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I am going to attempt in my amateur, non-doctor, manner to explain "Free Radicals," how they are destructive, and some methods to prevent them. Free Radicals can be atoms, ions, molecules, or fragments of molecules. The one thing they have in common is that they contain at least one electron that is "free" – that is not paired with another electron. (Pierce, James B., Ph.D. 19944. Heart Healthy Magnesium). This electron is unstable and highly reactive and to regain stability, must "steal" another electron from a neighboring molecule.
That neighbor is usually a cell membrane, or plasma, or a chromosome. As it steals an electron, it causes injury. This injury can lead to atherosclerosis, heart disease, cancer, tumor, diabetes, arthritis, aging, hypertension, anemia, emphysema, cataract, skin disease, weak immune system, and many more complications. Free radicals are molecules produced when your body breaks down food, or by environmental exposures like tobacco smoke, pollution and radiation. There are many man-made causes for Free Radicals – these are just a few we know of now. Sounds BAD! It happens in the body! Now, the "cure" or prevention. "Antioxidants!" Antioxidants are substances that may protect your cells against the effects of free radicals. Antioxidants are found in many foods. These include fruits and vegetables, nuts, grains, and some meats, poultry and fish.
How Antioxidants stabilize Free Radicals – The free radicals are superoxides, hydroxyl, hydrogen peroxides. Each orbit of an atom usually contains a pair of electrons per orbital. At its outermost orbital, when a fellow had been removed the remaining fellow turns the atom into a "Free Radical" that is highly unstable. An electron is removed by heat, or "by your drawing in cigarette smoke, submitting yourself to an X-ray examination, basking in the sun, eating a bologna sandwich, drinking a cocktail, etc. (Walker, M. M.P.D. 1984. The Miracle Healing Power of Chelation Therapy, page 139). PHILNoni has excellent antioxidants that mop up Free Radicals! I will be posting more about PHILNoni in the near future and some of the specific characteristics. 30ML in the morning and 30 ML in the evening will have amazing results.
My own testimony – I had a cut on the bottom of my foot for over 6 years – all the medicines, ointments, special socks, special shoes – nothing worked to heal the cut. After five weeks of PHILNoni – the cut disappeared! Now – there is a difference – I talked with Dr. Tito Contado – a former Professor at the University of the Philippines at Los Banos, and former Chief of the United Nations FAO Service of Agricultural Education and Extension. DR. Tito explained to me that PHILNoni, not only being an excellent and God-Given natural resource to fight Free Radicals – is also a BODY ENHANCER! There are supplements that when the body receives, if the body needs them uses them and discharges the rest. This happens with vitamins. With PHILNoni – it actually helps body functions – function! That is why my cut was repaired naturally by the body! There are so many wonderful all-natural, organic resources around the world. We have been fortunate to been involved with Dr. Tito as he developed PHILNoni – it is now available to you! I personally have used it – now so can you!
You can also order PHILNoni as well! This is brand NEW – although it has been around for centuries! Thank-you for your time and consideration. For those that are tired of taking pills, medicine – try some alternatives – see if they work! it has for me and countless others! I have to put a disclaimer here – check with your doctor first – although I didn't – I am not advocating completely unleashing from man-made medicines immediately – however, there are alternatives!


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… Scowling at injustice with a big, big sense of right and wrong
… Sulking every day on the way to school complaining to Mom about girls and telling her how much you hate them
… Singing your lungs out in the shower and doing the boogie woogie while at it
…And staging a water battle for German and American soldiers while you're at it
… Needing to look cool all the time
…Wanting – not a pet dog, a pet kitten, but a pet JAGUAR
… Making a mess with your Lego and turning a deaf ear to Mom's nagging about bricks hurting her feet (like, who cares?)
… Being extremely creative, with tons of mischief up your sleeve
…Wanting many moments of being all alone to create something massive
… Leading the pack
… Having no fear
… Having intense conversations with friends, and wanting to be with older boys
…. Monkeying with Mom and loving every minute of it
6 years ago, this boy entered my world. A leaking water bag and almost 14 laborious hours later, I met the gentlest, sweetest, baby who came to rock my world.
And rock my world he did. Every. Single. Day.
I love you, Son. Every. Single. Day.

subset: commoncrawl


text: Our dog Sophie, an almost 8 year old cairn terrier, has been very lethargic, eating snow, and losing weight the past couple of weeks. And her hair looks funky, not full and fluffy like normal - it's tufted and strange looking, almost greasy. She's peeing and BM-ing okay, but despite upping her food intake, she is always ravenous. Today she went to the vet to get checked out. One look at Sophie's gums and our vet said she was severely anemic. After some initial bloodwork, the vet said that Sophie's red blood cell count should be between 30-35 and her's is only at 13, less than half normal. By all rights, the vet said Sophie should've collapsed by now and was amazed at her stamina (because she's barking, walking and eating). This is where the confusion comes in - she has an appetite. They took a lot of blood and made her drink water by putting treats in it. They gave her two handfulls of treats and she ate them and was looking for more. They also took two stool samples from her. They have checked preliminarily for certain parasites and those tests came back negative. They also checked for a parasite that is related to lymes and since she has had lymes, they figured it was worth a shot. The final blood work will determine whether or not her body is battling something that is preventing her bone marrow from producing normal red blood cells or if it's not making it at all, which leaves us on the edge of our seat. If her body is not making it at all, they can try a few things, but basically, it means she likely has cancer and will decline rapidly. The vet said rapidly only because her symptoms to date came on so fast. on the other hand, if there is something attacking her bone marrow, they can try various antibiotics and steroids. The vet said we have to keep a close eye on her because she could collapse and we'd need to take her to the animal ER for a blood transfusion. We also have to feed her beef and poultry livers, as well as her normal vitamins and an iron supplement they gave me today. I cannot even tell you how astounded I am - easy child was with me and we were both dumbfounded by this news. Sophie is the dog who is all puppy - even at 8 years old; she's playful, full of energy, teases and barks at everything. She runs off, comes back, does giant donut rounds in the yard, burrows through the snow and is polite when you give her a treat. Never bites. And loves to snuggle. She is also kinda sort difficult child's dog. I am keeping a positive thought that this is something Sophie can overcome. Please throw up a prayer, if you will, for I do not want to lose Sophie and I especially do not want difficult child to lose Sophie. She's the blonde one in my avatar.


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Tag Archives for 2017-18
SHOW GUIDE: The Best Christmas Pageant Ever
It's almost time for our next production, The Best Christmas Pageant Ever, a new musical adaptation of the hilarious Christmas comedy classic story by Barbara Robinson! We've assembled some special information to help you and your family to get ready for the upcoming performance. Before you go to the show, you might want to take […]
Posted in Show Guide


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kill each other; we just like to kill stuff, period. In this regard, when a new MMO releases, we're always curious about the combat system. Age of Conan's combat system seems to be a big hit among players, which adds so much to the overall game experience.
So when we hear about the hit and damage system in Fallen Earth, and how it will work within the game, we're even more excited about this upcoming Icarus Studios title. Apparently, as we already know, there will be a first-person and third-person view mode, but these will also change while in aim mode. It sounds to us that the whole aiming process will be handled much as is done in Tabula Rasa, except not as much of the automatic or "sticky" aiming. There's also a good discussion on weapon types and weapon damage in this article, some of which gets fairly specific when it comes to what damage these weapon types will do exactly. The rest of the damage system seems fairly straightforward, with defense skills, attributes, resistances, mutations and special items all playing a role in how protected you will be from the various weapon damage.


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Salem Community College admissions qualifications
Think about what career is best suited for you, and where you want to study. Request more information now.
Think about what career is best suited for you, and where you want to study. Request more information now.
Think about what career is best suited for you, and where you want to study. Request more information now.
Find information about Salem Community College admissions qualifications, and registering for elective seminars and discussion sections. Which college degrees give you the best chances of entering a successful career path after graduation? Concentrating on Salem Community College admissions qualifications, for example, may give you additional focus in the admissions selection process. Check with the admissions office for assistance with prerequisites, course registration deadlines, and college degree program requirements. Please use the form above to request admissions info for Salem Community College.
Given the large number of colleges, universities, and vocational training programs, how can you make a wise choice, in oder to select the school that's best for you? There are literally 4,000 degree-granting institutions, and over 2,000 technical schools. Of the degree-granting schools, over 1,600 award associate degrees and 2,400 award bachelors degrees. By asking youself several important questions, you can narrow down the list to a reasonable size. Many students choose a college nearby, not only to save money on in-state tuition, but to remain close to family and friends. If you're looking further afield, ask yourself what you want to achieve long-term, and don't start college hoping you'll figure out what major to choose later.
By looking over the course catalog from any major university, you can get an idea which courses are going to be required in any given major. Try to remember how you felt when you took any related subjects during high school, and ask yourself if you really would look forward to several years of in-depth involvement and study. If you're lucky enough to know what direction you want to take, then college rankings can help you define your options, based on your GPA, class standing, extracurriculars, and test scores. Beyond major requirements within the admissions department, you may consider Georgia Regents University research seminar for complementary electives to your primary course of study.
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A TREATY designed to remove radical cleric Abu Qatada from the UK has moved another step toward to full ratification.
The agreement, which aims to allay fears that evidence extracted through torture will be used against the terror suspect at a retrial, has now been published in the Jordanian government's official gazette – leaving just a handful of legal moves remaining before the deportation process can begin.
In May, Qatada unexpectedly volunteered to leave the country as soon as the treaty is ratified.
Security minister James Brokenshire said: "The publication of the treaty in the Jordanian Official Gazette is welcome. While further steps remain, our focus is on seeing Abu Qatada on a plane to Jordan at the earliest opportunity."
The publication of the treaty in the gazette comes after both houses of the Jordanian parliament and the country's king approved the treaty, while the UK parliamentary scrutiny process has also completed.
However, Home Secretary Theresa May previously warned that, even when the treaty is fully ratified, it will not necessarily mean that Qatada will be on a plane to Jordan within days.
The case remains open to legal challenge.
For the past eight years, the government has been attempting to deport Qatada to Jordan, where he was convicted of terror charges in his absence in 1999.
The legal battle to remove him from the UK has cost the taxpayer more than £1.7 million since 2005.


