing on a TV screen at ≈×1000 magnification and storage of digitized images of the specimen during the experiment. The signals from the force and displacement transducer were visualized on the screen of a storage oscilloscope (model 5113, Tektronix) and on a chart recorder (WR3701, Graphtec). The signals after analog-to-digital conversion (Interface CED 1401 Plus) were fed into a personal computer and stored on the hard disk. For data storage, recall and analysis were performed using the software Spike 2 (CED) implemented on the computer (DEX 486,66).
Skinning, relaxing, preactivating (pCa 9.0), and maximally activating (pCa 4.45) solutions were prepared according to Bottinelli et al.20 Activating solutions of intermediate calcium concentrations were obtained by mixing appropriate quantities of two solutions having the same ionic composition as the maximally activating solution but differing in calcium concentration: one at pCa 9.0 and one at pCa 5.05. Free ionic concentration and ionic strength of all solutions were calculated using a computer program designed by Fabiato.22
Experimental Plan
Force-pCa relationships, Po, and Vo of a large population of fibers from FHC patients (FHC fibers, n=150) and from control subjects (control fibers, n=110) were determined. After the experiment, all single fibers were characterized on the basis of MHC isoform composition by SDS-PAGE and divided in five types: types 1, 2A, and 2B (pure types) and types 1–2A and 2A-2B (mixed types).20 Calcium sensitivity (pCa50%), Hill's slope coefficient, Po, and Vo of corresponding fiber types from FHC and control subjects were compared. A subset of type 2A and type 2A-2B FHC fibers and of type 2A control fibers whose force-pCa relationship had been determined were studied for TM isoform composition. After TM isoform identification, a more selective comparison between FHC fibers actually containing the Asp175Asn α-fast TM and control fibers was performed for all functional parameters studied. Finally, the relative amounts of the Asp175Asn α-fast TM and of the WT α-fast TM were determined by densitometric scanning using SDS-PAGE and Western blots of whole biopsy samples and of single fibers.
Experimental Procedure
The following procedures were used in all mechanical experiments: temperature was set at 12°C; the fiber segment was mounted in a chamber containing relaxing solution; sarcomere length and fiber diameter were measured at three different locations along the length of the specimen at ×320 magnification; sarcomere length, which was determined by counting striations in segments of known length, was set at 2.5 μm; and fiber length was measured using a stereomicroscope fitted over the apparatus at ×40 magnification. Cross-sectional area of the specimen was determined assuming a circular shape from the mean of the three diameters measured at ×320 magnification, without correction for swelling. For Po and Vo determinations, fibers were first transferred to preactivating solution for at least 2 minutes and then maximally activated (pCa 4.45) for ≈40 to 60 seconds. To determine Vo, slack-test maneuvers were used.23 Details of Vo determinations have been reported previously.20 24 To determine force-pCa curves, fibers were mounted in the chamber containing relaxing solution, whereas the six smaller chambers were filled with activating solutions containing progressively higher concentrations of calcium. The solution in the last chamber was the same activating solution (pCa 4.45) used to elicit maximal activation for Po and Vo determinations. The fiber was moved from one solution to the next one and kept in each solution long enough to allow full tension development. Before moving to the next chamber, a large (10% muscle length) and fast shortening, followed after ≈7 milliseconds by a fast reelongation to the initial length, was applied to carefully and reliably measure developed tension.
In order to obtain the force-pCa curve, tension was measured at each calcium concentration, normalized to tension developed at the highest calcium concentration (pCa 4.45), and plotted versus the pCa values. The resulting sigmoid curve was fitted by Hill's equation: relative tension=(K×C)n/[1+(K×C)n], where C=[Ca2+].
The parameters pK=−log K, corresponding to pCa50%, and n, corresponding to the slope (Hill's slope) of the curve, were determined. pCa50% and Hill's slope give an indication of sensitivity to calcium and of cooperativity between calcium binding sites, respectively.16 An example of an experimental force-pCa curve is given in the inset of Fig 5⇓.
Fiber Typing and Myosin Isoform Identification
Since the three main human fiber types (types 1, 2A, and 2B) are known to differ in contractile properties25 (ie, Vo, Po, and calcium sensitivity), a comparison of functional properties of isolated fibers is meaningful only between fibers of the same type.
