Jacques Derrida Essay Example | Topics and Well Written Essays - 3000 words

Jacques Derrida - Essay Example The book wa' tran'lated in Engli'h and publi'hed in U'A in the year 1970. (Gla', John , Leavey and Richard , 86-98) 'ection' of the book and what they contain:Thi' I' no common composition. The basic man would not have the option to gra'p the import of the substance' of thi' book. In any event, for the educated person', it I' an extreme perusing exerci'e. The fir't half of the book contain' the celebrated e''ay on De'carte' and Foucault. It focu'e' on the advancement of Derrida'' technique for decon'truction. Derrida cautiously explain' the conventional idea of 'ome nontraditional current' of present day thought. The 'econd half contain' Derrida'' wise analy'i' 'howing how and why metaphy'ical thinking mu't prohibit composing from it' origination of language. The'e e''ay' are on Artaud, Freud,Hegel, Bataille and Leve-'trau'- 'ort of Derrida'' reply to their contention'. Thi' I' the untran'latable definition of a metaphy'ical 'idea' which doe' not reject composing. We discover Derrida at deal with hey' 'y'tematic decon'truction of We'tern metaphy'ic'. The book'' fir't half, which incorporate' the praised e''ay on De'carte' and Foucault, 'how' the improvement of Derrida'' strategy for decon'truction. ... Howdy' better approach for' thinking, perusing and composing are ba'ed on the all out under'tanding of the old way', in their unadulterated and unadulterated structure. The part wi'e content' of the book are1. Power and 'ignification, 2. Cogito and the Hi'tory of Madne'', 3. Edmond Jab'' and the Que'tion of the Book,4. Brutality and Metaphy'ic': An E''ay on the Thought of Emmanuel Levina', 5. Gene'i' and 'tructure and Phenomenology, 6. La parole 'ouffl', 7. Freud and the 'cene of Writing, 8. The Theater of Cruelty and the Clo'ure of Repre'entation,9. From Re'tricted to General Economy: A Hegeliani'm without Re'erve 10. 'tructure, 'ign, and Play in the Di'cour'e of the Human 'cience', and 11. Ellip'i'. Jacque' Derrida wa' destined to 'ephardic Jewi'h parent' in Algeria in 1930. He did military 'ervice in France. He started hello there' 'tudie' in philo'ophy at the Ecole Normale 'uperieure in Pari' in 1952. Derrida went to Harvard on 'cholar'hip in 1956-57. What the writer I' pre'enting in the book:Derrida advocate' for the dismissal of We'tern metaphy'ic' 'tating and 'howing that composed word' didn't 'tand for the 'poken word'. 'poken word' then again didn't 'tand for thought. Thought proce'' doe' not 'tand for Truth or God. All idea proce'' end' at the obstruction called mind. The idea' can not have any exi'tence past the brain level. That I' the space of 'piritual experience, where thought' have no task to carry out becau'e at that level they are not there by any means. (Gla', John , Leavey and Richard , 86-98) How it ha' been used in the field of communication:Derrida'' commitment to the correspondence enterpri'e I' blended one - it I' negative and po'itive. A' for the po'itive 'ide, greetings' contention' add to the awarene'' of humankind'' normal apprehen'ion of being mi'under'tood. No essayist can

Oedipus The King: Images Of Blindness :: Oedipus Rex, Sophocles

Oedipus the King has numerous pictures of visual deficiency, both physical and visual impairment of the brain. The characters encompassing these pictures are Oedipus and Tiresias the prophet. At the point when the play starts Oedipus has vision and Tiresias can't see, yet before the finish of the play, it is clear who can truly observe and who is visually impaired.      When Oedipus first experiences Tiresias, the visually impaired prophet continues to tell Oedipus after much weight that Oedipus is the person who has brought the incredible plague downward on Thebes and that he is the killer he has sent the inhabitants of the city to discover and cast away. In spite of the fact that Tiresias is outwardly impeded, he can even now observe reality of the possibility. Notwithstanding, Oedipus is oblivious in regards to reality and promptly starts blaming Tiresias for being engaged with a scheme with Creon to topple him as ruler. Oedipus throws affronts at Tiresias about his visual deficiency saying â€Å"You have no quality, daze in your ears, your explanation and your eyes.† (374-375). Tiresias reacts by saying that the put-down Oedipus has heaved will after a short time return upon him. He likewise reveals to Oedipus that what has brought him enormity is the very karma that will destroy him. Tiresias says â€Å"Blind who could se e, a homeless person who was rich, through outside grounds he’ll proceed to point before him with a stick,† (460) inferring that in spite of the fact that Oedipus can see now, reality will in the end dazzle him and cause him to free all he has.      Oedipus stays incognizant in regards to reality until he can deny it no more. In the wake of hearing the declaration of the herder it is consummately obvious to Oedipus that he has satisfied the prescience by murdering his dad and wedding his mom, thusly bringing the incredible incident about the city of Thebes. After finding this, alongside finding Jocasta’s dead body, Oedipus blinds himself with the pins on her dress and yells that his eyes â€Å"would no longer observe the wrongs he had endured or had done, find in obscurity those he ought not have seen.

Are 0% APR Offers a Scam

Are 0% APR Offers a Scam Are 0% APR Offers a Scam? Are 0% APR Offers a Scam?Learn how these types of loans work to protect both your monthly balance and yourself from running into a bad credit situation.The higher a loan’s annual percentage rate, or APR, the more you will have to pay in interest and fees on that loan. That is why, all else being equal, you will want to find a loan with the lowest APR possible. By this logic, a loan with 0% APR would be the best loan of all â€" or so one might think.Anything that sounds too good to be true probably is, and that rule definitely applies when it comes to 0% APR loans. It’s all a matter of timing.The truth behind 0% APR loansThe only way to get a true 0% APR loan is to borrow money from a friend or family member who is willing to lend it to you at no cost. Any other so-called 0% APR loan or credit offer is just a temporary situation or will come with certain stipulations.“If you miss on-time payments or making the minimum payments, your 0% perk can be revoked,” says Jared Weitz, C EO and founder of United Capital Source Inc. “Even a single late payment can be reasonable cause for the bank to increase your interest rates. If your bank writes 0% APR in bold advertisement on the card, read the fine print. Not all transactions will fall within the percentage.”Of course, there are almost as many different kinds of 0% APR loans as there are loans in general.The partial field guide to 0% APR loansSince you should approach any 0% APR offer with a hefty dose of scrutiny and skepticism, it is important to have a sense of the different kinds of offer you are likely to encounter.Mike Scott, a senior mortgage loan originator with Independent Bank, offered a few examples of typical 0% APR offers:Automotive loans“The 0% APR offer is often in lieu of certain rebates. Depending on your credit, down payment, and the term of the loan, it may be better to take the rebate instead of the zero interest rate.”Credit card offers“Credit cards often will offer a 0% rate for n ine to 18 months, but many of them also charge a balance transfer fee of 3%. If you pay the 3% balance transfer fee, then the effective rate on the card is not 0%. If you pay the balance off in 12 months, the effective rate is 3% or more, since the assumption is that your balance is declining throughout the year. If you pay it off in 12 equal payments, the effective rate is actually 6%, since your mid-point balance is going to be half of the original transfer.”General purchases“They offer ‘no interest if paid off in 12 months’ or something similar. Many of those have a stipulation that, if you go past the offer term, you pay all of the interest that accrued during the term. In other words, if you take 13 months to pay off the balance rather than 12, you will pay the interest that would have accrued during the entire term.Using 0% APR offers to your advantageEven though many 0% APR offers may be trying to take advantage of you, there are some that you might be able to use to your advantage.“Many clients have transferred their balance to a card with an 18-month 0% offer in order to get ahead of their credit card payments,” says Nathalie Noisette, owner of Credit Conversion. “If the offer was only six months, it may not have been worth it. Another reason a card with an introductory rate of 0% may be good is if you plan on making a big purchase.”Of course if the card has a balance transfer fee, you will need to take that into account. Remember that most credit cards have a grace period, and if you pay the bill in full each month, you may be able to avoid paying any interest at all.Always follow the essential finance rule: Never sign anything unless you are absolutely sure what you will have to pay, when you will have to pay it, and if you can comfortably make those payments.ContributorsNathalie Noisette  is the founder of  Credit Conversion, a credit counseling and repair company located in Avon, MA. Credit Conversion uses principles of behavioral change to only allow clients to improve their credit score and understand the habits that lend to poor credit. Follow her  @credconversion.Mike Scott is currently a senior loan officer with 20 years of experience in the mortgage industry, specializing in low- and moderate-income lending, first time homebuyer programs, and down payment assistance. Before that, he worked for five years in consumer lending, mainly in the automotive industry. He has been a regional production manager for one of the big banks for several years before choosing to work for smaller, more responsive banks. Follow him  @IndependBank.Jared Weitz  has been in the financial services industry for more than 10 years. Due to his extensive work experience and deep network of close financial relationships, he handles a multitude of different finance options for his clients and contacts. Throughout the years, he has held positions in some of the largest business financing companies in the United States. Some of his ro les have been: underwriter, director of business development, managing partner, and currently, CEO of  United Capital Source, LLC. Follow him  @jaredweitz.

