text:
It only takes a minute to get started. Fill out our simple sign up form and login to the site.
This step is discretionary but will greatly enhance your chances of having members look at your profile. Write a few sentences about yourself, your hobbies and upload some good photographs of yourself.
Utilise our website's advanced search to pinpoint members who meet your criteria. Or just scan through listings and pinpoint members that appeal to you.
Found someone who you're attracted to? Send a letter to them or just a wink and see how you get along. With a little luck you'll be on your way to discovering your perfect match.
It doesn't take long


text:
Family tree▼
2 Dea. John Dam, b. 1610. 2d dea. Dover, gr.j. 1646, Com.t.e.s.c. 1661. … M. Elizabeth Pomfret who came in 1640, liv. 1682. He d. 27 Jan. 1689-90; will 19 May 1687-23 Mar. 1693-4 names 2 sons, 3 daus. Ch: John, by an earlier wife, ± 68 in June 1702. Elizabeth, b. 1 May 1649, m. Thomas Whitehouse. Mary, b. 4 May 1651, m. Joseph Canney(1), 2d 22 Nov. 1701 Wm. Harford. William, b. 14 Oct. 1653. Susannah, b. 14 Dec. 1661. Judith. b. 15 Nov. 1666, m. Thomas Tibbetts.
"The name was originally Dam. The Dams were freeholders in England from the time of Edward VI, and it is claimed that the majority of them became Puritans. It is also asserted that most of them left England during the seventeenth century, some seeking religious liberty in Holland, while others came to America for the same purpose.(I) John Dame or Dam, who belonged to a Cheshire family of the latter name, came to New England in 1633 with a company of colonists under the guidance of Captain Thomas "Wiggans", and settled in Dover, New Hampshire. He may have been a brother of Nicholas Dam [Noyes, Libby and Davis, GDMNH, p. 181, indicate that Nicholas Dam is a misreading of "Dunn"], whose name, together with those of John Dam and several others, appears in a petition presented to the governor of New Hampshire in 1689, but there is no further mention of Nicholas in the Dover records. John Dame was one of the first to receive a grant of land at the confluence of the Cocheco and Fresh Creek rivers, known as Dam Point, and he was also allotted land on Great Bay and at Bloody Point (now Newington). In 1675 he was chosen a deacon of the First Parish Church in Dover, and his death occurred January 27, 1690. His will was dated May 19, 1687, and proved March 23, 1693. He married Elizabeth Pomfret, daughter of Lieutenant William Pomfret and was the father of six children: John, Elizabeth, Mary, William, Susanna and Judith."
He is not included in the The Great Migration Study Project sketches through 1635, indicating that there is no record of him in New England before at least 1636
"JOHN, Dover 1640, or earlier, by w. Elizabeth had John; Elizabeth b. 1 May 1649; Mary, 4 Sept. 1651; William, 4 Oct. 1653; Susanna, 14 Dec. 1661; and Judith, lB Nov. 1666; was freem. 1672; deac. d. 27 Jan. 1690, in advanced age. Judith m. 6 July 1684, Thomas Tebbets, and d. bef. mid. age."

subset: commoncrawl


text:
Great Day Learns How to Prepare Firecracker Chicken
General Manager and Panda Express Chef John Yue stopped by the Great Day Kitchen on Wednesday morning to show us how they prepare their famous firecracker chicken.
Kim and Kopi also learned about the Chinese New Year, which is on February 3rd. Chef Yue also discussed why rabbits are so important this year.
On Thursday, February 3rd, you can taste the Firecracker Chicken at Panda Express for free! Just click on the following link to print out a coupon: Firecracker Chicken.


text:
BY now, beads of sweat have collected at the temples of high school juniors as they prepare for next month's college entrance exams. Given the fact that these students have practically grown up in cyberspace, and that many live in areas not served by traditional test tutorial companies like Kaplan and Princeton Review, one might assume that businesses are making real money from online test preparation.
But so far, that has not happened. For that, the companies can hold responsible -- who else? -- parents and teachers.
The online test-preparation industry has rolled out an array of services in recent years, looking to capitalize on several factors that, at first glance, make this market appear to be a lock. Not only is the population of high school students surging, but a higher percentage of students are seeking slots at four-year colleges that have generally held the line on enrollment. That has made competition among applicants stiffer and increased the importance of high test scores. Half of the 1.4 million students who take the SAT will rely on preparatory classes or tutorials, at a cost of $500 million, according to Eduventures Inc., a consulting company based in Boston. Of that, only about 5 percent, or $25 million will go to online test preparation.
''Generation Y is more than ready for this,'' Adam Newman, vice president for research at Eduventures, said. ''But it's the baby boomers who have to pay for it, and they're not convinced that students can perform as well as, or better than those taking the classroom-based prep courses if they go online.''
According to Seppy Basili, vice president for learning and assessment at Kaplan, parents have been slow to embrace online test preparation not because they doubt its pedagogical merits. ''They're concerned that their kids' time online hasn't been valuable time online,'' he said. ''I've heard this from parents all over the country.''Continue reading the main story
Test preparation companies offer online classes in a variety of flavors. Princeton Review's most popular online course, for $400, includes interactive multimedia lessons, with audio explanations of various academic principles. Offline, the company's courses cost $800 to $1000, depending on the region.
Online students, who are expected to complete the course in 40 to 60 hours, also receive eight practice tests that, when completed, yield explanations of correct answers, as well as personalized assignments for students to improve on weaknesses. Princeton Review coaches are available at all hours to answer basic questions from course participants online, while more detailed questions are answered via e-mail message.
The lessons ''are highly interactive,'' according to Drew Deutsch, a Princeton Review assistant vice president. ''Students are constantly clicking. It emulates the Socratic method we use in the classroom experience.''
Princeton Review also offers a more expensive online course that, for $700, includes eight live online classroom sessions led by a company instructor. The sessions, which feature both text chat and a virtual ''white board,'' will later this month include two-way audio between the instructor and the students. Budget-conscious (or time-pressed) students can also enroll in a $100 cram course, with two practice tests and about 20 online lessons.
Mr. Deutsch would not say how many of Princeton Review's students enroll in online courses, or how much of the company's revenues come from the online division, but he said that with annual growth of about 80 percent, ''it's one of the fastest growing areas of our business.''
Profit margins, meanwhile, ''are at least twice what they are for our classroom course business, because you don't have to provide rooms or instructors, and all the training associated with that.''
Profitable as they are, the online courses were never intended to displace the off-line courses as much as they were ''meant to expand the reach of our business,'' Mr. Deutsch said. In particular, online courses are targeted to students who are too busy to schedule classroom sessions, those who cannot afford the traditional courses, those who cannot stomach the idea of sitting in a classroom in nonschool hours, and students who live too far from the company's traditional classroom centers.
To help prove the effectiveness of online study, Princeton Review will release a study of its Internet courses this fall. ''We see similar score improvement in our Internet and classroom courses, which average 140 points combined in the math and verbal,'' Mr. Deutsch said. ''We expect the study to show numbers very close to that, if not higher.''
That study ''will certainly help, in that we can put it in marketing material,'' he said, ''but I don't know if it'll give us a significant increase in business.''
Mr. Basili at Kaplan said he was ''satisfied'' with the company's online test preparation revenues, which he would not disclose. ''They're not close to rivaling our traditional business yet,'' he said, ''but we're really excited about the growth.''
Part of the reason the Web classes have not caught on more quickly, Mr. Basili said, is that students are not as ready for online education as some believe. ''People get attached to how they traditionally learn, and this is not how they're learning in school.''
Ann Kirschner, president of Comma International, an education and technology consulting business, agreed. ''Their high school experience bears no resemblance to the rest of the way they communicate and learn, because the high school instructors haven't caught up,'' she said. ''Yes, classrooms have Web connections, and many have computers, but they're in the back of the room, literally and figuratively.''
''The classroom experience is magical, no doubt about it,'' said Ms. Kirschner, who taught at Princeton University before embarking in a career that eventually led her into online media and education ventures. ''But there's also no getting around the fact that the kids love this technology, and it can help make learning fun, and there's nothing trivial about making learning fun.''
SparkNotes (www.sparknotes.com), part of the Barnes & Noble Learning Network, will sell online practice SAT tests to 500,000 students this year alone, said Justin Kestler, the site's former editorial director, who is now a consultant with the company. The company provides the first practice test free, along with scores and a diagnostic report, then sells tests for $5 each, or $25 for an unlimited number.
The College Board, a nonprofit organization that administers the SAT, will also join the low-cost online preparation market this fall, in advance of the new SAT test to be offered next year. David Justus, a member of the College Board's product development team, said the organization had not set prices for the online course, but ''it'll be at a meaningful discount to the expensive commercial offerings now available.''
Lower income students will qualify for discounts from the base price of the online course, Mr. Justus said. ''The goal here is to broaden student access to SAT preparation, and to college,'' he said. ''We're hoping this will help level the playing field.''Continue reading the main story


text:
Five hundred local residents are needed to spend one day caring for the community on Friday, Sept. 12, by signing up for one of 31 volunteer projects happening across the region on United Way's Second Day of Caring, sponsored by Nationwide.
The event, which kicks off the fall fundraising campaign, will start at 8 a.m. with a breakfast and rally at Cal Expo and keynote presentation by Sacramento Kings president Chris Granger. Volunteer projects will begin at 10 a.m. in Yolo, Amador, El Dorado, Placer, and Sacramento counties.
"We're excited to watch companies, volunteers and nonprofits come together again for an amazing day transforming our community through volunteer projects," said Stephanie McLemore Bray, United Way president and CEO. "This is what United Way and the community do best — joining hands with people across the region to make a tangible difference."
The event also kicks off United Way's new five-year strategic plan designed to have local residents think of United Way as its first course of action when there is need in the community.
"We're creating even more pathways to success for individuals and families in our region so they can achieve their potential through education, financial stability and healthy living," Bray said. "We're going to do this by joining and building coalitions, having donors and volunteers speak out about their work, advocating at a higher level, mobilizing volunteers and continuing to fine-tune our money management so we can do as much as possible."
The organization is rolling out a new brand with a fire extinguisher box to show how United Way is the number one tool for meeting the community's most urgent needs.
"We all have a natural inclination to protect, help and give to those in need," Bray said. "If there were a fire, we would simply look for the fire extinguisher, break the glass and put out the fire. That's what United Way is here for: putting out the biggest fires in our community right now, which include low graduation rates, low financial stability and high obesity."
Sign up at: http://www.yourlocalunitedway.org/dayofcaring by Sept. 5. Last year, 362 volunteers completed 25 volunteer activities across five counties.
For more than 90 years, United Way California Capital Region has brought together nonprofits, businesses, donors, volunteers, community leaders and more to meet the community's greatest needs, give immediate aid and find lasting solutions for future generations.