At the end of the mechanical experiment, each fiber segment was immersed in a small test tube containing 20 μL of sample buffer21 and stored at −20°C. The composition in MHC isoforms, which is the most precise marker of fiber type, of each segment was determined later by PAGE (acrylamide concentration, 6%) after denaturation in SDS (SDS-PAGE). The method was derived from that of Danieli-Betto et al26 and was described in detail by Stienen et al.27 In the MHC region, three bands corresponding to three MHC isoforms (MHC-1, MHC-2A, and MHC-2B) could be separated. In relation to the presence of one or two bands in the MHC region, fibers were classified into one of the following five types: 1, 2A, and 2B (or pure fibers) and 1–2A and 2A-2B (or mixed fibers). Only ≈2 to 4 μL of the 20 μL of sample buffer containing the fiber (total protein content, ≈4 μg) was used for MHC identification.
Overexpression and Purification of Recombinant Human α-TM
A PCR product containing the human α-TM cDNA sequence28 was generated using primers that incorporate unique restriction sites: HTMF (5′-ACTGCAGAACATATGGCTAGCATGGACGCCA-TCAAG -3′) and HTMR (5′-GCAGAAAAGCTTTTATATGGAAGTCATATCGTT -3′).
The amplified product was digested using the enzymes Nde I and HindIII and subcloned into the Escherichia coli expression plasmid, pMW172,29 cut with the same enzymes. The HTMF primer also encodes a Met-Ala-Ser N-terminal extension, such that the final construct encodes human α-TM as a fusion protein. This two-residue extension functionally mimics the N-terminal acetylation that occurs in vivo30 ; the expressed WT α-TM is indistinguishable on SDS-PAGE from α-TM extracted from tissue.
The expression plasmid encoding the Asp175Asn mutant was assembled by PCR amplification of a 582-bp fragment containing the mutation using the primers HTMF and D175N (5′-TTCTGAGAGCTCAGCCCGCTCCTCTGCACGTTCCAGGTTGCTCTCAAT -3′) (mutated base is underlined). This fragment was then digested using Nde I and Sac I and ligated into a WT construct cut with the same enzymes. All PCR-generated fragments were sequenced in full. Both constructs were used to transform the E coli strain, BL21(DE3)pLysS. Cultures were grown, induced for 3 hours, and harvested, and the bacteria were lysed according to standard protocols.31 Both WT and mutant TM were expressed at a high level and were purified by a modification of the method described by Monteiro et al.30
TM Isoform Identification
TM isoform identification was performed by SDS-PAGE on 15% acrylamide concentration gels according to a procedure described by Giulian et al32 (Fig 1A⇓) and subsequent Western blot analysis (Fig 1B⇓). Identification of TM bands on gels was confirmed by comigration with commercially available purified TM (Sigma T-3640), which was separated in two major bands on SDS-PAGE (Fig 1A⇓, lane 2). Three WT TM isoforms were identified on the basis of their order of migration: β-TM (slowest migrating), α-slow TM, and α-fast TM (fastest migrating).18 33 Western blot analysis was performed after transferring proteins from unstained gels to nitrocellulose sheets (Fig 1B⇓). Transfer was obtained by electrophoresis and semidry transfer procedure34 by applying a current of 0.8 mA/cm2 for 6 hours. Nitrocellulose sheets were reacted first with a primary mouse monoclonal antibody against sarcomeric TM (Sigma T-9283) and then with a peroxidase-conjugated rabbit anti-mouse antibody (P 260, Dako). TM bands were visualized by an enhanced chemiluminescence method in which luminol was excited by peroxidase in the presence of H2O2.35 The identification of the Asp175Asn α-fast TM (Fig 1D⇓) was achieved by coelectrophoresis and subsequent Western blot analysis (Fig 1D⇓) of biopsy samples (Fig 1D⇓; lanes 1, 4, and 5) with the two purified recombinant human α-fast TMs (WT α-fast TM [lane 6] and Asp175Asn α-fast TM [lanes 2 and 3]).
TM isoform identification was performed on biopsy samples from the two FHC patients and the two control subjects. Attempts were also made to characterize TM isoform composition and the presence of the mutant Asp175Asn α-fast TM in all FHC fibers (types 1, 2A, and 2A-2B) and in all type 2A control fibers whose force-pCa relationships had been studied. TM isoform analysis was successfully achieved only for 48 (21 type 2A FHC fibers, 12 type 2A-2B FHC fibers, and 15 type 2A control fibers) of the 97 fast fibers analyzed. Because segments of fibers used for experiments were kept short (2 mm) to optimize mechanical behavior and because a fraction of the specimen was used for MHC identification, the low recovery of single fibers with TM isoform identification was likely due to a combination of technical difficulties and the small amount of proteins available. Determination of TM isoform distribution in type 1 fibers used for the experiment was less successful, possibly because of the smaller size and amount of protein in such fibers, and was abandoned. However, TM isoform composition was successfully determined in longer (5-mm) segments of 43 slow fibers, which were dissected for this purpose and not used for mechanical experiments.