Essay on Evolution Applied to Human Health and Medicine

Evolution is defined as a gradual process in which organisms become better adapted to their environment through gradual changes that occur from generation to generation. Throughout the history of life, the human species has changed to become better suited to the environment. All of the changes have ultimately resulted from mutations, which occur at the gene level. Pathogens such as bacteria or viruses that live inside of our cells have had a major influence upon our evolution (Parks, Panelli Weinstein, 2003). Pathogens have affected our evolution in two major ways, which I will focus on: antibiotic resistance and virulence. Virulence has also changed in regards to ways of reproduction and establishing a higher rate of fitness. Other†¦show more content†¦On the other hand, if a mutation increases the chances for survival, then it will be selected and will continue to be passed on through generations. Pathogens as well as their hosts can have mutations that increase their fitness. Pathogens are also in the presence of selection pressures: temperature, pH, and medicines such as antibiotics (Fitch, Leiter, Li Palese, 1991) and will, therefore, also evolve. As the human species experiences illnesses, doctors will prescribe medicine, usually an antibiotic, which will kill many of the pathogenic bacteria. In regard to pathogens, if a virus is attacking the body, an antibiotic will destroy the bacteria and leave the virus alone. Viruses or bacteria that survive medication will develop resistance to that medicine (Parkes, Panelli Weinstein, 2003). As a resistance is developed, they can also reproduce and pass the resistance trait to their offspring, increasing the level of fitness. There has been documentation of evolution resistance in bacterial populations and within individual large-scale human populations. Experimentally, there have been studies where the use of antibiotics was lessened and so was resistance in bacteria (Bull Wichman, 2001). This shows the correlation that as antibiotics are being used, bacteria and viruses are able to withstand the effects of medicine and can develop a resistance toShow MoreRelatedExamination of Clinical Psychology1665 Words   |  7 Pagesbeen dated back as far as 2500 B.C. In those days, approaches to examining mental health included the supernatural, holistic, religious and medical perspectives. Greek physician Hippocrates, also known as the father of ancient medicine, played a considerable role in the development of psychology. Hippocrates developed the theory of humors, which states that four humors, or bodily fluids, are the key to good health. These fluids were black bile, yellow bile, phlegm, and blood (Plante, 2010). Too muchRead MoreStudying Diet, Immunization, and Infection of Our Ancestors674 Words   |  3 PagesEvolutionary Medicine at the University of Zurich. She studies how humans co-evolved with their environment, diets and disease by looking at the skeletal and mummified remains of ancient humans. In her Ted X Talk given in Long Beach, California in March 2012 she provided explanations for the difficulties researchers are faced with, the description of the new implementation of Warinner and her team and the results that were found, Warinner shares with others how the evolutionary history of health and humanRead MoreThe Evolution Of Health Care1184 Words   |  5 PagesWhat is quality in health care? We can divide it into three elements: 1. Care by practitioners and other providers which means receive correct diagnosis and an appropriate strategy of care from practitioners; and convenience, comfort, quite, privacy, food etc. from care providers. 2. Care Implemented by patient that means patient should take care not only for him or herself but also for his or her family. 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Thau Bioethics 4242 12/3/16 Over the last 10,000 years, humans have evolved more than any other species that has ever set foot on planet Earth. As a population we have grown exponentially and made changes to our society that allow us to do things that have not been done before. In 1878, Thomas Edison recognized the ability of a bamboo filament that was connected to platina wires, to burn for significantRead MoreHuman Blueprint: Thoughts on Genetic Modification Essay examples734 Words   |  3 PagesHumans have strived for improvement in every facet of their lives, all the way from technology down to their diet. In this current day and age, humans are now capable of gene manipulation in their children, meaning mankind is taking a great step in familial modification. Yet even with our advancements in the medical field, we still fall to the ills of cancer, Alzheimer’s, or anything else on the laundry list of genetic disorders. With such opportunity to better improve our children, what mother orRead MoreWha t Is Good Medicine?1271 Words   |  6 PagesWhat is good medicine? Humans have been practicing medicine for over a million years. In order to understand how modern medicine gets to where is it today, it is very helpful to read about the history of medicine. According to the group parasites projects that we have done during the past semester, we studied different kinds of parasites and their influence on both patients and doctors aspects during different time periods. Through out the whole research, good medicines were found to have similarRead More A New Future: Gene Therapy Essay1434 Words   |  6 Pageswhere a person could change his or her genetic structure and redirect the future course of evolution in their child and themselves. Through gene therapy this is a very real possibility. In the future it could be just as easy to change your physical or mental health as it is to get flu shot now. But the affects of gene therapy are long lasting and could affect your future offspring as well as your own health. What is gene therapy? First discovered in the middle of the 1970s researchers wereRead MoreTechnology And The Growth And Expansion Of The Population And Society Essay1508 Words   |  7 Pageshave led to the development of different technologies. These technologies, have helped with the evolution of civilizations and have equipped people with a variety of tools to address problems that, until then, had no solution with the means available at that time. Since man appeared on earth there is technology. Technology is considered as a set of knowledge and applied techniques, which are used by human beings to try to transform the world, with the purpose of best suiting their needs. TechnologyRead MoreThe Pros And Cons Of Artificial Intelligence1245 Words   |  5 Pageskinds of functions that characterize human thought processes. The possibility of developing some such artifact has intrigued human beings since ancient times. With the growth of modern science, the search for AI has taken many directions: psychological and physiological research into the nature of human thought and cognitive processes, and the technological development of increasingly sophisticated computing systems that simulate various aspects of such human activities as reasoning, learning, and

Essay on Charles Dickens Great Expectations - 2265 Words

Charles Dickens Great Expectations Charles Dickens wrote Great Expectations in 1860-1861 when he was in London. It is set in the mid nineteenth century, in Kent, and London. The basic plot of Great Expectations is: Pip, a young orphan living with his sister and her husband in the marshes of Kent, sits in a cemetery one evening looking at his parents tombstones. Suddenly, an escaped convict springs up from behind a tombstone, grabs Pip, and orders him to bring him food and a file for his leg irons. Pip obeys, but the fearsome convict is soon captured anyway. The convict protects Pip by claiming to have stolen the items himself. One day his uncle takes him to Miss Havishams house to play. A few years later he is†¦show more content†¦The original ending had Pip and Estella meeting outside miss Havishams talking a while and then going there separate ways. Dickens based some of the book on his own experiences; apart from David Copperfield this story is the most autobiographical book he wrote. I will now compare the different circumstances of the two main characters two chapters: In chapter 1 we see Pip as a young boy visiting his parents graves. He is small and frightened by the convict. He is terrified by what the convict says will happen to him if he doesnt do what the convict wants. `You get me a file. He tilted me again. `And you get me wittles. He tilted me again. `You bring em both to me. He tilted me again. `Or Ill have your heart and liver out. He tilted me again. The convict has escaped from prison and is hiding in the graveyard where Pip is. He was dressed in all in coarse grey, with a great iron on his leg. A man with no hat, and with broken shoes, and with an old rag tied round his head. He was covered in mud and was soaked in water. A man who had been soaked in water, and smothered in mud, and lamed by stones, and cut by flints, and stung by nettles, and torn by briars; who limped, and shivered, and glared and growled; and whose teeth chattered in his head as he seized me by the chin. He was desperate for food and would do anything to get some. In chapter 39 we see Pip when he is 23 years old, living in London thanks to a secretShow MoreRelatedGreat Expectations By Charles Dickens1113 Words   |  5 Pagesadventures that the male characters go on. This seems to be relevant in a lot of movies and books like the story Great Expectations by Charles Dickens. In Great Expectations there are multiple female characters like Estella, Biddy, and Miss Havisham who all play a large part in the main character, Pip’s life. One of the first that we meet the character Estella in Charles Dickens’ Great Expectations is when Pip goes to Miss Havisham’s to play with her. The two kids play the game beggar my neighbor when EstellaRead MoreGreat Expectations By Charles Dickens1347 Words   |  6 Pagespoor status of the economy, social mobility does not seem to be occurring at high rates, with the poor getting poorer and rich getting richer. Despite this, social mobility is alive and well, and has been for centuries. In his novel, Great Expectations, Charles Dickens voices the concerns of many that lived in Victorian England during the 19th century by promoting such a desire to live life in a more prosperous social class. One of the most fundamental and reoccurring themes in the novel is that ofRead MoreGreat Expectations By Charles Dickens1426 Words   |  6 Pages Twelve-year-old Charles dickens gets ready for bed after a long day at the blacking house. These Victorian-aged memories will provide him with many ideas for his highly acclaimed novel Great Expectations. Set in 1830 England, Great Expectations is a coming-of-age story about a common innocent boy named Pip and his road to becoming a gentleman through the influence of others. Pip is influenced both positively and negatively by Estella, Herbert, and Magwitch. Estella left a huge impression on PipRead MoreGreat Expectations by Charles Dickens984 Words   |  4 PagesCharles Dickens utilizes his life for inspiration for the protagonist Pip in his novel Great Expectations. They both struggle with their social standing. Dickens loved plays and theatre and therefore incorporated them into Pip’s life. Dickens died happy in the middle class and Pip died happy in the middle class. The connection Dickens makes with his life to Pip’s life is undeniable. If readers understand Dickens and his upbringing then readers can understand how and why he created Pip’s upbringingRead MoreCharles Dickens Great Expectations943 Words   |  4 Pages This is true in many cases but none as much as in Great Expectations. In many ways the narrator/protagonist Pip is Charles Dickens in body and mind. While there are many differences between the story and Charles Dickens life there remains one constant. This constant is the way Pip as the narra tor feels, because these feelings are Dickens s own feelings about the life he lead. Since Great Expectations was written towards end of Charles Dickens life, he was wiser and able to make out the mistakesRead MoreGreat Expectations By Charles Dickens1375 Words   |  6 PagesGreat Expectations by Charles Dickens and The Talented Mr Ripley by Anthony Minghella present similar criticisms of society to a large extent. Both of these texts consider the criticisms of rich social contexts (wealth and status), societal morality (whether a society is good or not. Status [can lead to the wrong people being in a high position i.e. making bad decisions affecting the community/society] Appearance [society appears to be moral/good (if you’re from a higher status) {dickens criticisesRead MoreCharles Dickens Great Expectations1223 Words   |  5 PagesBeloved author Charles Dickens was born on February 7, 1812 in Portsmouth, England. Growing up in a life of poverty, his childhood hardshi ps provided the inspiration to write a myriad of classic novels including his 1861 seminole masterpiece, Great Expectations (â€Å"BBC History - Charles Dickens†). Great Expectations follows the life of an orphan named Pip, who’s perspective of the world is altered when he is attacked by an escaped convict in his parents’ graveyard in the town of Kent. Throughout hisRead MoreCharles Dickens Great Expectations1344 Words   |  6 Pagessomething that is not what they truly need? Often, they use social class to fill a void in their lives that can not be filled by materialistic possessions. Many people realize this, but it is often too late. Charles Dickens demonstrates the effects of social climbing in his novel, Great Expectations. This novel explores the connections and effects of human nature and society, which are the two most powerful forces that guide people’s decisions. Some may say that social climbing is good, but as will beRead MoreGreat Expectat ions by Charles Dickens1285 Words   |  6 PagesAP Great Book Assignment: Great Expectations The 544-page, Bildungsroman novel, Great Expectations, by Charles Dickens is considered a classic because it has stood the test of time, appealing to generation after generation of readers while still remaining relevant to them. Published in 1861, Dickens created a coming-of-age story that is similar to his other novel, David Copperfield, but Great Expectations is considered to have reflected parts of his own life. There are several parallels betweenRead MoreGreat Expectations By Charles Dickens924 Words   |  4 Pagesa character driven novel, or a mix of the two. In order for a novel to be character driven, it must revolve more around the characters’ individual thoughts, feelings, and inner struggles, rather than around the quest of the story. Great Expectations, by Charles Dickens, is a character driven novel. While the story does have a plot, it is not contingent upon that plot, but rather is reliant upon its characters and their natures. This is evident from the beginni ng of the novel. From the opening of