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Stanford Defense Atty Begs Judge For Cash
Right now, Stanford's personal assets are frozen because the receiver handling the collapse of his bank has linked his money to his alleged scheme. With no assets, though, Stanford has no money he can use to find any untainted money, attorney Stephen R. Cochell wrote...
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Article Side
Online Article Directory!
Cancellation of Reservation Possible when Travel Plan Changes
Indian railways , the fourth largest railway in the world running 114500 km track length, transporting over 30 million passengers and 2.8 million tons of freight every day employs a work force around 1.36 million to take care of its operational and maintenance activities. Indian rail reservation for passenger travel is handled both by Railways department and through one of its BPOs called Indian Railways Catering and Tourism Corporation Limited apart from Indian railways reservation counters.
Rail reservation is also done through Travel agents authorized by the Ministry of Railways and these agents collect a small fee for the services, over and above the train fare and railways reservation charges.
Rail reservation is normally for a specific person to travel and a rail ticket reserved for one person cannot be used by another person to travel. The ticket examiner verifies the identity of the person during travel.
Indian rail reservation can be done three months ahead of travel in all sections and the rail reservation can be cancelled at any point of time, depending upon the exigencies of circumstaces. However, the fare remitted for purchase of tickets is not fully refundable. Depending upon the time of cancellation, refunds are made after deducting cancellation charges, as described below:
Time of cancellation
24 hours before the departure time of the train
Within 24 hours, but before 4 hours of the departure time
Within 4 hours of the departure time
Cancellation charges
Standard fee of Rs 70 for AC I Class,, Rs 60 for AC 2 & 3 tier, Rs 40 for Sleeper and Rs 20 for sitting, per passenger
25% of the fare subject to the above minimum rates
50% of the fare subject to the above minimum rates
The above cancellation charges are applicable only for confirmed rail reservations, including reservation against cancellation type of tickets (RAC). For waitlisted tickets, only ticket stationery charges of Rs 20 is deducted.
In respect of E tickets after rail reservation chart preparation at the originating station or previous chart preparation station, cancellations are not permitted, but a TDR claiming permissible refund has to be filed online upon which Indian railways will order refund after verification of travel details.
Tickets bought at the railway reservation counters and i-tickets can be cancelled only at the counters. Likewise, E tickets can be cancelled online. E-tickets are print outs upon online rail reservations and i-tickets are regular railway tickets sent to the passenger upon online reservations.
Advance rail ticket booking through Indian rail reservation system is a good practice for traveling in comfort and without hassle , although you can choose to travel in unreserved compartments available in most of the trains.
Kylea Taylor - About Author:
Indian Rail Reservation - Information about indian railway reservation, railway reservation rules for the travellers, enquiry about railway resrvation, railway timetable schedule at Indianrailwayreservation.net.in
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- 74 years old
- Date of birth: Jan 26, 1942
- Place of birth:
Springfield, Missouri, United States
- Date of passing: May 21, 2016
- Place of passing:
Morristown, Tennessee, United States
|Forever loved, forever missed...
This memorial website was created in memory of our loved one, Jerry Stafford Sr, 74, born on January 26, 1942 and passed away on May 21, 2016. We will remember and love him forever.
"Thank you for all you have taught me, Rest in love and and in peace. I love you Dad."
"I very much miss the man I wished you had been, not the man you actually were."
"May your spiritual journeys be as eventful as your earthly adventures. May you be able to ascend on your own terms, but with grace and dignity. I wish you peace."
"If it werent for u I wouldnt be. And if I couldnt be, then I i would never see all the beauty in having a father such as you......I love you papa."
"May the Lord give Dad's beloved wife Sharon Stafford the strength she needs to get through this time of grieving....this time of healing....this time of prayer..."
"To my Dad...."
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38 of the Most Inspirational Leadership Quotes
No one can deny the power of a good quote.
No one can deny the power of a good quote.
Sticking your neck out and taking charge of your career is no trivial matter.
Knowing when to quit is a skill that can be learned.
It's hard to find the motivation to focus on the positive when positivity seems like nothing more than wishful thinking.
Developing a successful morning routine is essential.
What if I told you in just 10 seconds a day, you can sleep better, make more money, reduce stress and lose weight?
Smart people are more prone to silly mistakes because of blind spots in how they use logic.
Genuine people know who they are. They are confident enough to be comfortable in their own skin.
Even when it seems you're doing everything right, promotions can be few and far between.
A new personality type has emerged that puts the old introvert versus extrovert debate to rest. Find out if you're an Ambivert.
The ability to manage your emotions and remain calm under pressure has a direct link to your performance.
How you start your day has a huge impact on your energy level and self-control.
Mental strength is a choice and a discipline, not an innate quality bestowed upon the lucky.
A great leader has the incredible ability to bring out the best in you.
Most people wait too long to leave a bad job and their health and sanity suffer.
How you work is far more important than how much you work.


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A unique monumental site in Aragon. Uncastillo is 110 kilometres north of Zaragoza. Its centre is home to impressive historic-artistic heritage. Special mention should be made of the castle-fortress, with its "Torre del Homenaje" (keep), originally from the 11th century and restored in the 14th, where you will find the Tower Museum. Then there is the Gothic palace of Peter IV, dating from the same century, an excellent example of Gothic palace architecture, unique in Aragon. Santa María Church is a designated Cultural Property, with its cloister and Romanesque/Gothic decoration. San Juan Church is also designated Cultural Property and has 13th century murals. Finally we should make mention of the medieval exchange (13th century). It is also worth visiting the Los Bañales archaeological area, one of Aragon's main Roman sites. The beautiful surrounding countryside makes Uncastillo a good place for hiking. In summer, the Crafts Exhibition is held, where you will find stonework, wrought iron, woodwork and embroidery.
Turer du ikke bør gå glipp av: Uncastillo
På det neste kartet finner du alle besøkene vi har foreslått. Ikke gå glipp av noe!


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OK, I'm sure everyone in the state is going to be spouting out their opinions on this. As I live in the state I consider myself entitled to add in my own opinions here. Let me start by saying I appreciate the comments made by Beau Biden regarding his current position as Attorney General. I will list his … comments here:
Dear Friend,
As many of you know, since returning home from Iraq, I have been giving serious consideration to running for the United States Senate. I have received strong encouragement both here in Delaware and all across the country to undertake this effort – and this outpouring of support has truly been humbling.
I understand why people care so deeply about this election. The challenges we're facing as a country are extraordinarily difficult. The economy. Jobs. Health care. Energy. Education. Climate change. Financial regulation. Foreign policy. These are not only the issues of the moment – they're the issues that will determine our children's future. And as someone who has had the privilege of serving with the bravest men and women on this planet, I care deeply about how we treat our returning veterans and how we resolve our involvement in Iraq and Afghanistan.
I feel strongly about these issues. However, my first responsibilities are here in Delaware. I have a duty to fulfill as Attorney General – and the immediate need to focus on a case of great consequence. And that is what I must do. Therefore I cannot and will not run for the United States Senate in 2010. I will run for reelection as Attorney General.
One of the primary reasons I ran for Attorney General was to protect the most vulnerable among us: children. As the father of two young children, I can think of no worse crime than those committed by child predators. It is why one of my first actions and top priorities as Attorney General was the creation of the Child Predator Task Force.
The idea that any child can be a victim of abuse is horrific. The fact that it has engulfed an entire community is unspeakable. The pain and trauma suffered by the children, their parents, and the families, can't be measured. But justice can be done. And I am determined to see that it is.
I have a job to finish. And that's what I must do.
Thank you for all your support,
Beau Biden
You can view this at it's original site here
Now, I mean no offense to the man and know very little about his actions while in office. He states that the reason he became AG was to protect children. He was unable to stop the recent child predator we have all heard so much about recently as we all were. No one else in the state seems to be filled with the amount of guilt which he does to the point where they choose not to further their careers.
On the other hand, no one else in the state has a sitting Vice-President as a father.
Here is my honest opinion. I read the article in the News Journal and no matter what it says, the reason Biden isn't running has a great amount to do with the recent election in Massachusetts. I believe Beau was told by his Daddy that he was not allowed to lose whatever race he participated in this year. It would look horrible politically if Beau lost running for his own father's seat (one which he held for years). After the race in Boston he must have realized being a Democrat is not a shoe-in anymore. In fact, if anything, it may help you lose.
Now anyone and most likely everyone will come to this opinion. I realize this and that's why I feel no need to elaborate. Just wanted to get the word out there.
That being said, check out this blog: http://delmarvacenterright.wordpress.com/ which a friend of mine has started. I would have to say he is intelligent, even if I don't always agree with him 🙂 Well worth checking out!