To quantify the relative amount of Asp175Asn and WT α-fast TM, densitometric scanning of SDS-PAGE TM gels (Fig 1E⇑, upper trace) and Western blots of whole biopsy samples (Fig 1E⇑, lower trace) and single fibers (Fig 1F⇑, upper and lower traces) were also done. Densitometry and quantification of the area under densitometric peaks were performed by a computerized densitometer. Images of the gels were fed to a personal computer (Laris P-100 Computer Discount) through a digital video camera (MICAM, HRS, System-Sud) and a frame grabber (Matrox Graphics Inc), and densitometry was performed by an especially designed software (Eidosoft). Quantification of the relative amount of Asp175Asn and WT α-fast TM, performed on SDS-PAGE (Fig 1E⇑, upper trace) and on Western blots bands (Fig 1E⇑, lower trace) of the same biopsy samples, gave very similar results. The latter finding suggested that under the conditions used, such quantification could be reliably performed on Western blots, presumably because Asp175Asn α-fast TM and WT α-fast TM, which differ by only one amino acid, very likely have the same affinity for the antibody. This is important because quantification of the relative content in Asp175Asn and α-fast TM of single fibers was possible only on Western blots (Fig 1F⇑).
Statistical Analysis
Data were expressed as mean±SE. Statistical significance of the differences between means was assessed by ANOVA followed by the Student-Newman-Keuls test. A probability of <5% (P<.05) was considered to be statistically significant. Statistical analysis, linear regression analysis for slack-test determination, and nonlinear regression for force-pCa curve fitting were performed using a computer program (Prism, GraphPad software). Slopes were considered significantly different from zero at P<.05.
TM Isoform Composition of Whole Biopsy Samples and Single Fibers
Fig 1⇑ shows representative SDS-PAGE runs (Fig 1A⇑) and Western blot analysis (Fig 1B⇑) of whole biopsy samples of FHC patients and control subjects. In Fig 1A⇑, whole biopsy samples of a control subject (lane 1) and of a patient (lane 3) are comigrated in an SDS-PAGE gel. Purified TM is also comigrated to confirm TM identification (lane 2). In Fig 1B⇑, Western blot analysis of whole biopsy samples of the two patients (lanes 1 and 4) and of the two control subjects (lanes 2 and 6) are shown. It can be seen that (1) in control subjects, the three main TM isoforms (β, α-slow, and α-fast) could be separated, and no other band was stained by the antibody against TM in Western blot, and (2) in both patients, besides the three known TM isoforms, a fourth electrophoretic band that migrated faster than α-fast TM and that was stained by anti-TM antibody in Western blot analysis was identified.
With the TM bands in whole biopsy samples used as standards, TM isoform composition was successfully analyzed in a subset of fibers from FHC patients (FHC fibers) and control subjects (control fibers) whose force-pCa relations were determined. Fig 1C⇑ shows Western blot analysis of two single FHC fibers (lanes 2 and 3) and of a whole biopsy sample from a patient (lane 1); not only in the biopsy sample but also in both single fibers the aberrant TM band can be seen. The aberrant TM isoform is referred to as Asp175Asn α-fast TM because it has been shown to correspond to the mutant TM (see below), and the WT α-fast TM is referred to as WT α-fast TM. The Table⇓ shows the number of 2A and 2A-2B FHC fibers and 2A control fibers in which TM identification was successfully achieved and the number of fibers that contained each of the four TMs (Asp175Asn α-fast, WT α-fast, α-slow, and β). It can be seen that the newly identified extra TM band was present in the large majority of single type 2A and type 2A-2B fibers from FHC subjects and in none of the control fibers. α-Fast TM was expressed virtually in all type 2A and 2A-2B FHC fibers and in all 2A control fibers. Type 2A FHC and control fibers did not have the same α-slow and β-TM isoform composition. FHC fibers containing the mutant TM did not contain α-slow TM and mostly contained β-TM. Control fibers contained α-slow TM more frequently (9 of 15 fibers) and β-TM less frequently (8 of 15 fibers) than did FHC fibers. This point is relevant for functional comparisons and will be considered below. Type 2A-2B FHC fibers contained mostly Asp175Asn α-fast and WT α-fast TM and β-TM and did not contain α-slow TM.