Changes That Have Occurred To The National Curriculum Education Essay Free Essays

string(196) " National Literacy Strategy to all Primary Schools in England from September 1998 after puting marks for students at Key Stage 2 in English \( 80 % \) , Mathematics \( 75 \) , but non in Science\." ‘Since the debut of the National Curriculum in 1992, a figure of alterations have been made to its construction and execution. Identify and explicate these alterations and measure the part of the National Curriculum in the attempt to raise criterions in Education ‘ . By Keith Lyons This essay will depict, place and explicate the alterations that have occurred to the ‘National Curriculum ‘ ( NC ) since its construct in 1992. We will write a custom essay sample on Changes That Have Occurred To The National Curriculum Education Essay or any similar topic only for you Order Now It will besides measure these alterations and measure the benefits and troubles that it has experienced since 1992. The ‘Education Reform Act ‘ ( ERA ) came into consequence in 1988, this enabled Kenneth Baker the Conservative Minister of Education to implement the roll-out of the National Curriculum in 1992 within primary / secondary instruction. However, prior to 1992 there had been no National Curriculum and antecedently instructors had worked out their ain strategies of work that they deemed appropriate for their students. As a consequence of this the criterion of instruction across the state varied well and the methods employed to learn were wide-ranging. With the execution of the National Curriculum in 1992, duty was shifted off from instructors to centralized authorities over what was to be taught. The National Curriculum established a ‘set-framework ‘ of larning to enable kids to travel freely between schools as they would be larning from the same model. The National Curriculum ensured that schools taught a certain scope of topics, this consisted of 10 topics. The topics were divided into two sub-categories ( nucleus and foundation ) . The three chief nucleus topics were English, Maths and Science, and together with seven other foundation topics ( Art, Home Economics. Music, History, a Modern Foreign Language ( merely compulsory in secondary schools ) , Geography and Physical Education created the foundation of the National Curriculum. Compulsory National trials ( SATS ) were introduced at 7, 11 and 14 on nucleus topics. The consequences are published yearly in conference tabular arraies ( along with GCSE/A degrees and hooky statistics ) . Changes occurred to the National Curriculum from its origin. Rather than being embraced the National Curriculum was met with ill will from some instructors and most of the larger instructors brotherhoods. One of the chief unfavorable judgments of the National Curriculum by instructors and learning brotherhoods was that at first glimpse that it contained far excessively many topics and was considered to be excessively stiff, compared to what was antecedently taught. This meant that it was hard to learn the topic good as the pupils had to larn so many topics, and they presumed that it would be really hard for pupils. The National Curriculum was besides criticized for being highly ‘Eurocentric ‘ , as it was chiefly focused around European civilization, giving those from cultural backgrounds really small chance to larn about their roots. Certain subdivisions of the populace besides argued that the authorities deliberately fashioned the National Curriculum to take topics which they as a political party are against for political grounds, such as sociology, political relations, and environmental scientific disciplines ( they were ne’er contained in the National Curriculum ) . However, a benefit to the National Curriculum was that instructors now had to educate pupils in a scope of topics. This gave kids / pupils a better start for the accomplishments they would necessitate for subsequently in life. It was besides disputed that the national Curriculum helped to cut down the division of misss and boys topics, as both sexes were taught the same topics. This helped to cut down the stereotyping of gender. In 1993, instructors decided to boycott the national course of study proving agreements ( SATS ) after kicking about the work load. The Secretary of State for Education, in a move in the right way asked Sir Ron Dearing, who was the current Chairman of the School Curriculum and Assessment AuthorityHYPERLINK â€Å" hypertext transfer protocol: //www.bookrags.com/tandf/school-curriculum-and-assessment-tf # p2000a9f68830215001 † ( SCAA ) , to reexamine the national course of study. Dearing made an Interim Report in 1993 and a Final Report in 1994 ( The Dearing Report ) after a period of audience with instructors and the instructors brotherhoods. In his study his recommended on slimming down the course of study, and bettering its disposal. He besides recommended that the slimmed down national course of study should non be altered for five old ages and that national trials should be simplified without giving cogency or dependability. The revised course of study was implemented from August 1995. The 1992, Ofsted ( The Office for Standards in Education ) formed as portion of the major inspection and repair and centralization of the school system begun by the Education Reform Act 1988, which introduced the National Curriculum, extended testing in schools and the publication of conference tabular arraies. Ofsted reviews were carried out on school every 6 old ages. This alteration was introduced as it was believed criterions needed to be raised in schools. Many people thought Ofsted reviews were a good thought, if schools were neglecting, it was noticed and steps were put in topographic point in order to better these schools. However, Ofsted were besides frequently seen to be excessively rigorous on schools, doing it hard for schools to go through review extremely. Besides, it was felt that many instructors and schools would better their instruction criterions while they were being inspected. Some schools would besides direct some of their worst behaved pupils on school trips for the reviews. This defeated the object of Ofsted reviews as it was n’t the normal instruction criterions that were being tested. Recent reviews by Ofsted have revealed that although a good proportion of schools had improved since they were last checkered many were calming weakness. Figures released by Ofsted in 2009 show that 11 % of schools checked since last September were rated outstanding, while 9 % were non up to rub. [ Angela Harrison, BBC ] In the twelvemonth 2006-07, 14 % of those checked were outstanding and 6 % were â€Å" unequal † . [ Angela Harrison, BBC ] In 1997, the incoming ‘The New Labour Government ‘ came into power in the UK. They Introduced the National Literacy Strategy to all Primary Schools in England from September 1998 after puting marks for students at Key Stage 2 in English ( 80 % ) , Mathematics ( 75 ) , but non in Science. You read "Changes That Have Occurred To The National Curriculum Education Essay" in category "Essay examples" Previously a pilot undertaking had been tested during 1996, which involved schools in 14 Local Education Authorities. The scheme was planned for instructors to learn a day-to-day Literacy Hour, which followed a form of 30 proceedingss whole category instruction, so group work and reasoning with a plenary session. A figure of paperss have been published by the Department for Education and Employment ( DfEE ) to assist instructors raise criterions in literacy at the clip. The National Literacy Strategy model endeavoured to better criterions for all primary aged students. The scheme ‘s intent was to do certain that all students were having on a day-to-day footing dedicated one hr of literacy. The terminal consequence would give pupils the chance to develop accomplishments in reading, grammar, spelling, and unwritten work and assist raise criterions in instruction and acquisition. In the undermentioned twelvemonth of September 1999 The National Numeracy Strategy model was introduced and like the literacy scheme, aimed at raising criterions for all primary students. Similar to the National Literacy Strategy it prescribed a one hr day-to-day mathematics lesson for all students. The Five Year Review in 2000 set out the chief purposes and intents of the National Curriculum for the first clip. The four chief intents of the National Curriculum are: To set up an entitlement. To set up criterions. To advance continuity and coherency. To advance public apprehension. The National Curriculum has been put into topographic point in the hope that kids will accomplish and will hold an entitlement to larning irrespective of their background, be it race, gender, cultural or otherwise.A It besides makes outlooks for kids ‘s attainment explicit for all concerned and sets out national criterions for performance.A These criterions can so be used for mark scene, mensurating advancement and monitoring patterned advance. The Foundation Stage was introduced by the Labour authorities in 2000, to supply counsel for scenes which provide attention and instruction to pre-school kids ( aged 3 to 5 ) . It was named the Foundation Stage because â€Å" †¦ it lays the foundations for kids ‘s subsequently larning. † [ Neaum and Tallack, 2002 ] . It evolved from the Rumbold Report of 1990, which investigated the educational proviso for the under 5 ‘s, and found that there was a patchy, unplanned course of study which was unsatisfactory. In 2004 a reappraisal was carried out of Key Stage 4, from this reappraisal the debut of ‘Entitlement topics ‘ . The Curriculum Entitlement Framework provides students with entree to a wider scope of larning chances suited to their demands, aptitudes and involvements, irrespective of where they live or the school they attend. In 2007 the authorities abolished formal written Key Stage 1 SATS and replaced them with instructor recorded appraisals. The Key Stage 1 appraisals are really low key and completed by the student ‘s instructor over a period of a few hebdomads so they will be barely cognizant that an appraisal is taking topographic point. In 2008 a reappraisal of Secondary National Curriculum resulted in new Key Stage 3 and 4 Curriculum which was introduced in 2009. This updated portion of the course of study now offers Diplomas and other options to current GCSE and A-level scrutinies. In 2009 the so current Labour authorities announces that Key Stage 3 Sats scrutinies are to be abolished and that Sir Jim Rose will be carry oning a full reappraisal of the Primary National Curriculum. The findings of the study will be implemented from September 2011. In May 2010 a general election was held and ‘New Conservative ‘ / Liberal Democrat authorities came to power under David Cameron and Nick Clegg. The hereafter changes to the new primary national course of study which were put frontward by Sir Jim Rose to be implemented from September 2011 have been shelved, the authorities saying that it does non mean to continue with the new primary course of study. Alternatively they are committed to giving schools more freedom from unneeded prescription and bureaucratism. They have ever made clear their purpose to do alterations to the National Curriculum that will guarantee ‘a grim focal point on the rudimentss and give instructors more flexibleness than the proposed new primary course of study offered. The National Curriculum has undergone considerable alteration and development over the past 20 old ages and is still being altered and adjusted at the present twenty-four hours. Controversy still exists as to the attack instruction should follow for those over the age of 14 testing is thought to be to a great extent based on callback of cognition promoting a deficiency of accomplishment development activities. Many of the original topics that were compulsory no longer are, as it was felt the course of study was excessively full. In my sentiment, since the debut of the National Curriculum, I believe that it was the anchor to greatly bettering the criterions of instruction that kids receive in today ‘s society. This is because before the criterion of instruction pupils received was extremely based on category position and was typically biased towards the in-between / upper category households, another factor was your vicinity in the state. Teachers besides taught a scope of topics that they wished to learn as there were no set subjects so what you could be taught varied across the state. This led to many pupils go forthing school with limited cognition. With the debut of the Literacy and Numeracy hours Sats consequences have increased once more of the subsequent old ages but once more now have slowed to a arrest. As seen in the tabular array below. But on the impudent side holding felt that on the whole the National Curriculum is effectual it is besides slightly flawed. Children are ‘taught to the trial ‘ at a hurt to other topics, and so they are losing out on a wide and balanced course of study. Schools and learning staff will besides acknowledge that they are being forced to â€Å" learn to the trial † ; cutting out topics such as history, geographics and art to blow up their place on national conference tabular arraies or else expression as if they are neglecting. The national trials besides distort kids ‘s instruction as they are being offered a restricted timetable as instructors are forced to concentrate on the nucleus topics. In a figure of schools an accent on trials in English, Mathematics and Science limits the scope of work in these topics in peculiar twelvemonth groups, every bit good as more loosely across the course of study in some primary schools. Having said this, the positive points outweighed the negative greatly as it was the start towards the National Curriculum that we presently have today. How to cite Changes That Have Occurred To The National Curriculum Education Essay, Essay examples