text:
It has been a difficult week for the tennis world, particularly the British contingent.
The death of Elena Baltacha has cast a pall over everything and last night, after he squeaked out a three-set win over Nicolas Almagro at the Mutua Madrid Open, Andy Murray dedicated it to his late friend and compatriot.
As he left the court, he signed the TV camera lens, as is the custom, and then added the name "Bally" and a heart. "It's been a tough 10 days or so," Murray said. "I heard what was going on because my mum was very close to Bally and her husband, so I knew."
Loading article content
It was a tough couple of hours on court, too, as Murray looked on course to win in double-quick time then almost lost from a winning position and finally held on to stumble over the winning line 6-1, 1-6, 6-4. It was not pretty and it was not a match for the faint-hearted but he got the job done. After almost a month without playing a competitive match, winning was all that mattered.
For a set, Murray could do no wrong. Almagro was limping with a foot injury and, unable to run or move, he could only wallop any ball that happened to land near his racket. But after 40 minutes of one-way traffic, suddenly the momentum changed. Murray lost his rhythm, Almagro began to feel a little better and all at once it was the Scot who was taking a hiding.
"If I got ahead two-love at the start of that second set, then I think it could have been a bit more comfortable," Murray said. "But when I got broken there, he started playing much better."
He managed to put the brakes on Almagro in the third set. As the pair traded blows, it was a case of who would make the first mistake, and two errors from the Spaniard sealed it. One brought up match point and the next one handed victory to the world No.8, which will bring him back today to face Columbia's Santiago Giraldo, a 6-4, 6-3 winner over Jo-Wilfried Tsonga. "He's playing some good tennis," Murray said of Giraldo. "He likes the clay. I played him before at the French Open a couple years ago. I played a good match against him, but he's gotten a new coach. He's working with Fernando González, so it's exciting for him."
Meanwhile, reports of Rafael Nadal's demise appear to have been exaggerated. He may not have won a tournament on European clay this year but he is getting back into his stride and yesterday made mincemeat of Juan Monaco, one of his best friends on tour.
Nadal gave Monaco a 69-minute hiding to win 6-1, 6-0, a victory so one-sided that the Argentine thought he ought to pay his pal a few pesos for the master class. Nadal is back in action today against Jarkko Nieminen.


text:
Ed Young Television Devotional
Men and Women - The Final Four
Husbands, love your wives, just as Christ loved the church and gave himself up for her (Ephesians 5:25).
Because God made us, both male and female, in His image, God has stamped his masculine qualities on men and his feminine qualities on women. And one of the characteristics that God has stamped on men is that of initiative. Man is wired up to lead, to step out, and to close the deal.
God expects men to take spiritual initiative in the family and in the church. The husband should take the romantic initiative in dating and marriage (this includes romancing your wife after marriage!). Men should also take the moral initiative in the workplace and in society. But, most importantly, men should take the initiative in service and sacrifice, just as Christ served and sacrificed himself for his bride, the Church.
Men are initiative-takers because God is an initiative-taking God. 1 John 4:19 says, "We love because he first loved us." Men, lead the way this week in love, in forgiveness, in sacrifice, and in righteousness, just as God, in Christ, led the way for you.
FIFTY SHADES OF THEY From New York Times Best-Selling Author Ed Young
Fifty Shades of They gives you fifty simple, yet profound insights that will help any relationship thrive, from friendships to business partnerships to marriages. Based on biblical standards and the teaching of Ed Young, this book is written for anyone who is looking to give new life to their relationships.
For more information about this and other subjects, visit the Ed Young Television website: www.edyoung.com


text:
How to Survive the Apocalypse
September 23, 2017
Entropion is the turning in of an edge of an eyelid, causing the lashes to rub against the eye. It usually is seen on the lower eyelid.
Entropion can be present at birth (congenital).
In babies, it rarely causes problems because the lashes are very soft and do not easily damage the eye. In older people, the condition is usually caused by a spasm or weakening of the muscles surrounding the lower part of the eye.
Although rare in North America and Europe, trachoma infection can cause scarring of the inner side of the lid, which may cause entropion. Trachoma scarring is one of the three leading causes of blindness in the world.
Risk factors for entropion are:
Your health care provider can usually diagnose this condition by looking at your eyelids. Special tests are usually not necessary.
Artificial tears can keep the eye from becoming dry and may help you feel better. Surgery to correct the position of the eyelids usually works well.
The outlook is usually good if the condition is treated before eye damage occurs.
Dry eye and irritation may increase the risk of:
Call for an appointment with your health care provider if:
If you have entropion, the following should be considered an emergency:
Most cases cannot be prevented. Treatment reduces the risk of complications.
See your doctor if you have red eyes after visiting an area where there is trachoma (North Africa, South Asia).
Howard GR. Eyelid retraction. In: Yanoff M, Duker JS, eds. Ophthalmology . 3rd ed. St. Louis, Mo: Mosby Elsevier; 2008:chap 12.4.


text:
Drunk girl goes on rampage inside police station in Mumbai
Tue, Jun 28, 2016, 10:28 AM
A 21-year-old student assaulted three women constables and a police officer and pulled off wires attached to computers at Worli police station in Mumbai. Police brought her to the police station after she drove her vehicle over a divider in an inebriated state along with three male friends. Even, she used foul language against women constables and broke three mobile phones.


text: 9/28/08 9/28/08 POPSUGAR Fashion Bruno Pieters Paris Fashion Week: Bruno Pieters Spring 2009 September 28, 2008 by coutorture 0 Shares Chat with us on Facebook Messenger. Learn what's trending across POPSUGAR. View On One Page Photo 2 of 21 ADVERTISEMENT () Start Slideshow Share This Link Copy What's Your Reaction? Thanks for your reaction Don't forget to share this with your friends! ← Use Arrows Keys → 2 0 previous images -10 more images Previous Next Start Slideshow Paris Fashion Week Spring 2009Spring 2009Bruno PietersGalleryRunwayDesigner


text:
The pirate site plans to ignore the lawsuits from Elsevier and the American Chemical Society.
Gene Therapy Fixes Mouse Hearing
July 9, 2015|
Expressing a gene for a component of the inner ear's hair cells treated a form of genetic deafness.
July 9, 2015|
Expressing a gene for a component of the inner ear's hair cells treated a form of genetic deafness.
July 8, 2015|
A reanalysis calls into question a year-old claim that humans can decipher at least 1 trillion different scents.
July 7, 2015|
Circulating tumor cells, exosomes, and DNA can improve the diagnosis of many cancers. But are liquid biopsies ready for prime time?
July 6, 2015|
The results of a Phase 2 trial suggest that delivering normal copies of the gene that causes cystic fibrosis may slow lung decline.
July 1, 2015|
For assistance in solving crimes, a company has developed a service that will construct a face based on a genetic sample.
July 1, 2015|
Stoned, Anxious, The Deeper Genome, and Testosterone
July 1, 2015|
Meet the digestive tract–lacking oligochaete that has fueled Max Planck researcher Nicole Dubilier's interest in symbiosis and marine science.
July 1, 2015|
A chance discovery results in the first report of DNA-based gene silencing in an animal.
July 1, 2015|
A love of the ocean lured Nicole Dubilier into science; gutless sea worms and their nurturing bacterial symbionts keep her at the leading edge of marine microbiology.
June 29, 2015|
And too few insights gleaned from them


text:
found a new home!
Plenty of my friends are looking
for one too. Find a pet to adopt.
I am already neutered, up to date with shots, good with kids, and good with dogs.
Sonny - adoption pending, a Norwalk Papillon/Dachshund puppy was adopted! Here's how you can benefit by looking at adoption as an option to buying a Papillon/Dachshund for sale in Norwalk, or seeking a Papillon/Dachshund puppy from a breeder in Norwalk.


text:
Microsoft is beefing up its Zune music service right before the holiday season kicks off in an effort to take some market share away from Apple's iPod sales. The company has announced a new Zune Pass subscription model. Most other subscription models allow you the ability to simply access all the music you want, Microsoft's new subscription model is offering a little something extra.{ad}
Zune Pass subscribers will now be able to select 10 tracks per month to keep. Typically, in order to keep ten tracks you would have to buy them at an estimated value of US$10. That's one heck of a value considering the monthly subscription for Zune Pass is US$14.99 a month.
Microsoft launched the Zune media player and the corresponding Zune music service two years ago. The move was in response to Apple's domination of the music market with their iPod media player and accompanying iTunes music service. Microsoft believes that 90% of the music on the Zune music service will soon be in MP3 format. MP3 format allows the music to be played on more devices including many mobile phones and iPods since the format is DRM-free.
Read more at the Microsoft press release
Brian's Opinion
This is exactly the kinf od step Microsoft needs to take if they really want to offer some serious competition to Apple. Now don't get me wrong, I'm not living in a dream world where I believe that Microsoft is going to grab significant market share from Apple overnight. Apple's lead is too significant for that.
That's not to say that Microsoft won't be able to grab some market share from Apple, especially from iPod users who are looking for a subscription model to enjoy an unlimited amount of tracks per month. The Zune's added bonus is the ability to keep 10 tracks as well.
It's nice to see the music industry start to step up and welcome the idea of unique business models in order to sell more music. If they aren't willing to give a little the music industry will take the brunt of the downturn in the current economy.
The nice thing about Micorosoft's Zune Pass subscription is that it gives music lovers the ability to enjoy an unlimited amount of music at a fixed monthly rate. While in turn being able to keep a full CD worth of music at no additional cost every month.


text:
How trauma affects your brain — we're doing it again! The free HOW TRAUMA AFFECTS YOUR BRAIN webinar this week caused such a groundswell of response we've been overwhelmed by emails asking us to do an encore presentation. So, of course, I've agreed!
I have to admit, it was easy to say, yes, to the requests for an encore when the emails were so enormously positive.
Here are just a few examples:
That call last night was a GAME CHANGER for me…
It's extremely interesting, and I'm finding out why I reacted to things the way I did. Thanks for the good teaching. This brain research is amazing because it shows why people act the way they do.
It was such a great webinar… I will recommend it to my clients and other professionals. Everyone should hear it and gain the information your offer on understanding the brain and trauma.
You have provided so much healing information this evening to so many people!
Thank you for your great webinar. It was fantastic.
So, next Tuesday, April 1st, 2pm EDT, we'll host the whole event all over again. Are you in? Even if you can't make the event at that time, sign up because the replay will be available afterward. Sign up here:
With PTSD it's easy to experience symptoms and think you're crazy, the one to blame and at fault for the insomnia, anger/rage, nightmares, flashbacks and other PTSD symptoms that are part of your daily experience. But that's far from the truth. Your brain is designed to create those symptoms when it gets disrupted by trauma.
PTSD & the brain is a hot topic. With the recent advances in the field of neuroscience — and particularly related to neuroplasticity, the ability of your brain to change… again and again and again — the idea of how trauma affects the brain, and how healing can help the brain reverse those effects is exciting for survivors, their caregivers and every healing professional. Next Tuesday, April 1, 2pm EDT, join me for this FREE webinar, How Trauma Affects Your Brain. On the webinar we'll cover:
- 3 significant ways trauma changes your brain
- Why you can't "just get over it"
- How your brain is designed to create PTSD symptoms and why
- How your brain functions before, during and after trauma
- 6 things that stop your brain from properly functioning after a traumatic event
- 4 parts of your brain most important in relation to trauma, memory, PTSD symptoms and recovery
Even if you're halfway around the world and the time doesn't sync up for you, sign up anyway if you want to personally own this information: We'll send you the replay link after the event. Plus, I'll be giving away a free audio series to help you take what you learn and put it into action in your own recovery process.