Because attempts to determine TM isoform composition in slow fibers whose force-pCa relation was studied were mostly unsuccessful (see "Materials and Methods"), TM isoform composition was determined in 43 slow fibers dissected for this purpose and not used for mechanical experiments (Table⇑). Interestingly, on average, 30% (13 of 43) of the slow fibers contained the aberrant TM isoform; such fibers always contained the α-fast TM and β-TM and never the α-slow TM. On the other hand, the slow fibers that did not contain the aberrant TM contained α-slow TM and β-TMs but not α-fast TM.
Identification of the Mutant Asp175Asn α-Fast TM
To assess whether the extra TM expressed in whole biopsy samples and in most fast single fibers of the FHC patients was the Asp175Asn α-fast TM, two purified recombinant human α-fast TMs, one WT and one carrying the Asp175Asn mutation, were prepared (see "Materials and Methods"). Both purified proteins were run in SDS-PAGE with the biopsy samples. Fig 1D⇑ shows that the extra TM band of the FHC samples (fastest migrating band in lanes 1 and 4 where biopsy samples of the two patients were loaded) comigrated with the purified recombinant Asp175Asn TM (lanes 2 and 3), whereas the WT α-fast purified TM (lane 6) comigrated with the native α-fast TM (fastest migrating band in lane 5 where a control biopsy sample was loaded). Confirmation that the aberrant migration of the recombinant Asp175Asn TM protein reflects the impact of only the single residue substitution was provided by identical observations on SDS-PAGE electrophoresis of recombinant WT and Asp175Asn mutant chicken α-TM (data not shown).
Quantification of the Relative Amount of WT and Mutant TM
To determine the relative amount of the Asp175Asn and WT α-fast TM in whole biopsy samples, densitometric scanning was performed on three different SDS-PAGE gels of each of the two patients. An example of a densitometric trace of a gel of a whole bioptic sample from an FHC patient is shown in Fig 1E⇑ (upper trace). Approximately equal amounts of Asp175Asn (51.5±0.5% [mean±SE]) and WT (48.5±0.50% [mean±SE]) α-fast TMs were found in both patients. On the basis of considerations reported in "Materials and Methods," quantification was also performed on Western blots (Fig 1E⇑, lower trace), and very similar results were obtained (49.35±2.65% for Asp175Asn α-fast TM versus 50.15±3.15% for WT α-fast TM) (note that upper and lower traces of Fig 1E⇑ refer to same sample). This supports the quantification of the relative amount of Asp175Asn and WT α-fast TMs in single fibers expressing the mutant TM on Western blots, which also showed approximately equal amounts of Asp175Asn (53.31±1.29%) and WT (46.79±1.29%) α-fast TMs. Examples of densitometric scanning of Western blots of single fibers are shown in Fig 1F⇑ (upper and lower traces).
Functional Properties of Single Skeletal Fibers
Calcium sensitivity (pCa50%) was found to be significantly higher in type 1 and type 2A fibers from FHC patients (FHC fibers) than in corresponding fiber types from control subjects (control fibers) (Fig 2A⇓ and 2B⇓). Since few pure 2B fibers were found in control subjects (n=1), comparison was performed on mixed fibers containing both 2A and 2B MHC isoforms. Calcium sensitivity was again higher in FHC than control 2A-2B fibers, but this difference did not reach statistical significance (Fig 2C⇓).
Differences in Hill's slope between type 1, 2A, and 2A-2B fibers of FHC and control subjects were not statistically significant (Fig 3D⇓ through 3F⇓).
Fig 3⇑ shows the mean values of Po (Fig 3A⇑ through 3C⇑) and Vo (Fig 3D⇑ through 3F⇑) of types 1, 2A, and 2A-2B fibers of the FHC subjects compared with the control subjects. As expected,20 Vo and Po depended on MHC composition both in FHC patients and in control subjects: Vo of type 1 fibers was significantly lower than Vo of type 2A, and Vo of type 2A was significantly lower than Vo of type 2A-2B fibers; Po of type 1 was significantly lower than Po of type 2A and 2A-2B fibers. However, when Po and Vo of corresponding fiber types of FHC and control subjects were compared, no statistically significant differences were observed (although there was a trend toward increased Vo and lower Po in the FHC fiber groups). The very few fibers coexpressing MHC-1 and MHC-2A were not used for analysis.