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Maintaining brain Cu homeostasis is vital for normal brain function. The role of systemic Fe deficiency (FeD) or overload (FeO) due to metabolic diseases or environmental insults in Cu homeostasis in the cerebrospinal fluid (CSF) and brain tissues remains unknown. This study was designed to investigate how blood-brain barrier (BBB) and blood-SCF barrier (BCB) regulated Cu transport and how FeO or FeD altered brain Cu homeostasis. Rats received an Fe-enriched or Fe-depleted diet for four weeks. FeD and FeO treatment resulted in a significant increase (+55%) and decrease (-56%) in CSF Cu levels (p<0.05), respectively; however, neither treatment had any effect on CSF Fe levels. The FeD, but not FeO, led to significant increases in Cu levels in brain parenchyma and the choroid plexus. In situ brain perfusion studies demonstrated that the rate of Cu transport into the brain parenchyma was significantly faster in FeD rats (+92%) and significantly slower (-53%) in FeO rats than in controls. In vitro two chamber Transwell transepithelial transport studies using primary choroidal epithelial cells revealed a predominant efflux of Cu from the CSF to blood compartment by the BCB. Further ventriculo-cisternal perfusion studies showed that Cu clearance by the choroid plexus in FeD animals was significantly greater than control (p<0.05). Taken together, our results demonstrate that both the BBB and BCB contribute to maintain a stable Cu homeostasis in the brain and CSF. Cu appears to enter the brain primarily via the BBB and is subsequently removed from the CSF by the BCB. FeD has a more profound effect on brain Cu levels than FeO. FeD increases Cu transport at the brain barriers and prompts Cu overload in the CNS. The BCB plays a key role in removing the excess Cu from the CSF.
Copper (Cu) and iron (Fe) are both essential minerals for normal brain function. They play important roles as catalysts, gene expression regulators and second messengers. Both metals are highly reactive and can readily interact with oxygen to form toxic free radicals. The high reactivity of both metals, in part, explains why they are in such a high demand for many enzymatic processes and why they are strictly regulated in the body. These two metals are often conjugated with proteins in the serum and cellular compartments, such as transferrin and ferritin for Fe, and ceruloplasmin, albumin and metallothionein for Cu, to prevent the formation of reactive oxygen species (ROS) that cause the damage in the body.
The chemical homeostasis of the central nervous system (CNS) is maintained through the coordinated action of two major brain barrier systems, i.e., the blood-brain barrier (BBB) and the blood-CSF barrier (BCB). The BBB separates the blood circulation from the brain interstitial fluid, and the BCB separates the blood from the cerebrospinal fluid (CSF). Both barriers selectively transport essential nutrients, metals, and drug molecules into the CNS. During the past decades, there has been a substantial research effort on the understanding of the role of brain barriers in metal transport and toxicity (Zheng et al. 1991; 1996; 2001a; 2001b; 2003). Notable work by this group includes lead (Pb), iron (Fe), copper (Cu) and manganese (Mn) (Choi and Zheng, 2009; Shi and Zheng, 2007; Wang et al., 2006; 2008). While Fe transport across the brain barriers has received considerable attention over the years, Cu transport across these barriers has only started to receive attention recently.
Available experimental evidence suggests an intimate relationship between the metabolic roles of Cu and Fe in humans (Fairweather-Tait et al., 2004; Gambling et al., 2004,Sharp et al., 2004). Recent research has shown that intestinal Cu transport is enhanced in rats during Fe deficiency (FeD), indicating that Cu may play a role in various aspects of overall body Fe homeostasis (Collins et al., 2006). With FeD being one of the most common nutritional disorders in the world affecting up to two billion people worldwide (World Health Organization, 2007), there are clearly public health issues associated with imbalances in the nutritional supply of Fe and potentially Cu. In addition, both an excess and deficiency in Cu status is known to be detrimental to the CNS. For example, a mutation in the Cu exporter ATP7A in Menke's disease results in severe Cu deficiency in the brain. Affected individuals display psychomotor deterioration, extensive neurodegeneration in brain grey matter, and an ultimate failure to survive. In Wilson's disease, the mutated ATP7B, another form of Cu exporter in the liver, fails to eliminate excess Cu, leading to the overload of Cu in the brain (Stuerenburg et al., 2000).
A stable metal ion homeostasis is essential for brain's normal function as many metals are required as cofactors for a variety of enzymes. Abnormal Cu homeostasis both systemically and subcellularly, is believed to be associated with the pathogenesis of Parkinson's disease (Gagelli et al., 2006). Evidence has also suggested that FeD may play a critical role in the poor cognitive functioning and the pathogenesis of Alzheimer's disease (Sparks et al., 2003; Murray-Kolb and Beard, 2007; Carlson et al., 2008; Youdim et al., 2008). Carlson et al. (2008) report that FeD alters the expression of genes implicated in Alzheimer's disease. Most recently a study by Salustri et al. (2010) demonstrates that the cognitive function is inversely correlated with serum free Cu levels; as Cu levels rise, the cognitive function declines. While Fe and Cu interactions are known from the literature, the questions as to whether FeD altered Cu homeostasis in the brain and CSF and how exactly FeD affected Cu transport at the BBB and BCB were virtually unexplored.
Previous work from this laboratory suggests that Cu transport into the brain primarily occurs via the BBB as a free Cu ion and that the BCB may serve as a regulatory site for Cu in the CSF (Choi and Zheng, 2009). Cu may enter the brain through various Cu transporters located at the brain barriers such as Cu transporter-1 (Ctr1), divalent metal transporter-1 (DMT-1), ATP7A, and ATP7B (Choi and Zheng, 2009). However, the mechanism by which Cu is sequestered and transported by the brain barriers and the impact of Fe status on these processes remained largely unknown.
The purpose of this study was to (1) establish a relationship between Fe and Cu in the CNS following FeD or Fe overload (FeO); (2) determine whether such a relationship between brain Fe and Cu was maintained by Fe effect on Cu transport at the brain barriers; and (3) understand the direction of free Cu transport between the blood and CSF across the BCB under normal conditions. The study would enable us to understand the role of the brain barriers in regulating Cu homeostasis and the impact of FeO and FeD on CNS Fe and Cu homeostasis.
Chemicals and reagents were obtained from the following sources: copper chloride, sodium pyruvate, calcium chloride and HEPES from Sigma Chemical Company; Hank's balanced salt solution (HBSS), fetal bovine serum (FBS), Dulbecco's modified Eagle's medium (DMEM), and antibiotic-antimycin solution from Gibco (Grand Island, NY); epidermal growth factor (EGF) from Roche Applied Science (Indianapolis, IN); pronase and protease from Calbiochem (La Jolla, CA); and collagen-coated Transwell-COL inserts from Corning (Cambridge, MA). The appropriate rat chow was purchased from Dyets Inc. (Bethlehem, PA). All reagents were analytical grade, HPLC grade, or the best available pharmaceutical grade.
14C-sucrose (specific activity: 495 mCi/mmol) was purchased from Moravek Biochemicals, Inc. (Brea, CA) and Eco-lite-(+) scintillation cocktail from MP Biomedicals (Irvine, CA). 64CuCl2 (specific activity 15–30 mCi/μg) was obtained from Washington University at St. Louis, which was produced by cyclotron irradiation of an enriched 64Ni target by using methods reported (McCarthy et al., 1997).
Male Sprague Dawley rats were purchased from Harlan Sprague-Dawley Inc. (Indianapolis, IN). At the time of use the rats were 5 weeks old weighing 125-149 g. They were housed in a temperature-controlled room under a 12-h light/12-h dark cycle. The protocol using rats in this report was approved by Purdue Animal Care and Use Committee.
Study 1 (n=6 for each group) was performed to establish the normal physiological values of Cu and selected essential metals in the serum, CSF, brain parenchyma, and choroid plexus in order to investigate how FeO or FeD may affect these values. Rats were randomly divided into 3 groups: a control group that was fed with a normal diet (35 mg Fe/kg), an FeD group which received an FeD diet (3-5 mg Fe/kg), and an FeO group whose diet contained an excess amount of Fe (20 g carbonyl Fe/kg) (Dyets Inc.). Animals in each group took the respective diets and distilled-deionized water at libertum for 4 weeks; the dose paradigm was adapted from reports by Dallman et al. (1982) and Ryu et al. (2004). The body weight of the animals was recorded twice a week. After 4 weeks, animals were sacrificed and the appropriate tissues were collected for ICP-MS analysis.
Study 2 (n=5 for each group) was performed to determine the effect of Fe status on the unidirectional uptake rate of Cu (Kin) in brain capillaries, brain parenchyma, CP, and CSF. Free unbound 64Cu (5μM) was perfused in situ to brains.
Study 3 (n=4 for each group) was performed to determine the directional transport of Cu across the blood-CSF barrier under normal conditions. A primary choroidal epithelial cell culture model in a two-chamber Transwell system as previously established in the laboratory (Zheng et al., 1998) was used.
Study 4 (n=5-6 for each group) was performed to determine the effect of Fe status on the clearance of Cu by the CP from the CSF. A ventriculo-cisternal perfusion technique established in this laboratory (Wang et al. 2008) was used.
Serum Fe status was analyzed by quantifying total serum Fe, unsaturated Fe binding capacity (UIBC), total Fe binding capacity (TIBC), and transferrin saturation (TS, %) in serum. Total Fe and UIBC were determined using an Iron/TIBC testing kit (Pointe Scientific, Inc.). The TIBC was calculated as the sum of total serum Fe and UIBC. The TS were determined by dividing total serum Fe by TIBC.
For ICP-MS metal analysis, samples were transferred to a Teflon 96-well plate and digested with 0.15 mL of concentrated HNO3 (Mallinckrodt, AR Select grade) at 110°C for 4 h. Each sample was diluted to 1.45 mL with 18-MΩ water and analyzed on a PerkinElmer Elan DRCe ICP-MS. Indium (EM Science) was used as an internal standard.
A brain perfusion technique has been well established and used in this laboratory (Choi and Zheng, 2009; Deane et al., 2004). Rats from the control, FeD or FeO group were anesthetized with an intraperitoneal (i.p.) injection of ketamine/xylazine (75:10 mg/mL, 1 mL/kg), the neck shaved, and the animal placed on a warming pad. Following the ligation of the external carotid and common carotid arteries, the right internal carotid artery was cannulated with a polyethylene catheter tubing (PE-10) containing a 95% O2 – 5% CO2 saturated and continuously gassed 37°C Ringer's solution ( in 1000 mL: NaCl 7.31 g, KCl 0.356, NaHCO3 2.1 g, KH2PO4 0.166 g, MgSO4-7H20 0.3 g, glucose 1.5 g, sodium pyruvate 0.11 g, and CaCl2 0.278 g, pH 7.4) at a flow rate of 2.9 mL/min (Heidolph Pumpdrive 5201). The "hot" solution was kept in a separate syringe and contained 14C-sucrose (as a space marker) and 5 μM 64Cu in pre-gassed Ringer's solution, was perfused in the cannulated internal carotid artery via a second syringe pump (Harvard Compact Infusion Pump, Model 11 Plus) at a flow rate of 1 mL/min. The total flow rate of the perfusion was therefore 3.9 mL/min. To prevent recirculation of the rat blood, the left ventricle of the heart was cut at the start of the perfusion. This technique has been validated for CNS transport studies (Takasato et al., 1984; Smith, 1996; Zlokovic et al., 1986; Deane and Bradbury, 1990), and is well established in this laboratory (Deane et al., 2004; Choi and Zheng, 2009).
At the end of the perfusion (5 min), the Harvard syringe pump was switched off and the brain vascular system was washed for 1 min with the Ringer solution to remove Cu adsorbed to the luminal surface and the luminal content. Immediately after perfusion, a sample of CSF was collected from the cisterna magna, using a 25-guate butterfly needle (Becton Dickinson, Franklin Lakes, NJ). The brain was harvested and washed with ice-cold saline. The ipsilateral CP tissue was collected from the lateral brain ventricles and the ipsilaterally perfused cerebrum was used for capillary extraction.
The brain capillary separation was carried out as previously described (Preston et al., 1995; Triguero et al., 1990; Zlokovic, 1995; Deane et al., 2004; Choi and Zheng, 2009). Briefly, the brain was weighed and homogenized in 3 volumes of ice-cold buffer, with 7 strokes in a 7-mL grind pestle (Kontes, Vineland, NJ). The buffer contained (mmol/L) HEPES, 10; NaCl, 141; KCl, 4; MgSO4, 1; NaH2PO4, 1; CaCl2, 2.5; and glucose, 10 at pH 7.4. Dextran 70 was added to a final concentration of 15% (w/v) and the solution homogenized with 3 additional strokes. The homogenate was then spun at 5,400 ×g for 15 min at 4°C. The supernatant (brain parenchyma) and pellet (capillary-enriched fraction) were separated carefully, and counted for radioactivity (in-situ perfusion) or analyzed for metal content via ICP-MS.
64Cu uptake was expressed as a volume of distribution, Vd, and calculated as (C Tissue/CPerfusate or CCSF/CPerfusate), where CTissue or CCSF are d.p.m./g of brain tissue (e.g. cerebral capillaries, choroid plexus, brain parenchyma, etc.) or CSF, and CPerfusate are d.p.m./mL of the perfusion fluid. The unidirectional uptake rates, Kin (μL/s/g), corresponding to the slope of the uptake curve was determined using linear regression analysis of Vd against the perfusion time (T, s) as Vd=Kin T+Vi, where V =i is the ordinate intercept of the regression line. 64Cu uptake was corrected for residual radioactivity by deducting Vd for 14C-sucrose from the total 64Cu distributing volume (Choi and Zheng, 2009).
The method to culture primary choroidal cells has been well established in this laboratory (Zheng et al., 1998). CP tissues from 4-week old rats were dissected and digested in Hank's balanced salt solution (HBSS) containing 0.2% pronase at 37°C for 10-15 minutes. The digested cells were then washed twice with HBSS and resuspended in growth DMEM medium containing 10% FBS, 100 units/mL penicillin, 100 μg/mL streptomycin, 0.25 μg/mL amphotericin, 100 μg/mL gentamycin and 10 ng/mL epidermal growth factor (EGF). A 20-gauge needle was used to pass cells through 14-15 times to ensure adequate cell separation for seeding. Cells stained with 0.4% Trypan blue were counted under a light microscope before seeded on 35-mm Petri dishes (pre-coated with 0.01% collagen) at a density of 2-3 × 105 cells/mL. The cultured cells were maintained at 37°C with 95% air-5% CO2 in a humidified incubator without disturbance for at least 48 hrs. The growth medium was replaced 3 days after the initial seeding and every 2 days thereafter.
After cultured in dishes for 7-10 days, the cells were transferred to a Transwell transport device which is composed of two chambers. The inner chamber, also known as the apical chamber, is immersed in the outer chamber. When 64Cu was added to the inner chamber and the radioactivity measured in the outer chamber (i.e. transport from the inner to the outer chamber) the experiment was referred to as an efflux study. Likewise, the same experiment done from the outer chamber to the inner chamber was referred to as an influx study. Overnight medium incubation in the Transwell was to improve the cell attachment to the collagen coated membrane in the inner chamber. In the following day, an aliquot of 0.8-mL cell suspension (23 × 105/mL for initial seeding) was added to the inner chamber and 1.2 mL of medium was added to the outer chamber. The transepithelial electrical resistance (TEER), an indicator of the tightness of the barrier, was used to track the formation of a cellular monolayer between the inner and outer chambers. The TEER value was measured every other day by an Epithelial Volt-Ohmmeter (EVOM, World Precision Instruments, Sarasota, FL) until the resistance was 50-60 Ω-cm2. The same two-chamber system without cells was used as the blank and its background was subtracted from the measured TEER. When the cells grew to a confluence, the surface level of the medium in the inner chamber was roughly 2 mm above that of the outer chamber. This in vitro system mimicking the blood-CSF barrier has been used in our previous studies (Zheng et al., 1998; Crossgrove et al., 2004; Shi et al., 2005; Wang et al., 2008; Behl et al., 2009).
To investigate the directional transepithelial transport of Cu across the BCB, an aliquot of 64CuCl2 was mixed with 150 μL of 14C-sucrose (specific activity: 495 mCi/mmol) to a final concentration of 5 μM CuCl2 (specific activity 5 μCi/mL) and 1 μM with 0.5 μCi/mL 14C-sucrose in HBSS. All the transport parameters were conducted in an incubator kept at 37°C. For the influx study, 64Cu-containing HBSS was added to the outer chamber (donor) and an aliquot of medium was taken as the initial donor radioactivity. A series of time points (0, 5, 10, 15, 30, 45, 60, 75, 90 min, 2, 3, 4 h) were set for sample collection. At each time point, an aliquot of 10 μL of HBSS was taken from the inner (receiver) chamber and replaced with an equal volume of fresh HBSS. The efflux study was conducted in the exact same manner except the inner chamber was the donor chamber and the outer chamber was the receiver.
All samples were counted by an auto-Gamma 5000 Series Gamma Counter (Packard Instrument Company). To count 14C-sucrose the samples were mixed with Eco-lite cocktail and counted on a Packard Tr-Carb 2900 TR Liquid Scintillation Analyzer.
To determine the transport coefficient for 64Cu and 14C-sucrose across the cellular monolayer, the data within the linear range were used for linear regression analyses. The slope (d.p.m./mL-min) of each dataset was used to calculate the total and blank permeability coefficients in Eq. (1),
where PT represents the total permeability coefficient (cell monolayer + membrane + coating, cm/min); PB, the blank permeability coefficient (membrane + coating, cm/min); VR, volume of media in the receiver chamber; A, surface area of transport membrane (1.1 cm2); CD, concentration of radiolabeled compounds in donor chamber (d.p.m./mL); CR, concentration of radiolabeled compounds in receiver chamber (d.p.m./mL). The permeability coefficients of epithelial barrier (PE) are then obtained from the Eq. (2) (Shi and Zheng, 2005; Wang et al., 2008).
where PE is the permeability coefficient of epithelial barrier.
Control, FeD and FeO rats were anesthetized with an i.p. injection of ketamine/xylazine (75:10 mg/mL, 1 mL/kg) and immobilized in a stereotaxic device. A midline cutaneous incision was made from the forehead to the neck, exposing the top of the skull. A hole was drilled for insertion of a guide cannula (Plastics One Inc., Roanoke, VA). The cannula was inserted into the lateral ventricle according to the following parameters on three scales: 0.8 mm posterior to the bregma, 1.4 mm lateral to the midline, and 3.5 mm vertical from the surface of the skull (Wang et al., 2008). An internal guide cannula connected to PE50 tubing was inserted into the guide cannula for lateral ventricle perfusion controlled by the pump-driven syringe filled with artificial CSF (aCSF). Pre-gassed aCSF containing either 0.5 μCi/mL of 14C-sucrose or 2 μM of CuCl2 was delivered to the lateral ventricle at a rate of 28 μL/min controlled by a syringe pump (Harvard Compact Infusion Pump, Model 11 Plus). A 26G butterfly needle was inserted at an appropriate angle into the cisterna magna for collection of the perfusion outflow. Cisternal outflow samples were collected at 10-min intervals throughout the perfusion time (80 min). The CSF volume was determined by measuring its weight assuming the CSF density was 1 g/mL. Additional anesthesia was given to the rats as needed. Body temperature was maintained at 37°C using a heating pad during the perfusion. At the end of the perfusion, the animal was decapitated, the brain removed, and both lateral choroid plexus tissues were harvested. Samples were taken for liquid scintillation counting and ICP-MS.
All data are expressed as mean ± SEM unless stated otherwise. Statistical analyses of the differences between groups were carried out by one-way ANOVA, and the Pearson correlations were determined using SPSS 17.0 statistic package for Windows. Linear regression lines were plotted with Microsoft Excel 2007. The differences between the means were considered significant if P values were equal or less than 0.05.