text:
Dutar Canvas Tote Bags are an Idakoos specialtiy! Idakoos has many things to offer. Whether you want to get a Dutar Canvas Tote Bag for a special occasion, give a Dutar Tote Bag as a present or need to represent with a funny Custom Dutar Bag during Holidays or Family and Friends get-togethers, Idakoos is sure to have the right Dutar Canvas Tote Bag for you! Even if it turns out you can't find the right Custom Dutar Bag with the perfect design for your mother, father or children (or friends!); Idakoos offers all of its Dutar Canvas Tote Bags as Custom Dutar Tote Bags. Personalize any Dutar Canvas Tote Bag the way you want to ensure the perfect gift.
Showing 1 - 24 of 92 Canvas Tote Bag


text:
Will it come back? That question has been haunting public health officials in New York City and state since a surprise outbreak of the West Nile virus sickened more than 60 people late last summer and killed seven. No more cases of this rare illness were detected after temperatures started dropping in September, rendering the climate inhospitable to the mosquito that transmits the disease, most likely a subspecies of Culex pipiens. But nobody knew whether the virus would be able to survive the winter, either in mosquitoes or their eggs, or in birds, the virus's animal reservoir.
Now evidence suggests that the New York region may indeed be at risk again this summer. Over the past few months, the Centers for Disease Control and Prevention (CDC) and city and state researchers have been monitoring mosquito populations in sewers, abandoned hangars, swimming pool utility rooms, and other likely winter shelters. Last week, they announced that a sensitive polymerase chain reaction test had picked up traces of viral genetic material in three Culex samples in Fort Totten, an historic site in the New York borough of Queens.
Although the find suggests that the insects still harbor the virus and may start spreading it anew when they go looking for bird and human blood, researchers cannot be sure, because they only detected RNA fragments; another test that looks for live viruses came up negative. One interpretation is that the number of viral particles was low enough to escape detection, CDC officials say. Another possibility is that the virus may have become inactivated in the mosquitoes or killed as the specimens were processed. Attempts to isolate the virus using a different technique are under way.
Despite the uncertainty, the results have experts worried--most notably some entomologists, who question the city's and state's ability to wage an effective war against mosquitoes this spring and summer. With viral epidemics such as AIDS taking a huge toll, insect-borne diseases have been "pretty far back on the burner" in New York, says John Edman, director of the Center for Vector-Borne Disease Research at the University of California, Davis. He points out that New York state is still in the process of hiring personnel for new surveillance and control programs, making it difficult to mount an aggressive larvae control campaign in early spring, as experts recommended during a workshop in January. "I'm really worried," adds Yale medical entomologist Durland Fish. "In all honesty, I don't think they'll be able to mount an effective response."
But state officials dismiss those worries. "We have been working very diligently," says a spokesperson for the state Department of Health in Albany. "We will be prepared."


text:
Efforts by a small Colorado community that began with the best of intentions - rallying around a dying child - ended with many people feeling used and betrayed by one of their own. According to a CBS News story:
A Colorado mountain community rallied around a dying child with leukemia only to find out the whole thing was a hoax, and the woman who made up the story probably won't face any charges. So many people fell for the story and tried to help the little boy who didn't even exist.
The hoax began a few weeks ago when a 22-year-old woman named Briana Augustenbourg told co-workers about Alexander Jordan, a nine-year-old family friend dying of leukemia. The little boy had been courageous through his treatments, and was a fan of the local high school football team.
The team invited Jordan to attend their game, released balloons in his honor and signed a football for him. Soon the local newspaper reported on the community outpouring of support for the brave little boy, and even ran an obituary for him.
However, it was soon discovered that the whole thing was a cruel hoax. Alexander Jordan never existed, and a photo that had circulated of him was taken from a cancer web site of a sick (but alive) patient.
Local authorities seem puzzled by the apparent lack of motive, and because Augustenbourg did not explicitly request donations of money or services on behalf of Jordan or herself, the police are unlikely to charge her with any crime. Though many are outraged by her actions, the fact is that lying about having a disease isn't a crime.
Often cancer fakers do it for attention and sympathy, not because they are necessarily trying to scam people out of money. Some people really do have a disease, not cancer but a mental illness known as a factitious disorder. People with this disorder pretend to have an illness (usually a terminal one) and often go to great lengths to maintain the hoax.
Just as it's not illegal to lie about having a disease, it's not illegal to lie about military service, as the Supreme Court ruled earlier this year. Lying or exaggerating military service is fairly common, from claiming medals not earned to telling war stories or claiming to have been a part of elite military units such as the SEALs. In all these cases, the lies are legally protected speech. However if those fabrications are presented in order to steal money or get free services, that is illegal.
Even when laws are broken and money is taken under false pretenses, those who fake illnesses are usually let off lightly. For example, in 2009 a young Canadian woman named Ashley Kirilow made news for fighting bravely against breast, ovarian, brain, and liver cancers, and raised money for her treatments.
Kirilow and her supporters started a charity called Change for a Cure, which asked people to donate their spare change so she could donate the money to the University of Alberta for cancer research. She also opened Facebook and Twitter accounts for the organization, with the slogan, "Together we can change the world one penny at a time." Fundraisers were held for her, and a Toronto charity flew terminally ill Ashley to Disney World as her dying wish. Altogether over $20,000 was raised for her.
Kirilow eventually admitted that she never had cancer. She had shaved her head and eyebrows to fake the signs of chemotherapy, and had spent much of the money given to her on personal expenses. She was arrested and charged with fraud.
Kirilow later defended her actions, saying she faked having cancer because she wanted attention from her family: "Originally it was because I was alone and had no one who cared about me. I just wanted (my family) to change their crazy ways and love me and be a normal family."
Kirilow was spared jail time, instead sentenced to 15 months house arrest and ordered to repay the victims of her scam.
Unlike Kirilow, Alexander Jordan was a completely fictional person. This case is reminiscent of Anthony Godby Johnson, the "author" of a 1995 memoir titled "A Rock and a Hard Place," about a young boy who endured child abuse, street life and even AIDS. Billed as an inspiring autobiography of an extraordinarily courageous boy, it even featured an introduction by Fred Rogers (better known as TV's Mr. Rogers).
The book was later revealed to be completely fiction; Anthony Godby Johnson never existed.
The legality of publishing faked memoirs isn't clear, though when James Frey's 2005 autobiography "A Million Little Pieces" was exposed as a hoax, his publisher had to offer over $2 million in refunds to readers who bought the book under false pretenses.
Credit: Corbis Images


text:
Beautiful Cockatoo, very loveing, great addition to someone that can give her a good home. Loves everyone, $1200.00 or best offer, moveing soon must sell…$1,000
All major credit cards accepted () - Photos and contact information on Advertigo website.No Price Listed
Homer female is an amazon parrot. She is years old and she loves to talk. She comes with a large cage with a play area on top and an large outside the…No Price Listed
Valentino is friendly and loving. He has never had a bad day and has always been cherished and loved. He is used to other animals and birds being around…SOLD
Mango is a sweet baby girl. Mango is learning to talk and picks up words very well. She loves to cuddle and give kisses. She loves everyone and loves to…SOLD
Sweetest Macaw on the face of the earth. Emmett is a loving sweet boy who goes with the flow. He is learning to talk and such a Casa Nova. He has been…SOLD
I have 2 greencheek conures. One is cinnamon I think is what they call it and the other is just a regular. Asking 250.00 for the pair, have been together…SOLD
Not a hand tamed bird. He is a bit of a plucker, goes only to the gray. Have had him for over a year. Would make a good breeder or what ever you wanted…SOLD
Green cheek conures, got in a package deal last year. Have too many types of birds. Will take 225.00 for the pair or 125.00 each.SOLD
These are proven breeders, not pets. The female is green/ blue and the male is yellow.SOLD


text:
News that a Vacaville pilot was killed in a plane crash south of Paradise on Tuesday was greeted with shock Wednesday by the local airport community.
Butte County officials wouldn't confirm who was flying the plane, but did verify that it was a Champion 7GCAA, a small two-seat plane, registered to Fred Lewis, co-owner of AA Aerial Surveillance in Vacaville. The two people on the plane were killed, according to a report from the Federal Aviation Administration. The crash caused a fire that completely destroyed the plane and burned 21 acres before being brought under control.
Through word of mouth, the local pilot community confirmed Lewis' death in phone calls and e-mails. A memorial is planned for Lewis on Friday at Nut Tree Airport in Vacaville.
Local pilot Lloyd Tincher knew Lewis for more than 20 years and called him a good friend. He learned of Lewis' death Wednesday morning.
"He was a professional," Tincher said. "He was very outgoing and he knew everybody. He was a good person to know. You certainly weren't bored around him."
AA Aerial Surveillance co-owner Jerry Anderson would not confirm Lewis' death.
The circumstances of the crash are still unknown, but the FAA and the National Transportation Safety Board are investigating.
Contracted by Pacific Gas and Electric Co., the pilot was reportedly patrolling a natural gas transmission pipeline in the area.
Lewis was well known in the local aviation community. He was active in the effort to establish the Jimmy Doolittle Air and Space Museum at Nut Tree.
"I'm stunned to hear of this loss," said former Vacaville Mayor Len Augustine, who serves on the fundraising cabinet for the museum effort. "He was a great guy. A huge, huge loss."
Tom Kane, who served on the museum's board with Lewis, wrote in an e-mail that Lewis died doing what he loved.
"Chief Fred was an outspoken advocate for our Air Force and our heritage and brought great respect to all airmen and their families," Kane wrote.
He added that Lewis worked with the Collings Foundation and its Wings of Freedom tour to help recognize the contributions of airmen of every generation and instill a sense of patriotism in every citizen. Lewis was also the president of the Travis Heritage Center.
The profile on the company's website said that Lewis served from 1976 to 2007 in the U.S. Air Force, before he retired. He flew as a crewmember aboard C-5s, C-141s, KC-10s and VC-25s. He was a FAA designated examiner from 1989 to 2003. He also had logged time in B-17s, B-24s and B-25s. He was a commercial pilot, air frame and power plant mechanic, had logged more than 15,000 hours of flight experience in various aircraft and was operations qualified in pipeline patrol, the site noted.
Friday's memorial will be from 11 a.m. to 2 p.m. at the Copart hangar at the airport, 301 County Airport Road. Drinks and snacks will be provided. Those who wish to attend are asked to RSVP and send a favorite photo of Fred to Serafina Chavez by noon today to firstname.lastname@example.com.
The Paradise Post and Enterprise-Record, Chico, contributed to the report.
Follow Staff Writer Melissa Murphy at Twitter.com/ReporterMMurphy.