Therapists Role in Gastric Bypass Procedure free essay sample

The purpose of this paper is to determine the role of the psychologist when evaluating a patient seeking a gastric bypass procedure. To this date there is no standard avenue given to those conducting the interview. Due to the limited knowledge of long term success post-surgery a guideline is given for psychologists to follow. Continued research is needed to determine what may be needed in the interview that is not being addressed at this time. A Therapists Role in A Pre-Surgical Interview for a Gastric Bypass Procedure A huge problem in the United States for the average individual is obesity. While traditional weight loss programs are failing and more extreme methods are being used, psychologists are being called upon to determine if individuals are mentally fit to have a procedure called Bariatric surgery (ie, gastric bypass). This procedure is used for populations of people whom are extremely obese. Therefore extremely obese patients are turning to this unconventional method to lose weight. We will write a custom essay sample on Therapists Role in Gastric Bypass Procedure or any similar topic specifically for you Do Not WasteYour Time HIRE WRITER Only 13.90 / page Unlike traditional weight lose methods whose risks are low, gastric bypass procedures have high health risks involved. In order for this surgery to be successful patients need to reframe their thinking after surgery to continue to maintain the weight that is lost due to the procedure. There have been no clear assessment tools psychologists’ use for pre-op assessment but many psychologists are using a standard assessment to determine if the individual is a candidate for bar iatric surgery. Even though a patient may not want to undergo a psychological evaluation it is a necessary criterion in order to undergo this extreme weight loss procedure. The information a psychologist collects in the pre-op procedure is why they are seeking surgery, diet and weight history, what are their habits currently in regard to eating, educating patient that future lifestyle changes associated with behavior modification therapy, history and current social supports, and any psychiatric symptoms (current and past) (Snyder 2009). The psychologist’s 3 responsibilities during the assessment is to collect a history about the patient, act as an educator providing information to patient about the psychological aspect of the surgery, and the therapist motivating the patient to seek post surgical behavior modification treatment while helping the client deal with emotions that may come up during the evaluation (Snyder 2009). The psychologist’s initial question is the patient’s reason for surgery and what are they hoping to accomplish. The therapist therefore will determine the clients expectations to assess if the client has unrealistic expectations centered around the surgery results (ie. motivation due to only changing physical appearance). The results may suggest the patient may not be a candidate for surgery. Researchers have learned the motivation for surgery should be strictly for health reasons (Bauchowitz, A. , 2007). Patients that have unrealistic expectations regarding weight loss are prone to issues of depression when the surgery and outcome is not what they expected. The second area assessed is a review of the patients eating habits past and current. Researchers found that clients learned messages from family that were contributing factors to weight gain (ie. eat everything on your plate) (Gibbons, L. M. , et al. , 2006). Psychologists’ also determine if a client has had past failures with diets and if not the client is encouraged to try dieting before seeking surgery. The philosophy behind this way of thinking is due to getting used a healthy lifestyle with food intake. The role of the psychologist here is to educate the patient with what will be needed in the future to maintain their ideal weight. They are warned going back to old behaviors with eating behaviors and habits could result in gaining all their weight back. Also, psychologists help the patient if they are dealing with shame for failed diet attempts in the past. Another area of assessment is determining the patient’s current eating behavior. It is important for the therapist to determine the motivation for behavior modification and also seek out eating disordered clients. The patient is also questioned about their plan for exercise post surgery. The biggest finding about non-compliance among failures with post treatment was the lack of motivation for exercise (Snyder 2009). Psychologists also determine if a client is bulimic due to the dangers of a bulimic client can have with gastric bypass procedures. Psychologists at this point educate the patient on the importance of exercise for long term benefits of surgery (Snyder 2009). The psychologist also educates the patient to help them to understand what they are agreeing to when seeking gastric bypass. The therapist also should point out the consequences of going back to the past ifestyle with eating behavior (Snyder 2009). Another area of importance is the social network of family and friends the patient has when having surgery. The social network can make or break a patient’s success or failure. Therefore, it is important for the psychologist to understand the patient’s social network in the past and present. Negative consequences of the surgery (ie. affair) ca n lead to jealousy of the spouse and present sabotaging behavior of the spouse. Therefore it is important for a psychologist to address issues that could present themselves if applicable (Snyder 2009). One of the primary reasons an interview is conducted pre-surgery is to determine if there are any psychiatric disorders that might interfere with success of surgery. Symptoms of anxiety, depression, psychosis, mania, suicidal ideation, substance abuse, history of abuse, family history of mental disorders, and any treatment regarding mental health issues are assessed. An examination called a Mini-Mental Status is given to the patient to determine any psychiatric complications that can present themselves (Fabricatore, Crerand, Wadden , et al. 2006). The MMPI-2 is a psychological test given to pre-surgical candidates for a gastric bypass. This is a way to validate that the psychologist’s assessment interview are the same as compared to the MMPI-2. The psychologist should instruct the client to answer all questions honestly so the test results will be accurate. If the clients’ results show defensiveness with the test results the test may have to be taken again (Walfish, S. , 2007). The Millon Behavior Medicine Diagnostic allows for norms within the population that have undergone surgery. These two tests have been selected for psychologists to use even though there are other tests available. In conclusion, it is important for patients to be assessed before having surgery. The psychologist role is to understand the readiness of a patient and how emotional issues can challenge long term success. The psychologist’s role also is to educate the client on all possible issues that they may face post surgical. Overall, this demonstrates how important a pre-surgical evaluation is needed to increase success for the patient short and long term.

Whether vs. If

Whether vs. If Whether vs. If Whether vs. If By Maeve Maddox A reader writes: A colleague at work says the following sentence is incorrect: â€Å"Promotion depends on if the employee maintains a good attendance record.† I don’t see anything wrong with it. Is she just being disagreeable? I can’t say whether the colleague is being disagreeable or not, but I can say why she objects to the sentence. The conjunctions if and whether are often used interchangeably. Sometimes such usage can pass, but sometimes whether is the only choice. One rule often cited is that if does not follow a preposition. Corrected, the sentence is â€Å"Promotion depends on whether the employee maintains a good attendance record.† Seeking to avoid confusion, Sir Ernest Gowers added this entry to the 1963 edition of Fowler’s Modern English Usage: To avoid possible ambiguity it may be prudent to confine if to its proper duty of introducing the protasis of a conditional sentence, and not to use it as a substitute for though or whether or (with not) to introduce a possible alternative. Note: In the context of grammar, protasis is â€Å"the first or introductory clause in a sentence, especially the clause which expresses the condition in a conditional sentence.† The Chicago Manual of Style spells out two instances in which if should not be used in lieu of whether: 1. to introduce a noun clause in an indirect question the answer to which is either yes or no. INCORRECT: He asked if his tie was straight. CORRECT: He asked whether his tie was straight. The only possible answer is yes or no. 2. to avoid ambiguity. INCORRECT: Call me to let me know if you can come. CORRECT: Call me to let me know whether you can come. With if, two interpretations are possible: 1. Call regardless of your answer. 2. Call only if you will be coming. By using whether, the speaker makes it clear that a call is desired, regardless of whether the person is coming or not. Chicago includes three other notes on the if/whether dichotomy in the â€Å"Good usage versus common usage† section: determine whether; determine if. The first phrasing is irreproachable style; the second is acceptable as a colloquialism. The same is true of decide whether versus decide if. doubt that; doubt whether; doubt if. Doubt that conveys a negative sense of skepticism or questioning: â€Å"I doubt that you’ll ever get your money back.† Doubt whether also conveys a sense of skepticism â€Å"The official says that he doubts whether the company could survive.† Doubt if is a casual phrasing for doubt that. question whether; question of whether; question as to whether. The first phrasing is the best, the second is next best, and the third is to be avoided. Paul Brians (Common Errors in English Usage) acknowledges that if â€Å"can’t really be called an error,† but adds, â€Å"when you are discussing two alternative possibilities, whether sounds more polished.† A note by David Foster Wallace in the Oxford American Writer’s Thesaurus offers â€Å"a simple test†: If you can coherently insert an â€Å"or not† after either the conjunction or the clause it introduces, you need whether. He gives these examples: He didn’t know whether [or not] it would rain. YES She asked me straight out whether I was a fetishist [or not]. YES We told him to call if [or not] he needed a ride. NO If is used to express a conditional. Whether is used to introduce alternative possibilities. Want to improve your English in five minutes a day? Get a subscription and start receiving our writing tips and exercises daily! Keep learning! Browse the Grammar category, check our popular posts, or choose a related post below:30 Synonyms for â€Å"Meeting†Rules for Capitalization in Titles30 Words Invented by Shakespeare

How Do You Become a Pharmacy Technician

How Do You Become a Pharmacy Technician a pharmacy technician is responsible for assisting the pharmacist on duty to help patients or customers and fill prescriptions. you’ll need a keen eye for detail, a great deal of patience, and training that covers basic pharmacology, medication dispensing, and an understanding of pharmacy law and ethics. education and trainingyou will need a high school diploma or a ged equivalent to qualify for on-the-job training- many aspiring pharm techs find getting such  training is easier after post-secondary training at an online certification program or vocational school, community college, or technical institute. it may take anywhere from 3 months to a year.vocational school and community college coursework will emphasize commonly used mathematics, chemistry, and medication orientations to familiarize aspiring pharm techs with names, side effects, interactions, and dispensing protocols. some training programs even offer internships for hands-on experience, a great way to get to kno w a pharmacy before committing to the 6 year process of becoming a pharmacist.certificationsthe pharmacy technician certification board (ptcb) requires you to have a diploma and pass a certification exam. the national healthcareer association (nha) requires you to be 18 years old and have a high school diploma, a formal training program, and a year of work under your belt.you’ll be required to re-certify every 2 years because of the rate of change in healthcare policies and advances in the field. pursuing 20 hours of continuing education will keep you up-to-date. many pharm techs report that this process of staying current (along with the patience of keeping up with a busy pharmacy) are the most challenging parts of the job.employersmost pharm techs are found in hospitals, retail pharmacies, assisted living and education institutions, and veterinary offices. the bureau of labor statistics predicts employment growth of about 20 percent for the field through  2022- faster tha n the average growth rate for all occupations. those stats amounts to  70,700 new positions to be filled during that period.here are some of the biggest current employers in the field:salarythe average salary is $38,600 nationally, with rates increasing with seniority, specialty certifications, and competitive institutional pay scale policies. the metropolitan areas that offer the highest compensation are all located in california, including the metropolitan areas of oakland, napa, san francisco, san jose, and madera.sample job postingpharmacy technician,   orlando, flunder the direct supervision of the pharmacy services call center supervisor, the pharmacy call center technician is responsible for providing knowledgeable and courteous customer assistance and response to members, providers, and pharmacy call center technician inquiries.position qualifications/requirementseducation and training:  high school diploma or the equivalentexperience:  2 years pharmacy technician ex perience in a healthcare, long term care or retail environment; minimum 35 wpm typingrequired experience:  pharmacy tech, high school, national pharmacy technician certification: 3 yearslicenses, registrations or certifications:  national pharmacy technician certificationprincipal accountabilitieshandles calls and correspondence regarding pharmacy issues from providers/physicians and members. documents calls in inquiry tracking system. facilitates problem resolution and acts as customer advocate.performs initial research of pharmacy issues. reconciles plan information utilizing department-generated procedures and reference materials. utilizes internal pharmacy systems to update, modify and extract member information.assists network pharmacist in processing prescription claims. reviews claims for accuracy and adherence to contract guidelines maintained.assists with entering prior authorizations when required; provides follow up assistance for prior authorization requests, follows up with requests from physicians for denial letters, provides assistance in gathering historical claim information as required.discriminates between routine and significant matters; apprises management of issues which can negatively affect service and business. identifies provider/physician/member issues and refers to appropriate departments.maintains a balance of productivity, quality, and timeliness of job accountabilities.demonstrates flexibility and cooperation when faced with changing priorities to meet shifting needs.creates and supports an environment which fosters teamwork, cooperation, respect, and diversity.assists with training of new hires. attends required training as requested. develops and maintains desktop procedures.apply here: pharmacy technician