Both FeO and FeD treatment in diet resulted in the hematological changes one would expect to find in the serum of individuals who are overloaded or deficient with Fe, i.e., a nearly 2-fold increase in serum Fe in FeO animals compared to controls (p<0.05) and a marked, yet nearly significant decrease in serum Fe in FeD animals (p=0.072) (Fig. 1A). A more sensitive ICP-MS analysis revealed that FeD animals had roughly half the amount of Fe as compared to controls (p<0.05; Fig. 2A). The change in UIBC was as expected in FeO and FeD animals with FeO having significantly less iron binding capacity and FeD animals having greater Fe binding capacity (p<0.05; Fig. 1B). The TIBC was elevated in FeD animals and unchanged in FeO animals (p<0.05; Fig. 1C), while transferrin saturation was elevated in FeO animals and decreased in FeD (p<0.05; Fig. 1D). These results indicate that the animals were rendered either overloaded of deficient of Fe through the dietary regiment.
Quantitation of Fe levels in the serum, CSF, and brain parenchyma by ICP-MS analysis indicated that FeO treatment significantly elevated Fe concentrations in both the serum and brain parenchyma (Fig. 2A, C). Interestingly, treatment with FeD diet also resulted in a significant increase of Fe level in brain parenchyma (Fig. 2C), although serum Fe was indeed decreased in these animals (Fig. 2A). Neither FeO nor FeD treatment had any effect on Fe levels in the CSF (Fig. 2B).
Following FeO treatment, Cu concentrations in serum and CSF were significantly decreased by 97% (p<0.05), and 56% (p<0.02), respectively (Fig. 3A, B). In contrary, the Cu concentrations were significantly elevated in the brain, CSF and choroid plexus in FeD rats by 85 % (p<0.05), 52% (p<0.05), and 67% (p<0.05), respectively (Fig. 3B, C, D). Noticeably, the magnitude of changes in serum Cu levels was much greater in FeO rats (nearly 40 fold decrease) than in FeD rats. In contrast with CSF Fe levels, where both FeO and FeD had no detectable effect on CSF Fe, the CSF concentration of Cu was significantly lower in FeO rats and significantly higher in FeD rats than those in controls, suggesting a direct impact of systemic Fe homeostasis on Cu regulation by brain barrier systems and thereupon an altered Cu level in the CSF. Interestingly, both FeO and FeD treatments led to an elevated Cu concentration in brain parenchyma (Fig. 3C).
Further linear regression analysis revealed that CSF Cu levels were inversely associated with serum Fe levels (r=-0.665, p<0.01, Fig. 4A). However, no significant association was found between serum Fe and CSF (r=0.065, p=0.812, Fig. 4B) or in capillary depleted brain parenchyma (r=-0.164, p=0.543, Fig. 4C). An inverse association, which approached but did not reach statistical significance, was found between serum Fe and choroid plexus Cu (r=-0.419, p=0.106, Fig. 4D), whereas strong positive correlations were found between Fe and Cu in the choroid plexus (r=0.738, p<0.01, Fig. 5A) as well as in brain parenchyma (r=0.781, p<0.01, Fig. 5B).
The above results prompted us to further explore whether Fe status altered the transport rate of Cu across the brain barriers. Unidirectional rate constants (Kin) represent the rate of 64Cu entering a particular compartment after 5 min of perfusion via the intra-carotid artery. In FeO animals, the rate constant for 64Cu transported into brain parenchyma was significantly decreased, about 50% that of controls (Fig. 6A), while the rate constant to brain capillary was not changed in the same group of rats (Fig. 6B). Treatment with FeD greatly increased the transport of 64Cu into brain tissue; the rate constant was increased by nearly 2 fold in FeD animals compared to controls (Fig. 6A). Consistently, the Kin of 64Cu into the brain capillaries in FeD animals was nearly 5 times that of controls (Fig. 6B). The rates of Cu transport into the CSF and choroid plexus were also significantly increased (p<0.05) in FeD animals (Fig. 6C, ,6D).6D). These finding are consistent with our ICP-MS studies, suggesting an increased influx of Cu into the brain and CSF during FeD.
When 14C-sucrose, a leakage marker, was added to the donor chamber, the flux of 14C-sucrose in the receiver chamber in either direction, was nearly equal (Fig. 7A), suggesting an equal passive leakage of 14C sucrose molecules across the barrier no matter to which chamber sucrose was added. When 64Cu was added to either the inner or outer chamber to study the direction of Cu transport by the BCB, the primary choroidal epithelia appeared to transport Cu more readily in the direction of efflux (apical to basolateral) than influx, as the permeability rate constant (PE) for 64Cu was roughly 1.8 fold higher in the efflux study than in the influx study (Fig. 7A). To correct for non-specific leakage across the barrier system, the PE values of 64Cu were normalized by 14C-sucrose and expressed as a ratio of 64Cu to 14C-sucrose. After correcting for nonspecific leakage, Cu efflux (from the CSF to the blood) in the blood-CSF model was nearly 2 fold greater than that of the influx (p<0.01, Fig. 7B). These results suggested that the efflux of Cu across the blood-CSF barrier was a more favorable pathway for Cu transport than the influx.
Using an in situ ventriculo-cisternal brain perfusion technique, we investigated Cu clearance from the CSF from control, FeO, and FeD animals. Cu recovered from the cisternal effluent was expressed as the ratio of Cu concentration in the collected effluent to that in the perfusate. As a result of the choroid plexus being the major tissue responsible for removing materials present in the ventricular CSF, a higher recovery of Cu in the cisternal effluent suggests there is less in situ uptake of Cu by this tissue, while less recovery of Cu suggests a greater uptake of Cu by the choroid plexus. In the FeD animals, the Cu recovered in the cisternal effluent was lower than that of both the control and FeO animals (p<0.05), suggesting there was a greater clearance of Cu from the CSF in these animals (Fig. 8A). Since the CSF secretion rate, measured by the use of the space marker 14C-sucrose was not altered by either FeO or FeD (Fig. 8B), the increased Cu clearance from the CSF was apparently due to elevated Cu uptake by the choroid plexus.
ICP-MS metal analysis revealed numerous changes in metal concentrations as a result of FeO and FeD. Most significantly, we found that the metal concentrations in brain parenchyma and choroid plexus tissues varied as a result of Fe status much more readily than metals in the CSF or serum (Table 1). In the brain parenchyma FeO caused an elevation of Ca2+, Fe2+, Zn2+, Se2+, and Sr2+, while FeD caused increases in Ca2+, Mn2+, Fe2+, Cu2+, Sr2+, and a decrease in Ni2+ (p<0.05). In the choroid plexus, FeO caused only an elevation of Fe2+, while FeD caused an increase in Ca2+, Fe2+, Co2+, Ni2+, Cu2+, Zn2+, Se2+, and Rb2+ (p<0.05). It appeared that under the FeD condition, the fluctuation of metal concentrations was more significant in the choroid plexus than in the CSF, perhaps due to the regulatory function of the blood-CSF barrier to actively transport metals between the blood and CSF in order to keep the CSF metal levels constant and to protect the normal brain function.
In serum, FeO caused an increase in Fe2+, and decreases in Cu2+, Rb2+, and Sr2+ (p<0.05). FeD resulted in decreased Fe2+ and elevated Co2+, and Sr2+ levels in the CSF (p<0.05). There was also an increased serum Cu level although not significant (p=0.064). In the CSF, FeO caused decreases in Cu2+ and Sr2+ levels, and Fe D caused an increase in Cu2+ and Se2+ levels (p<0.05).
The results of this study clearly indicate that systemic Fe homeostasis has a significant impact on Cu homeostasis in blood, CSF, choroid plexus and brain parenchymal tissues. More specifically, our results showed the following characteristics of Fe-Cu interactions: (1) Fe levels in the CSF and brain parenchyma appeared to be more tightly regulated than Cu; (2) a decreased serum Fe level in FeD animals seemed likely to increase Cu levels in the choroid plexus and CSF; (3) changes in serum Fe levels affected more profoundly the Cu level in the CSF than in brain parenchyma; and (4) an increase in tissue concentrations of Cu in the choroid plexus and brain parenchyma was associated with the increased Fe level in the corresponding tissues.
A vast amount of evidence in literature has suggested that the homeostasis of Fe and Cu in the body is physiologically intertwined (Crowe et al., 1996; Garrick et al., 2003). Some investigators have suggested that this interaction may take place primarily in intestinal absorption (Arredono et al., 2000;Arredondo and Nunez, 2005; Linder et al., 2003). For example, using an immortalized intestinal epithelial line known as Caco-2 cells, Linder et al. (2003) report that inducing FeD in this in vitro model system causes an increase in both the uptake and retention of Cu. Arredondo et al. (2006) further demonstrate that Cu and Fe display a competitive inhibition with regards to intestinal absorption in Caco-2 cells. These findings are consistent with our own data obtained from the brain barrier systems that systemic FeD apparently caused Cu overload in brain tissues. Also, the serum Fe statuses of these treated animals were similar to those in Ryu et al. 2004, and Dallman et al. 1982, from which our dose paradigm was adopted. In addition, our data clearly demonstrated a strong relationship between Cu and Fe in the serum, CSF, brain parenchyma, and choroid plexus. Considering the overall importance of Cu in disease causation and progression of numerous neurodegenerative disorders, a disrupted Fe homeostasis due to either genetic defects or environmental alteration can perceivably impact significantly the Cu stability of the central nervous system (CNS).
How exactly the changes in Fe homeostasis affect the regulation of Cu in the brain is unknown. Previous studies from this laboratory support the view that both brain barriers play a major role in regulating brain Cu transport. It is the free Cu, but not protein-bound Cu, that represents the major Cu species being transported across the brain barriers (Choi and Zheng, 2009). The works by Erikson et al. (2004) and Garcia et al. (2007) explore the effect of FeD on divalent metal accumulation in different regions of the brain. FeD resulted in increased brain accumulation of manganese (Mn), zinc (Zn) and Cu in regions such as the globus pallidus, striatum, and substantia nigra (Erikson et al., 2004; Garcia et al., 2007). Our current data are in good agreement with these previous observations with respect to Cu transport by the brain barriers and the impact of Fe status on Cu homeostasis in the CNS. Interestingly, FeO also led to elevated Cu concentration in brain parenchyma in our study. It is well known that the balance of Fe levels are regulated by Cu-dependent extracellular Fe transport protein ceruloplasmin as well as Cu-dependent intracellular Fe transport protein hephaestin. Conceivably, FeO in the brain parenchyma may increase the cellular demand for more of these Cu-dependent Fe transport proteins so as to export the excess Fe to prevent Fe toxicity; this hypothesis, however, needs additional experimental testing. It is possible that this phenomenon is responsible for the positive correlations found between Cu and Fe in both the brain parenchyma and choroid plexus.
In this study we focused on the Cu influx via the BBB to brain parenchyma, as well as its efflux at the BCB by the choroid plexus. In brain parenchyma, the unidirectional uptake (Kin) of Cu was roughly 2 fold faster than the uptake rate to the CSF in control animals, indicating that Cu appeared to be transported at a greater rate to the brain parenchyma than to the CSF. Since the parenchyma and CSF are in direct contact with the BBB (parenchyma) and the BCB (CSF), respectively, and since there is no apparent barrier between brain parenchyma and the CSF, it is reasonable to suggest that Cu may enter brain parenchyma from the BBB; subsequently it may flow from the brain parenchyma to the CSF. Interestingly, our in situ studies indicated that the Kin for Cu in the choroid plexus, where the BCB locates, was nearly 100 fold faster than the uptake rate in the CSF. Thus, the role of the choroid plexus in regulating Cu in the CSF cannot be ignored.
As a result of the choroid plexus being in contact with both the CSF and blood, it is possible that the high accumulation of Cu in the choroid plexus could come from either the blood, CSF, or both. Subsequently the Cu in the CSF could be derived from either the choroid plexus, or the brain parenchyma. However, it is unlikely that the Cu is being transported from the blood to the choroid plexus and subsequently to the CSF, because the rate of transfer to the CSF was about a 3-4 fold increase in FeD rats, whereas it was only about a 1.5 fold increase in the plexus tissue. Thus, most of the excess Cu in the CSF must come from the interstitial fluid between neurons and glial cells in the brain parenchyma.
To further address the role of choroid plexus in Cu transport, we used a two-chamber Transwell culture system to determine the primary direction through which Cu was transported across the BCB. This in vitro system possesses a number of advantages: (1) it forms a biological barrier that mimics the in vitro BCB (Zheng et al., 1998); (2) the polarized cells on the Transwell membrane can be used to study the orientation of metal transport across the BCB; and (3) the system allows the metal to be added to either side of the barrier system. Using this in vitro BCB model system, we demonstrated that the rate of Cu transport from the CSF to the blood was greater than from the blood to the CSF. Additionally, ventriculo-cisternal perfusion studies provided in vivo evidence to support the role of the choroid plexus in the uptake of Cu from the CSF. Considering the fact that FeD increased Cu uptake at a greater rate in the BBB, brain parenchyma, and CSF, it was tempting to speculate that the BCB may increase the rate of Cu transport from the CSF to blood in an attempt to clear excess CSF Cu. Indeed, FeD animals did display a greater rate of Cu clearance from the CSF compared to either FeO or control animals, while 14C-sucrose clearance, a measure of BCB permeability, was unchanged. Thus both the in vitro and in vivo data from our current studies support the theory that the BCB's role in CNS Cu homeostasis is to efflux Cu from the CSF to the blood.
Our results demonstrated that FeD appeared to have a more profound effect than FeO on Cu homeostasis at the brain barriers. Our in vivo data showed that FeD increased the rate of Cu transport across the BBB, resulting in elevated Cu levels in all cerebral compartments. In the case of FeO, while the treatment decreased Cu transport in the brain parenchyma, the rates of Cu transport across both the BBB and BCB were unchanged. These seemed to suggest that the brain barriers are better adapted at controlling Cu homeostasis brought on by FeO than FeD. This was also demonstrated by unchanged Cu levels in the brain following FeO. Although FeO caused a significant decrease in serum and CSF Cu levels brain parenchyma Cu levels remained unchanged. It is plausible that the brain barriers and brain interstitial compartment are able to retain adequate Cu stores to perform necessary functions within the CNS. Interestingly, Fe levels in the brain parenchyma and choroid plexus were significantly increased in the FeD animals. This may have occurred as a result of the brain barriers and brain interstitial fluid attempting to maintain a stable Fe homeostasis. It is reasonable to suggest that the brain barriers are able to uptake and retain more Fe in an FeD state to prevent Fe deficiency in the brain. Our ICP-MS data also indicated that the overall metal ion homeostasis was impacted to a much greater extent in both the choroid plexus and brain parenchyma in FeD than in FeO animals (Table 1). While the rationale as to why the brain barrier systems as well as the CNS have a stronger response to FeD than FeO remains to be explored; it is possible the different response of Cu/Fe regulation to FeO and FeD may be the result of sensitivity of metal transporters to the abnormal Fe status.
Several transporters have been suggested to transport Cu across the brain barriers, including Ctr1, DMT1, ATP7A, and ATP7B (Li and Zheng, 2005; Choi and Zheng, 2009). Our recent data suggest a profound expression of DMT1 in the choroid plexus and the FeD-induced upregulation of DMT1 (Data not shown). However, the exact functions of these transporters at the brain barriers in regulating Cu transport and the impact of FeO and FeD on the expression of these transporters are currently still unknown. Studies to investigate Cu transporters localization, regulatory mechanism of their expression, and how the dysregulation of these transporters by FeO and FeD affects brain Cu homeostasis are currently underway in this laboratory.
In summary, the present work indicates that both the BBB and BCB contribute to maintain a stable Cu homeostasis in the brain. The BBB appears to be a more important route than the BCB in the transport of Cu into brain parenchyma; upon entering brain, Cu is utilized and released into the CSF via the interstitial fluid. Subsequently Cu is removed by the BCB from the CSF to blood. FeO increases Fe levels in the brain and serum, and decreases Cu levels in the serum and CSF. In contrast, FeD increases Cu transport at the brain barriers; consequently FeD prompts Cu overload in the CNS. Under the latter condition, the BCB in the choroid plexus plays a key role to remove excess Cu from the CSF so to maintain the Cu homeostasis in the CNS. Further investigation into the mechanism of Cu and Fe transport by the brain barriers under normal and disease states is well warranted.
The authors thank Dr. Yanshu (Sue) Zhang's technical assistance. The research reported in this manuscript was supported in part by a Ross Fellowship from the Purdue Research Foundation (ADM) and by NIH/National Institute of Environmental Health Sciences grant RO1-ES008146 and R21-ES017055.
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Question about Euro-Pro 8260 Mechanical Sewing Machine
I have a Euro Pro 8260 sewing machine. The long plastic thread holder that is used when to transfer thread to a bobbin broke off, can it be replaced. I tried to super glue it but it did not hold. Thank you, Michelle Levick
Posted by mlevick on
If you can provide a photo of the broken part and lication where it attaches to machine it would help greatly.
SOURCE: I have freestanding Series 8 dishwasher. Lately during the filling cycle water hammer is occurring. How can this be resolved
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Posted on Jan 02, 2017
SOURCE: needle will not pick up bobbin threat
I had the exact same problem. The timing is off. You can actually fix this yourself all you need is an allen wrench. If you look at the rod the needle is inserted into and follow it up to about 2 inches below the top of the machine there should be a clamp. use the allan wrench to loosen the clamp and adjust the needle height. I used this website as a guide to getting the timing correct. http://www.sewusa.com/Sewing_Machine_Repair/Repair_Pictures/timing1.gif
now if only I could figure out why my thread keeps getting jammed under the bobbin!
Posted on Feb 22, 2008
SOURCE: Euro-Pro 7133 - Thread is getting stuck underneath
I had the exact same problem!!!!finally i took to the store and they showed me that i was not threading the too string correctly. what you have to make sure that the needle is at its highest position and that you thread it through all the guides!
Posted on Mar 07, 2008
SOURCE: the bobbin thread on sewing machine will not catch
how do you retime
Posted on Aug 13, 2008
SOURCE: bobbin thread/feed dog eating fabric
This is a very common problem with really lightweight fabric and can often be cured (if doing straight stitching) by using a straight stitch needle plate and foot. The wider needle plate and foot openings on newer machines give the machine/thread a wider area to pull the fabric into the machine. By using a straight stitch plate/foot you remove the wide opening, eliminating the majority of the problem.
Also - try using a specially coated needle (Teflon or other non-stick finish). The thread feeds more smoothly through the needle and the needle more smoothly through the fabric, causing less drag into the needle plate opening.
Hope this helps. Don't hesitate to respond to this if you have further specific questions. We're always happy to help
Happy stitching,
Kim & Linnette
Posted on Dec 03, 2008
SOURCE: euro pro 385w problems
if your machine is like mine the bobbin is not filling right. My bobbin thread is very loose when it should be tight. I am also looking for bobbin solutions. If I wind the thread around the thread tension guide 2 or three times it keeps the thread tight going to the bobbin winder. I hope this helps
Posted on May 31, 2009
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text: NA. Near Fine. A beautiful stone book made of a beige-ish, vellum colored marble for the spine and corners and a grey/pink/white marble for the "boards. The faux book measures143 by 95 by 26 mm and weighs 33.6 ounces, or 2 pound, 1.6 ounces. The spine is nicely rounded, with two "bands", each comprised of two different light marbles -- in each, a light colored marble above and below a slightly widerband of the same marble used for the "boards". A microscopic nick on the upper spine extremity is the only very minor shortcoming we could find. (Inventory #: 005140)