Functional Properties of Single Fibers Related to Mutant TM Expression
The study of TM isoform composition of single fibers (see above) allowed division of type 2A and type 2A-2B fibers from FHC patients into two subgroups according to the presence or absence of the Asp175Asn TM band and a more selective comparison between only those FHC fibers containing the mutant TM and control fibers. Both type 2A (Fig 4A⇓) and type 2A-2B (Fig 4B⇓) fibers containing the mutant TM had significantly higher calcium sensitivity than did the corresponding fiber types from control fibers (Fig 4A⇓ and 4B⇓). Differences in pCa50% between FHC and control fibers were larger (0.09 pCa for type 2A and 0.10 pCa for type 2A-2B) when only FHC fibers expressing the aberrant fast migrating TM were considered (Fig 4A⇓ and 4B⇓) than when all fibers from FHC patients were pooled together regardless of their TM composition (0.06 pCa for type 2A and 0.09 pCa for type 2A-2B, Fig 2B⇑ and 2C⇑). Interestingly, pCa50% of the FHC type 2A fibers that did not express the Asp175Asn α-fast TM (Fig 4A⇓) was not distinguishable from pCa50% of control fibers and significantly lower than pCa50% of FHC type 2A fibers that expressed the mutant TM (Fig 4A⇓). The shift in the force-pCa curve that corresponds to the reported changes in pCa50% can be appreciated in Fig 5⇓, where mean force-pCa curves of type 2A fibers expressing Asp175Asn α-fast TM and type 2A control fibers are shown.
Type 2A FHC fibers that contained the Asp175Asn α-fast TM and type 2A control fibers did not have identical α-fast, α-slow, and β-TM content. To verify that the increased calcium sensitivity of FHC fibers containing the mutant α-fast TM was actually due to Asp175Asn mutation and not to coexpression of specific TM isoforms, two approaches were used. First, control fibers (all type 2A fibers) were grouped on the basis of the presence of β-TM, α-slow TM, and α-fast TM, and their calcium sensitivities were compared. No statistically significant differences were found among fibers containing β-TM (pCa50%=5.68±0.02, n=8), α-slow TM (pCa50%=5.69±0.02, n=9), and α-fast TM (pCa50%= 5.72±0.02, n=14) and between each group and the mean calcium sensitivity of all control fibers (pCa50%=5.71±0.02, n=28) pooled together, whereas all groups had statistically lower pCa50% than did FHC 2A fibers containing the Asp175Asn α-fast TM (pCa50%=5.80±0.02, n=15). Second, because FHC fibers containing Asp175Asn α-fast TM always contained WT α-fast TM, almost always the β-TM, and never the α-slow TM, the following, more selective, comparisons were also done: (1) the whole group of FHC fibers containing the Asp175Asn α-fast TM was compared only with control fibers containing the α-fast TM (pCa50%=5.72±0.02, n=14) and with a subset of such fibers expressing the α-fast but not the α-slow TM (pCa50%=5.70±0.03, n=6); (2) FHC fibers containing Asp175Asn α-fast TM, WT α-fast TM, and β-TM (pCa50%=5.80±0.04, n=6) were compared only with control fibers containing both α-fast TM and β-TM (pCa50%=5.70±0.03, n=6). In all cases, FHC fibers expressing the Asp175Asn α-fast TM had significantly higher calcium sensitivity than did the corresponding control groups.
No significant differences were observed between fibers containing the extra TM band and fibers that did not contain such a band regarding Hill's slope, Po, and Vo (data not shown).
We have taken advantage of the expression of α-fast TM in skeletal, as well as heart, muscle to determine whether the mutant Asp175Asn α-fast TM that causes FHC is expressed and incorporated and whether it alters contractile function. Aberrant electrophoretic migration of both mutant TMs extracted from patient samples and purified recombinant TM allowed direct identification and quantification of the mutant peptide in a way that has not previously been possible in FHC mutant proteins. The primary results of the present study are as follows: (1)the mutant TM is expressed in skeletal muscle of FHC patients carrying a Asp175Asn α-fast TM mutation; (2) Asp175Asn and WT TM are expressed and almost certainly incorporated in approximately equal amounts; (3) Asp175Asn α-fast TM is associated with an increased calcium sensitivity, whereas cooperativity between calcium binding sites, Po, and Vo were not detectably modified.