Assignment about ethics Example | Topics and Well Written Essays - 1000 words

About ethics - Assignment Example System 1 thinking is characterized by an unconscious and automatic way of thinking. This system has the universal cognition that occurs both in human beings and in animals, an aspect that is considered evolutionary. System one thinking is comprised of subsystems that work hand-in-hand within the same autonomy. System 1 thinking is responsible for instinctive behaviors that are programmed inherently. This system solves problems through the dependence of prior information that is associated with the system including automatic, holistic and fast cognitive ability (Kaufman & Singer, 2012). System 2 is recent compared to system 1. This system is characteristic of human beings only. This system is also understood as the explicit and rule-based system. It is slow, sequential, and domain-general and is performed in the central memory system. System 2 allows abstract hypothetical thinking that does not happen in system 1. This system functions with regard to logical standards and is rule based, demanding, analytic and controlled of cognitive capacity (Kaufman & Singer, 2012). The kind of individual ethical problem-solving that happens in business and organizations involves the application of system 1 and system 2 thinking. The use of instinctive information must be used then making an ethical decision. It also involves a holistic approach that will involve looking at the causes of the ethical problem and going towards the identification of a solution. System 2 is also significant because ethical problem solving in a business or an organization must be made using logic and reasoning. This happens in the sense that one has to think through an ethical dilemma or problem and go through certain processes that involve identification, ranking, developing an action plan, implementing it and reflecting on its success. An example of a system 1 response to an ethical dilemma in business is when

Should Government assist business Essay Example | Topics and Well Written Essays - 1500 words

Should Government assist business - Essay Example up tariff schemes and other import restrictions; assists in foreign operations; provides for a postal system; conducts and encourages research and development; pays out vast sums for goods, services, and transfer payments; and helps in maintaining a sound banking system (Isaacs & Slesinger, 1964, p. 10). Therefore, the purpose of this essay is to illuminate the benefits of this assistance provided by the US government to business on economy and also about the burden it implies on the government and its policies and then come to a point to decide as to should government assist small businesses in their effort to be a part of internationalization by entering into export market. Encouraging exports is a primary concern of most governments. In the United States, the Department of Commerce has many programs devoted to the development and nurturing of beginning exporters. Substantial resources are devoted to export promotion programs designed to increase the propensity of small companies to export. However, while useful politically throwing monetary resources at a problem can be very wasteful. In the era of government budgetary problems and fiscal frugality, program accountability is part of every politician and administrators agenda. In the export promotions sector, the need to spend money wisely has emerged as a key concern. Government assistance refers to the policies that a government puts forth to help the exporter conduct international business. Studies have shown that governments can either help or hinder the export process. Typically, they help by providing information, sale leads, tax incentives, insurance, and financing programs. Czinkota and Ricks (1981) and Reid (1984) found that government assistance could stimulate export activity by providing relevant information. Governments can also hinder export decisions via their foreign exchange rate policy. Bauerschmidt, Sullivan, and Gillespie (1985) found that a high U.S. dollar relative to foreign currency

Friedrich Nietzsche Essay example -- essays research papers

Friedrich Nietzsche Some call Friedrich Nietzsche the father of the Nazi party. Was Nietzsche's ideas twisted and warped by a needy country? Nietzsche himself despised the middle and lower class people. Was it Nietzsche's Will to Power theory that spawned one of the greatest patriotic movements of the twentieth century? These are some of the questions I had when first researching Friedrich Nietzsche for the following paper. Friedrich Nietzsche, at one time called "the arch enemy of Christianity"(Bentley, p.82), was born into a line of Protestant Clergyman on October 15, 1844. During Nietzsche's early years, he gave no indication that he would not follow in his families' clergy tradition. As a boy, Nietzsche considered himself a devout Lutheran. At age six(two years after his father passed away)Nietzsche, his mother and sister moved to the small town of Naumburg. When Nietzsche was twelve he wrote â€Å"I saw God in all his glory†(Bentley, p.82). Later his description of his own mental state was one of Gottergebenheit; â€Å" surrender to God†(Bentley, p.82). At a very early age Nietzsche had already displayed an aptitude for highly intellectual prowess. At fourteen, Nietzsche left his home of Naumburg and went to an exclusive boarding school at the nearby Schulpforta Academy. The school was famous for its grandeur of alumni that included â€Å"Klopstock and Fichte†(Brett-Evans, p.76). â€Å"It was here that Nietzsche received the thorough education in Greek and Latin that set him upon the road to classical philology.†(Brett-Evans, p. 76) On many occasions Nietzsche's zeal to prove himself at the Pforta school spurned legendary tales. One certain tale is when Nietzsche â€Å"could not bear to hear of the courage of Mucius Scaevol, who did not flinch when his hand was burnt off, without seizing a box of matches and firing them against his own hand.†(Bentley, p.84) At the age of twenty, Nietzsche left to attend Bonn University. By this time Nietzsche had come to think of himself as an â€Å"aristocrat whose great virtues are fearlessness and willingness to assume leadership.†(Bentley, p.85) Ironically, Nietzsche planned to study theology(to please his mother). At this time Nietzsche no longer believed in Christianity, because â€Å"with maturity he lost his heavenly father†(Bentley, p.86). In 1868 Nietzsche was a student in Leipzig. This is when he met Cosima and Richard Wagner. The... ...world domination is only possible by ignoring the greater part of what he wrote.†(Brett-Evans, p.81) Matter-of-factly, Nietzsche sternly despised anti-Semites. At certain times, there was not a harsher critic of racist German nationlism. But some questions arise out of these statements. What of the comments Nietzsche made concerning the â€Å"will-to-power† theory, the constant reference to the â€Å"superman†, and his sometimes vigorous patriotism? One of the most significant contributions Nietzsche made was in the area of psychology not philosophy. One of the â€Å"most significant conclusions he came to in this field was that traditional morality consists of different expressions for the same thing, that "good" actions and "bad" actions can ultimately derive from the same motive.†(Brett-Evans, p.80) In truth I believe that Friedrich Nietzsche was a visionary who was never able to replace his earthly Father or his heavenly Father. This led to his strange emotional relationship with women. His only friends were those women who he had failed relationships with and men who he quarreled with. In the end, Nietzsche died of syphilis that was allegedly contracted while in college.