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Aishwarya Rai Bachchan is busy promoting her film Sarbjit these days but the stunning actress saw some scenes from Abhishek Bachchan's Housefull 3 recently and loved it.
She told a leading news agency, ''The fact that it's a such a huge successful franchise ('Housefull 3'), it means it is succeeding in entertaining the audience. I have seen a couple of scenes of 'Housefull 3' as we were dubbing in same area, so Abhishek and Akshay called to see the scenes and it's a laugh riot.''
Click On VIEW PHOTOS to see some adorable pictures of Abhi & Aish
Aishwarya Rai Bachchan further added, "As an actor I have always been open to doing all kinds of cinema. Just as much serious is intense work, I have always said comedy is serious work as well for the artists, writers and director. It is wonderful when that kind of cinema evokes that kind of reaction. It means that they (Housefull 3 team) have managed to hit the spot," she said.
Also Read: Farhan Akhtar & Shraddha Kapoor Get Naughty At The Party, Leave Everyone Shocked!
''Both Abhishek's film Housefull 3 and my film Sarbjit are completely different. As actors, both Abhishek and I thoroughly enjoy this (comedy) kind of genre. We as actors don't do one kind of cinema and this is what we both share in common,'' she revealed.
Talking about comedy films, Aishwarya Rai Bachchan said, "It's a genre that is celebrated for what it is...it's out there, in the face, big picturisation for songs, etc. So that's the genre that the audience enjoy thoroughly. We are also audience when we see a film. Sometimes people get the humour (she does) and sometimes they don't.''
Meanwhile, Aishwarya Rai Bachchan is all set to woo the audiences with Sarbjit. The actress is playing the role of Sarabjit Singh's sister Dalbir Kaur in the movie.