Asp175Asn TM Expression
Although it is generally thought that in FHC, mutant contractile proteins are expressed and incorporated in the sarcomere, this is still a hypothesis based mainly on in vitro experiments.3 Resolution of mutant from WT protein has typically not been possible because of the subtle nature of most FHC mutations and the limited access to affected cardiac tissue. The incorporation of mutant protein in vivo has been demonstrated for only a single β-MHC mutation (Arg403Gln), since the mutant protein could be distinguished in extracts from skeletal muscle through loss of an endoproteinase Arg-C digest site.8 In the present study, besides the three main TM isoforms, a fourth TM band was found in skeletal muscle of patients carrying an Asp175Asn TM mutation. This aberrant TM could be identified as the Asp175Asn α-fast TM, since it comigrated with a purified recombinant human Asp175Asn α-fast TM. The SDS-PAGE separation of the mutant TM from the WT TM is not easily explained purely on the change in charge that follows the substitution of Asp with Asn. Presumably, the substitution in some way alters the conformation of the protein under the denaturing conditions of SDS-PAGE. Of note, a similar effect has been seen with the Glu180Gly TM mutation that also causes FHC (Drs Redwood and Watkins, unpublished data, 1997).
This is the first demonstration that mutant contractile proteins other than myosin are in fact expressed in FHC in vivo. In addition, given the direct way in which the mutant protein can be distinguished, these observations allow the first accurate quantification of the ratio of mutant and WT protein. The observation that Asp175Asn and WT α-fast TM were expressed in virtually identical amounts suggests that in vivo Asp175Asn α-fast TM expression is not downregulated.
Formal demonstration of incorporation of Asp175Asn α-fast TM in the sarcomere is not possible with immunohistological techniques in the absence of an antibody that distinguishes mutant from WT α-fast TM. However, our observations indicate that the Asp175Asn α-fast TM is almost certainly functionally incorporated in the thin filament. In skinned fibers, the plasma membrane has been removed, and all proteins that are soluble and/or not firmly bound to cytoplasmic structures leave the cell. In the FHC biopsies, the ratio of mutant-to-WT TM was the same in skinned fibers as in whole biopsy samples, indicating that all of the mutant TM is firmly bound. The observation of a functional effect corresponding to the presence of the mutant TM makes it extremely unlikely that mutant TM is unincorporated (and just attached to some contractile or cytoskeletal protein) but indicates that the protein is incorporated and functional.
The observation that some fast fibers that expressed WT did not express Asp175Asn α-fast TM is difficult to interpret given that overall expression was so close to 50% (and no purely mutant fibers were observed to make up the balance). Although this might suggest that in a minority of fibers Asp175Asn α-fast TM is not expressed and/or incorporated in the sarcomere, the apparent lack of mutant TM may simply reflect some technical limitations in single-fiber analysis.
Asp175Asn α-Fast TM Function
Type 1, type 2A, and type 2A-2B fibers from FHC patients had higher calcium sensitivity than did corresponding fiber types from control subjects. Differences were small, ≈0.05 U of pCa, but statistically significant for type 1 and type 2A fibers. Interestingly, differences almost doubled (≈0.1 U of pCa) and became more statistically significant when only FHC fibers containing the mutant TM were compared with control fibers. It is also of note that the few type 2A FHC fibers not expressing the mutant α-fast TM were indistinguishable from control fibers and had significantly lower calcium sensitivity than did the type 2A FHC fibers expressing the mutant TM. These observations strongly suggest that the expression of Asp175Asn α-fast TM causes the increased calcium sensitivity. This conclusion is in agreement with a preliminary report that suggested an increased calcium sensitivity of myofilaments from transgenic mice overexpressing Asp175Asn α-fast TM compared with myofilaments from control mice expressing WT α-fast TM.36
Our data suggest that it is very unlikely that the increased calcium sensitivity in the presence of Asp175Asn α-fast TM reflects secondary changes in expression of other contractile protein isoforms and is not a functional property of the mutant TM itself. Within fibers with homogeneous MHC isoform composition, calcium sensitivity is thought to be modulated by the expression of at least three main TM isoforms and several troponin isoforms.17 18 Although troponin isoforms were not studied, major changes in troponin expression induced by mutant TM expression seem unlikely given that expression of the mutant α-fast TM appeared to determine just subtle, if any, changes even in the expression of the other TM isoforms. Because the comparisons reported here between FHC and control fibers grouped on the basis of TM isoform composition (to control for the effects of differences in other TM isoforms) showed that in all cases FHC fibers expressing the Asp175Asn α-fast TM had significantly higher calcium sensitivity than did the corresponding control groups, the presence of Asp175Asn α-fast TM itself is by far the most likely determinant of the increased calcium sensitivity observed in FHC fibers. However, a minor role of possibly subtle TM isoform shifts cannot be ruled out.