Titration Journal

E r J. Biochem. 40,177-185 (1973) u. Intracellular Titration of Cyclic AMP Bound to Receptor Proteins and Correlation with Cyclic-AMP Levels in the Surviving Rat Diaphragm Lien DO KHAC,Simone HARBON Hubert J. CLAUSER and lnstitut de Biochimie, Universit6 de Paris-Sud, Orsay (Received April 9/July 17, 1973) Extracts prepared from rat diaphragms incubated with or without theophylline and/or epinephrine have been tested for their total cyclic AMP content and for their ability to bind exogenously added cyclic [â€Å"]AMP.Less cyclic [3H]AMP can be bound inthe extracts after theophylline and/or epinephrine treatment indicating that the rise in cyclic AMP level was accompanied by a n increase in the quantity of cyclic AMP bound intracellularly to the cyclic AMP-dependent protein kinases. Maximum cyclic AMP binding capacities, as measured by total cyclic AMP exchanges, were however identical in all cases. Accurate estimations of intracellular binding of cyclic AMP have been correlated with the level of cyclic AMP in the tissue : the reaction seems to obey simple saturation kinetics, a n apparent intracellular K d for cyclic AMP has been evaluated as 330 nM.The findings are consistent either with a real difference in the intracellular binding constant as compared to that measured in vitro (28 nM) or with the fact that the cyclic nucleotide in the cell may not all be available for the kinase protein receptors. They also suggest that the method described may prove useful for studying any possible intracellular control beyond the step of cyclic AMP synthesis.Regulation of cellular metabolism by adenosine 3†² :5†²-monophosphate (cyclic AMP) [I], its mediation through complex protein kinases [2,3] and the mechanism of the activation of these enzymes [4–61 have been well documented within the past years in the eukaryotic cell. Activation has been demonstrated to occur according to Equation (1) through a n interaction of cyclic AMP with the regulatory subuni t (R) of the enzyme, leading to a dissociation of this subunit from the catalytic subunit (C) which is thus activated. RC cyclic AMP + R cyclic AMP C . (1) + +However completely satisfactory correlations between the levels of intracellular cyclic AMP and its ultimate metabolic effects have been in many cases difficult to obtain. Striking examples for this situation are to be found in the results of Craig et al. [7] in rat diaphragm, of Stull and Mayer [8] in rabbit skeletal muscle concerning the regulation of phosphorylase activation, of Schaeffer et al. [9] and Miller et al. [lo] concerning regulation of glycogen metabolism in adrenalectomized rats, and of Harbon and Clauser [Ill This work is dedicated to Professor E. Lederer for his 65 th anniversary. Abbreviations.Cyclic AMP; adenosine 3†²: 5†²-monophosphate. in the rat uterus stimulated by prostaglandin El or E,. I n all these cases, cyclic AMP levels may be elevated without eliciting the expected metabolic responses. Two hypotheses have been formulated to explain these obvious discrepancies, either a decrease in the activation of the enzymes mediating cyclic AMP action within the cell, or a compartmentalization of the intracellular nucleotide. Hence it seems necessary to measure directly the degree to which the first step of the activation sequence (Equation 1)reflects the apparent intracellular cyclic AMP concentrations.This might be achieved by establishing in intact cells or tissues, correlations between the levels of intracellular cyclic AMP under welldefined physiological conditions, the extent to which it is bound to the specific receptor protein and the extent to which the complex protein kinases are in the active state. Satisfactory correlations between cyclic AMP levels and protein kinase activation have been recently established in various tissues by Corbin et al. [I21 and Soderling et al. [13].The present work was to investigate if correlations could also be obtained between intracell ular cyclic AMP levels and the amounts of intracellular cyclic AMP bound to receptor protein (R cyclic AMP) in the surviving rat diaphragm incubated with or without theophylline and epinephrine. The results reported demonstrate that – E r J. Biochem. 40 (1973) u. 178 Intracellular Titration of Cyclic AMP-Receptor Protein Binding precise titrations of endogenous cyclic AMP bound versus cyclic AMP present in the intact tissue may be obtained.An apparent Kd value for the intracehlar cyclic AMP binding is observed which differs widely from the K d of the same binding established in vitro [14-161. This method may prove to be useful for studying the modification of cyclic AMP binding under conditions where the formation and breakdown of cyclic AMP does not seem to be affected. A preliminary report of these results has been presented [17]. MATERIALS AND METHODS Cylic AMP was obtained from P L Biochemicals Inc. , theophylline and Tris from Merck (Darmstadt), Na,ATP 4 H,O, L-epinephri ne bitartrate from Calbiochem.Cellulose ester membrane filters (HA 0. 45 pm, 24 mm) were purchased from Millipore Corp. All reagents used were products of Prolabo (reagent grade). Cyclic [3H]AMP was a product of New England Nuclear Inc. , specific activity 24 Ci/ mmol. Animals were Wistar rats weighing about 200 to 300 g and fasted 24 h before the experiments. Tissue homogenizations were performed with an Ultra Turrax homogenizer. – The reaction mixture for the binding assay contained in a final volume of 250 p1, 20 mM TrisHC1 buffer pH 7. 5, 10 mM MgCI,, 6. 7 mM theophylline and cyclic [3H]AMP a t various concentrations as indicated.The reaction was initiated by the addition of a n aliquot of diaphragm extracts equivalent to 70- 150 pg protein. Method B. I n this case, cyclic [3H]AMPwas added to the homogenizing medium a t saturating concentrations up to 0. 2 p M a t 0 â€Å"C, centrifugation was carried out immediately and cyclic [3H]AMP bound measured directly on the extr act. Cyclic [3H]AMP bound to the proteins, under either condition, was determined after different incubation times at 0 â€Å"C: the reaction mixtures were then diluted to 3 m l with cold buffer (20mM TrisHC1, 10mM MgCl,, pH 7. 5) and passed through cellulose acetate Millipore filters (0. 45 pm).The filters were washed with 25ml of the same buffer, dried and counted in i 0 ml scintillation fluid, in a Packard Tri-Carb liquid scintillation spectrometer. Results were expressed as pmol cyclic AMP bound/mg protein ; the concentration of endogenous unlabelled cyclic AMP has been always taken into account for the estimation of the specific activity of cyclic [3H]AMP present in the incubation medium. Incubation Procedures The animals were killed by decapitation. The diaphragms were rapidly removed, freed from connective tissue, cut to small pieces, pooled and divided into equal parts. 200-250 mg tissue were preincubated in 2. ml Krebs-Ringer-bicarbonate buffer pH 7. 4, gas phase (95O/, O, , 5O//, CO,) for 30 min a t 37 â€Å"C, in the absence or presence of 10 mM theophylline. Incubations were then performed in the absence or presence of epinephrine (5 pM) for varying periods of time. Extraction of the Tissue Standard Binding Assays for Cyclic A M P Two methods have been deviced to extract the tissue and estimate the binding of exogenous cyclic [3H]AMP to the extracted proteins, both slightly modified from the method defined by Walton and Garren [15]. Method A . The tissue was homogenized a t 0 â€Å"C in 3 ml of one of the following solutions: 20 mM TrisHCl buffer pH 7. or 20 mM sodium acetate pH 7. 5 or 4 mM EDTA pH 6. 0. Theophylline (10 mM) was always present in the various homogenizing media in order to minimize any degradation of cyclio AMP by phosphodiesterase present in diaphragm extracts. A first centrifugation was carried out for 5 min a t 3000 x g , followed by a second one a t 50000 x g for 30min. The supernatants will be referred to as Tris extract, ac etate extract and EDTA extract. Assay for Cyclic-AMP Levels For cyclic AMP assay, the tissue was homogenized in 3 ml cold 7 trichloroacetic acid and centrifuged for 30 min a t 50000 xg.After addition of 0. 1 ml N HC1, the supernatants were extracted 7-8 times with twice their volume of cold ether and evaporated to dryness. Total levels of cyclic AMP in the tissue trichloroacetic acid extract were determined according to Gilman using a protein b a s e and the heatstable inhibitor prepared from rabbit skeletal muscle [161. I n some instances, cyclic AMP content was also evaluated in the Tris and acetate extracts. Proteins were precipitated by trichloroacetic acid and extracts processed as described above. Proteins in the extracts were determined according to Lowry et al. 18] using bovine serum albumin as a standard. RESULTS AXD DISCUSSION Total Cyclic-AMP Levels in Rat Diaphragm. Effects of Epinephrine and Theophylline In order to study the cyclic AMP binding capacity of rat diaphragm proteins and its possible rnodification under the influence of epinephrine, it seemed necessary to test the first effect of the catecholamine, viz. the rise in the tissue cyclic AMP level under our experimental conditions. Em. J. Biochem. 40 (1973) L. Do Khac, S. Harbon, and H. J. Clauser Table 1. Total cyclic A H P levels in trichloroacetic acid extracts of rat diaphragm.Effect of epinephrine and theophylline R a t diaphragms (200-250 mg) were preincubated for 30 rnin a t 37 â€Å"C in 2. 5 ml Krebs-Ringer-bicarbonate buffer (0, 95 °/0-C0, 50/0) in the absence or presence of 10mM theophylline, Incubation was then performed for 5 rnin with or without 5 pM epinephrine. The tissue was then homogenized in 7O/, trichloroacetic acid for cyclic AMP assay as described under Methods. Levels of cyclic AMP were expressed as pmol cyclic AMP/100mg wet tissue and as pmol cyclic AMP/mg soluble protein (as estimated by the Lowry procedure in the Tris extract.Values are means f S. E. M. of 5 di fferent experiments Incubation condit,ions Total cyclic AMP TheoDhvlline EDineDhrine pmo1/100 mg pmol/mg wet tissue soluble protein 41 f 8. 0 20. 5 f 4. 7 104 & 1. 1 52 & 0. 47 93 f 4. 5 46 & 2 350 f 21 170 f 10. 7 179 Table 3. Distribution of cyclic [3H]AMP-bindingfractions i n different hom. ogenutes from rat diaplwagms incubated with or without epinephrine Preincubation and incubation conditions as described in Table 2. Tissues were homogenized in 3 ml 20mM TrisHCI, p H 7. 5, 4 mM EDTA or 20 mM sodium acetate pH 7. and centrifuged for 5 rnin at 3000 x g, the supernatants were centrifuged once more at 500OOxg for 30 min yielding extract 1 and pellet 1. The sediment of the first centrifugation was resuspended in 1. 5 ml of the corresponding buffer and centrifuged at 500OOxg for 30 min giving extract 2 and pellet 2. Binding activity for cyclic rSH]AMP was measured in each fraction as described in the text under method A and was expressed as pmol cyclic AMP bound/l00 mg wet tissue Fr action Cyclic AMP bound in EDTA Acetate Tris extract extract, extract, 5 yM noepinoepino epinephrine nephrine nephrine – + + + + – :Lhrine pmo1/100 mg wet tissue Extract 1 Extract 2 Pellet 1 Pellet 2 15. 70 1. 47 0. 76 1. 49 14. 90 1. 54 0. 83 1. 50 15. 30 1. 35 0. 80 1. 10 9. 40 0. 80 0. 44 0. 39 Table 2. Cyclic A M P levels in different extracts obtained from epinephrine-treated and untreated rat diaphragms Preincubation with 10 mM theophylline and incubation conditions in the absence or presence of 5 pM epinephrine as in Table 1. Diaphragms were homogenized in three different solutions: cold 7O/, trichloroacetic acid, Tris-HC1 pH 7. 5 or acetate p H 7. 5 as described under methods.Centrifugation was carried out for 30 rnin at 50000 x g. Soluble Tris extract, acetate extract and their corresponding sediments were deproteinized by 7 o/o trichloroacetic acid before cyclic AMP assay Incubation with epinephrine None 5wM Total cyclic AMP in Trichloroacetic 20 mM acetate a cid extract pellet 57 280 – 20 mM Tris extract pellet 48 218 9. 5 26 extract pellet 45 242 pmo1/100 mg wet tissue – 8. 5 8. 3 As shown in Table 1, epinephrine (5 pM) in the absence of theophylline increases (by a factor of 2. 