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Manchester City maintains Premier League title push with 4-1 win over Southampton
London: Manchester City has turned on the pressure on their Premier League title rivals with a 4-1 victory over Southampton on Saturday at the Etihad.
The victory moved City to within a point of leaders Liverpool, who visit West Ham on Sunday having already played one game more than Manuel Pellegrini`s men.
According to the BBC, a penalty shot by Yaya Toure gave the hosts the lead three minutes into the game, although visiting striker Rickie Lambert equalised with a spot-kick of his own.
However, the report mentioned that goals from Samir Nasri and Edin Dzeko in first-half stoppage time, followed by a late effort from Stevan Jovetic, made it 14 wins from 15 home league games for City this season.
But the report added that there was bad news for watching England boss Roy Hodgson as Southampton striker Jay Rodriguez was carried off with what looked like a serious knee injury, just moments after Toure`s penalty shot.


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ORINDA — Bare walls carry endless possibilities for Maya Kobashigawa.
When she moved into an apartment in Bakersfield years ago where she worked as a news writer and producer, she recalled, "I wanted to decorate it but couldn't find anything for my wall so I painted an abstract."
And when she and her family moved to Lafayette 14 years ago, she saw great potential in the bare walls of her new home.
"When I see a space, I can envision an art piece there," she said. "I can create a specific piece for a room. A lot of it has to do with colors."
Kobashigawa's abstract paintings, currently on display at the Orinda Gallery, show her versatility using acrylics and mixed media on canvas. Abstract pieces need not be limiting or esoteric, she said.
Her path toward abstracts stemmed from an early age of artistic enlightenment; the fascination of viewing great works of art as a child left a huge impression on her.
"My father was from India so when we were young, we'd go visit museums and see incredible works of art and it instilled a passion in me," said Kobashigawa. "Traveling to various countries and visiting museums allowed me to appreciate all different kinds of art. We used to travel to India almost every year to visit family and on the way we would stop in another country for several days before continuing on to India. Every visit to a new country involved a trip to a museum. I learned to appreciate art from all different genres and countries."
The moment she first saw the Mona Lisa at the Louvre in Paris was such a vivid childhood memory. Other favorite museums include the Peggy Guggenheim museum in Venice.
While she majored in art history in college thinking she'd become a curator, Kobashigawa took a different path, venturing into broadcast journalism. But art had always been on her mind. Drawn to abstracts, she counts Russian painter Wassily Kandinsky and Jackson Pollock as huge stylistic influences.
Inspired by outdoor experiences, the artist conveys her connection to nature by exploring "the interplay of color and light" through her abstract artwork.
"My work is more focused on awakening an emotion from the viewer, and less concerned with the subject," she said.
In her piece Waterfall, she uses layers of fabric and cellophane to give a sense of depth, almost as if looking at a sculpture, she said.
"The streaks of green, yellow and blue are combined in a fluid, lyrical fashion that I hope evokes a memorable experience when viewing the piece," Kobashigawa said. "Perhaps the viewer can imagine the sound of water trickling down in Waterfall."
Liz Rosenberg said she was lucky to purchase Kobashigawa's painting Blue Skies at a school auction years ago.
"We've known the Kobashigawas since our kids were in preschool together and were honored to have a piece of Maya's art hanging in our home," Rosenberg said. "The painting is navy, yellow and white, which complemented the decor in our family room perfectly when we bought it. We subsequently moved to a new home and the piece came to rest in our 'playroom' which is the perfect spot for it with its bold colors and modern, fun vibe."
Kobashigawa gave a piece of her artwork — comprised of similar techniques as Waterfall — as a housewarming gift 16 years ago to her friend Shelley Powers.
"We've always thought that Maya's technique reminds us of Jackson Pollock's drip painting work," said Powers. "The interplay of all the colors and its 3D texture bring the canvas to life."
Also on display is a piece titled The Barnyard, created with different types of string, hay, cellophane and acrylic paints using the drip technique.
"A lot of the pieces can be hung horizontally or vertically," Kobashigawa said.
In Walking in the Woods, a mixed media piece, the artist wants the viewers to see fabric and all the detail up close.
Kobashigawa hopes that her work will encourage more people to appreciate abstract art. She said that while her work isn't meant to convey a specific message, she wishes her abstract art pieces stir some kind of emotion.
"Hopefully, they will have some kind of connection that will be a positive experience."
WHAT: Abstract art by Maya Kobashigawa
WHEN: Through October
WHERE: Orinda Library Gallery, 26 Orinda Way