TM isoform composition of type 1 fibers used for determination of calcium sensitivity was not studied because of technical limitations (small fiber size; see "Materials and Methods"), and the association between mutant TM and higher calcium sensitivity in slow fibers cannot be directly shown as for fast fibers. However, the finding that 30% of type 1 FHC fibers contained the mutant TM demonstrates that the mutant TM is expressed in a subset of slow FHC fibers and suggests than its presence is the most likely explanation of the higher calcium sensitivity not only of type 2A and type 2A-2B FHC fibers but also of type 1 FHC fibers. Interestingly, Asp175Asn α-fast TM was much more frequent in the slow fibers of one patient (50%) than in the slow fibers of the other one (10%), and calcium sensitivity was also significantly higher in the slow fibers of the former patient than in the slow fibers of the latter patient (data not shown). The fact that mutant and WT α-fast TMs were also found in slow FHC fibers is unlikely to be a feature of FHC patients themselves, since a specific pattern of association between fiber type and TM isoform expression has not been clearly shown in human muscle fibers so far.37 38 According to the only previous work in which α-fast TM, α-slow TM, and β-TM isoforms have been possibly identified in human muscle fibers,38 it is also not surprising that type 2A control fibers were found to contain all three TM isoforms.
At present, little can be concluded about the molecular mechanism at the basis of the observed increase in calcium sensitivity. The Asp175Asn mutation and one other (Glu180Gly) of the three known TM mutations linked to FHC occur at residues that have been highly conserved through evolution4 and that are in the region of the putative calcium-dependent troponin-T binding domain.39 Two recent articles have reported in vitro studies on purified Asp175Asn and Glu180Gln mutant α-TMs expressed in E coli. Bing et al40 have shown that in the in vitro motility assay in the presence of troponin and activating Ca2+ (pCa5), the mutant TMs give an increase in actin filament velocity compared with WT α-TMs. This suggests that the mutations in TM may be causing a change in the "on" state of the thin filament mediated by altered interaction of TM either with the troponin complex or actin. Golitsina et al41 have used myosin subfragment-1 to induce the binding of pyrene-labeled WT or FHC mutant TM to actin in the "on" state. The change in excimer fluorescence for WT TM was found to be different from that observed using either Asp175Asn or Glu180Gln TM, thus also suggesting that the FHC mutations affect the conformation of the TM in the "on" state, possibly because of an increase in local flexibility of the TM molecule at the level of the mutation. The Asp175Asn mutation, possibly supported by the head-to-tail assembly of adjacent TM molecules, apparently does not interfere with the TM role in determining cooperativity, since fibers containing the Asp175Asn α-fast TM were not different from control fibers regarding Hill's slope. The Asp175-Asn mutation does not appear to severely hinder the structural role of TM either, since force and shortening velocity, which heavily depend on the integrity of myofilament structure, were not impaired in fibers expressing the mutation. It should be pointed out, however, that some subtle change in force cannot be excluded on the basis of the present data because of the large variability in force of corresponding fiber types of healthy control subjects. Indeed, there were appreciable, but nonsignificant, trends toward increased velocity, but decreased force, in the FHC fibers.
Pathogenetic Mechanism of FHC
The present findings support the hypothesis that FHC is caused by mutations in contractile proteins that are expressed and incorporated in the sarcomere and, having abnormal function, interfere with the function of the WT proteins, ie, are consistent with a dominant-negative action of the mutation.3 However, at variance with most functional studies so far, the present results did not show an impairment of actin-myosin interaction and/or some major structural alteration following expression of the FHC mutation. Po and Vo in FHC fibers expressing Asp175Asn α-fast TM were not distinguishable from normal fibers. On the contrary, an increased calcium sensitivity was certainly associated with, and very likely determined by, the TM mutation. Such an increase should be even more pronounced in the heart, since α-fast TM is the dominant cardiac TM.42 Because the heart is not fully activated in vivo, the increase in calcium sensitivity could potentially enhance and not depress cardiac contractility. Therefore, it is not straightforward to attribute to the Asp175Asn mutation the pathogenetic mechanism of functional impairment and compensatory hypertrophy suggested for most mutations studied so far.