5) the total cyclic AMP content of rat diaphragm extracted by trichloroacetic acid.Theophylline alone (10 mM) had a stimulating effect, double; when both compounds were used together, the rise in cyclic AMP levels was 8- t o 9-fold, reaching 350pmol cyclic AMP/100 mg wet tissue. When cyclic AMP was assayed in either acetate or Tris extracts after deproteinization with trichloroacetic acid the values obtained were identical t o those found when the diaphragms were directly extracted with trichloroacetic acid ; hence almost none of the cyclic nucleotide in these extracts was associatcd with membrane-bound fractions (Table 2). Eur. J. Biochem. 0 (1973) Location of Cyclic AMP-Binding Fractions Table 3 shows the distribution of cyclic AMP binding activ ity in various fractions of three rat diaphragm homogenates measured by method A : in all cases more than goo/, of this activity was recovered in the 50000 x g supernatant, almost no cyclic AMP binding occurred in the pellets. Preincubation of the diaphragm with epinephrine did not modify the percentage distribution of the radioactive nucleotide between the supernatants and the pellets, hence subsequent experiments have been performed on the soluble extracts.On the other hand, in the case of epinephrine-treated diaphragms, less exogenous labelled cyclic AMP (about 50-60 °/0) was bound to the various fractions, indicating a decrease in the binding capacity of the extract as compared to the untreated diaphragm. Dilution by endogenous cyclic AMP cannot explain the effect of epinephrine, since allowance was made for this parameter (see Methods) ; the phenomenon was consistently reproducible and will be further substantiated and discussed below.The binding capacities of the various ext racts for cyclic E3H]AMP have also been verified in the absence of any free endogenous cyclic AMP after removal of the latter by filtration through Sephadex G 50 (1x 37 cm) columns, previously equilibrated with 20 mM Tris-HC1 buffer, pH 7. 5 a t 4 â€Å"C. I n these experiments, the detail of which w l not be reported in i l the present manuscript, the effect of epinephrine was still observed, when binding was measured on the main protein peak emerging with the void volume of the columns. When the corrections outlined in the 180 Intracellular Titration of Cyclic AMP-Receptor Protein Binding Z A 0. 51 / 0 20 40 60 Time ( m i n ) l / f r e e cyclic AMP (nM-‘) l / f r e e cyclic A M P (nM-‘) Fig. 1. The time wurse and cyclic-AMP-concentration dependence of cyclic A M P binding in rat-diaphragm extracts (method A ) . (A) Diaphragms were incubated for 30 min in the presence of 10 mM theophylline and extracted with Tris HCI buffer (method A). Cyclic AMP binding was estimated in the presence of various concentrations of cyclic E3H]AMP: 20nM ( 0 – 0 ) ; 60nM ( – ) 0 0 ; SO& (A-A); 100 nM ( –) #-. , a t 0 â€Å"C. The react,ion mixtures contained in a final volume of 2. 5 ml, 20 mM Tris-HC1 buffer, pH 7. , 10 mM MgCI,, 6. 5 mM theophylline. The reaction was initiated by the addition of 930 pg protein. At the indicated times, aliquots were pipetted, immediately diluted with cold 30 mM Tris-HC1buffer pH 7. 5,lO mM MgCl, and passed on the Millipore filters. Filters were washed with the same buffer, dried and counted. Binding activity is expressed as pmol cyclic AMP bound/mg protein. (B) Data obtained from similar experiments where binding for cyclic AMP was performed a t 0 â€Å"C, for 1 h, in the presence of cyclic [aHIAMP ranging from 12 nM to 110 &I. Double-reciprocal plot, according to Klotz [25] Fig. 2.Cyclic-AMP-Concentration dependence of cyclic A M P binding in rat-diaphragm extracts (method B ) . Binding assays were carried out as described under method B. Various concentrations of cyclic [3H]AMP ranging from 12nM to 200 nM were added directly to the homogenizing medium for preparing extracts from epinephrine treated (A-A) and untreated (0-0) rat diaphragms. Aliquot,s of the extracts were filtered through Millipore filters, dried and counted. Double-reciprocal plot, according to Klotz [25] present paper were applied to these figures, the results were essentially identical to those obtained with the unfiltered extracts.Specificity. Kinetics and Concentration Dependence of Exogenous Cyclic-AMP Binding in the Extracts Specificity of cyclic AMP binding has been assessed by dilution experiments of cyclic [3H]AMP (100 nM) with unlabelled nucleotides (adenine, AMP, ATP, cyclic AMP) a t molar concentrations equalling up t o 100 times cyclic [3H]AMP concentrations. I n no case, except with unlabelled cyclic AMP, the amount of radioactive material bound to proteins by either method A or B was significantly reduced (the details of these experiments are not reported).When various concentrations of cyclic [3H]AMP were added to diaphragm extracts (after homogenization and centrifugation) and the binding reaction (method A) carried out for different incubation times at 0 â€Å"C (Fig. I), it appears that saturation was obtained at a concentration of 80 nM for the cyclic nucleotide which essentially coincides with previously published data [14-161 and that binding equilibrium was reached a t p H 7. 5 and 0 â€Å"C after less than 60 min incubation. It has also been verified that with the protein concentration used (70-150 pg in 250 pl) binding of cyclic AMP was directly proportional to the amount of added proteins.From a reciprocal plot of cyclic AMP binding versus cyclic AMP concentration (inset of Fig. I), an apparent Kd of 33 nM can be calculated. When similar experiments were performed by adding various concentrations of cyclic [3H]AMP into the homogenizing medium (method B) and using diaphra gms which have been incubated in the presence and absence of epinephrine, the double-reciprocal plots of Fig. 2 were obtained. The apparent Kd values calculated with this method (45 nM) are in the same range as with method A.I n addition this figure shows that epinephrine treatment of the diaphragms does not modify this Kd but decreases the amount of exogenous cyclic AMP which can be bound to the extract proteins. By comparing exogenous cyclic AMP binding values obtained with methods A and B, it appears (Table 4) that when cyclic [3H]AMPwas added to the Eur. J. Biochem. 40 (1973) L. Do Khac, S. Harbon, and H. J. Clauser Table 4. Comparison of exogenous binding of cyclic [SII]AMP to diaphragm extracts by method A or method B. Rat diaphragms were incubated with theophylline in the absence or presancc of 5 p M epinephrine.Extracts in Tris-HC1 were prepared as described under method A for subsequent binding of cyclic [3H]AMP (100 nM), 1 h, a t 0 â€Å"C. A second series of extracts wer e prepared in the same way but in the prescnce of 100 nM cyclic [3H]ABIP in the homogenizing medium (method R); binding of cyclic [3H]AMP was measured in a n aliquot immediately after centrifugation at 0 â€Å"C (about 1 h after the end of incubation). Values are expressed as pmol bound cyclic AMP/mg protein. Numerals within brackets indicate number of experiments Method Cvclic A P bound with M 5 pM epinephrine no epinephrine pmol/mg protein 4 f 0. 22 (9) 4. 80 5 0. 2 (5) 181 6 t e . ;? 4 Q Q E A B 2 f 0. 13 (9) 3 f 0. 19 (5) 0 I I I 30 60 90 * Time (rnin) homogenization medium (extract B) higher binding values were obtained both with epinephrine-treated and untreated diaphragms, than with method A. This demonstrates that some additional binding of endogenous cyclic AMP occurred during the homogenization and fractionation procedures, which tends to decrease the amount of unoccupied binding sites available for exogenous cyclic [3H]AMP. Hence method B has been currently used to measu re exogenous cyclic AMP binding, since the values obtained with this method seem to reflect intracellular conditions more accurately.Fig. 3. Time course of cyclic [3H]AMP binding in extracts from rat diaphragms incubated in the absence or presence of theophylline orland epinephrine. Half rat diaphragms were preincubated in the absence (m, A ) or in the presence ( 0 , 0 ) of 10 m31 theophylline for 30 min at 37 â€Å"C. Epinephrine (5 pM) was added ( A , 0 )and incubation continued for 5min. Tissue was homogenized in 1. 5 ml Tris-HC1 buffer containing 200 nnf cyclic [3H]AMP and centrifuged at 5000xg for 10 min at 0 â€Å"C.Binding of cyclic [3H]AMP was measured in aliquots of the supernatant at the times indicated, through Millipore filtration, t = 0 corresponds to the onset of the extraction. Results are expressed as pmol cyclic AMP bound/ mg protein (without correction for cyclic AMP exchange) Effect of Theophylline and Epinephrine Treatment on the Binding of Exogenous Cyclic [3H ]AMP by Diaphragm Extracts Fig. 3 shows the results of a typical experiment in which diaphragms have been incubated in the absence or presence of theophylline and epinephrine. Homogenization has been performed according to method B, the centrifugation time of the homogenate kept to a inimum (10 min), and the binding capacity for cyclic [3H]AMP determined a t different times. As may have been expected, this cyclic [3H]AMP binding (which measures the residual binding capacities of the extracts) was, in the course of the whole titration period, inversely related t o the amount of endogenous cyclic AMP present in the relevant extracts (see Table 1). Hence the agents which increase the intracellular cyclic AMP level appear to decrease the amount of binding sites available for exogenous cyclic [3H]AMP, probably through an increase of endogenous cyclic AMP binding to the receptors.I n order to titrate endogenous binding of cyclic AMP accurately, experiments were designed to estiEm. J. Bioc hem. 40 (1973) mate the total binding capacities of the extracts through complete exchange of endogenously bound cyclic AMP with cyclic [3H]AMP, and also to estimate the actual amount of exchange occurring in the extracts between endogenous bound unlabelled cyclic AMP and exogenous cyclic [3H]AMP during the titration period. A precise knowledge of these two parameters is required for the determination of the binding sites occupied by endogenous cyclic AMP at the moment where the tissues are homogenized.Cyclic-AM P Exchange and Determination of Maximal Binding Capacities Total cyclic AMP exchange has been measured under the conditions defined by Wilchek et al. [19] for parotid gland and skeletal muscle : extracts from both treated and untreated diaphragms were f i s t incubated at 0 â€Å"C with cyclic [3H]AMP (100 nM) under binding conditions of method A and then allowed t o exchange with 1 pM unlabelled cyclic AMP at 20 â€Å"C in the presence of 100p. M ATP and 10mM MgCl,. Fig. 4 shows that almost complete exchange of the bound labelled nucleotide occurred within 30 min, 182Intracellular Titration of Cyclic AMP-Receptor Protein Binding 0 10 20 30 40 50 60 Time (min) 70 80 90 Fig. 4. Exchange of bound cyclic [SHIAMP. Extracts were prepared from epinephrine-treated ( + o ) and untreated (0-0) rat diaphragms. Binding of cyclic [3H]AMP was carried out a t 0 â€Å"C in a volume of 2. 5 ml with 500 pg proteins, and 100 nM cyclic r3H]AMP in Tris-HC1 buffer, MgCl, and theophylline a t the concentrations described for the standard binding assay. After 1-h incubation, 1 pM unlabelled cyclic AMP and 100 pM ATP were added and the mixture allowed to stand at 20 °C.At the different times indicated in the figure, aliquots corresponding t o 50 pg protein were pipetted, rapidly diluted with 20 mM Tris-HC1 buffer, 2. 5 mM MgC1, p H 7 5 and filtered through Millipore filters. The filters . were washed with the same buffer, dried and counted. Results are expressed as pmol/ mg protein 0 30 60 90 120 Time (rnin) 180 240 – Total binding capacities of the proteins could thus be measured by incubating the extracts first with 100 nM unlabelled cyclic AMP a t 0 â€Å"C and carrying on the exchange reaction in the presence of 1 pM cyclic I13H]AMP at 20 â€Å"C for 1-2 h ; the values obtained averaged 8. -9. 5 pmol cyclic [3H]AMP/mg soluble protein, both with epinephrine-treated and untreated diaphragms. These results were confirmed by direct assay of bound cyclic AMP: the extracts have been fully saturated with unlabelled 1pM cyclic AMP and filtered as described. After washing the Millipore filters, bound cyclic AMP was extracted by cold 7 O/, trichloroacetic acid and the cyclic nucleotide was directly assayed according to Gilman [16]. The average value was 9. 8 f 0. 4 pmol cyclic AMP bound per mg protein, which is of the same order of magnitude as the amount of bound cyclic [3H]AMP calculated above.Previously published data are in close agreement wi th these values. Walton and GarFen [15] reported maximal binding capacities of 9. 