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Pope Francis meets with Chilean President who has proposed legalizing abortion
"Madame, welcome.â?
"It's a pleasure, Your Holiness. I'm thrilled to be here.â?
This greeting marked the start of the Chilean President Michelle Bachelet's meeting with Pope Francis.
Their agenda was full. They discussed relations between Chile and the Holy See, education, and, according to a press release from the Vatican, "the preservation of human life.â?
Bachelet supports legalizing abortion in three instances: risk to the mother, unviablity of the pregnancy, or rape. After the meeting, the president spoke to the press but did not answer questions about whether Pope Francis discussed the proposed abortion law with her.
President of Chile
"It was a meeting where he had been extraordinarily caring and informative. He seemed supportive of making Chile a more just country, a more united country where our citizens can leave inequality behind and live a happier life.â?
She also said that they spoke about a possible papal trip to Chile, Argentina, and Uruguay in 2016. However, they did not set a concrete date.
At the end of the meeting, they exchanged gifts. The president gave Pope Francis an ornate rosary and a book about churches in Chile. The Pope gave her a Saint Martin medallion in return, which is his customary gift for world leaders. He also gave her a copy of Evangelii Gaudium.
Pope Francis asked her to pray for him, and the meeting ended with both of them smiling.
Up: MPI


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Nashville, TN
We are really enjoying the Nashville scene.The most amazing and different thing we saw was a tavern running on bicycle power! The picture on the right has a driver and a barkeep, otherwise, the 8 guys are peddling to make the car go. We saw them turn a corner, and they all had to stand up and peddle like mad. They were hooping and hollering and burning calories while they were drinking! Some of the pictures below are from !the Hall of Fame museum and it was a wonderful experience. It's amazing to hear and see some of the oldies from the 30s and 40s. We also went to Nashville's Honkey Tonk section – bar hopped all night, listening to all the bands. We must have been in 16 different places. We were thrilled with the quality of the musicians and the breath of their repertoires. There was one band in particular that would only take requests for $20 bills! Requests kept coming in for the Devil Went Down to Georgia, which the band said was their most requested song, and they were tired of playing it because it was so intense. They'd only play it for $100. So the crowd acquiesced and put in the cash. We thought, "Wow, 'Will sing for cash,'" is a hard way to make a living. We loved the fact that there were no cover charges at any of these bars. But of course we did drink a bit! (OK, more than a bit.) We had taken a shuttle from our RV site to downtown, so I didn't have to be designated driver! (Cheryl)