text:
conveyor belt
West Virginia mine safety officials on Thursday issued 253 violations against Massey Energy in their final report on the 2010 Upper Big Branch mining disaster that killed 29 men. Jeffrey Brown and NPR's Howard Berkes discuss the findings and prosecution efforts to reach higher into the ranks of Massey's upper management. Continue reading


text:
SF Giants Rumors: 2012 World Series Starts Tonight!
- By admin
- 24 October, 2012
- Comments Off on SF Giants Rumors: 2012 World Series Starts Tonight!
The stage is set for the start of the 2012 World Series. The Detroit Tigers are representing the American League and the National League is represented by your San Francisco … Continue Reading →
SF Giants Rumors: The Giants Win The Pennant!
It's been a wild and crazy post season for the San Francisco Giants, and it's not over yet! These guys have showed no quit, and a play with a ton … Continue Reading →
SF Giants Rumors: Vogelsong Delivers, Cain's Turn In Game 7, Etc.
The 2012 playoffs haven't gone as smooth as the San Francisco Giants would have liked, but they seem to strive with their backs to the wall. Ryan Vogelsong pitched … Continue Reading →
SF Giants Rumors: Vogelsong Brings It, Scutaro Injured On A Dirty Holliday Slide
- By admin
- 16 October, 2012
- 2 Comments
The National League Championship Series is all tied up at one game apiece. It's now a best of five game series, and the next three are in St. Louis. … Continue Reading →
History Comeback To Win The NLDS, Next Stop Is The NLCS!
The San Francisco Giants have just done the unthinkable! They managed to run the table in Cincinnati, and win three games in a row to advance to the National League … Continue Reading →
SF Giants Rumors – Crowds View of Uribe's Sac Fly
- By admin
- 25 October, 2010
- 3 Comments
Hey Giants fans is this the year? This year has been torture, but this group of guys sure make it fun! Below is a video that was sent to … Continue Reading →
SF Giants Rumors – Cody Ross and Dan Uggla Rumors
- By admin
- 20 October, 2010
- 2 Comments
The San Francisco Giants hottest hitter in the playoffs Cody Ross says he wants to finish his career with the Giants. Ross is in his final year of arbitration, … Continue Reading →
SF Giants Rumors – Game 1, 2 and 3 Matchups!
Here is a look at the numbers. The game one, two and three starters for the Philadelphia Phillies and the San Francisco Giants. … Continue Reading →
SF Giants Rumors – Lincecum Blister, Rotation, and NLCS Roster
- By admin
- 14 October, 2010
- 2 Comments
Giants manager Bruce Bochy was on KNBR this morning with Murph and Mac. When asked about the blister on Tim Lincecum's finger. Bochy said, Timmy is fine. He also … Continue Reading →


text:
TUSCALOOSA |When it comes to running backs, University of Alabama sophomore linebacker Dont'a Hightower usually doesn't pause, even when asked a question about how to attack one.
Sophomore Mark Ingram, though, is not just any running back, especially considering the driving nature of his 5-foot-10, 212-pound frame. Not only did it key his 728-yard freshman season, but made him the Crimson Tide's preferred ball carrier near the goal-line.
"What I do, is jump on his back," Hightower said. "I can't bend my knees enough to get low enough to hit him. He's too strong to try and bulldozer him. So I just try and jump on his back during practice, hold him up until I get some help over there."
Monday afternoon, when Coach Nick Saban unveiled the Crimson Tide's initial depth chart for the 2009--10 season, there were no surprises at the running back position where Ingram was listed first among four names - those who will likely take nearly every hand-off this season.
Behind him, junior Terry Grant and senior Roy Upchurch shared the No. 2 spot, with Trent Richardson the first name Saban mentioned when asked about what kind of impact the true freshmen might make this season.
Ingram's impact will likely depend on how well he completes the transition from spot player - with his 12 rushing touchdowns setting an Alabama freshman record last season - to every-down running back. Granted, he took 143 hand-offs in 14 games in 2008, but most of the workload went to Glen Coffee (233 carries, 1,417 yards).
"I just have to be more consistent, as far as making my reads on a regular basis and picking up pass protection, being the best player I can be," Ingram said last week.
As for the physical side of it, he added: "You're going to take few extra hits, but as long as you take care of your body I think you'll be all right."
The four-deep also signaled one other important facet, in addition to the starter, that like usual Saban won't hesitate to insert someone into the game, whether for fresh legs, a third-down back or change of pace. He might give each running back a different role.
Ask the Tide players and their own scouting reports are remarkably consistent: Upchurch is explosive; Grant is the speed guy; Richardson is the up-and-comer.
"Each one of us has something different to offer," Ingram said. "I think as a running back corps we're going to be pretty good.
"I like to do everything, run between tackles, run outside, break big runs, run short yardage, run routes, catch passes. I just consider myself an all-around back."
Yet running back was the position with the most uncertainly during fall camp.
Sophomore Jermaine Griffin was lost for the season with a knee injury. Upchurch had what he called a groin "tweak," but is back practicing with the team after missing the second of two scrimmages. Richardson sustained a sprained shoulder during the most recent scrimmage, adding injury to insult (not the other way around) after fumbling on the same play.
And Ingram?
He and standout sophomore receiver Julio Jones have been investigated by the university for enjoying an offseason fishing trip with a non-booster. Although the school has not reported any violations, it has been in contact with the National Collegiate Athletic Association and the Southeastern Conference and the matter has yet to be resolved mere days before the season opener.
Meanwhile, the player to take advantage was Grant, even though he went from 892 yards on 180 carries in 2007 to just 88 yards off 35 hand-offs last season. Teammates have been quietly praising his work ethic, while Hightower added "He'll run by you in a blink."
"Terry Grant has done an outstanding job, and did a very good job last year," Saban said. "I think he handled an adverse circumstance last year on our team as a guy who had gotten hurt and coming back from that injury sort of had some guys pass him up that he probably didn't count on. … Instead of going into a shell and accepting that circumstance he has worked as hard as anybody on our team and been as positive in energy and attitude as anyone on our team.
"We feel like both (Ingram and Grant) can be starters. He has speed and is a space player, and hopefully we can get him the ball so that he can make plays and give us another weapon on offense."
So the pecking order is set for now, with each of the four having something to prove. But against Virginia Tech, it appears that Ingram will get the first shot, and everyone already knows how tough he can be to stop.
"Every running back is going to have his own style," senior guard Mike Johnson said. "I think that it's something that's going to develop over time and Mark's going to have a lot of reps to do that. Mark's going to bring his own physical style to the table. He might be a little quicker and a lot more powerful (than Coffee). He's going to be a good runner between the tackles."


text:
Free Shipping on Live Orders over $99!
Synodontis Lace Catfish, Synodontis nigrita, is one of the most popular of this species, growing to a typical captive length of around four inches. Often mistaken for the Upside down catfish, it is a unique gray color with dark spots. It usually found singly, hiding out in caves during the day. It can make a good scavenger for an African Cichlid aquarium, though some older, established specimens may not tolerate other Synodontis cats. It needs to be treated as a smooth skin catfish when treating for parasite infestations. The diet of the Synodontis Lace Cat should include meaty foods (blood worms) and some vegetable based sinking pellets. If properly maintained, the this fish can live for many years.