8 pmol/mg protein for adrenal extracts, whereas Gilman [l6] found a total binding of 12pmol/mg protein in muscle extracts. The values for maximal cyclic AMP binding are very low as compared t o the total endogenous cyclic AMP present in the extract (46 pmol/mg protein with the theophylline-treated diaphragm and 170 pmol/mg protein with the epinephrine theophylline-treated diaphragm).It must be added that the binding proteins, saturated with cyclic AMP or not, were almost completely retained on the Millipore filters, and that endogenous cyclic AMP, not Fig. 5. T i m e course of cyclic A M P exchange under binding (0 â€Å"C) and exchange (20 â€Å"C} conditions. Extracts were prepared from epinephrine treated (0,A ) and untreated ( 0 , A) r a t diaphragms. Binding of cyclic AMP was performed as described in Fig. 2 in the presence of 100 nM cyclic AMP for 60 min at 0 â€Å"C. A t the end of the bindin g reaction 1 pM cyclic [3H]AMP was added t. the different extracts, in the absence (A, A ) or presence ( 0 , 0 ) of l00p. M ATP. The reaction mixtures were maintained a t 0 â€Å"C for 2 h and then at 20 â€Å"C (arrow) for 2 more hours. At the different times indicated on the figure, aliquots corresponding t o 70 pg protein were pipetted and treated as in Fig. 4. Results are expressed as cyclic rH]AMP bound in pmol/mg protein. bound to these fractions, was quantitatively recovered in the Millipore filtrates after trichloroacetic acid extraction. The extent t o which this â€Å"free† cyclic AMP may or not be bound to other proteins is presently not known.Cyclic-AMP Exchange under Binding Conditions The extent of cyclic AMP exchange under binding conditions (0 â€Å"C, 1 h, 100 nM cyclic AMP) must be controlled if corrections for simultaneous exchange have to be applied t o binding data: extracts of rat diaphragms treated with theophylline and theophylline epinephrine were first saturated with 1 O O n M unlabelled cyclic AMP (binding conditions) and then exchanged with 1 pM cyclic [3H]AMP but a t 0 â€Å"C. After 2 h, the temperature was raised to 20 â€Å"C and completion ofthe exchange measured after 1-2 h further incubation.Fig. 5 shows that a t 0 â€Å"C, within 1h incubation time, which are the conditions described above for the binding assay, about 200/, of total sites were exchangeable. Under these conditions, ATP and Mg ions slightly increase the exchange velocity. I n addition, this figure confirms that a t 20 â€Å"C total exchange capacities were identical for epinephrine-treated and untreated diaphragms ; hence initial + + Em. J. Biochem. 40 (1973) L. Do Khac, S. Harbon, and H. J. Clauser 183 Table 5. Relationship between intracellular cyclic A M P levels and cyclic AM P binding in extracts from diaphragm incubated under various conditions Diaphragms were incubated with or without 10 mM theophylline for 30 min at 37 â€Å"C, 5 pM epin ephrine was added where indicated and incubation continued for varying times. From each incubation, half a diaphragm was extracted by trichloroacetic acid for cyclic AMP estimation. The other half was homogenized with Tris-HC1buffer lOOnM cyclic [3H]AMP(method B) for exogenous cyclic AMP binding after 1 h a t 0 â€Å"C; maximal binding capacities were determined in the same extracts a t 20 â€Å"C in the presence of 1 pM cyclic [3H]AMP under conditions described for cyclic A P exchange.R. esults are expressed as pmol cyclic AMP/mg M protein. Endogenous binding values were calculated as the difference between maximal binding capacities ( A )and exogenousbinding ( B ) and corrected for the 200/, exchange + Incubation conditions Theophylline 10 mM Epinephrine 5t*M Time Cyclic AMP Total level Maximal binding Exogenous capacity binding (a) (b) Endogenous binding (a-b) corrected min pmol/mg protein – – – + + + + + + 0 2 10 30 5 5 20. 5 52 43 38 46 170 f 4. 7 & 0. 47 f2 f 10. 7 9. 6 f 0. 9 9. 4 f 0. 1 9. 20 9. 40 8. 9 5 0. 73 8. 9 & 0. 85 5. 35 f0. 40 4. 50 f 0. 133 4. 40 4. 70 4. 46 f 0. 20 2. 7 f0. 224 5. 31 6. 13 6 5. 5 5. 53 7. 77 differences in residual binding capacities reflect variations in the degree of saturation of the receptor proteins by endogenous cyclic AMP, rather than modifications of their maximal binding capacity. 1 Titration o Endogenous Cyclic-AMP Binding in Rat f Diaphragm. Effects of Theophylline and Epinephrine Since total binding capacities of the receptor proteins in the extracts and the amount of exogenous cyclic [3H]AMP bound by these extracts after homogenization may be estimated, it appears possible to calculate endogenous cyclic AMP bound in the intact organs, correcting for a 2001, exchange during the titration period.Table 5 summarizes the results of a series of experiments where diaphragms have been incubated under conditions which modify endogenous levels of cyclic AMP :in every case, half of the diaphragm was extracted with cold trichloroacetic acid (see Methods) for the assay of intracellular cyclic AMP levels: the second half was extracted according to method B for the estimation of exogenous cyclic [3H]AMP binding and of total cyclic AMP binding capacities. The endogenous cyclic AMP bound was calculated from the latter experimental data.This table definitely establishes that the average values obtained for the intracellular binding of endogenous cyclic AMP in the intact organ seem to correlate with its cyclic AMP levels. A reciprocal plot of intracellular binding versus intracellular cyclic AMP concentrations (Fig. 6) shows that this correlation fits simple saturation kinetics very accurately. I n the unstimulated diaphragm (no theophylline nor epinephrine added to the incubation medium) about 50 °/, of the available binding sites are occupied by endogenous cyclic AMP; this Eur. J. Biochem. 40 (1973) -0. 002 I 0. 002 l/Free cyclic AMP (nM-‘) 0 0. 004 . Fig. 6. Reciprocal plot of intracellular cyclic A M P levels and cyclic A M P binding in rat-diaphragm extracts. Data arc obtained from experiments performed as described in Table 5 and replotted according t o the Klotz equation. The intercept on the y axis yields a n estimate of the number of binding sites and the x intercept provides a n estimation of the intracellular apparent dissociation constant. Statistical analysis of the data were performed according to Cleland [26] using a Wang electronic calculator alue increases to almost goo/,, when the diaphragms have been fully stimulated with both theophylline and epinephrine. Various treatments with one of the agonists alone cause endogenous bindings ranging between these two extreme values. The apparent Kd value for intracellular binding according to this plot was estimated to 330 nM f 50, as compared to the apparent Kd (33-45 nM) when binding was assayed in the extracts (Fig. l and 2). Hence a difference of about one order of magnitude appears to obtain between the Kd values calculated within the cell and the 84 Intracellular Titration of Cyclic AMP-Receptor Protein Binding same constant measured with diaphragm homogenates. The double-reciprocal plot may also be used to calculate the intracellular maximal binding capacities, from its intercept with the ordinate axis. A value of 8. 9 pmol/mg protein was found which coincides with the values measured in the extracts by total cyclic [3H]AMP exchange. This discrepancy between the intracellular Kd and the Kd measured in vitro in a variety of tissue extracts including diaphragm may a t first sight seem surprising.It has however repeatedly been pointed out that cyclic AMP concentration even in the unstimulated cell was far in excess of the concentration which should result in almost maximal stimulation of protein kinases and compartmentalization of the nucleotide within the cell has usually been postulated to explain this contradiction [8,9,20]. The present work shows that despite these high intracellular concentrations of cyclic AMP, protein kinases could indeed not be fully activated, since under the same conditions, the receptor proteins appear not to be fully saturated with cyclic AMP. Concluding RemarksAs might have been expected from Equation (1) (if this reaction truly reflects intracellular conditions) a rise in cyclic AMP should be paralleled by an increase in the amount of cyclic AMP bound to receptor protein in the cell. The results reported show this indeed to be the case in the isolated rat diaphragm: when this tissue is stimulated by various agents which increase the level of cyclic AMP the amount of protein receptors endogenously saturated by cyclic AMP (R cyclic AMP) rises, as indicated in our experiments by a decrease in their ability to bind exogenously added cyclic [3H]AMP after tissue extraction.Maximal binding capacities for cyclic AMP do not seem to be affected under any circumstance. A parallel approach t o the study of this problem has been undertaken by Corbin et al. [12] and Soderling et al. [13] who investigated in adipose tissue under various stimulatory conditions, the state of activation of the catalytic subunit (C) by assaying the cyclic AMP dependence of the protein kinase in tissues extracts. These authors demonstrated that under well-defined xperimental conditions, there was a quantitative relationship between the intracellular level of cyclic AMP and the amount of the active C unit which could be separated from the complex protein kinase RC. However in their experiments high concentrations of NaCl had to be added to the extracts, since in its absence R and C tended to reassociate almost immediately, indicating that cyclic AMP is no longer bound to its receptor protein (R). The situation in various other tissue xtracts has been found to be analogous, except with skeletal muscle, where preliminary results obtained by the authors led them to suggest that the protein kinase subunits do not readily reassociate. T his seems also to be the case for the diaphragm, since under the conditions of the present work, it has been possible to titrate for R * cyclic AMP in the crude extracts even in the absence of high salt concentrations : acccurate estimations of intracelM a r binding of cyclic AMP have been obtained and correlated with the absolute amounts of the nucleotide present in the stimulated and unstimulated cell.The binding seems t o obey simple saturation kinetics but the apparent Kd of this binding is about10 times higher as compared with the crude extracts. These results may be explained by cyclic AMP compartmentalization within the cell ; in this case, however, the simple saturation kinetics would indicate that the various pools of the cyclic nucleotide attain equilibrium very rapidly.Or else, if cyclic AMP within the cell is not compartmentalized, and if the reaction described by Equation (1) may be applied, without any modification, to intracellular equilibria, a decrease in the appare nt Kd could be merely a consequence of the dilution (about 10-fold) of the protein components during extraction of the tissue, while cyclic AMP concentrations are maintained by the addition of exogenous cyclic [3H]AMP.However these two hypotheses are certainly oversimplified, since they do not take into account factors like the intracellular concentration of the heat-stable kinase inhibitor [21,22], ATP or Mg2+ [19,23], which are known to affect cyclic AMP binding either in crude extracts or with purified protein kinase preparations. It seems impossible to decide at present which of these interpretations is most likely to reflect true intracellular conditions. It is noteworthy that the apparent Kd estimated is close to the intracehlar cyclic AMP concentration of the nstimulated tissue, a fact which should account for maximal sensitivity of the regulatory mechanisms under physiological conditions. Hormonal controls at the level of cyclic AMP-receptor protein interaction have hitherto never been described; the data reported above provide a suitable means for investigating such problems. The authors are very much indebted to Mrs Ginette Delarbre for her excellent technical assistance and to Mrs Marie-ThBrBse Crosnier for preparing the manuscript. The present work has been performed thanks to two official grants of the C. N. R. S. Paris, France: ERA No 33 and ATP No 429. 914), to a grant obtained from the D. G. R. S. T. 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