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New Leaf Entertainment cartridges were prototypes intended to be used in a retail flash kiosk for the Sega Genesis.
Packaging Images
|Alternate Titles
|NewLeaf Entertainment
|Release Dates
|Release Prices
|Product IDs
|In Packages
New Leaf Entertainment Information
These cartridges are fairly rare. It's a prototype for a retail flash kiosk that a few companies tested back in the 90's.
In 1993, IBM and Blockbuster launched a joint endeavor called NewLeaf Entertainment. Consumers would be able to stroll into a retailer and order up a CD or video game manufactured on the spot. Retailers would win because it would help with inventory control. Content companies (record labels, game publishers, etc.) would benefit from being able to have their products always available to any paying customer. The idea was pretty unique at the time.
Content developers would supply the company with the data for the medium to be used, along with the packaging design. The customer would walk up and order it. The machine would then flash the cart and produce the appropriate packaging and manual for the game.
This specific cart was a prototype for the idea.
In the Fall and Winter season of 1994 in Columbia, South Carolina; 10 local Blockbuster stores participated in a test where 16-bit game carts would be available to order. The game manufacturing machine was called Game Factory. The whole process would take only 45 seconds from order to completion. The test seemed to have gone over well, according to an article in Billboard's January 1995 issue. Robert Carberry, acting CEO of NewLeaf at the time, said the next step would be to adapt to 32-bit games.
The idea was abandoned in 1995, more than likely thanks to major record labels not wanting an even playing field along with indie publishers. With the way everything was setup, distribution would have been just as wide for any label. You can't dominate a market if the little guys can get just as much shelf space now, can you?
A poster named RetailGeek (real name Jason Goldberg) later offered some more insights:
Hey… I can tell you what New Leaf was.
Back in the early 90's Blockbuster wanted to put databases of music in the stores and make CD's on demand for customers. The music labels didn't really want to sell their music that way (this was many years before iTunes), and so Blockbuster did a proof of concept for Sega video games. The company that developed the solution was called New Leaf Entertainment, and I used to work for them.
The idea was that stores could just carry one cartridge, and we'd burn whatever game you wanted to rent on it for the 3 days, then you returned the cartridge we could burn a different title on it for the next customer. We actually had to reverse engineer the cartridges (Sega didn't agree to work with us until after we developed a proof of concept demo). The cartridge you found was from our test market in Columbia, South Carolina where the system was deployed in 10 stores. (By the way, someone owes some serious late fees for that cartridge, lol).
The in-store burner was expensive in 1992, but it just took a couple mins to burn a title. The problem with the system was that every new big title that came out back then would have some new feature in it (more memory, etc…) that previous titles didn't have. So as soon as you made a standard blank cartridge, it was incompatible with the hottest most in demand titles. Then of course the whole world went to optical media. And Blockbuster gave up trying to get content owners to let is distribute it's content digitally.
Back in the day we made a promotional video with Dennis Miller.
Said video is below.
Over on his now offline blog<, Jason also wrote the following:
We got quite far actually. We built an extravagant demo center (to help persuade content owners and the media) that included high speed VHS Duplication, bleeding edge 4X CD-ROM burners, first generation digital color printers, and robotics to automate the fabrication and assembly of a complete piece of media with all its packaging. At the time we had research that indicated shoppers would still want the physical liner notes, etc… from a CD Jewell box. The demo center had motion sensors with theatrical lighting, the Dennis Miller video, etc… it was fun.
We did a live trial of video games in Columbia, South Carolina. We partnered with Sega, and had 10 Blockbuster stores carry single blank videogame SKU (a re-writable EPROM chip). The store could burn any Sega title on the EPROM, and then re-write it for the next customer. So we had perfect inventory for all Sega platform titles. It was well received and solved a lot of problems for the stores. (Although I think Blockbuster still thinks I have some overdue games from the trial).
Games were pretty rapidly evolving at that point, each new title required a bigger storage capacity EPROM than the last, etc… and of course the next gen consoles all went to optical media.
They are found memories, although it was too bad that content owners weren't willing to provide their product the way their consumers wanted to use it.
Later, he returned an email I sent with the following:
I ran the marketing group so wasn't a core technical resource, but I'm pretty sure the system wasn't using dial-up and I am certain that it didn't intend to use dial-up if deployed. I think the test market had local servers, that were updated via leased lines. In deployment, we hoped not to have local content, but have a pretty fast (but expensive back then) pipe to the stores. Security was a major concern which is why we didn't want a lot of local content sitting around. The theory was that leased lines to 4000 blockbuster stores would be practical long before the bandwidth was available to stream entertainment software direct to homes. (A major business objective of NewLeaf was to convince the stock market that Video on Demand wasn't' going to put Blockbuster out of business). Games were burned pretty quickly, so I'm guessing the comments in the forum that you linked about the system being slow were actually related to music or video. The common CD burners back then were 2X and (4X at the end) so a full CD could have taken 15-30 minutes to burn. We also bought a company that could make high-speed VHS tapes in store and even considered using the system to make rental tapes on VHS (which tool like 10 minutes).
Most of the technical resources were former IBM employees. Blockbuster and IBM actually formed a joint venture called Soundsational in 1990 to do the system for music and make a big public announcement. Unfortunately, they hadn't talked to any music studios yet, who promptly all panned the idea and said that Soundsational must be planning to steal the music, etc… since no music company had agreed to work with the system. The original Soundsational concept was also going to let customers make custom CD's sold per song, which the music studio's particularly hated. So the Soundsational name was burned, and Blockbuster and IBM restarted the company as "New Leaf" (literally a play on the fact that they had to turn over a "new leaf" after the PR blunder). New Leaf was 50% owned by Blockbuster and 50% owned by IBM. New Leaf's demo center was about 20 minutes from Blockbusters corporate HQ in Deerfield Beach, Florida. The office was on a street called Fairway Drive. Most of the engineers were ex-IBMers that worked for another startup company that was 50% owned by New Leaf and 50% owned by IBM, that company was named after the street our office was on, so they were called Fairway Technologies. In the pictures of the circuit board, you can see the Fairway Technology named silk-screened on the board. As I recall just as we were getting to launch with those 16 Meg cartridges, Maximum Carnage came out and required 32 Megs of RAM. That was the same year that Blockbuster launched it's first annual video game tournament, and Maximum Carnage was the title, I think the slogan was "Practice or Parish".
Sega USA was pretty open to working with us, but they were pretty much just a marketing firm. Sega Japan was open to the system but was very cautious. They were very impressed we were able to reverse engineer the cartridge, as at the time they felt their security would have prevented us from being able to do so.
It was sad that all the work never got used. When Viacom bought Blockbuster, the fear of video on demand kinda went away because Viacom owned so much content, so the appetite to keep spending on New Leaf went away. I left Blockbuster to move back to the West Coast, but I was led to believe that a firm called Digital On Demand bought some of the technology and patents from Blockbuster. Digital on Demand is still around and makes some retail kiosks called RedDotNet (http://www.digitalon-demand.com/). I met them a few years ago, but no one knew anything about the old Blockbuster tech.
That's pretty much all I remember.
Dennis Miller promo video
Video of a cartridge
running on a Genesis
Here is a scan of the Billboard January 1995 article on NewLeaf's endeavor.
External Links
Search for NewLeaf on eBay (Good luck on that one!)
Special Thanks / Sources
– Retail Geek / Jason Goldberg for all of the information he provided.
– Schiggidyd from Sega-Age for the picture of his cartridge's internals and video of the cartridge with Jungle Book.


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BSA volunteer Dave Holmes has been getting his hands dirty at Labor of Love for almost 20 years.
Holmes first moved to Baltimore in 1992, and met BSA Board Member Samuel Polakoff a few years later while in graduate school at the Johns Hopkins University. When Holmes started looking for ways to get involved in the local community, he reached out to Polakoff, who told him about Labor of Love.
Labor of Love, BSA's annual school clean-up day, originally focused on cleaning and painting. However, Holmes, a real estate developer and investor, saw other pressing needs, such as drippy faucets and electrical issues.
"Can I jump on this Sam?" he asked. "Take the ball and run with it," Polakoff replied.
They began by asking faculty and staff what were the school's most critical needs, and soon volunteers for Labor of Love tackled plastering, plumbing, electrical, and carpentry challenges as well.
"Baltimore School for the Arts produces amazing, talented young individuals and the building should be equal to them," said Holmes.
Holmes, who moved from volunteer to head organizer of the event four years ago, keeps an eye out every time he visits the campus for projects to add to next year's list. And he's always looking for new volunteers to join him.
"We see people come in at 9 am, and when they leave at 1 pm, you can tell they had a great time because they know they made a difference," said Holmes.
"But the best part is when the students come in on Monday morning and notice the difference."


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How to Stop Nagging Your Man
Online Dating Tips
How to Stop Nagging Your Man
We all know that every man hates a nagger girlfriend or wife. This is why as early as now you must practice more effective techniques to communicate with your man in an appropriate manner so that both of you can talk well without arguing or fighting about something.
You must learn to be quiet and patient sometimes regardless of how mad you are to your partner because nagging your man can only make the situation worse and your relationship will surely be at risk if you will not stop your unpleasant behavior to your man.
Nagging is never a good idea to any relationship. It will only make you more angry, hurt and frustrated if you will always repeat yourself of continuous nagging. And it's more difficult for you to be to communicate effectively to your partner because you let your temper to control you.
So instead of nagging him, try to talk to him patiently about the situation or reason of your madness in order to make your man understand his shortcomings or faults that can make him realize his mistakes without the need of your high pitch voice or shouting. If you will talk to your man nicely about what makes you angry and frustrated, he will surely listen to you and will definitely change his unpleasant behavior to a better one because you clearly explained your side and needs to him.
If you will practice good conversation to your loving boyfriend or husband no matter how furious you are, you are saving the relationship and respect to your partner. You must never forget that nagging is innately chauvinistic word, used for women but never for men. This is why you need to adjust your temper more patiently so you can avoid unwanted fights that can lead to hatred, unpleasant argument, etc.
The key is to open up with your partner about all the things that can affect your relationship and tell him about your needs. By doing this, your partner will surely anticipate your request and needs. Your burden issues will slowly fade away because you made a good strategy where your partner can understand you more.


text: Hi everyone, hoping someone can help me out very quickly with this issue! I have a 07 stander, 52 inch deck with the 19hp kawasaki FH580V engine. It has about 601 hrs on it. Earlier this spring we replaced the valve seal in the right side(right meaning standing behind the mower and to the the right). Today, however, I blew a tire halfway through a job and went back replace it when all of a sudden is began running on one cylinder. The intake valve seems to be stuck open(the same one that was replaced) and the rocker arm is loose and came off the pushrod and bent the pushrod. Any suggestions on a fix for this? A new head? I'm not sure if the piston is damaged yet or not, will check tomorrow tho. If it does need a new head, whats the price range? Or is this a thing with these engines and do you suggest getting an entire new/different engine? This is going to be a major hindrance if i can't get this resolved quickly. I'm a small time landscaper and this is my only mower Thanks everyone!


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People serving life terms for murders they committed as teenagers were looking to the U.S. Supreme Court on Tuesday for signals about whether they will have a chance to seek their freedom.
But the 75-minute high court argument in the case of a Louisiana man who has been imprisoned since 1963 ended with a distinct possibility that the justices could dismiss the case on technical grounds and perhaps take up another inmate's plea in the spring.
The court spent more time debating whether it has authority to hear the case of Henry Montgomery than it did considering the merits of his claim.
"We weigh in when we have jurisdiction," said Justice Antonin Scalia, suggesting that the court had no business hearing Montgomery's case.
Three years ago, the justices struck down automatic life sentences with no chance of release for teenage killers. The main question for the court Tuesday was whether that decision in Miller v. Alabama should be extended retroactively to Montgomery and hundreds of other inmates whose convictions are final.
The issue of juvenile sentencings also is under review at the California Supreme Court, with at least two Riverside County appeals on hold pending rulings in other cases before the state high court, which has asked attorneys to address the retroactive issue.
In the 5-4 U.S. Supreme Court decision in 2012, Justice Elena Kagan wrote for the majority that judges weighing prison terms for young offenders must take into account "the mitigating qualities of youth," among them immaturity and the failure to understand fully the consequences of their actions.
S. Kyle Duncan, arguing on behalf of the state of Louisiana, said the Alabama decision doesn't qualify as an extraordinary ruling that should be extended to older cases because it left open the imposition of life without parole for people under 18 if courts take account of a defendant's circumstances.
"In Miller this court was invited to categorically bar the penalty of life without parole for juveniles who commit murder, but it decided not to do so," Duncan said.
Kagan disagreed about the significance of the Alabama decision, noting that courts now have to consider less severe punishments. "What the court has done is to say, there have to be other options," she said.
If the court says it should not have taken up Montgomery's case, others are waiting, including one from Virginia. The inmate in that case, Shermaine Ali Johnson, was 16 years old when he raped and killed a woman in 1994.
In California, the state Supreme Court has asked attorneys to address retroactive application in juvenile cases when the defendant is serving a sentence that is the functional equivalent of life without the possibility of parole.
A law that went into effect Jan. 1, 2014, includes provisions for a parole suitability hearing after a maximum of 25 years for most juvenile offenders serving life sentences.
In one Riverside County case before the state high court, Joseph Navarro of Homeland was convicted in 2012 in a knife attack that killed two and wounded a third person, and was sentenced to 50 years to life in prison. In another case, teenager Javante Marquis Scott was sentenced to 120 years to life for his conviction in a 2009 attempted murder case involving three victims in Riverside.
The Associated Press and staff writer Gail Wesson contributed to this report.