text:
Three "blood substitutes," a diaspirin cross-linked human hemoglobin (DBBF-Hb), a bovine polymerized hemoglobin (PolyHbBv), and a human polymerized hemoglobin (O-R-PolyHbA0), that have undergone clinical trials are used in this study. Previously, we showed in the rat that coadministration of sodium selenite (Na2SeO3) and DBBF-Hb significantly decreased mesenteric venular leakage and epithelial disruption produced by DBBF-Hb alone but did not reduce mast cell degranulation unless given orally. The purpose of this study was to determine whether Na2SeO3 produced similar beneficial responses when used with PolyHbBv and O-R-PolyHbA0. In anesthetized Sprague-Dawley rats, the mesenteric microvasculature was perfused with PolyHbBv or O-R-PolyHbA0, with and without Na2SeO3 in the perfusate and suffusate, for 10 min, followed by FITC-albumin for 3 min, and then fixed for microscopy. Na2SeO3 did not reduce leak number or area in preparations perfused with PolyHbBv and only reduced leak number (but not significantly) in preparations perfused with O-R-PolyHbA0. Na2SeO3 significantly increased mesenteric mast cell degranulation and impaired epithelial integrity in animals treated with PolyHbBv. In vitro, Na2SeO3 significantly reduced the oxidation rate of DBBF-Hb in the presence of oxidants, had little effect on PolyHbBv, and increased the oxidation rate of O-R-PolyHbA0. These results suggest that Na2SeO3 moderates hemoglobin-induced damage, at least partly, through its redox interactions with the heme sites in the hemoglobin molecules studied and that accessibility of the heme site to Na2SeO3 governs those interactions.
- rat mesentery
- mast cell degranulation
- intestinal mucosal epithelium
cell-free hemoglobin (Hb)-based oxygen carriers, such as diaspirin cross-linked Hb (DBBF-Hb), PolyHbBv (Oxyglobin), and O-R-PolyHbA0 (Hemolink) have been proposed as blood substitutes for transfusions because of their plasma expansion and oxygen transport capabilities. In addition to their use after accidents or major surgery, such substitutes could also be employed to alleviate anemia in patients with hematocrits too high to qualify for blood transfusions. In fact, PolyHbBv (Oxyglobin) is currently approved by the Food and Drug Administration for veterinary use in the United States, and its human counterpart has recently been approved for clinical use in humans in South Africa. Hb-based blood substitutes have the added advantages that they can be easily purified, stored for relatively long periods of time, and used in patients of all blood types. However, a number of largely unresolved problems were found during preclinical trials and development of some of these Hb-based substitutes. These include cardiovascular and/or hemodynamic effects, gastrointestinal changes, immune cell activation, coagulation changes, oxidative stress, and decreased host resistance to overwhelming infection (20). A dose-response study performed on dogs by Biopure has shown that Oxyglobin increases arterial oxygen content in the face of normovolemic anemia and produces transient clinical signs (skin discoloration, discolored stools, nausea, vomiting). In addition, histopathology of Oxyglobin administration includes activation of tissue macrophages in multiple organs (Biopure Oxyglobin solution, package insert 2003, Biopure www.biopure.com/products/vetpackageinsert.cfm?cDID=2&cPglD=31#Contraindications). Hemolink has recently been withdrawn from phase III clinical trials in cardiac bypass grafting because it produced adverse cardiac events (www.hemosol.com/companynewsreleases.cfm?newsID=1870&companyID=24).
Previously, our laboratory showed that bolus injection of DBBF-Hb in rats increased venular leakage to bovine serum albumin (BSA) and produced mast cell degranulation in the rat mesentery (6) and also caused detachment of intestinal epithelial cells from each other and from the basement membrane (5, 11). Such changes are characteristic of an inflammatory response (7, 33). This is not surprising because mast cell degranulation, as observed after injection of DBBF-Hb, causes release of histamine and other inflammatory mediators, which then damage cell membranes. It is disadvantageous for a potential blood substitute to cause microvascular leakage because the substitute itself will rapidly leave the circulation and also because alterations in transvascular exchange of plasma proteins will disturb the fluid balance between blood and tissue. Increased microvascular leakage also changes the kinetics of delivery of intravascularly injected drugs and of endogenous enzymes and hormones to various tissues. When transfusions are needed, for example after hemorrhagic shock, it is important that regulation of microvascular exchange is not compromised. In another study, we showed that mesenteric microvascular leakage to BSA, mesenteric mast cell degranulation, and epithelial disruptions were significantly lower in animals treated with PolyHbBv compared with those treated with DBBF-Hb (9). However, even in animals treated with PolyHbBv, the average number of leaks per unit length of venule was still significantly greater than for controls that were perfused with HEPES-buffered saline with 0.5% BSA (HBS-BSA).
It has been hypothesized that the deleterious effects of intravascularly injected free modified Hbs are caused by their oxidation and subsequent formation of reactive oxygen species (1). In fact, we have shown that the antioxidant sodium selenite (Na2SeO3) was effective in significantly reducing the intestinal epithelial damage (8) and microvascular leakage (10) associated with bolus injection of DBBF-Hb in rats. Simoni et al. (28) also demonstrated that treatment with Na2SeO3 was very effective in the prevention of oxidative damage induced by Hb. In vitro, Na2SeO3 reduced the oxidation rate of DBBF-Hb while in the presence of oxidants (10). Thus it appears that Na2SeO3 moderates Hb-induced damage at least partly through its interactions with the Hb. The following study was performed to determine whether coadministration of Na2SeO3 would significantly reduce the microvascular damage caused by PolyHbBv and O-R-PolyHbA0 in the rat mesentery and whether site-specific modifications of Hbs can play a role in the interactions between Hb and selenium (Se). To aid in the interpretation of the results of the animal experiments, the interactions of Na2SeO3 with PolyHbBv and O-R-PolyHbA0 were investigated in vitro and compared with results obtained by using DBBF-Hb and cyanomet-DBBF-Hb (CNmet-DBBF-Hb) (8, 10), a Hb in which the cyanide (CN) groups are tightly bound to the heme such that the molecule is unable to participate in redox reactions. Effects of Na2SeO3 on the rates of autoxidation, oxidation, and oxygen affinity of the Hbs were measured. In addition, the effect of Na2SeO3 on percent reduction in ferric (met) forms of the modified Hbs were determined.
Hb Solutions
Glutaraldehyde-polymerized Hb (PolyHbBv) was purchased from Biopure (Boston, MA). Spectral analysis of the solution just before injection demonstrated the presence of 5–8% of the met form. This percentage of metHb in a modified Hb for clinical use is acceptable according to criteria initiated by the manufacturers of these products. PolyHbBv contains a heterogeneous mixture of polymeric (∼95%) and nonpolymeric (∼5%) species ranging in size from 32 to 500 kDa (24). Both the oxygen-carrying and redox properties of this Hb have been well documented (2, 16). O-R-PolyHbA0, a purified human Hb (HbA0) that is cross-linked with o-raffinose and oligomerized, was obtained from Hemosol Research (Toronto, ON, Canada). The O-R-PolyHbA0 solution that results from this procedure consists predominantly of polymers of HbA0 [32 kDa (5%), 64 kDa (33%), 128–600 kDa (63%), ≥600 kDa (3%)] (23). In these experiments both Hbs were diluted to 2 mg/ml with HBS-BSA, pH 7.4, and equilibrated with room air. However, the concentration of Hb used in these experiments was less than one-tenth of that found in blood, and, in addition, oxygen has a low solubility in water. Therefore, the tissues were not exposed to higher concentrations of oxygen than they would experience in vivo. All Hb solutions used in this study were free of red cell enzymes, such as superoxide dismutase or catalase.
Preexperimental Treatment of Rats
Animal experiments were conducted with approval from the University of Arizona IACUC. Male Sprague-Dawley rats, weighing 300–350 g, were obtained from Harlan Teklad (Madison, WI). Monthly serology, bacteriology, and parasitology evaluations had been performed on animals from each virus-free barrier at Harlan. The rats were transported to the animal facility at Tucson Veterans Administration Medical Center by truck. The animal facility is small with a low personnel activity, and monthly tests are performed on sentinel rats. On arrival, the animals were housed two per cage in a room (3 m × 4 m) deliberately chosen so as to be remote from noisy air vents, cage washers, and the like. The cages were 45 cm long and 24 cm wide and contained standard Harlan Sanichip bedding. Ten to 20 rats were housed in the room at any given time, and no other rats, apart from those participating in this study, were housed with them. A technician entered both rooms once a day to feed and tend to the rats. The temperature ranged between 72 and 74°F, and the humidity was kept between 55 and 60%. The rats were fed Harlan Tech Lab 485 rat chow and placed on a light cycle with lights on between 6:00 AM and 6:00 PM.
Sprague-Dawley rats were preanesthetized with 1 mg/kg body wt of the following mixture: ketamine hydrochloride (5 ml of 100 mg/ml), acepromazine maleate (2 ml of 10 mg/50 ml), and xylazine (8 ml of 20 mg/ml). This was followed by an intraperitoneal injection of pentobarbital sodium (30 mg/kg). In each rat, a tracheostomy was performed for artificial ventilation.
Design of Animal Experiments
Venular leakage and mast cell degranulation in mesentery. Experiments were performed to characterize the mesenteric microvascular leakage to albumin and mast cell degranulation, caused by intravenous injection of PolyHbBv or O-R-PolyHbA0, in the presence (6 rats per group) and absence (5 and 6 for PolyHbBv and O-R-PolyHbA0 experiments, respectively) of Na2SeO3. The number of animals per group was justified to be sufficient by utilizing a sample-size nomogram in conjunction with estimates of the difference in means that needed to be detected, and the mean standard deviation for each parameter (36). The mesenteric microvasculature was perfused for 10 min with PolyHbBv or O-R-PolyHbA0, with or without 2 μg/ml Na2SeO3. The same concentration of Na2SeO3 was also added to the HBS-BSA suffusate that was applied during the 10-min perfusion. In addition to the animals mentioned, four rats had their mesenteric microvasculature perfused with HBS-BSA instead of a modified Hb, and they served as controls. The results from these experiments were compared with those from a previous experiment to determine the effects of Na2SeO3 on microvascular leakage induced by DBBF-Hb (10). To ensure that there was no systematic difference between the two different batches of rats, two of the present rats were perfused with DBBF-Hb and the microvascular leaks were assessed. These animals gave leak numbers and areas that were similar to those of the previous study. For this reason, DBBF-Hb data from the previous study were directly compared with results obtained with PolyHbBv and O-R-PolyHbA0.
Epithelial integrity and goblet cell secretion in intestine. Two groups of rats (5 per group) were used in experiments to determine the effects of PolyHbBv, with and without Na2SeO3 (Sigma Chemical, St. Louis, MO), on the epithelial integrity of the intestinal mucosa. One group received 2 μg/ml Na2SeO3 in their drinking water. Se has previously been administered to rats in their drinking water at a dose of 0.1 μg/ml and has been shown to be effective in increasing antioxidant enzyme activities (37). In the present experiments, the rats drank an average of 32 ml water per day; therefore they were receiving 64 μg of Se daily. The rats were kept, as described, for 3 wk before experiments were performed on them. The experimental design was as follows: a 5-ml bolus of 10 mg/ml PolyHbBv was injected via the aorta in both groups of rats and allowed to circulate for 30 min before perfusion fixing of the small intestine and preparation of tissue samples for light and electron microscopy. The results from these experiments were compared with those from previous experiments (8) in which DBBF-Hb was used instead of PolyHbBv.
Mesenteric Preparation to Measure Microvascular Leakage and Mast Cell Degranulation
Surgical procedures. The animal procedure was similar to that described previously (31) and is summarized here. After anesthesia, the abdomen was opened and several contiguous well-vascularized mesenteric windows were selected and spread out flat over a Plexiglas platform. A mesenteric window is defined as the tissue extending between two adjacent feeding arterioles in the mesentery. Next, the superior mesenteric artery was cannulated, arterioles and venules surrounding the chosen windows were ligated, and the microvascular network was perfused with HBS-BSA and 1 U/ml heparin at 37°C, followed by 2 mg/ml PolyHbBv or O-R-PolyHbA0 in HBS-BSA, with or without Na2SeO3. The preparation was kept moist during the 10-min perfusion by dripping 20 ml HBS-BSA. The suffusate only contained Na2SeO3 if this was included in the perfusate. When the microvascular network was filled with PolyHbBv or O-R-PolyHbA0, the portal vein was clamped to prevent back-flow of blood, and the microvascular pressure was adjusted to 40 mmHg. After 10 min, the portal clamp was removed and the network was perfused with a fluorescent tracer, BSA labeled with FITC (Sigma Chemical). As soon as the vasculature of the windows was filled with FITC-albumin, as judged by the color, the pressure was adjusted to 40 mmHg, and the portal vein was clamped. After 1 min, the clamp was removed and 3 ml of fixative (3% formaldehyde in HBS) were perfused at a pressure of 100 mmHg. This pressure was necessary to ensure that the venules and veins received adequate perfusion. Next, the pressure was reduced to 40 mmHg, the portal vein was clamped, and fixation was continued at 4°C. After 1 h the mesenteric tissue was carefully excised and each window from the preparation was mounted between two thin glass coverslips by using aqueous mounting medium (Vectashield, Vector Laboratories, Burlingame, CA).
Assessment of venular leakage. An assessment of overall vascular leakage was made by measuring the number and area of regions with extravascular FITC-albumin. Slides were examined by use of a Zeiss Axioplan microscope with a ×10 objective, numerical aperture 0.6, fitted for epifluorescence. The resolution obtained with this system was such that the smallest leak that could be detected was 2 μm2 in area. The light source was a 100-W Hg lamp for epifluorescence and a halogen lamp for transmitted illumination. A video camera (Optronix 750D) was mounted at the camera port of the microscope. Ten images of leaky vessel networks from each mesentery, produced by epifluorescence with the appropriate FITC excitation and emission filters (λ = 488 nm and 515 nm, respectively), were viewed on a black-and-white monitor and also recorded on a video recorder. Each slide was only exposed to the excitation wavelength for 5 s. Recordings were also made of the networks under transillumination. Videotaped images were later analyzed by using an analog-to-digital converter and appropriate software (NIH Image) to measure the length and diameter of each venule, the number of leaks per venule, and the area of each leak. If a leak was positioned at a vascular junction, the leak area was divided by the number of venules involved. Data were pooled within each group and the following values were calculated: 1) average number of leaks per length of venule and 2) average leak area per micrometer of venule.