A recent study has suggested that not all FHC-linked mutations necessarily act through this common mechanism but that some might determine a "hypercontractile" state that could directly induce cardiac hypertrophy.11 In that study, in fact, recombinant troponin T carrying an Ile79Aln mutation determined an increase, and not the generally observed decrease, in the velocity of actin-TM filaments in an in vitro motility assay. Interestingly, the very rare cardiac MLC mutations have also been shown to cause an increase in velocity in a sliding filament assay.5
The present findings involving the Asp175Asn α-fast TM mutation might be taken to support the possibility of a mutation-induced hypercontractile state. However, it should be pointed out that not only the "hypercontractile" hypothesis but also the more common and better defined "hypocontractile" hypothesis are still somewhat tentative and incomplete. For technical reasons, in most studies the motility assay technique has been used to assess function of mutant proteins. Because velocity is determined in the absence of load and the heart always works under load in vivo, it is not clear what a decrease8 or increase11 in velocity in motility assays necessarily means in terms of force generation, or power output, under load. Force was found depressed only in two of the three β-MHC mutations studied by Lankford et al9 and in one troponin-T mutation (troponin-T truncation12 ). Moreover, care must be taken to transfer to a complex in vivo situation all findings reported so far that were not obtained in strictly physiological conditions. Even results involving human skinned fibers from living muscles (Reference 99 and present results), which can be considered the most physiological of the specimens used to date, should be considered with some caution, because the experiments were performed at low temperature (12°C to 15°C).
In conclusion, the present work provides the first quantitative evidence of equal expression and apparent incorporation of mutant and WT contractile protein into muscle in subjects with FHC. These findings strongly support the dominant-negative role of mutant TM in determining FHC. The Asp175Asn mutant TM interferes with the function of the WT protein and results in increased calcium sensitivity. This might imply an enhancement in cardiac performance.
Selected Abbreviations and Acronyms
|familial hypertrophic cardiomyopathy
|myosin heavy chain
|myosin light chain
|myosin binding protein C
|pCa value at which relative tension is 50% of maximum
|polymerase chain reaction
|isometric force
|maximum shortening velocity
The study was supported by grants from Telethon-Italy to Dr Bottinelli (grant 868) and Dr Coviello (grant 723) and from the British Heart Foundation and the Wellcome Trust to Prof Watkins. We would like to thank Giovanna Esposito (Department of Cardiovascular Medicine, Oxford) and Liliana Lobaido (Institute of Human Physiology, Pavia) for excellent technical assistance and Drs Rosetta Rossi and Cristina Zanardi (Institute of Human Physiology, Pavia) for help in analysis of single-fiber data.
Reprint requests to Dr Roberto Bottinelli, Istituto di Fisiologia Umana, Via Forlanini 6, 27100 Pavia, Italy.
- Received June 9, 1997.
- Accepted October 10, 1997.
- © 1998 American Heart Association, Inc.
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- A Mutant Tropomyosin That Causes Hypertrophic Cardiomyopathy Is Expressed In Vivo and Associated With an Increased Calcium SensitivityR. Bottinelli, D. A. Coviello, C. S. Redwood, M. A. Pellegrino, B. J. Maron, P. Spirito, H. Watkins and C. ReggianiCirculation Research. 1998;82:106-115, originally published January 23, 1998https://doi.org/10.1161/01.RES.82.1.106
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- A Mutant Tropomyosin That Causes Hypertrophic Cardiomyopathy Is Expressed In Vivo and Associated With an Increased Calcium SensitivityR. Bottinelli, D. A. Coviello, C. S. Redwood, M. A. Pellegrino, B. J. Maron, P. Spirito, H. Watkins and C. ReggianiCirculation Research. 1998;82:106-115, originally published January 23, 1998https://doi.org/10.1161/01.RES.82.1.106


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(b. Lucknow, 1918)
Eminent tabla player, born in a family of tabla exponents of Lucknow. He was a direct descendant of Miyā Bakhshu, Miyā Makshu, and Miyā Mohammad Khan of ...
Subjects: Indian Music.
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