Assessment of mast cell degranulation. After videotaping of the fluorescent leaks, the tissue was suffused with 1% toluidine blue for 20 s, rinsed with HBS-BSA, and then remounted. Toluidine blue was used to stain the mast cells to determine the numbers that had degranulated. Degranulated mesenteric mast cells, identified by the presence of intracellular granules released into the surrounding tissue, were counted within each circular ×20 microscopic field of view (field area 1.13 mm2). Rows of fields were counted systematically from left to right. Cells located in the periphery of the field were only counted if at least one-half of the cell area was within the field. The error of repeat counting was <2%. From 15 to 30 fields were counted for each mesenteric window.
Data analysis. Each parameter was compared between different groups by using one-way analysis of variance. If a significant difference was found between groups, pairs of groups were compared by using the Student's t-test with a P value <0.05 to determine statistical significance. All values are presented as means ± SE. The n used in the leakage studies was the number of venules examined per group, and the n used for the mast cell degranulation was the number of mesenteric fields of view examined per group.
Microscopy of Intestinal Mucosa to Assess Goblet Cell Secretion and Epithelial Damage
Surgical procedures. After anesthesia, a midline abdominal incision was made to expose the aorta. The aorta was cannulated just downstream from the superior mesenteric artery in a retrograde direction. The free end of the catheter tubing was connected to a reservoir of HBS-2% BSA, pH 7.4, at 37°C. A loop of intestinal ileum, close to the cecum, was pulled outside the body cavity and arranged on a Plexiglas pillar attached to the plastic stage on which the rat was situated. PolyHbBv (50 mg) in a 5-ml bolus of HBS-2% BSA was injected through a 0.2-μm filter via the aortic cannula and allowed to circulate with the blood for 30 min. With the assumption that the animal's blood volume in milliliters is equivalent to 8% of the animal's weight in grams, injection of this amount of blood substitute produced a concentration that was similar to that used in the mesenteric experiments (2 mg/ml). After 30 min, the aorta was clamped proximal to (upstream of) the superior mesenteric artery, and the intestinal circulation was perfused with Karnovsky's fixative in phosphate buffer, pH 7.4, at 4°C. When perfusion was complete, the inlet pressure was dropped to 40 mmHg and the portal vein was clamped. The animal was killed with an intravenous injection of Beuthanasia. Fixation continued for 60 min, after which the intestinal loop was excised and cut into several segments, each containing a Peyer's patch; these segments were washed in buffered saline.
Tissue preparation for histology. Tissue squares were immersed in diaminobenzidine (DAB) overnight in the dark to stain specific granules in immune cells and thus make the cells easier to identify. The DAB was prepared as follows (22): DAB (0.1 g) was added to 50 ml 0.1 M monobasic phosphate buffer, and the pH was adjusted to 7.2 very gradually with concentrated NH4OH. The solution became a light tannish-pink color. Next, the tissue squares were rinsed in distilled water. Meanwhile, 25 ml of DAB solution were added to 1.66 ml 3% H2O2 to give final concentration of 0.2%. The tissue was placed in this solution for 60 min and then rinsed three times in 0.15 M sodium cacodylate buffer. Finally, the tissue was dehydrated in increasing concentrations of ethanol and embedded in Spurr's resin. The pieces of tissue were oriented in the resin so that the blocks could be sectioned perpendicular to the villus plane. Thick sections (2 μm) were cut for light microscopy, mounted on slides, and stained with toluidine blue. Ultrathin sections were cut for electron microscopy (Phillips CM12). Before examination of the sections under electron microscopy, the grids were stained with lead citrate and uranyl acetate.
Data analysis. Thick sections were examined under light microscopy to count the numbers of secreting goblet cells, per villus cross section, in 30–60 villi per animal taken from four different regions of the tissue sample. Only villus sections that contained a central lacteal were included because these villi were centrally sectioned. For collection of data, the slides were coded and the code was not revealed until the data were analyzed, as previously described, with n as the number of villi in each group. Electron microscopy (×3,000) was used to assess the integrity of the epithelium (EI) in each villus cross section examined. Intact epithelium was assigned an EI of 1; epithelium with some cell-cell separation was assigned 2, and epithelial cells detaching from the basement membrane scored 3.
Effect of Na2SeO3 on the Oxidation of PolyHbBv and O-R-PolyHb
Autoxidation experiments. Autoxidation occurs when the ferrous iron (Fe2+), the only form of Hb that carries oxygen, undergoes spontaneous oxidation to the ferric (met) form (Fe3+). Autoxidation experiments for PolyHbBv and O-R-PolyHbA0 samples (50 μM) in Hanks' balanced salt solution (HBSS), pH 7.4, 37°C, in the presence of 500 μM of Na2SeO3 were carried out in the dark. Absorbance changes in the range of 490–640 nm due to the spontaneous oxidation of PolyHbBv or O-R-PolyHbA0 were recorded on a Perkin-Elmer Lambda 6 spectrophotometer. Multicomponent analysis was used to calculate the time-dependent changes in concentration of the oxy, met, and deoxy on the basis of known spectra of each species (21). First-order autoxidation rate constants were derived from a fit of the data during 10-h incubation to a single exponential expression by using a nonlinear least squares fitting program (Origin 6.1).
Enzymatic oxidation experiments. Chemically induced oxidation of PolyHbBv and O-R-PolyHbA0 by low and steady levels of H2O2 was achieved in vitro by use of the glucose-glucose oxidase system (GOX) as previously described (17). The reaction mixture was prepared containing 50 μM oxy-PolyHbBv and 10 mU/ml glucose oxidase in HBSS solution, pH 7.4, which also contained 5.6 mM glucose as substrate at 37°C. Under these conditions, the rate of H2O2 production by the GOX system was 1.5 μM H2O2/min (17). In another set of experiments, 500 μM Na2SeO3 was added to the reaction mixture and spectral changes in the Hb were monitored throughout the experiments.
Se-mediated reduction experiments. In these experiments, the met derivatives of PolyHbBv and O-R-PolyHbA0 (50 μM) were incubated in HBSS buffer, pH 7.4, 37°C with increasing concentrations of Se (125 μM to 1 mM). The reduction of the ferric iron of the Hbs induced by Se was monitored immediately after mixing of Hb solution and Se in a cuvette placed inside a diode-array spectrophotometer.
Effect of Na2SeO3 on the Oxygen Affinity of PolyHbBv and O-R-PolyHb
Oxygen equilibrium curves of PolyHbBv and O-R-PolyHbA0 in the presence of various concentrations of Na2SeO3 (250 μM to 1 mM) were obtained by using the Hemox analyzer (TCS Scientific, New Hope, PA). This instrument measures the oxygen tension with a Clark oxygen electrode (model 5331 oxygen probe; Yellow Springs Instruments, Yellow Springs, OH) and simultaneously uses a dual-wavelength spectrophotometer to calculate the Hb oxygenation. Oxygen equilibrium experiments were carried out in 0.1 M phosphate buffer, pH 7.4, and incubation times ranged from 10 to 20 min. The concentration of Hb samples was between 60 and 75 μM (heme) and the temperature was maintained at 37°C. To maintain the ferric (MetHb) content to a minimum level (<2%), 4 μl of the Hayashi enzymatic reduction system, which consists of a number red cell enzymes and cofactors, were included in the final solution (4 ml) (19). In another set of experiments, the Hayashi system was omitted from the Hb solutions to determine the effects of increasing concentrations of Se on methemoglobin build up throughout the experiments and consequently the oxygen affinities of PolyHbBv and O-R-PolyHbA0. Oxygen equilibrium parameters were derived by fitting the Adair equations to each oxygen equilibrium binding curve by the nonlinear least squares procedure included in the Hemox analyzer software (p50 PLUS, version 1.2) (2).
Mesenteric Preparation to Measure Microvascular Leakage and Mast Cell Degranulation
Distribution of leaks. Table 1 shows the percentage of venules containing leaks for the different groups. For preparations perfused with PolyHbBv without Na2SeO3, 7.7% of venules examined contained leaks. Most of the leaks (62/67; 93%) were <100 μm2 in area. This compares with a luminal surface area of individual endothelial cells that ranges from 400 to 900 μm2 (7). In preparations that were perfused with both PolyHbBv and Na2SeO3, 9.6% of venules examined contained leaks. PolyHbBv preparations, with or without Na2SeO3, resembled controls that were perfused with HBS-BSA when they were viewed by fluorescence microscopy, except that some of the larger venules showed a few small FITC-BSA leaks. Typical mesenteric networks after perfusion with PolyHbBv or with PolyHbBv and Na2SeO3, followed by FITC-BSA, can be seen in Fig. 1, A and B, respectively. For preparations perfused with O-R-PolyHbA0 without Na2SeO3, 9.6% of venules examined contained leaks. Although the majority of the leaks (41/59; 69%) were <100 μm2 in area, this percentage was lower than for PolyHbBv. In preparations that were perfused with both O-R-PolyHbA0 and Na2SeO3, 5.5% of venules examined contained leaks. Although the percentage of venules showing leaks after treatment with O-R-PolyHbA0 was relatively low, the individual leaks were, on average, significantly larger than those produced by Poly-HbBv [173 ± 42 (n = 59) vs. 23 ± 5 (n = 71)], both without Na2SeO3. Typical mesenteric networks after perfusion with O-R-PolyHbA0 with or O-R-PolyHbA0 and Na2SeO3, followed by FITC-BSA, can be seen in Fig. 2, A and B, respectively.
Quantification of leaks. The mean numbers and areas, respectively, of leaks per venule length for networks perfused with PolyHbBv or O-R-PolyHbA0 with and without Na2SeO3, are shown in Fig. 3. Results from previous experiments using DBBF-Hb or CNmet-DBBF-Hb are also included for comparison (10). In preparations perfused with PolyHbBv, Na2SeO3 did not reduce the number or area of leaks. PolyHbBv alone produced an average of 0.53 ± 0.14 × 10-3 leaks/μm venule length, and the combination of PolyHbBv and Na2SeO3 gave a value of 0.91 ± 0.22 × 10-3. A value of 0.91 × 10-3 leaks/μm is equivalent to one leak per 1,100 μm of venule length. A typical total sum of venule lengths measured in one preparation was about 15,000 μm, and so 0.91 ± 0.22 × 10-3 leaks/μm would be equivalent to 14 leaks per preparation. Corresponding leak areas per micrometer of venule length were 0.010 ± 0.003 and 0.020 ± 0.008 μm2/μm, respectively, which were not significantly different from each other. In preparations perfused with O-R-PolyHbA0, Na2SeO3 somewhat reduced the number of leaks, but the difference was not significant. O-R-PolyHbA0 alone produced an average of 0.59 ± 0.12 × 10-3 leaks/μm venule length, and the combination of O-R-PolyHbA0 and Na2SeO3 gave a value of 0.25 ± 0.05 × 10-3. Corresponding leak areas per micrometer of venule length were 0.092 ± 0.028 and 0.084 ± 0.032 μm2/μm, which were not significantly different from each other.
On the other hand, in a previous study (10) it was shown that in preparations perfused with DBBF-Hb, when Na2SeO3 was included in the perfusate and suffusate, the microvascular leak number was significantly reduced from 2.41 ± 0.33 × 10-3 leaks/μm venule length to 0.23 ± 0.09 × 10-3 leaks/μm venule length (P < 0.01), and leak area was reduced from 0.10 ± 0.03 μm2/μm to 0.012 ± 0.006 μm2 (P < 0.01). In rats perfused with CNmet-DBBF-Hb, without addition of Na2SeO3, the mean leak number per micrometer (0.36 ± 0.14 × 10-3) and the mean leak area per micrometer (0.005 ± 0.003 μm2/μm) were significantly lower than after DBBF-Hb perfusion and were similar to controls. Inclusion of Na2SeO3 in the perfusate and suffusate during the 10-min perfusion did not significantly affect the results. The corresponding values were 0.53 ± 0.32 × 10-3 and 0.007 ± 0.004.
Mast cell degranulation. Degranulated mast cells were easy to identify in mesenteric tissue stained with toluidine blue, because the granule contents were expelled into the surrounding tissue and were stained a dark red against a blue background. Intact mast cells formed compact, oval-shaped disks that also stained dark red. The number of mesenteric windows per group examined for mast cell degranulation was six. The mean numbers of degranulated mast cells per field of view for networks perfused with PolyHbBv (107 fields) or PolyHbBv and Na2SeO3 (92 fields) are shown in Fig. 4. Corresponding values for O-R-PolyHb alone (350 fields) and with Na2SeO3 (352 fields) are also shown. Results from previous experiments using DBBF-Hb or CNmet-DBBF-Hb are also included for comparison. In the PolyHbBv-perfused animals, Na2SeO3 significantly increased the number of degranulated mast cells per field of view from 3.21 ± 0.52 to 17.38 ± 2.02 (P < 0.01). Preparations perfused with O-R-PolyHbA0 showed very little mast cell degranulation, irrespective of whether Na2SeO3 was included in the perfusate and suffusate. The values were 0.43 ± 0.07 and 1.86 ± 0.32, without and with Na2SeO3, respectively. In preparations that were perfused with DBBF-Hb, addition of Na2SeO3 to the perfusate and suffusate did not significantly alter the already high mast cell degranulation (from 12.88 ± 1.61 to 13.86 ± 1.20). Mesenteric mast cell degranulation in animals whose mesenteric microvasculature was perfused with CNmet-DBBF-Hb (6.85 ± 0.78) was also not affected by inclusion of Na2SeO3 in the perfusate and suffusate (6.06 ± 0.72).
Microscopy of Intestinal Mucosa to Assess Goblet Cell Secretion and Epithelial Damage
Secreting goblet cells. The extent of goblet cell secretion after injection of PolyHbBv adjacent to and remote from Peyer's patches is shown in Fig. 5, A and B, respectively. Results from previous experiments using DBBF-Hb are included for comparison. Injection of PolyHbBv into animals that were not pretreated with Na2SeO3 produced an average of 2.27 ± 0.24 secreting goblet cells per villus near Peyer's patches and 1.21 ± 0.13 away from Peyer's patches. Corresponding values for animals pretreated with Na2SeO3 were 3.10 ± 0.25 and 2.40 ± 0.21, respectively. Thus pretreatment of animals with Na2SeO3 significantly increased goblet cell secretion in both locations. In contrast, in animals injected with DBBF-Hb, pretreatment with Na2SeO3 did not significantly alter goblet cell secretion near Peyer's patches (from 3.41 ± 0.25 without pretreatment to 3.11 ± 0.25 with pretreatment) or away from Peyer's patches (from 1.41 ± 0.17 to 1.16 ± 0.12).
Epithelial damage. Electron micrographs of interstitial mucosa from rats perfused with PolyHbBv looked very similar to those from another study in which rats were perfused with HBS-BSA instead of with a modified Hb (8). As seen in Fig. 6A, the epithelial intercellular junctions (arrows) were tight and the cells were closely adhering to the basement membrane. In animals that were treated with both PolyHbBv and Na2SeO3 (Fig. 6B), the epithelial cells were often partially separated from each other and showed thin, tortuous, cytoplasmic projections along their lateral membranes (arrows). Occasionally, cells were detached from the basement membrane. The average EI indexes for each group are depicted in Fig. 7. Injection of PolyHbBv into animals that were not pretreated with Na2SeO3 produced an average EI of 1.08 ± 0.03 near Peyer's patches and 1.07 ± 0.02 away from Peyer's patches. Corresponding values for animals pretreated with Na2SeO3 were 1.24 ± 0.05 and 1.23 ± 0.05. Treatment with Na2SeO3, before injection with PolyHbBv, significantly increased EI and thus reduced epithelial integrity, in both cases. In contrast, treatment with Na2SeO3, before injection with DBBF-Hb, had the opposite effect. Near Peyer's patches, the values for EI without and with Na2SeO3 were 1.22 ± 0.03 and 1.03 ± 0.02, respectively, and away from Peyer's patches the corresponding values were 1.37 ± 0.04 and 1.25 ± 0.04.
Effect of Na2SeO3on the Oxidation of PolyHbBv and O-R-PolyHbA0
We first monitored the effects of inclusion of 500 μM Na2SeO3 with PolyHbBv as it undergoes normal spontaneous oxidation under physiological conditions in HBSS buffer, pH 7.4, and 37°C for 6 h. The calculated rates of autoxidation of PolyHbBv with and without Na2SeO3 were only very slightly decreased by Na2SeO3. We then focused on the effects of Na2SeO3 on PolyHbBv oxidation under mild oxidative stress induced by low and steady supplies of H2O2, at a rate of 1.5 μM/min generated by the GOX system (17). The rate for the exponential decrease in oxy-PolyHbBv under these conditions was calculated to be 0.0454 min-1. However, in the presence of Na2SeO3, the rate of oxy-PolyHbBv decay was 0.0355 min-1, ∼22% slower than PolyHbBv/GOX medium alone. Similarly the rate of met-PolyHbBv formation in the presence of Na2SeO3 was 0.0445 min-1, ∼24% lower than 0.0584 min-1 calculated for PolyHbBv/GOX alone. Na2SeO3 had the opposite effect on the oxidation of O-R-PolyHbA0. Figure 8 shows typical time courses of the oxy-O-R-PolyHbA0 disappearance and met-O-R-PolyHbA0 buildup as a function of time in the presence of the GOX system and in the presence or absence of Na2SeO3. The rate for the exponential decrease in oxy-O-R-PolyHbA0 was calculated to be 0.03132 min-1. However, in the presence of Na2SeO3, the rate of oxy-O-R-PolyHbA0 decay was 0.03736 min-1, ∼19% faster than O-R-PolyHbA0/GOX medium alone. Similarly, the rate of met-O-R-PolyHbA0 formation in the presence of Na2SeO3 was 0.05270 min-1, ∼11% higher than 0.04736 min-1 calculated for O-R-PolyHbA0/GOX. Na2SeO3 had a more marked effect on suppressing the oxidation of DBBF-Hb under mild oxidative stress than it did with PolyHbBv. For DBBF-Hb, the rate of decrease in oxy-DBBF-Hb was reduced by 50% in the presence of Na2SeO3, and the increase in met DBBF-Hb formation was decreased by 70%. To investigate the potential direct redox interactions between modified Hb oxidation forms and Se, we measured the rates of reduction of the ferric iron of the modified Hbs in the presence and absence of Na2SeO3. These results were compared with those obtained from incubation of Se with the met form of DBBF (Fig. 9). It is clear that, as the concentration of Se increases, there was a corresponding increase in the reduction in the levels of metHb in PolyHbBv, O-R-PolyHbA0 and DBBF-Hb solutions, although the effect was most marked for DBBF-Hb and least marked for O-R-PolyHbA0. The rapid reduction in the met forms of Hbs, even at low Se concentration, is not surprising because Se occupies a higher position in the pecking order of free radicals than iron in terms of standard reduction potential and is therefore capable of donating electrons to the ferric iron of Hb (13). However, similar concentrations of Se showed no effects on the levels of metHb trapped by cyanide, confirming the need for an unliganded binding site for the redox communication to occur between Se and the ferric iron of PolyHbBv, O-R-PolyHbA0, and DBBF-Hb.
Effect of Na2SeO3 on the Oxygen Affinity of DBBF-Hb
Increasing concentrations of Na2SeO3 did not appear to affect the oxygen affinity of PolyHbBv, as demonstrated by the constancy of values of P50 and the cooperativity (Hill number) in the presence or absence of reducing enzymes. In the presence of enzymes, an average P50 value of 30.8 ± 0.4 Torr was obtained for PolyHbBv regardless of the Se concentrations used. This value is close to that calculated for Hb solutions without Se (control) (P50 = 31.21 Torr). We have previously obtained similar results using DBBF-Hb (10) and more recently for O-R-PolyHbA0; the average P50 value for DBBF was 29.4 ± 0.9 Torr in the presence of 125 μM to 1 mM Na2SeO3 and 29.0 Torr in the absence of Na2SeO3. For O-R-PolyHbA0 the average P50 value was 36.5 ± 1.9 Torr in the presence of 125 μM to 1 mM Na2SeO3 and 29.0 Torr in the absence of Na2SeO3.
This study has shown that the modified Hbs, DBBF-Hb and O-R-PolyHbA0, both produce microvascular leakage to FITC-BSA in the rat mesentery to a similar degree, as judged by total leak area per unit length of venule, that is significantly greater than for preparations perfused with HBS-BSA. On the other hand, PolyHbBv does not produce significant microvascular leakage. There is a growing experimental evidence to support the idea that modified Hbs, injected in vivo, can cause tissue damage and organ dysfunction by producing highly reactive oxygen species (8, 20, 28). Modified Hbs spontaneously autooxidize to produce metHb and superoxide anions (). Superoxide is also produced from the respiratory burst of phagocytes that are activated by Hb. Superoxide undergoes the dismutation reaction, in the presence of superoxide dismutase, which is contained within the cells, with consequent production of H2O2. The H2O2 is normally decomposed by its reaction with catalase or with glutathione peroxidase. However, these enzymes are inhibited by superoxide (12). It has been postulated that the excess H2O2 then reacts with metHb in vivo to initiate further oxidation cycles resulting in the formation of highly reactive ferryl Hb, in which the iron center is at higher oxidation state (Fe4+) (28). The interaction of ferryl Hb with H2O2 results in the formation of rhombic heme, which is considered to be one of the best measures of the toxicity of a blood substitute (23). The rhombic heme in which the geometry of the iron is distorted due in some instances to the chemical modification of the protein then initiates a cascade of oxidative side reactions resulting in the formation of free iron. Iron in turn catalyzes the production of the highly reactive hydroxyl radical. Interestingly, measured values for formation of rhombic heme were more than tenfold greater for DBBF-Hb and O-R-PolyHbA0 than for PolyHbBv (23), consistent with the relative degrees of microvascular injury produced by these substances as reported in the present study.
This study has demonstrated that coinjection of Na2SeO3 with modified Hbs in the microvasculature may significantly reduce, have no effect on, or somewhat enhance the tissue damage mediated by the Hb and that the result is dependent on the interaction of the Na2SeO3 with the particular modified Hb. Dietary supplemented Se, an antioxidant, has been shown to reduce Hb-mediated lipid peroxidation in the liver (28). Se is thought to act as an antioxidant in the body because it is a component of the enzyme glutathione peroxidase, which catalyzes removal of H2O2 (30). However, Simoni et al. (28) only found slightly increased concentrations of glutathione peroxidase in the livers of Se-supplemented rats, even though the tissue was resistant to Hb-induced damage. They hypothesized that the Se had been incorporated into other, nonglutathione peroxidase antioxidant cellular systems, such as seleno-amino acids (18, 35). As another possibility, a selenoprotein from the heart and muscle of lambs has been isolated that may be a Se-containing cytochrome with a possible participation in oxidation-reduction reactions (32). However, Simoni et al. concluded that the exact mechanism by which Se exerts its protective role against Hb-induced oxidative injury could not be proposed but that the concomitant injection of Se with Hb solutions seemed to be the most practical in the case of emergency situations.
In a more recent study, Chen and Lin (15) gave rats SeO2 daily for 14 days and observed increases in oxyHb in which iron is in its ferrous form and decreases in metHb in the blood. Because they also saw increased concentrations of glutathione peroxidase activity in the blood of rats fed SeO2, they concluded that this enzyme must be responsible for change in the oxy-met ratio of the Hb because it reduces the presence of H2O2. However, the results of the present study demonstrate that there is no need for glutathione peroxidase to be involved in this process; merely combining Na2SeO3 with DBBF-Hb, or to a lesser extent with PolyHbBv, in vitro, produced a similar result. Thus Se can directly alter the redox state of modified Hbs, such that the oxy-met ratio is increased, within a very short time period.
The results of the in vivo experiments described in the present study demonstrate that there is no need to pretreat rats previous to injection with modified Hbs to produce the beneficial effects of Se on the microvasculature. These results are consistent with the idea indicated by the in vitro experiments that Se exerts its beneficial effects by reacting directly with the Hb rather than by becoming incorporated into an enzyme that scavenges reactive oxygen species. This study has also shown, in three different modified Hbs, that the interaction of Se does not alter the oxygen-carrying capacities of the Hbs and thus does not compromise their function as Hb-based oxygen carriers. However, although Na2SeO3 significantly reduced the tissue damage produced by DBBF-Hb, it had no effect on damage induced by O-R-PolyHbA0, and its effect with Poly-HbBv was deleterious. These differing effects in vivo are consistent with the results of the in vitro experiments showing that Na2SeO3 does not affect the redox properties of PolyHbBv and O-R-PolyHbA0 as much as those of DBBF-Hb. For example, Na2SeO3 was not so effective at decreasing the oxidation of PolyHbBv and O-R-PolyHbA0, compared with DBBF-Hb, under mild oxidative stress or of increasing the rates of reduction of the met forms of PolyHbBv and O-R-PolyHbA0. It is possible that Na2SeO3 may alter the extent of formation of heme degradation products, such as rhombic heme, but further experiments are required to test this hypothesis.
The in vivo experiments demonstrated that, for PolyHbBv and O-R-PolyHbA0, Na2SeO3 actually increased mesenteric mast cell degranulation. For PolyHbBv, further experiments showed that goblet cell secretion and epithelial disruption were also increased with coadministration of Na2SeO3. These results are consistent with the evidence that the Na2SeO3 did not interact with the PolyHbBv and O-R-PolyHbA0 as effectively as it did with DBBF-Hb, and thus some Se was free to react with other components in the body. Our previous experiments in which Na2SeO3 was perfused in the microvasculature with phosphate-buffered saline, rather than with a modified Hb, or administered as a dietary supplement to animals that were not intravascularly injected with a modified Hb showed similar results (10). A possible explanation is that the Se activated neutrophils and macrophages to produce oxidants (3, 4). In addition, Na2SeO3 is metabolized by interaction with reduced glutathione, and reactive oxygen species are by-products of this reaction (27, 34). Reduced glutathione is present in erythrocytes and also in neutrophils (14) and macrophages (26). Reactions of the Se with immune cells, or with reduced glutathione, could be responsible for the effect of Na2SeO3 on preparations perfused with PolyHbBv or O-R-PolyHbA0. In cases in which DBBF-Hb is present, Na2SeO3 interacts with the DBBF-Hb, as shown by our in vitro results and thus may not be free to stimulate immune cells. Thus the interaction is synergistic and the prooxidant properties of both molecules are reduced. These experiments indicate that Se compounds will only be useful adjuncts to Hb-based blood substitutes for which it can be shown, in advance, that the oxidation state is significantly reduced by interaction with the Se.
The relatively small degree of microvascular leakage and mast cell degranulation accompanying perfusion with CNmet-DBBF-Hb, vs. DBBF-Hb, is consistent with the hypothesis that the tissue damage induced by Hbs is caused by their oxidation and accompanying formation of reactive oxygen species. As mentioned previously, CNmet-DBBF-Hb is a Hb in which the CN groups are tightly bound to the heme such that the molecule is unable to participate in redox reactions. The fact that Na2SeO3 did not significantly affect the microvascular leakage and mast cell degranulation that accompanied perfusion with CNmet-DBBF-Hb, whereas it increased these responses after HBS-BSA perfusion, suggests that the Na2SeO3 interacted with the CNmet-DBBF-Hb and thus was not free to stimulate immune cells.
Both the in vivo and the in vitro experiments in this study are consistent with a hypothesis called the Se link hypothesis that was introduced by Nyberg-Swenson (24). In this model, Se constitutes a protective system that prevents the direct transfer of electrons and perhaps also prevents contact between the substance being oxidized and oxygen, thus impeding the formation of oxygen compounds. If this hypothesis is applied to autooxidation of oxyHb, this would mean that the Se would be placed in such a position such that the transfer of one electron from the HbFe2+ to the attached O2 would be impeded, and so the metHb, or HbFe3+, and superoxide would not form. However, in the present study, Na2SeO3 had little effect on the autooxidation of the modified Hbs, and thus it is unlikely that Se formed such a link. In contrast, Na2SeO3 reduced the oxidation of DBBF-Hb and, to a lesser extent, PolyHbBv in the presence of H2O2. When HbFe2+ reacts with H2O2, it donates two electrons to the H2O2, to form ferryl Hb (HbFe4+) and OH-. The ferryl Hb then accepts one electron from another HbFe2+ molecule to form two molecules of metHb or HbFe3+ (16). Alternatively, a second H2O2 molecule reduces the ferryl Hb to metHb, and a superoxide radical is formed in the heme pocket, which then attacks the heme. If the Se link hypothesis were applied to the reaction to form ferryl Hb, this would mean that the Se would be positioned such that the Fe2+ would be separated from the H2O2 and hence unable to donate two electrons. The effect of Na2SeO3 in decreasing the rate of oxidation of oxyHb to metHb, demonstrated in the presence of H2O2, is consistent with the Se link hypothesis.
To summarize, the in vivo and in vitro experiments performed in this study demonstrate that Se can reduce the oxidative damage produced by some modified Hbs, but only if it interacts sufficiently with the Hb molecule to reduce its rate of oxidation. The fact that Se compounds can reduce oxidative damage by directly interacting with the modified Hb, and without changing its oxygen carrying capacity, makes them ideal for use in clinical situations. Because the effect is not dependent on incorporation of the Se into an enzyme system, there is no need to pretreat the patient; the Se compound can be coinjected with the modified Hb. This technique could also be used to improve the storage properties of blood substitutes. However, the differing efficacy of Na2SeO3 in reducing the tissue damage caused by the three Hb-based oxygen carriers used in the present study highlights the importance of knowing the redox chemistry of the blood substitute in question.
We thank Francine Wood of Center for Biologics Evaluation and Research, Food and Drug Administration, for technical assistance in carrying out some of the in vitro experiments. We thank the Southern Arizona Veterans Administration Healthcare System for allowing us to house our animals in their facility.
This work was supported by National Heart, Lung, and Blood Institute Grant HL-53047.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Copyright © 2004 the American Physiological Society


text:
Julie Travelino's Profile
Welcome to Too Chic & Little Shab! A custom design studio operated by me, Julie Travelino, I love designing invitations using fabulous high end materials to make every event extra special. Allow me to make you the perfect invitation!
Please visit me on my new website: www.toochiclittleshab.com
- Female
- Born on February 21
- Joined August 5, 2011
Favorite materials
Stardream Metallic cardstock, authentic Swarovski crystals and Rhinestone buckles
- Julie Travelino
- Chicago, IL


text:
Even this was avoidable.
View Single Post
November 12, 2011, 06:59 PM
Senior Member
Join Date: December 1, 2009
Location: Stillwater, OKlahoma
Posts: 8,203
I agree with Nnobby45 when he wrote:
Ah, but a gun accident. The above rules don't apply. The heartbroken adult must, in addition to the moral consequences, go to prison and pay the price for his negligence. The news media believes that, and so do (as I mentioned) members of our own gun community.
To say the gun accidents shouldn't happen is true to the same extent that other accidets shouldn't happen. Accidents have always happened and always will, even though that fact can't be used as an excuse.
In some states, negligence resulting in death of a child (non gun accident) is business as usual in the court system. The prosecuter has the descretion.
But gun accidents mandate charges and jail time upon conviction. Off to jail goes a heartbroken adult who was negligent only for an instant. Something that could happen to any of us. That gun owner faces mandatory prosecution not forth coming for other accidents doesn't sound fair to me.
I truly believe that as a society we are becoming a group of self-righteous prigs,,,
We seem to revel in the harsh punishment of even accidental offenders,,,
Our society is trending towards the belief that there are no accidents.
There is a quote attributed to Thomas Paine,,,
I believe it applies very well to this situation.
An avidity to punish is always dangerous to liberty.
It leads men to stretch, to misinterpret, and to misapply even the best of laws.
He that would make his own liberty secure must guard even his enemy from oppression.
And there's always the old,,,
"Let he who is without sin cast the first stone."
JM ns HO
Caje: The coward dies a thousand times, the brave only once.
Kirby: That's about all it takes, ain't it?
Combat: "A Silent Cry"
Aarond is good,,, Aarond is wise,,, Always trust Aarond!
(most of the time)
View Public Profile
Visit aarondhgraham's homepage!
Find More Posts by aarondhgraham
Page generated in
seconds with


text:
i'll tear that down!
Did You See This?
Some Would Consider Her To Be Fat! [Pics]
not to me though, she's PERFECT! A post shared by L e s l i e S i d o r a 🌼 (@lessance) [...]
I Posted Her Again Because You Said You Would Smash [Pics]
her name is cassandra!
Be Honest! Would You Hit Cassandra? [Pics]
Would You Call Her Thick? Or Fat? [Pics]
regardless to what you say, i'm smashing!